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.

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.

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.

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.

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.

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.

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.

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.

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.

Development of an immunochromatographic lateral flow test strip for detection of beta-adrenergic agonist Clenbuterol residues.

A rapid immunochromatographic lateral flow test strip was developed in a competitive format with the gold-conjugated monoclonal antibody to specifically determine the residues of Clenbuterol (CL), a beta-adrenergic agonist. The test strip is made up of a sample pad, a conjugate reagent pad, a test membrane containing a control line and a test line, and an absorbent pad. CL standard samples of 0, 0.1, 0.3, 0.9, 2.7, 8.1 ng/ml in swine urine were determined by the test strip. It was shown that detection limit of the test strip was as low as 0.1 ng/ml of CL and that the half of maximal inhibition concentration (IC50) in relative optical density was calculated to be 1.78+/-0.17 ng/ml under an optical density scanner. The sensitivity by eye measurement was 1.0 ng/ml. It takes 10 min to accomplish a test. Parallel analysis of urine samples from pigs fed with CL showed comparable results obtained from the test strip and GC-MS. Therefore, the test strip is very useful as a screening method for quantitative, semi-quantitative or qualitative detection of CL residues in swine urine.

Production and characterization of single chain Fv directed against beta2-agonist clenbuterol.

The single chain Fv (scFv) directed against beta2-agonist clenbuterol (CBL) was produced by using phage display technology. The heavy chain and light chain variable region genes (VH) VL) were amplified by the polymerase chain reaction (PCR) from CBL specific hybridoma cell lines 5D1 and assembled as a single chain Fv (scFv) fragment with linker peptide (Gly4Ser)3. Then the scFv DNA fragment was cloned into M13 phagemid vector pCANTAB5E and the anti-CBL antibody libraries were constructed. Phages displaying scFv were enriched by panning with CBL-ovalbumin (CBL-OVA) conjugate. After only one round of panning, antigen-positive recombinant phage clones were successfully selected by ELISA. The positive phage was used to infect Escherichia coli HB2151, and the expression of soluble scFv was then induced by IPTG. The scFv showed an improved sensitivity (with IC50 of 0.78 +/- 0.005 ng/mL (n = 4)) when compared with the parent monoclonal antibody (MAb) (with IC50 of 1.34 +/- 0.006 ng/mL (n = 4)) in competitive indirect ELISA (CI-ELISA). Cross-reactivity studies showed that the specificity of scFv was similar to that of MAb. The recombinant scFv prepared in this study could be potentially used instead of conventional antisera or MAb for development of a rapid and affordable immunoassay for the detection of residual CBL in biological matrices.

Studies on the antibody response of Lama glama--evaluation of the binding capacity of different IgG subtypes in ELISAs for clenbuterol and BSA.

Camelidae are known to produce three subtypes of immunoglobulin G (IgG), two of which are devoid of light chains. Two llamas (Lama glama) were immunised against clenbuterol-bovine serum albumin (BSA). Enzyme-linked immunosorbent assays (ELISAs) for clenbuterol and BSA on the basis of protein A-coated microtitration plates were established to investigate the titre development. Three subclasses of IgG (IgG(1): 29+66KDD, IgG(2): 52KDD, IgG(3): 56KDD) depending on their different binding properties to protein A and protein G could be separated chromatographically. Only IgG(1), which consists of conventional four-chain antibodies, bound to clenbuterol, whereas all forms of heavy-chain antibodies merely bound BSA.

Solid phase extraction of clenbuterol from plasma using immunoaffinity followed by HPLC.

An immuno-extraction column for clenbuterol has been prepared. Optimum conditions for the selective retention and elution of clenbuterol have been developed, based on a modification of our earlier work on morphine, chlortoluron and isoproturon. Clenbuterol could be retained on the immuno-column then eluted in one x one ml fraction using 50% methanol in phosphate buffered saline pH 2. On columns containing antisera (but not to clenbuterol) the clenbuterol was removed in the washing step. HPLC-UV determination gave clean traces. Day-to-day reproducibility was improved by precipitating the plasma proteins with acetonitrile.

Effects of active immunization against clenbuterol on the growth-promoting effect of clenbuterol in rats.

We examined the effect of active immunization against clenbuterol on the growth-promoting effect of clenbuterol in rats in two experiments. Six-week-old female Sprague-Dawley rats were immunized against clenbuterol conjugated to histone by diazotization and then received clenbuterol approximately 4 wk after the initiation of immunization. Antibody titers were determined using indirect ELISA with diazotized clenbuterol-BSA conjugate as an antigen in coating the microwells. Antibody titer increased during booster injections. No significant difference in titer value was observed between two doses of immunogen (.1 vs .5 mg). Competitive ELISA showed that terbutaline cross-reacted with anti-clenbuterol antibodies, and the cross-reactivity was 12%. Alprenolol, propranolol, phentolamine, epinephrine, norepinephrine, and L644,969 showed no affinity for anti-clenbuterol antibodies. The rats immunized against clenbuterol-histone conjugate had 11% lower body weight gain during the 23-d immunization period than the rats immunized against histone only. When clenbuterol was administered after the immunization, no significant difference in growth rate was observed between the rats immunized against clenbuterol-histone conjugate and rats immunized against histone only. No significant difference in muscle weight was observed between the two groups at the termination of the experiment. Results indicate that active immunization against clenbuterol before clenbuterol administration did not modify the growth-promoting effects of clenbuterol in rats.

[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.

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.