Eplet/Epitope analysis of Anti-HLA antibodies can be Used to determine positive MFI cut off of complement binding antibodies

Abstract

Aim Detection of HLA specific antibodies (Ab) plays a critical role in monitoring of transplant recipients (TR). Their concentration, epitope specificity, isotype and ability to bind complement represent the main components of analysis. In this study, we have demonstrated that HLA class 1 epitopes recognized by complement binding (C1q+) Ab differ from those recognized by non-complement binding (C1q−) Ab. Methods Analysis of anti-HLA Ab was performed in 21 TR DTT treated serum using single antigen bead (SAB) Luminex-IgG technology. Complement binding activity of Ab was investigated using Luminex-C1q (final concentration 150.0 μ g/ml) solid phase (One Lambda, Inc) assay. HLAMatchmaker computer algorithm was used for HLA-A/B epitope/eplet analysis. Data analysis was performed using ANOVA statistics. Results Difference between HLA eplet pattern(s) recognized by Ab was detected in 18 sera when HLA specificity (MFI values) of complement binding (C1q+) and non-complement binding (C1q−) Ab were compared. Limited epitope number of Ab reactivity was not observed in 2 sera, and one serum showed uncertain eplet pattern. Representative eplet specific Ab analysis in a single serum is shown in the Table 1. The largest MFI values of C1q negative Ab were used for subsequent statistical analysis. In the representative serum this value was 231. Using this approach we were able to determine the MFI values range (280–500) of positive cut off for Ab demonstrating C1q binding. Conclusions In 86% of sera (18/21) tested HLA epitope/eplet restricted pattern of Ab reactivity was observed. Functional diversity of reactivity patterns of anti-HLA Ab obtained with Luminex-IgG and Luminex-C1q assays has been demonstrated. C1q positive MFI cut off range of 280–500 can be used in order to better identify complement binding Ab in TR serum. Download : Download full-size image