When obtaining a plurality of hybridoma cell lines, it is necessary to pass various identifications in order to know which strain is most suitable. Common methods are:
(I) Ig type determination is usually carried out by anti-mouse Ig and subclass antibodies and monoclonal antibodies in culture medium by two-way agar diffusion method to determine its Ig type or Ig subtype.
(B) Specificity determination The substance associated with the corresponding antigen was subjected to various immunological tests with McAb to identify the specificity of McAb. This is directly related to the reliability of the McAb application.
(C) Antibody titer determination The titer is expressed by the dilution of the peritoneal effusion or culture solution. The higher the dilution, the higher the antibody titer. In the agglutination reaction, the titer of peritoneal effusion can reach more than 50,000; in ELISA, the titer of peritoneal effusion can reach more than 1 million. If the ELISA titer is less than 100,000, it will not reach high sensitivity for diagnostic assays and should be re-prepared.
(IV) epitope measurement by two-way agar diffusion method, two McAbs are mixed, added to the same well, and the corresponding antigen is placed in the well. If two precipitation lines appear, it can be proved that the two are McAbs resistant to different epitopes; In the ELISA double-antibody sandwich method, one McAb was coated with an antibody, and the other was labeled with an enzyme. The addition of an antigen, such as color development, proved that the two were resistant to different epitopes.
(5) Affinity determination The affinity of the antibody is greatest when the antigen and the binding site of the antibody are completely identical. This binding force is expressed as the ratio of the product of the antigen to the antibody concentration to the antigen-antibody complex concentration when the antigen-antibody reaction is balanced. If the affinity is too low, it will seriously affect the sensitivity of the assay.
(VI) Chromosome analysis The number of chromosomes of spleen cells of normal mice: 40, all of which are centromeres; the chromosomes of mouse myeloma cells: 62-68 for SP2/0 cells and 54-64 for NS-1. Most are aneuploidy, with central and sub-central centromeres. The chromosome number of hybridoma cells is close to the sum of the chromosome numbers of the two parental cells, and most of the structure is the centromere extrachromosomal. A small number of marker chromosomes should also appear. Hybridoma cells with a large number of chromosomes and a high concentration secrete high titers of antibodies.
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