Myco-TB – TB IgG
Contents
1. ELISA kit Positive Results S C 0 21 samples giving an absorbance greater than or equal to the Cut off value are considered initially reactive which indicates that IgG antibodies to Mycobacterium tuberculosis have probably been detected using this TB IgG ELISA kit Retesting in duplicates of any initially reactive sample is recommended Repeatedly reactive samples can be considered positive for IgG antibodies to Mycobacteria Borderline S C O 0 9 1 1 Samples with absorbance to Cut off ratio between 0 9 and 1 1 are considered borderline and retesting of these samples in duplicates is recommended to confirm the results Repeatedly positive samples can be considered positive for antibodies to MTb Analytical Specificity 1 No cross reactivity observed with samples from patients infected with HAV HIV HBV HCV CMV and TP 2 No interference was observed from rheumatoid factors up to 2000U ml 3 These assay performance characteristics are unaffected from elevated concentrations of bilirubin hemoglobin and triolein 4 Frozen specimens have been tested to check for interferences due to collection and storage LIMITATIONS 1 Non repeatable positive result may occur due to the general biological characteristics of the ELISA method The assay is designed to achieve very high performance characteristics of sensitivity and specificity and the indirect model minimizes the unspecific reactions due to interference with un2. each time into the wells 5 In case of manual washing we suggest to perform at least 5 cycles dispensing 350 400ul well and aspirating the liquid for 5times If poor results high background are observed increase the washing cycles or soaking time per well 6 In any case the liquid aspirated out the strips should be treated with a sodium hypochlorite solution final concentration of 2 5 for 24 hours before liquids are disposed in an appropriate way 7 The concentrated Washing solution should be diluted 1 to 20 before use For one plate mix 50 ml of the concentrate with 950ml of water for a final volume of 1000mI diluted Wash Buffer If less than a whole plate is used prepare the proportional volume of solution STORAGE AND STABILITY The components of the kit will remain stable through the expiration date indicated on the label and package when stored between 2 8 C do not freeze To assure maximum performance of this TB IgG ELISA kit protect the reagents from contamination with microorganism or chemicals during storage PRECAUTIONS AND SAFETY This kit is intended FOR RESEARCH USE ONLY The ELISA assay is a time and temperature sensitive method To avoid incorrect result strictly follow the test procedure steps and do not modify them 1 Do not exchange reagents from different lots or use reagents from other commercially available kits The components of the kit are precisely matched as to achieve optimal performa
3. MpressBio Myco TB TB IgG Catalog WT1096 IgG ANTIBODIES TO M TUBERCULOSIS ELISA KIT Two Step Incubation Indirect Principle INSTRUCTIONS FOR USE This kit is an enzyme linked immunosorbent assay for qualitative detection of antibodies to Syphilis in human serum or plasma For research use only SUMMARY It has been estimated that more than 1 7 million people were infected with Mycobacterium tuberculosis MTb in 1990 and this number increase every year due to new infections HIV and immigration Now with worldwide prevalence of 30 million cases and an incidence of 10 million new cases each year tuberculosis TB is one of the most important health threatening problems worldwide PRINCIPLE OF THE ASSAY This kit employs solid phase indirect ELISA assay for detection of IgG antibodies to MTb in two step incubation procedure Polystyrene microwell strips are pre coated with affinity purified MTb antigens including A60 antigen complex During the first incubation step MTb IgG specific antibodies if present will be bound to the solid phase pre coated antigen complexes The wells are washed to remove unbound serum proteins and rabbit anti human IgG antibodies anti lgG conjugated to horseradish peroxidase HRP are added During the second incubation step these HRP conjugated antibodies will be bound to any antigen lgG complexes previously formed and the unbound HRP conjugate is then removed by washing Chromogen solution
4. ate at 37 C for 15 minutes avoiding light The enzymatic reaction between the Chromogen A B solutions produces blue color in Positive control and TB IgG Positive sample wells Step 11 Stopping Reaction Using a multichannel pipette or manually add 50 ul Stop Solution into each well and mix by tapping the plate gently Intensive yellow color develops in Positive control and TB IgG Positive sample wells Step 12 Measuring the Absorbance Calibrate the plate reader with the Blank well and read the absorbance at 450nm If a dual filter instrument is used set the reference wavelength at 630nm Calculate the Cut off value and evaluate the results Note read the absorbance within 5 minutes after stopping the reaction INTERPERTATION OF RESULTS AND QUALITY CONTROL Each microplate should be considered separately when calculating and interpreting the results regardless of the number of plates concurrently processed The results are calculated by relating each sample s optical density OD value to the Cut off value C O of the plate If the Cut off reading is based on single filter plate reader the results should be calculated by subtracting the Blank well OD value from the print report values of samples and controls In case the reading is based on dual filter plate reader do not subtract the Blank well OD from the print report values of samples and controls 1 Calculation of Cut off value C O NC 0 15 NC the mea
