HEPATITIS B – anti HBs (Qualitative)
Contents
1. Blank microwell strips fixed on a white strip holder The plate is sealed in aluminum pouch with desiccant 8x12 12x8 well strips per plate Each well contains purified HBsAg The microwell strips can be broken to be used separately Place unused wells or strips in the plastic sealable storage bag together with the desiccant and return to 2 8 C NEGATIVE CONTROL 1 vial Yellowish liquid filled in a vial with green screw cap 1ml per vial Protein stabilized buffer tested non reactive for anti HBs 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 colored liquid filled in a vial with red screw cap 1ml per vial anti HBs diluted in protein stabilized buffer 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 colored liquid filled in a white vial with red screw cap 6 5ml per vial Horseradish peroxidase conjugated HBsAg Ready to use as supplied Once open stable for one month at 2 8 C STOCK WASH BUFFER 1 bottle Colorless liquid filled in a clear bottle with white screw cap 30ml per bottle pH 7 4 20 x PBS Contains Tween 20 as a detergent DILUTE BEFORE USE The concentrate must be diluted 1 to 20 with distilled deionized water before use Once diluted stable for one week at room temperature or for two weeks when stored at 2 8 C CHROMOGEN SOLUTION A2. 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 1hour at 121 C or treated with 10 sodium hypochlorite for 30minutes to decontaminate before any further steps for disposal The Stop solution 2M H2S0 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 such substances Materials Safety Data Sheet MSDS available upon request 21 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 ASSAY PROCEDURE Step 1 Step 2 Step 3 Step 4 Step 5 Step 6 Step 7 Reagents preparation Allow the reagents and samples to reach room temperature 18 30 C for at lest 15 30 minutes Check the Wash buffer concentrate for the presence of sa
3. 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 highly 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 performance
4. in very rare cases some HBV mutants or subtypes can remain undetectable 2 If 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 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 5 Samples tested using test kits from different manufacturer can give similar qualitative results but some samples can give discrepancies due to the antibodies diversity and the antigenic properties of HBsAg used in the assay 6 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 reteste
5. 1 vial Colorless liquid filled in a white vial with green screw cap 7ml 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 a black vial with black screw cap 7 ml per vial TMB solution Tetramethyl benzidine 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 a white vial with white screw cap 7ml per vial Diluted sulfuric acid solution 2 0 M H2804 Ready to use as supplied PLASTIC SEALABLE BAG 1 unit For enclosing the strips not in use CARDBOARD PLATE COVER 1 sheet 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 1 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 reader single wavelength 450nm or dual wavelength 450nm and 630nm 10 Microwell aspiration wash system SPECIMEN COLLECTION TRANSPORTATION
6. For Research Use Only MpressBio HEPATITIS B anti HBs Qualitative Catalog WB2396 Not for Diagnostic Use ANTIBODY TO HEPATITIS B VIRUS ANTIGEN ELISA One Step Incubation Double Antigen Sandwich Principle INSTRUCTIONS FOR USE This anti HBs ELISA kit is an enzyme linked immunosorbent assay for in vitro qualitative detection of antibodies to hepatitis B virus surface antigen anti HBs 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 disease which in some cases can lead to cirrhosis and carcinoma of the liver Classification of a hepatitis B infection requires the identification of several serological markers expressed during three phases incubation acute and convalescent of the infection Hepatitis B surface antigen HBsAg which appears shortly after infection is an important protein of the envelope structure of the virus HBsAg is a key serological marker for detection and diagnosis of HBV and is detectable in blood during the acute phase of the disease Clearance after treatment shows recovery while presence for more than half year after infection indica
7. 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 wells Calibrate the pipette frequently to assure the accuracy of samples reagents dispensing Always use different disposal pipette tips for each specimen 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 incubator 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 2 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
8. d In case of constant erroneous results classified as due to deterioration or instability of the reagents immediately substitute the 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 expiry date indicated on the kit box and reagent labels REFERENCES 1 Lewis T et al 1972 A Comparison of the frequency of hepatitis B Antigen and antibody in hospital and non hospital personnel New Engl J Med 289 647 2 Hadler S C et al 1986 Long term Immunogenicity and Efficacy of Hepatitis B vaccine in homosexual men New Engl J Med 315 209 3 Jilg W et al 1989 Vaccination against Hepatitis B Comparison of three different vaccination schedules J Infect Dis 160 766 4 Jilg W et al 1990 Hepatitis B vaccination strategy for booster doses in high risk population groups Progress in Hepatitis B Immunisation P Coursaget M J Tong eds Colloque INSERM 194 419 5 Engvall E and Perlmann P J Immunochemistry 8 871 874 1971 6 Engvall E and Perlmann P J lmmunol 109 129 135 1971 7 Remington J S and Klein J O In Infectious diseases of the fetus and newborn infant Sanders Philadelphia London Toronto 8 Volk W A In Essential of Medical Microb
