Fragment Binding In IgG And IgM Antibodies

Thu, 02/04/2010 - 12:10

Research facilities often require a supply of antibodies of all classes, in both their whole and fragmented forms. There are both pros and cons to using fragments in place of entire proteins. As IgG is the most prevalent class on our antibody database, we’ll cover that one first.

IgG consists of two heavy chains and two light chains, held together by hydrophobic interactions and disulphide bonds. Secondary (anti-IgG) antibodies are derived by injecting an animal of one species (e.g. a sheep) with whole Ig from another species (e.g. mouse) that produced the 1° Ab to the antigen. This results in polyclonal secondary Ab which reacts to both the H and L chains. It reacts to every possible primary epitope, resulting in the largest possible signal.

However, the same light chains are common in all the antibody classes, and since 1° Abs are a heterogynous mix, it’s possible the 2° Ab may cross-react with other classes of immunoglobulins. This is a problem in immunohistochemistry procedures, where heterogeneous tissue samples are used rather than cultured cells.

The upper part of the gamma (γ) heavy chains of IgG contains a terminal variable (V) region plus a constant (C) region. The V region binds to the antigen, meaning the same antibody type can be generated to show specificity to many different immunogens. γ - specific 2° antibodies only target fragments of the IgG molecule containing all or part of the gamma chain i.e. whole IgG, Fc, Fab and F(ab')2 fragments. Thus using γ – specific Abs removes L-chain cross-reactivity.

We at Novus Biologicals produce a wide range of gamma-chain specific secondary antibodies. They are prepared by affinity purification and adsorption to remove any antibodies that recognise light chains. This reduces the risk of non-specific binding and produces a clearer signal.


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