Becker C, Piironen T, Kiviniemi J, Lilja H, Pettersson K
Department of Clinical Chemistry, Lund University, University Hospital Malmö, S-205 02 Malmö, Sweden.
Clin Chem. 2000 Feb;46(2):198-206.
Human glandular kallikrein 2 (hK2) is expressed in the prostate and is present in serum from men with prostate cancer. Specific detection in serum is difficult mainly because of low concentrations and immunological cross-reactivity with prostate-specific antigen (PSA). Our objectives were to design an assay with improved analytical detection and functional sensitivity and nonsignificant cross-reactivity with PSA, and to characterize different immunoreactive forms of hK2.
In the assay, critical PSA epitopes were blocked with four monoclonal antibodies (MAbs) specific for PSA. Subsequently, hK2 was captured using a MAb against hK2 (5% cross-reactivity with PSA), and after washing, hK2 was detected by a europium-labeled MAb with identical affinity for hK2 and PSA.
The analytical detection limit was <10 ng/L, and functional sensitivity was 30 ng/L. Cross-reaction with PSA was <0.01%. Between-assay imprecision was 3.1% for 1600 ng/L hK2 and 4. 8% for 160 ng/L hK2; corresponding values for within-assay precision were 1.9% and 4.5%, respectively. Complexes of hK2-alpha(1)-antichymotrypsin (ACT) were detected in vitro with -6% bias compared with the free form of hK2. Gel filtration of patient samples showed that hK2 correlated in size mainly with free hK2; only 4-19% corresponded to hK2 possibly complexed with ACT or protein C inhibitor.
Our assay had extremely low cross-reactivity with PSA, provided a very low detection limit, and allowed close to equimolar detection of the free and complexed forms of hK2. Moreover, we found that free hK2 is the predominant immunoreactive form of hK2 in serum.
人腺激肽释放酶2（hK2）在前列腺中表达，存在于前列腺癌患者的血清中。血清中的特异性检测困难，主要原因是浓度低以及与前列腺特异性抗原（PSA）存在免疫交叉反应。我们的目标是设计一种分析检测和功能敏感性得到改善且与PSA无显著交叉反应的检测方法，并对hK2的不同免疫反应形式进行表征。
在该检测方法中，用四种针对PSA的单克隆抗体（MAb）阻断关键的PSA表位。随后，使用抗hK2的MAb捕获hK2（与PSA的交叉反应性为5%），洗涤后，用对hK2和PSA具有相同亲和力的铕标记MAb检测hK2。
分析检测限<10 ng/L，功能敏感性为30 ng/L。与PSA的交叉反应<0.01%。对于1600 ng/L的hK2，批间不精密度为3.1%，对于160 ng/L的hK2为4.8%；批内精密度的相应值分别为1.9%和4.5%。体外检测到hK2-α(1)-抗糜蛋白酶（ACT）复合物，与游离形式的hK2相比偏差为-6%。患者样本的凝胶过滤显示，hK2的大小主要与游离hK2相关；只有4%-19%对应于可能与ACT或蛋白C抑制剂复合的hK2。
我们的检测方法与PSA的交叉反应极低，检测限非常低，能够近乎等摩尔地检测游离和复合形式的hK2。此外，我们发现游离hK2是血清中hK2的主要免疫反应形式。
