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Topic:Immunoassay
Immunoassays are analytical techniques used to detect and quantify specific proteins, hormones, or other molecules in biological samples from horses. These assays rely on the specificity of antibodies to bind to target antigens, allowing for the measurement of various substances that can indicate health status or disease. In equine research, immunoassays are employed to assess biomarkers related to metabolic, infectious, and inflammatory conditions. Common applications include measuring hormone levels, detecting infectious agents, and monitoring immune responses. This page compiles peer-reviewed research studies and scholarly articles that explore the development, application, and validation of immunoassays in the context of equine health.
A retrospective study of increased plasma progestagen concentrations in compromised neonatal foals. Plasma progestagen concentrations were measured daily by radioimmunoassay (RIA) in 35 sick foals for the duration of their illness. The foals were divided into three groups on the basis of time to stand after birth. Foals were given intensive care treatment according to the severity of their illness. Plasma and urine concentrations of pregnenolone (P5) and pregnenediol (P5 beta beta) were measured by gas chromatography--mass spectrometry; plasma cortisol concentrations were measured by RIA and the foals' renal and respiratory status were assessed by creatinine clearance ratios and arterial oxy...
Involvement of endogenous opioids in the regulation of LH and testosterone release in the male horse. To investigate the involvement of endogenous opioids in the regulation of gonadotrophin release in male horses, effects of the opioid antagonist naloxone (0.5 mg kg-1 i.v.) on plasma LH and testosterone concentrations and the possible influence of season and of gonadal steroids were investigated. To determine quantitative as well as qualitative changes in gonadotrophin release, LH concentrations were measured by radioimmunoassay and by an in vitro bioassay. Experiments were performed in May, August and December. In stallions, basal LH secretion in May and August was significantly higher than i...
Short-term secretion patterns of corticotropin-releasing hormone, arginine vasopressin and ACTH as shown by intensive sampling of pituitary venous blood from horses. To characterize the short-term ACTH secretion pattern and to investigate factors regulating it, pituitary venous (PV) blood was collected using our nonsurgical method from 8 unperturbed horses every 20 or 30 s for approximately 1 h. In all but 1 horse, sampling occurred during the broad circadian maximum in plasma cortisol concentrations. Concentrations of corticotropin-releasing hormone (CRH; n = 7 horses), arginine vasopressin (AVP), ACTH and cortisol were measured by radioimmunoassay. In all horses, CRH, AVP and ACTH secretion patterns appeared irregular in time and amplitude. The mean (+/-...
Effects of the opioid antagonist naloxone on release of luteinizing hormone in mares during the anovulatory season. To investigate an involvement of endogenous opioids in the regulation of circannual changes in reproductive activity, effects of the opioid antagonist naloxone on the concentration of immunoreactive and bioactive luteinizing hormone (LH) in plasma were measured in mares during the anovulatory season. Naloxone (0.5 mg/kg i.v.) caused a significant increase (P < 0.05) in immunoreactive as well as bioactive LH concentration in plasma. The amplitude of the increase in LH concentrations measured with an in vitro bioassay was more pronounced than the amplitude of the increase in LH secretion dete...
Development of homologous radioimmunoassays for equine growth hormone and equine prolactin and their application to the detection of circulating levels of hormone in horse plasma. Highly purified and well-characterised preparations of equine prolactin and growth hormone from equine pituitary glands were employed to set up highly sensitive and specific homologous radioimmunoassays (RIA) for the measurement of hormone in horse plasma. The limit of sensitivity of the GH RIA was 1.2 ng/ml with mean intra- and inter-assay coefficients of variation (CV) of 6.6 and 10%, respectively. The sensitivity of the equine prolactin (ePRL) RIA was 0.5 ng/ml with mean intra and inter-assay CV of 9.1 and 15.6%, respectively. Dose-response curves of a crude pituitary gland extract and plas...
