An ti-glia din IgA kit. An ti-gliad in Ig G k it D I A G N O S T I C S. Enzyme-linked immunosorbent ass ay ki t For in vitro diagnostic use only

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1 Labmaster ELISA tests: Anti-Gliadin Ig A kit Anti-Gliadin Ig G kit Tro po nin I ELISA kit 1006 Labmaster TR-FIA test s: Enter ola cton e kit 2001 Equol kit Genistein kit Daidzein kit CD Labmaster ELISA-test An ti-glia din IgA kit or An ti-gliad in Ig G k it Enzyme-linked immunosorbent ass ay ki t For in vitro diagnostic use only Instructions for us e LABMASTER D I A G N O S T I C S PO BOX 71, FIN TU R KU, FINLAND TEL FAX INTENDED USE Lab ma st e r EL I SA-t est Anti-gliadin IgA kit is int e nd ed fo r th e determination of hu m an anti-gliadin IgA in serum. Lab ma st e r EL I SA-t est Anti-gliadin IgG kit is int e nd ed fo r th e determination of hu m an anti-gliadin IgG in serum. These tests have been designed to be used as sensitive screening tests for jejunal biopsy in patien t s with su sp e ct ed coe liac disea se. The mo nito r ing of the disappearance of gliadin antibodies during gluten-free diet can be used to indicate successful elimination of glut e n in th e die t. These tests are also useful in th e scr ee ning of dermatitis herpetiformis. 16 1

2 Sin ce g liadin antib od ies are formed also in some other conditio ns, e.g. cow`s milk alle rg y, Cro hn `s dise a se or oth er in flammatory intestinal diseases, the diagnosis of coeliac disease cannot be based solely on g liadin antib od ies, but an in testinal bio psy must always be taken to confirm th e dia g no sis. SUMMARY AND EXPLANATION OF THE ASSAY Coeliac dise a se is cha ra cte rize d by the pre sen ce of villou s atrophy in a jejunal biopsy specimen. The disease is caused by hyp er se n sitivit y to glut e n, and with d ra wa l of glu ten fr o m th e die t cures the patient. Hig h pre va lence ( %) of coeliac dise a se has bee n rep o rt ed in juve nile (t yp e I) diab e tes mellitus (1,2). The ove ra ll lifetime in cid en ce of coe liac dise a se has be en estima t ed 1:300-1: 2000 (3,4). On ce diag n osed coe liac pa tie nt s ar e advise d to follow gluten-free diet. St rict diet a ry com p lian ce is re co mm e nd ed to avoid long-term risks associated with coeliac dise a se - po or ge ne ra l health and growth, malabsorption, and the risk of developing int estin al lymp ho ma (5,6). Alth o ug h coe liac disease can present and con se qu e nt ly be dia g no se d at alm ost any sta g e in life, many patients are dia g no se d in ch ildhood. In children under 2 years of age coeliac disea se ma y pre se nt with classical symp toms, i.e. severe gastrointestinal symptoms with faecal fat excretion, anaemia, folate deficie n cy, and maln ut rit ion, but in older children and adults typical sym pt om s a re ra re ly se en. According to the crite ria of th e Eur o pe an So ciet y for Pe diat r ic Gastroenterology (ESPGAN) the diagnosis of coeliac dise a se in child re n must be co n firm e d by a th re e -b iopsy procedure to exclude tra nsie n t villous atrophy due to milk allergy or other causes (7). The ESPGAN procedure includes biopsies before and after a gluten-free diet an d aft e r ch allen ge. This is la b or ious and can be stressful to th e child, and therefore alternative dia g no st ic methods are needed. 2 During the la st few ye ar s it ha s beco me evid en t tha t th e determination of g liadin antib od ies in the serum offers a co n ve nie nt non-invasive laboratory method for this purpose (8,9,10). Mea su re m en t of bo th IgA and IgG antibodies is important in the detection of IgA-deficient coeliac pa tie nt s. In cr ea sed I ga g liadin antibodies ha ve also bee n cha ra cte ristic fo r adu lts with coeliac disea se (10). PRINCIPLES OF THE ASSA Y Lab ma st e r Elisa (En zym e- linked immunosorbent assay) tests of anti-gliadin IgA and anti-gliadin IgG are ba se d on th e san d wich technique. In the first incubation specific IgA and IgG antibo die s in patient se ru m rea ct with g liadin co at e d to a m icrotiter well. Af t er washing gliadin/iga and g liadin/igg -co m plexe s re ma in. In the second incubation inorganic pyr op ho sph at a se lab elle d anti-iga reacts with the bound IgA or inorganic pyrophosphatase labelled anti-i gg antibody reacts with the bound IgG (11). After incubation the nonrea ct ed lab elle d antibodies are washed away. In en zym e re a ct ion the in or ga nic p yr op ho sph at a se cle a ve s one pyrophosphate molecule to two ph o sp ha te molecules. The Colour Solution terminates the reaction and phosphate molecules form coloured complexes with ammonium molybdate and malachite gre en. The intensity of the for m ed colour is directly proportional to th e level of anti-gliadin IgA or anti-gliadin IgG in sample. KIT CONTE NTS Each La b ma st e r EL ISA -test Anti-gliadin IgA kit an d Ant i- gliadin IgG kit contains reagents for 96 wells, sufficient for blank, positive re f er en ce, neg a tive co nt ro l and 45 sa m ples in dup licate. The e xp iry date of th e com p lete pa ckag e is st a ted on th e out er label, and the expiry date of each component on its own label. Sto re at C. 3

