assay protocol

Antibody- description, structure, function, application

The term "antibody" dates to 1901. Antibodies are a family of glycoproteins that bind specifically to foreign molecules (antigens), can also be called immunoglobulin (Ig). The term immunoglobulin can be used to refer to any antibody-like molecule, regardless of its antigen-binding specificity. The frame of an immunoglobulin molecule is like a letter Y as showed in the picture below.


Antibody structure-related concepts:

Light chain and heavy chain:

IgG consists of two light chains and two heavy chains; each light chain pairs with a heavy chain, and each heavy chain pairs with another heavy chain.

a. The chains are linked by covalent inter-chain disulfide bonds and noncovalent interactions.

b. The two light chains (23 kD each) and two heavy chains (50 kD each) are identical.

Fab fragment and Fc fragment:

a. Fab fragments (50 kD) consist of the entire light chain and part of the heavy chain.

b. Fab fragments contain the antigen-binding sites.

c. The Fc fragment (50 kD) crystallizes in cold and does not bind antigen.

Variable region and constant region:

Each heavy and light chain consists of a large region that is constant in different types of antibodies (even from different species) and a similar-sized region that shows great variability in amino acid sequence among the chains. This large region is called the constant (C) region and the opposite end is called the variable (V) region.

Antibody category:

1. Divided by amino acid sequence or structure:

Antibodies are divided into classes by the antigenic determinants on their heavy chains. The antigenic determinants of antibodies show three levels of variability. The determinants (from general to specific) are called isotypic, allotypic, or idiotypic and classify antibody molecules into isotypes, allotypes, and idiotypes, respectively. Isotypic determinants, found on the C-region part of heavy chains, divide antibodies into five classes (or isotypes) called IgG, IgA, IgM, IgD, and IgE. Isotypic determinants also can be used to classify antibody light chains into κ or λ chains. Isotypic differences can be used to partition classes into subclasses. Allotypic determinants are carried by only some individuals within a given species and are inherited in a mendelian fashion. Idiotypic determinants are individual-specific and represent the antigen-combining site of an antibody.

For antibodies in human body, IgG is the major antibody in the blood, but it is able to enter tissue spaces and coat antigens, speeding antigen uptake. IgA concentrates in body fluids to guard the entrances of the body. IgM is the largest antibody; it tends to remain in the blood, where it can lead to efficient killing of bacteria. IgD remains membrane-bound and somehow regulates the cell's activation. IgE is found in trace amounts in the blood, but it still triggers allergies.

2. Divided by function:

Beginning with the immune response, depending on the nature of the antigen and the site of entry this response can take up to 14 days to resolve and leads to the generation of memory cells with a high specificity for the inducing antigen.

A primary antibody is one that directly recognizes and binds to a specific invading particle (antigen).

A secondary antibody is an antibody that binds to a primary antibody or a fragmented invader. They can be labeled with biotins for applications of detection, purification or cell sorting etc.