U.S. patent application number 10/034424 was filed with the patent office on 2002-10-17 for immunoassays in capillary tubes.
This patent application is currently assigned to IDEXX Laboratories, Inc.. Invention is credited to Jang, Larry Sheldon, Kumar, Amit, Leung, Danton Kai-Yu, Platshon, Mark Charles, Rocco, Richard Michele.
Application Number | 20020148728 10/034424 |
Document ID | / |
Family ID | 24762887 |
Filed Date | 2002-10-17 |
United States Patent
Application |
20020148728 |
Kind Code |
A1 |
Kumar, Amit ; et
al. |
October 17, 2002 |
Immunoassays in capillary tubes
Abstract
A fluorescent immunoassay employing the interior surface of a
capillary tube is provided. Devices to permit immunoassays using
one or more capillary tubes, an apparatus for use with the devices,
and a process for screening for analyte in a sample using the
devices and apparatus are also provided. Samples suspected of
containing analyte are added to a disposable self-contained sample
tray containing one or more sample wells, mixed with a reagent,
drawn into one or more spaced-apart capillary tubes held within a
disposable cartridge connected to an analytical apparatus, reacted
with a binding member on the surface of the capillary tube, washed
to stop the reaction, and dried by the apparatus. The capillary
tube is then exposed to a signal generation device to create a
fluorescence signal that is detected using a signal detector. The
apparatus determines the presence of the analyte and optionally
determines the amount of analyte present in the sample, and
presents the results to the operator.
Inventors: |
Kumar, Amit; (Milpitas,
CA) ; Jang, Larry Sheldon; (San Jose, CA) ;
Leung, Danton Kai-Yu; (Los Altos, CA) ; Rocco,
Richard Michele; (Sunnyvale, CA) ; Platshon, Mark
Charles; (Menlo Park, CA) |
Correspondence
Address: |
MCDONNELL BOEHNEN HULBERT & BERGHOFF
300 SOUTH WACKER DRIVE
SUITE 3200
CHICAGO
IL
60606
US
|
Assignee: |
IDEXX Laboratories, Inc.
|
Family ID: |
24762887 |
Appl. No.: |
10/034424 |
Filed: |
December 28, 2001 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10034424 |
Dec 28, 2001 |
|
|
|
08688043 |
Jul 29, 1996 |
|
|
|
5976896 |
|
|
|
|
08688043 |
Jul 29, 1996 |
|
|
|
09361391 |
Jul 26, 1999 |
|
|
|
09361391 |
Jul 26, 1999 |
|
|
|
08254302 |
Jun 6, 1994 |
|
|
|
5624850 |
|
|
|
|
Current U.S.
Class: |
204/451 ;
435/287.2 |
Current CPC
Class: |
G01N 33/94 20130101;
G01N 35/00 20130101; G01N 21/6428 20130101; G01N 2021/0346
20130101; G01N 2021/6482 20130101; G01N 33/54366 20130101; G01N
21/03 20130101; G01N 33/552 20130101 |
Class at
Publication: |
204/451 ;
435/287.2 |
International
Class: |
G01F 001/64; C12M
001/34 |
Claims
What is claimed is:
1. A cartridge for securely holding a plurality of spaced-apart
capillary tubes, which cartridge comprises a frame for holding the
tubes in a spaced-apart manner, wherein the frame has a pathway in
which each capillary tube can be aligned; and at least one region
in the frame to expose at least a portion of each capillary tube so
that an electromagnetic signal can contact a portion of each
tube.
2. The cartridge of claim 1, which further comprise a holder
positioned with the frame for sealingly holding one end of each of
the capillary tubes, the holder having a passageway therethrough
for each capillary tube and a port for each passageway for suitably
pumping fluid through each capillary tube.
3. The cartridge of claim 1, wherein the port is suitable for
attaching to a fluid source for pumping a fluid through the
port.
4. The cartridge of claim 1, wherein the holder also contains a
chamber in fluid communication with each passageway of each
capillary tube and connected to a single port.
5. The cartridge of claim 4, wherein the holder is defined by a cap
and a receptacle for sealingly holding one end of each of the
capillary tubes such that the cap in conjunction with the
receptacle defines the chamber that (a) connects each of the
capillary tube passageways in the receptacle to each other in fluid
communication and (b) leads to the single port for attachment to a
fluid source for pumping fluid through each capillary tube.
6. The cartridge of claim 5, wherein the frame is designed to hold
the capillary tubes in a radially spaced-apart manner so that the
proximal ends of the capillary tubes held by the holder converge
toward each other while the distal ends of the capillary tubes
diverge from each other.
7. The cartridge of claim 5, wherein the receptacle is
flexible.
8. The cartridge of claim 1, wherein the frame is substantially
flat and the region for exposing at least a portion of each
capillary tube comprises an opening that is accessible from both
the front and back sides of the frame.
9. The cartridge of claim 1, in combination with capillary tubes,
wherein a sample can be introduced into at least one capillary
tube, which sample can be subjected to a treatment selected from
the group consisting of reacting with a reaction component,
incubating for a period of time, washing with a fluid, optionally
repeating any one of reacting, incubating, and washing; and any
combination thereof.
10. The cartridge of claim 1, wherein the frame has protective tabs
extending beyond the frame in the capillary tube pathway distal
from the holder to protect the distal ends of the capillary tubes
from breakage.
11. A tray for holding multiple portions of a sample, which tray
comprises a reservoir sufficient to hold a quantity of fluid and a
shelf extending substantially perpendicularly outward from a
sidewall of the reservoir, the shelf having a plurality of
spaced-apart wells therein.
12. The tray of claim 11, wherein the sample is selected from the
group consisting of a fluid and a dry solid state material.
13. The tray of claim 11, wherein at least one of the wells has a
reagent therein so that a portion of a sample to be tested for an
analyte can be placed in each well.
14. The tray of claim 13, wherein the reagent is in a solid dry
state and adheres to the well.
15. The tray of claim 11, wherein at least one well has a retaining
ridge at the well opening having a diameter smaller than that of
the rest of well.
16. The tray of claim 11, wherein at least one well has a metallic
object held therein capable of being moved by a magnetic field.
17. The tray of claim 11, wherein each metallic object is loosely
held within each respective well such that it will not fall out
when the receptacle is tipped upside down.
18. The tray of claim 17, wherein the metallic object is held in
place by a retaining ridge at the well opening having a diameter
smaller than that of the rest of the well.
19. The tray of claim 18, wherein the metallic object is a washer
that is retained by the retaining ridge.
20. A process for screening for an analyte in a sample, which
process comprises importing a fluid mixture into a capillary tube
coated on at least a portion of its interior surface with a
substrate, wherein the fluid mixture comprises a sample suspected
of containing the analyte and a reagent comprising a
fluorescently-labeled conjugate that is (a) capable of binding to
the analyte or to the analyte and the substrate and (b) capable of
fluorescing when irradiated with an appropriate electromagnetic
signal; maintaining the fluid mixture in the capillary tube for a
time sufficient for binding to take place between the substrate and
the fluorescently-labeled conjugate; removing excess fluid mixture
from the capillary tube; externally irradiating the coated portion
of the capillary tube with an electromagnetic signal sufficient to
cause fluorescence of bound fluorescently labeled conjugate; and
detecting the resulting fluorescence to screen for the analyte.
21. The process of claim 20, wherein the capillary tube is dried
prior to detecting the resulting fluorescence.
22. The process of claim 21, wherein the capillary tube is dried by
spinning the capillary tube on a centrifuge, which is designed to
securely hold the capillary tube and spinning it for a sufficient
time and at a sufficient speed to dry the capillary tube.
23. The process of claim 22, wherein the capillary tube is held by
a cartridge designed to fit and be securely held on said
centrifuge.
24. The process of claim 21, wherein the means to dry the capillary
tube comprises aspirating the capillary tube with a stream of
gas.
25. The process of claim 20, wherein the binding in the capillary
tube involves binding a fluorescently-labeled conjugate to the
substrate of the interior surface of the capillary tube.
26. The process of claim 25, wherein the electromagnetic signal is
generated by a laser or a tungsten lamp.
27. The process of claim 20, wherein the detected fluorescence is
used to determine the amount of analyte present.
28. The process of claim 20, wherein data reduction and data
analysis of detected fluorescence is used to give a result.
29. The process of claim 28, wherein the result of the process is
presented as a digital display, a printout, a computer storable
file, an output to an external device, or any combination of the
foregoing.
30. The process of claim 27, wherein data reduction and data
analysis of detected fluorescence is used to give a result.
31. An apparatus for screening for at least one analyte in a
sample, which apparatus comprises a reservoir for a fluid; a
conduit to transport the fluid to a port; the port being positioned
to draw the sample thereto and to pump fluid therethrough; a means
to draw at least a portion of the sample to the port; a means to
pump the fluid through the port; a first section having connecting
means for a cartridge holding at least one capillary tube so that
one end of the capillary tube is in fluid communication with the
port; a second section having means to hold a tray having at least
one well to communicate with the other end of the capillary tube,
the second section also having a means to create a changing
magnetic field so that a magnetizable metallic object held within
the well of the tray is moved sufficiently to agitate a sample when
placed in the well; a means to hold the cartridge and capillary
tube to permit the capillary tube to be exposed to a signal
generation means; the signal generation means; and a signal
detection means positioned to detect a signal emitted from the
capillary tube as a result of exposure to the signal from the
signal generation means.
32. The apparatus of claim 31, wherein the capillary tube is dried
while in the cartridge, which drying occurs prior to detection of
the emitted signal.
33. The apparatus of claim 32, wherein the means to dry the
capillary tube comprises a centrifuge with a means to secure and
spin the cartridge for a sufficient time and at a sufficient speed
to dry the capillary tube, wherein the means to secure and spin the
cartridge is arranged such that each capillary tube can be
positioned in the path of the signal generation means so that the
signal detection means can collect the emitted signal.
34. The apparatus of claim 32, wherein the means to dry the
capillary tube comprises aspirating the capillary tube with a
stream of gas.
35. The apparatus of claim 31, where in the cartridge comprises a
frame for holding a plurality of capillary tubes in a spaced-apart
manner; at least one region in said frame to expose at least a
portion of each capillary tube to enable a signal from the signal
generating means to contact a portion of each tube; and a holder
located in the frame for sealingly holding one end of each of the
capillary tubes and designed so that fluid can pass
therethrough.
36. The apparatus of claim 35, wherein the holder is defined by a
cap and a receptacle such that the cap in conjunction with the
receptacle defines a chamber that connects each of the capillary
tubes to each other in fluid communication and leads to a chamber
port that can be associated with the first port.
37. The apparatus of claim 31, wherein the tray comprises, a
reservoir sufficient to hold a quantity of fluid, a shelf for
holding multiple portions of a sample, the shelf having a plurality
of spaced-apart wells therein, at least one of the wells having a
reagent therein so that a portion of a sample to be tested for an
analyte can be placed in each well.
38. The apparatus of claim 37, wherein at least one well in the
tray has a metallic object held therein for mixing the contents of
the well by creating an oscillating magnetic field under the
well.
39. The apparatus of claim 31, wherein the signal generation means
is selected from the group of a laser and a tungsten lamp.
40. The apparatus of claim 39, wherein the signal detection means
comprises a fluorescence detector.
41. The apparatus of claim 31, wherein the amount of the analyte is
determined from the signal emitted from the capillary tube.
42. The apparatus of claim 31, wherein data analysis of the signal
emitted from the capillary tube is used to give a result.
43. The apparatus of claim 42, wherein the apparatus also includes
a data presentation means that comprises a digital display, a
printout, a computer storable file, an output to an external
device, or any combination of the foregoing.
44. The apparatus of claim 41, wherein data analysis of the signal
emitted from the capillary tube is used to give a result.
45. A combination of a cartridge holding at least one capillary
tube, which combination comprises a capillary tube coated on at
least a portion of its interior surface with a substrate that is
capable of binding to a fluorescently-labeled conjugate and a frame
comprising a means for positioning the capillary tube in an
exposure region of the frame, wherein the exposure region permits
exposure of at least a portion of the coated capillary tube to an
external electromagnetic signal that is capable of causing bound
fluorescently-labeled conjugate to fluoresce.
46. A capillary tube comprising a substrate on at least a portion
of its interior surface, which substrate is capable of being bound
to a fluorescently-labeled conjugate.
47. The capillary tube of claim 46, wherein the tube is glass and
the substrate comprises a silane-based material bound to said
portion of the interior surface of the capillary tube.
48. The capillary tube of claim 47, wherein a protein conjugate is
bound to the silane-based material.
49. The capillary tube of claim 48, wherein the protein conjugate
comprises bovine serum albumin or human serum albumin.
50. The capillary tube of claim 49, wherein the protein conjugate
comprises bovine serum albumin conjugated to biotin.
51. The capillary tube of claim 50, wherein the protein conjugate
comprises neutravidin-4-amino-penicillinic acid bound to the bovine
serum albumin-biotin conjugate.
52. The capillary tube of claim 51, wherein the protein conjugate
comprises neutravidin-ceftiofur bound to the bovine serum
albumin-biotin conjugate.
53. The capillary tube of claim, 49, wherein the protein conjugate
comprises bovine serum albumin conjugated to cephapirin.
54. The capillary tube of claim 47, wherein the silane-based
material is represented by the formula R--Si(OR.sub.1).sub.3
wherein R is an alkyl or alkenyl of about 12 to about 20 carbon
atoms and R.sub.1 is an alkyl of one to four carbon atoms.
55. The capillary tube of claim 54, wherein R is a straight chain
alkyl of 18 carbon atoms and R.sub.1 is ethyl, namely
octadecyltriethoxy silane.
56. A process for preparing a glass capillary tube for use in a
fluorescent immunoassay, which process comprises coating at least a
portion of the internal surface of the capillary tube with a
substrate that is capable of binding to a fluorescently-labeled
conjugate.
57. The process of claim 56, wherein the coating step further
comprises coating at least a portion of the interior surface of the
capillary tube with a silane-based material and further comprises
binding a protein conjugate to the silane-based material, which
conjugate is capable of binding to a fluorescently-labeled
conjugate.
58. The process of claim 57, wherein the protein conjugate
comprises bovine serum albumin or human serum albumin.
59. The process of claim 58, wherein the protein conjugate
comprises bovine serum albumin conjugated to biotin.
60. The process of claim 59, wherein the protein conjugate
comprises neutravidin-4-amino-penicillinic acid bound to the bovine
serum albumin-biotin conjugate.
61. The process of claim 60, wherein the protein conjugate
comprises neutravidin-ceftiofur bound to bovine serum
albumin-biotin conjugate.
62. The process of claim 58, wherein the protein conjugate
comprises bovine serum albumin conjugated to cephapirin.
63. The process of claim 57, wherein the silane-based material is
represented by the formula R--Si(OR.sub.1).sub.3 wherein R is an
alkyl or alkenyl of about 12 to about 20 carbon atoms and R.sub.1
is an alkyl of one to four carbon atoms.
64. The process of claim 63, wherein R is a straight chain alkyl of
18 carbon atoms and R.sub.1 is ethyl, namely octadecyltriethoxy
silane.
Description
CROSS-REFERENCE
[0001] This is a continuation-in-part of co-pending U.S. patent
application Ser. No. 08/254,302 filed Jun. 6, 1994, which
application is incorporated herein by reference in its
entirety.
BACKGROUND
[0002] 1. Field of the Invention
[0003] This invention relates generally to immunoassays
(particularly solid-phase fluorescent immunoassays--SPFIAs), to
devices for detecting analytes by immunoassay using capillary
tubes, to an apparatus for use with such devices, and to manual,
semi-automated, and automated, methods for such testing.
[0004] 2. Background of the Invention
[0005] Many situations exist where qualitative, semi-quantitative,
or quantitative detection of the presence of an analyte in a sample
is desired. Situations where analyte detection is desirable arise
in diverse industries, including: 1) the health care industry,
e.g., in clinical and diagnostic medicine (e.g., in vitro
analysis); 2) the food processing and chemical industries, e.g. in
quality control for food production; and 3) the environmental
control industry, e.g. monitoring for the presence of various
pollutants in air, ground water, or soil.
[0006] Many assays using unique devices and protocols to detect the
presence of analytes through chemical and physical means have been
developed. Immunoassays make up one broad field of assays which
find use in the detection of analytes. In immunoassays, the
occurrence of binding events between specific binding pair members
is used as an indication of the presence of analyte in the sample.
Benefits of using immunoassays, as compared to non-immunoassays, in
analyte detection include high sensitivity, high specificity,
reliability, and relatively short assay times.
[0007] The binding events that are utilized in immunoassays often
occur at the surface of a solid support with one binding member
held at the surface of the solid support and the other binding
member in the sample. The time required for a particular
immunoassay to be completed will depend on the ability of the
binding member in the sample to reach and bind to the member on the
support surface. The ability of the binding member in the sample to
bind with its pair on the support surface is dependent on many
factors; such factors include the concentration of the binding
member on the support surface, and the surface to volume ratio of
the sample/support combination. One method to decrease the time
required for an immunoassay is to increase the concentration of a
binding member on a support surface. Another approach is to
increase the ratio of the surface area of the support relative to
the volume of the sample to be assayed.
[0008] Common immunoassays include radio immunoassays (RIA),
enzyme-linked immunosorbent assays (ELISA), and membrane based
assays, such as common home pregnancy tests. These immunoassays
have several disadvantages. A significant disadvantage of RIA is
the requirement for the use of hazardous radioactive isotopes. A
disadvantage of ELISA is the numerous steps of sample addition,
incubation, washing, addition of color reagent, addition of stop
reagent, and reading required to perform the assay; such
manipulations can be especially troublesome and a source of
significant error in the field. Also, enzyme reactions tend to be
temperature sensitive, which require temperature control. Unlike
RIA, in an which the label is directly detected, in an ELISA the
enzyme label is not directly detected. Instead, one must allow for
a detectable product to be produced. Further, ELISA protocols may
not be suited for all assays on all types of liquids, such as where
the liquid comprising the analyte of interest contains contaminants
which interfere with one or more individual steps in the assay,
e.g. enzyme activity, detectability of enzyme product, and the
like. Additionally, the safety concerns with RIA and the complexity
of ELISA typically require that they be performed by relatively
highly trained personnel and further require constant monitoring by
or interaction with the trained operator. A disadvantage with
membrane based assays is that they often provide poor quantitation
and sensitivity. Many of these assays also require long incubation
times, typically in the tens of minutes to hours, making analysis
of multiple samples time consuming and expensive.
[0009] Nevertheless, ELISA is a commonly used format. In ELISA,
binding events of interest are detected through the appearance of
detectable product produced by an enzyme acting on a substrate. The
formation of the detectable product can be amplified to the extent
required by increasing the concentration of the substrate and/or
increasing the reaction time. On this basis, there is an
opportunity to greatly increase the signal when only a few enzymes
becoming bound.
[0010] Conventionally, ELISA has been conducted in microtiter
plates consisting of wells. In an effort to improve performance,
ELISA has been demonstrated in capillary tubes. With ELISA
immunoassays conducted in capillary tubes, rapid quantitative
results are reported. At least one reported ELISA is described as
sensitive and able to detect small amounts of analyte. See e.g.
Chandler et. al., "A new enzyme immunoassay system suitable for
field use and its application in a snake venom detection kit"
Clinica Chimica Acta, 121:225-230 (1982). However, the
aforementioned disadvantages inherent in ELISA still exist.
[0011] Additionally, while ELISA assays have been performed in
capillary tubes in the laboratory, no successful products have been
developed. A primary reason for this is the difficulty of bringing
several solutions into and out of the capillary tubes and the
ability to effectively read the result.
