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Purification of placental alkaline phosphatase and its monoclonal antibody
Monoclonal antibodies (MAbs) to placental alkaline phosphatase (PAlP) were raised by using the hybridoma technique. Spleen cells from immunized mice were fused with the mouse myeloma line NS-1 using 50% polyethylene glycol and cultured in a selection medium. Antibodies were screened by the enzyme immunoassay using immobilized solid-phase antigen and anti-mouse immunogloblin Fab' (rabbit)-beta-D-galactosidase complex. Four double-cloned hybridomas were obtained. The cross-reactivity with different kinds of alkaline phosphatases of the raised MAbs was examined. At first, MAb 11-D-10, with which placenta was stained but liver and small intestine were not stained in indirect immunofluorescence, was selected. Then, the cross-reactivity of MAb 11-D-10 was further investigated by immunoblotting (Western blotting). MAb 11-D-10 reacted with PAlP but did not react with hepatic and intestinal alkaline phosphatase at all. The binding activity of 125I-labeled MAb 11-D-10 with different choriocarcinoma cell lines (SCH, BeWo, NaUCC-1, NaUCC-2, and NaUCC-3) was highest in SCH, then in the order of BeWo, NaUCC-3, NaUCC-1 and NaUCC-2, and correlated to the PAlP content of the cells (SCH greater than BeWo greater than NaUCC-3 greater than NaUCC-1 greater than NaUCC-2). Also the intensity of indirect immunofluorescence of the above cell lines with MAb 11-D-10 correlated with the PAlP content of each cell.
Monoclonal antibodies (MAbs) to placental alkaline phosphatase (PAlP) were raised by using the hybridoma technique. Spleen cells from immunized mice were fused with the mouse myeloma line NS-1 using 50% polyethylene glycol and cultured in a selection medium. Antibodies were screened by the enzyme immunoassay using immobilized solid-phase antigen and anti-mouse immunogloblin Fab' (rabbit)-beta-D-galactosidase complex. Four double-cloned hybridomas were obtained. The cross-reactivity with different kinds of alkaline phosphatases of the raised MAbs was examined. At first, MAb 11-D-10, with which placenta was stained but liver and small intestine were not stained in indirect immunofluorescence, was selected. Then, the cross-reactivity of MAb 11-D-10 was further investigated by immunoblotting (Western blotting). MAb 11-D-10 reacted with PAlP but did not react with hepatic and intestinal alkaline phosphatase at all. The binding activity of 125I-labeled MAb 11-D-10 with different choriocarcinoma cell lines (SCH, BeWo, NaUCC-1, NaUCC-2, and NaUCC-3) was highest in SCH, then in the order of BeWo, NaUCC-3, NaUCC-1 and NaUCC-2, and correlated to the PAlP content of the cells (SCH greater than BeWo greater than NaUCC-3 greater than NaUCC-1 greater than NaUCC-2). Also the intensity of indirect immunofluorescence of the above cell lines with MAb 11-D-10 correlated with the PAlP content of each cell.