DIAGNOSTIC KITS AND COMPONENTS

A complete range of assays for diagnosis of CELIAC DISEASE and DERMATITIS HERPETIFORMIS

Gluten Sensitive EnteropathyCELIAC DISEASEDERMATITIS HERPETIFORMIS

Disease background and diagnostics

REFERENCES

1. Guandalini S et al. Clin Appl Immun Rev 2002;2:293-305.2. Guandalini S et al. Clin Appl Immun Rev 2002;2:293-305.3. Fasano A, et al. Arch Intern Med. 2003; 163:286-92.4. Wolber R, et al. Gastroenterology 1990; 98:310-315.5. Catassi, et al. JAMA 2002;287:1413-1419.6. Arranz E, et al. Gastroenterology 1993;104:1263-72.7. Hill ID, et al. J. Pediatr Gastroenterol Nutr 2002; 35:S78-S88.8. Ferreira M et al. Gut. 1992; 33:1633-7.9. Chorzelski TP et al. Serologic Diagnosis of Celiac Disease.

CRC Press Boca Raton; 1990.10. Kapuscinska A et al. J Pediatric Gastroenterol Nutr; 1987, 6: 529-534.11. Rossi TM et al. J Pediatric Gastroenterol Nutr; 1988, 7: 858-863.12. Kumar V et al. Immun Invest; 1989 18: 533-544.13. Karpati S et al. Lancet; 1990, 336: 1335-1338.14. Hallström O. Gut; 1989, 30:1225-1232.15. Khoshoo V et al. J Pediatric Gastroenterol Nutr; 1988, 7: 864-866.16. Calabuig M et al. J Paediatric Gastroenterol Nutr; 1990, 10:435-442.17. Kumar V et al. Arch Dermatol; 1987, 123: 1179-1182.18. Rizzetto M et al. J Clin Path, 1973, 26:841-851.19. Seah PP et al. Lancet; 1971, 1:834-836.20. Seah PP et al. Gut; 1973, 14:311-315.21. Lancaster-Smith M et al. Br J Dermatol; 1975, 92:37-42. 22. Ljunghall K et al. Arch Dermatol; 1983, 119:970-974. 23. Vainio E et al. J Pediatr Gastroenterol Nutr; 1986, 51:375-379.24. Jones RA et al. J Cell Sci; 1997, 110:2461-2472.25. US Patent Number 6,703,208 B1.26. Savilahti E et al. Lancet 1973; I:320-322.27. Kumar V et al. J Pediatr Gastroenterol Nutr 1986; 5:730-734.28. Montgomery AMP et al. Gut 1988; 29:1564-1568.29. Weiss JB et al. J Clin Invest 1983; 72:96-101.30. Mearin ML et al. J Pediatr Gastroenterol Nutr 1984: 3:373-377.31. Levenson SD et al. Gastroenterology 1985; 89:1-5.32. Lerner A et al. J Pediatr Gastroenterol Nutr 1991; 12:407-409.33. Lebenthal E et al. J Pediatr 1983; 102:711-712.34. Kumar V et al. J Pediatr Gastroenterol Nutr 1984; 3:815.35. Tucker NT et al. J Pediatr 1988; 113:286-289.36. Unsworth DJ et al. Clin Exp Immunol 1981; 46:286-293.37. Bürgin-Wol A et al. J Pediatr 1983; 102:655-660.38. Kelly J et al. Arch Dis Child 1987; 62:469-473.

IFA

CODE DESCRIPTION DETERMINATIONS1114 ImmuGlo™ Endomysial antibody kit 48

6 well primate smooth muscle slides - IgA/IgG conjugate 1114A* ImmuGlo™ Endomysial antibody kit 48

6 well primate smooth muscle slides - IgA conjugate1114A-PDE ImmuGlo™ Endomysial antibody kit 48

6 well primate esophagus slides - IgA conjugate1114G-PDE ImmuGlo™ Endomysial antibody kit 48

6 well primate esophagus slides - IgG conjugate1115 ImmuGlo™ Reticulin antibody kit 48

6 well rat kidney slides - IgA/IgG conjugate2155-1 primate distal esophagus substrate slide 6 well2155-18 primate distal esophagus substrate slide 8 well2160 primate smooth muscle substrate slide 6 well2161 rat kidney substrate slide 6 well2161-8 rat kidney substrate slide 8 well2250 EMA positive control 0.5ml2251 ARA positive control 0.5ml2100 IgG conjugate with counterstain 5 ml2100x IgG conjugate 5ml2107 IgA conjugate with counterstain 5ml2107x IgA conjugate 5ml2113 IgA/IgG conjugate with counterstain 5ml2113x IgA/IgG conjugate 5ml

