Rapid characterization of hybridomas producing monoclonal antibodies against platelet β3 integrin using ELIspot

Abstract

Generally, B-cell responses against human platelet antigens are assessed by the serological detection of specific platelet antibodies, mostly against β3 integrin. However, this approach seems to be of low sensitivity, since platelet autoantibodies against αIIbβ3 are detected in only 50% of all patients with immune thrombocytopenia (ITP). In this study, a novel B-cell ELIspot method was established to characterize the specificity of mouse monoclonal antibodies (moabs) against human β3 integrin. Moabs produced by hybridomas were immobilized on membrane and bound antibodies were visualized as spots using biotinylated recombinant proteins αIIbβ3 or αvβ3 and the enzyme labeled streptavidin-substrate system. Three hybridomas, Gi5, Gi16 and AP3, designated previously as anti-αIIbβ3, anti-αIIb and anti-β3, respectively, were investigated. Hybridoma producing moab against CD177 was used as the negative control. Whereas AP3 reacted with αIIbβ3 and αvβ3, Gi5 only formed spots with αIIbβ3. Titration analysis showed that the number of spots correlated significantly with the number of seeded cells. Approximately 15 antibody producing hybridoma cells could be identified among 103 nonproducing B-cells. Furthermore, superior correlation with the total number of IgG producing cells was obtained. Analysis of the third hybridoma, Gi16 (anti-αIIb), showed only few spots with αIIbβ3, indicating that this hybridoma contained different clones (producer and non-producer). Significant increased number of spots could be identified after re-cloning of these clones by limiting dilution method. Our results demonstrate that this B-cell ELIspot assay can be used for the identification of a small number of hybridoma cells producing moabs against β3 integrin, verification of their monoclonality, productivity and for determining their specificity in the early state of workup steps. In the future, this approach may be useful to define B-cell clones in patients who developed platelet antibodies against different β3-integrins and to differentiate their diversities.