PLATELIA RUBELLA IgM TMB 96 TESTS 72922

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1 PLATELIA RUBELLA IgM TMB 96 TESTS QUALITATIVE DETECTION OF IgM ANTI-RUBELLA VIRUS IN HUMAN SERUM OR PLASMA BY ENZYME IMMUNOASSAY IVD All manufactured reagents are prepared according to our Quality System, starting from reception of raw material to the final commercialization of the product. Each lot is submitted to quality control assessments and is only released to the market, after conforming to pre-defined acceptance criteria. The records relating to production and control of each single lot are kept within Bio-Rad.

2 TABLE OF CONTENTS 1- CLINICAL VALUE PRINCIPLE OF THE TEST PRODUCT INFORMATION WARNINGS AND PRECAUTIONS SPECIMEN COLLECTION, PREPARATION, AND STORAGE ASSAY PROCEDURE MATERIALS REQUIRED NOT PROVIDED REAGENTS RECONSTITUTION STORAGE OF OPENED AND/OR RECONSTITUTED REAGENTS PROCEDURE INTERPRETATION OF RESULTS QUALITY CONTROL CALCULATION OF THE CUT-OFF VALUE CALCUL OF SAMPLE RATIO INTERPRETATION OF THE RESULTS TROUBLE SHOOTING GUIDE CALCULATION EXAMPLE LIMITATIONS OF THE PROCEDURE SPECIFIC PERFORMANCE CHARACTERISTICS COMPARATIVE STUDY SPECIFICITY STUDIES SENSITIVITY STUDIES PRECISION CROSS REACTIVITY PREVALENCE REFERENCES

3 1- CLINICAL VALUE Rubella is a viral illness with worldwide distribution. Rubella infection is predominantly a mild disease in children and adults. Clinical manifestations include a low-grade fever, headache, sore throat, and a generalized skin rash. However, Rubella infection during pregnancy is more serious, with well documented multiple congenital complications, including deafness, cataracts, mental retardation, and fetal death. Aggressive immunization of pre-school children has greatly reduced the incidence of Rubella epidemics, but a need still exists for accurate monitoring of immune status, especially for women of child-bearing age. Demonstration of Rubella IgG antibody in women prior to conception provides assurance of fetal protection from possible Rubella viral infection during pregnancy. Vaccination efficiency can also be determined by detection of Rubella IgG antibody in serum following immunization. Since the isolation of Rubella virus in 1962, the detection of specific Rubella antibodies has been of great interest due to the teratogenic risk of the virus. Several test methods have been developed in the past including serum neutralization, complement binding, and immunofluorescence. These assays are either difficult to perform in a routine laboratory setting, or yield unstable or irregular results. Hemagglutination inhibition techniques allow a rapid diagnosis of both the acute infected state and patient immune status. Engvall and Perlmann described the first enzyme immunoassay procedures in The development of the enzyme immunoassay procedure has contributed to an improved specificity and sensitivity in the detection of a wide variety of antigens and antibodies. Interpretations of successive serological tests, demonstrating the presence of IgM, the apparition or a significant increase in IgG antibody titer (doubling of the titer) in two serum samples obtained at a minimum interval of three weeks should be considered as evidence of exposition to Rubella virus even if clinical pathognomonic signs of this infection are not present. 2- PRINCIPLE OF THE TEST PLATELIA Rubella IgM TMB is an immunoenzymatic assay, with capture of the IgM antibodies on the solid phase (microplate wells) coated with anti human -chain antibodies. Test sera and calibrators are diluted and placed into the wells of the microplate. During this incubation, IgM present in the sample are captured by the solide phase. After incubation, IgG and serum proteins are removed by washings. 3

4 A mixture of the rubella antigen (Rub Ag) and anti-rubella monoclonal antibody labelled with peroxidase (MAB-Per) is deposited in each microplate well. During this second incubation, if the test sample contains anti-rubella IgM )A!/ s, these IgM!/ s captured on the solid phase fix the (Rub Ag - MAB-Per) complex. Non-fixed or excess Rub Ag and MAB-Per are eliminated by repeated washing at the end of this incubation period. The presence of the immune complex is revealed by the addition of substrate in each well. The enzymatic reaction is stopped with sulphuric acid. The result, the optical density obtained at 450/620 nm read over a cutoff value, indicates the presence or absence of anti-rubella virus IgM in the test serum. 3- PRODUCT INFORMATION For storage conditions and expiration date refer to the indications mentioned on the box. Label Reagents Quantity R1 Microplate Microplate (Ready to use): 1 12 strips with 8 break-away wells each, coated with anti human -chains antibodies. R2 Concentrated Concentrated Washing Solution (10x): 1 x 100 ml Washing TRIS-NaCl buffer (ph 7.4), 1 % Tween 20. Solution Preservative: 0.01% Thimerosal R3 Negative Negative control: 1 x 1ml Control TRIS-NaCl buffer (ph 8 )A!@ 0.2), bovine serum albumin, glycerol, E102 and E122. Preservative: 0.2% ProClin TM 300. R4a Cut-off Cut-off control (human): 1 x 1ml Control TRIS-NaCl buffer (ph 8 )A!@ 0.2), human serum IgM reactive against Rubella Ag, bovine serum albumin, glycerol, E102 and E122. Preservative: 0.2% ProClin TM 300 and < 0.01 % Thimerosal R4b Positive Positive control (human) : 1 x 1ml Control TRIS-NaCl buffer (ph 8 )A!@ 0.2), human serum IgM reactive against Rubella Ag,, bovine serum albumin, glycerol, E102 and E122. Preservative: 0.2% ProClin TM 300 and < 0.01 % Thimerosal 4

5 R6a Rubella Rubella Antigen (lyophilised): 4 x 3 ml Antigen Preservative: 0.01 % Thimerosal R6b Conjugate Concentrated Conjugate (50x): 1 x 0.3 ml Murine monoclonal antibody anti-rubella coupled with horseradish Peroxidase. Preservative: 0.01 % Thimerosal R7 Diluent Diluent (Ready to use): 2 x 80 ml TRIS-NaCl buffer (ph 7.7 )A!@ 0.15), bovine serum albumin, 0.1% Tween 20 and phenol red. Preservative: 0.01 % Thimerosal R8 Substrate TMB Substrate Solution (Ready to use): 1 x 60 ml Buffer Solution of citric acid and sodium acetate (ph 4.0), containing 0.015% H 2 O 2 and 4% DMSO. R9 TMB Concentrated TMB Chromogen (11x): 1 x 5 ml Chromogen: 0.25% tetramethylbenzidine (TMB) solution. R10 Stopping Stopping Solution (Ready to use): 1 x 28 ml Solution 1N sulfuric acid solution Plate Sealers 4 4- WARNINGS AND PRECAUTIONS The reliability of the results depends on correct implementation of the following Good Laboratory Practices: Do not use reagents after expiry date. Do not mix or associate reagents from different lots within a given test run. Washing Solution (R2), Substrate Buffer (R8), Chromogen (R9) and Stopping Solution (R10) are interchangeable between lots, provided the same lot is used within a given test run. Carefully reconstitute the reagents avoiding any contamination. Do not carry out the test in the presence of reactive vapors (acid, alkaline, aldehyde vapors) or dust that could alter the enzyme activity of the conjugate. The enzyme reaction is very sensitive to metal ions. Consequently, do not allow any metal element to come into contact with the various conjugate or substrate solutions. The diluted chromogen solution (substrate buffer + chromogen) must be colorless. Do not use the solution if a blue color appears within a few minutes after reconstitution. Prepare this solution in a clean disposable single use plastic or glass container that has first been pre-washed with 1N HCl, and rinsed thoroughly with distilled water and dried. Store the solution in the dark. 5

