U.S. patent application number 10/050848 was filed with the patent office on 2002-09-19 for detection and characterization of microorganisms.
This patent application is currently assigned to LARGE SCALE PROTEOMICS CORPORATION. Invention is credited to Anderson, N. Leigh, Anderson, Norman G..
Application Number | 20020132338 10/050848 |
Document ID | / |
Family ID | 22138250 |
Filed Date | 2002-09-19 |
United States Patent
Application |
20020132338 |
Kind Code |
A1 |
Anderson, Norman G. ; et
al. |
September 19, 2002 |
Detection and characterization of microorganisms
Abstract
A method for separating microorganisms, especially infectious
agents, from a mixture by two dimensional centrifugation on the
basis of sedimentation rate and isopycnic banding density, for
sedimenting such microorganisms through zones of immobilized
reagents to which they are resistant, for detecting banded
particles by light scatter or fluorescence using nucleic acid
specific dyes, and for recovering the banded particles in very
small volumes for characterization by mass spectrometry of viral
protein subunits and intact viral particles, and by fluorescence
flow cytometric determination of both nucleic acid mass and the
masses of fragments produced by restriction enzymes. The method is
based on the discovery that individual microorganisms, such as
bacterial and viral species, are each physically relatively
homogeneous, and are distinguishable in their biophysical
properties from other biological particles, and from non-biological
particles found in nature. The method is useful for distinguishing
infections, for identifying known microorganisms, and for
discovering and characterizing new microorganisms. The method
provides very rapid identification of microorganisms, and hence
allows a rational choice of therapy for identified infectious
agents. A particularly useful application is in clinical trials of
new antibiotics and antivirals, where it is essential to identify
at the outset individuals infected with the targeted infectious
agent.
Inventors: |
Anderson, Norman G.;
(Rockville, MD) ; Anderson, N. Leigh; (Washington,
DC) |
Correspondence
Address: |
ROTHWELL, FIGG, ERNST & MANBECK, P.C.
1425 K STREET, N.W.
SUITE 800
WASHINGTON
DC
20005
US
|
Assignee: |
LARGE SCALE PROTEOMICS
CORPORATION
Germantown
MD
|
Family ID: |
22138250 |
Appl. No.: |
10/050848 |
Filed: |
January 18, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10050848 |
Jan 18, 2002 |
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09571274 |
May 16, 2000 |
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6340570 |
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09571274 |
May 16, 2000 |
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09265541 |
Mar 9, 1999 |
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6254834 |
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60077472 |
Mar 10, 1998 |
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Current U.S.
Class: |
435/304.1 |
Current CPC
Class: |
G01N 15/042 20130101;
G01N 2015/0065 20130101; Y10T 436/255 20150115; Y10T 436/25375
20150115; B01L 3/5021 20130101; G01N 15/14 20130101; G01N 2015/047
20130101; Y10T 436/24 20150115; G01N 15/0255 20130101 |
Class at
Publication: |
435/304.1 |
International
Class: |
C12M 001/24 |
Goverment Interests
[0002] This invention was made with Government support under an
SBIR grants from NIH, Grant Nos. 1 R43 AI41819-01/02. The United
States government may have certain rights in the invention.
Claims
What is claimed is:
1. An ultracentrifuge tube comprising an upper region, a middle
region and a lower region wherein an inner diameter of said upper
region is larger than an inner diameter of said middle region,
wherein (i) an inner diameter of said middle region is larger than
an inner diameter of said lower region or (ii) the inner diameter
of said middle region is the same as the inner diameter of said
lower region, wherein that inner diameter is small enough to trap
an air bubble between two layers of aqueous liquid such that the
air bubble will keep said two layers of aqueous liquid separate so
long as said centrifuge tube is at rest, and wherein said lower
region has a closed bottom.
2. The ultracentrifuge tube of claim 1, wherein said inner diameter
of said lower region is smaller than 0.25 inch.
3. The ultracentrifuge tube of claim 1, wherein said lower region
is at least 5% of the total length of said tube.
4. The ultracentrifuge tube of claim 1, wherein the inner surfaces
are polished by vapor polishing.
5. The ultracentrifuge tube of claim 1, wherein the inner surfaces
are coated with adhering polymer to prevent adsorption of
biological particles.
6. The ultracentrifuge tube of claim 1, wherein said tube is
prepared from materials such that said tube can be centrifuged at
velocities high enough to band viruses in CsCl gradients without
said tube breaking.
7. The ultracentrifuge tube of claim 1, wherein said tube is
prepared from materials such that said tube can be centrifuged at
velocities high enough to band mycoplasmas in CsCl gradients
without said tube breaking.
8. The ultracentrifuge tube of claim 1, wherein said tube is
prepared from materials such that said tube can be centrifuged at
velocities high enough to band rickettsia in CsCl gradients without
said tube breaking.
9. The ultracentrifuge tube of claim 1, wherein said tube is
prepared from materials such that said tube can be centrifuged at
velocities high enough to band yeast in CsCl gradients without said
tube breaking.
10. The ultracentrifuge tube of claim 1, wherein said tube is
prepared from materials such that said tube can be centrifuged at
velocities high enough to band bacteria in CsCl gradients without
said tube breaking.
11. The ultracentrifuge tube of claim 1, wherein said tube is made
of polycarbonate.
12. The ultracentrifuge tube of claim 1, wherein said upper region,
middle region and lower region have outer diameters equal to each
other.
13. The ultracentrifuge tube of claim 1, wherein said upper region
has an outer diameter larger than an outer diameter of said lower
region.
14. The ultracentrifuge tube of claim 1, wherein said inner
diameter of said lower region is smaller than 0.1 inch.
15. The ultracentrifuge tube of claim 1, wherein said inner
diameter of said lower region is in the range 0.08-0.1 inch.
16. The ultracentrifuge tube of claim 1, wherein said inner
diameter of said lower region is in the range 0.039-0.08 inch.
17. The ultracentrifuge tube of claim 1, wherein said inner
diameter of said lower region is 0.064 inch.
18. An ultracentrifuge tube comprising an upper region, a middle
region and a lower region wherein an inner diameter of said upper
region is larger than an inner diameter of said lower region,
wherein said upper region is separated from said lower region by
said middle region having a decreasing diameter from said upper
region toward said lower region and wherein said lower region has a
closed bottom.
19. The ultracentrifuge tube of claim 18, wherein said middle
region comprises one or more serrations.
20. The ultracentifuge tube of claim 18, wherein said lower region
has an inner diameter small enough to trap an air bubble between
two layers of liquid such that the air bubble will keep said two
layers of liquid separate so long as said centrifuge tube is at
rest.
21. The ultracentrifuge tube of claim 18, wherein said inner
diameter of said lower region is smaller than 0.25 inch.
22. The ultracentrifuge tube of claim 18, wherein said lower region
is at least 5% of the total length of said tube.
23. The ultracentrifuge tube of claim 18, wherein the inner
surfaces are polished by vapor polishing.
24. The ultracentrifuge tube of claim 18, wherein the inner
surfaces are coated with adhering polymer to prevent adsorption of
biological particles.
25. The ultracentrifuge tube of claim 18, wherein said tube is
prepared from materials such that said tube can be centrifuged at
velocities high enough to band viruses in CsCl gradients without
said tube breaking.
26. The ultracentrifuge tube of claim 18, wherein said tube is
prepared from materials such that said tube can be centrifuged at
velocities high enough to band mycoplasmas in CsCl gradients
without said tube breaking.
27. The ultracentrifuge tube of claim 18, wherein said tube is
prepared from materials such that said tube can be centrifuged at
velocities high enough to band rickettsia in CsCl gradients without
said tube breaking.