Aromatase activity in the mare ovary during estrous cycle. Measurement of endogenous steroids and of their in vitro inhibitory effect. This present study was undertaken to clarify estrogen synthesis in the mare ovary. First of all, an evaluation of endogenous steroid contents was carried out in the follicular fluid and in the luteal tissue at different stages of the luteal phase. Radioimmunoassays were performed after separation and purification of each hormone by chromatography. High amounts of conjugated (0.9 mg/l) and unconjugated (4 mg/l) estradiol-17 beta were found in the follicular fluid of the large follicules (50 mm). These concentrations of estrogens decreased drastically in the luteal tissue, and only low levels of...
Pulsatile secretion of luteinizing hormone and progesterone in mares during the estrous cycle and early pregnancy. Existence of ultradian variation in serum progesterone concentration and the relation between progesterone and luteinizing hormone (LH) secretory patterns were investigated in nonpregnant and pregnant mares. Blood samples were taken every 15 minutes for a 24-hour period on day 8 of the estrous cycle and day 18 of pregnancy, respectively. Progesterone and LH concentrations were determined by radioimmunoassay. Progesterone was secreted in pulsatile manner in nonpregnant and pregnant mares. Luteinizing hormone also was secreted in a pulsatile manner in both groups of mares. There was little tempo...
Comparison of immunoaffinity chromatography combined with gas chromatography-negative ion chemical ionisation mass spectrometry and radioimmunoassay for screening dexamethasone in equine urine. A comparison of the sensitive analytical methods of radioimmunoassay (RIA) and immunoaffinity chromatography (IAC) combined with gas chromatography-negative ion chemical ionisation mass spectrometry for the specific and reliable screening of dexamethasone in equine post-race urine is presented. Results from analyses of samples collected from a mare during 144 hours post administration of 26 mg of dexamethasone sodium phosphate are described.
Fibrinogen as a ferritin-binding protein in horse plasma. Lower apparent concentrations of ferritin were observed in horse plasma than in serum using the enzyme-linked immunosorbent assay (ELISA). However, the ferritin concentrations in plasma and serum were increased to the same level on heating the samples at 75 degrees C for 15 min. These results suggest that horse plasma has specific ferritin-binding protein(s) which inhibit(s) the ferritin assay. The apparent ferritin concentrations in horse serum were markedly decreased by adding horse fibrinogen to the serum. It was also found that fibrinogen bound to spleen ferritin and inhibited the immunoas...
Furosemide magnifies the exercise-induced elevation of plasma vasopressin concentration in horses. The purpose of this study was to test the hypothesis that furosemide administration before exercise would cause greater increases in plasma arginine vasopressin (AVP) concentration in exercising horses than exercise alone. Six adult, clinically normal, unfit mares underwent three randomly ordered 60 minute standard exercise tests on an equine treadmill to examine the effect of furosemide administration on plasma AVP concentration. In one trial, furosemide (1 mg kg-1) was infused four hours before exercise (FUR-4) and a placebo (10 ml saline) was infused two minutes before exercise; in another ...
Twenty-four hour gastric pH monitoring and blood gastrin concentrations in fasted ponies. Gastric pH varied widely among eight fasted ponies, of which seven exhibited discrete episodes of spontaneous alkalinisation (SA). SA occurred at all times of the study and no significant variation in the summary variables of pH (median pH, mean pH and percentage of readings exceeding pH 4.0) was noted among the periods 0 to eight, eight to 16 and 16 to 24 hours. The occurrence of SA has significant implications for the performance of acid secretory studies in the fasted pony. There was, however, no significant correlation between pH and plasma gastrin concentration measured using a commercial...
Growth hormone secretion in the horse: unusual pattern at birth and pulsatile secretion through to maturity. A heterologous radioimmunoassay was developed and validated for the measurement of horse GH in plasma. It utilized recombinant-derived bovine GH as the radiolabelled ligand, a guinea-pig anti-porcine GH serum as first antibody and pituitary-derived horse GH as standard. Cross-reactivities were high with all of the pituitary and recombinant-derived GH preparations tested (49-140%) and very low (< 0.3%) with horse FSH, LH and prolactin. A synthetic analogue of GH-releasing factor(1-29) stimulated the expected pattern of GH release in foals. Plasma GH concentrations in foals were low at birth (< ...