6 Use a new pipette tip for each sample. Washing the microplate is a critical step in the procedure: follow the recommended number of washings cycles and make sure that all wells are completely filled and then completely emptied. Incorrect washings may lead to inaccurate results. Do not allow the microplate to dry between the end of the washings operation and the reagent distribution. Never use the same container to distribute conjugate and development solution. Check the pipettes and other equipment for accuracy and correct operation. 6 HEALTH AND SAFETY INSTRUCTIONS Wear disposable gloves when handling reagents. Do not pipette by mouth. Human origin material used in the preparation of the reagents has been tested and found non-reactive for hepatitis B surface antigen (HBs Ag), antibodies to hepatitis C virus (anti-hcv) and to human immunodeficiency virus (anti-hiv1 and anti-hiv2). Because no method can absolutely guarantee the absence of infectious agents, handle reagents of human origin and patient samples as if capable of transmitting infectious disease. Rubella Ag has been inactived by beta-propiolactone. Any material, including washings solution, that comes directly in contact with samples and reagents containing materials of human origin, should be considered as if capable of transmitting infectious disease. Avoid spilling, but if in the event of a spill with an acid, it must be neutralized with sodium bicarbonate, then cleaned with bleach at the concentration of 12 )A!c diluted to 1/10 and dried absorbent paper. The material used for cleaning must be discarded in a contaminated residue container. Patient samples, reagents containing human origin material, as well as contaminated material and products should be discarded after decontamination only. Do not introduce solutions containing sodium hypochlorite into the autoclave. Avoid skin and mucous membrane exposure to the substrate buffer, chromogene, and wash solution, since this can cause toxicity, irritation, or burn lesions.

7 CAUTION : Xi - Irritant ProClin TM 300 : < 0.2 % : IRRITANT PRODUCT R43 May cause sensitization by skin contact. S28/37 After contact with skin, wash immediately with plenty of water and soap. Wear suitable gloves. The Material Safety Data Sheet (MSDS) is available upon request. 5- SPECIMEN COLLECTION, PREPARATION, AND STORAGE 1. Serum or plasma (EDTA, citrate, and heparin) are the recommended sample types. 2. Observe the following recommendations for the handling, processing, and storing blood samples: - Collect all blood samples observing routine precautions for venipuncture. - For serum, allow samples to clot completely before centrifugation. - Keep tubes stoppered at all times. - After centrifugation separate the serum or plasma from the clot or red cells in a tightly stoppered storage tube. - The specimens can be stored at +2-8 )A!c C if screening is performed within 7 days. - If the assay will not be completed within 7 days, or for shipment of samples, freeze at )G!9 20 "x C, or colder. - Thaw samples only three times. - After thawing, previously frozen specimens should be thoroughly mixed prior to testing. 3. Samples containing 90 g/l albumin, 100 mg/l bilirubin, lipemic samples containing the equivalent of 36 g/l triolein (triglyceride), and hemolyzed samples containing up to 10 g/l hemoglobin do not affect the result. 4. Do not heat the samples. 6- ASSAY PROCEDURE 6.1 MATERIALS REQUIRED NOT PROVIDED Vortex mixer. Microplate reader equipped with 450 nm and 620 nm filters (*). Water bath or equivalent microplate incubator, thermostatically set at 37 )A!@ 1!c C (*). Manual, semi-automatic or automatic microplate washer (*). 7

8 Container for biohazard waste. Sodium hypochlorite (bleach) and sodium bicarbonate. Germ-free distilled or deionized water. Graduated cylinders of 25, 50, 100 and 1000 ml capacity. Disposable gloves. Goggles or safety glasses. Absorbent paper. Automatic or semi-automatic, adjustable or preset, pipettes or multipipettes to measure and dispense 10 to 1000 l and 1, 2 and 10 ml. Disposable tubes. (*) Consult our technical department for detailed information about the recommended equipment. 6.2 REAGENTS RECONSTITUTION R1: Cut the bag cm above the line. Return unused strips into the bag immediately. Check for dessicant. Re-seal the bag carefully and store it at +2-8 )A!c C. R2: Dilute 1/10 in distilled water. Prepare 350 ml for one plate of 12 strips if washings manually. R6a: Reconstitute the lyopilised antigen by adding 3 ml of diluent (R7) to one vial of antigen. Mix thoroughly. Waiting 15 minutes before mixing with R6b allows an homogenous rehydratation of the antigen. R6b: Prepare the necessary quantity of conjugate for one run by adding one volume of concentrated conjugate to 50 volume of diluent (R7). R6: Mix equal volumes of reconstituted R6a and R6b. R9: Dilute 1/11 with R8, example: 1 ml of R ml R STORAGE OF OPENED AND/OR RECONSTITUTED REAGENTS R1: Once the bag has been opened, the unused strips are stable for 1 month at )A!@ 2-8!c C in the resealed bag. R2: The diluted the solution is stable for up to 2 weeks at )A!@ 2-8!c C. The concentrated solution stored at )A!@ 2-25!c C, in absence of microbiol contamination is stable until the expiry date indicated on the label. R6a: Once diluted the solution is stable for up to 7 days at )A!@ 2-8!c C. 8

