Production and characterization of anti‐(mucin MUC1) single‐domain antibody in tobacco (Nicotiana tabacum cultivar Xanthi)

Abstract

Members of the Camelidae (camels, dromedaries, llamas, alpacas, guanacos and vicunas) are known to produce Igs (immunoglobulins) devoid of light chains and CH1s (constant heavy-chain domains). The antigen-specific binding fragments of these heavy-chain antibodies therefore comprise one single domain (the so-called 'VHH') and are of great importance in biotechnological applications. To evaluate the expression and biological activity of sdAbs (single-domain antibodies) in plants, which, on account of their small size and antigen-recognition properties, would have a major impact on antibody-engineering strategies, we constructed a pBI121-VHH gene encoding the recombinant sdAb fragments with specificity for a cancer-associated mucin, MUC1. Analysis of transgenic tobacco (Nicotiana tabacum cultivar Xanthi) plants by PCR and Western blotting demonstrated the expression of sdAb, while ELISA results with various MUC1 antigens and immunocytochemistry with cancerous cell lines confirmed that the activity of these molecules compared favourably with that of the parent recombinant antibodies. Protein purification was achieved by using sequential (NH4)2SO4 precipitation, gel filtration and immunoaffinity chromatography. Analysis of the purified VHH by ELISA indicated that the purified antibody fragments were able to react successfully with a MUC1-related peptide. These results reaffirm that the tobacco plant is a suitable host for the production of correctly folded VHH antibody fragments with diagnostic and therapeutic applications.

Characterization of single-domain antibodies against Foot and Mouth Disease Virus (FMDV) serotype O from a camelid and imaging of FMDV in baby hamster kidney-21 cells with single-domain antibody-quantum dots probes

“…The use of sdAbs can overcome these disadvantages and enable new functional studies. SdAbs are the smallest functional antibodies and are easily produced in prokaryotic and eukaryotic microorganisms [ 8 , 10 , 33 ]. In addition, they can be widely used in immunofluorescence techniques to capture antigens because of their high affinity [ 34 , 35 ].…”

“…Given their small size, high physicochemical stability, facile genetic manipulation, and capability to bind epitopes inaccessible to conventional antibodies, sdAbs are ideal antigen-binding elements [ 7 ]. In addition, sdAbs are highly expressed and easily purified in Escherichia coli [ 8 ], yeasts [ 9 ], mammalian cell lines, and plants [ 10 ]. Thus, these antibodies have various medical and biotechnological applications, such as in cancer therapy [ 11 , 12 ].…”

Nanobodies to Study G Protein–Coupled Receptor Structure and Function

“…Nanobodies, the recombinant variable domains of the heavy-chain antibodies, are monomeric single-domain proteins encoded by single-gene fragments and harbor the full antigen-binding capacity of the parental antibody (63). Nanobodies are produced easily in microorganisms (64–67), mammalian cell lines (68), and plants (69, 70) and exhibit superior stability compared to recombinant derivatives of conventional antibodies such as single-chain variable fragments (71). …”

Anti-VP6 VHH: An Experimental Treatment for Rotavirus A-Associated Disease

“…VHH consist of only one polypeptide chain and are expressed to a high level in microorganisms [24,25], mammalian cell lines, insect larvae [26] and plants [27,28] with low production costs [29], which represents an advantage for their application in massive treatment strategies. In the present work, we investigated llama-derived single-chain Ab fragments -VHH- directed to VP6 as a treatment against RVA-associated disease.…”