scholarly journals Immunofluorometric Point-of-Care Assays for the Detection of Acute Coronary Syndrome-Related Noncomplexed Pregnancy-Associated Plasma Protein A

2006 ◽  
Vol 52 (9) ◽  
pp. 1794-1801 ◽  
Author(s):  
Saara Wittfooth ◽  
Qiu-Ping Qin ◽  
Juha Lund ◽  
Ilkka Tierala ◽  
Kari Pulkki ◽  
...  
Keyword(s):  
Detection Limit ◽  
Plasma Protein ◽  
Specific Antibody ◽  
Point Of Care ◽  
Protein A ◽  
Serum Samples ◽  
Coronary Syndrome ◽  
A Subunit ◽  
The Difference ◽  
Eosinophil Major Basic Protein

Abstract Background: We recently reported that the pregnancy-associated plasma protein A (PAPP-A) form specifically related to acute coronary syndromes (ACS) is not complexed with the proform of eosinophil major basic protein (proMBP). The aim of this study was to develop rapid point-of-care immunoassays for the measurement of the noncomplexed PAPP-A. Methods: We developed immunofluorometric noncompetitive dry-reagent assays for total PAPP-A with 2 PAPP-A subunit-specific monoclonal antibodies and for PAPP-A/proMBP complex with 1 PAPP-A subunit-specific antibody and 1 proMBP subunit-specific antibody. The concentration of noncomplexed PAPP-A was determined as the difference of the results obtained with the 2 assays. Results: The assays were linear from 0.5 to 300 mIU/L. The analytical detection limit and functional detection limit (CV <20%) were 0.18 mIU/L and 0.27 mIU/L for total PAPP-A assay and 0.23 mIU/L and 0.70 mIU/L for PAPP-A/proMBP assay, respectively. The total assay imprecisions were <10%, and recoveries were 88%–107% for both assays. The mean difference (95% limits of agreement) between the new total PAPP-A assay and a previously reported total PAPP-A assay was −3.2% (−45.7% to 39.3%; n = 546; P = 0.0019). In serum samples from 159 non-ACS individuals, median concentrations (interquartile range) were 2.42 (1.14) mIU/L for total PAPP-A, 2.20 (1.18) mIU/L for PAPP-A/proMBP, and 0.18 (0.63) mIU/L for noncomplexed PAPP-A. Total PAPP-A and PAPP-A/proMBP, but not noncomplexed PAPP-A, correlated with age (r = 0.290, P = 0.0002; r = 0.230, P = 0.0035; r = 0.075, P = 0.3483, respectively). Conclusions: The new assays described revealed that noncomplexed PAPP-A is found only in negligible amounts in non-ACS samples.

scholarly journals Quantification of Proteolytically Active Pregnancy-Associated Plasma Protein-A with an Assay Based on Quenched Fluorescence

2007 ◽  
Vol 53 (5) ◽  
pp. 947-954 ◽  
Author(s):  
Claus Gyrup ◽  
Michael Christiansen ◽  
Claus Oxvig
Keyword(s):  
Proteolytic Activity ◽  
Plasma Protein ◽  
Basic Protein ◽  
Biological Function ◽  
Limit Of Detection ◽  
Protein A ◽  
Maternal Serum ◽  
Serum Samples ◽  
A Subunit ◽  
Eosinophil Major Basic Protein

Abstract 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, ∼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.

scholarly journals Molecular Distinction of Circulating Pregnancy-Associated Plasma Protein A in Myocardial Infarction and Pregnancy

2005 ◽  
Vol 51 (1) ◽  
pp. 75-83 ◽  
Author(s):  
Qiu-Ping Qin ◽  
Saara Kokkala ◽  
Juha Lund ◽  
Natalia Tamm ◽  
Liisa-Maria Voipio-Pulkki ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Pregnant Women ◽  
Molecular Mass ◽  
Plasma Protein ◽  
Specific Antibody ◽  
Protein A ◽  
Gel Filtration ◽  
Single Peak ◽  
Serum Samples ◽  
A Subunit

