The Experts below are selected from a list of 753 Experts worldwide ranked by ideXlab platform

Bruce D Hammock - One of the best experts on this subject based on the ideXlab platform.

  • production and characterization of a single chain variable fragment alkaline phosphatase fusion protein for Glycocholic Acid detection in a one step enzyme linked immunosorbent assay
    Analytical Methods, 2018
    Co-Authors: Xiping Cui, Ding Shen, Suqing Zhao, Yingshan Chen, Zhengyun Jiang, Bruce D Hammock
    Abstract:

    A single-chain variable fragment (scFv)-alkaline phosphatase (AP) fusion protein for Glycocholic Acid (GCA) was produced and characterized. The scFv gene with a 218 linker was generated by splicing by overlap extension (SOE)-polymerase chain reaction (PCR) and sequentially inserted into the expression vector pecan45 containing AP gene to express the scFv-AP fusion protein in Escherichia coli (E. coli). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses revealed that the fusion protein showed the expected molecular weight of about 80 kDa. Both the antibody binding capacity and AP enzyme activity of the scFv-AP fusion protein were validated by colorimetric analysis. One-step competitive direct enzyme-linked immunosorbent assay (ELISA) based on the scFv-AP fusion protein indicated that the average concentration required for 50% inhibition of binding (IC50) and limit of detection (LOD) for GCA were 216 ng mL−1 and 37.0 ng mL−1, respectively, and the linear response range extended from 71.0 to 657 ng mL−1. The cross-reactivity (CR) of the scFv-AP fusion protein was similar to those of its parental scFv antibody. The scFv-AP fusion protein was bifunctional, retaining both antibody binding specificity and AP enzyme activity. This work indicates that the production of the scFv-AP fusion protein in E. coli strain BL21(DE3)pLysS is feasible and suggests that it could be further used as convenient one-step detection probes for GCA.

  • biotinylated single chain variable fragment based enzyme linked immunosorbent assay for Glycocholic Acid
    Analyst, 2018
    Co-Authors: Xiping Cui, Natalia Vasylieva, Bogdan Barnych, Jun Yang, Ding Shen, Suqing Zhao, Zhengyun Jiang, Bruce D Hammock
    Abstract:

    Glycocholic Acid (GCA) has been identified as a novel selective and sensitive biomarker for hepatocellular carcinoma (HCC). In this work, a recombinant antibody, scFv-G11, which was shown previously to have selective reactivity for GCA, was labeled with biotin using a chemical and an enzymatic method, respectively. The enzymatic method proved superior giving sensitive scFv-biotin preparations. Based on biotinylated scFv against GCA and a biotin-streptavidin system for signal amplification, an indirect competitive biotin-streptavidin-amplified enzyme-linked immunosorbent assay (BA-ELISA) has been established for the sensitive and rapid detection of GCA. Several physiochemical factors that influenced assay performance, such as organic cosolvent, ionic strength, and pH, were studied. Under the optimized conditions, the indirect competitive BA-ELISA based on the obtained biotinylated scFv antibodies indicated that the average concentration required for 50% inhibition of binding (IC50) and the limit of detection (LOD) for GCA were 0.42 μg mL-1 and 0.07 μg mL-1, respectively, and the linear response range extended from 0.14 to 1.24 μg mL-1. Cross-reactivity of biotinylated scFv antibodies with various bile Acid analogues was below 1.89%, except for taurocholic Acid. The recoveries of GCA from urine samples via this indirect competitive BA-ELISA ranged from 108.3% to 131.5%, and correlated well with liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS), which indicated the accuracy and reliability of biotinylated scFv-based ELISA in the detection of GCA in urine samples. This study also demonstrates the broad utility of scFv for the development of highly sensitive immunoassays.

  • biotinylated single chain variable fragment based enzyme linked immunosorbent assay for Glycocholic Acid
    Analyst, 2018
    Co-Authors: Natalia Vasylieva, Bogdan Barnych, Jun Yang, Ding Shen, Qiyi He, Suqing Zhao, Zhengyun Jiang, Bruce D Hammock
    Abstract:

