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EPITOPE SPECIFICITIES OF ANTIBODIES REACTIVE WITH SINGLE HLA ANTIGENS IN LUMINEX ASSAYS

EPITOPE SPECIFICITIES OF ANTIBODIES REACTIVE WITH SINGLE HLA ANTIGENS IN LUMINEX ASSAYS. Aurora D, Balazs I, Davies A, Ebner D, Hariharan J, Jiang N, Mostecki J, Ray B, Zaslavskaia L TEPNEL LIFECODES CORPORATION, STAMFORD, CT, USA. Objective.

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EPITOPE SPECIFICITIES OF ANTIBODIES REACTIVE WITH SINGLE HLA ANTIGENS IN LUMINEX ASSAYS

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  1. EPITOPE SPECIFICITIES OF ANTIBODIES REACTIVE WITH SINGLE HLA ANTIGENS IN LUMINEX ASSAYS Aurora D, Balazs I, Davies A, Ebner D, Hariharan J, Jiang N, Mostecki J, Ray B, Zaslavskaia L TEPNEL LIFECODES CORPORATION, STAMFORD, CT, USA

  2. Objective • Identify the sensitizing HLA Class I and Class II epitopes in transplant cases.

  3. Procedure outline Sera samples used for this study were derived from transplanted patients positive for anti-HLA Class I and/or II Ab using LSA (Tepnel Lifecodes). Individual HLA single antigens were bound to latex beads. The pattern of reactivity of antibody eluted from each bead was compared with the reactivities predicted by the epitopes listed in HLAMatchmaker. Based on the Ag of the donor and the anti-HLA reactivity(ies), serum was incubated with different antigen-beads. Following incubation, anti-HLA bound to each antigen-bead was eluted and tested with the Lifecodes Single Antigen assay.

  4. Lifecodes Class 2 Single Antigen Panel (LSA2)

  5. Lifecodes Class 1 Single Antigen Panel (LSA1)

  6. Class I HLA Matchmaker eplets

  7. Class II HLA Matchmaker eplets

  8. Example of HLA Class I eplets Patient: A2; B27; B38 Donor: A3; A23; B7; B49

  9. Adsorption and Elution Experiment IgG eluted from single antigen A*0301 beads, incubated with LSA1 bead mixture.

  10. List of eplets consistent with the pattern of reactivity of the antibody eluted from single antigen A*0301 bead IgG eluted from A*0301 (eplet 66RNQ and 142MI)

  11. Elution Experiment Antibody eluted from single antigen A*2302 beads, incubated with LSA1 bead mixture.

  12. List of eplets consistent with the pattern of reactivity of the antibody eluted from single antigen A*2302 bead IgG eluted from A*2302 (eplet 142MI and 167DG)

  13. IgG eluted from B*0702 (eplet 80ERN)

  14. IgG eluted from B*4901 (eplet 12AMR and 163LW) IgG eluted from B*4701 (eplet 12AMR) Summary HLA-A Ab to 66RNQ, 142MI, 167DG HLA-B Ab to 12AMR, 80ERN, 163LW

  15. Transplant # 1 Example of HLA Class II eplets • Patient: DR1, 15; DR51; DQB5, 6 • Donor: DR4, 15; DR51, 53; DQB6, 8 • Post transplant HLA Ab: • DRB1*0401, 0701, 0901, 1001, 1401 • DRB4*0101; DQB*0301, 0302, 0303

  16. Specificity of eluted Class II Ab • DRB1*0401, 0701, 0901, 1001, 1401 • DRB4*0101; • DQB*0301, 0302, 0303 Adsorbed/elute w/DR*0401 or 4*0101; eplets: 12VKH, 70LRRA & 4Q Adsorbed/elute w/DQ*0302; eplets: 55PPP

  17. Transplant # 2 • Patient: DR11, 13; DR52; DQ2, 9 • Donor: DR7, 15; DR51, 53; DQ2, 6 • Post transplant HLA Ab: • DRB1*0901, 1001, 1401, 1501 • DRB4*0101; DRB5*0202; DQB*0503, 0601

  18. Specificity of eluted Class II Ab • Patient Ab: DRB1*0901, 1001, 1401, 1501 • DRB4*0101; DRB5*0202 • DQB*0503, 0601 Adsorbed/elute w/DR*0701 or 1501; eplets: 71RRA, 71QAA Adsorbed/elute w/DQ*0601; eplets: many

  19. Conclusions • Eplets, as defined in HLAMatchmaker, may be used to define the sensitizing epitopes of antibodies developed in transplant patients. • These type of studies could be used: • to define the most common epitopes involved in humoral rejection. • to understand the properties of Ab/Ag interaction for different epitopes and their effect in antidoy assays (e.g. relative sensitivity of individual Ag). • to determine the effect of competition among Ab, in a polyclonal Ab, for the same Ag. How does this affect relative signal?

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