Yingshan Chen

Development of a Highly Specific Fluorescence Immunoassay for Detection of Diisobutyl Phthalate in Edible Oil Samples.

The diisobutyl phthalate (DiBP) hapten containing an amino group was synthesized successfully, and the polyclonal antibody against 4-amino phthalate-bovine serum albumin (BSA) was developed. On the basis of the polyclonal antibody, a rapid and sensitive indirect competitive fluorescence immunoassay (icFIA) has been established to detect DiBP in edible oil samples for the first time. Under the optimized conditions, the quantitative working range of the icFIA was from 10.47 to 357.06 ng/mL (R(2) = 0.991), exhibiting a detection limit of 5.82 ng/mL. In this assay, the specific results showed that other similar phthalates did not significantly interfere with the analysis, with the cross-reactivity less than 1.5%, except for that of DiBAP. Thereafter, DiBP contamination in edible oil samples was detected by icFIA, with the recovery being from 79 to 103%. Furthermore, the reliability of icFIA was validated by gas chromatography-mass spectrometry (GC-MS). Therefore, the developed icFIA is suitable for monitoring DiBP in some edible oil samples.

A fluorescence polarization immunoassay method for detection of the bisphenol A residue in environmental water samples based on a monoclonal antibody and 4′-(aminomethyl)fluorescein

Based on a sensitive monoclonal antibody against bisphenol A (BPA) and a new tracer named BVA–AMF, a homogeneous fluorescence polarization immunoassay (FPIA) was developed and applied in the determination of bisphenol A in environmental water samples. BVA was selected as the hapten to couple with bovine serum albumin and the conjugate was used as the immunogen for the monoclonal antibody production. Three fluorescein-labeled BVA tracers with different structures (BVA–AMF, BVA–EDF, and BVA–lysFITC) were synthesized. Under the same optimal conditions, BVA–AMF showed the highest sensitivity for FPIA and the detection of BPA exhibited a limit of detection of 5.60 ng mL−1, an IC50 of 140 ng mL−1 and a dynamic range of 11.32–904.21 ng mL−1 approximately. In this assay, several similar compounds were shown of little significantly with the cross-reactivity being less than 0.15%. Four different kinds of water samples were analyzed, with recoveries being 87.91–114.28%. The detection standard curve of BPA exhibited a good linearity (R2 = 0.9913, n = 3). Compared with ELISA and HPLC methods, FPIA showed reliability and a high correlation with ELISA of 0.9964 and HPLC of 0.9971. The proposed immunoassay technique is suitable for detection of BPA in authentic environmental water samples.

Development of a double-antibody sandwich ELISA for rapid detection to C-peptide in human urine

&NA; C‐peptide level is recognized as an important indicator of diabetes diagnosis. A sensitive and specific double‐antibody sandwich enzyme‐linked immunosorbent assay for the detection of C‐peptide based on double antibody sandwich method was studied in this paper. The rabbit and hen were innunized with PLL‐C‐peptide and BSA‐C‐peptide respectively to obtain specific Yolk antibody (IgY) and polyclonal antibody used to construct the sandwich ELISA for the measurement of C‐peptide. The limit of detection was 0.51 &mgr;g/mL and the half maximal inhibitory concentration (IC50) was 3.26 &mgr;g/mL. The method developed in the study showed no evident cross‐reactivity with other similar analogs. The detection standard curve of C‐peptide exhibited a good linearity (R2 = 0.9896, n = 15). 17 types of the urine of diabetes patients on c‐peptide levels compared with the hospital type of diabetes information, with a conclusion of a high consistent rate. Therefore, the methods could be selectively used for rapid screening of C‐peptide in human urine, and the type of diabetes has some referential significance. Graphical abstract Figure. No caption available.

Development of a simple, rapid and high-throughput fluorescence polarization immunoassay for glycocholic acid in human urine

HIGHLIGHTSAn FPIA method was developed to quantify glycocholic acid in urine samples.The sensitivity difference among different tracers was studied.The new method performed satisfactory sensitivity and good recovery.The method was suitable for routine hospital laboratory. 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 306ng/mL. The working range of FPIA was 36 ˜ 2 600ng/mL and the limit of detection (LOD) was 9ng/mL. The developed FPIA was time‐saving that could be completed within 10min. 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

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.

Synthesis and structure–activity relationship of N4-benzylamine-N2-isopropyl-quinazoline-2,4-diamines derivatives as potential antibacterial agents

A series of N4-benzylamine-N2-isopropyl-quinazoline-2,4-diamine derivatives has been synthesized and tested for antibacterial activity against five bacterial strains. Twelve different substituents on the N4-benzylamine group have been investigated along with replacement of the quinazoline core (with either a benzothiophene or regioisomeric pyridopyrimidine ring systems). In order to develop structure activity relationships, all derivatives were tested for their antibacterial activities against Escherichia coli and Staphylococcus aureus via Kirby–Bauer assays and minimum inhibitory concentration assays. Eight of the most potent compounds against S. aureus and E. coli were also screened against one strain of methicillin-resistant S. aureus (MRSA), Staphylococcus epidermidis and Salmonella typhimurium to further examine their antibacterial activities. Lead compound A5 showed good activities with MICs of 3.9 μg mL−1 against E. coli, S. aureus and S. epidermidis and 7.8 μg mL−1 against MRSA. Selected front runners were also screened for their DMPK properties in vitro to assess their potential for further development.