ELISA (Enzyme linked immunosorbent Assay) : Types, procedure, Result, application

Enzyme linked immunosorbent Assay

 

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We use this technique to identify antigen and antibody.

Technique performed for quantitative and qualitative analysis.

Quantitative: for identifying the quantity or concentration of antigen or antibody in ELISA.

Qualitative: for identifying any pathogen particle or antigen, it should be bacterial or viral present there and identify the type of antibody.

There are four types of ELISA 

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1. Direct ELISA

2. Indirect ELISA

3. Sandwich ELISA

4. Competitive ELISA

In this article we discuss , direct, indirect,and sandwich Elisa but the competitive Elisa will be discussed in my next article.

Direct ELISA : we said it direct ELISA because there is a direct interaction between enzyme linked antibody and antigen.

Credits: Soleil Nordic 

We perform this technique for identifying antigen.

Procedure: firstly I said that for identifying antigen we perform direct Elisa technique here we take blood from the patient then isolate the serum from the blood. Antigen molecules are present in the serum.

We use here PBS buffer as the page in ionic concentration of PBS buffer is same as blood so the shape of the antigen remain same.

Then we apply the antigen in microtiter plate in one well.

Picture : microtiter plate.

Antigen will attach to the bottom side of the well.

Next step is called surface blocking.

we use BSA protein which is non reactive it does not react on antigen or antibody.

When we pour blocking reagent then they will attach to the site where antigens are not binding or the free surface on the well.

The enzyme linked antibody can bind antigen and as well as a side or surface of well. Hence we use blocking reagent as the block the free sides.

After the first step we perform washing because some antigens are not purely attached to the surface they can even remain flow in the well.

We perform the washing technique after adding antibody.

In second step antibodies can link to different enzyme, for determining the enzyme type we use substrate on the specific enzyme.

Which usually use horseradish peroxide or alkaline phosphatase.

Specific substrate react only specific enzyme and give a a certain colour.

After providing substrate with get a colour by which we can easily identify the enzyme type.

just assume that if a person has some infection or some disease on the basis of symptoms take his blood and perform direct ELISA.

If the primary antibody on the basis of the suspected symptom attached then we can say the patient is positive and the symptom was accurate as suspected and if not then the suspension is wrong

Indirect ELISA: Here direct interaction between enzyme linked antibody and antigen does not occur.

A primary antibody is found between antigen and secondary antibody. So we call it indirect ELISA.

We perform indirect Elisa for detection of antibody. 

now as you that if a person has some infection cause some disease like malaria we did not know properly.

On the basis of the symptoms we think that it should be malaria but data is not confirmed.

 

In this case we take blood from the patient and then isolate the serum,  there will some antibody present against the antigen of the infection. If the antibody get attach to the specific antigen then you will sure that the patient will positive if not then negative.

Step of indirect ELISA :

Can we perform microtiter plate which is sealed with 96 Wells. used and design attach to Wells with BSA as suspected disease. 

Then we pour the patient's antibody , if the antibody is specific on the given antigen and then antibody will bind.

If not mind then we see only say that the patient does not have any such disease that was suspected. 

The patient antibody is a primary antibody. We use secondary antibody with enzyme linked which can bind to primary antibody. after using substrate it will give colour on the basic of the enzyme linked secondary antibody then we can analyse the bacterial or viral strain present in the patient serum.

Sandwich ELISA : this technique is 2 to 5 times sensitive than direct ELISA. 

Sandwich Elisa typically required the use of matched antibody pairs, where is antibody is specific for a different, non overlapping part which is Epitope of the antigen molecule. first antibody known as capture antibody is coated to the Wells . The sample solution is then added to the Wells. A second antibody known as detection antibody follows in the step in order to the measure concentration of the sample.

Used for antigen detection.

The process is same as before, we use microtiter plate to perform this technique.

Coat capture antibody to the Wells and attach to the bottom side of the microtiter. 

In the second step we use blocking agent.

Third step we add the sample. 1 2 and 3 steps are performed to within washing as required by the buffer solution.

The protein of interest in mind to the capture antibody then again we perform washing technique as some antigens does not attach with the capture and they randomly floats.

Epitope: specific type of protein that can be found on the pathogen surface called epitope.

In the fourth step we perform secondary antibody on the specific epitope on the antigen. The secondary antibodies are enzyme test on basic of the enzyme we add substrate and as a result of the reaction we get colourful solution in the well. If that's happen then we say that the patient is positive.

The substrate and the enzyme that I have already mentioned before.

As a blocking agent we can use here nonfat dry milk or BSA. 

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                            Suggested by Dada .

This topic is very broad I Just here come to explain all this topic in a short and simple manner if I have forgot to mention any main point then comment in the comment box.