28. The ultracentrifuge tube of claim 18, wherein said tube is
prepared from materials such that said tube can be centrifuged at
velocities high enough to band yeast in CsCl gradients without said
tube breaking.
29. The ultracentrifuge tube of claim 18, wherein said tube is
prepared from materials such that said tube can be centrifuged at
velocities high enough to band bacteria in CsCl gradients without
said tube breaking.
30. The ultracentrifuge tube of claim 18, wherein said tube is made
of polycarbonate.
31. The ultracentrifuge tube of claim 18, wherein said upper
region, middle region and lower region have outer diameters equal
to each other.
32. The ultracentrifuge tube of claim 18, wherein said upper region
has an outer diameter larger than an outer diameter of said lower
region.
33. The ultracentrifuge tube of claim 18, wherein said inner
diameter of said lower region is smaller than 0.1 inch.
34. The ultracentrifuge tube of claim 18, wherein said inner
diameter of said lower region is in the range 0.08-0.1 inch.
35. The ultracentrifuge tube of claim 18, wherein said inner
diameter of said lower region is in the range 0.039-0.08 inch.
36. The ultracentrifuge tube of claim 18, wherein said inner
diameter of said lower region is 0.064 inch.
37. An ultracentrifuge tube comprising an upper centripetal region
having a cylindrical shape, a middle region having a cylindrical
shape and a lower centrifugal region having a cylindrical shape,
wherein an inner diameter of said upper region is larger than an
inner diameter of said lower region, wherein said upper region is
separated from said lower region by said middle region having a
decreasing diameter from said upper region toward said lower region
and wherein said lower region has a closed bottom.
38. The ultracentrifuge tube of claim 37, wherein said middle
region comprises one or more serrations.
39. The ultracentifuge tube of claim 37, wherein said lower region
has an inner diameter small enough to trap an air bubble between
two layers of liquid such that the air bubble will keep said two
layers of liquid separate so long as said centrifuge tube is at
rest.
40. The ultracentrifuge tube of claim 37, wherein said inner
diameter of said lower region is smaller than 0.25 inch.
41. The ultracentrifuge tube of claim 37, wherein said lower region
is at least 5% of the total length of said tube.
42. The ultracentrifuge tube of claim 37, wherein the inner
surfaces are polished by vapor polishing.
43. The ultracentrifuge tube of claim 37, wherein the inner
surfaces are coated with adhering polymer to prevent adsorption of
biological particles.
44. The ultracentrifuge tube of claim 37, wherein said tube is
prepared from materials such that said tube can be centrifuged at
velocities high enough to band viruses in CsCl gradients without
said tube breaking.
45. The ultracentrifuge tube of claim 37, wherein said tube is
prepared from materials such that said tube can be centrifuged at
velocities high enough to band mycoplasmas in CsCl gradients
without said tube breaking.
46. The ultracentrifuge tube of claim 37, wherein said tube is
prepared from materials such that said tube can be centrifuged at
velocities high enough to band rickettsia in CsCl gradients without
said tube breaking.
47. The ultracentrifuge tube of claim 37, wherein said tube is
prepared from materials such that said tube can be centrifuged at
velocities high enough to band yeast in CsCl gradients without said
tube breaking.
48. The ultracentrifuge tube of claim 37, wherein said tube is
prepared from materials such that said tube can be centrifuged at
velocities high enough to band bacteria in CsCl gradients without
said tube breaking.
49. The ultracentrifuge tube of claim 37, wherein said tube is made
of polycarbonate.
50. The ultracentrifuge tube of claim 37, wherein said upper
region, middle region and lower region have outer diameters equal
to each other.
51. The ultracentrifuge tube of claim 37, wherein said upper region
has an outer diameter larger than an outer diameter of said lower
region.
52. The ultracentrifuge tube of claim 37, wherein said inner
diameter of said lower region is smaller than 0.1 inch.
53. The ultracentrifuge tube of claim 37, wherein said inner
diameter of said lower region is in the range 0.08-0.1 inch.
54. The ultracentrifuge tube of claim 37, wherein said inner
diameter of said lower region is in the range 0.039-0.08 inch.
55. The ultracentrifuge tube of claim 37, wherein said inner
diameter of said lower region is 0.064 inch.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] The present application is a continuation application of
U.S. patent application Ser. No. 09/571,274 filed May 16, 2000,
which is a divisional of U.S. patent application Ser. No.
09/265,541, filed Mar. 9, 1999, each incorporated herein by
reference. The present application is further related to U.S.
provisional patent application Serial No. 60/077,472, filed Mar.
10, 1998, incorporated herein by reference, and claims priority
thereto under 35 USC .sctn.119(e).
BACKGROUND OF THE INVENTION
[0003] The present invention relates to the field of separating and
identifying microorganisms, particularly infectious agents, using
two-dimensional centrifugation and exposure to chemical and
enzymatic agents, combined with detection in density gradients
based on light scatter or fluorescence, counting by fluorescence
flow cytometry, and characterization of intact virions, bacteria,
proteins and nucleic acids by mass spectrometry, flow cytometry and
epifluorescence microscopy.
[0004] The publications and other materials used herein to
illuminate the background of the invention or provide additional
details respecting the practice, are incorporated by reference, and
for convenience are respectively grouped in the appended List of
References. Patents referenced herein are also incorporated by
reference.
[0005] In the prior art, diagnosis of viral and bacterial
infections has been done by culturing the causal agents in suitable
media or in tissue culture to obtain sufficient particles for
analysis, followed by identification based on which conditions
support growth, on reaction to specific antibodies, or based on
nucleic acid hybridization (Gao and Moore, 1996). Biological growth
can be omitted when the polymerase chain reaction (PCR) is used to
amplify DNA, however, PCR requires sequence-specific primers, and
is thus limited to known or suspected agents (Bai et al., 1997).
For all these methods, considerable time is required, and the
methods are useful for agents whose properties are known or
suspected. Existing methods do not provide means for rapidly
isolating and characterizing new infectious agents. Hundreds of
infectious agents are known, and it is infeasible to have available
reagents for an appreciable fraction of them.
[0006] Techniques for recovering infectious agents from blood,
urine, and tissues have been previously developed based on
centrifugation or filtration, but have not been widely used
clinically (Anderson et al., 1966; Anderson et al., 1967). The
highest resolution methods use rate zonal centrifugation to
separate fractions based on sedimentation rate (measured in
Svedberg units, S) and isopycnic banding density (measured in grams
per mL or .rho.). S-.rho. separations have been used to isolate
virus particles in a high state of purity from rat liver
homogenates, and have been used to isolate the equivalent of
approximately 20 virions per cell (Anderson et al., 1966). In these
studies, virus particles were detected by light scattering and
visualized by electron microscopy. The separations required complex
special equipment not generally available, one or more days of
effort, and they did not provide a definitive identification of the
bacterial or viral species separated.
[0007] It is important to show that candidate infectious particles
isolated by centrifugal methods actually contain nucleic acids. DNA
and RNA in both active and fixed bacterial and viral particles have
been stained with fluorescent dyes specific to nucleic acids, and
observed and counted by fluorescent microscopy and flow cytometry.
Many dyes are now known which exhibit little fluorescence in the
free state, but become highly fluorescent when bound to nucleic
acids. Some bind differentially to DNA or RNA or to different
specific regions, and some show different emission spectra
depending on whether bound to DNA or RNA. In this disclosure, dyes
referred to are fluorescent dyes. By differential fluorescence
spectroscopy ssDNA, dsDNA and RNA may be distinguished. See,
Haugland, 1996; Mayor and Diwan, 1961; Mayor, 1961; Hobbie et al.,
1977; Zimmerman, 1977; Perter and Feig, 1980; Paul, 1982; Suttle,
1993; Hirons et al., 1994; Hennes and Suttle, 1995; Hennes et al.,
1995.
