U.S. patent number RE44,031 [Application Number 13/439,400] was granted by the patent office on 2013-02-26 for antibody profiling sensitivity through increased reporter antibody layering.
This patent grant is currently assigned to Battelle Energy Alliance, LLC. The grantee listed for this patent is William A. Apel, Vicki S. Thompson. Invention is credited to William A. Apel, Vicki S. Thompson.
United States Patent |
RE44,031 |
Apel , et al. |
February 26, 2013 |
**Please see images for:
( Certificate of Correction ) ** |
Antibody profiling sensitivity through increased reporter antibody
layering
Abstract
A method for analyzing a biological sample by antibody profiling
for identifying forensic samples or for detecting the presence of
an analyte. In an embodiment of the invention, the analyte is a
drug, such as marijuana, Cocaine (crystalline tropane alkaloid),
methamphetamine, methyltestosterone, or mesterolone. The method
comprises attaching antigens to a surface of a solid support in a
preselected pattern to form an array wherein locations of the
antigens are known; contacting the array with the biological sample
such that a portion of antibodies in the sample reacts with and
binds to the antigens in the array to form immune complexes;
washing away antibodies that do form immune complexes; and
detecting the immune complexes, to form an antibody profile.
Forensic samples are identified by comparing a sample from an
unknown source with a sample from a known source. Further, an
assay, such as a test for illegal drug use, can be coupled to a
test for identity such that the results of the assay can be
positively correlated to the subject's identity.
Inventors: |
Apel; William A. (Jackson,
WY), Thompson; Vicki S. (Idaho Falls, ID) |
Applicant: |
Name |
City |
State |
Country |
Type |
Apel; William A.
Thompson; Vicki S. |
Jackson
Idaho Falls |
WY
ID |
US
US |
|
|
Assignee: |
Battelle Energy Alliance, LLC
(Idaho Falls, ID)
|
Family
ID: |
47721403 |
Appl.
No.: |
13/439,400 |
Filed: |
April 4, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
|
11101254 |
Apr 6, 2005 |
7682798 |
|
|
|
10017577 |
Jan 24, 2006 |
6989276 |
|
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60290256 |
May 10, 2001 |
|
|
|
Reissue of: |
11691096 |
Mar 26, 2007 |
7695919 |
Apr 13, 2010 |
|
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Current U.S.
Class: |
435/7.21;
424/130.1; 435/973; 422/1; 436/538; 422/430; 435/971; 435/975;
435/7.92; 435/7.1; 436/540; 435/7.93; 436/518 |
Current CPC
Class: |
G01N
33/94 (20130101); G01N 33/543 (20130101) |
Current International
Class: |
G01N
33/53 (20060101); G01N 31/00 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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86/02734 |
|
Oct 1985 |
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WO |
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90/05296 |
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Oct 1989 |
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WO |
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97/29206 |
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Feb 1997 |
|
WO |
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WO 98/31839 |
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Jul 1998 |
|
WO |
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98/38490 |
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Sep 1998 |
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WO |
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99/38985 |
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Jan 1999 |
|
WO |
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03/052422 |
|
Jun 2003 |
|
WO |
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2007/031874 |
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Mar 2007 |
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WO |
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Other References
Thompson et al., "Forensic Validation Study of Antibody Profiling
Identification," Frenzy--Forensic Science and Crime Scene
Technology, Conference and Expo, Washington, D.C., May 14-17, 2001.
cited by applicant .
Thompson et al., "Novel Assay for Drug and Identity Determination
in Body Fluids," American Academy of Forensic Sciences Annual
Meeting, Reno, Feb. 22-26, 2000. cited by applicant .
Thompson et al., 44 J. Agric, Food Chem., 1041-1046 (1996). cited
by applicant .
Thompson et al., Antibody profiling as an identification tool for
forensic samples, 3576 Investigation and Forensic Science
Technologies 52-59 (1999). cited by applicant .
Unger et al., MD FAAP, Individual-Specific Antibody Profiles as a
Means of Newborn Infant Identification, Journal of Perinatology
vol. 15, No. 2, 152-155 (1995). cited by applicant .
Unlu et al., "Difference gel electrophoresis: A single gel method
for detecting changes in protein extracts," Electrophoresis, vol.
18; 1997; pp. 2071-2077. cited by applicant .
Wong, S.S., Chemistry of Protein Conjugation and Cross-Linking (CRC
Press, entire book, 1991). cited by applicant .
Young et al., Yeast RNA Polymerase II Genes: Isolation with
Antibody Probes, 222 Science 778-782 (1983). cited by applicant
.
Agg et al., "Preliminary Investigations into Tris(2,2'-bipyridyl)
Ruthenium (III) as a Chemiluminescent Reagent for the Detection of
3,6-Diacetylmorphine (Heroin) on Surfaces," Journal of Forensic
Science, Sep. 2007, vol. 52, No. 5, pp. 1111-1114. cited by
applicant .
Ascher et al., Determination of the Etiology of Seroreversals in
HIV Testing by Antibody Fingerprinting, Journal of Acquired Immune
Deficiency Syndromes, 6:241-244 (1993) Raven Press, Ltd, NY. cited
by applicant .
Baird, Jeffrey, Forensic DNA in the Trial Court 19909-1992: A Brief
History, pp. 61-75. cited by applicant .
Bernard et al., "Micromosaic immunoassays," Analytical Chemistry
73, 8-12 (2001). cited by applicant .
Bernstein, R. M., Cellular Protein & RNA Antigens in Autoimmune
Disease, 2 Mol. Biol. Med., 105-120 (1984). cited by applicant
.
BioDiscovery, Inc. "ImaGene User Manual," version 7.2006. Retrieved
from http://yfgdb.princeton.edu/ImaGeneUserManual.pdf on Nov. 6,
2009. cited by applicant .
Boguslaski et al., Clinical Immunochemistry: Principles of Methods
and Applications (1984). cited by applicant .
Bowers, Larry D., Athletic Drug Testing, Sport Pharmacology, pp.
299-319 (1998). cited by applicant .
Cabilly, S., Combinatorial Peptide Library Protocols, Humana Press,
pp. 129-154 (1997). cited by applicant .
Cambridge Healthtech Institute's Fourth Annual, DNA Forensics
(Brochure) (2000). cited by applicant .
Caterino-de-Araujo et al., "Sensitivity of Two Enzyme-linked
Immunosorbent Assay Tests in Relation to Western Blot in Detecting
Human T-Cell Lymphotropic Virus Types I and II Infection among
HIV-1 Infected Patients from Sao Paulo, Brazil," Diagnostic
Microbiology and Infectious Disease, Mar. 1998, vol. 30, No. 3, pp.
173-182. cited by applicant .
Cone, E. J., Saliva Testing for Drugs of Abuse, 694 Ann. N. Y.
Acad. Sci. 91-127 (1995). cited by applicant .
Controversy Over Forensic DNA Analysis, Science in the Courtroom,
QC Researcher, pp. 924-925, Oct. 22, 1993. cited by applicant .
Cwirla et al, Peptides on Phage: A Vast Library of Peptides for
Identifying Ligands, 87 Proc. Nat'l Acad. Sci. USA 6378-6382
(1990). cited by applicant .
Derisi et al., "Exploring the Metabolic and Genetic Control of Gene
Expression on a Genomic Scale," Science, vol. 278; Oct. 24, 1997;
pp. 680-686. cited by applicant .
Devlin et al., Random Peptide Libraries: A Source of Specific
Protein Binding Molecules, Science, vol. 249, 404-406 & 336-337
(1990). cited by applicant .
Dow et al., "Automatic Multiparameter Fluorescence Imaging for
Determining Lymphocyte Phenotype and Activation Status in Melanoma
Tissue Sections," Cytometry, vol. 25; 1996, pp. 71-81. cited by
applicant .
Eisen, M. "ScanAlyze User Manual," 1999, Retrieved from
http://rana.lbl.gov/manuals/ScanAlyzeDoc.pdf on Nov. 6, 2009. cited
by applicant .
Fodor, P. S., 277 Science 393-395 (1997). cited by applicant .
Francoeur et al., 136 J. Immunol 1648 (1986). cited by applicant
.
Francoeur, Ann-Michele, Antibody Fingerprinting: A Novel Method For
Identifying Individual People and Animals, Miragen, Inc., 821-825
(1988). cited by applicant .
Good et al., Hydrogen Ion Buffers, 24 Methods Enzymology 53-68
(1972). cited by applicant .
Hemmila, I., Fluoroimmunoassays and Immunofluorometric Assays, 31
Clin. Chem. 359 (1985). cited by applicant .
International Preliminary Examination Report for International No.
PCT/US2002/039027, dated Mar. 19, 2004. cited by applicant .
International Preliminary Report on Patentability for International
No. PCT/US2008/065339, dated Dec. 1, 2009. cited by applicant .
International Preliminary Report on Patentability for International
No. PCT/US2008/054011, dated Sep. 29, 2009. cited by applicant
.
International Preliminary Report on Patentability for International
No. PCT/US2008/065321, dated Dec. 1, 2009. cited by applicant .
International Search Report and Written Opinion of the
International Searching Authority, PCT/US08/65339, International
Filing Date May 30, 2008. cited by applicant .
International Search Report and Written Opinion of the
International Searching Authority, PCT/US08/065321, International
Filing Date May 30, 2008. cited by applicant .
International Search Report and Written Opinion of the
International Searching Authority, PCT/US08/54011, International
Filing Date Feb. 14, 2008. cited by applicant .
International Search Report, dated Feb. 21, 2003. cited by
applicant .
Karch, S. B., Drug Abuse Handbook (CRC Press, 1998). cited by
applicant .
Kemeny et al., ELISA and Other Solid Phase Immunoassays (1988).
cited by applicant .
Kidwell et al, Testing for drugs of abuse in saliva and sweat, 713
J. Chrom. B 111-135 (1998). cited by applicant .
Lam et al., A New Type of Synthetic Peptide Library for Identifying
Ligand-binding Activity, 354 Nature 82-84 (1991). cited by
applicant .
Leland et al., Electrogenerated Chemiluminescense: An
Oxidative-Reduction Type ECL Reactions Sequence Using Triprophyl
Amine, 137 J. Electrochemical Soc. 3127-3131 (1990). cited by
applicant .
McCabe, John, DNA Fingerprinting: The Failings of Frye, pp.
455-481, date 1996. cited by applicant .
Miragen Antibody Profile Assay Advertisement, 1996 (U.S. Appl. No.
60/011,511). cited by applicant .
Parry, Tests for HIV and hepatitis viruses, 694 Annals N. Y Acad.
Sci. 221 (1993). cited by applicant .
Peat et al., Drug Abuse Handbook (CRC Press, Boca Raton, Fla.
1998). cited by applicant .
Persoon, T., Immunochemical Assays in the Clinical Laboratory, 5
Clinical Laboratory Science 31 (1992). cited by applicant .
Sanchez-Carbayo, Marta: "Antibody arrays: Technical considerations
and clinical applications in cancer," Clinical Chemistry, vol. 52,
No. 9, Sep. 2006, pp. 1651-1659. cited by applicant .
Santangelo et al., Cloning of Open Reading Frames and Promoters
from the Saccharomyces cerevisiae Genome: Construction of Genomic
Libraries of Random Small Fragments, 46 Gene 181-186 (1986). cited
by applicant .
Schramm et al., Drugs of Abuse in Saliva: A Review, 16 J. Anal.
Toxicology 1-9 (1992). cited by applicant .
Scott et al., Searching for Peptide Ligands with an Epitope
Library, Science, vol. 249, 386-390 (1990). cited by applicant
.
Stites et al, Basic and Clinical Immunology (1994). cited by
applicant .
Supplementary European Search Report from EP 08 72 9906 dated Mar.
25, 2010; 5 pages. cited by applicant .
Thompson et al., "A Novel Test for Detection of Drugs in the Body
That Also Provides the Identity of the Person Being Examined,"
ONDCP International Technology Symposium, Counterdrug Research and
Development: Technologies for the Next Decade, San Diego, Jun.
25-28, 2001. cited by applicant .
Thompson et al., "Antibody Profiling Technique for Rapid
Identification of Forensic Samples," CAT/NWAFS/SWAFS/SAT Combined
Professional Training Seminar, Las Vegas, Nov. 3-7, 1997. cited by
applicant .
Thompson et al., "Antibody Profiling Technique for Rapid
Identification of Forensic Samples," California Association of
Criminalists Fall Seminar, Irvine, California, Oct. 8-11, 1997.
cited by applicant .
Caterino-de-Araujo et al. (Diagnostic Microbiology and Infectious
Disease, Mar. 1998, vol. 30, No. 3., pp. 173-182). cited by
examiner .
Thompson et al. ("Antibody Profiling Technique for Rapid
Identification of Forensic Samples", California Association of
Criminalists Fall Seminar, Irvine California, Oct. 8-11, 1997).
cited by examiner .
A M. Francoeur et al., 136 J. Immunol 1648 (1986). cited by
applicant .
S. Cabilly, Combinatorial Peptide Library Protocols, Humana Press,
pp. 129-154 (1997). cited by applicant .
