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HDV-IgM

1. 3 Sample preparation Each sample must be diluted 1 10 with normal saline 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 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 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 5 cycles dispensing 350 400ul well and aspirating the liquid for 5 times 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 at a final concentration of 2 5 for 24 hours before liquids are wasted in an appropriate way The concentrated Washing solution should be diluted 1 to 20 b
2. develops in Positive control and HDV IgM Positive sample wells 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 INTERPRETATION 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 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 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 Example 1 Calculation of NC Well No B1 C1 D1 Negative controls OD value 0 02 0 012 0 016 NC 0 016 Nc is lower than 0 05 so take it as 0 05 2 Calculation of Cut off C O 0 05 x 2 1 0 105 If one of the
3. 995 64 259 286 4 Centers for Disease Control and Prevention Epidemiology and Prevention of Viral Hepatitis A to E An Overview 2000 5 Hepatitis Delta WHO CDS CSR NCS 2001 1 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 XpressBio
4. E DILUENT 1 vial Blue liquid filled in a white vial with blue crew cap 12ml per vial Protein buffer solution Preservatives 0 1 ProClin 300 STOCK WASH BUFFER 1 bottle Colorless liquid 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 a 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 a 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 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 timer 3 Appropriate waste c
5. For Research Use Only MpressBio HDV IgM Catalog WD6296 Not for Diagnostic Use IgM ANTIBODY TO HEPATITIS D VIRUS HDV ELISA KIT Two step Incubation Antibody Capture Principle INSTRUCTIONS FOR USE This HDV IgM kit is an enzyme linked immunosorbent assay ELISA for qualitative determination of IgM class antibodies to hepatitis D virus in human serum or plasma For Research Use Only SUMMARY Hepatitis D is caused by Hepatitis D virus Delta agent a defective 36 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 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 co infection or whether the newly infected HDV patient is also a chronic HBV carrier super infection The co infection with HDV can lead to severe acute hepatitis disease with low risk of chronic stage development Chronic HBV carrier patients super infected 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 Hepatitis D is based on detection of specific HDV antibodies anti HDV or antigens IgM anti HDV is the first antibody
6. 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 1 The absorbance of the Blank well which contains only Chromogens and Stop solution is less than 0 080 at 450 nm 2 The absorbance value OD of the Positive control must be equal to or greater than 0 800 at 450 630nm or at 450nm after blanking 3 The absorbance value OD 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 IgM class antibodies to HDV have been detected with this HDV IgM ELISA 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 IgM class antibodies to HDV have probably been detected with this HDV IgM ELISA kit Any initially reactive samples must be retested in duplicates Repeatedly reactive samples can be considered positive for IgM antibodies to HDV Borderline S CO 0 9 1 1 Samples with absorbance to Cut off ratio between 0 9
7. and 1 1 are considered borderline Retesting of these samples in duplicates is recommended Repeatedly reactive samples could be considered positive for IgM antibodies to HDV LIMITATIONS 1 Non repeatable positive result may occur due to the general biological and biochemical characteristics of the ELISA assays The test is design to achieve performance characteristics of high sensitivity and specificity However in very rare cases some HDV mutants or subtypes can remain undetectable Antibodies may be undetectable during the early stages of the disease and in some immunosuppressed individuals 2 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 3 The prevalence of the marker will affect the assay s predictive values VALIDITY Please do not use this kit beyond the expiration indicated on the kit box and reagent label 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 3 Lai MCC The molecular biology of hepatitis Delta virus Annual Review of Biochemistry 1
8. diately 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 and reactive chemical and substances like sodium hypochlorite acids alkalis etc Do not perform the assay in the presence of such substances ASSAY PROCEDURE Step 1 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 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 Mark three wells as Negative control Step 2 Step 3 Step 4 Step 5 Step 6 Step 7 Step 8 Step 9 Step 10 Step 11 e g B1 C1 D1 two wells as Positive control e g E1 F1 and one Blank e g A1 neither samples nor HRP Conjugate should be added into the Blank well Use only number of strips required for the test Specimen Dilution Before adding dilute each specimen 10 times with normal saline Adding Samples Dispense 100 ul of Sample diluent provided with the kit into each well except in the Positive Negative controls and Blank wells Add 10 wl of saline diluted samples into each well and 100 ul controls into their respective wells Mix by tapping the plate gently Note to avoid cros
