Adrenergic Modulation of Hematopoiesis.

Hematopoiesis produce every day billions of blood cells and takes place in the bone marrow (BM) by the proliferation and differentiation of hematopoietic stem cells (HSC). HSC are found mainly adjacent to the BM vascular sinusoids where endothelial cells and mesenchimal stromal cells promote HSC maintenance by producing a variety of factors. Other cell types that regulate HSC niches include sympathetic nerves, non-myelinating Schwann cells and a variety of mature hematopoietic cells such as macrophages, neutrophils, and megakaryocytes. This review will focus on the role of adrenergic signals, i.e. of catecholamines, in the regulation of the HSC niche. The available evidence is rather controversial possibly due to the fact that adrenergic receptors are expressed by many cellular components of the niche and also by the often neglected observation that catecholamines may be produced and released also by the BM cells themselves. In addition one has to consider that, physiologically, the sympathetic nervous system (SNS) activity follows a circadian rhythmicity as driven by the suprachiasmatic nucleus (SCN) of the hypothalamus but may be also activated by cognitive and non-cognitive environmental stimuli. The adrenergic modulation of hematopoiesis holds a considerable potential for pharmacological therapeutic approaches in a variety of hematopoietic disorders and for HSC transplantation however the complexity of the system demands further studies. Graphical Abstract Sympathetic nerve termini may release NE while mature BM cells may release norepinephrine (NE) and / or epinephrine (E). Both may bind to ß-adrenergic receptor (AR) expressed in nestin+MSC in the hematopoietic stem cell (HSC) niche and regulate the physiological trafficking of HSC by modulating the expression of CXCL12 and SCF. Both NE and E may also activate Lin - c-Kit+ Sca-1+ (LKS) cell via another AR. In addition, NE may also signal to α1-AR expressed in pre-B cells which by TGF-ß secretion might regulate proliferation of their lymphoid progenitors in an autocrine manner and/or inhibit myeloid progenitors.

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.

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 paper-based competitive lateral flow immunoassay for multi ß-agonist residues by using a single monoclonal antibody labelled with red fluorescent nanoparticles.

An ultrasensitive paper based lateral flow assay is described for rapid and simultaneous fluorometric detection of several ß-agonists including clenbuterol and its chemical analogues (mabuterol, brombuterol, cimaterol, cimbuterol, bromchlorbuterol and banbuterol). A nonspecific monoclonal antibody (mAb) against clenbuterol and its analogues was prepared and employed in a competitive immunoassay where mAb conjugated to fluorescent nanoparticles and free ß-agonists compete for the binding sites. This enables rapid screening for the 7 ß-agonists in a single run that takes about 8 min. Detection limits for the seven ß-agonists are <50 pg g-1 of pork. Recoveries ranged from 69.5% to 102.4%, and relative standard deviations were ±15%. The assay was applied to the analysis of both using spiked and unspiked pork for ß-agonists, and the results compare well to those obtained by HPLC-MS. Graphical abstractSchematic presentation of an ultra sensitive fluorescent nanoparticle based paper based assay for rapid detection of multi ß-agonists in pork tissue.

Four Hapten Spacer Sites Modulating Class Specificity: Nondirectional Multianalyte Immunoassay for 31 ß-Agonists and Analogues.

Immunoassay methods are important for monitoring ß-agonists illegally used for reducing animal fat deposition in livestock. However, there is no simultaneous screening surveillance immunoassay for detecting various ß-agonist chemicals that are possibly present in food. In this study, through the use of an R-(-)-salbutamol derivative as the immunizing hapten, an antibody recognizing 31 ß-agonists and analogues was generated for the first time. Three-dimensional quantitative structure-activity relationship (3D QSAR) revealed that strong steric and hydrophobic fields around the hapten spacer near C-2, as well as a chirality at C-1', dominantly modulated the class specificity of the raised antibody. However, a hapten spacer linked at C-2' or C-1 would lead to a narrow specificity, and the spacer charge at C-6 could affect the raised antibody specificity spectrum. A class specificity competitive indirect enzyme-linked immunosorbent assay (ciELISA) was established with an ideal recovery ranging from 81.8 to 118.3% based on the obtained antibody. With a good agreement to the HPLC/MS method, the proposed ciELISA was confirmed to be reliable for the rapid surveillance screening assay of ß-agonists in urine. This investigation will contribute to the rational design and control of the immunoassay specificity.

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.

Rapid determination of phenylethanolamine A in biological samples by enzyme-linked immunosorbent assay and lateral-flow immunoassay.

