Development of a peptide-based sandwich ELISA for human tissue prokallikrein with no cross-reactivity from mature kallikrein.
Human tissue prokallikrein is the enzymatically inactive zymogen of a serine proteinase involved in the liberation of vasoactive kinin peptides, and it is supposed that an impaired prokallikrein-to-kallikrein conversion is closely related to certain hypertensive and inflammatory disorders. Progress in understanding the biological role of the proenzyme has been limited by the absence of an accurate assay for the kallikrein precursor. We describe a sandwich enzyme-linked immunosorbent assay to measure human tissue prokallikrein using monospecific anti-peptide antibodies raised against propeptide derivatives. This method could detect a minimum concentration of 60 pg/ml prokallikrein and displayed no cross-reactivity or interference with mature tissue kallikrein. The intra- and inter-assay precision varied from 8-15%, respectively, indicating a reasonable reproducibility of the method. The level of prokallikrein was defined in different human urine samples, and the corresponding dilution curves showed good linearity. The mean recovery of added zymogen was 104%. Prokallikrein immunoassay is the first reported tool for the direct and sensitive quantification of the precursor of tissue kallikrein and should facilitate the precise determination of prokallikrein levels in a variety of biological specimen.
Mauer
S
S
Institute for Biochemistry, Darmstadt University of Technology, Germany.
Maidhof
A
A
Kemme
M
M
D000328:Adult; D003429:Cross Reactions; D004792:Enzyme Precursors / Q000276:immunology / Q000652:urine*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D007610:Kallikreins / Q000276:immunology / Q000652:urine*; D010446:Peptide Fragments / Q000276:immunology; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D004792:Enzyme Precursors; D010446:Peptide Fragments; D007610:Kallikreins; C052910:prokallikrein, tissue