Vitrotest Rubella-IgM ultra ELISA test-kit for qualitative determination
Contents
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3. CERO SSE SHEER t t f 9060 09090004909000450209 h 9 4 9 HHH EHH eee 22 4690060960649 600664949844244624a444 524 9 4 b 9 9 4 9 9 HEHEHE ns ruc ion or use re 9999009 6 9909004990099999 09094 99 99 ciMtitiiiitii 9999090900090 9000909009099 99 0099 9999 20099000902 2020220000 00090600 Oo NNEA RE EEERerE T 0 ty t ppt ap Geaa lintel eapo dl i Geen A 9o906490209049049 9 59949 b9 9 95 bh xo SSSSSCOCSESHSCE COSHH HOSE HSH Hee ere eee eeerrere 1 Intended use PRE EFIDSOEDEDD ED LE bled deo pred Mid dur e 9000009090000 00000950090450 9 9 5 9 9 iiciii BOSE SEEMS q REA pp dap eode prodiens pe ein eie s E 9 90649900994990994 999 99 9 9 515 64424114144144441444444 44 21 22 221 22 ee kw 2 Clinical value 990909090000000900000000000000009009000 9922 e 9 e t h 96606600000000409000099 20909400909 99 9 2 5 v5i Q8 q s REDS E EEO R EG Epi ch abcde peated odes Peale 66 9 6 n eesveeoeeceo eee eo eo eee eee ee ree ee ee eee 32 2222200022900 DED hhh ea heeded eeeesteeeereresss 3 Principle of the test e 9900900990009 0000609500450022 59 eto h 960006600006000000500090500 4500055 oo5ss5oc rris 4365 6 9 bdo pdreibdedediodciadicdoreodididipii 00 9 9 4 M 990909 9990909 09 9 9799 9 9 99 9 99 9 9 eee eee 32 SSESCSSCSCSCSCOSCSCEE OSCE OSS SSCS SHEESH HHH HEHEHE Hee aterials and equipment 4656 9 909090 06 09050060909 9 9505
4. Vitrotest Rubella IgM ultra are negative for anti HCV anti HIV1 2 anti T pallidum antibodies and HBsAg Nevertheless all controls and sera should still be regarded and handled as potentially infectious the liquid waste must be inactivated for example with the hydrogen peroxide solution at the final concentration of 6 for 3 hours at room temperature or with the sodium hypochlorite at the final concentration of 5 for 30 minutes or with other disinfectant agents the solid waste must be inactivated with autoclaving at 121 C for 1 hour do not autoclave the solutions that contain sodium azide or sodium hypochlorite avoid spilling of TMB solution and Stop reagent and any contact of these solutions with the skin or mucosa in case of spilling of solutions that do not contain acid e g sera rinse the surface with hydrogen 6 solution then dry with filter paper 6 Storage and stability Reagents of the kit are stable up to the expiry date on the label when stored at 2 8 C Transport the test kit at 2 8 C Disposable transportation at temperature not higher than 20 C during two days is allowed 7 Specimen collection The serum or plasma samples should be stored at 2 8 C not more then 3 days after collection It is possible to store them longer but frozen 20 to 70 C Before use frozen samples wait for 30 minutes for the reagents to stabilize at room temperature Mix thawed samples well to homogeneity Av
5. foaming After addition of serum color of the solution in wells changes from brown green to blue The procedure of dilution of samples and controls should be carried out immediately prior to analysis 9 Assay procedure 9 1 Take out from the protective packing the support frame and the necessary number of wells the number of investigated samples and five wells for calibrators Wells with calibrators must be included in each analysis 9 2 Complete the sera identification plan 9 3 Prepare washing solution refer to point 8 2 9 4 Conduct pre dilution of sera refer to point 8 3 9 5 Dispense 90 ul of sample diluent in each well 9 6 Dispense 10 ul of pre diluted controls and unknown samples into the wells in the following order A1 pre diluted 1 10 positive control B1 C1 and D1 pre diluted 1 10 negative control E1 and rest wells pre diluted 1 10 unknown samples Thus the final dilution of serum in wells of ELISA plate should be 1 100 Gently pipette the mix in wells avoiding foaming color of the solution in wells changes from yellow to green 9 7 Cover strips with an adhesive film and incubate for 30 min at 37 C 9 8 After completing the incubation remove the adhesive film carefully and wash the wells five times using the automatic washer or 8 channel pipette in the following order aspirate the content of wells strips into a liquid waste container fill the strip wells with a minimum of 300 microliters