G. M. Santangelo et al., Cloning of Open Reading Frames and
Promoters from the Saccharomyces cerevisiae Genome: Construction of
Genomic Libraries of Random Small Fragments, 46 Gene 181-186
(1986). cited by applicant .
S.S. Wong, Chemistry of Protein Conjugation and Cross-Linking (CRC
Press, entire book, 1991). cited by applicant .
N. E. Good & S. Izawa, Hydrogen Ion Buffers, 24 Methods
Enzymology 53-68 (1972). cited by applicant .
D. M. Kemeny & S. J. Challacombe, ELISA and Other Solid Phase
Immunoassays (1988). cited by applicant .
R. C. Boguslaski et al., Clinical Immunochemistry: Principles of
Methods and Applications (1984). cited by applicant .
D. P. Stites et al, Basic and Clinical Immunology (1994). cited by
applicant .
I. Hemmila, Fluoroimmunoassays and Immunofluorometric Assays, 31
Clin. Chem. 359 (1985). cited by applicant .
W. Schramm, et al., Drugs of Abuse in Saliva: A Review, 16 J Anal.
Toxicology 1-9 (1992). cited by applicant .
E. J. Cone, Saliva Testing for Drugs of Abuse, 694 Ann. N. Y. Acad.
Sci. 91-127 (1995). cited by applicant .
D. A. Kidwell et al, Testing for drugs of abuse in saliva and
sweat, 713 J. Chrom. B III-135 (1998). cited by applicant .
V. S. Thompson et al., Antibody profiling as an identification tool
for forensic samples, 3576 Investigation and Forensic Science
Technologies 52-59 (1999). cited by applicant .
S. B Karch. Drug Abuse Handbook (CRC Press. 1998). cited by
applicant .
M. Peat & A.E. Davis, Drug Abuse Handbook (CRC Press, Boca
Raton. Fla. 1998). cited by applicant .
Larry D. Bowers, Ph:D. Athletic Drug Testing, Sport Pharmacology,
pp. 299-319 (1998). cited by applicant .
Cambridge Healthtech Institute's Fourth Annual, DNA Forensics
(Brochure). cited by applicant .
Jeffrey Baird, Forensic DNA in the Trial Court 19909-1992: A Brief
History, pp. 61-75. cited by applicant .
Controversy Over Forensic DNA Analysis, Science in the Courtroom.
QC Researcher, pp. 924-925. cited by applicant .
John McCabe, DNA Fingerprinting: The Failings of Frye, 16 N. Ill.
U. L. Rev. 455 (1996). cited by applicant .
Thompson et al., "Novel Assay for Drug and Identity Determination
in Body Fluids," American Academy of Forensic Sciences Annual
Meeting, Reno. Feb. 22-26, 2000. cited by applicant .
Thompson et al., "Antibody Profiling Technique for Rapid
Identification of Forensic Samples," California Association of
Criminalists Fall Seminar, Irvine California, Oct. 8-11, 1997.
cited by applicant .
R. M. Bernstein, Cellular Protein & RNA Antigens in Autoimmune
Disease, 2 Mol. Biol. Med., 105-120 (1984). cited by applicant
.
P. S. Fodor, 277 Science 393 & 395 (1997). cited by applicant
.
S. E. Cwirla et al, Peptides on Phage: A Vast Library of Peptides
for Identifying Ligands, 87 Proc. Nat'l Acad. Sci. USA 6378-6382
(1990). cited by applicant .
K. S. Lam et al., A New Type of Synthetic Peptide Library for
Identifying Ligand-binding Activity, 354 Nature 82-84 (1991). cited
by applicant .
R. A. Young & R. W. Davis, Yeast RNA Polymerase II Genes:
Isolation with Antibody Probes, 222 Science 778-782 (1983). cited
by applicant .
Thompson and Maragos, Fiber-Optic Immunosensor for the Detection of
Fumonisin B1, 44 J. Agric, Food Chem., 1041-1046 (1996). cited by
applicant .
T. Persoon, Immunochemical Assays in the Clinical Laboratory, 5
Clinical Laboratory Science 31 (1992). cited by applicant .
J.K. Leland et al., Electrogenerated Chemiluminescense: An
Oxidative-Reduction Type ECL Reactions Sequence Using Triprophyl
Amine, 137 J. Electrochemical Soc. 3127-3131 (1990). cited by
applicant .
Miragen Antibody Profile Assay Advertisement. cited by applicant
.
David P. Ascher and Chester Roberts, Determination of the Etiology
of Seroreversals in HIV Testing by Antibody Fingerprinting, Journal
of Acquired Immune Deficiency Syndromes, 6:241-244 (1993) Raven
Press, Ltd, NY. cited by applicant .
Ann-Michele Francoeur, Antibody Fingerprinting: A Novel Method For
Identifying Individual People and Animals, Miragen, Inc., 821-825
(1988). cited by applicant .
Thomas F. Unger, PhD and Arthur Strauss, MD FAAP,
Individual-Specific Antibody Profiles as a Means of Newborn Infant
Identification, Journal of Perinatology vol. 15, No. 2, 152-155
(1995). cited by applicant .
James K. Scott and George P. Smith, Searching for Peptide Ligands
with an Epitope Library, Science, vol. 249, 386-390 (1990). cited
by applicant .
James J. Devlin, Lucy C. Panganiban, Patricia E. Devlin, Random
Peptide Libraries: A Source of Specific Protein Binding Molecules,
Science, vol. 249, 404-406 & 336-337 (1990). cited by applicant
.
Agg, Kent M., et al., "Preliminary Investigations into
Tris(2,2'-bipyridyl) Ruthenium (III) as a Chemilumincscent Reagent
for the Detection of 3,6-Diacetylmorphine (Heroin) on Surfaces,"
Journal of Forensic Science, Sep. 2007, vol. 52, No. 5, pp.
1111-1114. cited by applicant .
Derisi, Joseph L., et al., Exploring the Metabolic and Genetic
Control of Gene Expression on a Genomic Scale, Science, Oct. 24,
1997, pp. 680-686, vol. 278. cited by applicant .
Unlu, Mustafa, et al., Difference Gel Electrophoresis: A Single Gel
Method for Detecting Changes in Protein Extracts, Electrophoresis,
1997, pp. 2071-2077, vol. 18. cited by applicant .
Dow, Alasdair I., et al., Automatic Multiparameter Fluorescence
Imaging for Determining Lymphocyte Phenotype and Activation Status
in Melanoma Tissue Sections, Cytometry, 1996, pp. 71-81, vol. 25.
cited by applicant .
Bernard et al., "Micromosaic immunoassays," Analytical Chemistry
73, 8-12, (2001). cited by applicant .
Eisen, M. "ScanAlyze User Manual," 1999, Retrieved from
http://rana.lbl.gov/manuals/Scan/AlyzeDoc.pdf on Nov. 6, 2009.
cited by applicant .
Parry, Tests for HIV and hepatitis viruses, 694 Annals N.Y Acad.
Sci. 221 (1993). cited by applicant .
M. Peat & A.E. Davis, Drug Abuse Handbook (CRC Press, Boca
Raton, Fla. 1998). cited by applicant .
Larry D. Bowers, Ph.D. Athletic Drug Testing, Sport Pharmacology,
pp. 299-319 (1998). cited by applicant .
Cambridge Healthtech Institute's Fourth Annual, DNA Forensics
(Brochure), date 2000. cited by applicant .
Controversy Over Forensic DNA Analysis, Science in the Courtroom,
QC Researcher, pp. 924-925, date Oct. 22, 1993. cited by applicant
.
J.K. Leland et al., Electrogenerated Chemiluminescense: An
Oxidative-Reduction Type ECL Reactions Sequence Using Triprophyl
Amine. 137 J. Electrochemical Soc. 3127-3131 (1990). cited by
applicant .
Miragen Antibody Profile Assay Advertisement, date 1996, U.S. Appl.
No. 60/011,511. cited by applicant .
James J. Devlin, Lucy C. Pangamban, Patricia E. Devlin, Random
Peptide Libraries: A Source of Specific Protein Binding Molecules,
Science, vol. 249, 404-406 & 336-337 (1990). cited by
applicant.
|
Primary Examiner: Cook; Lisa
Attorney, Agent or Firm: Taylor English Duma LLP
Government Interests
CONTRACTUAL ORIGIN OF THE INVENTION
This invention was made with government support under Contract No.
DE-AC07-94ID13223, Contract No. DE-AC07-99ID13727, and Contract No.
DE-AC07-05ID14517 awarded by the United States Department of
Energy. The government has certain rights in the invention.
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation-in-part of .[.pending.]. U.S.
patent application Ser. No. 11/101,254, filed Apr. 6, 2005
.Iadd.now U.S. Pat. No. 7,682,798.Iaddend., which is a divisional
of U.S. patent application Ser. No. 10/017,577, filed Dec. 14,
2001, now U.S. Pat. No. 6,989,276, issued Jan. 24, 2006, which
claims priority to U.S. Provisional Patent Application Ser. No.
60/290,256, filed May 10, 2001, the entire disclosure of each of
which is incorporated by reference herein.
Claims
What is claimed is:
1. A method for analyzing biological material comprising
individual-specific antibodies, the method comprising: forming an
array comprising multiple antigens attached to a surface of a solid
support in a preselected location pattern; obtaining a sample of a
biological material having individual-specific antibodies and
contacting the array with the sample to bind at least a portion of
the individual-specific antibodies to the multiple antigens of the
array, to form immune complexes; washing the array containing the
immune complexes; detecting the immune complexes by the application
to the array of at least three separate antibodies; and identifying
the immune complexes on the array, to obtain an antibody
profile.
2. The method of claim 1, wherein forming an array comprises
attaching the multiple antigens to the solid support through a
covalent bond.
3. The method of claim 1, comprising obtaining a sample of a
biological material selected from the group of biological material
consisting of tissue, blood, saliva, urine, perspiration, tears,
semen, serum, plasma, amniotic fluid, pleural fluid, cerebrospinal
fluid, and combinations thereof.
4. The method of claim 1, wherein forming the array comprises
attaching multiple antigens to a solid support comprising glass or
silica.
5. The method of claim 1, wherein detecting the immune complexes
comprises treating the array such that the presence of immune
complexes at a location is characterized by a color change at the
location.
6. The method of claim 5, wherein detecting the immune complexes
comprises obtaining an output using a charge-coupled device and
wherein the color change comprises fluorescence or luminescence
emission.
7. The method of claim 1, wherein detecting the immune complexes
further comprises monitoring the array with solid state color
detection circuitry and comparing color patterns before and after
detecting the immune complexes.
8. The method of claim 1, wherein detecting the immune complexes
further comprises obtaining a color camera image before contacting
the array with the sample and after detecting the immune complexes,
and analyzing pixel information obtained from the color camera
image.
9. The method of claim 1, wherein detecting the immune complexes
further comprises scanning the array before and after contacting
the array with the sample, wherein the solid support is a surface
plasmon resonance chip.
10. The method of claim 1, wherein forming the array comprises
attaching a first subset of antigens configured for obtaining an
antibody profile and a second subset of at least one antigen
configured for assaying for a selected analyte in the sample.
11. The method of claim 10, wherein attaching the second subset of
at least one antigen comprises attaching at least one drug.
12. The method of claim 11, wherein attaching at least one drug
comprises attaching a drug selected from the group consisting of
marijuana, Cocaine (crystalline tropane alkaloid), methamphetamine,
amphetamine, heroin, methyltestosterone, mesterolone and
combinations thereof.
13. The method of claim 1, wherein obtaining a sample of a
biological material comprises obtaining the biological material
from a forensic sample.
14. The method of claim 13, further comprising comparing the
antibody profile obtained from the biological material from the
forensic sample to an antibody profile prepared from a biological
sample obtained from a crime suspect.
15. A method for analyzing biological material comprising
individual-specific antibodies, the method comprising: forming an
array comprising multiple antigens attached to a surface of a solid
support in a preselected location pattern; obtaining a sample of a
biological material having individual-specific antibodies and
contacting the array with the sample to bind at least a portion of
the individual-specific antibodies to the multiple antigens of the
array, to form immune complexes; washing the array containing the
immune complexes; contacting the immune complexes with primary
antibodies capable of binding the immune complex, wherein the
primary antibodies are from a different species than the
individual-specific antibodies; removing primary antibodies not
bound to the immune complexes; contacting the primary antibodies
bound to the immune complexes with secondary antibodies capable of
binding the primary antibodies, wherein the secondary antibodies
are from a different species than the individual-specific
antibodies and the primary antibodies; removing unbound secondary
antibodies; contacting the secondary antibodies bound to the
primary antibodies with tertiary antibodies capable of binding the
secondary antibodies, wherein the tertiary antibodies are from a
different species than the individual-specific antibodies, the
primary antibodies, and the secondary antibodies; removing unbound
tertiary antibodies; detecting the primary, secondary, or tertiary
antibodies; and identifying the immune complexes on the array, to
obtain an antibody profile.
