Different conformational forms of serum carnosinase detected by a newly developed sandwich ELISA for the measurements of carnosinase concentrations

Abstract

Serum carnosinase (CN-1) measurements are at present mainly performed by assessing enzyme activity. This method is time-consuming, not well suited for large series of samples and can be discordant to measurements of CN-1 protein concentrations. To overcome these limitations, we developed sandwich ELISA assays using different anti-CN-1 antibodies, i.e., ATLAS (polyclonal IgG) and RYSK173 (monoclonal IgG1). With the ATLAS-based assay, similar amounts of CN-1 were detected in serum and both EDTA and heparin plasma. The RYSKS173-based assay detected CN-1 in serum in all individuals at significantly lower concentrations compared to the ATLAS-based assay (range: 0.1-1.8 vs. 1-50 μg/ml, RYSK- vs. ATLAS-based, P<0.01). CN-1 detection with the RYSK-based assay was increased in EDTA plasma, albeit at significantly lower concentrations compared to ATLAS. In heparin plasma, CN-1 was also poorly detected with the RYSK-based assay. Addition of DTT to serum increased the detection of CN-1 in the RYSK-based assay almost to the levels found in the ATLAS-based assay. Both ELISA assays were highly reproducible (R: 0.99, P<0.01 and R: 0.93, P<0.01, for the RYSK- and ATLAS-based assays, respectively). Results of the ATLAS-based assay showed a positive correlation with CN-1 activity (R: 0.62, P<0.01), while this was not the case for the RYSK-based assay. However, there was a negative correlation between CN-1 activity and the proportion of CN-1 detected in the RYSK-based assay, i.e., CN-1 detected with the RYSK-based assay/CN-1 detected with the ATLAS-based assay × 100% (Spearman-Rang correlation coefficient: -0.6, P<0.01), suggesting that the RYSK-based assay most likely detects a CN-1 conformation with low CN-1 activity. RYSK173 and ATLAS antibodies reacted similarly in Western blot, irrespective of PNGase treatment. Binding of RYSK173 in serum was not due to differential N-glycosylation as demonstrated by mutant CN-1 cDNA constructs. In conclusion, our study demonstrates a good correlation between enzyme activity and CN-1 protein concentration in ELISA and suggests the presence of different CN-1 conformations in serum. The relevance of these different conformations is still elusive and needs to be addressed in further studies.