Abstract Background: In the blood of pregnant women, pregnancy-associated plasma protein A (PAPP-A) is present as a covalent complex with the proform of eosinophil major basic protein (proMBP). Recently, increased serum concentrations of PAPP-A have been found in acute coronary syndromes (ACS). The aim of this study was to investigate whether the circulating PAPP-A in ACS is the same as that in pregnancy. Methods: We developed two time-resolved immunofluorometric assays based on a relative epitope map constructed by the use of 17 monoclonal antibodies. One assay, which measured total PAPP-A, used two PAPP-A subunit-specific antibodies. The other assay, which measured PAPP-A/proMBP complex, used one proMBP subunit-specific antibody and one PAPP-A subunit-specific antibody. Serum samples from four patients with myocardial infarction (MI), three pregnant women in their first trimester, and one in her third trimester were fractionated by gel filtration on a Superose™ 6 precision column. The two assays were used to analyze fractions obtained by gel filtration as well as serum samples serially collected from four other MI patients. Results: Pregnancy-related PAPP-A was eluted as a single peak with a molecular mass of ∼700 kDa, whereas ACS-related PAPP-A was also eluted as a single peak but with a molecular mass of ∼530 kDa. Pregnancy-related PAPP-A was detected equally by the two assays, whereas increased ACS-related PAPP-A was detected only by the assay for total PAPP-A. Conclusions: Our results provide the first evidence that circulating ACS-related PAPP-A is different from circulating pregnancy-related PAPP-A in that it is not complexed with proMBP. These findings provide a solid foundation for the design of immunoassays to accurately measure atherosclerosis-associated plasma protein A in the circulation.

scholarly journals Studies on regulation of IGF (insulin-like growth factor)-binding protein (IGFBP) 4 proteolysis by pregnancy-associated plasma protein-A (PAPP-A) in cells treated with phorbol ester

2004 ◽  
Vol 379 (1) ◽  
pp. 57-64 ◽  
Author(s):  
Arun S. SIVANANDAM ◽  
Subburaman MOHAN ◽  
Hirohito KITA ◽  
Sanjay KAPUR ◽  
Shin-Tai CHEN ◽  
...  
Keyword(s):  
Growth Factor ◽  
Proteolytic Activity ◽  
Plasma Protein ◽  
Binding Protein ◽  
Protein A ◽  
Insulin Like Growth Factor ◽  
Factor Binding ◽  
Fold Reduction ◽  
Sds Page ◽  
Eosinophil Major Basic Protein

PAPP-A (pregnancy-associated plasma protein-A) is produced by hSFs (human skin fibroblasts) and hOBs (human osteoblasts) and enhances the mitogenic activity of IGFs (insulin-like growth factors) by degradation of IGFBP-4 (insulin-like growth factor-binding protein 4). PKC (protein kinase C) activation in these cells led to reduction in IGFBP-4 proteolysis. This study was undertaken to determine the mechanism by which activation of PKC suppresses IGFBP-4 proteolysis. Treatment of hSFs/hOBs with TPA (PMA; 100 nM) reduced IGFBP-4 proteolysis without significantly decreasing the PAPP-A level in the CM (conditioned medium). Immunodepletion of the proform of eosinophil major basic protein (proMBP), a known PAPP-A inhibitor, from CM of TPA-treated cells (TPA CM) failed to increase IGFBP-4 proteolytic activity. Transduction of hSFs with proMBP retrovirus increased the concentration of proMBP up to 30 ng/ml and led to a moderate reduction in IGFBP-4 proteolysis. In contrast, TPA treatment blocked IGFBP-4 proteolysis but failed to induce a detectable amount of proMBP in the CM. While proMBP overexpression led to the formation of a covalent proMBP–PAPP-A complex and reduced the migration of PAPP-A on SDS/PAGE, TPA treatment dose- and time-dependently increased the conversion of a ≈470 kDa PAPP-A form (PAPP-A470) to a ≈400 kDa PAPP-A form (PAPP-A400). Since unreduced PAPP-A400 co-migrated with the 400 kDa recombinant PAPP-A homodimer and since PAPP-A monomers from reduced PAPP-A470 and PAPP-A400 co-migrated on SDS/PAGE, conversion of PAPP-A470 to PAPP-A400 is unlikely to be caused by proteolytic cleavage of PAPP-A. Consistent with the data showing that the increase in the ratio of PAPP-A400/PAPP-A470 is correlated with the extent of reduction in IGFBP-4 proteolysis, partially purified PAPP-A400 exhibited a 4-fold reduction in IGFBP-4 proteolytic activity compared with PAPP-A470. These data suggest that a novel mechanism, namely conversion of PAPP-A470 to the less-active PAPP-A400, could account for the TPA-induced suppression of PAPP-A activity.