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8. 25cvo ee 06 9 50090945099 9 9 9 9 EEE Hee TS nm 9900099 0 6 06 4 9 9 9 9 9 9 rn c or beo 0084646445824 8 4 b 9 9 9 9 9 e 49900999999949 9999 9 99 ET M GO99P9 9 99 9 99 99999 29 99 9 9 itii PWePOSCCOSCSE COSCO SEEPS SEE EHH Eee ee SSSSCCHESSEEHREEEEESEEAEP ESSERE HEHEHE Hee PPSOSSOSSAES OSCARS HE S HEE ELISA test for the diagnosis of infectious diseases TS TY Y 24 4 36555928 001 2011 Vitrotest Rubella IgM ultra ELISA test kit for qualitative determination of IgM class antibodies to rubella virus 1 Intended use ELISA test kit Vitrotest Rubells IgM ultra is intended for qualitative determination of IgM class antibodies to rubella virus in human serum or plasma The test kit may be used both for the ELISA using automatic pipettes and standard equipment and for setting with the open system immunoenzymatic automated analyzer 2 Clinical value Rubella is a viral infectious disease characterized by skin rash generalized lymphadenopathy mild intoxication and short lived fever Usually the disease is rapid and mild Particular importance of rubella infection is connected with the risk of serious complications for the child that may develop with infection of the mother during pregnancy Given that the symptoms of the rubella disease are often few expressed and in 30 5096 of cases occur without noticeable symptom
9. 50 nm is possible Reserve blank well to adjust spectrophotometer in such analysis Only TMB substrate and stop reagent must be added in blank well 10 Calculation and interpretation of the results 10 1 Test validity conditions Calculate the mean optical density OD of negative control OD NC mean OD NC1 OD NC2 OD NC3 3 In order for an assay to be considered valid the following criteria must be met OD of the positive control is not lower than 1 2 optical unit OU OD of negative controls should be lower or equal to 0 15 OU OD of every negative control differs no more than twice from the mean value of negative control that is OD NCmean X 0 5 lt OD NCn lt OD NCmean x 2 0 If one of the negative controls does not respect this norm disregard and recalculate the mean using remaining values 10 2 Calculation of the results Calculate cut off by adding value 0 3 to the mean NC that is Cut off OD NCmean 0 3 Calculate the index of positivity IP for each sample IP ODsample Cut off 10 3 Interpretation of the results The samples with IP above 1 1 are considered positive IP gt 1 1 The samples with IP below 0 9 are considered negative IP lt 0 9 The samples with IP within 0 9 1 1 are considered indeterminate 0 9 IP 1 1 It is recommended to retest such samples in duplicate If the results are again within indeterminate it is necessary to test sera obtained after 2 4 weeks If you get undefine
10. 90090949 99 9 99 9 tvcitiittit SSSSSSSSS SSS sess sess SOCCOSSER OCHO RESEHE HEHEHE HH HH Oe eebestarsisteccccsesersectessces secs 10 Calculation and interpretation of the results ee r 64 99 9 qi bod erdebded pipe A 6909995 bt h 9999 90e0eeeveeeevoovevsv oseso oos ttt nnn i 226 99066 i4 eH PROC de a HR ddp ox 6 4600000 0950004500909 9599 2 4 9 97 2e 699909000090090944090404526042445 8424224 24 4242 5 5 11 Performance characteristics ret 99909099990909 909099 49 99 9 9 9 9 c cxiititi tt 0690909900099900999090909499094 59009 99 9 9 4 9 9 iicii ersebe43 TOP EOE SEE OH e peat 6 kv Lie 6 6 0680060659 99 094 5 999 9 9 t t 2s 4 9909 099909094949009949 9 9 094 295 4 94iirtitiz 12 Limits of the test 99999099090090909000909000e90909 999 99999999 e 9 6 9990909900000000000000000099000 9999 95 i EAT Hin iif t1 1b e eMe R f 64690949094 45450444 h 4 h 9 c 99099 9 9909094 9009949 909094 9 9 9 9 itiiiititit t e erence souces SOSSSCSSSSOSSESOSSEHESSSER ESSERE HEHEHE HHH ee 1120099029009 DE DE ag e dedehcdedbatestate TERT 6860606605060945299 9 9 9 9 s 0999909099 990949909090945295 249452524 cc Legend 990909009000090900009000090000090900999999999922 644 v9900909099 090 0909009009900064509 9 9 b cif nni SESOOOSHOOEEO
11. SSESESEEEE SHEP EHH HEHE ee ees TT 090609090500608448584 064064586094586064249559292292 4 v 5 ee 9990609999 0909409009949 9 5994 9 9 9 9 titiitiittli Pe 9 99 9 9 9 99 9909990 9 9 9 9 9 9 9 9 9 9 9 v4 9009099090009 50604049 50045 09 9 9 4 59 9 oe SESOSSESESHSEERESSEEESEEHEEEHE HHH HEHEHE HEHE 4 SPOSSSCSSSSESCOSSAES ESSE SESH bh9 9 3 HEHEHE HEE a 99 9 99 909 4 909 9949 9 9 9 49 9 9 9 8 5 9 w v 0909o 9 099 0909 0909 5 090 09 9 5 090945 0992924 9 29 92 4 5 9 8 war gt SSSCSCSCSSCCECOSCSCE CR CCE PSHE P HHO ee e 6060606606484 40448464584a484 58240 4 bh9 99499 HEHEHE eee H 909999599094 90090949 990945 9299 4 9 9 hi rh Rr es 99 9 999 909 9 99999 99 999 99 99 99 9 9 tivi oe VOSSSOSCOSSESOSCSEH OSCE OHHH 9 HEH HH HEE eee 640 444344455455455555S5ESoaSR See e a n Iooro900909000090000909000099090909 90900292222 8 8 9 8 4 eo 9 9 9999 999099499 9 99 9 9 9 9 9 tttctitttic oe 009 9099 9099909999 5 9995 999 9 994 4 49 5 600000000000000000000000900009099 ee d w90060006484948594 4 46095244844584484845 599 29992 5 5 ee 9o99 0990949009949 9 9 945 49 9 9 99 9 9 t 122522 99 99 99 099 99 9 9999 9 9 ree eee ee v 5 1 122024 99909090909 0000499509045 090909 9 9 4 0 959060600000000409090004509090