    Glycocholic Acid (GCA) has been identified as a novel selective and sensitive biomarker for hepatocellular carcinoma (HCC). In this work, a recombinant antibody, scFv-G11, which was shown previously to have selective reactivity for GCA, was labeled with biotin using a chemical and an enzymatic method, respectively. The enzymatic method proved superior giving sensitive scFv-biotin preparations. Based on biotinylated scFv against GCA and biotin-streptavidin system for signal amplification, an indirect competitive biotin-streptavidin-amplified enzyme-linked immunosorbent assay (BA-ELISA) has been established for sensitive and rapid detection of GCA. Several physiochemical factors that influenced assay performance, such as organic cosolvent, ionic strength, and pH, were studied. Under the optimized conditions, the indirect competitive BA-ELISA based on the obtained biotinylated scFv antibodies indicated that the average concentration required for 50% inhibition of binding (IC50) and the limit of detection (LOD) for GCA were 0.42 μg/mL and 0.07 μg/mL, respectively, and the linear response range extended from 0.14 to 1.24 μg/mL. Cross-reactivity of biotinylated scFv antibodies with various bile Acid analogues were below 1.89 %, except for taurocholic Acid. The recoveries of GCA from urine samples via this indirect competitive BA-ELISA ranged from 108.3 % to 131.5 %, and correlated well with liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS), which indicated the accuracy and reliability of biotinylated scFv-based ELISA in the detection of GCA in urine samples. This study also demonstrates the broad utility of scFv for the development of highly sensitive immunoassays.

  • development of an indirect competitive enzyme linked immunosorbent assay for Glycocholic Acid based on chicken single chain variable fragment antibodies
    Analytical Chemistry, 2017
    Co-Authors: Natalia Vasylieva, Panpan Wu, Bogdan Barnych, Jun Yang, Ding Shen, Qiyi He, Suqing Zhao, Bruce D Hammock
    Abstract:

    Glycocholic Acid (GCA) is an important metabolite of bile Acids, whose urine levels are expected to be a specific diagnostic biomarker for hepatocellular carcinoma (HCC). A high-throughput immunoassay for determination of GCA would be of significant advantage and useful for primary diagnosis, surveillance, and early detection of HCC. Single-chain variable fragment (scFv) antibodies have several desirable characteristics and are an attractive alternative to traditional antibodies for the immunoassay. Because chicken antibodies possess single heavy and light variable functional domains, they are an ideal framework for simplified generation of recombinant antibodies for GCA detection. However, chicken scFvs have rarely been used to detect GCA. In this study, a scFv library was generated from chickens immunized with a GCA hapten coupled to bovine serum albumin (BSA), and anti-GCA scFvs were isolated by a phage-displayed method. Compared to the homologous coating antigen, use of a heterologous coating antigen ...

  • Development of an Indirect Competitive Enzyme-Linked Immunosorbent Assay for Glycocholic Acid Based on Chicken Single-Chain Variable Fragment Antibodies
    2017
    Co-Authors: Xiping Cui, Natalia Vasylieva, Bogdan Barnych, Jun Yang, Ding Shen, Suqing Zhao, Shirley J. Gee, Bruce D Hammock
    Abstract:

    Glycocholic Acid (GCA) is an important metabolite of bile Acids, whose urine levels are expected to be a specific diagnostic biomarker for hepatocellular carcinoma (HCC). A high-throughput immunoassay for determination of GCA would be of significant advantage and useful for primary diagnosis, surveillance, and early detection of HCC. Single-chain variable fragment (scFv) antibodies have several desirable characteristics and are an attractive alternative to traditional antibodies for the immunoassay. Because chicken antibodies possess single heavy and light variable functional domains, they are an ideal framework for simplified generation of recombinant antibodies for GCA detection. However, chicken scFvs have rarely been used to detect GCA. In this study, a scFv library was generated from chickens immunized with a GCA hapten coupled to bovine serum albumin (BSA), and anti-GCA scFvs were isolated by a phage-displayed method. Compared to the homologous coating antigen, use of a heterologous coating antigen resulted in about an 85-fold improvement in sensitivity of the immunoassay. This assay, under optimized conditions, had a linear range of 0.02–0.18 μg/mL, with an IC50 of 0.06 μg/mL. The assay showed negligible cross-reactivity with various related bile Acids, except for taurocholic Acid. The detection of GCA from spiked human urine samples ranged from 86.7% to 123.3%. These results, combined with the advantages of scFv antibodies, indicated that a chicken scFv-based enzyme-linked immunosorbent assay is a suitable method for high-throughput screening of GCA in human urine

Suqing Zhao - One of the best experts on this subject based on the ideXlab platform.