3 R EAGENTS Specific r eagents : Anti-gliadin IgA kit : Anti-gliadin IgG kit : Positiv e ref e re nc e s er um Pos itiv e ref e re nc e s er um Wash Concentrate One bottle 40 ml A 25-fold co n ce ntra te of Tris-HCl b uffer ed ( p H 7.75) Contains 0.95 % sodium azide as pre s er va tive. s alt s olu tion with Twe en 2 0. pre dilu ted and re ad y for u se p re dilu ted and re ad y for use DO NOT DILUTE DO NOT DILUTE containing anti-gliadin IgA containing anti-gliadin IgG One vial 2.0 ml One vial 2.0 ml Negative control serum Negative control serum pre dilu ted and re ad y for u se p re dilu ted and re ad y for use DO NOT DILUTE DO NOT DILUTE One vial 2.0 ml One vial 2.0 ml The se s o lutio ns c on trols are in Tris-HCl b uffer ed s a lt solution with bo vine albumin, inert re d d ye, a nd 0.05 % sodium azide as pre s er va tive. Tracer Solution Tracer Solution Enzyme-lab elle d anti-iga Enzyme-lab elle d anti-igg Ready for use. Ready for use. One vial 12 ml O ne vial 12 ml Contains IgA specific anti- serum Contains IgG specific anti- serum (rabbit) in Tris-HCl b uffer ed salt ( ra bb it) in Tris-HCl b uffer ed salt solution with bovine ser u m s olutio n with bovine serum albumin, inert re d d ye, a nd 0.05 % a lbumin, inert re d d ye, a nd 0.05 % sodium azide as pre s er va tiv e. s od ium azide as pre s er va tive. se ru m Substrate Concentrate One bottle 0.4 ml A 200 -fo ld Tris-HCl b uffer ed ( p H 9.0 ) co nc e ntra te of inorganic pyr o ph os p ha te. Contains 0.02 % Thime ro s al a s pre se r va tive. Substrate Buffer One bottle 20 ml Tris-HCl b uffer ed ( p H 9.0 ) so lution for substrate Thime ro s al a s pre se r va tive. Colour Solut ion Ready for use. One bottle 10 ml d ilution. C on tain s Colour and stop solution for en z yme r ea ctio n. NOTE: Contains Sulfuric acid (12 %, c or rosiv e) and st aining c olour. Gliadin coated microtitration strips One Plate (12 x 8 wells) 0.02 % C ommon reagents : Sample D ilution Solution Ready for use. Two bottles 50 ml ea ch Tris-HCl b uffer ed ( p H 7.75), sa lt solution with bovine serum albumin, in e rt r ed dy e, and 0.05 % sodium azide as pre s er va tive. 4 M ATERIALS REQUIRED BUT NOT SUPPLIED WITH THE KIT Lab ma st e r EL I SA-t est req u ires the following items. 1. Microtit er plat e/ st r ip re ad ing photometer, which ca n me a su re absorbance at 620 nm wavelength. (Wa velen gt h s be t we en nm are possible to use ). 2. Precision pipettes for dispe n sing m icroliter volu me s. 5

