Generation of polyclonal anti-idiotype antibodies

An idiotype is a unique antigen-binding site in an antibody and is made up of several epitopes in the variable region. Antibodies raised against the idiotype of a given antibody are called anti-idiotypic antibodies or commonly anti-Ids. These anti-Ids are highly specialized reagents that are produced against human monoclonal antibody (mAb) drug therapeutics. Anti-Id antibodies are generally used for the detection, identification and analysis of therapeutic drugs or as a positive control in immunogenicity assays to support preclinical and clinical development.

Anti-Ids can be generated through polyclonal or monoclonal approaches depending on the needs of the project. They can be produced in a variety of host species such as rodents, rabbits, sheep, goats or even primates. In a majority of situations, polyclonal anti-Id antibodies serve their purpose, but in some cases, monoclonal anti-Ids are warranted.

At Labcorp, we generate polyclonal anti-Ids in various host species using a customized protocol against human mAb therapeutics. This involves preparing Fab fragments from full-length antibodies, wherever applicable, for immunization and monitoring antibody response in an ELISA format against the drug molecule. In some instances, where the fragmentation of antibody is not possible due to absence of proper enzyme recognition sites, full-length antibody is used for immunization. Once the antibody response reaches optimal titers, antiserum is collected and anti-Id antibodies are purified using a two-step procedure. The first step is to purify the total anti-drug antibodies (ADAs) using a positive affinity column to which the drug molecule is covalently coupled. The second step is to remove all the cross-reacting antibodies using a normal human IgG or corresponding isotype coupled to a negative affinity column. This process will result in the enrichment of drug-molecule-specific anti-Id antibodies with minimal cross-reactivity to normal human IgG. The final anti-Id antibody is characterized using standard parameters such as protein concentration, purity and specificity.

The following is an example of a typical project where a polyclonal anti-Id antibody was generated against a human mAb therapeutic.

Anti-Therapeutic Antibody Titers in Rabbit Serum as Measured by ELISA

Specificity of Anti-Id Antibody Titers in Purified Antibody as Measured by ELISA

The above results highlight the successful generation of a polyclonal anti-Id antibody. This reagent specifically recognizes the therapeutic drug molecule while having minimal cross-reactivity with normally circulating human IgG as evidenced by >350-fold selectivity. The antibody reagent was successfully used in both a preclinical and clinical setting to develop a very sensitive ADA assay and as a capture reagent in a PK assay.

Need help generating anti-Id antibodies against your therapeutic for your ADA or PK assays? Whether your project warrants a polyclonal or monoclonal approach, contact us today to discuss your project needs.