12. Zimmerman L Chapter 11 Rubella VPD Surveillance Manual 3rd edition 2002 P 11 1 11 12 ni I A ul Uli For ail Legend Interpretation of notation conventions For in vitro diagnostic use Batch code Catalogue number Production date Expiry date Storage temperature limitation Meant for lt n gt tests Protect from direct solar radiation Attention Consult instruction for use Manufacturer and its address Consult instructions for use questions and suggestions regarding the kit contact the manufacturer Ramintek Innovation Production Company 03039 Ukraine Kiev 7 October 40th Anniversary Av of 227 registered address 07300 Vishgorod Kiev region 19 Sholudenko Str factual address Tel 380 44 222 76 72 Scheme of the assay Vitrotest Rubella IgM ultra Keep reagents at room temperature 18 25 C during 30 minutes Prepare washing solution dilute 20x concentrate washing solution Tw20 with distilled water 1 20 1 19 For example 4 ml of solution 76 ml of water Complete the sera identification plan Prepare pre dilution 1 10 of sera in wells of plates for pre dilution add 90 ul of sample pre diluent brown green and 10 pl of samples or controls After dispensing of serum the color in well switches from brown green to blue Dispense 90 pl of sample diluent yellow and add 10 ul of pre diluted controls and patient samples into the wells A1 positive control B1 C1 D1 negat
13. d results assume that the serum is negative 11 Performance characteristics 11 1 Specificity and sensitivity Specificity and sensitivity of the test Vitrotest Rubella IgM ultra were evaluated using commercial panel Anti Rubella Mixed Titer Performance Panel PTR201 production SeraCare Life Sciences USA which consists of 5 samples containing IgM antibodies to rubella virus and 20 sera not containing specific IgM antibodies In the test Vitrotest Rubella IgM ultra all samples of panel PTR 201 were determined correctly according to passport data In the study of specificity of the test Vitrotest Rubella IgM ultra using 154 sera negative for antibodies to rubella virus all 154 sera were found negative In comparative studies of the test Vitrotest Rubella IgM ultra and another commercial test with CE marking were analyzed 12 sera containing IgM antibodies to rubella virus including four sera obtained from vaccinated persons at 24 28 days after vaccination all of these samples were found positive in both test system 11 2 Accuracy Intra assay reproducibility Coefficient of variation CV was calculated in 32 repetitions of two sera with different level of specific antibodies on two series of test kits Serum Ne OD mean CV CV2 S10 0 898 7 4 5 9 S7 1 732 6 5 5 8 Inter assay reproducibility Coefficient of variation CV for two sera with different level of specific antibodies was calculated for three days in three ELISA
14. ive control E1 and other wells patient samples Cover wells with an adhesive film incubate for 30 min at 37 C Wash wells five times Dispense 100 pl of conjugate solution violet into the wells Cover wells with adhesive film incubate for 30 min at 37 C Wash wells five times Dispense 100 pl of TMB substrate solution into the wells Incubate the plate for 30 min in the dark at room temperature 18 25 C Add 100 ul of stop reagent in each well Read optical density at 450 620 nm Calculate the cut off of the assay Vitrotest Rubella IgM ultra Cut off OD NC mean 0 3 Calculate the index of positivity IP for patient samples zp 2 of patient sample cut off Interpret the results according to the table IP value Result IPsample gt 1 1 positive 0 9s IPsample lt 1 1 indeterminate IPsampie lt 0 9 negative
15. ntigen on the solid phase After washing out unbound components anti specific conjugate of anti IgM monoclonal antibodies with horseradish peroxidase added to the wells it binds to immune complexes in the solid phase Unbound components are washed out The immune complex formed in the wells are visualized by adding chromogen solution of 3 3 5 5 tetramethylbenzidine TMB As a result of the reaction solution in wells where immune complexes were formed would be painted in blue The reaction is stopped by adding stop reagent blue colored wells become yellow The result of the analysis is determined by spectrophotometric reading at 450 620 nm 4 Materials and equipment 4 1 Contents of the kit ELISA plate 