[0012] Therefore, there is a significant need for a fast, reliable,
accurate immunoassay that requires minimal interaction with the
operator. There is also a need for an immunoassay that can screen
several similar or different samples sequentially or simultaneously
for the same analyte or which can screen for different analytes in
the same sample or in a plurality of aliquots of the same
sample.
OBJECTS OF THE INVENTION
[0013] An object of this invention is to provide a simple,
semi-automated method for detecting and quantifying an analyte in a
sample.
[0014] It is a further object of this invention to provide a
solid-phase fluorescence immunoassay (SPFIA) that has fewer
manipulations than a comparable enzyme-linked immunosorbent assay
(ELISA) for detecting an analyte in a sample.
[0015] A further object of this invention is to provide a rapid
SPFIA that independently and semi-quantitatively or quantitatively
assays for a plurality of analytes from a plurality of samples or
aliquots from the same sample.
[0016] A further object of this invention is to provide a reliable
SPFIA that requires minimal manipulation of equipment by an
operator performing the immunoassay.
[0017] A still further object of this invention is to provide a
SPFIA that independently and semi-quantitatively or quantitatively
assays for a plurality of analytes from a plurality of samples in
less than about 5 minutes.
[0018] A still further object of this invention is to provide a
unique capillary tube suitable to be used to achieve the
aforementioned objects of this invention.
[0019] A still further object of this invention is to provide a
method for preparing a capillary tube to be used to achieve the
aforementioned objects of this invention.
[0020] A still further object of this invention is to provide a
uniquely-designed cartridge for carrying at least one capillary
tube (and preferably more than one) that can be used to achieve the
aforementioned objects of this invention.
[0021] A still further object of this invention is to provide a
tray having a reservoir and a plurality of wells for holding a
plurality of aliquots of a sample, which tray can be used in
conjunction with the cartridge-held capillary tubes to assist in
achieving the aforementioned objects.
[0022] A still further object of this invention is to provide an
apparatus to be used in conjunction with the cartridge-held
capillary tubes and sample tray to perform the SPFIA of this
invention and to further assist in achieving the aforementioned
objects of this invention.
[0023] Other objects will be apparent to one of ordinary skill in
the art upon reading the follow specification and claims.
SUMMARY
[0024] The present invention provides devices for screening for one
or more analytes in a sample comprising capillary tubes, a
cartridge, and a sample tray which can combine to form a portable
and disposable testing kit, and an apparatus and process for
screening for one or more analytes, and a method for preparing
capillary tubes for use with the method for screening for one or
more analytes, that are directed to the disadvantages of the prior
art and address heretofore unmet needs previously discussed.
[0025] One aspect of the present invention is a cartridge for
securely holding a plurality of spaced-apart capillary tubes. The
cartridge comprises a frame for holding the tubes in a spaced-apart
manner, wherein the frame has a pathway in which each capillary
tube can be aligned; and at least one region in the frame to expose
at least a portion of each capillary tube so that an
electromagnetic signal can contact a portion of each tube.
[0026] Another aspect of the present invention is a tray for
holding multiple portions of a sample. The tray comprises a
reservoir sufficient to hold a quantity of fluid and a shelf
extending substantially perpendicularly outward from a sidewall of
the reservoir, the shelf having a plurality of spaced-apart wells
therein.
[0027] Another aspect of the present invention is a process for
screening for an analyte in a sample. The process comprises
importing a fluid mixture into a capillary tube coated on at least
a portion of its interior surface with a substrate, wherein the
fluid mixture comprises a sample suspected of containing the
analyte and a reagent comprising a fluorescently-labeled conjugate
that is (a) capable of binding to the analyte or to the analyte and
the substrate and (b) capable of fluorescing when irradiated with
an appropriate electromagnetic signal; maintaining the fluid
mixture in the capillary tube for a time sufficient for binding to
take place between the substrate and the fluorescently-labeled
conjugate; removing excess fluid mixture from the capillary tube;
externally irradiating the coated portion of the capillary tube
with an electromagnetic signal sufficient to cause fluorescence of
bound fluorescently labeled conjugate; and detecting the resulting
fluorescence to screen for the analyte.
[0028] Still another aspect of the present invention is an
apparatus for screening for at least one analyte in a sample. The
apparatus comprises a reservoir for a fluid; a conduit to transport
the fluid to a port; the port being positioned to draw the sample
thereto and to pump fluid therethrough; a means to draw at least a
portion of the sample to the port; a means to pump the fluid
through the port; a first section having connecting means for a
cartridge holding at least one capillary tube so that one end of
the capillary tube is in fluid communication with the port; a
second section having means to hold a tray having at least one well
to communicate with the other end of the capillary tube, the second
section also having a means to create a changing magnetic field so
that a magnetizable metallic object held within the well of the
tray is moved sufficiently to agitate a sample when placed in the
well; a means to hold the cartridge and capillary tube to permit
the capillary tube to be exposed to a signal generation means; the
signal generation means; and a signal detection means positioned to
detect a signal emitted from the capillary tube as a result of
exposure to the signal from the signal generation means.
[0029] Another aspect of the present invention is a combination of
a cartridge holding at least one capillary tube. The combination
comprises a capillary tube coated on at least a portion of its
interior surface with a substrate that is capable of binding to a
fluorescently-labeled conjugate and a frame comprising a means for
positioning the capillary tube in an exposure region of the frame,
wherein the exposure region permits exposure of at least a portion
of the coated capillary tube to an external electromagnetic signal
that is capable of causing bound fluorescently-labeled conjugate to
fluoresce.
[0030] Another aspect of the present invention is a capillary tube
comprising a substrate on at least a portion of its interior
surface, which substrate is capable of being bound to a
fluorescently-labeled conjugate.
[0031] Still another aspect of the present invention is a process
for preparing a glass capillary tube for use in a fluorescent
immunoassay. The process comprises coating at least a portion of
the internal surface of the capillary tube with a substrate that is
capable of binding to a fluorescently-labeled conjugate.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] FIG. 1 is a perspective view of a preferred embodiment of
the invention showing cooperation between a cartridge containing
capillary tubes and a sample tray and cartridge holder combination
when in use.
[0033] FIG. 2 is a perspective view of a preferred embodiment of
the invention showing cooperation between the sample tray and
cartridge during storage.
[0034] FIG. 3a is a perspective view of the obverse side of the
fully assembled cartridge of a preferred embodiment of the
invention showing elements of the design and the capillary tubes
held therein.
[0035] FIG. 3b is a perspective view of the reverse side of the
fully assembled cartridge of a preferred embodiment of the
invention showing elements of the design and the capillary tubes
held therein.
[0036] FIG. 3c is a perspective view of the reverse side of the
fully assembled cartridge of a preferred embodiment of the
invention showing elements of the design and the capillary tubes
held therein, and particularly showing the distal portions of
passageways for holding the capillary tubes.
[0037] FIG. 3d is a top view of the obverse side of the fully
assembled cartridge of a preferred embodiment of the invention
showing elements of the design and the capillary tubes held
therein.
[0038] FIG. 3e is a top view of the reverse side of the fully
assembled cartridge of a preferred embodiment of the invention
showing elements of the design and the capillary tubes held
therein.
[0039] FIG. 3f is view of the top or proximal end of the fully
assembled cartridge of a preferred embodiment of the invention
showing elements of the design and particularly details of the
design of the top of the cap that forms a component of the
cartridge.
[0040] FIG. 3g is a view of the bottom or distal end of the fully
assembled cartridge of a preferred embodiment of the invention
showing elements of the design and the capillary tubes held
therein.
[0041] FIG. 3h is a side view of the fully assembled cartridge of a
preferred embodiment of the invention showing elements of the
design and the capillary tubes held therein.
[0042] FIG. 4 is an exploded view of the various components of a
preferred embodiment of the invention showing cooperation among
elements.
[0043] FIG. 5 is a perspective view showing a distal orientation of
the reverse side of a frame that forms a component of a preferred
embodiment of the cartridge of the invention.
[0044] FIG. 6 is a perspective view showing a proximal orientation
of the reverse side of the frame that forms a component of a
preferred embodiment of the cartridge of the invention.
[0045] FIG. 7 is an exploded view of the top side of the holder
that forms a component of a preferred embodiment of the cartridge
of the invention showing cooperation between a receptacle and a
cap.
[0046] FIG. 8 is an exploded view of the underside of the holder
that forms a component of a preferred embodiment of the cartridge
of the invention showing cooperation between the receptacle and the
cap.
[0047] FIG. 9 is a perspective view of a preferred embodiment of
the sample tray of the invention showing sample wells, a reservoir,
and a cartridge storage compartment.
[0048] FIG. 10 is a perspective view of a preferred embodiment of
the sample tray of the invention showing the various components of
the cartridge storage compartment.
[0049] FIG. 11 is a perspective view of an alternative embodiment
of the sample tray.
[0050] FIG. 12 is a perspective view of a preferred embodiment of
an apparatus for use with preferred embodiments of the devices for
determining the presence of an analyte, particularly showing the
relative position of the sample tray and cartridge.
[0051] FIG. 13 is a transparent view of a preferred embodiment
depicted in FIG. 12, showing critical internal components.
[0052] FIG. 14a is a block diagram of a preferred embodiment of the
apparatus showing cooperation among the various electronic
parts.
[0053] FIG. 14b is a block diagram, corresponding to FIG. 14a, of a
preferred embodiment of the apparatus showing component
designations known to those of ordinary skill in the art.
[0054] FIG. 15 is a simplified diagram of the solid-phase
fluorescence immunoassay (SPFIA) principle of a preferred
embodiment of the invention employing a coating of a capture
binding member comprising an antigen in a competitive fluorescence
immunoassay.
[0055] FIG. 16a is a representative plot of concentration in parts
per billion to normalized fluorescence for calibration standards
comprising known amounts of Penicillin G.
[0056] FIG. 16b is a representative plot of concentration in parts
per billion to normalized fluorescence for calibration standards
comprising known amounts of Amoxicillin.
[0057] FIG. 16c is a representative plot of concentration in parts
per billion to normalized fluorescence for calibration standards
comprising known amounts of Ampicillin.
[0058] FIG. 16d is a representative plot of concentration in parts
per billion to normalized fluorescence for calibration standards
comprising known amounts of Cloxacillin.
[0059] FIG. 17a is a representative plot of concentration in parts
per billion to normalized fluorescence for calibration standards
comprising known amounts of Cephapirin.
[0060] FIG. 17b is a representative plot of concentration in parts
per billion to normalized fluorescence for calibration standards
comprising known amounts of Ceftiofur.
DETAILED DESCRIPTION OF PRESENTLY PREFERRED EMBODIMENTS
[0061] Definitions
[0062] The following definitions are provided to help interpret the
disclosure and claims of this application.
[0063] Amount: The term amount as used herein refers to the
concentration or quantity of analyte in a sample either relatively
or absolutely.
[0064] Antibody: The term antibody as used herein refers to a
protein that recognizes a particular epitope on its homologous
antigen. The antibody can be a capture binding member of a
substrate comprising the surface of a capillary tube or it can be a
moiety of a fluorescently-bound conjugate. An antibody can be a
polyclonal antibody, a monoclonal antibody or a genetically
engineered molecule capable of binding the corresponding member of
a specific binding pair.
[0065] Antigen: The term antigen as used herein refers to any
substance that binds specifically to an antibody, as defined
herein. An antigen can be a capture binding member of a substrate
comprising the surface of a capillary tube, or it can be a moiety
of a fluorescently-bound conjugate, or it can comprise an
analyte.
[0066] Analyte: The term analyte as used herein, refers to any
substance or chemical constituent of a sample that is being
analyzed. Analytes detectable by an immunoassay can be any analyte
capable of being recognized and bound by a specific binding pair
member. An analyte can be one of the binding members of a
homologous antibody-antigen binding pair, that is, the analyte can
comprise an antibody or an antigen in an immunoassay.
[0067] Capillary tube: A capillary tube as used herein comprises a
high surface to volume ratio. A capillary tube can have any
suitable shape wherein the longitudinal cross section of the
internal walls can be defined by a cylinder, oval, square,
rectangle, or any suitable polygon and dimensions that are
appropriate for use in this invention, as described
hereinafter.
[0068] Conjugate: The term conjugate as used herein refers to a
compound that comprises two substances, wherein one of the
substances is coupled to the other. For example, a first conjugate
can be coupled to a third substance to make a second conjugate
comprising a first conjugate and a third substance, or a first
conjugate can be coupled to a second conjugate to make a third
conjugate comprising two conjugates. Coupling can be covalent or
non-covalent.
[0069] Fluorescent label: The term fluorescent label as used herein
refers to a substance which, when stimulated by an appropriate
electromagnetic signal or radiation, will absorb the radiation and
emit a signal that persists only so long as the stimulating
radiation is continued, i.e. it fluoresces.
[0070] Fluorometer: The term fluorometer as used herein, refers to
an instrument for measuring fluorescence. Generally it comprises a
signal generation means (i.e., a source of electromagnetic
radiation of a suitable wavelength to cause a fluorescent label to
fluoresce), a signal detection means comprising a fluorescence
detector, and an appropriate filter or filters.
[0071] Immunoassay: The term immunoassay as used herein, refers to
a technique that makes use of the specific binding between an
antigen and its homologous antibody, either polyclonal or
monoclonal, in order to analyze for an analyte in a sample. If the
immunoassay comprises use of a fluorescent label that can be
detected when excited by an appropriate electromagnetic signal, the
immunoassay is a fluorescent immunoassay or FIA.
[0072] Reagent: The term reagent as used herein refers to a
substance that participates in a chemical reaction or physical
interaction. A reagent can comprise an active component, that is, a
component that directly participates in a chemical reaction (e.g.
covalent binding) or physical interaction (e.g. non-covalent
binding), such as a fluorescently-labeled conjugate, and other
materials or compounds directly or indirectly involved in the
chemical reaction or physical interaction. It can include a
component inert to the chemical reaction or physical interaction,
such as catalysts, stabilizers, buffers, and the like.
[0073] Specific binding: As used herein specific binding refers to
the chemical recognition between two substances that results in the
coupling of those substances. Such coupling can include, but is not
limited to, covalent or non-covalent interaction. For example, the
specific binding between an antigen and its corresponding antibody
is of greater affinity than the non-specific binding of either
specific binding pair member and a non-corresponding substance.
[0074] Specific binding pair: As used herein specific binding pair
refers to two substances that specifically bind to each other.
[0075] Specific binding pair member: Specific binding pair member,
as used herein, refers to one member of a specific binding pair.
For example, a conjugate, a hapten, an antigen or an antibody can
be a specific binding pair member.
[0076] Substrate: The term substrate as used herein refers to a
material to which another material binds or can be attached. Such a
material is generally a surface of a material (e.g., the interior
surface of a capillary tube), a solid material on a surface, or a
first solid material on a second solid material. Generally a
substrate can comprise a capture binding member of a binding member
pair that can bind to a second member of the binding pair. For
example, a substrate can be an antibody conjugated with a substance
that binds to the interior surface of a capillary wall, wherein the
antibody would be considered a capture binding member of a binding
pair. The corresponding antigen conjugated with a fluorescent label
would be the second binding member.
[0077] Introduction
[0078] In one broad aspect, this invention can be considered to be
an immunoassay that employs the interior surface of a capillary
tube as a solid phase substrate that can bind a
fluorescently-labeled conjugate and detect an analyte in a sample.
Another aspect of this invention includes a capillary tube coated
on at least a portion of its interior surface with a substrate that
is capable of binding with a fluorescently-labeled conjugate. This
unique capillary tube provides, at least in part, the basis for
other aspects of the invention, such as a combination of the
capillary tube with a cartridge and a process for screening for an
analyte in a sample using the capillary tube. Another aspect of the
invention is a process for preparing the capillary tube of the
invention. Still other aspects of the invention include a uniquely
designed sample tray for use with the cartridge-capillary tube
combination and an apparatus for performing the screening process
of this invention. These and other aspects of the invention will be
discussed in greater detail hereinafter.
[0079] Types of Analytes to be Detected
[0080] The immunoassay of this invention is useful for screening
for analytes in numerous industries including, but not limited to,
health care, food processing, chemical, environmental control, and
the like. Thus, the types of samples include various fluids
suspected of containing a target analyte such as blood, plasma,
urine, saliva, and the like; food products such as milk, wine,
beer, and the like; chemical streams, waste streams from chemical
plants, rivers, and the like. In some situations, the sample may
need to be pre-treated prior to the immunoassay. Where the sample
is initially complex, solid, or viscous, it may need to be
extracted, dissolved or diluted in order to obtain a sample having
the appropriate characteristics for use in the immunoassay.
Further, the sample should be one in which binding complexes formed
in the subject immunoassay are stable. Binding complexes of the
subject immunoassay will generally be stable at pH values ranging
from about 5 to about 9. The pH value of the sample may be
adjusted, if necessary, to be about 7 by diluting with an
appropriate buffer.
[0081] A wide range of analytes can be detected using the subject
method. Detectable analytes can be any analyte capable of being
recognized and bound by a specific binding pair member. The analyte
can be an antigen, and antigen receptor (e.g. an antibody), or
hapten. Analytes of interest include naturally occurring and
synthetic small organic compounds, proteins, saccharides, nucleic
acids and the like. Illustrative analytes include compounds used in
the raising of domestic animals (e.g. antibiotics and food
supplements), food additives, naturally occurring contaminants,
dyes, microorganisms and their toxins, and the like. Other analytes
include physiologically active compounds, pathogenic markers found
in physiological fluids, toxins, surface membrane proteins,
cytokines, antibodies, human lymphocyte antigen proteins, hormones,
natural and synthetic drugs, proteins of bacteria, fungi and
viruses, and the like. Other analytes include compounds found in
the environment, such as pesticides, herbicides, organic components
of waste discharges, and the like.
[0082] Examples of specific analytes detectable in the immunoassay
of this invention include compounds from the sulfa family drugs
such. as sulfamethazine, sulfadimethozine, sulfathiazole,
sulfaquinoline and others; tetracycline, gentamicin,
chloramphenicol, aflatoxin, digoxin, and salmonella. Still other
analytes include, hydrocarbons, such as benzene, toluene,
ethyl-benzene, and xylenes.
[0083] The immunoassay is particularly valuable for detecting
analytes, such as antibiotics in milk samples, and is presently
preferred to be used for these compounds. Antibiotics are
administered to cows for the prevention and treatment of
infections, such as mastitis, and to enhance animal growth and milk
production. Antibiotics are also abused through off-label, illegal
administration in an attempt to quickly bring a sick animal back
into the producing herd.
[0084] For the purpose of maintaining a safe and healthy food
supply, it is important to identify, monitor and minimize the
existence of antibiotic residues in milk and milk products. Since
milk and milk products are widely consumed, antibiotic residues
should be avoided for several reasons: (i) Some antibiotic residues
can cause allergic reactions in sensitive consumers (approximately
5 to 10% of the population is hypersensitive to penicillin and
other antibiotics), (ii) Small concentrations of antibiotic
residues can aid in the selection of resistant strains of pathogens
that are harmful to humans, and (iii) Antibiotic residues can
interfere with starter cultures used in the production of processed
milk products such as cheese and yogurt. Consequently, the United
States Food and Drug Administration (US FDA) has established
safe/tolerance levels for antibiotic residues in milk and milk
products, shown in Table I.
[0085] The antibiotics that are most commonly administered to
lactating cows, and consequently those antibiotics that are usually
found as contaminants in milk and mil-products are from the
.beta.-lactam group (penicillin G, ampicillin, cloxacillin,
cephapirin, ceftiofur, and amoxicillin). The structures for these
compounds are available by consulting the "Merck Index"--Eleventh
Edition.
