The Generation and Detection of Murine Antibodies Specific for DNA-Binding Proteins Present in Trophoblast Cell Nuclei Using an Enzyme-Linked Immunosorbent Assay
Lamont, Jeffrey P.
MetadataShow full item record
Monoclonal antibodies are useful tools for the purification and characterization of proteins expressed in specific tissues and cellular organelles. In this study, DNA-binding proteins from nuclear extracts of trophoblast cells are the target immunogens. Gene expression of MHC class II proteins is repressed in trophoblasts and transcription factors that bind their respective gene promoters are believed to be involved. This paper describes the first stages in the development of monoclonal antibodies specific for trophoblast HLA-DR gene promoter binding proteins from trophoblast cells. Several mice were immunized and given boosting injections with proteins at several stages of purification. The proteins appeared in two electrophoretic gel bands. Mouse sera were screened using an enzyme-linked immunosorbent assay (ELISA) developed specifically for this purpose. The ELISA was able to detect antibody amounts ranging from 1 ng to 300 ng. From the first ELISA screen, five mice were chosen to be given subsequent boosting injections and rescreened. One mouse was eventually identified as a possible candidate after testing positive when screened against purified forms of the DNA-binding proteins. The myeloma fusion and hybridoma culture for monoclonal antibody preparation that is to follow is expensive and time-consuming, so it was recommended that this mouse be given further boosting injections before proceeding with the production of monoclonal antibodies.