Lameness and performance evaluation in ambulatory practice.

Lameness and performance evaluation can be one of the most rewarding aspects of equine veterinary practice. There is a misconception that it depends on new sophisticated and expensive diagnostic modalities, when the reality is that knowing where and when to use these modalities form the real art of equine lameness practice. The most expensive ultrasound machine in the world is not very diagnostic if applied to the wrong limb. The art of lameness practice is vested in knowledge of horsemanship, an understanding of anatomy and function, and inquiring senses to sort out what the horse and his handlers are telling you. The ability to listen to both and figure out a horse's lameness problem will be one of the most valuable services experiences the equine ambulatory clinician can provide for his or her clientele.

Surface plasmon resonance signal enhancement for immunoassay of small molecules.

Sensitive detection of small molecules using surface plasmon resonance (SPR) presents significant challenges as the antigen cannot serve as a signal generator because of its low mass; efficient binding of the target requires the binding event to be spaced from the sensor surface through a specialist linker conjugation. Competitive immunoassay of steroid hormones can be performed by conjugation through a rationally designed linker system at positions distant from existing antigenic functional groups. The binding signal from the primary antibody can then be further enhanced by sequential addition of secondary antibody or conjugated gold nanoparticles which can produce 13-fold signal enhancements through both their mass and co-operative plasmon coupling.

Matrix effects on an antigen immobilized format for competitive enzyme immunoassay of salivary testosterone.

Matrix interferences in salivary testosterone enzyme immunoassays are important in the development of direct ELISA. An alternative format for sensitive enzyme immunoassay of testosterone was developed using immobilization of the antigen as part of a protein conjugate using oligoethylene glycol linker to project the antigen, with color development via enzyme labeled secondary antibody. This technique gave the required sensitivity for detection of testosterone from male saliva with a limit of detection (LOD) of 8.9 pg/mL (31 pmol/L). Application of the immunoassay directly in human saliva gave suppression of binding signals for the samples, indicating clear matrix interference with antibody binding. A wide variety of treatments were used in an attempt to overcome this effect, including use of both synthetic saliva and stripped human saliva for standard preparation, use of bovine serum albumin (BSA) to reduce non-specific binding of contaminants, pH adjustment of samples and/or standards and use of different antibodies. None of these techniques proved effective, causing either substantial suppression or enhancement of signal, and dilution was not possible because of the very low physiological concentrations. Complete removal of the saliva medium by chemical extraction was the only technique studied that could overcome this problem. These issues have not been explored previously for direct ELISA of salivary testosterone using alternative assay formats and have implications for the design of small molecule plate-based immunoassays in this medium.

Ultrasensitive detection of testosterone using conjugate linker technology in a nanoparticle-enhanced surface plasmon resonance biosensor.

A rationally designed oligoethylene glycol linker conjugate to testosterone was synthesised and covalently immobilized on a surface plasmon resonance (SPR) biosensor surface. The sensing surface was stable for more than 330 binding and regeneration cycles allowing a high degree of re-use. This surface was then used in the development of an ultrasensitive immunobiosensor system for testosterone in buffer utilizing both secondary antibody and gold nanoparticle signal enhancement. The mechanism for the increased sensitivity results from increased binding mass and a gold plasmon coupling effect. The addition of a secondary antibody with an attached gold nanoparticle increased the signal sensitivity of the assay 12.5-fold compared with primary antibody alone. In the enhanced format the assay had limits of detection (LOD) of 3.7 pgml(-1) with standard in running buffer, and 15.4 pgml(-1) in a stripped human saliva matrix. This immunobiosensor system has sufficient sensitivity to measure testosterone across the broad physiologically relevant range in male saliva (29-290 pgml(-1)) in under 13 min allowing monitoring of testosterone in near real-time.

Rapid ultrasensitive measurement of salivary cortisol using nano-linker chemistry coupled with surface plasmon resonance detection.

Cortisol detection in saliva is of great interest for the diagnosis of various disease states and the monitoring of stress in humans. Currently, measurements are performed predominantly by radioimmunoassay (RIA) which is expensive, labour intensive, uses hazardous radioisotopes and involves extensive delays in obtaining results. A rationally designed cortisol-linker conjugate allowing high assay sensitivity was employed as a coating antigen in a microfluidic surface plasmon resonance (SPR) biosensor immunoassay for the ultrasensitive and rapid detection of salivary cortisol. Detection of cortisol is by competitive immunoassay using a secondary antibody for signal enhancement. The method requires no chemical extraction or complex sample pre-treatment despite high saliva viscosity. The cortisol assay was optimized for maximum sensitivity in buffer before being adapted for the salivary matrix, where it showed a limit of detection of 49 pg/mL. The results showed good correlation to RIA (r = 0.94). The biosensor assays showed an inter-assay coefficient of variation (CV) of 13.5% and recoveries close to 100%. The covalently immobilized sensor surface provided stable responses for more than 140 binding and regeneration cycles, enabling re-use. Cortisol in saliva was detected across the physiologically relevant range using the SPR immunobiosensor by employing a rationally designed assay format including signal enhancement for maximum sensitivity. The system can handle saliva matrix effects by use of chemical treatment during the assay to reduce non-specific binding to sensor surfaces. This sensor system provides an automated, high sensitivity analytical tool capable of yielding results in approximately 15 min. This biosensor could potentially be used for active stress-monitoring and in the diagnosis of disease.

Rapid saliva processing techniques for near real-time analysis of salivary steroids and protein.

