Abstract
An immunosorbent affinity chromatography was applied to the purification step of human placental alkaline phosphatase (P-ALP).
The anti-P-ALP antibody was prepared from a rabbit and coupled with CNBr-Sepharose 4B, which was packed in a mini-column. A crude extract of P-ALP was applied and washed with 0.2M Tris-HCl buffer (pH 8.0) to excrude contaminated substances. Then, the purified P-ALP was eluted from the column with 0.2M Na2CO3 solution (pH 11.4) and examined its enzyme activity.
Sera and body fluids of patients with malignancy containing carcinoplacental alkaline phosphatase (CPALP) were also examined. The existence of CPALP was easily detected as yellow-colored change of the column by adding carbonate-bicarbonate buffer containing p-nitrophenylphosphate as the substrate.
Thus, a micro-affinity chromatography column method for the detection of CPALP is possible.
