HEPATITIS B – HBsAg
Contents
1. LIMITATIONS 1 Non repeatable positive result can occur due to the general biological and biochemical characteristics of ELISA assays The test is designed to achieve performance characteristics of high sensitivity and specificity However in very rare cases some HBV mutants or subtypes may remain undetectable Antigens may be undetectable during the early stages of the disease and in some immunosuppressed individuals 2 Any positive result must be interpreted in conjunction with patient clinical information and other laboratory testing results 3 Common sources for mistakes 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 4 The prevalence of the marker will affect the assay s predictive values VALIDITY As indicated in the labellings Never use this kit Beyond the Expiration REFERENCES 1 Stevens C E P E Taylor and M J Tong 1988 Viral hepatitis and liver disease Alan R Riss New York N Y 142 Stevens C E P E Taylor M J Tong P T Toy G N Vyas P V Nair 2 J Y Weissman and S Krugman 1987 Yeast recombinant hepatitis B vaccine Efficacy with hepatitis B immune globulin in prevention of perinatal hepatitis B virus transmission JAMA 257 2612 2616 143 Stevens C E P T Toy P E Taylor2. Allow the reagents and samples to reach room temperature 18 30 C before use Shake reagent gently before use Return to 2 8 C immediately after use Do not touch the bottom exterior of the wells fingerprints or scratches may interfere with microwell reading When reading the results ensure that the plate bottom is dry and there are no air bubbles inside the wells 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 Avoid assay steps long time interruptions Assure same working conditions for all the wells Calibrate the pipette frequently to assure the accuracy Use different disposal pipette tips for each specimen and reagents in order to avoid cross contaminations Never pipette solutions by mouth The use of automatic pipettes and disposable tips is recommended Assure that the incubation temperature is 37 C inside the incubator When adding samples avoid touching the well s bottom with the pipette tip When reading the absorbance with a plate reader it is recommended to determine the absorbance at 450nm and with reference at 630nm All specimens from human origin should be considered as potentially infectious Strict adherence to GLP Good Laboratory Practice regulations can ensure the personal safety Never eat drink smoke or apply cosmetics in the assay laboratory The pipette tips vials
3. strips and sample containers should be collected and autoclaved for 1hour at 121 C or treated with 10 sodium hypochlorite for 30 minutes to decontaminate before any further steps for disposal The Stop solution 2M H2SO 4 is a strong acid Corrosive Use it with appropriate care Wipe up spills immediately or wash with water if come into contact with the skin 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 these substances ASSAY PROCEDURE Step 1 Step 2 Step 3 Reagents preparation Allow the reagents to reach room temperature 18 30 C 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 19 with distilled or deionized water Use only clean vessels to dilute the 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 Use only number of strips required for the test Adding Sample and HRP Conjugate Add 50 ul of Positive control Negativ
4. tested non reactive for HBsAg Preservatives 0 1 ProClin 300 Ready to use as supplied Once open stable for one month at 2 8 C POSITIVE CONTROL 1 vial Red color liquid filled in vial with red screw cap 1ml per vial HBsAg diluted in protein stabilized buffer containing preservatives 0 1 ProClin 300 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 7 ml per vial Horseradish peroxidase conjugated anti HBs antibodies Ready to use as supplied Once open stable for one month at 2 8 C STOCK WASH BUFFER 1 bottle Colorless liquid filled in blank bottle with white screw cap 30 ml per bottle PH 7 4 20 x PBS Containing Tween 20 as a detergent DILUTE BEFORE USE The concentration must be diluted 1 19 with distilled deionized water before use Once diluted stable for one week at room temperature or for one month at 2 8 C CHROMOGEN SOLUTION A 1 vial Colorless liquid filled in white vial with green screw cap 7 ml per vial Urea peroxide solution Ready to use as supplied Once open stable for one month at 2 8 C CHROMOGEN SOLUTION B 1 vial Colorless liquid filled in the black vial with black screw cap 7 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 wh
5. the remaining two values If more than one NEGATIVE 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 a The absorbance of the Blank well is less than 0 08 at 450 b The absorbance value OD of the Positive control must be equal to or greater than 1 800 after blanking c The absorbance value OD of the Negative control must be less than 0 100 after blanking 3 Interpretations of results S the individual absorbance OD of each specimen Negative Results S C O lt 1 samples giving an absorbance less than the Cut off value are considered negative which indicates that no hepatitis B surface antigen has been detected with this HBsAg 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 HBV surfaces antigen has probably been detected with this HBsAg ELISA kit Borderline Samples with absorbance to Cut off ratio between 0 9 and 1 00 are considered borderline samples and retesting is recommended Repeatedly positive samples can be considered positive for HBsAg
6. BsAg Patient s serum or plasma sample is added to the microwell together with a second antibody conjugated with horseradish peroxidase HRP and directed against a different epitope of HBsAg During incubation the specific immunocomplex formed in case of presence of HBsAg in the sample is captured on the solid phase After washing to remove sample serum proteins and unbound HRP conjugate Chromogen solutions containing tetramethylbenzidine TMB and urea peroxide are added to the wells In presence of the antibody antigen antibody HRP sandwich 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 can be measured and is proportional to the amount of antigen in the sample Wells containing samples negative for HBsAg remain colorless COMPONENTS 96 Tests 12 wells MICROWELL PLATE 1 plate Blank microwell strips fixed on white strip holder Eight 12 well strips per plate Each well contains monoclonal antibodies reactive to HBsAg anti HBs The plate is sealed in aluminium pouch with desiccant 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 to 2 8 C NEGATIVE CONTROL 1 vial Yellowish liquid filled in vial with green screw cap 1 ml per vial Protein stabilized buffer
