From IgG monoclonals to IgM-like molecules.

Abstract

One problem in blood group testing is that IgG monoclonal antibodies, in contrast to IgM, do not usually agglutinate erythrocytes. One of the reasons is the high zeta potential induced by the negative charge of the cell surface. During the last few years, we have produced a series of human monoclonal antibodies by the conventional fusion technique directed against antigens of the Rh blood group system. Some of these monoclonals, especially those directed against Rh-subgroups such as the c-antigen, were mainly of the IgG-subtype and unsuitable for agglutination tests. We have therefore tried to establish a molecular biological method to make IgM-like molecules from IgG monoclonals. From the c-antigen specific human hybridoma BS 240 (IgG subtype), we isolated mRNA that was transcribed into cDNA and then amplified by PCR using family specific primers. The heavy and light chain products were cloned into the pHen vector containing a DNA linker fragment, a myc-tag for identification and a His-tag for purification. After transformation in E.coli and phage rescue with helper phage, the culture supernatant was screened for antigen positive recombinant phage antibodies as a first control for specificity using c-antigen positive erythrocytes and anti-M13 antibodies as bridging antibodies (Coombs technique). Erythrocytes being negative for the c-antigen served as a negative control. After changing the culture conditions, soluble single chain fragments (scFv) were obtained from the periplasmatic extract. Specificity was shown using the c-antigen positive and negative erythrocytes and the 9E10 antibody (anti-myc) as a bridging antibody. To obtain IgM-like molecules, DNA coding for the specific scFv was cloned into the vector pSTE containing DNA coding for the monomer of core streptavidin. After expression, purification and refolding of the monomer, the core streptavidin combines to form tetrameric structures, termed scFv::strep, that are able to bind biotin as shown using ELISA plates coated with biotinylated BSA. Binding was detected with 9E10 and a peroxidase conjugated secondary antibody. In the agglutination assay, the construct was able to agglutinate c-antigen positive erythrocytes but not the negative erythrocytes. These experiments show that it is possible to construct IgM-like agglutinating molecules from cells containing secreting IgG antibodies. Experiments employing human antibody libraries instead of hybridoma cell lines are now in progress.