Construction, expression and functional analysis of anti-clenbuterol codon-optimized scFv recombinant antibody.

The construction, expression and functional analysis of codon-optimized single-chain variable fragment (coscFv) against clenbuterol (CBL) prepared from the Escherichia coli system is described. First, the ionic concentration for coscFv expression was optimized through single-factor experiments. Then, the extraction conditions of inclusion bodies were optimized, and coscFv was affinity-purified. Finally, the functional analysis of coscFv was elucidated by indirect competitive enzyme-linked immunosorbent assay (icELISA) and molecular docking. After optimizing the ionic concentration, the yield of coscFv increased from 21.69% to 23.26%. The molecular weight of coscFv was determined to be approximately 27 kDa according to the SDS-PAGE and Western blot assay. The percentage of coscFv was as high as 43.9% after the inclusion bodies were extracted, washed, and dissolved. Functional analysis indicated that the coscFv recognized CBL, and the 50% inhibition average concentration of CBL (IC50) was 4.22 ± 0.01 (n = 3) ng/mL. The binding site between coscFv and CBL consisted of Asp33H, Met34H, Ser50H, Arg52H, Tyr57H, Leu59H, Asp99H, and Tyr93L. Our study confirms that coscFv can bind with CBL through the key amino acid residues and can be used to sensitively detect CBL.

Comparison of three sample addition methods in competitive and sandwich colloidal gold immunochromatographic assay.

Immunochromatographic assays (ICAs) are mainstream point-of-care diagnostic tools in disease control, food safety, and environmental monitoring. However, the important issue pertaining to the influence of sample addition methods on the detection performance of ICAs has not been addressed, and related information is still lacking. Herein, we selected the well-accepted gold nanoparticles (AuNPs) as visual labels. AuNP-based ICA was then used to explore the effects of three sample addition methods (i.e., dry, wet, and insert) on the analytical performance of ICAs by using competitive and sandwich models. Under optimized conditions, the competitive ICA with clenbuterol as an analyte showed a negligible difference (p > 0.05) in the detection performance of the three methods in ideal phosphate buffered saline solution. However, the wet method demonstrated the worst performance in pork samples (p < 0.05). The sandwich ICA strip with human chorionic gonadotropin as an analyte revealed the significantly different analytical performances of the three approaches in phosphate buffer (PB) solution and spiked serum (p < 0.05). Two independent linear correlations were observed with the increase in target concentration. However, for the wet method in the PB solution and serum, the first linear correlation was at a relatively narrow target concentration range, and the second linear correlation was at a wider concentration range compared with those for the dry and insert methods. Our findings demonstrated that sample addition methods slightly influence competitive ICAs (p > 0.05) but remarkably affect sandwich ICAs (p < 0.05). We believe that this study can further explain the differences in detection results for the same target analyte in actual ICA detection. The results may serve as a reference in the rational selection of the appropriate sample addition method for succeeding ICA works.

A silica nanoparticle based 2-color immunochromatographic assay for simultaneous determination of clenbuterol and ractopamine.

An immunochromatographic assay (ICA) is presented that can be applied to simultaneous detection of clenbuterol (CLE) and ractopamine (RAC). It is making use of two red and blue silica nanoparticles (SiNPs) that act as labels for encoding the antibodies. This design permits multiplexed analysis in a single test line and does not require an external source for photoexcitation. Anti-CLE was labeled with red SiNPs, and anti-RAC with blue SiNPs. The capture antigens CLE-BSA and RAC-BSA were placed onto the conjugate pad and the test line of the test strip, respectively. Under bare eye examination, no cross-colored lines or nonspecific bioconjugate adsorption were observed, and the visible limit of detections for CLE (red) and RAC (blue) are 3 and 2 ng‧mL-1, respectively. This design allows for multiplexed detection with reduced device dimensions and costs, and with easy integration and manufacturing. Conceivably, the method may be extended to simultaneous determination of numerous other analytes. Graphic abstract The principle of qualitative detection strategy of multiplex immunochromatographic assay for clenbuterol (CLE) and ractopamine (RAC) is schematically illustrated. Depending on the type and ratio of organic dyes, the color of colored silica nanoparticle can be tuned from red to purple and even to black (lower right corner).

A voltammetric immunosensor for clenbuterol based on the use of a MoS2-AuPt nanocomposite.

