Mouse HBsAg(Hepatitis B Virus Surface Antigen) ELISA Kit Catalogue No.: EM0002 Size: 96T Reactivity: Mouse Application: This immunoassay kit allows for the qualitative determination of HBsAg in Mouse serum or plasma. Storage: 4 for 6 months. NOTE: FOR RESEARCH USE ONLY. Kit Components Item Specifications(96T) Storage Micro ELISA Plate(Dismountable) 8 12 4 /-20 Lyophilized HBsAg Positive Control(8ng) 4 vial 4 /-20 HB Negative Control 2ml 4 HBsAg Positive Control dilution buffer 10ml 4 Biotin- detection antibody (Concentrated) 120ul 4 Antibody dilution buffer 10ml 4 HRP-Streptavidin Conjugate(SABC) 120ul 4 (shading light) SABC dilution buffer 10ml 4 TMB substrate 10ml 4 (shading light) 1 / 8
Stop solution 10ml 4 Wash buffer (25X) 30ml 4 Plate Sealer Product Description 5pieces 1 copy Background The hepatitis B surface antigen (HBsAg) is the first marker that appears in the blood following infection with hepatitis B virus (HBV) some days or weeks before clinical symptoms manifest. It is a lipoprotein polypeptide which constitutes the external envelope of the HB virus. The detection of HBsAg in Mouse serum or plasma indicates an ongoing HBV infection, either acute or chronic. Testing of additional HBV markers is needed to define the specific disease state. HBsAg assays are used not only to diagnose HBV infections but also to monitor the course of the disease and the efficacy of antiviral therapy. Hepatitis B surface antigen ELISA kit is a fast test for the qualitative detection of the presence of HBsAg in serum or plasma (heparin, citrate or EDTA) specimen. The test utilizes monoclonal and polyclonal (anti-guinea pig) antibodies to selectively detect elevated levels of HBsAg in serum or plasma. Sample which are non-reactive by Hepatitis B surface antigen ELISA kit are considered negative for HBsAg. Sample with positive reaction should be retested in duplicate. In case of a reactive repeat reaction, the specimen should be confirmed for HBsAg reactivity with validated confirmatory reagents. Only confirmed positive sample are considered to contain HBsAg. Principle of the Assay This kit was based on sandwich enzyme-linked immune-sorbent assay technology. Anti- HBsAg antibody was pre-coated onto 96-well plates. And the biotin conjugated anti- HBsAg antibody 2 / 8
was used as detection antibodies. The controls, test samples and biotin conjugated dete ction antibody were added to the wells subsequently, and wash with wash buffer. HRP-Streptavidin was added and unbound conjugates were washed away with wash buffer. TMB substrates were used to visualize HRP enzymatic reaction. TMB was catalyzed by HRP to produce a blue color product that changed into yellow after adding acidic stop solution. The optical density of developed color is read with a suitable photometer at 450nm with a selected reference wavelength within 650 nm. Precautions for Use 1. To inspect the validity of experiment operation and the appropriateness of sample dilution proportion, pilot experiment using standards and a small number of samples is recommended. 2.After opening and before using, keep plate dry. 3. Before using the Kit, spin tubes and bring down all components to the bottom of tubes. 4. Storage TMB reagents avoid light. 5. Washing process is very important, not fully wash easily cause a false positive. 6. Duplicate well assay is recommended for both standard and sample testing. 7. Don t let Micro plate dry at the assay, for dry plate will inactivate active components on plate. 8. Don t reuse tips and tubes to avoid cross contamination. 9. Avoid using the reagents from different batches together. Material Required But Not Supplied 1.Microplate reader (wavelength: 450nm) 2.37 incubator 3.Automated plate washer 3 / 8
4.Precision single and multi-channel pipette and disposable tips 5.Clean tubes and Eppendorf tubes 6.Deionized or distilled water Manual Washing Discard the solution in the plate without touching the side walls. Clap the plate on absorbent filter papers or other absorbent material. Fill each well completely with 350ul wash buffer and soak for 1 to 2 minutes, then aspirate contents from the plate, and clap the plate on absorbent filter papers or other absorbent material. Repeat this procedure two more times for a total of THREE washes. Automated Washing Aspirate all wells, then wash plate THREE times with 350ul wash buffer. After the final wash, invert plate, and clap the plate on absorbent filter papers or other absorbent material. It is recommended that the washer be set for a soaking time of 1 minute. Sample Collection and Storage Isolate the test samples soon after collecting, then, analyze immediately (within 2 hours). Or aliquot and store at -20 for long term. Avoid multiple freeze-thaw cycles. Serum: Coagulate the serum at room temperature (about 1 hours). Centrifuge at approximately 1000 g for 15 min. Analyze the serum immediately or aliquot and store at - 20. Plasma: Collect plasma with heparin or EDTA as the anticoagulant. Centrifuge for 15min at 2-8 C at 1500 x g within 30 min of collection. For eliminating the platelet effect, suggesting that further centrifugation for 10 min at 2-8 C at 10000 x g. Analyze immediately or aliquot and store frozen at -20. 4 / 8
