U.S. patent application number 11/035047 was filed with the patent office on 2006-02-23 for test device and method for colored particle immunoassay.
Invention is credited to David E. Charlton, Neal W. Miller.
Application Number | 20060040405 11/035047 |
Document ID | / |
Family ID | 22787518 |
Filed Date | 2006-02-23 |
United States Patent
Application |
20060040405 |
Kind Code |
A1 |
Charlton; David E. ; et
al. |
February 23, 2006 |
Test device and method for colored particle immunoassay
Abstract
Disclosed is a test cell and a method for detection of a
preselected ligand in a liquid sample such as a body fluid. The
test cell includes an elongate outer casing which houses an
interior permeable material capable of transporting an aqueous
solution and defining a sample inlet, a test volume, and a
reservoir volume. The reservoir volume is disposed in a section of
the test cell spaced apart from the inlet and is filled with
sorbent material. The reservoir acts to receive liquid transported
along a flow path defined by the permeable material and extending
from the inlet and through the test volume. In the test volume is a
test site which includes a first protein having a binding site
specific to a first epitope of the ligand immobilized in fluid
communication with the flow path. The test site can be observed
through a window of the casing.
Inventors: |
Charlton; David E.;
(Allentown, NJ) ; Miller; Neal W.; (Lebanon,
PA) |
Correspondence
Address: |
CHURCH & DWIGHT CO., INC.
LAW DEPT. - PATENTS
469 NORTH HARISON STREET
PRINCETON
NJ
08543-5297
US
|
Family ID: |
22787518 |
Appl. No.: |
11/035047 |
Filed: |
January 12, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09441875 |
Nov 17, 1999 |
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11035047 |
Jan 12, 2005 |
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08886088 |
Jul 2, 1997 |
5989921 |
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09441875 |
Nov 17, 1999 |
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07995331 |
Dec 23, 1992 |
5714389 |
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08886088 |
Jul 2, 1997 |
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07702450 |
May 16, 1991 |
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07995331 |
Dec 23, 1992 |
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07211582 |
Jun 27, 1988 |
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07702450 |
May 16, 1991 |
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Current U.S.
Class: |
436/501 |
Current CPC
Class: |
B01L 2300/0681 20130101;
Y10S 436/817 20130101; Y10T 436/255 20150115; Y10S 436/81 20130101;
G01N 33/54366 20130101; Y10S 435/97 20130101; G01N 33/743 20130101;
Y10S 435/81 20130101; G01N 33/76 20130101; G01N 33/54313 20130101;
B01L 3/5023 20130101; Y10S 436/814 20130101; Y10S 435/805 20130101;
B01L 2400/0406 20130101; B01L 2300/0825 20130101; Y10S 436/818
20130101 |
Class at
Publication: |
436/501 |
International
Class: |
G01N 33/566 20060101
G01N033/566 |
Claims
1. A method of detecting a ligand in a liquid sample, the method
comprising the steps of: A. transporting along a flow path in a
test cell a solution, including a liquid sample suspected to
contain a ligand and a conjugate, into contact with a test site
visible through a window in a wall of said test cell, said test
site having immobilized thereon a first protein having a binding
site specific to a first epitope on the ligand, said conjugate
comprising colored particles coupled to a second protein selected
from the group consisting of proteins having a binding site
specific to a second epitope on the ligand and proteins which bind
with said first protein in competition with the ligand, and B.
continuing transport of said solution to progressively produce at
said test site a complex comprising said ligand for a time
sufficient to visually determine through said window whether a
color is developed at said test site:
2. The method of claim 1 wherein said test cell comprises a
filtration means for filtering said liquid sample, said method
comprising the additional step of transporting the sample through
said filtration means before said sample contacts said test
site.
3. The method of claim 1 wherein the cross-sectional area of said
flow path is restricted about said test site whereby ligand is
localized at said test site during flow of solution thereby.
4. The method of claim 1 comprising the additional steps of
transporting said solution into contact with a control site visible
through a window in a wall of said test cell and comparing the
color of said test site and control site.