An ultrasensitive immunosensor for the direct detection of the illegally used livestock feed clebuterol (CLB) is described. It is based on the use of a glassy carbon electrode modified with an MoS2-AuPt nanocomposite and on biotin-streptavidin interaction. The use of MoS2-AuPt accelerates electron transfer, and this leads to a sharp increase in the electrochemical signal for the electrochemical probe hydrogen peroxide. Differential pulse voltammetry was used to record the current signal at a peak potential of -0.18 V (vs SCE). Under optimal conditions, the electrode has a linear response in the 10 pg·mL-1 to 100 ng·mL-1 CLB concentration range and a 6.9 pg·mL-1 detection limit (based on the 3σ criterium). This immunosensor is sensitive, highly specific and acceptably reproducible, and thus represents a valuable tool for the determination of CLB in pork. Graphical abstract Schematic of a voltammetric immunosensor for the determination of clenbuterol (CLB) based on the use of a nanocomposite prepared from molybdenum disulfide and a gold-platinum alloy (MoS2-AuPt), and making use of the biotin-streptavidin system.

Development of an ELISA to detect clenbuterol in swine products using a new approach for hapten design.

This research outlines the application of an enzyme-linked immunosorbent assay (ELISA) for the analysis of clenbuterol in animal products. Our assay showed good sensitivity for clenbuterol (0.4 ng/g or 0.4 ppb) and low detection limit (0.09 ng/g or 0.09 ppb). A low cross-reactivity for other ß2-agonist drugs such as salbutamol, terbutaline, and epinephrine led to formatting an ELISA kit considered to have a high specificity for clenbuterol. A survey of Ho Chi Minh City pork market was conducted as part of the validation of our ELISA. ELISA results showed a surprisingly high value of contamination. However, it will be necessary to conduct a more statistically valid replicated survey with evaluation by other instrumental methods to obtain a definite conclusion. This ELISA kit will be used to monitor growth promoter residues in Vietnam's animal products.

Coupling purification and in situ immobilization process of monoclonal antibodies to clenbuterol for immunosensor application.

Clenbuterol (CL), which promotes the growth of muscular tissue and the reduction of body fat in pigs and cattle, has been confirmed to be a potential hazard to human health. In this study, a monoclonal antibody to clenbuterol (CL mAb) from a hybridoma culture supernatant was purified by an aqueous two-phase system (ATPS) at different polyethylene glycol (PEG) concentrations, PEG molecular weights, pH values, and NaCl concentrations. Then the CL mAb was immobilized in situ by directly adding polystyrene microspheres (PSMSs) into a PEG phase containing CL mAb. Using the immobilized antibody, an immunosensor was constructed to detect the CL residues in pork samples. The results showed that using an ATPS composed of 15% (w/w) PEG6000, 15% (w/w) phosphate, and 15% (w/w) NaCl at pH 8.0, the partition coefficient was 7.24, the activity recovery was 87.86%, and the purification fold was 2.88. The PEG-CL mAb-PSMS retained approximately 98% of its initial activity after 30-ml phosphate buffer (PBS) washings. After 30days of storage, the CL mAb-PSMS lost nearly 75% of its activity, whereas the PEG-CL mAb-PSMS retained as much as 95% of its initial activity. Furthermore, the constructed immunosensor obtained recoveries of 90.5 to 102.6% when applied to pork samples spiked with CL.

[Purification of monoclonal antibody to clenbuterol and its biology identity].

OBJECTIVE: To identify the self-preparation monoclonal antibody which target to clenbuterol, and set up the standard curve to clenbuterol (CL) detection. METHODS: The affinity constants and activity of the monoclonal antibody which target to CL were determined by ELISA. ELISA was also used to confirm whether the monoclonal antibody had any across-reaction with BSA and CL analogues. The rat ascites which contains the monoclonal antibody target to CL was purified by (NH4)2SO4 salt-out method and further by affinity column. At last, the CL detection standard curve which based on indirect competition ELISA was established. RESULTS: The ELISA experiment showed that the antibody titer was 10(6) and the monoclonal antibody affinity constants was 2.90 x 10(10) L/mol. The result of the indirect competition ELISA confirmed that the monoclonal antibody had no cross-reaction with BSA and a few kind of CL analogue. CL detection standard curve based on indirect competition ELISA was established, which R2 was 0.9812, and the lowest detectable limit was 1.0 ng/ml. CONCLUSION: The standard curve based on indirectly competitioning ELISA was established. The self-preparation monoclonal antibody which target to CL has high affinity and high specific to CL, which had established the foundation to the advanced development of the CL immune test paper and CL ELISA kit.

Codon modification for the DNA sequence of a single-chain Fv antibody against clenbuterol and expression in Pichia pastoris.

