SELF TRANSGLUTAMINASE-BASED ONE-MINUTE ONSITE BLOOD TEST FOR CELIAC DISEASE

1. SELFTRANSGLUTAMINASE-BASED ONE-MINUTE ONSITE BLOOD TEST FOR CELIAC DISEASE Ilma Korponay-Szabó, Tiina Raivio, Katri Kaukinen, Judit B.Kovács, Pekka Collin, Kaija Laurila, Jukka Partanen, Markku Mäki Pediatric Research Centre and Dept. of Internal Medicine, University of Tampere, Finland, University of Debrecen, Debrecen, Hungary Heim Pál Children’s Hospital, Budapest, Hungary

2. BACKGROUND AND AIMS • Circulating antibodies against type-2(tissue) tansglutaminase (TG) are good non-invasive markers of gluten-sensitive enteropathy. • Antibodies against TG are currently measured in specialized laboratories using ELISA or immunofluorescent method (endomysial antibody test, EMA). • The testing is expensive and the tournaround time of results is considerably long. →Quick, easy to perform, economical andwidely accessible celiac tests are needed

3. TG Y Y Y Y Y Hemolysis TG Y Y TG Y Y Y TG PRINCIPLE OF THE TESTING Whole blood samples contain endogeneous TG antigen inside normal red blood cells. RBC self-TG can be used as antigen to detect anti-TG antibodies present in the plasma of the same sample. Formation of self-TG/anti-TG complexes Label (anti-IgA) Capture of the labelled complexes by TG-binding protein

4. TG Y Y Y Y Y Hemolysis TG Y Y TG Y Y Y TG PRINCIPLE OF THE TESTING Whole blood samples contain endogeneous TG antigen inside normal red blood cells. RBC self-TG can be used as antigen to detect anti-TG antibodies present in the plasma of the same sample. Formation of self-TG/anti-TG complexes TG+/+ TG-/- RBC Label (anti-IgA) Nunc Immunstick™ surface (Maxisorp) Sensitive and specific for CD Color reaction entirely dependent on the presence of RBC-TG (no color when using TG-/- mouse RBC+serum

5. TG Y Y Y Y Y Hemolysis TG Y Y TG Y Y Y Y Y Y Y Y Y Y TG TG TG TG PRINCIPLE OF THE TESTING Whole blood samples contain endogeneous TG antigen inside normal red blood cells. RBC self-TG can be used as antigen to detect anti-TG antibodies present in the plasma of the same sample. Formation of self-TG/anti-TG complexes Capture of the labelled complexes by TG-binding protein line Label (anti-IgA) Y Lateral flow Y

6. The lateral flow system Biocard™ Celiac Disease Test AniBiotech, Vantaa, Finland

7. PATIENTS & METHODS Clinical diagnosis in the controls • A/ Retrospective cohort (n=224) • GI patients undergoing endoscopy • Gold standard: histology • Evaluation on stored whole blood • by blinded observers • 117 untreated celiac patients 12 also after • 1y on diet • 107 non-celiac controls • age: 1,8-72 years, median, 14 • Comparison with serum antibody measurements • EMA (indirect immunofluorescence on • monkey esophagus) • TG-Ab ELISA (Celikey™, Pharmacia)

8. PATIENTS & METHODS B/ Prospective cohort (n=168) - Symptomatic patients(n=69) (GI, iron deficiency, short stature, dental enamel defects, chronic fatigue) - Relatives of CD subjects (n=72) - Healthy volunteers (n=27) Testing in the outpatient office or at home Gold standard: EMA/TG-Ab (kappa-statistics) Subjects with positive results were referred for small-bowel biopsy

9. TEST RESULT (at 5 minutes) Positive Negative Celiac antibody detection Control line Biocard™ Celiac Disease Test AniBiotech, Vantaa, Finland

10. RESULTS Retrospectively studied patients / Correlation with histology: *Anti-TG antibodies deposited in the gut mucosa: 3 Partial villous atrophy in the past: 1

11. Agreement of lateral flow test results with standard laboratory antibody tests from serum 0.88 Biocard / TG-ELISA 0.89 Biocard / EMA Kappa statistics 0.40 0.75 excellent good poor Inter-observer agreement on 28 samples: same result

12. Prospectively studied patients: (fresh blood samples) Concordant: 98.8% % 3.7 6.9 13.9 57.6 Concordant: 99.4%

13. Prospectively studied patients: 35 /168 had a jejunal biopsy performed 26 new celiac cases were diagnosed 68 g/l = lowest hemoglobin level among CD patients Self TG-based rapid test result : +

14. Lateral flow test results before and after a gluten-free diet for one year Ten of the 12 rapid test+ coeliac patients reevaluated after one year on diet had negative lateral flow test result. The two patients still positive had TG-antibody ELISA levels near the cut-off (4.6 and 4.2 U/ml). Cut-off TG-Ab ELISA positive lateral flow negative lateral flow

15. ADVANTAGES • Results are available within 5 minutes onsite. • The testing is simple and easytoperform from capillary blood. • No recombinant / purified TG2 antigen is needed. • No laboratory equipment is needed. • No blood centrifugation is needed. • Suitable for home use

16. LIMITATIONS • Not meant for final diagnosis (biopsy is still required) • May be negative if the subject had adopted a gluten-free diet before testing • Not suitable to show decrease or increase in antibody concentrations (plus/minus test) • Immunoglobulin deficiency (IgA)

17. CONCLUSIONS • The self TG-based rapid celiac antibody test was highly accurate in detecting new celiac cases. • Its sensitivity was comparable to that of EMA testing and TG-antibody detection by ELISA. • The rapid test was able to show the clearence of antibodies after a prolonged gluten-free diet. • Immediate availability of results may help physicians to speed up the diagnostic process and to evaluate dietary compliance.

18. POTENTIAL APPLICATIONS of celiac antibody detection at the pointofcare • Case finding among symptomatic patients in the office or ward • Screening of subjects at-risk for celiac disease • family members, autoimmune patients, pregnant women • Population screening studies • Diet monitoring in the office or by home testing (longterm surveillance)