Pig main histocompatibility complex II kit instruction manual

Pig main histocompatibility complex II kit instruction manual

This article is provided by: Qiao Yu biological professional and technical staff!

This kit is for research use only.

examination range:

48T 25 ng/L -800 ng/L

purpose of usage:

This kit is used to determine the content of major histocompatibility complex II (MHC-II) in pig serum, plasma and related liquid samples.

Experimental principle

The kit uses a double antibody sandwich method to determine the level of major histocompatibility complex II (MHC-II) in pigs. The microporous plate was coated with purified porcine major histocompatibility complex class II (MHC-II) antibody to prepare a solid phase antibody, and the major histocompatibility complex II was sequentially added to the micropores of the coated monoclonal antibody. (MHC-II), which binds to the HRP-labeled major histocompatibility complex class II (MHC-II) antibody 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 color depth is positively correlated with the major histocompatibility complex class II (MHC-II) in the sample. The absorbance (OD value) was measured by a microplate reader at a wavelength of 450 nm, and the concentration of the main histocompatibility complex II (MHC-II) in the sample was calculated from the standard curve.

Kit composition

Specimen requirements

1. 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.

2. Samples containing NaN3 could not be detected because NaN3 inhibited horseradish peroxidase (HRP) activity.

Steps

1. Dilution of standard: This kit provides one original standard, which can be diluted in a small tube according to the following chart.

2. Adding samples: blank holes are respectively set (the blank control wells are not added with the sample and the enzyme standard reagent, and the other steps are the same), the standard holes, and the sample holes to be tested. Accurately load 50 μl of the standard on the enzyme-labeled plate, add 40 μl of the sample dilution to the well to be tested, and then add 10 μl of the sample to be tested (the final dilution of the sample is 5 times). Add the sample to the bottom of the well of the microplate, try not to touch the wall of the well, and shake gently to mix.

3. Incubation: The plate was sealed with a sealing film and incubated at 37 ° C for 30 minutes.

4. Dosing: 20 times concentrated washing solution diluted with distilled water 20 times and used

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: 50 μl of enzyme labeling reagent was added to each well, except for blank wells.

7. Incubation: The operation is the same as 3.

8. Washing: The operation is the same as 5.

9. Color development: Add 50 μl of the developer to each well, then add 50 μl of the developer, 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 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.

Precautions:

1. 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.

2. 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.

3. The sampler should be used for each step, and the accuracy should be corrected frequently to avoid test errors. The loading time is controlled within 5 minutes. If the number of specimens is large, it is recommended to use a gun.

4. Please make a standard curve at the same time for each measurement and make a double hole. If the content of the substance to be tested in the specimen is too high (the OD value of the sample is larger than the OD value of the standard pore), please first dilute the sample dilution with a certain multiple (n times) and then measure it. When calculating, multiply the total dilution factor ( ×n×5).

5. The sealing film is intended for single use only to avoid cross-contamination.

6. Please keep the substrate away from light.

7. Strictly follow the instructions of the manual, the test results must be based on the microplate reader reading.

8. All samples, washings and various wastes should be treated as infectious materials.

9. The different batch components of this reagent must not be mixed.

10. In the case of an English manual, the English manual shall prevail.

Storage conditions and expiration date

1. The kit is stored at: 2-8 °C.

2. Validity: 6 months

Natural Protein

Proteins are large biomolecules, or macromolecules, consisting of one or more long chains of amino acid residues. Proteins perform a vast array of functions within living organisms, including DNA replication, catalyzing metabolic reactions, responding to stimuli, and transporting molecules from one location to another.