The expression efficiency was improved for the recombinant single-chain variable fragment (scFv) against clenbuterol (CBL) obtained from mouse and expressed in the methylotrophic yeast Pichia pastoris GS115, by redesigning and synthesizing the DNA sequence encoding for CBL-scFv based on the codon bias of P. pastoris. The codons encoding 124 amino acids were optimized, in which a total of 156 nucleotides were changed, and the G+C ratio was simultaneously decreased from 53 to 47.2 %. Under the optimized expression conditions, the yield of the recombinant CBL-scFv (41 kDa) antibodies was 0.223 g L⁻¹ in shake culture. Compared to the non-optimized control, the expression level of the optimized recombinant CBL-scFv based on preferred codons in P. pastoris demonstrated a 2.35-fold higher yield. Furthermore, the recombinant CBL-scFv was purified by Ni-NTA column chromatography, and the purity was 95 %. The purified CBL-scFv showed good CBL recognition by a competitive indirect enzyme-linked immunoassay. The average concentration required for 50 % inhibition of binding and the limit of detection for the assay were 5.82 and 0.77 ng mL⁻¹, respectively.

Quantum dots based electrochemiluminescent immunosensor by coupling enzymatic amplification for ultrasensitive detection of clenbuterol.

An ultrasensitive electrochemiluminescence (ECL) immunosensor based on CdSe quantum dots (QDs) has been designed for the detection of clenbuterol. The immunosensor was fabricated by layer by layer and characterized with atomic force microscopic images (AFM) and electrochemical impedance spectra (EIS). In oxygen-saturated pH=9.0 Tris-HCl buffer, a strong ECL emission of QDs could be observed during the cathodic process due to the H2O2 product from electrochemical reduction of dissolved oxygen. Upon the formation of immunocomplex, the second antibody labeled with horseradish peroxidase was simply immobilized on the electrode surface. The ECL emission decreased since steric hindrance of the immunocomplex slowed down the electron-transfer speed of dissolved oxygen, and also could be greatly amplified by an enzymatic cycle to consume the self-produced coreactant. Using clenbuterol as model analyte, the ECL intensity was determined by the concentration of competitive immunoassay of clenbuterol with a wide calibration in the range of 0.05 ng mL(-1) to 1000 ng mL(-1), and a low detection limit was 0.02 ng mL(-1). The immunosensor shows good stability and fabrication reproducibility. It was applied to detecting practical samples with the satisfactory results. This immunosensing strategy opens a new avenue for detection of residue and application of QDs in ECL biosensing.

Sensitive detection of small molecules by competitive immunomagnetic-proximity ligation assay.

A novel detection method of small molecules, competitive immunomagnetic-proximity ligation assay (CIPLA), was developed and described in this report. Through the proximity effect caused by special proximity probes we prepared, small molecules can be detected using only one monoclonal antibody. CIPLA overcomes the obstacle that the proximity ligation assay (PLA) cannot be used in small molecular detection, as two antibodies are unable to combine to one small molecule due to its small molecular structure. Two small molecular compounds, clenbuterol (CLE) and ractopamine (RAC), were selected as targets for CIPLA. The limit of detection (LOD) reached 0.01 ng mL(-1), which was 10-50-fold lower than ELISA. With 5 orders of magnitude of the dynamic range achieved, the excellent sensitivity and broad dynamic range of CIPLA are noted. It can be applied widely in the sensitive detection of many other small molecular materials such as pesticides, additives in food, drugs, and biological samples, which have great significance in both theoretical and practical aspects.

Highly sensitive detection of clenbuterol using competitive surface-enhanced Raman scattering immunoassay.

In this report, we present a novel approach to detect clenbuterol based on competitive surface-enhanced Raman scattering (SERS) immunoassay. Herein, a SERS nanoprobe that relies on gold nanoparticle (GNP) is labeled by 4,4'-dipyridyl (DP) and clenbuterol antibody, respectively. The detection of clenbuterol is carried out by competitive binding between free clenbuterol and clenbuterol-BSA fastened on the substrate with their antibody labeled on SERS nanoprobes. The present method allows us to detect clenbuterol over a much wider concentration range (0.1-100 pg mL(-1)) with a lower limit of detection (ca. 0.1 pg mL(-1)) than the conventional methods. Furthermore, by the use of this new competitive SERS immunoassay, the clenbuterol-BSA (antigen) is chosen to fasten on the substrate instead of the clenbuterol antibody, which could reduce the cost of the assay. Results demonstrate that the proposed method has the wide potential applications in food safety and agonist control.