5. The method of claim 4 wherein said control site comprises a
negative control site free of said first protein.
6. The method of claim 4 wherein said control site comprises a
positive control site having immobilized thereon an authentic
sample of said ligand.
7. The method of claim 1 comprising the step of mixing said
conjugate with said liquid sample prior to step A.
8. The method of claim 1 wherein said conjugate is disposed in said
flow path, said method comprising the additional step of
transporting said liquid into solubilizing contact with said
conjugate prior to contact with said test site.
9. The method of claim 1 wherein said first and second proteins
comprise antibodies and at least one of said proteins is a
monoclonal antibody.
10. The method of claim 1 wherein said first protein has a binding
site specific to an epitope of human chorionic gonadotropin.
11. The method of claim 1 wherein said first protein has a binding
site specific to an epitope of human progesterone.
12. The method of claim 1 wherein said second protein has a binding
site specific to a second epitope on the ligand, and when said
sample contains said ligand, the complex produced in step B
comprises said ligand bound to both said first and second proteins,
and color is produced by aggregation of said colored particles at
said test site.
13. The method of claim 1 wherein said second protein binds with
said first protein in competition with the ligand, and when said
sample contains said ligand, the complex produced in Step B
comprises said ligand bound to said first protein, and when said
sample is free of said ligand, the complex produced in step B
comprises said conjugate bound to said first protein, and color is
produced by aggregation of said colored particles at said test
site.
14. A test cell for detecting a ligand in a liquid sample, the test
cell comprising an elongate casing for housing a permeable material
and defining a liquid sample inlet, a reservoir volume, a test
volume interposed between said inlet and reservoir volume, and a
window through said casing at said test volume, permeable material
capable of transporting an aqueous solution disposed within said
casing and defining a flow path extending from said sample inlet
through said test volume and into communication with said reservoir
volume, a first protein having a binding site specific to a first
epitope on said ligand, said first protein being immobilized at a
test site, disposed within said test volume in fluid communication
with said flow path and visible through said window, and a sorbent
material in said reservoir volume for drawing liquid sample along
said flow path and into contact with said test site.
15. The cell of claim 14 further comprising a liquid sample
filtering means disposed in said flow path between said inlet and
said test site.
16. The cell of claim 15 wherein said filtering means is defined by
a portion of said permeable material.
17. The cell of claim 14 wherein the cross sectional area of said
flow path is restricted about said test site so that ligand in
liquid passing therealong is localized at said test site.
18. The cell of claim 14 wherein said casing defines a second
window through said casing and said cell further comprises a
control site in fluid communication with said flow path visible
through said second window.
19. The cell of claim 18 wherein said control site comprises a
negative control site free of said first protein.
20. The cell of claim 19 wherein said control site comprises latex
particles disposed in contact with said permeable material.
21. The cell of claim 18 wherein said control site comprises a
positive control site having immobilized thereon an authentic
sample of said ligand.
22. The cell of claim 14 further comprising a conjugate disposed in
said flow path between said test site and said inlet; said
conjugate comprising colored particles coupled to a second protein
selected from the group consisting of proteins having a binding
site specific to a second epitope on the ligand, and proteins which
bind with said first protein in competition with the ligand.
23. The cell of claim 14 wherein said test site comprises an
antibody fixed to latex particles disposed in contact with said
permeable material.
24. The cell of claim 14 wherein said first protein binds with an
epitope of human chorionic gonadotropin.
25. The cell of claim 14 wherein said first protein binds with an
epitope of human progesterone.
26. The cell of claim 22 wherein at least one of said first and
second proteins is a monoclonal antibody.
Description
BACKGROUND OF THE INVENTION
[0001] This invention relates to assays for ligands, e.g.,
antigens, in a liquid sample such as a body fluid. More
particularly, the invention relates to a method and apparatus for
the detection of a ligand in a body fluid such as urine using a
conjugate comprising colored particles and a novel flow-through
test cell.
