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A Radioimmunoassay for Placental-type Alkaline Phosphatase¹

Department of Medical Oncology, Charing Cross Hospital (Fulham), St. Dunstan's Road, London, W6 8RF, England

Rabbit antiserum was raised against preparations of placental alkaline phosphatase (AP) extracted from human term placentae. The antiserum precipitated AP from the placenta and from the intestine, but it was inactive against AP from bone and liver. The concentration of antiserum required to precipitate intestinal AP was about 10 times higher than that necessary to precipitate placental AP.

Placental AP was labeled with iodide-¹²⁵I and was subjected to electrophoresis on polyacrylamide gel before use. A specific activity of about 100 µCi/µg protein was obtained. The isotopically labeled AP behaved somewhat like the unlabeled enzyme during electrophoresis and when subjected to Sephadex gel filtration. Both preparations were quantitatively and specifically bound by the antiserum when examined on polyacrylamide electrophoresis.

A radioimmunoassay of the solid-phase type was developed with the use of antiserum-coated plastic tubes. The assay was specific for placental-type AP when various phosphatase preparations and sera were tested, and it detected a minimum level of 20 ng protein per ml, which is equivalent to 0.03 enzyme unit/ml.

Sera from patients in the third trimester of pregnancy contained 60 to 320 ng of placental AP per ml, by radioimmunoassay. Radioimmunoassay and enzymatic assays gave comparable estimates of placental AP activity in the serum. Serum samples from approximately 100 patients with various forms of cancer were examined for placental-type AP by radioimmunoassay, by an enzymatic assay for heat-stable AP of comparable sensitivity, or by both methods. Placental-type AP was not detected in any instance.

Previous studies had revealed a placental-type AP in the serum of a patient with a bile duct adenocarcinoma. Both an AP purified from this tumor and the patient's serum gave parallel dose-response slopes to placental AP in the radioimmunoassay.

¹ Supported by grants from the Medical Research Council and the Charing Cross Hospital Clinical Research Sub-Committee.

Received 12/20/71. Accepted 7/28/72.