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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.
Running and shipping elevate plasma levels of beta-endorphin-like substance (B-END-LI) in thoroughbred horses. A specific RIA for beta-endorphin (B-END) was developed to measure horse plasma levels of B-END-like material (B-END-LI) during exercises and shipping. Three exercise speeds and durations were: trot at 260-300 m/min for 10 min; slow gallop at 390-420 m/min for 5 min and fast gallop at 700-800 m/min for 2 min. Blood samples were taken from 4 horses before, immediately after, 30 and 60 min after exercise. Trotting increased plasma B-END-LI from a basal level of 109 +/- 7 pg/ml to 172 +/- 22 at the end of exercise and returned to 127 +/- 17 and 107 +/- 10 pg/ml at 30 and 60 min after exercise. Si...
Radioimmunoassay for parathyroid hormone in equids. Radioimmunoassay for parathyroid hormone (PTH) in equids was performed on blood samples from healthy equids and equids with hypercalcemia and hypophosphatemia. The assay was validated for equine carboxy-terminal PTH. Manipulation of serum ionized Ca in healthy equids by infusing Na2 EDTA and CaCl2 produced an expected increase and decrease, respectively, in measurable immunoreactive PTH. Intra-assay and interassay coefficients of variation were 2.6% and 11.7%, respectively. The range of PTH valves for healthy mature horse mares and geldings maintained on pasture was less than 0.27 ng/ml to 0.9...
Radioimmunoassay of thromboxane B2 in horse plasma. A radioimmunoassay for thromboxane B2 (TXB2) in unextracted horse plasma was evaluated. Sensitivity of the assay was 14.0 (SD 5.6) pg ml-1 of plasma. Interassay and intra-assay variation were 21.3 per cent and 4.3 per cent, respectively. The percentage of tracer bound in unextracted plasma in the absence of TXB2 was often higher than that in buffer. Therefore standard curves were obtained using standards diluted in plasma from horses treated with aspirin or in charcoal treated TXB2-free plasma. Standard curves determined in plasma and buffer were parallel. This assay was used to determine the ...
Effects of month and age on prolactin concentrations in stallion serum. Prolactin concentrations in stallion serum were measured by a newly developed radioimmunoassay based on anti-dog prolactin serum and radiolabelled horse prolactin. Samples of serum from a total of 444 stallions were obtained at a commercial abattoir monthly from April to the following March. Ages of stallions were estimated from eruption and wear patterns of incisors. In the analysis of variance, both month (P less than 0.01) and age (P less than 0.05) were significant sources of variation whereas there was no interaction between these factors. Monthly means for prolactin concentrations were g...
Use of push-pull perfusion techniques in studies of gonadotrophin-releasing hormone secretion in mares. Push-pull perfusion was used to study GnRH secretory ability of the hypothalamus in anoestrous, transitional, dioestrous and oestrous Pony mares. The technique involved placement of a concentric (tube within a tube) cannula into the area of the medial basal hypothalamus and perfusing a carrier medium (artificial cerebrospinal fluid) through the inner tube whilst aspirating from the outer tube so that the flow rate within the hypothalamic tissue was essentially constant. The perfusion rate was 0.5 ml/10 min and samples were collected at 10-min intervals for 10-15 h. The carrier medium, which co...
Circadian, circhoral and seasonal variation in patterns of gonadotrophin secretion in geldings. Blood samples were obtained from 5 mixed-breed, long-term castrated geldings during five 24-h periods between May 1984 and April 1985. Blood samples were collected, beginning at 09:00 h, at 15-min intervals for 8 h and hourly for the remaining 16 h. Plasma concentrations of LH and FSH were determined by RIA. Seasonal changes in hormone concentrations and frequency and amplitude of secretory pulses were evaluated. No diurnal variation in either LH or FSH secretion was observed: however, marked circhoral fluctuations in LH and FSH secretion were noted. Mean LH and FSH concentrations in these lon...