  • development of enzyme free single step immunoassays for Glycocholic Acid based on palladium nanoparticle mediated signal generation
    Analytical and Bioanalytical Chemistry, 2021
    Co-Authors: Xiping Cui, Ding Shen, Huiyi Yang, Yingshan Chen, Sergei A Eremin, Suqing Zhao
    Abstract:

    Palladium nanoparticles (PdNPs) are composed mainly of inert noble metals, and their outstanding properties have attracted wide attention. PdNPs are not only capable of mimicking the oxidase-like characteristics of natural bio-enzymes, but they also present a clear black band in the test zone. In this work, the synthesized PdNPs promoted a transformation of colorless tetramethylbenzidine (TMB) to a blue oxidation product of TMB, providing a Km value of 0.09 mM for TMB, and revealing the good catalytic performance of the synthesized PdNPs. For both signal generation and amplification, PdNPs effectively replaced natural bio-enzymes as a new labeling tag. Thus, the PdNP-based enzyme-free single-step immunoassays were successfully developed for efficient and sensitive detection of Glycocholic Acid (GCA). Under optimal conditions, a noticeable linear relationship was identified by the enzyme-linked immunosorbent assay (ELISA) over a range of 8-2390 ng/mL, while the visual limit of detection (vLOD) in the constructed lateral flow immunoassay (LFA) was 10 ng/mL for GCA. The recovery rate in spiked urine samples obtained by the ELISA ranged from 84.2 to 117.9%, which was consistent with the results in LFA. The present work demonstrates the potential of PdNPs as labeling matrices in enzyme-free single-step immunoassays.

  • enhanced performance of a surface plasmon resonance based immunosensor for the detection of Glycocholic Acid
    Analytical Methods, 2021
    Co-Authors: Yanxiong Fang, Ding Shen, Huiyi Yang, Yingshan Chen, Xiping Cui, Sergei A Eremin, Yingying Zhong, Suqing Zhao
    Abstract:

    The concentration of Glycocholic Acid (GCA) in urine and blood is an important biomarker for liver cancer. Monitoring of GCA depends to a large extent on the availability of appropriate analytical techniques. In this work, based on the immobilization of GCA-OVA onto the sensor chip surface, a label-free competitive inhibition immunoassay for the determination of GCA with the surface plasmon resonance (SPR) technique was developed. The proposed SPR immunosensor is simple to prepare, recyclable and exhibits excellent sensitivity to GCA (a linear range of 13.3–119.4 ng mL−1 and a limit of detection (LOD) of 2.5 ng mL−1), which was 14 times lower than that of the traditional immunoassay. Excellent recoveries and correlation between these two methods were observed (R2 = 0.995). Hence, it can be proved that the SPR immunosensor could be used to achieve rapid and sensitive quantitative detection of GCA in real urine samples and meet clinical needs.

  • prussian blue nanoparticles with peroxidase mimicking properties in a dual immunoassays for Glycocholic Acid
    Journal of Pharmaceutical and Biomedical Analysis, 2020
    Co-Authors: Huiyi Yang, Ding Shen, Yingshan Chen, Xiping Cui, Chunguo Zhang, Huanxin Xiao, Sergei A Eremin, Yanxiong Fang, Suqing Zhao
    Abstract:

    Abstract Prussian Blue nanoparticles (PBNPs) were utilized in a lateral flow immunoassay (LFA) and in an indirect competitive nanozyme-linked immunosorbent assay (icELISA), respectively, for their intense blue color and peroxidase (POx) -like activity. The PBNPs with good POx-like activity was linked to the antibody. Under the optimal parameters, both the PBNP-icELISA and PBNP-LFA perform very well. The icELISA has an IC50 value of 190 ng/mL, the working range extends from 29 to 1200 ng/mL, and the limit of detection is 22 ng/mL. The visual cut-off limit is 10 ng/mL. The dual immunoassay was used to quantify Glycocholic Acid in spiked human urine. Excellent recoveries and correlation between the two methods were observed.

  • development of a simple rapid and high throughput fluorescence polarization immunoassay for Glycocholic Acid in human urine
    Journal of Pharmaceutical and Biomedical Analysis, 2018
    Co-Authors: Xiping Cui, Ding Shen, Yingshan Chen, Sergei A Eremin, Zhengyun Jiang, Suqing Zhao
    Abstract:

    Abstract In this paper, a simple, rapid and high-throughput fluorescence polarization immunoassay (FPIA) based on polyclonal antibodies (PAb) is described for the determination of Glycocholic Acid (GCA) in human urine. Three fluorescein-labeled GCA (tracers) with different structures and spacer bridges were synthesized and purified by thin-layer chromatography (TLC). The structure effect of tracers on the assay was investigated and the sensitivity of best tracer in the optimized FPIA demonstrated an IC50 value of 306 ng/mL. The working range of FPIA was 36 ∼ 2 600 ng/mL and the limit of detection (LOD) was 9 ng/mL. The developed FPIA was time-saving that could be completed within 10 min. Human urine samples spiked with GCA were analyzed by this method, followed by confirmation with commercial enzyme immunoassay analysis (EIA). Excellent recoveries and correlation between these two methods were observed (R2 = 0.996), suggesting the developed FPIA could be applied to screening of GCA in human urine samples without complicated cleanup.

