Immunofluorometric assay for sensitive and specific measurement of human prostatic glandular kallikrein (hK2) in serum.
Immunofluorometric assay for sensitive and specific measurement of human prostatic glandular kallikrein (hK2) in serum.
复制标题
免疫荧光测定法用于灵敏、特异地测量血清中人前列腺激肽释放酶 (hK2)。
作者：
T. Piironen;J. Lövgren;M. Karp;Riitta Eerola;Å. Lundwall;B. Dowell;T. Lövgren;H. Lilja;K. Pettersson
前列腺特异性抗原（PSA）与人前列腺激肽释放酶（hK 2）的一级结构有79%的同源性。当我们使用重组hK 2蛋白时，23种单克隆抗PSA IgG（单克隆抗体，MAb）中只有7种与hK 2发生交叉反应，这使得我们能够设计用于特异性检测hK 2的新型免疫荧光MAb-MAb测定法。在第一次孵育中，加入过量的不与hK 2交叉反应的MAb 2 H11，以防止游离和复合PSA在随后的免疫反应中反应。在第二次孵育中，通过与MAb 2 H11重叠的表位与hK 2交叉反应的生物素化MAb H50仅用于将hK 2结合至链霉亲和素包被的微量滴定威尔斯孔。在第三步中，Eu标记的单克隆抗体H117，它与hK 2交叉反应，检测固定化的hK 2。用重组hK 2校准hK 2测定。检测限为0.1 μ g/L，与重组PSA的交叉反应性≤ 0.7%。在来自334名总PSA浓度范围为1至3400微克/L的男性的血清样品中测量hK 2的浓度。大多数样品（57%）的hK 2浓度低于检测限。hK 2相对于总PSA的比例在79%中为0-2%，在14%中为2-5%，在4%中为5-10%，并且在3%的样品中>10%。10个血清样品的凝胶过滤与增加hK 2浓度显示一个单一的hK 2免疫反应峰的表观分子大小约为30 kDa，对应于重组hK 2和游离PSA。
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.