Biomedical subjects

D R Grattan

Publications and source records attributed to D R Grattan.

15 recordsLinked to original sources

PubMed1996

GABAergic neuronal activity and mRNA levels for both forms of glutamic acid decarboxylase (GAD65 and GAD67) are reduced in the diagonal band of Broca during the afternoon of proestrus.

There is considerable evidence that GABAergic neurons play an important role in the regulation of gonadotropin-releasing hormone (GnRH) secretion, and that these neurons may mediate the feedback actions of gonadal steroids on GnRH neurons. The aim of the present study was to investigate whether endogenous changes in ovarian steroid secretion during the estrous cycle influenced GABAergic neuronal activity in the preoptic region of the hypothalamus, and in other steroid-sensitive brain regions. Intact, adult female rats were sacrificed at various times during the days of metestrus or proestrus. GABAergic neuronal activity was estimated by measuring the rate of accumulation of GABA in microdissected brain regions after pharmacological inhibition of GABA degradation. Concentrations of mRNA for both forms of glutamic acid decarboxylase (GAD65 and GAD67) were quantified in microdissected brain regions by a microlysate ribonuclease protection assay. In the diagonal band of Broca at the level of the organum vasculosum of the lamina terminalis (DBB(ovlt)), GABAergic neuronal activity was significantly reduced during the afternoon of proestrus compared with the morning of either proestrus or metestrus. In the lateral septal nucleus, GABAergic neuronal activity was significantly increased in the afternoon of proestrus compared with the morning. There were no significant effects of time of day or day of estrous cycle in the medial preoptic nucleus, median eminence, ventromedial nucleus, suprachiasmatic nucleus, medial septal nucleus, hippocampus (CA1 region), or cingulate cortex. In the DBB(ovlt), mRNA levels for both GAD65 and GAD67 were significantly reduced in the afternoon of proestrus compared with the afternoon of metestrus. By contrast, there was no change in GAD65 and GAD67 mRNA levels in the cingulate cortex at any of the times examined. These results demonstrate that GABAergic neuronal activity, and mRNA levels for both GAD65 and GAD67, are reduced in the DBB(ovlt) during the afternoon of proestrus. These results support the hypothesis that decreased GABAergic neuronal activity in this region plays a major permissive role in the generation and maintenance of the estrogen-induced LH surge.

Animals↗

PubMed1996

Hormonal and neurotransmitter regulation of GnRH gene expression and related reproductive behaviors.

Gonadotropin-releasing hormone (GnRH), having a highly conserved structure across mammalian species, plays a pivotal role in the control of the neuroendocrine events and the inherent sexual behaviors essential for reproductive function. Recent advances in molecular genetic technology have contributed greatly to the investigation of several aspects of GnRH physiology, particularly steroid hormone and neurotransmitter regulation of GnRH gene expression. Behavioral studies have focused on the actions of GnRH in steroid-sensitive brain regions to understand better its role in the facilitation of mating behavior. To date, however, there are no published reports which directly correlate GnRH gene expression and reproductive behavior. The intent of this article is to review the current understanding of the way in which changes in GnRH gene expression, and modifications of GnRH neuronal activity, may ultimately influence reproductive behavior.

Animals↗

PubMed1996

Antiandrogen microimplants into the rostral medial preoptic area decrease gamma-aminobutyric acidergic neuronal activity and increase luteinizing hormone secretion in the intact male rat.