12 strips of 8 wells with the possibility of separation of the wells with immobilized antigen of Toxoplasma gondii Positive control 1 vial containing 0 3 ml solution of specific immunoglobulis pink Negative control 1 vial containing 0 5 ml negative human serum yellow Sample pre diluent 1 bottle containing 12 ml buffer with detergent and preservatives brown green Sample diluent 1 bottle containing 12 ml buffer with detergent and preservatives yellow Conjugate solution 1 bottle containing 12 ml buffer solution of monoclonal antibodies to human IgM conjugated with horseradish peroxidase with stabilizers and preservatives violet Ready to use Washing solution Tw20 20x 1 bottle containing 50 ml 20 fold c
16. of washing solution for each well respect the soak time of a minimum of 30 seconds aspirate the solution of all wells the residual volume of solution after aspiration must be lower than 5 microliters repeat the washing step four more times after the last aspiration blot the microplate by turning it upside down on absorbent paper 9 9 Dispense 100 ul of conjugate solution per well Cover strips with an adhesive film incubate for 30 min at 37 C 9 10 After completing the incubation remove the adhesive film carefully and wash the wells five times as described above refer to point 9 8 9 11 TMB solution is ready to use TMB substrate solution with hydrogen peroxide TMB solution should be colorless protect TMB substrate solution from straight sun rays To add TMB solution only new tips must be used carefully select a TMB solution from the vial and without touching the bottom and walls of the hole plate add 100 ul TMB solution per well 9 12 Incubate the strips for 30 minutes at room temperature of 18 25 C in the dark Do not use adhesive film in this incubation 9 13 Add 100 ul of stop reagent in each well Respect the same distribution sequence than for the TMB substrate solution 9 14 Read the optical density of every strip well in dual wavelength reading at 450 620 nm within the 5 minutes of stopping the reaction Pay attention to the cleanness of the wells bottom outside Measurement in the single wave procedure at 4
17. oid repeated freezing thawing Samples containing aggregates must be Clarified by centrifugation Do not use samples with contaminated hyperlipeamic and hyperhaemolysed sera 8 Reagent preparation It is very important to bring all reagents of the Vitrotest Rubella IgM ultra kit to room temperature 18 25 C for 30 minutes before use 6 1 ELISA plate preparation Before opening the ELISA plate allow it to stabilize at room temperature for 30 minutes to avoid water condensation inside the wells Open the vacuum bag and remove necessary amount of wells Immediately after removal of wells the remaining strips should be resealed with zip lock and stored at 2 8 C Microplate in thus packed bag is stable for 3 month 6 2 Washing solution The vial contains 50 ml of a concentrated 20X buffer with detergent Dilute the washing solution 1 20 1 19 with distilled or deionised water then mix For example for 4 ml of concentrate 76 ml of distilled water is enough for one strip Crystals in the solution disappear by warming up to 37 C for 15 20 min The diluted washing solution can be stored at 2 8 C not more than 7 days 6 3 Pre dilution of samples and controls Pre dilute samples and controls by 10 times with sample pre diluent For this purpose in the required number of wells of plate for pre dilution of sera comes in a set dispense 90 ul of sample pre diluent and add 10 ul of sera and controls Gently pipette the mix in wells avoiding
18. oncentrated phosphate buffer with Tween 20 colourless TMB Solution 1 bottle containing 12 ml of TMB solution and hydrogen peroxide with stabilizers and preservatives colourless Stop reagent 1 vial containing 12 ml of 0 5M sulphuric acid solution colourless Plate for pre dilution of serum 12 strips of 8 wells Adhesive film 2 sheets of adhesive film for covering the plates during incubation Sera identification plan 1 sheet of paper for noting the schemes of samples distribution Instruction for use one copy of user manual 4 2 Additional reagents materials and equipment In order to conduct the analysis the following additional reagents materials and equipment are required deionized or distilled water filter paper graduated cylinders of 10 200 and 1000 ml capacity disposable gloves hydrogen peroxide solution 6 disposable glassware for preparing the reagents bottles and trough timer mono and multi channel automatic adjustable pipettes capable of delivering volumes of 20 200 and 1000 microliters and tips for them thermostat for 37 C container for solid waste container for liquid waste automatic or semi automatic washer mono or multi channel reader for microplates at 450 620 695 nm 12 Please consult us about the adaptation of your equipment 5 Reservations and safety precautions 9 1 Reservations do not use e
