Basic knowledge of immunoassay

ELISA is an immunoassay. Immunoassay (IA) is a method for measuring specimens using immunological techniques. In clinical tests, antibodies or antigenic substances in body fluids are mainly detected by antigen-antibody reactions.

1.1 Antigen Antigen is a substance that can cause a specific immune response in the body. After the antigen enters the body, it can stimulate the body to produce antibodies and cause cellular immunity. In immunoassays, antigen refers to a substance that can bind to an antibody. Antigens that can cause antibodies in the body are mostly proteins with a molecular weight greater than 5000. The small molecule compound that can cause the body to produce specific antibodies after binding to the large molecule protein is called hapten. For example, certain hormones and drugs. The reactivity of an antigen depends on an antigenic determinant, or epitope. An antigen molecule can carry different determinants.

1.2 Antibodies

1.2.1 Structure of antibodies Antibodies are immunoglobulins (Ig) that can specifically bind to antigens. Ig is divided into five categories, namely IgG, IgA, IgM, IgD and IgE. Ig related to immunoassay is mainly IgG and IgM. Ig consists of two light chain (L) and two heavy chain (H) monomers. The light chain of Ig is the same, 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 called γ (gamma) chain and μ (mu) chain, respectively. The structure of IgG is shown in the figure.

â‘  Papaya enzyme cleavage site â‘¡ Pepsin cleavage site

The order of amino acids at the N-terminus of the heavy and light chains varies with various antibodies and is called the variable region, which is represented by VH and VL, respectively. The two constitute the antigen binding of the antibody
The site, which only matches the corresponding antigenic determinant, and specifically binds (see figure), is the structural basis for the specific binding of antibodies to antigens.

IgG can be broken down into three segments by papain, and two identical segments are called antigen binding fragments (Fab). Each Fab preserves the ability to bind antigen, but there is only one antigen binding site, which is monovalent and does not appear to aggregate or precipitate after binding to the antigen. The other segment is called the Fc segment, has no antibody activity, but has the unique antigenicity of IgG.

IgG can be broken down into two fragments by pepsin, a Fab duplex, called F (ab) 2, which can bind to two identical antigens; the other fragment is similar to Fc, and is subsequently broken down into small molecule peptides with no biological activity.
IgM is a pentamer composed of five monomers, contains 10 heavy chains and 10 light chains, and has 10 antigen binding valences. Due to the influence of spatial position, it only shows five antigen binding valences. The molecular weight of IgM is about 900,000 and the molecular weight of IgG is about 50,000.

After being infected by microorganisms, the body first produces IgM antibodies, and then produces IgG antibodies. After a period of time, the amount of IgM antibodies gradually decreases and disappears, while IgG antibodies can exist for a long time and can last for several years after the disease is cured.

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

1.2.2 Antibody production After the body is stimulated by antigens, B lymphocytes produce corresponding antibodies. The serum containing antibodies is called antiserum (antiserum). Each line of B cells only produces antibodies against a certain epitope. If multiple antigens or antigens containing multiple antigenic determinants are injected into the body, multiple lines of B cells will produce corresponding multiple antibodies, all of which are present in the immune serum. Antisera used in immunoassays are generally prepared by immunizing rabbits, sheep or horses with antigens. Antibody-producing B cells can be fused with proliferative tumor cells into hybridoma cells in vitro. Monoclonal antibody (McAb or Mab), which is a single hybridoma cell isolated and cultured in vivo or in vitro. Monoclonal antibodies are directed against only one epitope and have high specificity. Monoclonal antibodies are usually prepared by immunizing mice with antigen. Immune spleen cells (containing antibody-producing B cells) were fused with mouse tumor cells, and hybridoma cells were isolated and inoculated into the abdominal cavity of mice. The ascites produced contained high concentrations of monoclonal antibodies.

1.3 Antigen-antibody reaction

1.3.1 Reversibility
The process of combining antigen and antibody to form an antigen-antibody complex is a dynamic balance, and its reaction formula is: Ag + Ab → Ag · Ab. The affinity of antibody is the inherent binding force between antigen and antibody, which 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 point of high-affinity antibodies and the determinant of the antigen are very suitable in space configuration, the two are firmly bound and not easy to dissociate. The dissociated antigen or antibody can maintain the original structure and activity, so affinity chromatography can be used to purify the antigen or antibody. In antisera, specific IgG antibodies only make up a very small portion of total IgG. Specific antibodies extracted by affinity chromatography, called pure affinity chromatography antibodies, can be used in immunoassays for better results.

1.3.2 Optimal proportion
The amount of antibody added to the constant amount of antibody in increasing amounts to form an antibody complex (precipitation) is shown in Figure 1-4. The peak part of the curve is the most suitable range of antigen-antibody ratio, which is called the zone of equivalence. Before and after the equivalence band are the excess antibody band and the excess antigen band. If the antigen or antibody is extremely excessive, no precipitate is formed, which is called a zone phenomenon in the immunoassay. An excess of antibody is called a prezone, and an excess of antigen is called a postzone. When using immunological methods to determine the antigen, there should be enough antibody in the reaction system, otherwise the measured amount will be less than the actual content, or even false negative.