[0002] Many types of ligand-receptor assays have been used to
detect the presence of various substances, often generally called
ligands, in body fluids such as urine. These assays involve antigen
antibody reactions, synthetic conjugates comprising radioactive,
enzymatic, fluorescent, or visually observable metal sol tags, and
specially designed reactor chambers. In all these assays, there is
a receptor, e.g., an antibody, which is specific for the selected
ligand or antigen, and a means for detecting the presence, and
often the amount, of the ligand-receptor reaction product. Most
current tests are designed to make a quantitative determination,
but in many circumstances all that is required is a
positive/negative indication. Examples of such qualitative assays
include blood typing and most types of urinalysis. For these tests,
visually observable indicia such as the presence of agglutination
or a color changes are preferred.
[0003] Even the positive/negative assays must be very sensitive
because of the often small concentration of the ligand of interest
in the test fluid. False positives can also be troublesome,
particularly with agglutination and other rapid detection methods
such as dipstick and color change tests. Because of these problems,
sandwich assays and other sensitive detection methods which use
metal sols or other types of colored particles have been developed.
These techniques have not solved all of the problems encountered in
these rapid detection methods.
[0004] It is an object of this invention to provide a rapid,
sensitive method for detecting ligands in body fluids. Another
object is to provide an assay which has high sensitivity and fewer
false positives than conventional assays. A further object is to
provide a test cell for detection of low levels of ligands in body
fluids. Another object is to provide an assay system which involves
a minimal number of procedural steps, and yields reliable results
even when used by untrained persons.
[0005] These and other objects and features of the invention will
be apparent from the following description, drawing, and
claims.
SUMMARY OF THE INVENTION
[0006] The invention features a method and test cell for the
detection of a preselected ligand in a liquid sample such as a body
fluid.
[0007] The test cell useful in the practice of the invention has an
elongate outer casing which houses an interior permeable material,
e.g., glass fiber, capable of transporting an aqueous solution by
capillary action, wicking, or simple wetting. The casing defines a
sample inlet, and interior regions which, for ease of description,
can be designated as a test volume and a reservoir volume. The
reservoir volume is disposed in a section of the test cell spaced
apart from the inlet, and preferably is filled with sorbent
material. The reservoir acts to receive liquid transported along a
flow path defined by the permeable material and extending from the
inlet and through the test volume. In the test volume is a test
site comprising a first protein having a binding site specific to a
first epitope of the ligand immobilized in fluid communication with
the flow path, e.g., bound to the permeable material or to latex
particles entrapped in or bonded to the permeable material. A
window such as a hole or transparent section of the casing permits
observations of the test site through the casing wall.
[0008] In a preferred embodiment, the flow path is restricted or
narrowed in the test area, thereby channeling and concentrating
fluid-flow into contact with the test site. It is also preferred
that the test cell include a solution filtering means disposed in
the flow path between the sample inlet and the test site. The
filtration means can comprise a separate, conventional filter
element disposed within the casing of the test cell in fluid
communication with the permeable material defining the flow path,
but preferably is defined simply by a portion of the permeable
material itself. The provision of such a filtration means in the
test cell has the effect of removing by entrapment from impure
samples, such as urine samples, a portion of the particulates and
nonspecific interfering factors which sometimes cause false
positive readings.
[0009] The method of the invention requires the use of a conjugate
comprising a second protein bound to colored particles such as a
metal sol or colloid, preferably gold. The conjugate can take two
distinct forms, depending on whether the assay is designed to
exploit the "sandwich" or "competitive" technique.
[0010] In the case of the sandwich technique, the second protein
comprises a site which binds to a second epitope on the ligand.
This type of conjugate reacts with the ligand to form a complex in
the liquid sample. The complex is detected by visual observation of
color development at the test site in the test cell. At the test
site, the ligand bound with the conjugate reacts with the
immobilized first binding protein to form a "sandwich" of the first
protein, ligand, second protein, and colored particles. This
sandwich complex is progressively produced at the test site-as
sample continuously passes by, filling the reservoir. As more and
more conjugate is immobilized, the colored particles aggregate at
the test site and become visible through the window, indicating the
presence of ligand in the liquid sample.