[0008] Isolated nucleic acid molecules of the dimensions found in
bacteria and viruses have been counted and their mass estimated
using fluorescence flow cytometry for molecules in solution, and
epifluorescence microscopy of immobilized molecules (Hennes and
Suttle, 1995, Goodwin et al., 1993). In both instances, the size of
fragments produced by restriction enzymes can be estimated, and the
molecules identified by reference to a database listing the sizes
of fragments of known DNA molecules produced by different
restriction enzymes (Hammond et al., U.S. Pat. No. 5,558,998; Jing
et al., 1998).
[0009] Using specific fluorescently-labeled antibodies, specific
identifications may also be made. These studies are time consuming,
and require batteries of specific antibodies, together with
epifluorescent microscopy or fluorimeters.
[0010] Matrix-Assisted Laser-Desorption-Ionization Time-of-Flight
Mass Spectrometry (MALDI-TOF-MS) currently allows precise
measurements of the masses of proteins having molecular weights of
over 50,000 daltons. Individual virion proteins have been
previously studied by mass spectrometry (Siuzdak, 1998); however,
resolution of complete sets of viral subunits from clinically
relevant preparations of intact viruses, and the demonstration that
precise measurements could be made of their individual masses, have
not been previously reported. While single protein mass
measurements can reliably identify many proteins, when a set of
proteins from a virus or bacterial cell are known, detection of
such a set provides more definitive identification. Methods are
currently also being developed which allow partial sequencing of
proteins or enzymatically produced peptide fragments and thus
further increase the reliability of identifications. For
MALDI-TOF-MS currently used methods require a picomole or more of
protein, while electrospray mass spectrometry currently requires
5-10 femtomoles. The detection limits with mass spectrometry,
especially MALDI, depend on getting a sample concentrated and on to
a very small target area. Sensitivity will increase as ultramicro
methods for concentrating and transferring ever smaller-volume
samples are developed. See, Claydon et al., 1996; Fenselau, 1994;
Krishmanurthy et al., 1996; Loo et al., 1997; Lennon and Walsh,
1997; Shevchenko et al., 1996; Holland et al., 1996; Liang et al.,
1996.
[0011] Centrifugal methods for concentrating particles from large
into small volumes have been in use for decades. Using microbanding
centrifuge tubes which have a large cylindrical volume and cross
section which tapers gradually in a centrifugal direction down to a
small tubular section, particles may be concentrated or banded in a
density gradient restricted to the narrow tubular bottom of the
tube, or may be pelleted. The basic design of such tubes are well
known by those skilled in the arts. See, Tinkler and Challenger,
1917; Cross, 1928; ASTM Committee D-2, 1951; Davis and Outenreath,
U.S. Pat. No. 4,624,835; Kimura, U.S. Pat. No. 4,861,477; Levine et
al., U.S. Pat. No. 5,342,790; Saunders et al., U.S. Pat. No.
5,422,018; Saunders, U.S. Pat. No. 5,489,396. The original tubes of
this type were called Sutherland bulbs and were used to determine
the water content of petroleum (The Chemistry of Petroleum and Its
Substitutes, 1917, ASTM Tentative Method of Test for Water and
Sediment by Means of Centrifuge, ASTM Designation: D 96-50T, 1947).
Slight modifications of the basic design are described in U.S. Pat.
Nos. 4,106,907; 4,624,835; 4,861,477; 5,422,018, 5,489,396. Such
tubes have been made of glass or plastic materials, and the use of
water or other fluids to support glass or plastic centrifuge tubes
in metal centrifuge shields has long been well known in the art.
However, centrifuge tubes disclosed in the prior art which include
a shape similar to that of the microbanding centrifuge tubes of the
instant invention could not withstand the centrifugal forces
required to band viral particles in gradients. Conventional
centrifuge tubes, or tubes derivative from the Sutherland design
have been used for density gradient separations, and for
separations in which wax or plastic barriers are used which
position themselves between regions of different density to allow
recovery of these fractions without mixing. There has been no
previous discussion of barriers which prevent mixing of step
gradient components at rest, but which barriers are centrifuged
away from the gradient during rotation. Nor have tube closures for
high-speed thin-walled swinging-bucket centrifuge tubes been
described, whose exterior surfaces can be disinfected after the
tubes are loaded.
[0012] The efficient stabilization of very shallow density
gradients in centrifugal fields is well known, and is utilized in
analytical ultracentrifugation to cause a sample layer to flow
rapidly to the centripetal surface of a gradient without mixing
using a synthetic boundary cell (Anderson, U.S. Pat. No.
3,519,400). Hence, light physical barrier disks between step
gradient components can be moved away from the gradient by
centrifugal force without appreciably disturbing the gradient,
provided that they are made of porous, woven or sintered materials
having a physical density less than that of the sample layer, such
as polyethylene or polypropylene.
[0013] Many authors have noted that viruses and bacteria are often
resistant to the actions of detergents and enzymes which will
digest or dissolve contaminating particles of biological origin,
and efforts have been made to classify infectious agents on the
basis of their differential sensitivities. These differences have
not previously and conveniently been incorporated in a method for
detecting and quantifying infectious agents. See, Gessler et al.,
1956; Theiler, 1957; Epstein and Hold, 1958; Kovacs, 1962;
Planterose et al., 1962; Gard and Maaloe, 1959. Density differences
between different species of virus and bacteria are well known, but
have not been previously exploited for purposes of
identification.
[0014] Infectious particles exhibit a wide range of isopycnic
banding densities ranging from approximately 1.17 g/ml to 1.55
g/ml, depending on the type of nucleic acid present, and the ratios
between the amount of nucleic acid, protein, carbohydrate, and
lipid present. While such banding density differences are well
known, no attempt has been previously made to systematically
measure them and use the data to classify infectious agents.
[0015] The present invention is directed to an integrated system
for concentrating, detecting and characterizing infectious agents
using separations based on sedimentation rate and banding density,
spectral analysis of emitted fluorescent light to distinguish DNA
from RNA, differentiation of viral and bacterial particles from
other particles by sedimentation through zones of solubilizing
enzymes or reagents, determination of the isopycnic banding
densities of infectious particles by reference to the positions of
synthetic density standardization particles, particle detection
using fluorescent dyes for DNA or RNA, further concentration of
banded particles by pelleting, transfer of concentrated particles
to mass spectrometer targets for protein mass determination and
analysis, counting of concentrated particles by epifluorescent
microscopy and fluorescence flow cytometry, and identification of
bacterial or viral nucleic acids by restriction fragment length
polymorphism analysis using either immobilized nucleic acid
molecules, or ultrasensitive fluorescence flow cytometry. These
methods are especially useful in characterizing biological samples
which have low titres of virus and which contain viruses which are
not culturable.
[0016] Furthermore, all current methods used to detect and
characterize infectious agents, including use of fluorescent
antibodies, detection of agent-associated enzymes, culture to
increase agent mass, PCR amplification, restriction fragment length
polymorphism analysis, hybridization to probes immobilized on
chips, histochemical analysis, and all forms of microscopy
including electron microscopy, are vastly improved by
preconcentration of the microorganisms using the methods of the
present invention.
[0017] These techniques have not previously been assembled into one
operational system capable of routine field, hospital, and clinical
laboratory use. The present application describes innovations and
inventions which make such a system feasible. For work with
potentially lethal agents, the system will be assembled in
containment, and at least partially automated.