9 R6: Once diluted the solution is stable for up to 6 hours at room temperature ( )A!c C) and 5 days at +2-8!c C. R9: Once diluted the solution is stable for up to 6 hours in the dark, at room temperature ( )A!c C). 6.4 PROCEDURE Strictly follow the assay procedure. Use the calibrators with each run to validate the assay results. Before use, allow reagents to reach room temperature ( )A!c C) 1. Document the sample distribution and identification plan. 2. Dilute R2 as directed (Refer to section 6.2). 3. Take the carrier tray and the strips (R1) out of the protective pouch. 4. Reconstitute the lyopilised antigen by adding 3 ml of diluent (R7) to one vial of antigen. Mix thoroughly. 5. Dilute test sample and the controls in diluent R7 to give a 1/101 dilution (10 L sample + 1 ml diluent R7). Vortex. 6. Wash microplate strips once with diluted R2. Invert microplate and gently tap on absorbent paper to remove remaining liquid. 7. For manual performance of the test, add 200 L of 1/101 diluted controls (R3, R4a, R4b) and of 1/101diluted test samples (S1, S2 etc )A!- ) to the wells as suggested below: A R3 S5 B R4a S6 C R4a S7 D R4b S8 E S1 S9 F S2 S10 G S3 S11 H S4 S12 8) Cover the microplate with an adhesive plate sealer, pressing firmly onto the plate to ensure a tight seal. Incubate the microplate in a thermostat controlled water-bath or microplate incubator at 37 )A!@ 1!c C for 1 hour )A!@ 5 minutes. 9

10 9. Prepare conjugate solution R6 (refer to section 6.2). Mix thoroughly. 10.At the end of the first incubation period, remove the adhesive plate sealer. Aspirate the contents of all wells into a container for biohazard waste (containing sodium hypochlorite). Wash microplate 3 times. Invert microplate and gently tap on absorbent paper to remove remaining liquid. 11.Add 200 l of the diluted R6 to all wells. The solution must be shaken gently before use. 12.Cover the microplate with adhesive plate sealer; pressing firmly onto the plate to ensure a tight seal. Incubate the microplate in a thermostat controlled water-bath or microplate incubator at 37 )A!@ 1!c C for 1 hour!@ 5 minutes. 13.Remove the adhesive plate sealer, aspirate all wells, and wash 4 times as described above. Invert microplate and gently tap on absorbent paper to remove remaining liquid. 14.Prepare the R8 + R9 solution (refer to section 6.2) and add 200 L to each well. Allow the reaction to develop in the dark for 30 minutes at room temperature ( )A!c C). Do not use the adhesive plate sealer during this incubation. 15.Add 100 L of R10 to each well. 16.Carefully wipe the plate bottom. Read the optical density at 450/620 nm using a plate reader within 30 minutes after stopping the reaction (the strips must always be kept away from light before reading) 17.Check all results for agreement between the spectrophotometric and visual readings and against the plate and sample distribution, and identification plans. 7- INTERPRETATION OF RESULTS 7.1 )G!9 QUALITY CONTROL Include all the calibrators for each test run. For the assay to be considered valid, the following criteria must be met. Optical density values: - OD R3 < OD R4a > OD R4b >

11 Ratio : - Mean OD R4a / OD R3 > 4 - OD R4b / Mean OD R4a > 1.5 If the quality control criteria are not met, the test run should be repeated. 7.2 )G!9 CALCULATION OF THE CUT-OFF VALUE (CO) CO = mean of OD R4a. 7.3 )G!9 CALCULATION OF THE SAMPLE RATIO Sample ratio = sample OD / CO (mean of OD R4a). 7.4 )G!9 INTERPRETATION OF THE RESULTS Ratio Result Interpretation > 1.00 Positive Results to be confirmed by quantitative examination of the IgG antibodies on the same sample. Follow evolution: confirm days after the first control on a second serum sample )A!\ 1.00 Doubtful Result to be confirmed 10 )G!9 15 days later > 0.80 after the first control )A!\ 0.80 Negative Probably no recent infection due to Rubella 7.5 )G!9 TROUBLE SHOOTING GUIDE Non validated or non repeatable reactions are often caused by: Inadequate microplate washings, Contamination of negative samples by serum or plasma with a high antibody titer, Contamination of the development solution by oxidizing agents (bleach, metal ions...), Contamination of the stopping solution. 7.6 )G!9 CALCULATION EXAMPLE Note: The following data are given as an example and should not be used in place of actual results. 11

12 Results Well contents OD (450/620 nm) Ratio and results R Negative R4a R4a R4b Positive Serum Positive Serum Doubtful Optical density: - OD R3 : < OD R4 a : > OD R4 b : > Ratios: - Means of OD R4a / OD R3 : > 4 - OD R4b / mean of OD R4a : > 1.5 Quality control: Conform Cut-off value: CO = LIMITATIONS OF THE PROCEDURE 1. Diagnosis of recent infection by the Rubella virus can only be established on the basis of a combination of clinical observations and serological data. 2. The result of a single serum sample does not constitute sufficient proof for diagnosis of recent infection. 3. To confirm recent primary Rubella infection, evaluate the specimens using a specific test for Rubella IgG antibodies. 4. Anti-Rubella IgM should be determined as part of the serological follow-up of pregnant women, as the appearance of anti-rubella IgG may be slightly delayed with respect to that of anti-rubella IgM. 12

13 9- SPECIFIC PERFORMANCE CHARACTERISTICS The performances characteristics of Platelia Rubella IgM TMB summarized below have been established on 2 different sites using samples from pregnant women, blood donors and samples from commercial panels COMPARATIVE STUDY A total of 737 samples from pregnant women, blood donors, or samples known for being positive for other serology (CMV, HSV, EBV, Measles, Toxoplasmosis), or positive to autoantibodies or rheumatoid factors, and samples from seroconversion or vaccination panels were tested in parallel with the Platelia Rubella IgM TMB and another available commercial EIA test. A third EIA technique was used to resolve the discrepant results. Platelia Rubella IgM TMB Result Negative Doubtful Positive Total Other EIA test Negative Doubtful Positive Total After resolution of discrepant results Platelia Rubella IgM TMB Result Negative Doubtful Positive Total Other EIA test Negative Doubtful Positive Total For calculations, doubtful results have not been included : Relative concordance : 728/ % ( ) (CI 95%) Relative Specificity: 675/ % ( %) (CI 95%) Relative Sensitivity : 53/ % SPECIFICITY STUDIES The specificity of Platelia Rubella IgM TMB was evaluated on 252 specimens from blood donors and on 202 samples from pregnant women comparatively to another available commercial EIA test. A third EIA technique was used to resolve the discrepant results. The specificity was 98.9% (449/454) on this population. 13

