This kit is for research use only Intended application Quantitative determination of β-amyloid 1-40 (Aβ1-40) in human serum, plasma or other related fluids by ELISA. Experimental principle Coat the microplate with purified antibody to make a solid-phase carrier. Add specimens or standards, biotinylated anti-Aβ1-40 antibody, and HRP-labeled avidin to the monoclonal antibody-coated microwells in sequence. After washing, color was developed with the substrate TMB. TMB is converted into blue under the catalysis of peroxidase, and into the final yellow under the action of acid. The color depth is positively correlated with Aβ1-40 in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the sample concentration was calculated. Kit composition and reagent preparation 1. ELISA plate: one piece (96 wells) Standard product (lyophilized product): 2 bottles, each bottle is diluted with the sample diluent to 1ml before use, and left to stand for more than 10 minutes after being capped, and then repeatedly inverted / rubbed to help dissolve, its concentration is 300pg / ml, After serial dilutions, 300pg / ml, 150pg / ml, 75pg / ml, 37.5pg / ml, 18.8pg / ml, 9.4pg / ml, 4.7pg / ml, and the sample dilution is directly used as the standard concentration of 0pg / ml ml, prepared within 15 minutes before use. For example, to prepare a 150 pg / ml standard: Take 0.5 ml of 300 pg / ml of the above standard and add it to an Eppendorf tube containing 0.5 ml of sample diluent. 2. Sample diluent: 1 × 20ml / bottle. 3. Test the diluent A: 1 × 10ml / bottle. 4. Test dilution B: 1 × 10ml / bottle. 5. Detection solution A: 1 × 120ul / bottle (1: 100) is diluted with the detection diluent A1: 100 before use, prepared according to the pre-calculated total amount required for each experiment before dilution (100ul per well), In actual preparation, more 0.1-0.2ml should be prepared. For example, 1ul detection solution A plus 99ul detection dilution A is prepared in proportion, mix gently and prepare within one hour before use. 6. Test solution B: 1 × 120ul / bottle (1: 100) is diluted with test diluent B1: 100 before use. The dilution method is the same as that of Test Solution A. 7. Substrate solution: 1 × 10ml / bottle. 8. Concentrated washing solution: 1 × 30ml / bottle, each bottle is diluted 25 times with distilled water. 9. Stop solution: 1 × 10ml / bottle (2NH2SO4). Collection and preservation of specimens 1. Cell culture supernatant: collect the supernatant after centrifugation, and store the specimen at -20 ℃, and avoid repeated freezing and thawing. 2. Serum: Please leave the specimen at room temperature for 2 hours or overnight at 4 ° C and centrifuge at 1000xg for 20 minutes. Take the supernatant for testing, or store the specimen at -20 ° C, but avoid repeated freezing and thawing. 3. Plasma: EDTA or heparin can be used as an anticoagulant. Centrifuge the sample at 2-8 ° C1000xg for 15 minutes within 30 minutes after collection, or store the sample at -20 ° C, but avoid repeated freezing and thawing. Note: The above specimens should be stored at 4 ° C for less than 1 week. For long-term storage, please store at -20 ° C in a sealed state, but it should not exceed 3 months; specimen hemolysis will affect the final test results, so hemolysis specimens should not be used Detection; specimens with high blood lipids do not require special treatment and can be directly detected. Steps Before starting the experiment, please configure all reagents in advance. When the reagents or samples are diluted, they should be mixed well. Try to avoid foaming when mixing. A standard curve should be made for each test. If the sample concentration is too high, dilute with sample diluent to make the sample meet the detection range of the kit. 1. Add sample: set blank hole, standard hole and sample hole to be tested respectively. Add 100ul of sample diluent to the blank well, and 100ul of the standard or the sample to be tested in the remaining well. Be careful not to have air bubbles. Add the sample to the bottom of the well of the microtiter plate. The target plate is covered with a cover or film and reacted at 37 ° C for 120 minutes. To ensure the validity of the experimental results, please use a new standard solution for each experiment. 2. Discard the liquid and spin dry without washing. Add 100ul of detection solution A working solution to each well (take 1ul of detection solution A plus 99ul of detection dilution A to prepare, mix gently and prepare within one hour before use), 37 ℃, 60 minutes. 3. After incubating for 60 minutes, discard the liquid in the hole, spin dry, wash the plate 3 times, soak for 1-2 minutes each time, 350ul / per hole, spin dry (you can also pat the liquid in the hole to pat dry). 4. Add 100ul of testing solution B working fluid (same as testing A working fluid) to each well at 37 ℃ for 60 minutes. 5. After incubating for 60 minutes, discard the liquid in the hole, spin dry, wash the plate 5 times, soak for 1-2 minutes each time, 350ul / per hole, spin dry (you can also pat the liquid in the hole to pat dry). 6. Add 90ul of substrate solution to each well in sequence, and develop color at 37 ° C in the dark (within 30 minutes, at this time, the first 3-4 wells of the standard product have a visible blue gradient, and the latter 3-4 wells have no obvious gradient) , You can terminate). 7. Add 50ul of stop solution to each well in sequence to stop the reaction (in this case, the blue color turns to yellow). The order of adding the stop solution should be the same as that of the substrate solution. In order to ensure the accuracy of the experimental results, the termination solution should be added as soon as possible after the substrate reaction time expires. 