1.3.3 Specificity
The binding of antigen-antibody essentially occurs only between the antigenic determinant of the antigen and the antigen-binding site of the antibody. Since the two have a complementary relationship in chemical structure and spatial configuration, the antigen-antibody reaction is highly specific. For example, the surface antigen (HBsAg), e antigen (HBeAg) and core antibody (HBcAg) in hepatitis B virus are derived from the same virus, but only bind to their corresponding antibodies, but not react with the other two antibodies. The specificity of the antigen-antibody reaction allows immunoassays to measure a specific substance in a very complex protein compound (such as serum) without first isolating the analyte.

But this specificity is not absolute. If the two compounds have partially the same structure, cross-reactions can occur in the antigen-antibody reaction. For example, chorionic gonadotropin (hCG) and luteinizing hormone (LH) are both composed of α and β subunits, their structural differences are in β subunits, and the α subunits of the two are the same. The antiserum obtained by immunizing animals with hCG contains anti-α-hCG and anti-β-hCG antibodies. The anti-α-hCG antibodies will cross-react with LH. In clinical testing, if anti-hCG antiserum is used as a diagnostic reagent for pregnancy to detect hCG in urine, it can only be used for tests with high hCG concentration, otherwise women ’s physiological excretion of trace LH into urine will cross-react . Therefore, in the practice of early pregnancy diagnosis (sensitivity should reach 50mIu / mlhCG), anti-β-hCG specific for hCG must be used to avoid cross-reaction with other hormones.

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

1.4 The application of immunoassay in clinical testing Because various antigen components, including small molecule haptens, can be used to prepare specific antisera or monoclonal antibodies, using this antibody as a reagent can detect the corresponding antigen in the specimen, Therefore, the application range of immunoassay is extremely wide, and it can be used to determine in clinical tests:
1) Various proteins in body fluids, including very small amounts of proteins such as alpha-fetoprotein, etc.
2) Hormones, including small molecular weight steroid hormones.
3) Antibiotics and drugs.
4) Pathogen antigens, HBsAg, HBeAg, etc.
5) In addition, the purified antigen can also be used to detect antibodies in the specimen, such as anti-HBs.

1.5. Labeled immunoassay is as described above. Immunoassay is a very sensitive measurement method. The precipitate or turbidity directly measured after the antigen-antibody reaction can reach a sensitivity of 5 ~ 10μg / ml. However, in clinical tests, some The content of the analyte in the specimen is much lower than this level, so it is necessary to find a way to increase the sensitivity. The labeled immunoassay is to label the antigen or antibody in the detection reagent with a substance that can be measured in a trace amount, and to increase the sensitivity by measuring the label. In radioimmunoassay and enzyme immunoassay, the markers are radionuclide and enzyme, respectively. Finally, the radioactivity and enzyme activity are used to calculate the amount of analyte. The sensitivity can be increased by hundreds to thousands of times compared with the direct determination of the precipitate. In labeling immunoassays, an excess of labeling reagent is generally added to ensure a thorough reaction with the analyte. Taking labeled antibody (Ab *) for detection of antigen (Ag) as an example, the reaction formula is as follows: Ag + Ab ※ → AgAb ※ + Ab ※. There are Ab * and free and Ab * bound to Ag in the reaction product. If the label is measured without separating the two, the measured result will be the sum of the two. Therefore, the separation of free label and bound label is an important step in labeling immunoassay. Various means are available, and solid-phase supports are one of them. For example, if the antigen or antibody is coated on a solid phase carrier, and then directly reacts with the labeled antigen or antibody, the bound label is fixed on the carrier, and the free label remains in the solution. In this way, free Ab * can be removed by washing, and the measurement of the bound label can be performed on the solid phase.

1.6. Enzyme immunoassay Enzyme immunoassay (enzyme immunoassay) can be divided into homogeneous (homogenous) and heterogeneous (heterogenous) two types. In homogeneous EIA, the label can be directly determined without the separation of free and bound label. For example, under certain conditions, the enzyme in the enzyme-labeled antigen-antibody complex formed after the antigen-antibody reaction loses its activity on the substrate, so the measured enzyme activity directly reflects the free enzyme label. Homogeneous EIA is rarely used in clinical testing. Heterogeneous EIA requires separation of free and bound markers. As mentioned earlier, solid-phase supports can be used as a means of separation. This solid-phase enzyme immunoassay method was called enzyme-linked immunosorbent assay (enzyme linked immunosorbent assay), or ELISA for short, when it was first established in 1971. It has been translated as an enzyme-linked immunosorbent assay or an enzyme label in China and has been used.