User`s manual
Contents
1. 0338 3aninawv 0 36 03368 03348 0338 0338 03348 904 304 304 DNAN SOd 3MIS 1O8 ONDIVHS ONDIVHS 8 108 MNMOG 108 MNMOQ dSV VOIBBA IVINOZRIOH BBA W 8 BALO 45 45 481 0 1 13 L7Md OVMd LO LV14 3WVN 31 14 0 50 59A Z pouew 0 7 0 IM sz eo Pouow 855 661 eo PH 645 1 8 L LO PH WINI SIDE BIN STOAD mu 3B9ADN MOH 3AL dSv MS uoyouy don aw SNOS oun 38 8 108 MOH acy sow m 351 32WVN 0 1 1 50 Z pouow zs L 0r Md sv 0 1 sz 008 0 pouow 1 48 5 8059 6 9 8 L 10 WNI SIDE BIN STOAD awn GB9ADN MOH 3AL dSv MS 1 aw SNOS avus 108 MOH nOn yaa asy 330W PRO ave 351 32WVN S3l313W Vl Vd 393HSVM 31V1dO32IW 10 9 CALCULATION AND INTERPRETATION OF THE RESULTS The results are given in Optical Densities ODs after reading of the microplate at 450 620 nm 1 Qualitative determination For the quali2. E4 E 5 52 5 F 5 52 6 G S5 5 EZ 55 1 8 10 11 12 13 14 15 16 17 For the qualitative assay dilute the R3 R4a and R4b controls and the unknown sera to 1 100 R reagent e g 10 pl of sample in 990 pl of dilution solution For the quantitative assay prepare the quantification standards refer to chapter 5 3 and dilute the R3 controls and the unknown sera to 1 100 in R reagent e g 10 pl of sample in 990 yl of dilution solution Distribute 100 pl of diluted samples controls and Quantification standards in the correspondent microplate wells according to the pre established distribution plan Cover the microplate with adhesive film cut the sheet if necessary Press firmly all over the plate to ensure a tight seal Incubate the strips at 37 2 C for 60 minutes 5 minutes Prepare the wash solution R2 refer to chapter 5 Prepare the conjugate solution R7 as described in chapter 5 before the end of the first incubation Remove the adhesive film Perform 3 wash cycles Optimal washing conditions are obtained with PWAO PWA1 or 1575 Bio Rad plate washers with program TSE 3 Do not let the microplate stand for more than 5 minutes after the last wash cycle Dry by inversion on absorbent paper before the following step Distribute 100 yl of the conjugate solution R7 into each well Cover with a new adhesive film and incubate f
3. 8 C Do not use expired reagents Do not mix reagents from different lots within a given test run with the exception of generic reagents R2 R8 R9 R10 Before use wait 30 minutes for the reagents to adjust to the laboratory temperature Thoroughly reconstitute reagents avoiding any contaminations Do not perform the test in the presence of reactive vapors acids alkalis aldehydes or dust that could alter enzyme activity of the conjugate Use glassware thoroughly washed and rinsed with deionized water or preferably disposable material Do not leave the microplate more than 5 minutes between the end of washing operation and the reagent distribution Check the accuracy of the pipettes and the good operation of the apparatus used Never use the same container to distribute conjugate and the development solution The enzyme reaction is very sensitive to metal or metallic ions Consequently do not allow any metal element to come in contact with the various conjugate or substrate solution The development solution substrate buffer chromogen must be colourless The appearance of a blue colour within a few minutes after reconstitution of the development solution indicates that the reagent can not be used and must be replaced Preparation of this solution can be made in a clean disposable plastic tray or glass container that has first been pre washed with 1N and rinsed thoroughly with distilled water and dried Store
4. EU ml and the 56 4 EU ml Optical Density mean values In this case the unknown sample titre is found using the Standard curve On the Standard curve find the value corresponding to the OD reading of the sample on the y axis and draw a horizontal line to the standard curve At the point of the intersection with the standard curve draw a vertical line to the x axis Read the corresponding concentration in EU ml IF the OD value of the unknown sample is higher than the mean Optical Density value for the S standard a precise quantification cannot be performed If a precise quantification is required proceed to 1 10 dilution or more of the sample and perform the assay again in order to have an Optical Density in the interval of the