RESUMEN
Recombinant antibody libraries based on chicken immunoglobulin genes are potentially valuable sources of phage-displayed scFvs for use in veterinary diagnostics and research. To add diversity to the scFv repertoire, we expanded the library to include genes from the ostrich, indigenous to southern Africa. The libraries described in this chapter are based on the chicken and ostrich variable heavy and light chain immunoglobulin genes joined by a short flexible linker cloned in the phagemid vector pHEN1. The resulting phagemids produce either scFvs displayed on the surface of the fusion phage subsequent to co-infection with helper phage or soluble scFvs following IPTG induction. This chapter provides detailed and proven methods for the construction of such libraries.
Asunto(s)
Struthioniformes , Animales , Pollos , Anticuerpos , Cadenas Ligeras de Inmunoglobulina , Genes de InmunoglobulinasRESUMEN
Mycolic acids (MA) are major, species-specific lipid components of Mycobacteria and related genera. In Mycobacterium tuberculosis, it is made up of alpha-, methoxy- and keto-MA, each with specific biological functions and conformational characteristics. Antibodies in tuberculosis (TB) patient sera respond differently towards the three MA classes and were reported to cross-react with cholesterol. To understand the antigenicity and cholesterol cross-reactivity of MA, we generated three different chicken -derived phage-displayed single-chain variable fragments (scFv) that reacted similarly towards the natural mixture of MA, but the first recognized all three classes of chemically synthetic MAs, the second only the two oxygenated types of MAs and the third only methoxy MA. The cholesterol cross-reactivity was investigated after grafting each of the three scFv types onto two configurations of constant chain domains-CH1-4 and CH2-4. Weak but significant cross-reactivity with cholesterol was found only with CH2-4 versions, notably those two that were also able to recognize the trans-keto MA. The cholesteroid nature of mycobacterial mycolic acids therefore seems to be determined by the trans-keto MA subclass. The significantly weaker binding to cholesterol in comparison to MA confirms the potential TB diagnostic application of these antibodies.
Asunto(s)
Colesterol/inmunología , Mycobacterium tuberculosis/inmunología , Ácidos Micólicos/inmunología , Anticuerpos de Cadena Única/inmunología , Animales , Proteínas Aviares/inmunología , Pollos , Colesterol/química , Reacciones Cruzadas , Ensayo de Inmunoadsorción Enzimática , Células HEK293 , Humanos , Estructura Molecular , Ácidos Micólicos/síntesis química , Ácidos Micólicos/química , Unión Proteica , Ingeniería de Proteínas , Proteínas Recombinantes , Sensibilidad y EspecificidadRESUMEN
Recombinant antibody libraries based on chicken immunoglobulin genes are potentially valuable sources of phage displayed scFvs for use in veterinary diagnostics and research. The libraries described in this chapter are based on chicken variable heavy and light chain immunoglobulin genes joined by a short flexible linker cloned in the phagemid vector pHEN1. The resulting phagemids produce either scFvs displayed on the surface of the fusion phage subsequent to co-infection with helper phage, or soluble scFvs following IPTG induction. This chapter provides detailed and proven methods for the construction of such libraries.
Asunto(s)
Proteínas Aviares/genética , Pollos/genética , Biblioteca de Genes , Anticuerpos de Cadena Única/genética , Animales , Proteínas Aviares/inmunología , Pollos/inmunología , Anticuerpos de Cadena Única/inmunologíaRESUMEN
Two chicken single-chain variable region antibody fragments (scFvs) that recognised the 65 kDa heat-shock protein (HSP65) of Mycobacterium bovis were selected from a large semi-synthetic phage displayed library. Both recognised HSP65 in indirect enzyme-linked immunosorbent assay (ELISA) and immunoblots and retained their activity during storage. Neither, however, could function as the capture reagent in a sandwich ELISA when immobilised on polystyrene. To establish whether they could be engineered for general use in immunotests, the genes coding for these scFvs were subcloned in expression vectors that contained sequences encoding chicken IgY heavy-chain constant region domains. This resulted in larger bivalent constructs which more closely resembled IgY molecules. The engineered fragments were evaluated in ELISAs and gold-conjugated immunochromatographic tests (ICTs). In contrast to their previous behaviour as scFvs, the modified fragments (designated "gallibodies") could be used for immunocapture in ELISA and could be readily conjugated to colloidal gold nanoparticles. A sandwich ICT that could detect recombinant HSP65 was also devised. Although converting the recombinant single-chain monomeric antibody fragments to bivalent immunoglobulin-like molecules did not entirely 'standardise' the behaviour of the scFvs, this approach remains potentially useful for developing practical, robust, immunodiagnostic reagents.