Phenylethanolamine A (PA) is a ß-adrenergic agonist, which was first used in animal husbandry as a growth promoter in China in 2010. In this study, a monoclonal-antibody (mAb)-based indirect competitive enzyme-linked immunosorbent assay (icELISA) and lateral-flow immunoassay (LFA) for the detection of PA in swine urine and pork were developed. The immunogen was prepared by linking PA hapten with carrier protein via a diazotization method. The IC50 value of the optimized icELISA was 0.44 ng mL(-1). The limits of detection of the icELISA for PA in swine urine and pork were 0.13 ng mL(-1) and 0.39 ng g(-1), respectively. The recoveries of PA from spiked swine urine and pork were in the range 82.0-107.4 % and 81.8-113.3%, respectively, with the coefficients of variation in the range 4.1-16.2% and 1.2-6.3%, respectively. The mAbs had negligible cross reactivity with 10 other ß-agonists. In contrast, the LFA had a cut-off level of 5 ng mL(-1) (g) in swine urine and pork, and the results could be achieved within 5 min. Ten blind samples of swine urine were analyzed simultaneously by icELISA, LFA, and ultra-high-performance liquid chromatography-tandem mass spectrometry, and the results of the three methods agreed well. Therefore, the combination of two immunoassays provides an effective and rapid screening method for detection of PA residues in biological samples.

A sensitive and quantitative fluorescent multi-component immuno-chromatographic sensor for ß-agonist residues.

A sensitive and quantitative fluorescent multi-component immuno-chromatographic sensor was developed for detection of three ß-agonizts: clenbuterol, ractopamine and salbuterol. A competitive immune strategy between antibody conjugated fluorescent beads and ß-agonist or their antigens was employed. Each monoclonal antibody specifically recognizes it is corresponding ß-agonist in the conjugating zone. The unreacted antibodies were captured by ß-agonist antigens immobilized at three test lines in nitrocellulose membrane reaction zone. This enables simultaneous detection of 3 ß-agonizts in one single test without any further sample preparation. The test results can be obtained within 10 min. Limit of detections for clenbuterol, ractopamine and salbuterol were 0.10 ng/mL, 0.10 ng/mL and 0.09 ng/mL, respectively. Recoveries ranged from 70.0% to 100.5% and relative standard deviations were below 15%. The assay was evaluated using spiked and real samples and the results were compared with LC-MS/MS. The developed novel assay method provides a low cost, sensitive and rapid approach for on site detection of ß-agonizts.

Affinity-based biosensors in sport medicine and doping control analysis.

Affinity-based biosensors (ABBs) have started to be considered in sport medicine and doping control analysis because they are cheap, easy to use and sufficiently selective analytical devices, characterized by a reversible interaction with the analyte under investigation allowing the use of the same sensor for multiple analyses. In this review we describe the main categories of substances reported in the World Anti-Doping Agency Prohibited List and how ABBs may contribute to their detection. Although several ABBs proposed in the last few years display limit of detections that are in principle matching the World Anti-Doping Agency requirements, their application in the framework of 'traditional' antidoping tests seems quite unlikely, mainly because of the still insufficient selectivity especially in the case of 'pseudo-endogenous' compounds, and on the lack of complete information regarding potential matrix effects in real samples and following their routine use. At the same time, ABBs could contribute to fill a significant information gap concerning complementary evidence that can be obtained from their use 'on the spot', as well as to preselect a risk population of individuals to be targeted for a full antidoping test; while in sport medicine they could contribute to obtaining analytical information of physiological relevance from the measurement of specific parameters or markers before, during and after physical exercise.

Ru(phen)3(2+) doped silica nanoparticle based immunochromatographic strip for rapid quantitative detection of ß-agonist residues in swine urine.

A Ru(phen)3(2+) doped silica nanoparticle based immunochromatographic strip was developed for the rapid and quantitative detection of five common ß-agonist (salbutamol (SAL), cimbuterol, terbutaline, clenbuterol, and brombuterol) residues in swine urine. The broad spectrum monoclonal antibodies generated by immunizing BALB/c mice with salbutamol conjugated cationic bovine serum albumin. The fluorescence intensities (FIs) of the strip on the test line (FIT) and control line (FIC) were determined using a strip reader. Parameters that influenced the antibody and antigen interaction on the test strip were investigated by recording FIT and FIC values, and the concept of FIT/FIC ratio was used to offset the inherent heterogeneity of the test strips and the effect of the sample matrix. Under optimal conditions, the linear range for the quantitative detection of SAL was 0.6-5.0 ng/ml with a half maximal inhibitory concentration at 1.78 ng/ml. The limit of detection for real swine urine was 0.43 ng/ml. The recovery rates of the intraassay for spiked urine at SAL concentrations of 0.8, 1.5, and 3.5 ng/mL were 88.06%±3.75%, 95.77%±5.33%, and 94.06%±7.43%, whereas those for the interassay were 84.69%±5.0%, 95.06%±9.3%, and 88.34%±7.71%, respectively. The developed quantitative method exhibited excellent agreement with a commercially available competitive enzyme-linked immunosorbent assay kit for SAL-spiked urine samples, with a correlation of coefficient of 0.95 and a slope of 0.99 (n=36). The results indicated that the developed test strip enables sensitive, reproducible, and easily implementable screening for the rapid and quantitative detection of ß-agonist residues in swine urine.