4 3. Te st tu be s for sa mp le dilutions (2-3 ml) and a bottle to dilute wash concentrate. 4. A multichannel pipette or dispenser (microliter volume s) fo r dispensing the su bst ra te solutio n an d the colour solution 5. Distilled wa ter 6. Automatic platesh a ke r Automatic platewa she r is usef u l an d imp ro ves t e st results. SPECIMEN COLLEC TION Collect blood by venipuncture, allow to clot and separate the serum by cen t rifu ga t ion. Sa mp les can be sto r ed at C fo r on e wee k an d at -20 C for at least one year. WARNINGS AND PREC AUT IONS For in vit ro diag no stics use. These kits contain reagents man u fact u re d fr o m hu m an blo o d components. The sou r ce ma terials have been tested by immunoassay for hepatitis B surface antigen and antibodies to HIV virus and found to be negative. Ne ve r theless all recommended pre ca ut ions for the handling of blood derivative sh ou ld be ob se rved. Please refer to the U.S. Department of He alt h an d Hum an Se rvice s (Bethesda, MD., USA) pu blica t ion No. (CDC) on lab or a tory safety procedures or any ot he r local or national regulation. Handle all patient specimens as potentially in fectious. Reagents contain sodium azide (NaN 3 ) and thimerosal (so dium e thylmercurithiosalicylate) as preservative. Sod ium azide may react with lead and copper plumbing to formhighly explosive metal azides. On disposal, flush with a large volume of water to pre ve nt a zide bu ild- u p. Ple a se re fer to the decontamination procedures as outlin ed by U.S. Cen ters for Disease Control (Atlanta, GA.,USA) PREPARATION REAGENTS ASSAY PR EPA RATION Wash Solutio n Pou r th e 40 ml of W a sh C o nc en trate in to a clean contain e r an d d ilute 25- fold by adding 960 ml distilled water to giv e a bu ffe re d wash solution. Sub stra te Solution Pre pa re th e n ee de d v olume concentrate to 2 ml of substrate buffer for ev er y (10 µl+2 ml /2 strips ; 60 µl + 12 ml/ whole plate). o f su b stra te solutio n b y dilu tin g 1 0 µl strip to be use d same day. o f s ub s trate 2. DILUTION OF SAMPLES Dilute serum samples to sample dilution solution 1/100 with calibrated d ilutor or pip ette s ( e g. 10 µ L se r um µl dilu tion solutio n o r 20 µl s er u m µl dilution solution ) NOTE: The s a me s ample dilutions c an be use d w ith both anti-gliadin IgA and anti-gliadin IgG kits. NOTE: Positiv e re fe r ence s a nd ne ga tiv e cont r ol a r e pre dilut ed a nd re ady f or use, DO NOT DILUTE THEM. 3. PREPARATION OF MICROTITER STRIPS Transfer the re qu ired nu mbe r of mic ro titration strips to a strip frame. (Return re ma ining strips to the plastic bag and reseal) Wash each strip three times with the wa sh solution. Do not wash more strips tha n ca n be easily h an dle d with in 30 minutes. ASSAY PROCEDURE Perform ea ch de terminatio n in du plica te for both refere n ce s and unknowns. Blank, positive reference and negative co nt ro l sho u ld be run wit h each assay. All re ag en t s an d sam ple s mu st be br ou gh t to room temperature ( C) before use. 7