Standard curve Note An interpretation and conversion tool of OD values into titres is available on the CD Rabies QT ELISA BIORAD Vers 2007 12 A XLS for users who are not equipped with Bio Rad readers General comprehension of the results Results in Optical Density Titre for the unknown for the unknown sample sample X Interpretation of the result High seroconversion level If a precise titre is required the OP sample gt S AmA EU gml sample must be diluted before repeating the assay 53 lt OD sample lt S6 X in EU ml 0 5 4 EU ml Sufficient seroconversion level Insufficient seroconversion level according to PLATELIA RABIES II test 5 lt OD sample lt 53
5. X in EU ml 0 125 0 5 EU ml OD sample lt ST Undetectable seroconversion 10 LIMITATIONS OF THE TEST Insufficient or undetectable seroconversion can possibly be reported in recently vaccinated animals due to delayed immune response Similarly to the official neutralising antibody titration method and in accordance with regulations EC No 998 2003 effective July 3 2004 the PLATELIA RABIES II test should be carried out on samples taken at least thirty days after vaccination 11 EQUIPMENT AND MATERIAL REQUIRED BUT NOT SUPPLIED Equipment Microplate reader equipped with 450 and 620 nm filters Microplate incubator set at 37 C 2 C Manual semi automatic or automatic microplate washer Vortex mixer Contact us for detailed information about Bio Rad instruments validated by our technical departments Material Automatic or semi automatic adjustable or preset pipettes or micropipettes to measure and deliver 10 to 1000 pl and 1 2 and 10 ml Graduated cylinders of 25 ml 50 ml 100 ml and 1 000 ml capacity Disposable tubes Distilled or deionized water Sodium hypochloride bleach Absorbent paper Goggles or safety glasses Disposable latex gloves Container for biohazard waste 12 PRECAUTIONS 1 Precautions The quality of the results depends on correct implementation of the following good laboratory practices Reagents must be stored at 2 C to
6. mostly transmitted through the saliva via bites from rabid animals Immunization and control programs for the protection of animal populations from rabies virus have resulted in the elimination of the disease in several Western European countries and in some countries in South America As of July 3 2004 a new European regulation applies to international movements of domestic carnivores from rabies infected to rabies free countries Domestic carnivores are allowed to travel if their serum contain at least 0 5 IU ml International Units ml of rabies neutralising antibodies Anti rabies antibodies titration has several practical applications Individual serology for international trade purposes anti rabies antibodies are measured in specialized laboratories in order to determine the degree of immunity of vaccinated animals cats and dogs A level of antibody equal to or superior to 0 5 IU ml is considered by OIE experts as an acceptable seroconversion level above which the vaccination could be considered as successful Confirmation of vaccinated status during a vaccination campaign control of the anti rabies antibodies enables indirect assessment of the effectiveness of oral vaccination campaign in wildlife foxes PRINCIPLE OF THE PLATELIA RABIES ASSAY PLATELIA RABIES II is an immuno enzymatic technique for the detection of rabies virus anti glycoprotein antibodies This assay can be carried out on the serum of several spe
7. the solution in the dark Use a new pipette tip for each sample Do not change the assay procedure Washing of the wells is a crucial step in the procedure respect the recommended number of washing cycles and make sure that all wells are completely filled then emptied Inadequate washing can give incorrect results 2 Hygiene and safety instructions Generally hygiene conditions biosafety measures and good laboratory practices must be in agreement with recommendation of regular authorities of the country All the reagents of the kit are intended for in vitro veterinary diagnostic use dog cat and fox sera Wear disposable gloves when handling reagents and samples Thoroughly wash your hands after sample and reagent handling Do not pipette with the mouth Consider any material directly in contact with the samples and the reagents as well as the wash solutions as infectious material Avoid spilling samples or solutions containing samples Spills may be rinsed with bleach diluted to 10 Chl If the contaminating fluid is acid spills must be initially neutralized with sodium bicarbonate then cleaned with bleach and dried with absorbent paper The material used for cleaning must be discarded after decontamination Samples and contaminated material and products should be discarded after decontamination either by immersion in bleach at the final concentration at 5 of sodium hypochloride for 30 min or by aut
