What is Conjugation?

 

Conjugation is the process of attaching an antigen to a larger molecule that will ensure that the antigen will activate an immune response that produces antibodies. ProSci’s antibody developers will discuss the chemical properties of your antigen and generate a plan that produces the best antibodies for your assays.

Why is Conjugation Necessary?

B Lymphocytes produce antibodies against foreign antigens that they encounter. However, the antigen must be large enough that the immune system will mount a humoral response – meaning a response involving antibodies secreted by B cells. Thus, in order to produce an antibody to a specific antigen, the researcher must ensure that the antigen is part of a molecular complex that is sufficiently large to activate a humoral response.

Common Conjugates

The two most common conjugates, also known as carrier proteins which antigens attach to, are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). These two molecules are highly immunogenic, meaning they are good at activating humoral response that creates antibodies directed against the antigen. Another conjugate is ovalbumin (OVA), the main protein found in egg whites. OVA is soluble in polar reagents, making it a strategic carrier for antigens that are also polar.

Strategies for Exposing Specific Parts of the Antigen to the Immune System

The N-terminal and C-terminal of polypeptides contain distinct chemical moieties that are good for different types of reactions that result in covalent bonds. Furthermore, the side-chains of the amino acid residues in the polypeptide also have chemical moieties that can be exploited to form desired covalent bonds with a carrier protein. This strategy allows antibody developers to make sure that the antigen is oriented on the carrier protein in such a way that the antibodies produced by an immune reaction will recognize the desired epitope on the antigen. Common chemical cross-linkers used to influence the orientation of antigens on carrier proteins include glutaraldehyde, carbodiimide, benzidine, and succinimide esters.

Extra Steps to Isolate Antibodies That Bind Only to the Antigen

Since antigens attached to carrier proteins are engulfed and digested along with carrier protein, the antibodies that the immune reaction produces will consist of antibodies that bind to different combinations of antigen-carrier fragments. This is problematic of an assay requires a pure solution of antibodies that bind only to the antigen. Extra purification steps need to be done in order to isolate the desired antibodies from those that recognize the carrier protein only or a fragment that contains both antigen and carrier.