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Clinical Chemistry 48: 1689-1695, 2002;
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(Clinical Chemistry. 2002;48:1689-1695.)
© 2002 American Association for Clinical Chemistry, Inc.

Development and Evaluation of an ELISA for Human Trefoil Factor 3

Else Marie Vestergaard1a, Steen Seier Poulsen4, Henning Grønbæk2, Rune Larsen5, Anette Mertz Nielsen6, Kirsten Ejskjær1, Jes Thorn Clausen3, Lars Thim3 and Ebba Nexø1

Departments of
1 Clinical Biochemistry and
2 Internal Medicine V (Gastroenterology and Hepatology), Aarhus University Hospital, DK-8000 Aarhus C, Denmark.
3 Novo Nordisk, Department of Protein Chemistry, Novo Alle, DK-2880 Bagsværd, Denmark.

4 Department of Medical Anatomy B, University of Copenhagen, DK-2200 N Copenhagen, Denmark.

5 Department of Medicine M, Division of Gastroenterology, Glostrup University Hospital, DK-2600 Glostrup, Denmark.

6 Department of Medicine I, Division of Gastroenterology, Bispebjerg Hospital, DK-2400 Copenhagen, Denmark.

aAddress correspondence to this author at: Department of Clinical Biochemistry, Aarhus University Hospital, Nørrebrogade 44, DK-8000 Aarhus C, Denmark. Fax 4589493060; e-mail else.marie.vestergaard{at}dadlnet.dk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Trefoil factor 3 (TFF3), or intestinal trefoil factor (1)(2), belongs to a small family of three mucin-associated peptides (TFF1, TFF2, and TFF3). The botanical name comes from the existence of one or more 38- or 39-amino acid domains in which six cysteine residues form three disulfide bonds to create a characteristic three-leafed structure (3)(4). Because of this compact structural motif, the peptides are extremely stable toward proteolytic digestion (3)(5), acid, and heat degradation (6). TFF3 exists in a 60-amino acid monomeric form (6.7 kDa) and a 118-amino acid dimeric form (13.1 kDa) (5). In addition to TFF3, the mammalian trefoil factor family includes the breast cancer-associated peptide pS2 (now named TFF1) (7)(8) and spasmolytic polypeptide SP (also named TFF2) (6)(9)(10). Trefoil factors are expressed in several tissues of the body but most pronouncedly in the gastrointestinal tract. Under normal circumstances, TFF1 and TFF2 are primarily located in the stomach (11)(12), whereas TFF3 is predominantly present in the mucus cells of the small and large intestine (2). In the gastrointestinal tract, up-regulation of the expression of all three TFF members occurs at the site of damage in conditions such as peptic ulcer or inflammatory bowel disease (4)(13). In addition, trefoil peptides are aberrantly secreted by a variety of tumors (14).

A large body of evidence now supports the involvement of trefoil peptides in mucosal defense and restitution in the gastrointestinal tract. In vitro studies have shown that trefoil peptides promote the migration of epithelial cells independently of the transforming growth factor-ß pathway (15). In addition, in cooperation with mucins, TFF3 protects the intestinal cell monolayers from a variety of injurious agents (16). In vivo studies have shown that oral administration of TFF3 in rats protects gastric mucosa from injury (17), and transgenic mice that overexpress trefoil peptides are protected from nonsteroidal antiinflammatory drug-induced injury (18), whereas TFF3-null mice exhibit impaired mucosal healing (19).

The potential role of TFF3 in the gastrointestinal tract has led to an increasing interest in assays able to measure this peptide in humans.

The present study describes the first ELISA for TFF3 in human serum and presents preliminary clinical data, which indicate that patients with inflammation and/or ulceration of the upper gastrointestinal tract have increased serum concentrations of TFF3.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
participants and specimens
We obtained blood for preparation of serum from the following groups: (a) 10 individuals (4 men and 6 women) recruited from among the employees of the hospital (blood samples were taken after an overnight fast and 2 h after a full breakfast); (b) 7 healthy women who did not use hormonal contraception for at least 3 months before blood samples were collected (the women were 32–40 years of age and had regular ovulatory menstrual cycles of 25–35 days, and blood was collected four times throughout one menstrual cycle); (c) 300 healthy blood donors receiving no hormonal medication [150 women and 150 men; age groups, <50 years (n = 195) and >=50 years (n = 105)]; (d) 12 consecutive male patients with endoscopically confirmed esophagitis, gastric ulcer, or duodenal ulcer and a control group consisting of 7 consecutive male patients with no endoscopic signs of inflammation or ulceration (clinical data on the patients studied are shown in Table 1 , and blood was collected either just before the endoscopy or within 2 h after, except for 2 cases where blood was drawn 24 and 48 h after the endoscopy); (e) 3 male patients with severe exacerbation of chronic inflammatory bowel disease restricted to the colon only (1 patient with Crohn disease and 2 with ulcerative colitis) as determined by colonoscopy and x-ray of the small bowel to exclude affection. For this last group, baseline blood samples were collected before the start of intravenous high-dose methylprednisolone treatment and again after 2–4 days and after 1 week. After clinical improvement, the intravenous treatment was replaced with oral prednisolone by day 7 with tapering of the daily dose. Blood samples were obtained after 6 weeks, when the prednisolone dose was approximately one-half the initial dose, and again after 12 weeks when prednisolone treatment was stopped.