Effects of sample collection and handling on concentration of osteocalcin in equine serum. A commercially available radioimmunoassay kit for measurement of human osteocalcin was validated for use in horses. For accurate measurement of equine serum osteocalcin, blood samples may be collected at a temperature between 20 and 25 C, then centrifuged within 90 minutes; serum may be stored at -20 C in plastic tubes for up to 26 weeks. Serum may be thawed and refrozen up to 5 times without significant change in measured equine serum osteocalcin concentration. Assay sensitivity was 0.16 ng/ml. Recovery of bovine osteocalcin standard added to equine serum was linear. Intra-assay coefficient o...
Preliminary investigations into factors that affect plasma aldosterone concentrations in horses. The effect of feeding diets with low, adequate and high sodium contents on plasma aldosterone concentrations in horses and ponies was evaluated using human immunoassay kits. The effect of moderate to high intensity exercise of up to six minutes duration on plasma aldosterone concentrations in three thoroughbred horses was also investigated. On an adequate sodium diet plasma aldosterone concentrations increased to a peak around four hours after feeding. Little daily variation was found in the pre-feeding aldosterone concentrations over three days. Feeding additional salt resulted initially in n...
Airborne dust and aeroallergen concentration in a horse stable under two different management systems. Airborne dust concentration (ADC) was measured in 2 different horse management systems using an Andersen cascade impactor in the box-stall, and a personal Marple cascade impactor attached to the halter to measure ADC in the breathing zone. The levels of aeroallergens implicated in chronic obstructive pulmonary disease were measured by radioallergosorbent-inhibition immunoassay. A conventional management system (System C) utilising hay feed and straw bedding, and a recommended environment (System R) utilising wood shaving bedding and a complete pelleted diet were studied. In the stall, total an...
Quantification of histamine in plasma and pulmonary fluids from horses with chronic obstructive pulmonary disease, before and after ‘natural (hay and straw) challenges’. A commercial radioimmunoassay kit was used to quantify histamine concentrations of plasma, bronchoalveolar lavage fluid (BALF) and pulmonary epithelial lining fluid (PELF) of normal horses and horses with chronic obstructive pulmonary disease (COPD), before and after 'natural (hay and straw) challenge' (NC). There were no significant changes in the concentrations of histamine in plasma or BALF at 0.5 or 5 h after NC, but the PELF histamine concentration of COPD affected horses was significantly increased at 5 h, but not at 0.5 h, following NC. As the histamine concentrations of whole BALF lysa...
Effect of surgical removal of endometrial cups on concentrations of chorionic gonadotrophin and subsequent fertility in the mare. Seven pregnant mares underwent general anaesthesia, laparotomy, hysterotomy and removal of a 50-day conceptus. Eversion of the uterine horn through the hysterotomy site allowed direct visualisation and electrosurgical removal of endometrial cup tissue from 5 randomly selected mares (Nos 1-5), while cup tissue in 2 mares (Nos 6 and 7) was left intact. Two pregnant mares served as unoperated controls (Nos 8 and 9). Efforts to re-establish pregnancy were initiated 20 days after surgery. Serum samples collected before surgery and during the post-operative period were analysed for concentration of ...
Immunochemical studies of equine chorionic gonadotropin (eCG), eCG alpha, and eCG beta. The equine (e) placental glycoprotein hormone eCG plays a critical though not completely understood role during the first trimester of gestation in mares. In the present work, we have developed immunoradiometric assays (m-IRMAs) for detection of eCG, eCG alpha, and eCG beta using combinations of monoclonal antibodies (mAbs) specific for epitopes that reside on free and/or combined subunits. The free eCG alpha m-IRMA was based on AHT20 mAb, specific for the free alpha-subunit of all species, and 125I-labeled ECG01 mAb, which recognizes both free and combined alpha-subunit from equine and primat...
