P088 Multidimensional identification of anti-HLA antibodies

Abstract

Aim Although single-antigen-bead Luminex technology has revolutionised the detection of anti-HLA antibodies, it is still difficult to characterise with precision the specificity of these antibodies. In order to improve this characterisation we have tried to the expose the various properties of different antibodies by using different methods with the hope that they could be told apart from each other. Methods Sera with known presence of anti-HLA antibodies were analysed using two different methods: (A) the standard One Lambda single-antigen bead protocol, and (B) a modification of this protocol by Dr. Robert Liwski (Dalhousie University, Halifax, Nova Scotia). Each bead was assigned two values, one by each method, creating a 2-dimensional vector space in which each bead is represented by a vector. The results of each sample were represented in a vector space. It would be possible to use more than two methods to create a higher order multidimensional vector space. Results The 2-dimensional vector space for each sample revealed distinct clusters of reactivity, which could not be separated by any single method by itself. These clusters of reactivity closely corresponded to known epitopes. For example, the DRB1 specificities defined by {181}:(M), {13}:(R) and {57–58}:(AA) in one sample were clearly distinct as separate clusters, whereas in any single method they were all clustered together. Actually the corresponding DRB1 alleles with the {181}:(M) isoform (DRB1 ∗ 07, ∗ 09, ∗ 10) formed a separate cluster from the DRB5 alleles, suggesting, contrary to current belief, that there are actually two distinct antibodies at work here. Conclusions – Current methods of antibody detection and identification are not precise in regard to measurements of levels of reactivity. – We should not be looking at reactivity levels, but at specificity patterns and reactivity clusters. – These reactivity clusters are better revealed by using different analytical methods simultaneously.