Antigen-antibody reaction in ELISA

Antibody structure

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 μ (mu) chains, respectively.

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 antigen-binding sites that constitute the antibody only match the corresponding antigenic determinants and specifically bind, which is the structural basis for the specific binding of the antibody to the 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, called the Fc segment, has no antibody activity but has antigenicity specific for IgG.

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

Antigen-antibody reaction

Reversibility

The process by which an antigen binds to an antibody to form an antigen-antibody complex is a dynamic equilibrium whose reaction formula is:

Ag+Ab→Ag·Ab

The affinity of an antibody is the intrinsic binding between antigen and antibody and can be expressed by the equilibrium constant K:

K=[Ag·Ab]/[Ag][Ab]

The degree of dissociation of Ag·Ab is related to the K value. The antigen binding site of the high affinity antibody and the determinant of the antigen are very suitable in the spatial configuration, and the two are firmly combined and are not easily dissociated. The dissociated antigen or antibody retains its original structure and activity, so affinity chromatography can be used to purify the antigen or antibody. In antisera, specific IgG antibodies account for only a very small fraction of total IgG. A specific antibody extracted by affinity chromatography, which is called an affinity chromatography pure antibody, can be used in an immunoassay to obtain a better effect.

2. Optimal ratio

The amount by which an increasing amount of antigen is added to a constant amount of antibody to form an antibody complex (precipitate) can be plotted as a curve. The peak portion of the curve is the most appropriate range of antigen-antibody ratios, called the zone of equivalence. Before and after the equivalence band are the excess antibody bands and the excess antigen bands. If the antigen or antibody is extremely excessive, no precipitate is formed, which is called a zone phenomenon in immunoassay. The excess antibody is called the prezone and the excess of the antigen is called the postzone. When the antigen is determined by immunological methods, there should be sufficient amount of antibody in the reaction system, otherwise the measured amount will be less than the actual content, and even a false negative.

3. Specificity

Binding of an antigen-antibody occurs essentially only between the antigenic determinant of the antigen and the antigen binding site of the antibody. The antigen-antibody reaction is highly specific due to the complementary relationship between the two in chemical structure and spatial configuration. For example, surface antigen (HBAg), e antigen (HBeAg) and core antigen (HBcAg) in hepatitis B virus are derived from the same virus but only bind to their corresponding antibodies, but not to the other two antibodies. This specificity of the antigen-antibody reaction allows the immunoassay to determine a particular substance in a very complex protein compound (eg, serum) without first separating the analyte.

But this specificity is not absolute. If the two compounds have partially identical structures, cross-reactivity can occur in the antigen-antibody reaction. For example, both chorionic gonadotropin (hCG) and luteinizing hormone (LH) are composed of two subunits, α and β, and their structures differ in the β subunit, while the α subunits of both are homogeneous. The antisera obtained by immunizing animals with hCG contain anti-α-hCG and anti-β-hCG antibodies, and the anti-α-hCG antibody will cross-react with the α-enzyme position in LH. In clinical tests, if anti-hCG antiserum is used as a pregnancy diagnostic reagent to check hCG in urine, it can only be used in tests with higher hCG concentration, otherwise the trace LH that women physiologically excrete into the urine will cross-react with it. . Therefore, anti-β-hCG specific to hCG must be used in the diagnosis of early pregnancy (sensitivity should reach 50 mIu/ml hCG) to avoid cross-reaction with other hormones.

4. Sensitivity

When measuring the content of a substance in serum, the sensitivity of chemical colorimetry is mg/ml, the sensitivity of enzyme reaction assay is about 5~10 μg/ml, gel diffusion method and turbidity in immunoassay. The sensitivity of the method is similar to that of the enzyme reaction method. The sensitivity of labeled immunoassays can be increased by thousands of times to levels of ng/ml. For example, HBsAg can be determined by radioimmunoassay or enzyme immunoassay with a sensitivity of up to 0.1 ng / ml.

The application of immunoassay in clinical testing:

Since various antigenic components, including small molecule haptens, can be used to prepare specific antisera or monoclonal antibodies, the antibody can be used as a reagent to detect the corresponding antigen in the specimen, and thus the immunoassay has a wide range of applications. Can be used in clinical tests to determine:

1) Various proteins in body fluids, including proteins with very little content such as alpha-fetoprotein.

2) Hormones, including small molecular weight steroid hormones.

3) Antibiotics and drugs.

4) Pathogen antigens, HBsAg, HBeAg, etc.

5) In addition, antibodies in the specimen, such as anti-HBs, etc., can also be detected using purified antigen.

5. Labeled immunoassay

As mentioned above, immunoassay is a very sensitive assay. The antigen or antibody is directly measured to form a precipitate or turbidity with a sensitivity of 5-10 μg/ml. However, in clinical tests, some analytes are tested. The amount in the specimen is much lower than this level, so look for ways to increase sensitivity. The labeled immunoassay is to label the antigen or antibody in the detection reagent with a substance that can be measured in a minute amount, and to increase the sensitivity by measuring the label. In radioimmunoassay and enzyme immunoassay, the markers are radionuclides and enzymes, respectively. Finally, the radioactivity and enzyme activity are measured to calculate the amount of the analyte, and the sensitivity can be increased by several hundred to several thousand times compared with the direct determination of the precipitate. In labeling immunoassays, an excess of labeling reagent is typically added to ensure complete reaction with the analyte. Take the labeled antibody (Ab ※) as an example to detect the antigen (Ag). The reaction formula is as follows:

Ag+ Ab※ →AgAb※+Ab※

Among the reaction products, there are Ab* and free and Ab* bound to Ag, and if the two are not separated, the label is measured, and the measured result is the sum of the two. Therefore, the separation of free markers from binding markers is an important step in labeling immunoassays. A solid phase carrier can be one of a variety of means. If the antigen or antibody is coated on a solid support and then directly reacted with the labeled antigen or antibody, the bound label is immobilized on the support and the free label is left in solution. In this way, the free Ab* can be removed by washing, and the measurement of the binding label can be carried out on a solid phase.

6. Enzyme immunoassay

Enzyme immunoassays can be divided into two types, homogenous and heterogenous. The marker can be directly assayed in the homogeneous EIA without isolation of the free and bound label. For example, under certain conditions, the enzyme in the enzyme-labeled antigen-antibody complex formed by the antigen-antibody reaction loses its activity on the substrate, and thus the measured enzyme activity directly reflects the free enzyme label. Homogeneous EIA is less used in clinical testing. Heterogeneous EIA requires separation of the free and bound label first. As mentioned previously, the solid support can be used as a means of separation. This solid-phase enzyme immunoassay was originally called enzyme-linked immunosorbent assay (ELISA) when it was first established in 1971. It has been translated into enzyme-linked immunosorbent assay in China, although the meaning is not completely accurate. Conventional.

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