Chicken Newcastle Disease Antibody (ND-Ab) Enzyme-Linked Immunosorbent Assay (ELISA) Kit Instruction Manual This reagent is for research use only. This kit is used to determine Newcastle disease antibody (ND-Ab) in chicken serum, plasma and related liquid samples. content. Experimental principle: This kit uses the double antigen sandwich method to determine the level of Newcastle disease antibody (ND-Ab) in the specimen. A solid phase antigen was prepared by coating the microplate with purified chicken Newcastle disease (ND) antigen, and Newcastle disease antibody (ND-Ab) was sequentially added to the antigen-coated micropores, and then combined with HRP-labeled chicken Newcastle disease (ND) antigen. The antigen-antibody-enzyme-labeled antigen complex was formed, and after thorough washing, the substrate TMB was added for color development. TMB is converted to blue under the catalysis of HRP enzyme and converted to the final yellow color by the action of an acid. The color depth is positively correlated with the chicken Newcastle disease antibody (ND-Ab) in the sample. The absorbance (OD value) was measured by a microplate reader at a wavelength of 450 nm, and the concentration of Newcastle disease antibody (ND-Ab) in the sample was calculated by a standard curve. Kit composition: Kit composition 48-well configuration 96-well configuration Storage instructions 1 part 1 part sealing film 2 pieces (48) 2 pieces (96) Sealed bag 1 1 enzyme label coated plate 1 × 48 1 × 96 2 -8 ° C preservation standard: 72ng / L 0.5ml × 1 bottle 0.5ml × 1 bottle 2-8 ° C preservation standard dilution 1.5ml × 1 bottle 1.5ml × 1 bottle 2-8 ° C preservation enzyme standard reagent 3 ml × 1 bottle 6 ml × 1 bottle 2-8 ° C Preservation sample dilution 3 ml × 1 bottle 6 ml × 1 bottle 2-8 ° C Preservation developer A solution 3 ml × 1 bottle 6 ml × 1 bottle 2-8 ° C preservation Reagent B liquid 3 ml × 1 bottle 6 ml × 1 bottle 2-8 ° C Preservation solution 3 ml × 1 bottle 6 ml × 1 bottle 2-8 ° C Preservation concentrated washing solution (20ml × 20 times) × 1 bottle (20ml × 30 times) × 1 bottle 2-8 ° C preservation sample processing and requirements: 1. Serum: room temperature blood solidification for 10-20 minutes, centrifugation for about 20 minutes (2000-3000 rev / min). The supernatant is carefully collected, and if precipitation occurs during storage, it should be centrifuged again. 2. Plasma: EDTA or sodium citrate should be selected as an anticoagulant according to the requirements of the specimen. After mixing for 10-20 minutes, centrifuge for about 20 minutes (2000-3000 rpm). The supernatant is carefully collected, and if a precipitate forms during storage, it should be centrifuged again. 3. Urine: Collect with a sterile tube and centrifuge for about 20 minutes (2000-3000 rpm). The supernatant is carefully collected, and if a precipitate forms during storage, it should be centrifuged again. The chest and ascites and cerebrospinal fluid are referred to. 4. Cell culture supernatant: When detecting secreted components, collect them in a sterile tube. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. When the components in the cells were detected, the cell suspension was diluted with PBS (pH 7.2-7.4), and the cell concentration reached about 1 million/ml. By 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. Tissue specimen: After cutting the specimen, weigh the weight. Add a certain amount of PBS, pH 7.4. It was quickly frozen and stored in liquid nitrogen for use. The specimen still maintains a temperature of 2-8 ° C after melting. A certain amount of PBS (pH 7.4) was added, and the specimen was homogenized by hand or homogenizer. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. One part of the package is to be tested, and the rest is frozen for use. 6. The specimens should be extracted as soon as possible after collection, and the extraction should be carried out according to the 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 horseradish peroxidase (HRP) activity. Operation steps 1. Dilution and loading of standard products: 10 wells of standard wells are placed on the enzyme labeling board, 100 μl of standard is added to the first and second holes, and then added in the first and second holes. 50 μl of the standard dilution, mix; then add 100 μl from each of the first and second wells to the third and fourth wells, and then add 50 μl of the standard dilution to the third and fourth wells. Then, 50 μl of each of the third and fourth wells is discarded, and 50 μl of each is added to the fifth and sixth wells, respectively, and 50 μl of the standard dilution is added to the fifth and sixth wells, respectively. After mixing, 50 μl from each of the fifth and sixth holes is added to the seventh and eighth holes, respectively, and then 50 μl of the standard dilution is added to the seventh and eighth holes, respectively, and mixed from the seventh. 