ELISACODE DESCRIPTION DETERMINATIONS1117A ImmuLisa™ Gliadin Ab IgA ELISA 961117G ImmuLisa™ Gliadin Ab (AGA) IgG ELISA 961117S* ImmuLisa™ Gliadin Ab (AGA) Screen ELISA 961144 ImmuLisa™ hu tTG Ab IgA ELISA 965144A Celiac tTG IgA Enhanced ELISA 961144G ImmuLisa™ hu tTG Ab IgG ELISA 965144G Celiac tTG IgG Enhanced ELISA 961144S ImmuLisa™ hu tTG Ab IgA/IgG ELISA 965159A ImmuLisa™ Celiac G+ (Gliadin) IgA ELISA 965159G ImmuLisa™ Celiac G+ (Gliadin) IgG ELISA 96* For research use only in the US

Gluten Sensitive Enteropathy (GSE) isan autoimmune disorder that mayoccur in genetically susceptible indi-viduals triggered by the ingestion ofgluten containing grains such as wheat,barley and rye. Patients experience animmune response against gluten as itpasses through the intestines. GSEincorporates Celiac Disease (CD), acondition characterized by malabsorb-tion resulting from in ammatoryinjury to the small intestinal mucosa,and Dermatitis Herpetiformis (DH),characterized by blistering of the skinand itchy rash on the head, elbows,knees, lower back and buttocks. Of thetwo conditions CD is more common,with prevalence as high as 1 in 130,however, studies have found the preva-lence of CD to be highly variable frompopulation to population1-3. e variedsymptoms and disparate criteria usedin diagnosis of CD seem to be the

cause. Classical symptoms of CDinclude diarrhea, weight loss and mal-nutrition, but only a small percentageof patients with CD present with thesesymptoms.

e proper treatment for GSE is agluten-free diet. Once the gluten isremoved from the diet, the immuneresponse declines, the intestinalmucosa normalizes and symptomsabate. It is important to establish thecorrect diagnosis so that treatment canbe implemented. Failure to diagnoseCD at an early stage has deleteriouse ects on quality of life and may pre-dispose an individual to long termcomplications such as splenic atrophyand intestinal lymphoma. One studyhas shown incidence of lymphomainvolving the gastrointestinal tract inpatients with CD to range from 3.6percent to 40 percent4. In another

recent study, CD was found to be asso-ciated with signi cantly elevated riskfor intestinal lymphoma, especiallynon-Hodgkin’s5.

Histological examination of the smallintestine biopsy remains the gold stan-dard for diagnosing CD. However, cer-tain patients with latent or even activeCD may have normal histopathology6.Serological methods of diagnosis arecommonly used to screen and supportdiagnosis of CD and DH. e revisedEuropean Society of PaediatricGastroenterology and Nutrition (ESPGAN) criteria for diagnosis of CD include only a single biopsy withclear cut remission of clinical symp-toms on GFD. Positive serology at thetime of diagnosis with disappearanceon GFD contributes to the diagnosis7.

focus on celiac disease and dermatitis herpetiformis

IMAGES FROM LEFT TO RIGHT: WHEAT, ONE SOURCE OF GLUTEN; NORMAL INTESTINAL VILLI; DAMAGED VILLI CHARACTERISTIC OF CD; TISSUE TRANSGLUTAMINASE MOLECULE.

United StatesIMMCO Diagnostics, Inc.60 Pineview DriveBuffalo, NY 14228+1.716.691.0091

800.537.8378+1.716.691.0466 Faxinfo@immco.comwww.immco.com

©2010 IMMCO Diagnostics, Inc.ML 001 REV 3/10

CanadaNova Century Scientific, Inc.An IMMCO Diagnostics Company5022 South Service RoadBurlington, Ontario L7L 5Y7

800.615.5072800.639.9006 Faxinfo@novacentury.comwww.novacentury.com

IMMUNOFLUORESCENCE ASSAYS [IFA]

ENDOMYSIAL ANTIBODIES [EMA]

Since 1983, IMMCO has specialized inserological diagnosis of CD and DH.Serology offers a less invasive alterna-tive to biopsy when considering a diag-nosis. Our laboratory was the first todescribe and offer the endomysial anti-body (EMA) test. Studies indicate thatEMA results correlate with biopsy ingreater than 99% of CD cases and inapproximately 70% of DH cases8. Ofthe various antibody markers of CDand DH, EMA of the IgA isotype seem to be the most sensitive and specific.EMA of the IgG class occur in CDpatients with high titer IgA class EMAor those CD patients with IgA defi-ciency.

With adherence to a gluten-free diet,EMA levels rapidly decrease. A glutenchallenge or a failure to maintain agluten-free diet leads to the appearanceor an increase in EMA. Patients on agluten free diet >9 months experiencesignificantly reduced or negative EMAtiters if they adhere to their dietaryrestrictions 9-13.

EMA are detected on primate smoothmuscle tissues. IMMCO offers twosuch substrates for EMA detection byindirect immunofluorescence: primatesmooth muscle (bladder) and primatedistal esophagus. These tissues havebeen selected for optimal reactions,providing a balance of maximum sen-sitivity and minimum background.