14 9.3 - SENSITIVITY STUDIES The sensitivity of Platelia Rubella IgM TMB was studied on 16 samples from pregnant women comparatively to another available commercial EIA test. A third EIA technique was used to resolve the discrepant results. Comparatively to another available commercial EIA test and after control with a third EIA test, the sensitivity of Platelia Rubella IgM TMB on this population is 100 % (11/11) ; 2 samples doubtful with Platelia and negative with the second EIA test but positive with the third test, seem to be due to residual IgM, anti-rubella IgG ratio being high ; these two samples were not included in the final count. 3 samples were found negative with the 3 EIA tests. 60 samples from 9 vaccination follow up panels were also evaluated. All the positive samples (34) were found at the same time between the available EIA and Platelia Rubella IgM TMB CROSS REACTIVITY 156 specimens known to be positive for the following serology (CMV, EBV, HSV, VZV, Mumps, Measles, toxoplasmosis) and 39 samples positive for rheumatoid factors, autoantibodies and heterophilic antibodies as well as samples from patients with myeloma were tested with the Platelia Rubella IgM TMB tests. All the samples were found negative (195/195) PREVALENCE The rate of positivity for anti Rubella IgM antibodies in the north of France (Lille area) is 4%. 14 Number of sample < Sample repartition histogramm on a blood donor population (n = 264) [ [ [ [ [ [ [ [ [ [ Ratio [ [ [ [ [ [ [0.9-1[ > 1

15 9.6 - PREVALENCE Intra assay repeatability : In order to evaluate intra-assay repeatability, 3 positive samples and one negative sample were tested 30 times during the same assay. The ratio (S/CO) of the sample optical density (OD) to the cut-off calibrator (R4a) OD was determined for each sample. The mean OD ratio, standard deviation (SD), and percent coefficient of variation (%CV) for each of the four specimens are listed in the table below. Intra Assay Data Summary N=30 Neg Low Pos Med Pos High Pos Ratio (S/CO) Mean SD %CV Intra assay repeatability : In order to evaluate inter-assay reproducibility, each of four specimens (one negative and 3 positive samples) was tested in duplicate, two runs a day, over a twenty-day period. The ratio (S/CO) of the sample optical density (OD) to the cut-off calibrator (R4a) OD was The mean OD ratio, standard deviation (SD), and percent coefficient of variation (%CV) for each of the four specimens are listed in the table below. Inter Assay Data Summary Neg Low Pos Med Pos High Pos Ratio (S/CO) Mean SD %CV

16 10- REFERENCES 1- CLOPET H., DENOYEL G.A., IgM sp )A (& cifiques de la rub (& ole. Rev. Inst. Pasteur (Lyon) 1978, 11 (1), ECHEVARRIA J.M., SAINZ C., DE ORY F., NAJERA R., Evaluation of commercial methods of enzymeimmunoassay (EIA) for the measurement of Rubella-specific IgM. Journal Virology Methods, 1985 ; 11, GRAVELL M., DORSETT P.H., GUTTENSON O., LEY A.C. Detection of antibody to rubella virus by ELISA. J. Inf. Dis ; 136, S300-S KURTZ J.B, MALIC A., Rubella specific IgM detected by an antibody capture assay/elisa technique. J. Clin. Pathol ; 34, LUCAS G., DELETOILLE P., DELAGNEAU J.F., COIGNARD S., GRANGEOT-KEROS L., Evaluation of a commercially available kit for detection of specific Rubella IgM by capture enzyme immunoassay. 4th European Congress of Clinical Microbiology, April 17th - 20th, 1989 Nice 307/PP MORTIMER P.P., TEDDER R.S., HAMBLING M.W., SHAFI M.S., BURKHARDT F., SCHULT U., Antibody capture immunoassay for anti-rubella IgM, J. Hyg ; 86, PATTISON J.R., DANE D.S., MACE J.E., Persistance of specific IgM after naturai infection with rubella virus. Lancet 1975 ; 1, TEDDER R.S., AYAO J.L., ANDERSON M.J. The production of monoclonal antibodies to rubella hemagglutinin and their use in antibody-capture assays for rubella specific IgM. J. Hyg ; 88,

17 PLATELIA RUBELLA IgM TMB 96 TESTS DTECTION QUALITATIVE DES IgM ANTI-VIRUS DE LA RUBOLE DANS LE SRUM OU LE PLASMA HUMAIN PAR TECHNIQUE IMMUNOENZYMATIQUE IVD Tous les produits fabriqu )A (& s et commercialis (& s par la soci (& t (& Bio-Rad sont plac )A (& s sous un syst (( me d'assurance qualit (& de la r (& ception des mati (( res premi )A (( res jusqu' ( la commercialisation des produits finis. Chaque lot de produit fini fait l'objet d'un contrle de qualit )A (& et n'est commercialis (& que s'il est conforme aux crit )A (( res d'acceptation. La documentation relative ( la production et au contrle de chaque lot est conserv )A (& e par le fabricant.

18 TABLE DES MATIERES 1- INTRT CLINIQUE PRINCIPE DU TEST COMPOSITION DE LA TROUSSE PRCAUTIONS ET SCURITE CHANTILLONS MODE OPRATOIRE MATRIEL NCESSAIRE MAIS NON FOURNI RECONSTITUTION DES RACTIFS CONSERVATION DES RACTIFS OUVERTS ET/OU RECONSTITUS PROCDURE CALCUL ET INTERPRTATION DES RSULTATS CONTRLE DE QUALIT CALCUL DE LA VALEUR SEUIL CALCUL DU RATIO DES CHANTILLONS INTERPRTATION DES RSULTATS EXPERTISE DES CAUSES D )A!/ ERREUR EXEMPLE DE CALCUL LIMITES DU TEST PERFORMANCES TUDES COMPARATIVES TUDES DE SPCIFICIT TUDES DE SENSIBILIT PRCISION RACTIVITE CROISE PRECISION RFRENCES

