PATIENTS AND HEALTHY CONTROLS

We examined serum samples from 70 untreated CD patients (30 males and 40 females, median age 12 years (range 2–60)) diagnosed between June 1997 and October 1999, following the revised ESPGHAN criteria for CD.12 Controls comprised 58 first degree relatives of CD patients and 74 subjects suffering from various diseases (10 Crohn's disease, four ulcerative colitis, one lactose intolerance, one Zolliger-Elison syndrome, two epilepsy, 12 anaemia, 30 failure to thrive, 10 diarrhoea, three arthralgia, and one gastritis). These groups consisted of 79 males and 53 females (median age 7 years (range 1–47)). In addition, healthy control sera were taken from a group of 64 blood donors (35 males and 29 females, median age 30 years (range 19–54)).

From January 1999 onwards, as well as testing sera, we also consecutively collected and examined whole blood samples from 17 untreated coeliacs (11 males and six females, median age 12 years (range 2–60)), 13 CD patients (five males and eight females, median age 9 years (range 3–35)) who had been on a gluten free diet for at least two years, and 22 patients tested as patient controls (seven Crohn's disease, five anaemia, three failure to thrive, two diarrhoea, two ulcerative colitis, two epilepsy, one arthralgia; 10 males and 12 females, median age 27 years (range 25–47)).

ANTIENDOMYSIUM ANTIBODY ASSAY

Serum IgA antiendomysium antibodies were measured by means of indirect immunofluorescence using cryostat sections of human umbilical cord, as previously described.13 Briefly, the sections were incubated with the subject's serum, diluted 1:5, for 30 minutes. After washing, sections were incubated with fluorescein labelled goat antihuman IgA antibodies for 30 minutes. The slides were washed, mounted in aqueous mounting medium, and examined by fluorescent microscopy.

HUMAN RECOMBINANT TRANSGLUTAMINASE

We cloned h-tTG cDNA from the intestinal biopsy of a patient with active CD using primers specific for the coding regions of the gene.14 cDNA was cloned into an expression vector (pET28b-Novagen, Madison, Wisconsin, USA), expressed, and purified under non-denaturising conditions using IMAC (Qiagen, Valencia, California, USA).

ELISA

Serum IgA and IgG anti-h-tTG antibodies were determined, as reported recently.9 Microtitre plates (EIA/RIA 2580, Costar, Cambridge, Massachusetts, USA) were coated with 1 mg of h-tTG in 100 ml of 0.05 M Tris buffered saline (TBS) with 5 mM CaCl₂ , pH 7.5. The plates were washed three times with 0.05 M TBS, 0.01 M EDTA, and 0.1% Tween 20 (TTBS). Serum samples diluted 1:100 in TTBS were incubated for one hour at room temperature. The plates were washed and incubated for one hour at room temperature with either 1:3000 phosphatase conjugated antihuman IgA (Sigma A-3062, St Louis, Missouri, USA) or 1:7000 antihuman IgG (Sigma A-8542) diluted in TTBS. The immune reaction was developed by adding substrate solution and the absorbance read in a microplate reader at 405 nm until the positive control serum reached an optical density value of 2 for IgA and 1.5 for IgG. Results were expressed as a percentage of the positive control serum. Normal values were taken as <13% for IgA and <30% for IgG, which represented a value >2 SD above the mean of 100 healthy subjects (60 males, 40 females, aged 1–16 years).

DOT IMMUNOBINDING ASSAY

Squares of Immobilon-P membrane (Millipore, IPVH00010) were wetted in 100% methanol for 15 seconds. They were washed with distilled water for two minutes and h-tTG was absorbed as a spot (0.1 μg/spot) onto the squares. After 15 minutes, the Immobilon-P free binding sites were blocked with phosphate buffered saline (PBS)-3% Tween 20 for 30 minutes and the squares were washed, allowed to dry, and stored at 4°C. The coated membranes were found to remain stable for at least one year. Serum samples or one drop (30 μl) of whole blood samples were diluted 1:50 in PBS-0.3% Tween 20 and incubated for five minutes at room temperature. The squares were washed three times with PBS-0.3% Tween 20 and incubated for five minutes at room temperature with phosphatase conjugated antihuman IgA and IgG immunoglobulins (Sigma A-3062, A-8542) diluted 1:2500 in PBS-0.3% Tween 20. The squares were then washed and the immunocomplexes were revealed by substrate solution. The strips were dried and examined, and the results expressed as a colorimetric reaction.

The immunological tests were performed by four operators (VB, AT, CT, IB) who were unaware of the clinical and laboratory findings of the subjects tested.

HLA-DQ TYPING

The known susceptibility alleles for CD were determined by polymerase chain reaction with allele specific primers identifying DQ2 and DQ8, carried out with a Dynal Classic SSP DQ kit (Dynal AS, Oslo, Norway).