Abstract
Shiga toxin 1 produced by enterohaemorrhagic Escherichia coli is an AB(5) toxin that is involved in the life-threatening haemolytic-uraemic syndrome. The B subunits (Stx1B) are cell-binding subunits. We previously established mouse hybridoma cell line producing IgA and IgG monoclonal antibodies (mAbs) against Stx1B. Here, we cloned cDNAs encoding each of the heavy, light and joining (J) chains from the hybridoma cell lines by means of the 5' rapid amplification of cDNA ends (RACE) PCR method. Upon assignment of the variable regions of the heavy and light chains to known germline sequences, we found substantial somatic hypermutation in the complementarity-determining regions in both the IgA and IgG mAbs. We also established a hybrid-IgG/IgA heavy chain having variable regions of the IgG mAb by means of recombinant PCR methods. Upon transient expression of the hybrid-IgG/IgA heavy, IgG-associated light and J chains in COS-1 cells, the translated dimeric hybrid-IgG/IgA bound to immobilized Stx1B, as revealed on ELISA. The production of dimeric hybrid-IgG/IgA was revealed on immunoblot analysis. The dimeric hybrid-IgG/IgA inhibited the binding of digoxigenin-conjugated Stx1B to natural ligands (CD77) displayed on Burkitt's lymphoma cell line Ramos. These results indicate that the replacement of variable regions resulted in the production of more useful recombinant dimeric IgA against Stx1B.
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