Ranibizumab ELISA Kit
Ranibizumab ELISA Kit
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Product Details
Product Specification
| protein | Ranibizumab | ||||||||||||||||||||||||||||||||||||
| Usage |
1. Self-prepared test equipment required for the experiment: 1, plate reader (450nm) 2, high-precision sampler and gun head: 0.5-10uL、5-50uL、20-200uL、200-1000uL 3, 37℃ Incubator 4, Distilled water or deionized water 2. Sample processing and requirements: The detection range of the kit is not equivalent to the concentration range of the test substance in the sample. It is recommended to estimate the concentration of the test substance in the sample through relevant literature before the experiment and determine the actual concentration of the sample through pre-experiment. If the concentration of the test substance in the sample is too high or too low, please dilute or concentrate the sample appropriately. If the sample tested is not among the sample types listed in the instructions, it is recommended to conduct pre-experiments to verify its detection effectiveness. Serum: Whole blood samples collected in serum separation tubes were placed at room temperature 2 Hour or 2-8℃ Overnight, then 1000×g Centrifugation 20 Minutes, take the supernatant, or place the supernatant in -20℃ Or -80℃ Store, but repeated freezing and thawing should be avoided. Plasma: use EDTA Or heparin as an anticoagulant to collect a sample, and collect the sample after collection 30 Within minutes 2-8℃1000×g Centrifugation 15 Minutes, take the supernatant to detect, or place the supernatant in -20℃ Or -80℃ Store, but repeated freezing and thawing should be avoided. Tissue homogenate: With pre-cooled PBS(0.01M,pH=7.4) The tissue is flushed to remove residual blood (lysed red blood cells in the homogenate can affect the test results), and the tissue is weighed and cut into pieces. Combining the shredded tissue with the corresponding volume PBS (Generally according to 1:9 Weight to volume ratio, such as 1g The tissue samples correspond to 9mL Of PBS The specific volume can be appropriately adjusted according to the experimental needs and recorded. Recommended in PBS Add protease inhibitor) into a glass homogenizer, and thoroughly grind or homogenize on ice Machine Grinding. For further lysis of tissue cells, the homogenate can be sonicated, or freeze-thawed repeatedly. Finally, the homogenate was mixed in 5000×g Centrifugation 5~10 Minutes, take the supernatant for detection. Cell culture supernatant: Please 1000×g Centrifugation 20 Minutes, take the supernatant to detect, or place the supernatant in -20℃ Or -80℃ Store, but repeated freezing and thawing should be avoided. Cell lysate: Precooling for adherent cells PBS Gently washed, followed by trypsinization, 1000×g Centrifugation 5 Cells were collected after minutes; The suspended cells can be collected directly by centrifugation. The collected cells were pre-cooled with PBS Washing 3 Times, every 1×10^6 Added to cells 150-200μLPBS Resuspension (recommended at PBS Adding a protease inhibitor; If the content is very low, it can be appropriately reduced PBS Volume) and the cells were disrupted by repeated freeze-thaw or sonication. The extracts were mixed in 2-8℃, 1500×g Centrifugation 10 Minutes, take the supernatant for detection. Other biological samples: 1000×g Centrifugation 20 Minutes, take the supernatant to detect. Sample Appearance: The sample should be clear and transparent, and the suspended solids should be centrifuged to remove. Sample Preservation: After sample collection, if 1 Those tested within weeks can be stored in 4℃, if it cannot be detected in time, please pack it according to the one-time usage amount and freeze it in -20℃（1 Within months), or -80℃（6 Test within months) to avoid repeated freezing and thawing. Hemolysis of the sample will affect the final test result, so hemolyzed samples are not suitable for this test. 3. Sample dilution plan: Please estimate the concentration range of the sample in advance. If your test sample needs to be diluted, refer to the dilution plan as follows: Dilution 100 Times: One step dilution. Take 5μL Sample to 495μL Within universal diluent, do 100 Double dilution; Dilution 1000 Times: Two-step dilution. Take 5μL Sample to 95μL Within universal diluent, do 20 Dilute, and then take 5μL20 Double dilute sample to 245μL Within universal diluent, do 50 Double dilution, total dilution 1000 Times; Dilution 100000 Times: Three-step dilution. Take 5μL Sample to 195μL Within universal diluent, do 40 Dilute, and then take 5μL40 Double dilute sample to 245μL Within universal diluent, do 50 Time dilution, and finally take 5μL 2000 Double dilute sample to 245μL Within universal diluent, do 50 Double dilution, total dilution 100000 Times; The amount of liquid taken during each dilution step is not less than 3μL, the dilution factor is not more than 100 Times. Each step of dilution should be mixed evenly to avoid foaming. 