CAPTIA TM Syphilis (T. Pallidum)-G

Transcription

1 CAPTIA TM Syphilis (T. Pllidum)-G Pour d'utres lngues Für ndere Sprchen Pr otrs lengus Per le ltre lingue Dl innych języków Tests Tests INTENDED USE Syphilis (T. pllidum)-g is n enzyme immunossy for the qulittive detection of IgG ntiodies to T. pllidum in serum specimens, to e used in conjunction with non-treponeml testing to provide serologicl evidence of infection with T. pllidum ( the gent of syphilis) Syphilis (T. pllidum)-g is lso intended for testing of serum or plsm specimens to screen lood nd/or plsm donors to exclude history of syphilis. For in vitro Dignostic Use Only For Professionl Use Only Wrning: A positive result is not useful for estlishing dignosis of syphilis. In most situtions, such result my reflect prior treted infection; negtive result cn exclude dignosis of syphilis except for incuting or erly primry disese. INTRODUCTION Syphilis is disese, usully sexully trnsmitted, cused y infection with the spirochete Treponem pllidum (T.pllidum). Infection is systemic from the outset nd the disese is chrcterized y periods of ltency, often in excess of twenty yers. These fetures, together with the fct tht T. pllidum cnnot e isolted in culture men tht serologicl techniques ply mjor role in the dignosis of syphilis nd tretment follow-up. 1 The procedures most commonly used to screen for ntiodies to T. pllidum in clinicl dignostic lortories re sed upon their rection with non-treponeml lipoidl ntigens (the regin tests). Regin tests, such s the RPR or VDRL, cn e used to test seril dilutions of the serum specimen. The end point vlues from sequentilly otined serum smples decline following successful tretment until fter period of severl months the ptient will usully ecome regin test non-rective. Clinicl dignostic serum specimens which re rective in regin tests re typiclly confirmed using treponeml tests such s the Microhemgglutintion-T. pllidum () or the Fluorescent Treponeml Antiody-Asorption (FTA-ABS) test. In contrst to the non-treponeml tests, treponeml test rectivity will persist following tretment in pproximtely 85% of the cses often for the life of the ptient. 2 Any ser giving rective or equivocl results on initil treponeml sed ssys must e supplemented with quntittive non-treponeml test (such s RPR or VDRL) to distinguish from ctive disese nd ssist in ruling out flse positives. Donors of lood nd/or plsm for trnsfusion re screened for T. pllidum ntiodies using either regin or treponeml tests. The detection of T. pllidum ntiodies is used to help identify donors who present n incresed risk of trnsmitting lood-orne infections. Syphilis-G is treponeml test for T. pllidum IgG clss ntiodies. 3,4 The enzyme immunossy formt llows use of microplte reder which elimintes sujective interprettion of results nd the test procedure cn e utomted for high-volume testing. PRINCIPLE OF THE ASSAY Microtitrtion wells coted with T. pllidum ntigens re exposed to test specimens which my contin specific ntiodies. After n incution period, unound components in the test smple re wshed wy. Specificlly-ound IgG rects with conjugted horserdish peroxidse (HRP) monoclonl ntiody (ma) during second incution period. Following second wsh cycle, specificlly-ound enzyme conjugte is detected y rection with TMB (tetrmethylenzidine). The enzymtic rection is stopped using 1 N Sulfuric cid. The ssy is mesured spectrophotometriclly to indicte the presence or sence of IgG treponeml ntiodies. MATERIALS SUPPLIED Pr outrs língus Για τις άλλες λώσσες För ndr språk For ndre språk KIT PRESENTATION Syphilis-G regents supplied in this pck re for in vitro dignostic use only. 1. Coted microwell strips. Plstic microtitrtion wells coted with whole-cell sonicted T. pllidum ntigens, strin Nichols. (96T: one plte; 960T: ten pltes) 2. High titre rective control (HTR): contining humn serum diluted in uffer contining <0.1% sodium zide s preservtive. (96T: one ottle, 1.5 ml; 960T: two ottles, 1.5 ml ech) 3. Low Titre rective control (LTR): contining humn serum diluted in uffer contining <0.1% sodium zide s preservtive. (96T: two ottles, 1.8 ml ech; 960T: four ottles, 1.8 ml ech) 4. Non-rective control (N): contining humn serum diluted in uffer contining <0.1% sodium zide s preservtive. (96T: one ottle, 1.5 ml; 960T: two ottles, 1.5 ml ech) 5. Conjugte. HRP leled ma in uffer contining 0.01% romonitrodioxne s preservtive (96T: one ottle, 20 ml; 960T: five ottles, 20 ml ech) 6. Wsh Buffer (conc.x 20). Phosphte uffered sline ph , contining 0.05% Tween 20. (96T: one ottle, 100 ml; 960T: two ottles, 500 ml ech) 7. Smple Diluent. Phosphte uffered sline, ph , contining protein stilizer, 0.05% Tween 20 nd < 0.1% ProClin 300 s preservtive (96T: one ottle, 115 ml; 960T: two ottles, 500 ml ech) 8. CAPTIA Sustrte Solution: Redy to use. Slight lue colortion. Tetrmethylenzidine (< 0.1%TMB) ph The regent should remin closed when not in use. If llowed to evporte, precipitte my form in the regent wells. (96T: one ottle, 15 ml; 960T: ten ottles, 15 ml ech) 9. Plte Selers ADDITIONAL REQUIREMENTS FOR MANUAL PROCESSING 1N (0.5M) Sulfuric cid. Add 2.77 ml concentrted H2SO4 (18M/36N) to ml distilled or deionized wter. Mix well. Disposle tip micropipettes to deliver 5 L, 10 L, 25 L, 100 L nd 200 L (multichnnel pipette preferred for dispensing regents into microtiter pltes). Distilled or deionized wter. 37 C incutor. Disposle tues of pproximtely 1.0 ml cpcity, for performing specimen dilutions. Types which cn e rcked in configurtion comptile with Microtitrtion pltes re recommended. Clen, disposle glss/plstic test tues pproximte cpcities 5 ml nd 