Porcine HBsAg ELISA Kit
Porcine HBsAg ELISA Kit
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Product Specification
| Usage |
Experimental equipment required for the experiment: 1. Microplate reader (450nm) 2. High-precision pipette and gun tips: 0.5-10uL, 5-50uL, 20-200uL, 200-1000uL 3. 37℃ constant temperature box 4. Distilled water or deionized water Sample processing and requirements: Serum: Place the whole blood sample collected in the serum separation tube at room temperature for 2 hours or at 4℃ overnight, then centrifuge at 1000×g for 20 minutes, and take the supernatant, or store the supernatant at -20℃ or -80℃, but avoid repeated freezing and thawing. Plasma: Collect the specimen using EDTA or heparin as an anticoagulant. Centrifuge the specimen at 1000 × g for 15 minutes at 2-8°C within 30 minutes of collection. The supernatant can be assayed or stored at -20°C or -80°C, but avoid repeated freezing and thawing. Tissue homogenization: Rinse the tissue with pre-chilled PBS (0.01M, pH 7.4) to remove residual blood (lysed red blood cells in the homogenate will affect the measurement results). Weigh the tissue and mince it. Add the minced tissue to the appropriate volume of PBS (generally a 1:9 weight-to-volume ratio, e.g., 1 g of tissue sample to 9 mL of PBS. The specific volume can be adjusted according to experimental needs and recorded. It is recommended to add protease inhibitors to the PBS) in a glass homogenizer and grind thoroughly on ice. To further lyse tissue cells, the homogenate can be sonicated or repeatedly frozen and thawed. Finally, centrifuge the homogenate at 5000 × g for 5-10 minutes, and the supernatant can be assayed. Cell lysis solution: Gently wash adherent cells with pre-cooled PBS, then digest with trypsin, and collect the cells after centrifugation at 1000×g for 5 minutes; suspended cells can be directly collected by centrifugation. Wash the collected cells 3 times with pre-cooled PBS, add 150-200uL PBS for every 1×10^6 cells to resuspend (it is recommended to add protease inhibitors to PBS; if the content is very low, the PBS volume can be appropriately reduced) and break the cells by repeated freezing and thawing or ultrasound. Centrifuge the extract at 2-8℃, 1500×g for 10 minutes, and take the supernatant for detection. Cell culture supernatant: Centrifuge at 1000×g for 20 minutes, take the supernatant for detection, or store the supernatant at -20℃ or -80℃, but avoid repeated freezing and thawing.Other biological fluids: Centrifuge at 1000xg for 20 minutes and take the supernatant for testing. Preparation before testing: 1.Please take the reagent kit out of the refrigerator 10 minutes in advance and allow it to equilibrate to room temperature. 2.Preparation of positive and negative control working solutions: Add 1 mL of universal diluent to each freeze-dried control, let stand for 15 minutes until completely dissolved, and then gently mix. 3.Preparation of biotinylated antibody working solution: 15 minutes before use, centrifuge the concentrated biotinylated antibody at 1000×g for 1 minute. Dilute the 100× concentrated biotinylated antibody to a 1× working concentration using universal diluent (e.g., 10uL concentrated solution + 990uL universal diluent). Use on the same day. 4.Preparation of enzyme conjugate working solution: 15 minutes before use, centrifuge the 100x concentrated enzyme conjugate at 1000×g for 1 minute. Dilute the 100x concentrated HRP enzyme conjugate to a 1x working concentration with universal diluent (e.g., 10uL concentrate + 990uL universal diluent). Use on the same day. 5.Preparation of 1× washing solution: Take 10ml of 20× washing solution and add it to 190ml of distilled water (the concentrated washing solution taken out of the refrigerator may have crystals, which is normal. It can be left at room temperature and shaken evenly. Wait until the crystals are completely dissolved before reconstitution). Operation steps: 1.After equilibration at room temperature for 10 minutes, remove the required strips from the aluminum foil bag and seal the remaining strips in a ziplock bag and place them back at 4°C. 2.Sample Addition: Add 100µl of sample or positive and negative controls to the corresponding wells. Add 100µl of universal diluent to the blank wells. Cover with a film sealer and incubate at 37°C for 1 hour. (Recommendation: Dilute the test sample at least 1-fold with universal diluent before adding it to the ELISA plate to minimize matrix effects. When calculating sample concentration, multiply by the dilution factor. It is recommended to run replicates for all test samples and positive and negative controls.) 