8. 79 BIDERFIELD P GHETIE V and SJOQUIST J 1975 Demonstration and assaying of IgG antibodies in tissues and on cells by labelled staphylococcal protein A J Immunol Meth 6 249 259 BOURHY H SUREAU P 1989 Comparative field evaluation of the fluorescent antibody test virus isolation from tissue culture and enzymes immunodiagnosis for rapid laboratory diagnosis of rabies J Clin Microbiol 27 519 523 BRIGGS D J SMITH J S MUELLER F L SCHWENKE J DAVIS R D GORDON C R SCHWEITZER K ORCIARI L A YAGER P A RUPPRECHT C E 1998 A comparison of two serological methods for detecting the immune response after rabies vaccination in dogs and cats being exported to rabiesfree areas Biologicals 26 347 355 CLIQUET F AUBERT M SAGNE L 1998 Development of a fluorescent antibody virus neutralization test FAVN test for the quantitation of rabies neutralising antibody J Immunol Methods 212 79 87 CLIQUET F L SAGNE J L SCHEREFFER M F A AUBERT 2000 ELISA test for rabies antibody titration in orally vaccinated foxes sampled in the fields Vaccine 18 3272 3279 CLIQUET F VERDIER Y SAGNE L AUBERT M SCHEREFFER J L SELVE M WASNIEWSKI M SERVAT A 2003 Neutralising antibody titration in 25 000 sera of dogs and cats vaccinated against rabies in France in the framework of the new regulations that offer an alternative to quarantine Rev Sci Tech Off Int Epiz 22 3 857 866 CLIQUET F M LLER
9. AU P 1986 The influence of the type of immunosorbent on rabies antibody EIA advantages of purified glycoprotein over whole virus J Biol Standard 14 95 102 SERVAT A CLIQUET F 2006 Collaborative study to evaluate a new ELISA test to monitor the effectiveness of rabies vaccination in domestic carnivores Virus Res 120 17 27 SERVAT FEYSSAGUET M BLANCHARD l BOUE F CLIQUET F 2007 A quantitative indirect ELISA to monitor the effectiveness of rabies vaccination in domestic and wild carnivores J Immunol Methods 318 1 2 1 10 STANTIC PAVLINIC M HOSTNIK P LEVICNIK STEZINAR S ZALETEL KRAGELJ L 2006 Vaccination against rabies and protective antibodies comparison of ELISA and fluorescent antibody virus neutralization FAVN assays Veterinarski Archiv 76 4 281 289 WRIGHT C WILLIAM KJ SJSODAHL J BURTON D R and DWEK 1977 The interaction of protein A and the Fc fragment of rabbit immunoglobulin G as probed by complementfixation and nuclear magnetic resonance studies Bioch J 167 661 668 Manual of standards for diagnostic tests and vaccines OIE 2000 Chapters 1 1 3 and 2 2 US Catalogue number US Manufacturer R f rence catalogue Fabricant N mero de cat logo E Fabricante I Numero di catalogo I Produttore D Bestellnummer D Hersteller US Batch code US Expiry date YYYY MM DD Code du lot Date de
10. PLATELIA RABIES II KIT Ad Usum Veterinarium 192 TESTS Ref 355 0180 KIT FOR IN VITRO DETECTION AND TITRATION OF IgG ANTI RABIES VIRUS GLYCOPROTEIN IN DOGS CATS AND FOXES SERUM All manufactured and commercialized reagents are under complete quality system starting from reception of raw materials to the final commercialization of the product Each lot is submitted to a quality control and released on the market when conforming to the acceptance criteria Fitness for purpose validated and certified by OIE Registration number 20070101 User s manual TABLE OF CONTENTS 1 INTENDED USE 2 INTEREST OF PLATELIA RABIES KIT 3 PRINCIPLE OF THE PLATELIA RABIES ASSAY 4 COMPOSITION OF THE PLATELIA RABIES KIT 5 PREPARATION OF THE REAGENTS STORAGE SHELF LIFE 7 SAMPLES 8 ASSAY PROTOCOL 9 CALCULATION AND INTERPRETATION OF THE RESULTS 10 LIMITATIONS OF THE TEST 11 EQUIPMENT AND MATERIAL REQUIRED BUT NOT SUPPLIED 12 PRECAUTIONS 13 LITERATURE 1 N INTENDED USE The PLATELIA RABIES kit is an in vitro diagnostic ELISA test allowing detection and titration of IgG anti rabies virus glycoprotein in animal dogs cats and foxes serum INTEREST OF THE PLATELIA RABIES II KIT Rabies is one of the oldest viral diseases afflicting humans and animals Rabies virus is a highly neurotropic virus usually causing fatal encephalitis in mammals The virus is
