Mycoplasma hyopneumoniae (MP) ELISA detection kit

This reagent kit can only be used for scientific research and cannot be used for medical diagnosis

pig（Porcine）Mycoplasma pneumoniae（MP）ELISA detection kit

Instruction Manual

Detection Principle

The reagent kit adopts a double antibody one-step sandwich enzyme-linked immunosorbent assay（ELISA）。Pre packagedbyMycoplasma pneumoniae（MP）The antibody is wrapped in micropores and sequentially added to the specimen, standardHRP labeled detection antibodies were incubated and washed. Using substrate TMB for color development, TMB is converted to blue under the catalysis of peroxidase and to the final yellow under the action of acid. The depth of color and the sampleMycoplasma pneumoniae（MP）submitPositive correlation. Using an enzyme-linked immunosorbent assay (ELISA) readerMeasure the absorbance (OD value) at a wavelength of 450nm and calculate the sample concentration.

Sample collection, processing, and preservation methods

1. Serum: Use test tubes free of pyrogens and endotoxins, avoid any cell irritation during the operation, collect blood, centrifuge at 3000 rpm for 10 minutes, and quickly and carefully separate serum and red blood cells.

2. Plasma: anticoagulant with EDTA, citrate or heparin. Centrifuge at 3000 rpm for 30 minutes and collect the supernatant.

3. Cell supernatant: Centrifuge at 3000 rpm for 10 minutes to remove particles and polymers.

4. Tissue homogenate: Crush the tissue by adding an appropriate amount of physiological saline. Centrifuge at 3000 rpm for 10 minutes and collect the supernatant.

5. Storage: If the sample is not tested in a timely manner after collection, please divide it into batches according to a single dose, freeze it at -20 ℃, avoid repeated freezing and thawing, thaw it at room temperature, and ensure that the sample is evenly filled and thawed.

Personal belongings

1. ELISA reader（450nm）

2. High precision sampler and gun head:0.5-10uL、2-20uL、20-200uL、200-1000uL

3. 37 ℃ constant temperature box

Operation precautions

1. The reagent kit is stored in2-8 ℃, equilibrate at room temperature for 20 minutes before use. The concentrated washing solution taken out of the refrigerator may crystallize, which is a normal phenomenon. Heating in a water bath should dissolve the crystals before use.

2. The Flat noodles not used in the experiment should be immediately put back into the self sealing bag, sealed (low-temperature drying) and stored.

3. The concentration isThe S0 standard sample with 0 can be considered as a negative control or blank; When operating according to the instructions, the sample has already been diluted 5 times, and the actual concentration of the sample is obtained by multiplying the final result by 5.

4. Strictly follow the time, liquid dosage, and sequence indicated in the instructions for incubation operation.

5. Shake all liquid components thoroughly before use.

Kit components

Note: Standard product（S0-S5) Concentrationin turnFor:0、1.25、2.5、5、10、20 ng/mL

Preparation of reagents

20 x dilution of washing buffer: Dilute distilled water at a ratio of 1:20, which means adding 19 parts of distilled water to 1 part of 20 x washing buffer.

Washing method

1. Hand washed board: Shake off the liquid in the holes, fill each hole with detergent, and let it standAfter 1 minute, shake off the liquid in the hole and pat dry on absorbent paper. Wash the plate 5 times in this way.

2. Automatic washing machine: Inject washing solution into each hole350μL， Soak for 1 minute and wash the board 5 times.

Mycoplasma hyopneumoniae (MP) ELISA detection kitoperating steps

1. Balance from room temperatureAfter 20min, take out the required Flat noodles from the aluminum foil bag, and seal the remaining Flat noodles with a self sealing bag and put it back at 4 ℃.

2. Set standard and sample wellsAdd standard samples of different concentrations to each standard well50μL；

3. Add sample well firsttest sample10μL， plusSample diluent40μL；Blank hole not added

4. Except for blank holes,Add horseradish peroxidase to each well of the standard and sample wells（100 μ L of HRP labeled detection antibody was used to seal the reaction well with a sealing plate membrane, and incubated at 37 ℃ in a water bath or constant temperature incubator for 60 minutes.

5. Discard the liquid, pat dry on absorbent paper, fill each hole with detergent, and let it stand1min， Discard the detergent, pat dry on absorbent paper, and repeat washing the board 5 times (or use a board washing machine).

6. Add substrate to each wellA. Incubate 50 μ L each at 37 ℃ in the dark for 15 minutes.

7. Add termination solution to each wellMeasure the OD values of each well at a wavelength of 450nm within 15 minutes using 50 μ L.

result judgment

Draw standard curve: inIn the Excel worksheet, use the standard concentration as the horizontal axis and the corresponding OD value as the vertical axis to draw a linear regression curve of the standard. Calculate the concentration values of each sample according to the curve equation.

Mycoplasma hyopneumoniae (MP) ELISA detection kitTest kit performance

1. Accuracy: Correlation coefficient between standard linear regression and expected concentrationR value, greater than or equal to 0.9900.

2. Sensitivity: The maximum detection concentration is less than0.1 ng/mL.

3. Specificity: Does not cross react with other soluble structural analogues.

4. Repeatability: The coefficient of variation within and between boards is less than15%.

5. Storage:Store at 2-8 ℃, away from light and moisture.

6. Validity period:6 months

Disclaimers

1. The reagent kit is for research use only and should not be used for clinical trials ormousePhysical experiments, otherwise all consequences arising shall be borne by the experimenter, and our company shall not be responsible.

2. Strictly follow the instructions for operation. If the experimenter violates the instructions, the consequences shall be borne by the experimenter.