Piironen T, Lövgren J, Karp M, Eerola R, Lundwall A, Dowell B, Lövgren T, Lilja H, Pettersson K
Department of Biotechnology, University of Turku, Finland.
Clin Chem. 1996 Jul;42(7):1034-41.
Prostate-specific antigen (PSA) and human prostatic glandular kallikrein (hK2) have 79% identity with the primary structure. When we used recombinant hK2 protein, only 7 of 23 monoclonal anti-PSA IgGs (monoclonal antibodies, MAbs) cross-reacted with hK2, which enabled us to design a novel immunofluorometric MAb-MAb assay for the specific detection of hK2. In the first incubation, an excess of MAb 2H11, which does not cross-react with hK2, is added to prevent both free and complexed PSA from reacting in subsequent immunoreactions. In the second incubation, biotinylated MAb H50, which cross-reacts with hK2 by an epitope overlapping with MAb 2H11, served to bind only hK2 to the microtitration wells coated with streptavidin. In the third step, Eu-labeled MAb H117, which cross-reacts with hK2, detected the immobilized hK2. The hK2 assay was calibrated with recombinant hK2. The detection limit of the assay was 0.1 microgram/L, and the cross-reactivity with recombinant PSA was < or = 0.7%. The concentration of hK2 was measured in serum samples from 334 males with total PSA concentrations ranging from 1 to 3400 microgram/L. Most of the samples (57%) had hK2 concentrations below the detection limit. The proportions of hK2 relative to total PSA were 0-2% in 79%, 2-5% in 14%, 5-10% in 4%, and >10% in 3% of the samples. Gel filtration of 10 serum samples with increased hK2 concentrations showed a single peak of hK2 immunoreactivity with an apparent molecular size of approximately 30 kDa, corresponding to that of recombinant hK2 and free PSA.
前列腺特异性抗原（PSA）与人前列腺腺激肽释放酶（hK2）的一级结构有79%的同源性。当我们使用重组hK2蛋白时，23种单克隆抗PSA IgG（单克隆抗体，MAb）中只有7种与hK2发生交叉反应，这使我们能够设计一种新型免疫荧光MAb-MAb检测法来特异性检测hK2。在第一次温育中，加入过量的不与hK2交叉反应的单克隆抗体2H11，以防止游离和复合的PSA在随后的免疫反应中发生反应。在第二次温育中，生物素化的单克隆抗体H50通过与单克隆抗体2H11重叠的表位与hK2发生交叉反应，仅将hK2结合到包被有链霉亲和素的微量滴定孔中。在第三步中，与hK2发生交叉反应的铕标记单克隆抗体H117检测固定化的hK2。hK2检测法用重组hK2进行校准。该检测法的检测限为0.1微克/升，与重组PSA的交叉反应率≤0.7%。在334名总PSA浓度范围为1至3400微克/升的男性血清样本中测量hK2浓度。大多数样本（57%）的hK2浓度低于检测限。在79%的样本中，hK2相对于总PSA的比例为0-2%，14%的样本为2-5%，4%的样本为5-10%，3%的样本>10%。对10份hK2浓度升高的血清样本进行凝胶过滤，显示hK2免疫反应性的单峰，表观分子大小约为30 kDa，与重组hK2和游离PSA的分子大小一致。