gamma-Aminobutyric acid (GABA)ergic neurons terminating in the rostral hypothalamus are stimulated by testosterone. To investigate whether this action is mediated locally through androgen receptors in the rostral hypothalamus, bilateral microcannulas (28 gauge) containing the androgen receptor antagonist, hydroxyflutamide (HF), were stereotaxically implanted into the rostral medial preoptic area (rMPA) just dorsal to the major population of GnRH cell bodies. Two days later, blood samples were collected for assay of LH, and animals were killed for determination of GABAergic neuronal activity in tissue dissected from the site of the implanted cannulas. Animals were decapitated either without treatment or 60 min after inhibition of GABA degradation by aminooxyacetic acid (100 mg/kg, ip). The rate of GABA accumulation in the tissue after aminooxyacetic acid treatment was used as a measure of GABA turnover. Levels of messenger RNA for both forms of glutamic acid decarboxylase (GAD65 and GAD67), the rate-limiting enzyme responsible for GABA synthesis also were measured by a microlysate ribonuclease protection assay. LH levels were significantly increased (1.8-fold) in HF-treated animals compared with controls. In the MPA, beneath the implant cannulas, GABA turnover was significantly reduced in HF-treated rats. There was no effect of treatment in the frontal cortex, which was used as a control region. Surprisingly, levels of messenger RNA for both GAD65 and GAD67 were significantly increased in HF-treated rats. The results indicate that GABAergic neurons terminating in the rostral hypothalamus are tonically stimulated by testosterone acting by means of androgen receptors localized in this region. These findings support the working hypothesis that androgen-sensitive GABAergic neurons in the rMPA mediate the negative feedback action of testosterone on GnRH secretion in the male rat.

Androgen Antagonists↗

PubMed1995

Absence of short-loop autoregulation of prolactin during late pregnancy in the rat.

Animals bearing intrahypothalamic anterior pituitary (AP) grafts exhibit a central hyperprolactinemia, and thus, serve as a model for the study of short-loop feedback regulation of prolactin (PRL) secretion. We investigated the effects of intrahypothalamic AP grafts on PRL secretion during late pregnancy (n = 7). A further group of five rats was injected with the dopamine against bromocriptine during late pregnancy and five control rats were treated with the bromocriptine-vehicle only. A nocturnal surge in plasma PRL concentrations was observed in the vehicle-injected control animals, peaking at 212 +/- 11 ng/ml at 0300 h on the day of parturition. Despite the central hyperprolactinemia due to the grafts, a similar PRL surge was observed in grafted animals, peaking at 205 +/- 35 ng/ml at 0300 h on the day of parturition. Bromocriptine treatment completely blocked the nocturnal surge of PRL. These results suggest that short-loop feedback autoregulation of PRL secretion becomes less responsive or nonfunctional in the last 24 h of pregnancy in the rat. This apparent change in sensitivity of the autofeedback mechanism may be an important physiological mechanism to allow the hypersecretion of PRL during lactation.

Animals↗

PubMed1995

Orchidectomy and NMDA increase GnRH secretion as measured by push-pull perfusion of rat anterior pituitary.

Using push-pull perfusion to measure concentrations of gonadotropin-releasing hormone (GnRH) in the extracellular fluid of the anterior pituitary gland of the male rat, we have measured GnRH release at specific times before and after castration and in response to acute administration of N-methyl-D-aspartate (NMDA). After castration (7 days), mean GnRH levels were substantially increased (4.3-fold) compared with intact controls (0.94 +/- 0.16 vs. 0.22 +/- 0.08 pg/10 min, respectively, P < 0.05) due to an increase in both the frequency and amplitude of GnRH pulses. Testosterone partially reduced GnRH release (0.62 +/- 0.10 pg/10 min). NMDA induced a rapid increase in plasma luteinizing hormone (LH) in both intact and castrated rats and increased GnRH concentrations in the perfusion samples (P < 0.05). There was no change in LH release induced by two doses of injected GnRH (5 and 25 ng/100 g body wt) 2 days after castration, but by 6 days after castration the response to both doses was significantly increased. These results demonstrate that GnRH release in the male rat is acutely increased by NMDA and is chronically increased after orchidectomy. Increased pituitary sensitivity to GnRH also contributes to the hypersecretion of LH after castration, particularly at longer times after removal of testosterone negative feedback.

Animals↗

PubMed1994

Castration-induced decrease in the activity of medial preoptic and tuberoinfundibular GABAergic neurons is prevented by testosterone.

