Markers for the Diagnosis of Celiac Disease

Abstract

The invention relates to isolated antibodies that bind to specific peptides and to their use in the diagnosis of celiac disease.

Claims

1. An isolated antibody that binds to a peptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and/or SEQ ID NO: 4.

2. Method for the detection of pathogens associated with celiac disease, preferably in foods, comprising the steps of: i) a sample is contacted in vitro with the isolated antibody of claim 1, and ii) detecting binding of the isolated antibody to a pathogen in the sample, thereby detecting said pathogen.

3. A kit for performing the method of claim 2, wherein said kit comprises the isolated antibody of claim 1.

4. The kit of claim 3, wherein the isolated antibody of claim 1 is immobilized on a support.

5. The kit according to claim 3, wherein the kit is in the form of an ELISA or strip test.

Description

FIGURES

[0041] FIG. 1 shows the sensitivities of commercially available anti-gliadin IgA and IgG ELISAs as well as anti-tTG IgA and IgG ELISAs compared to a CD3 ELISA and indicates the % rate of correctly classified celiac patient sera as a measure for the sensitivity of each test.

[0042] FIG. 2 shows the sensitivities of commercially available anti-gliadin IgA and IgG ELISAs as well as anti-tTG IgA and IgG ELISAs compared to CDPtTG ELISA and tTG ELISA, using the CDPtTG ELISA protocol, and indicates the % rate of correctly classified celiac patient sera as a measure for the sensitivity of each test.

[0043] FIG. 3 shows sensitivity overlaps between the individual forms of ELISA used and indicates the percentage of celiac sera not recognized by the quoted tests that were tested positive with the respective assay.

[0044] FIG. 4 shows that CD3-specific antibodies isolated from patient sera can specifically recognize CDPtTG in a Western blot.

EXAMPLES

[0045] 1. Preparation and Purification of CD3, tTG and CDtTG

[0046] CD3 of SEQ ID NO: 4 was prepared by synthesis and biotinylated at the C-terminal lysine residue.

[0047] Human tTG2 of SEQ ID NO: 5, hereinafter referred to as tTG, was cloned in a vector with removable 6xHis tag for recombinant expression and expressed according to a standard protocol, isolated using NINTA column purification, and the 6×His was removed.

[0048] CDPtTG of SEQ ID NO: 6, hereinafter referred to as CDtTG, was likewise cloned in a vector with C-terminal 6xHis tag for recombinant expression and expressed according to a standard protocol, isolated using NINTA column purification, and the His tag was removed.

[0049] 2. ELISA for CD3, tTG (Homemade) and CDtTG

[0050] To detect CD3-specific antibodies, a CD3 peptide EL ISA was developed wherein biotinylated CD3 of SEQ ID NO: 4 was initially coupled on a neutravidin-coated microtiter plate, followed by the protocol below:

[0051] Neutravidin-CD3 peptide ELISA

[0052] The wells of the microtiter plate were initially blocked with PBS/5% MP buffer overnight. Thereafter, the biotinylated peptides were applied using 500 pmol/well in PBS buffer each time. After incubation for 2 h, the CD3/neutravidin-coated plates were washed 4 times with PBS/0.1% Tween and incubated for 1 h with patient serum at a dilution of 1:800 in PBS/2% MP. Thereafter, washing was repeated 4 times, followed by application of peroxidase-conjugated second Ab at a dilution of 1:5000. Incubation was also for 1 hour. Finally, washing was repeated 4 times, followed by application of the substrate. The reaction with development of a blue color was quenched after 5 min using 0.5 M sulfuric acid. The resulting yellow color was measured photometrically at a measurement wavelength of 450 nm versus a reference wavelength of 620 nm using an ELISA reader and visualized with the aid of the Magellan software. Each solution was applied using 100 μl per well. Blocking and the single washing steps were carried out using 300 μl per well each time. The microtiter plates with peptide, patient serum and 2n.sup.d Ab were incubated with agitation at RT.