1TABLE I U.S.F.D.A. Safe/tolerance levels for .beta.-lactam drugs
in milk Drug Safe/Tolerance Level (ppb).sup.a Penicillin G 5
Amoxicillin 10 Ampicillin 10 Cloxacillin 10 Cephapirin 20 Ceftiofur
50 .sup.appb = parts per billion, 1 ppb is equal to 1 ng/mL.
[0086] The Immunoassays
[0087] Depending on the type of analyte to be detected, different
immunoassay formats can be employed. A particular immunoassay
format can be modified depending on the nature of the analyte, the
nature of the sample, and the like. In general, immunoassays useful
in the subject method are based on the formation of complexes
between specific binding pair members. Common to each immunoassay
used in the subject method will be a substrate comprising a first
binding pair member. The immunoassay also employs a second binding
pair member comprising a fluorescently-labeled conjugate that can
couple to an analyte or to both the first binding pair member and
an analyte. Throughout this specification, the term "capture" is
used with binding member or specific binding pair member. It refers
to a specific binding pair member comprising a substrate on the
interior surface of a capillary tube, although it is not limited to
such use if indicated otherwise.
[0088] Both sandwich and competitive immunoassay formats can be
employed in the subject method. The particular immunoassay format
employed will depend on the particular analyte characteristics, the
sample characteristics, the available reagents, and the like.
[0089] In a sandwich immunoassay, a fluorescently-labeled conjugate
is employed that is a specific binding member, wherein the
fluorescently-labeled conjugate binds to the analyte at a site
other than the site to which the other binding member, which is on
the interior surface of the capillary tube. The
fluorescently-labeled conjugate is mixed with a sample and the
resulting mixture is drawn up into the capillary tube. The analyte
will bind to the fluorescently-labeled conjugate and to the other
binding member moiety of the substrate, so that the amount of
fluorescent label bound to the capillary tube wall will be directly
proportional to the amount of analyte present.
[0090] In one type of a competitive immunoassay, a
fluorescently-labeled conjugate binding member binds directly to an
analyte or to the substrate, via an analog of the analyte present
on the substrate. Thus, the analyte and the substrate "compete" to
bind with the fluorescently-labeled conjugate binding member. In
this format, both the substrate and the analyte have a binding
region (also referred to as an epitopic region) that bind to the
fluorescently-labeled conjugate binding member. The moiety of the
substrate can be a ligand, an antibody, or binding fragment
thereof, typically analogous to an epitopic region of the analyte.
Here the amount of fluorescent label bound to the interior wall of
the capillary tube is inversely proportional to the amount of
analyte present.
[0091] In another type of a competitive immunoassay, a
fluorescently-labeled conjugate competes with the analyte for
binding sites to the capture binding member of the substrate. In
this format, the capture binding member can comprise an antibody
with both the analyte and the fluorescently-labeled conjugate
comprising homologous antigen binding members. In the absence of
analyte, the fluorescently-labeled conjugate will not have
competition for binding sites on the capture binding member of the
substrate. Thus, the amount of fluorescent label bound to the
interior wall of the capillary tube is inversely proportional to
the amount of analyte present.
[0092] With this in mind, it can be seen that an aspect of this
invention is a process for screening for an analyte in a sample,
which process comprises importing a fluid mixture into a capillary
tube having a substrate comprising a capture binding member wherein
the fluid mixture comprises a sample suspected of containing the
analyte and a reagent comprising a fluorescently-labeled conjugate
that is (a) capable of binding to the analyte or to the analyte
when bound by the capture binding member and to the substrate; (b)
capable of fluorescing when irradiated with an appropriate
electromagnetic signal. The process further comprises maintaining
the fluid mixture in the capillary tube for a time sufficient for
binding to take place between the substrate and the
fluorescently-labeled conjugate; removing excess fluid mixture from
the capillary tube; externally irradiating the coated portion of
the capillary tube with an electromagnetic signal sufficient to
cause fluorescence of bound fluorescently-labeled conjugate; and
detecting the resulting fluorescence to screen for the analyte.
[0093] In a competitive immunoassay, a particularly convenient
protocol is where fluorescently-labeled conjugate is first mixed
with a liquid sample suspected of containing the analyte to provide
a substantially homogeneous mixture and then the sample is taken
into the capillary tube. The fluorescently-labeled conjugate can be
a solid, or preferably, a buffered solution, and as appropriate,
can serve to dilute the sample and provide the appropriate pH.
[0094] Once the sample suspected of containing the analyte has been
mixed with the appropriate fluorescently-labeled conjugate, the
resulting mixture is introduced into the interior of a capillary
tube that has been interiorly coated with an appropriate capture
binding pair member moiety of a substrate appropriate for the
analyte being assayed. Preferably, a sample is introduced by
capillary force, although an external force such as suction or
positive pressure can also be used. In the example referred to
above, an antibiotic in milk, an antigen analogous to an antibiotic
of interest, is coated on the interior surface of the capillary
tube. The entire interior surface or a part thereof can be coated.
However, enough of the surface must be coated so that a binding
reaction can take place between the fluorescently-labeled conjugate
and the capture binding member moiety of the substrate such that
the capillary tube can be read by irradiating it and detecting the
resulting fluorescence. Generally, sample volumes introduced into
the capillary tube will range from about 2 to about 20 .mu.l,
usually about 5 to about 15 .mu.l, more usually about 5 to about 10
.mu.l.
[0095] After the sample portion has been introduced into the
capillary tube, the sample is incubated for a sufficient time
period for binding to occur, that is to form complexes between
members of specific binding pairs, e.g. a fluorescently-labeled
conjugate binding member and the substrate comprising the bound
antigen. The incubation step will typically occur at room
temperature, although temperatures in the range of about 10.degree.
C. to about 50.degree. C. can be employed. Incubation times will
typically range from about 0.5 to about 5 minutes, usually about
0.5 to about 3 minutes, and more usually about 2 minutes.
Frequently, the time necessary for introducing a wash solution into
the capillary tube will suffice for the incubation.
[0096] For the most part, the subject methods will depend solely on
the capillary tube and the fluorescently-labeled conjugate for
carrying out the immunoassay. However, in some situations more
complex protocols can be employed. For example, instead of having
the conjugate binding member labeled directly, one can indirectly
label the binding member. Where the binding member is an antibody,
one can use a fluorescently-labeled anti-antibody, so as to have a
universal fluorescent reagent. One can have a situation where one
adds both a fluorescently-labeled conjugate and its reciprocal
binding member, where the conjugate competes with the analyte for
the reciprocal binding member. The capillary tube can comprise a
capture binding member that captures the reciprocal binding member.
For example, the reciprocal binding member can be an antibody and
the capillary tube can be coated with Protein A or G, so as to
capture all antibodies.
[0097] After an incubation step, any fluorescently-labeled
conjugate free in the medium is preferably removed from the
capillary tube. Removal of unbound fluorescently-labeled conjugate
is conveniently accomplished through introduction of a washing
fluid that displaces unbound fluorescently-labeled conjugate from
the capillary tube. A variety of wash fluids can find use for the
washing step. The pH of the wash fluid will be a pH in which the
binding pair complexes are stable. Typically, the pH will range
from 5 to 9, usually 6 to 8, and more usually about is 7. Depending
on the nature of the fluorescent label of the conjugate, wash
solutions which enhance the fluorescence of the conjugate label can
be employed. For example, the fluorescence of a particular
fluorescent label can be enhanced in slightly alkaline or basic
solution. In such a case, a buffer having a pH above 7, but usually
less than 9, can be employed. Exemplary wash fluids comprise water,
buffers, such as phosphate, phosphate buffered saline (PBS), saline
solutions, carbonate buffers, and the like. The wash fluid can be
introduced into the capillary tube using any convenient means.
Usually the wash fluid will be introduced into the capillary tube
using the same means as the means used for introduction of the
sample. The wash solution can be taken up a number of times,
usually not more than about 6, more usually not more than about 2,
or the wash solution can be forced through the capillary tube using
a syringe, pump or other device.
[0098] After the washing step where the unbound labeled conjugate
is washed from the capillary tube, the presence of
fluorescently-labeled conjugate remaining bound to the capture
binding member on the substrate on the capillary tube surface is
detected in a detection step. The detection step can be conducted
immediately after the wash step, or can be delayed for a period of
time, if necessary. While the detection step can be conducted with
wash fluid in the capillary tube, preferably the capillary tube can
be dried prior to the detection step to minimize the possibility of
interference in the ensuing detection step. The drying may be done
by any appropriate means such as centrifuging, air drying, vacuum
drying and the like. Preferred techniques are discussed
hereinafter. If the detection step is to be delayed, the capillary
tube can be stored for a reasonable period of time under ambient or
reduced temperature conditions.
[0099] Many different fluorescent labels can be employed in the
subject immunoassays. Suitable fluorescent labels should be capable
of conjugation with antigens, haptens or antibodies in order to be
used in the fluorescently-labeled conjugate. Selection of the
fluorescent label is based on synthetic convenience, emission
maximum, quantum efficiency, stability under the assay conditions,
and the like, but the fluorescent label is not critical to the
invention, so long as there is a minimum quantum yield to provide
the desired sensitivity. A large number of commercially available
fluorescent labels can be employed. Illustrative fluorescent labels
include fluorescein-isothiocyanate (FITC), rhodamine, Texas Red,
phycoerythrin, Cy-5.RTM. and allophycoerythrin, and particularly,
fluorescent labels that fluoresce above about 550 nm, more
particularly, fluorescent labels that fluoresce above 600 nm, and
efficiently absorb light having absorption above 500 nm; more
particularly, 650 nm, such as Cy-5.RTM.. The fluorescent labels can
be conjugated to form the fluorescently-labeled conjugate using any
convenient method. See e.g. Harlow & Lane, Antibodies (1988) pp
353-358.
[0100] When fluorescently-labeled conjugates are used, detection is
accomplished by first irradiating a region of the capillary tube
comprising the detection region, followed by measuring the
resultant emitted fluorescent signal. Any convenient irradiation
means can be employed for providing the appropriate wavelength.
Exemplary irradiation means include lasers, light emitting diodes,
tungsten lamps and the like. The wavelength of light used in the
stimulation means will depend on the particular fluorescent label.
Generally, the irradiation light wavelengths will range from 300 to
900 nm, usually from about 350 to 800 nm, and more usually from
about 450 to 800 nm. For example, where Cy-5.RTM.is the fluorescent
label, the wavelength of the irradiation light will range from 630
to 650 nm. The fluorescence from the fluorescently-labeled
conjugates present in the capillary tube will be measured.
Measuring the emitted signal is accomplished by detecting the
photons emitted in the detection region. Means for measuring
fluorescence are commercially available and any convenient
fluorescence detector can be used. Various photodiodes,
photomultipliers, and the like, can be employed, and in some
instances a visual detection will suffice, if a
fluorescently-labeled conjugate is used that fluoresces in the
visible spectrum.
[0101] Depending upon whether a competitive or sandwich immunoassay
is employed, and the reagents employed, the fluorescence intensity
will be directly or inversely proportional to the amount of analyte
in the sample. Where one is interested in a qualitative result or a
semi-quantitative result, such as determining whether the amount of
analyte is above a predetermined threshold, versus determining the
concentration of analyte, the amount of fluorescently-labeled
conjugate is selected to provide a clear signal as compared to the
absence of analyte or analyte below the predetermined value. Thus,
one can use an amount of conjugate which will be substantially
absent in the detection region in the absence of analyte and
provide an intense signal at the lowest concentration that one
would anticipate to be encountered of analyte in the sample or vice
versa.
[0102] For quantitation, the resultant electrical signal can be
accurately measured using appropriate hardware and software. The
area from which the fluorescence is measured is controlled to
provide for consistent values. Controls can be employed, where the
signal to concentration of the analyte is determined, so that the
signal can be directly related to the concentration of analyte in
the immunoassay sample. In this manner, both the presence and the
amount of analyte in the sample can be determined.
[0103] As discussed hereinafter, generally only a small amount of
the sample (less than about 1 ml) is mixed with the conjugate. As
discussed in greater detail hereinafter, this is done preferably in
a small well of a sample tray using a means of agitation. It may be
important that the mixing and incubation step and the subsequent
incubation step in the method of this invention be carried out for
the same defined period of time to minimize variations from test to
test in a series. Thus, it may be preferred to automate the steps
of the method to eliminate operator error to the greatest extent
possible. This will be discussed in greater detail in the following
discussion of preferred devices and apparatus depicted in the
figures.
[0104] Thus, from the preceding discussion, it can be seen that
solid-phase fluorescence immunoassay (SPFIA) takes advantage of the
specificity of a binding pair affinity, e.g., antibody-antigen
recognition, and incorporates a suitable fluorescent label for
detection. FIG. 15 diagrams the principle of the SPFIA. A sample
(e.g., milk) is mixed with a known amount of a
fluorescently-labeled antibody which is specific to an analyte
(e.g., a .beta.-lactam antibiotic). An essentially instantaneous
binding reaction will occur between the antibody and the analyte in
the sample. A solid support, e.g., the inside wall of a glass
capillary tube as a substrate comprising an antigen analogous to
the analyte attached thereto will then be exposed to the sample.
When the sample is removed from the capillary tube and the
capillary tube is examined with a fluorometer, and any fluorescence
is detected. In the milk example shown in FIG. 15, a substrate
incubated with milk samples with high concentrations of an
antibiotic will have fewer free fluorescently-labeled antibody
molecules to react with the antigen substrate, thus yielding lower
fluorescence signals. Conversely, milk samples with low
concentrations of antibiotic will have more free antibody
molecules, thus more fluorescent conjugates will bind to the
substrate and will provide higher fluorescence signals.
Mathematically, the measured fluorescence signal is inversely
proportional to the concentration of analyte in the sample for
these competitive assays. Thus, in FIG. 15 the analyte is the
antibiotic and the capture binding member moiety of the substrate
on the interior capillary tube surface is an antigen (e.g., an
analog of the analyte) that is bound by the fluorescently-labeled
antibody conjugate.
[0105] The Capillary Tube and its Preparation
[0106] An important aspect of this invention is the unique
capillary tube coated on at least a portion of its interior surface
with a substrate that is capable of binding with a
fluorescently-labeled conjugate. A wide variety of capillary tubes
designed for diverse uses are known in the art and are suitable for
use in the subject invention. Generally, capillary tubes used in
the subject method are made of a substance that allows irradiation
light and fluorescence emissions to be transmitted across the
capillary tube wall. Thus, when an electromagnetic signal from an
external source contacts the capillary tube wall, the signal must
go through the wall. The resulting fluorescence must then be
transmitted out through the wall. Materials from which suitable
capillary tubes can be formed include glasses, such as soft glass,
silicate glass and fused silica. Other materials include plastics,
such as polystyrene, polyethylene, polypropylene, polyvinyl
chloride (PVC), and the like. Other materials may be apparent to
one of skill in the art upon reading this disclosure. A presently
preferred embodiment is a borosilicate glass capillary tube, such
as the one available from Drummond Scientific, Broomall, Pa.
[0107] Capillary tubes suitable for use in the subject invention
can have a wide variety of dimensions, as long as liquid media are
effectively drawn up by suction or capillary force. The capillary
tubes can have cross-sections which are circular, square,
rectangular, oval, and the like. Typically, the capillary tubes
will have circular cross-sections. The inner diameters of suitable
capillary tubes can range from about 0.1 micrometers (.mu.m) to
about 1 millimeter (mm), usually about 0.3 .mu.m to about 1.0 mm
and more usually about 0.50 .mu.m to about 1 mm, preferably about
0.65 mm for a milk immunoassay. The outer diameter of a suitable
capillary tube can be about 1.0 mm to about 1.5 mm. The length of
suitable capillary tubes will typically be about 10 mm to about 150
mm. Usually the length will be at least about 15 mm, more usually
at least about 25 mm, up to about 250 mm for ease of handling.
While the capillary tubes of this invention may be interconnected
by a planar sheet, generally they are free standing, individual
capillary tubes.
[0108] For glass capillary tubes, to enhance binding of a substrate
to the glass, the surface can be coated with a material to enhance
a binding capability on the interior surface. At least one region
of the interior surface of the capillary tube, a detection region,
will be coated with a suitable substrate that will include a member
of a binding pair. The member of a binding pair coated on the
surface, at the detection region, binds directly or indirectly
through an intermediate binding agent to a fluorescently-labeled
conjugate. Depending on the particular method used to coat the
capillary tube, the region can encompass the entire interior
surface of the capillary tube or can be limited to a portion of the
interior surface of the capillary tube. The region of the interior
surface comprising a capture binding member coated thereon will
typically range from about 10 to 100% of the interior surface, and
will usually range from about 30 to about 100% of the interior
surface. Preferably more than about 80% of the interior surface is
coated. Conveniently, the entire capillary tube, or one end
thereof, can be immersed in the coating media to be coated. When
immersed at one end, the coating medium can be brought up into the
capillary tube by any suitable means, e.g. by capillary force,
conveniently to a predetermined height, which can be indicated by a
scoring or other designation on the capillary tube. Alternatively,
the liquid can be pumped or sucked into a portion of or the whole
length of the capillary tube. This means that all or a portion of
the external surface can be coated with the capture binding member
as well.
[0109] Depending on the particular immunoassay format used in the
subject method, a variety of agents can serve as the binding
members. In general, a binding member of a pair should complex or
bind to its complementary binding pair member in the subject
immunoassays with sufficient affinity to withstand wash procedures
used in the subject method. Typically, the affinity between the
binding member and its complementary binding pair will be at least
about 10.sup.6 L/mol, frequently at least about 10.sup.8 L/mol or
higher. One member of a binding pair will bind or complex directly
or indirectly to a fluorescently-labeled conjugate. For example, in
a competitive immunoassay format, the capture binding member (e.g.,
the analyte analog on the interior surface of the capillary) binds
directly to a fluorescently-labeled conjugate. In a sandwich
immunoassay format, the capture binding member will complex to the
fluorescently-labeled conjugate indirectly through the analyte.
Illustrative binding members include receptors, such as antibodies
and binding fragments thereof, e.g. F(ab) and F(ab).sub.2
fragments), lectin, ligands, such as antigens, haptens or other
reciprocal binding members; and conjugates comprising ligands and
receptors, bonded to the fluorescent label.
[0110] Instead of coating a region of the interior with a capillary
tube with a substrate containing a single capture binding member,
the interior surface of the capillary tube can be coated with two
or more capture binding members at the same or different sites. In
this embodiment, each different capture binding member will be
involved in the detection of a different analyte in the sample.
When the binding members are at the same site, the fluorescent
label associated with each analyte can be independently determined,
e.g. different emission maximum wavelength, at least about 10 nm
different, and/or different delay time for emission. Thus, one can
assay for a multiplicity of analytes simultaneously.
[0111] To coat the internal wall of capillary tubes for use in the
subject method, capillary tubes are contacted with a solution
comprising the capture binding member. For coating the internal
capillary tube surface with the substrate solution, a variety of
techniques can be employed, depending in part on the nature of the
substrate and the nature of the internal wall. With most proteins,
particularly antibodies, albumins and globulins, the proteins stick
to the surface without covalent bonding, and are stable under the
conditions of the immunoassay.
[0112] In preparing the subject capillary tubes, as indicated for
proteins above, it can be sufficient to contact capillary tubes
with untreated surfaces to a solution comprising the binding
reagent. The binding solution is usually a buffered solution having
from about 10.sup.-7 to 10.sup.-3 g protein/ml. Typically, the
protein binding member will be an antibody or fragment thereof for
direct assays. For indirect assays, the protein will typically be
an analog of the analyte. For the most part, the protein binding
member will be an antibody or fragment thereof. Methods of stably
coating glass and plastic surfaces are well known. See e.g. Harlow
& Lane, Antibodies: A laboratory manual, Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y. (1988). In many instances,
where the binding member is not a protein, it can be conjugated to
a protein, leaving the binding sites available for binding to the
complementary binding member. For example, haptens can be
conjugated to a protein that will not interfere with the
immunoassay. In this way, an otherwise non-binding analyte or a
mimetic or analog thereof can be directly bound to the internal
surface of the capillary tube without having to functionalize the
surface so as to provide covalent binding of the otherwise
non-binding substance.
