Analysis of TCRAV and TCRBV Repertoires in Healthy Individuals by Microplate Hybridization Assay

ABSTRACT: We have developed an adaptor ligation PCR-based microplate hybridization assay (MHA) for analysis of T cell receptor α chain variable region (TCRAV) and T cell receptor β chain variable region (TCRBV) repertoires. Forty three TCRAV and thirty eight TCRBV-specific probes were immobilized onto microplate wells in water-soluble carbodiimide. After hybridization of 5′-biotinylated PCR products, quantitative ELISA was carried out and followed by automated colorimetric reading. The conditions for immobilization and hybridization were optimized using representative TCRBV-specific probes. The sensitivity of MHA allows us to detect as low as 40 pg of biotinylated PCR products. The frequencies of individual V segments obtained by MHA were consistent with those obtained by FACS analysis and reverse dot blot assays. Analysis of the entire TCRAV and TCRBV repertoires could be done using a single 96-well plate, and completed in less than 6 h. Simplicity and reproducibility of this method make it suitable for routine laboratory use.