Instructions for Human Leucylaminopeptidase (LAP) Elisa Kit This kit is for research use only. Detection range: 96T 80pg / mL-2400pg / mL purpose of usage: This kit is used to determine the content of leucine aminopeptidase (LAP) in human serum, plasma and related liquid samples. Experimental principle Instructions for Human Leucylaminopeptidase (LAP) Elisa Kit This kit uses the double antibody sandwich method to determine the level of human leucine aminopeptidase (LAP) in the specimen. Purified human leucine The amidopeptidase (LAP) antibody is coated on the microplate to make a solid-phase antibody, and leucine is added sequentially to the monoclonal antibody-coated microwells Base peptidase (LAP), then combined with HRP-labeled leucine aminopeptidase (LAP) antibody to form antibody-antigen-enzyme labeled antibody After thorough washing, the compound is added with TMB to develop color. TMB is converted into blue under the catalysis of HRP enzyme Under the effect of the conversion into the final yellow. The color depth is positively correlated with the Leucylaminopeptidase (LAP) in the sample. With enzymes The standard instrument measures the absorbance (OD value) at a wavelength of 450 nm, and calculates the human leucine aminopeptidase (LAP) in the sample through the standard curve concentration. Kit composition 1 30 times concentrated washing solution 20ml × 1 bottle 7 stop solution 6ml × 1 bottle 2 Enzyme label reagent 6ml × 1 bottle 8 standard (4800 pg / mL) 0.5ml × 1 bottle 3 Enzyme label coated plate 12 wells × 8 strips 9 standard dilutions 1.5ml × 1 bottle 4 Sample diluent 6ml × 1 bottle 10 instructions 1 copy 5 Developer A solution 6ml × 1 bottle 11 2 sealing film 6 Developer B liquid 6ml × 1 / bottle 12 1 sealed bag Specimen requirements 1. Specimens are extracted as soon as possible after collection, and extraction is performed according to relevant literature, and experiments should be conducted as soon as possible after extraction. If not Perform the test immediately. The specimen can be stored at -20 ℃, but repeated freezing and thawing should be avoided 2. The samples containing NaN3 cannot be detected because NaN3 inhibits horseradish peroxidase (HRP) activity. Steps 1. Dilution of standard products: this kit provides one original standard product, the user can dilute in a small test tube according to the following chart release. 2400pg / mL No. 5 standard 150μl original standard added 150μl standard dilution 1200pg / mL No. 4 standard 150μl No. 5 standard added 150μl standard dilution 600pg / mL No. 3 standard 150μl No. 4 standard added 150μl standard dilution 300pg / mL No. 2 standard 150μl No. 3 standard added 150μl standard dilution 150pg / mL No. 1 standard 150μl No. 2 standard added 150μl standard diluent 2. Add sample: set up blank wells (the blank control wells do not add samples and enzyme reagents, the rest of the operation is the same), standard well Sample hole to be tested. Add 50μl of the standard on the enzyme-coated plate accurately, and add 40μl of sample diluent to the sample well. Then add 10μl 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 microtiter plate Do not touch the wall of the hole, shake gently to mix. 3. Incubation: seal the plate with the sealing film and incubate at 37 ° C for 30 minutes. 4. Mixing solution: Dilute 30 times concentrated washing liquid with distilled water 30 times and reserve 5. Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing liquid, let stand for 30 seconds and then discard, so Repeat 5 times and 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: add 50μl of developer A to each well, and then add 50μl of developer B, mix gently, and develop color at 37 ℃ in the dark 10 minutes. 10. Termination: Add 50μl of stop solution to each well to terminate the reaction (at this time, the blue color turns to yellow). 11. Determination: Measure the absorbance (OD value) of each well in sequence with the blank air conditioner at zero and 450nm wavelength. Determination should be terminated Within 15 minutes after the solution. Summary of operating procedures: Calculation Taking the concentration of the standard as the abscissa and the OD value as the ordinate, draw a standard curve on the coordinate paper, according to the sample The corresponding concentration of OD value is found by the standard curve; multiplied by the dilution factor; or the standard concentration and OD value are used to calculate the standard The linear regression equation of the quasi-curve, the OD value of the sample is substituted into the equation, the sample concentration is calculated, and then multiplied by the dilution factor, This is the actual concentration of the sample. Precautions 1. The kit should be taken out of the refrigerated environment and equilibrated at room temperature for 15-30 minutes before it can be used. After use, the slats should be stored in sealed bags. 2. Crystals may be precipitated in the concentrated washing liquid, which can be heated and dissolved in the water bath during dilution, and the results will not be affected during washing. 3. The sample adder should be used in each step of sample addition, and its accuracy should be regularly checked to avoid test errors. One sample loading time is best Control within 5 minutes, if the number of specimens is large, it is recommended to use a volley gun to add samples. 4. Please make the standard curve at the same time of each measurement, it is better to make the complex hole. If the content of the test substance in the specimen is too high (sample OD value Is greater than the OD value of the first well of the standard product), please dilute it with a certain multiple (n times) of the sample diluent before measuring. When calculating, please multiply the total dilution factor (× n × 5). 5. The sealing film is limited to one-time use to avoid cross contamination. 6. Please keep the substrate away from light. 7. Strictly follow the instructions, and the test results must be determined based on the reading of the microplate reader. 8. All samples, washing liquid and various wastes should be treated as infectious agents. 9. The components of different batches of this reagent shall not be mixed. Storage conditions and validity period 1. Kit storage :; 2-8 ℃. 2. Validity: 6 months manual of human leucine aminopeptidase (LAP) Elisa kit
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