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

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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

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Objective

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

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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.
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.

• The pattern of reactivity of antibody eluted from
  each bead was compared with the reactivities
  predicted by the epitopes listed in
  HLAMatchmaker.

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Lifecodes Class 2 Single Antigen Panel (LSA2)

DP antigens     DQ antigens     DR antigens
DPB10101 DPA10103 DQB10202 DQA10201 DRB10101
DPB10101 DPA10201 DQB10202 DQA10302 DRB10102
DPB10101 DPA10202 DQB10202 DQA10501 DRB10103
DPB10101 DPA10301 DQB10402 DQA10302 DRB10401
DPB10201 DPA10103 DQB10401 DQA10401 DRB10701
DPB10301 DPA10103 DQB10402 DQA10401 DRB10801
DPB10401 DPA10103 DQB10301 DQA10302 DRB10901
DPB10401 DPA10201 DQB10301 DQA10501 DRB11001
DPB10401 DPA10202 DQB10302 DQA10201 DRB11101
DPB10401 DPA10301 DQB10302 DQA10302 DRB11201
DPB10402 DPA10103 DQB10303 DQA10302 DRB11301
DPB10402 DPA10301 DQB10303 DQA10401 DRB11303
DPB10501 DPA10103 DQB10503 DQA10104 DRB11401
DPB10501 DPA10201 DQB10503 DQA10201 DRB11501
DPB10501 DPA10202 DQB10601 DQA10104 DRB11502
DPB10501 DPA10301 DQB10601 DQA10201 DRB11601
DPB11301 DPA10201 DQB10601 DQA10501 DRB10301
DPB11401 DPA10201     DRB10303
DPB11701 DPA10201     DRB30101
DPB11801 DPA10103     DRB30202
DPB11901 DPA10201     DRB30301
DPB11901 DPA10301     DRB40101
DPB12801 DPA10202         DRB50101
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HLA-A     HLA-B     HLA-C
A0101 A3601   B0702 B4002(B61)   Cw0102
A0201 A4301   B0703 B4101   Cw0210
A0203 A6601   B0801 B4201   Cw0303
A0206 A6602   B1302 B4402   Cw0304
A0301 A6801   B1401(B64) B4403   Cw0401
A1101 A6802   B1402(B65) B4501   Cw0403
A1102 A6901   B1501(B62) B4601   Cw0501
A2302 A7401   B1502(B75) B4701   Cw0602
A2402 A7405   B1503(B72) B4801   Cw0701
A2403 A8001   B1512(B76) B4901   Cw0702
A2501     B1513(B77) B5001   Cw0704
A2601     B1516(B63) B5101   Cw0801
A2902     B1518(B71) B5201   Cw0802
A3001     B1801 B5301   Cw1202
A3101     B2703 B5401   Cw1402
A3201     B2705 B5501   Cw1502
A3301     B2708 B5601   Cw1601
A3303     B3501 B5701   Cw1701
A3402     B3508 B5801   Cw1801
A3601     B3701 B7301    
A4301     B3801 B7801    
A6601     B3901 B8101    
A6602     B4001(B60) B8202    
Lifecodes Class 1 Single Antigen Panel (LSA1)
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Class I HLA Matchmaker eplets
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Class II HLA Matchmaker eplets
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Example of HLA Class I eplets
Patient A2 B27 B38
Donor A3 A23 B7 B49
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Adsorption and Elution Experiment
IgG eluted from single antigen A0301 beads,
incubated with LSA1 bead mixture.
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List of eplets consistent with the pattern of
reactivity of the antibody eluted from single
antigen A0301 bead
IgG eluted from A0301 (eplet 66RNQ and 142MI)
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Elution Experiment
Antibody eluted from single antigen A2302 beads,
incubated with LSA1 bead mixture.
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List of eplets consistent with the pattern of
reactivity of the antibody eluted from single
antigen A2302 bead
IgG eluted from A2302 (eplet 142MI and 167DG)
13
IgG eluted from B0702 (eplet 80ERN)
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IgG eluted from B4901 (eplet 12AMR and 163LW)
IgG eluted from B4701 (eplet 12AMR)
Summary HLA-A Ab to 66RNQ, 142MI, 167DG HLA-B Ab
to 12AMR, 80ERN, 163LW
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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
• DRB10401, 0701, 0901, 1001, 1401
• DRB40101 DQB0301, 0302, 0303