SUMMARY OF THE INVENTION
[0018] The overall objective of this invention is to develop a
physical system for rapidly identifying infectious disease agents
without growing them, and for discovering new infectious agents.
The process is based on the thesis that infectious agents
constitute a unique group of particles which can be isolated by
physical and chemical means from other natural particles and
identified by their physical parameters using centrifugal means,
fluorescence, and mass spectrometry. The system will allow rapid
clinical distinction between viral and bacterial infections,
identification of specific agents with the aim of providing
specific therapy, and the rapid discovery of new infectious agents.
In addition the system will make it feasible to develop and test
new antibiotics and antiviral agents in man by measuring the
effects of these agents on bacterial and viral loads. At present
the development of new antiviral drugs is severely hindered by
inability to define populations of individuals in the early stages
of infection who might benefit from treatment.
[0019] In accordance with the present invention, an ultracentrifuge
tube is provided which comprises upper, middle and lower regions of
successively smaller diameters. In one embodiment, the tube has an
upper region for receiving a sample, a funnel-shaped middle region
and a lower narrow tubular microbanding region. The diameter of the
lower region may be 0.25 inch or less, preferably 0.1 inch or less,
more preferably 0.1 to 0.08 inch, and most preferably 0.08 to 0.039
inch. Smaller diameter microbanding regions are feasible and are
within the province of this disclosure. The length of the lower
region is typically between 5% to 25% of the length of the tube. In
one aspect, the tube may also include a seal with a central opening
which can be plugged and unplugged.
[0020] In accordance with the present invention, a bucket is
provided for holding an ultracentrifuge tube. The bucket comprises
upper and lower regions which may be of successively smaller
diameters, may have inserts to successively decrease the inner
diameter, or may be of uniform internal diameter, and may further
comprise a third region which attaches the bucket to a rotor.
[0021] Further in accordance with the present invention, a method
is provided for concentrating microorganisms. As used herein, the
term "microorganisms" is intended to include viruses, myoplasmas,
rickettsia, yeast and bacteria. The method comprises
ultracentrifugation of a sample containing the microorganism in an
ultracentrifuge tube described herein. The ultracentrifugation may
include the formation of density gradients and/or the staining of
the microorganism(s). In one aspect, the staining can be used to
distinguish the DNA or RNA content of a virus. The banding of the
microorganisms upon ultracentrifugation can be used to identify the
microorganisms.
[0022] In a further aspect of the invention, the concentrated
microorganisms are further characterized by conventional techniques
such as mass spectrometry, flow cytometry, optical mapping,
isopycnic banding densities, fluorescence, restriction enzyme
analysis, genome size, enzymatic or chemical
resistance/susceptibility, immunochemistry and the like. In another
aspect of the invention, the amount or titre of the microorganisms
can be determined.
[0023] In accordance with the present invention, a system is
provided for measuring fluorescence from a sample in a centrifuge
tube. In one embodiment, the system includes a centrifuge tube, a
light source, such as a laser, and a detector to detect light
passing through the sample or emitted from the sample upon light
passing through it. Optical filters select and separate the
exciting and emitted wavelengths of light.
[0024] The internal surfaces of the tubes and especially the funnel
portion must be very smooth in order to prevent small virus
particles from being retarded by surface irregularities, and in
addition, the surfaces must be treated so that infectious particles
are not adsorbed. Polishing of plastic surfaces is done by brief
exposure to a solvent vapor. For example, polycarbonate is polished
by brief exposure to heated methylene chloride gas. Plastic
surfaces are modified to prevent adsorption of infectious particles
by exposure to dilute solutions of proteins such as bovine serum
albunin or gelatin, or to charged polymers such as heparin or
derivatives of heparin. Both of these procedures are well known to
those practiced in the arts.
[0025] Further in accordance with the present invention, a system
is provided for counting particles concentrated in a small volume.
The system includes a container in which the particles are
concentrated, a capillary tube, two pumps, means for moving the
container relative to the capillary tube, a flow cell, a light
source and detector. Alternatively, fluid may be moved by gas
pressure instead of pumps.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIG. 1 shows an S-rho plot for a typical tissue and for
representative viruses.
[0027] FIGS. 2A-2C are a diagrammatic representation of one
embodiment of a centrifugal microbanding tube and its use.
[0028] FIGS. 3A-3G show alternative embodiments of microbanding
tubes and use in a rotor (3F).
[0029] FIGS. 4A and 4B illustrate a centrifuge swinging bucket
design that allows higher speed fractionation of large sample
volumes.
[0030] FIG. 5 illustrates a complete system including vertical
monochromatric laser illumination, goniometer and X-Y stage for
supporting and positioning microbanding tube, microbanding tube
with banded viruses particles, and camera system.
[0031] FIG. 6 illustrates a complete system including interference
filter light sources, light pipe illumination, digital data
acquisition, and CRT data presentation.
[0032] FIG. 7 illustrates a method for recovering banded virus
particles using a micropipette, and counting them by flow
cytometry.
[0033] FIGS. 8A-8C illustrate details of band recovery.
[0034] FIGS. 9A-9F illustrate one embodiment of a closure for
swinging bucket rotor centrifuge tubes and detection of a sample
with respect to the tubes.
DETAILED DESCRIPTION OF THE INVENTION
[0035] The invention is directed to methods of identifying and
measuring the presence of microbial agents such as bacteria and
viruses in biological samples. The methods include centrifugation
steps to purify the microbial agents in a very small volume. The
agents are then assayed by means such as isopycnic banding density,
fluorescence or mass spectrometry.
[0036] It is an object of this invention to develop integrated
systems and methods in which suspensions containing microorganisms,
including infectious agents, are stained with one or more
fluorescent dyes, in which a step or continuous gradient is
automatically formed during centrifugation, in which the
microorganisms are centrifuged away from the stain-containing
suspending medium and are washed free of external stain, are
concentrated in a gradient of very small cross section, separated
according to their isopycnic banding densities, their banding
densities determined, and the microorganisms detected by
fluorescence.
[0037] It is a further object of this invention to concentrate
microorganisms, including infectious agents, into microbands by a
factor of 1-5,000.
[0038] It is a further object of this invention to expose the
microorganisms, such as infectious agents, to reagents including
detergents, surfactants, enzymes, or organic solvents contained in
distinct zones in a density gradient to dissolve or disassemble
contaminating particles to prevent them from banding with the
microorganisms, and to separate stained particles from the free
stain of the initial sample volume.
[0039] It is a further object of this invention to use one or more
dyes which bind differentially to RNA, single stranded DNA or
double stranded DNA to allow these to be distinguished by their
fluorescent spectra.
[0040] It is a further object of this invention to provide for the
concentration of banded microorganisms, for example infectious
agents, by resuspending the banding gradient, which is typically
0.04 mL, to approximately 4 mL in water or a very dilute buffer,
and pelleting the microorganisms one or more times to provide a
concentrated pellet free of gradient materials for mass
spectrometric analysis, for counting by epifluorescent microscopy
or by flow cytometry.
[0041] It is a further object of this invention to provide means
for the diagnosis of infectious diseases which minimize exposure of
laboratory personnel to infectious agents.
[0042] It is an additional object of this invention to provide
means for preparing nucleic acids from small quantities of
microorganisms, including infectious agents, to determine the
masses of intact nucleic acid molecules, and for characterization
of fragments produced by restriction enzymes using either flow
cytometry or epifluorescence microscopy.
[0043] It is an additional object of this invention to determine
the banding densities of the microorganisms, such as infectious
agents, accurately by reference to the positions of calibrated
particles added to the gradients.
