HEPATITIS B – HBV NRAg
Contents
1. PRINCIPLE OF THE ASSAY This kit uses sandwich ELISA method in which polystyrene microwell strips are pre coated with monoclonal antibodies specific to HBV pre S1 antigen and core antigen Patient s serum or plasma sample is added to the microwell together with a second monoclonal antibody conjugated to horseradish peroxidase HRP Conjugate During incubation the specific immunocomplex formed in case of presence of pre S1 antigen and core antigen in the sample is captured on the solid phase After washing to remove sample and unbound HRP Conjugate Chromogen solutions containing Tetramethylbenzidine TMB and urea peroxide are added into the wells In presence of the antibody antigen antibody 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 antigen captured in the wells and to the sample respectively Wells containing samples negative for HBV pre S1 antigen and core antigen remain colorless COMPONENTS 96 Tests MICROWELL PLATE 1 plate Blank microwell strips fixed on white strip holder The plate is sealed in aluminium pouch with desiccant 8x12 12x8 well strips per plate Each well contains monoclonal antibodies reactive to HBV pre S1 antigen and core antigen The microwell strips can b2. 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 each time into the wells 5 In case of manual washing we suggest to perform at least 5cycles 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 app
3. For Research Use Only MpressBio HEPATITIS B HBV NRAg Catalog WB2996 Not for Diagnostic Use HEPATITIS B VIRUS NUCLEIC ACID RELATED ANTIGEN ELISA KIT Double Antibody Sandwich Principle INSTRUCTIONS FOR USE This kit is an enzyme linked immunosorbent assay ELISA for qualitative detection of HBV pre S1 antigen and core antigen in hepatitis B virus nucleic acid related antigen HBV NRAg 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 virus particle is composed of outer membrane proteins S protein pre S1 protein pre S2 protein and core shell HBV pre S1 antigen and core antigen are not only the marker of HBV infection also the marker of viral replication and they are highly correlated with HBV DNA 31 collectively known as hepatitis B virus nucleic acid related antigen HBV NRAg
4. 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 15 minutes 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 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 12 NC the mean absorbance value for three negative controls Example 1 Calculation of NC Well No Bi Ci 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 12 0 136 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
5. e broken to be used separately Place unused wells or strips in the plastic sealable storage bag together with 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 HBeAg Preservatives 0 1 ProClin 300 Ready to use as supplied Once open stable for one month at 2 8 C HBV PRE S1 ANTIGEN POSITIVE CONTRO 1 vial Red colored liquid filled in a vial with red screw cap 0 5ml per vial Recombinant non infective HBV PreS1 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 HBV CORE ANTIGEN POSITIVE CONTROL 1 vial Red colored liquid filled in a vial with red screw cap 0 5ml per vial Recombinant non infective HBcAg 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 SPECIMEN DILUENT 1 vial Blue liquid filled in a white vial with blue screw cap 6ml 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 colored liquid filled in a white vial with red screw cap 12ml per vial Horseradish peroxidase conjugated anti HBV pre S1 anti HBc and anti HBs antibodies Ready to use as supplied Once open stable for on
6. e month at 2 8 C STOCK WASH BUFFER 1 bottle Colorless liquid filled in a clear bottle with white screw cap 50ml per bottle PH 7 4 20 x PBS Containing 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 at 2 8 C CHROMOGEN SOLUTION A 1 vial Colorless liquid filled in a white vial with green screw cap 6ml 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 6ml 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 in a white vial 6ml 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 use CARDBOARD PLATE COVER 3 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 Disposable gloves and timer Appropriate waste containers for potentially contaminated materials Disposable V shaped troughs Dispensing system and or pipette sing
7. ith absorbance to Cut off ratio between 0 9 and 1 1 are considered borderline samples and retesting is recommended Repeatedly reactive samples can be considered positive for HBeAg LIMITATIONS 1 Non repeatable positive result may occur due to the general biological and biochemical characteristics of ELISA method The kit is designed to achieve performance characteristics of very high sensitivity and specificity However in very rare cases some HBV mutants or subtypes could 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 Any positive result must be interpreted in conjunction with patient clinical information and other laboratory testing results 4 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 The prevalence of the marker will affect the assay s predictive values This kit is intended ONLY for testing of individual serum or plasma samples Do not use it for testing of cadave
8. le or multichannel disposable pipette tips Absorbent tissue or clean towel Dry incubator or water bath 37 0 5 C e Microshaker for dissolving and mixing conjugate with samples Microwell plate reader single wavelength 450nm or dual wavelength 450nm and 630nm Microwell aspiration wash system 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 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