16. A method for detecting a selected drug in a biological sample
comprising individual-specific antibodies and identifying a source
of the biological sample, the method comprising: immobilizing
multiple antigens in a pre-selected pattern on a solid support;
immobilizing a detectable amount of a selected drug on the solid
support, to form an array; providing an antibody-enzyme conjugate
comprising an antibody configured to bind the selected drug and an
enzyme that is capable of converting a colorigenic substrate into a
colored product; contacting the array with a biological sample, to
bind at least some of the multiple antigens with
individual-specific antibodies in the biological sample, to form
immune complexes; contacting the array with the antibody-enzyme
conjugate, wherein the antibody-enzyme conjugate competitively
binds to (i) the selected drug immobilized on the array, to form an
immobilized antibody-enzyme conjugate, and (ii) any selected drug
that may be present in the biological sample, to form a soluble
drug-antibody-enzyme conjugate; washing the solid support, to
remove at least the soluble drug-antibody-enzyme complexes;
contacting the solid support with a colorigenic substrate to
convert the colorigenic substrate to a colored product using the
immobilized antibody-enzyme conjugate; determining an amount of the
colored product present, wherein the amount of the colored product
may be inversely correlated with an amount of the selected drug in
the biological sample; and detecting the immune complexes
immobilized on the solid support by the application to the solid
support of at least three separate antibodies to form an antibody
profile characteristic of the source of the biological sample.
17. The method of claim 16, further comprising comparing the
antibody profile to one or more candidate antibody profiles from
candidate sources, wherein a match of the antibody profile to the
one or more candidate antibody profiles identifies the source of
the biological sample.
18. The method of claim 16, wherein immobilizing a detectable
amount of the selected drug on the solid support comprises
selecting the selected drug from the group consisting of marijuana,
Cocaine (crystalline tropane alkaloid), methamphetamine,
amphetamine, heroin, methyltestosterone, mesterolone and
combinations thereof.
19. The method of claim 16, wherein contacting the array with a
biological sample comprises obtaining a biological sample from a
source selected from the group consisting of tissue, blood, saliva,
urine, perspiration, tears, semen, serum, plasma, amniotic fluid,
pleural fluid, cerebrospinal fluid, and combinations thereof.
20. The method of claim 16, comprising obtaining the biological
sample from saliva.
21. The method of claim 16, comprising immobilizing multiple
antigens from a HeLa cell.
22. The method of claim 16, comprising immobilizing multiple
antigens from a random peptide library.
23. The method of claim 16, comprising immobilizing multiple
antigens from an epitope library.
24. The method of claim 16, comprising immobilizing multiple
antigens from a random cDNA expression library.
25. The method of claim 16, comprising immobilizing multiple
antigens on the solid support, wherein the solid support comprises
at least one substance selected from the group of substances
consisting of glass, silicon, silica, polymeric material,
poly(tetrafluoroethylene), poly(vinylidene difluoride),
polystyrene, polycarbonate, polymethacrylate, ceramic material, and
hydrophilic inorganic material.
26. The method of claim 16, comprising immobilizing multiple
antigens on the solid support, wherein the solid support comprises
a hydrophilic inorganic material selected from the group consisting
of at least one of alumina, zirconia, titania, nickel oxide.
27. The method of claim 16, wherein providing the antibody-enzyme
conjugate comprises the antibody conjugated to alkaline
phosphatase.
28. The method of claim 16, wherein providing the antibody-enzyme
conjugate comprises providing the antibody conjugated to
horseradish peroxidase.
29. The method of claim 16, wherein detecting the immune complexes
immobilized on the solid support by the application to the solid
support of at least three separate antibodies comprises: contacting
the immune complexes with primary antibodies capable of binding the
immune complex, wherein the primary antibodies are from a different
species than the individual-specific antibodies; removing primary
antibodies not bound to the immune complexes; contacting the
primary antibodies bound to the immune complexes with secondary
antibodies capable of binding the primary antibodies, wherein the
secondary antibodies are from a different species than the
individual-specific antibodies and the primary antibodies; removing
unbound secondary antibodies; contacting the secondary antibodies
bound to the primary antibodies with enzyme-conjugated tertiary
antibodies capable of binding the secondary antibodies, wherein the
enzyme-conjugated tertiary antibodies are from a different species
than the individual-specific antibodies, the primary antibodies,
and the secondary antibodies; removing unbound enzyme-conjugated
tertiary antibodies; and detecting bound enzyme-conjugated tertiary
antibodies, to detect the immune complexes on the solid
support.
30. The method according to claim 1, wherein the at least three
separate antibodies comprise: a first antibody capable of binding
the immune complex; a second antibody capable of binding the first
antibody; and a third antibody capable of binding the second
antibody.
.Iadd.31. A method for analyzing biological material, the method
comprising: contacting an array comprising multiple antigens
attached to a surface of a support in a preselected pattern of
antigen locations with a sample of a biological material having
individual-specific antibodies to bind at least a portion of the
individual-specific antibodies to one or more of the multiple
antigens of the array, to form immune complexes on the array;
detecting the immune complexes on the array by the application to
the immune complexes on the array of at least three separate
antibodies; and using the detected immune complexes on the array,
to obtain an antibody profile..Iaddend.
.Iadd.32. The method of claim 31, wherein contacting the array
comprising multiple antigens comprises contacting multiple antigens
attached to the support through covalent bonds..Iaddend.
.Iadd.33. The method of claim 31, wherein contacting the array with
a sample of biological material comprises contacting the with a
sample of biological material selected from the group consisting of
tissue, blood, saliva, urine, perspiration, tears, semen, serum,
plasma, amniotic fluid, pleural fluid, cerebrospinal fluid, and
combinations thereof..Iaddend.
.Iadd.34. The method of claim 31, wherein contacting the array
comprising multiple antigens attached to a surface of a support
comprises contacting the array comprising multiple antigens
attached to a surface of a support comprising glass or
silica..Iaddend.
.Iadd.35. The method of claim 31, wherein detecting the immune
complexes comprises treating the array to cause a color change at a
location responsive to the presence of at least one immune complex
at the location..Iaddend.
.Iadd.36. The method of claim 35, wherein detecting the immune
complexes comprises obtaining an output from at least one immune
complex using a charge-coupled device and wherein the color change
comprises fluorescence or luminescence emission..Iaddend.
.Iadd.37. The method of claim 31, wherein detecting the immune
complexes further comprises monitoring the array with solid state
color detection circuitry and comparing color patterns on the array
before and after detecting the immune complexes..Iaddend.
.Iadd.38. The method of claim 31, wherein detecting the immune
complexes further comprises obtaining a color camera image of the
array before contacting the array with the sample and after
detecting the immune complexes on the array, and analyzing pixel
information obtained therefrom..Iaddend.
.Iadd.39. The method of claim 31, wherein detecting the immune
complexes further comprises scanning the array before and after
contacting the array with the sample, wherein the support is a
surface plasmon resonance chip..Iaddend.
.Iadd.40. The method of claim 31, wherein contacting an array
comprising multiple antigens comprises contacting an array
comprising a first subset of antigens configured for obtaining an
antibody profile and a second subset of at least one antigen
configured for assaying for a selected analyte in the sample of a
biological material having individual-specific
antibodies..Iaddend.
.Iadd.41. The method of claim 40, wherein contacting an array
comprising a first and a second subset of antigens comprises
contacting an array comprising a second subset of antigens
comprising at least one drug..Iaddend.
.Iadd.42. The method of claim 40, wherein contacting a array
comprising a first and a second subset of antigens comprises
contacting a array comprising a second subset of antigens
comprising at least one drug selected from the group consisting of
marijuana, Cocaine (crystalline tropane alkaloid), methamphetamine,
amphetamine, heroin, methyltestosterone, mesterolone and
combinations thereof..Iaddend.
.Iadd.43. The method of claim 31, wherein contacting the array with
the sample of biological material comprises contacting the array
with a forensic sample..Iaddend.
.Iadd.44. The method of claim 43, further comprising comparing the
antibody profile obtained from the forensic sample to an antibody
profile obtained from a biological material sample obtained from a
subject..Iaddend.
.Iadd.45. A method for analyzing biological material, the method
comprising: contacting an array comprising multiple antigens
attached to a surface of a support in a preselected pattern of
antigen locations with a sample of a biological material having
individual-specific antibodies to bind at least a portion of the
individual-specific antibodies to one or more of the multiple
antigens of the array, to form immune complexes on the array;
contacting the immune complexes with primary antibodies capable of
binding to immune complexes on the array, wherein the primary
antibodies are from a different species than the individual
specific antibodies; removing primary antibodies not bound to
immune complexes on the array; contacting the primary antibodies
bound to the immune complexes with secondary antibodies capable of
binding to the primary antibodies, wherein the secondary antibodies
are from a different species than each of the individual specific
antibodies and the primary antibodies; removing secondary
antibodies not bound to primary antibodies; contacting the
secondary antibodies bound to the primary antibodies with tertiary
antibodies capable of binding to the secondary antibodies, wherein
the tertiary antibodies are from a different species than each of
the individual specific antibodies, the primary antibodies, and the
secondary antibodies; removing tertiary antibodies not bound to
secondary antibodies; and detecting a presence of at least some of
primary, secondary, and tertiary antibodies on the array; and using
the detected at least some primary, secondary and tertiary
antibodies on the array, to obtain an antibody
profile..Iaddend.
.Iadd.46. A method for detecting a selected drug in a biological
sample and identifying a source of the biological sample, the
method comprising: contacting an array comprising multiple antigens
attached to a surface of a support in a preselected pattern of
antigen locations with a sample of a biological material having
individual-specific antibodies to bind at least a portion of the
individual-specific antibodies to one or more of the multiple
antigens of the array, to form immune complexes on the array;
wherein the array comprises a first subset of antigens configured
for obtaining an antibody profile and a second subset of at least
one antigen comprising at least one drug; contacting the array with
an antibody-enzyme conjugate comprising an antibody configured to
bind the at least one drug and an enzyme that is capable of
converting a colorigenic substrate into a colored product, wherein
the antibody-enzyme conjugate competitively binds to (i) the at
least one drug of the array, to form an immobilized antibody-enzyme
conjugate, and (ii) any selected drug that may be present in the
biological sample, to form a soluble drug-antibody-enzyme
conjugate; removing at least the soluble drug-antibody-enzyme
conjugate; contacting the support with a colorigenic substrate to
convert the colorigenic substrate to a colored product using the
immobilized antibody-enzyme conjugate; determining an amount of the
colored product present, wherein the amount of the colored product
may be inversely correlated with an amount of the selected drug in
the biological sample; and detecting the immune complexes on the
array by an application to the array of at least three separate
antibodies to obtain an antibody profile characteristic of a source
of the biological sample..Iaddend.
.Iadd.47. The method of claim 46, further comprising comparing the
antibody profile to one or more candidate antibody profiles from
candidate sources, wherein a match of the antibody profile to the
one or more candidate antibody profiles identifies the source of
the biological sample..Iaddend.
.Iadd.48. The method of claim 46, wherein contacting an array
comprising a first subset of antigens and a second subset of at
least one antigen comprises contacting an array comprising a second
subset of at least one antigen comprising at least one drug
selected from the group consisting of marijuana, Cocaine
(crystalline tropane alkaloid), methamphetamine, amphetamine,
heroin, methyltestosterone, mesterolone and combinations
thereof..Iaddend.
.Iadd.49. The method of claim 46, wherein contacting an array with
a sample of biological material comprises contacting an array with
a sample of biological material selected from the group consisting
of tissue, blood, saliva, urine, perspiration, tears, semen, serum,
plasma, amniotic fluid, pleural fluid, cerebrospinal fluid, and
combinations thereof..Iaddend.
.Iadd.50. The method of claim 46, wherein contacting the array with
a sample of biological material comprises contacting the array with
saliva..Iaddend.
.Iadd.51. The method of claim 46, wherein contacting an array
comprising a first subset of antigens and a second subset of at
least one antigen comprises contacting an array comprising a first
subset of antigens configured for obtaining an antibody profile
comprising multiple antigens from a HeLa cell..Iaddend.
.Iadd.52. The method of claim 46, wherein contacting an array
comprising a first subset of antigens and a second subset of at
least one antigen comprises contacting an array comprising a first
subset of antigens configured for obtaining an antibody profile
comprising multiple antigens from a random peptide
library..Iaddend.
.Iadd.53. The method of claim 46, wherein contacting an array
comprising a first subset of antigens and a second subset of at
least one antigen comprises contacting an array comprising a first
subset of antigens configured for obtaining an antibody profile
comprising multiple antigens from an epitope library..Iaddend.
.Iadd.54. The method of claim 46, wherein contacting an array
comprising a first subset of antigens and a second subset of at
least one antigen comprises contacting an array comprising a first
subset of antigens configured for obtaining an antibody profile
comprising multiple antigens from a random cDNA expression
library..Iaddend.
.Iadd.55. The method of claim 46, wherein contacting an array
comprising multiple antigens attached to a surface of a support
comprises contacting an array comprising multiple antigens attached
to a surface of a support comprising at least one substance
selected from the group of substances consisting of glass, silicon,
silica, polymeric material, poly(tetrafluoroethylene),
poly(vinylidenedifluoride), polystyrene, polycarbonate,
polymethacrylatem, ceramic material, and hydrophilic inorganic
material..Iaddend.
.Iadd.56. The method of claim 46, wherein contacting an array
comprising multiple antigens attached to a surface of a support
comprises contacting an array comprising multiple antigens attached
to a surface of a support comprising a hydrophilic inorganic
material selected from the group consisting of at least one of
alumina, zirconia, titania, nickel oxide..Iaddend.
