Instructions for Human Bullous Pemphigoid Antibody (BP) ELISA Kit

Human Bullous Pemphigoid Antibody (BP) ELISA Kit Instructions This reagent is for research use only: This kit is used to determine the level of bullous pemphigoid antibody (BP) in human serum, plasma and related liquid samples . Experimental principle: This kit uses double antigen sandwich enzyme-linked immunoassay (ELISA) to determine the human bullous pemphigoid antibody (BP) in the specimen. Microporous plates are coated with purified human bullous pemphigoid antibody (BP) antigen to make a solid phase antigen, which can be combined with bullous pemphigoid antibody (BP) in the sample and washed to remove unbound The antibodies and other components are then combined with HRP-labeled bullous pemphigoid antibody (BP) antigen to form an antigen-antibody-enzyme-labeled antigen 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 absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm and compared with the CUTOFF value to determine the presence or absence of human bullous pemphigoid antibody (BP) in the specimen. 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 negative control 0.5ml × 1 bottle 0.5ml × 1 bottle at -8 ℃ Store positive control 0.5ml × 1 bottle 0.5ml × 1 bottle at 2-8 ℃ Store enzyme reagent 3 ml × 1 bottle 6 ml × 1 at 2-8 ℃ Store the sample diluent at 2-8 ° C 3 ml × 1 bottle 6 ml × 1 bottle Store the developer A at 2-8 ° C 3 ml × 1 bottle 6 ml × 1 bottle Store the developer B at 2-8 ° C 3 ml × 1 bottle 6 ml × 1 bottle 2-8 ° C storage stop solution 3ml × 1 bottle 6ml × 1 bottle 2-8 ° C storage concentrated washing solution (20ml × 20 times) × 1 bottle (20ml × 30 times) × 1 bottle Sample handling and requirements for storage at 2-8 ° 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). Operation steps: Numbering: Number the samples corresponding to the microwells in sequence, each plate should be set with 2 wells for negative control, 2 wells for positive control, and 1 well for blank control (the blank control well does not add sample and enzyme reagents, the rest of the operations are the same) Add sample: add 50μl of negative control and positive control to the negative and positive control wells respectively. Then add 40μl of sample diluent to the sample well, and then add 10μl of the sample to be tested. 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, shake gently to mix, and incubate: seal the plate with the sealing film and incubate at 37 ℃ for 30 minutes. Mixing solution: Add 30 times (20 times of 48T) concentrated washing liquid to distilled water to 600ml, and then stand by washing: carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing liquid, let it stand for 30 seconds and then discard Go, repeat this 5 times, pat dry. Enzyme addition: Add 50μl of enzyme label reagent to each well, except for blank wells. Incubation: The operation is the same as 3. Washing: The operation is the same as 5. Color development: add 50μl of developer A to each well, and then add 50μl of developer B, mix gently and stop at 37 ° C for 15 minutes. Stop the reaction by adding 50μl of stopper solution to each well Color stand to yellow). Measurement: The absorbance (OD value) of each well was measured in sequence with the blank air conditioner at zero and 450 nm wavelength. The measurement should be carried out within 15 minutes after adding the stop solution. Judgment of results: Test validity: average of positive control wells ≥1.00; average of negative control wells ≤0.10 cut-off value (CUT OFF) calculation: cut-off value = average of negative control wells +0.15 negative judgment: OD value of samples
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Manual of human bullous pemphigoid antibody (BP) ELISA kit.zip
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