Different conformational forms of serum carnosinase detected by a newly developed sandwich ELISA for the measurements of carnosinase concentrationsAdelmann, K., Frey, D., Riedl, E., Koeppel, H., Pfister, F., Peters, V., Schmitt, C. P., Sternik, P., Hofmann, S., Zentgraf, H. W., Navis, G., van den Born, J., Bakker, S. J. L., Kraemer, B. K., Yard, B. A. & Hauske, S. J., Jul-2012, In : Amino Acids. 43, 1, p. 143-151 9 p.
Research output: Contribution to journal › Article › Academic › peer-review
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 mu g/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 x 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.
|Number of pages||9|
|Publication status||Published - Jul-2012|
- Carnosinase, ELISA, Conformation, Antibodies, Plasma, BETA-ALANINE SUPPLEMENTATION, CENTRAL-NERVOUS-SYSTEM, IDENTIFICATION, ABSORPTION, ACTIVATION, SECRETION, FATIGUE, TISSUE, CNDP1, CELLS