Immunofluorometric assay for sensitive and specific measurement of human prostatic glandular kallikrein (hK2) in serum

• Published in: Clinical chemistry (Baltimore, Md.) Vol. 42; no. 7; pp. 1034 - 1041
• Main Authors: Piironen, T, Lovgren, J, Karp, M, Eerola, R, Lundwall, A, Dowell, B, Lovgren, T, Lilja, H, Pettersson, K
• Format: Journal Article
• Language: English
• Published: Washington, DC Am Assoc Clin Chem 01.07.1996; American Association for Clinical Chemistry
• Subjects: Antibodies, Monoclonal - immunology; Antibody Specificity; Biological and medical sciences; Chromatography, Gel; Epitopes - immunology; Female; Fluoroimmunoassay - methods; Fluoroimmunoassay - statistics & numerical data; Humans; Investigative techniques, diagnostic techniques (general aspects); Kallikreins - analysis; Kallikreins - chemistry; Male; Medical sciences; Molecular Weight; Pathology. Cytology. Biochemistry. Spectrometry. Miscellaneous investigative techniques; Prostate-Specific Antigen - blood; Recombinant Proteins; Reference Values; Sensitivity and Specificity; Tissue Kallikreins; Urinary system; Human; Prostate disease; Serine endopeptidases; Antigenic determinant; Enzyme; Exploration; Tumoral marker; Monoclonal antibody; Immunological method; Prostate specific antigen; Tissue kallikrein; Clinical biology; Hydrolases; Tumor; Recombinant protein; Immunofluorescence; Male genital diseases; Proteinases; Prostate
• ISSN: 0009-9147; 1530-8561
• DOI: 10.1093/clinchem/42.7.1034

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.