RETICULIN ANTIBODIES [ARA]

A number of studies have noted thepresence of anti-reticulin antibodies in patients with CD and DH9, 14-17. The detection of ARA is performedprimarily by indirect immunofluores-cence. ARA by definition are detectedon rodent tissue.

In 1973, Rizzetto and Doniach18 identi-fied five different ARA reaction pat-terns. Of these, the R1 pattern, charac-terized by peri-glomerular, peri-tubu-lar, and peri-vascular staining of mouse

or rat kidney is associated with GSE.This typical ARA staining patternappears in the image array above.

Both IgG and IgA immunoglobulinclass ARA occur in GSE. IgA ARA arethe more specific and sensitive marker.Specificity of IgA ARA has beenreported from 59% - 100% with sensitivity ranging from 30-95% 9, 11, 14,15,18-23. IgG ARA occur less frequently,either in conjunction with IgA classARA or in CD patients who are IgAdeficient9.

IMMCO EMA Correlation with BiopsyINITIAL GLUTEN GLUTEN

TEST DIAGNOSIS FREE DIET CHALLENGEPOSITIVE NEGATIVE POSITIVE

BIOPSY 133 122* 133IgA EMA 132 132 129IgG EMA 1 1 1TOTAL EMA 133 133 130

*NO SECOND BIOPSY IN 11 CASES

TISSUE TRANSGLUTAMINASE[tTG] ANTIBODIES

Tissue Transglutaminase (tTG) hasbeen identified as the endomysialautoantigen in CD. tTG belongs to afamily of calcium dependent acyltransferase that catalyze the cross-link-ing of proteins post transitionally.Antibodies to tTG are present in over95% of celiac patients and antibodylevels seem to correlate with the pres-ence or absence of gluten in the diet24.The advantage of the anti-tTG anti-body assay is that it is easily automat-able and less subjective than EMA. For

this reason, many laboratories haveopted to use tTG antibody methods forhigh volume testing.

The ImmuLisa™ human tTG assaysincorporate a patented technology toenhance sensitivity and specificity25.These methods allow detection of bothIgA and IgG antibody isotypes, facili-tating proper diagnosis of CD even incases of IgA deficiency. A total tTG(IgA/IgG) antibody assay is also avail-able for screening applications. Thecorrelation between well standardizedtTG and EMA results is quite strong asindicated in the table below.

GLIADIN ANTIBODIES (AGA)

Studies of anti-gliadin antibodies (AGA)are commonly used in the diagnosis ofCD. Both IgA and IgG gliadin anti-bodies can be detected in the sera ofpatients with GSE by ELISA. IgGgliadin tests can help establish a diag-nosis of CD in IgA deficient patientswith CD. Studies show 1-2% of patientsare IgA deficient.

Published literature suggests that AGAmay be used to monitor compliancewith a prescribed gluten-free diet.Compliance causes antibody levels todrop and while IgA AGA drop quickly,IgG AGA taper off to normal levelsover a period of months to years26-38.

IMMCO has developed next genera-tion assays for detection of gliadin anti-bodies: the ImmuLisa™ Celiac G+ IgAand IgG ELISAs. Celiac G+ incorpo-rate proprietary gliadin peptides thatsignificantly improve sensitivity andspecificity. In combination withImmuLisa™ tTG antibody assays, theCeliac G+ systems provide the totalsolution in CD diagnosis.

from the inventors of the endomysial antibody assay, the gold standard in CD serology

simple, objective and automatable tests to support diagnosis of GSE

IMAGES FROM LEFT TO RIGHT: EMA ON SMOOTH MUSCLE, EMA ON ESOPHAGUS, ARA ON KIDNEY, FLUORESCENCE MICROSCOPE.

SENSITIVITY SPECIFICITY SENSITIVITY SPECIFICITY

IMMCO New Kit 99% 96% 100% 100%

Competitor 97% 95% 100% 100%

Table 1: Comparative Study of tTG ELISAs

HUMAN tTG CD PATIENTS CHILDRENANTIBODY ELISA

IGA

IMMCO OFFERS A CHOICE

OF SUBSTRATES OPTIMIZED

FOR DETECTION OF EMA

IMMCO HUMAN tTG ASSAYS

INCORPORATE A PATENTED

TECHNOLOGY TO ENHANCE

SENSITIVITY AND SPECIFICITY.

SENSITIVITY SPECIFICITY SENSITIVITY SPECIFICITY

IMMCO Celiac G+ 84% 96% 91% 97%

Competitor 64% 92% 90% 98%

Table 2: Comparative Study of Gliadin Peptide ELISAs

GLIADIN IgA IgG

ELISA

IMMUNOFLUORESCENCE ASSAYS [IFA]

ENZYME LINKED IMMUNOSORBENT ASSAYS [ELISA]

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