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.

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. 
 
     
     
       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.   
     
     
       32. The method of claim 31, wherein contacting the array comprising multiple antigens comprises contacting multiple antigens attached to the support through covalent bonds. 
     
     
       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. 
     
     
       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. 
     
     
       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. 
     
     
       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. 
     
     
       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. 
     
     
       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. 
     
     
       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. 
     
     
       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. 
     
     
       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. 
     
     
       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. 
     
     
       43. The method of claim 31, wherein contacting the array with the sample of biological material comprises contacting the array with a forensic sample. 
     
     
       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. 
     
     
       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.   
     
     
       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.   
     
     
       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. 
     
     
       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. 
     
     
       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. 
     
     
       50. The method of claim 46, wherein contacting the array with a sample of biological material comprises contacting the array with saliva. 
     
     
       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. 
     
     
       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. 
     
     
       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. 
     
     
       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. 
     
     
       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. 
     
     
       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. 
     
     
       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. 
     
     
       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. 
     
     
       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.   
     
     
       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.