19 1- INTRT CLINIQUE La rub )A (& ole est une maladie d!/(& tiologie virale r (& pandue dans le monde entier. Elle est presque toujours b )A (& nigne voire inapparente chez l!/ enfant ou l )A!/ adulte. Les manifestations cliniques en sont une (& ruption cutan (& e g )A (& n (& ralis (& e sur tout le corps, une fi (( vre mod (& r (& e, des maux de t (: te et parfois des maux de gorge. Cette maladie est grave dans le cas de l )A!/ infection chez la femme enceinte ; en effet, les s (& quelles de l!/ infection virale chez le ftus peuvent )A (: tre: surdit (&, cataracte, arri (& ration mentale chez l )A!/ enfant ( natre; il y a parfois mort du ftus. La vaccination des enfants d )A!/ ge scolaire a r (& duit de mani (( re significative l )A!/ incidence des (& pid (& mies de rub (& ole. Le d (& pistage syst (& matique des femmes s )A (& ron (& gatives pour la rub (& ole reste cependant tout ( fait d )A!/ actualit (&. La pr (& sence d!/ anticorps de classe IgG anti-virus rub (& oleux chez une femme enceinte ant )A (& rieurement ( sa grossesse assure la protection du ftus lors d )A!/ une (& pid (& mie de rub (& ole. L!/ efficacit (& de la vaccination est d )A (& montr (& e en v (& rifiant la pr (& sence d!/ anticorps de classe IgG anti-virus de la rub )A (& ole apr (( s celle-ci. L )A!/ isolement du virus de la rub (& ole a (& t (& r (& alis (& en 1962 ; la d (& tection des anticorps sp )A (& cifiques de ce virus est importante du fait du risque t )A (& ratog (( ne li (& ( une primo-infection rub (& oleuse en d (& but de grossesse. Les premi )A (( res m (& thodes utilis (& es pour la d (& tection des anticorps furent le test de neutralisation, la r )A (& action de fixation du compl (& ment ou la technique d )A!/ immunofluorescence; elles sont de r (& alisation d (& licate et leur reproductibilit )A (& difficile ( obtenir. Plus r (& cemment, les techniques d )A!/ inhibition de l!/ h (& magglutination ont permis un diagnostic rapide de l )A!/ infection aigu ainsi que de l!/(& tat immunitaire des patients. En 1971, Engvall et Perlmann d )A (& crivaient les premiers tests immunoenzymatiques. Le d )A (& veloppement de telles m (& thodes a contribu (& ( am (& liorer la sp )A (& cificit (& et la sensibilit (& des techniques de recherche des antig (( nes et des anticorps, en particulier dans le domaine du diagnostic au laboratoire de la rub )A (& ole. L )A!/ interpr (& tation de tests s (& rologiques successifs, d (& montrant la pr (& sence d )A!/ anticorps de classe IgM, l!/ apparition ou une augmentation significative de titre des IgG (un doublement du titre) entre deux pr )A (& l (( vements espac (& s d )A!/ au moins trois semaines, est en faveur d!/ une exposition au virus de la rub )A (& ole m (: me lorsque les signes cliniques pathognomoniques de cette infection ne sont pas pr )A (& sents. 19

20 2- PRINCIPE DU TEST PLATELIA Rubella IgM TMB est un dosage immunoenzymatique avec capture des IgM s )A (& riques sur la phase solide (cupule de microplaque) recouverte d )A!/ anticorps anti-chaine humaine. Les contrles et )A (& chantillons ( tester sont dilu (& s et distribu (& s dans les cupules de la microplaque. Durant cette incubation, les IgM pr )A (& sentes dans l )A!/(& chantillon sont capt (& es par la phase solide. Les IgG et les autres prot (& ines du s )A (& rum sont (& limin (& es par les lavages pratiqu (& s ( la fin de l!/ incubation. Un m )A (& lange d!/ antig (( ne du virus rub (& oleux (Ag Rub) et d!/ anticorps monoclonal anti-ag Rub marqu )A (& ( la peroxydase (AcM-Per) est d (& pos (& dans toutes les cupules de la microplaque. Durant cette deuxi )A (( me incubation, si l )A!/(& chantillon test (& contient des IgM anti-rub, ces IgM capt (& es sur la phase solide fixent le complexe (Ag Rub-AcM-Per). L )A!/ Ag Rub et l!/ AcM-Per non fix )A (& s ou en exc (( s sont (& limin (& s par les lavages, pratiqu (& s ( la fin de l )A!/ incubation. La pr (& sence du complexe immun est r (& v (& l (& e par l!/ addition dans chaque cupule d )A!/ une solution de r (& v (& lation enzymatique. La r (& action enzymatique est arr )A (: t (& e par l!/ addition d!/ une solution d!/ acide. Le r )A (& sultat, la lecture en densit (& optique ( 450/620 nm, interpr (& t (& par rapport )A ( une valeur seuil, permet de confirmer ou d!/ infirmer la pr (& sence d )A!/ IgM anti-virus rub (& oleux dans l!/(& chantillon test (&. 3- COMPOSITION DE LA TROUSSE Se reporter aux indications donn )A (& es sur la boite concernant les conditions de stockage et la date d )A!/ expiration de la trousse. Etiquetage Nature des r )A (& actifs Pr (& sentation R1 Microplate Microplaque (pr )A (: t- ( -l!/ emploi) : 1 12 barrettes de 8 cupules s )A (& cables sensibilis (& es par des anticorps anti-chaines humaines sous sachet ferm )A (&. R2 Concentrated Solution de lavage concentr )A (& e (10x) : 1 x 100 ml Washing Tampon TRIS-NaCl (ph 7,4), 1 % Tween 20. Solution Conservateur : 0,01% Thimerosal. R3 Negative Contrle n )A (& gatif : 1 x 1ml Control Tampon TRIS-NaCl (ph 8 )A!@ 0.2), s (& rum albumine bovine, glyc )A (& rol, E102 et E122. Conservateur : 0,2% ProClin TM

21 R4a Cut-off Contrle Valeur Seuil (humain) : 1 x 1ml Control Tampon TRIS-NaCl (ph 8 )A!@ 0.2), s (& rum humain IgM r )A (& actif vis- ( -vis des Ag rub (& oleux, s (& rum albumine bovine, glyc )A (& rol, E102 et E122. Conservateur : 0,2% ProClin TM 300 et < 0,01 % Thimerosal R4b Positive Contrle positif (humain) : 1 x 1ml Control Tampon TRIS-NaCl (ph 8 )A!@ 0.2), s (& rum humain IgM r )A (& actif vis- ( -vis des Ag rub (& oleux, s (& rum albumine bovine, glyc )A (& rol, E102 et E122. Conservateur : 0,2% ProClin TM 300 et < 0,01 % Thimerosal R6a Rubella Antig )A (( ne rub (& oleux (lyophilis (& ) : 4 x 3 ml Antigen Conservateur : 0,01 % Thimerosal R6b Conjugate Conjugu )A (& (50x) : 1 x 0,3 ml Anticorps monoclonal d )A!/ origine murine anti-rub (& ole coupl )A (& ( la peroxydase. Conservateur : 0,01 % Thimerosal R7 Diluent Diluant (pr )A (: t- ( -l!/ emploi) : 2 x 80 ml Tampon TRIS-NaCl (ph 7,7 )A!@ 0,15), s (& rum albumine bovine, 0,1% Tween 20 et rouge de phenol. Conservateur : 0,01 % Thimerosal R8 Substrate Tampon Substrat TMB (pr )A (: t- ( -l!/ emploi) : 1 x 60 ml Buffer Solution d )A!/ acide citrique et d!/ ac (& tate de sodium ph 4,0, contenant 0,015% d )A!/ H 2 O 2 et de 4% de DMSO. R9 TMB Chromog )A (( ne TMB Concentr (& (11x) : 1 x 5 ml Chromogen: Solution contenant 0,25% tetramethylbenzidine (TMB) R10 Stopping Solution d )A!/ arr (: t (pr (: te- ( -l!/ emploi) : 1 x 28 ml Solution Solution d )A!/ acide sulfurique 1N Films adh )A (& sifs 4 4- PRCAUTIONS ET SCURIT La qualit )A (& des r (& sultats d (& pend du respect des bonnes pratiques de laboratoire suivantes : Ne pas utiliser les r )A (& actifs apr (( s la date d'expiration. Ne pas m )A (& langer, ni associer au cours d!/ une m (: me s (& rie, des r (& actifs provenant de trousses portant des num )A (& ros de lots diff (& rents. Avant utilisation, attendre 15 minutes que les r )A (& actifs s!/(& quilibrent ( la temp )A (& rature ambiante (18-30!c C). Il est possible d )A!/ utiliser d!/ autres lots de solution de lavage (R2), de tampon substrat (R8), de chromog )A (( ne (R9) et de solution d!/ arr (: t (R10), 21

