Enzyme-linked immunosorbent assay (ELISA) is one of the most specific and straightforward assays for detecting biomolecules. ELISA is a useful tool for measuring changes in biological processes in vivo or in vitro and its test samples can include plasma, serum, saliva, urine, tissue culture media, cells lysates and other materials. The four main types are direct ELISA, indirect ELISA, sandwich ELISA, and competitive ELISA. In the following guide, we review the different types of ELISA to fit your research needs.
Direct ELISA is a plate-based immunosorbent assay intended for the detection and quantification of a specific analyte from a complex biological sample. Among the four different ELISA formats, direct ELISA is the simplest and fastest method, but this method has certain disadvantages (please refer to our another guide: How to choose the appropriate ELISA kit).
In the direct ELISA, the antigen of interest in fluid phase is immobilized to a microtiter plate through passive absorption. A blocking buffer is added to saturate all unbound sites followed by the binding of enzyme-labeled antibody. Antibody in direct ELISA can be monoclonal or polyclonal. A substrate is added to generate a color change or emit light indicating the presence of antigen. Direct ELISA is useful for qualitative or quantitative antigen detection in a sample, antibody screening, and epitope mapping. Since only one antibody is involved, there is no cross reactivity with secondary antibody, and the assay can be performed in less amount of time compared to other ELISA methods.
The reason why this method is called the indirect method is that what determines and separates the antigen to be measured is not the primary antibody, but another antibody that is placed in the medium. In this method, the test sample is added to the antigen-coated wells and the plates are incubated. During this incubation, the antibodies formed against the antigens in the test sample produce an antigen–antibody complex. In order to render the antigen–antibody complex visible, a secondary antibody that recognizes the antibody and that is tagged with the enzyme is added. Then the substrate of the enzyme is added to the medium to produce color and the concentration is determined. This method utilized to identify antigens is used more commonly in endocrinology. The magnitude of signal production is proportional to the amount of antigen in the sample. Use of a secondary detection reagents has significant advantages over direct ELISA, namely signal amplification.
In Sandwich ELISA, the wells are coated with a capture antibody and blocked. The sample is added to the microplate wells coated with the antibody; then, the plate is incubated for some time and washed. Washing removes the unbound antigens. Following the washing step, antibodies that are tagged with the enzyme specific to the antigen are added and incubated. After incubation and washing, enzyme substrate is added to the medium and coloration is ensured. Coloration shows a positive result, while lack of coloration indicates lack of enzymes, or a negative result. As the relevant molecules is stuck between two antibody molecules, this method is called Sandwich ELISA.
The competitive enzyme-linked immunosorbent assay (cELISA; also called an inhibition ELISA) is designed so that purified antigen competes with antigen in the test sample for binding to an antibody that has been immobilized in microtiter plate wells. After the wells are washed and enzyme substrate is added, the resulting coloration enables quantifying the concentration. There is an inverse proportion between the analyte concentration and the intensity of resulting coloration. To put it differently, when the amount of the antigen or the antibody analyzed in the serum is low, high absorbance is obtained, while greater quantities produce low absorbance.
Alpha Lifetech Inc. is becoming a choice for sourcing ELISA kits for customers throughout the world. We provide all the components you need to build a ELISA. You can search for the ELISA kit you need by target name, research area, etc. In addition, we can offer customized services for your special requirements, providing protein expression, antibody production, ELISA construction, conduction of assays, results analysis and other one-stop technical services.
Whether you are ordering our product or talking to one of our technical service representatives, our staff is dedicated to providing professional assistance. Please feel free to contact us for more information.