5 Anti-gl i ad in Ig A ki t : Ant i-gl i ad in IgG kit : 1. Pipette 1. Pipette 100 µl sample dilution solution 100 µl sample dilution solution in (blank) in duplicate, (blank) du plica te, 100 µl anti-gliadin IgA po sitiv e 100 µl anti-gliadin IgG positive reference (POS REF) in du p licate, reference (POS REF) in d up licate, and a nd 100 µl negative control (NEG CON) 100 µl negative control (NEG CON) in duplicate. in duplicate. NOTE: Anti-gliadin IgA kit and NOTE: Anti-gliadin IgA kit and anti-gliadin IgG kit ha ve a nt i-gliadin IgG kit ha ve separate positive refe re nce s, s epar at e positive refe re nce s, DO NOT MIX THEM! DO NOT MIX THEM! Pip ette Pip ette 100 µl diluted patient serum 100 µl diluted patient serum samples (un kn ow n s - ) in samples (unk no wn s - ) in duplicate acc o rd ing to t he d up licate according to the follo win g ta b le. follo win g ta b le A blank blank 6th B C D E F G POS REF NEG CON 1st 2nd 3rd 4th POS REF NEG CON 1st 2nd 3rd 4th etc. 6th etc. 2. Incubate th e pla te fo r 1 hour with 2. In c ub ate the plate for 1 hour with continu o us sha kin g at room c on tinu o us s ha king at room temperature. temperature. 3. After th e inc ub a tion ste p, a sp irate 3. Afte r th e in c ub ation ste p, aspirate and wash eac h s trip 6 times with a nd wash each strip 6 times with washing solution. Use auto ma tic washing solution. U se auto ma tic platewasher if possible. platewasher if possible. 4. Add 100 µl anti-iga tr ac er 4. Add 100 µl anti-igg tr ac er solution to each well. solution to each well. 5. Incubate th e pla te for 1 hour with 5. In c ub ate the plate for 1 hour with continuous shaking at room continuous shaking at room temperature. temperature. 6. Wash each strip 6 times according 6. Wash each strip 6 times according to step 3. to step 3. Following procedure is the same in both k its: 7. Pip ette 100 µ L s ub str ate so lutio n ( pr ep a re d s ame da y a cc o rd ing to th e instructions above) to each well. NOTE: U s e multicha nnel pipe tt e or dis pe ns e r and r emar k t he or de r of pipetting substrate to wells. Incubate the plate for exactly 20 minutes with shaking at ro o m te mp e ra ture. 8. Add 50 µl colour solution to each well in the same order as the substrate solution, us e multich a nn el pipe tte o r d ispe ns e r (mix th e c olou r s olution g en tly prior to u se ). Sh ak e the fr ame g en tly with p late sh a ke r for 5 minu tes and a llow to sta nd for 10 minutes befo re measuring. NOTE: Do not touc h the surf a ce of subst ra t e s olution with c olour dis pe ns ing unit. 9. Mea su re th e a bs or ba n ce o f wells with microtiter plate/strips rea ding ph oto me ter using wavelength 620 nm ( nm). Use bla n k fo r zero adjustment. 10. Calculate the results. H 5th 5th 8 9

6 Calculation of results : Calculate the mean absorbance of each pair of wells. (Diminish the mean absorbance of sample dilution so lutio n [b lank] fro m th e abso rb an ce of other wells, if yo ur re ad e r do es no t allow ze ro adjustment with the blank wells). Exp re ss th e me a n ab so r ba nce of the negative co nt ro l and the mean absorbance of unknown samples as arbitrary units (AU), i. e. as percentage of the positive reference. Sensitivity a nd specificity : (> 2years) Cut o ff value 25 AU 5 0 AU IgA IgG IgA + IgG Sensitivity 93.4% 82.9% 96.4% Specificity 81.2% 77.8% 89.8% Sensitivity 82.4% 63.6% 89.3% Specificity 95.5% 87.5% 97.2% Mea n ab sor ba n ce of Negative con t ro l x 100 = Level of neg a tive control in AU Mea n ab sor ba n ce of Positive reference Mea n ab sor ba n ce of Sam ple x 100 = Level o f unknown sample in AU Mea n ab sor ba n ce of Positive reference Interpretation of results : Anti-gliadin IgA kit and Anti-gliadin IgG kit: Following table can be used as a guideline to establish cut-off values for the two age groups. The cut-off values are determined to give optimal diagnostic sensitivity and specificity both for anti gliadin- IgA and -IgG antibodies. Cut-off values: AGE ( ye a rs ) N eg ativ e W ea k po s itive Positive < 2 < > 100 > 2 < > The se re su lts are based on a study in Finland with 144 patients with coeliac dise a se con f irmed with jejunal biopsy and 176 normal serums in Finland. EXPECTED VALUES Goo d pr a ctice dicta t es t h at each laboratory establishes it s own exp ecte d range of values. LIM ITATION OF PROCEDUR E Sin ce g liadin antib od ies are formed also in some other conditions, e.g. cow`s milk alle rg y, Cro hn `s dise a se or oth er inflammatory in testinal disea se s, th e dia gn o sis of coe liac dise a se cannot be based solely on g liadin antib od ies, but an in testinal bio psy must always be ta ke n to confirm the diagnosis. PROCEDURAL NOTES 1. A th o ro ug h und er sta nd ing of this package insert is necessary for successful use of the Labmaster Elisa-test anti-gliadin IgA and anti-gliadin IgG. Th e rea g en ts su pp lie d with th is kit ar e intended for use as an integral unit. Do no t mix id en ti ca l rea g en ts from kits having different lot numbers. Do not use kit reagents after expiry da te printed on th e kit la be l. 11