  • production and characterization of a single chain variable fragment alkaline phosphatase fusion protein for Glycocholic Acid detection in a one step enzyme linked immunosorbent assay
    Analytical Methods, 2018
    Co-Authors: Xiping Cui, Ding Shen, Suqing Zhao, Yingshan Chen, Zhengyun Jiang, Bruce D Hammock
    Abstract:

    A single-chain variable fragment (scFv)-alkaline phosphatase (AP) fusion protein for Glycocholic Acid (GCA) was produced and characterized. The scFv gene with a 218 linker was generated by splicing by overlap extension (SOE)-polymerase chain reaction (PCR) and sequentially inserted into the expression vector pecan45 containing AP gene to express the scFv-AP fusion protein in Escherichia coli (E. coli). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses revealed that the fusion protein showed the expected molecular weight of about 80 kDa. Both the antibody binding capacity and AP enzyme activity of the scFv-AP fusion protein were validated by colorimetric analysis. One-step competitive direct enzyme-linked immunosorbent assay (ELISA) based on the scFv-AP fusion protein indicated that the average concentration required for 50% inhibition of binding (IC50) and limit of detection (LOD) for GCA were 216 ng mL−1 and 37.0 ng mL−1, respectively, and the linear response range extended from 71.0 to 657 ng mL−1. The cross-reactivity (CR) of the scFv-AP fusion protein was similar to those of its parental scFv antibody. The scFv-AP fusion protein was bifunctional, retaining both antibody binding specificity and AP enzyme activity. This work indicates that the production of the scFv-AP fusion protein in E. coli strain BL21(DE3)pLysS is feasible and suggests that it could be further used as convenient one-step detection probes for GCA.

Ding Shen - One of the best experts on this subject based on the ideXlab platform.

  • development of enzyme free single step immunoassays for Glycocholic Acid based on palladium nanoparticle mediated signal generation
    Analytical and Bioanalytical Chemistry, 2021
    Co-Authors: Xiping Cui, Ding Shen, Huiyi Yang, Yingshan Chen, Sergei A Eremin, Suqing Zhao
    Abstract:

    Palladium nanoparticles (PdNPs) are composed mainly of inert noble metals, and their outstanding properties have attracted wide attention. PdNPs are not only capable of mimicking the oxidase-like characteristics of natural bio-enzymes, but they also present a clear black band in the test zone. In this work, the synthesized PdNPs promoted a transformation of colorless tetramethylbenzidine (TMB) to a blue oxidation product of TMB, providing a Km value of 0.09 mM for TMB, and revealing the good catalytic performance of the synthesized PdNPs. For both signal generation and amplification, PdNPs effectively replaced natural bio-enzymes as a new labeling tag. Thus, the PdNP-based enzyme-free single-step immunoassays were successfully developed for efficient and sensitive detection of Glycocholic Acid (GCA). Under optimal conditions, a noticeable linear relationship was identified by the enzyme-linked immunosorbent assay (ELISA) over a range of 8-2390 ng/mL, while the visual limit of detection (vLOD) in the constructed lateral flow immunoassay (LFA) was 10 ng/mL for GCA. The recovery rate in spiked urine samples obtained by the ELISA ranged from 84.2 to 117.9%, which was consistent with the results in LFA. The present work demonstrates the potential of PdNPs as labeling matrices in enzyme-free single-step immunoassays.

  • enhanced performance of a surface plasmon resonance based immunosensor for the detection of Glycocholic Acid
    Analytical Methods, 2021
    Co-Authors: Yanxiong Fang, Ding Shen, Huiyi Yang, Yingshan Chen, Xiping Cui, Sergei A Eremin, Yingying Zhong, Suqing Zhao
    Abstract:

    The concentration of Glycocholic Acid (GCA) in urine and blood is an important biomarker for liver cancer. Monitoring of GCA depends to a large extent on the availability of appropriate analytical techniques. In this work, based on the immobilization of GCA-OVA onto the sensor chip surface, a label-free competitive inhibition immunoassay for the determination of GCA with the surface plasmon resonance (SPR) technique was developed. The proposed SPR immunosensor is simple to prepare, recyclable and exhibits excellent sensitivity to GCA (a linear range of 13.3–119.4 ng mL−1 and a limit of detection (LOD) of 2.5 ng mL−1), which was 14 times lower than that of the traditional immunoassay. Excellent recoveries and correlation between these two methods were observed (R2 = 0.995). Hence, it can be proved that the SPR immunosensor could be used to achieve rapid and sensitive quantitative detection of GCA in real urine samples and meet clinical needs.

