Ultramarine blue nanoparticles as a label for immunochromatographic on-site determination of ractopamine.

A competitive immunochromatographic assay (ICA) is presented and used for on-site determination of ractopamine (RAC). Ultramarine blue nanoparticles were directly separated from ultramarine blue industrial products by centrifugation (< 10,000 rpm and > 4000 rpm) and used as visible labels in ICAs. The ultramarine blue nanoparticles were coated by polyacrylic acid (PAA), which provides carboxyl groups on the surface of ultramarine blue nanoparticles. An anti-RAC monoclonal antibody (mAb) was covalently immobilized on the carboxyl-modified ultramarine blue nanoparticle surface via diimide-activated conjugation between the carboxyl groups on the ultramarine blue nanoparticle surface and the amino groups of the antibodies. RAC and BSA-modified RAC competitively bind to the anti-RAC mAb on the ultramarine blue nanoparticles. The blue band in the test line is generated by the accumulation of ultramarine blue nanoparticles and is negatively associated with the RAC content. Under optimal conditions, the visual limit of detection (vLOD) of this ICA for RAC is 2.0 ng mL-1, 2.0 ng mL-1, and 1.0 ng mL-1 in phosphate-buffered saline (PBS), feed samples, and pork samples, respectively. The ultramarine blue nanoparticle-based ICA also shows no cross activity with salbutamol, clorprenaline, clenbuterol, or terbutaline. Graphical abstract Schematic representation of the ultramarine blue nanoparticles immunochromatographic assay for detection of ractopamine (RAC) based on competitive method. The ultramarine blue nanoparticles were screened from commercial ultramarine pigments for the first time and used to detect ractopamine.

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).

New haptens and antibodies for ractopamine.

In this work, three unreported immunizing haptens of ractopamine (RAC) were synthesized and used to produce highly sensitive and specific polyclonal antibody. The spacer arms of haptens for coupling to protein carrier were located on different position of RAC with different length. High affinity polyclonal antibodies were obtained and characterized in terms of titer and sensitivity by using enzyme-linked immunosorbent assay (ELISA). The best antibody employed in a heterologous competitive ELISA exhibited an IC50 value as low as 0.12ngmL(-1) and could not recognize other 10 ß-agonists including clenbuterol and salbutamol. The heterologous competitive ELISA was preliminary applied to swine urine and the results showed the new antibody was sufficiently sensitive and specific, and potentially used for the detection of RAC at trace level in real samples.

Highly selective and sensitive detection of ß-agonists using a surface plasmon resonance sensor based on an alkanethiol monolayer functionalized on a Au surface.

Immunosensor surfaces for surface plasmon resonance (SPR) have been constructed using a functionalized succinimidyl propanethiol monolayer as a linker to immobilize ß-agonist protein conjugates on a Au surface. Because ß-agonist is a small molecule, an indirect competitive inhibition immunoassay was used for detection. The lowest detection limits for ractopamine and salbutamol were 10 ppt (10 pg mL(-1)) and 5 ppt (5 pg mL(-1)), respectively. The fabricated immunosensor surface can be used again for detection after regeneration in 0.1 M sodium hydroxide. It was found that the same sensor surface could be reused for performing over 100 rapid immunoreactions. Moreover, one immunosensing-regeneration cycle requires only 600 s. The fabricated immunosensor surfaces were characterized using SPR and scanning tunneling microscopy observation. In the kinetic study of the indirect competitive immunosensing inhibition, the affinity constant (K1) of salbutamol antibody was smaller than the K1 of ractopamine antibody. Compared to a previous study of clenbuterol detection, it was concluded that the high K1 was coupled with low sensitivity. In the selectivity study, both immunosensor surfaces provided >90% of confidence level for the specific detection of ß-agonist compounds. The fabrication of highly selective and sensitive sensor surfaces for detecting ß-agonist compounds was confirmed.

Antibody purification using affinity chromatography: a case study with a monoclonal antibody to ractopamine.