.Iadd.57. The method of claim 46, wherein contacting the array with
an antibody-enzyme conjugate comprises contacting the array with an
antibody-alkaline phosphatase conjugate..Iaddend.
.Iadd.58. The method of claim 46, wherein contacting the array with
an antibody-enzyme conjugate comprises contacting the array with an
antibody-horseradish peroxidase conjugate..Iaddend.
.Iadd.59. The method of claim 46, wherein detecting the immune
complexes on the array by the application to the array of at least
three separate antibodies comprises: contacting the immune
complexes on the array with primary antibodies capable of binding
the immune complex on the array, wherein the primary antibodies are
from a different species than the individual specific antibodies;
contacting the primary antibodies bound to the immune complexes on
the array with secondary antibodies capable of binding the primary
antibodies, wherein the secondary antibodies are from a different
species than both the individual specific antibodies and the
primary antibodies; contacting the secondary antibodies hound to
the primary antibodies with enzyme-conjugated tertiary antibodies
capable of binding the secondary antibodies, wherein the
enzyme-conjugated tertiary antibodies are from a different species
than all of the individual specific antibodies, the primary
antibodies, and the secondary antibodies; and detecting bound
enzyme-conjugated tertiary antibodies, to detect the immune
complexes on the array..Iaddend.
.Iadd.60. The method according to claim 46, wherein detecting the
immune complexes on the array by an application to the array of at
least three separate antibodies comprises detecting the immune
complexes on the array by an application to the array of a first
antibody capable of binding to the immune complex; a second
antibody capable of binding to the first antibody; and a third
antibody capable of binding to the second antibody..Iaddend.
Description
FIELD OF THE INVENTION
This invention relates to assaying biological samples. More
particularly, the invention relates to methods for analyzing
biological samples comprising antibody profiling. In an embodiment
of the invention, the analyzing of biological samples comprises a
combination of antibody profiling for characterizing
individual-specific antibodies in the biological samples and
simultaneous assay of an analyte in the biological samples.
BACKGROUND
Many methods are known for identifying individual or biological
samples obtained from such individuals. For example, blood typing
is based on the existence of antigens on the surface of red blood
cells. The ABO system relates to four different conditions with
respect to two antigens, A and B. Type A individuals exhibit the A
antigen; Type B individuals exhibit the B antigen; Type AB
individuals exhibit both the A and B antigens; and Type O
individuals exhibit neither the A nor the B antigen. By analyzing a
sample of a person's blood, it is possible to classify the blood as
belonging to one of these blood groups. While this method may be
used to identify one individual out of a small group of
individuals, the method is limited when the group of individuals is
larger because no distinction is made between persons of the same
blood group. For example, the distribution of the ABO blood groups
in the U.S. is approximately 45% O, 42% A, 10% B, and 3% AB. Tests
based on other blood group antigens or isozymes present in body
fluids suffer from the same disadvantages as the ABO blood typing
tests. These methods can exclude certain individuals, but cannot
differentiate between members of the same blood group.
A variety of immunological and biochemical tests based on genetics
are routinely used in paternity testing, as well as for determining
the compatibility of donors and recipients involved in transplant
or transfusion procedures, and also sometimes as an aid in the
identification of humans and animals. For example, serological
testing of proteins encoded by the human leukocyte antigen (HLA)
gene locus is well known. Although a good deal of information is
known concerning the genetic makeup of the HLA locus, there are
many drawbacks to using HLA serological typing for identifying
individuals in a large group. Each of the HLA antigens must be
tested for in a separate assay, and many such antigens must be
assayed to identify an individual, an arduous process when
identifying one individual in a large group.
In the past decade, DNA-based analysis techniques, such as
restriction fragment length polymorphisms (RFLPs) and polymerase
chain reaction (PCR) have rapidly gained acceptance in forensic and
paternity analyses for matching biological samples to an
individual. RFLP techniques are problematic, however, due to the
need for relatively large sample sizes, specialized equipment,
highly skilled technicians, and lengthy analysis times. For
forensic applications, there is often not enough sample available
for this type of assay, and in remote areas the necessary equipment
is often not available. In addition, this technique can take from
two to six weeks for completion and can result in costly delays in
a criminal investigation. Moreover, the cost of RFLP analysis can
be prohibitory if screening of many samples is necessary. PCR
techniques have advantages over RFLP analysis of requiring much
smaller sample sizes and permitting more rapid analysis, but they
still require specialized equipment and skilled technicians, and
they are also expensive.
U.S. Pat. No. 4,880,750 and U.S. Pat. No. 5,270,167 disclose
"antibody profiling," or "AbP," as a method that purportedly
overcomes many of the disadvantages associated with DNA analysis.
Antibody profiling is based on the discovery that every individual
has a unique set of antibodies present in his or her bodily fluids.
R. M. Bernstein et al., "Cellular Protein and RNA Antigens in
Autoimmune Disease," 2 Mol. Biol. Med. 105-120 (1984). These
antibodies, termed "individual-specific antibodies" or "ISAs," have
been found in blood, serum, saliva, urine, semen, perspiration,
tears, and body tissues. A. M. Francoeur, "Antibody Fingerprinting:
A Novel Method for Identifying Individual People and Animals," 6
Bio/Technology 821-825 (1988). ISAs are not associated with disease
and are thought to be directed against cellular components of the
body. Every person is born with an antibody profile that matches
the mother's antibody profile. T. F. Unger and A. Strauss,
"Individual-Specific Antibody Profiles as a Means of Newborn Infant
Identification," 15 J. Perinatology 152-155 (1995). The child's
antibody profile gradually changes, however, until a stable unique
pattern is obtained by about two years of age. It has been shown
that even genetically identical individuals have different antibody
profiles. An individual's profile is apparently stable for life and
is not affected by short-term illnesses. A. M. Francoeur, supra.
Few studies have been conducted on individuals with long-term
diseases. Preliminary results, however, indicate that, although a
few extra bands may appear, the overall pattern remains intact.
This technique has been used in the medical field to track patient
samples and avoid sample mix-ups. In addition, the technique has
been used in hospitals in cases where switching of infants or
abduction has been alleged. The method has a number of advantages
over DNA techniques, including low cost, rapid analysis (two hours
from the time the sample is obtained), and simplicity (no special
equipment or training is necessary). In addition, this method will
potentially work on samples that contain no DNA.
WO 97/29206 discloses a method for identifying the source of a
biological sample used for diagnostic testing by linking diagnostic
test results to an antibody profile of the biological sample. By
generating an antibody profile of each biological sample, the
origin of the biological sample is identified.
Many assays are now available that use the attachment of specific
nucleic acid probes or other biological molecules to surfaces such
as glass, silicon, polymethacrylate, polymeric filters,
microspheres, resins, and the like. In a configuration where the
surface is planar, these assays are sometimes referred to as
"biochips." Initially, biochips contained nucleic acid probes
attached to glass or silicon substrates in microarrays. These DNA
chips are made by microfabrication technologies initially developed
for use in computer chip manufacturing. Leading DNA chip
technologies include an in situ photochemical synthesis approach,
P. S. Fodor, 277 Science 393-395 (1997); U.S. Pat. No. 5,445,934;
an electrochemical positioning approach, U.S. Pat. No. 5,605,662;
depositing gene probes on the chip using a sprayer that resembles
an ink-jet printer; and the use of gels in a solution-based
process. Arrays of other types of molecules, such as peptides, have
been fabricated on biochips, e.g., U.S. Pat. No. 5,445,934.
While the known methods for using antibody profiling are generally
suitable for their limited purposes, they possess certain inherent
deficiencies that detract from their overall utility in analyzing,
characterizing, and identifying biological samples. For example,
the known methods rely on fractionation of antigens by
electrophoresis and then transfer of the fractionated antigens to a
membrane. Due to differences in conditions from one fractionation
procedure to another, there are lot-to-lot differences in the
positions of the antigens on the membrane such that results
obtained using membranes from one lot cannot be compared with
results obtained using membranes from another lot. Further, when
colorimetric procedures are used for detecting immune complexes on
the membrane, color determination can be subjective such that
results may be interpreted differently by different observers.
In view of the foregoing, providing a method for analyzing
biological samples, wherein lot-to-lot differences in reagents and
subjectivity do not affect interpretation of results, would be a
significant advancement in the art. More particularly, it would be
advantageous to provide a method for analyzing biological samples
by antibody profiling in a biochip format such that analysis would
be amenable to automation.
BRIEF SUMMARY OF THE INVENTION
One embodiment of the invention comprises a method for analyzing
biological material including individual-specific antibodies,
comprising: forming an array of multiple antigens by attaching the
multiple antigens to the surface of a solid support in a
preselected pattern such that the respective locations of the
multiple antigens are known; obtaining a sample of the biological
material and contacting the array with the sample such that a
portion of the individual-specific antibodies contained in the
sample reacts with and binds to antigens in the array to form
immune complexes; washing the solid support containing the immune
complexes such that antibodies in the sample that do not react with
and bind to the antigens in the array are removed; and detecting
the immune complexes and determining the locations thereof such
that an antibody profile is obtained. In one embodiment, detecting
the immune complexes may be performed by exposing the immune
complexes to a first additional antibody that recognizes and binds
to the individual-specific antibodies. In a further embodiment, one
more additional antibodies that recognize the first additional
antibody or each other may be used.
According to embodiments of the invention, the detecting of the
immune complexes comprises treating the solid support having immune
complexes attached thereto such that the presence of immune
complexes at a location is characterized by a color change as
compared to the absence of immune complexes at the location. In one
embodiment, the process of detecting the immune complexes further
comprises monitoring the solid support with solid state color
detection circuitry for comparing the color patterns before and
after contacting the array with the sample. In another embodiment,
the process of detecting the immune complexes further comprises
obtaining a color camera image before and after contacting the
array with the sample and analyzing pixel information obtained
therefrom. In still another embodiment of the invention, the solid
support is a surface plasmon resonance chip and the detecting of
the immune complexes further comprises scanning the surface plasmon
resonance chip before and after contacting the array with the
sample and comparing data obtained therefrom. In yet another
embodiment of the invention, the detecting of immune complexes
comprises obtaining an image using a charge-coupled device to
detect the color change comprising fluorescence emission.
In yet another embodiment of the invention, the method is used as a
test for the use of drugs. Still another embodiment of the
invention comprises analysis of an antibody profile obtained from a
forensic sample and comparison with an antibody profile obtained
from a sample from a criminal suspect or a victim of crime.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows illustrative antibody profiles obtained from saliva
samples according to the procedure of Example 1.
FIG. 2 shows comparisons of paired saliva and blood antibody
profiles from five individuals according to the procedure of
Example 1.
FIG. 3 shows antibody profiles obtained from saliva samples from a
single individual after contamination with various adulterants
according to the procedure of Example 1.
FIG. 4 shows illustrative results obtained from immunoassay of
Cocaine (crystalline tropane alkaloid) in saliva samples according
to the procedure of Example 1.
FIG. 5 shows illustrative results obtained from immunoassay of
methamphetamine in saliva samples according to the procedure of
Example 1.
FIG. 6 shows illustrative results of immunodetection of Cocaine
(crystalline tropane alkaloid) on a PVDF membrane: strip 5, 0
.mu.g/ml Cocaine (crystalline tropane alkaloid); strip 6, 0.1
.mu.g/ml Cocaine (crystalline tropane alkaloid); strip 7, 10
.mu.g/ml Cocaine (crystalline tropane alkaloid); and strip 8, 1000
.mu.g/ml Cocaine (crystalline tropane alkaloid).
FIG. 7 shows illustrative results of immunodetection of
methamphetamine on a PVDF membrane: strip 1, 0 .mu.g/ml
methamphetamine; strip 2, 0.1 .mu.g/ml methamphetamine; strip 3, 10
.mu.g/ml methamphetamine; strip 4, 1000 .mu.l/ml
methamphetamine.
FIG. 8 shows antibody profiles from three different individuals:
one strip of each pair contains no drugs; and the other strip of
each pair contains 1000 .mu.g/ml of Cocaine (crystalline tropane
alkaloid) and of methamphetamine.
FIG. 9 shows antibody profiles for different amounts of serum using
a two-stage antibody layering process. Strip A was exposed to 50
microliters of serum; strip B was exposed to 10 microliters of
serum; strip C was exposed to 5 microliters of serum; strip D was
exposed to 3 microliters of serum; strip E was exposed to 1
microliter of serum; strip F was exposed to 0.5 microliter of
serum; strip G was exposed to 0.1 microliter of serum; and strip H
was exposed to 0 microliter of serum.
FIG. 10 shows antibody profiles for different amounts of serum
using a three-stage antibody layering process. Strip A was exposed
to 50 microliters of serum; strip B was exposed to 25 microliters
of serum; strip C was exposed to 15 microliters of serum; strip D
was exposed to 7.5 microliters of serum; strip E was exposed to 10
microliters of serum; strip F was exposed to 2.5 microliters of
serum; strip G was exposed to 1 microliter of serum; strip H was
exposed to 0.5 microliter of serum; strip I was exposed to 0.1
microliter of serum; and strip J was exposed to 0 microliter of
serum.