  • prussian blue nanoparticles with peroxidase mimicking properties in a dual immunoassays for Glycocholic Acid
    Journal of Pharmaceutical and Biomedical Analysis, 2020
    Co-Authors: Huiyi Yang, Ding Shen, Yingshan Chen, Xiping Cui, Chunguo Zhang, Huanxin Xiao, Sergei A Eremin, Yanxiong Fang, Suqing Zhao
    Abstract:

    Abstract Prussian Blue nanoparticles (PBNPs) were utilized in a lateral flow immunoassay (LFA) and in an indirect competitive nanozyme-linked immunosorbent assay (icELISA), respectively, for their intense blue color and peroxidase (POx) -like activity. The PBNPs with good POx-like activity was linked to the antibody. Under the optimal parameters, both the PBNP-icELISA and PBNP-LFA perform very well. The icELISA has an IC50 value of 190 ng/mL, the working range extends from 29 to 1200 ng/mL, and the limit of detection is 22 ng/mL. The visual cut-off limit is 10 ng/mL. The dual immunoassay was used to quantify Glycocholic Acid in spiked human urine. Excellent recoveries and correlation between the two methods were observed.

  • development of a simple rapid and high throughput fluorescence polarization immunoassay for Glycocholic Acid in human urine
    Journal of Pharmaceutical and Biomedical Analysis, 2018
    Co-Authors: Xiping Cui, Ding Shen, Yingshan Chen, Sergei A Eremin, Zhengyun Jiang, Suqing Zhao
    Abstract:

    Abstract In this paper, a simple, rapid and high-throughput fluorescence polarization immunoassay (FPIA) based on polyclonal antibodies (PAb) is described for the determination of Glycocholic Acid (GCA) in human urine. Three fluorescein-labeled GCA (tracers) with different structures and spacer bridges were synthesized and purified by thin-layer chromatography (TLC). The structure effect of tracers on the assay was investigated and the sensitivity of best tracer in the optimized FPIA demonstrated an IC50 value of 306 ng/mL. The working range of FPIA was 36 ∼ 2 600 ng/mL and the limit of detection (LOD) was 9 ng/mL. The developed FPIA was time-saving that could be completed within 10 min. Human urine samples spiked with GCA were analyzed by this method, followed by confirmation with commercial enzyme immunoassay analysis (EIA). Excellent recoveries and correlation between these two methods were observed (R2 = 0.996), suggesting the developed FPIA could be applied to screening of GCA in human urine samples without complicated cleanup.

  • production and characterization of a single chain variable fragment alkaline phosphatase fusion protein for Glycocholic Acid detection in a one step enzyme linked immunosorbent assay
    Analytical Methods, 2018
    Co-Authors: Xiping Cui, Ding Shen, Suqing Zhao, Yingshan Chen, Zhengyun Jiang, Bruce D Hammock
    Abstract:

    A single-chain variable fragment (scFv)-alkaline phosphatase (AP) fusion protein for Glycocholic Acid (GCA) was produced and characterized. The scFv gene with a 218 linker was generated by splicing by overlap extension (SOE)-polymerase chain reaction (PCR) and sequentially inserted into the expression vector pecan45 containing AP gene to express the scFv-AP fusion protein in Escherichia coli (E. coli). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses revealed that the fusion protein showed the expected molecular weight of about 80 kDa. Both the antibody binding capacity and AP enzyme activity of the scFv-AP fusion protein were validated by colorimetric analysis. One-step competitive direct enzyme-linked immunosorbent assay (ELISA) based on the scFv-AP fusion protein indicated that the average concentration required for 50% inhibition of binding (IC50) and limit of detection (LOD) for GCA were 216 ng mL−1 and 37.0 ng mL−1, respectively, and the linear response range extended from 71.0 to 657 ng mL−1. The cross-reactivity (CR) of the scFv-AP fusion protein was similar to those of its parental scFv antibody. The scFv-AP fusion protein was bifunctional, retaining both antibody binding specificity and AP enzyme activity. This work indicates that the production of the scFv-AP fusion protein in E. coli strain BL21(DE3)pLysS is feasible and suggests that it could be further used as convenient one-step detection probes for GCA.

Xiping Cui - One of the best experts on this subject based on the ideXlab platform.

