Direct ELISA (Sandwich ELISA) Test for Antigen Detection

Enzyme-linked immunosorbent assay (ELISA) is an extremely sensitive test to detect specific antibodies or antigens. Direct ELISA is a test for detecting antigens using specific immobilized antibodies.

Direct ELISA uses monoclonal antibody pairs; one antibody, called the capture antibody, binds to the plate and the antigen. The other antibody, called the detector antibody, also binds the antigen and is conjugated with an enzyme.

This method of utilizing paired antibodies is also referred as a sandwich ELISA. The capture and detector antibodies bind two different epitopes or regions of the antigen.

Principle of Sandwich ELISA

A known antibody is adsorbed inside the well in a microtiter plate. After rinsing to remove excess antibodies, the sample suspected of containing antigen is added.

Sandwich ELISA

Next, an enzyme-linked antibody (detector antibody) that can react with the antigen is added. The enzyme is involved in generating a specific color. If an antigen is present in the well, the enzyme-linked antibody binds to it and is retained.

The colorless substrate for the enzyme is then added. Development for color (after enzyme-substrate reaction) indicates the presence of the antigen. The intensity of the color generated indicates the amount of antigen present in the sample, which can be determined quantitatively using an ELISA reader.

Direct ELISA Procedure

Antigen-specific antibody (capture antibody) is attached to a solid phase surface. (i.e., Antibody directed against a specific infectious agent in question is firmly fixed to a solid matrix, either the inside of the walls of a microdilution tray or the outside of a spherical plastic or metal bead or some other solid matrix)

antibody attached

Test specimen is added. If the antigen is present in the fluid to be tested, stable antigen-antibody complexes are formed; unbound antigen is thoroughly removed by washing.

antigen and antibody

The second antibody (detector antibody) against the sought antigen is added to the system. This antibody has been complexed to an enzyme such as alkaline phosphatase or horseradish peroxidase.

ag ab and eab

The antigen on the solid matrix binds the second antibody, forming a sandwich with antigen in the middle. Unbound labeled antibody is removed by washing.

A chromogenic enzyme substrate is added. The hydrolysis of the enzyme-substrate causes the color change and completes the action. The color developed is proportional to the amount of antigen present in the test specimen.

development of color

Results and Interpretation 

The intensity of the color is directly proportional to the amount of the antigen present in the same.  The amount of antigen present in the test sample can be measured using the ELISA reader.

References and further reading

  1. Murthy, N., Nair, K. M., & Bhaskaram, P. (1995). A direct ELISA technique to detect antibodies against polioviruses. Indian journal of biochemistry & biophysics, 32(5), 249–253.
  2. Kohl, T. O., & Ascoli, C. A. (2017). Direct Competitive Enzyme-Linked Immunosorbent Assay (ELISA). Cold Spring Harbor protocols, 2017(7), pdb.prot093740. 
  3. Souvras, M., Montagnon, B., Fanget, B., van Wezel, A. L., & Hazendonk, A. G. (1980). Direct enzyme linked immunosorbent assay (ELISA) for quantification of poliomyelitis virus D-antigen. Developments in biological standardization, 46, 197–202. 

Acharya Tankeshwar

Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. As an asst. professor, I am teaching microbiology and immunology to medical and nursing students at PAHS, Nepal. I have been working as a microbiologist at Patan hospital for more than 10 years.

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