scholarly journals e0440 Pregnancy-associated plasma protein-a polymorphisms and the risks of acute coronary syndrome

Heart ◽  
2010 ◽  
Vol 96 (Suppl 3) ◽  
pp. A137-A137
Author(s):  
C. Tian ◽  
W. Qin ◽  
W. Zheng ◽  
T. Liu
Keyword(s):  
Acute Coronary Syndrome ◽  
Plasma Protein ◽  
Protein A ◽  
Coronary Syndrome

scholarly journals Pregnancy-Associated Plasma Protein A Induces Inflammatory Cytokine Expression by Activating IGF-I/PI3K/Akt Pathways

2019 ◽  
Vol 2019 ◽  
pp. 1-12 ◽  
Author(s):  
Weiping Li ◽  
Hongwei Li ◽  
Li Zhou ◽  
Zijian Wang ◽  
Bing Hua
Keyword(s):  
Signaling Pathway ◽  
Inflammatory Cytokines ◽  
Plasma Protein ◽  
Inflammatory Cytokine ◽  
Protein A ◽  
Dependent Manner ◽  
Coronary Syndrome ◽  
Raw264.7 Macrophages ◽  
Igf I

Pregnancy-associated plasma protein A (PAPP-A) was previously reported to be an inflammatory biomarker and a prognostic marker of acute coronary syndrome (ACS) and involved in the process of atherosclerosis and plaque rupture. However, the role of PAPP-A in inflammation is poorly understood. In this study, we aimed to investigate the role of PAPP-A in macrophage activation and inflammatory cytokine production. RAW264.7 macrophages were treated with or without PAPP-A. Reverse-transcriptase quantitative real-time PCR (RT-qPCR) and Western blot were performed to detect gene and protein expressions. The concentration of monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) in culture supernatants was determined by ELISA. Results showed that PAPP-A significantly stimulated the expression of MCP-1, TNF-α, and IL-6 at both transcriptional and translational levels in a dose-dependent and time-dependent manner. The secretion of these inflammatory cytokines by macrophages was also increased after PAPP-A treatment. Moreover, PAPP-A activated the IGF-I/PI3K/Akt signaling pathway in macrophages. The PAPP-A-mediated upregulation of MCP-1, TNF-α, and IL-6 mRNA and protein levels were strongly inhibited by PI3K inhibitors or IGF-IR siRNA, indicating that the upregulation of MCP-1, TNF-α, and IL-6 could involve the IGF-I/PI3K/Akt pathway. Together, this study demonstrates that PAPP-A activates the macrophage signaling pathway (IGF-I/PI3K/Akt), which drives the expression and production of inflammatory cytokines known to contribute to the initiation and progression of ACS. These findings indicate that PAPP-A may play a proinflammatory role in the pathophysiology of ACS and serve as a potential therapeutic target.

Radioimmunoassay for pregnancy-associated plasma protein A.

1982 ◽  
Vol 28 (1) ◽  
pp. 50-53 ◽  
Author(s):  
M J Sinosich ◽  
B Teisner ◽  
J Folkersen ◽  
D M Saunders ◽  
J G Grudzinskas
Keyword(s):  
Detection Limit ◽  
Amniotic Fluid ◽  
Plasma Protein ◽  
Early Pregnancy ◽  
Twin Pregnancy ◽  
Protein A ◽  
Trophoblastic Disease ◽  
Coefficients Of Variation ◽  
Highly Sensitive

Abstract A specific and highly sensitive radioimmunoassay for determination of pregnancy-associated plasma protein A in human serum is described. The minimum detection limit for this protein was 2.9 micrograms/L. The within- and between-assay coefficients of variation were 4.0 and 4.5%, respectively. The circulating protein was detected within 32 days of conception in eight normal pregnancies and within 21 days in a twin pregnancy. Circulating concentrations in the mother at term were consistently higher (10-fold) than in matched amniotic fluid; none was detected in the umbilical circulation. This protein was also detected in the circulation of patients with hydatiform mole. This assay will permit investigations into the clinical evaluation of measurements of the protein during early pregnancy and trophoblastic disease.

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