FIG. 11 shows side-by-side antibody profiles of 3 microliters of
serum where strip A is developed with a three-stage antibody
layering process and strip B is developed with a two-stage antibody
layering process.
FIG. 12 shows densitometry data from strips A and B of FIG. 11. The
top line is strip A and the lower line is strip B.
DETAILED DESCRIPTION OF THE INVENTION
Before the present methods for analyzing biological samples are
described in detail, it is to be understood that this invention is
not limited to the particular configurations, process acts, and
materials disclosed herein as such configurations, process acts,
and materials may vary somewhat. It is also to be understood that
the terminology employed herein is used for the purpose of
describing particular embodiments only and is not limiting since
the scope of the present invention will be limited only by the
appended claims and equivalents thereof.
The publications and other reference materials referred to herein
to describe the background of the invention and to provide
additional detail regarding its practice are hereby incorporated by
reference. The references discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
the inventors are not entitled to antedate such disclosure by
virtue of prior invention.
It must be noted that, as used in this specification and the
appended claims, the singular forms "a," "an," and "the" include
plural referents unless the context clearly dictates otherwise.
Thus, for example, reference to a method for analyzing a biological
sample from "an animal" includes reference to two or more of such
animals, reference to "a solid support" includes reference to one
or more of such solid supports, and reference to "an array"
includes reference to two or more of such arrays.
In describing and claiming the present invention, the following
terminology will be used in accordance with the definitions set out
below.
As used herein, "comprising," "including," "containing,"
"characterized by," and grammatical equivalents thereof are
inclusive or open-ended terms that do not exclude additional,
unrecited elements or method steps. "Comprising" is to be
interpreted as including the more restrictive terms "consisting of"
and "consisting essentially of."
As used herein, "consisting of" and grammatical equivalents thereof
exclude any element, step, or ingredient not specified in the
claim.
As used herein, "consisting essentially of" and grammatical
equivalents thereof limit the scope of a claim to the specified
materials or steps and those that do not materially affect the
basic and novel characteristic or characteristics of the claimed
invention.
As used herein, "solid support" means a generally or substantially
planar substrate onto which an array of antigens is disposed. A
solid support can comprise any material or combination of materials
suitable for carrying the array. Materials used to construct these
solid supports need to meet several requirements, such as (1) a
presence of surface groups that can be easily derivatized, (2)
inertness to reagents used in the assay, (3) stability over time,
and (4) compatibility with biological samples. For example,
suitable materials include glass, silicon, silicon dioxide (i.e.,
silica), plastics, polymers, hydrophilic inorganic supports, and
ceramic materials. Illustrative plastics and polymers include
poly(tetrafluoroethylene), poly(vinylidene difluoride),
polystyrene, polycarbonate, polymethacrylate, and combinations
thereof. Illustrative hydrophilic inorganic supports include
alumina, zirconia, titania, and nickel oxide. An example of a glass
substrate would be a microscope slide. Silicon wafers used to make
computer chips have also been used to make biochips. See, for
example, U.S. Pat. No. 5,605,662.
As used herein, "array" means an arrangement of locations on the
solid support. The locations will generally be arranged in
two-dimensional arrays, but other formats are possible. The number
of locations can range from several to at least hundreds of
thousands. The array pattern and spot density can vary. For
example, using a commercially available GMS 417 Arrayer from
Genetic MicroSystems, Inc. (Woburn, Mass.), the spot size and
density can be selected by the user. With spots of 150 .mu.m
diameter and 300 .mu.m center-to-center spacing, more than 1000
spots can be placed in a square centimeter and more than 10,000
spots can be placed on a standard microscope slide. With 200 .mu.m
center-to-center spacing, these numbers increase to 2500 per square
centimeter and more than 25,000 per slide.
As used herein, "colorigenic" refers to a substrate that produces a
colored product upon digestion with an appropriate enzyme. Such
colored products include fluorescent and luminescent products.
A first act in the present method is to prepare an array of
antigens by attaching the antigens to the surface of the solid
support in a preselected pattern such that the locations of
antigens in the array are known. As used herein, an antigen is a
substance that is bound by an antibody. Antigens can include
proteins, carbohydrates, nucleic acids, hormones, drugs, receptors,
tumor markers, and the like, and mixtures thereof. An antigen can
also be a group of antigens, such as a particular fraction of
proteins eluted from a size exclusion chromatography column. Still
further, an antigen can also be identified as a designated clone
from an expression library or a random epitope library.
In one embodiment of the invention, antigens are isolated from HeLa
cells as generally described in A.M. Francoeur et al., 136 J.
Immunol. 1648 (1986). Briefly, HeLa cells are grown in standard
medium under standard tissue culture conditions. Confluent HeLa
cell cultures are then rinsed, preferably with phosphate-buffered
saline (PBS), lysed with detergent, and centrifuged to remove
insoluble cellular debris. The supernate contains approximately
10,000 immunologically distinct antigens suitable for generating an
array.
There is no requirement that the antigens used to generate the
array be known. All that is required is that the source of the
antigens be consistent such that a reproducible array can be
generated. For example, the HeLa cell supernate containing the
antigens can be fractionated on a size exclusion column,
electrophoretic gel, density gradient, or the like, as is well
known in the art. Fractions are collected, and each fraction
collected could represent a unique set of antigens for the purpose
of generating the array. Thus, even though the antigens are
unknown, a reproducible array can be generated if the HeLa cell
antigens are isolated and fractionated using the same method and
conditions.
Other methods, such as preparation of random peptide libraries or
epitope libraries are well known in the art and may be used to
reproducibly produce antigens (e.g., J. K. Scott and G. P. Smith,
"Searching for Peptide Ligands with an Epitope Library," 249
Science 386 (1990); J. J. Devlin et al., "Random Peptide Libraries:
A Source of Specific Protein Binding "Molecules," 249 Science
404-406 (1990); S. E. Cwirla et al., "Peptides on Phage: A Vast
Library of Peptides for Identifying Ligands," 87 Proc. Nat'l Acad.
Sci. USA 6378-6382 (1990); K. S. Lam et al., "A New Type of
Synthetic Peptide Library for Identifying Ligand-binding Activity,"
354 Nature 82-84 (1991); S. Cabilly, "Combinatorial Peptide Library
Protocols" (Humana Press, 304 pp. 129-154, 1997); and U.S. Pat. No.
5,885,780). Such libraries can be constructed by ligating synthetic
oligonucleotides into an appropriate fusion phage. Fusion phages
are filamentous bacteriophage vectors in which foreign sequences
are cloned into phage gene III and displayed as part of the gene
III protein (pIII) at one tip of the virion. Each phage encodes a
single random sequence and expresses it as a fusion complex with
pIII, a minor coat protein present at about five molecules per
phage. For example, in the fusion phage techniques of J. K. Scott
and G. P. Smith, supra, a library was constructed of phage
containing a variable cassette of six amino acid residues. The
hexapeptide modules fused to bacteriophage proteins provided a
library for the screening methodology that can examine
>10.sup.12 phages (or about 10.sup.8-10.sup.10 different clones)
at one time, each with a test sequence on the virion surface. The
library obtained was used to screen monoclonal antibodies specific
for particular hexapeptide sequences. The fusion phage system has
also been used by other groups, and libraries containing longer
peptide inserts have been constructed. Fusion phage prepared
according to this methodology can be selected randomly or
non-randomly for inclusion in the array of antigens. The fusion
phages selected for inclusion in the array can be propagated by
standard methods to result in what is virtually an endless supply
of the selected antigens.
Other methods for producing antigens are also known in the art. For
example, expression libraries can be prepared by random cloning of
DNA fragments or cDNA into an expression vector (e.g., R. A. Young
and R. W. Davis, "Yeast RNA Polymerase II Genes: Isolation with
Antibody Probes," 222 Science 778-782 (1983); G. M. Santangelo et
al., "Cloning of Open Reading Frames and Promoters from the
Saccharomyces cerevisiae Genome: Construction of Genomic Libraries
of Random Small Fragments," 46 Gene 181-186 (1986)). Expression
vectors that could be used for making such libraries are
commercially available from a variety of sources. For example,
random fragments of HeLa cell DNA or cDNA can be cloned into an
expression vector, and then clones expressing HeLa cell proteins
can be selected. These clones can then be propagated by methods
well known in the art. The expressed proteins are then isolated or
purified and can be used in the making of the array.
Alternatively, antigens can be synthesized using recombinant DNA
technology well known in the art. Genes that code for many viral,
bacterial, and mammalian proteins have been cloned, and thus large
quantities of highly pure proteins can be synthesized quickly and
inexpensively. For example, the genes that code for many eukaryotic
and mammalian membrane-bound receptors, growth factors, cell
adhesion molecules, and regulatory proteins have been cloned and
are useful as antigens. Many proteins produced by such recombinant
techniques, such as transforming growth factor, acidic and basic
fibroblast growth factors, interferon, insulin-like growth factor,
and various interleukins from different species, are commercially
available.
In most instances, the entire polypeptide need not be used as an
antigen. For example, any size or portion of the polypeptide that
contains at least one epitope, i.e., antigenic determinant or
portion of an antigen that specifically interacts with an antibody,
will suffice for use in the array.
The antigens, whether selected randomly or non-randomly, are
disposed on the solid support to result in the array. The pattern
of the antigens on the solid support should be reproducible. That
is, the location and identity of each antigen on the solid support
should be known. For example, in a 10.times.10 array one skilled in
the art might place antigens 1-100 in locations 1-100,
respectively, of the array.
The proteins may placed in arrays on the surface of the solid
support using a pipetting device or a machine or device configured
for placing liquid samples on a solid support, for example, using a
commercially available microarrayer, such as those from Cartesian
Technologies, Inc. (Irvine, Calif.); Gene Machines (San Carlos,
Calif.); Genetic MicroSystems, Inc. (Woburn, Mass.); GenePack DNA
(Cambridge, UK); Genetix Ltd. (Christchurch, Dorset, UK); and
Packard Instrument Company (Meriden, Conn.).
Relevant methods to array a series of protein antigens onto a
surface include non-contact drop-on-demand dispensing and inkjet
technology. Commercially available instruments are available for
both methods. Cartesian Technologies offers several nanoliter
dispensing instruments that can dispense liquid volumes from 20 mL
up to 250 .mu.L from 96-, 384-, 1536-, 3456-, and 9600-well
microtiter plates and place them precisely on a surface with
densities up to 400 spots/cm.sup.2. The instruments will spot onto
surfaces in a variety of patterns. As the name implies, inkjet
technology utilizes the same principles as those used in inkjet
printers. MicroFab Technologies, Inc. (Plano, Tex.), offers a
ten-fluid print head that can dispense picoliter quantities of
liquids onto a surface in a variety of patterns. An illustrative
pattern for the present application would be a simple array ranging
from 10.times.10 up to 100.times.100.
There are a number of methods that can be used to attach proteins
or other antigens to the surface of a solid support. The simplest
of these is simple adsorption through hydrophobic, ionic, and van
der Waals forces. This method is not optimal, however, since the
proteins tend to detach from the surface over time. One suitable
attachment chemistry involves the use of bifunctional organosilanes
(e.g., Thompson and Maragos, 44 J. Agric. Food Chem. 1041-1046
(1996)). One end of the organosilane reacts with exposed --OH
groups on the surface of the chip to form a silanol bond. The other
end of the organosilane contains a group that is reactive with
various groups on the protein surface, such as --NH.sub.2 and --SH
groups. This method of attaching proteins to the chip results in
the formation of a covalent linkage between the protein and the
chip. Other suitable methods that have been used for protein
attachment to surfaces include arylazide, nitrobenzyl, and
diazirine photochemistry methodologies. Exposure of the above
chemicals to UV light causes the formation of reactive groups that
can react with proteins to form a covalent bond. The arylazide
chemistry forms a reactive nitrene group that can insert into C--H
bonds, while the diazirine chemistry results in a reactive carbene
group. The nitrobenzyl chemistry is referred to as caging chemistry
whereby the caging group inactivates a reactive molecule. Exposure
to UV light frees the molecule and makes it available for reaction.
Still other methods for attaching proteins to solid supports are
well known in the art, (e.g., S. S. Wong, Chemistry of Protein
Conjugation and Cross-Linking CRC Press, 340, 1991).
Following attachment of the antigens on the solid support in the
selected array, the solid support should be washed by rinsing with
an appropriate liquid to remove unbound antigens. Appropriate
liquids for washing include phosphate buffered saline (PBS) and the
like, i.e., relatively low ionic strength, biocompatible salt
solutions buffered at or near neutrality. Many of such appropriate
wash liquids are known in the art or can be devised by a person
skilled in the art without undue experimentation (e.g., N. E. Good
and S. Izawa, "Hydrogen Ion Buffers," 24 Methods Enzymology 53-68
(1972)).
The solid support is then processed for blocking of nonspecific
binding of proteins and other molecules to the solid support. This
blocking step prevents the binding of antigens, antibodies, and the
like to the solid support wherein such antigens, antibodies, or
other molecules are not intended to bind. Blocking reduces the
background that might swamp out the signal, thus increasing the
signal-to-noise ratio. The solid support is blocked by incubating
the solid support in a medium that contains inert molecules that
bind to sites where nonspecific binding might otherwise occur.
