Chicken Newcastle Disease Virus (NDV) Enzyme Linked Immunoassay (ELISA) Kit User Manual

Chicken Newcastle Disease Virus (NDV) Enzyme Linked Immunoassay (ELISA) Kit Instructions for Use This reagent is for research use only: This kit is used to determine the content of Newcastle Disease Virus (NDV) in human serum, plasma and related liquid samples. Experimental principle: This kit uses the double antibody sandwich method to determine the level of Newcastle Disease Virus (NDV) in the specimen. Microporous plates were coated with purified chicken Newcastle disease virus (NDV) antibody to make solid phase antibodies. Newcastle disease virus (NDV) was added to the microwells coated with mAb in turn, followed by HRP-labeled Newcastle disease virus (NDV) antibody The combination forms 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 color depth is positively correlated with Newcastle Disease Virus (NDV) in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the concentration of Newcastle Disease Virus (NDV) in the samples was calculated by a standard curve. 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 standard at -8 ° C: 1800 ng / L 0.5ml × 1 bottle 0.5ml × 1 bottle 2-8 ° C store standard dilution 1.5ml × 1 bottle 1.5ml × 1 bottle 2-8 ° C store enzyme label reagent 3 ml × 1 bottle of 6 ml × 1 bottle of sample diluent 3 ml at 2-8 ° C × 1 bottle of 6 ml × 1 bottle of 2-8 ° C storage of developer A solution 3 ml × 1 bottle of 6 ml × 1 bottle of 2-8 ° C Storage Developer B solution 3 ml × 1 bottle 6 ml × 1 bottle 2-8 ° C Storage stop solution 3ml × 1 bottle 6ml × 1 bottle 2-8 ° C Store 20 times concentrated washing solution 20ml × 1 bottle 30ml × 1 bottle 2 Sample processing and requirements for storage at -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), 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). Procedure 1. Dilution and loading of standard products: set 10 standard wells on the enzyme-coated plate, add 100 μl of standard products in the first and second wells, and then add in the first and second wells. 50μl of standard diluent, mix well; then take 100μl from the first well and the second well respectively to the third and fourth wells, then add 50μl of standard diluent to the third and fourth wells respectively, mix Evenly; then take 50μl each in the third and fourth wells and discard, then add 50μl each to the fifth and sixth wells, and then add 50ul of standard dilution solution to the fifth and sixth wells respectively, mix After mixing, take 50μl from the fifth and sixth wells and add them to the seventh and eighth wells respectively. Then add 50μl of the standard dilution solution to the seventh and eighth wells respectively. Take 50μl from the eighth well to the ninth and tenth wells, and then add 50μl of the standard dilution solution to the ninth and tenth wells respectively. After mixing, take 50μl from the ninth and tenth wells and discard. (After dilution, the amount of sample added to each well is 50 μl, and the concentration is 1200 ng / L, 800 ng / L, 400 ng / L, 200 ng / L, 100 ng / L. 2. Sample addition: set blank wells ( Blank control wells do not add samples and enzyme labeling reagents, the rest of the operation is the same), the sample well to be tested. Add 40μl of sample diluent to the sample wells to be tested on the enzyme coated plate, then add 10μl of the sample to be tested The dilution is 5 times.) 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, and gently shake to mix. 3. Incubation: seal the plate with a sealing film and incubate at 37 ° C for 30 minutes 4. Mixing solution: dilute the 20-fold concentrated washing solution with distilled water 20-fold and set aside. 5. Washing: carefully peel off the sealing film, discard the liquid, spin dry, fill each well with the washing solution, and let stand for 30 seconds Discard, repeat this 5 times, pat dry. 6. Add enzyme: add 50μl of enzyme label reagent to each well, except the blank well. 7. Incubation: The operation is the same as 3. 8. Washing: The operation is the same as 5. 9. Color development: Add 50μl of developer A to each well, and then add 50μl of developer B, mix gently, and develop at 37 ° C in the dark for 15 minutes. 10. Termination: add 50μl of stopper solution to each well to terminate Reaction (blue to yellow at this time). 11. Determination: The absorbance (OD value) of each well is measured in sequence with a blank air conditioner at zero, 450 nm wavelength. The measurement should be performed within 15 minutes after the addition of the stop solution. Note: 1 ．Remove the kit from the refrigerated environment and equilibrate it at room temperature for 15-30 minutes before use. If the enzyme-coated plate is unopened after opening, the strip should be stored in a sealed bag. 2. Concentrated washing solution may be Crystals are precipitated, which can be heated and dissolved in a water bath during dilution, and the results are not affected during washing. 3. The sampler should be used at each step of sample addition, and the accuracy should be regularly checked to avoid experimental errors. It is easy to control within 5 minutes. If there are a large number of specimens, it is recommended to use a rifle to add samples. 4. Please make a standard curve at the same time for each measurement. It is best to do a re-hole. If the content of the substance to be tested in the specimen is too high (the sample OD value is greater than OD value of the first well of the standard well), please dilute with a certain multiple (n times) of the sample diluent before measuring, and finally multiply by the total dilution multiple (× n × 5) when calculating. 5. Seal the membrane only It is limited to one-time use to avoid cross-contamination. Please keep it away from light. 7. Perform the operation in strict accordance with the instructions, and the test results must be determined by the reading of the microplate reader. 8. All samples, washing liquid and various wastes should be treated as infectious agents. 9. This reagent The components of different batches shall not be mixed. 10. If there is any difference with the English instructions, the English instructions shall prevail. Calculation: The concentration of the standard is taken as the abscissa, the OD value is the ordinate, and the standard curve is drawn on the coordinate paper, according to the sample The OD value of the corresponding concentration is found by the standard curve; then multiplied by the dilution factor; or the linear regression equation of the standard curve is calculated from the concentration of the standard and the OD value, and the OD value of the sample is substituted into the equation to calculate the sample concentration, then Multiplied by the dilution factor is the actual concentration of the sample. (This picture is for reference only) Kit performance: 1. The linear regression coefficient R of the sample and the expected concentration is 0.990 or more. 2. The batch and batch see should be less than 9% and 11% respectively. Detection range: 70ng / L -1500 ng / L Storage conditions and expiration date: 1. Storage of the kit :; 2-8 ℃. 2. Validity: 6 months

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