Immunofluorometric assay for sensitive and specific measurement of human prostatic glandular kallikrein (hK2) in serum
(1996) In Clinical Chemistry 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)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/3965255
- author
- Piironen, T. ; Lövgren, Janita LU ; Karp, M. ; Eerola, R. ; Lundwall, Åke LU ; Dowell, B. ; Lovgren, T. ; Lilja, Hans LU and Pettersson, K.
- publishing date
- 1996
- 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
- Clinical Chemistry
- volume
- 42
- issue
- 1
- pages
- 41 - 1034
- publisher
- American Association for Clinical Chemistry
- external identifiers
-
- scopus:0029902282
- ISSN
- 0009-9147
- language
- English
- LU publication?
- no
- additional info
- 7
- 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
- 2016-04-04 13:29:12
- date last changed
- 2022-01-30 00:19:07
@article{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 < 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.}}, 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.}}, issn = {{0009-9147}}, 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}}, language = {{eng}}, number = {{1}}, pages = {{41--1034}}, publisher = {{American Association for Clinical Chemistry}}, series = {{Clinical Chemistry}}, title = {{Immunofluorometric assay for sensitive and specific measurement of human prostatic glandular kallikrein (hK2) in serum}}, url = {{http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=8674186}}, volume = {{42}}, year = {{1996}}, }