10 ml. Rnge of stndrd, clen volumetric lortory glsswre consisting of, t lest, 15 ml nd 100 ml ekers, 1 ml, 5 ml, nd 10 ml glss pipettes. Asorent pper towels. Automtic microtitrtion plte wsher or lortory wsh ottle. Microtitrtion plte reder with 450 nm filter. Ltex gloves, sfety glsses nd other pproprite protective grments. Biohzrd infectious wste continers. Sfety pipetting devices for 1 ml or lrger pipettes. Timer. Plte Shker. Vcuum line with trp. AUTOMATIC, OR SEMI-AUTOMATIC PROCESSING Syphilis-G my e used with vriety of utomtic or semi-utomtic processors/liquid hndling systems. It is essentil tht ny such system is qulified, efore it is used routinely, y demonstrting tht the Syphilis-G results otined using the utomtic processor re equivlent to those otined for the sme specimens using the mnul test method. Susequently the utomtic processor should e periodiclly requlified y the end user. STORAGE AND STABILITY All regents should e stored t 2-8 C nd should not e used eyond the expirtion dte on the lel. Once opened, microtitrtion strips my e stored t 2-8 C until the expirtion dte on the lel, provided tht desiccted conditions re mintined. Unused strips should e returned to their originl foil pouch long with the schet of desiccnt. Opened pouches should e securely reseled y folding over the open end nd securing it with dhesive tpe. Microwell strips not needed for the ssy my e returned to the plte pouch nd seled, nd then used t lter time. Strips from different pltes cn only e mixed to ssemle full or prtil pltes if they re from the sme plte lot nd hve come from pltes tht hve previously een tested with kit controls nd yielded vlid runs. When ssemling plte tht contins strips from newly opened, previously untested plte, one of these strips should e plced t the eginning of the plte nd tested with the kit controls. The concentrted Wsh Buffer contins no preservtives nd, therefore, working-strength Wsh Buffer should not e stored for longer thn 4 weeks t 2-8 C. It is recommended tht Wsh Buffer e freshly diluted efore ech ssy. If the working strength uffer ecomes visily cloudy or develops precipitte during the 4 weeks, do not use it. Indictions of Deteriortion Syphilis-G my e considered to hve deteriorted if: 1. The kit fils to meet the required criteri for vlid test (see INTERPRETATION). 2. Regents ecoming visily cloudy or develop precipitte. Note: Concentrted wsh uffer, when cold, normlly develops crystlline precipittes which redissolve on heting t 37 C. 3. The Sustrte Solution is lue. This is likely to e cused y chemicl contmintion of the Sustrte Solution or the continer. Plese note Sustrte Solution when supplied hs slight lue colortion. SAFETY WARNING AND PRECAUTIONS For In Vitro Dignostic Use Only. For Professionl Use Only The control ser in this kit contin < 0.1% sodium zide s preservtive. Sodium zide cn rect with led nd copper pluming to form potentilly explosive metl zides. Upon disposl, flush with lrge volume of wter to prevent zide uild-up. Cution: All lood products should e treted s potentilly infectious. Source mteril from which kit control ser were derived ws found negtive when tested in ccordnce with current FDA recommendtions. No known test methods cn offer ssurnce tht products derived from humn lood will not trnsmit infectious gents. The Treponem pllidum ntigen hs een inctivted during the production processes. Nevertheless, treponeml ntigen-coted microtitrtion wells should e hndled using the norml precutions ccorded to potentilly infectious mteril. Sulfuric cid is corrosive. Avoid contct with skin nd eyes. If splshing onto skin or eyes occurs, rinse the ffected re with copious quntities of wter nd seek medicl ttention. PROCEDURAL 1. This kit should e used in strict ccordnce with the instructions in the Pckge Insert Pge 1 of 7 EN

2 2. enzyme immunossy kits contin regent systems which re optimized nd lnced for ech kit lot. Do not interchnge regents from kits with different lot numers. Do not interchnge vil cps or stoppers either within or etween kits. 3. Do not use Syphilis-G kits fter the expirtion dte printed on the outer crton lel. 4. Do not cross-contminte regents. Alwys use fresh pipette tips when drwing from stock regent ottles. 5. Alwys use clen, preferly disposle, glsswre for ll regent preprtion. 6. Allow foil to wrm to room temperture efore opening. This voids condenstion on the inner surfce of the g which my contriute to deteriortion of coted strips intended for future use. 7. Regents should e dispensed with the tip of the micropipette touching the side of the well t point out mid-section. Follow mnufcturer's recommendtions for utomtic processors. 8. Alwys keep the upper surfce of the microtitrtion strips free from excess fluid droplets. Regents nd uffer overspill should e lotted dry on completion of the mnipultion. 9. Do not llow the wells to completely dry during n ssy. 10. Disposl or decontmintion of fluid in the wste reservoir from either the plte wsher or trp for vcuum line in the mnul system should e in ccordnce with guidelines set forth in the Deprtment of Lor, Occuptionl Sfety nd Helth Administrtion, occuptionl exposure to lood-orne pthogens; finl rule (29 CFR 1910,1030) FEDERAL REGISTER, pp ,12/6/ Automtic or semi-utomtic EIA processors or liquid hndling systems should e qulified specificlly for use with Syphilis-G y demonstrtion of equivlence to the mnul processing methods. 12. Consistent with good lortory prctice, it is recommended tht ll pipetting devices (mnul or utomtic) re regulrly clirted ccording to the mnufcturer's instructions. 