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

Piironen, T.; Lövgren, Janita LU ; Karp, M.; Eerola, R.; Lundwall, Åke LU ; Dowell, B.; Lovgren, T.; Lilja, Hans LU and Pettersson, K. (1996) In Clin Chem 42(1). p.41-1034
Abstract
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,... (More)
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. (Less)
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author
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Prostate-Specific Antigen/blood, Molecular Weight, Male, Kallikreins/*analysis/chemistry, Humans, Fluoroimmunoassay/*methods/statistics & numerical data, Female, Epitopes/immunology, Comparative Study, Gel, Chromatography, Monoclonal/immunology, Antibodies, Antibody Specificity, Recombinant Proteins, Reference Values, Research Support, Non-U.S. Gov't, Sensitivity and Specificity, Tissue Kallikreins
in
Clin Chem
volume
42
issue
1
pages
41 - 1034
external identifiers
  • Scopus:0029902282
language
English
LU publication?
no
id
7fec8718-8bbd-45e7-ada4-34ee2748587d (old id 3965255)
alternative location
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=8674186
date added to LUP
2013-08-11 14:30:12
date last changed
2016-10-13 04:55:23
@misc{7fec8718-8bbd-45e7-ada4-34ee2748587d,
  abstract     = {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 &lt; 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 &gt;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.},
  author       = {Piironen, T. and Lövgren, Janita and Karp, M. and Eerola, R. and Lundwall, Åke and Dowell, B. and Lovgren, T. and Lilja, Hans and Pettersson, K.},
  keyword      = {Prostate-Specific Antigen/blood,Molecular Weight,Male,Kallikreins/*analysis/chemistry,Humans,Fluoroimmunoassay/*methods/statistics & numerical data,Female,Epitopes/immunology,Comparative Study,Gel,Chromatography,Monoclonal/immunology,Antibodies,Antibody Specificity,Recombinant Proteins,Reference Values,Research Support,Non-U.S. Gov't,Sensitivity and Specificity,Tissue Kallikreins},
  language     = {eng},
  number       = {1},
  pages        = {41--1034},
  series       = {Clin Chem},
  title        = {Immunofluorometric assay for sensitive and specific measurement of human prostatic glandular kallikrein (hK2) in serum},
  volume       = {42},
  year         = {1996},
}