Open sandwich ELISA with V(H)-/V(L)-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 was approximately 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.
Suzuki
C
C
Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, Japan.
Ueda
H
H
Tsumoto
K
K
Mahoney
W C
WC
Kumagai
I
I
Nagamune
T
T
D000469:Alkaline Phosphatase / Q000235:genetics / Q000276:immunology*; D000595:Amino Acid Sequence; D001483:Base Sequence; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D007128:Immunoglobulin Fragments / Q000276:immunology*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000276:immunology*; D007147:Immunoglobulin Light Chains / Q000235:genetics / Q000276:immunology*; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology*; D007700:Kinetics; D008969:Molecular Sequence Data; D009113:Muramidase / Q000235:genetics / Q000276:immunology*; D016296:Mutagenesis; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology
D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; C055966:immunoglobulin Fv; D000469:Alkaline Phosphatase; D009113:Muramidase