IVD R&D staff review of antibody basics

IVD R&D staff review of antibody basics

Antibodies, on the one hand, are the raw materials for the development of immunodiagnostic reagents, and on the other hand are the objects for the detection of immunodiagnostic reagents. I used to think that I had been exposed to antibodies since I was a student. I knew the antibodies very thoroughly. However, I discovered recently that the antibodies are not that simple. Therefore, come out of the teaching of immunology, do a review and study of antibodies.

Antibodies are classified into secretory immunoglobulins (serum, body fluids and exudates) and membrane immunoglobulins (mIg, BCR). The IVD industry's main test samples are blood, urine, and spinal fluid, and are mainly secretory immunoglobulins.

Antibodies, on the one hand, are the raw materials for the development of immunodiagnostic reagents, and on the other hand are the objects for the detection of immunodiagnostic reagents. I used to think that I had been exposed to antibodies since I was a student. I knew the antibodies very thoroughly. However, I discovered recently that the antibodies are not that simple. Therefore, come out of the teaching of immunology, do a review and study of antibodies.

Antibodies are classified into secretory immunoglobulins (serum, body fluids and exudates) and membrane immunoglobulins (mIg, BCR). The IVD industry's main test samples are blood, urine, and spinal fluid, and are mainly secretory immunoglobulins.

The "class" and "type" of antibodies: The type and type of antibodies are not the same, not universal, heavy chains determine "class", and light chains determine "type"

Class: The antigenic differences that determine the different Ig classes are present in the constant region (CH) of the heavy chain. According to the difference in the antigenicity of CH, that is, the amino acid composition, arrangement, spatial configuration, and number of disulfide bonds, the H chain is divided into five classes of μ, γ, α, δ, and ε chains, and the L chain constitutes an intact Ig molecule. . The subcategory mainly depends on the amino acid composition of the hinge region and the difference in the number of disulfide bonds.

Type: It is determined that the difference in antigenicity of Ig type is determined by the amino acid composition, arrangement, and spatial configuration of the constant region (CL) of the L chain, and is classified into two types, kappa and lambda. The subtypes can be divided into four subtypes λ1, 2, 3, and 4 according to the difference in the individual amino acids in the lambda light chain constant region.

Antigenicity of Antibodies: Isotypes, Allotypes, and Idiotypes

Isoforms: Immunization of heterologous animals with Ig will induce the production of isotype antibodies against this Ig. The antigenic markers shared by all healthy individuals in the same species are mainly concentrated in the constant region of Ig. Indirect methods for detection of anti-gamma chain antibodies used by IgG and anti-microchain antibodies used for capture, as well as the use of various secondary antibodies, are based on the antigenicity of the antibody's isotype.

Allotypes: Animals immunized with Ig to the same different species usually induce antibodies against that Ig, ie, allotype antibodies. The generation of allotypes is due to differences in the few amino acids in the constant regions of Ig molecules between different individuals, and can be inherited stably.

Idiosyncratic type: specificity of the antigen on the Ig V region, can induce the corresponding antibody in xenogeneic, allogeneic and self-in vivo, called anti-idiotypic antibody. In the process of Ig formation, a variety of V-region gene combinations are generated, and during the process of antibody affinity maturation, various changes in the variable regions are also made, making it a unique antigen-specific marker of Ig.

Various types of immunoglobulin properties:

IgA: The daily human IgA synthesis is approximately 66 mg/Kg body weight, more than all other Ig sums, but its half-life is only 6 days, so the blood IgA content is less than IgG, accounting for only about 10% of the blood Ig, but IgA is the major immunoglobulin in the mucosal surface and exudates. The human IgA monomer has a molecular weight of 160 kDa and contains two subclasses of IgA1 and IgA2, of which IgA2 also has two isotypes of IgA2m(1) and IgA2m(2). The IgA1 and IgA2 heavy chains differ by only 22 amino acids, and their major structural difference is that IgA2 lacks 13 amino acids rich in O-glycosylation sites in the hinge region.

IgA can be in monomeric form and can also form dimers, trimers, and tetramers with the help of the J chain. Secretory IgA are all multimers. Blood and mucosal tissues have different IgA. The IgA in the blood is mainly monomer, and the ratio of IgA1 to IgA2 is 10:1. It is synthesized by plasma cells that are activated and differentiated into lymphocytes and enter the bone marrow. The majority of IgA produced in the intestinal mucosa is a J chain-containing dimer, synthesized by the plasma cells of the lamina propria, and can produce about 5 g per day, with a ratio of IgA1 to IgA2 of 2:3. Most of the IgA generated is The pIgR is transported to the mucosal surface, and a part of it enters the blood via the portal vein. After entering the liver, the pIgR expressed by the liver cells is transported to the bile duct and then to the small intestine. IgA can function as a neutralizing antibody.