INTRODUCTION: Point-of-care (POC) measurements using saliva samples have immense potential to assess systemic health and wellbeing, but sample viscosity and contaminants can affect analyses. We sought a portable clean-up method for whole saliva appropriate for use with POC measurement techniques such as biosensors. METHODS: Whole saliva from each of 13 male subjects was split into 5 fractions. Each fraction was treated with a different clean-up process: a freeze-thaw-centrifuge (FTC) step; centrifugation alone; or passage through a Mini-UniPrep polyethersulfone filter, cotton Salivette, or foam Oracol device. Following clean-up, each subject's treated saliva fractions were assayed for cortisol, testosterone, dehydroepiandrosterone (DHEA), and protein concentrations. The effects of clean-up methods on nonspecific binding (NSB) in a biosensor were also assessed. RESULTS: Compared with FTC, no analytes were affected by centrifugation alone. Cotton Salivettes significantly altered all analytes, with increases in cortisol (+64%), testosterone (+126%), and DHEA (off-scale) levels, and decreased protein (-21%) and biosensor NSB (-75%). Oracol foam devices decreased DHEA levels by 28%. Mini-UniPrep filtration decreased testosterone (-45%) and DHEA (-66%) concentrations while increasing cortisol (+40%). CONCLUSION: No method was optimal for all analytes, highlighting the need for validation of saliva treatment methods before their adoption in rapid POC analyses.

Gold nanohole array substrates as immunobiosensors.

A gold nanohole array is functionalized with a cortisol thiol derivative, and binding to a monoclonal antibody is conveniently detected using the sensitive shift in the 1060 nm transmission peak of the array. Detection is also enhanced 3-fold by the application of a secondary antibody-gold nanoparticle conjugate. This regenerable response represents a more sensitive shift than that obtained previously for higher affinity binding and opens the way to application of nanohole arrays in immunobiosensing of important biomolecules.

Direct ring conjugation of catecholamines and their immunological interactions.

Catecholamine derivatives were synthesized with potential applications as coating antigens in biosensors or in the raising of specific antibodies. Thioether-bridged derivatives of the catecholamines dopamine, norepinephrine, and epinephrine that attach carboxylic acid functionalities directly to the aromatic ring via an easily incremented linker chain were synthesized by an electrochemical method. These derivatives were purified by convenient ion-exchange chromatography, exact positions of conjugation determined by NMR, and a dopamine derivative immobilized in situ in a BIAcore surface plasmon resonance (SPR) biosensor and its antibody binding studied in comparison with immobilization via the catecholamine primary amine. Binding of an antibody raised to an amine-conjugated protein conjugate showed clear distinction between conjugations at different positions on the catecholamine, illustrating the importance of rational conjugate design in immunosensing of the catecholamines.

Estrogen conjugation and antibody binding interactions in surface plasmon resonance biosensing.

Thioether-linked 3-mercaptopropionic acid derivatives of 17beta-estradiol and estrone were formed at the A-ring 4-position of the steroids by substitution of their 4-bromo analogues. The carboxylic acid terminal was used to link to an oligoethylene glycol (OEG) chain of 15-atoms in length. The OEG derivative of 17beta-estradiol was then in situ immobilized on a carboxymethylated dextran-coated gold sensor surface used to detect refractive index changes upon protein binding to the surface by surface plasmon propagation in a BIAcore surface plasmon resonance (SPR) instrument. Two other estradiol-OEG derivatives with Mannich reaction linkage at the 2-position and hemisuccinate linkage at the 3-position were also immobilized on the sensor surfaces for comparison. Binding performance between these immobilized different positional conjugates and monoclonal anti-estradiol antibody, raised from a 6-position conjugate, clearly demonstrated that both 2- and 4-conjugates, not conjugated through existing functional groups, gave strong antibody bindings, whereas the 3-conjugate through an existing functional group (3-OH) gave very little binding (2% compared to the 2-conjugate). Both 2- and 4-position conjugates were then applied in a highly sensitive estradiol SPR immunoassay with secondary antibody mediated signal enhancement that gave up to a 9.5-fold signal enhancement of primary antibody binding, and a detection limit of 25 pg/mL was achieved for a rapid and convenient flow-through immunoassay of estradiol.

Sensitivity enhancement of surface plasmon resonance biosensing of small molecules.

Surface plasmon resonance (SPR) biosensor formats using gold nanoparticle or protein signal amplification for the sensitive assay of small molecules were developed using progesterone as a model compound. Progesterone was immobilized to a dextran surface in the Biacore biosensor through in situ covalent immobilization using an oligoethylene glycol linker attached to the 4 position of the steroid. This surface produced stable antibody binding for in excess of 1100 assay cycles. Using this surface, assays were developed for progesterone using 10- and 20-nm gold-streptavidin labels attached to biotinylated monoclonal antibody in both label prebinding and sequential binding formats. Prelabeling formats gave no signal enhancement but produced assays with limits of detection of 143 pg/ml, compared with approximately 1 ng/ml in previous studies. Sequential binding formats gave signal enhancements of 2.2-fold over the monoclonal antibody and a limit of detection of 23.1 pg/ml. It was found that secondary antibody labeling gave 8.1-fold signal enhancements and a limit of detection of 20.1 pg/ml, whereas use of secondary antibody-25 nm gold complexes provided more signal enhancement (13-fold) and a further improvement in limit of detection of 8.6 pg/ml.