[0011] In the case of the competitive technique, the second protein
binds with the first protein in competition with the ligand. The
second protein comprises, for example, an authentic sample of the
ligand or a fraction thereof which has comparable affinity for the
first protein. As the liquid sample is transported in contact with
the test site, ligand, if any, and the conjugate compete for sites
of attachment to the first protein. If no ligand is present,
colored particles aggregate at the test site, and the presence of
color indicates the absence of detectable levels of ligand in the
sample. If ligand is present, the amount of conjugate which binds
at the test site is reduced, and no color, or a paler color,
develops.
[0012] In one embodiment of the invention, the test liquid is mixed
with the conjugate outside the test cell. In another embodiment,
the conjugate is disposed in freeze-dried or other preserved form
on the permeable material between the inlet and the test site, and
the sample liquid resolubilizes the conjugate as it passes along
the flow path.
[0013] Color development at the test site may be compared with the
color of one or more standards or internal controls to determine
whether the development of color is a true indication of the
presence or absence of the ligand, or an artifact caused by
nonspecific sorption.
[0014] In one embodiment employing the sandwich technique, the
standard consists of a negative control site, preferably disposed
adjacent the test site, and visible through a second window
proximate the first. The negative control site preferably is
prepared identically to the test site, except immobilization of the
first binding protein is omitted. Therefore, although the conjugate
will reach the control site, it aggregates due only to non-specific
binding. If the test site is not appreciably more intense in color
than the control site, the assay is considered negative.
[0015] In another embodiment, the assay and test cell may include a
positive control. Thus, when exploiting the sandwich technique, the
cell may have an authentic sample of the ligand immobilized at a
control site. If no color develops at this control site, the assay
is considered inconclusive. When exploiting the competitive
technique, the development of color at the positive control site
means the assay results are inconclusive.
[0016] Broadly, the method of the invention involves the use of a
test cell of the type described above to achieve an easily
readable, sensitive, reproducible indication of the presence of a
ligand, e.g., human chorionic gonadotropin (hCG), in a test sample
such as a human urine sample. The method involves the step of
transporting the sample and a conjugate comprising a protein bound
to a metal sol or other colored particle along a flow path and in
contact with a test site comprising immobilized binding protein
specific to an epitope of the ligand, and preferably also in
contact with a control site. Preferably, the colored particle
comprises a gold sol; the flow path in the region of the test site
is reduced in cross-section relative to other parts of the flow
path; the sample is passed through a filtration means after it
enters the test cell but before it contacts the test site; and the
test site comprises latex particles entrapped or otherwise fixed in
the flow path having the immobilized protein on their surface. In
the practice of the process, either the conjugate is premixed with
the sample, or the conjugate is disposed in preserved form, e.g.,
lyophilized, in the flow path between the inlet and the test site.
In either case, placement of the test cell in the sample, or
application of the sample to the inlet, initiates flow, and the
result is read by observing color development a the test site, or
by comparing the color of the test site and control site.
[0017] The use of the colored particle detection system in
combination with the filtration means, the concentrating effect of
flow of the sample, and the ease of comparison between the colors
of the test and control sites, together enable construction of a
family of extremely sensitive assay systems which minimize false
positives and can be used effectively by untrained persons.
BRIEF DESCRIPTION OF THE DRAWING
[0018] FIG. 1 is a cut-away, schematic, top view of an embodiment
of a test cell useful in explaining the test cell and process of
the invention;
[0019] FIG. 2 is a cross-sectional side view of the test cell of
FIG. 1;
[0020] FIG. 3 is a perspective view of a currently preferred test
cell constructed in accordance with the invention;
[0021] FIG. 4A is a cross-sectional, top view of the test cell of
FIG. 3;
[0022] FIG. 4B is a cross-sectional, side view of the test cell of
FIG. 3 taken at line 4B-4B of FIG. 4A;
[0023] FIG. 5 is a cross sectional view of the cell of FIG. 3 taken
at line 5-5 of FIG. 4B; and
[0024] FIG. 6 is a perspective view of another embodiment of a test
cell constructed in accordance with the invention.
