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An antibody is defined as an immunoglobulin (glycoprotein) capable of specific combination with the antigen that caused its production in a susceptible animal. They are produced in response to the invasion of foreign molecules in the body. Antibodies exist as one or more copies of a Y-shaped unit, composed of four polypeptide chains. Each Y contains two identical copies of a heavy chain, and two identical copies of a light chain, named as such by their relative molecular weights. The top of the Y shape contains the variable region, which is the antigen binding site.
The light chains of any antibody can be classified as either a kappa (κ) or lambda (λ) type (based on small polypeptide structural differences); however, the heavy chain determines the subclass of each antibody.
Antibody structure |
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Heavy chains
There are five types of mammalian Ig heavy chain denoted by the Greek letters: α, δ, ε, γ, and μ. The type of heavy chain present defines the class of antibody; these chains are found in IgA, IgD, IgE, IgG, and IgM antibodies, respectively. Distinct heavy chains differ in size and composition; α and γ contain approximately 450 amino acids, while μ and ε have approximately 550 amino acids.
Each heavy chain has two regions, the constant region and the variable region. The constant region is identical in all antibodies of the same isotype, but differs in antibodies of different isotypes. Heavy chains γ, α and δ have a constant region composed of three tandem (in a line) Ig domains, and a hinge region for added flexibility; heavy chains μ and ε have a constant region composed of four immunoglobulin domains. The variable region of the heavy chain differs in antibodies produced by different B cells, but is the same for all antibodies produced by a single B cell or B cell clone. The variable region of each heavy chain is approximately 110 amino acids long and is composed of a single Ig domain.
Light chains
In mammals there are only two types of light chain, lambda (λ) and kappa (κ). A light chain has two successive domains: one constant domain and one variable domain. The approximate length of a light chain is 211 to 217 amino acids. Each antibody contains two light chains that are always identical; only one type of light chain, κ or λ, is present per antibody in mammals. Other types of light chains, such as the iota (ι) chain, are found in lower vertebrates like Chondrichthyes and Teleostei.
Fab and Fc regions
Direct-conjugated antibodies are labeled with an enzyme or fluorophore in the Fc region. The Fc region also anchors the antibody to the plate in ELISA procedures and is also seen by secondary antibodies in immunoprecipitation, immunoblots and immunohistochemistry. These three regions can be cleaved into two F(ab) and one Fc fragments by the proteolytic enzyme papain, or into just two parts: one F(ab)2 and one Fc at the hinge region by the proteolytic enzyme pepsin. Fragmenting IgG antibodies is sometimes useful because F(ab) fragments (1) will not precipitate the antigen; and (2) will not be bound by immune cells in live studies because of the lack of an Fc region. Often, because of their smaller size and lack of crosslinking (due to loss of the Fc region), Fab fragments are radiolabelled for use in functional studies. Interestingly, the Fc fragments are often used as blocking agents in histochemical staining.
Antibody isotypes
| View high resolution pdf showing antibody isotypes In mammals, antibodies can be divided into five isotypes or classes: IgG, IgM, IgA, IgD and IgE, based on the number of Y units and the type of heavy chain. Heavy chains of IgG, IgM, IgA, IgD, and IgE, are known as gamma, mu, alpha, delta, and epsilon, respectively. |
The isotypes differ in their biological properties, functional locations and ability to deal with different antigens, as depicted in the table below:
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Chicken IgY
There are several advantages to choosing chickens, rather than rabbit or goats to produce your polyclonal antibodies.
1. Chickens are not mammals and therefore are more able to make high-avidity antibodies to mammalian antigens (especially highly conserved mammalian proteins).
2. To our knowledge, it is the most ethical way to produce polyclonal antibodies. There is no need to bleed the chicken. Simply collect the eggs. Our hens are kept in flocks of six in coops that have outside runs. Our unique egg identification system allows our chickens to live freely.
3. A single chicken can produce an enormous amount of antibody, up to 3 g of IgY per month, which is 10-20 times the amount of a rabbit. Furthermore, compared to rabbits, chickens produce antibody much quicker, high-titre antibody is available from eggs as early as day 25.
4. Fc region of chicken IgY is sufficiently different from mammalian IgG:
- Reduces background by not binding to mammalian rheumatoid factors or other naturally occurring anti-mammalian antibodies (e.g. HAMA)
- Does not activate mammalian complement systems
- Does not bind to mammalian Fc receptors
- Does not bind to Staphylococcal protein A or protein G
5. By having the immunoglobulin Y (IgY) packaged conveniently in eggs, one can collect and store eggs over a long period of time and retroactively purify the IgY from the eggs of desired titre/avidity.
6. It is cheaper to feed and house chickens than rabbits.
7. IgY is a stable antibody sharing the following characteristics with mammalian IgG:
- Divalent
- Degraded by papain to yield divalent Fab fragment
- May be enzyme-labeled, biotinylated and gold–labeled by standard procedures
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