人血清抗酒石酸酸性磷酸酶免疫分析方法的建立。

Kraenzlin M E, Lau K H, Liang L, Freeman T K, Singer F R, Stepan J, Baylink D J

Department of Medicine, Loma Linda University, California.

J Clin Endocrinol Metab. 1990 Aug;71(2):442-51. doi: 10.1210/jcem-71-2-442.

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 cross-reactivity 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.

一种抗酒石酸酸性磷酸酶（TrACP），有人认为它与破骨细胞的TrACP非常相似，已从一名毛细胞白血病患者的脾脏中部分纯化出来。纯化过程包括羧甲基琼脂糖凝胶、磷酸纤维素、Sephacryl S - 200和苯基琼脂糖凝胶色谱法。用豚鼠制备了效价至少为1:6000的多克隆抗体。用抗血清对胎鼠胫骨进行免疫组织化学染色显示，只有破骨细胞的溶酶体被染色，而成骨细胞未被染色。用抗血清开发了一种酶联免疫吸附测定（ELISA）。该抗血清与以下物质无交叉反应：1）来自正常人及牛脾脏的部分纯化酸性磷酸酶（ACP）；2）人成骨细胞提取物中的ACP；3）纯化的牛骨基质TrACP；4）市售前列腺ACP。然而，含有大量真正破骨细胞的巨细胞骨肿瘤提取物显示出大量交叉反应物质，在ELISA中其稀释情况与部分纯化的毛细胞白血病TrACP平行。市售血清条带5b TrACP也与部分纯化的毛细胞白血病TrACP平行取代。免疫印迹研究表明，该抗血清能与我们实验室最近纯化的纯毛细胞白血病脾脏条带5 TrACPs发生反应，而非免疫豚鼠血清则不能。ELISA在代谢性骨病患者血清中的初步应用表明，正常健康个体血清中有可测量的免疫反应性物质，患有佩吉特病或甲状旁腺功能亢进症（这些疾病骨转换率应较高）的患者血清中该免疫反应性物质水平升高。相比之下，一名急性淋巴细胞白血病患者的血清破骨细胞TrACP水平正常。总之，1）我们已表明毛细胞白血病脾脏TrACP与破骨细胞TrACP以及血清条带5b TrACP具有显著的免疫相似性；2）ELISA有望成为一种用于骨吸收的灵敏且特异的检测方法。