Fluorophor-linked immunosorbent assay: a time- and cost-saving method for the characterization of antibody fragments using a fusion protein of a single-chain antibody fragment and enhanced green fluorescent protein.
A novel assay, referred to as fluorophor-linked immunosorbent assay (FLISA), for the characterization of single-chain antibody fragments (scFvs) is described. The principle of the method is the fusion of an scFv to enhanced green fluorescent protein (EGFP). The scFv domain, which binds to the immobilized hapten, can be detected by measuring the fluorescence of the EGFP domain. The time-consuming binding of secondary antibodies and enzyme reaction, necessary for enzyme-linked immunosorbent assays (ELISAs) are not required. Consequently, the assay time of 1.5 h needed to complete the FLISA is much shorter than that of comparable ELISAs, which require about 5 h. This renders the FLISA suitable for applications where a short assay time is essential, such as screening of mutant libraries of scFvs in directed evolution experiments or monitoring of the amount of functionally expressed recombinant protein during production processes. In contrast to a comparable ELISA, the FLISA showed no saturation when determining the relative amount of functional scFv. The amount of the soluble fraction of cell extracts from Escherichia coli expressing the fusion protein and the normalized fluorescence signal showed a linear correlation with R(2)>0.99. The usefulness of a competitive FLISA for the detection of analytes is shown exemplarily by the detection of s-triazines with the s-triazine-specific scFv K411B.
Oelschlaeger
Peter
P
Institute of Technical Biochemistry, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany.
Srikant-Iyer
Srividhya
S
Lange
Stefan
S
Schmitt
Jutta
J
Schmid
Rolf D
RD
D000918:Antibody Specificity; D002138:Calibration; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005470:Fluorometry / Q000379:methods*; D049452:Green Fluorescent Proteins; D006241:Haptens / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D007128:Immunoglobulin Fragments / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D007163:Immunosorbent Techniques; D008164:Luminescent Proteins / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D057127:Single-Chain Antibodies; D014227:Triazines / Q000302:isolation & purification
D017931:DNA Primers; D006241:Haptens; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; D014227:Triazines; C410726:scFV green fluorescent protein, recombinant; D049452:Green Fluorescent Proteins