  • development of enzyme free single step immunoassays for Glycocholic Acid based on palladium nanoparticle mediated signal generation
    Analytical and Bioanalytical Chemistry, 2021
    Co-Authors: Xiping Cui, Ding Shen, Huiyi Yang, Yingshan Chen, Sergei A Eremin, Suqing Zhao
    Abstract:

    Palladium nanoparticles (PdNPs) are composed mainly of inert noble metals, and their outstanding properties have attracted wide attention. PdNPs are not only capable of mimicking the oxidase-like characteristics of natural bio-enzymes, but they also present a clear black band in the test zone. In this work, the synthesized PdNPs promoted a transformation of colorless tetramethylbenzidine (TMB) to a blue oxidation product of TMB, providing a Km value of 0.09 mM for TMB, and revealing the good catalytic performance of the synthesized PdNPs. For both signal generation and amplification, PdNPs effectively replaced natural bio-enzymes as a new labeling tag. Thus, the PdNP-based enzyme-free single-step immunoassays were successfully developed for efficient and sensitive detection of Glycocholic Acid (GCA). Under optimal conditions, a noticeable linear relationship was identified by the enzyme-linked immunosorbent assay (ELISA) over a range of 8-2390 ng/mL, while the visual limit of detection (vLOD) in the constructed lateral flow immunoassay (LFA) was 10 ng/mL for GCA. The recovery rate in spiked urine samples obtained by the ELISA ranged from 84.2 to 117.9%, which was consistent with the results in LFA. The present work demonstrates the potential of PdNPs as labeling matrices in enzyme-free single-step immunoassays.

  • enhanced performance of a surface plasmon resonance based immunosensor for the detection of Glycocholic Acid
    Analytical Methods, 2021
    Co-Authors: Yanxiong Fang, Ding Shen, Huiyi Yang, Yingshan Chen, Xiping Cui, Sergei A Eremin, Yingying Zhong, Suqing Zhao
    Abstract:

    The concentration of Glycocholic Acid (GCA) in urine and blood is an important biomarker for liver cancer. Monitoring of GCA depends to a large extent on the availability of appropriate analytical techniques. In this work, based on the immobilization of GCA-OVA onto the sensor chip surface, a label-free competitive inhibition immunoassay for the determination of GCA with the surface plasmon resonance (SPR) technique was developed. The proposed SPR immunosensor is simple to prepare, recyclable and exhibits excellent sensitivity to GCA (a linear range of 13.3–119.4 ng mL−1 and a limit of detection (LOD) of 2.5 ng mL−1), which was 14 times lower than that of the traditional immunoassay. Excellent recoveries and correlation between these two methods were observed (R2 = 0.995). Hence, it can be proved that the SPR immunosensor could be used to achieve rapid and sensitive quantitative detection of GCA in real urine samples and meet clinical needs.

  • prussian blue nanoparticles with peroxidase mimicking properties in a dual immunoassays for Glycocholic Acid
    Journal of Pharmaceutical and Biomedical Analysis, 2020
    Co-Authors: Huiyi Yang, Ding Shen, Yingshan Chen, Xiping Cui, Chunguo Zhang, Huanxin Xiao, Sergei A Eremin, Yanxiong Fang, Suqing Zhao
    Abstract:

    Abstract Prussian Blue nanoparticles (PBNPs) were utilized in a lateral flow immunoassay (LFA) and in an indirect competitive nanozyme-linked immunosorbent assay (icELISA), respectively, for their intense blue color and peroxidase (POx) -like activity. The PBNPs with good POx-like activity was linked to the antibody. Under the optimal parameters, both the PBNP-icELISA and PBNP-LFA perform very well. The icELISA has an IC50 value of 190 ng/mL, the working range extends from 29 to 1200 ng/mL, and the limit of detection is 22 ng/mL. The visual cut-off limit is 10 ng/mL. The dual immunoassay was used to quantify Glycocholic Acid in spiked human urine. Excellent recoveries and correlation between the two methods were observed.

  • development of a simple rapid and high throughput fluorescence polarization immunoassay for Glycocholic Acid in human urine
    Journal of Pharmaceutical and Biomedical Analysis, 2018
    Co-Authors: Xiping Cui, Ding Shen, Yingshan Chen, Sergei A Eremin, Zhengyun Jiang, Suqing Zhao
    Abstract:

    Abstract In this paper, a simple, rapid and high-throughput fluorescence polarization immunoassay (FPIA) based on polyclonal antibodies (PAb) is described for the determination of Glycocholic Acid (GCA) in human urine. Three fluorescein-labeled GCA (tracers) with different structures and spacer bridges were synthesized and purified by thin-layer chromatography (TLC). The structure effect of tracers on the assay was investigated and the sensitivity of best tracer in the optimized FPIA demonstrated an IC50 value of 306 ng/mL. The working range of FPIA was 36 ∼ 2 600 ng/mL and the limit of detection (LOD) was 9 ng/mL. The developed FPIA was time-saving that could be completed within 10 min. Human urine samples spiked with GCA were analyzed by this method, followed by confirmation with commercial enzyme immunoassay analysis (EIA). Excellent recoveries and correlation between these two methods were observed (R2 = 0.996), suggesting the developed FPIA could be applied to screening of GCA in human urine samples without complicated cleanup.

