HEPATITIS D – IgG
Contents
1. request 20 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 Reagents preparation Allow the reagents 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 Wash buffer Step 2 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 Setp 3 Adding Sample Diluent Dispense 100 ul of Sample diluent provided with the kit into each well except in the Blank well Step 4 Adding Sample Add 10ul specimen 10pl Step 5 Negative control and 10yl Positive control into their respective wells Note Use a separate disposal pipette tip for each specimen Negative Positive Control as2. 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 Use 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 u
3. Csontrols OD value 0 02 0 012 0 016 NC 0 016 2 Calculation of Cut off C O 0 016 0 12 0 136 2 Quality control range 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 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 IgG class antibodies to HDV have been detected with this kit Positive Results S C 0 21 Samples giving an absorbance greater than or equal to Cut off value are considered initially reactive which indicates that lgG class antibodies to HDV have probably been detected with this HDV IgG ELISA kit Any initially reactive samples should be retested in duplicates Repeatedly reactive samples could be considered positive for IgG antibodies to HDV Borderline S CO 0 9 1 1 Samples with absorbance to Cut off ratio between 0 9 and 1 1 are considered borderline Retesting of these samples in duplicates is recommended Repeatedly reactive samples could be considered positive for IgG antibodies to HDV LIMITATIONS 1 Non repeatable positive result ma
4. DV IgM 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 HDV 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 10 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 Cut off value C O NC 0 12 NC the mean absorbance value for three negative controls Example 1 Calculation of NC Well No B1 Ci D1 Negative
5. For Research Use Only MpressBio HEPATITIS D IgG Catalog WD6196 Not for Diagnostic Use ANTIBODIES TO HEPATITIS D VIRUS ELISA KIT Two Step Incubation Indirect Principle INSTRUCTIONS FOR USE This kit is an enzyme linked immunosorbent assay ELISA for qualitative determination of IgG antibodies to hepatitis D virus HDV in human serum or plasma For Research Use Only SUMMARY Hepatitis D is a liver diseases caused by Hepatitis D virus Delta agent a defective 86 nm 43 nm enveloped RNA virus which requires co infection with Hepatitis B virus HBV for its replication Transmitted percutaneously or sexually through contact with infected blood or blood products HDV is associated with the most severe forms of chronic and acute hepatitis in many Hepatitis B HBsAg positive patients Since the infection with HDV requires infection with HBV the development of the disease depends on whether the two viruses infect simultaneously coinfection or whether the newly infected HDV patient is also a chronic HBV carrier superinfection The co infection with HDV can lead to severe acute hepatitis disease with low risk of chronic stage development Chronic HBV carrier patients superinfected with HDV are at risk to develop chronic HDV disease which can lead to cirrhosis in 70 80 of the patients The serological diagnosis of HDV is based on detection of specific HDV antibodies anti HDV or antigens The anti HDV IgG antibodies a
6. colored liquid filled in a white vial with blue crew cap 12 ml per vial Protein buffer solution Preservatives 0 1 ProClin 300 STOCK WASH BUFFER 1 bottle Colorless liquid filled in a clear bottle with white screw cap 50ml per bottle pH 7 4 20x 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 when stored at 2 8 C CHROMOGEN SOLUTION A 1 vial Colorless liquid filled in 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 7ml 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 7 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 use 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 1 Freshly distilled or deionized water 2 Disposable gloves and time
7. d 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 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 as
8. pirated 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 50ml of the concentrate with 950ml of water for a final volume of 1000m 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 HDV IgG 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 performance during testing 2 Make sure that all reagents are within the validity indicated on the kit box and are of the same lot 10 11 12 13 14 15 16 17 Never use reagents beyond the expiry date stated on reagents labels or on the kit box CAUTION CRITICAL STEP Allow
9. r 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 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 shoul