22 22 que ceux fournis dans la trousse, sous r )A (& serve d!/ utiliser un seul et m (: me lot au cours d )A!/ un m (: me essai. Reconstituer ou diluer soigneusement les r )A (& actifs en (& vitant toute contamination. Ne pas r )A (& aliser le test en pr (& sence de vapeurs r (& actives (acides, alcalines, ald )A (& hydes) ou de poussi (( res qui pourraient alt (& rer l!/ activit (& enzymatique du conjugu )A (&. La r )A (& action enzymatique est tr (( s sensible ( tous m (& taux ou ions m )A (& talliques. Par cons (& quent, aucun (& l (& ment m (& tallique ne doit entrer en contact avec les diff )A (& rentes solutions contenant le conjugu (& ou la solution substrat. La solution de r )A (& v (& lation (tampon substrat + chromog (( ne) doit (: tre incolore. L )A!/ apparition d!/ une coloration bleue dans les minutes suivant la reconstitution indique que le r )A (& actif est inutilisable et doit (: tre remplac )A (&. Pour cette pr (& paration, utiliser de pr (& f (& rence des r (& cipients et du mat )A (& riel de distribution en plastique ( usage unique ou de la verrerie pr )A (& alablement lav (& e ( l!/ acide chlorhydrique 1N, rinc (& e ( l )A!/ eau distill (& e et s (& ch (& e. Conserver cette solution ( l!/ abri de la lumi (( re. Utiliser un cne de distribution neuf pour chaque s )A (& rum. Le lavage des cupules est une )A (& tape essentielle de la manipulation: respecter le nombre de cycles de lavage prescrits, et s )A!/ assurer que toutes les cupules sont compl )A (( tement remplies, puis, compl (( tement vid )A (& es. Un mauvais lavage peut entraner des r (& sultats incorrects. Ne pas laisser la microplaque s )A (& cher entre la fin du lavage et la distribution des r )A (& actifs. Ne jamais utiliser le m )A (: me r (& cipient pour distribuer le conjugu (& et la solution de r )A (& v (& lation. V )A (& rifier l!/ exactitude des pipettes et le bon fonctionnement des appareils utilis )A (& s. CONSIGNES D )A!/ HYGIENE ET SCURIT Porter des gants )A ( usage unique lors de la manipulation des r (& actifs. Ne pas pipeter )A ( la bouche. Le mat )A (& riel d!/ origine humaine utilis (& dans la pr (& paration des r (& actifs a )A (& t (& test (& et trouv (& non-r (& actif en antig (( ne de surface du virus de l )A!/ h (& patite B (Ag HBs), en anticorps dirig (& s contre le virus de l!/ h (& patite C (anti-hcv), en anticorps dirig )A (& s contre les virus de l!/ immunod (& ficience humaine (anti-hiv1 et anti-hiv2). Du fait qu )A!/ aucune m (& thode ne peut

23 garantir de faon absolue l )A!/ absence d!/ agents infectieux, consid (& rer les r )A (& actifs d!/ origine humaine, ainsi que tous les (& chantillons de patients, comme potentiellement infectieux et les manipuler avec les pr )A (& cautions d )A!/ usage. L )A!/ antig (( ne rub (& oleux a (& t (& inactiv (& par la b (: ta-propiolactone. Consid )A (& rer le mat (& riel directement en contact avec les (& chantillons et les r )A (& actifs d!/ origine humaine, ainsi que les solutions de lavage comme des produits contamin )A (& s. Eviter les )A (& claboussures d!/(& chantillons ou de solution les contenant. Les surfaces souill )A (& es seront nettoy (& es par de l!/ eau de javel dilu (& e ( 10%. Si le liquide contaminant est un acide, les surfaces souill )A (& es seront neutralis )A (& es au pr (& alable avec du bicarbonate de soude, puis nettoy )A (& es avec de l!/ eau de javel et s (& ch (& es avec du papier absorbant. Le mat )A (& riel utilis (& pour le nettoyage devra (: tre jet (& dans un conteneur sp )A (& cial pour d (& chets contamin (& s. Les )A (& chantillons, les r (& actifs d!/ origine humaine ainsi que le mat (& riel et les produits contamin )A (& s seront (& limin (& s apr (( s d (& contamination. Ne pas introduire dans l )A!/ autoclave de solutions contenant de l )A!/ hypochlorite de sodium. Eviter tout contact du tampon substrat, du chromog )A (( ne et de la solution d )A!/ arr (: t avec la peau et les muqueuses (risques de toxicit (&, d'irritation et de brlures). ATTENTION : ProClin TM 300 : < 0,2 % : PRODUIT IRRITANT R43 peut entraner une sensibilisation par contact avec la peau. S28/37 apr )A (( s contact avec la peau, se laver imm (& diatement et Xi - Irritant abondamment avec de l )A!/ eau et du savon. Porter des gants appropri )A (& s. La fiche de donn )A (& es de s (& curit (& est disponible sur demande. 5- CHANTILLONS 1. Les tests sont effectu )A (& s sur des (& chantillons de s (& rum ou de plasma (collect )A (& avec des anticoagulants comme l!/ EDTA, l!/ h (& parine et le citrate) 2. Respecter les consignes suivantes pour le pr )A (& l (( vement, le traitement et la conservation de ces )A (& chantillons de sang: - Pr )A (& lever un (& chantillon de sang selon les pratiques en usage. 23