[0113] In the case of a preferred capillary tube, i.e., glass, it
is preferred to first coat the interior surface with an agent which
enhances the binding of the protein to the surface. Thus, where the
binding member does not provide for stable binding to the interior
surface of the capillary tube, the surface is activated or
functionalized to provide covalent or non-covalent binding of the
binding member to the capillary tube surface. The particular
technique used in treating the capillary tube surface will depend
on the composition of the capillary tube and the binding member,
e.g. the functional groups available on the binding member for
reaction. With surfaces such as plastics, e.g. polystyrene and
polyethylene, the surface can be functionalized to provide for
reactive amino, carboxy, thio, sulfonyl, hydroxy or other
functional groups, by acylation, nitration and reduction, oxidation
with ozone, chlorosulfonation, and the like. The specific
functional group provided on the capillary tube surface will depend
on the binding member. If the binding member does not naturally
comprise a useful available functional group, the binding member
can be modified, so as to provide for a functional group that will
react with the activated surface, e.g. amino with carboxy, thiol
with activated olefin, hydroxy with an activated halogen, and the
like. For non-covalent binding of the binding member to the
surface, a hydrophobic surface may be provided, that is, a surface
that has a long chain alkyl or alkenyl group attached, e.g.,
through a silicon attaching group.
[0114] For treating the surfaces of glass capillary tubes, the
surface can be "functionalized" by using a silicon-based compound
having as one part of the compound a silicon moiety that reacts
with the glass surface of the capillary tube and the other part of
the compound being carbon-based that provides a suitable functional
group, e.g. alkyl, alkenyl, amino, carboxy, sulfonyl, thiol,
activated olefin, such as maleimido, and the like, that will bind
with the binding member either covalently or non-covalently (e.g.,
by van der Waals' forces).
[0115] Generally, the coating is done using a silicon-based
material that provides a basis for covalently or non-covalently
forming a suitable substrate on the interior surface. Suitable
silicon-based materials include silanes or siloxanes that bind
through the silicon to the interior glass surface and provide a
surface extended into the cylinder of the capillary tube to which
an appropriate substrate is bound. This is seen in FIG. 15.
Examples of suitable siloxane materials include aminoalkylsiloxanes
and alkyl or alkenyltrialkoxysilanes. Conveniently,
aminoalkylsiloxanes known in the art can be used, where the
aminoalkyl group is of from about 2 to 6 carbon atoms and the
alkoxy groups are of from about 1 to 6 carbon atoms. Preferably the
silane based material is represented by the formula
R--Si(OR.sub.1).sub.3, wherein R is an alkyl or alkenyl of about 12
to about 20 carbon atoms and R.sub.1 is an alkyl of one to four
carbon atoms. A particularly preferred silane-based material is a
compound represented by the formula R--Si(OR.sub.1).sub.3, wherein
R is a straight chain alkyl of 18 carbon atoms and R.sub.1 is
ethyl. Preferably octadecyltriethoxy silane is chosen as the silane
coating material, this is available through Pierce Chemical Company
as AquaSil.RTM..
[0116] After the binding member has been bound to the surface of
the capillary tube, non-specific active sites or "hot spots" can
remain on the capillary tube surface. These active sites must be
occupied or blocked prior to use of the capillary tube in the
subject method. Otherwise, non-specific adsorption of the various
assay reagents can occur. Non-specific adsorption should be avoided
because it can result in non-specific binding of the label to the
surface. Blocking of active sites can be achieved by contacting the
internal capillary tube surface with a wide variety of blocking
solutions. Coating the capillary tube surface with the blocking
solution can be achieved using the methods described above for
coating the surface with the binding member. The blocking solution
of choice will depend on the particular immunoassay being
conducted. See e.g., Harlow and Lane, Antibodies (1988) 497, supra.
Illustrative blocking solutions include Blotto, Blotto/Tween,
Tween, BSA and Horse Serum.
[0117] Generally, silane-based coating is carried out by incubating
an appropriate capillary tube in a solution containing the silane
based material dissolved in an appropriate solvent, such as water,
for a period of time and at a temperature sufficient to allow
binding of the silane material to the internal surface of the glass
capillary tube. An appropriate temperature range is about
10.degree. C. to about 50.degree. C., with ambient temperature
being preferable. The binding will take place within about 10
minutes, but the capillary tubes can be incubated for an hour or
more. The capillary tube is then dried via a suitable means such as
by purging with nitrogen or other suitable gas or by
centrifugation. Any residual moisture can be removed by incubating
the capillary tube in an oven at a suitable temperature, such as
about 110.degree. C. to about 140.degree. C., for about an hour
and/or under a vacuum. After incubation, the capillary tube will be
allowed to cool to room temperature before treating it with the
next coating. Once the glass is alkylated by the silane material it
is susceptible to non-covalent binding with sticky proteins. A
substrate comprising a capture binding member, i.e. a protein
conjugated to a capture binding member of an antigen-antibody
binding pair, can be coupled to the silanization substrate layer.
Alternatively, a protein conjugated to another protein substance
that permits binding to a third substrate comprising the capture
binding member can be applied. When coating a protein or conjugated
protein to the silanization layer, a longer incubation time will
usually be required, typically between about 1 to about 24 hours
for a similar period of time. After coating with appropriate
substrate layers, a final coating with an innocuous protein
blocking solution is applied to cover "hot spots" previously
discussed comprising exposed alkyl groups. Alternatively, the
blocking step can occur after the attachment of the second layer,
but before attachment of a third layer in cases where a third layer
is used. The coated and blocked capillary tubes are typically
incubated in the blocking solution for at least 1 hour and as long
as 24 hours. The capillary tube is then substantially dried. A wash
solution, such as deionized water, is then passed through the
capillary tube to remove any substrate material not bound to the
surface of the capillary tube or to another substrate. The
capillary tube is then substantially dried by any appropriate
means. It is advantageous to aid removal of residual water by
incubating the capillary tube in a vacuum oven at the highest
temperature possible that would not denature any bound protein,
e.g. about 37.degree. C. for at least one hour. Once the capillary
tubes have been prepared, they can be used immediately or
conveniently stored for use at a later time. Capillary tubes can be
stored for use at a later time at ambient or reduced temperature
conditions. Example 9, Example 10, and Example 11, describe in
detail, preparation of capillary tubes for use with a preferred
embodiment of the invention.
[0118] Another aspect of this invention comprises a process for
preparing a glass capillary tube for use in a fluorescent
immunoassay, which process comprises coating at least a portion of
the internal surface of the capillary tube with a substrate that is
capable of binding to a fluorescently-labeled conjugate. In a
preferred aspect, the process comprises coating at least a portion
of the interior surface of the capillary tube with a silane-based
material and binding a protein conjugate to the silane-based
material, which conjugate is capable of binding to a
fluorescently-labeled conjugate. Protein conjugates that are
particularly useful include bovine serum albumin (BSA) and human
serum albumin (HSA), the former being preferred.
[0119] A Cartridge for Use with Capillary Tubes
[0120] Capillary tubes prepared by the methods described above can
be used individually or in combination for an immunoassay in
accordance with the method discussed above. The assay can be
performed manually or automatically, as discussed hereinafter. One
end of a single capillary tube can be introduced into a sample and
sample drawn up by any convenient means, e.g. capillary action or
active pumping, to provide an appropriately sized sample in the
capillary tube. Alternatively, a plurality of capillary tubes
coated with appropriate binding members can be used to permit a
plurality of immunoassays on one sample or a single immunoassay on
multiple samples to screen for one or multiple analytes. The
capillary tubes can be held in a cartridge that permits sequential
or simultaneous use and/or that permits interaction with other
devices and/or apparatus to effectuate an immunoassay of this
invention.
[0121] Thus another aspect of this invention is a combination of a
cartridge holding at least one capillary tube, which cartridge
comprises a capillary tube coated on at least a portion of its
interior surface with a substrate that is capable of binding to a
fluorescently-labeled conjugate and a frame comprising a means to
position the capillary tube in a region of the frame that permits
exposure of at least a portion of the coated capillary tube to an
external electromagnetic signal that is capable of causing any
bound fluorescently-labeled conjugate to fluoresce. The
fluorescence is then detected with an appropriate signal detection
means.
[0122] Another related aspect of this invention is a cartridge for
securely holding a plurality of spaced-apart capillary tubes,
wherein the cartridge has a plurality of passageways for aligning
the capillary tubes therein and at least a portion of the cartridge
permits exposure of the capillary tubes to an external
electromagnetic signal that is capable of causing a
fluorescently-labeled conjugate on the internal surface to
fluoresce. The cartridge preferably has a holder with a passageway
therethrough and a port for permitting fluid to be passed through
each capillary tube. The cartridge can be manufactured by methods
known to those of ordinary skill in the art, and is preferably
injection molded. The cartridge is particularly suitable for
cooperating with a unique sample tray and with a semi-automated
apparatus for fluorescence immunoassays, as will be discussed in
more detail hereinafter.
[0123] A detailed description of a preferred embodiment of the
cartridge is provided with the description of the drawings and
alternative embodiments are provided thereafter.
[0124] A Sample Tray for Use in Immunoassays
[0125] Capillary tubes as prepared above can be used with
commercially available containers suitable for independently
housing the reagents and waste of immunoassays, such as microtiter
wells or plates. Alternatively, the capillary tubes can be used
with a sample tray designed to contain a reagent and means to mix
fluid held within the wells.
[0126] Another aspect of this invention, therefore, is a tray for
holding multiple portions of a sample, which tray comprises a
reservoir sufficient to hold a quantity of fluid, and a plurality
of spaced apart wells therein. Preferably, the sample tray also
independently houses a metallic washer for mixing any fluid held
within the wells, and has a means to prevent the metallic washer
from falling out of the well, preferably a retaining ridge with a
diameter smaller than the external diameter of the metallic washer.
The sample tray may be manufactured by any number of methods known
to those of ordinary skill in the art and is preferably injection
molded.
[0127] A preferred aspect of this invention is the combination of
the sample tray and cartridge to form a disposable immunoassay
testing kit. In this aspect, the sample tray has a section for
securely holding the cartridge therein for storage and
transportation. Details of the sample tray and cartridge and sample
tray combination are provided in the description of the
figures.
[0128] An Apparatus for Performing the Process for Screening for
Analytes
[0129] The methods of the present invention for screening for
analytes can be conducted manually. The steps of washing and drying
can also be conducted manually. The capillary tube can also be used
in accordance with the invention with a commercially available
fluorometer.
[0130] However, it is preferred that the methods be conducted by an
automated or semi-automated apparatus. Since, incubation times are
relatively short for the SPFIA in capillary tubes, a greater
probability exists that operator timing will be off and thus
reproducibility compromised. Therefore, an immunoassay apparatus
for use with the immunoassays discussed above should have the
ability to manipulate one or more capillary tubes and a sample
tray, to control the flow of and house fluids, to carry out a
substantial portion of the steps of the immunoassay, including the
step of detecting the and analyzing the florescent signal. The
ability to conduct a plurality of immunoassays on a plurality of
samples also increases efficiency. Furthermore, quantitative
analysis is possible with an integrated, semi-automated,
immunoassay.
[0131] Another aspect of the present invention, therefore, is an
apparatus for determining the presence of at least one analyte in a
sample, which apparatus comprises a reservoir, a means to control
the flow of fluid contained within the reservoir or in a sample
tray, a first section to attach the cartridge of the present
invention and similarly the sample tray of the present invention, a
means to dry the capillary tubes, a fluorometer to measure the
level of fluorescence, and a means to analyze the resulting signal
and report a qualitative or semi-quantitative result and optionally
a quantitative result.
[0132] The apparatus can cooperate with the cartridge and sample
tray combination of the present invention and certain variations
thereof. In a typical immunoassay using the cartridge and sample
tray combination in cooperation with the apparatus of the present
invention, a sample is added to one or more wells in the sample
tray. The sample tray is placed on the apparatus and a magnetic
mixer located therein agitates the metal washers of the sample tray
to mix the contents of the well comprising a fluorescently-labeled
conjugate reagent and the sample so as to permit the analyte in the
sample to bind to the fluorescently-labeled conjugate. After the
sample and reagent are properly mixed, the apparatus positions the
sample tray under the cartridge located thereon and draws the
mixture into one or more capillary tubes, e.g., via suction. The
mixture is incubated in the capillary tubes for a sufficient time
for a suitable amount of fluorescently-labeled conjugate to bind to
the capture binding member of the substrate, on the surface of the
capillary tube. After incubation, the apparatus passes a wash
solution through the capillary tube to remove any unbound material.
The resulting waste fluid is emptied into the reservoir of the
sample tray by the apparatus. The apparatus then notifies the
operator to position the cartridge on the centrifuge to spin dry
the capillary tubes.
[0133] The apparatus then positions the cartridge proximate to a
fluorometer. The capillary tubes are exposed to an electromagnetic
signal through an exposure opening located on the cartridge and the
emitted fluorescence is subsequently detected by a fluorescence
detector. The detected signal can be processed for qualitative,
semi-quantitative, or quantitative analysis depending on the
controls used, and on the analysis software employed.
[0134] The level of quantitation possible using the apparatus with
the devices of the present invention depends on the affinity of the
capture binding member as previously discussed, detector
sensitivity, mathematics used to analyze the signal, and whether
standards and/or controls are used and if so on what kinds of
standards and/or controls. Generally, affinity of about 10.sup.6
L/mol can provide sensitivity in the parts per million range and
affinity of about 10.sup.9 L/mol can provide sensitivity in the
parts per billion range.
[0135] The most basic form of quantitation is the determination of
the presence of an analyte. For this to occur, the concentration of
analyte in the sample must be above some lower limit of
quantitation for the immunoassay. Generally, a semi-quantitative
result is reported. A bar code that accompanies the cartridge and
sample tray kit contains information regarding the level of
fluorescence that corresponds to the boundary between a pass an a
fail as indicated by acceptance levels such as the safe/tolerance
levels shown in Table I. Typically, each lot of reagent will have a
different associated critical level of fluorescence due to, among
other things, variations in the binding affinity of the capture
binding member substrate. The apparatus will measure the level of
fluorescence and compare it to the pass/fail level for the specific
immunoassay corresponding to the concentration of interest.
[0136] More quantitative analysis is possible with the present
invention. FIGS. 16a-d to FIGS. 17a-b show plots of normalized
fluorescence versus concentration of analyte in parts per billion
(ppb) for the .beta.-lactam antibiotics used with the present
invention. Normalized fluorescence corresponds to the level of
fluorescence emitted by a fluorescent label bound to the surface of
a capillary tube containing analyte as a percentage of the level of
fluorescence emitted by a fluorescent label bound to the surface of
a capillary tube with no analyte, i.e. a blank. Since concentration
is inversely proportional to the level of fluorescence for a
comparative assay, the curves formed by the plurality of
concentration points have a negative slope. If a sample is run on
the apparatus, the resulting fluorescence signal can be compared
against the fluorescence signal generated by a blank run in
parallel with the sample. The resulting percentage can be plotted
on the appropriate graph and a relative concentration of analyte in
sample can be determined.
[0137] Generally, the apparatus will calculate a semi-quantitative
result by using a pass/no-pass level of fluorescence or a
quantitative result by plotting a normalized level of fluorescence
versus concentration, and calculating a least-squares best fit of a
line corresponding to the curves. Thus a multilevel calibration
curve can be used, wherein quantitative determination of the amount
of analyte in a sample is possible when such concentration is
interpolated within the linear range of the best fit polynomial.
Even greater quantitative results are possible when standards are
prepared to run in parallel with a sample immunoassay, wherein the
same lot of fluorescent label is used for both the sample conjugate
and blank conjugate.
[0138] Since milk production is regulated by the FDA, immunoassays
for .beta.-lactam antibiotics are similarly regulated. The FDA
typically requires that positive immunoassay results be confirmed.
Consequently positive and negative controls should be run with
these immunoassays. One convenient method is the use of a control
cartridge comprising passing, failing, and blank concentrations of
analyte. This control cartridge can be run immediately following a
positive test result with the immunoassay.
DETAILED DESCRIPTION OF THE FIGURES
[0139] With a detailed description of the various aspects of the
present invention provided, a detailed description of preferred
embodiments of the devices and apparatus for use with the method
for screening for analytes as well as a detailed description of a
preferred embodiment of the method as used with the devices and
apparatus is now given. While a description of preferred
embodiments is given in considerable detail, it should not be
construed as limiting to those descriptions discussed hereinafter
and variations thereof discussed hereafter. Other variations of the
described embodiments can occur to one of ordinary skill in the art
that fall within the scope of the present invention.
[0140] With reference to terminology, it will be noted in the
detailed description of the various aspects of this invention that
portions of the devices are referred to as "top", "bottom",
"obverse", "reverse", "proximal" and "distal" portions. This is
done wholly for convenience and to relate the description to the
diagrammatic representations in the drawings. It will be
appreciated that the devices can function in any position or
orientation and it is within the scope of this invention to have
them do so.
[0141] FIG. 1 is a front perspective view of the devices of a
preferred embodiment of the invention showing cooperation between
the components during use. The devices, which are preferably
portable and disposable, comprise a cartridge 1, for sealingly
holding four radially spaced apart antigen coated capillary tubes
2, where a reaction takes place; and a sample tray 3 for containing
sample, a fluorescently-labeled conjugate reagent, and other fluid,
such as liquid waste; and for providing storage of the cartridge 1.
The sample tray 3, is positioned under the cartridge 1 by an
apparatus discussed hereafter so as to permit the cartridge 1 to
cooperatively draw fluid into each capillary tube 2 from the sample
tray 3. The radial orientation of the capillary tubes 2 functions
to ensure that in cooperation with an exposure opening 7 each
capillary tube 2 is presented to a signal detection means,
discussed hereafter, at the same angle and to permit each capillary
tube 2 to be submitted to centrifugal force parallel to its
longitudinal axis while secured within the cartridge.
[0142] FIG. 2 is a perspective view of the devices of a preferred
embodiment of the invention showing cooperation between the
components of the devices during storage when not in use. The
cartridge 1 slides into a pair of retaining shelves 83, 84
discussed hereafter and snaps in place so as to be secured by the
retaining shelves 83, 84 and a pair of fastening clips 75, 76
discussed hereafter (also see FIG. 10).
[0143] FIG. 3a to FIG. 3h show the fully assembled cartridge 1 from
perspectives showing all sides of the cartridge 1 and particularly
showing details of the various design features.
[0144] FIG. 4 is an exploded view of the devices of a preferred
embodiment of the invention showing cooperation among the elements
of the cartridge 1 and sample tray 3. The cartridge 1 comprises a
frame 4 and a holder 5 for securing the capillary tubes 2 within
the frame 4. The frame 4 comprises a rigid substantially flat
rectangular body preferably made of polystyrene, four radially
oriented passageways 6 for containing four glass capillary tubes 2
preferably coated with antigen (so as to conduct a competitive
assay), an exposure opening 7 for permitting a beam of light to
contact the capillary tubes 2 when secured within the cartridge 1,
and four protective tabs 8 for protection of the tips of the
capillary tubes 2.
