Mycoplasmal enzyme-linked immunosorbent assay ( ELISA )
Kit instruction manual
This reagent is for research use only   Purpose: This kit is used to detect Mycoplasmal levels in hamster ovary cells.
Experimental principle :
This kit uses double antibody sandwich enzyme-linked immunosorbent assay (ELISA) to determine Mycoplasmal in specimens. Mycoplasma with purified hamster (Mycoplasmal) antibody-coated microtiter plate, made solid-phase antibody, can mycoplasma (Mycoplasmal) combining the sample, after washing to remove unbound antibodies and other components, and then HRP labeled M. ( The Mycoplasmal antibody binds to form an antibody-antigen-enzyme-labeled antibody 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 Mycoplasmal in the specimen.
Kit composition :
Kit composition | 48 hole configuration | 96-well configuration | save |
Instruction manual | 1 copy | 1 copy | |
Sealing film | 2 pieces (48) | 2 pieces (96) | |
sealed bag | 1 | 1 | |
Enzyme label coated plate | 1×48 | 1×96 | Store at 2-8 ° C |
Negative control | 0.5ml × 1 bottle | 0.5ml × 1 bottle | Store at 2-8 ° C |
Positive control | 0.5ml × 1 bottle | 0.5ml × 1 bottle | Store at 2-8 ° C |
Enzyme standard reagent | 3 ml × 1 bottle | 6 ml × 1 bottle | Store at 2-8 ° C |
Sample diluent | 3 ml × 1 bottle | 6 ml × 1 bottle | Store at 2-8 ° C |
Developer A solution | 3 ml × 1 bottle | 6 ml × 1 bottle | Store at 2-8 ° C |
Developer B solution | 3 ml × 1 bottle | 6 ml × 1 bottle | Store at 2-8 ° C |
Stop solution | 3ml × 1 bottle | 6ml × 1 bottle | Store at 2-8 ° C |
Concentrated washing solution | (20ml × 20 times) × 1 bottle | (20ml × 30 times) × 1 bottle | Store at 2-8 ° C |
Sample processing and requirements :
1. Serum: The blood is naturally coagulated for 10-20 minutes at room temperature and centrifuged for about 20 minutes (2000-3000 rpm). 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 have 2 holes for negative control, 2 holes for positive control and 1 well for blank control (no blank sample and enzyme standard reagent for blank control well, the other steps are the same)
2. Loading: 50 μl of negative control and positive control were added to the negative and positive control wells, respectively. Then, add 40 μl of the sample dilution 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, and shake it gently.
3. Incubation: The plate was sealed with a sealing film and incubated at 37 ° C for 30 minutes.
4. Liquor: 30 (48 times of 20T) concentrated washing solution plus distilled water to 600ml for use
5. Washing: Carefully remove the sealing film, discard the liquid, dry it, fill each well with the washing solution, let stand for 30 seconds, then discard it, 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: The operation is the same as 3.
8. Wash: Operate 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, and color at 37 ° C for 15 minutes.
10. Termination: 50 μl of stop solution was added to each well to stop the reaction (the blue color turned yellow).
11. Measurement: The absorbance (OD value) of each well was measured in sequence 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.
The result is judged:
Test validity: mean value of positive control well ≥ 1.00; mean value of negative control ≤ 0.10
CUT OFF calculation: critical value = negative control well average + 0.15
Negative judgment: sample OD value < critical value (CUT OFF) is negative for Mycoplasmal
Positive judgment: sample OD value ≥ critical value (CUT OFF) is Mycoplasmal positive
Precautions
1. The operation is carried out in strict accordance with the instructions. The components of the different batches of this reagent shall not be mixed.
2. The kit should be taken out from the refrigerated environment and should be used at room temperature for 15-30 minutes before use. If the enzyme label is unsealed after opening, the slats should be stored in a sealed bag.
3. Concentrated washing liquid may crystallize out. When diluted, it can be heated and dissolved in a water bath. The washing will not affect the result.
4. The sealing film is intended for single use only to avoid cross-contamination.
5. Please keep the substrate away from light.
6. The test results must be determined by the microplate reader. When using dual-wavelength detection, the reference wavelength is 630nm.
7. All samples, washings and various wastes should be treated as infectious materials. The stop solution is 2M sulfuric acid and must be used safely.
Storage conditions and expiration date
1. The kit is stored at: 2-8 °C.
2. Validity: 6 months
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