13. Cre must e tken to ensure tht specimens re dispensed correctly to ech test well. If specimen is indvertently not dded to well, the result for tht well will e non-rective, regrdless of the ctul result of the specimen. METHOD FOR USE SPECIMEN COLLECTION AND STORAGE Syphilis-G is intended for use with serum, EDTA or citrted plsm smples. If serum specimens re to e stored, they my e stored t 2-8 C for up to five dys. However, if storge periods greter thn five dys re nticipted, the serum specimens should e stored frozen t -20 C or elow. Specimens which hve een frozen nd thwed should e thoroughly mixed efore testing. If plsm specimens re to e stored, they my e stored t 2-8 C for up to 48 hours. Plsm specimens should not e frozen due to firin clot formtion. Optiml performnce of the Trinity Biotech ELISA kit depends upon the use of fresh serum/plsm smples (cler, non-hemolyzed, nonlipemic, non-icteric). If serum specimens re to e shipped, they should e pckged nd leled in complince with federl nd interntionl regultions covering the trnsporttion of clinicl specimens nd etiologic gents. Serum specimens my e shipped mient, refrigerted (2-8 C) on wet ice or frozen (-10 C or colder) on dry ice. Upon rrivl, if serum specimens re to e stored, they my e stored t 2 to 8 C for up to five dys fter collection or frozen (-20 C or colder). The NCCLS provides recommendtions for storing lood specimens (Approved Stndrd Procedures for the Hndling nd Processing of Blood Specimens, H 18-A. 1990). RINSE CYCLE Efficient rinsing to remove uncomplexed smple components is fundmentl requirement of enzyme immunossy procedures. Syphilis-G utilizes two stndrd five-rinse cycles. Automtic plte wshers my e used providing they meet the following criteri: 1. All wells re completely spirted. 2. All wells re filled to the rims (350 L) during the rinse cycle. 3. Wsh uffer is dispensed t good flow rte. 4. The microtitrtion plte wsher must e well mintined to prevent contmintion from previous use. Mnufcturer s clening procedures must e followed diligently. 5. Vlidted y end user For ech rinse cycle, the mchine should e set to five consecutive wshes. On completion of the cycle, invert the microtitrtion plte nd tp firmly on sorent pper towels. Check for ny residul wsh uffer in the wells nd lot dry the upper surfce of the wells with pper towel. Alterntively, the following mnul system my e employed: 1. Aspirte well contents using vcuum line fitted with trp. 2. Fill ll wells to the rim with wsh uffer dispensed from squeeze-type lortory wsh ottle. 3. Aspirte ll wells. 4. Repet Steps 2 nd 3, four times. 5. Invert the microtitrtion plte nd tp firmly on sorent pper towels. PREPARATION FOR THE ASSAY Specimen Dilution Test specimens should e tested following 1:21 dilution with Smple Diluent. This dilution cn e performed off-plte in tues, or directly in the microtitrtion well. Tue dilution is recommended for dignostic l use. In-well dilution is recommended for high volume (lood nks) screening. Tue method This is most ccurtely performed in disposle tues of ml cpcity, which cn e rrnged in 8 x 12 - plce rcks tht conform to microtitrtion plte geometry. This fcilittes lter trnsfer of diluted specimens from the tues to the test microtitrtion plte using multichnnel micropipette. Dispense 1.0 ml of Smple Diluent into ech tue. Add 50 L of test specimen nd mix 6 to 8 times. Use fresh disposle micropipette tip for ech serum specimen. In-well method Dispense 200 L of Smple Diluent into ll the wells designted for test specimens. Dispense 10 L of ech specimen into its designted well. Pump mixture in nd out of the micropipette tip 3 times to ensure full delivery. When dispensing of test specimens into the microtitrtion plte is complete nd the working strength controls hve lso een dispensed, mix the well contents thoroughly y shking on mechnicl microtitrtion plte shker for 5-10 seconds, or y mixing in-proe if utomtic EIA processors re used. Thorough mixing is essentil. Kit Controls Note: The kit controls provided re working strength nd do not require dilution. The kit controls should e included on ech microtitrtion plte. The Low Titre Rective Control should lwys e tested in duplicte. Additionlly, it is recommended tht well chrcterized low titre rective specimen, or n independent reference smple, diluted in Smple Diluent, e included in ech microplte set-up. Wsh Buffer The 20 x concentrted Wsh Buffer my hve developed crystlline deposits during prolonged storge t 2-8 C. These should e redissolved y stnding the ottle in 37 C wter th until the crystls dispper. Prepre working-strength Wsh Buffer y diluting 1 prt concentrte with 19 prts of distilled or deionized wter. If kit is likely to e utilized over period in excess of 4 weeks, then it is recommended tht only enough stock concentrte e diluted sufficient for immedite needs (see STORAGE AND STABILITY). Ech row of 8 wells my e dequtely wshed with 150 ml of working strength uffer. ASSAY PROCEDURE 1. Allow ll regents to rech room temperture (18-25 C). 2. The kit controls should e included on ech microtitrtion plte. The Low Titre Rective Control should lwys e tested in duplicte. 3. Select sufficient microtitrtion well strips to ccommodte test specimens nd controls. Fit the strips into the holding frme. Lel wells ccording to specimen: identify using the letter/numer cross-reference system molded into the plstic. 