Open sandwich ELISA with V-H-/V-L-alkaline phosphatase fusion proteins

The Sandwich ELISA is a widely used technique to measure antigen concentrat ion. Recently, a novel ELISA based on the interchain interaction of separat ed V-H and V-L chains from a single antibody variable region (Fv) was propo sed (Open Sandwich ELISA). Since it employs a single antibody recognizing o ne epitope, the assay requires, in essence, only one cycle of incubation an d washing steps. To demonstrate this directly, we have constructed a recomb inant 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) w as also constructed and the proteins were obtained with an E. coli expressi on/secretion system. Open Sandwich ELISAs were performed using microtiter p lates with immobilized V-L or V-H fragment, and V-H-PhoA or V-L-PhoA, respe ctively, as the detection reagent which was simultaneously added to each we ll with samples. As a result, HEL concentrations in the samples were determ ined after one round of incubation and washing steps, with a signal generat ed in a direct relationship to the concentration of HEL added to the reacti on mixture. The minimum detectable HEL concentration Mras similar to 10 ng/ ml, which was almost equal to the value previously obtained with plate-immo bilized V-L and V-H fragment displayed on M13 phage. When the active-site m utant V-H-PhoA(D101S) was employed instead of V-H-PhoA and reacted at an op timum pH of 10, a significant enhancement in signal was attained. (C) 1999 Elsevier Science B.V. All rights reserved.