A sensitive micro-platelet ELISA technique for screening anti-HLA antibodies

Abstract

An enzyme-linked immunosorbant assay (ELISA) in a Terasaki plate (Micro-Platelet ELISA), using 30 000 platelets per well, 2 μL primary antibody (anti HLA antiserum) and 5 μL of secondary antibody (1:2000) are described. Platelets from 30 selected HLA tissue typed cell panel individuals were used to characterize anti HLA A and B antibodies. The first half of the Terasaki tray had platelets in sequence to characterize anti HLA antibodies, while the second half contained anti HLA B antibodies. Results revealed that the HLA specificities of the sera identified by micro-platelet ELISA and microlymphocytotoxicity were concordant. Moreover, split antigens of broader specificities were identified in the Platelet ELISA technique. The advantages of micro-platelet ELISA technique were: (i) it does not require viable/frozen lymphocytes, (ii) reading is very simple and macroscopic, (iii) specificity of the serum is identified with accuracy within the same day, (iv) avoids inter-cell, inter-day variations, (v) complement is not required, (vi) requires only 1/50 volume of the reagents required by conventional ELISA in microtitre plates, and (vii) using platelets isolated from 5 mL of peripheral blood, fifteen thousand sera can be tested. This technique is, thus, very simple, cost effective, and very much suitable for any developing HLA laboratory, which is in the process of developing indigenous HLA reagents.