How does ELISA work? How can we identify AIDS through it?

 

You must be thinking that ELISA is a very complicated test that is used mostly to identify very dangerous viruses like AIDS and Hepatitis B. But this is not the case; in fact,  we identify various common bacterial infections through it, i.e., Lyme disease, brucellosis, and syphilis, as and as simple fungal infections like Candida (yeast infection). We can also identify autoimmune diseases like type 1 diabetes,where our immune system attacks its own pancreas. 

 We can also detect and identify hormone levels, like follicle-stimulating hormone (FSH) and testosterone. And we can also detect the presence of non-medical drugs like amphetamines and cocaine. In some cases, it can also identify cancer, i.e., prostate-specific antigens for prostate cancer.

So How does it work?

First, ELISA is a laboratory test,that detects and count antibodies and  antigens in bodily fluid samples. Samples can be Blood, Plasma, Pee,Saliva and CSF.

ELISA stands for "Enzyme-Linked immunosorbent assay", so its an assay (test) in which we are doing some antigen(Ag) and antibody(Ab) stuff, but that is with the help of a enzyme. And the enzyme's name is horseradish peroxidase (HRP). We also add some coloring agents (chromogens) like tetramethylbenzidine to identify the reaction i.e if color comes out then Positive otherwise its Negetive. And a Substrate i.e H2O2  which will break after the reaction with antigen-antibody complex, and react with the coloring agent and show colors.

ELISA plate contains wells, 12*8=96.
At a time 96 testing can be done.

Mainly, ELISA is of 4 types, on th basis of how it is detecting the antigens. or antibodies 

1. Direct 
2. Indirect
3. Sandwich
4. Competitive 

Lets understand one by one,

1.Direct ELISA 

It is used in the detection of antigens.

First, we add the patient's sample (Ag) to the assay plate. Then we add known antibodies (possible diseases), enzymes (HRP), and substrates (H2O2. Now add the coloring substance (tetramethylbenzidine).

If the result is positive, then color comes out.

The intensity of color is directly proportional to Ag concentration.

2.Indirect ELISA 

It is used in the detection of antibodies. 

In this case, we first add a known antigen, followed by the patient's antibody.

And now we add the antibody+ HRP enzyme to the complex. Then the substrate (H2O2) and coloring agent.

If the colors come out, then they are positive; otherwise, they are negative.

The intensity of the color is directly proportional to the antibody concentration.

3.Sandwich ELISA

It's the most common type of ELISA performed. Because of its high specificity and sensitivity. But it's a little challenging to develop.

In this type, we measure antigen in patients.

We have to make a sandwich, so first we add known antibodies, then antibodies from the patient's sample, and now we again add antibody+ enzyme complex .And Substrate (H2O2), and at last we put our color agent.

If color comes out, then it's positive, and if it doesn't, then it's negative. 

The intensity of color is directly proportional to the antigen concentration. 

4.Competitive ELISA

It is also used for antigen detection when the antigen is too small to sandwich between two antibodies. So, basically, it is used for small molecules. 

It's a little complicated, so I am going to make points:

• First, we add a known antibody and the patient's antibody to a slide or testube and make a mixture of the Ag-Ab complex.
• Now add the patient's Ag in the ELISA well. 
• Then add the mixture to the antigen-coated well.
• Then add known antibodies + enzymes.
• Add the substrate and coloring agent.
• Here's the twist: if color comes out, the result is negative, and if color doesn't come out, the result is positive. 

The intensity of color is inversely proportional to antibody concentration. 

So,why,

Because if an Ag-Ab complex has formed in the slide, it should not be reacting in the well. If it is reacting, the added antibodies in the complex might not be the right ones.

 

Now, Lets take the example of HIV infection/AIDS:

In the detection of HIV infection or AIDS, both indirect and direct ELISA methods can be used. However, the most widely used ELISA method for HIV detection is the indirect ELISA. Here's how it works:

1. In this method we detects HIV antibodies in a patient's blood sample. Here's how it works:

• Coating: The wells of a microplate are coated with HIV antigens, typically from viral proteins such as gp120 or gp41.

• Sample Incubation: The patient's blood sample is added to the wells and incubated. If the patient has been infected with HIV, their blood may contain HIV antibodies that will bind to the antigens coated on the microplate.

• Washing: After incubation, the microplate is washed to remove any unbound substances.

• Secondary Antibody: A secondary antibody, specific to human antibodies and conjugated with an enzyme [e.g., horseradish peroxidase (HRP)], is added to the microplate. This secondary antibody binds to any HIV antibodies that are already bound to the coated antigens.

• Substrate and Coloring agent addition: A substrate solution (H2O2)containing a colorless substrate is added to the microplate with coloring agent. If HIV antibodies are present in the patient's blood sample, the enzyme linked to the secondary antibody will catalyze a reaction that produces a colored product.

• Color Development: The intensity of the color developed is proportional to the amount of HIV antibodies present in the patient's blood sample.

Thus , this is ELISA and how it is used in HIV detection.

If you want to know more, see my other blogs.

Here is a recommendation,  if you know about our immune system, and how it protect us from viruses like HIV, 

How our immne system protect our body?

Thank you