A modified hybridoma technique for production of monoclonal antibodies having desired isotypes (1)
Red blood cells contain high zeta potential on their surface, which prevents them from being agglutinated by short-length IgG mAb. The polymeric IgM molecules are able to span distances between red blood cells in a manner that cannot be achieved by the IgG molecules. IgG mAb has been demonstrated to have higher affinity than IgM.
From the immunized mice, according to surface immunoglobulin expression, B cells expressing IgM and IgG were isolated from the spleen cells using the MACS system. The isolated cell fractions were then fused with myeloma cells for generation of hybridomas. After cultivation in HAT selective medium, 359 wells (37%) of the seeded wells contained hybridomas. All 359 hybridoma culture supernatants were screened for their antibody isotypes. Eighty percentage of the tested hybridomas produced IgM isotype antibody and 11% produced IgG isotype antibody. Four percentage of the tested hybridomas were non-IgG or IgM produc- ing cells and 6% produced both IgM and IgG isotypes. Seven percentage of the generated hybridomas were positive to HbF. Among of these, 5% were IgM and 2% were IgG isotype.
In three independent experiments, using each cell fraction in cell fusion, resulted in percentage yields of 32 ± 9%,35 ± 19% and 29 ± 14%(mean ± SD)of seeded wells contained hybridomas obtained from IgM+, IgG+ and IgM-/IgG- cell populations.
By using IgG+ cells, in three independent experiments, the percent- ages of hybridomas producing IgG isotype antibody ranged from 86 to 11% (41 ± 40%; mean ± SD). By this fusion, more than 50 hybridoma clones producing HbF specific IgG mAbs were obtained. In contrast, when IgM+ cells were used for cell fusion, the majority of the obtained hybridomas produced the IgM isotype antibody (85 ± 7%). From the IgM? cell fusion, a total of more than 50 hybridoma clones producing HbF specific mAbs and having the IgM isotype were obtained.
