Dog RV-IgG ELISA Kit
Dog RV-IgG 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 Buffer: Gently wash adherent cells with pre-chilled PBS, then trypsinize and collect them by centrifugation at 1000×g for 5 minutes. Suspension cells can be collected directly by centrifugation. Wash the collected cells three times with pre-chilled PBS and resuspend them in 150-200 μL of PBS per 1×10^6 cells (it is recommended to add protease inhibitors to the PBS; if the cell count is very low, the PBS volume can be reduced appropriately). Disrupt the cells by repeated freeze-thaw cycles or sonication. Centrifuge the extract at 1500×g for 10 minutes at 2-8°C, and remove the supernatant for analysis. Cell culture supernatant: Centrifuge at 1000×g for 20 minutes, remove the supernatant, or store at -20°C or -80°C, but avoid repeated freeze-thaw cycles. Other biological fluids: Centrifuge at 1000×g for 20 minutes, and remove the supernatant for analysis.Please take the test kit out of the refrigerator 10 minutes in advance and equilibrate it 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 HRP-secondary antibody working solution: 15 minutes before use, centrifuge 100× concentrated HRP-secondary antibody at 1000×g for 1 minute, and dilute 100× concentrated HRP-secondary antibody to 1× working concentration with universal diluent (e.g. 10uL concentratedReduction solution + 990uL universal diluent), ready for use. 4.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 from the refrigerator may have crystals, which is normal. It can be placed at room temperature and shaken evenly until the crystals are complete.Prepare after it is completely dissolved). 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.Add sample: add 100uL of sample or positive and negative control into the corresponding wells respectively.Add 100uL of universal diluent to the white wells. Cover with the sealing film and incubate at 37℃ for 60 minutes. (Recommendation: Dilute the sample to be tested with universal diluent at least 1 times before adding it to the ELISA plate for testing. This will reduce the risk ofTo minimize the impact of matrix effects on test results, the sample concentration should be multiplied by the corresponding dilution factor when calculating the final sample concentration. It is recommended to set up duplicate wells for all test samples and positive and negative controls during the test). 3.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 washing process 3 times (you can also use a plate washer to wash the plate). 4.Add HRP-secondary antibody: add 100uL HRP-secondary antibody working solution to each well, cover with sealing film and incubate at 37℃ for 30 minutes. 5.Wash the plate: discard the liquid and follow the washing method in step 3, wash the plate 5 times 6.Add substrate: Add 90uL substrate (TMB) to each well, cover with sealing film, and incubate at 37℃ in the dark for 15 minutes. 7.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. Experiment Result evaluation: 1.The conditions for the test results to be valid are: The average of the positive control wellsODValue＞0.20, average OD of negative control wellsValue < 0.20. 2.Test samplesS/PValue ≥ 0.2When the sample is positive, it is judged as positive;S/PValue < 0.2, 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 anti-rabies virus IgG (RV-IgG) capture antibody. 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 anti-rabies virus IgG (RV-IgG) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||
| Source | Dog | |||||||||||||||||||||||||||||||
| Synonym | Dog Anti-Rabies Virus IgG ELISA Kit | |||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||
| Composition |
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| Background | The rabies virus G protein is a trimer of approximately 67 kDa. It is the primary antigen that induces the production of virus-neutralizing antibodies (VNA) and can induce immunity against lethal rabies virus infection. The G protein also contains virulence determinants. The G gene was the first rabies virus gene to be cloned and sequenced. | |||||||||||||||||||||||||||||||
| 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 |