[0044] For ease of description, the invention will be described
with reference to viruses as the microorganisms. It will be
understood that the invention is also applicable to other
microorganisms, including mycoplasmas, yeast and bacteria. The
invention is particularly suited for the identification of
infectious agents, and will be described in this context.
[0045] FIG. 1 is a graph depicting the sedimentation coefficients
and isopycnic banding densities of subcellular organelles and
viruses to illustrate the concept of the "Virus Window" (Anderson,
1966). It is evident that viruses have a relatively narrow range of
sedimentation coefficients and banding densities and may be
isolated from a tissue homogenate or from blood in a high state of
purity using high resolution S-.rho. separation systems. For a
complete description of high resolution S-.rho. centrifugal methods
and of centrifuge development for virus isolation, refer to
National Cancer Institute Monograph 21, The Development of Zonal
Centrifuges and Ancillary Systems for Tissue Fractionation and
Analysis, U.S. Department of Health, Education and Welfare, Public
Health Service, 1966. This work describes S-.rho. separation theory
and systems, and the use of colored plastic beads of graded
densities as density markers.
[0046] In practice, a blood sample or tissue homogenate is
centrifuged to sediment all particles having sedimentation rates
higher than that of the particle or particles to be analyzed. For
viruses, that means particles of circa 10,000 S and above are
discarded. The supernatant after such a separation is then used as
the sample for second dimension isopycnic banding separations
carried out in microbanding tubes as described here, using
centrifugation conditions which will sediment and isopycnically
band all known infectious particles.
[0047] One picomole of virus would contain 6.022.times.10.sup.11
viral particles, while 6.times.10.sup.9 virions would contain 1
picomole of a viral coat protein present in 100 copies per virion.
Quantitative polymerase chain reaction (PCR) has been used to
demonstrate that in many infectious diseases >10.sup.8 virus
particles are present per mL of plasma or serum. Hence, if the
virions from a 5-10 mL biological sample containing 10.sup.8
virions/mL are concentrated to a microliter or two, and then
applied to a very small target area, individual viral proteins can
be detected using MALDI-TOF-MS (Krishmanurthy et al., 1996; Holland
et al., 1966). Using electrospray techniques, samples containing
10.sup.6 virus particles/mL can be detected, while with flow
cytometry and immobilized DNA epifluorescence microscopy, even
fewer particles are required (Hara et al., 1991; Hennes and Suttle
1995). The application of these methods to bacteria may require a
preseparation of proteins to reduce the complexity of the sample.
In mass spectrometry, detection has been by charged ion detection,
and the limitations of such detection have set the upper limits to
the size of proteins and nucleic acids that can be detected. Mass
spectrometric methods have now been described which allow masses of
biological particles above 100,000 daltons to be measured.
[0048] In order to work with such levels of virus, the virus must
be concentrated into a very small volume. This concentration is
accomplished during the second dimension of centrifugation (the
isopycnic banding step) by banding the virus using a centrifuge
tube specially designed to concentrate the virus into a microband
after passage through gradient layers that wash the particles and
expose them to selected reagents. An example of such a microbanding
centrifuge tube is shown in FIGS. 2A-2C. FIG. 2A illustrates
diagrammatically a hollow transparent centrifuge tube 1 with an
upper sample volume 2, grading into a serrated funnel region 3
having successively tapered and parallel-wall sections 4-6,
constricting down to a narrow tubular microbanding region 7. The
serrated funnel region 3 is an improvement over centrifuge tubes
which simply taper from top to bottom without including a serrated
region. By serrations is meant, for example, concentric rings or
edges or lips. These rings, edges or lips are preferably continuous
around the inner diameter of the centrifuge tube, but this is not
required. For example, three projections from the inner wall of the
centrifuge tube spaced equally around the diameter could be used to
hold a disk in place. The term serrations is meant to include such
possibilities but does not include a straight tapering with no
rings, edges, lips or projections on the inner surface of the
centrifuge tube. The serrations can be used as rests onto which
disks can be placed to separate two or more layers of liquid.
Although disks can be placed into tubes which simply taper without
serrations, the disks in such tapered tubes can be easily tipped up
on one edge by pushing down on the opposing edge. This would cause
a premature mixing of the layers which are to be separated by the
disks. The serrated region allows disks to lie flat and prevents
the disks from being accidentally tipped up. As an example, the
centrifuge tube may be 3.45 inches from top to bottom, have an
outer diameter at the top of 0.562 inch, and have an inner diameter
in the bottom microbanding region 7 of 0.064 inch. Such a tube is
suitable for use in an SW41 Ti (Beckman) rotor. The inside surface
of the tube is preferably polished using conventional techniques,
including vapor polishing, so that the virus particles do not stick
to the wall of the tube. Additionally, the internal surfaces of the
tubes may be coated with a protein or polymer to prevent particle
adhesion, as is well known in the art.
[0049] FIG. 2B illustrates how the tube is loaded at rest with a
series of fluids of decreasing physical density. The tube shown
comprises a series of serrations onto which can be laid disks to
separate one layer of fluid from the next layer of fluid. Liquid 8
is denser than any particle to be recovered, and is used to
partially fill the microbanding region 7. When a less dense fluid 9
is pipetted in with a micropipet an air bubble 10 (wherein by air
is meant atmospheric air or another gas) may be left to keep the
fluids 8 and 9 separate. Similarly when the first overlay fluid 11
is introduced, air bubble 12 may be left in place, thus keeping the
three liquids separate until centrifugation is commenced. A tube
with an inner diameter of 0.064 inches in microbanding region 7 is
suitable for allowing an air bubble to be left in place to separate
two layers of liquid. Alternatively, the air bubbles may be left
out and the fluids allowed to diffuse together to create a density
gradient. Fluid 11 is covered with a light porous plastic disk 13,
preferably of sintered polyethylene or polypropylene, which fits in
place in the first serration. A fluid 14, less dense than fluid 11,
which may contain one or more reagents, is then introduced, and
covered with disk 15, followed by even less dense liquid 16 which
is covered with disk 17. The entire system is stable until
centrifuged. Before use the sample layer 18, which has a density
less than that of fluid 16 is then added up to level 19. The tubes
are then centrifuged at high speed in metal shields, typically with
water or other liquid added to the shields. In addition, the tubes
may be supported by fitting adapters which fill the space between
the tubes and the shields, and water may be added to fill any
spaces between the tubes, adapters and shields to provide
additional support. Optionally the tubes may be capped (as shown in
FIGS. 9A-9F, described in further detail below), to minimize the
chances of operator infection.
[0050] FIG. 2C illustrates diagrammatically a tube after
centrifugation. The porous separation disks 13, 15, and 17 have
risen to the top of the tube, and sample layer 18 is cleared of
virus, and the original step gradient has changed, by diffusion,
into one of a series of shallow gradients. In addition, gas bubbles
10 and 12 have also moved centripetally, and fluids 8 and 9 have
come into contact to form a steep gradient by diffusion. As
centrifugation proceeds, the slope of this gradient diminishes,
producing a banding gradient of a width suitable for banding the
infectious agents. For cesium chloride gradients, the densities
typically range from 1.18 to 1.55 g/ml. These gradient steps may
not only contain reagents to dissolve non-viral particles, but also
serve to wash excess fluorescent dye away from the particles. For
example, various detergents or enzymes such as proteases may be
added either to the sample layer 18 or to other layers such as 14
or 16. Fluorescent dyes may also be present in these regions. The
free dye will not enter the lower, more dense regions in which the
virus bands and therefore the centrifugation will purify the
viruses from all of the reagents which may be present in the upper,
less dense layers. After centrifugation, the microbanding region of
the tube contains the upper portion of the banding gradient 27,
banded virus 28 (including any dye bound to the virus or viral
nucleic acid) and lower dense portion of the banding gradient 29,
and the gradient formed between them by diffusion.