The application of antibodies to small molecules in the field of bioanalytics requires antibodies with stable biological activity and high purity; thus, there is a growing interest in developing rapid, inexpensive and effective procedures to obtain such antibodies. In this work, a ractopamine (RAC) derivative, N-4-aminobutyl ractopamine (ABR), was synthesized for preparing new specific affinity chromatography to purify a murine monoclonal antibody (mAb) against RAC from ascites. The performance of the new specific chromatography was compared with four other purification methods in terms of recovery, purity and biological activity of mAb. These four purification methods were prepared by using specific ligands (RAC and RAC-ovalbumin) and commercial ligands (protein G and protein A), respectively. The results showed that the highest recovery (88.1%) was achieved using the new chromatography; in comparison, the recoveries from the other methods were all below 70%. The purity of the mAbs from the new chromatography was 88.3%, while, the highest purity of 97.6% was from protein G chromatography and the lowest purity of 84.7% was from protein A chromatography. The biological activity of the purified mAb from all of the chromatography methods was comparable in enzyme-linked immunosorbent immunoassay (ELISA).

Extracellular adenosine controls NKT-cell-dependent hepatitis induction.

Extracellular adenosine regulates inflammatory responses via the A2A adenosine receptor (A2AR). A2AR deficiency results in much exaggerated acute hepatitis, indicating nonredundancy of adenosine-A2AR pathway in inhibiting immune activation. To identify a critical target of immunoregulatory effect of extracellular adenosine, we focused on NKT cells, which play an indispensable role in hepatitis. An A2AR agonist abolished NKT-cell-dependent induction of acute hepatitis by concanavalin A (Con A) or α-galactosylceramide in mice, corresponding to downregulation of activation markers and cytokines in NKT cells and of NK-cell co-activation. These results show that A2AR signaling can downregulate NKT-cell activation and suppress NKT-cell-triggered inflammatory responses. Next, we hypothesized that NKT cells might be under physiological control of the adenosine-A2AR pathway. Indeed, both Con A and α-galactosylceramide induced more severe hepatitis in A2AR-deficient mice than in WT controls. Transfer of A2AR-deficient NKT cells into A2AR-expressing recipients resulted in exaggeration of Con A-induced liver damage, suggesting that NKT-cell activation is controlled by endogenous adenosine via A2AR, and this physiological regulatory mechanism of NKT cells is critical in the control of tissue-damaging inflammation. The current study suggests the possibility to manipulate NKT-cell activity in inflammatory disorders through intervention to the adenosine-A2AR pathway.

Highly sensitive near-simultaneous assay of multiple "lean meat agent" residues in swine urine using a disposable electrochemiluminescent immunosensors array.

Nowadays, ß(2)-agonists are abused illegally as "lean meat agents" for food-producing animals, and cause increasing food-safety accidents in some countries. Due to their hazard to the human health, "lean meat agents" are banned in most countries and required to be routinely monitored. We herein report a disposable electrochemiluminescent immunosensors array for near-simultaneous assay of multiple ß(2)-agonist residues in swine urine, by using ractopamine and salbutamol as the models. In this investigation, a screen-printed carbon electrodes array was assembled and acted as the substrate of the immunosensors array. Then the immunosensors array was constructed by site-selectively immobilizing the antigens of ractopamine and salbutamol on the working electrodes of array. After the competitive immuno-binding, with the aid of a homemade single-pore-four-throw switch, the electrochemiluminescent signals of the two ß(2)-agonists were sequentially detected using a non-array detector. The limits of detection for ractopamine and salbutamol were 8.5 and 17pg/mL, respectively, which were much lower than those of the most previous reports. Compared with other routine methods based on chromatography and ELISA, this method is more suitable for screening of multiple ß(2)-agonists in quantities of samples, owing to its merits of low cost, user-friendliness and high throughput, and shows great promise in food safety and agonist surveillance.

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.

Cross-reactivities of various phenethylamine-type designer drugs to immunoassays for amphetamines, with special attention to the evaluation of the one-step urine drug test Instant-View™, and the Emit® assays for use in drug enforcement.

