How to learn the Elisa experiment

The basis of Elisa is the binding of antigens or antibodies and the enzyme markers of antigens or antibodies.
The antigens or antibodies that bind to the surface of the solid carrier remain immunological activity, and the antigen or antibody that is labeled with the enzyme retains its immunological activity and retains the activity of the enzyme.
By adding the substrate after the reaction of the enzyme, the content of the material in the sample can be determined according to the depth of the color, and the qualitative or quantitative analysis is conducted.

Due to the high catalytic efficiency of enzyme, the result of immune reaction is amplified and the measurement method is highly sensitive.

• Step 1: determine the experimental method

1. Direct ELISA
The antigen is directly fixed on the solid carrier, and the first class antibody of the enzyme is added to determine the total antigen. The specificity of this antibody is very important.
Advantages: brief operation procedures, because no need to use two reactance to avoid interaction.
Disadvantages: an enzyme is used in the test, but not every antibody is suitable for marking, and the cost is relatively high.

2. indirect ELISA
This method is similar to the direct method. The difference is that the primary antibody has no enzyme marker, and the secondary antibody labeled by the enzyme is used to identify the primary antibody to determine the antigen volume.
Advantages: the two reactance can enhance the signal, and there are various options to make different analysis.
Non-enzymatic primary antibodies retain the most immune response.
Disadvantages: high probability of interaction.

3. sandwich ELISA
The tested antigen is between two antibodies, one of which is fixed to the solid carrier, which is to catch the antibody.
Another is the detection of antibodies, which can be used to determine the amount of antigen directly after the enzyme is labeled.
Or unmarked, the amount of antigen is determined by the secondary antibody labeled by the enzyme.
These two antibodies must be carefully selected to avoid interaction or competing antigen-binding sites.
Advantages: high sensitivity, high specificity, antigens need not be purified beforehand.
Disadvantages: antigen must have more than two antibody binding site.

4.competitive ELISA
The antigen in the sample (free) antigen and the purification and fixed on the solid phase carrier antigen (fixed antigen) competition with the same antibody, when the free antigen in the sample, the more you can combine the antibodies, and fixed antigen can combined into fewer antibodies, and vice versa.
After cleaning steps, wash free antigen and antibody of complex, leaving only a fixed antigen and antibody compounds, compared with only fixed antigen, in comparison with the control group the results of the samples can be calculated according to the color difference in the content of antigen.
Advantages: can be applicable to less pure samples, and the data reproducibility is very high.
Disadvantages: overall sensitivity and specificity are poor.

Our most commonly used method is double antibody sandwich method, so let's take this as an example.

• Step 2: experiment

Double antibody sandwich method to detect unknown antigen:

1. The package is: use a 0.05 M PH9 牰 carbonate package is buffer antibodies will be diluted to the protein content of 1 ~ 10 mu g/ml.
In each add 0.1 ml, the reaction of polystyrene board hole 4 ℃ for the night.
Next day, discard the solution in the hole and wash the buffer three times, three minutes each time.
(wash, same as).

2. Add: add certain diluted influenza virus samples of 0.1 ml in the above has been the response of pore package, incubated with the 37 ℃ for 1 hour.
Then wash.
(blank hole, negative control hole and positive control hole at the same time).

3. Enzymatic antibody: in each reaction hole, add the fresh diluted enzyme antibody (after titration) 0.1 ml.
37 ℃ 0.5 ~ 1 h incubation and washing.

4. Add chromogenic substrate liquid: to add temporary reaction hole configuration of TMB substrate solution 0.1 ml, 37 ℃ for 10 ~ 30 minutes.

5. Termination reaction: add 2M sulfuric acid 0.05 ml in each reaction hole.

6. Results: on a white background, direct observation with the naked eye: hole the deeper the color reaction, the stronger the degree of positive, negative reaction to colorless or very shallow, on the basis of assumes the color depth, expressed as a "+" and "-".
Also measurable value: o. D on ELISA detector, at 450 nm (if to ABTS color, the 410 - nm), bore to ck after zero measuring the o. D value, if is greater than the negative control stipulated by the OD value of 2.1 times, is positive.

Detection of unknown antibodies by double-antibody sandwich:

The response pattern is similar to the dual antibody sandwich method.
The specific antigen is used to bind and prepare the enzyme binding to detect the corresponding antibody.
In contrast to the indirect method, the enzyme antigen is substituted for the enzyme antibody.
In this method, the specimen is not diluted and can be directly used for determination, so its sensitivity is relatively higher than the indirect method.
This law is often used in anti-hbs detection in hepatitis b markers.
The key of this law is the preparation of enzyme antigen, which should be found according to the different antigen structure.

• Step 3: experiment

1. When the test is formally tested, the test conditions should be controlled by the positive control and negative control, and the sample should be made in duplicate to ensure the accuracy of the experimental results.
Sometimes the bottom is higher, indicating that there is a non-specific reaction, which can be closed with sheep serum, rabbit serum or BSA.

2. In ELISA, the selection of experimental conditions is important, including:
(1) selection of solid phase vectors: many substances can be used as solid carriers.
At present, 40 holes polystyrene concave plate is commonly used.
No matter what kind of carrier, can be filtered before use: use the same antigen package is, in the same reaction under experimental conditions, the color reaction was observed whether uniformity, thus determine its adsorption performance is good.

(2) selection of antibodies (or antigens) : when the antibody (or antigen) is adsorbed on the surface of the solid carrier, the purity is better and the PH is generally required between 9.0 and 9.6.
Adsorption temperature, time and the amount of protein also has a certain influence, more commonly used 4 ℃ 18 ~ 24 hours.
Package was the optimal concentration of protein: require titration with different protein concentration (0.1, 1.0, and 10 mu g/ml) after the package is in the other test conditions at the same time, observe the OD value of the positive samples.
The concentration of the maximum protein and minimum protein concentration is selected.
For most proteins, it is usually 1 ~ 10 mu g/ml.

(3) selection of the concentration of enzyme marker antibody: the initial titration of the initial titration with direct ELISA method (see an enzyme marker antibody part).
Then fixed conditions or take other "phalanx method" (package is, waiting for inspection reference sample product and enzyme labelled antibody respectively different dilution degrees) titration in formal experimental system accurately the working concentration.

(4) the substrate of the enzyme and the choice of hydrogen donor: the choice of hydrogen donor is low price, safe, obvious color reaction, and colorless itself.
The author should use non-carcinogenic, highly sensitive hydrogen donor, such as TMB and ABTS, which are currently satisfactory for hydrogen supply.
After a period of time, a strong acid or alkali should be added to terminate the reaction.
Usually the substrate is used for 10-30 minutes.
The liquid must be made fresh, especially if H2O2 is added before use.

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