Production and characterization of monoclonal antibodies directed against native epitopes of NhaA, the Na+/H+ antiporter of Escherichia coli.
Monoclonal antibodies (mAbs) recognizing native epitopes are an important tool for functional and structural studies of proteins, yet they have rarely been used with transport proteins. In an attempt to raise monoclonal antibodies against the NhaA Na+/H+ antiporter of Escherichia coli we encountered difficulties in the screening procedure, which is based on the standard enzyme-linked immunosorbent assay (ELISA). Here we report a rapid and efficient method of screening for anti-NhaA mAbs which recognize native epitopes of the antiporter. The method is based on the use of His-tagged protein, Ni2+-nitrilotriacetic acid coated plates and non-denaturing conditions in the assay. With this procedure four mAbs were obtained, three of which recognize the NhaA in its native conformation and one preferentially recognizes the denatured form. The latter mAb is Western blot positive, the others are Western blot negative and bind the detergent solubilized NhaA as assayed by gel filtration chromatography. Competition experiments show that the native epitopes are common to both the His-tagged and the wild-type protein. We suggest that in the standard ELISA the NhaA protein is not presented to the antibody in the native conformation whereas the His tag based protocol favors this presentation. Indeed, we could remarkably improve the low reactivity of the standard ELISA by coating the plates with anti-NhaA mAb and use it to present NhaA ('sandwich' ELISA or two antibodies assay). Remarkably, two of the mAbs (5H4, 2C5) which bind native NhaA inhibit drastically the deltapH driven 22Na uptake mediated by His-tagged NhaA reconstituted in proteoliposomes. Hence, these mAbs afford a new tool to study the structure/function relationship of the antiporter.
Padan
E
E
Division of Microbial and Molecular Ecology, The Hebrew University of Jerusalem, Israel.
Venturi
M
M
Michel
H
H
Hunte
C
C
D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000032:analysis / Q000276:immunology; D015153:Blotting, Western; D002850:Chromatography, Gel; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis*; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005260:Female; D007074:Immunoglobulin G; D007145:Immunoglobulin kappa-Chains; D008081:Liposomes; D051379:Mice; D008807:Mice, Inbred BALB C; D011510:Proteolipids; D017923:Sodium-Hydrogen Exchangers / Q000032:analysis* / Q000276:immunology*
D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D000939:Epitopes; D029968:Escherichia coli Proteins; D007074:Immunoglobulin G; D007145:Immunoglobulin kappa-Chains; D008081:Liposomes; C102839:NhaA protein, E coli; D011510:Proteolipids; D017923:Sodium-Hydrogen Exchangers; C020485:proteoliposomes