Widely Used Commercial ELISA Does Not Detect Precursor of Haptoglobin2, but Recognizes Properdin as a Potential Second Member of the Zonulin Family

Abstract

BackgroundThere 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.MethodsSerum 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.ResultsAs 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-haptoglobin2 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.ConclusionOur study suggests that the zonulin ELISA does not recognize pre-haptoglobin2, rather structural (and possibly functional) analog proteins belonging to the mannose-associated serine protease family, with properdin being the most likely possible candidate.

Zonulin in serum as a biomarker fails to identify the IBS, functional dyspepsia and non-coeliac wheat sensitivity

“…One possible explanation may lie in the specificity of the ELISA in serum. Recent studies suggest a high level of cross-reactivity between zonulin and the serum protein properdin, which may impede the assay sensitivity 10. While we did observe a significantly higher mean value of zonulin in IBS and FD versus CD, the low sample size of CD in our study warrants caution when interpreting these findings.…”

Changes in serum zonulin in individuals with morbid obesity after weight-loss interventions: a prospective cohort study

“…The method might measure another protein e.g. properdin, with structural similarities to zonulin [12,33,34]. The current study adds to the evidence that the protein measured by Immundiagnostik ELISA is associated with the diet, gut microbial dysbiosis, diarrhoea and several biomarkers of metabolic disease.…”

Intraindividual Variation in Markers of Intestinal Permeability and Adipose Tissue Inflammation in Healthy Normal-Weight to Obese Adults

“…13). Furthermore, a recent analysis suggests that not only is serum zonulin concentration highly variable from day to day and even within a single day (23), but a widely used, commercially available ELISA kit from Immundiagnostik (supplied by ALPCO) does not actually detect the zonulin protein alone, but rather several structurally similar proteins (24). All studies, including our own, which utilized this assay to measure plasma zonulin, must now be interpreted with this in mind.…”