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58-OR
S52
58-OR
Abstracts
DETECTION OF MICA ANTIBODIES AND EPITOPE ANALYSIS USING LUMINEX MICA SINGLE ANTIGEN BEADS Miyuki Ozawa,1 Nadim El-Awar,1 Jar-How Lee,1 Paul I. Terasaki.2 1Research, One Lambda, Inc., Canoga Park, CA, USA; 2Terasaki Foundation Laboratory, Los Angeles, CA, USA Aim: Recent reports have shown that antibodies against MHC Class I-related Chain A (MICA) antigens affect transplant survival. With the newly developed Luminex Single Antigen MICA beads, we tested 1,564 post-transplant sera for MICA antibodies. In studying the frequency of MICA antibodies, we also observed cross-reactive patterns similar to those of HLA antibodies. Here we report the use of Single Antigen MICA beads to determine the specificities of MICA antibodies and to show whether the antibody specificities can be attributed to shared unique amino acid sequences, or epitopes. Methods: Soluble MICA*001, *002, *004, *007, *012, *018, *019, and *027 antigens purified from recombinant cell lines were coated onto Luminex beads. Post-transplant sera from 1,379 kidney, 122 liver, and 52 heart patients were tested with these beads for the presence of MICA antibodies and cross-reactive patterns examined. To investigate whether the multiple specificities were caused by more than one antibody or by one antibody directed against one epitope, sera were absorbed with a single-allele MICA recombinant cell line, then eluted and tested on Luminex MICA single antigen beads. Scope: MICA antibodies were found in 263 (16.8%) of total 1,564 post-transplant patient sera. While about 38% of the detected antibodies were directed against only one MICA antigen, more than 60% of antibodies showed multiple specificities, such as antibody against MICA*001, *012, and *018, or antibody against MICA*004 and *027. Figure 1 shows an example of absorption/elution experiment to identify possible target amino acids of the multi-specific antibodies. When patient serum ALB77 positive to MICA*001, *012, and *018 was absorbed with a single cell line MICA*018, the eluate reacted to all three beads *001, *012, and *018. This suggests that this serum contains one MICA antibody which reacts to a shared unique amino acid, possibly the threonine (T) at position 24, as illustrated in Figure 2. Conclusions: With the new MICA single antigen Luminex beads, we were able to screen a large number of patient sera and to study MICA cross-reactive patterns that are similar to HLA antibodies. In addition, absorption and elution tests made it possible to identify some of the MICA epitopes. Since MICA antibodies have now been shown to affect transplant outcome, it would be of great value to test for MICA antibodies and their specificities, pre and post transplantation.
58-OR
Abstracts
DETECTION OF MICA ANTIBODIES AND EPITOPE ANALYSIS USING LUMINEX MICA SINGLE ANTIGEN BEADS Miyuki Ozawa,1 Nadim El-Awar,1 Jar-How Lee,1 Paul I. Terasaki.2 1Research, One Lambda, Inc., Canoga Park, CA, USA; 2Terasaki Foundation Laboratory, Los Angeles, CA, USA Aim: Recent reports have shown that antibodies against MHC Class I-related Chain A (MICA) antigens affect transplant survival. With the newly developed Luminex Single Antigen MICA beads, we tested 1,564 post-transplant sera for MICA antibodies. In studying the frequency of MICA antibodies, we also observed cross-reactive patterns similar to those of HLA antibodies. Here we report the use of Single Antigen MICA beads to determine the specificities of MICA antibodies and to show whether the antibody specificities can be attributed to shared unique amino acid sequences, or epitopes. Methods: Soluble MICA*001, *002, *004, *007, *012, *018, *019, and *027 antigens purified from recombinant cell lines were coated onto Luminex beads. Post-transplant sera from 1,379 kidney, 122 liver, and 52 heart patients were tested with these beads for the presence of MICA antibodies and cross-reactive patterns examined. To investigate whether the multiple specificities were caused by more than one antibody or by one antibody directed against one epitope, sera were absorbed with a single-allele MICA recombinant cell line, then eluted and tested on Luminex MICA single antigen beads. Scope: MICA antibodies were found in 263 (16.8%) of total 1,564 post-transplant patient sera. While about 38% of the detected antibodies were directed against only one MICA antigen, more than 60% of antibodies showed multiple specificities, such as antibody against MICA*001, *012, and *018, or antibody against MICA*004 and *027. Figure 1 shows an example of absorption/elution experiment to identify possible target amino acids of the multi-specific antibodies. When patient serum ALB77 positive to MICA*001, *012, and *018 was absorbed with a single cell line MICA*018, the eluate reacted to all three beads *001, *012, and *018. This suggests that this serum contains one MICA antibody which reacts to a shared unique amino acid, possibly the threonine (T) at position 24, as illustrated in Figure 2. Conclusions: With the new MICA single antigen Luminex beads, we were able to screen a large number of patient sera and to study MICA cross-reactive patterns that are similar to HLA antibodies. In addition, absorption and elution tests made it possible to identify some of the MICA epitopes. Since MICA antibodies have now been shown to affect transplant outcome, it would be of great value to test for MICA antibodies and their specificities, pre and post transplantation.