Rapid determination of clenbuterol in urine by a competitive bead immunoassay based on Luminex technology.

A new competitive bead immunoassay (CBIA) based on Luminex technology for detecting clenbuterol in urine was reported. The carboxylated fluorescent beads were directly coated with clenbuterol derivatives without carrier protein spacer. Clenbuterol antibody was biotinylated, which was used for clenbuterol detection in combination with the functionalized bead and streptavidin-phycoerythrin (SAPE). The effects of spacer on the CBIA method were investigated. The results indicated that the presence of small molecular spacer between bead and hapten improved the assay sensitivity and the hydrophilic spacer (glycine) was better than the hydrophobic spacer (m-aminobenzoic acid) for this CBIA method. The study affirms the importance of the judicious choice of hapten derivatives in the CBIA method for detecting small molecule drug based on Luminex technology. The method could be used for clenbuterol detection in livestock urine and possible for the simultaneous detection of multiple veterinary drugs.

Expression and purification of an anti-clenbuterol single chain Fv antibody in Escherichia coli.

Recombinant antibodies with desirable characteristics that can replace polyclonal or monoclonal antibodies are important for enzyme-linked immunosorbent assay (ELISA) of residues of clenbuterol (CBL), an illicit veterinary drug. Here, we report our work on expression and purification of a mouse-derived anti-CBL single chain Fv (scFv) antibody in Escherichia coli (E. coli). An expression plasmid pBV220-CBL was constructed and transformed into E. coli BL21 (DH3) strain cells. After induction by temperature, the 6x His-tagged anti-CBL scFv antibodies were expressed with the yield of 31%. The solubilized inclusion bodies were extracted, denatured and then purified by Ni-NTA column chromatography. The purified recombinant target protein was analyzed by high performance liquid chromatography, SDS-PAGE and Western blotting, respectively. The results showed the prepared anti-CBL scFv antibodies posed HRP-anti-His-tag antibody-recognized activity and their purity was up to 96%. Moreover, an indirect competitive ELISA based on the anti-CBL scFv antibodies revealed that the limit of detection for CBL was 0.5 ng/ml and the linear range was 1.5-10.6 ng/ml. Taken together, these findings suggest that the prepared recombinant antibody can be used for future immunoassay detection for CBL.

Production and characterization of a single-chain Fv antibody-alkaline phosphatase fusion protein specific for clenbuterol.

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.

Preparation of anti-salbutamol antibody based on a new designed immunogen and development of a heterologous indirect ELISA for detection of salbutamol residue.

To synthesize salbutamol immunogen and develop an enzyme immunoassay (ELISA), a new salbutamol immunogen was synthesized using 4-aminobenzoic acid as a linker to connect hapten with carrier protein. An enzyme immunoassay based on the antibody prepared was developed and applied to detect salbutamol residue spiked in swine liver. An unusual coating antigen, clenbuterol-ovalbumin (OVA) conjugate instead of salbutamol-OVA conjugate, was used in the immunoassay and the results were discussed based on the structures of related compounds. The antibodies showed high sensitivity in the heterologous assay when using clenbuterol-OVA as a coating antigen, with an IC50 value of 8.97 ng mL(-1) toward salbutamol. The antibodies prepared showed high cross-reactivity with clenbuterol (107%) and were promising for the simultaneous determination of salbutamol and clenbuterol residues in food and food products. Recovery rates from the salbutamol-spiked swine liver samples were in the range of 70%-99%, while the intra-assay and inter-assay coefficients of variation were <13.3% and <14.3%, respectively. In summary, the antibodies of salbutamol have been successfully prepared. Sensitive and stable analysis for the detection of salbutamol residues in swine liver was obtained based on the competitive ELISA methods developed in this study.

A novel CdSe/CdS quantum dot-based competitive fluoroimmunoassay for the detection of clenbuterol residue in pig urine using magnetic core/shell Fe3O4/Au nanoparticles as a solid carrier.

Quantum dots (QDs) are semiconductor fluorescent nanoparticles, which can be used for food safety or environmental monitoring with high sensitivity. This work demonstrates the feasibility of detecting clenuterol residue in pig urine using CdSe/CdS quantum dots as fluorescent labels based magnetic core/shell Fe3O4/Au nanoparticles (MCFN) as solid carriers. The detection of clenbuterol is carried out by a fluoroimmunoassay-based biosensor using competitive binding between conjugated clenbuterol antigen-CdSe/CdS QDs and free clenbuterol with immobilized clenbuterol antibodies on MCFN. This assay method allows for clenbuterol determination in a linear working range of 0.5-20000 pg mL(-1). It would provide a simple, rapid, and ultra-sensitive detection method for clenbuterol or other biomolecular analysis.