5. e CARDBOARD PLATE COVER 2 sheets To cover the plates during incubation and prevent evaporation or contamination of the wells PACKAGE INSERTS 1 copy ADDITIONAL MATERIALS AND INSTRUMENTS REQUIRED BUT NOT PROVIDED Freshly distilled or deionized water 2 Disposable gloves and timer 3 Appropriate waste containers for potentially contaminated materials 4 Disposable V shaped troughs 5 Dispensing system and or pipette single or multichannel disposable pipette tips 6 Absorbent tissue or clean towel 7 Dry incubator or water bath 37 0 5 C 8 Microshaker for dissolving and mixing conjugate with samples 9 Microwell plate reader single wavelength 450nm or dual wavelength 450nm and 630nm 10 Microwell aspiration wash system SPECIAMEN COLLECTION TRANSPORTATION AND STORAGE 1 Sample Collection Either fresh serum or plasma samples can be used for this assay Blood collected by venipuncture should be allowed to clot naturally and completely the serum plasma must be separated from the clot as early as possible as to avoid hemolysis of the RBC Care should be taken to ensure that the serum samples are clear and not contaminated by microorganisms Any visible particulate matters in the sample should be removed by centrifugation at 3000 RPM for at least 20 minutes at room temperature or by filtration on 0 22u filters Plasma samples collected into EDTA sodium citrate or heparin may be tested but hig
6. ent Add 100uI Specimen Diluent into each Step 4 Note Step 5 well except in the blank Adding Sample Add 10ul of Specimen and 10ul of Positive and Negative controls into their respective wells Use a separate disposal pipette tip for each specimen Negative Control and Positive Control as to avoid cross contamination Mix by tapping the plate gently Incubating Cover the plate with the plate cover and incubate for 30 minutes at 37 C It is recommended to use water tank to assure the temperature stability and humidity during incubation If dry incubator is used do not open the door frequently Step 6 Washing After the end of the incubation remove and Step 7 Step 8 Step 9 discard the plate cover Wash each well 5 times with diluted Wash buffer Each time allow the microwells to soak for 30 60 seconds After the final washing cycle turn the strips plate onto blotting paper or clean towel and tap the plate to remove any remainders Adding HRP Conjugate Add 100 ul HRP Conjugate to each well except the Blank HRP Conjugate Incubating Cover the plate with the plate cover and incubate for 30 minutes at 37 C Washing After the end of the incubation remove and discard the plate cover Wash each well 5 times with diluted Wash buffer as in Step 6 Step 10 Coloring Dispense 50ul of Chromogen A and 50ul Chromogen B solution into each well including the Blank and mix by tapping the plate gently Incubate the pl
7. he reagents with new ones 2 If after mixing of the Chromogen A and B solutions into the wells the the color of the mixture turns blue within few minutes do not continue carrying out the testing and replace the reagents with fresh ones VALIDITY Please do not use this kit beyond the expiration indicated on the kit box and reagent labels REFERENCES 1 Haslav K Boosting effects in BCG vaccinated guinea pigs of tuberculin skin tests on subsequent skin tests and on the in vitro lymphocyte transformation response 1983 International Symposium on BCG Vaccines and Tuberculins Budapest Hungary Develop biol Standard 58 517 521 S Karger Basel 1986 2 Zou YL Zhnag JD Chen MH Shi GQ Prignot J Cocito C 1194 Serological analysis of pulmonary and extrapulmonary tuberculosis with enzyme linked immunosorbent assay for anti A60 immunoglobulings C I D 19 1084 1091 3 Wirrmann C 1990 Public health applications of serological tests for tuberculosis Study of the incidence of inapparent infectious employees of an alsacian supermarket Eur J Epidemol 6 304 308 4 Harboe M 1981 Antigens of PPD old tuberculin and autoclaved M bovis BCG studied by cross immunoelectrophoresis Am Rev Respir 5 Dis 124 80 87 6 Anda Biologicals Publications P O BOX 458 Thurmont MD 21788 USA Tel 301 228 2444 Fax 301 560 6570 Toll Free 888 562 8914 www xpressbio com info xpressbio com XpressBio Life Science Prod
8. hly lipaemic icteric or hemolized samples should not be used as they could give erroneous results in the assay Do not heat inactivate samples This can cause sample deterioration 2 Transportation and Storage Store samples at 2 8 C Samples not required for assaying within 3 days should be stored frozen 20 C or lower Multiple freeze thaw cycles should be avoided For shipment samples should be packaged and labeled in accordance with the existing local and international regulations for transport of clinical samples and ethological agents SPECIAL INSTRUCTIONS FOR WASHING 1 A good washing procedure is essential to obtain correct and precise analytical data 2 It is therefore recommended to use a good quality ELISA microplate washer maintained at the best level of washing performances In general no less than 5 automatic washing cycles with dispensing of 350 400ul well are sufficient to avoid false positive reactions and high background all wells turn yellow 3 To avoid cross contaminations of the plate with sample or HRP conjugate after incubation do not discard the content of the wells but allow the plate washer to aspirate it automatically 4 Anyway we recommend calibrating the washing system on the kit itself in order to match the declared analytical performances Assure that the microplate washer s liquid dispensing channels are not blocked or contaminated and sufficient volume of Wash buffer is dispensed