24 - Pour les s )A (& rums, laisser le caillot se former compl (( tement avant centrifugation. - Conserver les tubes ferm )A (& s. - Apr )A (( s centrifugation, extraire le s (& rum ou le plasma du caillot ou des h )A (& maties et le conserver en tube ferm (&. - Les )A (& chantillons seront conserv (& s ( +2-8!c C si le test est effectu (& dans les 7 jours. - Si le test n )A!/ est pas effectu (& dans les 7 jours, ou, pour tout envoi, les )A (& chantillons seront congel (& s ( -20!c C (ou plus froid). - Congeler / d )A (& congeler les (& chantillons trois fois seulement. - Apr )A (( s d (& cong (& lation les (& chantillons devront (: tre soigneusement homog )A (& n (& is (& s (vortex) avant le test. 3. Les r )A (& sultats ne seront pas affect (& s par les (& chantillons contenant 90 g/l d )A!/ albumine ou 100 mg/l de bilirubine, les (& chantillons lip (& miques contenant l )A!/(& quivalent de 36 g/lde trioleine (triglyc (& ride) ou les )A (& chantillons h (& molys (& s contenant 10 g/l d!/ h (& moglobine. 4. Ne pas chauffer les )A (& chantillons. 6- MODE OPRATOIRE MATRIEL NCESSAIRE MAIS NON FOURNI Agitateur type vortex. Appareil de lecture* pour microplaques ( )A (& quip (& de filtres 450/620nm). Bain-marie, ou incubateur sec*, pouvant )A (: tre thermostat (& ( 37!@ 1!c C. Syst )A (( me de lavage, automatique*, semi-automatique* ou manuel pour microplaque. Conteneur de d )A (& chets contamin (& s. Hypochlorite de sodium (eau de javel) et bicarbonate de soude. Eau distill )A (& e ou d (& sionis (& e st (& rile. Eprouvettes gradu )A (& es de 25, 50, 100 et ml. Gants )A ( usage unique. Lunettes de protection. Papier absorbant. Pipettes, multipipettes, automatiques ou semi-automatiques, r )A (& glables ou fixes pouvant distribuer de 10 )A ( 1000 L et 1, 2 et 10 ml Tubes )A ( usage unique. (*) Nous consulter pour une information pr )A (& cise concernant les appareils valid )A (& s par nos services techniques.

25 6.2 RECONSTITUTION DES RACTIFS R1: Couper le sachet )A ( l!/ aide de ciseaux ou d!/ un scalpel 0,5 ( 1 cm audessus du ZIP. Replacer imm )A (& diatement dans le sachet les barrettes non utilis )A (& es. V (& rifier la pr (& sence du dispositif dessicant. Refermer soigneusement le sachet et le replacer )A ( +2-8!c C. R2: Diluer 10 fois la solution dans l )A!/ eau distill (& e. Pr (& voir environ 350 ml pour une plaque de 12 barrettes pour un lavage manuel. R6a: Reprendre le contenu d )A!/ un flacon par 3 ml de diluant (R7) et bien homog )A (& n (& iser. Une attente de 15 minutes environ avant le m (& lange avec le R6b permet d'assurer une r )A (& hydratation homog (( ne de l!. antig (( ne. R6b: Pr )A (& parer la quantit (& de conjugu (& n (& cessaire ( la r (& alisation d!/ une s )A (& rie en diluant un volume de conjugu (& concentr (& 50 x dans 50 volumes de diluant (R7). R6: M )A (& langer volume ( volume R6a et R6b reconstitu (& s. R9: Diluer 11 fois dans R8 (exemple : 1 ml de R ml de R8). 6.3 CONSERVATION DES RACTIFS OUVERTS ET/OU RECONSTITUS R1 : Apr )A (( s ouverture, les barrettes conserv (& es dans le sachet bien referm (& sont stables pendant 1 mois )A ( +2-8!c C. R2 : Apr )A (( s dilution, la solution de lavage se conserve 2 semaines ( +2-8!c C. Apr )A (( s ouverture, la solution de lavage concentr (& e conserv (& e ( +2-25!c C, en absence de contamination microbienne est stable jusqu )A!/( la date de p )A (& remption indiqu (& e sur l!/(& tiquette. R6a : Apr )A (( s reconstitution, le r (& actif se conserve 7 jours ( +2-8!c C. R6 : Apr )A (( s reconstitution, la solution de travail du conjugu (& se conserve 6 heures )A ( temp (& rature ambiante et 5 jours ( +2-8!c C. R9: Apr )A (( s reconstitution, le r (& actif conserv (& ( l!/ obscurit (& est stable pendant 6 heures )A ( temp (& rature ambiante (+18-30!c C). 6.4 PROCDURE Suivre strictement le protocole propos )A (&. Utiliser les s )A (& rums de contrle ( chaque mise en uvre du test pour valider la qualit )A (& du test. Avant utilisation, laisser tous les r )A (& actifs atteindre la temp (& rature ambiante ( )A!c C). 25

26 1) Etablir soigneusement le plan de distribution et d )A!/ identification des )A (& chantillons. 2) Pr )A (& parer la solution de lavage dilu (& e (R2) (se r (& f (& rer au chapitre 6.2). 3) Sortir le cadre support et les barrettes (R1) de l )A!/ emballage protecteur. 4) Reprendre le contenu d )A!/ un flacon R6a par 3 ml de diluant (R7) et bien homog )A (& n (& iser. 5) Diluer les )A (& chantillons ( tester et les contrles au 1/101 (( dans la solution R7 (10 l de s )A (& rum + 1 ml Diluant R7). Bien homog (& n (& iser (vortex). 6) Laver une fois toutes les cupules de la microplaque avec la solution R2 dilu )A (& e. S (& cher la plaque par retournement sur une feuille de papier absorbant. 7) Pour une utilisation manuelle du test, distribuer 200L des contrles dilu )A (& s au 1/101 (R3, R4a, R4b) et des (& chantillons dilu (& s au 1/101 (E1, E2, etc.) dans les cupules comme sugg )A (& r (& ci-dessous : A R3 E5 B R4a E6 C R4a E7 D R4b E8 E E1 E9 F E2 E10 G E3 E11 H E4 E12 8) Couvrir la microplaque d )A!/ un film adh (& sif en appuyant bien sur toute la surface pour assurer l )A!/(& tanch (& it (&. Puis, incuber la microplaque au bainmarie thermostat )A (& ou dans un incubateur sec de microplaques pendant 1 heure )A!@ 5 minutes ( 37!@ 1!c C. 9) Pr )A (& parer la solution de conjugu (& (R6) (se r (& f (& rer au chapitre 6.2). Bien homog )A (& n (& iser. 10)A la fin de la premi )A (( re incubation, retirer le film adh (& sif, aspirer le contenu de toutes les cupules dans un conteneur pour d )A (& chets contamin (& s (contenant de l )A!/ hypochlorite de sodium) et proc (& der ( 3 lavages. S )A (& cher la plaque par retournement sur une feuille de papier absorbant. 26

