Yanping Li
Nanchang University
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Featured researches published by Yanping Li.
Talanta | 2015
Yanwei Ji; Meiling Ren; Yanping Li; Zhibing Huang; Mei Shu; Hongwei Yang; Yonghua Xiong; Yang Xu
Immunochromatographic test strips (ICTS) are commonly limited to higher concentrations of analytes. This limitation stems from the relatively low sensitivity of conventional gold nanospheres (AuNSs with a diameter of 20 nm) to emit detectable brightness values. The larger multi-branched gold nanoflowers (AuNFs) with a higher optical brightness as well as good colloidal stability exhibit significant improvements over conventional AuNSs for enhanced sensitivity of ICTS. In this study, blue AuNFs with an average diameter of 75±5 nm were synthetized and employed as a signal amplification probe for ultrasensitive and quantitative detection of aflatoxin B1 (AFB1) in rice. A portable optical strip reader was used to record the optical densities of test and control lines of the strip. Under the optimal conditions, the AuNF based ICTS system accurately detected AFB1 linearly and dynamically over the range of 0.5-25 pg/mL with a half maximal inhibitory concentration at 4.17 pg/mL. The inhibitory concentration was achieved 10 times lower than that of the traditional AuNS based ICTS systems (41.25 pg/mL). The limit of detection for AFB1 in rice extract was achieved at 0.32 pg/mL. In summary, AuNFs are a novel probe that exhibited excellent sensitivity in the ICTS system and could be used for ultrasensitive detection of other analytes in food safety monitoring, and even medical diagnostics.
Analytical Chemistry | 2013
Zhen-yun He; Qinghua He; Yang Xu; Yanping Li; Xing Liu; Bo Chen; Da Lei; Cheng-hao Sun
With the advantage of replacing mycotoxins and their conjugates, mimotopes have been applied to immunoassays, the most common of which were seleted from random phage displayed peptide libraries. However, these mimotopes were limited by the diversities of the peptide libraries. The aim of this study was to demonstrate that a variety of mimotopes can be obtained by constructing a second-generation peptide library. Using mycotoxin ochratoxin A as a model system, a dodecapeptide mimotope was isolated after panning the second-generation peptide library. The half inhibition concentration of the chemiluminescent enzyme-linked immunosorbent assay setup with this mimotope was 0.04 ng/mL, and the linear range was 0.006-0.245 ng/mL. The mimotope was also used to develop a qualitative dipstick assay with a cutoff level of 1 ng/mL. The method not only presents a high sensitivity but also contributes to the development of mimotope-based assays for mycotoxins avoiding the need of synthesizing toxic mycotoxin conjugates.
Journal of the Science of Food and Agriculture | 2014
Long Zou; Yang Xu; Yanping Li; Qinghua He; Bo Chen; Dan Wang
BACKGROUND Fumonisin B1 (FB1 ) is a cancer-promoting mycotoxin produced by Fusarium species and one of the major food-borne toxins in corn and corn products. The objective of this study was to produce a single-chain variable fragment (scFv) antibody for determination of FB1 in corn samples. RESULTS Anti-FB1 monoclonal antibodies were obtained via the hybridoma technique. Specific heavy- and light-chain variable fragments were amplified with degenerate primers and constructed into scFv antibody fragments by splice overlap extension polymerase chain reaction with linker sequences. The resulting scFv DNA fragments were cloned into the phagemid pHEN1for selection and identification of functional scFv fragment by phage display. Prokaryotic expression vector pET22b-scFv was constructed to prepare anti-FB1 scFv antibody for establishment of indirect competitive ELISA. The detection capability (CCβ) of the scFv-based ELISA was 15.00 µg kg(-1), and the limit of detection was 8.32 µg kg(-1). The recoveries and coefficients of variation were 86.74-107.34% and 9.72-14.03%, respectively. In addition, the determined results of 30 naturally contaminated corn samples by the scFv-based ELISA are in agreement with the findings of high-performance liquid chromatography (R(2) = 0.97). CONCLUSION This scFv-based ELISA could be used as an efficient screening method for routine monitoring the residues FB1 in corn samples.