  • production and characterization of a single chain variable fragment alkaline phosphatase fusion protein for Glycocholic Acid detection in a one step enzyme linked immunosorbent assay
    Analytical Methods, 2018
    Co-Authors: Xiping Cui, Ding Shen, Suqing Zhao, Yingshan Chen, Zhengyun Jiang, Bruce D Hammock
    Abstract:

    A single-chain variable fragment (scFv)-alkaline phosphatase (AP) fusion protein for Glycocholic Acid (GCA) was produced and characterized. The scFv gene with a 218 linker was generated by splicing by overlap extension (SOE)-polymerase chain reaction (PCR) and sequentially inserted into the expression vector pecan45 containing AP gene to express the scFv-AP fusion protein in Escherichia coli (E. coli). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses revealed that the fusion protein showed the expected molecular weight of about 80 kDa. Both the antibody binding capacity and AP enzyme activity of the scFv-AP fusion protein were validated by colorimetric analysis. One-step competitive direct enzyme-linked immunosorbent assay (ELISA) based on the scFv-AP fusion protein indicated that the average concentration required for 50% inhibition of binding (IC50) and limit of detection (LOD) for GCA were 216 ng mL−1 and 37.0 ng mL−1, respectively, and the linear response range extended from 71.0 to 657 ng mL−1. The cross-reactivity (CR) of the scFv-AP fusion protein was similar to those of its parental scFv antibody. The scFv-AP fusion protein was bifunctional, retaining both antibody binding specificity and AP enzyme activity. This work indicates that the production of the scFv-AP fusion protein in E. coli strain BL21(DE3)pLysS is feasible and suggests that it could be further used as convenient one-step detection probes for GCA.

Natalia Vasylieva - One of the best experts on this subject based on the ideXlab platform.

  • biotinylated single chain variable fragment based enzyme linked immunosorbent assay for Glycocholic Acid
    Analyst, 2018
    Co-Authors: Xiping Cui, Natalia Vasylieva, Bogdan Barnych, Jun Yang, Ding Shen, Suqing Zhao, Zhengyun Jiang, Bruce D Hammock
    Abstract:

    Glycocholic Acid (GCA) has been identified as a novel selective and sensitive biomarker for hepatocellular carcinoma (HCC). In this work, a recombinant antibody, scFv-G11, which was shown previously to have selective reactivity for GCA, was labeled with biotin using a chemical and an enzymatic method, respectively. The enzymatic method proved superior giving sensitive scFv-biotin preparations. Based on biotinylated scFv against GCA and a biotin-streptavidin system for signal amplification, an indirect competitive biotin-streptavidin-amplified enzyme-linked immunosorbent assay (BA-ELISA) has been established for the sensitive and rapid detection of GCA. Several physiochemical factors that influenced assay performance, such as organic cosolvent, ionic strength, and pH, were studied. Under the optimized conditions, the indirect competitive BA-ELISA based on the obtained biotinylated scFv antibodies indicated that the average concentration required for 50% inhibition of binding (IC50) and the limit of detection (LOD) for GCA were 0.42 μg mL-1 and 0.07 μg mL-1, respectively, and the linear response range extended from 0.14 to 1.24 μg mL-1. Cross-reactivity of biotinylated scFv antibodies with various bile Acid analogues was below 1.89%, except for taurocholic Acid. The recoveries of GCA from urine samples via this indirect competitive BA-ELISA ranged from 108.3% to 131.5%, and correlated well with liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS), which indicated the accuracy and reliability of biotinylated scFv-based ELISA in the detection of GCA in urine samples. This study also demonstrates the broad utility of scFv for the development of highly sensitive immunoassays.