Secretion of luteinizing hormone and follicle stimulating hormone in intact and ovariectomized mares in summer and winter. Sequential samples of blood were drawn via jugular catheters every 15 min for 24 h from four mares in each of five reproductive states: intact anestrous mares in winter, intact diestrous mares in summer, intact estrous mares in summer, ovariectomized mares in winter and ovariectomized mares in summer. Estrous mares were sampled on d 4 or 5 of estrus and diestrous mares on d 10 or 11 of diestrus. Each sample of plasma was assessed for concentrations of luteinizing hormone (LH) and follicle stimulating hormone (FSH) in two independent radioimmunoassays. A computer program was developed that dete...
Effects of age, season and active immunization against estrogen on serum prolactin concentrations in stallions. Prolactin concentrations in stallion serum were measured by radioimmunoassay based on antiserum generated against equine prolactin and radioiodinated canine prolactin. Prolactin concentrations in serum collected from 152 stallions at a slaughterhouse were higher (P less than .01) in summer than in winter (5.7 +/- .15 vs 2.0 +/- .17 ng/ml). Moreover, there was an effect of age (P less than .02) in the analysis of variance; there was no interaction between age and season. In general, prolactin concentrations increased with age up to 3 to 5 years. Samples of serum collected from five control and ...
Release of LH, FSH and GnRH into pituitary venous blood in mares treated with a PGF analogue, luprostiol, during the transition period. Nine mares received cannulae to collect blood from the pituitary venous outflow in the intercavernous sinus (ICS) and the jugular vein; in 4 mares, only jugular cannulae were used. Those 4 mares and 3 of the mares with cannulae in both positions received 7.5 mg luprostiol i.m. and 1 mare with both cannulae was treated with 3.75 mg uprostiol i.v. Blood samples were kept before and after treatment at 2-, 5- or 10-min intervals and concentrations of LH, FSH and GnRH were determined by RIA. Treatments resulted in an immediate sharp rise of LH and FSH in ICS and jugular blood samples within 2-10 mi...
Comparison by three different radioimmunoassay systems of the polymorphism of plasma FSH in mares in various reproductive states. FSH was measured in the pituitary, and in pituitary venous and jugular blood collected at frequent intervals from mares in various reproductive states, using 3 validated and highly specific radioimmunoassay systems based on different antibodies, 'o', 'h' and 'e'. In the pituitary, 4 forms of FSH were found which differed in isoelectric point and relative potency in the 3 assays. In jugular blood, mean FSH concentrations and short-term patterns depended on the assay used and the reproductive state of the mare. In pituitary venous blood, although FSH concentrations were greatly elevated above ju...
Transitory changes of hormones in the plasma of parturient pony mares. Frequent blood samples were collected from 8 pony mares before, during and after labour, parturition and placental expulsion and assayed for progesterone, oestradiol, androstenedione and LH concentrations by radioimmunoassay. A significant (P less than 0.05) decrease in progesterone, oestradiol and in the progesterone:oestradiol ratio was not detected until 0.5 h after foaling. Androstenedione concentrations rose before and peaked at parturition and then declined. A significant (P less than 0.05) rise in LH was detected 0.5 h after parturition. This LH peak was not detected in one mare and she...
Plasma prolactin concentrations in non-pregnant mares at different times of the year and in relation to events in the cycle. Plasma prolactin concentrations were measured in mares using an homologous radioimmunoassay. An annual rhythm in plasma prolactin was found, with concentrations higher during the summer than during the winter. In addition to this seasonal pattern, occasional high concentrations of prolactin were seen when concentrations were otherwise basal. Blood samples taken from mares during an oestrous cycle in October-November showed that prolactin values were basal for most of the cycle, with a marked rise in prolactin shortly before the onset of oestrus. This prolactin peak was associated with an incre...