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.

Determination of beta-adrenergic agonists by hapten microarray.

The use of highly active beta-agonists as growth promoters is not appropriate because of the potential hazard for human and animal health. To investigate the residue level of these beta-agonists, hapten microarrays were employed for clenbuterol (CLB), ractopamine (RAC) and salbutamol (SAL) residue analysis. CLB, RAC and SAL conjugates were immobilized on the slides, which were precoated by agarose film to construct hapten microarrays, and then the corresponding monoclonal antibodies of these beta-agonists and the standards or samples were introduced for indirect competitive immunoassay. Finally, Cy3-labeled secondary antibody was employed to indicate the antigen-antibody complex. The fluorescence intensity of each spot was imaged and recorded, and the calibration curve of each analyte was obtained by plot fluorescence intensity against different standard concentrations. Compared to the ELISA, the hapten microarray method was more sensitive, which got the detection limits 0.09 microg/L for CLB, 0.50 microg/L for RAC, and 0.01 microg/L for SAL. What's more, with the recovery rate between 96.5% and 106.4%, and the coefficient of variation below 10%, the proposed hapten microarray method was shown to be both quantitative and reproducible.

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.

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.

Modulation of protective immunity against herpes simplex virus via mucosal genetic co-transfer of DNA vaccine with beta2-adrenergic agonist.

Cholera toxin, which has been frequently used as mucosal adjuvant, leads to an irreversible activation of adenylyl cyclase, thereby accumulating cAMP in target cells. Here, it was assumed that beta(2)-adrenergic agonist salbutamol may have modulatory functions of immunity induced by DNA vaccine, since beta(2)-adrenergic agonists induce a temporary cAMP accumulation. To test this assumption, the present study evaluated the modulatory functions of salbutamol co-administered with DNA vaccine expressing gB of herpes simplex virus (HSV) via intranasal (i.n.) route. We found that the i.n. co-administration of salbutamol enhanced gB-specific IgG and IgA responses in both systemic and mucosal tissues, but optimal dosages of co-administered salbutamol were required to induce maximal immune responses. Moreover, the mucosal co-delivery of salbutamol with HSV DNA vaccine induced Th2-biased immunity against HSV antigen, as evidenced by IgG isotypes and Th1/Th2-type cytokine production. The enhanced immune responses caused by co-administration of salbutamol provided effective and rapid responses to HSV mucosal challenge, thereby conferring prolonged survival and reduced inflammation against viral infection. Therefore, these results suggest that salbutamol may be an attractive adjuvant for mucosal genetic transfer of DNA vaccine.

Modulation of protective immunity against herpes simplex virus via mucosal genetic co-transfer of DNA vaccine with beta2-adrenergic agonist

Cholera toxin, which has been frequently used as mucosal adjuvant, leads to an irreversible activation of adenylyl cyclase, thereby accumulating cAMP in target cells. Here, it was assumed that beta2-adrenergic agonist salbutamol may have modulatory functions of immunity induced by DNA vaccine, since beta2-adrenergic agonists induce a temporary cAMP accumulation. To test this assumption, the present study evaluated the modulatory functions of salbutamol co-administered with DNA vaccine expressing gB of herpes simplex virus (HSV) via intranasal (i.n.) route. We found that the i.n. co-administration of salbutamol enhanced gB-specific IgG and IgA responses in both systemic and mucosal tissues, but optimal dosages of co-administered salbutamol were required to induce maximal immune responses. Moreover, the mucosal co-delivery of salbutamol with HSV DNA vaccine induced Th2-biased immunity against HSV antigen, as evidenced by IgG isotypes and Th1/Th2-type cytokine production. The enhanced immune responses caused by co-administration of salbutamol provided effective and rapid responses to HSV mucosal challenge, thereby conferring prolonged survival and reduced inflammation against viral infection. Therefore, these results suggest that salbutamol may be an attractive adjuvant for mucosal genetic transfer of DNA vaccine.

Obtainment of polyclonal antibodies to clenbuterol with the use of colloidal gold.

The effectiveness of an in vivo synthesis of antibodies to clenbuterol with the use of gold nanoparticles as a carrier was evaluated. For comparison, conjugates of clenbuterol with bovine serum albumin were used in immunization. The serum titer was determined by an ELISA. The antibodies were tested by an immunodot assay with immunogold markers. With both techniques we obtained specific and relatively high-titer antibodies to clenbuterol. It was found that the antibodies obtained with the use of gold nanoparticles were not inferior in titer to those obtained to the conjugates of clenbuterol with the protein but surpassed them in specificity.

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.