10. r stopping the reaction with sulfuric acid The amount of color intensity can be measured and it is proportional to the amount of antibody captured in the wells and to the amount of antibody in the sample respectively Wells containing samples negative for HDV IgG 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 purified HDV antigens 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 Blueish liquid filled in a vial with green screw cap 0 5 ml per vial Protein stabilized buffer tested non reactive for anti HDV 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 0 5 ml per vial Anti HDV 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 12 ml per vial Horseradish peroxidase conjugated anti HDV antibodies Ready to use as supplied Once open stable for one month at 2 8 C SAMPLE DILUENT 1 vial Blue
11. re indicator of past or current delta infection High titers of the antibodies may be indicative of chronic or active infection During HBV HDV co infection detectable concentrations of anti HDV IgG appear after the tenth week of exposure to the viruses and clearance during convalescence indicates recovery During HDV superinfection detectable levels of antibodies appear four to six weeks after exposure and failure to clear indicates possible progression to long chronic carrier stage PRINCIPLE OF THE ASSAY This kit is based on solid phase two step incubation indirect ELISA method Polystyrene microwell strips are pre coated with recombinant HDV antigens During the first incubation step anti HDV specific antibodies if present will be bound to the solid phase pre coated HDV antigens The wells are washed to remove unbound serum proteins and anti human IgG antibodies anti IgG conjugated to the enzyme horseradish peroxidase HRP Conjugate are added During the second incubation step these HRP conjugated antibodies will be bound to any antigen antibody IgG complexes previously formed and the unbound HRP conjugate is then removed by washing Chromogen solutions containing Tetramethylbenzidine TMB and urea peroxide are added to the wells and in presence of the antigen antibody anti lgG HRP immunocomplex the colorless Chromogens_ are hydrolyzed by the bound HRP conjugate to a blue colored product The blue color turns yellow afte
12. sed 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 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 H2SO is a strong acid Corrosive Use it with appropriate care Wipe up spills immediately or wash with water if come into 18 19 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
13. tested 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 expiration indicated on the kit box and reagent labels REFERENCES 1 Purcell RH and Gerin JL Hepatitis Delta virus In Fields Virology 3 ed Philadelphia Lippincott Raven 1996 2 Hadziyannis_ SJ Hepatitis delta an overview In Rizzetto M Purcel RH Gerin JL and Verme G eds Viral hepatitis and liver disease Turin Edizoni Minerva medica 1997 z 3 Lai MCC The molecular biology of hepatitis Delta virus Annual Review of Biochemistry 1995 64 259 286 4 Centers for Disease Control and Prevention Epidemiology and Prevention of Viral Hepatitis A to E An Overview 2000 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
14. 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 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 Remove and discard the plate cover Wash each well 5 times with diluted Washing buffer Each time allow the microwells to soak for 30 60 seconds After the final washing cycle turn down the plate onto blotting paper or clean towel and tap it to remove any remaining liquids Step 7 Adding HRP Conjugate Add 100ul of antibody HRP Conjugate to each well except the blank Step 8 Incubating 2 Cover the plate with the plate cover and incubate for 30 minutes at 37 C as Step 5 Step 9 Washing 2 Remove and discard the plate cover Wash each well 5 times with diluted Washing buffer Each time allow the microwells to soak for 30 60 seconds After the final washing cycle turn down the plate onto blotting paper or clean towel and tap it to remove any remaining liquids as step 6 Step 10 Coloring Add 50ul of Chromogen A and 50ul Chromogen B solution into each well cover the plate with plate cover and mix gently Incubate at 37 C for 15 minutes avoiding light The enzymatic reaction between Chromogen solutions and the HRP Conjugate produces blue color in Positive control and H
15. y occur due to the general biological and biochemical characteristics of ELISA assays 2 The test is design to achieve very high performance characteristics of sensitivity and specificity However antibodies may be undetectable during the early stages of the disease and in some immunosuppressed individuals 3 The prevalence of the marker will affect the assay s predictive values 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 5 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 This is a qualitative assay and the results cannot be use to measure antigens 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 re
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