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A low-cost bifunctional immunochromatographic colorimetric biosensor was developed that can be read visually or by using an optical density scanner. Five test lines (T lines) coated with different antigens were set on a nitrocellulose (NC) membrane to indicate the concentration of analyte. This method was applied for the detection of dexamethasone. The corresponding detection range was 0.1-9 ng mL -1 , and the detection limit for dexamethasone in food supplements and cosmetic samples was 2.0 μg kg -1 . For visual inspection of the colour the quantitative relative error range between the proposed method and liquid chromatography was -62 to -25%, with a detection time of only 10 min. More accurate assay results were obtained by using an optical density scanner with the relative error range of -31 to 20%. The results indicated that the proposed method has the potential of application for rapid and efficient screening of dexamethasone in cosmetics and food supplements. Graphical abstract.

Phthalate acid esters (PAEs) contamination raised concerns as a result of migration from food packaging and environmental exposure. Because of the adverse effects of PAE reported in humans, the aim of this study was to examine the ability to screen for the detection these chemicals as an indicator of potential exposure. Too develop a sensitive screening test to determine PAE, a specific polyclonal antibody against phthalic acid (PA), the hydrolysate of PAEs, was used as a marker of total PAEs. This method involved the use of 4-aminophthalic acid (APA) as an immunizing hapten to generate antibody. Subsequently, this antibody conjugated with labeled gold nanoparticles (GNPs) was then used to develop an immunochromatographic assay (ICA) for visually detecting PA. After establishing optimal assay conditions, the ICA strip detected visually PA at 3 μg/ml rapidly in less than 5 min. Further, this assay exhibited reliable specificity for PA with no apparent cross-reactivity with structurally related PAEs. A significant correlation between data obtained with the ICA strip and high-performance liquid chromatography (HPLC) analysis was achieved using cooking oils as model spiked samples. The proposed use of ICA offers an effective tool for rapid on-site screening for total PAEs in cooking oils.

To deepen our understanding of the physiochemical principles that govern hapten-antibody recognition, ofloxacin enantiomers were chosen as a model for epitope prediction of small molecules. In this study, two monoclonal antibodies (mAbs) mAb-WR1 and mAb-MS1 were raised against R-ofloxacin and S-ofloxacin, respectively. The enantioselective mAbs have a high sensitivity and specificity, and the enantioselectivity is not affected by heterologous coating format reactions. The epitopes of the ofloxacin isomers were predicted using the hologram quantitative structure-activity relationship (HQSAR) and comparative molecular field analysis (CoMFA) approaches. The results consistently show that the epitope of the chiral hapten should be primarily composed of the oxazine ring and the piperazinyl ring and mAbs recognize the hapten from the side of this moiety. The enantioselectivity of mAbs is most likely due to the steric hindrance caused by the stereogenic center of the epitope. Modeling of chiral hapten-protein mimics reveals that ofloxacin isomers remain upright on the surface of the carrier protein. Suggestions to improve the enantioselectivity of antibodies against ofloxacin isomers were also proposed. This study provided a simple, efficient, and general method for predicting the epitopes of small molecules via molecular modeling. The epitope predictions for small molecules may create a theoretical guide for hapten design.

Coumaphos, an organophosphorus pesticide (OP) used worldwide, has raised serious public concerns due to its positive association with major types of cancer. Herein, a novel method for attomolar coumaphos detection was developed on the basis of an electrochemical displacement immunoassay coupled with oligonucleotide sensing. An optimized displacement immunoassay was constructed to improve the binding efficiency of an antigen-antibody pair, and a guanine-rich single-strand DNA label, in combination with oligonucleotide sensing, was used to amplify the detection signal with "direct" relationship to the analyte. As a result, coumaphos was sensitively determined from the enhanced catalytic cycle of guanine-Ru(bpy)(3)(2+) by chronoamperometry. The limit of detection (LOD) was down to 0.18 ng L(-1) (S/N = 3), which is equal to 49.6 amol in a sample solution of 100 μL. In comparison with conventional methods, the proposed method has the lowest LOD and better accessibility to high-throughput sensing systems. Besides, it can complete the whole analysis process in under 50 min and exhibits good performance of excellent selectivity to the OPs. With regard to the advantages of rapidity, convenience, low cost, and ease of operation, the proposed method has provided a promising platform capable of fast and in-field OP detection, which may make the system promising for potential applications in the detection of other small molecules.