[0051] FIGS. 3A-3G illustrate alternative embodiments of tubes
useful for microbanding of viruses and bacteria and all have a
serrated internal construction which allows one or more light
barriers to be positioned and retained at rest. Tubes shown in
FIGS. 3A-3D and 3G are designed to be centrifuged in swinging
bucket rotors so that the tubes are horizontal during
centrifugation and vertical at rest. The tube shown in FIG. 3A is
the more conventional design with a sample reservoir 31, a serrated
funnel region 32, and a microbanding section 33. The tube shown in
FIG. 3B is similar to that of FIG. 3A, but it is supported in a
centrifuge shield by a support insert 34 which may be of plastic or
metal. The tubes shown in FIGS. 3B-3E fill a rotor chamber
completely. The tube of FIG. 3C has an opaque bottom section 35
which absorbs scattered light, while that shown in FIG. 3D has a
bulbous section 36 at the bottom of the microbanding tube 37 to
contain an excess volume of the fluid forming the dense end of the
gradient, thus stabilizing the gradient. The tube shown in FIG. 3E
is designed to be centrifuged in an angle head rotor as shown in
FIG. 3F, and has a linearly continuous wall 38 along one side
positioned in the rotor so that particles may readily slide down to
microbanding region 40. The tube shown in FIG. 3G illustrates how a
very large microbanding tube may be fabricated.
[0052] FIGS. 4A-4B illustrate how the tube of FIG. 3G may be
centrifuged at higher speed than tubes having a constant radius
from top to curved bottom. This is accomplished by using a metal,
plastic or carbon fiber shield 45 which matches the dimensions of
tube 46. The shield has a cap 47 and the shield or bucket swings on
integral attachment 48, as is conventionally done in high speed
swinging bucket rotors. Tip 49 of the shield is much smaller
diameter than the upper section of the shield, has much less mass
swinging at its maximum radius, and hence can reach much higher
speeds than is the case with shields of uniform internal diameter.
This makes possible isolation of trace amounts of virus from much
larger volumes than would otherwise be the case. During
centrifugation using rotor 50 driven by drive 51, shield and tube
52 assume a horizontal position as shown.
[0053] The microbanded viruses can be analyzed at this stage or
they can be collected, diluted, and further processed. To analyze
the microbanded viruses at this stage, they can be detected by a
system as shown in FIG. 5. For example, the isopycnic banding step
or an earlier step may have included a fluorescent dye or
fluorescent dyes within the solution with which the virus was mixed
or through which the virus was centrifuged. Dyes are known with
which intact viruses may be stained and which can distinguish
between RNA, DNA, single stranded nucleic acid and double stranded
nucleic acid thereby allowing one to detect the presence or absence
of an infectious agent, and further to determine which type of
virus one has purified. The apparatus of FIG. 5 can be used to
analyze these stained particles.
[0054] A scanning and detection system is illustrated schematically
in FIG. 5 where microbanding tube 60 is held in a vertical position
on mount 61 supported by goniometers 62 and 63 which are in turn
supported by X-Y movements 64 and 65 in such a manner as to align
and center the microbanding section of tube 60 with respect to
laser beam 66. Laser beam 66 is generated by a laser 67, which may
be an argon ion laser producing coherent light at 458, 488, 496,
502, and 515 nm. The beam passes through an interference or other
filter 68 to isolate one wavelength, and is reflected down into the
microbanding tube by dichroic mirror 69. The fluorescent banded
particle zone 70 is photographed or electronically scanned by
camera 71 through emission filter 72. The entire system may be
enclosed to eliminate stray light, and filters 68 and 72 may be
replaced by filter wheels (not shown) to optimize detection using
fluorescent dyes which absorb and emit at different wavelengths, or
to distinguish ssDNA, dsDNA and RNA by differences in the spectra
of emitted fluorescent light. Electronic shutters may be attached
to the laser to minimize sample exposure to light and to the camera
to control exposure. The goniometers and X-Y movements may also be
motor driven and remotely controlled, and the entire system may be
controlled by a computer (not shown).
[0055] FIG. 6 illustrates a different version of the scanning
system which can cover all of the visible spectrum and on into the
near ultraviolet. Microbanding tube 80 is aligned in a fixed
support between transparent intensity equilibrators 81 and 82
attached to light pipes 83 and 84 which are in turn attached to
intensity equilibrator 85 illuminated through filter 86 by
condensing lens 87 and light source 88. Filter 86 is one of a set
attached to filter wheel 89 indexed by motor 90. The result is
uniform illumination from two sides of one or more bands 91, 92 and
93. The image is captured through emission filter 94 by digital
camera 95 and the image stored, processed and displayed by computer
96 on CRT 97. Filter 94 may be replaced by a filter wheel identical
to 89 and 90 so that, with both an excitation filter wheel and an
emission filter wheel and a wide spectrum light source such as a
xenon lamp or a halogen lamp, a wide variety of combination of
exciting and emitting light may be chosen, which in turn makes
possible use of a wide variety of fluorescent dyes. Both
fluorescent light and light scatter at a chosen wavelength may be
employed for particle detection. This arrangement facilitates
distinction between ssDNA, dsDNA and RNA.
[0056] The processed image 98 may be displayed to show a picture of
the tube and contained bands 99, 100 and 101. The amount of light
from each band may be integrated and displayed as peaks 102, 103,
and 104, and in addition the integrated values may be displayed
digitally (not shown). The entire system including shutters on the
light source and camera (not shown), filter movement and
positioning, and focusing of the camera may be digitally controlled
by computer 96.
[0057] Display bands 99 and 101, representing centrifuge tube bands
91 and 93 may be fluorescent or non-fluorescent density marker
beads of known density, and the virus band 92 represented by
display band 103. The banding density of the virus may be
determined by interpolation from the positions of the density
markers. When non-fluorescent density markers are used, these are
detected by scattered light using identical filters at positions 86
and 94. A second image using suitable and different filters is then
captured which is comprised solely of fluorescent light. The two
images are electronically inter-compared and the physical density
of the infectious agent determined by interpolation.
[0058] At this stage, the virus can be identified as being a DNA
virus or an RNA virus, and if a DNA virus it can be determined
whether it is single stranded or double stranded. Furthermore, the
density of the virus can be determined. This data can be used to
help identify the type of virus which has been purified.
Nevertheless, it may be desirable or necessary to gather more data
to fully determine what the exact virus is and also to determine
the original viral titre.
[0059] FIG. 7 illustrates diagrammatically counting of individual
fluorescent particles recovered from a tube 110 containing zones of
banded virus 111 and 112 after all fluid above the banding gradient
has been removed and replaced. The tube is placed in a tube holder
113, and an overlay of deionized water or very dilute buffer 114 is
introduced above the gradient supplied through tube 115 to replace
the volume drawn up in the probe 117. The tube may be closed at the
top by a plastic closure 116. The capillary probe 117 is held
stationary, and the microbanding tube 110 is slowly raised under
it. The tube holder 113 is part of a precision drive mechanism 118
and associated stepping motor 119 that moves the tube holder
vertically at a very slow and controllable rate. A slow steady
stream of fluid is drawn into constriction 120 which is centered in
sheath stream 121 provided by pump 122. The result is a constant
flow of fluid through flow cell 123 with a fine virus containing
stream in the center, elongated and extended by the flowing sheath.
