Selection Of Special ScFv Antibody Against The Furazolidone By Vitro Display Technology

The researches have found that furazolidone is small molecule which is carcinogenic,teratogenic and mutagenic.It cannot be quickly and effectively degraded and will take great risk to human health.Therefore,research and development with independent intellectual property rights,rapid and sensitive detection of furazolidone immunoassay is an urgent need to address the task.The total RNA extracted from the hybridoma cells was used to synthesize first strand cDNA,through which the full set of heavy-chain and light-chain variable region were amplified by PCR,and the single chain gene of VH-Linker-VL fragment was produced by overlapping PCR.The diazotization method were used to prepare furazolidone-protein coupled antigen.Then the antigen was used to pan specific ScFvs against furazolidone by ribosome display.After screening,the ScFvs were used to study its affinity and specificity by indirect competitive ELISA.The main results are as follows:(1)The diazotization method was used to prepare furazolidone-protein coupled antigen.Coupling products were detected by UV and HPLC methods,indicating that furazolidone-protein were successfully coupled.(2)The total RNA extracted from the hybridoma cells used to construct antibody library.After 1%agarose gel electrophoresis,the DNA strap of VH and VL can be distinguished easily which around 400 bp and 600 bp.After ligated with the linker(Gly3Ser)4,the size of ScFvs were lkb.(3)In each round of ribosome display,the ScFvs library was used for in vitro transcription and translated in rabbit reticulocyte lysate system to produce antibody-ribosome-mRNA complexes.The coated antigen were used to pan the ARM complexes.After washing,the remained ARM complexes were dissociated and the released mRNA was recovered by RT-PCR.(4)The ScFv from the third selected transformed into E.coli Rosetta(DE3)linking with express vector pET-28a(+)after digestion.After transformation,100 clones from the third selected antibody library were tested by ELISA.It is showed that about 20%of the isolated clones from selected library showed high binding activity.Among them,one clone showed the highest affinity was used for next study.(5)The affinity and specificity of the clone was tested by indirect competitive ELISA.The furazolidone detection range was 10~100 ng/mL,the IC50 was 13.01 ng/mL,the lowest detection limit was 1.28 ng/mL,and the ScFv had shown almost no cross reactivity with the other nitrofurans and their metabolites.This method had good reproducibility,with an average error of 3.83%.In testing samples the recovery for furazolidone addition was 73.38～84.52%,and coefficient of variation was 6.81～9.41%.