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Instruction Manual of Canine Hepatitis B Virus (HBV) Enzyme Linked Immunoassay (ELISA) Kit
Canine Hepatitis B Virus (HBV) Enzyme Linked Immunoassay (ELISA) Kit Instructions for Use This reagent is for research purposes only: This kit is used to detect canine serum, plasma, cell culture fluid and related liquid samples (HBV) level. Experimental principle: This kit uses double antibody sandwich enzyme-linked immunoassay (ELISA) to determine the canine hepatitis B virus (HBV) in the specimen. Coat the microplate with purified canine hepatitis B virus (HBV) antibody to make a solid-phase antibody, which can be combined with the hepatitis B virus (HBV) in the sample. It is combined with HRP-labeled hepatitis B virus (HBV) antibody to form an antibody-antigen-enzyme-labeled antibody 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 canine hepatitis B virus (HBV) 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 Sample handling and requirements for storage at 2-8 ° C: 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), 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. 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 for 30 minutes at 37 ℃ 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 of positive control wells ≥1.00; average of negative control wells ≤0.10 cut-off value (CUT OFF) calculation: cut-off value = average of negative control wells +0.15 negative judgment: OD value of samples
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