Talanta | 2015
Mei Shu; Yang Xu; Dan Wang; Xing Liu; Yanping Li; Qinghua He; Zhui Tu; Yulou Qiu; Yanwei Ji; Xianxian Wang
Nanobodies that are small and thermally stable, as well as have high expression level, are leading alternative to produce anti-idiotypic antibodies. These antibodies have the advantage of replacing mycotoxins and their conjugates for immunoassays. In this work, anti-fumonisin B1 (FB1) monoclonal antibody (mAb) was used as the target for biopanning from a naïve alpaca nanobody (Nb) phage display library. After three cycles of panning, one anti-idiotypic nanobody (Ab2β Nb) was isolated and subjected to a Nb-ELISA for the detection of FB1. Surface plasmon resonance was used to analyze the reaction kinetics between the Ab2β Nb and anti-FB1 mAb. The developed assay showed a half inhibitory concentration (IC50) of 0.95±0.12 ng/mL, a limit of detection of 0.15 ng/mL, a linear range of 0.27-5.92 ng/mL, and a low cross-reactivity toward FB2 of 4.93%. The sensitivity was enhanced approximately 20-fold compared with that of the chemosynthetic FB1-BSA conjugates. The equilibrium dissociation constant (KD) measured for Ab2β Nb: anti-FB1 mAb was 164.6 nM. The assay was compared with conventional ELISA (the commercial ELISA kit), and the results indicated the reliability of Ab2β Nb replacing the antigen-carrier protein conjugates. The use of biotechnology in developing the surrogate is an ideal strategy for replacing conventional synthesized antigens.
Talanta | 2016
Jing Chen; Qinghua He; Yang Xu; Jinheng Fu; Yanping Li; Zhui Tu; Dan Wang; Mei Shu; Yulou Qiu; Hongwei Yang; Yuanyuan Liu
Immunoassay for cancer biomarkers plays an important role in cancer prevention and early diagnosis. To the development of immunoassay, the quality and stability of applied antibody is one of the key points to obtain reliability and high sensitivity for immunoassay. The main purpose of this study was to develop a novel immunoassay for ultrasensitive detection of cancer biomarker alpha-fetoprotein (AFP) based on nanobody against AFP. Two nanobodies which bind to AFP were selected from a phage display nanobody library by biopanning strategy. The prepared nanobodies are clonable, thermally stable and applied in both sandwich enzyme linked immunoassay (ELISA) and immuno-PCR assay for ultrasensitive detection of AFP. The limit detection of sandwich ELISA setup with optimized nanobodies was 0.48ng mL(-1), and the half of saturation concentration (SC50) value was 6.68±0.56ng mL(-1). These nanobodies were also used to develop an immuno-PCR assay for ultrasensitive detection of AFP, its limit detection values was 0.005ng mL(-1), and the linear range was 0.01-10,000ng mL(-1). These established immunoassays based on nanobodies were highly specific to AFP and with negligible cross reactivity with other tested caner biomarkers. Furthermore, this novel concept of nanobodies mediated immunoassay may provide potential applications in a general method for the ultrasensitive detection of various cancer biomarkers.
Food Chemistry | 2015
Yang Xu; Liang Xiong; Yanping Li; Yonghua Xiong; Zhui Tu; Jinheng Fu; Xiao Tang
Anti-idiotypic antibodies (AIds) can mimic antigen molecules and can thus offer an alternative to conventional antigens in immunoassays. In this study, citrinin (CIT) was chosen as a target analyte, and an anti-idiotypic single-domain antibody (VHH) was selected from a naïve alpaca VHHs library to serve as a surrogate for CIT hapten. The phage-displayed VHH was used as a signal-amplification carrier to develop an indirect competitive phage enzyme-linked immunosorbent assay (P-ELISA) for the sensitive detection of CIT. The half-inhibition concentration (IC50) of P-ELISA was 10.9 μg/kg, which was 9-fold better than that of conventional ELISA (IC50=102.1 μg/kg). Results on P-ELISA analysis of naturally contaminated samples were also consistent with those obtained by conventional ELISA. In conclusion, the proposed P-ELISA demonstrates the potential use of phage-displayed anti-idiotypic VHH as surrogate for small molecules and signal-amplification carrier to improve assay performance for more sensitive analyte detection in food safety monitoring.
Talanta | 2016
Xianxian Wang; Qinghua He; Yang Xu; Xing Liu; Mei Shu; Zhui Tu; Yanping Li; Wei Wang; Dongmei Cao
Immunoassay is frequently used to analyze mycotoxin contamination. However, the introduction of mycotoxins or their conjugates in conventional immunoassay threatens the safety of individuals and the environment. The variable domain of heavy-chain antibodies (VHHs) can be used as alternative compounds to produce anti-idiotypic antibodies, which work as non-toxic surrogate reagents in immunoassay. In this work, anti-zearalenone (ZEN) monoclonal antibody (mAb) was used as the target for biopanning anti-idiotypic VHH from a naïve alpaca VHH phage display library. After four panning cycles, one anti-idiotypic VHH phage clone (Z1) was isolated and the Z1 based phage ELISA for ZEN showed a half inhibitory concentration (IC50) of 0.25±0.02ng/mL, a linear range of 0.11-0.55ng/mL, and a limit of detection (LOD) of 0.08ng/mL. Furthermore, the phage particles of Z1 were also applied to immuno-polymerase chain reaction (PD-IPCR), which supplied both the detection antigens and deoxyribonucleic acid (DNA) templates. Compared with that of phage ELISA, the LOD of Z1 based PD-IPCR was 12-fold improved, with a detection limit of 6.5pg/mL and a linear range of 0.01-100ng/mL. The proposed method was then validated with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Results showed the reliability of PD-IPCR for the determination of ZEN in cereal samples. The use of anti-idiotypic VHH phage as non-toxic surrogate and signal-amplification function of PCR make it a promising method for actual ZEN analysis in cereals.