IgD: The human serum IgD content is 3 to 40 μg/mL and the half-life is about 2.8 days. IgD is a glycoprotein with a molecular weight of 184 kDa. The glycosyl radical is 9% of its molecular weight. IgD is only monomeric. The heavy chain consists of a variable region and three constant region domains. Due to the presence of a longer hinge region, Easily degraded by enzymes.

IgD is a hallmark of the development, differentiation and maturation of B cells. Many B cells co-express membrane IgD and membrane IgM. When B cells are transformed into memory cells, the expression level of membrane IgD is decreased until disappearance.

IgE: mainly secreted by the respiratory and intestinal epithelial plasma cells, IgE content in the blood is very low, normal blood IgE content of about 50ng/mL, blood IgE half-life of only two days, allergic patients IgE levels can rise High several times. Most of the body's IgE binds to tissue cells, and the half-life of cell-bound IgE can be extended to several weeks.

IgE has a molecular weight of 190 kDa and is only monomeric and its heavy chain consists of 4 constant regions. The degree of glycosylation of IgE is high, with glycosyl content accounting for 13% of its molecular weight, and 7 potential glycosylation sites. Under pathological conditions, some people will have abnormally high reactivity to antigens, leading to the occurrence of Type I hypersensitivity.

IgG:IgG is widely distributed in vivo and is the main immunoglobulin in blood and tissue fluids. Human blood IgG1 content 9mg/mL, IgG2 content 3mg/mL, IgG3 content 1mg/mL, IgG4 content 0.5mg/mL. IgG1, 2,4 have a molecular weight of 146 kDa and a half-life of about 3 weeks; IgG3 has a molecular weight of 165 kDa and a half-life of only one week.

IgG is mainly produced in the re-immune response of B cells through affinity maturation. In general, the affinity of IgG for antigen is much higher than that of IgM, and it is the main antibody for re-immune response.

Mouse IgG is divided into four categories: IgG1, IgG2a, IgG2b, and IgG3.

IgM: The IgM monomer has a molecular weight of 180 kDa and a sugar content of 10% to 12%. Each heavy chain of IgM contains 4 constant regions. IgM molecules are less flexible due to lack of hinge region. The IgM monomer is expressed on the naive B cells together with IgD mainly in the form of membrane proteins.

The concentration of IgM in the blood is 1.5 mg/mL, mostly pentamers with a molecular weight of 970 kDa, in addition to a small amount of tetramers and hexamers. The pentamer IgM is formed by the J chain and the disulfide bond between the monomer and the monomer.

There are two major sources of IgM in the body, one being the primary immune response to B-2 cells to foreign antigens. Although IgM is also produced in reinfection, it is not the main immunoglobulin. Another source of IgM is the polyclonal activation of B-1 cells induced by viruses and bacteria. They are often targeted to the sugar component of the pathogen's surface.

The initial antibody produced by the body during the primary immune response is always IgM. At this time, since the somatic mutation of B cells has not yet occurred, the IgM formed has a low affinity. However, IgM is a pentamer and has 10 antigen binding sites (actually pentavalent) with high affinity.

Appendix: The three stages of immune response

The body's response to infectious agents includes three stages:

Innate immunity period (0-4h): When pathogenic microorganisms or foreign substances enter the body, some existing effectors are involved in the immune system to identify and clear the pathogens. When infectious agents cannot be removed, they enter the innate immune response period.

Innate immune response period (4-96h): The innate immune response period is an early inducing response that occurs within 4 to 96 hours of bacterial invasion. Phagocytic cells recognise and activate pathogens, phagocytose and clear pathogens; secrete a series of cytokines that cause inflammatory reactions; Activated NK cells and other innate immune effector cells, jointly perform the removal of infection primers. When the infectious agent cannot be removed, it enters the adaptive immune response period. B1 cells can be recognized and combined with polysaccharide antigens common to the surface of some bacteria and activated. In short time (48h), low-affinity IgM-based antibodies can be produced.

Adaptive immune response period (>96h): The adaptive immune response phase is an late induction response that occurs 96 hours after bacterial invasion. Uncleared infectious agents are presented to T cells to initiate specific T cell immune responses. B cells Direct recognition of the antigen induces a specific humoral immune response. Each B cell generally expresses IgM at the beginning, and first secretes IgM in an immune response, but can then be expressed to produce IgG, IgA, or IgE. There are mainly 1 types of antigens that affect the Ig class conversion: B cell responses to TI-1 antigen do not cause antibody type conversion, generally only induced IgM class antibodies; response to TI-2 antigen mainly produces IgM, but also can produce IgD . Responses to TD antigens can be type-transformed. In general, viruses and bacteria mainly induce the conversion of antibodies to IgG; parasites and allergens mainly induce the conversion of antibodies to IgE. The help of Th cells, the cytokines secreted by Th cells also play an important role in the type conversion of Ig. 3 Related to the anatomical location of B cells.