Examples of suitable blockers include bovine serum albumin, human
albumin, gelatin, nonfat dry milk, polyvinyl alcohol, TWEEN.RTM.
20, and various commercial blockers, such as SEABLOCK.TM.
(trademark of EastCoast Bio, Inc., North Berwick, Me.) and
SUPERBLOCK.RTM. (trademark of Pierce Chemical Co., Rockford, Ill.)
blocking buffers.
Following washing for removal of unbound antigens from the array
and blocking, the solid support is contacted with a liquid sample
to be tested. The sample can be from any animal that generates
individual-specific antibodies. For example, humans, dogs, cats,
mice, horses, cows, and rabbits have all been shown to possess
ISAs. The sample can be from various bodily fluids and solids,
including blood, saliva, semen, serum, plasma, urine, amniotic
fluid, pleural fluid, cerebrospinal fluid, and mixtures thereof.
These samples are obtained according to methods well known in the
art. Depending on the detection method used, it may be required to
manipulate the biological sample to attain optimal reaction
conditions. For example, the ionic strength or hydrogen ion
concentration or the concentration of the biological sample can be
adjusted for optimal immune complex formation, enzymatic catalysis,
and the like.
As described in detail in U.S. Pat. No. 5,270,167 to Francoeur,
when ISAs are allowed to react with a set of random antigens, a
certain number of immune complexes form. For example, using a panel
of about 1000 unique antigens, about 30 immune complexes between
ISAs in a biological sample that has been diluted twenty-fold can
be detected. If the biological sample is undiluted, the total
number of possible detectable immune complexes that could form
would be greater than 10.sup.23. The total number of possible
immune complexes can also be increased by selecting "larger"
antigens, i.e., proteins instead of peptides) that have multiple
epitopes. Therefore, it will be appreciated that depending on the
antigens and number thereof used, the dilution of the biological
sample, and the detection method, one skilled in the art can
regulate the number of immune complexes that will form and be
detected. The set of unique immune complexes that form and fail to
form between the ISAs in the biological sample and the antigens in
the array constitute an antibody profile.
Methods for detecting antibody/antigen or immune complexes are well
known in the art. The present invention can be modified by one
skilled in the art to accommodate the various detection methods
known in the art. The particular detection method chosen by one
skilled in the art depends on several factors, including the amount
of biological sample available, the type of biological sample, the
stability of the biological sample, the stability of the antigen,
and the affinity between the antibody and antigen. Moreover, as
discussed above, depending on the detection methods chosen, it may
be required to modify the biological sample.
While these techniques are well known in the art, examples of a few
of the detection methods that may be used to practice the present
invention are briefly described below.
There are many types of immunoassays known in the art. The most
common types of immunoassay are competitive and non-competitive
heterogeneous assays, such as enzyme-linked immunosorbent assays
(ELISAs). In a non-competitive ELISA, unlabeled antigen is bound to
a solid support, such as the surface of the biochip. Biological
sample is combined with antigens bound to the reaction vessel, and
antibodies (primary antibodies) in the biological sample are
allowed to bind to the antigens, forming the immune complexes.
After the immune complexes have formed, excess biological sample is
removed and the biochip is washed to remove nonspecifically bound
antibodies. The immune complexes may then be reacted with an
appropriate enzyme-labeled anti-immunoglobulin (secondary
antibody). The secondary antibody reacts with antibodies in the
immune complexes, not with other antigens bound to the biochip.
Secondary antibodies specific for binding antibodies of different
species, including humans, are well known in the art and are
commercially available, such as from Sigma Chemical Co. (St. Louis,
Mo.) and Santa Cruz Biotechnology, Inc. (Santa Cruz, Calif.). After
a further wash, the enzyme substrate is added. The enzyme linked to
the secondary antibody catalyzes a reaction that converts the
substrate into a product. When excess antigen is present, the
amount of product is directly proportional to the amount of primary
antibodies present in the biological sample. The product may be
fluorescent or luminescent, which can be measured using technology
and equipment well known in the art. It is also possible to use
reaction schemes that result in a colored product, which can be
measured spectrophotometrically.
In other embodiments of the invention, the secondary antibody may
not be labeled to facilitate detection. Additional antibodies may
be layered (i.e., tertiary, quaternary, etc.) such that each
additional antibody specifically recognizes the antibody previously
added to the immune complex. Any one of these additional (i.e.,
tertiary, quaternary, etc.) may be labeled so as to allow detection
of the immune complex as described herein.
Sandwich or capture assays can also be used to identify and
quantify immune complexes. Sandwich assays are a mirror image of
non-competitive ELISAs in that antibodies are bound to the solid
phase and antigen in the biological sample is measured. These
assays are particularly useful in detecting antigens having
multiple epitopes that are present at low concentrations. This
technique requires excess antibody to be attached to a solid phase,
such as the biochip. The bound antibody is then incubated with the
biological samples, and the antigens in the sample are allowed to
form immune complexes with the bound antibody. The immune complex
is incubated with an enzyme-linked secondary antibody, which
recognizes the same or a different epitope on the antigen as the
primary antibody. Hence, enzyme activity is directly proportional
to the amount of antigen in the biological sample. D. M. Kemeny and
S. J. Challacombe, "ELISA and Other Solid Phase Immunoassays"
(1988).
Typical enzymes that can be linked to secondary antibodies include
horseradish peroxidase, glucose oxidase, glucose-6-phosphate
dehydrogenase, alkaline phosphatase, .beta.-galactosidase, and
urease. Secondary antigen-specific antibodies linked to various
enzymes are commercially available from, for example, Sigma
Chemical Co. and Amersham Life Sciences (Arlington Heights,
Ill.).
Competitive ELISAs are similar to noncompetitive ELISAs except that
enzyme linked antibodies compete with unlabeled antibodies in the
biological sample for limited antigen binding sites. Briefly, a
limited number of antigens are bound to the solid support.
Biological sample and enzyme-labeled antibodies are added to the
solid support. Antigen-specific antibodies in the biological sample
compete with enzyme-labeled antibodies for the limited number of
antigens bound to the solid support. After immune complexes have
formed, nonspecifically bound antibodies are removed by washing,
enzyme substrate is added, and the enzyme activity is measured. No
secondary antibody is required. Because the assay is competitive,
enzyme activity is inversely proportional to the amount of
antibodies in the biological sample.
Another competitive ELISA can also be used within the scope of the
present invention. In this embodiment, limited amounts of
antibodies from the biological sample are bound to the surface of
the solid support as described herein. Labeled and unlabeled
antigens are then brought into contact with the solid support such
that the labeled and unlabeled antigens compete with each other for
binding to the antibodies on the surface of the solid support.
After immune complexes have formed, nonspecifically bound antigens
are removed by washing. The immune complexes are detected by
incubation with an enzyme-linked secondary antibody, which
recognizes the same or a different epitope on the antigen as the
primary antibody, as described above. The activity of the enzyme is
then assayed, which yields a signal that is inversely proportional
to the amount of antigen present.
Homogeneous immunoassays can also be used when practicing the
method of the present invention. Homogeneous immunoassays may be
preferred for detection of low molecular weight compounds, such as
hormones, therapeutic drugs, and illegal drugs that cannot be
analyzed by other methods, or compounds found in high
concentration. Homogeneous assays are particularly useful because
no separation step is necessary. R. C. Boguslaski et al., "Clinical
Immunochemistry: Principles of Methods and Applications"
(1984).
In homogeneous techniques, bound or unbound antigens are
enzyme-linked. When antibodies in the biological sample bind to the
enzyme-linked antigen, steric hindrances inactivate the enzyme.
This results in a measurable loss in enzyme activity. Free antigens
(i.e., not enzyme-linked) compete with the enzyme-linked antigen
for limited antibody binding sites. Thus, enzyme activity is
directly proportional to the concentration of antigen in the
biological sample.
Enzymes useful in homogeneous immunoassays include lysozyme,
neuraminidase, trypsin, papain, bromelain, glucose-6-phosphate
dehydrogenase, and .beta.-galactosidase. T. Persoon,
"Immunochemical Assays in the Clinical Laboratory," 5 Clinical
Laboratory Science 31 (1992). Enzyme-linked antigens are
commercially available or can be linked using various chemicals
well known in the art, including glutaraldehyde and maleimide
derivatives.
Prior antibody profiling technology involves an alkaline
phosphatase labeled secondary antibody with
5-bromo-4-chloro-3'-indolylphosphate p-toluidine salt (BCIP) and
nitro-blue tetrazolium chloride (NBT), both of which are
commercially available from a variety of sources, such as from
Pierce Chemical Co. (Rockford, Ill.). The enzymatic reaction forms
an insoluble colored product that is deposited on the surface of
the membrane strips to form bands wherever antigen-antibody
complexes occur. This method is suboptimal in a biochip format
since it is difficult to quantify and since colorimetric methods
are typically less sensitive than assays based on fluorescence or
luminescence.
Fluorescent immunoassays can also be used when practicing the
method of the present invention. Fluorescent immunoassays are
similar to ELISAs except the enzyme is substituted for fluorescent
compounds called fluorophores or fluorochromes. These compounds
have the ability to absorb energy from incident light and emit the
energy as light of a longer wavelength and lower energy.
Fluorescein and rhodamine, usually in the form of isothiocyanates
that can be readily coupled to antigens and antibodies, are most
commonly used in the art. D. P. Stites et al., "Basic and Clinical
Immunology" (1994). Fluorescein absorbs light of 490 nm to 495 nm
in wavelength and emits light at 520 nm in wavelength.
Tetramethylrhodamine absorbs light of 550 nm in wavelength and
emits light at 580 nm in wavelength. Illustrative
fluorescence-based detection methods include ELF-97 alkaline
phosphatase substrate (Molecular Probes, Inc., Eugene, Oreg.);
PBXL-1 and PBXL-3 (phycobilisomes conjugated to streptavidin)
(Martek Biosciences Corp., Columbia, Md.); FITC (fluorescein
isothiocyanate) and Texas Red labeled goat anti-human IgG (Jackson
ImmunoResearch Laboratories, Inc., West Grove, Pa.); and
B-Phycoerythrin and R-Phycoerythrin conjugated to streptavidin
(Molecular Probes, Inc.). ELF-97 is a nonfluorescent chemical that
is digested by alkaline phosphatase to form a fluorescent molecule.
Because of turnover of the alkaline phosphatase, use of the ELF-97
substrate results in signal amplification. Fluorescent molecules
attached to secondary antibodies do not exhibit this
amplification.
Phycobiliproteins isolated from algae, porphyrins, and
chlorophylls, which all fluoresce at about 600 nm, are also being
used in the art. I. Hemmila, "Fluoroimmunoassays and
Immunofluorometric Assays," 31 Clin. Chem. 359 (1985); U.S. Pat.
No. 4,542,104. Phycobiliproteins and derivatives thereof are
commercially available under the names R-phycoerythrin (PE) and
QUANTUM RED.TM. from, for example, Sigma Chemical Co.
In addition, Cy-conjugated secondary antibodies and antigens are
useful in immunoassays and are commercially available. Cy3, for
example, is maximally excited at 554 nm and emits light at between
568 nm and 574 nm. Cy3 is more hydrophilic than other fluorophores
and thus has less of a tendency to bind nonspecifically or
aggregate. Cy-conjugated compounds are commercially available from
Amersham Life Sciences.
Illustrative luminescence-based detection methods include CSPD.RTM.
and CDP star alkaline phosphatase substrates (Roche Molecular
Biochemicals, Indianapolis, Ind.); and SUPERSIGNAL.RTM. horseradish
peroxidase substrate (Pierce Chemical Co., Rockford, Ill.).
Chemiluminescence, electroluminescence, and
electrochemiluminescence (ECL) detection methods are also
attractive means for quantifying antigens and antibodies in a
biological sample. Luminescent compounds have the ability to absorb
energy, which is released in the form of visible light upon
excitation. In chemiluminescence, the excitation source is a
chemical reaction; in electroluminescence the excitation source is
an electric field; and in ECL an electric field induces a
luminescent chemical reaction.
Molecules used with ECL detection methods generally comprise an
organic ligand and a transition metal. The organic ligand forms a
chelate with one or more transition metal atoms forming an
organometallic complex. Various organometallic and transition
metal-organic ligand complexes have been used as ECL labels for
detecting and quantifying analytes in biological samples. Due to
their thermal, chemical, and photochemical stability, their intense
emissions and long emission lifetimes, ruthenium, osmium, rhenium,
iridium, and rhodium transition metals are favored in the art. The
types of organic ligands are numerous and include anthracene and
polypyridyl molecules and heterocyclic organic compounds. For
example, bipyridyl, bipyrazyl, terpyridyl, and phenanthrolyl, and
derivatives thereof, are common organic ligands in the art. A
common organometallic complex used in the art includes
tris-bipyridine ruthenium (II), commercially available from IGEN,
Inc. (Rockville, Md.) and Sigma Chemical Co.
Advantageously, ECL can be performed under aqueous conditions and
under physiological pH, thus minimizing biological sample handling.