Food Additives and Contaminants Part A-chemistry Analysis Control Exposure & Risk Assessment | 2015
Yanping Li; Xiao Tang; Wei Wu; Yang Xu; Zhibing Huang; Qinghua He
Citrinin, a fungal secondary metabolite of polyketide origin, is moderately nephrotoxic to vertebrates, including humans. Citrinin is synthesised by condensation of acetyl-CoA and malonyl-CoA. Six genes involved in the citrinin biosynthesis, including pksCT, ctnA and ctnB, have been cloned in Monascus purpureus. The pksCT gene encodes a polyketide synthase; ctnA is a regulatory factor; and ctnB encodes an oxidoreductase. When the three genes were respectively disrupted, the disruption strains drastically decreased citrinin production or barely produced citrinin. Ten new genes have been discovered in Monascus aurantiacus besides the above six genes. One of these gene displayed the highest similarity to the β-carbonic anhydrase gene from Aspergillus oryzae (74% similarity) and was designated ctnG. To learn more about the citrinin biosynthetic pathway, a ctnG-replacement vector was constructed to disrupt ctnG with the hygromycin resistance gene as the selection marker, then transformed into M. aurantiacus Li AS3.4384 by a protoplast-PEG method. The citrinin content of three disruptants was reduced to about 50%, meanwhile pigment production decreased by 23%, respectively, over those of the wild-type strains. ctnG was deduced to be involved in the formation of malonyl-CoA as a common precursor of red pigments and citrinin. Therefore, the disruption of the ctnG gene decreased citrinin and pigment production. M. aurantiacus Li AS3.4384 can produce higher concentrations of citrinin than other strains such as M. purpureus and M. ruber. Establishing the function of citrinin biosynthetic genes in M. aurantiacus is helpful in understanding the citrinin synthetic pathway and adopting some strategies to control contamination. Graphical Abstract
Analytical Biochemistry | 2016
Zhui Tu; Qi Chen; Yanping Li; Yonghua Xiong; Yang Xu; Na Hu; Yong Tao
Listeria monocytogenes (LM), one of the eight species belonging to the genus Listeria, is pathogenic for both humans and animals. In this study, two novel LM-specific clones, designated L5-78 and L5-79, were isolated from a phage display antibody library that was derived from the variable domain of heavy-chain antibodies (VHHs) of non-immunized alpaca. These two clones were expressed, purified, and characterized. Results showed that both isolated VHHs recognize three serotypes (1/2a, 1/2b, and 4b), which are responsible for more than 95% of documented human listeriosis cases. The recombinant VHHs possess high thermal stability, pH tolerance, and urea resistance. A sandwich enzyme-linked immunosorbent assay (ELISA) based on the VHH clone L5-79 and a monoclonal antibody was developed to detect LM in pasteurized milk, with a detection limit of 1 × 10(4) colony-forming units (CFU)/ml. These findings indicated that the species-specific VHHs could be directly isolated from the non-immunized library with a properly designed panning strategy and VHH could be a new source for possible diagnosis/detection of foodborne pathogens in food because it was shown to be highly specific and stable.
Biotechnology Letters | 2010
Bohua Wang; Yang Xu; Yanping Li
Ma-pyrG was cloned from Monascus aurantiacus AS3.4384 using degenerate PCR with primers designed with an algorithm called CODEHOP, and its complete sequence was obtained by a PCR-based strategy for screening a Monascus fosmid library. Ma-pyrG encodes orotidine-5′-phosphate decarboxylase (OMPdecase), a 283-aminoacid protein with 81% sequence identity to that from Aspergillus flavus NRRL 3357. A pyrG mutant strain from M. aurantiacus AS3.4384, named UM28, was isolated by resistance to 5-fluoroorotic acid after UV mutagenesis. Sequence analysis of this mutated gene revealed that it contained a point mutation at nucleotide position +220. Plasmid pGFP-pyrG, bearing the green fluorescent protein gene (GFP) as a model gene and Ma-pyrG as a selection marker, were constructed. pGFP-pyrG were successfully transformed into UM28 by using the PEG method.