4. If the tue dilution method hs een used, dispense 100 L of ech diluted serum or plsm specimen into the correspondingly pre-leled wells. Mix the diluted specimen 6 to 8 times using the 100 L micropipette, prior to trnsfer. Use fresh micropipette tip for ech diluted specimen. Dispense 100 l. of the kit control into the designted wells. Alterntively use the in-well dilution method descried ove. If this method is used, dispense 200 L of the kit control into the designted wells. If the in-well dilution method hs een used, gitte the plte on mechnicl microtitrtion plte shker for 5-10 seconds or mix in-proe if utomtic EIA processor is used. 5. Sel the strips nd holding frme with plte seler nd incute t 37(± 1) C for 60 (± 5) minutes. 6. Aspirte diluted specimen from the wells nd wsh the microtitrtion plte s descried in the Rinse Cycle section. 7. Dispense 100 L conjugte into ech well, sel the strips with plte seler nd incute t 37(± 1) C for 60 (± 5) minutes. 8. Aspirte the conjugte from the wells nd wsh the microtitrtion plte s descried in the Rinse Cycle section. 9. Without dely, dispense 100 L of sustrte solution into ech well. A multichnnel pipette should e used for est results. Leve t room temperture (18-25 C) protected from direct sunlight, for 30 (±2) minutes. 10. Stop the rection y dding 100 L of stop solution (1 N sulfuric cid) to ech well. Mix on plte shker for 5 to 10 seconds, or tp lightly. This is to ensure tht the lue solution chnges to uniform yellow color in ech well. Ensure tht the undersides of the wells re dry nd tht there re no ir ules in the well contents. 11. Within 30 minutes of dding the cid, red the sornce vlues t 450 nm using microtitrtion plte reder lnked on ir unless the mnufcturer specificlly recommends otherwise. ASSAY VALIDATION INTERPRETATION A Syphilis-G ssy should e considered vlid if: The sornce of the Non-rective Control (N) is less thn or equl to The sornce vlue of the High Titre Rective Control is greter thn or equl to 0.8. The men sornce vlue of the Low Titre Rective Control (LTR) is greter thn or equl to 2.5 x the sornce of the Non-rective Control. TROUBLESHOOTING ADVICE If the kit controls fil to give the sornce vlues indicted ove, the following points should e considered: 1. Ensure tht the detils given in Procedurl hve een reviewed. 2. For High Asornce Non-rective Control (>0.25): Ensure tht the wshing procedure detiled in Methods for Use: Rinse Cycle ws followed. If using n utomtic wsher, ensure ll inlet nd outlet proes re not locked nd tht ll wells re eing filled nd spirted fully. 3. For Low Asornce High Titre Rective Control (<0.8): Check tht the correct incution conditions (time nd temperture) were chieved. Pge 2 of 7 EN

3 Ensure tht ll residul wsh uffer hs een removed from the wells fter use. 4. For LTR/N rtio <2.5, check ll points detiled in (1) to (3) of this section. After considertion of the points ove, repet the ssy. If the kit controls gin fil to vlidte, contct your supplier for further dvice. ANALYSIS Clculte the men sornce vlue of the duplicte Low Titre Rective Controls. This is the cut-off vlue for Syphilis-G nd ws derived from clinicl trils s the vlue giving optimum discrimintion etween specimens which re rective or non-rective for ntiodies to T. pllidum s chrcterized y rnge of stndrd serologicl techniques. Specimen sornce vlues within 10% of the men of the Low Titre Rective Controls should e considered equivocl results. A specimen my e considered rective for IgG ntiodies to T. pllidum if it gives n sornce vlue greter thn the men of the Low Titre Rective Controls nd outside the equivocl rnge. A specimen my e considered non-rective for IgG ntiodies to T. pllidum if it gives n sornce vlue less thn the men of the Low Titre Rective Controls nd outside the equivocl rnge. It is often useful to scrie numericl vlue to specimen which represents its Syphilis-G rectivity so tht comprisons cn e mde etween different ssys. Such vlue is derived y expressing the sornce of the test specimen s rtio of the men sornce of the kit Low Titre Rective Controls. For exmple: Test serum sornce = 0.75 Men LTR* sornce = 0.30 Antiody Index 0.75 = *LTR - Low Titre Rective Control An Antiody Index etween 0.9 nd 1.1 should e considered equivocl. An Antiody Index greter thn or equl to 1.1 is rective result nd n Index less thn or equl to 0.9 is non-rective result. A Syphilis-G rective result (following rective nontreponeml test result in the dignostic ppliction) indictes current or pst infection with T. pllidum. A non-rective result indictes sence of infection t ny time more thn 2-3 weeks previous to drwing the specimen. (See Limittions of Use). SYPHILIS-G USED AS A CLINICAL LAB TEST. Initilly rective or equivocl results should e repet tested in duplicte. If the repet test is gin equivocl fresh serum specimen should e tested. The Syphilis-G is treponeml ssy, therefore ptients with previously treted syphilis will e positive on the ssy. The test cn not distinguish etween present nd pst infection. Any ser giving rective or equivocl results on initil testing must e supplemented with quntittive nontreponeml test (such s RPR nd VDRL) to distinguish ctive disese nd ssist in ruling out flse positives. The following tle is used for result interprettion: Non-Treponeml Result (NT) Treponeml Result Cpti Syphilis - G Report/interprettion for ll except neontes or infnts. 2 nonrective negtive (nonrective) No serologicl evidence of infection with T. Pllidum (incuting or erly primry syphilis cnnot e excluded). rective negtive (nonrective) Current infection unlikely, proility of iologicl flse positive secondry to other medicl conditions (ferile diseses, immuniztions, IV DU, utoimmune diseses, etc.). Recommend repet testing (nontreponeml nd treponeml y other test method). nonrective positive (rective) Proly pst infection or potentil crossrectivity with other spirochetes/relted ntigens; dditionl testing pproprite to clinicl findings/history; 1 possiility of flse negtive nontreponeml (NT) due to prozone nd lte syphilis or neurosyphilis. rective positive (rective) Presumptive evidence of current infection (or indequtely treted infection, persistent infection, reinfection, or iologicl flse positive if prior history); dditionl testing consistent with clinicl ssessment. 