ABSTRACT
<p><b>OBJECTIVE</b>To prepare monoclonal antibodies (mAbs) against enoyl-CoA hydratase 1 (ECH1).</p><p><b>METHODS</b>Normal human liver tissues were homogenized, and the mitochondria were isolated by differential centrifugation. The total mitochondrial proteins were used to immunize BALB/c mice to prepare mAbs by routine hybridoma technique. The mAbs were characterized by ELISA, Western blotting and immunohistochemistry. The specificity of the antibody was identified by mass spectrometry (MS) following immunoprecipitation (IP) and confirmed by Uni-ZAP expression library screening.</p><p><b>RESULTS</b>One clone of the hybridoma BGB095 secreting specific mAb against ECH1 was obtained. The mAb was identified to belong to Ig subclass IgG1 and could be used in ELISA, Western blotting, immunohistochemistry, and immunoprecipitation.</p><p><b>CONCLUSION</b>A hybridoma cell line stably secreting specific mAb against ECH1 has been established. The specific mAb against ECH1 can be of great value for functional and distribution studies of ECH1.</p>
Subject(s)
Animals , Humans , Mice , Antibodies, Monoclonal , Allergy and Immunology , Antibody Specificity , Blotting, Western , Cell Line , Enoyl-CoA Hydratase , Allergy and Immunology , Enzyme-Linked Immunosorbent Assay , Immunohistochemistry , Liver , Cell Biology , Metabolism , Mice, Inbred BALB C , Mitochondria , MetabolismABSTRACT
The study was aimed to generate monoclonal antibodies (mAbs) against homo sapiens UDP-glucose pyrophosphorylase 2 (UGP2). Normal human liver tissues homogenized, and cytosolic proteins isolated by centrifugation were used to immunize BALB/c mice to generate mAbs by hybridoma technique. The mAbs were identified by ELISA, Western blot, and immunohistochemistry assay. The antibody specificity was confirmed by Uni-ZAP expression library screening. The results indicated that one hybridoma BAD062 secreting specific mAb against UGP2 was established. The Ig subclass of this mAb was IgG(2b) (kappa), and it could be used in ELISA, Western blot, immunohistochemistry assay. The antigen recognized by BAD062 mAb was localized in the hepatocyte cytoplasm, with molecular weight of 56 kD in the cytosolic proteins of human liver tissue. The BAD062 mAb was further confirmed by immunoscreening of Uni-ZAP XR liver cDNA expression library. It is concluded that a hybridoma cell line stably secretes specific mAb against UGP2. This mAb reacted with UGP2 in ELISA, Western blot, immunohistochemistry assay, and would be very useful for the UGP2 studies.