Note: Samples to be used within 5 days may be stored at 4,otherwise samples must be stored at -20 ( 1 month) or -80 ( 2 months) to avoid loss of bioactivity and contamination. Hemolyzed samples are not suitable for use in this assay. Reagent Preparation and Storage 1. Reconstitution of the Lyophilized HBsAg Positive Control Positive Control should be prepared no more than 3 month prior to the experiment. Add 1 ml of HBsAg Positive Control dilution buffer into one Lyophilized HBsAg Positive Control tube, keep the tube at room temperature for 10 min and mix thoroughly. Note: The reconstitution of the Lyophilized HBsAg Positive Control should be at 4 for up to 3 month. Or store at -20 for up to 6 month. Avoid repeated freeze-thaw cycles. 2. Preparation of Biotin- detection antibody working solution prepare within 1 hour before the experiment. 1). Calculate the total volume of the working solution: 0.1 ml / well quantity of wells. (Allow 0.1-0.2 ml more than the total volume) 2). Dilute the Biotin- detection antibody with Antibody dilution buffer at 1:100 and mix thoroughly.( i.e. Add 1 μl of Biotin conjugated detection antibody into 99 μl of Antibody dilution buffer. ) 3. Preparation of HRP-Streptavidin Conjugate (SABC) working solution: prepare within 30min before the experiment. 1). Calculate the total volume of the working solution: 0.1 ml / well quantity of wells. (Allow 0.1-0.2 ml more than the total volume) 2). Dilute the SABC with SABC dilution buffer at 1:100 and mix thoroughly. (i.e. Add 1 μl of SABC into 99 μl of SABC dilution buffer.) 5 / 8
Assay Procedure Before adding to wells, equilibrate TMB substrate for 30 min at 37. 1. Wash plate 2 times before adding standard, sample and control (zero) wells! 2. Add 100 µl of each control or Samples to appropriate wells of the microtiter plate (2 Negative Controls and 2 Positive Controls) 3. Seal the plate with a cover and incubate at 37 for 60 min. 4. Remove the cover and discard the plate content, and wash plate 2 times with Wash Buffer. Do NOT let the wells dry completely at any time. 5. Add 0.1 ml of Biotin- detection antibody work solution into the above wells (controls, test sample). Add the solution at the bottom of each well without touching the side wall. 6. Seal the plate with a cover and incubate at 37 for 60 min. 7. Remove the cover, and wash plate 3 times with Wash buffer. 8. Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37 for 30 min. 9. Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min. 10. Add 90 μl of TMB substrate into each well, cover the plate and incubate at 37 in dark within 15-30 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the Positive Controls, Negative Controls wells show no obvious color. 11. Add 50 μl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately. 12. Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution. 6 / 8
Data Analysis Calculation of Results Calculation of the NCx (Mean Absorbance of Negative Control) Example: Sample No. Absorbance 1 0.052 2 0.025 3 0.023 The optical density 0.052 is twice higher than 0.025, which is abnormal. In this case we ignore the number 0.052. NCx = (0.023 + 0.025) / 2 = 0.024 NCx should be 0.1, otherwise, the test is invalid. Calculation of the PCx (Mean Absorbance of Positive Control) Example: Sample No. Absorbance 1 1.432 2 1.508 PCx = (1.432 + 1.508) / 2 = 1.470 PC should be 0.6, otherwise, the test is invalid. Calculation of the P - N Value P - N = PCx NCx Example: NCx = 0.024 PCx = 1.470 P - N = 1.470-0.024 = 1.446 P - N Value must be 0.5, otherwise, the test is invalid. Calculation of the Cutoff Value Cutoff Value = NCx + 0.025 Example: Cutoff Value = 0.024 + 0.025 = 0.04 7 / 8
Note: 1. Sample with absorbance values LESS than the Cutoff Value are NON-REACTIVE and are considered NEGATIVE for HBsAg. 2. Sample with absorbance value GREATER than or EQUAL to the Cutoff Value are considered INITIALLY REACTIVE. The original sample must be retested in duplicate. 3. If both absorbance values in the retest are less than the cutoff value, the sample are considered NEGATIVE for HBsAg. 4. If in the retest at least one of the two absorbance values is GREATER than or EQUAL to the Cutoff Value then the sample are considered as repeated HBsAg positive. The repeated positive specimen shall be confirmed with certain valid confirmatory reagents. 8 / 8