1 nonrective not done Current infection unlikely; effectively treted infection if previous dignosis nd tretment; cnnot exclude incuting or erly primry syphilis; cnnot exclude ltent or neurosyphilis. not done negtive (nonrective) Current or pst infection unlikely; cnnot exclude incuting or erly syphilis. 1 Quntittive nontreponeml testing; clinicl history; repeted (sequentil) serologicl testing for chnges in titer. HIV-infected individuls my hve delyed serorectivity or negtive serology. SYPHILIS-G USED AS A PRIMARY BLOOD OR PLASMA DONOR SCREEN FOR BLOOD BANKS AND PLASMAPHARESIS CENTERS. Initilly rective or equivocl results should e repet tested in duplicte. Where plsm specimens hve een tested initilly, serum specimens re preferred for retesting. If the repet test is gin rective or equivocl the specimen should e referred for confirmtion to Syphilis Reference Lortory. Specimens which give repet equivocl results y should e considered rective until confirmtory testing (RPR, VDRL) hs een done. In prctice, equivocl results re found in less thn 0.5% of specimens tested (see Tril 9 in Performnce Chrcteristics). SYPHILIS-G USED AS A DIAGNOSTIC CONFIRMATORY TEST Specimens giving equivocl results should e retested in duplicte. If the result is gin equivocl, fresh serum specimen should e tested. When retesting ser giving equivocl results on initil testing, the smple should e tested using nontreponeml test nd nother treponeml test. A second serum smple should lso e otined (one drwn t lter dte) if results of the nontreponeml nd treponeml tests do not resolve the dignosis. EXPECTED VALUES The percentge of specimens rective y Syphilis-G is dependent upon the popultion from which the specimens were derived. It cn e expected tht specimens derived from 'high risk' ptients (e.g. those ttending genitourinry clinics) will show higher rectivity rte thn those derived from low risk popultion (e.g. lood donors). Also the numer of specimens rective will e dependent upon the type of lortory crrying out the testing - Reference Lortory testing smples tht my lredy hve een screened for syphilis y serologicl ssy will hve higher incidence of rective specimens thn routine Clinicl Lortory testing specimens for the first time. From the results presented in the section Performnce Chrcteristics it could e expected tht Clinicl Lortory testing referred smples nd routine specimens including those from genitourinry clinics my hve rectivity rte of pprox. 4.5% (61/1321 -Tril 1). From the dt presented in Tril 9, it could e expected tht Blood Testing Center would otin pprox. 0.8% (73/9323) rective specimens using Syphilis G. LIMITATIONS OF USE 1. Results from Syphilis-G should e considered in the context of ll ville clinicl nd lortory dt. 2. A Syphilis-G non-rective result does not preclude the possiility of: very recent infection (within the lst 2-3 weeks) with T. pllidum. n old, successfully cured infection with T. pllidum (for exmple >10 yers previous). 3. Syphilis-G my e rective with ser from ptients with Yws (T. pllidum suspecies pertenue) or Pint (T. crteum). 4. Detection of treponeml ntiodies my indicte recent, pst, or successfully treted syphilis infections, therefore, the test cnnot e used to differentite etween ctive nd cured cses. 5. When performing clinicl l ssys, ny ser giving rective or equivocl results must e supplemented with quntittive nontreponeml test (such s RPR nd VDRL) to distinguish ctive disese nd ssist in ruling out flse positives. The Syphilis-G is treponeml ssy, therefore ptients with previously treted syphilis will e positive on the ssy. 6. The use of Syphilis-G s n initil screening test for lood donors my result in higher numers of rective donors who my not e currently infected compred to screening donors with stndrd, nontreponeml ssys. It is recommended tht Syphilis-G repet rective specimens e tested y methods cple of indicting the current disese sttus of the donor e.g. RPR nd VDRL tests. 7. AIDS ptients with impired immunity nd who re coinfected with syphilis my rect flsely nonrective in treponeml nd nontreponeml tests. 8. Rective treponeml IgG ntiody test results usully remin rective for lifetime, therefore ntiody indices cnnot not e used to determine response to therpy. PERFORMANCE CHARACTERISTICS CLINICAL LAB TEST APPLICATION Comprison with other serologicl tests. Syphilis-G hs een evluted t numer of independent clinicl lortories. Serum specimens routinely referred for syphilis serology were nlyzed y Syphilis-G in prllel with hemgglutintion () nd VDRL tests. Specimens giving rective results y ny test were further tested y the FTA-ABS procedure. The tles elow summrize results from initil testing t two tril centers. In ll cses rective is defined s serum specimen which gives rective result y either the or VDRL test nd which is confirmed y the FTA-ABS test. Tril 1 Totl 1321 Specimens SYPHILIS-G T. pllidum ntiody sttus R N Rective 60 1 d Non-rective Reltive Sensitivity 98.4% Reltive Specificity 99.3% N = Non-rective R = Rective c % 99.5% VDRL e NA N/A Note: Equivocls scored s rective. Confirmed untreted primry infection, rective y Syphilis-G. Includes 7 specimens in equivocl rnge. c On repet testing confirmed s non-specific gglutintion, therefore re inconclusive. d Wekly rective cse of treted ltent syphilis. e The proportion of this specimen group representing cses of successfully treted syphilis, which would not normlly e VDRL rective, is not known. Tril 2 Totl Specimens T. pllidum ntiody sttus SYPHILIS-G R N VDRL Pge 3 of 7 EN