Comparison of two reference preparations for horse chorionic gonadotrophin in four in-vivo and in-vitro assays. A number of horse chorionic gonadotrophin (CG) preparations of different purities and from diverse sources have been compared in radioimmuno-, radioreceptor, in-vitro cell culture, and in-vivo assays. The relative activities of the great majority of the preparations tested were consistent in the 4 assay systems. Moreover, their relative activities in the 4 assays were consistent with those found for unfractionated plasmas. These preparations were therefore considered to represent the native form of hormone. The second International Reference Preparation (IRP2) was among the few preparations ex...
Steroid secretion by different cell types of the horse conceptus. Horse conceptuses were recovered non-surgically at Day 12-Day 15 and were dissociated with collagenase. Separation of the cells on a 31.8% Percoll gradient gave two bands of cells and indirect evidence suggests that the low density cells (LDC) are endoderm and the higher density cells (HDC) are trophectoderm. Each band was incubated for 24 h in Minimum Essential Medium and concentrations of oestradiol and progesterone in the medium were measured by radioimmunoassay (RIA). The LDC secreted predominately progesterone (log oestradiol/progesterone = -0.994 +/- 0.141; N = 15) whereas the HDC secret...
Endotoxin-induced production of thromboxane and prostacyclin by equine peritoneal macrophages. Equine peritoneal macrophages were isolated and cultured in vitro to assess their ability to produce thromboxane (TxA2) and prostacyclin (PGI2) in response to endotoxin. Peritoneal macrophages (2.5 x 10(6)/ml) were incubated in tissue culture media, containing 1) no additive (nonstimulated control), 2) endotoxin (0.5 to 100 ng/ml) or 3) the calcium ionophore, A23187 (0.95 microM) for two and six h. Concentrations of the stable metabolites of TxA2 and PGI2 thromboxane B2 (TxB2) and 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha), in the incubation media were determined by radioimmunoassay. Th...
Changes in the hypothalamic-hypophyseal axis of mares associated with seasonal reproductive recrudescence. Four groups of mares, representing anestrus (AN; n = 8), early transition (ET; n = 7), late transition (LT; n = 8) and estrus (EST; n = 12) were used to examine changes in the hypothalamus and anterior pituitary during the period of transition from winter anestrus into the breeding season. Mares were of mixed breeding, between the ages of 3 and 20 years, and had shown normal patterns of estrous behavior and ovulation during the breeding season previous to this experiment. Hypothalamic content of gonadotropin-releasing hormone (GnRH) and anterior pituitary content of luteinizing hormone (LH) an...
[Pregnancy diagnosis in thoroughbred mares using radioimmunoassay (RIA), enzyme immunoassay (kit) and ultrasound echography (scanning)]. On the basis of progesterone determination on plasma or blood after RIA and the kit method respectively and consecutive scanning performed on a total number of 31 mares the following features were demonstrated: The overall material shows that in 20 mares (64.5%) embryonic vesicles were demonstrated. Of these mares 16 have conceived after service in the 1st estrous cycle and 3 mares in the 2nd estrous cycle. 18 mares were scanned in the time interval 13th-26th day after the latest service, while 2 mares were scanned on day 46 and 41 respectively. A total of 14 scanning positive mares were exami...
Molecular cloning and physical characterization of integrated equine infectious anemia virus: molecular and immunologic evidence of its close relationship to ovine and caprine lentiviruses. Molecular clones of the integrated form of the genome of equine infectious anemia virus (EIAV), the etiologic agent of a naturally occurring, worldwide disease of horses, were obtained. The restriction map of a full-length genome was determined. Additional evidence for the close evolutionary relationship between EIAV and a prototype lentivirus (caprine arthritis encephalitis virus) was acquired by Southern blotting and immunological analyses. An interspecies radioimmunoassay was developed in which EIAV and ovine and caprine lentiviruses could be detected equally well. These studies make availa...