9. kin or eyes ProClin 300 used as a preservative can cause sensation of the skin The enzymatic activity of the HRP conjugate might be affected from dust reactive chemical and substances like sodium hypochlorite acids alkalis etc Do not perform the assay in the presence of such substances Materials Safety Data Sheet MSDS available upon request If using fully automated microplate processing system during incubation do not cover the plates with the plate cover The tapping out of the remainders inside the plate after washing can also be omitted Step 1 Reagents preparation Allow the reagents and samples to reach room temperature 18 30 C for at least 15 30minutes Check the Wash buffer concentrate for the presence of salt crystals If crystals have formed in the solution resolubilize by warming at 37 C until crystals dissolve Dilute the stock wash Buffer 1 to 20 with distilled or deionized Step 2 water Use only clean vessels to dilute the Wash buffer Numbering Wells Set the strips needed in strip holder and number sufficient number of wells including three Negative control e g B1 C1 D1 two Positive control e g E1 F1 and one Blank A1 Neither samples nor HRP Conjugate should be added into the Blank well If the results will be determined by using dual wavelength plate reader the requirement for use of Blank well could be omitted Use only number of strips required for the test Step 3 Adding Dilu
10. known matters in sample and the anti human IgG antibodies Antibodies may be undetectable during the early stages of the disease and in some immunosuppressed individuals 2 Common sources for mistakes are kits beyond the expiry date bad washing procedures contaminated reagents incorrect assay procedure steps insufficient aspiration during washing failure to add samples or reagents equipment timing volumes sample nature and quality 3 The prevalence of the marker will affect the assay s predictive values 4 lf after retesting of the initially reactive samples the assay results are negative these samples should be considered as non repeatable false positive and interpreted as negative As with many very sensitive ELISA assays false positive results can occur due to the several reasons most of which are related but not limited to inadequate washing step 5 This is a qualitative assay and the results cannot be used to measure antibodies concentrations INDICATIONS OF INSTABILITY OR DETERIORATION OF THE REAGENTS 1 Values of the Positive or Negative controls which are out of the indicated Quality control range are indicator of possible deterioration of the reagents and or operator or equipment errors In such case the results should be considered as invalid and the samples must be retested In case of constant erroneous results classified as due to deterioration or instability of the reagents immediately substitute t
11. n absorbance value for three negative controls Example 1 Calculation of NC Well No B1 C1 D1 Negative controls OD value 0 02 0 012 0 016 NC 0 016 2 Calculation of Cut off C O 0 016 0 15 0 166 If one of the Negative Control values does not meet the Quality Control Range specifications it should be discarded and the mean value is calculated again using the remaining two values If more than one control OD value does not meet the Quality Control Range specifications the test is invalid and must be repeated 2 Quality Control Range The test results are valid if the Quality Control criteria are verified It is recommended that each laboratory must establish appropriate quality control system with quality control material similar to or identical with the patient sample being analyzed 1 The OD value of the Blank well which contains only Chromogens and Stop solution is less than 0 080 at 450 nm 2 The OD value of the Positive control must be equal to or greater than 0 800 at 450 630nm or at 450nm after blanking 3 The OD value of the Negative control must be less than 0 100 at 450 630nm or at 450nm after blanking 3 Interpretations of results S the individual absorbance OD of each specimen Negative Results S C O lt 1 samples giving absorbance less than the Cut off value are negative for this assay which indicates that no IgG antibodies to Mycobacterium tuberculosis have been detected with this TB IgG
