Human Anti-Hepatitis C Virus Antibody, Anti-HCV ELISA Kit

HCV Kit

Unopened Kit: Store at2 - 8oC. Do not use past kit expiration date. Opened/ReconstitutedReagents: TMB Solution A, TMB Solution B, TMB Stop Solution, WashBuffer, HRP-conjugate antibody The above mentionedreagents should be stored for up to 1 month at 2 - 8oC. Microplate Wells: Returnunused wells to the foil pouch containing the desiccant pack, reseal alongentire edge of zip-seal. May be stored for up to 1 month at 2 - 8oC.

anti-HCV employssolid phase, indirect ELISA method for detection of antibodies to HCV intwo-step incubation procedure. Polystyrene microwell strips are pre-coatedwith recombinant, highly immunoreactive antigens corresponding to the coreand the non-structural regions of HCV (third generation HCV ELISA). Duringthe first incubation step, anti-HCV specific antibodies, if present, will bebound to the solid phase pre-coated HCV antigens. The wells are washed toremove unbound serum proteins, and rabbit anti-human IgG antibodies(anti-IgG) conjugated to the enzyme horseradish peroxidase (HRP-Conjugate)are added. During the second incubation step, these HRP-conjugated antibodieswill be bound to any antigen-antibody(IgG) complexes previously formed andthe unbound HRP-conjugate is then removed by washing. Chromogen solutionscontaining Tetramethylbenzidine (TMB) and urea peroxide are added to thewells and in presence of the antigen-antibody-anti-IgG (HRP) immunocomplex, the colorless TMB are hydrolyzed by the bound HRP conjugate to a blue-coloredproduct. The blue color turns yellow after stopping the reaction withsulfuric acid. The amount of color intensity can be measured and it isproportional to the amount of antibody captured in the wells, and to theamount of antibody in the sample respectively. Wells containing samplesnegative for anti-HCV remain colorless.

1. Validatedmicroplate reader. 2. Eppendorf Tubesfor dilution for samples and standards. 3. Deionized ordistilled water. 4. Validatedadjustable micropipettes, single and multi-channel. 5. Automaticmicrotiter plate washer or manual vacuum aspiration equipment. 6. 37C incubator.

Centrifuge theserum, plasma or cell culture supernatant samples for 10 minutes at 1, 000×g.Remove particulates and assay immediately or aliquot and store samples at-20C or -80oC. Avoid repeated freeze-thaw cycles.

Each microplateshould be considered separately when calculating and interpreting results ofthe assay, regardless of the number of plates concurrently processed. Theresults are calculated by relating each sample’s optical density (OD) valueto the Cut-off value (C.O.) of the plate. If the Cut-off reading is based onsingle filter plate reader, the results should be calculated by subtractingthe 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 theBlank well OD from the print report values of samples and controls. 1. Calculation ofCut-off value: Cut-off value(C.O.) = *Pc × 0.13+*Nc *Nc = the meanabsorbance value for three negative controls *Pc = the meanabsorbance value for three positive controls Important: The ODvalue of the positive control should be lower than 0.1, If the mean OD valueof the negative control is lower than 0.02, take it as 0.02. The OD value ofthe positive control should be higher than 0.5, If the mean OD value ofthe positive control is higher than 2.0, take it as 2.0. If one of theNegative control values does not meet the Quality control rangespecifications, it should be discarded and the mean value is calculated againusing the remaining two values. If more than one negative control OD valuedoes not meet the Quality control range specifications, the test is invalidand must be repeated. 2. Quality controlrange: The test results arevalid if the Quality Control criteria are verified. It is recommended thateach laboratory must establish appropriate quality control system withquality control material similar to or identical with the patient samplebeing analyzed 1) The OD value ofthe Positive control must be equal to or greater than 0.500 at 450/630 nm, orat 450 nm after blanking. 2) The OD value ofthe Negative control must be less than 0.100 at 450/630 nm or at 450 nm afterblanking. 3. Interpretationsof the results: (S = the individualoptical density (OD) of each specimen) Negative Results(S/C.O.<1): samples giving an optical density less than theCut-off value are negative for this assay, which indicates that no antibodiesto hepatitis C virus have been detected with anti-HCV ELISA kit. Therefore, the patient is probably not infected with HCV. Positive Results(S/C.O.>=1): samplesgiving an optical density greater than, or equal to the Cut-off value areconsidered initially reactive, which indicates that antibodies to hepatitis Cvirus have probably been detected using anti-HCV ELISA kit. Retesting induplicates of any initially reactive sample is recommended. Repeatedlyreactive samples could be considered positive for antibodies to HCV andtherefore the patient is probably infected with hepatitis C virus. Blood unitpositive for HCV antibodies should be immediately discarded. Borderline: Sampleswith optical density O.D.<=Cut-off×2 are consideredborderline and retesting of those samples in duplicates is recommended.Repeatedly positive samples could be considered positive for hepatitis Cvirus infection. Follow-up andsupplementary testing of any anti-HCV positive samples with other analyticalsystem (e.g. RIBA, WB) is required to confirm the diagnosis.

human serum or plasma

1. Non-repeatablepositive result may occur due to the general biological characteristics ofELISA assays. The assay is design to achieve very high performancecharacteristics of sensitivity and specificity and the “indirect model”minimizes the unspecific reactions, which can occur due to interferencebetween unknown meters in sample and the rabbit anti-human IgG used as aconjugate. Antibodies may be undetectable during the early stages of thedisease and in some immunosuppresed individuals. 2. Positive resultsmust be confirmed with another available method. Any positive result must beinterpreted together with the patient clinical information and otherlaboratory results. 3. Common sourcesfor mistakes: kits beyond the expiry date, bad washing procedures, contaminated reagents, incorrect assay procedure steps, insufficientaspiration during washing, failure to add samples or reagents, equipment, timing, volumes, sample nature and quality. 4. The prevalence ofthe marker will affect the assay’s predictive values. 5. This kit isintended ONLY for testing of individual serum or plasma samples. Do not useit for testing of cadaver samples, saliva, urine or other body fluids, orpooled (mixed) blood. 6. This is aqualitative assay and the results cannot be use to measure antigensconcentrations.

1. Alter HJ. (1978)You will wonder where the yellow went: A 15-year retrospective ofposttransfusion hepatitis. In: Moore SB, ed. Transfusion-Transmitted ViralDiseases. Alington, VA. Am. Assoc. Blood Banks, pp. 53-38. 2. Alter HJ., Purcell RH, Holland PV, et al. (1978) Transmissible agent in non-A, non-Bhepatitis. Lancet I: 459-463. 3. Choo Q-L, WeinerAJ, Overby LR, Kuo G, Houghton M. (1990) Hepatitis C Virus: the majorcausative agent of viral non-A, non-B hepatitis. Br Med Bull 46: 423-441. 4. Engvall E, Perlmann P. (1971) Enzyme linked immunosorbent assay (ELISA): qualitativeassay of IgG. Immunochemistry 8:871-874.