Development of a colloidal gold-based lateral-flow immunoassay for the rapid simultaneous detection of clenbuterol and ractopamine in swine urine.

A multianalyte lateral-flow immunochromatographic technique using colloidal gold-labeled polyclonal antibodies was developed for the rapid simultaneous detection of clenbuterol and ractopamine. The assay procedure could be accomplished within 5 min, and the results of this qualitative one-step assay were evaluated visually according to whether test lines appeared or not. When applied to the swine urines, the detection limit and the half maximal inhibitory concentration (IC(50)) of the test strip under an optical density scanner were calculated to be 0.1 +/- 0.01 ng mL(-1) and 0.1 +/- 0.01 ng mL(-1), 0.56 +/- 0.08 ng mL(-1), and 0.71 +/- 0.06 ng mL(-1), respectively, the cut-off levels with the naked eye of 1 ng mL(-1) and 1 ng mL(-1) for clenbuterol and ractopamine were observed. Parallel analysis of swine urine samples with clenbuterol and ractopamine showed comparable results obtained from the multianalyte lateral-flow test strip and GC-MS. Therefore, the described multianalyte lateral-flow test strip can be used as a reliable, rapid, and cost-effective on-site screening technique for the simultaneous determination of clenbuterol and ractopamine residues in swine urine.

[Preparation of clenbuterol monoclonal antibody with subtractive immunization method].

AIM: To obtain Clenbuterol monoclonal antibodies. METHODS: Clenbuterol complete antigen was prepared with diazotization method. BALB/c mice was immunized with subtractive immunization, Clenbuterol monoclonal antibody was prepared with rule hybridoma technique. RESULTS: The mice obtained tolerance to BSA by subtractive immunization. The rate of the hybridoma cell with positive reaction which had obtained was 8.2%, and the specific clenbuterol monoclonal antibody was obtained at last. CONCLUSION: Monoclonal antibodies to micromolecule contaminant be prepared by subtractive immunization, could decrease the workload in the bolting of monoclonal antibodies, and increase the chance to obtain the antibody of expected.

[Construction and expression of anti-clenbuterol single chain Fv recombinant vector].

To construct the recombinant vector pBV220-scFv and express anti-clenbuterol (CBL) scFv antibody in Escherichia coli, we amplified the scFv gene using plasmid pCANTABSE-CBL as a template, recombined it with pPICZalphaA, then amplified the scFv-His-tag gene from plasmid pPICZalphaA-scFv and linked it with expression plasmid pBV220. We identified the recombinant plasmid by restrictive enzyme digestion, PCR amplification and sequence analysis. Finally, we transformed the recombinant vector into E. coli DH5alpha that was temperature-induced and expressed recombinant protein. We identified the recombinant protein by SDS-PAGE, Western blotting and indirect competitive ELISA. The results show that recombinant plasmid pBV220-scFv contained the inserted fragment with highest homology about 99.8%. The expression of scFv induced by temperature show 37 kD Mw and anti-His-tag mAb recognized-activity by SDS-PAGE and Western blotting respectively, and could competitively combine with CBL, the IC50 is 4.55 ng/mL. The recombinant plasmid pBV220-scFv is constructed and expresses the scFv gene of CBL in E. coli successfully. This study suggests the corresponding immunoassay methods could be set up by the recombinant scFv.

Rapid determination of hazardous compounds in food based on a competitive fluorescence microsphere immunoassay.

Development of a microsphere-based competitive fluorescence immunoassay for the determination of hazardous low-molecular-weight compounds in food is described. In this method, antigens are covalently bound to carboxy-modified microspheres to compete monoclonal antibody with low-molecular-weight compounds in food samples; mouse IgG/fluorescein isothiocyanate conjugate is used as the fluorescent molecular probe. Thus, the hazardous low-molecular-weight compounds are quantified using a multiparameter flow cytometer. This method has been evaluated using clenbuterol as a model compound. It has a sensitivity of 0.01 ng/mL with dynamic range of 0.01-100 ng/mL, and the concentration of clenbuterol providing 50% inhibition (IC(50)) is 1.1 ng/mL. The main advantages of this method are its high efficiency, biocompatibility, and selectivity, as well as ultralow trace sample consumption and low cost.