Related Experiment Videos
Pitfalls in the use of ELISA to screen for monoclonal antibodies raised against small peptides
S C Smith1, N McIntosh, K James
1Department of Child Life and Health, University of Edinburgh, Scotland, UK.
Small peptides are often conjugated to large carrier proteins such as thyroglobulin to increase their immunogenicity. Antibodies are raised against peptide, thyroglobulin and contaminants, such as immunoglobulins or BSA in the thyroglobulin preparation, but anti-contaminant antibodies will not be revealed if immune serum is diluted in buffer containing immunoglobulin-contaminated BSA. This may lead to the development of an inappropriate hybridoma-screening assay which detects more anti-contaminant than anti-peptide antibodies if ELISA plates are coated with a capture antibody or blocked with BSA. Dilution of culture supernatants in buffer containing BSA and Tween 20 minimises the risk of false positive results and makes plate-blocking unnecessary. The high affinity peptide-specific antibodies in immune serum, which are more readily detected than lower affinity monoclonal antibodies, may result in an inadequately sensitive hybridoma-screening ELISA.
Small peptides are often conjugated to large carrier proteins such as thyroglobulin to increase their immunogenicity. Antibodies are raised against peptide, thyroglobulin and contaminants, such as immunoglobulins or BSA in the thyroglobulin preparation, but anti-contaminant antibodies will not be revealed if immune serum is diluted in buffer containing immunoglobulin-contaminated BSA. This may lead to the development of an inappropriate hybridoma-screening assay which detects more anti-contaminant than anti-peptide antibodies if ELISA plates are coated with a capture antibody or blocked with BSA. Dilution of culture supernatants in buffer containing BSA and Tween 20 minimises the risk of false positive results and makes plate-blocking unnecessary. The high affinity peptide-specific antibodies in immune serum, which are more readily detected than lower affinity monoclonal antibodies, may result in an inadequately sensitive hybridoma-screening ELISA.