Development of a homologous equine relaxin radioimmunoassay. Equine relaxin (eRlx) immunoactivity has previously been measured in the mare during pregnancy using the porcine relaxin (pRlx) RIA (pRlx-RIA). This was not the optimal system for measurement of eRlx because the dose-response curve obtained with equine plasma was not parallel to the pRlx standard curve. A homologous eRlx-RIA has been developed and used to measure relaxin immunoactivity during pregnancy and parturition in the mare. Highly purified eRlx was used for the generation of antiserum in rabbits, preparation of tracer, and as assay standards. A double antibody eRlx RIA (eRlx-RIA) was de...
Concentrations of prolactin, luteinizing hormone and follicle stimulating hormone in pituitary and serum of horses: effect of sex, season and reproductive state. Pituitary and serum from 86 male or female horses of various reproductive states were collected in the normal breeding season (summer) and in the nonbreeding season (winter) at a commercial slaughterhouse. Concentrations of prolactin (PRL), luteinizing hormone (LH) and follicle stimulating hormone (FSH) were measured by radioimmunoassay. Concentrations of pregnant mare serum gonadotropin and reproductive steroids in serum and gross appearance of the reproductive tract and gonads were used to catagorize reproductive state. Concentrations of PRL were higher (P less than .01) in summer than in wi...
Radioimmunoassay screening for etorphine in racing horses. A commercially available radioimmunoassay kit was used to screen for the presence of etorphine in post-race urines from horses racing in Kentucky. Most horse urines contained small amounts of materials which reacted positively in this immunoassay. These materials are apparently endogenous to the horse and were called apparent etorphine equivalents. The levels of these apparent etorphine equivalents in post-race urines from 70 horses were estimated. Their modal level averaged 0.1 ng/ml, the population distribution was log normal, and individual horses showed levels of up to 0.8 ng/ml.
Isolation and characterization of three forms of luteinizing hormone from the pituitary gland of the horse. Three isoforms of equine luteinizing hormone (eLH-A, eLH-B and eLH-C) have been isolated from horse pituitary glands. Separation was achieved on the basis of charge heterogeneity by ion-exchange chromatography. These charge differences were apparent after final purification, as determined by electrophoretic mobility on polyacrylamide disc gels (RF = 0.14, 0.19 and 0.26 for eLH-A, -B and -C, respectively). Apparent size differences were also noted between the isohormones by gel filtration on Sephadex G-100. Ve/Vo ratios for eLH-A, -B and -C were 1.72, 1.54 and 1.47, respectively. All 3 isoforms...
Impaired estrogen production by Leydig cells of the naturally retained testis in unilaterally cryptorchid boars and stallions. Estrogen production in vitro was compared for Leydig cells from cryptorchid and scrotal testes in boars and stallions. Animals with natural and experimental cryptorchidism were used. Purified Leydig cells were prepared from testes of mature animals by collagenase treatment and Percoll density gradients. After incubation for 3 hours (1 X 10(6) cells), estrone sulfate and estrone in the media were measured by direct radioimmunoassay. Androstenedione and testosterone in media extracts also were determined. Cells from the abdominal testis of unilateral cryptorchid boars and stallions showed impair...
Effects of flunixin meglumine, phenylbutazone and a selective thromboxane synthetase inhibitor (UK-38,485) on thromboxane and prostacyclin production in healthy horses. The efficacy of three agents which alter the metabolism of arachidonic acid was investigated in normal, conscious horses. A dose response evaluation was made of flunixin meglumine and phenylbutazone, two cyclo-oxygenase inhibitors, and of a selective thromboxane synthetase inhibitor, UK-38,485. Radioimmunoassay of thromboxane B2 (TxB2) and 6-keto prostaglandin F1 alpha (PGF1 alpha) was used to assess the concentrations of thromboxane A2 (TxA2) and prostacyclin (PGI2) respectively, in serum. Flunixin was the most potent inhibitor of serum TxB2 and 6-keto PGF1 alpha production. UK-38,485 also de...
