ELISA for Ebola virus-Ab Kit Instructions for Use This reagent is for research only Purpose: This kit is used to determine the level of Ebola virus-Ab in monkey serum, plasma and related liquid samples. Experimental principle: The kit uses double antigen sandwich enzyme-linked immunoassay (ELISA) to determine the level of monkey Ebola virus-Ab in the specimen. Coated microtiter plates with purified monkey Ebola virus antigen to make a solid-phase antigen, which can be combined with the monkey Ebola virus antibody (Ebola virus-Ab) in the sample, after washing to remove unbound antibody and other components And then combined with HRP-labeled Ebola virus antigen to form an antigen-antibody-enzyme-labeled antigen complex, after thorough washing, the substrate TMB is added for color development. TMB is converted into blue under the catalysis of HRP enzyme, and into the final yellow under the action of acid. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm and compared with the CUTOFF value to determine the presence or absence of Ebola virus-Ab in the specimen. Kit composition: kit composition 48 well configuration 96 well configuration storage instructions 1 copy 1 copy sealing film 2 pieces (48) 2 pieces (96) 1 sealed bag 1 enzyme coated plate 1 × 48 1 × 96 2 Store negative control 0.5ml × 1 bottle 0.5ml × 1 bottle at -8 ℃ Store positive control 0.5ml × 1 bottle 0.5ml × 1 bottle at 2-8 ℃ Store enzyme reagent 3 ml × 1 bottle 6 ml × 1 at 2-8 ℃ Store the sample diluent at 2-8 ° C 3 ml × 1 bottle 6 ml × 1 bottle Store the developer A at 2-8 ° C 3 ml × 1 bottle 6 ml × 1 bottle Store the developer B at 2-8 ° C 3 ml × 1 bottle 6 ml × 1 bottle 2-8 ° C storage stop solution 3ml × 1 bottle 6ml × 1 bottle 2-8 ° C storage concentrated washing solution (20ml × 20 times) × 1 bottle (20ml × 30 times) × 1 bottle Store at 2-8 ℃ Sample processing and requirements: 1. Serum: room temperature blood coagulates naturally for 10-20 minutes, centrifuged for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully and centrifuge again if a precipitate appears during storage. 2. Plasma: EDTA or sodium citrate should be selected as the anticoagulant according to the requirements of the specimen, mixed for 10-20 minutes, and centrifuged for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. If a precipitate forms during storage, it should be centrifuged again. 3. Urine: collected in a sterile tube and centrifuged for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. If a precipitate forms during storage, centrifuge again. Pleural and ascites, cerebrospinal fluid reference implementation. 4. Cell culture supernatant: When detecting secreted components, collect with a sterile tube. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. When detecting the components inside the cells, dilute the cell suspension with PBS (PH7.2-7.4), and the cell concentration will reach about 1 million / ml. Through repeated freezing and thawing, the cells are destroyed and the intracellular components are released. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. If a precipitate forms during storage, it should be centrifuged again. 5. Organize the specimen: after cutting the specimen, weigh it. Add a certain amount of PBS, PH7.4. Quickly freeze and save with liquid nitrogen for later use. After the specimen melts, it still maintains a temperature of 2-8 ° C. Add a certain amount of PBS (PH7.4) and homogenize the specimen with a manual or homogenizer. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. After aliquoting, a portion is to be tested, and the rest is frozen for future use. 6. The specimen should be extracted as soon as possible after collection. The extraction should be carried out according to relevant literature. The experiment should be carried out as soon as possible after extraction. If the test cannot be performed immediately, the specimen can be stored at -20 ° C, but repeated freezing and thawing should be avoided. 7. Samples containing NaN3 cannot be detected because NaN3 inhibits the activity of horseradish peroxidase (HRP). Operation steps: 1. Numbering: Number the samples corresponding to the microwells in sequence. Each plate should be provided with 2 wells for negative control, 2 wells for positive control, and 1 well for blank control. Same) 2. Add sample: add negative control and positive control 50μl to the negative and positive control wells respectively. Then add 40μl of sample diluent to the sample well, and then add 10μl of the sample to be tested. Add the sample and add the sample to the bottom of the well of the microplate, try not to touch the wall of the well, shake gently to mix, 3. Incubate: seal the plate with the sealing film and incubate at 37 ℃ for 30 minutes. 4. Mixing solution: add 30 times (20 times of 48T) concentrated washing solution to distilled water to 600ml and reserve for use. 5. Washing: Carefully remove the sealing film, discard the liquid, spin dry, fill each well with washing solution, and let stand Discard after 30 seconds, repeat this 5 times, pat dry. 6. Add enzyme: add 50μl of enzyme label reagent to each well, except blank well. 7. Incubation: The operation is the same as 3. 8. Washing: The operation is the same as 5. 9. Color development: 50μl of developer A is added to each well, and then 50μl of developer B is added, shake gently to mix, avoid light at 37 ℃ Color development for 15 minutes 10. Termination: Add 50μl of stop solution to each well to stop the reaction (in this case, the blue will turn to yellow). 11. Determination: Measure the absorbance (OD value) of each well in sequence with the blank air conditioner at zero and 450 nm wavelength. The measurement should be carried out within 15 minutes after adding the stop solution. Judgment of results: Test validity: average value of positive control wells ≥1.00; average value of negative control ≤0.10 cut-off value (CUT OFF) calculation: cut-off value = average value of negative control wells +0.15 negative judgment: sample OD value <cut-off value (CUT OFF) is positive for Ebola virus-Ab monkey negative: the sample OD value ≥ critical value (CUT OFF) is positive for Ebola virus-Ab monkey 1. The operation is strictly in accordance with the instructions. The components of different batches of this reagent must not be mixed. 2. The kit should be equilibrated at room temperature for 15-30 minutes before being taken out of the refrigerated environment. If the enzyme label coated plate is unopened, the strip should be stored in a sealed bag. 3. Crystals may be precipitated in the concentrated washing liquid, which can be heated and dissolved in a water bath during dilution, and the results will not be affected during washing. 4. The sealing film is limited to one-time use to avoid cross-contamination. 5. Please keep the substrate away from light. 6. The result of the test must be determined by the reading of the microplate reader. When using dual-wavelength detection, the reference wavelength is 630nm. 7. All samples, washing liquids and various wastes should be treated as infectious agents. The stop solution is 2M sulfuric acid, and you must pay attention to safety when using it. Storage conditions and validity period Kit storage :; 2-8 ℃. 2. Validity: 6 months Shanghai Yuping Biological Technology Co., Ltd. mainly deals with ELISA kits of various brands and grades, with quality assurance and perfect after-sales service. And provide free generation testing. Serving universities and immunology research units. 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