Sheep Bluetongue Antibody (BLU Ab) ELISA Kit Instructions

Sheep Bluetongue Antibody (BLU Ab) Enzyme-Linked Immunosorbent Assay (ELISA) Kit Instruction Manual This reagent is for research use only. This kit is used to detect serum levels of blue tongue disease (BLU Ab) in sheep serum. Experimental principle: This kit uses double antigen sandwich enzyme-linked immunosorbent assay (ELISA) to determine the amyloidosis antibody (BLU Ab) in the specimen. The microporous plate is coated with purified amphioxus antigen to prepare a solid phase antigen, which can be combined with blue tongue disease antibody (BLU Ab) in the sample, washed to remove unbound antigen and other components and then labeled with HRP. The bluetongue antigen combines to form an antigen-antibody-enzyme-labeled antigen complex, which is 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 absorbance (OD value) was measured by a microplate reader at a wavelength of 450 nm, and compared with the CUTOFF value to determine the presence or absence of the amniotic blue tongue antibody (BLU Ab) in the specimen. Kit composition: Kit composition 48-well configuration 96-well configuration Storage instructions 1 part 1 part sealing film 2 pieces (48) 2 pieces (96) Sealed bag 1 1 enzyme label coated plate 1 × 48 1 × 96 2 -8 °C preservation negative control 0.5ml × 1 bottle 0.5ml × 1 bottle 2-8 ° C Preservation positive control 0.5ml × 1 bottle 0.5ml × 1 bottle 2-8 ° C Preservation enzyme standard reagent 3 ml × 1 bottle 6 ml × 1 Bottle 2-8 °C Preservation Sample Diluent 3 ml × 1 bottle 6 ml × 1 bottle 2-8 ° C Preservation developer A solution 3 ml × 1 bottle 6 ml × 1 bottle 2-8 ° C Preservation developer B solution 3 Ml × 1 bottle 6 ml × 1 bottle 2-8 ° C Preservation solution 3 ml × 1 bottle 6 ml × 1 bottle 2-8 ° C Preservation concentrated washing solution (20ml × 20 times) × 1 bottle (20ml × 30 times) × 1 bottle 2-8 ° C preservation sample processing and requirements: 1. Serum: room temperature blood solidification for 10-20 minutes, centrifugation for about 20 minutes (2000-3000 rev / min). The supernatant is carefully collected, and if precipitation occurs during storage, it should be centrifuged again. 2. Plasma: EDTA or sodium citrate should be selected as an anticoagulant according to the requirements of the specimen. After mixing for 10-20 minutes, centrifuge for about 20 minutes (2000-3000 rpm). The supernatant is carefully collected, and if a precipitate forms during storage, it should be centrifuged again. 3. Urine: Collect with a sterile tube and centrifuge for about 20 minutes (2000-3000 rpm). The supernatant is carefully collected, and if a precipitate forms during storage, it should be centrifuged again. The chest and ascites and cerebrospinal fluid are referred to. 4. Cell culture supernatant: When detecting secreted components, collect them in a sterile tube. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. When the components in the cells were detected, the cell suspension was diluted with PBS (pH 7.2-7.4), and the cell concentration reached about 1 million/ml. By 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. Tissue specimen: After cutting the specimen, weigh the weight. Add a certain amount of PBS, pH 7.4. It was quickly frozen and stored in liquid nitrogen for use. The specimen still maintains a temperature of 2-8 ° C after melting. A certain amount of PBS (pH 7.4) was added, and the specimen was homogenized by hand or homogenizer. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. One part of the package is to be tested, and the rest is frozen for use. 6. The specimens should be extracted as soon as possible after collection, and the extraction should be carried out according to the 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 horseradish peroxidase (HRP) activity. Operation steps: 1. No.: The corresponding micropores of the sample are numbered sequentially. Each plate should be set with 2 holes of negative control, 2 wells of positive control and 1 well of blank control (the blank control well is not added with sample and enzyme standard reagent, and the other steps are operated. The same) 2. Loading: Negative control and positive control 50 μl were added to the negative and positive control wells, respectively. Then, add 40 μl of the sample diluent to the sample well to be tested, and then add 10 μl of the sample to be tested. Add the sample to the bottom of the well of the microplate. Try not to touch the wall of the well. Gently shake and mix. 3. Incubate: Cover with a sealing plate and incubate at 37 °C for 30 minutes. 4. Solution: Add 30 (48 times of 20T) concentrated washing solution to distilled water to 600ml and then use it. 5. Wash: carefully remove the sealing film, discard the liquid, dry, fill each well with washing liquid, and let stand. Discard after 30 seconds, repeat 5 times, and pat dry. 6. Add enzyme: Add 50 μl 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: Add 50 μl of developer A to each well, then add 50 μl of developer B, gently shake and mix, avoid at 37 °C Color development for 15 minutes 10. Termination: 50 μl of stop solution was added to each well to terminate the reaction (when the blue color turned yellow). 11. Measurement: The absorbance (OD value) of each well was measured sequentially with a blank air conditioner of zero and a wavelength of 450 nm. The measurement should be carried out within 15 minutes after the addition of the stop solution. Outcome determination: Test validity: mean value of positive control well ≥ 1.00; mean value of negative control ≤ 0.10 Critical value (CUT OFF) calculation: critical value = average value of negative control well + 0.15 Negative judgment: OD value of sample

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