4. Preparations before testing: 1, please advance 10 Minutes remove the kit from the refrigerator and equilibrate to room temperature. 2, Standard gradient working solution preparation: join 1mL Universal Diluent into Lyophilized Standard, let stand 15 Minutes until it is completely dissolved and then gently mix (the concentration is 1000ng/mL）And then according to the following concentrations: 1000ng/mL、500ng/mL、250ng/mL、125ng/mL、62.5ng/mL、31.25ng/mL、15.625ng/mL、0ng/mL The dilution was performed. Double dilution method: Take 7 branch EP Tubes, each tube is added 500uL Universal diluent,1000ng/mL Pipette from the standard working solution 500uL To the first EP Mix evenly in a tube 500ng/mL Standard Working Solution, according to this step, absorb and mix evenly in turn. The last tube is directly used as a blank hole, There is no need to draw liquid from the penultimate tube, as shown in the figure below.  3, Biotin- Preparation of antibody working solution: Before use 15 Minutes will concentrate Biotin- Antibody to 1000×g Centrifugation 1 Minutes, with a universal diluent 100× concentrate Biotin- The antibody is diluted into 1× Working concentration (ex: 10uL Concentrate +990uL Universal Diluent), used on the same day. 4, Preparation of enzyme conjugate working solution: Before use 15 Minutes will 100× Concentrated enzyme conjugate in 1000×g Centrifugation 1 Minutes, with a universal diluent 100× The concentrated enzyme conjugate is diluted into 1× Working concentration (Example: 10μL Concentrate +990μL Universal diluent), now available for use. 5, 1× Wash solution preparation: Take 10mL 20× Wash liquid to 190mL In distilled water (the concentrated washing liquid taken out from the refrigerator may have crystals, which is a normal phenomenon. It can be placed at room temperature and prepared after the crystals are completely dissolved). 5. Operation steps: 1, Equilibration from room temperature 10 After minutes, remove the required slats from the aluminum foil bag, and seal the remaining slats with ziplock bag and put them back 4℃. 2, Add samples: respectively add samples or different concentration standards according to 50μl Each well is added to the corresponding well, and the blank well is added 50μL Universal dilution followed by each well 50μL VEGFA Antigen working solution, after covering with sealing membrane 37℃ Incubation 60 Minutes. (Recommendation: It is recommended to set up double holes for all samples and standards to be tested during testing). 3, Plate washing: discard the liquid and add to each well 300μL1x Wash liquid, stand 1 Minutes, throw off the washing liquid, pat dry on absorbent paper, and repeat washing the plate 3 Times (plate washing machine can also be used to wash the plate) 4, Add biotin antibody working solution: add per well 100μL Biotin antibody working solution, after covering the sealing film 37℃ Incubation 60 Minutes. 5, Plate washing: discard the liquid and repeat steps 3 Repeat plate washing 3 Times (plate washing machine can also be used to wash the plate) 6, Adding enzyme conjugate working solution: adding enzyme conjugate working solution to each well 100μL, after covering the sealing film 37℃ Incubation 30 Minutes. 7, Plate washing: discard the liquid and repeat steps 3 Repeat plate washing 5 Times (the plate can also be washed with a plate washing machine). 8, Add substrate: add substrate per well (TMB)90μL Covered with a sealing film, 37℃ Incubation protected from light 15 Minutes. 9, Add stop solution: add stop solution to each well 50μL, immediately in 450nm The values of each well were measured at the wavelength. VI. Calculation of experimental results: Result judgment: 1, calculate the average of the standard and sample replica well OD Value and subtract the blank hole's OD Values as correction values. Taking concentration as the abscissa, OD Value is ordinate, draw the standard curve of the four-parameter logic function on the double logarithmic coordinate paper. 2, If the sample OD If the value is higher than the upper limit of the standard curve, the test should be retested after appropriate dilution and multiplied by the corresponding dilution factor when calculating the sample concentration.  7. Kit performance:1, Repeatability: the coefficient of variation in the plate is less than 10%, the interplate coefficient of variation is less than 10%. 2, Recovery rate: added to selected healthy serum, plasma and cell culture supernatant 3 Of different concentration levels Ranibizumab, calculate the recovery.