Arterial-venous difference in atrial natriuretic peptide concentration during exercise in horses. Six nontrained mares were subjected to steady-state, submaximal treadmill exercise to examine the effect of exercise on the plasma concentration of atrial natriuretic peptide (ANP) in arterial, compared with mixed venous, blood. Horses ran on a treadmill up a 6 degree grade for 20 minutes at a speed calculated to require a power equivalent to 80% of maximal oxygen uptake (VO2MAX). Arterial and mixed venous blood samples were collected simultaneously from the carotid and pulmonary arteries of horses at rest and at 10 and 20 minutes of exercise. Plasma was stored at -80 C and was later thawed; A...
Dose-response effects of gonadotropin-releasing hormone on plasma concentrations of gonadotropins and testosterone in fertile and subfertile stallions. Five fertile and five subfertile stallions were treated with a single intravenous injection of saline the first week followed by a single intravenous injection of varying doses of gonadotropin-releasing hormone (5, 10, 25, 100, 500 micrograms) given in a randomized fashion over the next 5 weeks during the nonbreeding season. Blood samples were collected periodically before and after treatment for analysis of luteinizing hormone, follicle stimulating hormone, and testosterone content by radioimmunoassay. Before treatment, semen samples were collected every other day for 3 weeks for analysis of ...
Substance P in the synovial membrane and fluid of the equine middle carpal joint. This preliminary study was designed to determine whether the neurotransmitter substance P was present in the middle carpal synovial membrane of the normal horse and whether the neuropeptide could be identified in the synovial fluid of normal horses and those with joint diseases. Immunocytochemistry on middle carpal synovial membrane biopsies from fresh cadavers was used to demonstrate substance P-containing neural elements. Substance P was most abundant in the subintimal portion of the membrane, with occasional filaments coursing via synovial fronds to the intimal portion. Radioimmunoassay tec...
Decreased airway mucosal prostaglandin E2 production during airway obstruction in an animal model of asthma. Heaves is a respiratory disorder of horses and ponies characterized by bouts of acute airway obstruction and airway hyperresponsiveness. We measured prostaglandin E2 (PGE2) and 15-hydroxyeicosatetraenoic acid (15-HETE) production in vitro in tracheal epithelium obtained from six affected horses at the time of acute airway obstruction as compared with six matched control horses. Strips of epithelium and subepithelial tissue were prepared and stimulated with A23187, histamine, and bradykinin. The PGE2 and 15-HETE in media from strips was quantitated by radioimmunoassay. 15-HETE above the limits ...
Increase in plasma progestagen concentrations in the mare after foetal injection with CRH, ACTH or betamethasone in late gestation. Maternal plasma progestagen concentrations increase about 20 days before parturition. The major contributors to the increase are reduced metabolites (ie 5 alpha-pregnanes). Precocious increases (ie less than 310 days of gestation) in these metabolites may occur in abnormal pregnancies. The effects of CRH, ACTH or betamethasone administered to the foetus at gestational ages ranging from about 250 to 320 days were examined. Sixteen healthy pony mares were used for foetal injection employing aseptic techniques. Water or normal saline were used as controls. Maternal plasma progestagen concentratio...
Synovial and serum levels of triamcinolone following intra-articular administration of triamcinolone acetonide in the horse. Seven mature thoroughbred horses, weighing between 400 and 541 kg, were each injected intra-articularly into three joints with 6 mg/joint of triamcinolone acetonide (Vetalog). The fourth joint, the control, was injected with saline. Synovial fluid was taken from all four legs of the horses on days 1, 2, 3, 4, 5, 6, 7, 8, 11, and 15 following the injections. Triamcinolone acetonide was assayed by a radioimmunoassay. Blood was collected at 1, 2, 4, 6, 12 h and on days 1, 2, 3, 4, 5, 6, 7, 8, 11, and 15 following injection of either triamcinolone or saline. Both cortisol and triamcinolone were as...