[0145] FIG. 5 and FIG. 6 show perspective views of what can be
referred to as the reverse side of the frame 4. In a preferred
embodiment shown, the frame forms a substantially flat and hollow
rectangle about 4 centimeters to about 10 centimeters, preferably
about 5.6 centimeters long along a side 9 and a wall 10; about 1
centimeter to about 8 centimeters wide, preferably about 2.7
centimeters wide along two side walls 11, 12; and about 2
millimeters to about 1.3 centimeters high, preferably about 8
millimeters high along the three walls 10, 11, 12. The four
passageways 6 originate from a rectangular protruding segment 13
extending from the wall 10. The protruding segment 13 preferably
extends about 2 millimeters from the long wall 10 along the
horizontal plane of the frame 4 as shown in FIG. 5 and is the same
width as the long wall 10, about 3.7 centimeters long. Located on
each length wise edge of the protruding segment 13 are three clips
14 for securely attaching the holder 5 discussed hereafter to the
protruding segment 13. Extending perpendicularly from the
protruding segment 13 are two guide pins 15, 16, a first guide pin
15 and a segmented second guide pin 16, each for guiding a
receptacle 35 and cap 36 discussed hereafter into place on the
frame 4. The first guide pin 15 has an external diameter between 2
millimeters and 5 millimeters, preferably about 3 millimeters; and
is about 1.5 millimeter to about 5 millimeters long or of a
sufficient length so that the end of the first guide pin 15 is
flush with the top of the cap 36, when the two are cooperatively
engaged. The segmented second guide pin 16 has essentially the same
length as the first guide pin 15 and an external diameter similar
to that of the first guide pin 15 at the base of the segmented
second guide pin 16 and up to a ledge 17 located around the
perimeter of the segmented second guide pin 16 at about the halfway
point along the longitudinal axis of the segmented second guide pin
16. From the ledge 17 to the end of the segmented second guide pin
16, the external diameter of the segmented second guide pin 16 is
between about 1.5 millimeters to about 4.5 millimeters, preferably
about 2.5 millimeters. In any case of this preferred embodiment,
the segmented second guide pin 16 should have a ledge 17 that
defines two distinct external diameters that are complementary to
the internal diameter of a corresponding segmented guide shaft 50
discussed hereafter located on the cap 36. The function of the
different designs of the two guide pins 15, 16, is to permit only
one orientation of engagement with the cap 36.
[0146] As shown in FIG. 6, along the 8 millimeter wide side of the
protruding segment 13 are four equally spaced openings 18, each
about 5 millimeters in diameter, for receiving the capillary tubes
2 and receptacle 35 combination discussed hereafter. The openings
18 lead into each passageway 6 which comprises a receiving chamber
19 and a narrow shaft 20 shown in FIG. 5 and FIG. 6. For the
purpose of description, the passageways can be divided into two
pairs, an inner pair and an outer pair. The axis of the inner pair
extend radially toward the long side 9 preferably at about a 5
degree angle from an imaginary line perpendicular to the center of
the plane defined by the long wall 10. The axis of the outer pair
extend radially toward the long side 9 preferably at about an 18
degree angle from an imaginary line perpendicular to the center of
the plane defined by the long wall 10. The angles at which the
capillary tubes 2 will extend from an imaginary line perpendicular
to the center of the plane defined from the long wall 10 depend on
the dimensions of the cartridge 1, but should be such that the
capillary tubes 2 are radially orientated so as to ensure
consistent exposure of each capillary tube 2 to a signal detection
means, discussed hereafter, when the cartridge 1 is placed in a
circular centrifuge, also discussed hereafter, and to provide
uniform centrifugal force to each tube. That is, when an imaginary
line is drawn along the longitudinal axis of each capillary tube 2
when positioned in the cartridge 1, one end of each of the lines
should converge to a central point and the arc formed by the
combination of the other ends of the lines should form part of a
circle. The receiving chambers 19 of the inner pair follow the axis
angle, are of the same diameter as the openings 18 or are slightly
tapered, and are preferably about 4.5 millimeters deep. The
receiving chambers 19 of the outer pair also follow the axis angle,
are of the same diameter as the openings 18 or are slightly
tapered, and are preferably about 6.5 millimeters deep. The
function of the different depths of the receiving chambers 19 is to
cooperatively engage the receptacle discussed hereafter. The inner
receiving chamber 19 and shaft 20 combinations are about 1.5
centimeters to about 2 centimeters, preferably about 1.75
centimeters long, and the outer receiving chamber 19 and shaft 20
combinations are about 1.3 centimeters to about 1.8 centimeters,
preferably about 1.6 centimeters long. The lengths of the
combinations of the inner and outer receiving chambers 19 and
narrow shafts 20 are determined by the radial orientation of the
passageways 6 and a curve formed by an exposure opening 7, which
curve functions to ensure that each radially oriented capillary
tube 2 positioned within the frame 4 is equally exposed to a signal
detection means discussed hereafter when the cartridge 1 is
positioned in a centrifuge discussed hereafter. Each shaft 20 is of
a thickness sufficient for slidingly fitting a capillary tube 2
therein. Each receiving chamber 19 and shaft 20 combination is
cross-sectionally open along the top plane of the frame 4. This
results in a cross-sectional parabolic shape for the shaft 20 that
is about 5 millimeters deep. The cross-sectional cut is a byproduct
of the design of a tool used to manufacture the frame 4. However,
the cross-sectional cut of the shaft 20 also functions to expose
any damage to a capillary tube 2 held therein. At the end of the
shafts 20 are openings 21, as shown in FIG. 5, through which the
capillary tubes 2 pass for presentation to the exposure opening 7.
As discussed previously, the exposure opening 7 forms a curve that
corresponds to the radial orientation of the capillary tubes 2 so
as to ensure equal exposure of the capillary tubes 2 to a signal
detection means discussed hereafter. The exposure opening 7 is of a
width such that preferably about an 8 millimeter portion of each
capillary tube 2 is exposed along the curve formed by the exposure
opening 7 when secured within the frame 4 by the holder 5 discussed
hereafter. The long side 9 comprises a lip preferably about 1.5
millimeters deep and preferably about 3.2 millimeters high wherein
the `L` formed by the lip faces towards the reverse side of the
frame 4. The dimensions of the lip are such that the long side 9 is
provided sufficient structural support to make it substantially
rigid. In this preferred embodiment, the lip extends into the frame
4 on either end about 3.2 millimeters to a curved end of the two
side walls 11, 12. At the top of the curved ends of each of the two
side walls 11, 12, is a semicircular resting ridge 22, 23, which
extends preferably about 1.6 millimeters from the edge of the two
side walls 11, 12 along the same plane. The resting ridges 22, 23
function to ensure that the cartridge 1 does not rock when laid on
a flat surface with the reverse side of the frame 4 facing toward
the surface. The function of the resting ridges 22, 23 is necessary
to consistently orient the capillary tubes 2 at a proper angle to a
signal detection means when the cartridge 1 containing the
capillary tubes 2 is inserted into a centrifuge, discussed
hereafter, which permits exposure to the signal detection means
discussed hereafter. Along the long side 9 are four openings 24 for
receiving the distal ends of the capillary tubes 2 and extending
from the long side 9 at each receiving opening 24 are the tabs 8
for protecting the tips of the capillary tubes 2. Each tab 8
extends preferably about 3.2 millimeters from the long side 9 at
the same angle as the corresponding capillary tube 2, which extends
through the receiving opening 24 on the long side 9 such that the
tip of each tab 8 is substantially flush with the tip of each
capillary tube 2. As depicted in FIG. 4, the front of the frame 4
has a bevel 25, 26 at either end of the exposure opening 7
positioned about 8 millimeters from the corners formed by the lip
of the long side 9. Each bevel 25, 26 runs parallel to the long
side 9, extends from each side wall 11, 12 to the exposure opening
7, and slopes at approximately a 45 degree angle for about 3
millimeters toward the obverse side of the frame 4 and toward the
long wall 10. The bevels 25, 26 function to permit the long side 9
to support the distal ends of the capillary tubes via the receiving
openings 24 such that they are parallel to the plane of the frame 4
while minimizing the use of plastic and additionally function to
help ensure proper exposure of each capillary tube 2 to the signal
generation and detection means discussed hereafter.
[0147] Turning again to FIG. 5 and FIG. 6, a pair of parabolically
shaped female shafts 27, 28 can be seen. The female shafts 27, 28
comprise a parabolic element 29, 30, with the apex of the parabola
pointing toward the long wall 10 and a bendable clip 31, 32 with a
lip 33, 34. Each female shaft 27, 28, is preferably located about
6.4 millimeters from the long wall 10 and proximate to each side
wall 11, 12 and extends away from the reverse side of the frame 4
for about 8 millimeters at a 90 degree angle. The bendable clips
31, 32 in combination with the lips 33, 34 function as a locking
mechanism to attach the cartridge 1 via the female shafts 27, 28 to
an apparatus discussed hereafter designed for use with the
cartridge 1.
[0148] FIG. 4 shows the four capillary tubes 2 preferably coated
with antigen. Each capillary tube 2 is preferably made of
transparent silicon dioxide glass, more preferably borosilicate
glass, and is about 10 millimeters to about 250 millimeters,
preferably about 20 millimeters to about 150 millimeters, and more
preferably about 35 millimeters long with an internal diameter of
about 0.5 millimeters to about 1 millimeters, preferably about 0.65
millimeters. It should be noted that when the sample of a preferred
embodiment is milk, an internal diameter of the capillary tube 2
smaller than a preferred value of 0.65 millimeters can be
insufficient for the immunoassay, since aggregations of materials
in the milk can clog the capillary tubes 2 and interfere with fluid
flow. The capillary tubes 2 frictionally fit into the receptacle
discussed hereafter at one end and slidingly fit into the
passageways 6 of the frame 4 on the other end.
[0149] FIG. 7 is an exploded view of the top side of the holder 5,
and FIG. 8 is an exploded view of the underside of the holder 5 for
holding four capillary tubes 2 to be inserted into the frame 4. The
holder 5 comprises a receptacle 35 and a cap 36. The receptacle 35
is preferably made of flexible Krayton.RTM. and comprises a flat
rectangular support 37 preferably about 8 millimeters wide by about
3 centimeters long and about 1.6 millimeters thick, two guide
openings 38, 39, and four collars 40 connected to the support 37.
The collars 40 function to frictionally hold the capillary tubes 2
for insertion into the passageways 6 of the frame 4 with aid from
the guide openings 38, 39 which slidingly engage the guide pins 15,
16 located on the protruding segment 13 of the frame 4. The
diameter of the guide openings 38, 39 is about the same as the
external diameter of the first guide pin 15 located on the
protruding segment 13 of the frame 4, preferably about 3.4
millimeters. Each collar 40 has a narrow passageway 41 therein with
an internal diameter sufficient to permit a capillary tube 2 to be
inserted and held by friction therein, a cone 42 that functions as
a guide for inserting the capillary tubes 2, and an opening 43 to
permit insertion of the capillary tubes 2. The combination
receptacle 35 and capillary tubes 2 slide into the frame 4 via the
four openings 18 on the protruding segment 13. The collars 40 are
of dimensions such that they can slidingly fit into the
corresponding receiving chambers 19 of the frame 4 as referenced
previously. For the purpose of description, the collars 40 can be
divided into two pairs corresponding to the inner and outer pairs
of the passageways 6 within the frame 4. The inner pair of collars
40 are preferably about 4.8 millimeters long and extend radially
away from the center of the support 37 at the same angle as the
corresponding inner pair of passageways 6 of the frame 4. The outer
pair of collars 40 are preferably about 6.4 millimeters long and
extend radially away from the center of the support 37 at the same
angle as the corresponding outer pair of passageways 6 of the frame
4. The varying lengths of the collars function to extend the outer
pair of radially oriented capillary tubes 2 such that the distal
ends of all the capillary tubes 2 form a line parallel to the long
side 9 of the frame 4 so that each capillary tube 2 penetrates each
sample well discussed hereafter at an equivalent depth. Each collar
40 is preferably about 3.2 millimeters in diameter and flares out
at the cone 42 about 3 millimeters from the end to the opening 43
which has an external diameter of about 4.8 millimeters and an
internal diameter of about 3.2 millimeters.
[0150] FIG. 7 and FIG., 8 show the rectangular cap 36, preferably
made of rigid polystyrene, for securing the receptacle 35 and
capillary tubes 2 combination in the frame 4. The cap 36 can be of
any dimension suitable to cooperatively engage the receptacle 35
and protruding segment 13 of the frame 4, but preferably comprises
two long walls 44, 45, about 3.8 centimeters long by about 5
millimeters high; two short walls 46, 47 about 4.8 millimeters high
by about 9.5 millimeters wide at the top sloping down to about 8
millimeters wide at the bottom; and a top 48 about 3.8 centimeters
long by about a 9.5 millimeters wide. Located proximate to the
short walls 46, 47 are two asymmetrical guide shafts 49, 50, a
first guide shaft 49 and a segmented second guide shaft 50, each
for slidingly engaging the guide pins 15, 16, respectively, on the
protruding segment 13 of the frame 4 in combination with the guide
openings 38, 39 of the receptacle 35. The first guide shaft 49
extends above the top 48 to form a ridge about 0.5 millimeters to
about 3 millimeters, preferably about 1.5 millimeters high; has an
internal diameter of between about 2.1 millimeters to about 5.1
millimeters, preferably about 3.1 millimeters or an internal
diameter such that it slidingly engages the first guide pin 15
located on the protruding segment 13 of the frame 4; and is
preferably between about 1.5 millimeters to about 5 millimeters
deep or of a sufficient depth such that the top of the first guide
pin 15 is flush with the top of the first guide shaft 49 when the
two are cooperatively engaged as referenced previously. The
segmented second guide shaft 50 extends above the top 48 to form a
ridge about 0.5 millimeters to about 3 millimeters, preferably
about 1.5 millimeters high; has an internal diameter of between
about 2.1 millimeters to about 5.1 millimeters, preferably about
3.1 millimeters or of a sufficient diameter to slidingly engage the
base of the segmented second guide pin 16 located on the protruding
segment 13 of the frame 4, and extending from the end facing the
space formed by the four walls 44, 45, 46, 47 to a ledge 51 located
at about a half way point of the segmented second guide shaft 50.
From the ledge 51 to the top end of the segmented second guide
shaft 50, the segmented second guide shaft 50 has an internal
diameter between about 1.7 millimeters to about 4.7 millimeters,
preferably about 2.7 millimeters or a sufficient internal diameter
to slidingly engage the upper portion of the segmented second guide
pin 16 located on the protruding segment 13 of the frame 4. The
segmented second guide shaft 50 is preferably between about 1.5
millimeters to about 5 millimeters deep or of a sufficient depth
such that the top of the segmented second guide pin 16 located on
the protruding segment 13 of the frame 4 is flush with the top of
the segmented second guide shaft 50 when the two are cooperatively
engaged as referenced previously. In any case, the segmented second
guide shaft 50 should have a ledge 51 that defines two distinct
internal diameters such that the segmented second guide shaft 50 is
complementary to the corresponding segmented second guide pin 16
located on the protruding segment 13 of the frame 4 and can
slidingly engage therein. The position of the asymmetrical guide
shafts 49, 50 is such that they properly align with the guide pins
15, 16 and guide openings 38, 39. The difference of the dimensions
of the two asymmetrical guide shafts 49, 50, functions to permit
only one orientation for engaging the holder comprising the cap 36
and receptacle 35 with the frame 4 as previously discussed. That
is, the segmented second guide shaft 50 can only engage the
segmented second guide pin 16 located on the protruding segment 13
of the frame 4 and cannot engage the first guide pin 15 similarly
located. This ensures that the opening of a single port 54,
discussed hereafter, located on the cap 36 faces away from the
obverse side of the frame 4 when the holder 5 and the frame 4 are
fully assembled to form the cartridge 5. Attached to the top 48 of
the cap 36 is a trough 52 preferably about 2.2 centimeters long by
about 1.6 millimeters wide and preferably about 1.6 millimeters
deep and opens into the space created by the four walls 44, 45, 46,
47. The trough 52 is designed such that when it contacts the
support 37 of the receptacle 35, it forms a sealed chamber so that
the capillary tubes 2 are in fluid communication with each other.
The seal is provided by an narrow oval ridge 53, shown in FIG. 8,
that encircles the trough 52 about 2 millimeters from the edges of
the trough 52 in this preferred embodiment, but can be any
appropriate distance around the trough 52; and about 3 millimeters
from the ends of the trough 52 and presses against the support 37
of the receptacle 35. Attached to the trough 52 at its center is a
single port 54, shown in FIG. 7, with an internal diameter
preferably about 3.2 millimeters which can be connected to a luer
fitting discussed hereafter for pumping fluid through the chamber
formed by the trough 52 and receptacle support 37. The single port
54 is about 8 millimeters long and is oriented such that its
passage runs parallel to the horizontal plane of the top 48 while
leading away from the trough 52 at a perpendicular angle as shown
in FIG. 7. An opening 55 connecting the single port 54 to the
trough 52 is located at the center of the trough 52 as shown in
FIG. 7. Each longitudinal wall 44, 45 of the cap 36 contains three
equally spaced rectangular openings 56 which fit over the three
equally spaced clips 14 located on each of the lengthwise edges of
the protruding segment 13 located on the frame 4 as discussed
previously. The cooperation between the openings 56 and the clips
14 functions to secure the cap 36, receptacle 35, and capillary
tubes 2 combination over the protruding segment 13 of the frame 4;
with the aid of the guide pins 15, 16, the guide openings 38, 39,
and guide shafts 49, 50; and with additional aid of the angle of
the long cap walls 44, 45 formed by the short cap walls 46, 47. The
combination frame 4, capillary tubes 2, receptacle 35, and cap 36
therefore comprise the cartridge 1 in this preferred
embodiment.
[0151] FIG. 9 and FIG. 10 are perspective views of a preferred
embodiment of the sample tray 3 which is preferably made of
polypropylene. The sample tray 3 comprises four equally spaced
wells 57 for holding aliquots of a fluorescently-labeled conjugate
reagent and/or a sample, a reservoir 58 for holding fluid, such as
liquid waste; and a molded cartridge storage compartment 59. The
sample tray 3 forms a square preferably about 6 centimeters on all
sides. The wells 57 are supported by a shelf 60. The shelf 60
extends from a wall 61 running the full width of the tray 3, and
attached to two side walls 62, 63. The wall 61 from which the shelf
61 extends is about 6 centimeters long and preferably about 9.5
millimeters high. The. two side walls 62, 63 are each preferably
about 2.5 centimeters long and preferably about 1.4 centimeters
high. The shelf 60 originates from the long wall 61 preferably
about 1.6 millimeters below the top edge of the wall 61 such that a
lip 64 is formed to aid in retention of any escaped liquid. The
shelf 60 extends from the wall 61 at a slight downward angle that
functions to guide escaped liquid to the reservoir 58 discussed
hereafter and extends for about 9.5 millimeters and attaches to a
wall 67 perpendicular to it which drops from it to the floor 68 of
the reservoir 58 discussed hereafter. Each well 57 located on the
shelf 60 has a parabolic shape with an internal diameter preferably
about 8 millimeters and a depth preferably about 4.7 millimeters.
Held within at least one well 57 is a metallic object comprising a
stainless steel washer 65 for mixing. By passing a varying magnetic
field under the washer 65, a dried fluorescently-labeled conjugate
reagent resting on the bottom of the well 57 and optionally adhered
to the bottom of the well 57, is reconstituted when a solution
(optionally containing a sample) is added to the well 57. About a
4.8 millimeter diameter retaining ridge 66 is located along the
opening to each well 57 and functions to prevent the washer 65 from
falling out of the well 57.