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Specificity of eluted Class II Ab
DRB10101 --- --- --- ---
DRB10102 --- --- --- ---
DRB10103 --- --- --- ---
DRB10301 --- --- --- ---
DRB10303 --- --- --- ---
DRB10401 --- 12VKH 34HQ ---
DRB10402 --- 12VKH 34HQ ---
DRB10405 --- 12VKH 34HQ ---
DRB10701 4Q   --- ---
DRB10801 --- --- --- ---
DRB10901 4Q   ---  
DRB11001 ---   --- 70LRRA
DRB11101 --- --- --- ---
DRB11201 --- --- --- ---
DRB11301 --- --- --- ---
DRB11303 --- --- ---  
DRB11401 --- --- --- 70LRRA
DRB11501 --- --- --- ---
DRB11502 --- --- --- ---
DRB11601 --- --- --- ---
DRB30101 --- --- --- ---
DRB30202 --- --- --- ---
DRB30203 --- --- --- ---
DRB30301 --- --- --- ---
DRB40101 4Q   --- 70LRRA
DRB50101 --- --- --- ---
DRB50202 --- --- --- ---
DRB10401, 0701, 0901, 1001, 1401 DRB40101
DQB0301, 0302, 0303
DQB10202 ---
DQB10301 55PPP
DQB10302 55PPP
DQB10303 55PPP
DQB10401 ---
DQB10402 ---
DQB10501 ---
DQB10503 ---
DQB10601 ---
Adsorbed/elute w/DR0401 or 40101 eplets
12VKH, 70LRRA 4Q Adsorbed/elute w/DQ0302
eplets 55PPP
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Transplant 2

• Patient DR11, 13 DR52 DQ2, 9
• Donor DR7, 15 DR51, 53 DQ2, 6
• Post transplant HLA Ab
• DRB10901, 1001, 1401, 1501
• DRB40101 DRB50202 DQB0503, 0601

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Specificity of eluted Class II Ab
DRB10101 71QRA
DRB10102 71QRA
DRB10103 71DEA
DRB10301 ---
DRB10303 ---
DRB10401 ---
DRB10402 71DEA
DRB10405 71QRA
DRB10701 ---
DRB10801 ---
DRB10901 71RRA
DRB11001 71RRA
DRB11101 ---
DRB11201 ---
DRB11301 71DEA
DRB11303  
DRB11401 71RRA
DRB11501 71QAA
DRB11502 71QAA
DRB11601 ---
DRB30101 ---
DRB30202 ---
DRB30203 ---
DRB30301 ---
DRB40101 71RRA
DRB50101 ---
DRB50202 71QAA
Patient Ab DRB10901, 1001, 1401,
1501 DRB40101 DRB50202 DQB0503, 0601
DQB10202 --- --- --- --- --- --- --- --- --- --- --- --- ---
DQB10301 --- --- --- --- --- --- --- --- --- --- --- --- ---
DQB10302 --- --- --- --- --- --- --- --- --- --- --- --- ---
DQB10303 --- --- --- --- --- --- --- --- --- --- --- --- ---
DQB10401 --- --- --- --- 55PRL   --- 70ED --- --- --- --- ---
DQB10402 --- --- --- --- 55PRL   --- 70ED --- --- --- --- ---
DQB10501 --- 14GL 30HYV 52PQ 55PRP   --- 70GA 84EV 87AY 90AGI 116I 125SQ
DQB10503 --- 14GL 30HYV 52PQ 55PRP 56RPD --- 70GA 84EV 87AY 90AGI 116I 125SQ
DQB10601 3P --- 30YDV 52PQ 55PRP 56RPD 67DIT --- 84EV 87AF 90AGI --- 125GQ
Adsorbed/elute w/DR0701 or 1501 eplets 71RRA,
71QAA Adsorbed/elute w/DQ0601 eplets many
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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?