[0025] Like reference characters in the respective drawn figures
indicate corresponding parts.
DESCRIPTION
[0026] The invention provides a test cell for conducting a sandwich
or competitive immunoassay, and a process which utilizes the test
cell and a conjugate comprising colored particles. As disclosed
below, various features of the process and test cell of the
invention cooperate to enable untrained personnel reliably to assay
a liquid sample for the presence of extremely small quantities of a
particular ligand while avoiding false positives and simplifying
test procedures. The invention is ideal for use in over-the-counter
assay test kits which will enable a consumer to self diagnose, for
example, pregnancy, venereal disease, and other disease, infection,
or clinical abnormality which results in the presence of an
antigenic marker substance in a body fluid, including determination
of the presence of metabolites of drugs or toxins. The assay
process and the cell are engineered specifically to detect the
presence of a preselected individual ligand present in a body or
other fluids.
[0027] Broadly, the test cell and process of the invention can be
used to detect any ligand which has heretofore been assayed using
known immunoassay procedures, or known to be detectable by such
procedures, using polyclonal or monoclonal antibodies or other
proteins comprising binding sites for ligands. Various specific
assay protocols, reagents, and analytes useful in the practice of
the invention are known per se, see, e.g., U.S. Pat. No. 4,313,734,
columns 4-18, and U.S. Pat. No. 4,366,241, columns 5-40.
[0028] The combination of features believed to be responsible for
the excellent sensitivity and reproducibility of assays constructed
in accordance with the invention is the use of the novel test cell
which serves to concentrate ligand from a test sample at a test
site in the cell, and the use of a metal sol or other colored
particle as a marker system which permits direct visual observation
of color development. False positives are reduced while maintaining
excellent sensitivity by including in the test cell a negative
control or control site whose color is compared with the test site,
and by including a filtration means which limits the introduction
to the test site of contaminants from the sample.
[0029] The assay is conducted by simply placing the inlet of the
test cell in contact with a liquid test sample. One then merely
waits for the test sample to pass through the cell and into
reactive contact with the test site (and optionally one or more
control sites) visible through a window or windows in the cell's
exterior casing. In one embodiment, the conjugate is mixed with the
sample and incubated briefly before the test cell is inserted. In
another embodiment, the conjugate is disposed in preserved form in
the flow path within the cell. If the ligand is present in the
sample, it passes through the inlet and the interior of the cell
along the flow path past the test and control sites, where, in the
sandwich embodiment, it reacts with immobilized binding protein,
e.g., antibody, at the test site, and perhaps also non-specifically
at the control site. A "sandwich" forms at the test site comprising
immobilized binding protein-ligand binding protein-colored
particle. The presence of the sandwich complex and thus the ligand
is indicated by the development of color caused by aggregation of
the metal sol particles at the test site. A deeper color at the
test site than at the negative control site is a positive
indication of the presence of the ligand.
[0030] By providing a reservoir of sorbent material disposed beyond
the test and control sites, a relatively large volume of the test
liquid and any ligand it contains can be drawn through the test
area to aid sensitivity. Optionally, the region of the flow path in
the test cell defining the test and control sites is restricted in
cross-sectional area relative to other regions of the flow path.
This feature produces a "bottle-neck" effect wherein all ligand in
the entire volume of sorbed sample must pass through the restricted
flow area immediately about the test site where it will be
immobilized by reaction with binding protein.
[0031] From the foregoing, it will be apparent that the success of
the test procedure is dependent on ligand present in the sample
reacting with the conjugate, or on reproducible competition between
the ligand and the conjugate for sites of attachment at the test
site. In accordance with the invention, as noted above, the assays
can be conducted by premixing the conjugate with the liquid sample
prior to introduction into the elongate test cell. Alternatively,
the conjugate may be disposed in preserved form, e.g.,
freeze-dried, in the flow path within the test cell upstream of the
test and control sites. In this case, the cell is placed directly
in the liquid sample solution without premising. Ligand, if any,
passing up through the cell and entrained within the liquid moves
into contact with the conjugate forming an immune complex or
initiating competition in situ as flow continues. This latter
technique has the advantage that it eliminates a manipulative step
in the assay procedure, and accordingly a possible source of
error.