[0152] The reservoir 58 is defined by a wall 67 which drops about 5
centimeters from the edge of the shelf 60, as discussed previously,
to a floor 68; the portions of the side walls 62, 63 extending from
the shelf 60, and a molded center wall 69 which rises preferably
about 1.3 centimeters from the floor 68 such that it is about flush
with the tops of the side walls 62, 63 and also forms part of the
cartridge storage compartment 59 discussed hereafter. The reservoir
58 functions to contain fluid, discussed previously, passed through
the capillary tubes 2 via the single port 54 on the cap 36 of the
cartridge 1. The volume of the reservoir 58 is defined by the
length and height of the wall 67 and distance from the wall 67 to
the molded center wall 69 and should be of sufficient dimensions to
contain an appropriate multiple of the combined volume of fluid
capable of being contained by all the capillary tubes 2 contained
within the cartridge 1.
[0153] FIG. 9 and FIG. 10 also show the molded cartridge storage
compartment 59 which forms part of the sample tray 3. The
compartment 59 is designed to form a molded contour around the
cartridge 1 and securely retain the cartridge 1 within the sample
tray 3 for storage and transportation as shown in FIG. 2. The
compartment 59 is defined on its periphery by the molded center
wall 69, a rear wall 70 opposite the center wall, two partial walls
71, 72, and a floor 73. The center wall 69 is molded to conform to
the dimensions defined by the portion of the cartridge 1 comprising
the long wall 10 of the frame 4, and the cap 36 secured on the
protruding segment 13 of the frame 4 as shown in FIG. 2. At the
center of the center wall 69 is a curved section 74 forming a half
circle which curves toward the shelf 60 and is of such dimensions
that it can slidingly receive the portion of the cartridge 1
defined by the single port 54 wherein the arc defined by the curve
of the curved section 74 is substantially similar to the arc of the
curve defined by the single port 54 such that the curved section 74
substantially forms a uniform contact with the periphery of the
single port 54. The center wall 69 extends linearly on either side
of the curved section 74, bends perpendicularly on either side
towards the rear wall 70 to form a mold that encases the cap 36 of
the cartridge 1, and bends again perpendicularly on either side
towards and attaches to the two side walls 62 and 63. In this
manner the center wall 69 forms a mold that substantially forms a
uniform contact with. the surfaces of the cartridge formed by the
side of the cartridge 1 containing the holder 5. Located near to
either edge of the curved section 74 and facing towards the rear
wall 70 are a pair of fastening clips 75, 76 for securely holding
the cartridge 1 within the storage compartment 59 in cooperation
with the pair of retaining shelves 83, 84 discussed hereafter. The
fastening clips 75, 76 are positioned at a height above the floor
73 such that they will engage the edge of the cap 36 comprising the
corner formed by the front wall 44 and top 48 of the cap 36 of the
cartridge 1 when the cartridge is positioned within the compartment
59 of the sample tray 3 with the reverse side facing the floor 73
as shown in FIG. 2. The two partial walls 71, 72 extend from the
rear wall 70 for about 1 centimeter and are of the same height as
the rear wall 70. At each corner formed by the partial walls 71, 72
and the rear wall 70 is a column 77, 78 extending from the floor 73
to the edge of the walls 70, 71, 72. Each column 77, 78 is adjacent
to each corner at two sides, forms a side 79, 80 extending
perpendicularly away from each of the partial walls 71, 72 and
towards each other for a distance about the same as the distance
from each of the corners formed by side 9 on the frame 4 of the
cartridge 1 to the base of the outer pair of protective tabs 8, and
angles towards the rear wall 70 at an angle corresponding to the
angle of the outer pair of protective tabs 8 to form an angled side
81, 82. The columns 77, 78 function to hold the side of the
cartridge 1 comprising the long side 9 of the frame 4 and
protective tabs 8 extending from the long side 9, whereby the tips
of the protective tabs 8 rest against the rear wall 70 and the
corners of the long side 9 of the cartridge 1 rest against the
column sides 79, 80 and the partial sides 71, 72 as shown in FIG.
2. Extending from each of the corners formed by the partial walls
71, 72 and the column sides 79, 80 is a narrow retaining shelf 83,
84 that forms a square with sides of equal width to the adjacent
column side 79, 80 and positioned at a height above the floor 73
such that the obverse planer portion adjacent to the corners
nearest the protective tabs 8 of the cartridge 1 rest against the
underside of each of the retaining shelves 83, 84 so as to secure
the cartridge 1 within the storage compartment 59 of the sample
tray 3 as discussed previously and as shown in FIG. 2. Connecting
the base of each of the corners formed by central wall 69 and short
walls 62, 63 to the ends of the partial walls 71, 72 is a curved
ledge 85, 86 that functions to aid removal of the cartridge 1 from
the storage compartment 59. The curved ledges 85, 86 curve inward
to form an arc complementary to the shape of human finger and rises
above the floor 73 to a height sufficient for portions of the edges
of the side walls 11, 12 of the frame 4 of the cartridge 1 to rest
against the curved shelves 85, 86 when secured within the storage
compartment 59 of the sample tray 3 as shown in FIG. 2. Extending
inward from the rear wall 70 and along converging axis are three
braces 87, 88, 89 for providing support for the cartridge 1 while
positioned in the storage compartment 59 of the sample tray 3 by
bracing against the obverse junctions of the receiving chambers 19
of the frame 4 and the edge of the lip formed by the long side 9 of
the frame 4. The braces 87, 88, 89 are of dimensions that permit
distal contact of the receiving chamber 19 junctions while
providing a path for the narrow shafts 20. In a preferred
embodiment shown, the three braces 87, 88, 89 comprise three narrow
walls, but can also comprise three stubs along the rear wall 70 and
three stubs to contact the junctions previously described. Along
the outer edges of each of the external walls 62, 63, 70, 71, 72 is
a lip 90 about 3 millimeters wide that aids the physical support of
the sample tray 3.
[0154] FIG. 11 is a perspective view of another embodiment of a
sample tray 103. The sample tray 103 comprises four equally spaced
wells 157 supported on a shelf 160 for holding aliquots of a
fluorescently-labeled conjugate reagent and/or a sample as in a
preferred embodiment discussed above, and a reservoir 158, for
holding fluid, such as liquid waste; defined by the shelf 160,
three walls 162, 163, 170, and a floor 173. The sample tray 103
forms a square preferably of the same dimensions as a preferred
embodiment of the tray 3 discussed previously. The two walls 162,
163 that run perpendicular to the shelf 160 are preferably about
1.4 centimeters high and the wall 170 opposite the shelf 160 is
preferably about 9.5 millimeters high. Extending along the top edge
of walls 162, 163, 170 is about a 3 millimeter lip 190 which aids
physical support of the sample tray 103. The four wells 157 are
located along the shelf 160 in an equally spaced manner, are
preferably of the same dimensions as the wells 57 of a preferred
embodiment previously discussed. The wells 157 preferably contain a
stainless steel washer or disc 165 for mixing a dried reagent
resting on the bottom of the well 157, optionally, the reagent can
be coated on the washer. The reagent is reconstituted when a
solution optionally containing a sample is added to the well 157.
Wells contain a retaining ridge 166 similar to the retaining ridge
66 of a preferred embodiment previously discussed. The shelf 160 is
preferably of the same dimensions as the shelf 60 of a preferred
embodiment discussed above with a wall 167 extending to the floor
173 preferably similar to the corresponding wall 67 of a preferred
embodiment discussed above.
[0155] In this alternative embodiment depicted in FIG. 11, the
reservoir 159 is defined by the three walls 162, 163, 170, the
shelf 167, and the floor 173 and, as stated above, similarly
functions to contain fluid, such as liquid waste, as does the
reservoir 59 of a preferred embodiment discussed above. Located
preferably about 3.2 centimeters from the edge of the shelf 160 is
a bevel 195 extending across the reservoir 159 from one side to the
other 162, 163. The bevel 195 is preferably about 4.7 millimeters
wide and slopes at about a 45 degree angle to form a second shelf
196 about 3.2 millimeters above the floor 173 and about 1
centimeter deep. The bevel 195 functions to prevent fluid,
typically liquid, from being splashed against the rear wall 170
when the sample tray 103 is tipped. The cartridge 1 can also be
stored in this embodiment of the sample tray 103 by laying it flat
on the bottom 173 such that the protective tabs 8 face the rear
wall 170. The cartridge 1 can be secured within the sample tray 103
by a plastic cover or by similar means.
[0156] FIG. 12 is a perspective view of a preferred embodiment of
the apparatus 200 for use with the cartridge 1 and sample tray 3
combination of the present invention. FIG. 13 is a transparent view
of a preferred embodiment of the apparatus 200 showing the critical
internal components. Referring to FIGS. 12 and 13, the apparatus
200 comprises a prep station 201, a centrifuge 202 for drying the
capillary tubes 2, a signal generation and detection station 203
for measuring a signal, an alphanumeric keypad controller 204 for
permitting interaction between an operator and the apparatus 200, a
liquid crystal display (LCD) 205 for displaying input and output
messages and analytical results produced by a computing device
discussed hereafter, and a printer 206 for printing a result.
[0157] Referring again to FIG. 12 and FIG. 13 the prep station 201
comprises a reservoir 207 for containing a fluid, a conduit 208 for
transporting the fluid to a port 209, a syringe 210 for drawing
sample to the port 209 and for pumping fluid from the reservoir 207
through the port 209; a first section comprising a previously
referenced luer fitting 211 for attaching the single port 54 of the
cartridge 1 to and for permitting fluid communication with the port
209 of the apparatus 200; a second section comprising a tray holder
212 for holding the sample tray 3, and a magnetic mixer 213 for
mixing fluid in the wells 57. Magnetic mixer 213 is positioned
under the tray holder 212 in such a manner as to align with the
wells 57 of the sample tray 3 when the sample tray 3 is positioned
in the tray holder 212. The centrifuge 202 is housed in a
protective compartment 214 and comprises a disc capable of securely
engaging the cartridge 1. The electromagnetic signal generation and
detection station 203 comprises a signal generation means 215, such
as laser or tungsten lamp suitable to emit an electromagnetic
signal at an appropriate wavelength to cause a fluorescing compound
in the sample-reagent mixture to become excited and fluoresce, and
a signal detection means 216, such as a photon detector for
detecting fluorescence, which photon detector can comprise a
photomultiplier, phototube, photocell, or silicon diode, and
preferably comprises a silicon diode.
[0158] FIG. 14a is a block diagram of the apparatus of a preferred
embodiment of the invention showing cooperation among the
electronic parts and FIG. 14b is a similar block diagram of the
apparatus showing designations known to those of ordinary skill in
the art of the various components. A logic and motor control
printed circuit assembly (PCA) 300, hereafter referred to as a
control PCA, functions to control and interface with the various
components of the apparatus 200. A 40 wire ribbon cable 301
connected to two 40 position ribbon headers 302, 303 functions to
connect the control PCA 300 to a prep station breakout PCA 304 for
controlling the various components of the prep station 201.
[0159] The electrical components connected to the prep station PCA
304 comprise the following:
[0160] a syringe motor 305 for actuating and accurately controlling
movement of the syringe 210, which syringe motor 305 is connected
to the prep station PCA 304 via a first 8 position amplitude
modulator 306;
[0161] a mixer motor 307 for actuating the magnetic mixer 213,
which mixer motor 307 is connected to the prep station PCA 304 via
a second 8 position amplitude modulator 308;
[0162] a lift motor 309 for moving the tray holder 212 to properly
position the sample tray 3 under the cartridge 1, which lift motor
309 is connected to the prep station PCA 304 via a third 8 position
amplitude modulator 310;
[0163] a valve 311 for controlling the direction of flow of the
fluid contained in the reservoir 207, which valve 311 is connected
to the prep station PCA 304 by 6 position amplitude modulator
312;
[0164] a syringe sensor 313 for monitoring the position of the
syringe 210, which syringe sensor 313 is connected to the prep
station PCA 304 via a fourth 8 position amplitude modulator 314; a
mixer sensor 315 for monitoring the position of the magnetic mixer
213, which mixer sensor 315 is connected to the prep station PCA
304 via a fifth 8 position amplitude modulator 316; and
[0165] a lift sensor 317 for monitoring the position of the lift
motor 309, which lift sensor 317 is connected to the prep station
PCA 304 via a sixth 8 position amplitude modulator 318.
[0166] A 16 position amplitude modulator 319 connects the control
PCA 300 to three additional PCA cards, a laser PCA 320 for
controlling the operation of the signal generation means, which
laser PCA 320 is connected to the control PCA 300 via a 6 position
molex pocket header 321; a preamplifier PCA 322 for amplifying a
photogenerated signal before processing by the control PCA 300,
which preamplifier PCA 322 is connected to the control PCA 300 via
a 5 position molex pocket header 323; and an barcode PCA 324 for
interfacing with a commercially available barcode wand discussed
hereafter, which barcode PCA 324 is connected to the control PCA
300 via a 4 position molex pocket header 325. An 18 position
amplitude modulator 326 connects the various electrical components
of the centrifuge 202 to the control PCA 300. The electrical
components of the centrifuge 202 comprise a rotor motor 327 for
rotating the centrifuge 202, a rotor door sensor 328 for detecting
the opening of the door of the protective compartment 214, and a
rotor home sensor 329 for aiding with determination of the position
of the cartridge 1 while in the centrifuge 202 so as to permit
proper presentation of the cartridge 1 to the signal generation and
detection station 203. A 50 wire ribbon cable 330, connected to two
50 position ribbon headers 331, 332, connects a peripheral adapter
PCA 333 that functions to interface with the alphanumeric keypad
204, the printer 206, the liquid crystal display 205, and an audio
buzzer 334 for providing audible feedback to the operator about the
functional status of the apparatus 200. A panel connector PCA 335
located at the rear of the apparatus 200 interfaces with an
external DC power input connector 336, an RS-232 port 337, and a
barcode wand input connector 338. The panel connector PCA 335 is
connected to the control PCA 300 via a 10 wire ribbon cable 339
connected at each end to a 10 position ribbon header 340, 341; and
via a power cable 342. Main power to the apparatus 200 is supplied
via serial cooperation among a 110 input plug 343, a power entry
module 344, a signal transformer 345, and an AC input cable 346 as
shown in FIG. 14a and 14b. Two 2 position mini-fit molex connectors
347 connect the AC power cord 346 and the DC power cord 342 to the
control PCA 300. The automated non-operator dependent portion of
the apparatus 200 system software is provided by an erasable
programmable read only memory (EPROM) module 348 connected to the
control PCA 300 via a 30 position edge connector 349.
[0167] A Typical Immunoassay Using Preferred Embodiments of the
Present Invention
[0168] FIG. 15 is a simplified representation of the solid-phase
fluorescence immunoassay (SPFIA) of a preferred embodiment of the
present invention. In a typical immunoassay using a preferred
embodiment of the present invention, the cartridge 1 and sample
tray 3 are placed on the prep station 201 by the operator as shown
in FIG. 12, then 100 microliters of a sample possibly containing an
analyte of interest, such as milk suspected of containing
.beta.-lactam antibiotics, is added to one or more wells 57 located
in the sample tray 3 which contain a dried stabilized reagent
comprising an antibody to the analyte of interest, which antibody
is conjugated with a highly fluorescent label, such as a cyanine
dye (Cy-5.RTM.), fluorescein isothiocyanate (FITC), rhodamine,
Texas Red, phycoerythrin, and allophycoerythrin or any other
compound with a sufficient quantum yield or efficiency to produce
appropriate fluorescence and that will not interfere with any
reactions of the immunoassay; or with a bio-luminescent compound. A
barcode, provided with the cartridge 1 and sample tray 3
combination kit, is then scanned using a standard commercially
available barcode wand associated with the barcode wand input
connector 338 to provide calibration information to the apparatus
200. For example, the barcode can contain the pass/fail threshold
level of fluorescence for a particular immunoassay. The operator
then inputs additional information via the alphanumeric keypad
controller 204 and subsequently starts the apparatus 200. The
sample and fluorescently-labeled conjugate reagent are then mixed
while in the well 57 by the application of a magnetic field
provided by the magnetic mixer 213 located under the tray holder
212 of the apparatus 200, such that the metallic washer 65 flips,
rotates or agitates to appropriately mix the sample and conjugate
reagent combination for a sufficient time for the
fluorescently-labeled conjugate and the analyte to bind. The
cartridge 1 is connected via the single port 54 on the cap 36 of
the cartridge 1 to the luer fitting 211 of the prep station 201 of
the apparatus 200 and positioned above the sample tray 3 with the
obverse side of the cartridge 1 facing the reservoir 58. Within the
cartridge 1 each capillary tube 2 has at least a portion of the
internal surface coated with antigen analogous to or mimetic of the
analyte of interest. The sample tray holder 212, and thereby the
sample tray 3, is lifted by the lift motor 309 so that the tips of
the capillary tubes 2 contact each sample and fluorescently-labeled
conjugate reagent mixture in the wells 57. The sample tray 1 is
oriented in a manner which permits the cartridge 1 to be positioned
such that the capillary tubes can penetrate the sample and
fluorescently-labeled conjugate reagent mixture at a sufficient
depth and at a sufficient angle to draw a sufficient amount of the
sample and fluorescently-labeled conjugate reagent mixture into
each capillary tube 2. The sample and fluorescently-labeled
conjugate reagent mixture is then drawn into the capillary tube via
capillary action and suction applied by the syringe 210 to the
single port 54 on the cap 36 of the cartridge 1, and is then
allowed to incubate for a period of time in one or more capillary
tubes 2 during which a percentage of fluorescently-labeled
conjugate not bound to the analyte, will bind to the antigen
comprised by the substrate comprising the capillary tube 2 walls.
If there is a relatively large amount of analyte in the sample,
very little fluorescently-labeled conjugate will bind to the
substrate of the capillary tube 2 walls and if there is a
relatively small amount of analyte in the sample a large amount of
fluorescently-labeled conjugate will bind to the substrate of
capillary tube 2 walls.
[0169] After the incubation period, a liquid residing in the
reservoir 207 of the apparatus 200 is pumped by the syringe 210
through each capillary tube 2 via the luer fitting 211 and the
single port 54 located on the cap 36. The liquid then enters the
chamber created by the trough 52 and receptacle support 37 and
subsequently enters each capillary tube 2. The liquid stops the
reaction and washes excess fluid out of each capillary tube 2.
Prior to or simultaneous with the pumping of the liquid into each
capillary tube 2, the lift motor 309 orients the sample tray holder
212, and thereby the sample tray 3, such that the capillary tubes 2
are positioned over the reservoir 58 within the sample tray 3, so
that the fluid pumped out of each capillary tube 2 is expelled into
the waste reservoir 58 of the sample tray 3. The sample tray 3 is
then discarded and the cartridge 1 is inserted into and dried by
the centrifuge 202 located on the apparatus 200 for use with the
cartridge 1 and sample tray 3 combination. After drying and while
in the centrifuge 202, the cartridge 1 is positioned in front of
the signal generation means 215, such that the exposure opening 7
of the frame 4 permits the signal generation means 215 to emit a
signal that substantially penetrates the capillary tube 2. The
signal detection means 216 positioned adjacent to the signal
generation means 215 then detects any fluorescence emitted by the
bound conjugate containing the fluorescent label. This signal is
then processed by the computing portion of the apparatus 200 and
the presence of analyte, alternatively the amount of analyte
present is determined where analyte concentration is inversely
proportional to the level of fluorescence for this preferred
embodiment (a competitive assay). FIGS. 16a-d to FIGS. 17a-b show
plots of normalized fluorescence versus concentration of six
.beta.-lactam antibiotics in parts per billion at different levels
of concentration. A least squares polynomial fit of the curves
generated by the multiple points can be used by the apparatus 200
to provide a quantitative result, wherein the computing portion of
the apparatus 200 interpolates the amount of analyte in the sample
by plotting the observed level of fluorescence on the appropriate
calibration line for the particular analyte and identifying the
corresponding concentration for that level of fluorescence.
