1. Coeliac Disease Study Group SELF TRANSGLUT RAPID COELIAC ANT BY LATERAL F Ilma Korponay-Szabó 1,2, Tiina Raivio 2, Pekka Collin 3, Kaija 1 University of Debrecen, Debrecen, Centre and Dept. of 3 Internal Medicine, Finland, 4 Heim Pál Children’s

2. Coeliac Disease Study Group AMINASE-BASED IBODY DETECTION LOW METHOD Katri Kaukinen 3, Judit B.Kovács 4, Katri Kaukinen 3, Judit B.Kovács 4, Laurila 2, Markku Mäki 2 Laurila 2, Markku Mäki 2 Hungary, 2 Paediatric Research Hungary, 2 Paediatric Research University of Tampere, Tampere, University of Tampere, Tampere, Hospital, Budapest, Hungary Hospital, Budapest, Hungary

3. Coeliac Disease Study Group

4. Circulating antibodies against type-2(tissue) tansglutaminase (TG) are characteristic activity markers of gluten-sensitive enteropathy. Their detections is an important diagnostic tool in finding new patients and evaluating treatment efficacy.Circulating antibodies against type-2(tissue) tansglutaminase (TG) are characteristic activity markers of gluten-sensitive enteropathy. Their detections is an important diagnostic tool in finding new patients and evaluating treatment efficacy. Antibodies against TG are currently measured in specialised laboratories using ELISA or immunofluorescent method (endomysial antibody test, EMA). The testing is expensive and patients and doctors have to wait for the results.Antibodies against TG are currently measured in specialised laboratories using ELISA or immunofluorescent method (endomysial antibody test, EMA). The testing is expensive and patients and doctors have to wait for the results. Future → quick, easy to perform, economical Future → quick, easy to perform, economical and widely accessible coeliac tests and widely accessible coeliac tests AIM of this study was to develop and evaluate a rapid inexpensive test for the use in the doctor’s office, at the bedside or in the home of the patients.AIM of this study was to develop and evaluate a rapid inexpensive test for the use in the doctor’s office, at the bedside or in the home of the patients. BACKGROUND AND AIMS

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

6. Coeliac Disease Study Group The lateral flow system

7. Coeliac Disease Study Group Samples from 109 subjects on normal diet 55 untreated patients with coeliac disease diagnosed according to the ESPGHAN criteria (32 children, 23 adults) age: 1,8-69 years 54 disease controls with normal mucosa at jejunal biopsy (23 children, 31 adults) age: 2,6-72 years All control subjects were negative for EMA. Samples on a gluten-free diet for one year obtained from 11 of the coeliac patients PATIENTS Clinical diagnoses of the controls Dyspepsia37 GOR5 IBD3 Cong. sucrase deficiency3 FAP2 Recurrent abdominal pain2 Swachman-Diamond sy1 Nonspecific rash1 All54

8. Coeliac Disease Study Group METHODS The rapid antibody test based on the use of self-TG antigen was developed into a lateral flow system.The rapid antibody test based on the use of self-TG antigen was developed into a lateral flow system. Initial evaluation was carried out with whole blood samples anticoagulated with EDTA or citrate.Initial evaluation was carried out with whole blood samples anticoagulated with EDTA or citrate. Results were read after 5 minutes by a blinded observer who was unaware of the clinical status of the patients.Results were read after 5 minutes by a blinded observer who was unaware of the clinical status of the patients. Lateral flow results were compared with the laboratory detection of coeliac antibodies from serum collected at the same time.Lateral flow results were compared with the laboratory detection of coeliac antibodies from serum collected at the same time. –TG antibody ELISA (Celikey™, Pharmacia, cut-off 5 U/ml) –EMA: indirect immunfluorescent method on monkey oesophagus (serum dilution 1:2.5)

9. Coeliac Disease Study Group TEST RESULT Positive Negative Control line Coeliac antibody detection (at 5 minutes)

10. Coeliac Disease Study Group 53 out of the 55 samples from untreated coeliac patients were positive (96.4 %) at 5 minutes with lateral flow. Most samples were clearly positive already at one minute after application of the sample.53 out of the 55 samples from untreated coeliac patients were positive (96.4 %) at 5 minutes with lateral flow. Most samples were clearly positive already at one minute after application of the sample. Three of the 54 controls showed a positive result, and 4 additional controls showed a very faint line at the test area.Three of the 54 controls showed a positive result, and 4 additional controls showed a very faint line at the test area. Lateral flow rapid test Lateral flow rapid test Sensitivity 96.4 % Sensitivity 96.4 % Specificity 87.0 % Specificity 87.0 % RESULTS

11. Coeliac Disease Study Group Comparison of lateral flow test results with conventional laboratory tests 100 92% Lateral flow+ Lateral flow- EMA+532 EMA-747 Total6049 10092% Lateral flow+ Lateral flow- TG-Ab+ 532 TG-Ab- 747 Total6049 Concordant test results with EMA/ELISA (red): 92% of all untreated samples

12. Coeliac Disease Study Group Nine of the 11 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). Comparison of lateral flow test results before and on a gluten-free diet positive lateral flow negative lateral flow

13. Coeliac Disease Study Group Results are available within 5 minutes onsite.Results are available within 5 minutes onsite. The testing is simple, cheap and easy-to- perform from capillary blood.The testing is simple, cheap and easy-to- perform from capillary blood. No recombinant / purified TG2 antigen is needed.No recombinant / purified TG2 antigen is needed. No laboratory equipment is needed.No laboratory equipment is needed. No blood centrifugation is needed.No blood centrifugation is needed. ADVANTAGES

14. Coeliac Disease Study Group The self TG-based rapid coeliac antibody test is comparably sensitive as EMA and TG- antibody detection by ELISA.The self TG-based rapid coeliac antibody test is comparably sensitive as EMA and TG- antibody detection by ELISA. The rapid test is able to show the clearence of antibodies (seroreversion) on a gluten- free diet.The rapid test is able to show the clearence of antibodies (seroreversion) on a gluten- free diet. Immediate availability of results may help physicians to speed up the diagnostic process and to evaluate dietary compliance.Immediate availability of results may help physicians to speed up the diagnostic process and to evaluate dietary compliance. CONCLUSIONS

15. Coeliac Disease Study Group  Case finding among symptomatic patients in the office or at the bedside  Screening of at-risk groups family members, autoimmune patients, pregnant women etc. family members, autoimmune patients, pregnant women etc.  Population screening studies  Diet monitoring in the office or at home of coeliac antibody detection at the point of care POTENTIAL APPLICATIONS