Techniques for improving the specificity of sandwich enzyme-linked immunosorbent assay-based drug screening.

ELISA assays are commonly used for drug screening by racing laboratories but are known to suffer from limited specificity. Inaccurate ELISA screening results are typically produced by non-specific antibody interactions or by the retention of chromogenic material in the sample well due to sample degradation. While confirmation of drug positives can be achieved by mass spectrometry, the follow-up of inaccurate ELISA screening results represents an unnecessary cost in staff time and reagents. This is particularly true in the case of rhEPO screening using sandwich ELISA assays, where the confirmation method requires up to 3 days to perform. While most racing laboratories purchase commercial ELISA kits, these products can be customised to provide increased specificity for enhanced screening of positive samples. The specificity of commercial sandwich ELISA kits can be improved by a variety of mechanisms including the addition of competing analyte specific antibodies, substitution of capture antibodies or by performing ELISA analysis with and without capture antibodies. Non-specific signals in difficult matrices such as canine urine can also be reduced by the addition of BSA solutions to the ELISA plate prior to the addition of samples.

Detection of gonadotropin-releasing hormone and its analogues in equine and canine urine by high-resolution data-independent acquisition.

Gonadotropin-releasing hormone (GnRH) and its synthetic analogues are considered banned substances by the racing industry. GnRH is used as a pharmaceutical to regulate the female oestrous cycle, but the hormone is also thought to increase the production of testosterone in male animals. Using liquid chromatography in conjunction with high-resolution mass spectrometry (LC-HRMS) and data-independent acquisition (DIA), a method is presented for the detection of intact and truncated peptides of GnRH and its analogues down to the low picogram level in equine urine. The study of the catabolism of GnRH and analogues in plasma, combined with the analysis of urine from administration studies, reveals a common pattern of peptide catabolites that can be used to guide the design of MS-based screens for this class of drugs. This culminated in the successful detection of the peptide in two out-of-competition canine urine samples.

Defining the specificity of recombinant human erythropoietin confirmation in equine samples by liquid chromatography-tandem mass spectrometry.

The proteotypic human EPO peptides YLLEAK (T4), SLTTLLR (T11), TITADTFR (T14), and VYSNFLR (T17) are often used to confirm the presence of recombinant human EPO (rhEPO) in equine samples. Each of these peptides contains one or more isomeric leucine or isoleucine amino acids, raising the possibility that a simple leucine/isoleucine substitution could lead to a false identification when compared with a rhEPO reference standard. To examine this possibility variants of these four peptides were analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). These studies indicate that confirmation of rhEPO in equine samples by immuno-affinity capture and LC-MS/MS analysis is true and accurate. It was also found that chromatography played a greater role in determining LC-MS/MS specificity than tandem mass spectrometry and that, in the case of more hydrophilic peptides, the accuracy of peptide identification could be enhanced by the inclusion of 13 C and 15 N labelled peptide internal standards.

Extraction of α-neurotoxins from equine plasma by receptor based affinity purification.

Venoms were first identified as potential doping agents by the racing industry in 2007 when three vials of cobra venom were seized during an inspection of a stable at Keeneland Racecourse in the USA. Venoms are a complex mixture of proteins, peptides, and other substances with a wide range of biological effects, including inhibiting the transmission of nervous and muscular impulses. As an example of this, cobratoxin, an α-neurotoxin found in cobra venom, is claimed to be an effective treatment for pain. Recent analysis of seized samples identified venom from two different species of snake. Proteomic analysis identified the first sample as cobra venom, while the second sample, in a vial labeled "Conotoxin", was identified as venom from a many banded krait. Cobratoxin, conotoxins, and bungarotoxins (a component of krait venom) are all α-neurotoxins, suggesting a common application for all three venom proteins as potential pain blocking medications. Using a peptide based on the nicotinic acetylcholine receptor, a one-step affinity purification method was developed for the detection of α-neurotoxins in plasma.

A method for confirming CJC-1295 abuse in equine plasma samples by LC-MS/MS.

