Transfection antibody screening using transfected

Because the fMet-Leu-Phe receptor is present only at low levels in neutrophils (-12 x 10 15 g of receptor per cell), it has proved difficult to purify and characterise. Researchers have therefore turned to molecular cloning techniques to gain insight into the molecular structure of this receptor. This approach itself has not been easy because, in the absence of an antibody that specifically binds to the receptor, or else without some amino acid sequence data that can be used to synthesise oligonucleotide probes, cDNA libraries cannot be screened to isolate relevant clones. Therefore, experimental systems in which functional fMet-Leu-Phe receptors are expressed on the surfaces of transfected cells have been used. Two main systems have been utilised expression of mRNA injected into Xenopus laevis oocytes and cDNA cloning into the COS-cell expression vector. 

MAbs produced by immunizing with receptor transfectants must be screened by flow cytometry—first against receptor transfectants to select for immunore-active MAbs to the immunogen and then against the parental cell line to eliminate those immunoreactive antibodies recognizing determinants other than the transfected receptor. Mice will make antibodies to cell surface determinants other than the receptor of interest even when a syngeneic mouse cell line is used as the parental line for transfectants. 

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