4 Rective Non-rective Reltive Sensitivity Reltive Specificity N = Non-rective % 100% R = Rective % 97.1% N/A N/A Note: Equivocls scored s rective. Includes 2 serum specimens which, on repet testing, were confirmed s cusing non-specific gglutintion, therefore re inconclusive. The proportion of this specimen group representing cses of successfully treted syphilis, which would not normlly e VDRL rective, is not known. Tril 3 Rection with serum specimens clssified ccording to stge of disese. The following tle summrizes Syphilis-G results using serum specimens tken from ptients t vrious stges of the disese nd following tretment. Dignosis ws sed on clinicl history together with serologicl dt from VDRL nd FTA-ABS tests. 3 Numer Rective y Test Syphilis Ctegory Numer of Specimens Syphilis-G VDRL FTA-ABS Untreted Primry Secondry Erly Ltent Lte Ltent Neurosyphilis Congenitl Reinfection Treted Unchrcterized Dt Not Aville Totl: 168 Sensitivity: 165/168 = 98.2% * Clculted from untreted cses only Tril 4 77/96 = 80.2%* 96/96 = 100%* The Centers For Disese Control nd Prevention nlyzed serum smples from high risk popultion using Syphilis-G, the RPR nd FTA-ABS tests. Serum smples were from ptients with primry, secondry or ltent infections nd ptients with no history of syphilis known to give flse rective rections in nontreponeml tests. RPR FTA-ABS Syphilis G STAGE PRIMARY untreted treted SECONDARY untreted treted LATENT untreted treted NONSYPHILIS N=non-rective R=rective These serum smples were otined from individuls without syphilis, ut who hd diseses known to cuse flse rective results in the nontreponeml tests. Sensitivity: RPR FTA-ABS Syphilis-G untreted 11/12=92% 12/12=100% 12/12=100% treted 17/19=90% 19/19=100% 19/19=100% overll 28/31=90% 31/31=100% 31/31=100% Specificity Inpproprite 18/18=100% 17/18=94% Agreement of CAPTIA" Syphilis-G with FTA-ABS: 48/49= 97.96% Agreement of RPR with FTA-ABS 40/49= 81.63% Tril 5 Comprison with the RPR test 585 serum specimens routinely sumitted to clinicl serology lortory, were tested y RPR nd Syphilis-G. Serum specimens yielding rective or equivocl results y either method were further tested y the FTA-ABS procedure. Of the 585 specimens tested, only 69 were initilly rective, nd then tested in the FTA-ABS procedure. The remining 516 serum specimens were non-rective in oth the RPR test nd Syphilis-G. RPR Crd Syphilis G FTA-ABS Numer with Pttern N N N 7 N R R 15 R R R 37 N 3 N E R 2 N E N 1 N N R 1 N 3 N=non-rective R=rective E=Equivocl Agreement etween Syphilis-G nd the FTA -ABS test results Syphilis-G Rective Syphilis-G Non-rective FTA-ABS Rective 54 1 FTA-ABS Non-rective 4 10 Equivocl results scored s rective. Agreement = 64/69 or 92.7% Tril 6 Clinicl Sensitivity A pnel of frozen retrospective chrcterized ser otined from the CDC (Centers for Disese Control) were ssyed on the Syphilis-G y Trinity Biotech. The pnel consists of 100 ser with clinicl dignosis of Syphilis with different stges of disese. Treted nd untreted ptients re included in ech disese stge. The following tle illustrtes the performnce of the ssy with the serum pnel. The dt is presented for informtion on the ssy with chrcterized serum pnel nd does not infer n endorsement of the ssy y the CDC. Results of the CDC Serum Pnel on the Cpti Syphilis G Disese + E - Totl % Positive Stge Primry Treted % Primry Untreted % Secondry Treted % Secondry Untreted % Lte Treted % Lte Untreted % Totl % Tril 7 Clinicl Sensitivity A pnel of frozen retrospective chrcterized ser were ssyed on the Cpti Syphilis-G y pulic helth lortory in the United Kingdom. The pnel consists of 200 ser with clinicl dignosis of Syphilis with different stges of disese. Treted nd untreted ptients re included in ech disese stge. The following tle illustrtes the performnce of the ssy with the serum pnel. Results of the chrcterized Serum Pnel on the Cpti Syphilis G Disese + E - Totl % Positive Stge Primry Treted % Primry Untreted % Secondry Treted % Secondry Untreted % Lte Treted % Lte Untreted % Totl % Provided elow is Summry Tle of Trils 3, 4, 6 nd 7 of dt otined from collections which were chrcterized y syphilis disese sttes: Summry Tle from Trils 3, 4, 6, 7 Ptient Ctegory # Ptients RPR/VDRL + - Syphilis-G Results + +/- - Syphilis-G Rective nd RPR or VDRL Rective RPR or VDRL Rective nd FTA Rective Untreted Syphilis Primry Secondry Ltent Treted Syphilis Primry Secondry Ltent FTA not performed on 8 smples 2 FTA not performed on 4 smples 3 FTA not performed on 7 smples 4 FTA not performed on 2 smples Pge 4 of 7 EN