  • biotinylated single chain variable fragment based enzyme linked immunosorbent assay for Glycocholic Acid
    Analyst, 2018
    Co-Authors: Natalia Vasylieva, Bogdan Barnych, Jun Yang, Ding Shen, Qiyi He, Suqing Zhao, Zhengyun Jiang, Bruce D Hammock
    Abstract:

    Glycocholic Acid (GCA) has been identified as a novel selective and sensitive biomarker for hepatocellular carcinoma (HCC). In this work, a recombinant antibody, scFv-G11, which was shown previously to have selective reactivity for GCA, was labeled with biotin using a chemical and an enzymatic method, respectively. The enzymatic method proved superior giving sensitive scFv-biotin preparations. Based on biotinylated scFv against GCA and biotin-streptavidin system for signal amplification, an indirect competitive biotin-streptavidin-amplified enzyme-linked immunosorbent assay (BA-ELISA) has been established for sensitive and rapid detection of GCA. Several physiochemical factors that influenced assay performance, such as organic cosolvent, ionic strength, and pH, were studied. Under the optimized conditions, the indirect competitive BA-ELISA based on the obtained biotinylated scFv antibodies indicated that the average concentration required for 50% inhibition of binding (IC50) and the limit of detection (LOD) for GCA were 0.42 μg/mL and 0.07 μg/mL, respectively, and the linear response range extended from 0.14 to 1.24 μg/mL. Cross-reactivity of biotinylated scFv antibodies with various bile Acid analogues were below 1.89 %, except for taurocholic Acid. The recoveries of GCA from urine samples via this indirect competitive BA-ELISA ranged from 108.3 % to 131.5 %, and correlated well with liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS), which indicated the accuracy and reliability of biotinylated scFv-based ELISA in the detection of GCA in urine samples. This study also demonstrates the broad utility of scFv for the development of highly sensitive immunoassays.

  • development of an indirect competitive enzyme linked immunosorbent assay for Glycocholic Acid based on chicken single chain variable fragment antibodies
    Analytical Chemistry, 2017
    Co-Authors: Natalia Vasylieva, Panpan Wu, Bogdan Barnych, Jun Yang, Ding Shen, Qiyi He, Suqing Zhao, Bruce D Hammock
    Abstract:

    Glycocholic Acid (GCA) is an important metabolite of bile Acids, whose urine levels are expected to be a specific diagnostic biomarker for hepatocellular carcinoma (HCC). A high-throughput immunoassay for determination of GCA would be of significant advantage and useful for primary diagnosis, surveillance, and early detection of HCC. Single-chain variable fragment (scFv) antibodies have several desirable characteristics and are an attractive alternative to traditional antibodies for the immunoassay. Because chicken antibodies possess single heavy and light variable functional domains, they are an ideal framework for simplified generation of recombinant antibodies for GCA detection. However, chicken scFvs have rarely been used to detect GCA. In this study, a scFv library was generated from chickens immunized with a GCA hapten coupled to bovine serum albumin (BSA), and anti-GCA scFvs were isolated by a phage-displayed method. Compared to the homologous coating antigen, use of a heterologous coating antigen ...

  • Development of an Indirect Competitive Enzyme-Linked Immunosorbent Assay for Glycocholic Acid Based on Chicken Single-Chain Variable Fragment Antibodies
    2017
    Co-Authors: Xiping Cui, Natalia Vasylieva, Bogdan Barnych, Jun Yang, Ding Shen, Suqing Zhao, Shirley J. Gee, Bruce D Hammock
    Abstract:

    Glycocholic Acid (GCA) is an important metabolite of bile Acids, whose urine levels are expected to be a specific diagnostic biomarker for hepatocellular carcinoma (HCC). A high-throughput immunoassay for determination of GCA would be of significant advantage and useful for primary diagnosis, surveillance, and early detection of HCC. Single-chain variable fragment (scFv) antibodies have several desirable characteristics and are an attractive alternative to traditional antibodies for the immunoassay. Because chicken antibodies possess single heavy and light variable functional domains, they are an ideal framework for simplified generation of recombinant antibodies for GCA detection. However, chicken scFvs have rarely been used to detect GCA. In this study, a scFv library was generated from chickens immunized with a GCA hapten coupled to bovine serum albumin (BSA), and anti-GCA scFvs were isolated by a phage-displayed method. Compared to the homologous coating antigen, use of a heterologous coating antigen resulted in about an 85-fold improvement in sensitivity of the immunoassay. This assay, under optimized conditions, had a linear range of 0.02–0.18 μg/mL, with an IC50 of 0.06 μg/mL. The assay showed negligible cross-reactivity with various related bile Acids, except for taurocholic Acid. The detection of GCA from spiked human urine samples ranged from 86.7% to 123.3%. These results, combined with the advantages of scFv antibodies, indicated that a chicken scFv-based enzyme-linked immunosorbent assay is a suitable method for high-throughput screening of GCA in human urine