Open sandwich ELISA with VH-/VL-alkaline phosphatase fusion proteins

The Sandwich ELISA is a widely used technique to measure antigen concentration. Recently, a novel ELISA based on the interchain interaction of separated V-H and V-L chains from a single antibody variable region (Fv) was proposed (Open Sandwich ELISA). Since it employs a single antibody recognizing one epitope, the assay requires, in essence, only one cycle of incubation and washing steps. To demonstrate this directly, we have constructed a recombinant gene fusion encoding the V-H chain of an anti-hen egg lysozyme (HEL) antibody HyHEL-10 and Escherichia coli alkaline phosphatase (V-H-PhoA). The same type of gene fusion using V-L chain instead of V-H chain (V-L-PhoA) was also constructed and the proteins were obtained with an E. coli expression/secretion system. Open Sandwich ELISAs were performed using microtiter plates with immobilized V-L or V-H fragment, and V-H-PhoA or V-L-PhoA, respectively, as the detection reagent which was simultaneously added to each well with samples. As a result, HEL concentrations in the samples were determined after one round of incubation and washing steps, with a signal generated in a direct relationship to the concentration of HEL added to the reaction mixture. The minimum detectable HEL concentration Mras similar to 10 ng/ml, which was almost equal to the value previously obtained with plate-immobilized V-L and V-H fragment displayed on M13 phage. When the active-site mutant V-H-PhoA(D101S) was employed instead of V-H-PhoA and reacted at an optimum pH of 10, a significant enhancement in signal was attained. (C) 1999 Elsevier Science B.V. All rights reserved.