Sensitive Western immunoblot detection of the TrpC3 biomarker
in human sera requires the development, affinity purification and
validation of highly specific synthetic peptide antibodies

Antibody Development

Larial's platform for synthetic peptide antibody development is an important facet of its successful biomarker screening program. Often, high affinity probes of sufficient purity are either not commercially available or their use cannot be properly validated in particular applications that require high detection sensitivities. Peptide conjugate immunogens are prepared using an improved, water-soluble KLH carrier that permits high reaction stoichiometries and ensures sufficient peptide conjugation that will elicit a good humoral antibody response. Maleimide coupling efficiencies are carefully monitored by an enzymatic process to ensure optimal conjugation stoichiometries are achieved. Antibodies are purified from animal sera using a proprietary process that first involves evaluating the affinities of immune serum globulins to a variety of peptide targets immobilized on biosensor surfaces. Peptides with reduced binding affinities are conjugated to a BSA carrier immobilized on cyanogen bromide-activated Sepharose 4B. Elution of antibodies using milder conditions to dissociate immune complexes with peptide conjugates enables more kinetically active reagents to be prepared with peptide affinities and specificities more closely resembling immune globulins present in crude antisera. Yields are also greatly improved as higher affinity antibodies are more readily recovered from affinity columns. Double affinity purification of serum antibodies using Larial's technology produces highly purified, functional probes capable of detecting very low amounts of circulating and tissue antigens. Direct chemical or biological tagging of the affinity-purified antibodies also permits signal enhancement without a correspondingly increased background. Synthetic peptide antibodies properly prepared and validated in this fashion provide excellent assay performance and are stable for many years.