Over-expression and purification of recombinant nanobodies and preparation of conjugated second antibody against them

The antigen binding fragments of camelids heavy chain antibodies are comprised in one single domain (VHH). The crystal structure of an isolated VHH indicated that it is a problate particle of 2.5 nm in diameter and <4 nm high, and has been referred to as a nanobody. The very close similarity of these molecules to human VH3 illustrates the potential application of these novel products as an immunodiagnostic immunotherapeutic reagent. One of the key components in roduction, characterizationand application of nanobodies in detection is the anti-nanobody HRP conjugate. In this study, we reported high expression and purification of some nanobodies against tumor markers. The nanobodies genes were sub-cloned into a pSJF9 vector to over-express the protein coupled with fusion tags in E. coli TG1. The expressed nanobodies were purified by immobilized metal affinity romatography (IMAC). Described here is the preparation, purification and haracterization of anti-nanobody antibody HRP conjugate for use in the various nanobody detection systems. Analysis by SDS-PAGE and Western blotting demonstrated the integrity of the purified nanobodies. Because of application of several biochemical modifications, the produced anti-nanobodies HRP conjugate have efficient sensitivity and specificity.