What is ELISA?
ELISA stands for Enzyme Linked Immunosorbent Assay. It is a type of immunoassay used to identify either the antigen or antibody. This study antigens and antibodies — quantitative or qualitative analysis is done. Samples routinely used in ELISA include plasma, serum, cell lysates, tissue lysates, and cell culture, etc. What does quantitative analysis mean? As the name suggests, quantity means the concentration of antigen or antibody, so this analysis is done to identify the concentration or antigen or antibody. On the other hand, qualitative analysis is done to see if the antigen is viral, or bacterial. If the antigen is viral then what kind of viral strain is present? In terms of antibody – what kinds of antibodies are present due to a particular kind of infection? This article discusses direct ELISA in detail with practical examples.
Types of ELISA
- Simple /Direct ELISA
- Indirect ELISA
- Sandwich ELISA
- Competitive ELISA
Simple /Direct ELISA –
This technique is used for the identification of antigens. This is called direct ELISA because there is a direct interaction between enzyme-linked antibodies and antigens. The antibodies used in ELISA technique are enzyme-linked antibodies. On the other hand, antigens could either be due to viral or bacterial infection. And the nature of antigens could be peptide, proteins or hormones. So, direct ELISA technique is used to find the source of antigens – whether the antigen is viral or bacterial or what kind of strain of virus is involved in the infection – direct ELISA is performed.
Extraction of Blood Serum from the Patient
In direct ELISA, blood is taken from the patient and then serum is extracted from the blood. Serum contains antigen molecules. The antigens present in serum are maintained in a buffer solution (PBS buffer). Why PBS buffer is used because its pH and ionic concentration is almost equal to the human blood pH and ionic concentration. This helps maintain the shape of the antigens. After that the antigens are applied in a well – antigens get attached to the bottom of the mcirotiter well. After that washing is done to remove extra antigens that are free-floating in the well.
Surface Blocking with BSA Proteins
The next step is surface blocking. This step is performed because when antigens are applied in a well – there is no guarantee that all the antigens get attached to the bottom of the well right? For surface blocking BSA (Bovine Serum Albumin) protein is used. BSA is a non-reactive protein and therefore, it will neither react with antigens or antibodies. When the blocking agent is poured in the well – it gets attached to the place where antigens are not present or we can say the space between two antigens. And blocking is also important because the enzyme-linked antibodies being used in this technique are very expensive because they are designed in the market. So, if we apply enzyme-linked antibodies without using the blocking agent – there is a probability that in addition to binding to the antigens – these enzyme-linked antibodies would also to the surface of the well where antigens are not present – this would give us a false reading.
After surface blocking, enzyme-linked antibodies are added to the well. These enzyme-linked antibodies get attached to the antigens in the well. The enzyme linked to the antibodies could be Horseradish peroxidase or alkaline phosphatase. After adding enzyme-linked antibodies – washing is done to remove extra enzyme-linked antibodies that didn’t bind with antigens. The next step is the addition of enzyme specific substrate in the well. The enzyme and substrate reacts with each other and a colored product is formed in each well. That colored product is then analyzed.
Now, we take a practical example to understand how direct ELISA works. Suppose, there is a patient showing dengue symptoms. And we expect that this patient might be suffering from dengue. So, we want to know what strain is causing dengue to the patient. First of all, we extract blood serum from dengue and follow the direct ELISA steps mentioned above. It is important to mention that we take dengue specific antibodies to perform direct ELISA. If the patient is having dengue antigens – the enzyme-linked antibodies would bind to antigens in the well and a particular color is produced after addition of enzyme-specific substrate in the well. And if the patient is not suffering from dengue, then the dengue antibodies would not bind with antigens and no color would produce in the well. It means the patient is not having dengue, he or she might be suffering from some other infections.
Conclusion:
ELISA is based on the principle of antigen-antibody interaction. ELISA is a widely used diagnostic test to identify specific substances, mostly antigens present in the blood serum such as HIV viruses. The presence of antigen is detected by antibodies synthesized against the pathogen.