[0032] Referring to the drawing, FIGS. 1 and 2. illustrate
schematically an embodiment of a test cell 5 constructed in
accordance with the invention useful in explaining its principles
of construction. It comprises an outer, molded casing 10 which
defines a hollow, elongate enclosure filled with a permeable,
sorbent material 12. Casing 10 also defines a test liquid inlet 14
and a pair of circular openings 16, 18 comprising windows through
which sorbent material 12 is visible.
[0033] Sorbent material 12 and the interior of casing 10 together
define a flow path passing generally from left to right in FIGS. 1
and 2. When the test cell is placed with inlet 14 disposed within
or otherwise in contact with a liquid sample, the liquid is
transported by capillary action, wicking, or simple wetting along
the flow path through upstream flow section 20, test volume 22, and
into reservoir volume 24, generally as depicted by the arrows. The
flow section 20 of the flow path disposed inwardly of the inlet 14
serves as a filter which can remove from impure test samples
particulate matter and interfering factors. The provisions of such
a filtration means 20 downstream of the inlet 14 is believed to
contribute to the success of the system and its ability to avoid
false positives.
[0034] Disposed within sorbent material 12 is a band 26 of
dehydrated conjugate, e.g., antibody-metal sol. As the liquid
sample moves past band 26, the conjugate is entrained in the
liquid, reconstituted, and reacts or competes with ligand, if
present, dissolved in the liquid sample. Of course, conjugate band
26 may be eliminated, and the conjugate added to the test liquid
prior to introduction of the cell 5 as previously noted.
[0035] Within the volume of sorbent material 12 disposed directly
beneath circular openings 16 and 18 in casing 10 is disposed,
respectively, control site 16' and test site 18'. In the drawing,
the control and test site are illustrated as being disposed
serially along the flow path. Alternatively, the control and test
site or sites may be disposed side by side or in other spacial
relationships.
[0036] Test site 18' comprises a preselected quantity of antibody
against an epitope of the ligand to be detected immobilized in
place within the flow path. Its detailed chemical structure can
vary widely. Control site 16' is preferably identical in size and
chemical makeup to test site 18', excepting that the immobilized
antibody present at the test site 18' is omitted at the control
site 16'. Thus, any nonspecific aggregation of, e.g.,
ligand-conjugate or free conjugate, which occurs at test site 18'
also will occur at control site 16'. A deeper color at test site
18' as compared with control site 16' is a positive indication of
ligand in the sample in the sandwich assay.
[0037] The invention is not limited by the precise nature of the
test site 18' and corresponding control site 16', and in fact,
control site 16' may be entirely eliminated if a reduction in
sensitivity can be tolerated. Generally, antibody or other binding
protein may be immobilized at test site 18' using adsorption,
absorption, or ionic or covalent coupling, in accordance with
methods known per se. A currently preferred formulation for test
site 18' is to immobilize monoclonal antibody against an epitope of
the ligand on latex beads, and then to entrap or otherwise link the
beads in sorbent material 12 at region 18'. Control site 16' is
fabricated identically, except that the latex beads contain non
specific immunoglobulin, e.g., immunoglobulin from bleedings from
an animal that has not been immunized.
[0038] Disposed beyond test volume 22 is a reservoir volume 24
comprising a relatively large mass of sorbent or supersorbent
material. The purpose of reservoir volume 24 is to assure that a
reasonably large amount of test liquid is drawn through test volume
22. Increasing the volume of reservoir 24 can have the effect of
increasing the sensitivity of the assay procedure, as it results in
an increase in the amount of ligand passing through the test area
22. Suitable sorbents include commercial materials of the type
available, for example, from The Dow Chemical Company of Midland,
Mich., and the Chemical division of American Colloid, Arlington
Heights, Ill. These materials can absorb many times their weight in
water and are commonly used in disposable diapers. They comprise
lightly crosslinked polyacrylate salts, typically alkali metal
salts.
