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Widely used commercial ELISA does not detect preHP-2, but recognizes properdin as a potential second member of the zonulin family

Lucas Scheffler, Alyce Crane, Henrike Heyne, Anke Tönjes, Dorit Schleinitz, Christian H. Ihling, Michael Stumvoll, Rachel Freire, Maria Fiorentino, Alessio Fasano, Peter Kovacs, John T. Heiker
doi: https://doi.org/10.1101/157578
Lucas Scheffler
1Leipzig University Medical Center, IFB Adiposity Diseases, Leipzig, Germany
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Alyce Crane
1Leipzig University Medical Center, IFB Adiposity Diseases, Leipzig, Germany
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Henrike Heyne
1Leipzig University Medical Center, IFB Adiposity Diseases, Leipzig, Germany
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Anke Tönjes
2Divisions of Endocrinology and Nephrology, University of Leipzig, Leipzig, Germany
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Dorit Schleinitz
1Leipzig University Medical Center, IFB Adiposity Diseases, Leipzig, Germany
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Christian H. Ihling
3Department of Pharmaceutical Chemistry and Bioanalytics, Institute of Pharmacy, Martin-Luther-University Halle-Wittenberg, Halle, Germany
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Michael Stumvoll
2Divisions of Endocrinology and Nephrology, University of Leipzig, Leipzig, Germany
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Rachel Freire
4Mucosal Immunology And Biology Research Center, Massachusetts General Hospital – Harvard Medical School, Boston, MA U.S.A.
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Maria Fiorentino
4Mucosal Immunology And Biology Research Center, Massachusetts General Hospital – Harvard Medical School, Boston, MA U.S.A.
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Alessio Fasano
4Mucosal Immunology And Biology Research Center, Massachusetts General Hospital – Harvard Medical School, Boston, MA U.S.A.
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Peter Kovacs
1Leipzig University Medical Center, IFB Adiposity Diseases, Leipzig, Germany
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  • For correspondence: jheiker@uni-leipzig.de peter.kovacs@medizin.uni-leipzig.de
John T. Heiker
5Institute of Biochemistry, Faculty of Biosciences, Pharmacy and Psychology, University of Leipzig, Leipzig, Germany
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  • For correspondence: jheiker@uni-leipzig.de peter.kovacs@medizin.uni-leipzig.de
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Abstract

BACKGROUND There is increasing evidence for the role of impaired intestinal permeability in obesity and associated metabolic diseases. Zonulin is an established serum marker for intestinal permeability and identical to pre-haptoglobin2. Here, we aimed to investigate the relationship between circulating zonulin and metabolic traits related to obesity.

METHODS Serum zonulin was measured by using a widely used commercial ELISA kit in 376 subjects from the metabolically well-characterized cohort of Sorbs from Germany. In addition, haptoglobin genotype was determined in DNA samples from all study subjects.

RESULTS As zonulin concentrations did not correlate to the haptoglobin genotypes, we investigated the specificity of the zonulin ELISA assay using antibody capture experiments, mass spectrometry and Western blot analysis. Using serum samples that gave the highest or lowest ELISA signals, we detected several proteins that are likely to be captured by the antibody in the present kit. However, none of these proteins corresponds to pre-haptoglobin2. We used increasing concentrations of recombinant pre-haptoglobin 2 and complement C3 as one of the representative captured proteins and the ELISA kit did not detect either. Western blot analysis using both the polyclonal antibodies used in this kit and monoclonal antibodies rose against zonulin showed a similar protein recognition pattern but with different intensity of detection. The protein(s) measured using the ELISA kit was (were) significantly increased in patients with diabetes and obesity and correlated strongly with markers of the lipid and glucose metabolism. Combining mass spectrometry and Western blot analysis using the polyclonal antibodies used in the ELISA kit, we identified properdin as another member of the zonulin family.

CONCLUSIONS Our study suggests that the zonulin ELISA does not recognize pre-haptoglobin 2, rather structural (and possibly functional) analogue proteins belonging to the mannose-associated serine protease family, with properdin being the most likely possible candidate.

Footnotes

  • Nonstandard abbreviations: HP, haptoglobin; T2D, type 2 diabetes; WHR, waist-to-hip ratio.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-ND 4.0 International license.
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Posted January 25, 2018.
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Widely used commercial ELISA does not detect preHP-2, but recognizes properdin as a potential second member of the zonulin family
Lucas Scheffler, Alyce Crane, Henrike Heyne, Anke Tönjes, Dorit Schleinitz, Christian H. Ihling, Michael Stumvoll, Rachel Freire, Maria Fiorentino, Alessio Fasano, Peter Kovacs, John T. Heiker
bioRxiv 157578; doi: https://doi.org/10.1101/157578
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Widely used commercial ELISA does not detect preHP-2, but recognizes properdin as a potential second member of the zonulin family
Lucas Scheffler, Alyce Crane, Henrike Heyne, Anke Tönjes, Dorit Schleinitz, Christian H. Ihling, Michael Stumvoll, Rachel Freire, Maria Fiorentino, Alessio Fasano, Peter Kovacs, John T. Heiker
bioRxiv 157578; doi: https://doi.org/10.1101/157578

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