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  		Table 1. Clinical data on the patients studied.

The local scientific ethics committee approved all procedures. All participants gave written or oral consent.

trefoil peptides, antibodies, and calibrators
We used human TFF3, cloned, expressed, and purified as described (5), for preparation of calibrators and for production of rabbit antibodies. The dimeric form of TFF3 was used (5). The antibodies were produced in two rabbits, 2241A and 2241B. Before immunization, the TFF3 was coupled to ovalbumin under standard conditions. The rabbits were each immunized with 100 µg of human recombinant TFF3 dimer mixed with Freund’s complete adjuvant the first two times and thereafter with Freund’s incomplete adjuvant. The immunizations were carried out every second week. The {gamma}-globulin fraction of serum obtained from each of the two rabbits (2241A and 2241B) was purified by chromatography (20). We prepared calibrators from a stock solution of 1 g/L (76.1 µmol/L) human recombinant TFF3. We assigned the values of the calibrators based on amino acid analysis. This stock solution was diluted in polystyrene test tubes with assay buffer (0.1 mol/L sodium phosphate, pH 8.0, containing 1 g/L human albumin; Behringwerke) to obtain calibrators ranging from 3 to 100 pmol/L. We used the assay buffer as zero calibrator.

development of the elisa
The ELISA was based on an immobilized antibody that captures the analyte and a biotinylated detection antibody that reacts with horseradish peroxidase-avidin and produces a color reaction that increases with increasing concentration of the analyte. The ELISA was established in three steps: (a) the {gamma}-globulin fraction from each of the two rabbits was tested in a checkerboard design to select the optimal combination of capture and detection antibody; (b) the optimal dilution of the detection antibody was established; and (c) the characteristics of the assay were studied.

Coating of the ELISA plates and preparation of the biotinylated antibody were performed essentially as described previously for epidermal growth factor (21). Briefly, Maxisorp F-96 immunoplates (Life Technologies) were coated by adding 0.19 µg of anti-human TFF3 IgG (2241A and 2241B) in 100 µL of 15 mmol/L sodium carbonate, 35 mmol/L sodium bicarbonate, pH 9.6, to each well. We incubated the plates for 20 h at 4 °C before emptying the wells and adding 200 µL of 1 mol/L ethanolamine, pH 8–9, to each well. The plates were incubated for 20 h at 4 °C and subsequently stored at -20 °C. For evaluation as detection antibody, the same anti-human TFF3 IgGs were biotinylated. Briefly, 1 mL of IgG (1 g/L) that had been dialyzed overnight against 0.1 mol/L sodium bicarbonate, pH 8.3, was mixed gently for 4 h at room temperature in the dark with 50 µL of 4.4 mmol/L biotinamidocaproate N-hydroxysuccimide ester (Sigma) dissolved in dimethyl sulfoxide (Merck). Subsequently, we added 50 µL of 0.1 mol/L L-lysine (Fluka) and, after 15 min, 50 µg of rabbit {gamma}-globulin (Calbiochem). The sample was dialyzed for 72 h against 10 mmol/L sodium phosphate buffer containing 145 mmol/L NaCl, and 1 g/L sodium azide was added to the buffer during the last 24 h. We stored the biotinylated IgG at -20 °C and diluted it in assay buffer before use. For each assay, plates were washed three times with 10 mmol/L sodium phosphate buffer, pH 7.4, containing 145 mmol/L NaCl, 1 g/L bovine albumin (Sigma), and 1 g/L Tween 20 (washing buffer). Calibrator, control, or sample (100 µL) was then added in duplicate and incubated for 30 min. After the plates were washed three times, 100 µL of biotinylated antibody was added, and the plates were incubated for 30 min. After the plates were washed three times, 100 µL of horseradish peroxidase-avidin (Dakopatt) diluted 1:2000 in 10 mmol/L sodium phosphate, pH 7.4, 400 mmol/L NaCl, and 0.2 g/L lysozyme (Sigma) was added to each well, and the plates were again incubated for 30 min, followed by three washes. The color reaction was developed by the addition of 100 µL of TMB Microwell Substrate System (Kirkegaard and Perry Laboratories) to each well and incubation at room temperature for ~10 min. The reaction was stopped by the addition of 100 µL of 1 mol/L phosphoric acid to each well. The color developed was measured photometrically at 450 nm and corrected for the absorbance at 620 nm. Two serum pools diluted 1:5 (to ~12 and 40 pmol/L TFF3) were used as daily controls.

