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Development of an Immunoassay for Human Serum Osteoclastic Tartrate-Resistant Acid Phosphatase^*

MARIUS E. KRAENZLIN, K.-H. WILLIAM LAU, LIAN LIANG, TIMOTHY K. FREEMAN, FREDERICK R. SINGER, JAN STEPAN and DAVID J. BAYLINK

Departments of Medicine and Biochemistry, Loma Linda University (M.E.K., K.-H.W.L, T.K.F., J.S., D.J.B.), and Mineral Metabolism Unit, Jerry L. Pettis Memorial Veterans Hospital (M.E.K., K.-H. W.L.,T.K.F.,J.S., D.J.B.) Loma Linda, California 92357
The Department of Medicine, University Southern California (L.L., F.R.S.) and Orthopedic Hospital (L.L., F.R.S.) Los Angeles, California 90033

Address all correspondence and requests for reprints to: K.-H. William Lau, Ph.D, Mineral Metabolism (151), Jerry L. Pettis Memorial Veterans Hospital, 11201 Benton Street, Loma Linda, California 92357.

A tartrate-resistant acid phosphatase (TrACP), which has been suggested to be very similar to the osteoclastic TrACP, was partially purified from the spleen of a patient with hairy cell leukemia. The purification procedure consisted of carboxymethyl-Sepharose, phosphocellulose, Sephacryl S-200, and phenyl-Sepharose chromatographies. Polyclonal antibodies were generated in guinea pigs with a titer of at least 1:6000. Immunohistochemical staining of fetal rat tibia with the antisera revealed that only the lysosomes of osteoclasts, but not osteoblasts, were stained. An enzyme-linked immunosorbent assay (ELISA) was developed with the antisera. There was no crossreactivity with 1) partially purified acid phosphatases (ACPs) from normal human and beef spleens, 2) ACPs in extracts of human osteoblastic cells, 3) purified bovine bone matrix TrACP, or 4) commercial prostatic ACP. However, extracts of giant cell bone tumors, containing large amounts of bona fide osteoclasts, showed large amounts of cross-reactive material, which diluted in parallel with the partially purified hairy cell leukemic TrACP in the ELISA. Commercial serum band 5b TrACP also displaced in parallel with the partially purified hairy cell leukemic TrACP. Immunoblotting studies revealed that the antiserum, but not nonimmune guinea pig serum, reacted with the homogeneous hairy cell leukemia splenic band 5 TrACPs, which were recently purified by our laboratory. Preliminary application of the ELISA to sera of patients with metabolic bone diseases revealed that normal healthy individuals had measurable amounts of the immunoreactive material, and patients with Paget’s disease or hyperparathyroidism, who should have high bone turnover, had elevated levels of this immunoreactive material in their sera. In contrast, the level of serum osteoclastic TrACP in a patient with an acute lymphatic leukemia was normal. In summary, 1) we have shown that hairy cell leukemia splenic TrACP shares significant immunological similarity with the osteoclastic TrACP and with the serum band 5b TrACP, and 2) the ELISA holds promise for a sensitive and specific assay for bone resorption.

^* This work was supported in part by the Department of Medicine, Loma Linda University, and research grants from the V.A., the Arthritis Foundation, Southern California Chapter, and the Loma Linda University Research Foundation.

Current address: Saint Vincent’s Institute of Medical Research, Fitzray, Victoria 3065, Australia.

Current address: Bone Center, Cedars-Sinai Medical Center, Los Angeles, California 90048.

Current address: Department of Internal Medicine 3, Charles University Faculty of Medicine, U nemocnice 1, CS-12821 Prague 2, Czechoslovakia.

Received December 21, 1989.

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