Antibodies and Intracellular Antibodies

Antibody Structures and Types

Antibodies, also known as immunoglobulins (Igs), are large, complex, multifunctional proteins produced by the immune system in response to invading foreign antigens, such as microorganisms and viruses. As such, they play a critical role in the immune system's defense against infection and diseases.

In humans, there are five classes of Igs including IgM, IgG, IgA, IgD, and IgE. All classes of human antibodies exist as one or more copies of a Y-shaped unit, composed of four peptide chains with two identical heavy and light chains. The N-terminus of each heavy chain combines with one of the light chains to create two antigen-binding domains, called fragment antigen-binding (Fab) domains (Fig.1). The C-termini of the two heavy chains combines to form a fragment crystallization (Fc) domain. The Fc domain is important for antibody interactions with effector cells (such as macrophages) and for activation of the complement cascade. The four polypeptide chains are linked together by covalent disulfide and non-covalent bonds.

Figure 1. Five main classes (left) and structures of antibodies (right). (Raina, 2009)

Intracellular Antibodies Structures and Types

Intracellular antibody, also called intrabody, is an antibody or a fragment of an antibody that is expressed within a designated intracellular compartment and can be directed to a specific target antigen present in various subcellular locations including the cytosol, nucleus, endoplasmic reticulum (ER), mitochondria, peroxisomes, plasma membrane and trans-Golgi network (TGN) through in-frame fusion with intracellular trafficking/localization peptide sequences.

Intrabodies can be expressed in any shape or form, such as intact IgG molecules, Fab fragment, scFv antibodies, single domain intrabodies, or bispecific tetravalent intrabodies, and the like. Although intrabodies can be expressed in different forms, the most common form is scFv fragment, which is made by linking antigen-binding variable domains of heavy and light chain with an interchain linker, most often the 15 amino acid linker (GGGGS) between the variable heavy and variable light chains.

Antibody Technology

• Polyclonal & monoclonal antibody (PcAb & mAb) technology and production

In PcAbs production, laboratory animals are usually injected with a target antigen to activate many different B cells, which can produce antibodies against multiple epitopes. Therefore, they are considered as a mixture of antibodies that can react with multiple epitopes on the surface of the antigen. PcAbs are generally tolerant of small changes in the antigen, such as heterogeneity of glycosylation, polymorphism, or slight denaturation, but have poor specificity because they contain a wide variety of antibodies.

In mAb production, a B cell produces only one specific sequence of an antibody against a given antigen. Therefore, mAb is a collection of identical antibodies secreted by a single B-cell clone and is specific for only one epitope. mAbs are produced by the fusion of B cells with immortal cells to produce hybridomas, which produce many copies of the exact same antibody. Their high specificity makes mAb very useful in highly targeted immunoassay and diagnostic applications.

• Recombinant antibody technology and production

Recombinant antibodies are generated by cloning antibody genes into expression vectors and do not involve the use of hybridomas. These antibodies can be cloned from any species using suitable oligonucleotide primers. Recombinant antibodies can be produced in many forms, such as scFv fragment, Fab fragments, single-domain antibody and bispecific antibody. In addition, they can be expressed in a variety of hosts, including bacteria, insect cells, yeast, and mammalian cells. Unlike mAbs produced by hybridoma technology, recombinant antibodies maintain high specificity and low immunogenicity for a longer period of time.

Antibodies and Intracellular Antibodies Applications

Some important applications of antibodies in medicine and biomedical research are discussed below:

• Diagnosis
• Antibodies are very useful in medical diagnostics. Many biochemical assays enable the detection of specific antibodies for the diagnosis of diseases.
• Most immunodiagnostic techniques (such as ELISAs) use multiple antibodies to detect specific antigens that can cause infectious diseases.
• In clinical immunology, levels of different classes of Igs help analyze the patient's antibody profile.
• The increase in certain Igs is a useful indicator in the diagnosis of many diseases, e.g., the elevation of IgM is an indication of viral hepatitis.
• Antibodies that can bind to human chorionic gonadotropin are used in over the counter pregnancy test kits.

• Therapy
• Antibodies are used to treat immunodeficiency diseases such as hypogammaglobulinemia, and ready-made antibodies can be administered to patients to induce passive immunity.
• mAbs are widely used to treat a variety of diseases, such as multiple sclerosis, rheumatoid arthritis, psoriasis, and several different cancers including colorectal cancer and breast cancer. To date, the FDA has approved about a dozen mAbs for cancer treatment. Clinical trials are underway to develop more mAbs to help treat more types of cancer.

• Biomedical Research

Advances in biotechnology have made it possible to produce antibodies on a large scale. The high specificity and sensitivity of antibodies make them very important in biomedical research.

• Western blotting: In this technique, proteins are separated by electrophoresis and then transferred to a blotting paper, which is exposed to labeled antibodies to detect the proteins.
• Immunosorbent assays: ELISA is a very popular technique for detecting and quantifying specific antigens. This assay utilizes the high specificity of antibodies to different target antigens. Direct ELISA uses mAbs to detect specific antigens in solution, while indirect ELISA uses primary and secondary antibodies to detect antigens.
• Immunohistochemistry/immunocytochemistry: Both are techniques for determining the presence and location of proteins in situ. In these techniques, primary antibodies are used to bind to target antigens and conjugated secondary antibodies are used to detect the antigen-primary antibody complex.
• Immunoprecipitation (IP) assays: In IP assays, antibodies help label and precipitate target antigens from aqueous solutions. Agarose beads firstly bind to the Fc fragment of the antibody and then allow centrifugation of the antibody-antigen complex.
• Flow cytometry (FC): Antibodies are widely used in FC for intracellular analysis. In this method, single-cell suspensions are stained with highly specific fluorochrome-labeled antibodies and can be easily detected.

Reference

1. Raina, M.; et al. (2009). Environmental Microbiology (Second Edition). Academic Press. 2009.