Chicken secretory immunoglobulin A (sIgA) enzyme-linked immunosorbent assay (ELISA) kit Instructions for use This kit is for research use only. Drug Name: Generic Name: Chicken Secretory Immunoglobulin A (sIgA) Enzyme-Linked Immunoassay Kit Use: This kit is used to determine secretory immunoglobulin A (sIgA) in chicken serum, plasma, or other tissue fluids. content. Experimental principle This kit uses the double antibody sandwich method to determine the level of chicken secretory immunoglobulin A (sIgA) in the specimen. The microplate was coated with purified anti-chicken sIgA antibody to prepare a solid phase antibody, and sIgA was sequentially added to the microcapsules of the coated monoclonal antibody, and then combined with HRP-labeled goat anti-chicken antibody to form an antibody-antigen-enzyme label. The antibody complex was thoroughly washed and then added to the substrate TMB 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 depth of the color is positively correlated with the secretory immunoglobulin A (sIgA) in the sample. The absorbance (OD value) was measured by a microplate reader at a wavelength of 450 nm, and the concentration of chicken secretory immunoglobulin A (sIgA) in the sample was calculated by a standard curve. Kit composition 1 20 times concentrated washing solution 30ml × 1 bottle 7 Stop solution 6ml × 1 bottle 2 Enzyme standard reagent 6ml × 1 bottle 8 standard product (2700ng / L) 0.5ml × 1 bottle 3 enzyme label coating plate 12 holes × 8 9 standard dilutions 1.5ml × 1 bottle 4 sample dilution 6ml × 1 bottle 10 instructions 1 part 5 color reagent A liquid 6ml × 1 bottle 11 sealing film 2 sheets 6 coloring agent B liquid 6ml × 1 Bottle 12 sealed bag 1 specimen requirement 1. Specimen processing: (1) serum or plasma: can be directly used for detection (2) tissue: select fresh tissue, take 2g tissue and cut into small pieces (2 ~ 5mm size) with scissors. Then, the tissue pieces were added to a 10 mL centrifuge tube, 2 mL of sample physiological saline was added, and the mixture was incubated for 5 hours at room temperature (while vortex mixing occasionally), homogenized, 2500 rpm/separated for 10 minutes, and 1 ml of the supernatant was taken for use. (3) Culture: 2 ml of physiological saline was added to the culture, and the mixture was incubated for 1 hour at room temperature (with occasional vortex mixing), and then centrifuged for 2 minutes at 2500 rpm for 10 minutes, and the supernatant was taken for testing. (4) After extracting according to the relevant literature, take the supernatant for testing. Samples containing NaN3 could not be detected because NaN3 inhibited horseradish peroxidase (HRP) activity. 4. Specimens can be stored at -20 ° C, but repeated freezing and thawing should be avoided. 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, and the concentrations were (1800ng/L, 1200ng/L, 600ng/L, 300ng/L, 150ng/L). 2. Loading: blank holes (blank control holes) No sample and enzyme labeling 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 microplate and mix gently by shaking. 3. Incubate: Cover with a sealing plate and incubate for 30 minutes at 37 ° C. 4. Dosing: 20 times The concentrated washing solution is diluted 20 times with distilled water and used for 5. Washing: Carefully remove the sealing film, discard the liquid, dry it, fill each well with washing liquid, let stand for 30 seconds, discard it, repeat 5 times, pat dry 6. Add enzyme: add 50μl of enzyme labeling reagent to each well, except for blank wells 7. Incubation: operation is the same as 3. 8. Washing: operation is the same as 5. 9. Color development: first add coloring agent A50μl to each well, then Add 50 μl of color developer B, gently shake and mix, and develop color at 37 ° C for 15 minutes. 10. Termination: 50 μl of stop solution per well, stop the reaction (blue at this time) Vertical turn yellow. 11. Determination: The absorbance (OD value) of each well is measured sequentially with blank air conditioner zero and 450 nm wavelength. The measurement should be performed within 15 minutes after adding the stop solution. Calculate the concentration of the standard as the abscissa. The OD value is the ordinate, the standard curve is drawn on the coordinate paper, the corresponding concentration is found from the standard curve according to the OD value of the sample; multiplied by the dilution factor; or the straight line of the standard curve is calculated by using the concentration of the standard substance and the OD value. Regression equation, the OD value of the sample is substituted into the equation, the sample concentration is calculated, and multiplied by the dilution factor, which is the actual concentration of the sample. Cautions 1. The kit should be taken out from the refrigerated environment and equilibrated at room temperature for 15-30 minutes. If it is used, if the enzyme label is unsealed, the slats should be stored in a sealed bag. 2. The concentrated washing liquid may crystallize out. When diluted, it can be heated and dissolved in the water bath. Results 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 sample loading time within 5 minutes. If the number of specimens is large, it is recommended to use the gun to load the sample. 4. Please make a standard curve at the same time of each measurement. It is best to make a double hole. If the content of the test substance in the sample is too high (the OD value of the sample is larger than the OD value of the first hole of the standard product hole), please first dilute with the sample diluent. After a certain multiple (n times), measure again. When calculating, please multiply by the total dilution factor (×n×5). 5. Strictly follow the instructions. The test result must be based on the microplate reader. 6. Substrate Please keep it away from light. The sealing film can only be used once to avoid cross-contamination. 7. All samples, washing liquid and various wastes should be treated as infectious materials. 8. Different batch components of this reagent should not be mixed. If the English manual is different, the English manual shall prevail. Detection range: 100ng/L – 2000ng/L Specification: 96 person/box storage condition and expiration date 1. The kit is stored at: 2-8 °C. 2. Validity: 6 months
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