[0170] It should be noted that in a presently preferred embodiment
of the present invention, the operator only carries out the steps
of placing the sample tray 3 in the sample tray holder 212, adding
sample to the sample tray 3, optionally scanning the barcode,
starting the apparatus 200, moving the cartridge 1 to the
centrifuge 202, and discarding the cartridge 1 and sample tray 3.
The apparatus 200, performs all the other steps of the immunoassay
discussed above.
[0171] Alternative Embodiments and Variations of Preferred
Embodiments
[0172] Although the present invention has been described in
considerable detail with reference to a preferred embodiments
thereof, other embodiments are possible. For example variations of
either one or all of the immunoassay chemistry used, the cartridge
and sample tray design, and the design of the apparatus for use
with the cartridge and tray combination are possible within the
scope of the present invention. A preferred variation for the
immunoassay chemistry is simply to reverse the label and binding
member in the competitive fluorescence immunoassay of a preferred
embodiment of the invention. That is, the fluorescently-labeled
conjugate reagent can contain an antigen analogous to or mimetic of
the analyte, wherein the antigen is conjugated with the fluorescent
label; and the antibody to the analyte is coated onto the surface
of the capillary tubes. In this case, the fluorescently-labeled
antigen conjugate competes with the analyte antigen for binding
sites on the antibody coated on the surface of the capillary tubes.
As in the competitive fluorescence immunoassay of a preferred
embodiment of the present invention, the level of fluorescence will
be inversely proportional to the amount of analyte in the
sample.
[0173] In addition to the competitive fluorescence immunoassay of
preferred embodiments described above, a non-competitive sandwich
fluorescence immunoassay can also be used. In this case, the sample
is mixed with a fluorescently-labeled conjugate reagent and drawn
into the capillary tube. The analyte binds to the conjugate and to
a capture binding member on the substrate which comprises a surface
of the capillary tube. The capillary tube is washed, optionally
dried, and exposed to a signal generation means and signal
detection means to measure the level of fluorescence. In a sandwich
immunoassay such as this, the amount of fluorescence is directly
proportional to the amount of analyte in the sample.
[0174] While an immunoassay described above as a preferred
embodiment is a competitive inhibition immunoassay employing a
fluorescent label, other immunoassay methods can also be employed.
These include ELISA sandwich immunoassays wherein an analyte sample
is mixed with free antibody to the antigen and subsequently washed
and further mixed with an enzyme linked antibody specific to a
different epitope on the analyte. Any free antibody is then washed
away and an enzyme substrate is added to complex with the enzyme
mixture. Detection of the analyte can then be effectuated
colorimetrically or via other means whereby the level of the signal
is positively correlated to the concentration of analyte
detected.
[0175] As noted above, variations on the immunoassay can include
different labels, such as enzymes for use in a non-competitive
immunoassay. Similarly, an enzyme label can be used in a
competitive immunoassay whereby detection of a signal generated by
a UV lamp occurs via a UV absorbance detector and the absorbance
reading is negatively correlated with the concentration of analyte
in the sample. Alternatively RIA immunoassays known to those of
ordinary skill in the art can be used with an appropriate
embodiment of the present invention.
[0176] The dimensions of the cartridge and sample tray can vary
from that already described depending on the particular application
and use. All that is required are dimensions appropriate for use
with an apparatus comprising any analytical instrument sufficient
to effectuate the chosen immunoassay. For example, these dimensions
include a length of capillary tube sufficient to contain an amount
of a substrate and sample mixture, a cartridge comprising a region
suitable to expose at least a portion of each tube to an emitted
signal, and a sample tray with wells therein sufficient to contain
sample and reagent solution. Furthermore, a reservoir can
optionally be included with a waste disposal means comprising a
vacuum system connected to the cartridge for instance, wherein the
evacuation product of the capillary tube contents are contained in
a separate container or chamber.
[0177] In many cases, the apparatus, the cartridge or tube design
will depend on the type of immunoassay employed, instrumentation
with which the subject interacts, and conditions under which the
subject is used. Immunoassays that can be adapted for use with the
present invention or variations thereof include ELISA, RIA, and FIA
as discussed previously, but can also include other bioassays,
including but not limited to various other immunoassays utilizing
different labels, such as enzymes, isotopes, fluorescent and
bio-luminescent compounds, physical constructs, reactants; and
other bioassays appropriate for use in capillary tubes.
Instrumentation can include custom or commercially available
analytical instruments including, but not limited, to
spectrophotometers, chromatographs for collection fractions from
the immunoassay, counters, densitometers, diagnostic instruments,
and forensic instruments; and other instruments necessary for use
with a particular embodiment of the present invention. Conditions
for use include, but are not limited to, clinical laboratories, use
in the field, such as outdoors and/or in agricultural, dairy, and
industrial settings; and in research or crime laboratories.
Alternative embodiments of the present invention can thus be used
to adapt to the above uses, conditions for use, and cooperation
with other devices.
[0178] The cartridge can comprise substantially one piece with the
coated capillary tubes frictionally sealed within the cartridge.
Alternatively, the capillary tubes can be sealed within the
cartridge by a means other than by friction, such as an adhesive
sealant. The capillary tubes can be spaced apart linearly rather
than radially. In this case, drying preferably occurs via a means
other than by centrifugation, such as by air drying, by purging
with a gas, such as nitrogen; or by vacuum. The cartridge can have
a plurality of chambers each independently connected to each
capillary tube. In this case an even greater diversity of
immunoassays can be carried out in each capillary tube, including
the use of different detectable events and detection means.
Protection of the capillary tubes can also be accomplished by
various means, such as use of a hinged cover that can be optionally
positioned over the tips of the capillary tubes and moved away from
the capillary tubes when fluid is drawn or added to the capillary
tubes, or the capillary tubes can be retractable into and out of
the cartridge. The region of the cartridge for exposing each
capillary tube to a signal detection means can also be designed so
that only one side of the cartridge is open since, in the case of
fluorescence immunoassays, the signal generation means and signal
detection means can be adjacent to each other. Additionally, a
clear plastic cover can be located over the exposure opening 7 to
protect the capillary tubes from damage.
[0179] One embodiment of a one piece design is a cartridge wherein
the capillary tubes are linearly spaced apart within the cartridge
with a central opening for exposing them to a signal detection
means. On one side of the capillary tubes can be a receiving
chamber for receiving a sample, such as milk. A syringe on the
other side of the capillary tubes can be used to draw sample into
the capillary tubes. To conduct the immunoassay sample in the
receiving chamber is placed in contact with the capillary tubes by
passageways that connect to individual reagent chambers in fluid
communication with each capillary tube. The reagent chambers can
contain reagent such that backward engagement of the syringe can
draw sample into these chambers and stop to permit mixture of
sample and reagent in each chamber by magnetic or other means
discussed herein or known to those of ordinary skill in the art.
Backward engagement of the syringe can then continue and be used to
draw the reagent sample mixture from the mixing/reagent chamber and
into the capillary tubes for incubation and an immunoassay reaction
to occur. The same syringe can be used to push a fluid into the
capillary tubes via a passage connected to the distal end of each
capillary tube, wherein a third chamber containing wash fluid for
instance, can be opened via a valve or puncture means such that
forward engagement of the syringe forces the fluid into the
passageway and through each capillary tube. In this embodiment a
drying step would not be required.
[0180] The cartridge can also be of an entirely different shape.
For example, the cartridge can comprise a disc wherein the
capillary tubes are spaced radially throughout the disc and a
sample can be added to chambers at the periphery of the disc in
such a manner that sample can be drawn into each capillary tube and
expelled into the first chamber by pumped fluid added to the tubes
via a port at the center of the disc; or other variations of a
circular cartridge.
[0181] Similarly, a circular disc can be used wherein the sample is
added to a first chamber near the center of the disc. Upon
application of centrifugal force, the sample, which can also react
with a reagent in the first chamber, can be drawn into capillary
tubes or passageways radially spaced apart within the disc wherein
one or more second chambers at the periphery of the disc and
fluidly attached to the capillary tubes can collect excess fluid or
provide for further reaction steps. Variations of this embodiment
can include additional capillary passageways and/or chambers.
Alternatively the cartridge can be shaped like a pie wedge such
that a plurality of cartridges can fit together to form a circle
for drying in a centrifuge or for permitting reactions to occur as
previously described.
[0182] Another embodiment is a "Gatling gun" design wherein the
capillary tubes are spaced apart along the outside of the
longitudinal side of a cylinder or by a disc or plurality of discs
containing openings to hold a plurality of spaced apart capillary
tubes in a circular manner. In this case the cylinder can rotate to
present a particular tube to a sample source or to a fluid source
for washing, adding reagent, or other desired fluid to each tube.
The cylinder can also rotate to present a capillary tube to a
signal generation and detection means. Alternatively, the cylinder
can cooperate with a multiwelled circular sample tray for serial or
simultaneous sample removal and/or washing and expelling of fluid
and even multiple signal generation and detection means. The
circularly spaced apart capillary tubes can encircle a central
syringe that is in fluid communication with the capillary tubes and
draws fluid into the capillary tubes and/or advances fluid from the
capillary tubes into any chambers and/or reaction sites on the
surface of the capillary tubes.
[0183] The cartridge can also comprise a substantially rectangular
shape where a plurality of capillary tubes are proportionately
spaced apart and arranged in two rows; e.g. tubes are spaced in a
cartridge that has an exposure opening on each side, which thereby
permits simultaneous detection of analyte in two or more samples.
In this embodiment, two pairs of signal generation and signal
detection means can be simultaneously used, wherein the cartridge
is designed such that each row of capillary tubes can be
simultaneously or independently contacted by signal generation and
signal detection means when mounted on an appropriately designed
apparatus, such as an analytical instrument.
[0184] The cartridge can also be made of different types of plastic
such as, polyvinyl chloride, polyethylenes, polyurethanes,
polystyrenes, polypropylenes, and other plastic materials. The
capillary tubes can also optionally be made of plastic such as
polyvinyl chloride, polyurethanes, polystyrenes, polypropylenes,
polyethylenes and other plastic materials commonly used to form
capillary tubes or tubing. Preferably, the chosen material not
interfere with either the chemistry of the immunoassay or any
analytes or products thereof, or the signal generation and
detection means. Preferably the chosen material provides for an
inexpensive and therefore disposable cartridge design.
[0185] The sample tray can also vary from preferred and an
alternative embodiment described above. It can have more than 4
sample wells, larger or smaller sample wells, and even multiple
waste reservoirs or other chambers. Alternatively the waste
reservoir can comprise a sponge or similar absorbent material. It
can also comprise a vacuum system fluidly attached to a waste
reservoir as previously discussed, and of various materials, with
preference to inexpensive materials as discussed previously for the
cartridge.
[0186] Variations of preferred embodiments of the apparatus for use
with a preferred cartridge and sample tray combination as disclosed
herein are also possible within the scope of the present invention.
An important aspect of an alternative embodiment of the apparatus
for use with a preferred embodiment of the cartridge and sample
tray is that embodiments of the apparatus are able to communicate
with the cartridge and sample tray, that the apparatus is equipped
with appropriate electronics and/or optics for a given immunoassay,
and that the apparatus is capable of performing or interfaced with
an instrument capable of performing an automated immunoassay and of
computing qualitative, semi-quantitative, and/or quantitative
results. Another aspect of the apparatus for use with a preferred
embodiment of the cartridge and sample tray combination is that the
apparatus house or control a suitably substantial portion of the
means to move fluid in and out of the wells, capillary tubes, and
chambers.
[0187] Aspects of an alternative embodiment of the apparatus for
use with alternative embodiments of the cartridge and sample tray
will vary depending on the design of alternative embodiments of the
cartridge and sample tray, but preferably effectuate an immunoassay
in capillary tubes, and is preferably relatively simple, rapid,
reliable, and requires minimal operator interaction.
[0188] Several variations of a preferred embodiment of the
apparatus of the present invention can be employed for use with a
fluorescence immunoassay or other immunoassays. A vacuum system can
be employed in place of a syringe within or in cooperation with the
apparatus to remove fluid from the capillary tubes and/or the
sample tray. Similarly, the means to mix the sample and reagent can
be other than by magnetic field such as agitation by vibration or
by physical mixing.
[0189] Various types of spectroscopic hardware can also be
employed. Such variations are dictated by the immunoassay, analyte
of interest, and other criteria and would be obvious to one of
ordinary skill in the art. These variations include, but are not
limited to, use of different signal generation means, including,
but not limited to, argon lamps, xenon lamps, hydrogen lamps,
deuterium lamps, tungsten lamps, nernst glower, nichrome wire,
globar, and hollow cathode lamps or other appropriate signal
generation means capable of providing emitted signals covering
appropriate wavelengths in one or more regions of ultraviolet,
visible, near infrared, infrared, and far infrared light; various
wavelength selectors including, but not limited to, filters,
including interference filters and glass absorption filters, and
monochromators, including prism monochromators, such as fluorite
prism, fused silica or quartz prism, glass prism, sodium chloride
prism, and potassium bromide prism; and gratings; and various
signal detection means including, but not limited to,
photomultipliers, phototubes, photocells, silicon diodes, and
semiconductors.
[0190] Variations to the signal processing means can also be
employed to provide quantitative results, such as constant
automatic calibration of the signal generation and detection means,
use of a more sophisticated analog to digital converter, use of
multi-level calibration curves, e.g. a least squares fit of curves
such as those shown in FIGS. 16a-d to FIGS. 17a-b, and data
reduction and analysis that includes statistical analysis. The
apparatus can also include a computer disc drive for loading a
computer disc for storing a result provided by the signal
processing or computing means in a computer storable file. The
result can be transmitted via the RS-232 port on the apparatus to
an external computing or storage device for more sophisticated data
reduction and analysis. It should be noted that use of an erasable
programmable read-only memory (EPROM) module, as in a preferred
embodiment of the apparatus of the present invention, makes it
relatively simple to modify the software of the apparatus to adapt
it to various immunoassay formats, levels of data reduction and
analysis, and interaction with external and/or internal devices
and/or components.
[0191] Alternative embodiments of the apparatus of the present
invention can also include various levels of automation. One
embodiment of the apparatus of the present invention can be
designed such that only instrument control is possible with the
stand alone apparatus and all data reduction and analysis is
performed by a computer connected or networked to the apparatus via
a port, such as RS-232, IEEE-488 (HP-IB), contact closure, and the
like. Similarly, control of the apparatus can be automated by a
connection to a computer as previously described where all control
inputs are directed by the computer and initiated by an operator or
alternatively by a computer program.
[0192] Additionally, a preferred embodiment of the apparatus of the
present invention or variations of it can be interfaced with
laboratory robots, such as cylindrical, Cartesian, and articulated
robots and the like; to enable complete automation of the
immunoassay. The robotics device can be programmed to remove sample
from a central container and add it to each well and alternatively
to perform any pre-assay sample processing and preparation
necessary, such as filtration, extraction, dilution, removal of
certain components, and other manipulations depending on the
sample. The robotics device can also place the sample tray and
cartridge on the apparatus and subsequently move the cartridge to
the centrifuge or other device and dispose of the sample tray. The
robotics device can be integrated into an alternative embodiment of
the apparatus of the present invention or it can be a commercially
available robot for use in the laboratory and known to those of
ordinary skill in the art of the present invention.
EXAMPLES
Example 1
Capillary-Tube Surface Preparation
[0193] The surface of borosilicate, glass capillary tubes (Drummond
Scientific, Broomall, Pa.) was treated with a silanizing reagent in
accordance with the process for Aquasil.RTM. silanization coating
in accordance with Example 9, coating with a protein substrate
conjugated to a reactive protein, blocking the capillary tubes, and
drying and incubating. The lengths of individual capillary tubes
were 3.5 centimeters (cm) with an inner diameter of 0.65
millimeters (mm) and an outer diameter of 1 mm. By using these
capillary tubes, one achieves a high surface area to volume ratio
and minimizes the use of reagents. Other capillary tube dimensions
can be used. The high surface area to volume ratio allowed for a
short two-minute incubation. Tubes having other inner diameter
dimensions were also examined, however, the 0.65 mm inner diameter
tubing was found to be most useful with fresh, raw milk samples,
which often contain fat globules that can be as large as several
hundred micrometers (.mu.m). Such fat particles can potentially
clog the capillary tube channels if tubes of smaller inner diameter
were used. Additionally, the transport of reactants to the interior
surface of the tube, where the measurable binding reaction occurs,
is strictly through diffusion. Therefore, potential problems due to
irreproducible agitation are eliminated.
Example 2
Synthesis of Antigen Conjugates for Coating Capillary Tubes
[0194] The antigen conjugates, for coating onto the surface of the
capillary tubes, were prepared by binding the appropriate
.beta.-lactam drug, such as Penicillin G, Ampicillin, Cloxacillin,
Cephapirin, Ceftiofur, Amoxicillin and the like; to a carrier
protein either bovine serum albumin (BSA) or a polypeptide
copolymer consisting of lysine and alanine subunits (Sigma Chemical
Co., St. Louis, Mo.). The covalent linking of antigen to carrier
protein was accomplished through the use of conventional
homobifunctional or heterobifunctional linkers, such as
Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),
Bis(sulfosuccinimidyl) suberate, and
1-Ethyl-3-(3-Dimethylaminopropyl)-ca-
rbodiimideHydrochloride(Pierce Chemical Co., Rockford, Ill.) in
accordance with the process provided by the manufacturer.
Example 3
General Procedure for Coating Antigen-Conjugate on the Surface of
Capillary Tubes
[0195] After the silanizing surface treatment, the capillary tubes
were incubated for from about 30 minutes to 24 hours in a buffered
solution of the antigen-conjugate (20-40 .mu.g/ml). The incubation
temperature was usually 4-7.degree. C., although occasionally room
temperature incubation was used. The capillary tubes were removed,
washed with distilled water, dried in a stream of compressed air,
and then incubated in a solution of bovine-serum albumin (0.1% BSA
in PBS-phosphate buffered saline-pH=7.2, 0.05% Proclin 300
(Supelco) a biocide) for 1 hour at room temperature. The purpose of
this incubation with BSA was to block any solution regions of the
surface that were not coated by the antigen-conjugate. The tubes
were again removed, washed with distilled water and dried in a
stream of compressed air (20-30 psi). They were stored in the dark,
at room temperature in a sealed, foil pouch with an indicating
desiccant package. Maintaining dry and dark conditions were
important to maintain the stability of the coated capillary
tubes.
Example 4
Preparation of Fluorescent-Labeled Antibody Conjugates
[0196] a. Antibody Production.
[0197] Antibodies to the antibiotics in the penicillin family were
produced by immunizing goats with a conjugate of keyhole limpet
hemocyanin (KLH) (Sigma Chemical) and ampicillin (Sigma).
Ampicillin was used because it has an amino-group available
conjugation. The resulting bleeds were screened for
cross-reactivity with penicillin G, ampicillin, cloxacillin and
amoxicillin (Sigma). The cross-reactivity of this antibody with
penicillin G and ampicillin was comparable, while the
cross-reactivity for cloxacillin and amoxicillin was approximately
50% less. This antibody also exhibited less than 1%
cross-reactivity with ceftiofur (UpJohn Inc.) and cephapirin
(Sigma). Antibodies to ceftiofur were developed by immunizing goats
with KLH-ceftiofur. A monoclonal to cephapirin was developed using
conventional techniques with KLH-cephapirin as the conjugate.
