Production of anti-D human monoclonal antibody (IgM) using human monoclonal hybridoma technique

Abstract

Background: At a present, the National Blood Centre, Thai Red Cross Society and Japanese Red Cross Society are co-operating in researches about rare blood group reagent production by using human monoclonal hybridoma technique.

Objective: To produce human hybridoma secreting anti-D monoclonal antibody from human B lymphocytes.

Methods: Human B lymphocytes secreting anti-D were isolated from buffy coated or whole blood by ficoll- hypaque and were transformed with Epstein-Barr virus (EBV). EBV-transformed secreting cell lines were fused with human myelomas (JMS-3) by using 50% polyethylene glycol (PEG). Cultured supernatants were tested for their ability to agglutinate Rh (D) antigen red blood cells. In order to select secreting clones, the limiting dilution was performed. After cell proliferation, culture supernatants were tested again for their specificity with panel cells, tested with different Rh (D) phenotypes and antibody titer measurement was done by
testing two flow dilutions that all step must be tested both of room temperature and indirect antiglobulin technique. However, thoroughly serologytesting would be done.

Results: The production 1 stable cell line secreted anti-D (1B97C9) is achieved with antibody titer tested with R1r cells is 1:128. Different Rh (D) phenotypes testing results are 100% reacted with D positive cells and 100% non-reacted with D negative cells in parallel with a present lot monoclonal anti-D.

Conclusion: The cell line secrete anti-D reagent is isolated partial D antigen specificity.