Plasma renin activity and aldosterone and vasopressin concentrations during incremental treadmill exercise in horses. Six untrained mares were subjected to incremental treadmill exercise to examine exercise-induced changes in plasma renin activity (PRA) and plasma aldosterone (ALDO) and plasma arginine vasopressin (AVP) concentrations. Plasma renin activity, ALDO and AVP concentrations, and heart rate (HR) were measured at each step of an incremental maximal exercise test. Mares ran up a 6 degree slope on a treadmill set at an initial speed of 4 m/s. Speed was increased 1 m/s each minute until HR reached a plateau. Plasma obtained was stored at -80 C and later was thawed, extracted, and assayed for PRA and AL...
Seasonal effects on seminal quality, plasma hormone concentrations, and GnRH-induced LH response in fertile and subfertile stallions. Seasonal effects on hormonal and seminal parameters in subfertile stallions have not been well documented and could provide information that is needed to understand the underlying endocrine mechanisms associated with testicular dysfunction. Such information may be useful in developing diagnostic tools to identify those stallions who are candidates for treatment. This investigation characterizes and compares the effects of season on endocrine function and seminal quality in fertile and subfertile stallions. Eight fertile and six subfertile stallions between the ages of 5 and 18 years were injec...
Reduced endotoxin-induced production of tumor necrosis factor activity by equine peritoneal macrophages exposed to the dual inhibitor of arachidonic acid metabolism, SK & F 86002. The purpose of this study was to determine if a structurally novel dual inhibitor of arachidonic acid metabolism, SK & F 86002, would inhibit the endotoxin-induced production of tumor necrosis factor (TNF) activity by equine peritoneal macrophages. Equine peritoneal macrophages were variously pretreated for 0, 0.5 and 2 h with SK & F 86002 at 10(-9) to 10(-4) molar final concentrations or were left untreated. Then, the macrophages were cultured in vitro in the presence of endotoxin (5 ng/mL). Supernatant media were collected after 4 h and stored at -70 degrees C until assayed for TNF a...
Thromboxane and prostacyclin production in ponies with colonic volvulus. Effects of 1 hour of colonic volvulus and 3 hours of reperfusion on concentrations of thromboxane (TXB2) and prostacyclin (6-keto-PGF1 alpha) in portal, pulmonary arterial, and jugular blood were determined by radioimmunoassay to assess the site of production and clearance of these eicosanoids from the circulation in 5 anesthetized ponies. Colonic volvulus had no significant effect on mean arterial pressure or TXB2 concentrations, but significantly (P less than 0.05) increased 6-keto-PGF1 alpha concentrations in all blood samples. Immediately after colonic reperfusion, all eicosanoid concentra...
Hormonal contraception of feral mares with Silastic rods. Homogeneous Silastic rods containing ethinylestradiol (EE) (1.5 or 4 g), estradiol-17 beta (E) (4 g) or progesterone (P) (6 g) were implanted into feral mares (Equus caballus) between 4- and 10-yr-old. Six treatment groups (greater than or equal to 10 mares/group) of non-pregnant mares received 36 g P and 12 g E (P+E), 36 g P and 8 g EE (P+HEE), 1.5 g EE (LEE), 3 g EE (MEE, 8 g EE (HEE) or control-implanted mares (CI). CI received implants containing no steroid. Two groups of pregnant mares received P+HEE or HEE. Stallions were placed with the mares 15 to 26 mo after implanting. Blood was coll...
PGE2-independent immunosuppressive activity of horse trophoblast tissue. It has been proposed that PGE2 is an important immunosuppressant acting at the fetal-maternal interface during pregnancy. We have previously shown that horse conceptus-conditioned medium suppresses lymphocyte proliferation. This experiment was designed to determine if horse conceptus-derived immunosuppressive activity could be attributed to PGE2 production by the trophoblast tissue. Trophoblast tissue from 21-day-old conceptuses was cut into equal sections and cultured in the presence or absence of the prostaglandin inhibitor, indomethacin. Following culture, immunosuppressive activity and the...