11. R6 Quantification Concentrations obtained by serial standards dilutions of the R4b Positive control 6 RAb diluted to 1 100 4 EU ml S5 56 diluted to 1 2 2 EU ml S4 55 diluted to 1 2 1 EU ml 53 54 diluted to 1 2 0 5 EU ml 52 53 diluted to 1 2 0 25 EU ml 5 52 diluted to 1 2 0 125 EU ml 6 STORAGE SHELF LIFE As supplied all the components of the PLATELIA RABIES kit are stored at 2 to 8 C and can be used until the expiry date indicated on the kit Reagent R2 10 X can be stored at 2 C to 25 C before reconstitution The shelflives of the reagents after preparation are as follow REAGENT REMARKS SHELF LIFE After opening the sealed foil lined bag the unused strips should immediately be put 1 month at back in the bag and the bag 2 C to 8 C again sealed The desiccant should remain in the foil bag RI Microplate R2 Diluted 2 weeks at wash solution 2 C to 8 C Diluted The diluted conjugate solution R7 conjugate should preferably be used 8 hours 91 s d 2 C to 8C solution immediately Reconstituted 6 hours at room temperature R8 R9 development 18 C to 30 C always solution protected from light 7 SAMPLES The PLATELIA RABIES test has been developed for application to animal dogs cats and foxes serum The assay is carried out on sera after a 1 100 dilution in R reagent The samples are stored at 2 to 8 C if th
12. T MUTINELLI F BROCHIER B AUBERT M and all 2003 Standardisation and establishment of a rabies ELISA test in European laboratories for assessing the efficacy of oral fox vaccination campaigns Vaccine 21 2986 2993 CLIQUET F Mc ELHINNEY L M SERVAT A BOUCHER J M LOWINGS J P GODDARD T MANSFIELD K L FOOKS A R 2004 Development of a qualitative indirect ELISA for the measurement of rabies virus specific antibodies from vaccinated dogs and cats J Vir Methods 117 1 8 20 2 22 23 ENGVALL E and PERLMANN P 1971 Enzyme linked immunosorbent assay ELISA Quantitative assay of immunoglobulin G Immunochemistry 8 871 874 FEYSSAGUET M DACHEUX L AUDRY L COMPOINT A MORIZE JL BLANCHARD BOURHY 2007 Multicenter comparative study of a new ELISA PLATELIA RABIES Il for the detection and titration of anti rabies glycoprotein antibodies and comparison with the rapid fluorescent focus inhibition test on human samples from vaccinated and nonvaccinated people Vaccine 25 12 2244 51 FORSGREN A and SJOQUIST J 1966 Protein A from S aureus Pseudo immune reaction with human g globulin J Immunol 97 822 827 GRASSI M WANDELER A I PETERHANS 1989 Enzymelinked Immunosorbent Assay for Determination of the Envelope Glycoprotein of Rabies Virus J Clin Microbiol 27 5 899 902 PERRIN P VERSMISSE P DELAGNEAU J F LUCAS G ROLLIN P E and SURE
13. cies of animals dog cat and fox The test is based on the use of a solid phase enzyme immunoassay technique referred to as an indirect ELISA A microplate is coated with rabies glycoprotein extracted from the inactivated and purified virus membrane The enzymatic conjugate consists of a protein A from Staphylococcus aureus coupled with peroxidase Positive controls calibrated against OIE standard allow the qualitative or quantitative determination of anti rabies antibody titre in the serum Implementation of the test comprises the following reaction steps The unknown sera as well as the calibrated Positive Controls or the Quantification Standards are distributed in the glycoprotein coated wells of the microplates During incubation of one hour at 37 C anti rabies antibodies present in the sample bind to the glycoprotein coated to the microplate wells After incubation unbound antibodies and other serum proteins are removed by washings 2 The conjugate protein A labelled with peroxidase is added to the microplate wells During a second incubation of one hour at 37 C the labelled protein A binds to the anti rabies antibody antigen complexes attached to the microplate wells The unbound conjugate is removed by washings 3 presence of immune complex is demonstrated by the addition of a solution containing a peroxidase substrate and a chromogen initiating a colour development reaction 4 After 30 min incubation at room
