[Preparation and characterization of non-agglutinating mAbs against membrane antigen on human erythrocytes]

To prepare monoclonal antibodies (mAbs) against membrane antigens on human erythrocytes and characterize their properties.

BALB/c mice were immunized with the membrane antigens of human type O erythrocytes. The splenocytes of the immunized mice were fused with Sp2/0 myeloma cells by hybridoma technique. The antibodies to common antigen on human erythrocytes were screened by hexadimethrine bromide (polybrene) test tube method and then the agglutinating antibodies (complete antibodies) were weeded out by slide hemagglutination assay. The hybridoma cells secreting non-agglutinating antibodies (incomplete antibodies) were cloned by limiting dilution method. The stability of the obtained hybridoma cells and the properties of the mAbs were identified.

One hybridoma cell 2E8 was obtained, which secreted non-agglutinating antibody. The mAb 2E8 belonged to IgG1, could agglutinate H antigen, and had no species cross-agglutination reaction. The titers of culture supernatant and ascites of 2E8 were 1:1,024 and 1:64x10(6), respectively. When the affinity of mAb 2E8 was evaluated by agglutination reaction, erythrocytes began to agglutinate after 7 seconds and the clots exceeded 1 mm(2) in 3 minutes.

The non-agglutinating mAb against H antigen was prepared successfully. The mAb 2E8 has good titer, affinity and specificity, which lays the foundation for preparation of bispecific antibodies (BsAb).