Conventional antibody
Conventional antibodies or fully sized antibodies are glycoproteins called immunoglobulins that are produced by the reaction of plasma cells with foreign molecules or antigens. The most basic function of antibodies is to bind specific antigens and stimulate immune responses to protect the body from infection. Antibodies include several subtypes, and here are mainly antibodies that describe IgG and IgM subtypes. Antibodies to IgG and IgM subtypes are widely used in research, diagnosis and treatment.
Conventional antibody structure
The basic unit structure of an intact antibody consists of four peptide chains, including two heavy chains and two light chains, joined together by disulfide bonds. The antibody is shaped like a Y letter, and the hinge region of the Y structure is elastic. Each peptide chain has a constant region (very conserved across all antibodies) and a variable region (specific in one antibody). The symbol of the light chain variable region is VL, and the symbol for the light chain constant region is CL (Fig. 1 left). Similarly, the variable and constant regions of the heavy chain are designated as (VH) and (CH), respectively. Carbohydrates usually bind to the CH2 region of the heavy chain. The Fc segment includes only the constant region (CH) of the heavy chain, but the Fab segment (Fab) that binds to the antigen includes a variable region of the constant region and the heavy chain and a variable region (VH and VL) of the light chain. The Fv region (variable fragment) contains only two variable regions.
Conventional antibody application
Conventional antibodies have been used in research to detect target proteins by Western blotting, immunohistochemistry, and enzyme-linked immunosorbent assays for decades. Full-size antibodies are also used in clinical tests, such as pregnancy tests and the detection of HIV in the blood by ELISA. In addition, conventional intact antibodies are also used in the treatment of diseases. For example, infliximab is an antibody that recognizes tumor necrosis factor and is used to treat intestinal diseases and rheumatoid arthritis. Trastuzumab or Herceptin is an antibody that binds to epithelial growth factor II and is used to treat metastatic breast cancer. In addition, there are many antibodies, including Muromomab, that are used in basic therapies after organ transplantation to prevent graft rejection.
Advantages of using conventional antibodies include the ability of the Fc region to activate the body's immune response and bind to the target molecule to destroy it. Disadvantages of using intact antibodies include the inability to penetrate into certain tissues due to their large size. The ability of the Fc segment to activate some immune responses that are harmful to the patient is a disadvantage of clinically applied intact antibody therapy. The Fc region usually causes some non-specific binding and is detrimental to the application of antibody detection.
Antibody fragment
A fragment of an antibody can be obtained by chemical reagents and genetic engineering methods. The chemical reagent fragments are produced by disrupting the disulfide bond in the hinge region or by digesting the antibody with proteases, including pepsin and papain. Genetically engineered fragments provide a large number of fragments, each with specific binding regions and functional traits.
Fab, Fab', (Fab') 2, and Fv
The antibody is subjected to chemical treatment and protease digestion to obtain an antigen-binding fragment (Fab) derived from the variable regions of the IgG and IgM subtype antibodies. The antibody portion from which the Fabs fragment is removed is the Fc fragment and consists of the constant region of the heavy chain. Antigen-binding fragments include Fab, Fab', (Fab') 2, and Fv. These fragments are capable of binding antigen, but they lack the Fc segment, which includes constant regions 2 and 3 of the heavy chain. When the antibody was digested with papain, two separate F(ab) fragments were isolated from the Fc region. However, after digestion with pepsin, a F(ab')2 fragment with a small portion of the Fc hinge region was isolated from the antibody. Although the separation of antibody fragments by chemical means can produce many useful diagnostic and therapeutic tools, it is very time consuming and requires a large amount of antibody as a raw material.
The monovalent F(ab) fragment has only one antigen-binding region, whereas the multivalent F(ab')2 fragment has two antigen-binding regions that are joined together by disulfide bonds. The F(ab')2 fragment produces two monovalent Fab' fragments and a free thio group that can be used for the binding of other molecules.
The Fv fragment is the smallest fragment of the product after enzymatic analysis of IgG and IgM type antibodies. Fv fragment antigen binding region, which consists of VH and VC regions, but they lack the CH1 and CL regions (Figure 1 right panel). VH and VL are combined in the Fv fragment by non-covalent bonds.
ScFv, bispecific antibody, trispecific antibody, tetraspecific antibody, double-scFv, mimi antibody, Fab2, Fab3
The genetic engineering method is capable of producing a single-chain variable region (ScFv), which is an Fv-type fragment that includes VH and VL regions linked together by a flexible polypeptide (Fig. 1 right). If the binding region has a length of at least 12 residues, the ScFv fragment is the monoclonal antibody. Different forms of Fv molecules can be created by manipulating the length of the V-domain and the hinge region. The resulting scFv molecule whose linker is a 3-11 residue cannot be folded into a functional Fv domain. Together with other scFv molecules, these molecules create a bivalent, bispecific antibody. If the length of the linker is less than 3 residues, the scFv molecules can interact to produce a trispecific or tetraspecific antibody. Multivalent scFvs have a stronger affinity for antigen binding than corresponding monovalent antibodies. The Mini antibody is a scFv-CH3 fusion protein that is loaded into a bivalent dimer. Bis-scFv fragments are bispecific. Miniaturized ScFv fragments can be produced by two different variable regions, allowing these Bis-scFv molecules to simultaneously bind two non-existing epitopes. Genetic methods can be used to generate bispecific Fab dimers (Fab2) and trispecific Fab trimers (Fab3). These antibody fragments are capable of binding to 2 (Fab2) or 3 (Fab3) different antibodies at the same time.
Camel/shark source antibodies and Nanobodies
In addition to conventional antibodies, camelids and sharks also contain specific heavy chain antibodies (hcAbs) that are composed entirely of heavy chain homodimers lacking the light chain. The Fab portion of these antibodies is referred to as VHH (the variable domain of the heavy chain antibody) and is the smallest antigen binding region found in nature. Nanobodies are VHH-derived recombinant domains and are capable of binding antigen. They are very stable and can be easily mass produced by traditional simple systems, such as bacteria (but conventional antibodies with light and heavy chains are difficult to express in bacterial systems) and are therefore promising tools for research and therapeutic applications. Especially in the field of super-resolution microscopy, mass spectrometry and targeted protein degradation. Nanobodies can be delivered to living cells by covalent attachment to the polypeptide, or can be expressed and recognized directly in vivo, but conventional antibodies with both light and heavy chains cannot be used for living cells. For example, anti-GFP Nanobodies are used to develop electromagnetic control systems that study neuronal activity in vivo. When the anti-RFP or GFP Nanobody binds to the far red dye Atto, a fluorescent signal amplification of 118 times more than GFP or RFP can be obtained, which can be used to generate whole body mouse neuronal connections. They can also be used in structural studies to stabilize the active state of proteins. AAV expression vectors have been shown to produce universal influenza vaccines with linked Nanobodies against four different influenza strains. Recombinant anti-mouse and anti-rabbit IgG secondary Nanobodies have the potential to replace widely used polyclonal secondary antibodies. Nanobodies have a unique ability to cross the blood-brain barrier; however, they are often processed and cleared out of the body very quickly. Nanobodies can be used for specific purposes, such as (co)immunoprecipitation or real-time fluorescent protein tracing of intracellular targets in living cells.
Cattle Long CDR3H
Approximately 10% of bovine immunoglobulins contain a long CDR3H region with multiple cysteine residues that are believed to contribute to antibody diversity.
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