title: Cloning of scFv from hybridomas using a rational strategy: Application as a receptor to sensitive detection microcystin-LR in water.
abstract: Single chain variable fragment (scFv), containing of heavy and light chains (VH and VL) joined by a short peptide linker, has been used widely for immunodetection. Nevertheless, cloning functional variable genes is still a bottle neck for the scFv generation technology. Here, a rational strategy for cloning and selecting variable region genes from an anti-microcystin-LR hybridoma was devised, then the functional VH and VL genes were recloned and assembled to scFv using splicing overlap extension PCR. The resulting scFv gene was recombinantly expressed as a soluble scFv-alkaline phosphatase fusion protein (scFv-AP) by vector PLIP6/GN. Then an indirect competitive chemiluminescent enzyme immunoassay (ic-CLEIA) for detection of microcystin-LR was developed. The half-maximum inhibition concentrations (IC50) and limits of detection (LODs, IC15) were 0.81 ± 0.04 μgL(-1) and 0.13 ± 0.03 μgL(-1), respectively. With the mean coefficient of variation lowing 8%, the mean recovery in intra-assay and inter-assay were 100.06% and 96.46%, The proposed strategy should be useful for generation scFv in a rapid and simple way.
authors.affiliation: Hebei North University, Food Safety Research Centre of Hebei North University, Zhangjiakou, 075000, China; College of Animal Science, Inner Mongolia Agricultural University, Hohhot, 010018, China.
authors.forename: Xiuyuan
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Hebei North University, Food Safety Research Centre of Hebei North University, Zhangjiakou, 075000, China. Electronic address: hekuo7825@163.com.
authors.forename: Kuo
authors.initials: K
authors.lastname: He
authors.affiliation: Hebei North University, Food Safety Research Centre of Hebei North University, Zhangjiakou, 075000, China.
authors.forename: Ruiping
authors.initials: R
authors.lastname: Zhao
authors.affiliation: Hebei North University, Food Safety Research Centre of Hebei North University, Zhangjiakou, 075000, China.
authors.forename: Lixia
authors.initials: L
authors.lastname: Wang
authors.affiliation: Hebei North University, Food Safety Research Centre of Hebei North University, Zhangjiakou, 075000, China.
authors.forename: Yandan
authors.initials: Y
authors.lastname: Jin
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics; D003001:Cloning, Molecular; D006825:Hybridomas; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000276:immunology; D007147:Immunoglobulin Light Chains / Q000235:genetics / Q000276:immunology; D008387:Marine Toxins; D052998:Microcystins / Q000032:analysis* / Q000276:immunology; D016133:Polymerase Chain Reaction; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology*; D014874:Water Pollutants, Chemical / Q000032:analysis*
chemical_list: D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D008387:Marine Toxins; D052998:Microcystins; D057127:Single-Chain Antibodies; D014874:Water Pollutants, Chemical; D000469:Alkaline Phosphatase; C057862:cyanoginosin LR
keywords: Functional heavy and light chains; Microcystin-LR; Orbitrap mass spectrometry; Single-chain variable fragment