CJC-1295 is a peptide-based drug that stimulates the production of growth hormone (GH) from the pituitary gland. It incorporates a functional maleimido group at the C-terminus that allows it to covalently bind plasma proteins such as serum albumin. These CJC-1295-protein conjugates have a much greater half-life compared to the unconjugated peptide and are capable of stimulating GH production for more than six days in humans after a single administration. Conjugated CJC-1295 is difficult to detect in blood by mass spectrometry due to its low abundance, high molecular weight, and conjugation to a range of different protein substrates. Previously we described a screening procedure for the detection of CJC-1295 in equine plasma using an immuno-PCR assay. Here we demonstrate the confirmation of CJC-1295 in equine plasma by LC-MS/MS after immuno-affinity capture and tryptic digestion. Using this method, CJC-1295 was identified down to concentrations as low as 180 pg/mL in 1 mL of equine plasma.

An immuno polymerase chain reaction screen for the detection of CJC-1295 and other growth-hormone-releasing hormone analogs in equine plasma.

CJC-1295 is a 30 amino acid peptide-based drug that stimulates the release of growth hormone (GH) from the pituitary gland. It is unique among performance-enhancing peptides due to the presence of a reactive maleimidopropionic acid group that covalently links the peptide to free thiols on the surface of plasma proteins. Once conjugated, CJC-1295 remains active in the bloodstream for significantly longer than non-conjugated peptide-based drugs that are rapidly excreted. Conjugation of CJC-1295 to plasma proteins prevents its detection by top-down mass-spectrometry-based peptide screening protocols as it effectively becomes a macromolecular protein with an undefined molecular weight. Using a pair of monoclonal antibodies raised against the CJC-1295 peptide, we present an immuno-polymerase chain reaction (I-PCR) assay that is capable of detecting the CJC-1295-protein conjugate at concentrations down to 0.8 pg/mL. Detection of endogenous equine GHRH necessitated a screening threshold for CJC-1295 in equine plasma of 50 pg/mL. The effectiveness of the assay for controlling the illicit use of CJC-1295 was confirmed in equine blood samples after administration in thoroughbred race horses.

The detection of a synthetic Interleukin-1 receptor antagonist peptide in a seized product from a racing stable.

A synthetic Interleukin-1 receptor antagonist peptide with the sequence Acetyl-Phe-Glu-Trp-Thr-Pro-Gly-Tyr-Trp-Gln-Pro-Tyr-Ala-Leu-Pro-Leu-OH has been identified in a vial seized during a stable inspection. The use of peptide-based Interleukin-1 receptor antagonists as anti-inflammatory agents has not been previously reported, making this peptide the first in a new class of sports doping peptides. The peptide has been characterized by high-resolution mass spectrometry and a detection method developed based on solid-phase extraction and liquid chromatography - triple quadrupole mass spectrometry. Using in vitro and in vivo models to study the properties of the peptide after administration, the peptide was shown to be highly unstable in plasma and was not detected in urine after administration in a rat. The poor stability of the peptide makes detection challenging but also suggests that it has limited effectiveness as an anti-inflammatory drug. Copyright © 2015 John Wiley & Sons, Ltd.

Identification of recombinant human EPO variants in greyhound plasma and urine by ELISA, LC-MS/MS and western blotting: a comparative study.

The recombinant human erythropoietins epoetin alfa (Eprex®), darbepoetin (Aranesp®) and methoxy polyethylene glycol-epoetin beta (Mircera®) were administered to greyhounds for 7, 10 and 14 days respectively. Blood and urine samples were collected and analysed for erythropoietin by ELISA, LC-MS/MS and western blotting. Limits of confirmation in plasma for western blotting and LC-MS/MS methods ranged from a low of 2.5mIU/mL, and closely matched the sensitivity of ELISA screening.

A high-throughput LC-MS/MS screen for GHRP in equine and human urine, featuring peptide derivatization for improved chromatography.

The growth hormone releasing peptides (GHRPs) hexarelin, ipamorelin, alexamorelin, GHRP-1, GHRP-2, GHRP-4, GHRP-5, and GHRP-6 are all synthetic met-enkephalin analogues that include unnatural D-amino acids. They were designed specifically for their ability to stimulate growth hormone release and may serve as performance enhancing drugs. To regulate the use of these peptides within the horse racing industry and by human athletes, a method is presented for the extraction, derivatization, and detection of GHRPs from equine and human urine. This method takes advantage of a highly specific solid-phase extraction combined with a novel derivatization method to improve the chromatography of basic peptides. The method was validated with respect to linearity, repeatability, intermediate precision, specificity, limits of detection, limits of confirmation, ion suppression, and stability. As proof of principle, all eight GHRPs or their metabolites could be detected in urine collected from rats after intravenous administration.