The production and characterization of an anti-clenbuterol single-chain Fv antibody (CBLscFv)-bacterial alkaline phosphatase (AP) fusion protein are described. The CBLscFv and the phoA gene of Escherichia coli strain K12 chromosomal DNA were cloned by PCR and sequentially inserted into the expression vector pBV220 to express the CBLscFv-AP fusion protein in E. coli strain BL21(DE3)pLysS. SDS-PAGE and western blot analyses revealed that the fusion protein showed a molecular weight of 73 kDa and bound with the antibacterial AP monoclonal antibody. Determination of enzymatic activity indicated that k(cat) and K(m) values of the fusion protein were 113.60 s(-1) and 29.82 microM, respectively. Competitive direct enzyme-linked immunosorbent assay based on the obtained fusion protein indicated that the average concentration required for 50% inhibition of binding (IC(50)) and the limit of detection for CBL were 4.74 +/- 0.003 (n = 3) and 0.54 +/- 0.004 (n = 3) microg/l, respectively, and the linear response range extended from 1.13 to 69.68 microg/l. Cross-reactivity studies showed that the fusion protein did not cross-react with CBL analogs. The present findings indicate that the production of the CBLscFv-AP fusion protein in E. coli strain BL21(DE3)pLysS is feasible and suggest that it could be further used to develop a one-step ELISA for the specific detection of CBL.

Having the benefits of low cost, excellent stability and tolerance to extreme conditions, nanozymes are a potential alternative of horseradish peroxidase (HRP) or other enzymes for bioanalytical chemistry, especially immunoassays. CeO2 nanoparticles have oxidase-mimicking activity and can avoid the use of unstable H2O2. For robust assays, the effect of proteins on the activity of CeO2 needs to be carefully studied. Herein, we studied the adsorption and desorption of bovine serum albumin (BSA) from CeO2. The CeO2 nanoparticles exhibited a higher protein adsorption capacity compared to the other tested metal oxide nanoparticles. Although the oxidase-like activity of CeO2 was inhibited by BSA, low concentrations of phosphate and fluoride ions boosted the activity of protein-capped CeO2. CeO2 was still active under strong acidic conditions and at high temperature, while HRP lost its activity. For immunoassay development, we covered CeO2 with an amine-modified silane for covalent conjugation to antibodies. A one-step indirect competitive ELISA for fenitrothion was developed, and an IC50 value of 35.6 ng mL−1 and a limit of detection of 2.1 ng mL−1 were obtained. [ABSTRACT FROM AUTHOR]

Immunoassay direct for 3-amino-5-morpholinomethyl-2-oxazolidone (AMOZ), the metabolite of furaltadone, based on a specific antibody was usually unavailable due to its small molecule weight. In this work, a hapten with a double bond and an active carboxyl group was designed and then conjugated to peptide dendrimer to obtain novel multiple haptens. The haptens and multiple haptens were coupled to bovine serum albumin as immunogens. Polyclonal antibodies showed binding to free AMOZ in a heterologous competitive indirect enzyme-linked immunosorbent assay were obtained. The assay showed IC50 (half maximal inhibitory concentration) of 4.1 μg/kg and limit of detection of 0.2 μg/kg for AMOZ. Recoveries of AMOZ from grass carp were tested from 83.1% to 117.0%, with the coefficient of variation below 12%. As no laborious derivatisation procedure was employed, the proposed assay should be useful for the screening of AMOZ for a large number of samples. [ABSTRACT FROM AUTHOR]

To produce specific antibodies against leuco-malachite green (LMG), 15 haptens were synthesized and characterized, and conjugated to carrier protein for immunization. One antigen with excellent reactogenicity and immunogenicity was discovered. Specific monoclonal antibody with high sensitivity for LMG in competitive indirect enzyme-linked immunosorbent assay (ciELISA) was screened and selected. After the screening of a series of heterologous coating antigen and optimization of working conditions, the proposed ciELISA showed the 50% inhibition value (IC50) of 1.16 ng mL−1and the limit of detection of 0.06 ng mL−1for LMG. The average recoveries of LMG from spiked fish samples ranged from 78.0% to 101.0%, with coefficients of variation below 15%. Good correlation (R2 = 0.9977) was obtained between the results of ciELISA analysis and those of standard liquid chromatography–tandem mass spectrometry analysis. The proposed ciELISA is ideal for the rapid and sensitive detection of LMG with a low cost and high throughput. [ABSTRACT FROM PUBLISHER]

In this study, ofloxacin stereoisomers were chosen as a simple model to investigate the stereospecific recognition of chiral haptens and antibodies. Three polyclonal antibodies were studied and showed a relatively high enantioselectivity and an excellent sensitivity. Comparative molecular field analysis and comparative molecular similarity indices analysis were employed to investigate the chiral recognition between the antibody and the ofloxacin enantiomer, and all the models yielded high correlation and predictive ability. It was found that the chiral discrimination was probably caused by steric hindrance; the antibody stereospecificity could be ascribed to the variation of the R1 and R3 groups of quinolones; the common structure of the quinolones is also essential in the hapten–antibody recognition. The recognition between the chiral haptens and the antibodies was co-affected by multiple interaction forces, and those forces were defined explicitly at the sub-structural level. An illustrative enhanced model with good simplicity and universality was also developed for a better understanding of the stereospecific recognition of ofloxacin enantiomers and antibodies for the first time. This work provides insights into the stereospecific recognition of chiral haptens and antibodies. [ABSTRACT FROM AUTHOR]

