The Foundation And Clinical Study Of Antigenicity Of MICA In Transplantation Immunity

Objective: The Recombinant Protein were made using Baculovirus-insect expressionsystem and were prepared to the study of the correlation with antibody-mediated immune.To monitor the anti-MICA antibody in serum of pre-transplant and post-transplant recipientand analyze the significance of de novo anti-MICA specific antibody combined with theMICA typing in allograft dysfunction. To contrast the consensus among three differentspecificity MICA antibody kits in order to predict the clinical application value.Methods:In the first part, use Baculovirus-insect expression system to product the recombinantMICA08protein according MICA gene frequency. Firstly, the transfer vectorpFastBac-MICA*008was transformed into E.coil DH10Bac and obtained theBacmid-MICA*008which was identified by blue-white selection and confirmed by DNAsequencing. Secondly, the Bacmid-MICA*008was transfected into SF9cells byCellfection reagent. Then, the recombinant soluble MICA08protein was expressed andpurified by nickel-affinity agarose, analyzing the protein by Western blot and Coomassieblue staining.In the second part, the Luminex assay was used to detect the anti-MICA antibody of82recipient before transplantation and at1~4years after transplantation. The specificity ofanti-MICA antibody was distinguished according to MICA gene typing, in order to analyzethe relationship between anti-MICA specific antibody with renal function. Compare thestatistical significance of serum creatinine between negative antibody group andanti-MICA antibody group at1~4years after transplantation.In the third part, detect the anti-MICA specific antibody in16serum samples from16thInternational HLA and Immunogenetics workshop using Luminex assay. The resultsgenerated using three different kits were analyzed and the kits were named kit-A,-B and-C.Result: In the first part, the transformed Bacmid-MICA*008was blasted and confirmedsequencing. The P3supernatants were harvested by centrifugation, and the recombinantsoluble MICA08protein was purified by nickel-affinity agarose with a yiele of1.5mg/L.the protein was identified by Western blot and Coomassie blue staining showed a specificband about37kDa, which was consistent with the target protein.In the second part, the study showed the anti-MICA antibodies existed in12.2%ofrecipients before transplantation. There were three recipients in10patients withanti-MICA antibody which had de novo antibody. The anti-MICA specific antibody of theothers were not changed. One recipient’ result were found to change from negative topositive after transplantation. The donor specific antibody was detected in two recipientsafter transplantation. Another two recipients were detected non-donor specific antibody.The value of serum creatinine in recipients with anti-MICA antibody was higher thanrecipients with negative. The statistical significance existed at36~48months aftertransplantation.In the third part, S04sample was negative and the others fifteen samples werepositive. S10sample were attained complete consensus by all the laboratories. TheMICA-G1group (includes MICA01,02,07,12,17and18antibodies) and the MICA-G2group (includes MICA04,06,08/27,09and19antibodies) were ranged according to theMICA public epitopes. Five samples were recognized MICA-G1group specific antibodiesin laboratories using Kit-A and Kit-B, The others five samples recognized MICA-G2group specific antibodies in laboratories using Kit-A and Kit-B. The consensus ofMICA08/27(86.67%,13/15) was the best in ten MICA specific antibodies types using anyone reagent kits. But MICA19(40%,6/15)was worst. The results of thirteen samplesreached80%consensus for laboratories using same Kit-B reagent.Conclutions:1: The Baculovirus-insect expression system can product therecombinant MICA08protein and the nickel-affinity agarose can purify recombinantMICA08protein from P3supernatants.2: The specificity of anti-MICA antibody can bedistinguished by Luminex assay. The donors and patients had mismatch of HLA and MICAalleles were primary causes for creation de novo antibodies. The patients with pre-existingand de novo anti-MICA Antibody was related with allograft dysfunction. The DSAanti-MICA antibody was proven to have a strong factor for allograft rejection.3: There arecomplete consensus to judgment positive or negative result for MICA antibodies, but the MICA specific antibodies analysis maybe have comparatively large differences amonglaboratories. We suggested the laboratory choose wider MICA specific antibodies reagentor more MICA antibody detection kits to test.