Development and Evaluation of an ELISA for Human Trefoil Factor 3

Abstract

Background: The three trefoil factors (TFF1, TFF2, and TFF3) are small peptides believed to cross-link mucous glycoproteins and to play a role in the maintenance and repair of the gastrointestinal mucosa. To define the physiologic and potential diagnostic values of TFF3, assays able to measure TFF3 are warranted. Methods: An ELISA was developed that uses two antibodies from rabbits immunized with recombinant human TFF3 and a calibrator (3–100 pmol/L) prepared from recombinant human TFF3. Results: The ELISA had a detection limit of 3.0 pmol/L. The imprecision (CV) was 5–9% for mean concentrations of 13–65 pmol/L, corresponding to serum concentrations of 65–330 pmol/L. There was no cross-reaction toward human TFF1 and TFF2 (40 nmol/L). Neither food intake nor the menstrual cycle influenced the values of TFF3 significantly. The central 95% reference interval for TFF3 in serum from healthy blood donors (n = 300) was 91–250 pmol/L and showed no variation with age and limited variation with sex. TFF3 was increased in serum from patients (n = 12) with inflammation and/or ulceration of the upper gastrointestinal tract (P <0.05), whereas in serial measurements of serum from three patients with severe exacerbation of chronic inflammatory bowel disease restricted to the colon, normal concentrations and only minor variations during treatment and tapering were observed. Conclusions: The ELISA measures TFF3 in human serum and represents a specific and precise method for measurement of TFF3, which will be of value for further studies of TFF3 in health and disease.

Trefoil factor peptides in serum and sputum from subjects with asthma and COPD

“…The results for the control group were in accord with our previously published works . TFF1 was the most abundant of the peptides in serum.…”

“…We found that subjects with COPD had highly significant increases in both sputum levels and serum levels of TFF1 and TFF3 and increased level of TFF2 in serum. Compared with similar investigations related to diseases in the GI tract, in which serum TFF levels in diseased groups increased by less than 50% , the increases in the COPD group were substantially higher both in serum and in sputum. No changes in TFFs were detected in the group of patients with asthma.…”

“…Their expression is generally increased in diseases with mucosal inflammation, which has been demonstrated mainly in the GI where the expression levels of all three peptides were up‐regulated locally in the diseased mucosa; this local up‐regulation was reflected in increased levels of the peptides in serum . In healthy individuals, TFF1 and TFF2 are mainly expressed in the stomach and duodenum, whereas TFF3 is the peptide that is most universally expressed in the body; however, TFF1 is the most abundant of the peptides in serum .…”

Trefoil factor peptide 3 is positively correlated with the viscoelastic properties of the cervical mucus plug

“…Extractions were kept at À80°C until analysis. The TFF1, TFF2, and TFF3 concentrations were assessed on the 1:100 extractions after a further 100-fold dilution employing in-house enzyme-linked immunosorbent assay methods (19,20). Furthermore, hemoglobin concentrations in the extracts were determined using the routine method of the laboratory (Cobas6000, Roche A/S Hvidovre, Copenhagen, Denmark; http://www.roche.dk/home/produkter.html).…”

“…The Trefoil factor peptides (TFFs) constitute a family of small (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22) kDa) molecules containing one (TFF1 and TFF3) or two (TFF2) trefoil domains (7). TFFs are most abundantly expressed in the gastrointestinal tract, yet they can be detected in virtually all tissues containing mucin-secreting cells including the endocervix and the uterus (7,8).…”