ELISA (Enzyme-Linked Immunosorbent Assay) technique is a technique used to detect the presence of a specific antigen or antibody in a sample. Direct ELISA is a type of ELISA used to detect the presence of an antigen in a sample. In this technique, antigens are immobilized on a solid surface such as a microtiter plate, and specific antibodies are added to the surface to bind to the antigen. Bound antibody is then detected using an enzyme-labeled secondary antibody, and enzymatic activity is measured using a chromogenic substrate.
The principle of direct ELISA is based on the specific binding of an antigen to a capture antibody immobilized on a solid surface. The antigen is then detected using an enzyme-labeled detection antibody that specifically binds the antigen. The enzyme-labeled detection antibody is then detected using a chromogenic substrate, resulting in a color change that can be quantified using spectrophotometry.
Figure 1. Direct ELISA.
Now, let's go over the basic steps of a direct ELISA. First, a solid surface is coated with a capture antibody. Then, to reduce non-specific binding of the sample to the surface, unbound sites on the surface are blocked with a blocking agent such as BSA, casein, or milk powder. Next, a sample containing the antigen of interest is added to the coated surface and allowed to bind to the capture antibody. Afterwards, an enzyme-labeled detection antibody specific for the antigen is added to the surface and allowed to bind the captured antigen. Finally, enzyme activity is measured by adding a chromogenic substrate specific for the enzyme-labeled detection antibody. Enzymes convert substrates into colored products, and the color change is measured at specific wavelengths using a spectrophotometer.
Direct ELISA is a simple and straightforward detection method that requires fewer steps than other ELISA methods. It is also a very sensitive technique and can detect even very low concentrations of antigen. This is a quantitative technique that determines the exact concentration of antigen in a sample. Furthermore, it is a specific technique that only detects the antigen of interest and not other antigens or contaminants present in the sample. It's even versatile, being able to work with a variety of sample types, including serum, plasma, saliva, and urine.
However, direct ELISA can be affected by non-specific binding of the sample to the surface or the antibodies used in the assay, resulting in false positive or false negative results. Direct ELISA for sample detection needs higher requirements on the quality of the antibodies used. However, high quality antibodies are generally expensive and difficult to obtain. In addition, it is not suitable for the detection of complex antigen mixtures.
Nonetheless, direct ELISA has a wide range of applications in research, clinical diagnostics, and biotechnology. In clinical diagnostics, it is used to detect infectious diseases such as HIV, hepatitis, and Lyme disease by detecting the presence or absence of specific antigens in patient samples. In food and environmental sample testing, it is commonly used for the detection of foodborne pathogens such as Salmonella, E. coli and Listeria. In addition, it is also used quantitatively to quantify therapeutic protein or antibody concentrations during drug development and production, and to develop new immunoassay kits to detect specific antigens or antibodies in samples. In a word, direct ELISA, as a relatively direct ELISA detection method, is applied to the qualitative and quantitative detection of various molecules.