5 Tril 8 RPR Positive nd FTA Negtive Ser Two sites (Pulic Heth Ls locted in New York nd Mrylnd) tested 25 frozen retrospective ser on the Syphilis G tht were RPR positive on initil screen nd FTA negtive on confirmtion. The following tle summrizes the results. Syphilis Positive Syphilis Equivocl Syphilis Negtive % Negtive Site % Site 2 0 2* 23 92% *The two equivocls were negtive with repet testing. Tril 9 Blood nd Plsm Donor ScreeningAppliction Comprison with the RPR test Syphilis-G ws evluted t 2 mjor US lood centers nd plsm center, in comprison with the RPR test. A totl of 9,323 donors were tested s plsm specimens y Syphilis-G nd s serum specimens y the RPR test. 152 of the specimens were known rective from previous serologicl testing which did not include the FTA-ABS test. 4,274 of these donors were dditionlly tested s serum specimens using Syphilis-G. Initilly-rective specimens were repet tested in duplicte. Repet-rective specimens were confirmed using n FTA-ABS test. The following tles compre the Syphilis-G nd RPR efore nd fter reconcilition of discordnt results y the FTA test. Tests Plsm Specimens: Syphilis-G, RPR nd FTA-ABS Results RPUMBER FTA-ABS Syphilis-G Initil Repet Rective Non-rective R R E R E N N R N N TOTALS N=non-rective R=rective E=Equivocl Of 241 smples tht were repet rective in either or oth Syphilis-G nd RPR tests, 196 were FTA rective, nd 45 were FTA non-rective. Syphilis-G (Tests Plsm Specimens) RPR Rectives nd Discordnts Reconciled y FTA-ABS Rective Non-rective Rective Non-rective Rective Non-rective * * % Agreement % - Reltive Sensitivity 133/156 = 85.26% 195/196 = % Reltive Specificity 9082/9167 = 99.01% 9104/9127 = % * includes 9082 specimens which were nd RPR concordnt non-rective nd therefore not tested y FTA-ABS. 65/85 were FTA rective 22/23 were FTA non-rective. Equivocls were scored s rective. In this study, there were 25 specimens (0.27%) initilly equivocl y nd 16 specimens (0.17%) repet equivocl. Tests Serum Specimens. Syphilis-G, RPR nd FTA-ABS Results RPUMBER FTA-ABS Syphilis-G Initil Repet Rective Non-rective R R E R E N N R N N TOTALS N=non-rective R=rective E=Equivocl Of 191 smples tht were repet rective in either or oth tests ( nd RPR), 167 were FTA-ABS rective nd 24 were FTA-ABS non-rective. (Tests Plsm Specimens) RPR Rectives nd Discordnts Reconciled y FTA-ABS Rective Non-rective * * % Agreement % - Reltive Sensitivity 119/136 = 87.5% 167/167 = 100 % Reltive Specificity 4083/4138 = 98.67% 4100/4107 = % * includes 4083 specimens which were nd RPR concordnt non-rective nd therefore not tested y FTA-ABS 51/55 rective y FTA All non-rective y FTA Equivocls were scored s rective. In this study, there were 18 specimens (0.42%) initilly equivocl y nd 14 specimens (0.33%) repet equivocl. Comprison with n utomted (hemggiutintion) test Syphilis-G ws evluted t two mjor US lood centers, in comprison with commercilly ville utomted system (PK-TP test). A totl of 6,196 donors were tested s plsm specimens y Syphilis-G. 2,156 of these donors were dditionlly tested s serum specimens y Syphilis-G. The initil tests were performed with plsm specimens, nd repet tests were performed with serum specimens. Specimens which were repet rective in either test were confirmed using n FTA-ABS test. The following tles summrize the results of ll tests nd compre nd efore nd fter reconcilition of discordnt results y the FTA-ABS tests. Tests Plsm Smples: Syphilis-G, nd FTA-ABS Results RPUMBER FTA-ABS Syphilis-G Initil Repet Rective Non-rective R R R E E R E E E N N R N E N N TOTALS N=non-rective R=rective E=Equivocl Of 124 smples tht were repet rective in either or oth tests ( nd ), 105, were FTA-ABS rective nd 19 were FTA-ABS non-rective (Tests Plsm Specimens) Rectives nd Discordnts Reconciled y FTA-ABS Rective Non-rective * * % Agreement % - Reltive Sensitivity 93/98 = 94.90% 104/105 = 99.05% Reltive Specificity 6072/6098 = 99.57% 6076/6091 = 99.75% * includes 6072 specimens which were nd concordnt non-rective nd therefore not tested y FTA-ABS 11/26 FTA Rective 4/5 FTA Non-Rective Equivocls were scored s rective. In this study there were 18 specimens (0.29%) initilly equivocl y, nd 9 specimens (0.15%) repet equivocl. Tests Serum Smples: Syphilis-G, nd FTA-ABS Results NUMBER FTA-ABS Syphilis-G Initil Repet Rective Non-rective R R R E E R E E E N N R N E N N TOTALS N=non-rective R=rective E=Equivocl Of 98 smples tht were repet rective in either or oth tests ( nd ), 95 were FTA-ABS rective, nd 3 were FTA-ABS non-rective. (Tests Plsm Specimens) Rectives nd Discordnts Reconciled y FTA-ABS Rective Non-rective * * % Agreement 99.63% - Reltive Sensitivity 90/94 = 95.74% 94/95 = 98.95% Reltive Specificity 2058/2062 = 99.81% 2061/2061 = 100% * includes 2058 specimens which were nd concordnt non-rective nd therefore not tested y FTA-ABS. All rective y FTA 3/4 non-rective y FTA Equivocls were scored s rective. In this study, there were 10 specimens (0.46%) initilly equivocl y, nd 9 specimens (0.42%) repet equivocl. Pge 5 of 7 EN

6 Syphilis-G: comprison of performnce with plsm nd serum specimens A totl of 4,274 serum/plsm pirs were tested t 2 mjor US lood centers nd plsm center using Syphilis-G. Results re summrized in the following tle. 4,258 pirs (99.62%) gve concordnt results on initil testing (equivocl scored s positive). Eleven (11) specimen pirs gve discordnt results on repet testing. Of these 11 repet discordnt pirs, the FTA-ABS test confirmed the plsm result for 6 pirs, nd the serum results for the remining 5 pirs Syphilis-G Numer FTA-ABS Serum Plsm Initil Repet Rective Non-rective Rective Rective Rective Equivocl Rective Non-rective I I I Equivocl Rective Equivocl Equivocl Equivocl Non-rective Non-rective Rective Non-rective Equivocl Non-rective Non-rective Totl Tested Totl Rective SPECIFICITY AND CROSS-REACTIVITY The following tle summrizes Syphilis-G results from serum specimens tken from sujects with no known history or serologicl evidence of syphilis. This Group included serum specimens representing other disese sttes nd/or chrcteristics known to cuse flse rectives in other serologicl tests for syphilis. CATEGORY OF SPECIMEN n Syphilis-G Rective* Norml nte-ntl HBsAg Rective 68 1 ALT Ser from HBV Vccines 11 0 HIV 1/2 Antiody Rective 32 0 HCV Antiody Rective HTLV 1 Antiody Rective 34 0 Heterophile Antiody Glndulr Fever) 17 0 Rective Rheumtoid Fctor R 33 1 Systemic Lupus E 22 1 Autoimmune/Connective Tissue Disese 16 0 Regin Test