7. For Research Use Only MpressBio HEPATITIS B HBsAg Catalog WB2296 Not for Diagnostic Use SURFACE ANTIGEN HBsAg ELISA One Step Incubation Double Antibody Sandwich Principle INSTRUCTIONS FOR USE This kit is an enzyme linked immunosorbent assay ELISA for qualitative detection of HBsAg in human serum or plasma For research use only SUMMARY Hepatitis B virus HBV is an enveloped double stranded DNA virus belonging to the Hepadnaviridae family and is recognized as the major cause of blood transmitted hepatitis together with hepatitis C virus HCV Infection with HBV induces a spectrum of clinical manifestations ranging from mild inapparent disease to fulminant hepatitis severe chronic liver diseases which in some cases can lead to cirrhosis and carcinoma of the liver Hepatitis B surface antigen or HBsAg previously described as Australia antigen is the most important protein of the envelope of Hepatitis B Virus The surface antigen contains the determinant a common to all known viral subtypes and immunologically distinguished in two distinct subgroups ay and ad HBV has 10 major serotypes and four HBsAg subtypes have been recognized adw ady ayw and ayr HBsAg can be detected 2 to 4 weeks before the ALT levels become abnormal and 3 to 5 weeks before symptoms develop PRINCIPLE OF THE ASSAY This HBsAg ELISA kit uses polystyrene microwell strips pre coated with monoclonal antibodies specific to H
8. T Lee and H Y Yip 1992 Prospects for control of hepatitis B virus infection implications of childhood vaccination and long term protection Pediatrics 90 Suppl 170 173 Hurie M B E E Mast and J P Davis 1992 Horizontal transmission of hepatitis B virus infection to U S born children of Hmong refugees Pediatrics 89 269 273 Szmuness W C E Stevens E J Harley E A Zang W R Olesko D C Williams R Sadovsky J M Morrison and A Kellner 1980 Hepatitis B vaccine demonstration of efficacy in a controlled trial in a high risk population in the U S N Engl J Med 303 833 841 Bhatnagar P K E Papas H E Blum D R Milich D Nitecki M J Karels and G N Vyas 1982 Immune response to synthetic peptide analogues of hepatitis B surface antigen specific for the a determinant Proc Natl Acad Sci USA 79 4400 4404 XpressBio 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
9. amples collected into EDTA sodium citrate or heparin may be tested but highly lipaemic icteric or hemolized samples should not be used as they can give false results in the assay Do not heat inactivate samples This can cause sample deterioraration 2 Transportation and Storage Store samples at 2 8 C Samples not required for assay within 3 days should be stored frozen 20 C or lower Avoid multiple freeze thaw cycles 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 of 350 400ul well are sufficient to avoid false positive reactions and high background 3 To avoid 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 liquid dispensing channels are not blocked or contaminated and sufficient volume of Wash buffer is dispensed each time into the wells 5 In case of manual washing we suggest to carry out at least 5 cycles dispensing 350 400ul well and aspirating the liquid for 5 times If poor r
10. bsorbance 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 INTERPRETATION OF RESULTS AND QUALITY CONTROL Each microplate must be considered separately when calculating and interpreting results of the assay regardless of the number of plates concurrently processed The results are calculated by relating each sample 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 must 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 Cut off value C O NC X 2 1 NC the mean 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 value C O 0 016 0 05 0 066 Quality Control Range Note If the optical density OD value of ONE of the NEGATIVE CONTROLS is ABOVE the stated quality control range it should be discarded and the mean value is calculated again using
11. e control and specimen into their respective wells Note Use a separate disposal pipette tip for each specimen Negative Control and Positive Control to avoid cross contamination Add 50 ul HRP Conjugate to each well except the Blank and mix by tapping the plate gently Step 4 Incubating Cover the plate with the plate cover Step 5 Step 6 Step 7 and incubate for 60 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 Washing At the end of the incubation remove and 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 down onto blotting paper or clean towel and tap the plate to remove any remainders 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 plate at 37 C for 30 minutes avoiding light The enzymatic reaction between the Chromogen solutions and the HRP Conjugate produces blue color in Positive control and HBsAg Positive sample wells Stopping Reaction Using a multichannel pipette or manually add 50 wl Stop Solution into each well and mix gently Intensive yellow color develops in Positive control and HBsAg Positive sample wells Step 8 Measuring the A
12. esults high background are observed increase the washing cycles or soaking time per well 6 In any case the liquid aspirated out the strips must be treated with a sodium hypochlorite solution at a final concentration of 2 5 for 24 hours before liquids are wasted in an appropriate way 7 The concentrated Washing solution must be diluted 1 19 before use For one plate mix 30 ml of the concentrate with 570 ml of water 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 HBsAg 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 time and temperature sensitive 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 10 11 12 13 14 15 16 17 components of the kit are precisely matched for optimal performance of the tests Make sure all the reagents are within the validity indicated on the kit box and of the same lot Never use reagents beyond their expiry date stated on labels or boxes
13. ite vial with yellow screw cap 7 ml per vial Diluted sulfuric acid solution 2 0M H2804 PLASTIC SEALABLE BAG 1 unit For enclosing the strips not in use CARDBOARD PLATE COVER To cover the plates during incubation and prevent evaporation 1 sheet or contamination of the wells PACKAGE INSERTS 1 copy ADDITIONAL MATERIALS AND INSTRUMENTS REQUIRED BUT NOT PROVIDED 1 Freshly distilled or deionized water 2 Disposable gloves and timer 3 Appropriate waste containers for 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 potentially SPECIMEN 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 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 round per minutes for 20 minutes at room temperature 18 30 C or by filtration on 0 22u filters Plasma s
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