[0053] CDtTG, tTG EL ISA protocol:

[0054] To detect tTG2- or CDPtTG2-specific antibodies, a specific ELISA was developed wherein recombinant tTG of SEQ ID NO: 5 and CDtTG of SEQ ID NO: 6 were initially coupled to a MaxiSorb microtiter plate (Nunc) and the following protocol was subsequently used: [0055] A) Coupling buffer: 100 mM Tris, 10 mM NaCl, pH 7.8 [0056] B) Wash buffer: 50 mM Tris-HCl, 150 mM NaCl, 10 mM EDTA, 0.1% Tween 20, pH 7.4 [0057] C) Saturation buffer: 50 mM Tris-HCl, 150 mM NaCl, 0.5% BSA, 3% sucrose, pH 7.4 [0058] D) Serum dilution buffer: 50 mM Tris-HCl, 150 mM NaCl, 0.5% Tween 20, pH 7.4

[0059] Procedure:

[0060] Coating on MaxiSorb plates from Nunc: Coating quantity: CDPtTG and tTG were each used at a concentration of 0.5 μg/well.

[0061] Coating volume: 100 μl/well. All tTGs were suitably diluted in coupling buffer A.

[0062] For coating, the plates were incubated at 40 ° C. overnight.

[0063] Blank and 2.sup.nd Ab controls were carried along in ELISA implementation. The OD values of blank and 2.sup.nd Ab controls were subtracted in ELISA evaluation.

[0064] After coating the plates were washed with 3×300 μl/well wash buffer B. Each wash step corresponds to 600 rpm on the ELISA shaker for 3 min. Thereafter, the plates were blocked with 300 μl/well saturation buffer for 2 h at RT. The sera were diluted 1:800 in serum dilution buffer D and 100 μl/well directly placed on the plates after blocking. Incubation at RT with shaking; washing with 5×300 μl/well wash buffer B. 2.sup.nd Ab: [0065] <hlgA>HRP from Dako was diluted 1:1500 in wash buffer B, and 100 μl/well was used; [0066] <hlgG>-HRP from Dako at a dilution of 1:5000 likewise in wash buffer B, and 100 μl/well was used.

[0067] Incubation for 1 h at RT with shaking. Washing with 4×300 μl/well wash buffer B. Allowing reaction with 100 μl/well TMB substrate (SeramunBlue fast) for 5 min. Thereafter, quenching with 100 μl/well quenching soln. (0.5 M H.sub.2SO.sub.4) and evaluation in ELISA reader at 450 nm.

[0068] 3. Cohort of Celiac Patients Sera

[0069] In the course of the present work, human patient sera from 91 patients with positive celiac diagnosis, varying age, sex and pathological characteristics were employed. All sera used were obtained from the Department of Clinical Rheumatology at the Charité (Berlin). In addition, 80 sera from normal donors were processed as control group and compared with the group of autoimmune diseases in this work.

[0070] 4. Comparison of Sensitivities Obtained for CD3 ELISA, tTG ELISA, CDtTG ELISA and Commercially Available Gliadin and tTG ELISAs

[0071] The sera specified under Section 3 were then used in the ELISA tests specified under Section 2. In parallel, the same sera were also examined with commercially available celiac ELISA tests which react to either gliadin-specific antibodies or antibodies specific to tTG2. To this end, commercial anti-htTG IgA or IgG ELISAs from Generic Assays GmbH (Germany) and commercial anti-gliadin IgA and IgG ELISAs from Generic Assays GmbH (Germany) were used.

[0072] As shown in FIG. 1, the CD3-specific ELISA was found to be more sensitive in correct classification of celiac patient sera compared to the commercially available anti-tTG and anti-gliadin ELISAs.

