An alternative strategy for high throughput generation and characterization of monoclonal antibodies against human plasma proteins using fractionated native proteins as immunogens

Abstract

Construction of a monoclonal antibody (mAb) bank containing a vast variety of antibodies against human tissue proteins is important for proteomic research. A novel strategy of subtractive immunization using fractionated native proteins was developed for high throughput generation of mAb against human plasma proteins. By this novel approach, the bottleneck of antigen preparation can be overcome by combining repeated immunization of animals with subtracted fractions of plasma or tissue proteins and identification of target antigen by immunoprecipitation/mass spectrum strategies. Plasma freshly collected from healthy adults was pooled and three fractions were prepared by size exclusion chromatography. Mice were immunized with the fractionated plasma proteins, and 205 strains of hybridomas secreting mAb were obtained after two-round subtractive immunizations and cell fusions. In the first round, 110 strains of hybridomas were established, in which 77 strains secreting mAb were identified against 10 human plasma high-abundant proteins. In the second round, plasma fraction I was absorbed with mAb against IgM, IgG, ceruloplasmin and haptoglobin. The absorbed fraction I was used as immunogen for the second round immunization and cell fusion. Ninety-five strains of hybridomas secreting mAb were obtained. Although the target antigens of mAb from 82 strains of hybridomas were identified as IgM, IgA, alpha2-macroglobulin and fibrinogen, about 85% antibodies obtained from this round were identified as new antibodies when compared with mAb obtained in the first round immunization with plasma fraction I. The results suggest that subtractive immunization with fractionated plasma proteins followed by identification of antigens with immunoprecipitation/mass spectrum may be an effective approach for rapid preparation of mAb against high-and medium-abundant plasma or tissue proteins.

Mitochondrial Complexes in Trypanosoma brucei

“…We have used similar approaches to isolate and characterize enzymatically active editosomes from T. brucei mitochondria (10,29). Thus, mAbs for an organism of interest from banks that are generated using shotgun immunization with subcellular fractions or subtractive immunization (30) as well as previously incompletely characterized mAbs can be valuable tools for important proteomics studies.…”

High-Throughput Antibody Generation Using Multiplexed Immunization and Immunogen Array Analysis

“…New ways to improve antibody generation such as phage display and multiplexed immunization techniques have attracted attention in recent years as they enable rapid generation of antibody fragments, protein scaffolds, or nucleic acid scaffolds. [4][5][6][7] However, one common problem is that the number of antibody candidates produced is much higher than the traditional single-antigen/single-antibody generation approach. For example, a pilot study was performed by Schofield et al 8 using a phage display technology in which thousands of recombinant antibodies were screened with enzyme-linked immunosorbent assay (ELISA), and sensitive detection was demonstrated using a bead-based system.…”

“…Then the antibody hybridomas were generated by fusing the spleen cells with myeloma partner SP2/0 cells based on standard protocols. 4 …”

High throughput monoclonal antibody generation by immunizing multiple antigens

“…After 10 d, mice were boosted with half amount (5 μg each) of the same antigens, emulsified with Freund's incomplete adjuvant. Three days after boosting, spleen cells of immunized mice were fused with myeloma partner Sp2/0 cells following standard protocols [4]. A portion of the fusion cells from the 10-day immunization were cultured in a liquid medium and the rest were cultured in a semi-solid medium to compare the efficacy of hybridoma generation in each medium.…”

“…Numerous attempts to increase the speed of monoclonal antibody (mAb) generation and throughput have been reported. Ning et al [4] immunized mice with fractioned plasma proteins and identified the corresponding antigens by immunoprecipitation/mass spectrum. Ju et al [5] immunized mice with fractionated mitochondrial proteins and identified the mAb specificity using cDNA expression library screening combined with Mass spectrum.…”