12. nce during testing 2 Make sure that all reagents are within the validity indicated on the kit box and are of the same lot Never use reagents beyond the expiry date stated on reagents labels or on the kit box 3 CAUTION CRITICAL STEP Allow the reagents and samples to stabilize at room temperature 18 30 C before use Shake reagent gently before and return to 2 8 C immediately after use 4 Use only sufficient volume of sample as indicated in the procedure steps Failure to do so may cause in low sensitivity of the assay 5 Do not touch the bottom exterior of the wells fingerprints or scratches may interfere with microwell reading 6 When reading the results ensure that the plate bottom is dry and there are no air bubbles inside the wells 7 Never allow the microplate wells to dry after the washing step Immediately proceed to the next step Avoid the formation of air bubbles when adding the reagents 8 Avoid assay steps long time interruptions Assure same working conditions for all wells 9 Calibrate the pipette frequently to assure the accuracy of samples reagents dispensing Always use different disposal pipette tips for each specimen 10 11 12 13 14 15 16 17 18 19 20 21 and reagents as to avoid cross contaminations Never pipette solutions by mouth The use of automatic pipettes is recommended Assure that the incubation temperature is 37 C inside the incubat
13. or When adding samples avoid touching the wells bottom with the pipette tip When reading the results with a plate reader it is recommended to determine the absorbance at 450nm or at 450nm with reference at 630nm All specimens from human origin should be considered as potentially infectious Materials from human origin may have been used in the kit These materials have been tested with tests kits with accepted performance and found negative for antibodies to HIV HCV TP and HBsAg However there is no analytical method that can assure that infectious agents in the specimens or reagents are completely absent Therefore handle reagents and specimens with extreme caution as if capable of transmitting infectious diseases Strict adherence to GLP Good Laboratory Practice regulations can ensure the personal safety Never eat drink smoke or apply cosmetics in the assay laboratory Bovine derived sera may have been used in this kit Bovine serum albumin BSA and fetal calf sera FCS are derived from animals from BSE TSE free geographical areas The pipette tips vials strips and sample containers should be collected and autoclaved for 1 hour at 121 C or treated with 10 sodium hypochlorite for 30minutes to decontaminate before any further steps for disposal The Stop solution 2M H2SO is a strong acid Corrosive Use it with appropriate care Wipe up spills immediately or wash with water if come into contact with the s
14. s containing Tetramethylbenzidine TMB and urea peroxide are added to the wells In presence of the antigen lgG anti lgG HRP immunocomplex the colorless Chromogens are hydrolyzed by the bound HRP conjugate to a blue colored product The blue color turns yellow after stopping the reaction with sulfuric acid The amount of color intensity can be measured and is proportional to the amount of antibody captured in the wells and to the sample respectively Wells containing samples negative for IgG antibodies to MTb remain colorless COMPONENTS MICROWELL PLATE 1 plate Blank microwell strips fixed on white strip holder The plate is sealed in aluminum pouch with desiccant 8x12 12x8 well strips per plate Each well contains affinity purified MTb antigens The microwell strips can be broken to be used separately Place unused wells in the plastic sealable storage bag together with the desiccant and return at 2 8 C NEGATIVE CONTROL 1 vial Colorless liquid filled in vial with green screw cap 0 5ml per vial Protein stabilized buffer tested non reactive for MTb IgG antibodies Preservatives 0 1 ProClin 300 Ready to use as supplied Once open stable for one month at 2 8 C POSITIVE CONTROL SERUM 1 vial Red liquid filled in vial with red screw cap 0 5ml per vial MTb antibodies diluted in protein stabilized buffer containing preservatives 0 1 ProClin 300 Ready to use as supplied Once open stable for one month a
15. t 2 8 C SPECIMEN DILUENT 1 vial Blue liquid filled in a white vial with blue screw cap 12 ml per vial Protein stabilized buffer casein and sucrose solution Ready to use as supplied Once open stable for one month at 2 8 C HRP CONJUGATE REAGENT 1 vial Red liquid filled in a white vial with red screw cap 12ml per vial Horseradish peroxidase conjugated rabbit anti human IgG antibodies Ready to use as supplied Once open stable for one month at 2 8 C STOCK WASH BUFFER 1 bottle DILUTE BEFORE USE Colorless liquid 50 ml per bottle PH 7 4 20 x PBS Containing Tween 20 as a detergent The concentrate must be diluted 1 20 with distilled deionized water before use Once diluted stable for one weeks at room temperature or for two weeks at 2 8 C CHROMOGEN SOLUTION A 1 vial Colorless liquid filled in white vial with green screw cap 6 ml per vial Urea peroxide solution Ready to use as supplied Once open stable for one month at 2 8 C e CHROMOGEN SOLUTION B 1 vial Colorless liquid filled in the black vial with black screw cap 6 ml per vial TMB solution Tetramethylbenzidine dissolved in citric acid Ready to use as supplied Once open stable for one month at 2 8 C STOP SOLUTION 1 vial Colorless liquid filled in white vial with yellow screw cap 6 ml per vial Diluted sulfuric acid solution 2 0M H2804 Ready to use as supplied PLASTIC SEALABLE BAG 1 unit For enclosing the strips not in us
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