Key Difference – Direct vs Indirect ELISA
An enzyme-linked immunoassay (ELISA), also known as enzyme immunoassay, is a serological test which detects antibodies in the blood. It is used as a diagnostic tool to find out whether the patient has been exposed to a particular type of virus or another infectious agent (antigen) and whether the body has produced antibodies against the infection. ELISA can also identify the past and current infections. Hence, ELISA is often used as a prescreening test by the doctors prior to in-depth analysis of diseases. This test can be performed in the laboratory by taking a blood sample from the patient. There are two types of ELISA test: direct ELISA and indirect ELISA. The key difference between direct and indirect ELISA is that indirect ELISA is more sensitive and requires the addition of a secondary antibody while direct ELISA is less sensitive and only uses a primary antibody.
CONTENTS
1. Overview and Key Difference
2. What is Direct ELISA
3. What is Indirect ELISA
4. Side by Side Comparison – Direct vs Indirect ELISA
5. Summary
What is Direct ELISA?
ELISA is a disease diagnostic test performed to identify the presence of particular antigens or antibodies in the blood. It is done as a plate assay. It uses antibodies linked with easily assayed enzymes. Antigens’ presence in the serum sample bind with specific antibodies linked with enzymes. In the final step, a specific substrate is added to react with the enzyme. The enzyme converts the substrate into a coloured or signal-producing product. The colour change in the chemical substrate reveals the presence of a particular antibody in the serum sample. Direct ELISA test uses only a primary antibody linked to the enzyme. Upon binding to the antigen, it quickly changes the colour, indicating the presence of the infectious agent in the blood. However, the intensity of the signals is weaker in direct ELISA since the epitopes are limited to binding of the antigens. Hence, direct ELISA is less sensitive compared to indirect ELISA.
Figure 01: Direct ELISA Test
What is Indirect ELISA?
ELISA can be performed using two types of antibodies namely; primary antibody and secondary antibody. Indirect ELISA tool uses both types of antibodies to amplify the signals for better detection. Indirect ELISA technique is performed as follows.
- Plates are incubated with antigens and washed to block nonspecific binding.
- Then primary antibodies are added and washed.
- Enzyme-linked secondary antibody are added and washed.
- A substrate is added and allowed to react with enzymes.
- Signals are detected, and the presence or absence of the specific antigen in the sample is identified.
In indirect ELISA test, several secondary antibodies can bind to a single primary antibody. Secondary antibodies are linked with easily assayed enzymes. Therefore, one binding can make a strong signal due to more than one interactions. Hence, indirect ELISA is more sensitive than direct ELISA. However, indirect ELISA can make nonspecific signals due to cross reactions of the secondary antibodies.
Figure 02: Indirect ELISA Test
What is the difference between Direct and Indirect ELISA?
Direct vs Indirect ELISA |
|
| Direct ELISA is less sensitive compared to indirect ELISA. | Indirect ELISA is more sensitive. |
| Time Taken | |
| Direct ELISA test is a quick process. | Indirect ELISA is time-consuming. |
| Use of Antibodies | |
| Only one type of antibodies (Primary antibodies) are used in direct ELISA. | Primary and secondary antibodies are used for indirect ELISA. |
| Link with Enzymes | |
| Primary antibodies are linked with enzymes. | Secondary antibodies are linked with enzymes. |
| Cross-Reactivity of Second Antibodies | |
| Direct ELISA eliminates the cross-reactivity of second antibodies. | Indirect ELISA is affected with cross-reactivity of second antibodies. |
| Signals | |
| Signals are weak compared to indirect ELISA. | Signals are amplified in indirect ELISA. Hence, it is easy to detect. |
Summary – Direct vs Indirect ELISA
ELISA is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a blood sample of a patient. It can be performed via two processes known as direct or indirect ELISA. Direct ELISA test uses only primary antibodies to detect the antigen while indirect ELISA uses both primary and secondary antibodies. In direct ELISA, primary antibodies are labelled whereas in indirect ELISA secondary antibodies are labelled. This is the difference between direct and indirect ELISA.
References
1.”The enzyme-linked immunosorbent assay (ELISA).” Bulletin of the World Health Organization. U.S. National Library of Medicine, 1976. Web. 29 Mar. 2017
2. Loon, A. M van, J. T Van Der Logt, F. W. Heessen, and J. Van Der Veen. “Enzyme-linked immunosorbent assay that uses labeled antigen for detection of immunoglobulin M and A antibodies in toxoplasmosis: comparison with indirect immunofluorescence and double-sandwich enzyme-linked immunosorbent assay.” Journal of Clinical Microbiology. U.S. National Library of Medicine, June 1983. Web. 29 Mar. 2017
Image Courtesy:
1. “ELISA diagram” By Cavitri – Own work (CC BY 3.0) via Commons Wikimedia
2. “Indirect ELISA” By Cawang – Own work (CC0) via Commons Wikimedia