Immunofluorometric assay for sensitive and specific measurement of human prostatic glandular kallikrein (hK2) in serum.
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.
Piironen
T
T
Department of Biotechnology, University of Turku, Finland. timo.piironen@utu.fi
Lövgren
J
J
Karp
M
M
Eerola
R
R
Lundwall
A
A
Dowell
B
B
Lövgren
T
T
Lilja
H
H
Pettersson
K
K
D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D002850:Chromatography, Gel; D000939:Epitopes / Q000276:immunology; D005260:Female; D015200:Fluoroimmunoassay / Q000379:methods* / Q000706:statistics & numerical data; D006801:Humans; D007610:Kallikreins / Q000032:analysis* / Q000737:chemistry; D008297:Male; D008970:Molecular Weight; D017430:Prostate-Specific Antigen / Q000097:blood; D011994:Recombinant Proteins; D012016:Reference Values; D012680:Sensitivity and Specificity; D020840:Tissue Kallikreins
D000911:Antibodies, Monoclonal; D000939:Epitopes; D011994:Recombinant Proteins; D007610:Kallikreins; D020840:Tissue Kallikreins; D017430:Prostate-Specific Antigen