Characteristics of cells derived from the girdle region of the pre-implantation blastocyst of the donkey. The establishment of a monolayer culture of cells derived from the girdle region of a 34-day-old donkey conceptus is described. These cells have had over 100 repeated passages in culture. Low levels of pregnant mares' serum gonadotrophin (PMSG, eCG) could be detected in the cells by indirect immunofluorescence using some monoclonal anti-eCG antibodies, but the cells did not secrete eCG as measured by radioimmunoassay or inhibition of haemagglutination. There was marked nuclear polymorphism with binucleate and occasional multinucleate cells. The cells were strongly reactive with wheatgerm agglu...
Extraction, radioiodination, and in vivo catabolism of equine fibrinogen. Equine fibrinogen was isolated and aliquots were stored frozen at -70 C before radiolabeling with 125I (half-life = 60.2 days; gamma = 35 keV, using monochloroiodine reagent. Radioiodination efficiencies were 49% to 53%, resulting in a labeled product with 98% protein-bound activity and 91% clottable radioactivity. In 6 equine in vivo investigations, plasma half-lives of 125I-labeled fibrinogen were from 4.1 to 5.2 days, corresponding to a mean daily plasma elimination rate of approximately 15%.
Rapid extraction, radioiodination, and in vivo catabolism of 125I-labeled fibrinogen in the horse. Two methods were analyzed for the rapid extraction of equine fibrinogen from fresh plasma, using ammonium sulfate-sodium phosphate buffer. Fibrinogen from each of these 2 methods was then radiolabeled with 125I (half-life = 60.2 days, gamma = 35 keV), using monochloroiodine reagent. Mean protein-bound activity was 98.5% and mean clottable radioactivity was 94.1%. Radiolabeled fibrinogen administered IV to 15 horses had an overall mean (+/- SD) plasma half-life of 4.95 +/- 0.44 days.
Flunixin meglumine given in small doses: pharmacokinetics and prostaglandin inhibition in healthy horses. The pharmacokinetics and inhibition of prostaglandin synthesis in conscious horses given various dosages of flunixin meglumine were studied. Plasma concentrations of flunixin were measured by high-performance liquid chromatography, and serum thromboxane B2 and 6-keto prostaglandin F1 alpha were quantitated by radioimmunoassay. Within the dosage range studied, linear pharmacokinetics were achieved. After IV administration of flunixin (1.1 mg/kg, 0.25 mg/kg, 0.1 mg/kg), significant suppression of serum thromboxane generation persisted for 12, 4, and 3 hours, respectively. Repeated administration...
Studies on equine recurrent uveitis. I: Levels of immunoglobulin and albumin in the aqueous humor of horses with and without intraocular disease. A radioimmunoassay was developed for detection of immunoglobulin in the aqueous of normal horses and horses with intraocular diseases. Levels of albumin were detected by radial immunodiffusion. Results of assays on samples from normal eyes from which aqueous was obtained by paracentesis under anesthesia were 32.10 +/- 21.50 microgram/ml for IgG, 0.05 +/- 0.01 microgram/ml for IgM, 0.04 +/- 0.02 microgram/ml for IgA and 34.0 +/- 38.0 microgram/ml for albumin. Results in 138 normal eyes sampled post mortem were 41.56 +/- 38.65 microgram/ml for IgG, 0.18 +/- 0.43 microgram/ml for IgM, 0.46 +/- 1....
Combined pharmacokinetics of gentamicin in pony mares after a single intravenous and intramuscular administration. Healthy mature pony mares (n = 6) were given a single dose of gentamicin (5 mg/kg of body weight) IV or IM 8 days apart. Venous blood samples were collected at 0, 5, 10, 20, 30, and 45 minutes and at 1, 1.5, 2, 2.5, 3, 4, 6, 8, 10, 12, 18, 24, 30, 36, 40, and 48 hours after IV injection of gentamicin, and at 10, 20, 30, and 45 minutes and at 1, 1.5, 2, 2.5, 3, 4, 6, 8, 10, 12, 18, 24, and 30 hours after IM injection of gentamicin. Gentamicin serum concentration was determined by a liquid-phase radioimmunoassay. The combined data of IV and IM treatments were analyzed by a nonlinear least-square...
