As we all know, enzyme-linked immunosorbent assay( ELISA ) is a technique that uses antigen-antibody interaction to quantify proteins, hormones, antibodies or viral antigens. Up to now, more than 200 research papers related to ELISA and antibody detection have been published in journals around the world every year. Among them, there are more than 70 papers in which indirect ELISA method is applied to antibody detection (Figure 1). After decades of exploration and improvement, there are four ELISA techniques.In other words, Direct ELISA, Indirect ELISA, Sandwich ELISA, and Competitive ELISA are all different methods. ELISA technology is widely used in medical diagnosis, plant pathology and industrial research and development, driving the development of the human health industry. Today we learn about the indirect ELISA method. Medicilon's Macromolecular Drugs Bioanalysis Department uses ELISA, ECL, IP, Co-IP, qPCR, FACS, Elspot, enzymology and other methods to support the early development, preclinical and clinical assessment of biopharmaceuticals, including proteins, antibodies (monoclonal antibodies, bi- or multi-specific antibodies and antibody fragments), ADCs, polypeptides, nucleic acids, vaccines and cell gene therapy drugs.
Lindström and Wager published the indirect ELISA method for the first time in 1978. Compared with the direct ELISA method, it uses an enzyme-labeled secondary antibody for detection. Because the primary antibody has multiple binding epitopes, the detection sensitivity is improved. And accuracy, just like in a dark room, the ELISA direct method has only one 50W bulb, while the ELISA indirect method can connect two 50W bulbs in parallel, so the brightness of the room is different. The ELISA indirect method is operated with ELISA. The difference between the direct method is the additional incubation of the secondary antibody.
The first step is to passively adsorb the antigen to the surface of the porous plate, wash and seal the excess area, add the analytical sample containing the antibody to be tested, incubate until the specificity is fully combined, then wash and incubate the secondary antibody, and finally add the substrate for color reaction detection, so this method is mostly used for quantitative analysis of the antibody.Obviously, the specificity of the reaction is increased compared to the direct method, and it is also more economical, because there is no need to design the enzyme-labeled antibody for each specific antigen, and of course the introduction of the second antibody also has the effect of cross-activity, which may cause some background noise.
The difference between the direct method and the indirect method of ELISA
As we all know, the direct method of ELISA only requires one detection antibody, while the detection process of the indirect ELISA method is divided into two steps, incubating twice, and two antibodies are required to complete the specific binding. Now let’s take a look at the overall method. The difference helps us better understand and use.
Selection criteria for ELISA kits
The accuracy, precision and specificity of the ELISA kit determines the quality of the product, so when we participate in the evaluation of an ELISA kit, we need to carefully read the product report to see if it is qualified and high-quality.
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