[0039] Polyclonal antisera and indeed monoclonal antibodies or
fractions thereof having specific binding properties and high
affinity for virtually any antigenic substance are known and
commercially available or can be produced from stable cell lines
using well known cell fusion and screening techniques. The
literature is replete with protein immobilization protocols. See,
for example, Laboratory Techniques in Biochemistry and Molecular
Biology, Tijssen, Vol. 15, Practice and Theory of Enzyme
immunoassays, chapter 13, The Immobilization of Immunoreactants on
Solid Phases, pp. 297-328, and the references cited therein.
[0040] Metal sols and other types of colored particles useful as
marker substances in immunoassay procedures are also known per se.
See, for example, U.S. Pat. No. 4,313,734, Feb. 2, 1982, to
Leuvering, the disclosure of which is incorporated herein by
reference. For details and engineering principles involved in the
synthesis of colored particle conjugates see Horisberger,
Evaluation of Colloidal Gold as a Cytochromic Marker for
Transmission and Scanning Electron Microscopy, Biol. Cellulaire,
36, 253-258 (1979); Leuvering et al, Sol Particle Immunoassay, J.
Immunoassay 1 (1), 77-91 (1980), and Frens, Controlled Nucleation
for the Regulation of the Particle Size in Monodisperse Gold
Suspensions, Nature, Physical Science, 241, pp. 20-22 (1973).
[0041] The cell can take various forms. It will usually comprise an
elongate casing comprising interfitting parts made of polyvinyl
chloride, polypropylene, or other thermoplastic resin. Its interior
flow path will contain a relatively inert material or a combination
of materials suitable for transporting the liquid. In some
circumstances it may be preferable to use a material of higher
sorptivity as the reservoir, promoting the flow of liquid, and a
different material for remaining portions of the flow path.
[0042] From the foregoing it should be apparent that the advantages
in reproducibility, sensitivity, and avoidance of false positives
of assay systems constructed in accordance with the invention are
traceable to a combination of features of the invention. In use,
the test cell of the invention and the metal sol particles used as
a marker together cooperate to result in an increase in color
intensity progressively as ligand complexed with conjugate is
trapped at the test site by the immobilized binding protein. This
approach can be utilized to design assays and test cells for
essentially any antigenic material.
[0043] The invention will be further understood from the following
non-limiting examples.
EXAMPLE 1
[0044] The currently preferred test device embodying the invention
is shown in FIGS. 3, 4A, 4B, and 5. A modification of the device
depicted in FIG. 3 is shown in FIG. 6, and includes a second
control site 19 in addition to control site 16' and test site 18',
as well as a stand 21 useful for maintaining the test cell in an
incline position with the reservoir downhill. When a test sample is
applied to inlet 14, gravity as well as sorption aids in
transporting the sample along the flow path.
[0045] As shown in FIGS. 3, 4A, 4B, and 5, the preferred test cell
of the invention differs from the exemplary device discussed above
and shown in FIGS. 1 and 2 in certain of its more specific internal
features. Specifically, the casing comprises a pair of interfitting
polymeric parts including a U-shaped top part 10 which, when the
device is assembled, interfits with lower part 10'. Top and bottom
parts 10 and 10' may be connected through a hinge region 11. The
bottom section 10' defines a pair of channels 28 above which is
disposed a strip of glass fiber paper 13 (available commercially
from Eaton Dikeman, Grade 111, or Whatman, Grade GFA). Test liquid
applied through inlet 14 soaks along the paper strip 13 which
defines the flow path and a filtering means region 20, as well as a
positive control site 16' and test site 18' visible through windows
16 and 18 consisting of openings through upper mating member 10.