27 11)Distribuer 200 l de la solution de travail de conjugu )A (& (R6) dans toutes les cupules. Cette solution doit )A (: tre agit (& e avant emploi. 12)Couvrir la microplaque d )A!/ un film adh (& sif neuf en appuyant bien sur toute la surface pour assurer l )A!/(& tanch (& it (&. Puis, incuber la microplaque au bain-marie thermostat )A (& ou dans un incubateur sec de microplaques pendant 1 heure )A!@ 5 minutes ( 37!@ 1!c C. 13)A la fin de la deuxi )A (( me incubation, retirer le film adh (& sif, aspirer le contenu de toutes les cupules dans un conteneur pour d )A (& chets contamin )A (& s et proc (& der ( 4 lavages. S (& cher la plaque par retournement sur une feuille de papier absorbant. 14) Pr )A (& parer la solution de r (& v (& lation enzymatique (R8+R9) (se r (& f (& rer au chapitre 6.2). En distribuer rapidement 200 L dans toutes les cupules. Laisser la r )A (& action se d (& rouler ( l!/ obscurit (& pendant 30 minutes ( temp )A (& rature ambiante (+18-30!c C). Lors de cette incubation, ne pas utiliser de film adh )A (& sif. 15) Ajouter 100 l de solution d )A!/ arr (: t (R10). 16) Essuyer soigneusement le dessous des plaques. Lire la densit )A (& optique )A ( 450/620 nm ( l!/ aide d!/ un lecteur de plaques dans les 30 minutes qui suivent l )A!/ arr (: t de la r (& action. Les barrettes doivent toujours (: tre conserv )A (& es ( l!/ abri de la lumi (( re avant la lecture. 17) S )A!/ assurer avant la transcription des r (& sultats, de la concordance entre la lecture et le plan de distribution et d )A!/ identification des plaques et des )A (& chantillons. 7- CALCUL ET INTERPRTATION DES RSULTATS 7.1 )G!9 CONTRLE DE QUALIT Utiliser les contrles sur chaque microplaque pour chaque essai. Pour valider la manipulation, les crit )A (( res suivants doivent (: tre respect (& s : Valeurs des densit )A (& s optiques : - DO R3 < 0,150 - DO R4a > 0,300 - DO R4b > 0,600 Rapports : - Moyenne des DO R4a / DO R3 > 4 - DO R4b / moyenne des DO R4a > 1,5 Si ces sp )A (& cifications ne sont pas respect (& es, recommencer la manipulation. 27

28 7.2 )G!9 CALCUL DE LA VALEUR SEUIL (VS) VS = moyenne des DO R4a. 7.3 )G!9 CALCUL DU RATIO DES CHANTILLONS Ratio de l )A!/(& chantillon = DO de l!/(& chantillon / VS (moyenne des DO R4a) 7.4 )G!9 INTERPRETATION DES RSULTATS Ratio R )A (& sultat Interpr (& tation > 1,00 Positif R )A (& sultats ( confirmer par un test quantitatif des IgG sur le m )A (: me pr (& l (( vement. Evolution )A ( suivre : nouvel examen sur un 2 (( me pr (& l (( vement recommand )A (& 10 )G!9 15 jours environ apr )A (( s le 1er examen. )A!\ 1,00 Douteux R (& sultats ( confirmer par un nouveau test 10 )G!9 15 jours > 0,80 environ apr )A (( s le 1 er examen. )A!\ 0,80 N (& gatif Pas d!/ infection rub (& olique r (& cente probable. 7.5 )G!9 EXPERTISE DES CAUSES D!e ERREUR L )A!/ origine des r (& actions non valid (& es ou non reproductibles est souvent en relation avec les causes suivantes : Lavage insuffisant des microplaques Contamination des )A (& chantillons n (& gatifs par un s (& rum ou un plasma contenant un titre )A (& lev (& d!/ anticorps. Contamination ponctuelle de la solution de r )A (& v (& lation par des agents chimiques oxydants (eau de javel, ions m )A (& talliques...) Contamination ponctuelle de la solution d )A!/ arr (: t. 7.6 )G!9 EXEMPLE DE CALCUL NB: Les donn )A (& es suivantes ne constituent qu!/ un exemple et ne doivent pas )A (: tre utilis (& es ( la place des donn (& es obtenues par l!/ utilisateur. R )A (& sultats Contrles & S )A (& rums DO (450/620 nm) Ratio et R (& sultats R3 0,080 0,11 N )A (& gatif R4a 0,709 1 R4a 0,697 1 R4b 2,280 3,24 Positif S )A (& rum 1 1,181 1,67 Positif S )A (& rum 2 0,597 0,85 douteux 28

29 Valeurs des densit )A (& s optiques : - DO R3 : < 0,150 - DO R4 a : > 0,300 - DO R4 b : > 0,600 Rapports : - Moyenne des DO R4a / DO R3 : > 4 - DO R4b / moyenne des DO R4a : > 1,5 Contrle de qualit )A (& : Conforme Valeur Seuil : VS = 0, LIMITES DU TEST 1) Seul un ensemble de donn )A (& es cliniques et s (& rologiques permet le diagnostic d )A!/ une infection r (& cente. 2) Le r )A (& sultat d!/ un seul dosage ne constitue pas une (& preuve suffisante pour le diagnostic d )A!/ une infection r (& cente. 3) Pour confirmer une infection r )A (& cente par le virus de la rub (& ole, ces )A (& chantillons de s (& rums devront (: tre test (& s par une technique sp (& cifique des anticorps de classe IgG anti-virus de la rub )A (& ole. 4) La recherche des IgM anti-virus de la rub )A (& ole peut faire partie du suivi s )A (& rologique de la femme enceinte, l!/ apparition des IgG anti-virus de la rub )A (& ole pouvant (: tre l (& g (( rement retard (& e comparativement aux IgM. 9- PERFORMANCES Les performances de Platelia Rubella IgM TMB r )A (& sum (& es ci-dessous ont )A (& t (& (& tablies sur 2 sites diff (& rents en utilisant des (& chantillons provenant de femmes enceintes, de donneurs de sang et de panels de s )A (& rums disponibles commercialement ETUDE COMPARATIVE 737 )A (& chantillons provenant de femmes enceintes, donneurs de sang et )A (& chantillons connus pour (: tre positifs pour d!/ autres pathologies infectieuses (infections )A ( CMV, HSV ou EBV ; rougeole ou toxoplasmose) ou pour pr )A (& senter des autoanticorps ou des facteurs rhumatodes ainsi que des )A (& chantillons de s (& roconversions ou de suivis de vaccination ont )A (& t (& test (& s en parall (( le avec Platelia Rubella IgM TMB et un autre test EIA commercialis )A (&. Une troisi (( me technique EIA a (& t (& utilis (& e pour l!/(& tude des discordants. 29