Construction of antibody single variable region-alkaline phosphatase fusion proteins for novel one step detection method

摘要

We have constructed recombinant gene fusion encoding VH of an anti-hen egg lysozyme (HEL) antibody HyHEL-10, and Escherichia coli alkaline phosphatase (V_H(HEL)-PhoA). The same type of gene fusion using VL instead of VH (V_L(HEL)-PhoA) was also constructed. The recombinant fusion proteins were expressed in E. coli with good yields, purified via metal based affinity resin followed by anion exchange chromatography. VL and VH fragments of HyHEL-10 were produced using an E. coli secretion system. Then a novel immunoassay based on the interchain interaction of antibody Fv region (Open Sandwich ELISA) was performed using an immobilized VL fragment and V_H(HEL)-PhoA. HEL concentration was determined in one step with a signal generated in a direct relation to the concentration of HEL added to the reaction mixture. Minimum detectable HEL concentration was less than 10ng/ml, which was approaching to that of the standard ELISA.