9. efore use For one plate mix 50ml 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 HDV IgM 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 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 components of the kit are precisely matched for optimal performance of the tests 2 Make sure that all 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 10 11 12 13 14 15 16 Allow the reagents and samples to reach room temperature 18 30 C before use Shake reagent gently before use Return at 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 pla
10. lyzed 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 antibody in the sample Wells containing samples negative for HDV IgM 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 anti IgM antibodies anti u chain 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 0 5 ml per vial Protein stabilized buffer tested non reactive for HDV IgM 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 liquid filled in a vial with red screw cap 0 5 ml per vial Purified anti HDV IgM antibodies 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 liquid filled in a white vial with red screw cap 12 ml per vial Horseradish peroxidase conjugated HDV antigens Ready to use as supplied Once open stable for one month at 2 8 C SAMPL
11. ontainers 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 11 Normal saline solution for dilution of the samples 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 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 can give false results in the assay Do not heat inactivated samples This can cause sample deterioraration 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 Avoid multiple freeze thaw cycles
12. s contamination use a separate disposable pipette tip for each specimen Negative Control or Positive Control 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 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 3 times with diluted Wash buffer Each time allow the microwells to soak for 30 60 seconds After the last washing cycle turn down the strips plate onto blotting paper or clean towel and tap the plate to remove any remainders Adding HRP Conjugate Add 100ul of HRP Conjugate Reagent into each well except for the blank Incubating Cover the plate with the plate cover and incubate for 30 min at 37 C as Step 4 Washing Aspirate the liquid and rinse each well 5 times with wash buffer as step 5 Coloring Add 50ul or one drop of Chromogen A and 50ul or one drop Chromogen B solution into each well including the Blank and mix gently Incubate the plate 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 HDV IgM Positive sample wells Stopping Reaction Using a multichannel pipette or manually add 50 ul Stop solution into each well and mix gently Intensive yellow color
13. te 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 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 do not touch the well s bottom with the pipette tip When measuring 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 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 strips and sample containers should be collected and autoclaved for not less than 2 hours at 121 C or treated with 10 sodium hypochlorite for 30 minutes to decontaminate before any further steps of disposal The Stop Solution contains 2M H2SO Use it with appropriate care Wipe up spills imme
14. to appear in acute infection IgM anti HDV is transient and rapidly replaced by IgG anti HDV However persistence of IgM anti HDV_ will indicate chronic HDV infection which continues to replicate During HBV HDV co infection detectable concentrations of HDV anybodies appear after the tenth week of exposure and clearance during convalescence indicates recovery Decreasing or low titers of IgM suggest early recovery during HDV co and acute super infection while constantly elevated level of IgM indicates possible progression to chronic carrier stage PRINCIPLE OF THE ASSAY This HDV IgM ELISA kit is a solid phase two step incubation antibody capture assay in which polystyrene microwell strips are pre coated with antibodies directed to human IgM anti u chain The patients serum plasma sample is diluted and during the first incubation step any IgM antibodies will be captured in the wells After washing out all the other components of the sample and in particular IgG antibodies the specific HDV IgM captured on the solid phase is detected by the addition of purified HDV antigens conjugated to horseradish peroxidase HRP During the second incubation step the conjugated antigens will specifically react only with the specific HDV IgM antibodies and after washing to remove unbound conjugates Chromogen solutions are added into the wells In presence of the anti y HDV IgM HDV antigen HRP immunocomplex the colorless Chromogens are hydro

HDV-IgM

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