7 2. Do no t mix re ag en ts from An ti -g liadin IgA and Antigliadin IgG kits. Ho we ve r the sa me sa mp le dilutio ns ca n be used wit h bo t h kit s. 3. Re ag e nt s sho uld be allowed to rea ch ro om te mp e ra ture ( C) prior to the sample preparation. Fro ze n spe cim en s should be brought to room temperature slowly and they should be mixed gently by hand. Do not vortex or vigorously mix pa ti e nt spe ci me n s. 4. In th e wa shing pr oced u re all we lls should be filled with washing solution (approx. 300 µl ). Wait 2-3 se co n ds and th en em pt y all wells pr o pe rly. Re pe a t th is as many times as mentioned in the assay p r oced u re. 5. After washing th e str ips, ch e ck th at the we lls are dry. If the re is moistu re left, inver t the plate and tap firmly ag ain st abso rb en t paper. 6. Ma ke su re yo u o nly use the re ag en t s fr o m th is kit and pro pe r distille d or d eion ized water. As in th is test system the mea su re m en t is ba se d on pho sp ha t e mo lecules formed, all con ta mi n at ion by ph o sp ha t e e. g. a ph o sp ha te bu ff e r ba se d washing solution from other kits or home made washing solution wil l lead to malfunction of this kit. PER FORMANCE CHARA CTERISTIC S Precision: Int ra -a s sa y ( within run n=1 0) precision is shown below. Anti-gliadin IgA kit Sample AU %C V Anti-gliadin IgG kit Sample AU %CV Int er -a s sa y ( be tw ee n r un n= 10 ) pre cis ion is shown below. Anti-gliadin IgA kit Sample AU %CV Anti-gliadin IgG kit Sample AU % C V

8 REFEREN CES : 1. Mäki M, H älls tröm O, Huupponen T, Vesikari T, Vis ak or p i JK. Increased pre va lence of coeliac dise a se in dia be tes. Arch Dis Child 1984, 59, WARRANTY The performance data presented here were obtain e d u sin g t h e assay procedure indicated. Any change or modification of the procedure not re co mm e nd ed b y Lab ma st e r Oy may af fect the results, in which event Labmaster Oy disclaims all warranties. 2. Collin P, Salmi J, H älls tröm O, Oksa H, Oksala H, Mäk i M, R eu na la T. H igh fre qu en c y of c oe liac dise a se in a du lt patients with type-i diabetes. Scand J Gastroe n terol 1989, 24, Halle rt C, G otth ar d R, N or rb y K, W alan A. O n the p r ev alen c e of ad ult c oe liac dis ea se in Sw ed en. Sca nd J Ga str oe nter o l 1981, 16, Mylotte M, Eg an -Mitc he ll B, McCarthy CF, McNicholl B. In cide n ce o f c oe liac dis ea se in the West of Ireland. Br Med J 1973, 1, Co ok e WT, Ho lmes GKT. Coeliac dise a se. Lo n do n: Ch ur ch ill L ivingstone: Walker-Smith J. D isea s es o f the s mall in testin e in c h ildh o od. 3r d e d. Sev en oa k s: Butterworths, 1988, McN eish AS, H ar ms H K, R ey J, Shmer ling DH, Vis ak or p i JK, W alk er -Smith JA. The d iagno sis o f c oe liac dise a se : a commentary o n the cu rr en t p ra c tice s o f th e membe rs of th e Eu ro p ea n Soc iety for Pediatric Gastroe n terolog y an d N utr ition (ESPGAN). Arch Dis Child 1979; 54, Savilahti E, Viander M, Perkkiö M, Vainio E, Kalimo K, Reunala T. IgA anti-gliadin antibodies : a mar ke r o f muc os al da ma g e in c h ildh oo d c oe liac dise a se. La n ce t 1983; 1, Stå hlbe r g M- R, Sav ilahti E, Via nd er M. An tibo die s to g liadin by ELISA as a scr ee nin g te s t fo r c hild h oo d ce lia c d isea se. J Ped iatr Gastroe n terol Nutr 1986, 5, Volta U, L en zi M, L az za ri R, C as sa ni F, C ollina A, Bian ch i FB, Pis i E. An tibodies to g liadin detecte d by immun ofluo re s ce nc e a nd a micr o- ELISA me thod : markers of activ e c hild h oo d an d adult coelia c dise as e. Gu t 1985, 26, Baykov AA, Kahsno VN, Avaeva SM. Ino rg an ic pyr op ho s ph ata se as a label in heterogenous en zy me immu n oa ss ay. Anal. Bioc he m 1988; 171 :

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