14. e detection is carried out within 24 hours or can be stored at 20 C for 6 months The samples can be submitted to 3 cycles of freezing and thawing Previously frozen samples should be thoroughly mixed after thawing prior to testing NB Eliminate if necessary by centrifuging the fibrin particles or aggregates in suspension that can provide false positive results 8 ASSAY PROTOCOL Strictly follow the recommended protocol Use the negative and the positive controls for each test run and on each microplate in order to validate the quality of the detection in the qualitative assay If quantification is performed the negative control and the Quantification standards should be deposited on each plate In both cases follow the recommended microplate set up Use good laboratory practices 1 Remove the microplate rack and the required number of rows R1 from the protective packaging Replace the unused rows with the desiccated bag in the microplate sachet and hermetically close it N Carefully establish the sample distribution and identification plan as follow Microplate set up for Qualitative assay 1 2 3 4 10 11 12 A R3 E3 B R3 54 R4a 55 D R4a R4b E7 F R4b E8 G El E9 H E2 E10 Microplate set up for Quantitative assay A R3 54 El 5 R3 54 E2 E10 R4a 53 E3 R4a 53
15. ean of the two 51 ODs corresponds to 0 125 EU ml 52 mean of the two 52 ODs corresponds to 0 25 EU ml etc The signals of the standards must increase in the following way 51 lt 52 lt 53 lt 54 lt 5 lt 56 0 7 lt 53 8496 ODs lt 1 3 The ratio between 53 mean 53 standard and R4a mean R4a must be between 0 7 and 1 3 b Interpretation of the results The Threshold Value is equal to the mean of the two OD of the 53 Quantification standard 53 The S3 Quantification standard corresponds to the seroconversion threshold value 0 5 EU ml The quantity of anti rabies antibodies in a sample is determined by comparing the optical density of the sample to a standard curve Sera titres are expressed as Equivalent units per ml EU ml unit equivalent to the international units defined by seroneutralization Drawing the standard curve For manual data reduction use graph paper and plot the mean values of the OD readings of the Quantification standards S1 to S6 on the vertical y axes Plot the corresponding concentrations in EU ml on the horizontal x axes Draw a series of line segments which goes through the 6 points For automated data reduction a point to point function is used to construct a curve from the OD readings obtained for the standards A quantitative result for the anti rabies antibodies titre for an unknown sample can be delivered if the OD value for the sample ranges between the S1 0 125
16. er expelling any air then store at 2 C to 8 C Sample diluent R6 Stop solution R10 2 Reagents to be reconstituted Wash solution R2 Dilute the solution 1 10 in distilled water example 100 ml of reagent R2 in 900 ml of distilled water 500 ml are required for a plate Negative control R3 Dilute the solution 1 100 in R 0 5 EU ml Positive control R4a Dilute the R4a positive control 1 100 in R6 4 EU ml Positive control R4b Dilute the R4b positive control 1 100 in R6 Conjugate R7 The solution is concentrated 10 times To prepare diluted conjugate solution add 1 volume of concentrated conjugate to 9 volumes of prepared 1X wash solution R2 11 ml is required for a full microplate Enzymatic development solution R8 R9 Dilute reagent R9 to 1 11 in the reagent R8 example 0 1 ml of reagent R9 in 1 ml of reagent R8 bearing in mind that 11 ml of enzymatic revelation solution is sufficient for 1 microplate Homogenize gently Avoid using a Vortex agitator 3 Preparation of the Quantification Standards for a quantification assay Each quantification assay using PLATELIA RABIES kit should include 6 Quantification standards from S1 to 56 The calibrated R4b Positive control 4 EU ml corresponds to the S6 Quantification standard Serial dilutions out of the R4b reagent allow the preparation of 55 to 5 Quantification standards The dilutions are performed using the sample diluent reagent
