over–expression and purification of recombinant nanobodies and preparation of conjugated second antibody against them

purpose: 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 vhs illustrates the potential application of these novel products as an immunodiagnostic immunotherapeutic reagent. one of the key components in production, characterization and application of nanobodies in detection is the anti-nanobodiy hrp conjugate. materials and methods: here, we report 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 chromatography (imac). described here is the preparation, purification and characterization of anti-nanobody antibody hrp conjugate for use in the various nanobody detection systems. results: 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. conclusion: by setting the temperature, time and inducer reagent the nanobodies were produced in optimum yield. we concluded that the hrp conjugated anti-nanobody can detect nanobodies in various detection procedures with great sensitivity and accuracy.