Flse Rective 66 0 Lyme's Disese 34 1 Genitl Herpes 10 0 Acute Leptospirosis 10 0 Ser from Intrvenous Drug 39 0 Hypergmmgloulinemi Miscellneous** 18 0 Totl Specimens 1690 Totl Representing Known Disese 688 Totl Rective/FTA Non-Rective 8 * FTA Non-rective ** Miscellneous included 2 specimens from ptients with rthritis nd scleroderm, nd one specimen ech from ptients with lzheimer; rthrlgi; spergillosis; coelic disese; colitis C4 depressed; gout; immune complex infection; mcrogloulinemi; myelom (unspecified); myelom IgA; myelom IgG; myelom light chin; nephrotic syndrome nd cute renl filure. REPRODUCIBILITY The reproduciility of Syphilis-G ws evluted concurrently t 3 seprte US lood/plsm centres. Ech centre tested 6 stndrd serum smples, replicted x 3 in ech ssy, on ech of 5 dys. The serum smples comprised: 2 x high titre rective serum specimens; 2 x low titre (ner the cut-off) rective serum specimens nd 2 x non-rective serum specimens. Results re summrized in the following tle: SAMPLE NUMBER PARAMETER Intr-ssy Men ntiody index: 15 runs: 3 sites Rnge of within run %CV: 3 sites Inter-ssy Rnge of inter-ssy %CV from 5 runs: 3 sites The reproduciility of Syphilis-G ws evluted t two seprte Pulic Helth Ls. Ech centre tested 6 stndrd serum smples, replicted x 3 in ech ssy, on ech of 5 consecutive dys. The sme 6 smples were then replicted x 3 in ech ssy, seprted y one week intervls for five weeks. The lst two ssys were ech performed with different lots of kits. The serum smples comprised: 2 x high titre rective serum specimens; 2 x low titre (ner the cut-off) rective serum specimens nd 2 x non-rective serum specimens. Results re summrized in the following tle: SAMPLE NUMBER PARAMETER Intr-ssy Men ntiody index: 20 runs: 2 sites Rnge of within run %CV: 2 sites Inter-ssy Rnge of inter-ssy %CV from 20 runs: 2 sites Smple #3 ws equivocl 4/60 times Smple #4 ws equivocl 10/60 times All other ser remined in the sme sttus 60/60 times CAPTIA SYPHILIS-G SUMMARY OF ASSAY PROCEDURE Note: Red the full product instruction leflet efore strting the ssy. This summry is for quick reference only. 1. Dilute 1 prt Wsh Buffer concentrte with 19 prts distilled wter. 150 ml is sufficient to wsh 1 x 8 well row. 2. Dilute the test ser y dding 50 L to 1000 L (1.0 ml) Dilution Buffer III in disposle tues. Do NOT dilute kit controls. Incution One: 3. Dispense into leled wells I00 L of the (i) diluted test ser (ii) Kit Low Titre Rective Control IN DUPLICATE (iii) Kit High Titre Rective Control (iv) Kit Non Rective Control 4. Sel the strips with plte seler. Incute t 37(±1) C for 60(±5) minutes. 5. Aspirte the ser from the wells. Wsh the plte five times. Ensure there is no residul fluid in the wells Incution Two: 6. Pipette 100 L working strength conjugte into ech well. 7. Sel the strips with plte seler. Incute t 37(±1) C for 60 (±5) minutes. 8. Aspirte the conjugte from the wells. Wsh the plte five times. Ensure there is no residul wsh uffer in the wells. Incution Three: 9. Dispense 100 L of sustrte solution into the wells. 10. Incute t room temperture for 30 (±2) minutes. 11. Add 100 L 1N sulfuric cid to ech well. Tp the plte or mix on plte shker for 5 to 10 seconds until the lue solution chnges to uniform yellow. 12. Within 30 minutes, lnk plte reder on ir nd red the sornce of Kit Controls nd test ser t 450 nm. The sfety dt sheet is ville upon request. WARNING: Smple Diluent contins < 0.1% ut > 0.05% ProClin 300, iocidl preservtive tht my cuse sensitiztion y skin contct; prolonged or repeted exposure my cuse llergic rection in certin sensitive individuls. WARNING H317: My cuse n llergic skin rection. P280: Wer protective gloves / protective clothing / eye protection / fce protection. P302 + P352: IF ON SKIN: Wsh with plenty of sop nd wter. P333 + P313: If skin irrittion or rsh occurs: Get medicl dvice/ ttention. P501: Dispose of contents nd continer in ccordnce to locl, regionl, ntionl nd interntionl regultions. Prepred in ccordnce with requirements for EEC lel. EINECS REFERENCES 1. World Helth Orgniztion Technicl Report Series. No.674 (1982) Treponeml infections. 2. Schroeter A.L., Lucs J.B., Price E.V., nd Flcone V.H. (1972). Tretment for erly syphilis nd rectivity of serologic tests. Journl of the Americn Medicl Assocition, 221: Leferve J.C., Bertrnd M.A. nd Burtud F.L.(1990). Evlution of the CAPTIA Enzyme lmmunossys for Detection of lmmunogloulins G nd M to Treponem pllidum in Syphilis. Clinicl Microiology, 28, Young H., Moyes A., McMilln A. nd Roertson D.H. (1989). Screening for treponeml infection y new enzyme immunossy. Genitourinry Medicine, 65: U.S. Deprtment of Helth nd Humn Services. Biosfety in microiologicl nd iomedicl lortories. HHS Puliction (NH) , Wshington U.S. Government Printing Office, My World Helth Orgniztion Lortory Biosfety Mnul, Genev, World Helth Orgniztion, Advisory Committee on Dngerous Pthogens. Ctegoriztion of pthogens ccordirig to hzrd nd ctegories of continment. London, HMSO, Second Edition, Pge 6 of 7 EN

7 KIT Ctlog No ORDERING INFORMATION Item Cpti Syphilis-G Test Kit Cpti Syphilis-G Test Kit Cpti Syphilis-G Test Kit Quntity 96 Tests 960 Tests Mnufctured CONTROL + + High titre rective control (HTR) Authorized Representtive CONTROL + Low Titre rective control (LTR) Consult ccompnying documents CONTROL - Non-rective control (N) REF Product Numer LOT Lot CAL Clirtor C.F. Coefficient Fctor Use y RNG Rnge Cution, consult ccompnying documents Store t 2-8ºC STD Stndrd IVD For In Vitro Dignostic use Store t 2-30ºC or Hzrd Trinity Biotech USA Jmestown, NY Tel Fx: Trinity Biotech plc Bry Co. Wicklow, Irelnd Tel Fx /2013 Pge 7 of 7 EN