17. lue greater than the High Seroconversion Value originate from individuals who have highly seroconverted after vaccination according to PLATELIA RABIES II test Seroconverted OD R4b OD R4a lt OD sample lt lower than or equal to the High Samples with OD value greater than or equal to the Threshold Value and Seroconversion Value originate from individuals who have seroconverted after vaccination according to PLATELIA RABIES II test Not seroconverted OD sample lt OD R4a Samples with OD value lower than the Threshold Value originate from individuals presenting a level of seroconversion insufficient to assess the efficiency of the vaccination according to PLATELIA RABIES test Quantitative determination For the quantitative determination include all the standards and controls 51 to 56 R3 and R4a for each test run a Conditions of validation Criteria Validation The absorbance of each individual negative control must OD 838 lt 0 05 be lower than 0 05 The test must be repeated if at least one value lies outside of this limit 0 300 lt OD R4a i lt 1 200 Each individual value of the R4a positive control optical densities must be between 0 300 and 1 200 The test must be repeated if at least one of the R4a OD value lies outside of this limit 1 lt 52 lt 53 lt 54 lt 55 lt 5 Calculate the mean OD 5 to 56 in the following way ST m
18. ml calibrated positive control RA Glycine buffer containing BSA and dog serum with anti 1 vial rabies IgG Yellow coloured 0 6 ml Preservative ProClin 300 0 1 4 EU ml Positive control 4 EU ml calibrated positive control RAb Glycine buffer containing BSA and dog serum with anti 1 vial rabies IgG Blue coloured 0 6 ml Preservative ProClin 300 0 1 Sample diluent Ready to use TRIS EDTA buffer for sample 2 vial R6 dilution Red coloured ie Preservative ProClin 300 0 1 E d Conjugate Solution containing Protein A Peroxidase and 1 vial R7 purified bovine protein Concentrated 10 times Green 3 ml coloured Preservative ProClin 300 0 1 a Peroxydase substrate buffer Solution of citric acid vial R8 and sodium acetate containing 0 015 H202 60 and 4 dimethylsulfoxide DMSO 1 R9 Chromogen Tetramethylbenzidine TMB solution 0 25 R10 Stop solution 1 N sulphuric acid solution p Adhesive films for microplates 6 5 PREPARATION OF REAGENTS Note Before use allow reagents to reach room temperature 18 to 30 C for 30 minutes Homogenize the reagents by gently mixing before opening 1 Ready to use reagents Microplates R1 Prior to use let the microplate adjust to room temperature 18 C to 30 C in its protective packaging to avoid any water condensation in the wells Any unused strips should be immediately returned to the bag tightly close the bag aft
19. oclaving at 121 C for 2 hours minimum Caution Do not introduce solutions containing sodium hypochloride into the autoclave Chemicals should be handled and disposed of in accordance with Good Laboratory Practice Reagents containing 0 1 ProClin 300 are classified as irritating preparations according to European legislation Xi 0 1 ProClin 300 R 43 May cause sensitisation by skin contact S 28 37 After contact with skin wash immediately with plenty of water Wear suitable gloves 13 LITERATURE Ts 10 11 12 13 ATANASIU P SAVY V and PERRIN P 1977 Epreuve immuno enzymatique pour la recherche rapide des anticorps antirabiques Ann Inst Pasteur Microbiol 128 A 489 498 ATANASIU P SAVY V and GIBERT C 1978 Rapid immunoenzymatic technique for titration of rabies antibodies IgG and IgM results Med Microbiol Immunol 166 201 208 ATANASIU P and PERRIN P 1979 Microm thode immuno enzymatique de titrage des anticorps antirabiques utilisation de la glycoprot ine rabique et de la prot ine A conjugu es la p roxydase Ann Inst Pasteur Microbiol A 257 268 ATANASIU P PERRIN P and DELAGNEAU J F 1980 Use of an enzyme immunoassay with protein A for rabies antigen and antibody determination Develop Biol Standard 46 207 215 AVRAMEAS 5 and TERNYNCK T 1971 Peroxidase labelled antibody and Fab conjugates with enhanced intracellular penetration Immunochemistry 8 1175 11