A high throughput screen for 17 Dermorphin peptides in equine and human urine and equine plasma.

The Dermorphins are a family of peptides that act as potent agonists of the opioid µ receptor. Originally identified as a seven amino acid peptide on the skin of the South American Phyllomedusa frog, peptide chemists have since developed a large number of Dermorphin variants, many with superior opioid activity to the original peptide. Dermorphins are unique among the peptide opioid agonists as they appear to have a limited ability to cross the blood brain barrier, producing effects on both the central and peripheral nervous systems. It is this ability of Dermorphins to provide central anaesthesia after intravenous or subcutaneous administration that allows their use as analogues of the opioid class of drugs. Recently, illicit use of the Dermorphin peptide in the racing industry has shown the need for an analytical method to control the use of these peptides. We present a high-throughput liquid chromatography-tandem mass spectrometry screen for 17 Dermorphin peptides in equine urine and plasma with limits of detection down to 5 pg/mL. The peptide extraction technique is also suitable for use in human urine.

High throughput solubility determination with application to selection of compounds for fragment screening.

The development and application of a high throughput aqueous solubility assay is reported. Measurements for up to 637 compounds can be made in a fully automated experiment. Results from this assay were used to quantify risk of unacceptable solubility as a function of lipophilicity for neutral fragment-like compounds. Assessment of risk of unacceptable solubility was combined with experimental solubility measurement to select compounds for inclusion in a fragment-screening library.

The unfolded protein response transducer Ire1p contains a nuclear localization sequence recognized by multiple beta importins.

The Ire1p transmembrane receptor kinase/endonuclease transduces the unfolded protein response (UPR) from the endoplasmic reticulum (ER) to the nucleus in Saccharomyces cerevisiae. In this study, we analyzed the capacity of a highly basic sequence in the linker region of Ire1p to function as a nuclear localization sequence (NLS) both in vivo and in vitro. This 18-residue sequence is capable of targeting green fluorescent protein to the nucleus of yeast cells in a process requiring proteins involved in the Ran GTPase cycle that facilitates nuclear import. Mutagenic analysis and importin binding studies demonstrate that the Ire1p linker region contains overlapping potential NLSs: at least one classical NLS (within sequences 642KKKRKR647 and/or 653KKGR656) that is recognized by yeast importin alpha (Kap60p) and a novel betaNLS (646KRGSRGGKKGRK657) that is recognized by several yeast importin beta homologues. Kinetic binding data suggest that binding to importin beta proteins would predominate in vivo. The UPR, and in particular ER stress-induced HAC1 mRNA splicing, is inhibited by point mutations in the Ire1p NLS that inhibit nuclear localization and also requires functional RanGAP and Ran GEF proteins. The NLS-dependent nuclear localization of Ire1p would thus seem to be central to its role in UPR signaling.

Mitochondrial development during life cycle differentiation of African trypanosomes: evidence for a kinetoplast-dependent differentiation control point.

Life cycle differentiation of African trypanosomes entails developmental regulation of mitochondrial activity. This requires regulation of the nuclear genome and the kinetoplast, the trypanosome's unusual mitochondrial genome. To investigate the potential cross talk between the nuclear and mitochondrial genome during the events of differentiation, we have 1) disrupted expression of a nuclear-encoded component of the cytochrome oxidase (COX) complex; and 2) generated dyskinetoplastid cells, which lack a mitochondrial genome. Using RNA interference (RNAi) and by disrupting the nuclear COX VI gene, we demonstrate independent regulation of COX component mRNAs encoded in the nucleus and kinetoplast. However, two independent approaches (acriflavine treatment and RNA interference ablation of mitochondrial topoisomerase II) failed to establish clonal lines of dyskinetoplastid bloodstream forms. Nevertheless, dyskinetoplastid forms generated in vivo could undergo two life cycle differentiation events: transition from bloodstream slender to stumpy forms and the initiation of transformation to procyclic forms. However, they subsequently arrested at a specific point in this developmental program before cell cycle reentry. These results provide strong evidence for a requirement for kinetoplast DNA in the bloodstream and for a kinetoplast-dependent control point during differentiation to procyclic forms.