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

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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.

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Acknowledgments

This study was supported by grants from the Deutsche Forschungsgemeinschaft (Graduiertenkolleg 880 to E.R. and F.P. and DFG SA 2143/1-1 to S.J.H.) and the Deutsche Diabetes Gesellschaft (Projektförderung 2008 to E.R.).

Conflict of interest

The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. 5th Medical Clinic, Clinical Faculty Mannheim, University of Heidelberg, Theodor-Kutzer-Ufer 1-3, 68167, Mannheim, Germany

    Katja Adelmann, Dirk Frey, Hannes Koeppel, Paula Sternik, Stephanie Hofmann, Bernhard K. Krämer, Benito A. Yard & Sibylle J. Hauske

  2. Department of Pathology, University Medical Centre Mannheim, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany

    Eva Riedl & Frederick Pfister

  3. First Department of Pediatrics, University Hospital Heidelberg, Heidelberg, Germany

    Verena Peters & Claus P. Schmitt

  4. Department of Tumor Virology, German Cancer Research Center, Heidelberg, Germany

    Hans Walter Zentgraf

  5. Department of Internal Medicine, Division of Nephrology, University Medical Center Groningen, Groningen, The Netherlands

    Gerjan Navis, Jacob van den Born & Stephan J. L. Bakker

Authors
  1. Katja Adelmann
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  2. Dirk Frey
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  3. Eva Riedl
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  4. Hannes Koeppel
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  5. Frederick Pfister
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  6. Verena Peters
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  7. Claus P. Schmitt
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  8. Paula Sternik
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  9. Stephanie Hofmann
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  10. Hans Walter Zentgraf
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  11. Gerjan Navis
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  12. Jacob van den Born
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  13. Stephan J. L. Bakker
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  14. Bernhard K. Krämer
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  15. Benito A. Yard
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  16. Sibylle J. Hauske
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Corresponding author

Correspondence to Benito A. Yard.

Additional information

K. Adelmann and D. Frey contributed equally to this work.

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Adelmann, K., Frey, D., Riedl, E. et al. Different conformational forms of serum carnosinase detected by a newly developed sandwich ELISA for the measurements of carnosinase concentrations. Amino Acids 43, 143–151 (2012). https://doi.org/10.1007/s00726-012-1244-8

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  • DOI: https://doi.org/10.1007/s00726-012-1244-8

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