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ELISA is an immunoassay. Immunoassay (IA) is a method of measuring specimens using immunological techniques. Detection of antibodies or antigenic substances in body fluids by antigen-antibody reaction in clinical tests

1.1 antigen

An antigen is a substance that causes a specific immune response in the body. When the antigen enters the body, it can stimulate the body to produce antibodies and cause cellular immunity. In immunoassay, an antigen refers to a substance that binds to an antibody. Most of the antigens that cause antibody production in the body are proteins with a molecular weight of more than 5,000. Small molecule compounds, when combined with macromolecular proteins, cause the body to produce specific antibodies, called haptens. For example, certain hormones, drugs, and the like. The reactivity of an antigen depends on an antigenic determinant, or epitope. An antigen molecule can carry different determinants.

1.2 antibody

1.2.1 Structure of the antibody

An antibody is an immunoglobulin (Ig) that specifically binds to an antigen. Ig is divided into five categories, namely IgG, IgA, IgM, IgD and IgE. The Ig associated with immunoassays is primarily IgG and IgM. Ig consists of two light chain (L) and two heavy chain (H) monomers. The light chain of Ig is the same, and there are two types of κ (kappa) and λ (Lambda). The five types of Ig have different heavy chain structures, which determines their antigenicity. The heavy chains of IgG and IgM are referred to as gamma (gamma) chains and mu (mu) chains, respectively. The structure of IgG is shown in the figure.

1 papain cleavage site 2 pepsin cleavage site

The amino acid arrangement order of the N-terminus of the heavy chain and the light chain varies depending on various antibodies, and is called a variable region, and is represented by VH and VL, respectively. The two constitute the antigen binding site of the antibody, which only matches the corresponding antigenic determinant and occurs.

Specific binding (see figure) is the structural basis for antibody-specific binding to antigen.

IgG can be broken down into three segments by papain, two of which are called antigen-binding fragments (Fab). Each Fab retains the ability to bind antigen, but has only one antigen binding site, is monovalent, and does not agglutinate or precipitate upon binding to the antigen. The other segment is called the Fc segment and has no antibody activity, but has IgG specificity.

Antigenicity.

IgG can be broken down into two fragments by pepsin, a Fab duplex, called F(ab')2, which binds to two identical antigens; another fragment resembles Fc, which is subsequently broken down into small peptides without biological activity. .

IgM is a pentameric consisting of five monomers, containing 10 heavy chains and 10 light chains, with 10 antigen binding valencies, and exhibits only five antigen binding valencies due to spatial location. IgM has a molecular weight of about 900,000 and an IgG molecular weight of about 150,000.

After the organism is infected with a microorganism, an IgM antibody is first produced, and then an IgG antibody is produced. Over time, the amount of IgM antibodies is gradually reduced and disappears, while IgG antibodies can persist for many years and can persist for several years after the disease has healed.

IgM antibodies are generally protective antibodies and are immunogenic. Therefore, the determination of IgM antibodies has a high clinical diagnostic value for certain infectious diseases such as hepatitis A.

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