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

Abstract

Keywords: diabetes -lipid metabolism -pre-Haptoglobin 2 -ELISA -intestinal permeability 25 -properdin -obesity -metabolic diseases 26 27 Nonstandard abbreviations: HP, haptoglobin; T2D, type 2 diabetes; WHR, waist-to-hip 28 ratio.Abstract 30 BACKGROUND. There is increasing evidence for the role of impaired intestinal 31 permeability in obesity and associated metabolic diseases. Zonulin is an established serum 32 marker for intestinal permeability and identical to pre-haptoglobin2. Here, we aimed to 33 investigate the relationship between circulating zonulin and metabolic traits related to obesity. 34METHODS. Serum zonulin was measured by using a widely used commercial ELISA kit in 35 376 subjects from the metabolically well-characterized cohort of Sorbs from Germany. In 36 addition, haptoglobin genotype was determined in DNA samples from all study subjects. 37 RESULTS.As zonulin concentrations did not correlate to the haptoglobin genotypes, we 38 investigated the specificity of the zonulin ELISA assay using antibody capture experiments, 39 mass spectrometry and Western blot analysis. Using serum samples that gave the highest or 40 lowest ELISA signals, we detected several proteins that are likely to be captured by the 41 antibody in the present kit. However, none of these proteins corresponds to pre-haptoglobin2. 42 We used increasing concentrations of recombinant pre-haptoglobin 2 and complement C3 as 43 one of the representative captured proteins and the ELISA kit did not detect either. Western 44 blot analysis using both the polyclonal antibodies used in this kit and monoclonal antibodies 45 rose against zonulin showed a similar protein recognition pattern but with different intensity 46 of detection. The protein(s) measured using the ELISA kit was (were) significantly increased 47 in patients with diabetes and obesity and correlated strongly with markers of the lipid and 48 glucose metabolism. Combining mass spectrometry and Western blot analysis using the 49 polyclonal antibodies used in the ELISA kit, we identified properdin as another member of 50 the zonulin family. 51 CONCLUSIONS. Our study suggests that the zonulin ELISA does not recognize pre-52 haptoglobin 2, rather structural (and possibly functional) analogue proteins belonging to the 53 mannose-associated serine protease family, with properdin being the most likely possible 54 candidate. 55 56 The "intestinal barrier" is an established term, defined as a functional entity separating the gut 57 lumen from the inner host, and consisting of mechanical, humoral, immunological, muscular 58 and neurological elements. Intestinal barrier dysfunction is a characteristic feature of 59 pathological states such as inflammatory bowel disease, celiac disease, nonalcoholic 60 steatohepatitis and ulcerative colitis (1, 2). There is also emerging evidence for the role of 61 impaired intestinal permeability in metabolic diseases including obesity and type 2 diabetes 62 (T2D) (3-5). It has been hypothesized that gut bacteria and bacterial endotoxins may disrup...

Serum zonulin as a marker of intestinal mucosal barrier function: May not be what it seems

“…Consequently, we are unable to make valid interpretations of our results with respect to our study cohorts (Fig 2). However, we found this assay to have a different capture antibody than the Immundiagnostik assay, which differs from conclusions made by Scheffler et al In both primary and follow-up publications, the authors list a table with studies that use the Immundiagnostik assay with a note that “the kit sold by other companies (e.g., ALPCO) is the same as the Immundiagnostik kit” [20, 21]. Our results do not agree with this claim, as there was poor strength of relationship between both commercial assays (Fig 3) and the immunoprecipitation protein products of both assays differed (Fig 4).…”

“…Dose-responses or signals to high concentrations were not observed; as such, the inability of recombinant zonulin to bind to captured antibodies was confirmed. In support of our ongoing observations, a publication was concurrently released which claimed that the Immundiagnostik assay was not detecting zonulin as advertised, but complement C3 instead [20]. Using an adapted protocol, the captured serum antigens as well as other components from both assays were immunoprecipitated then analysed by mass spectrometry.…”

“…To collect the target antigens of CUSABIO and Immundiagnostik zonulin commercial assays, protocols for the immunoprecipitation of the serum antigen-immobilised antibody complex were developed as previously described with modifications [20]. Undiluted serum samples with high purported zonulin levels, as determined by each respective commercial assay, were selected as antigen sources.…”

Analysis of Faecal Zonulin and Calprotectin Concentrations in Healthy Children During the First Two Years of Life. An Observational Prospective Cohort Study

“…ELISA is commonly used in laboratories to assess the integrity of the intestinal barrier. Scheffler [37] suggested that a serum zonulin (pre-HP2) test (Immundiagnostik ELISA kit) may simultaneously detect a variety of proteins that resemble zonulin structurally and perhaps functionally. This would mean the existence of a zonulin protein family instead of one protein structure regulating intestinal permeability.…”

Perfluoroalkyl Substances are increased in patients with Late-Onset Ulcerative colitis and induce Intestinal Barrier defects ex vivo in Murine Intestinal tissue

“…30 In the current study, we used the Ussing chamber technology to assess barrier function, while the previous report relied on zonulin levels, for which the validity is currently questioned. 31,32 The increased variability seen in the FITC flux values may have contributed to the absence of statistical significance.…”