A rapid, specific, and sensitive fluorescence polarization immunoassay (FPIA) method was developed to detect residues of furazolidone, a forbidden drug in feed. An immuno-hapten was designed for monoclonal antibody preparation. Furthermore, seven novel tracers were synthesized and the 'heterology tracer' could improve the sensitivity of FPIA significantly. An optimized FPIA method was established with a cross-reactivity of less than 0.1%, IC50 of 5.5 ng/mL. The limits of detection (LOD) calculated from feed samples were 0.5-0.9 ng/mL. Recoveries of fortified samples at levels of 5, 20, and 50 ng/mL ranged from 79.0% to 85.0%. The coefficients of variation were less than 12%. [ABSTRACT FROM AUTHOR]

Phage display method was used to clone anti-carbofuran (CBF) single chain Fv (scFv) gene. The heavy chain and light chain variable region genes were amplified by the polymerase chain reaction from the CBF-specific hybridoma cell lines 5D3 and assembled as a scFv DNA fragment with linker peptide (Gly4Ser)3. The scFv DNA fragment was cloned into M13 phagemid vector pCANTAB5E and the anti-CBF antibody libraries were then constructed. After one round of panning with CBF-ovalbumin (CBF-OVA) as a conjugate, antigen-binding positive recombinant phage clones were successfully selected by enzyme-linked immunosorbent assay (ELISA). The positive phages were used to infect Escherichia coli HB2151 cells and the expression of the soluble scFv antibodies was then induced by IPTG. The scFv antibody was about 31 kDa by SDS-PAGE and showed HRP-anti-E-tag antibody-recognized activity by Western blotting. The indirect competitive ELISA (icELISA) showed that the recombinant scFv antibody could competitively combine with CBF, with the IC50 value of 1.07 ng/mL. The cross reactivity studies showed that the anti-CBF scFv antibody, similar to the parent monoclonal antibody, poses high specificity to CBF and has little reactivity to the analogs. Taken together, these findings suggest that the recombinant scFv antibody can be used for further developing immunoassay method for CBF. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [ABSTRACT FROM AUTHOR]

Abstract: The incorporation of melamine into food products is banned but its misuse has been widely reported in both animal feeds and food. The development of a rapid screening immunoassay for monitoring of the substance is an urgent requirement. Two haptens of melamine were synthesized by introducing spacer arms of different lengths and structures on the triazine ring of the analyte molecular structure. 6-Aminocaproic acid and 3-mercaptopropionic acid were reacted with 2-chloro-4,6-diamino-1,3,5-triazine (CAAT) to produce hapten 1 [3-(4,6-diamino-1,6-dihydro-1,3,5-triazin-2-ylamino) hexanoic acid] and hapten 2 [3-(4,6-diamino-1,6-dihydro-1,3,5-triazin-2-ylthio) propanoic acid], respectively. The molecular structures of the two haptens were identified by 1H nuclear magnetic resonance spectrometry, mass spectrometry and infrared spectrometry. An immunogen was prepared by coupling hapten 1 to bovine serum albumin (BSA). Two plate coating antigens were prepared by coupling both haptens to egg ovalbumin (OVA). A competitive indirect enzyme-linked immunosorbent assay (ciELISA) was developed to evaluate homogeneous and heterogeneous assay formats. The results showed that polyclonal antibodies with high titers were obtained, and the heterogeneous immunoassay format demonstrated a better performance with an IC50 of 70.6ngmL−1, a LOD of 2.6ngmL−1 and a LOQ of 7.6ngmL−1. Except for cyromazine, no obvious cross-reactivity to common compounds was found. The data showed that the hapten synthesis was successful and the resultant antisera could be used in an immunoassay for the rapid and sensitive detection of this banned chemical. [Copyright &y& Elsevier]

Schematic diagram of Database-assisted workflow to prescreen scFv from hybridomas and the development of one-step CLEIA based on scFv-AP. • Database-assisted workflow to prescreen scFv from hybridomas was presented. • The rearrangement between functional and the aberrant light chain was identified. • Characterization of scFv candidates was conducted by one-step ELISA. • A sensitive competitive CLEIA was developed based on scFv-AP fusion protein. • The developed CLEIA was applied for determination of total MG in tilapia fish. To determine malachite green (MG) and its major metabolite, leucomalachite green (LMG) residual levels in tilapia fish, chemiluminescent enzyme immunoassay (CLEIA) was developed based on a single-chain variable fragment (scFv)-alkaline phosphatase (AP) fusion protein. At first, V H and V L gene sequences were cloned from hybridoma cell lines secreting monoclonal antibody against LMG, and then thoroughly by database-assisted sequence analysis. Finally, the productive V H and V L were assembled to an intact scFv sequence and engineered to produce scFv-AP fusion protein. The fusion protein was further identified as a bifunctional reagent for immunoassay, then a sensitive one-step CLEIA against LMG was developed with a half-maximal inhibitory concentration (IC 50) and limit of detection (LOD) of 1.3 and 0.04 ng/mL, respectively. The validation results of this novel competitive CLEIA was in line with those obtained by classical HPLC method for determination of total MG in spiked and field incurred samples. [ABSTRACT FROM AUTHOR]