Technology Platform
High-throughput Screening Platform For Murine Monoclonal Antibodies
1. Introduction of Core Technology
SGE BIOTECH has iteratively upgraded the traditional hybridoma screening method, breaking through many limitations of the original technology, and comprehensively improving the throughput and efficiency of antibody screening. Specifically, using the self-developed antibody targeting membrane anchoring technology, the antibody is specifically anchored on the surface of the hybridoma cell that secretes the antibody, and then using antigen fluorescent staining and flow cytometry sorting, the hybridoma that secretes a positive antibody can be Tumor cells are sorted into 96-well plates or 384-well plates to directly obtain antibody monoclonal cells. This operation avoids the cumbersome subcloning work of traditional hybridoma technology, greatly saves time and cost, and at the same time increases the throughput of antibody screening 2~3 orders of magnitude.
In addition, we have developed a potent mouse immune adjuvant that can immunize mice in a short period of time and obtain ultra-high serum titers, resulting in the production of high peaks and diversity of antibody sequences in mice. At the same time, the efficiency of a single mouse fusion to obtain a million-order hybridoma is realized on the electrofusion. Theoretically, through the combination of strong immunity and excellent electrofusion, more than 95% of the antibody sequences that recognize the target antigen in mice can be covered. After the fusion of the number of millions of hybridomas by flow sorting, can be obtained in a short period of time 100 to 500 strains of strong positive antibodies, these antibodies are effectively used for subsequent antibody activity assessment, from which the target biological activity of the antibody molecules.
2. Antibody screening process
3. Technical features
➢ Good animal immunity effectSelf-developed powerful animal immune adjuvant, can effectively protect the antigen structure and significantly improve the serum titer antibody titer;
➢ High screening throughput: Deliverable according to project needs100-500 strainsELISA positive monoclonal hybridoma supernatant for subsequent screening and evaluation;
➢ Short screening cycle: 10 weeks faster delivery of hybridoma supernatant.
Perfect testing and evaluation system: It can complete antibody Elisa detection, FACS detection, affinity detection, antibody epitope grouping analysis, antibody neutralization activity & inhibition activity analysis and other analysis and detection.
4. Comparison of different antibody screening platforms
5. Screening Case 1
➢ Mouse Immunization
The target immunization method of this project is FC fusion recombinant protein as antigen, and mice are immunized four times. The immune adjuvant is PAP-1 powerful immune adjuvant independently developed by SGE BIOTECH. The last immune blood collection test shows that the serum titer is more than 1: 128000, the immune effect meets the requirements, and subsequent fusion is carried out.
➢ Hybridoma fusion and ELISA detection
Take the spleen of 4 mice with high titer, mix and grind and prepare single cells, mix them with SP20 cells, and use an electrofusion instrument to fuse. The fused cells are cultured in HAT medium. After several days of culture, the number of surviving clones is more than 4 million. The surviving hybridoma cells and cells were stained with FACS antigen and sorted into 18 96-well plates for single cells. After one week of culture, ELSIA identification was carried out, about 1300 ELSIA-positive monoclonal hybridomas were obtained.
➢ Blocking ELISA test
Of the 1300 positive hybridoma supernatants screened above, 94 hybridoma supernatants had a strong ability to inhibit the binding of antigens to their own receptors.
6. Screening Case 2
In order to verify the diversity of antibody sequences screened by the platform, the following project is to immunize mice with PAP-1, a powerful immune adjuvant independently developed by SGE BIOTECH. The antibody sequences screened by the high-throughput screening platform have both ELISA binding and FACS binding capabilities, totaling 205 antibodies. Through sequence sequencing and statistical analysis of the length of CDR3 and the frequency of germline use, the antibody diversity developed by the platform is good.
Affinity detection
Affinity detection of some antibodies, the technology platform can screen high affinity murine monoclonal antibodies