19. performance in eight repetitions for each analysis Serum Ne OD mean CV S10 0 931 7 1 S7 1 820 5 2 12 Limits of the test A positive result in the test Vitrotest Rubella lgM ultra is an evidence of presence in patient of antibodies of class IgM specific to rubella virus IgM class antibodies present in the blood during acute rubella infection in children and adults regardless of severity of clinical manifestations and in infants with congenital rubella infection For diagnosis should take into account both results of laboratory tests and clinical manifestations of the disease For the purpose of leveling of false positive results caused by the presence in samples of human blood serum autoantibodies specific for immunoglobulins G rheumatoid factor in the test is used a special block component that prevents the formation of immune complexes with anti human antibodies on the solid phase Reference sources 1 Center for Disease Control and Prevention Rubella and Congenital rubella syndrome United States 1994 1997 MMWR 1997 V 46 P 350 354 2 Knox E G Theoretical aspects of rubella vaccination Rev Infect Dis 1985 V 7 P 194 197 3 Tischer A Gerike E Immune response after primary ad revaccination with different combined vaccines against measles mumps rubella Vaccine 2000 V 18 P 1382 1392 4 WHO Guidelines for surveillance of CRS and Rubella WHO 1999 92 p 5
20. s laboratory diagnostic methods are acquiring particular importance After vaccination as well as in primary infection with rubella virus the first there are specific immunoglobulins IgM after infection they are recorded in 3 5 days after the appearance of rash and after vaccination antibodies of this class begin to appear in the blood at the third or fourth week Specific IgG antibodies begin to be produced by the body on 7 10 th day since the clinical manifestations reaching a maximum after 4 5 weeks and remained at a high level for a long time Reliable evidence of acute rubella infection is the presence in serum of specific antibodies of class IgM which are also considered evidence of congenital rubella in infants IgG antibodies against rubella virus can be considered a sign of a previous rubella infection because the presence of these antibodies in serum indicates the end of an active infection Determining the level of specific IgG antibodies makes it possible to estimate the intensity of immunity after previous infection or vaccination and predict the possibility of disease in the case of re infection 3 Principle of the test Principle of the test of Vitrotest Rubella IgM ultra kit is based on an indirect enzyme immunoassay technique The solid phase is made of strip microplate coated with the purified antigen of rubella virus During incubation of samples in wells of ELISA plate specific to rubella virus antibodies are bound to the a
21. xpired reagents do not use in the analysis and do not mix components of different lots and components of test kits with different nosology do not use reagents of other manufacturers in composition with the Vitrotest sets Note possible to use washing solution Tw20 20X Sample pre diluent TMB solution and Stop reagent with other series that are different from those attached to the test kit These reagents are used in other test systems of Ramintek IPC Please consult us for details close reagent vials after use only with their appropriate caps strictly follow to the washing procedure requirements described in this instruction control the filling and full aspiration of the solution in the wells use a new distribution tip for each serum and reagent protect kit reagents from straight sun rays TMB solution must be colourless or light blue before its use If solution is dark blue or yellow it cannot be used Avoid any contact of the TMB solution with metals or metal s ions Use glassware thoroughly washed and rinsed with deionized water use only disposable pipette tips for adding TMB substrate into plate s wells never use the same glassware for conjugate solution and chromogen 5 2 Safety precautions all reagents included in the kit are intended for in vitro diagnostic use wear disposable gloves when perform analysis do not pipette by mouth the controls of
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