tff3 elisa validation procedures
To test possible cross-reactivity, we prepared recombinant human TFF1 and TFF2 at a concentration of 40 nmol/L in assay buffer.

We obtained blood for preparation of plasma (EDTA) and serum from 35 apparently healthy blood donors to examine whether the two matrices could be used interchangeably.

To assess recovery, we added recombinant TFF3 to seven serum samples in sufficient amounts to increase the concentration of total TFF3 by 18 and 57 pmol/L and analyzed the supplemented and unsupplemented samples. We determined percentage of recovery by comparing the amount of added TFF3 with the amount measured after subtracting the endogenous TFF3 concentrations.

A pool of human serum diluted 1:5 with assay buffer (S1) was prepared and diluted to contain 80% (S2), 60% (S3), 40% (S4), and 20% (S5) of S1. On the basis of ELISA measurements, we assigned a TFF3 concentration of 39 pmol/L to S3, and the concentrations in the other pools were calculated from this value. Aliquots of each sample were frozen at -20 °C until analyzed. The samples were analyzed in duplicate in 20 independent runs over a period of 6 months, using four different batches of coated microtiter plates.

molecular sieve chromatography
Fresh serum samples (0.1 mL) from two apparently healthy individuals were subjected to size-exclusion chromatography on a Superdex 75 HR 10/30 column (Amersham Pharmacia Biotech) using a SMART system (Amersham Pharmacia Biotech). The column was preequilibrated and eluted with 0.05 mol/L Tris-HCl, pH 7.2, 9.0 g/L NaCl at 0.5 mL/min with collection of 0.5-mL fractions. We determined distribution of TFF3 immunoreactivity by TFF3 ELISA.

statistical analysis
We used the SPSS program package for statistical analysis. We calculated the total and within-day variances by nested ANOVA; we used regression analysis to analyze linearity and a paired t-test to compare results for plasma and serum TFF3 and serum TFF3 concentrations before and after the ingestion of a meal. We analyzed differences between groups nonparametrically by the Mann–Whitney U-test.

Testing for outliers was performed as described by Solberg (22). Four points were identified and removed by use of the range test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
tff3 elisa
We used an assay format with a capture antibody immobilized on microtiter plates and a biotinylated detection antibody visualized via an avidin-coupled enzymatic reaction.

We tested two polyclonal rabbit anti-recombinant human TFF3 as both capture and detection antibodies in three different combinations according to a previously described method (23). Both antibodies performed almost equally well as capture and detection antibodies. We chose 2241A and 2241B as capture and detection antibodies, respectively. We used 0.2 µg of IgG per well as capture antibody, and in the final design of the assay, detection antibody was titrated to optimize the analytical range of the assay and the incubation time needed to develop the color reaction. For the assay, we used 0.05 µg/well of the detection antibody, which allowed for a color development of ~10 min.

The concentration of the calibrators ranged from 3 to 100 pmol/L (Fig. 1 ). The signal obtained for the lowest calibrator was 1.8–2.6 times higher than the signal obtained for the zero calibrator (mean ± SD, 2.0 ± 0.2; n = 19). On the basis of these results, we judged the detection limit of the assay to be <=3.0 pmol/L. Serum samples were diluted 1:5 in assay buffer before analysis. Values outside the linear range of the calibration curve were rejected.



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  		Figure 1. TFF3 ELISA calibration curve.

An ELISA was developed that uses two rabbit antibodies against human TFF3 as capture (2241A) and detection (2241B) antibodies and recombinant human TFF3 as calibrator. Shown is a typical calibration curve covering TFF3 concentrations of 3.0–100 pmol/L.

linearity and imprecision
We tested linearity and imprecision by analyzing five serially diluted plasma samples (S1–S5) with concentrations of 13–59 pmol/L two times per assay in a total of 20 assays over a period of 6 months. We calculated the relative concentrations of the samples based on the assumption that S3 had a concentration of 39 pmol/L. Linear regression analysis of the data yielded an intercept of 3.06 ± 0.712 pmol/L and a slope of 0.876 ± 0.017; Sy|x was 3.036, and the correlation coefficient was 0.983.