9. g 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 Important If the mean OD value of the negative control is lower than 0 05 take it as 0 05 If higher than 0 05 see the Quality Control Range Example 1 Calculation of NC Well No Bi C1 D1 Negative Controls OD value 0 02 0 012 0 016 NC 0 016 the NC is lower than 0 05 so take it as 0 05 2 Calculation of Cut off value Cut off C O 0 05 x 2 1 0 105 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 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 1 The OD value of the Blank well which contains only Chromogens and Stop solution is less than 0 080 at 450 nm 2 T
10. he 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 the 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 antibodies to hepatitis B virus surface antigen have probably not been detected with this kit Positive Results S C O 21 samples giving an absorbance greater than or equal to the Cut off value are initially reactive which indicates that antibodies to HBV surfaces antigen have been detected using this anti HBs ELISA kit Retesting in duplicates of any reactive samples is recommended 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 HBsAg LIMITATIONS 1 Non repeatable positive result may occur due to the general biological of the ELISA assays This assay is designed to achieve very high performance characteristics of sensitivity and specificity and the sandwich model minimizes the unspecific reactions due to interference with unknown matters in sample However
11. iology 2nd ed pp 729 G B Lippincott Company Philadelphia New York S Jos Toronto XpressBio Express Biotech International 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
12. k for 30 60seconds After the final washing cycle turn down the plate onto blotting paper or clean towel and it plate to remove any remaining liquids Coloring Dispense 50u 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 15minutes avoiding light The enzymatic reaction between the Chromogen solutions and the HRP Conjugate produces blue color in Positive control and in anti HBs positive sample wells Stopping Reaction Using a multichannel pipette or manually add 50ul Stop Solution into each well Intensive yellow color develops in Positive control and anti HBs Positive sample wells Step 8 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 5minutes after stopping the reaction INTERPRETATION OF RESULTS AND QUALITY CONTROL Each microplate should 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 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 subtractin
13. lt 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 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 e g 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 Adding Sample and HRP Conjugate Add 50ul of Positive control Negative control and Specimen into their respective wells Note Use a separate disposal pipette tip for each specimen Negative and Positive Control as to avoid cross contamination Add 50 HRP Conjugate to each well except the Blank and mix by tapping the plate gently Incubating Cover the plate with the plate cover and incubate for 60 minutes at 37 C It is recommended to use thermostat controlled water tank to assure the temperature stability and humidity during the 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 soa
14. s 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 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 dilute 1 to 20 before use For one plate mix 30 ml of the concentrate with 570ml of water for a final volume of 600ml 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
15. tes possible progression to long chronic carrier stage During the acute phase of the infection strong immunological response develops and increasing titers of HBsAg neutralizing antibodies anti HBs are marker for recovery PRINCIPLE OF THE ASSAY For detection of anti HBs this kit uses antigen sandwich ELISA method where polystyrene microwell strips are pre coated with recombinant HBsAg Patient s serum or plasma sample is added to the microwells together with a second HBsAg conjugated to Horseradish Peroxidase HRP Conjugate In case of presence of anti HBs in the sample the pre coated and conjugated antigens will be bound to the two variable domains of the antibody and during incubation the specific immunocomplex formed is captured on the solid phase After washing to remove sample serum proteins and unbound HRP Conjugates Chromogen solutions containing Tetramethylbenzidine TMB and urea peroxide are added to the wells In presence of the antigen antibody antigen HRP sandwich complex 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 anti HBs remain colorless COMPONENTS 96 Tests MICROWELL PLATE 1 plate
16. the expiration date indicated on the label and package when stored between 2 8 C do not freeze To assure maximum performance of this anti HBs ELISA kit during storage protect the reagents from contamination with microorganism or chemicals 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 performance 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 10 11 12 13 14 15 16 17 18 19 20 fingerprints or scratches may interfere with microwell reading When reading the results ensure that the plate bottom is dry and there are no air
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