A second pump 124 withdraws fluid upward at a constant rate from
the flow cell, which rate is greater than the rate at which piston
pump 122 injects fluid into sheath 121. The difference in the rates
of pumps 124 and 122 is made up by the fluid coming through
capillary probe 117. The fluid coming through capillary probe 117
is a mixture of virus plus fluid from the overlay which is
introduced via tube 115.
[0060] The flow cell 123 is illuminated by laser beam 125 produced
by laser 126, that passes through exciting filter 127. Emitted
light is isolated by emission filter 128 and detected by a
photomultiplier 129. The output from the photomultiplier 129 is
integrated at intervals by computer 130, and the integrated signal
vs. time is displayed on CRT 131. When two viral bands are present,
two peaks such as 132 and 133, are displayed. Depending on the
number of fluorescent particles present, the signal generated from
a band may be integrated into a peak, or, if the suspension is
sufficiently dilute, the particles may be counted individually, the
values binned, and the integrated results displayed.
[0061] In order to count the particles as just described, it is
necessary that the virus particles are greatly diluted as they pass
through flow cell 123. FIG. 8 illustrates diagrammatically how the
problem of making an initial dilution of a very small-volume virus
band for counting individual particles is accomplished. FIG. 8A
shows a tube 110 as in FIG. 7, with a section indicated which is
shown enlarged in FIG. 8B, which in turn shows the section of that
panel enlarged in FIG. 8C. As the movement upward of the
microbanding tube causes the capillary tube to move toward the tube
bottom, the difference in pumping rates of the two pistons attached
to the flow cell causes fluid to flow up the capillary where it is
diluted as described by the combined action of pumps 122 and 124 of
FIG. 7. However, the amount of fluid drawn into the capillary 117
is much greater than the volume of fluid effectively displaced from
the banding gradient by the relative movements of the capillary and
the microbanding tube. This volume is replaced by fluid flowing
into tube 115 though cap 116 which is initially allowed to flow in
until the tube 110 is full. This fluid is much less dense than the
density of the fluid at the top of the gradient in the microbanding
region, and causes minimal disturbance in the gradient. As shown in
FIG. 8B, the capillary 117 slowly approaches virus band 144, and,
as shown in FIG. 8C, a small amount of gradient liquid 145 is
diluted by a larger amount of supernatant fluid 114 as it flows up
the capillary. In this manner, a sharp band of virus particles 144
is diluted and moves through the flow cell as, volumetrically, a
larger band, but with little effective loss of resolution. This
technique provides the dilution necessary to make counting of
individual virus particles feasible and accurate. The amount of
dilution can be controlled such that the concentration of
microorganisms in the capillary tube is less than one-half or
one-tenth, or one-hundredth, or one-thousandth, or one
ten-thousandth, or one-millionth, or one-billionth of the
concentration in the band in the lower region of said tube.
[0062] In addition to counting the particles or determining the
titre of a virus, the amount of DNA in the virus or other microbe
can be determined for individual particles. In this aspect of the
invention, the amount of DNA in the particles is measured by flow
fluorescence analysis (Goodwin et al., 1993) or epifluorescence
analysis (Jing et al., 1998). In this manner, yeast, bacteria,
mycoplasm and virus can be distinguished as groups, For example, it
is known that viruses contain 5-200.times.10.sup.3 bases or base
pairs, E. coli, a typical bacterium, contains 4.times.10.sup.6 base
pairs, while a typical yeast cell contains 1.3.times.10.sup.7 base
pairs. Thus, an estimate of the amount of DNA or RNA present allows
the class of an infectious agent to be determined.
[0063] Thus, the size of a genome can be determined. In this
embodiment of the invention, the genome is extracted from the
microorganism band and immobilized on a solid support. The
immobilized DNA is stained and electronically imaged using an
epifluorescence microscope (Jing et al., 1998). The length of the
individual nucleic acid molecules can then be measured.
[0064] The technique of microbanding is useful not merely for
staining the virus with dyes and being able to count the virus
particles. Once the viruses from a biological sample have been
highly purified and concentrated by the two dimensional
centrifugation technique as described above using microbanding
centrifuge tubes, the viruses are amenable for use in many other
assays.
[0065] When an infectious agent is banded in a microbanding tube,
the band may also be judiciously removed using a capillary needle
in a volume of a few microliters, diluted to 5 mL or more with very
dilute buffer or deionized water to dilute the gradient materials
by a factor as large as 1,000, and then pelleted in a fresh
microbanding tube. The supernatant may then be carefully withdrawn
by a suction capillary, and the virus or other agent resuspended in
approximately 1 microliter using a syringe made, for example, of
fine Teflon.RTM. tubing fitted with a very small stainless steel
wire plunger to fit. The sample may then be transferred to a mass
spectrometer target, mixed with a matrix dye, and used for matrix
assisted laser desorption ionization time of flight mass
spectrometry (MALDI-TOF-MS) to determine directly the masses of
viral coat proteins or of bacterial cell proteins. Technology
described for sample concentration may also be applied, without a
matrix dye, to electrospray or other mass spectrometric analysis
systems, including the detection of intact viral mass.
[0066] A system similar to that shown in FIG. 7 may also be used to
produce the equivalent of molecular restriction fragment length
maps of DNA molecules using restriction enzymes. For this work,
virus or bacterial particle bands may be diluted and sedimented as
described, after which the DNA may be extracted using detergents or
other reagents well known in the art, treated with a restriction
enzyme and a fluorescent dye, and the fragment sizes determined by
flow cytometry (Goodwin et al., 1993; Hammond et al., U.S. Pat. No.
5,558,998). Extracted DNA may also be immobilized on a solid
support, stained with a fluorescent dye, and photographed using an
epifluorescence microscope to determine the length of DNA
molecules. The preparation may then be treated with a restriction
endonuclease, and the number and lengths of the oligonucleotide
fragments determined (Jing et al., 1998). These data are then
compared with a database listing the expected fragment lengths for
different viral or bacterial species to identify each agent. DNA
fragment lengths may also be determined by gel electrophoresis.
[0067] Fluorescence labeled antibodies may also be added to the
particle suspension studied, and the presence or absence of the
label in isopycnically banded particles determined. This approach
is useful for specific identifications, and the use of a set of
antibodies labeled with dyes having different and unique spectral
characteristics allows the presence or absence of a series of
agents to be determined. Alternatively antibodies labeled with
chelators for rare earth's such as Europium and Terbium may be
employed, in which case delayed fluorescence is measured.
[0068] Serum or plasma typically has a physical density between
1.026 and 1.031. Viruses typically have banding densities between
1.17 and 1.55 in cesium chloride, and at much lower densities in
iodinated gradient materials such as Iodixanol or sucrose (Graham
et al., 1994). The intermediate wash and reagent layers between the
sample and the banding gradient must therefore have densities less
than the density of the lightest virus to be banded. Buffers used
to dissolve gradient material for virus isolation include 0.05 M
sodium borate, and 0.02 M sodium cyanide, both of which prevent
bacterial growth.
[0069] With human serum or plasma, centrifugation sufficient to
remove platelets and other particles having sedimentation
coefficients of approximately 10.sup.4 S is used before banding of
virus particles.
[0070] The banding density of virus particles depends on the
nucleic acid/protein ratio, and the presence or absence of lipids
and lipoproteins. Hence attachment of specific identifying
antibodies labeled with fluorescent dyes should not only allow
identification by fluorescence but by a banding density change.
