Aminopterin

Antibody Structure Immunoglobulins (Ig) or antibodies are important components of the immune system. They can specifically bind antigens through antigen-binding fragment(Fab) sites and activate other cells of the immune system through Fc regions to mediate immune responses. They can be classified as polyclonal antibodies — multiple antibodies that recognize multiple epitopes of an antigen — and monoclonal antibodies (mAbs). The latter are highly specific antibodies that recognize only a particular epitope of an antigen. Over the years, monoclonal antibodies have become invaluable for basic research and are widely used in immunohistochemistry, flow cytometry, immunoblotting and related techniques. In addition, in the last 20 years, monoclonal antibodies have become an important part of cancer therapy. Techniques for preparing human monoclonal antibodies Human monoclonal antibodies can be prepared by hybridoma cell techniques and single-cell sorting techniques. Hybridoma technique The hybridoma technique is a well-established method that is often used to prepare monoclonal antibodies. It is still used in many laboratories. The method involves fusing (hybridizing) antibody-producing B cells with myeloma cell lines, which are then cultured in a selective medium in which only the fused cells producing the specific monoclonal antibody can survive. How does it work? B cells are first isolated from the spleens of mice immunized with a specific antigen, and these B cells can generate a specific monoclonal antibody against that antigen, which is enriched to obtain an antigen-specific population of B cells. These B cells are fused (i.e., by chemical or viral-mediated fusion) with a selected myeloma B-cell line that lacks the gene encoding hypoxanthine-guanine-phosphoribosyltransferase (HGPRT) and is unable to produce antibodies on its own. Both types of B cells are plated into a selection medium (HAT medium) containing hypoxanthine, aminopterin, and thymidine. Because myeloma cells are unable to divide in this medium, B cells that can produce specific antibodies die after a few generations of division. In contrast, only hybridoma cells can divide and replicate. After screening for hybridoma clones capable of producing the target monoclonal antibodies, these monoclonal antibodies can be isolated from the culture medium supernatant. Single Cell Sorting Techniques One of the effective methods for preparing human monoclonal antibodies is flow cytometry-based single-cell sorting of B-cells, PCR amplification of Ig genes (both heavy- and light-chain genes included), and subsequent in vitro cloning of antibody expression vectors. How does it work? The strategy for producing human monoclonal antibodies via single B-cell sorting is based on PCR amplification of human IgH, Igκ and Igλ from single-cell cDNA. Heavy and light chains (κ and λ) are obtained by PCR amplification with specific primers for the sense and antisense strands, respectively. After amplification, the PCR products are cloned into specific expression vectors to express the heavy and light chains. The vectors are subsequently transfected into a human cell line to express the human monoclonal antibody, which can be isolated by culture medium supernatant.