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URL: http://antibodyregistry.org/AB_2861192

Proper Citation: Claus Oxvig, Aarhus University Cat# Conover_mAb-PA, RRID:AB_2861192

Target Antigen: PAPP-A

Host Organism: mouse

Clonality: monoclonal

Comments: "PAPP-A knockout mice [24] were immunized with human PAPP-A complex purified from human pregnancy serum [48]. Initial injections of 50 µg were given subcutaneously in Freund's Complete Adjuvant (MP Biomedical, 0855828). After one month, each mouse was given a booster injection in Freund's Incomplete Adjuvant (MP Biomedical, 0855829). Based on screening of tail blood on plates coated with the antigen, good responders were selected for final boosting following one additional month: For four consecutive days, mice received approximately 160 µg of the antigen intraperitoneally. On the fifth day, the mice were sacrificed and the spleens removed aseptically. Spleen cells were centrifuged, aliquoted and stored in liquid nitrogen. Fusion with SP2/0 cells was performed with one aliquot of the spleen cells. Culture supernatants of picked clones were screened on antigen coated plates, and positive clones were re-cloned. The clones were then screened for recognition of purified human C-terminal PAPP-A fragment, PAPP-A(1133-1547), coated onto plastic, and for lack of recognition of PAPP-A mutant D1484A. The latter PAPP-A mutant was immobilized to the wells by using polyclonal rabbit anti-PAPP-A [48]. Selected clones were cultured for antibody production and purification on protein A Sepharose (GE Healthcare). These clones were finally screened for their ability to inhibit PAPP-A (human and murine) cleavage of IGFBP-4 (see above). A kit (IsoQuick Strips, Sigma Aldrich, cat. no. I9285) was used for isotyping. Sufficient amounts of the mAb 1/41 antibody was produced and purified by a commercial service provider (BioServ UK Ltd., Sheffield, UK)."