Quantification of proteolytically active pregnancy-associated plasma protein-A with an assay based on quenched fluorescence.
BACKGROUND
Maternal serum concentrations of pregnancy-associated plasma protein-A (PAPP-A, pappalysin-1, EC 3.4.24.79) are used to predict the occurrence of Down syndrome. In pregnancy, PAPP-A primarily circulates as a covalent 2:2 complex with the proform of eosinophil major basic protein (proMBP), which inhibits the proteolytic activity of PAPP-A. At term, however, approximately 1% of PAPP-A exists as an active, uncomplexed dimer with proteolytic activity directed specifically toward insulin-like growth factor binding protein (IGFBP)-4 and IGFBP-5. No assays have been developed that allow quantification of PAPP-A proteolytic activity.


METHODS
We developed a sensitive and specific immunocapture assay for PAPP-A activity based on intramolecular quenched fluorescence. We used a 26-residue synthetic peptide derived from IGFBP-4 in which specific positions on each side of the PAPP-A cleavage site were substituted with 3-nitrotyrosine and o-aminobenzoic acid.


RESULTS
The assay detected the activity of recombinant PAPP-A as well as PAPP-A in serum samples from pregnant women. The limit of detection (mean signal of blank plus 3 SD) of the active PAPP-A subunit was 13 pmol/L, and the intra- and interassay CVs were <10% and <15%, respectively. Interestingly, the fraction of active PAPP-A decreased gradually from week 7 to week 19 of pregnancy.


CONCLUSIONS
This method allows the measurement of PAPP-A enzymatic activity and because of its specificity it is relevant to the study of the biological function of PAPP-A. The method may also be useful in the diagnosis of pregnancy disorders.
Gyrup
Claus
C
Department of Molecular Biology, University of Aarhus, Aarhus C, Denmark.
Christiansen
Michael
M
Oxvig
Claus
C
D000911:Antibodies, Monoclonal; D005260:Female; D005453:Fluorescence; D006801:Humans; D006868:Hydrolysis; D007118:Immunoassay; D018974:Insulin-Like Growth Factor Binding Protein 4 / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D010455:Peptides / Q000737:chemistry; D011247:Pregnancy; D011261:Pregnancy Trimester, First; D011262:Pregnancy Trimester, Second; D011266:Pregnancy-Associated Plasma Protein-A / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D011296:Prenatal Diagnosis / Q000379:methods*; D021122:Protein Subunits / Q000032:analysis; D011994:Recombinant Proteins / Q000032:analysis; D012680:Sensitivity and Specificity; D014443:Tyrosine / Q000031:analogs & derivatives / Q000737:chemistry; D062367:ortho-Aminobenzoates / Q000737:chemistry
D000911:Antibodies, Monoclonal; D018974:Insulin-Like Growth Factor Binding Protein 4; D010455:Peptides; D021122:Protein Subunits; D011994:Recombinant Proteins; D062367:ortho-Aminobenzoates; C031385:anthranilic acid; C002744:3-nitrotyrosine; D014443:Tyrosine; D011266:Pregnancy-Associated Plasma Protein-A