Hybridoma Cell Screening Overview

In the production of monoclonal antibodies using hybridoma technology, there are two hybridoma screenings in order to obtain function-guaranteed antibodies and precision antibody replication. The first screen is for the selection of hybridoma cells, which is the selective culture of hybridoma cells, and the second screen is for the further selection of hybridoma cells producing specific antibodies, which is the cloning of screened positive clones.

The First Screening

In the process of B cell fusion with tumor cells, there are many types of cells, including unfused B cells, unfused tumor cells, "B cell-B cell" fusion cells, "tumor cell-tumor cell" fusion cells, "B cell-tumor cell" fusion cells (hybridoma cells), and "B cell-multiple tumor cells" fusion cells. Therefore, it is necessary to accurately screen and culture the "B cell-tumor cell" type fusion cells.

At present, the most widely used method for screening "B cell-tumor cell" type fusion cells is to use HAT as a selective medium for screening. The components of the HAT medium are hypoxanthine, aminopterin, and thymidine. Hypoxanthine acts to complete the rescue pathway of hypoxanthine nucleotides, and aminopterin is a folate antagonist that blocks the major pathway of DNA synthesis. Thymidine serves as a "nucleotide precursor" for DNA synthesis by the donor cell via the salvage pathway.

Principles of HTA medium selection

Cellular DNA synthesis includes two pathways: primary synthesis and rescue synthesis. Among them, the main synthesis is the synthesis of nucleotides from amino acids and other small molecular compounds and further DNA synthesis from nucleotides. In this process, dihydrofolate reductase is involved as an important coenzyme.

The rescue pathways are hypoxanthine-guanine phosphoribosyl transferase (HGPRT) and thymidine kinase (thymidine kinase). HGPRT and TK are indispensable for the synthesis of nucleotides from hypoxanthine and thymine into DNA.

The fused tumor cells are HGPRT-deficient cell lines selected by the toxic medium. This process involves screening tumor cells with 8-azoguanine, which can participate in the rescue pathway of DNA synthesis, but the resultant DNA cannot perform the functions of normal DNA, so the cells die. The surviving cells are tumor cells that could not synthesize DNA in the rescue pathway. The defective tumor cells are fused with B cells, and the fused cells can synthesize nucleotides through both the primary and rescue pathways.

HTA medium selection results

Unfused B cells, "B cell-B cell" type fusion cells: unable to proliferate indefinitely and died in 5-7 days.
"B cell-multiple tumor cell" type fusion cells: unstable, barely viable.
Myeloma cells and "tumor cell to tumor cell" type fusion cells: the main pathway of DNA synthesis is blocked by aminopterin in the culture medium, and because of their lack of HGPRT, the DNA rescue synthesis pathway is blocked, they could not proliferate in HAT medium and died in 5-7 days.
Hybridoma cells have the ability to the unlimited proliferation of myeloma cells and can use HGPRT in B cells to synthesize H into purine nucleotides and eventually synthesize DNA with T, allowing them to grow and propagate in vitro for a long time.

The Second Screening

In the process of monoclonal antibody production, because of the different specificities of B cells, the hybridoma cells first screened by HAT culture medium also produce different antibodies, including specific antibody-secreting cells, unrelated antibody-secreting cells, and antibody-non-secreting cells. Therefore, the hybridoma cells must be screened a second time to select the hybridoma cells that can produce the specific antibody, and then cloned—that is, the cells in the culture wells are propagated from a single cell to become a monoclonal clone, and their antibody secretion is uniform. After the second screening, we could further validate the obtained hybridoma cell lines by hybridoma sequencing. The methods for screening hybridoma cells secreting specific monoclonal antibodies include direct inoculation, limited dilution, and soft AGAR culture. One of the most commonly used methods is limited dilution.

In the limiting dilution method, hybridoma cells are diluted multiple times and seeded on a porous cell culture plate, so that each well contains no more than one cell, and then they are allowed to proliferate in culture. Then the antibody secreted by the cells in the supernatant of each well is detected. At this time, the cells in the positive wells are not guaranteed to be from a single cell, therefore it is best to proceed with restricted dilution. Generally, it needs to be repeated 3-4 times until the proliferating cells in each well are assured to be monoclonal cells.

More Details on Hybridoma Animal Selection

Function-guaranteed Hybridoma Screening Introduction
Precision Antibody Replication with Hybridoma Introduction
Hybridoma Isotype Identification and Switching Introduction

At present, high-throughput hybridoma screening has been developed, which can achieve hybridoma screening more conveniently and accurately. Creative Biolabs is willing to share the key points and experience of hybridoma cell screening with you to smooth your development project.

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