8. Measure the optical density (OD value) of each well in sequence using an enzyme-linked instrument at a wavelength of 450 nm. Test within 15 minutes after adding stop solution. Note: 1. Leave one hole for each experiment as a blank zero-adjusting hole (different from the blank hole). No reagent is added to this hole, only the substrate solution and 2NH2SO4 are added at the end. Use this hole to adjust the OD value to zero when measuring. 2. Incubate in strict accordance with the prescribed time and temperature to ensure accurate results. All reagents must reach room temperature before use. Refrigerate the reagent immediately after use. 3. Incorrect plate washing can lead to inaccurate results. Before adding test solution B or substrate, make sure to absorb the liquid in the well as much as possible. Do not let the microwells dry out during incubation. In order to prevent the sample from evaporating, place the reaction plate in a closed box covered with a damp cloth during the test. The microplate is covered with a cover or film. After the plate is washed and dried, please add the next step reagent immediately. Avoid drying the micropores. 4. Eliminate the residual liquid and fingerprints on the bottom of the board, otherwise it will affect the OD value. 5. Substrate coloring liquid should be colorless or very light color. Substrate liquid that has turned blue cannot be used. 6. Avoid direct light exposure during storage and incubation. 7. Store unused microplates or reagents at 2-8 ° C. Standard products, working solution A, and working solution B should be configured and used according to the required amount. Do not reuse the diluted standard, test solution A working solution or test solution B working solution. 8. It is recommended to set a double-hole test when testing samples to ensure the accuracy of the test results. Plate washing method Manual plate washing method: absorb (not to touch the wall) or shake off the liquid in the enzyme plate; place a few layers of water on the experimental table Paper, with the microtiter plate down and vigorously pat several times; inject at least 0.3ml of the recommended wash buffer into the well and soak for 1-2 minutes, as needed Yes, repeat this process several times. Specificity The kit can detect recombinant or natural human Aβ1-40 at the same time, and has no cross-reactivity with other related proteins. Calculation Taking the concentration of the standard as the abscissa (logarithmic coordinate) and the OD value as the ordinate (ordinary coordinate), draw a standard curve on semi-logarithmic coordinate paper, and find the corresponding concentration from the standard curve according to the OD value of the sample; Multiply by the dilution factor; or use the standard concentration and OD value to calculate the linear regression equation of the standard curve, substitute the OD value of the sample into the equation, calculate the sample concentration, and multiply by the dilution factor to obtain the actual concentration of the sample. Precautions 1. The washing process is very important, inadequate washing is easy to cause false positives. 2. It is best to control the sampling time within 5 minutes. If there are many specimens, it is recommended to use a volley gun to add samples. 3. Please make a standard curve at the same time of each measurement, it is best to make a complex hole. 4. If the content of the substance to be tested in the specimen is too high, please dilute it and then determine it. When calculating, please multiply by the dilution factor. 5. When preparing standard products and testing solution working fluid, please prepare with corresponding diluent, not to be confused. 6. Please keep the substrate away from light. examination range: 4.7pg / ml-300pg / ml Minimum detection limit: 2.4pg / ml Explanation 1. Avoid exposing the reagent to strong light during storage and incubation. All reagent bottle caps must be tightly closed to prevent evaporation and contamination. Reagents should be protected from microbial contamination, because the interference of proteolytic enzymes will lead to erroneous results. 2. Aspirate the reagents carefully and strictly observe the given incubation time and temperature. Please note that when drawing samples / standards, enzyme conjugates or substrates, if the time interval between the first well and the last well is too large, it will result in different "pre-incubation" time, which obviously Affect the accuracy and repeatability of the measured value. Moreover, insufficient washing will affect the test results. 3. Storage of the kit: -20 ℃ (only for some reagents when not in use for a long time, see the label for details); 2-8 ℃ (when used frequently). 4. Validity: 6 months 5. Salt will be precipitated from the concentrated washing liquid, which can be heated and dissolved in the water bath when diluted. 6. The newly opened enzyme-linked plate well may contain a little water-like substance. This is normal and will not have any impact on the experimental results. 7. There may be inconsistencies between the Chinese and English instructions. Please refer to the English instructions. 8. All samples should be managed, and the samples and testing devices should be processed according to the prescribed procedures. Gemstone Jewelry,Gemstone Set Jewelry,Semi Precious Stone Set Jewelry,Crystal Women Jewelry JOYA GIFT CO.,LIMITED , http://www.joyagifts.com
Automatic plate washing: If there is an automatic plate washing machine, it should be used in the formal experiment process after being used skillfully.