9. 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 advisable 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 controls 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 hepatitis B virus e antigen has been detected with this HBeAg ELISA kit Positive Results S C 0 21 Samples giving an absorbance greater than or equal to the Cut off value are initially reactive which indicates that HBV e antigen has probably been detected using this HBeAg ELISA kit Any initially reactive samples should be retested in duplicates Repeatedly reactive samples could be considered positive for HBeAg Borderline S CO 0 9 1 1 Samples w
10. over The tapping out of the remainders inside the plate after washing can also be omitted ASSAY PROCEDURE Step 1 Reagents preparation Allow the reagents and samples to reach room temperature 18 30 C for at least 15 30 minutes 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 water Use only clean vessels to dilute the buffer Step 2 Numbering Wells Set the strips needed in strip holder and number sufficient number of wells including three Negative controls e g B1 C1 D1 two Positive controls e g E1 F1 and one Blank e g A1 neither Sample Diluent 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 Diluent Add 50ul Specimen Diluent into each well except the blank Step 4 Adding Samples Add 50ul of Positive controls Negative controls and Specimen into their respective wells Note to avoid cross contamination use a separate disposable pipette tip for each specimen Negative or Positive Control Step 5 Incubating 1 Cover the plate with the plate cover and incubate for 60 minutes at 37 C It is recommended to use
11. owever 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 1hour at 121 C or treated with 10 sodium hypochlorite for 30minutes to decontaminate before any further steps for disposal 18 19 20 21 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 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 If using fully automated microplate processing system during incubation do not cover the plates with the plate c
12. pe antigen in serum samples comparison with two commercial assays for monitoring hepatitis B virus DNA J Viral Hepat 7 387 92 Petit M A F Zoulim F Capel S Dubanchet C Dauguet and C Trepo 1990 Variable expression of preS1 antigen in serum during chronic hepatitis B virus infection an accurate marker for the level of hepatitis B virus replication Hepatology 11 809 14 Yuan Q S X Ge Q Yan Y Zhao J H Xiong J Zhang and N S Xia 2007 Establishment of a new combined enzyme immunoassay for detection of HBV preS1 and core antigens and the consistency with HBV DNA test Bing Du Xue Bao 23 252 7 Express Biotech International 503 Gateway Drive West Thurmont MD 21788 USA Tel 301 228 2444 Fax 301 560 6570 Toll Free 888 562 8914 www xpressbio com info xpressbio com XpressBio
13. r samples saliva urine or other body fluids or pooled mixed blood This is a qualitative assay and the results cannot be use to measure antigens concentrations INDICATIONS OF INSTABILITY OR DETERIORATION OF THE REAGENTS 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 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 Kimura T A Rokuhara A Matsumoto S Yagi E Tanaka K Kiyosawa and N Maki 2003 New enzyme immunoassay for detection of hepatitis B virus core antigen HBcAg and relation between levels of HBcAg and HBV DNA J Clin Microbiol 41 1901 6 Le Guillou D B J C Duclos Vallee F Eberle F Capel and M A Petit 2000 Evaluation of an enzyme linked immunosorbent assay for detection and quantification of hepatitis B virus PreS1 envelo
14. ropriate way 7 The concentrated Washing solution should be diluted 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 the expiration date indicated on the label and package when stored between 2 8 C do not freeze To assure maximum performance of this HBeAg 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 10 11 12 13 14 15 16 17 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 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 U
15. se only sufficient volume of sample as indicated in the procedure steps Failure to do so may cause in low sensitivity of the assay 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 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 well s 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 H
16. 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 Step 6 Washing 1 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 plate down onto blotting paper or clean towel and tap it as to remove any remaining liquids Step 7 Adding HRP Conjugate Add 100l HRP Conjugate into each well except in the Blank Step 8 Incubating 2 Cover the plate with the plate cover and incubate for 30 minutes at 37 C Step 9 Washing 2 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 cover the plate with plate cover and mix by tapping the plate gently Incubate the plate at 37 C for 15 minutes avoiding light The enzymatic reaction between the Chromogen solutions and the HRP Conjugate produces blue color in Positive control and positive sample wells Step 11 Stopping Reaction Remove and discard the plate cover Using a multichannel pipette or manually add 50ul Stop Solution into each well and mix gently Intensive yellow color develops in Positive control and positive sample wells
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