Method for enriching rat plasma cells and establishing plasma cell hybridoma

Abstract

The invention discloses a method for enriching rat plasma cells and establishing plasma cell hybridoma, which comprises the following steps: S1, developing a specific anti-rat CD138 monoclonal antibody by taking rat CD138 as a target spot and utilizing a hybridoma technology; s2, carrying out enrichment on the rat plasma cells by using an anti-rat CD138 antibody, wherein the anti-rat CD138 antibody contains EZ-Link NHS-PEG4-biotin; s3, treating the mouse myeloma cells and the rat plasma cells, and performing cell electrofusion on the treated mouse myeloma cells and the treated rat plasma cells; and S4, transferring the fusion cells into an HAT screening culture medium, culturing for 1-2 hours in a CO2 incubator, then paving the fusion cells in a 96-hole cell culture plate, culturing, and screening hybridoma antibodies 7-14 days later. The method provided by the invention has the advantages that the enrichment degree is high, the rat plasma cell hybridoma fusion technology is improved, the hybridoma antibody positive rate is increased by 10-20 times, the fusion efficiency and the bioavailability of the rat plasma cells are greatly improved, the number of plates is low, and the manpower and material resource cost is saved.

Description

technical field [0001] The present invention relates to the development of anti-rat CD138 antibody, the application of rat plasma cell enrichment, the research and development of new drug of rat hybridoma monoclonal antibody and the research and development of new drug of rat monoclonal antibody, in particular to a kind of enrichment of rat plasma cells and establishment of plasma cells Hybridoma method. Background technique [0002] Hybridoma technology was invented by Kohler and Milstein in 1975, for which they won the 1984 Nobel Prize in Physiology and Medicine. This technology uses splenocytes from immunized mice to fuse with myeloma cells to produce hybridoma cells that stably secrete antibodies. Hybridoma monoclonal antibodies are widely used in life science fields such as disease diagnosis, disease treatment and biological applications due to their high specificity and high affinity. Currently, therapeutic antibodies approved by the US Food and Drug Administration (...

AI Technical Summary

Problems solved by technology

However, in the existing hybridoma technology, there are still the following key technical bottlenecks: 1. The number of mixed B cells in rat spleen and lymph nodes is large, while the content of plasma cells that actually secrete antibodies is extremely low, accounting for only 0.1-6%; 2. A large number Unrelated cells are fused with mouse myeloma cells to produce a large number of "ineffective hybridoma" cells; 3. Each fusion requires a large number of mixed B cells, resulting in a large number of traditional fusion plating (at least 80-100 96-well cell culture plates)

Therefore, the high-throughput screening of antibodies requires a lot of work, and the cost of manpower, material resources, time and capital is extremely high; 4. A large number of "invalid hybridomas" are produced, which seriously interferes with the plasma cell hybridomas that actually secrete specific antibodies, greatly increasing the number of new drugs. The challenge of antibody screening and specific antibody production even led to the final failure of the project

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