Elisa Kit Test Requirements

1. Make a good comparison

In the formal test, the test conditions should be controlled with positive control and negative control respectively, and the samples to be tested should be made in duplicate to ensure the accuracy of the experimental results. Sometimes the background is high, indicating a non-specific reaction, which can be blocked with goat serum, rabbit serum or BSA.

2. Selection of Experimental Conditions

In ELISA, it is important to choose various experimental conditions, including:

(1) Selection of solid phase carrier: Many substances can be used as solid phase carrier, such as polyvinyl chloride, polystyrene, polyacrylamide and cellulose. It can be in the form of well plates, test tubes, beads, etc. Commonly used are 40-well polystyrene concave well plates. Regardless of the carrier, it can be screened before use: it is coated with the same amount of antigen, and the reaction is carried out under the same experimental conditions to observe whether the color reaction is uniform, and thus determine whether the adsorption performance is good.

(2) Selection of coated antibody (or antigen): When the antibody (or antigen) is adsorbed on the surface of the solid phase carrier, the purity is required to be good, and the pH is generally required to be between 9.0 and 9.6 during adsorption. The adsorption temperature, time and protein amount also have a certain influence, generally 4 ℃ for 18 to 24 hours. The optimal concentration of protein coating needs to be titrated: after coating with different protein concentrations (0.1, 1.0 and 10 μg/ml, etc.), and other test conditions are the same, observe the OD value of positive samples. Choose the concentration with the highest OD value and the least amount of protein. Typically 1-10 μg/ml for most proteins.

(3) Selection of the working concentration of the enzyme-labeled antibody: First, titrate the initial titer by the direct ELISA method (see the enzyme-labeled antibody section). Then fix other conditions or adopt the "square matrix method" (the coating material, the reference substance of the sample to be tested and the enzyme-labeled antibody are at different dilutions) to accurately titrate the working concentration in the formal experimental system.

(4) Selection of enzyme substrates and hydrogen donors: The selection of hydrogen donors requires cheap, safe, and obvious color reaction, but itself is colorless. Some hydrogen donors (such as OPD, etc.) have potential carcinogenic effects and should be protected. Those with conditions should use non-carcinogenic and high-sensitivity hydrogen donors, such as TMB and ABTS, which are relatively satisfactory hydrogen donors. After the substrate acts for a period of time, strong acid or strong base should be added to stop the reaction. Usually the substrate action time is 10-30 minutes. The substrate solution must be freshly prepared, especially H2O2 should be added immediately before use.

3. Import packaging:

The detection range and sensitivity are different. When the dosage is small, sub-packaging can be used. In the early stage, its durability is not very good.

The OD value of the highest point of the standard curve of the kit was all 2.0±0.2, and the OD value of the zero well was controlled below 0.10.

The standard curve correlation coefficient R≥0.98 of the kit.

The reproducibility of the kit is good, and the coefficient of variation within and between plates is <9 %.

The components of the reagents are all given 20% surplus to ensure that the detection of 48/96 wells is completed and to avoid the situation that the amount of reagents may be insufficient in the fractional detection.

The dilution of the detection antibody and enzyme conjugate is appropriate (both 1:30), which is convenient for the test operator to do the dilution work accurately and ensures the repeatability of the test results.

Interleukin-6 is a cytokine that has been shown to be a B-cell differentiation factor. It is an antibody-forming system, and its physiological properties, such as induction of acute protein synthesis in hepatocytes and growth stimulation based on synergy with interleukin-3, It has also attracted much attention. And it has been confirmed that there is a stable correlation between interleukin-6 and pathology. For example, it is reported that the growth factor of various diseases is interleukin-6, and myeloma cells can synthesize interleukin-6 and express interleukin-6 receptors. In addition, interleukin-6 plays an important role in a variety of inflammatory diseases and autoimmune diseases. This kit can detect interleukin-6 in mouse serum and cell matrix supernatant with high sensitivity. Principle This kit uses two specific antibodies, After washing the plate, add TMB substrate solution to develop color, the intensity of color development is proportional to the amount of interleukin-6 in mice. Detection range 10.94 700 pg/mL Expected use.