The total imprecision (CV) was 5.3–8.5%, and the intraassay imprecision was 1.8–3.2% (Table 2 ).


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  		Table 2. Total and intraassay imprecision (CV) for measurement of TFF3 by ELISA.1

recovery, stability, and interfering substances
When recombinant TFF3 dimer was added to serum samples in sufficient amounts to increase the total TFF3 concentration by 18 and 57 pmol/L, the final concentrations measured by the ELISA averaged 84% ± 4.8% and 92% ± 5.1% of those predicted.

We examined the effect of storage by six repeated measurements of five samples for a period of 57 days. Between analyses, we kept samples at -20 °C. No systematic changes in serum TFF3 values were observed (data not shown).

No cross-reactivity was observed for the two other trefoil peptides, TFF1 and TFF2. Concentrations of 40 nmol/L in the assayed samples, corresponding to a serum concentration of 200 nmol/L, were below the detection limit of the assay. The concentration of 40 nmol/L is ~1000-fold higher than the expected mean concentrations of TFF1 and TFF2 based on data from ELISAs that we are currently developing in our laboratory.

A comparison of the results obtained for 35 samples showed significantly higher values (P <0.001) for serum than for plasma. Serum values were on average 24% higher than the values obtained for plasma. We did not explore this difference further, but chose to perform the rest of the studies with serum.

molecular forms of tff3 in serum
To evaluate the distribution and molecular mass of the immunoreactivity detected in human serum, we fractionated two samples with TFF3 concentrations of 180 and 260 pmol/L by molecular sieve chromatography and assayed the fractions for TFF3. Only one major peak at ~6.6 kDa, corresponding to the reported molecular mass of monomeric TFF3, was detected (Fig. 2 ).



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  		Figure 2. Gel filtration of immunoreactive TFF3.

Two serum samples (solid lines) were fractionated by use of a SMART system on a precalibrated Superdex 75 HR 10/30 column. Fractions were analyzed by TFF3 ELISA. The column was calibrated with molecular mass calibrators from Amersham Pharmacia Biotech, eluting at fractions 12 (67 kDa), 14 (43 kDa), 18 (25 kDa), and 20 (13.7 kDa). Recombinant human TFF3 dimer (· · ·) and monomer (- - -) eluted at fractions 19 and 21, respectively.

biological variation
To examine the influence of recent food intake on TFF3 concentrations in serum, we measured TFF3 concentration in nine healthy volunteers after an overnight fast and 2 h after breakfast. After food intake, we observed a significant but small decrease in TFF3 (average, 5%; range, 4.9–12.8%). We judged the decline to be of limited value and concluded that strict control of the timing of blood collection for measurements of TFF3 is of minor importance.

We examined serum TFF3 in four samples collected from each of seven volunteers during the menstrual cycle. No significant differences were observed between TFF3 concentrations in samples collected during menstruation, the follicular phase, ovulation, or the luteal phase.

serum tff3 in blood donors
The reference interval for TFF3 in serum from healthy blood donors is shown in Table 3 . Like TFF1, the expression of TFF3 in breast cancer cells is regulated by estrogen (24), and therefore blood donors using any hormonal medication were excluded from the study. None of the donors used nonsteroidal antiinflammatory drugs during the week before blood sampling. The values obtained for women were significantly higher than the values obtained for men, but we judged the absolute difference in the values obtained to be of minor importance. We observed no significant age-related differences for men or women. We suggest a reference interval of 91–250 pmol/L.


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  		Table 3. Serum TFF3 in blood donors.

serum tff3 in patients with gastrointestinal diseases
To make an initial screening of possible alterations in TFF3 as a function of diseases in the gastrointestinal tract, we examined a limited number of patients with various gastrointestinal diseases.

The serum TFF3 concentrations were significantly higher in patients with inflammation and/or ulceration of the upper gastrointestinal tract (n = 12) than in the control group of patients suspected to have upper gastrointestinal diseases but without endoscopically found signs of inflammation and/or ulceration (n = 7; P <0.05) and were also significantly higher than the values obtained for the group of healthy male blood donors (P <0.01; Fig. 3 ).



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  		Figure 3. Serum concentrations of TFF3 in male blood donors (I; n = 149), controls (II; n = 7), and patients with endoscopically confirmed inflammation and/or ulceration of the upper gastrointestinal tract (III; n = 12).

