| Genetically engeering antibodies are artificial constructions obtained by recombining the antibody molecule or synthesizing the gene sequence through gene recombination technology and then the antibody DNA was transfrmed into cells for expression. The genetically engeering antibodies keep the binding properties of the classical monoclonal antibodies. As one of the most remarkable molecules of genetically engeering antibodies, ScFv (single chain variable fragment) only has the variable heavy and light chain region, but keeps the binding properties of classical antibody. The use of ScFvs is a new strategy for developing improved immunodetection tests. ScFvs can be produced by conventional hybridoma, phage display technology or ribosome display technology, and the ribosome display has some obvious advantages than others, such as simple method for construction of library and selection of antibody, no panning pressure, and improved antibody affinity by introducing mutation.Pesticides have made major contributions to agriculture and disease control but widespread use has created serious concerns regarding their effects on the environment and on human health. Organophosphorus (OP) pesticides are among the most widely used in horticulture, agriculture, and forestry. In high doses, OP pesticides can cause respiratory, myocardial, and neuromuscular impairments, necessitating high-sensitivity detectors for environmental and biological samples. Current methods, such as gas chromatography (GC) and high-performance liquid chromatography (HPLC), have been used successfully, with high sensitivity and reliability for analysis of many pesticides including OP pesticides. These methods are expensive and time-consuming, however, and can only be performed by skilled analysts at off-site laboratories. Therefore, there is a growing demand for more economical, rapid, and portable methods for detection of OP pesticide residues. Immunoassays such as enzyme-linked immunosorbent assay (ELISA) have the potential to become an alternative or a complementary method because they have proven to be sensitive, fast, and cost-effective tools for determining trace amounts of chemicals such as pesticides. immunoassay techniques rely largely on the availability of high-quality antibodies against target analytes.Ribosome display has been used for selection and evaluation of ScFvs against bacteria, viruses, hormones like progesterone, various drugs, and proteins, but not against OP pesticides. Here, we describe the isolation and characterization of ScFvs against fenitrothion, one of the most widely used OP pesticides. These recombinant antibody fragments were selected from a ScFv DNA library and expressed as secreted ScFvs in E. coli.The main results are as follows:①The hapten of fenitrothion was covalently attached to bovine serum albumin (BSA) or ovalbumin (OVA) by diazotization . The antigen with BSA was used as the immunogen (FE-BSA), while the hapten-OVA (FE-OVA) conjugate was used as the coating antigen for affinity selection and ELISA②The fenitrothion-BSA was used to immune the BALB/c mice and the titer of immunized mouse was 2500-fold.③The spleen of the mouse with the highest titer was isolated and RNA was extracted to construct the FcVs cDNA library. The size of amplificated VH was 400 bp and that of k-chain was 700 bp. The VH and VL were ligated with the linker (Gly3Ser)4 and the assembled ScFvs were of expected 1.1 kb size. The ScFv DNA was cloned into PMD18-T, and nine clones were selected randomly for sequencing. The result demonstrated that each CDR of ScFvs was different and the amine acid sequences of the CDRs were diversity. It indicated that the constructed library was diversity and could be used to selection of specific ScFvs.④In each round of ribosome display, the VH/k-DNA library was used for in vitro transcription with T7 RNA polymerase and the mRNA transcripts was translated in rabbit reticulocyte lysate system to produce antibody–ribosome–mRNA (ARM) complexes. The ARM complexes were then added to the microtiter plates coated fenitrothion-OVA and incubated. After washing, the remained ARM complexes were dissociated and the released mRNA was recovered by RT-PCR.⑤DNA outputs from the unselected and the third selected antibody library were ligated with express vector pPOW3.0 and then cloned into E.coli DH5αfor expression. After transformation, 50 clones from the unselected libraries and 100 clones from the third selected were picked randomly and their soluble proteins were expressed. The cell extracts from these clones were tested by indirect ELISA with the coating antigen FE-OVA. All tested clones from the unselected library showed little conjugation activity to FE-OVA, but about 25% of the isolated clones from the selected library showed high binding activity . Furthermore, none of the clones exhibited significant binding activity to (unconjugated) OVA, indicating that the recombinant antibodies against fenitrothion were enriched by ribosome display.⑥After screening, three clones (ScFv-AF50, AF93 and AF132) were chosen for further study. The affinity and specificity of the three ScFvs were tested by CI-ELISA. The IC50 values for fenitrothion with ScFv-AF50, ScFv-AF93 and ScFv-AF132 were 1.6, 3.4, and 2.2 ng/ml, respectively. The cross-reactivity with other organophosphorus (OP) pesticides was below 0.1% except for parathion-methyl (≤2.8%). The negligible cross-reactivity indicated that the soluble ScFv fragment antibodies of the three positive clones had high specificity to hapten of fenitrothion. The equilibrium dissociation constants (KDs) determined by Biacore analysis for ScFv-AF50, ScFv-AF93 and ScFv-AF132 were 4.56×10-10 M, 1.42×10-9 M and 2.66×10-10 M, respectively⑦The amplified DNA fragments coding for the ScFv-AF50, ScFv-AF93, and ScFv-AF132 were characterized by sequence analysis. All three sequences had different amino acid substitutions, and as expected, most of these were in the complementarity-determining regions (CDRs). The VH and VL gene families of the ScFvs were designated based on the international immunogenetics (IMGT) database. The light chains of all three clones belonged to the same Vk 4 family. The heavy chains of ScFv-AF50 and ScFv-AF132 belonged to the VH4 gene family while the ScFv-AF93 heavy chain belonged to the VH1 gene family. Sequencing alignment by the basic local alignment search tool (BLAST) demonstrated that the VH of the clones ScFv-AF50 and ScFv-AF93 had very similar sequences (97% homology) and that the VL of ScFv-AF50 and ScFv-AF132 shared 89% homology.⑧Rice and cucumber were spiked with fenitrotion and analyzed by both CI-ELISA and GC. The ScFv-AF132 clone was used in the CI-ELISA as it possessed the highest affinity. Recoveries of fenitrothion from food samples ranged from 80.6-108% for CI-ELISA and 64-92% for GC. Both methods demonstrated acceptable sensitivity, but the ELISA was superior, especially when the extraction solution contained fenitrothion concentrations near the IC50 of AF132.
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