50 μl of the eighth well was added to the ninth and tenth holes, and 50 μl of the standard dilution was added to the ninth and tenth holes, respectively, and 50 μl of each of the ninth and tenth holes was discarded. (The amount of each well was 50μl after dilution, the concentration was 48ng/L, 32ng/L, 16ng/L, 8ng/L, 4ng/L. 2. Loading: blank holes were set separately (blank control holes are not added) Sample and enzyme standard reagent, the other steps are the same), the sample hole to be tested. Add 40 μl of the sample diluent to the sample hole to be tested on the enzyme label coated plate, and then add 10 μl of the sample to be tested (the final dilution of the sample is 5 times) Add the sample to the bottom of the well of the ELISA plate, try not to touch the wall of the well, and shake gently to mix. 3. Incubation: After sealing with a sealing film, incubate at 37 ° C for 30 minutes. 4. Dosing : Dilute 30 (48 times of 48T) concentrated washing solution with distilled water 30 (20 times of 48T) and use. 5. Wash: carefully remove the sealing film, discard the liquid, dry, and fill each well. The solution is allowed to stand for 30 seconds and then discarded. This is repeated 5 times and patted dry. 6. Add enzyme: 50 μl of enzyme labeling reagent per well, except for blank wells. 7. Incubation: operation is the same as 3. 8. Washing: operation 5. 9. Color: Add 50μl of coloring agent to each well, then add 50μl of coloring agent, gently shake and mix, and color at 37°C for 15 minutes. 10. Termination: Add per well 50 μl of stop solution, the reaction was terminated (at this time, the blue color turned yellow). 11. Measurement: The absorbance (OD value) of each well was measured in sequence with blank air conditioner zero and 450 nm wavelength. The measurement should be performed within 15 minutes after the addition of the stop solution. Note: 1. The kit should be taken out from the refrigerated environment and should be used at room temperature for 15-30 minutes before use. If the enzyme label is not used up after opening, the slats should be stored in a sealed bag. The washing liquid may be crystallized. When it is diluted, it can be heated and dissolved in the water bath. The washing will not affect the result. 3. The sampler should be used for each step, and the accuracy should be corrected frequently to avoid the test error. It is best to control the loading time within 5 minutes. If the number of specimens is large, it is recommended to use a gun to load the sample. 4. Please make a standard curve at the same time for each measurement. It is best to make a double hole. If the sample is too high in the sample. (The OD value of the sample is larger than the OD value of the first hole of the standard well), please first dilute the sample dilution with a certain multiple (n times) and then measure it. When calculating, multiply the total dilution factor (×n×5). The sealing film is for one-time use only to avoid Fork contamination. 6. Keep the substrate away from light. 7. Strictly follow the instructions. The test results must be based on the reading of the microplate reader. 8. All samples, washing liquid and various wastes should be in accordance with the infectious materials. Disposal. 9. The different batch components of this reagent should not be mixed. 10. If it differs from the English manual, the English manual shall prevail. Calculation: the concentration of the standard is the abscissa, the OD is the ordinate, and the drawing is on the coordinate paper. A standard curve is obtained, and the corresponding concentration is determined from the standard curve according to the OD value of the sample; multiplied by the dilution factor; or the linear regression equation of the standard curve is calculated by using the concentration of the standard substance and the OD value, and the OD value of the sample is substituted into the equation. Calculate the sample concentration and multiply by the dilution factor, which is the actual concentration of the sample. (This figure is for reference only) Kit performance: 1. Sample linear regression and expected concentration correlation coefficient R value is 0.92 or more. 2. Within and within the batch should be less than 9% and 15% detection range: 2ng / L -50ng / L Storage conditions and expiration date: 1. Kit storage:; 2-8 ° C. 2. Validity: 6 months
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