20. or 60 minutes 5 minutes at 37 2 C Prepare the enzymatic development solution R8 R9 as described in chapter 5 just before use Remove the adhesive film perform 5 wash cycles Optimal washing conditions are obtained with PW40 PW41 1575 Bio Rad plate washers with programs TSE 5 Do not let the microplate stand for more than 5 minutes after the last wash cycle Dry by inversion on absorbent paper before the following step Away from direct light quickly distribute 100 pl of the enzymatic development solution R8 R9 into each well and incubate the plate in darkness at room temperature 18 to 30 C for 30 minutes 5 minutes NB Do not use adhesive film during this incubation Add 100 pl of the stop solution R10 to each well according to the same sequence and same distribution rate as for the enzymatic development solution Thoroughly wipe the bottom of the plate Read the optical density at 450 620 nm Dual wavelength mode within 30 minutes after stopping the reaction the strips must always be protected from light before reading Before recording the results check that the reading complies with the distribution and identification plan for the plates and samples L 6 9 6 9 8 9 6 Fel 0 vl
21. peremption AAAA MM JJ LOT C digo de lote Estable hasta AAAA MM DD l Codice del lotto l Da utilizzare prima del AAAA MM GG D D Verwendbar bis JJJJ MM TT US Storage temperature limitation US Consult Instruction for use Limites de temp ratures de stockage gt Consulter le mode d emploi Temperatura limite i E Consulte las instrucciones de uso I Limiti di temperatura di conservazione Consultare le istruzioni per uso D Lagertemperatur D Siehe Gebrauchsanweisung Made and distributed in France by Bio Rad 3 Boulevard Raymond Poincar 92430 Marnes La Coquette France Tel 33 1 47 95 60 00 Fax 33 1 47 41 91 33 04 2009 Code 862196
22. tative determination include all the controls R3 R4a and R4b for each test run a Conditions of validation Criteria Validation The absorbance of each individual negative control must OD R3 i lt 0 05 be lower than 0 05 The test must be repeated if at least one value lies outside of this limit 0 300 lt OD lt 1 200 Each individual value of the R4a positive control OD must be between 0 300 and 1 200 The test must be repeated if at least one of the R4a OD value lies outside of this limit 1 500 lt OD lt 3 500 Each individual value of the R4b positive control optical densities must be between 1 500 and 3 500 However a maximum of one individual aberrant value can be eliminated when its optical density is lower than 1 500 or higher than 3 500 The test must be repeated if the two R4b positive control OD lie outside of this limit b Interpretation of the results The Threshold Value is equal to the mean of the two R4a Positive controls OD R4a and corresponds to the seroconversion threshold value at 0 5 EU ml High seroconversion Value is equal to the mean of the two R4b Positive control OD R4b or to the single R4b positive control if one aberrant value has been eliminated Optical Density of each sample is compared to this High seroconversion and Threshold Value Criteria Result Seroconverted OD sample gt OD R4b Validation Samples with OD va
23. temperature the enzymatic reaction is stopped by addition of a solution H2SO4 1N The optical density reading obtained with a spectrophotometer set at 450 620 nm is proportional to the amount of anti rabies antibodies present in the samples A standard curve is constructed using the Quantification standards 51 to S6 obtained by serial dilutions of R4b calibrated Positive Controls The optical density values for the unknown samples are compared with the Positive Controls Sera titres in quantification tests are obtained after a direct reading on the standard curve and are expressed as Equivalent units ml EU ml unit equivalent to the international units defined by seroneutralization 4 COMPOSITION OF THE PLATELIA RABIES ASSAY All reagents are exclusively for in vitro veterinary use Enough reagents are provided for 2 x 96 assays On each microplate 90 samples can be analysed if a qualitative test is performed Up to 80 samples can be quantified precisely for rabies antibodies if a quantification system is used LABELLING TYPE OF REAGENT PRESENTATION R Microplate 12 strips of 8 wells sensitized with the rabies 2 plates virus glycoprotein R2 Wash solution 10 fold concentrated Tris NaCl buffer 1 vial Preservative ProClin 300 0 01 250 ml R3 Negative control Non reactive control TRIS EDTA 1 vial Preservative ProClin 300 0 1 0 6 ml 0 5 EU ml Positive control 0 5 EU
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