Cross-reactivities of 76 kinds of phenethylamine-type designer drugs and related compounds to the urine drug tests Instant-View ™ (IV) (the Methamphetamine (MA) test, the Amphetamine 300 test, and the MDMA test) have been investigated. An on-site urine test kit consisting of these three IV tests has been evaluated for the on-site screening of MA users, and the kit has been found to have satisfactory specificity for drug enforcement purposes by separately detecting both MA and its metabolite amphetamine. The cross-reactivity profiles of Emit(®) II Plus Amphetamines Assay, Emit(®) II Plus Ecstasy assay, and Emit(®) d.a.u.(®) Amphetamine Class assay have also been investigated and discussed.

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.

Development and identification of monoclonal antibodies against ractopamine.

Ractopamine was reacted with two carrier proteins, human serum albumin and bovine thyroglobulin, as immunogen and coating antigen, respectively. Using a conventional immunization protocol, we generated a stable murine monoclonal antibody toward ractopamine, which had high affinities. The clone was found to be of IgG(2a) subclass with kappa light chain. An indirect competitive enzyme-linked immunosorbent assay for the determination of ractopamine has been optimized and characterized. The sensitivity, estimated as the IC(50) value, was 21.25 ng/mL, with a practical working range between 2.9 and 450 ng/mL. The limit of detection was 1.5 ng/mL. Moreover, other phenethanolamine beta-agonists showed low cross-reactivity with the monoclonal antibody. In addition, the indirect competitive enzyme-linked immunosorbent assay for the detection of ractopamine in animal feed was also developed using this antibody.

Time-resolved fluoroimmunoassay for ractopamine in swine tissue.

This study describes the development and validation of a time-resolved fluoroimmunoassay (TR-FIA) for screening ractopamine (RAC) in swine tissue. The method is based on the direct competitive-type immunoassay using europium-labeled anti-RAC monoclonal antibody as a tracer and RAC-ovalbumin as a solid-phase antigen. When RAC was spiked at levels of 1-10 microg kg(-1), recoveries ranged from 88.2 to 118.5% for swine liver and muscle with coefficients of variation from 7.1 to 20.5%. The detection limit was 0.1 microg kg(-1). The proposed TR-FIA method was applied to the determination of RAC in an actual residue study and the applicability was confirmed by liquid chromatography-tandem mass spectrometry.

Production and characterization of a monoclonal antibody against the beta-adrenergic agonist ractopamine.

A monoclonal antibody was generated toward the beta-adrenergic agonist ractopamine hydrochloride ¿(1R,3R),(1R, 3S)-4-hydroxy-alpha-[[[3-(4-hydroxyphenyl)-1-methylpropyl]amino]methy l]benzenemethanol hydrochloride¿. Ractopamine-glutarate-keyhole limpet hemocyanin (KLH) was used as the antigen for antibody generation in mice. Clone 5G10, secreted antibody with isotype IgG1kappa, was used for the development of an immunoassay. The selected antibody was specific for racemic ractopamine with an IC(50) of 2.69 +/- 0.36 ng/mL (n = 15). Antibody binding toward ractopamine was stereoselective with (1R,3R)-ractopamine having an IC(50) of 0.55 +/- 0.09 ng/mL (n = 3). IC(50) values for the (1S, 3R)-, (1S,3S)-, and (1R,3S)-ractopamine stereoisomers were 2.00 +/- 0.37, 140 +/- 23, and 291+/- 32 ng/mL (n = 3), respectively. Phenethanolamine beta-agonists showed low cross-reactivity. Studies using a series of ractopamine metabolites and ractopamine analogues demonstrated structural requirements for the antibody binding. A free phenolic group on the N-butylphenol moiety was required for high-affinity binding because methoxylated analogues and metabolites glucuronidated at this phenol generally had IC(50) values greater than 200 ng/mL. Ractopamine analogues methoxylated or glucuronidated at the ethanolamine phenol had IC(50) values of 0.7-2.6 ng/mL. Lack of a benzylic hydroxyl group was of less importance to antibody binding than was the correct stereochemical orientation (3R) of ractopamine's N-phenylalkyl group. In conclusion, a highly specific monoclonal antibody to ractopamine hydrochloride was developed that could be of potential utility in screening assays.