[0073] As shown in FIG. 2, ELISAs based on recombinant tTG2 were likewise superior to the commercially available anti-tTG and anti-gliadin ELISAs with respect to sensitivity. The CDPtTG-based ELISA was by far the most sensitive ELISA for correct classification of celiac patient sera. Thus, in total, the presence of the peptide sequence of SEQ ID NO: 1 according to the invention results in a celiac ELISA with improved sensitivity compared to commercial ELISAs and an anti-tTG ELISA carried out under the same conditions as the CDPtTG ELISA.

[0074] Thus, it was shown that peptides comprising the peptide sequence of SEQ ID NO: 1 according to the invention are informative and can be used in the diagnosis of celiac disease. Also, it was shown that an ELISA based on a peptide comprising the peptide according to the invention of SEQ ID NO: 1 has improved sensitivity.

[0075] FIG. 3 illustrates overlaps in the sensitivities of the CD3 ELISA with the commercially available anti-gliadin and anti-tTG ELISAs. It was found that the CD3 ELISA can detect sera from celiac patients that were not recognized by one or more or even all of the commercially available ELISA tests used. Thus, the use of a celiac assay based on a peptide comprising the peptide sequence of SEQ ID NO: 1 provides a valuable contribution to complete serological detection and diagnosis of celiac disease patients.

[0076] 5. CD3-Specific Antibodies from Patient Sera

[0077] Affinity Purification of CD3 Antibodies

[0078] For isolation and purification of the affinity-specific antibodies against the CD3 peptide from patient sera, the biotinylated variant of the CD3 peptide was coupled to avidin or neutravidin microtiter plates, followed by using a pool of 24 strongly positive celiac sera with minor nonspecific background binding for immune complex formation. Initially, the microtiter plate (MTP) was blocked with

[0079] PBST/5% MP for 1 h at RT. This was followed by 3 washings with PBS wash buffer, and the wells of the 96-well plate was coated with 5000 pmol/well CD3 peptide, which corresponds to a 10-fold capacity per well. After incubation at RT for 1 hour, the plate was washed 3 times with wash buffer. Thereafter, the sera were applied to the streptavidin plate or neutravidin plate, to which end each patient serum was initially diluted 1:100 in PBST/2% MP. Subsequently, the sera were pooled and placed in the wells using a volume of 100 μl/well. This was followed by incubation for 1 hour at RT. Thereafter, the contents of all wells were removed and recombined and used in another purification cycle or as a control for the affinity-specific purification effect in another purification cycle with the negative control peptide, Bor21 in the present case. After removal of the sera the plate was washed 4 times with wash buffer. The wells were subsequently spiked with 150 μl/well glycine elution buffer and incubated on the shaker for 10 min.

[0080] Finally, the elution buffer was resuspended several times and removed from the wells and immediately combined with 1:10 1 M Tris-HCl pH 8.0 buffer. This was immediately followed by rebuffering by ultracentrifugation in AmiconFalcons with a membrane cutoff of 55 kDa, using 5× a 5-fold volume of PBS buffer as counterbuffer. As control of success the batch was tested in the CD3 ELISA with positive and negative control sera. The quantities of antibodies thus obtained were stored at 4 ° C. in PBS and used in further immunological tests.

[0081] All wash steps were performed using a volume of 300 μl/well. Furthermore, all incubation steps were carried out at RT on the ELISA plate shaker at a rotational speed of 600 rpm.

[0082] As shown in FIG. 4, the CD3 peptide antibodies thus isolated react specifically with CDPtTG in a Western blot experiment. From this it follows that the antigen in the CD3 peptide is not a conformational antigen, but can also be detected in denatured state by CD3 peptide antibodies isolated from patient sera. Consequently, peptides comprising the peptide according to the invention of SEQ ID NO: 1 are not only suitable for use in diagnostic tests focusing on antigens in native state, but also for test formats wherein the antigen is used in denatured state, such as Western blot assays, protein arrays, Luminex bead arrays and/or protein chip assays.