3.Add biotinylated antibody: Remove the ELISA plate and discard the liquid without washing. Add 100uL of biotinylated antibody working solution directly to each well, cover with sealing film and incubate at 37℃ for 1 hour. 4.Wash the plate: discard the liquid, add 300uL 1x washing solution to each well, let it stand for 1 minute, shake off the washing solution, pat dry on absorbent paper, and repeat this process 3 times (you can also use a plate washer to wash the plate). 5.Add enzyme conjugate working solution: Add 100uL of enzyme conjugate working solution to each well, cover with sealing film and incubate at 37℃ for 30 minutes. 6.Wash the plate: discard the liquid and follow the washing method in step 4, washing the plate 5 times. 7.Add substrate: Add 90uL of substrate (TMB) to each well, cover with sealing film, and incubate at 37℃ in the dark for 15 minutes. 8.Add stop solution: Take out the ELISA plate and add 50uL of stop solution directly to each well. Immediately measure the OD value of each well at a wavelength of 450nm. Experimental Result judgment: 1. The conditions for the test results to be valid are: The average of the positive control wellsODValue＞0.5, average OD of negative control wellsValue <0.2. 2. Test samplesS/PValue ≥ 0.2When the sample is positive, it is judged as positive; the sample is testedS/PValue < 0.2When , it is judged as a negative sample. |
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| Theory | This kit uses a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). Sample, standard, biotin-labeled detection antibody, and HRP conjugate are sequentially added to microwells pre-coated with hepatitis B virus surface antigen (HBsAg) capture antibodies. After incubation and washing, the sample is developed using the substrate TMB. TMB converts to blue under the catalysis of HRP and to yellow under the action of acid. The intensity of the color is positively correlated with the amount of hepatitis B virus surface antigen (HBsAg) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||
| Source | Porcine | |||||||||||||||||||||||||||||||
| Synonym | Pig hepatitis B virus surface antigen ELISA Kit | |||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||
| Composition |
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| Background | Hepatitis B virus surface antigen (HBsAg), also known as Australia antigen or Australia antigen, refers to the surface antigen of the hepatitis B virus. A positive antigen test indicates that the patient has been infected with the hepatitis B virus, which can be acute or chronic. The hepatitis B virus is an enveloped virus, and the surface proteins of its viral envelope serve as surface antigens. | |||||||||||||||||||||||||||||||
| General Notes | 1. Strictly adhere to the specified incubation time and temperature to ensure accurate results. All reagents must be at room temperature (20-25°C) before use. Refrigerate reagents immediately after use. 2. Improper plate washing may result in inaccurate results. Ensure that all liquid in the wells is aspirated thoroughly before adding substrate. Do not allow the wells to dry out during incubation. 3. Remove any residual liquid and fingerprints from the bottom of the plate, as this will affect the OD value. 4. The substrate developer solution should be colorless or very light in color. Do not use substrate solution that has turned blue. 5. Avoid cross-contamination of reagents and specimens to prevent erroneous results. 6. Avoid direct exposure to strong light during storage and incubation. 7. Do not expose any reagents to bleaching solvents or the strong fumes emitted by bleaching solvents. Any bleaching agent will destroy the biological activity of the reagents in the kit. 8. Do not use expired products, and do not mix components with different product numbers and batches. 9. Recombinant proteins from sources other than the kit may not be compatible with the antibodies in this kit and will not be recognized. 10. If there is a possibility of disease transmission, all samples should be managed properly and samples and testing devices should be handled according to prescribed procedures. |
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| Storage Temp. | If the unopened kit is stored at 4°C, the shelf life is 6 months. | |||||||||||||||||||||||||||||||
| Test Range | Qualitative testing | |||||||||||||||||||||||||||||||
| Applications | Serum, plasma, tissue homogenates, cell lysates, cell culture supernatants and other biological fluids |