The paper strip 13 overlaps into reservoir volume 24, which is
defined by a cavity between the interfitting top and bottom mating
members 10 and 10'. The cavity in this case is filled with sorbent
blotting paper 12 comprising the sorbent reservoir. A suitable
paper is sold as Grade 12A absorbent paper, a cellulose product
available from Schleicher and Schuell. In one preferred embodiment,
the dimensions of the glass fiber paper 13 were approximately one
quarter inch by three inches, and those of the absorbent material
12 approximately two inches by five thirty seconds of an inch on
each side. A number of these devices were produced and further
treated to adapt them to detect pregnancy by assay of urine.
[0046] Test site 18' in each device was fabricated as a spot within
fiber paper 13 using the following technique. Latex beads available
commercially and comprising polystyrene particles 0.3 micron in
diameter were passively coated with purified rabbit anti-human
chorionic gonadotropin. The polyclonal antibody was purified using
conventional techniques from bleedings of a rabbit previously
immunized with human chorionic gonadotropin in a manner know per
se. Equal parts of a latex (0.6% by weight) having a continuous
phase of glycine buffer, pH=8.3, and a 1 mg/ml antibody solution in
the same buffer were mixed and incubated at 37.degree. C. 15
microliters of this solution, comprising approximately 0.6% solids,
were added, one drop at a time, to the glass fiber paper 13 to
produce spot 18' after the devices had been assembled. The spots
were then allowed to dry at 37.degree. C. The control site 16' was
produced identically to the test site disclosed immediately above,
excepting that rabbit polyclonal non-immune gamma globulin was used
in place of the anti-hCG gamma globulin.
[0047] Metal sol particles were prepared in accordance with the
method of Frens, Controlled Nucleation for the Regulation of the
Particle Size in Mono Dispersed Gold Solutions (1973), supra.
Briefly, the gold sol was prepared by reducing a 4% solution of
gold chloride with 1% sodium citrate to produce gold particles of
approximately 18 nm in diameter. The particles were made
immunochemically reactive by admixture with a monoclonal antibody
specific for human chorionic gonadotropin with no detectable
cross-reactivity with human leutinizing hormone. The antibody was
purchased from Charles River Labs, and is produced using standard
techniques including purification from ascites using HPLC ion
exchange chromatography. It was added to the gold sol as a 10 ug/ml
solution in borate buffer, pH-6. The bound antibody fraction is
separated from the free fraction by either density centrifugation
or gel filtration chromatography. Additional details of the
currently preferred procedure for making the antibody sol conjugate
are disclosed by Leuvering et al, J. Immunoassay (1980) supra.
Individual batches of the latex and the conjugate are titrated to
optimize activity so that a suitable amount of latex is applied to
the test site and a suitable amount of conjugate is used in
conducting the test.
Test Protocol
[0048] To a 10.times.50 mm test tube of lyophilized gold sol
antibody conjugate is added 0.5 ml urine sample containing a known
quantities of hCG. The samples comprised hCG standards purchased
from Sigma Chemical Company diluted in processed, hCG negative
urine. The contents of the tube are mixed by shaking in a
horizontal motion until the lyophilized antibody is dissolved. The
device depicted in FIGS. 3-5 is then inserted into the tube, and
the results are read after the entire fluid volume has been
absorbed.
[0049] The results of this qualitative procedure follows:
TABLE-US-00001 Color of Color of mIU hCG Control Spot Reagent Spot
0 grey grey 25 grey pink hue 50 grey pink 100 grey rose 150 grey
rose >150 grey dark rose
[0050] The pink color clearly visible at 50 mIU of human chorionic
gonadotropin means that the test can detect pregnancy one day after
a missed menstrual period. In initial stages of testing,
approximately 5 negative samples from various sources have been run
with no false positives or even border-line cases. It is
anticipated that the commercial device will have less than 1% false
positives.
[0051] Non-limiting examples of materials which may be assayed in
accordance with the invention in addition to the human chorionic
gonadotropin noted above include human leutinizing hormone,
progesterone, estrogen, and streptococcus.
[0052] Other embodiments are within the following claims.
* * * * *