J. K. Leland et al., "Electrogenerated Chemiluminescence: An
Oxidative-Reduction Type ECL Reactions Sequence Using Triprophyl
Amine," 137 J. Electrochemical Soc. 3127-3131 (1990); WO 90/05296;
and U.S. Pat. No. 5,541,113. Moreover, the luminescence of these
compounds may be enhanced by the addition of various cofactors,
such as amines.
In practice, a tris-bipyridine ruthenium (II) complex, for example,
may be attached to a secondary antibody using strategies well known
in the art, including attachment to lysine amino groups, cysteine
sulfhydryl groups, and histidine imidazole groups. In a typical
ELISA immunoassay, secondary antibodies would recognize ISAs bound
to antigens, but not unbound antigens. After washing nonspecific
binding complexes, the tris-bipyridine ruthenium (II) complex would
be excited by chemical, photochemical, and electrochemical
excitation means, such as by applying current to the biochip (e.g.,
WO 86/02734). The excitation would result in a double oxidation
reaction of the tris-bipyridine ruthenium (II) complex, resulting
in luminescence that could be detected by, for example, a
photomultiplier tube. Instruments for detecting luminescence are
well known in the art and are commercially available, for example,
from IGEN, Inc. (Rockville, Md.).
Solid state color detection circuitry can also be used to monitor
the color reactions on the biochip and, on command, compare the
color patterns before and after the sample application. A color
camera image can also be used and the pixel information analyzed to
obtain the same information.
Still another method involves detection using a surface plasmon
resonance (SPR) chip. The surface of the chip is scanned before and
after sample application and a comparison is made. The SPR chip
relies on the refraction of light when the molecules of interest
are exposed to a light source. Each molecule has its own refraction
index by which it can be identified. This method requires precise
positioning and control circuitry to scan the chip accurately.
Yet another method involves a fluid rinse of the biochip with a
fluorescing reagent. The microlocations that combine with the
biological sample will fluoresce and can be detected with a
charge-coupled device (CCD) array. The output of such a CCD array
is analyzed to determine the unique pattern associated with each
sample. This approach avoids the problems associated with scanning
technologies. Speed is not a factor with any of the methods since
the chemical combining of sample and reference takes minutes to
occur.
Moreover, array scanners are commercially available, such as from
Genetic MicroSystems, Inc. The GMS 418 Array Scanner uses laser
optics to rapidly move a focused beam of light over the biochip.
This system uses a dual-wavelength system including high-powered,
solid-state lasers that generate high excitation energy to allow
for reduced excitation time. At a scanning speed of 30 Hz, the GMS
418 can scan a 22.times.75-mm slide with 10-.mu.m resolution in
about four minutes.
Software for image analysis obtained with an array scanner is
readily available. Available software packages include IMAGENE.RTM.
(BioDiscovery, Los Angeles, Calif.); ScanAlyze (available at no
charge; developed by Mike Eisen, Stanford University, Palo Alto,
Calif.); De-Array (developed by Yidong Chen and Jeff Trent of the
National Institutes of Health; used with IP Lab from Scanalytics,
Inc., Fairfax, Va.); Pathways (Research Genetics, Huntsville,
Ala.); GEM Tools (Incyte Pharmaceuticals, Inc., Palo Alto, Calif.);
and Imaging Research (Amersham Pharmacia Biotech, Inc., Piscataway,
N.J.).
Once interactions between the antigens and ISAs have been
identified and quantified, the signals may be digitized. The
digitized antibody profile serves as a signature that identifies
the source of the biological sample. Depending on the biochip used,
the digitized data may take numerous forms. For example, the
biochip may comprise an array with 10 columns and 10 rows for a
total number of 100 microlocations. Each microlocation contains at
least one antigen. After the biological sample containing the ISAs
is added to each microlocation and allowed to incubate,
interactions between antigens and ISAs in the biological sample are
identified and quantified. In each microlocation, an interaction
between the antigen at that microlocation and the ISAs in the
biological sample either do or do not result in a quantifiable
signal. In one embodiment, the results of the antibody profile are
digitized by ascribing each one of the 100 microlocations a
numerical value of either "0," if a quantifiable signal was not
obtained, or "1," if a quantifiable signal was obtained. Using this
method, the digitized antibody profile comprises a unique set of
zeroes and ones.
The numerical values "0" or "1" may, of course, be normalized to
signals obtained in internal control microlocations so that
digitized antibody profiles obtained at a later time can be
properly compared. For example, one or several of the
microlocations will contain a known antigen, which will remain
constant over time. Therefore, if a subsequent biological sample is
more or less dilute than a previous biological sample, the signals
can be normalized using the signals from the known antigen.
It will be appreciated by one skilled in the art that other methods
of digitizing the antibody profile exist and may be used. For
example, rather than ascribing each microlocation with a numerical
value of "0" or "1," the numerical value may be incremental and
directly proportional to the strength of the signal.
By digitizing the antibody profile signals, the biochemical results
can be entered into a computer and quickly accessed and referenced.
Within seconds of having the antibody profile digitized, a computer
can compare a previously digitized antibody profile to determine
whether there is a match. If a matching antibody profile is in the
database, a positive identification of the source of the biological
sample can be made. Thus, the method of the present invention can
both discriminate and positively identify the source of a
biological sample.
In an embodiment of the invention, the present method is used for
forensic analysis for matching a biological sample to a criminal
suspect. Forensic samples obtained from crime scenes are often
subject to drying of the samples, small sample sizes, mixing with
samples from more than one individual, adulteration with chemicals,
and the like. The present method provides the advantages of rapid
analysis, simplicity, low cost, and accuracy for matching forensic
samples with suspects. For example, the forensic sample and a
sample from one or more suspects are obtained according to methods
well known in the art. Antibody profiles for each of the samples
are prepared, as described herein. The antibody profiles are then
compared. A match of antibody profiles means that the forensic
sample was obtained from the matching suspect. If no match of
antibody profiles is obtained, then none of the suspects was the
source of the forensic sample.
In another embodiment of the invention, the present method is used
for drug testing of individuals. For example, in many work places
it is a condition of obtaining or maintaining employment to be free
of illegal drug use. The presence of illegal drugs in the
bloodstream of a person can be detected by the present method by
antibody capture or similar methods. Moreover, as described in WO
97/29206, the drug test and the identity of the sample can be
correlated in a single test. Drug tests are also important in
certain animals, such as horses and dogs involved in racing.
The present invention is further described in the following
examples, which are offered by way of illustration and are not
limiting of the invention in any manner.
EXAMPLE 1
The law enforcement community has demonstrated several needs
associated with drug testing of suspects including dealing with
privacy issues associated with sample collection, maintenance of
sample chain of custody, prevention of sample adulteration by the
suspect, and facilitating more rapid turnaround time on sample
analyses. Current drug testing protocols utilize urine samples and,
occasionally, blood samples. Invasion of privacy is a continuing
problem with urine samples since it is necessary to observe the
individual providing the sample to maintain the chain of custody
and eliminate the possibility of sample switching or adulteration.
Urine samples are also not a good indicator of the current level of
intoxication since many drug metabolites continue to be excreted
into urine for days or weeks after the drugs are initially taken.
While blood samples do not suffer from these problems, collecting
blood is an invasive procedure requiring special facilities and
trained personnel that may not always be available when the need
arises. It is necessary for law enforcement personnel to maintain
strict chain of custody for all samples collected to ensure that
mishandling or deliberate tampering do not occur. A break or even a
perceived break in the chain of custody can result in evidence
being dismissed outright or given little weight.
Embodiments of the present invention solve these issues in several
ways. First, incorporation of the antibody profiling identification
assay into the drug test makes identification of the sample donor
integral to the test and eliminates the need for complex chain of
custody procedures. Second, a saliva-based drug test is better than
a urine test because drug levels in saliva can be readily
correlated with drug levels in blood (W. Schramm et al., "Drugs of
Abuse in Saliva: A Review," 16 J. Analy. Toxicology 1-9 (1992); E.
J. Cone, "Saliva Testing for Drugs of Abuse," 694 Ann. N.Y. Acad.
Sci. 91-127 (1995)), therefore providing a better indicator of
current drug use (D. A. Kidwell et al., "Testing for Drugs of Abuse
in Saliva and Sweat," 713 J. of Chromatography B: Biomedical
Sciences and Applications 111-135 (1998)). Saliva samples from a
suspect can also be collected easily in view of a law enforcement
officer without invasion of privacy or with invasive methods.
Finally, the present test is easy to use and can be quickly
performed by law enforcement personnel on site, instead of
requiring the days to weeks necessary at distant centralized
laboratories. V. S. Thompson et al., "Antibody Profiling as an
Identification Tool for Forensic Samples," 3576 Investigation and
Forensic Science Technologies 52-59 (1999).
In this example, an antibody-based test is provided for two common
illicit drugs (Cocaine (crystalline tropane alkaloid) and
methamphetamine). These drugs are among the most commonly abused,
and their use is on the rise. S. B. Karch, Drug Abuse Handbook (CRC
Press, 1998); L. D. Bowers, "Athletic Drug Testing, 17 Sports
Pharmacology," 299-318 (1998).
Materials and Methods. Goat anti-rabbit IgG antibodies conjugated
to alkaline phosphatase were obtained from Jackson ImmunoResearch
(West Grove, Pa.). Rabbit anti-human IgA antibodies were purchased
from U.S. Biological (Swampscott, Mass.). SEABLOCK.TM., nitro-blue
tetrazolium chloride/5-bromo-4-chloro-3'-indolylphosphate
p-toluidine salt (NBT/BCIP), p-nitrophenyl phosphate disodium salt
(PNPP), EZ-LINK.RTM. maleimide activated alkaline phosphatase kits,
and FREEZYME.TM. conjugate purification kits were obtained from
Pierce Chemical (Rockford, Ill.). Monoclonal antibodies against
benzoylecgonine and methamphetamine, and bovine serum albumin (BSA)
conjugates of methamphetamine and benzoylecgonine were purchased
from O.E.M. Concepts (Toms River, N.J.). Cocaine (crystalline
tropane alkaloid) and methamphetamine hydrochloride salts were
obtained from Sigma-Aldrich (St. Louis, Mo.). Antibody Profiling
strips were purchased from Miragen, Inc. (Irvine, Calif.). Strips
used for the combined drug-AbP test were produced according to the
protocol of A. M. Francoeur, "Antibody Fingerprinting: A Novel
Method for Identifying Individual People and Animals," 6
Bio/Technology 822-825 (1988). Saliva samplers from Saliva
Diagnostic Systems, Inc. (Vancouver, Wash.), OraSure Technologies,
Inc. (Bethlehem, Pa.), and Sarstedt, Inc. (Newton, N.C.), were used
to collect saliva samples from volunteers.
A saliva-based AbP assay was developed through modification of an
earlier protocol designed for processing blood samples. T. F. Unger
and A. Strauss, "Individual-Specific Antibody Profiles as a Means
of Newborn Infant Identification," 15 J. Perinatology 152-155
(1995). Briefly, 500 .mu.l of saliva sample diluted with 1.0 ml of
PBST (50 mM phosphate buffered saline, 0.2% TWEEN.RTM. 20) was
incubated with an AbP strip overnight for a minimum of 16 hours,
and excess sample was washed off with PBST. Next, the strip was
incubated successively with 100 ng/ml rabbit anti-human IgA for 1
hour and 100 ng/ml goat anti-rabbit IgG-alkaline phosphatase
conjugate for 30 minutes with washes in between incubations. The
strip was washed again with PBST and a precipitation substrate for
alkaline phosphatase, NBT/BCIP, was added to allow development of
bands on the strip.
The SALIVA SAMPLER.TM. (Saliva Diagnostic Systems) and the
SALIVETTE.TM. (Sarstedt, Inc.) saliva collection systems were
examined for compatibility with the AbP assay. The SALIVA
SAMPLER.TM. system comprises a cotton pad attached to a plastic
handle. A window in the handle turns blue when sufficient sample
has been collected. The pad is placed in a preservative buffer
after collection. The SALIVETTE.TM. is a cotton roll placed in the
mouth for about 10 minutes and then centrifuged in a plastic tube
to collect a sample. Both types of samplers were placed in the
gingival crevice of the mouth for sample collection. The quality of
samples as a function of storage time at temperatures of
-20.degree. C., 4.degree. C., and 25.degree. C. was assessed by
performing AbP on samples collected with both samplers.
Five volunteers participated in studies to compare blood AbP
patterns with those obtained from saliva samples. Protocols for use
of human subjects were conducted in accordance with the Idaho
National Engineering and Environmental Laboratory Institutional
Review Board. Blood samples were collected in tubes containing the
anticoagulant EDTA and were used immediately. Saliva was collected
using the SALIVA SAMPLER.TM. saliva collection system. Paired blood
and saliva samples were analyzed using the blood protocol of Unger
and Strauss, supra, and the saliva AbP test described above.
Four additional volunteers participated in a saliva adulteration
study to assess the effects of various foods and beverages on the
AbP assay. The volunteers were given butterscotch and lemon hard
candy, sugar and sugar-free gum, sugar and sugar-free cola, and
milk chocolate. After eating the above, they were asked to collect
saliva samples using the provided saliva samplers. Volunteers were
also asked to consume alcohol, drink coffee, eat a food of their
choice, and brush their teeth prior to giving samples. A volunteer
who was a smoker provided a sample after smoking a cigarette.