3, linear dilution: respectively in the selected 4 Healthy serum, plasma and cell culture supernatant were added with high concentration Ranibizumab, linearity was assessed by dilution within the standard curve kinetic range.
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| Sensitivity | 8.67ng/mL | ||||||||||||||||||||||||||||||||||||
| Theory | This kit uses competitive enzyme-linked immunosorbent assay (ELISA). Sample/standard, recombinant human VEGFA antigen, biotin-labeled goat anti-human VEGFA polyclonal antibody, and HRP enzyme conjugate were sequentially added to microwells pre-coated with Ranibizumab, incubated and washed in the middle, and colored by substrate TMB, which was converted to blue under the catalysis of peroxidase (HRP) and final yellow under the action of acid. There was a negative correlation between the depth of color and Ranibizumab in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450nm, and the sample concentration was calculated. | ||||||||||||||||||||||||||||||||||||
| Source | General purpose | ||||||||||||||||||||||||||||||||||||
| Synonym | Ranibizumab ELISA Kit | ||||||||||||||||||||||||||||||||||||
| Detection Type | Competition Law | ||||||||||||||||||||||||||||||||||||
| Composition |
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| Background | Ranibizumab is a monoclonal antibody fragment (Fab) made from the same mother mouse antibody as bevacizumab. It is an anti-angiogenic drug that has been approved for the treatment of "wet" age-related macular degeneration (AMD, also known as ARMD), diabetic retinopathy, and macular edema caused by branch retinal vein occlusion or central retinal vein occlusion. | ||||||||||||||||||||||||||||||||||||
| General Notes | 1. Carry out incubation in strict accordance with the specified time and temperature to ensure accurate results. All reagents must reach room temperature 20-25 °C prior to use. Store reagents in refrigeration immediately after use. 2. Incorrect plate washing may lead to inaccurate results. Make sure to drain the liquid from the wells as much as possible before adding the substrate. Do not allow the wells to dry out during incubation. 3. Eliminate the residual liquid and fingerprints at the bottom of the plate, otherwise it will affect the OD value. 4. The substrate color development solution should be colorless or very light in color, and the substrate solution that has turned blue cannot be used. 5. Avoid cross-contamination of reagents and specimens to avoid wrong results. 6. Avoid direct exposure to strong light during storage and incubation. 7. Any reaction reagent cannot come into contact with the bleaching solvent or the strong gas emitted by the bleaching solvent. Any bleaching component will destroy the biological activity of the reaction reagents in the kit. 8. Expired products cannot be used, and components with different item numbers and batch numbers cannot be mixed. 9. Recombinant proteins from sources other than the kit may not match the antibodies in this kit and are not recognized. 10. If the disease may be spread, all samples should be managed well, and the samples and testing devices should be handled according to the prescribed procedures. |
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| Storage Temp. | Unopened kit, stored at 4 °C, shelf life 6 months. | ||||||||||||||||||||||||||||||||||||
| Test Range | 15.62-1000ng/mL | ||||||||||||||||||||||||||||||||||||
| Applications | Serum, plasma, tissue homogenate, cell culture supernatant, cell lysate, other biological samples |