Development of an immunoassay for the beta-adrenergic agonist ractopamine.

Antibody generated from ractopamine-hemiglutarate-KLH was used to develop a ractopamine ELISA. The antibody showed good sensitivity in phosphate buffer, with an IC50 of 4.2 ng/ml (ppb) toward ractopamine and 16.2 ng/ml toward glucuronides of ractopamine conjugated to the phenethanolamine phenol of ractopamine. Phenylbutylamine phenol glucuronides of the (RS, SR) ractopamine diastereoisomers showed about 4% cross-reactivity, but the glucuronide of the (RR, SS) diastereoisomers conjugated at the same phenolic group showed no detectable reactivity with the antibody. The antibody generally had cross-reactivity towards compounds with bis-phenylalkyl amine structures rather than compounds with simple branched N-alkyl substituents. For example, the antibody showed little or no cross reactivity towards clenbuterol, isoproterenol, metaproterenol, and salbutamol, but cross-reacted with dobutamine. The system demonstrated a matrix effect similar to other enzyme immunoassays, dilution of urine decreased but did not eliminate the matrix effect.

Evaluation of ractopamine cross-reactivity in several commercially available beta-agonist enzyme immunoassay kits.

Several enzyme immunoassay test kits are commercially available for screening of matrices including cattle feed, tissues, faeces and urine for the presence of beta-agonists. The kits utilized for this evaluation offer sensitivity as low as 0.1 ng ml-1 (assay solution concentration, not test sample). Evaluation of ractopamine hydrochloride (LY31537) solutions at concentrations as high as 1000 ng ml-1, using six different kits from four different manufacturers, showed cross-reactivity of less than 0.5%.

Measurement of the class III antidysrhythmic drug, UK-68,798, in plasma by radioimmunoassay.

A sensitive radioimmunoassay (RIA) for the specific determination of 1-(4-methanesulphonamidophenoxy)-2-[N-(4-methanesulphonamido -phenethyl)-N- methylamino]ethane (UK-68,798), a novel class III antidysrhythmic agent, in human plasma is described. Specific antisera were raised in sheep using desmesyl-UK-68,798-succinate-ovalbumin conjugate as the antigenic hapten carrier protein. The antisera produced exhibited high specificity for UK-68,798 compared with known metabolites from animals, other antidysrhythmic agents and co-administered drugs. Good correlation was found in a comparison of the RIA method with a high-performance liquid chromatography (HPLC) method (r = 0.997) and a 10-fold lower limit of determination was observed for the RIA method compared with the HPLC method (0.05 and 0.5 ng ml-1, respectively). The RIA method was applied to the analysis of UK-68,798 in plasma obtained from human volunteers receiving the compound.

[Effect of ethanol and the alpha-adrenoblockader IEM-611 on the antigenic spectrum of water-soluble brain proteins of rats]. / Vliianie étanola i al'fa-adrenoblokatora IEM-611 na antigennyi spekr vodorastvorimykh belkov mozga krys.

The composition of water-soluble antigens in the brain of rats preferring ethanol or water was studied. A significant difference was found in the content of two antigens in rats preferring water or ethanol. Administration of IEM-611 changed the antigen content ot the level characteristic for animals preferring water.

Methodical investigation of the production of antibodies towards 3,4-dimethoxyphenylethylamine.

In this study the synthesis of various antigens from 3,4-dimethoxyphenylethylamine (3,4-DMPEA) is described. Antigen A was formed by coupling the acylated side chain of 3,4-DMPEA to the protein, while antigen B was synthesized by introducing an acylated amino group to the benzene ring and linking this reactive group to the protein. With antigen A an antiserum with a titer of 1 : 16000 could be raised, while with antigen B coupled to human albumin and bovine gamma-globulin the antiserum had a titer of 1 :1000 and 1 : 4000. Antibodies formed by immunization with antigen A exhibited a high specificity for substituents of the benzene ring, but were less specific for substituents of the side chain. Antibodies formed by treatment with the antigen B were specific for both kinds of substituents. So far, it has not been possible to harvest any specific antibodies towards the corresponding antigens of dopamine.