Baseline samples were also collected from the volunteers.
Monoclonal antibodies against methamphetamine and benzoylecgonine
were conjugated to alkaline phosphatase using the Pierce
EZ-LINK.TM. maleimide activated alkaline phosphatase kit according
to the manufacturer's protocols. Unconjugated antibody was
separated from the antibody-enzyme conjugate using the FREEZYME.TM.
conjugate purification kit according to the manufacturer's
protocols.
Competitive enzyme linked immunosorbent assays (ELISAs) were
developed for both Cocaine (crystalline tropane alkaloid) and
methamphetamine. The BSA conjugates of methamphetamine or
benzoylecgonine were diluted in 50 mM carbonate buffer, pH 9.6, and
50 .mu.l was added to each well of a 96-well microtiter plate. The
plate was incubated overnight at 4.degree. C. to allow the
conjugates to bind to the well surfaces. The plate was then washed
with PBST to remove excess BSA conjugate. Next, 50 .mu.l of either
Cocaine (crystalline tropane alkaloid) or methamphetamine solution
in the concentration range from 0 to 1000 .mu.g/ml was added to the
plate and 50 .mu.l of either monoclonal anti-benzoylecgonine or
anti-methamphetamine conjugated with alkaline phosphatase was
added. During this step, the immobilized BSA drug conjugate
competed with the free drug in solution for binding sites on the
antibodies. After the competition reaction was complete, the
unbound antibodies and free drug were washed away. Finally, 100
.mu.l of soluble alkaline phosphatase substrate (PNPP) solution was
added to the wells to react with the alkaline phosphatase bound to
the well surfaces through the anti-drug antibodies. The reaction
was stopped after 20 to 30 minutes by addition of 25 .mu.l of 3 M
NaOH, and the absorbance of each well was read at 405 nm using a
Tecan Spectra microplate reader.
Polyvinylidene fluoride (PVDF) membrane is used in the manufacture
of the Miragen AbP strips, and was used to assess the feasibility
of binding the Cocaine (crystalline tropane alkaloid) and
methamphetamine-BSA conjugates to its surface. The PVDF membrane
was cut into strips the same size as those used in the AbP assay.
Four strips were prepared for each drug and 10 .mu.l spots of
either drug-BSA conjugate were placed at three locations on each
strip for analysis in triplicate. The strips were dried at
35.degree. C. for one hour prior to use. Non-specific binding sites
on the strips were blocked with PBST containing 1 mg/mL BSA for one
hour and then rinsed with PBST. Cocaine (crystalline tropane
alkaloid) and methamphetamine solutions were prepared in PBST at
concentrations of 0, 0.1, 10, and 1000 .mu.g/ml. Next, 750 .mu.l of
Cocaine (crystalline tropane alkaloid) or methamphetamine solution
was added to the strips and another 750 .mu.l of
anti-benzoylecgonine or anti-methamphetamine antibodies conjugated
with alkaline phosphatase were added and allowed to incubate for
one hour. During this time a competitive reaction between the free
and the immobilized drug for antibody binding sites took place. The
strips were washed to remove unbound antibodies and drugs and the
NBT/BCIP substrate was added. The strips were allowed to develop
for 15 minutes.
A combined AbP-drug assay was prepared by placing 10 .mu.l spots of
both methamphetamine and benzoylecgonine-BSA conjugate onto the
blank bottom portion of the AbP strip and allowing them to dry for
one hour at 35.degree. C. Saliva samples from three individuals
were collected using ORASURE.RTM. samplers. Half of the saliva
sample was spiked with 1000 .mu.g/ml of Cocaine (crystalline
tropane alkaloid) or methamphetamine. The strips were blocked with
PBST containing 1.0 mg/ml BSA for one hour and rinsed with PBST.
Next, 500 .mu.l of spiked or unspiked saliva was added to the
strips along with alkaline phosphatase conjugated
anti-benzoylecgonine and anti-methamphetamine antibodies and
allowed to incubate over night at room temperature. The strips were
washed with PBST and the AbP assay was conducted as described
above.
Results and Discussion. The saliva-based AbP assay was optimized
through variation of reagent concentrations, sample volumes, and
incubation times. Illustrative results of antibody profiles
obtained from saliva samples are shown in FIG. 1. Compared to the
blood-based AbP assay, the saliva assay takes much longer (18 hours
versus two hours) and requires a ten-fold larger amount of sample.
This is due to the 100-fold lower levels of total antibody present
in saliva as compared to blood. Parry, "Tests for HIV and Hepatitis
Viruses," 694 Annals. N.Y. Acad. Sci. 221 (1993).
The stability of antibodies present in the saliva samples collected
using the SALIVA SAMPLER.TM. or the SALIVETTE.TM. systems was
determined by storage at -20.degree. C., 4.degree. C., and
25.degree. C. and AbP testing of samples daily over the period of
one week to see if there were any changes in the patterns observed.
Fresh saliva samples from either sampler gave the best results. The
stability over time of samples collected with the SALIVA
SAMPLER.TM. system was superior to samples collected with the
SALIVETTE.TM. system at all temperatures. The preservative storage
buffer provided with the SALIVA SAMPLER.TM. system appears to
prevent antibody degradation due to bacterial contamination, while
the SALIVETTE.TM. sampler includes no preservative.
The samples collected with the SALIVA SAMPLER.TM. system and
maintained at room temperature showed no change in pattern over a
five-day period. This result is in contrast to the results obtained
with samples stored in a refrigerator, which showed marked
deterioration even after a few hours of storage. It is not clear
why this occurred. Frozen samples also showed some deterioration
due to damage caused by freeze-thaw cycles, but prolonged storage
at freezing temperatures resulted in no further degradation. Since
SALIVA SAMPLER.TM. saliva collection systems had superior storage
properties and were easier to use, they were used for the
adulteration studies.
Blood AbP patterns were compared to saliva AbP patterns to
determine if the ISAs present in those samples were the same. The
results showed that the patterns obtained from the two different
samples differed markedly (FIG. 2). This result was somewhat
surprising since saliva is a filtrate of blood, and it was expected
that the ISAs present in saliva would be the same as those present
in blood. The different patterns probably resulted from the isotype
of antibody examined in each case. In blood IgG antibodies were
analyzed since they are the most prevalent. In saliva, IgA
antibodies are more prevalent and were analyzed. After the above
result was obtained, saliva samples were also analyzed for IgG
antibodies to determine if those patterns would be the same as
those from the blood patterns. However, this was unsuccessful due
to the extremely low levels of IgG antibodies present in
saliva.
The saliva adulteration studies showed that virtually no changes
occurred in the antibody profiles when any of the adulterants were
present (FIG. 3). In some cases a band might be darker or lighter,
but there appeared to be no missing or additional bands present.
Since this was a preliminary study, the adulterants examined were
easily obtainable items that might be used during the course of
ordinary life. However, as a quick search of the Internet reveals,
there are many proposed methods to beat urine-based drug tests
including ingestion of substances and/or adulteration of samples
with various substances that are being sold by these sites. The
adulteration results shown here are promising since it appears that
the AbP test is not affected by foods that may be commonly consumed
before taking a saliva test.
Immunoassay tests for both Cocaine (crystalline tropane alkaloid)
and methamphetamine were developed using a direct competitive
assay. An anti-benzoylecgonine antibody was used for the Cocaine
(crystalline tropane alkaloid) assay; however, this antibody gave
the same response to Cocaine (crystalline tropane alkaloid) as to
benzoylecgonine (the primary metabolite of Cocaine (crystalline
tropane alkaloid)) so it did not affect the results of the assay.
In this assay, a drug present in a sample competes for binding
sites on enzyme labeled antibodies with a BSA-conjugated drug
immobilized to the surface of a well of a microtiter plate. In
samples with large drug concentrations, most of the antibody-enzyme
conjugate will bind to the drug in solution and will be washed away
during the final step. Therefore, there will be very little enzyme
present in the microtiter plate and the amount of color development
will be low. Conversely, if there is no drug in the sample, the
antibodies will bind to the immobilized drugs and stay in the wells
after the wash step, resulting in strong color development. This
results in a signal that is inversely proportional to the drug
concentration (FIGS. 4 and 5). The linear range for Cocaine
(crystalline tropane alkaloid) detection was from 0.1 to 5 .mu.g/ml
and for methamphetamine was from 0.1 to 10 .mu.g/ml. This range
covers the cutoff values for these drugs (0.3 and 1.0 .mu.g/ml,
respectively) currently set by the Substance Abuse and Mental
Health Services Administration. M. Peat and A. E. Davis, Drug Abuse
Handbook (CRC Press, Boca Raton, Fla. 1998).
Using the optimum concentrations of BSA-drug conjugates determined
during the ELISA studies, the drug assays were conducted on the
PVDF membranes. Because of the inverse relationship of the
immunoassay to drug concentration, a dark spot was observed when
the concentration of drugs was low, and spots gradually disappeared
as the drug concentration increased (FIGS. 6 and 7).
Since the drug test on the PVDF membranes were promising, the
feasibility of combining the two drug tests with the AbP assay was
assessed. Antibody profile patterns from the three individuals did
not change regardless of whether the drug was present or not (FIG.
8). This result shows that the presence of the drugs did not
interfere with the reagents used to perform the antibody profiling
assay.
EXAMPLE 2
In this example, the procedure of Example 1 is followed except that
fractionated HeLa cell antigens are immobilized on a PVDF membrane
in a predetermined pattern as a two-dimensional array.
Additionally, Cocaine (crystalline tropane alkaloid) and
methamphetamine are immobilized on the membrane as additional spots
on the array. After development of color as described, results are
substantially similar to those of Example 1.
EXAMPLE 3
In this example, the procedure of Example 2 is followed except that
the array is immobilized on a glass slide.
EXAMPLE 4
Assay strips were prepared as in Example 1 and pre-blocked in PBS.
The strips were then exposed to various amounts of serum ranging
from 50 .mu.l to 0.1 .mu.l for 20 minutes. The strips were then
placed into a bleach solution (0.5% v/v sodium hypochlorite) before
washing four times with PBS.
In the case of strips undergoing a two-stage antibody layering
process, the strips were exposed to a rabbit anti-human IgG for 12
minutes before being washed four times with PBS. These strips were
then exposed to a goat anti-rabbit IgG conjugated to alkaline
phosphatase for 12 minutes. The strips were then washed and the
color developed as outlined in Example 1. Results of two antibody
layers can be seen in FIG. 9 wherein a readable pattern with some
bands missing is visible down to 1 microliter of serum and a
complete pattern is visible at 3 microliters of serum.
In the case of strips undergoing a three-stage antibody layering
process, the strips were exposed to a rabbit anti-human IgG for 12
minutes before being washed four times with PBS. These strips were
then exposed to a goat anti-rabbit IgG for 12 minutes. The strips
were then exposed to a donkey anti-goat IgG conjugated to alkaline
phosphatase for 12 minutes. The strips were then washed and the
color developed as outlined in Example 1. Results of two antibody
layers can be seen in FIG. 10 wherein a readable pattern with some
bands missing is visible down to 0.5 microliter of serum and a
complete pattern is visible at 1 microliter of serum.
A comparison of FIGS. 9 and 10 shows that a three-stage antibody
layering process has increased sensitivity in providing a readable
pattern of individual-specific antibodies over the two-layer
process.
EXAMPLE 5
Assay strips were prepared as in Example 1 and pre-blocked in PBS.
The strips were then exposed to three microliters of serum ranging
for 20 minutes. The strips were then placed into a bleach solution
(0.5% v/v sodium hypochlorite) before washing four times with
PBS.
In the case of the strip undergoing a two-stage antibody layering
process, the strips were exposed to a rabbit anti-human IgG for 12
minutes before being washed four times with PBS. These strips were
then exposed to a goat anti-rabbit IgG conjugated to alkaline
phosphatase for 12 minutes. The strips were then washed and the
color developed as outlined in Example 1.
In the case of the strips undergoing a three-stage antibody
layering process, the strips were exposed to a goat anti-human IgG
for 12 minutes before being washed four times with PBS. These
strips were then exposed to a rabbit anti-goat IgG for 12 minutes.
The strips were then exposed to a donkey anti-rabbit IgG conjugated
to alkaline phosphatase 12 minutes. The strips were then washed and
the color developed as outlined in Example 1.
Results of the two-stage and three-stage antibody layering
processes can be viewed side by side in FIG. 11. Strip A is the
three-layer strip and strip B is the two-layer strip. As can be
seen, a much stronger signal and sensitivity were obtained with the
three-layer process.
A densitometry study of the two strips was performed and the
results are presented in FIG. 12, where the three-layer strip is
the top line and the two-layer strip is the bottom line. The height
of the lines indicates the intensity of the bands. As can be seen
in FIG. 12, the three-layer process has an increased readout for
specific bands without a proportional increase in the background
noise. Thus, the three-layer process has increased sensitivity over
the two-layer process.
While this invention has been described in certain embodiments, the
present invention can be further modified within the spirit and
scope of this disclosure. This application is therefore intended to
cover any variations, uses, or adaptations of the invention using
its general principles. Further, this application is intended to
cover such departures from the present disclosure as come within
known or customary practice in the art to which this invention
pertains and which fall within the limits of the appended
claims.
* * * * *
References