Median TFF3 concentration was increased in patients with inflammation/ulceration of upper gastrointestinal tract compared with male blood donors (P <0.01) and controls (P <0.05). Solid lines depict the median TFF3 concentration. Dashed lines depict the reference interval for the healthy blood donors. One concentration was above the values shown (M; 1700 pmol/L).

None of the patients with severe acute activity in chronic inflammatory bowel disease of the colon had baseline TFF3 values outside the reference interval before the start of high-dose prednisolone treatment. In addition, no significant fluctuations of TFF3 (serial measurements during a 6-week period) were observed on treatment with prednisolone and clinical improvement (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have developed a specific and reproducible ELISA for quantification of the trefoil peptide TFF3 in serum samples from humans. The assay has made it possible to report, for the first time, that TFF3 is present in human serum at a concentration of ~200 pmol/L. The concentration is surprisingly high considering the observation that this 6.7-kDa peptide is circulating in its free form. From animal studies it is known that another trefoil peptide, TFF2, has a very short half-life and that it is freely filtered in the kidney (25). Anticipating that the same applies to TFF3 in human serum, a concentration of 200 pmol/L reflects a daily release to the circulation of at least 40 nmol of TFF3.

The origin of circulating TFF3 is unknown, but several suggestions can be made. TFF3 is expressed in female genitalia (24), and in vitro studies have shown that, like TFF1, estrogen regulates the expression of TFF3 (14). For that reason, we anticipated that we would observe, in addition to variations in TFF3 during the menstrual cycle, considerably higher TFF3 concentrations in women than in men as well as an age-related decrease in serum TFF3 concentrations in women. The lack of fluctuations in serum TFF3 under these circumstances suggested that circulating TFF3 was derived from cells unaffected by estrogens.

TFF3, like TFF2, is expressed in the gastrointestinal tract. TFF2 in gastric juice shows dramatic diurnal variations with the highest concentration during the night, whereas the TFF2 concentration decreases on ingestion of food (26). In addition, in vitro assays have demonstrated that TFF3 expression is regulated by intestinal secretagogues (27). On the basis of these observations, we expected that the TFF3 concentration would vary as a function of food ingestion. We observed a small but significant decrease after breakfast, but we judged the absolute change of 5% to be of minor importance. We do not know whether this observation demonstrated a less pronounced fluctuation with food intake for TFF3 than for TFF2 or whether it indicated that there is no connection between the TFF3 concentration in the gastrointestinal tract and in serum.

The trefoil peptides are usually expressed in a site-specific pattern within the gastrointestinal epithelium: TFF1 throughout the stomach, TFF2 in the distal stomach and the duodenum, and TFF3 in the small and large intestines (1)(11)(12)(13)(28)(29). This ordered expression of the trefoil peptides is disrupted in ulcerative and inflammatory conditions, with TFF3 being aberrantly overexpressed in the stomach in areas of ulceration (24). In a rat model of stomach ulceration, TFF3 was expressed late after ulcer induction and remained increased long after the injury (30). In the present study, the increased TFF3 concentrations in sera from patients with inflammation and/or ulceration of the upper gastrointestinal tract may reflect increased production of TFF3 as a local response to the mucosal damage.

The present study also presents results from serial measurements of serum TFF3 in three patients on five occasions during an acute attack of chronic inflammatory bowel disease and during high-dose prednisolone treatment and tapering. Despite severe ulceration of the colonic mucosa, none of the patients demonstrated TFF3 serum concentrations above the upper reference interval during the observation period. This finding may reflect the presence of diffuse atrophy of the colonic mucosa and consequently a reduced ability to secrete TFF3 as a response to injury. Development of novel therapies with the addition of recombinant TFF3 may improve mucosal protection and repair in this group of patients.

In the gastrointestinal tract, two important functions of trefoil peptides have been identified: protection and healing of the epithelium. A functional role of trefoil peptides outside the gastrointestinal tract has not been demonstrated. The origin and possible function of TFF3 in serum are unknown, but considering the hypothesis that trefoils can induce effects on the gastrointestinal epithelium via basolateral receptors, which is based on the observations in animal studies that subcutaneously administered trefoil peptides are effective in much lower doses than oral doses (25)(31), indicate that TFF3 in serum may function in a receptor-mediated system.

In conclusion, we have developed an assay for quantification of TFF3 and have shown that TFF3 is present in serum. Circulating TFF3 was not regulated by estrogens, but was influenced by diseases in the upper gastrointestinal tract. Availability of this method should facilitate future investigation of the physiologic roles and potential diagnostic values of TFF3.


   Acknowledgments
 
This work was supported by grants from The Aarhus University Foundation and The Danish Medical Research Council. We warmly acknowledge the technical assistance of Inger Marie Jensen.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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