Optimisation of sandwich ELISA based on monoclonal antibodies for the specific measurement of pregnancy-associated plasma protein (PAPP-A) in acute coronary syndrome.
OBJECTIVE
PAPP-A has become the principal biochemical serum marker in first trimester screening for Down syndrome, the original data being based on results of a radioimmunoassay (RIA). Recent observations using sandwich ELISA technology have proposed PAPP-A as a potential marker in patients with acute coronary syndrome (ACS). The aims of the present study were to demonstrate (i) the importance of antibody specificity, (ii) the potential pitfalls in changing assay technology, (iii) the importance of strict definition of technology, and (iv) the application of a well-defined assay technology on sera from patients with ACS.


METHODS
Candidate monoclonal antibodies (Mab) were identified by immunohistochemistry, Western blot and the absence of positive signals (ELISA) with normal, non-pregnant serum as antigen source. The ELISA technology was standardized against the original PAPP-A RIA and the WHO reference preparation (WHO 78/610). Results different from those obtained by the original RIA led to ELISA modifications with respect to dilution buffer and enzymatic digestion of the Mab.


RESULTS
The first generation ELISA revealed serum measurements from a pool of non-pregnant (n=103) individuals which, compared to the RIA, seemed to be false positive. The false positive reaction was abolished by addition of bovine serum (BS) to the dilution buffer. Subsequent analysis of individual sera (n=103) indicated that 7/103 were still false positive. This reaction was eliminated by introduction of F(ab')(2)-fragment of the indicator antibody. This modified ELISA revealed that serum PAPP-A levels in ACS were statistically significantly higher than in controls (p<0.001). Moreover, serum PAPP-A in ACS patients with ST-segment elevation (STEMI) were higher (p<0.001) compared to patients without ST-segment elevation (NSTEMI). Immunohistochemical analysis failed to identify PAPP-A in the atherosclerotic plaques.
Rossen
Marie
M
Immunology and Microbiology, University of Southern Denmark, Odense, Denmark.
Iversen
Kasper
K
Teisner
Ane
A
Teisner
Børge
B
Kliem
Anette
A
Grudzinskas
Gedis
G
D000208:Acute Disease; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity / Q000276:immunology; D050197:Atherosclerosis / Q000097:blood / Q000378:metabolism; D015153:Blotting, Western; D003327:Coronary Disease / Q000097:blood* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D011247:Pregnancy; D011266:Pregnancy-Associated Plasma Protein-A / Q000378:metabolism*; D011863:Radioimmunoassay; D013577:Syndrome
D000911:Antibodies, Monoclonal; D011266:Pregnancy-Associated Plasma Protein-A