Enzyme immunoassay studies indicated that the cross-reactivity of
the ceftiofur antibody to the other five .beta.-lactam drugs of
interest was less than 1%. The cross-reactivity of the cephapirin
antibody to the other five .beta.-lactam antibiotics was less than
0.1%.
[0198] b. Fluorescently-Labeled Antibody Conjugate Production.
[0199] Monoclonal and polyclonal antibodies were prepared using
conventional purification techniques as taught by C. Schmidt, 1989,
"The Purification of Large Amounts of Monoclonal Antibodies,"
Journal of Biotechnology, Volume 11, pp 235-252. Using the
fluorescent label Cy-5.RTM., Cy-5.RTM.-antibody conjugates were
prepared using the protocols previously published and provided by
the manufacturer (Biological Detection Systems, Pittsburgh, Pa.).
The Cy-5.RTM. fluorescent label, available with an NHS ester
functionality, was linked to amino groups of the antibody.
Purification and isolation of the conjugated dye was performed
through chromatography. Spectroscopic examination indicated that
the number of Cy-5.RTM. dye molecules that were bound to each
antibody molecule was usually between two and four. The ratio of
antibody molecule to Cy-5.RTM. molecule is controlled by modifying
reaction conditions such as time of conjugation and/or ratio of
Cy-5.RTM. concentration to antibody concentration in the reaction
mixture. Batch to batch studies indicated that there was no
significant increase in fluorescence intensity with increase in the
number of Cy-5 .RTM. molecules per antibody molecule. In fact, the
fluorescence intensity often decreased when the
fluorophore/antibody ratio was greater than four. This phenomenon
was especially apparent when using monoclonal antibodies. It is
unclear at this time whether this decrease was due to increased
quenching with high loading of the fluorophore or due to decreased
antibody binding affinity resulting from inactivation of the
antibodies' recognition sites.
Example 5
Process for Analysis for Analytes in Samples Immunoassay
Protocol
[0200] (a) A measured amount of fluorescently-labeled conjugate,
comprising antibody-Cy-5.RTM. conjugate (preferably dried, e.g.
lyophilized or air dried,) was combined with a sample of milk in a
small container, such as the well of a microtiter-plate. The raw
milk sample and antibody conjugate were mixed on a vibratory shaker
for 10 seconds.
[0201] (b) The solution from (a) was then sipped into the
appropriate capillary tube through the use of a manifold device,
such as a modified pipettor or an embodiment of the cartridge of
the present invention.
[0202] (c) The solution was then incubated, while in the capillary
tube, for a time sufficient to react with the antigen attached to
an interior surface of the tube, e.g. for 2 minutes.
[0203] (d) All reagents were then washed out of the capillary tubes
with flowing distilled water from the end opposite from which the
solution was brought in. The reagents were then dried with a stream
of compressed air (20-30 psi).
[0204] (e) The fluorescence intensity of the tubes was then
measured using a fluorometer equipped with a 3 mW
semiconductor-diode laser (.lambda.max=635 nm, TOLD 9521 (s),
Toshiba, Japan) signal generation means, appropriate optics to
filter the signal, and a current response of a silicon p-i-n
junction diode to generate an analog photocurrent. The resulting
photocurrent was amplified, converted from an analog to a digital
signal, and processed on a computer to determine the presence of
analyte and semi-quantitatively measure an amount of analyte in a
sample.
[0205] Processing via a computer involved comparing the
fluorescence level of the detected signal with dose response curves
as shown in FIG. 16a-16d and FIG. 17a-17b. A normalized
fluorescence signal is located on the y-axis and the relative
concentration in parts per billion was located on the x-axis. Table
I shows U.S.F.D.A. safe/tolerance levels for the six .beta.-lactam
drugs in milk.
Example 6
Preparation of a Capillary Tube for Use in a Competitive
Immunoassay to Detect Cephapirin in Milk
[0206] A borosilicate glass capillary tube having a length of 65 mm
and an inner diameter of 0.6 mm (Drummond Scientific) was washed
with distilled water and then dried under a stream of air until
substantially all of the distilled water had evaporated from the
surfaces of the capillary tube. The capillary tube was then
incubated in a 2.5% aminopropyl triethoxysilate ethanol solution
for 20 minutes at 80.degree. C.
[0207] The incubated capillary tube was then washed with distilled
water and dried in a stream of air. The dried capillary tube was
then incubated for 2 hours at 120.degree. C. After incubation, the
capillary tube was cooled to room temperature.
[0208] The capillary tube was then incubated in the presence of a
buffer solution comprising a conjugate of Cephapirin-Bovine Serum
Albumin (BSA) conjugate. The cephapirin-BSA conjugate was prepared
by combining 65 mg Cephapirin, 46 mg EDC
(1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide) and 26 mg of N-HS
(N-hydroxysuccinimide) (Pierce Chemical) in a 16.times.100 mm glass
tube. 1 ml DMF (N,N dimethylformamide) (Fisher Scientific) was
added to the tube and the contents of the tube were stirred at room
temperature for 30 minutes. 100 mg BSA was dissolved in 3 ml 20 mM
potassium phosphate buffer, pH 7.2, in a separate 16.times.100
glass tube. The Cephapirin solution was combined with the BSA
solution and stirred at room temperature for 1 hour. The
Cephapirin-BSA conjugate was purified with a gel filtration column
(Sephadex G-25) using 20 mM potassium phosphate buffer, pH 7.2, as
the equilibration and elution buffer. The capillary tubes were
incubated in the resultant Cephapirin-BSA conjugate for 3 hours,
during which time the Cephapirin-BSA conjugate became bound to the
capillary tube surface. After incubation, the capillary was washed
with distilled water and dried under a stream of air.
[0209] The capillary tube was then incubated at 25.degree. C. for 2
hrs in a blocking solution of 10% sucrose, 0.1% bovine serum
albumin and 0.05% proclin 300 in order to block any free binding
sites of the capillary tube surface not occupied by the antigen.
After incubating the capillary tube in the blocking solution, the
capillary tube was washed with distilled water and dried under a
stream of air.
Example 7
Preparation of Anti-Cephapirin-Cy-5 .RTM. Conjugate
[0210] 1 milligram of activated Cy-5 .RTM. dye (Biological Detector
Systems) dissolved in 0.2 ml of 20 mM potassium phosphate buffer,
pH 7.2 was combined with 2 ml of a Cephapirin antibody solution (4
mg/ml Cephapirin antibody in 20 mM potassium phosphate buffer at pH
7.2). Cy-5.RTM. dye/Cephapirin solution was then stirred at room
temperature for 1 hour. The resultant Cy-5 .RTM.-Cephapirin
conjugate was purified on a gel filtration column (Sephadex G-25),
where 20 mM potassium phosphate buffer, pH 7.2, was used as both
the equilibration and elution buffer.
Example 8
Manual Immunoassay of Milk Suspected of Comprising Cephapirin
[0211] About 4 to 5 .mu.l of antibody conjugate solution, as
prepared in Example 7, was added to 0.5 ml of milk suspected of
comprising Cephapirin, resulting in an overall conjugate
concentration in the milk of 10 .mu.l/ml. The milk conjugate
mixture was then incubated for a few seconds.
[0212] One end of the prepared capillary tube from Example 6 was
dipped into the incubated milk. A 10 .mu.l plug of milk was taken
up into the capillary tube under capillary force. Milk also coated
the exterior of the capillary tube up to about 20 The capillary
tube was then turned upside down so that the 10 .mu.l plug of
liquid moved down the capillary tube into a region in which the
outer surface of the capillary tube had not contacted the milk. The
sample plug was incubated in the capillary tube for 1 minute to
allow the reaction to proceed to provide a detectable fluorescent
signal for a positive sample.
[0213] The plug was then washed from the capillary tube with 100
.mu.l of distilled water. After washing the capillary tube, the
capillary tube was dried with a stream of air from an air gun.
[0214] The capillary tube was then irradiated with a laser, where
the wavelength of light form the laser was 632.8 nm. Upon
irradiation, a highly intense emitted signal having a wavelength of
667 nm was detected. The intensity of the emitted signal indicated
that no cephapirin was present in the assayed milk.
Example 9
Method for Coating 4-Amino-Penicillinic Acid on a Capillary
Tube
[0215] AquaSil.RTM. Silanization Coating
[0216] One or more glass capillary tubes was immersed in a 0.2%
AquaSil.RTM.-octodecyltriethoxy silane (Pierce Chemical #42797)
deionized water solution in a manner that ensured a complete
coating on the desired portions of the capillary tubes and then
incubated in the solution for 40.+-.30 minutes at room temperature.
The capillary tube was then placed in the centrifuge and
centrifuged at 1000 rpm for 10.+-.5 minutes to spin dry the
capillary tube. Alternatively, the capillary tube may be air dried,
dried with a gas, or an suitable means to sufficiently dry the
capillary tube.
[0217] The AquaSil.RTM. coated capillary tube was then placed into
a 125.+-.10.degree. C. oven for 60.+-.10 minutes. Next, the
capillary tube was allowed to cool to room temperature and
subsequently stored in a desiccated, foil ziplock bag at room
temperature until further coating was required. The capillary tube
is stable for long periods in this storage condition.
[0218] BSA-Biotin Capillary Tube Coating
[0219] The AquaSil.RTM. coated capillary tube was immersed into a
BSA-biotin (bovine serum albumin) solution consisting of a stock
BSA-biotin solution (Sigma #A-3294) at 200 .mu.g/mL diluted 1:200
with 20 mM phosphate buffered saline (PBS)-pH 7.2. 100 mL of the 20
mM PBS-pH 7.2 solution can be prepared by mixing 268 g potassium
phosphate-dibasic (K.sub.2HP0.sub.4) (Fisher #P284-3), 60.2 mg
potassium phosphate-monobasic (KH.sub.2PO.sub.4H.sub.2O), and 875
mg sodium chloride (Fisher #P285-3) where the pH was
7.2.+-.0.1.
[0220] After it was ensured that all desired areas were contacted
with solution, the BSA-biotin coated capillary tube was then
incubated in solution for 1-24 hours at room temperature. After
incubation, the BSA-biotin coated capillary tube was centrifuged at
1000 rpm for 10.+-.5 minutes to spin dry the capillary tube. The
BSA-biotin coated capillary tube was then stored in a desiccated,
foil ziplock bag at 4.degree. C. until further coating was
required. The capillary tube was stable for long periods in this
storage condition.
[0221] Bovine Serum Albumin Blocking of Capillary Tubes
[0222] The BSA-biotin coated capillary tube was immersed in a 0.1%
BSA/10% sucrose blocking solution. The 100 ml of the 0.1% BSA/10%
sucrose blocking solution can be prepared by mixing 10 g sucrose
(Fisher #S-53) with 80 mL deionized water, mixing the solution to
dissolve the sucrose, adding 100 mg BSA (Sigma #A-3294) to the
sucrose solution, slowly mixing the sucrose-BSA solution, adding
0.05 mL of ProClin 300 (Supelco #4-8127), and adding additional
deionized water to bring the volume to 100 mL.
[0223] After it was ensured that all desired areas were contacted
the BSA-Sucrose blocked capillary tube was then incubated for 1-24
hours at room temperature. After incubation the capillary tube was
centrifuged at 1000 rpm for 10.+-.5 minutes to spin dry the
capillary tube.
[0224] The BSA-biotin and BSA-Sucrose coated capillary tube was
then stored in a desiccated, foil ziplock bag at 4.degree. C. until
further coating was required. The capillary tube is stable for long
periods at this condition.
[0225] NeutrAvidin-4-Amino-Penicillinic Acid Capillary Coating
[0226] The BSA-biotin/BSA-Sucrose coated capillary tube was
immersed in a solution of 40 .mu.g/mL
neutravidin-amino-penicillinic acid (NAV-APA) conjugate, which
NAV-APA conjugate solution was prepared by diluting stock NAV-APA
conjugate solution diluted to 40 .mu.g/mL with 20 mM PBS-pH
7.2.
[0227] After it was ensured that all the desired portions of the
capillary tubes were contacted with solution the NAV-APA coated
capillary tube was then incubated for 1-24 hours at room
temperature. After incubation, the NAV-APA coated capillary tube
was centrifuged at 1000 rpm for 10.+-.5 minutes to spin dry the
capillary tube.
[0228] The NAV-APA coated capillary tube was then immersed in a
container containing deionized water such that all surfaces of the
capillary tube were contacted by the water. The NAV-APA coated
capillary tube was then centrifuged at 1000 rpm for 10.+-.5 minutes
to spin dry the capillary tube.
[0229] The NAV-APA coated capillary tube was then placed in a
vacuum oven heated to 37.degree. C. The oven was evacuated to 24
in. Hg. The coated capillary tube was heated under vacuum for 60
minutes. The oven was then turned off, the vacuum line closed, and
vacuum pump turned off. The NAV-APA coated capillary tube was then
kept in the evacuated oven over night.
[0230] The AquaSil.RTM./BSA-biotin/NAV-APA coated capillary tube
can be kept with the baffled insert in a desiccated, foil ziplock
bag or the capillary tube can be decanted into a sealable,
desiccated container and placed in a foil ziplock bag and stored at
4.degree. C. until ready for use in a manual version of the
immunoassay or for assembly of the cartridges of the present
invention.
Example 10
Method for Coating a Ceftiofur Capillary Tube
[0231] One or more capillary tubes was prepared with the
Aquasil.RTM. capillary coating procedure, the BSA-Biotin capillary
coating procedure, and the BSA blocking procedure as described in
Example 9.
[0232] The BSA-biotin and BSA-Sucrose coated capillary tube was
immersed in a 20 .mu.g/mL neutravidin-ceftiofur conjugate solution
such that all desired portions of the capillary tube were contacted
by the conjugate solution, which conjugate solution was prepared by
diluting stock NAV-Ceftiofur conjugate solution to 20 .mu.g/mL in
20 mM PBS-pH 7.2.
[0233] The NAV-Ceftiofur coated capillary tube was then incubated
for 1-24 hours at room temperature. After incubation, the
NAV-Ceftiofur coated capillary tube was centrifuged at 1000 rpm for
10.+-.5 minutes to spin dry the capillary tube.
[0234] The NAV-Ceftiofur coated capillary tube was then immersed in
a container containing deionized water such that all desired
portions of the capillary tube were contacted with the deionized
water.
[0235] The NAV-Ceftiofur coated capillary tube was then centrifuged
at 1000 rpm for 10.+-.5 minutes to spin dry the capillary tube.
[0236] The NAV-Ceftiofur coated capillary tube was then placed in a
vacuum oven heated to 37.degree. C. The oven was evacuated to 25
in. Hg. The coated capillary tube was heated under vacuum for 60
minutes. The oven was then turned off, the vacuum line closed, and
vacuum pump turned off. The NAV-Ceftiofur coated capillary tube was
then kept in the evacuated oven over night.
[0237] The AquaSil/BSA-biotin/NAV-Ceftiofurcoated capillary tube
can be kept with the baffled insert in a desiccated, foil ziplock
bag or the capillary tube can be decanted into a sealable,
desiccated container and placed in a foil ziplock bag and stored at
4.degree. C. until ready for use in a manual version of the
immunoassay or for assembly of the cartridges of the present
invention.
Example 11
Method for Coating Cephapirin Capillary Tubes
[0238] One or more glass capillary tubes was prepared with the
AquaSil coating procedure of Example 9. The AquaSil coated
capillary tube was then immersed in a 25 .mu.g/mL BSA-Cephapirin
coating solution such that all desired portions of the capillary
tube were contacted with the coating solution, which BSA-Cephapirin
coating solution was prepared by diluting stock BSA-Cephapirin
conjugate solution to 25 .mu.g/mL with 20 mM PBS-pH 7.2.
[0239] The BSA-Cephapirin coated capillary tube was then incubated
for 1-24 hours at room temperature. After incubation the capillary
tube was centrifuged at 1000 rpm for 10.+-.5 minutes to spin dry
the capillary tube.
[0240] The BSA-Cephapirin coated capillary tube was then stored in
a desiccated, foil ziplock bag at 4.degree. C. until further
coating was required. The capillary tube is stable for long periods
at this condition.
[0241] The BSA-Cephapirin coated capillary tube was then coated
with the BSA blocking solution described in Example 9. The
BSA-Cephapirin coated and BSA-sucrose blocked capillary tube was
then immersed in a container containing deionized water such that
all desired portions of the capillary tube were contacted with the
water.
[0242] The BSA-Cephapirin coated and BSA-sucrose blocked coated
capillary tube was then centrifuged at 1000 rpm for 10.+-.5 minutes
to spin dry the capillary tube.
[0243] The BSA-Cephapirin coated and BSA-sucrose blocked capillary
tube was then placed in a vacuum oven heated to 37.degree. C. The
oven was evacuated to 25 in. Hg. The coated capillary tube was
heated under vacuum for 60 minutes. The oven was then turned off,
the vacuum line closed, and vacuum pump turned off. The
NAV-Ceftiofur coated capillary tube was then kept in the evacuated
oven over night.
[0244] The AquaSil.RTM./BSA-Cephapirin/BSA-sucrose coated capillary
tube can be kept with the baffled insert in a desiccated, foil
ziplock bag or the capillary tube can be decanted into a sealable,
desiccated container and placed in a foil ziplock bag and stored at
4.degree. C. until ready for use in a manual version of the
immunoassay or for assembly of the cartridges of the present
invention.
Example 12
Method of the Immunoassay Used With the Devices and Apparatus of
the Present Invention
[0245] The immunoassay was begun by placing preferred embodiments
of the cartridge and sample tray on a preferred embodiment of the
apparatus of the present invention (also called the Parallux
Processor Idetek, Sunnyvale, Calif.) and adding 100 microliters of
raw, unpasteurized milk into each well of the sample tray. A
barcode with calibration information for the particular kit was
scanned. Appropriate identification information was entered via the
keypad, and the apparatus began running the immunoassay. Movement
of magnets by the magnetic mixer under the tray agitated the metal
washers in the sample tray-wells for 20 seconds so the dry reagent
comprising a fluorescent label in each well was homogeneously
dissolved in the sample milk. After mixing, the sample tray was
raised by the lift motor so that the tips of the capillary tubes in
the cartridge were immersed in the milk. A syringe pump was
actuated to draw milk into the capillary tubes, where it remained
for an incubation period of two minutes. Upon completion of
incubation, the syringe pump was used to wash out all contents of
the tubes into the tray. The wash solution came from a reservoir in
the apparatus. The sample tray was discarded, and the cartridge was
placed on the centrifuge (called a read station). The centrifuge
spun at 3000 rpm for 20 seconds to eject any remaining wash
solution from the tubes. This step was followed by exposure to a
diode laser beam for measurement of fluorescence. The fluorescence
signals from each tube were collected, amplified, converted to a
digital signal and processed.
[0246] The cartridge used in this Example contained reagents for
three separate immunoassays. The fourth tube and corresponding well
were blank. The first immunoassay was for cephapirin, the second
for ceftiofur, and the third for the penicillin family of drugs
(penicillin G, ampicillin, cloxacillin, and amoxicillin).
Therefore, a single cartridge was able to screen for six
analytes.
[0247] For semi-quantitative immunoassays, the fluorescence signals
were compared to a value that had been input by the barcode. Any
measured fluorescence value above the barcoded value resulted in a
pass, and any measured value equal to or below the barcoded value
resulted in a fail.
[0248] For quantitative immunoassays, the raw fluorescence signals
were plotted as a function of spiked concentration in raw milk to
produce a multi-level standard curve as shown below, e.g., for the
Penicillin G immunoassay.
[0249] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be obvious that certain changes and
modifications can be practiced within the scope of the appended
claims. Therefore, the spirit and scope of the appended claims
should not be limited to the description of preferred versions
contained herein
* * * * *