Plastic Cutting Board,Marble Cutting Board,Personalised Chopping Board,Olive Cutting Board Yangjiang Yado Kitchen Industry Co., Ltd. , https://www.yadokitchen.com
purpose of usage:
Experimental principle
Kit composition
Specimen requirements
Steps
Calculation and result judgment:
Precautions
Storage conditions and validity period
Xiamen Huijia Biotechnology Co., Ltd. is a professional agent for many well-known brands in the international life science field. Committed to the sales and promotion of various ELISA kits, immunohistochemical kits, primary and secondary antibodies, cytokines, biological reagents, pipettes, and consumables.
Instruction manual of Mycoplasma bovis ELISA kit
Instruction manual of Mycoplasma bovis ELISA kit
This kit is for research use only.
48T
This kit is used to determine the expression of M. bovis in bovine serum, plasma and related liquid samples.
This kit uses the double antibody sandwich method to determine the expression of M. bovis in the specimen. Coat the microplate with purified M. bovis antibody to make a solid-phase antibody, which can be combined with M. bovis in the sample, washed to remove unbound antigen and other components, and then combined with HRP-labeled M. bovis to form an antibody- Antigen-enzyme labeled antibody complex, after thorough washing, add substrate TMB to develop color. 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 M. bovis in the specimen.
1
20 times concentrated washing liquid
20ml × 1 bottle
7
Stop solution
3ml × 1 bottle
2
Enzyme reagent
3ml × 1 bottle
8
Positive control
0.5ml × 1 bottle
3
Enzyme coated plate
12 holes × 4 strips
9
Negative control
0.5ml × 1 bottle
4
Sample diluent
3ml × 1 bottle
10
Instructions
1 serving
5
Developer A liquid
3ml × 1 bottle
11
Sealing film
2 sheets
6
Developer B liquid
3ml × 1 / bottle
12
sealed bag
1
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 the test cannot be performed 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.
1. Numbering: serially number the corresponding microwells of the sample, 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 reagent, the rest of the steps are the same)
2. Add sample: add negative control and positive control 50μl 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 to the bottom of the well of the microplate, try not to touch the wall of the well, gently shake to mix,
3. Incubation: Seal the plate with a sealing plate and incubate at 37 ° C for 30 minutes.
4. Mixing solution: dilute 20 times concentrated washing liquid with distilled water 20 times and reserve
5. Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with the washing liquid, let it stand for 30 seconds and then discard, repeat this 5 times, 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, then add 50μl of developer B, mix gently, and develop at 37 ° C in the dark for 15 minutes.
10. Termination: Add 50μl of stop solution to each well to stop the reaction (at this time the blue will turn to yellow).
11. Determination: Measure the absorbance (OD value) of each well 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.
Test effectiveness: the average of positive control wells ≥1.00; the average of negative control wells ≤0.10
Calculation of critical value (CUT OFF): critical value = average value of negative control wells + 0.15
Negative judgment: the sample with OD value <critical value (CUT OFF) is negative for M. bovis
Positive judgment: the sample with OD value ≥ critical value (CUT OFF) is positive for Mycoplasma bovis
.
1. The operation is strictly in accordance with the instructions. The components of different batches of this reagent must not be mixed.
2. The kit should be equilibrated at room temperature for 15-30 minutes before being taken out of the refrigerated environment. If the enzyme label coated plate is unopened, the strip should be stored in a sealed bag.
3. Crystals may be precipitated in the concentrated washing liquid, which can be heated and dissolved in a water bath during dilution, and the results will not be affected during washing.
4. The sealing film is limited to one-time use to avoid cross-contamination.
5. Please keep the substrate away from light.
6. The test results must be determined based on the reading of the microplate reader. When using dual wavelength detection, the reference wavelength is 630nm
7. All samples, washing liquids and various wastes should be treated as infectious agents. The stop solution is 2M sulfuric acid, and you must pay attention to safety when using it.
1. Kit storage :; 2-8 ℃.
2. Validity: 6 months