[0071] To assist in identifying particles by density, fluorescent
particles of known density may be included in the sample as shown
in FIG. 6. These particles may include known fixed and
fluorescently stained virus or bacterial particles of known banding
density, or very small fluorescently labeled or non-fluorescently
labeled plastic beads. When polystyrene latex particles are coated
with antibodies, their banding densities are increased appreciably,
and the density may be further increased by reaction of the
antibody-coated particles with the antigen for which the antibodies
are specific (Anderson and Breillatt, 1971). Antibody-coated
fluorescent polystyrene beads may therefore be used not only to
locate virus particles but to identify them.
[0072] The stains which are currently most useful are described in
the Handbook of Fluorescent Probes and Research Chemicals, R. P.
Haugland, ed., Molecular Probes, Inc., Eugene, Oreg. (1996), which
lists the abbreviated dye names, their chemical names, absorption
and emission maxima, and the filter combinations most used.
[0073] For work with human pathogens, safe operation and
containment are important (Cho et al., 1966). Use of swinging
bucket rotors, while optimal from a physico-chemical point of view,
require extensive manipulation and have more parts than an
angle-head rotor. The tubes described in FIG. 3E are designed to be
used in angle-head rotors, and allow sedimenting particles to
travel along a wall at one unchanging angle. Such rotors are easier
to use and handle in containment than are swinging bucket rotors,
however, sedimentation in an angle head rotor is far from ideal.
Hence, the development of methods and devices for safely working
with swinging bucket rotors is important.
[0074] High speed centrifuge tubes are notoriously difficult to
seal effectively and are a potential source of infection to
laboratory personnel. In practice, nearly all high speed swinging
bucket rotor tubes are not themselves sealed, but are enclosed in a
metal bucket which is sealed with a metal cap which does not seal
the tube. The centrifuge tubes are therefore open when loaded,
moved to the centrifuge rotor, inserted, and removed. It is very
difficult to decontaminate the outside of an open tube containing a
density gradient without disturbing the gradient. In the present
application it is desirable to be able to effectively seal the
plastic tubes is such a manner that the outside surfaces can be
cleaned with a suitable disinfectant before the tubes are inserted
into centrifuge shields, and to be able to handle them safely until
they are scanned.
[0075] Sealing is done, as shown in FIG. 9A, by inserting an
annular ring seal 151, having a physical density less than that of
water, into tube 152. Ring 151 is slightly tapered so that it fits
very tightly into tube 152, and has a center hole which can be
plugged and unplugged. In one embodiment, the center hole is
threaded to accept a short, plastic flat-head screw. Initially two
gradient components, including a lighter solution 153 and a denser
solution 155, are introduced to the bottom microbanding region with
a small air bubble 154 between, as previously described. As shown
in FIG. 9B, the solution 156 containing the infectious agent or
other particles is then introduced through tube 157, leaving bubble
158 to separate the sample from the upper gradient solution. The
volume of sample introduced does not completely fill the centrifuge
tube, leaving space 159 empty. The tube is then sealed, as shown in
FIG. 9C with, for example, a plastic flat head screw 160, leaving
air bubble 160 in place. The outside of the tube is then sterilized
by immersing it in a disinfectant such as a sodium hypochlorite or
hydrogen peroxide solution, followed by a water wash and gentle
drying--all with the tube in an upright position. After
centrifugation, as shown in FIG. 9D, the plastic upper seal has
been driven downward by centrifugal force a small distance as the
entrapped air rises around the plastic seal. However, since the
seal has a density less than water, it is retained at the top of
the liquid sample, leaving a small lip 160 which may be grasped
with a hemostat to remove the tube from the centrifuge shield.
During centrifugation, the infectious particles are sedimented out
of liquid 163 and produce band 164 in the gradient. The screw in
the ring seal is then removed, and, as shown in FIG. 9E, the
supernatant liquid 165, which may contain a fluorescent dye, is
removed through tube 166, leaving meniscus 167. As shown in FIG.
9F, a laser beam 168, entering the tube from above, is then aligned
with the tube, causing the banded infectious agent to emit
fluorescent light for detection as previously described.
[0076] When a step gradient containing various reagents in addition
to those used for isopycnic banding is employed, as illustrated in
FIG. 2, the discs used to separate the several solutions rise to
the top and would not allow the use of vertical laser illumination
as shown in FIG. 9F without removing the seal and the discs. In
this instance, side illumination, as illustrated in FIG. 6 would be
employed.
[0077] The laser or delayed fluorescence systems can be completely
contained, the mechanical operations done remotely through small
stepping motors, and the tubes moved in and out of the contained
system under remote control.
[0078] These techniques can be combined with mass spectrometry and
fluorescence-based restriction fragment mapping to allow rapid
diagnosis and identification of infectious agents. However, the
estimates of the masses of individual proteins are generally taken
from published sequence data, and do not include numerous
posttranslational modifications. Mass spectrometric data bases must
be created to include actual mass measurements of different
microorganisms. In addition, virion protein mass measurements will
allow the detection of many genetic variants. However, for many
studies of microorganisms, including development of data bases, the
key problem has been the development of methods for systematically
providing highly concentrated and purified microsamples of
microorganisms from patient samples, natural waters, and from
tissue culture fluids. This problem is solved by the present
invention.
[0079] The present invention is described by reference to the
following Example, which is offered by way of illustration and are
not intended to limit the invention in any manner. Standard
techniques well known in the art or the techniques specifically
described below were utilized.
EXAMPLE
[0080] To illustrate the use of microbanding tubes, experimental
studies have been carried out using a small single-stranded
non-pathogenic DNA virus .phi.X 174. Approximately 10.sup.10 virus
particles which had been purified by isopycnic banding in CsCl in a
microbanding tube such as shown in FIG. 2A were suspended in 5 mL
of 0.05 M borate buffer, were pelleted in a microbanding tube at
35,000 rpm in a swinging bucket rotor, and were resuspended in
approximately 3 .mu.L for analysis. The analyses were done on a
PerSeptive Biosystems DESTR instrument with an extraction delay
time set at 150 nseconds using a matrix of sinapinic acid. Bovine
insulin (Mw=5,734.59) and horse heart apomyoglobin (Mw=16,952) were
used as 1 and 2 pmole standards. The results are shown in Table 1.
The .phi.X 174 masses for virion capsid proteins F, G, H and J are
calculated from published sequence data. The differences between
the calculated and experimental values for F and H are probably due
to posttranslational modifications. The probability that an
unrelated virus could have subunits of the same masses listed is
vanishingly small. However, even more definitive protein
identifications can be made by treating viral proteins with
proteolytic enzymes such as trypsin and determining the masses of
the peptide fragments produced. Computer programs are available
which calculate the sizes of fragments of proteins of known
sequence by well characterized enzymes. Such programs include
Protein Prospector (available from the University of California,
San Francisco) and ProFound (available from Rockefeller
University).
1TABLE 1 Mass Spectrometric Analysis of .phi.X 174 Virion Proteins
Calculated Experimental Protein Mass Mass Mass Difference %
Difference F 48,351.53 48,407.4 +55.9 0.12% G 19,046.73 19,046.7
0.0 0.00% H 34,419.25 34,466.1 +46.9 0.14% J 4,095.78 4,097.03 +1.2
0.03%
[0081] This example demonstrates that highly purified and
concentrated suspensions of microorganisms can be isolated from
biological samples such as, but not limited to, patient samples
such as plasma, urine, feces and tissues, natural water and tissue
culture fluids. This example further demonstrates that such
purified and concentrated microorganisms can then be identified,
for example, using mass spectrometry to identify viruses.
[0082] While the invention has been disclosed in this patent
application by reference to the details of preferred embodiments of
the invention, it is to be understood that the disclosure is
intended in an illustrative rather than in a limiting sense, as it
is contemplated that modifications will readily occur to those
skilled in the art, within the spirit of the invention and the
scope of the appended claims.
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