Methods of detecting donor-specific antibodies and systems for practicing the same

1. A method for determining the presence or absence of donor specific antibodies (DSAs) in a biological sample, the method comprising:

forming a mixture by combining a donor cellular sample with a biological sample from a recipient under conditions sufficient for donor specific antibodies (DSAs), if present, to bind to donor cell surface antigen (Ag) to form DSA-Ag complex comprising donor cells bound to DSAs via the donor cell surface Ag;

contacting the mixture with beads comprising monoclonal antibody immobilized on a surface of the beads, that specifically binds the donor cell surface Ag in the DSA-Ag complex;

adding under lysis conditions, to the mixture contacted with the beads, a detectably-labeled antibody that specifically binds the DSAs in the DSA-Ag complex bound to the beads, wherein the lysis conditions are sufficient to lyse the donor cells associated with the DSA-Ag complex; and

detecting the presence or absence of the detectably-labeled antibody bound to the DSAs in the DSA-Ag complex bound to the beads to determine the presence or absence of the DSAs in the biological sample from the recipient.

2. The method according to claim 1 , wherein the detecting is semi-quantitative.

3. The method according to claim 1 , wherein the immune antibody is an alloantibody.

4. The method according to claim 1 , wherein the immune antibody is an autoantibody.

5. The method according to claim 1 , wherein the immune antibody is a complement fixing antibody (CFAb).

6. The method according to claim 1 , wherein the detecting comprises detecting a fluorescence emission.

7. The method according to claim 1 , wherein the detecting comprises flowing the complex through a flow cytometer.

8. The method according to claim 1 , wherein the detecting comprises detecting the complex by an enzyme-linked immunosorbent assay (ELISA).

9. The method according to claim 1 , wherein the detectably-labeled antibody comprises a detectable label attached to the antibody or an antigen binding fragment thereof.

10. The method according to claim 1 , wherein the detectable label comprises a fluorochrome, a chromophore, an enzyme, a linker molecule, a biotin molecule, an electron donor, an electron acceptor, a dye, a metal, or a radionuclide.

11. The method according to claim 1 , wherein the detectable label comprises a fluorophore selected from the group consisting of: indocarbocyanine (C3), indodicarbocyanine (C5), Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Texas Red, Pacific Blue, Oregon Green 488, Alexa fluor-355, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor-555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, JOE, Lissamine, Rhodamine Green, BODIPY, fluorescein isothiocyanate (FITC), carboxy-fluorescein (FAM), Allophycocyanin (APC), phycoerythrin (PE), rhodamine, dichlororhodamine (dRhodamine), carboxy tetramethylrhodamine (TAMRA), carboxy-X-rhodamine (ROX), LIZ, VIC, NED, PET, SYBR, PicoGreen, and RiboGreen.

12. The method according to claim 1 , wherein the donor specific antibody is an antibody produced by the recipient in response to receiving a transplant or transfusion from one or more donors.

13. The method according to claim 1 , wherein the Ag is a Human Leukocyte Antigen (HLA).

14. The method according to claim 1 , wherein the biological sample comprises serum, blood, saliva, or plasma.

15. The method according to claim 1 , comprising obtaining the cellular sample from the donor prior to forming the mixture, wherein the cellular sample comprises lymphocytes.

16. The method according to claim 1 , comprising obtaining the biological sample from the recipient prior to forming the mixture, wherein the biological sample comprises serum.

17. The method according to claim 1 , wherein the cellular sample from the donor comprises nucleated cells.

18. The method according to claim 1 , wherein the cellular sample from the donor comprises from 0.001×10 6 to 2.0×10 6 cells.

19. The method according to claim 1 , wherein the cellular sample from the donor comprises fewer than 0.2×10 6 cells.

20. The method according to claim 1 , wherein the cellular sample from the donor comprises fewer than 0.1×10 6 cells.

21. The method according to claim 1 , wherein the cellular sample from the donor comprises fewer than 0.5×10 5 cells.

22. The method according to claim 1 , wherein the cellular sample from the donor comprises about 25,000 to 200,000 cells.

23. The method according to claim 1 , wherein the average bead diameter is from 0.1 to 20 microns.

24. The method according to claim 1 , wherein the average bead diameter is 5 microns or less.

25. The method according to claim 1 , wherein the average bead diameter is between 2.5 to 5 microns.

26. The method according to claim 1 , wherein the beads are agarose beads, latex beads, magnetic beads, or polystyrene beads.

27. The method according to claim 1 , wherein the method is performed in 12 hours or less.

28. The method according to claim 1 , wherein the method is performed in 8 hours or less.

29. The method according to claim 1 , wherein the lysis conditions comprise administering a lysis buffer comprising tracer, detergent, and DNase.

30. The method according to claim 1 , comprising generating a report indicating whether donor specific antibodies are present in the biological sample from the recipient.

31. The method according to claim 30 , wherein generating the report is performed by a computer.

32. The method according to claim 31 , wherein the report is displayed to an output device at a location remote to the computer.

33. The method according to claim 1 , wherein the recipient is a candidate for receiving a transplant or a transfusion from the donor.

34. The method according to claim 33 , wherein the biological sample is serum, blood, or plasma.