We recently determined that castration specifically decreased GABA turnover in discrete rostral and mediobasal hypothalamic structures. This study aimed to investigate whether testosterone could stimulate GABAergic neuronal activity in these hypothalamic GABAergic neurons in the castrate rat, and to compare the effects of episodic testosterone replacement with the constant levels provided by subcutaneous testosterone implants. Animals were divided into 4 experimental groups: intact, 48 h castrate, 48 h castrate+testosterone capsules (2 x 30 mm Silastic implants, 1.57 mm ID, 3.18 mm OD) and 48 h castrate+testosterone injections (100 micrograms/injection s.c., every 8 h). GABA concentrations were measured in 4 microdissected brain regions either before or 60 min after inhibition of the GABA degrading enzyme, GABA transaminase, by injection of aminooxyacetic acid (AOAA, 100 mg/kg i.p.). The rate of GABA accumulation in the tissue following injection of AOAA was used as an index of GABAergic neuronal activity. Castration resulted in a 10-fold increase in serum LH concentrations compared with intact rats. Either mode of testosterone administration completely prevented this castration-induced LH rise. In the diagonal band of Broca at the level of the organum vasculosum of the lamina terminalis, the medial preoptic nucleus and in the median eminence, GABA turnover was significantly reduced by castration to approximately 50% that of intact rats. Either testosterone implants or testosterone injections prevented this castration-induced decrease in GABA turnover, such that the turnover rates were not significantly different from intact rats. There was no effect of castration with or without testosterone replacement in the cingulate cortex.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Prolactin- and testosterone-induced inhibition of LH secretion after orchidectomy: role of preoptic and tuberoinfundibular gamma-aminobutyric acidergic neurones.

The inhibitory amino acid neurotransmitter gamma-aminobutyric acid (GABA) may play an important role in the regulation of LH-releasing hormone secretion. The present study examined the effect of prolactin on GABAergic neuronal activity in microdissected brain regions of the orchidectomized rat, to determine whether inhibition of LH secretion after castration by acute hyperprolactinaemia was associated with prolactin-induced changes in GABAergic neuronal activity. The effects of prolactin were contrasted with the effects of testosterone on hypothalamic GABAergic neurones after orchidectomy. GABA concentrations were measured by high pressure liquid chromatography in eight microdissected brain regions in untreated rats and 60 min after inhibition of the GABA catabolic enzyme GABA transaminase by injection of amino-oxyacetic acid (AOAA). The rate of GABA accumulation in microdissected brain regions following injection of AOAA was taken as an index of GABAergic neuronal activity. Rats were divided into seven experimental groups: intact controls, 2 days after castration, 2 days after castration with prolactin treatment (2.5 mg ovine prolactin injected s.c. every 12 h, starting at the time of castration), 2 days after castration with testosterone replacement (30 mm silicone elastomer implant containing crystalline testosterone), 6 days after castration, 6 days after castration with prolactin treatment, and 6 days after castration with testosterone replacement. Both 2 and 6 days after castration, plasma LH was markedly elevated above levels in intact rats, and AOAA-induced GABA accumulation was significantly decreased in the diagonal band of Broca at the level of the organum vasculosum of the lamina terminalis, in the medial preoptic nucleus and in the median eminence. Hyperprolactinaemia significantly reduced LH levels 2 days but not 6 days after castration. GABAergic neuronal activity, however, was not significantly affected by prolactin at either time. Testosterone replacement blocked the postcastration elevation in plasma LH and prevented the castration-induced suppression of GABAergic neuronal activity both 2 and 6 days after castration. There were no castration- or hormone-induced changes in GABAergic neurones observed in the medial or lateral septum, caudate nucleus, cingulate cortex or arcuate nucleus. These results demonstrate that the activity of GABAergic neurones terminating in the rostral hypothalamus and the median eminence is positively regulated by testosterone, and that these steroid-sensitive GABAergic neurones may be important in the negative-feedback control of LH secretion. Inhibition of LH secretion by hyperprolactinaemia, however, may not be mediated by changes in GABAergic neuronal activity.

Animals↗

PubMed1993

Regional variation in gamma-aminobutyric acid turnover: effect of castration on gamma-aminobutyric acid turnover in microdissected brain regions of the male rat.

This study compared the turnover of GABA neurons in different brain areas of the male rat and examined the effect of castration on GABA turnover in regions of the brain associated with the control of gonadotropin secretion. To estimate GABA turnover, GABA was quantified by HPLC in microdissected brain regions 0, 30, 60, 90, and 120 min after inhibition of GABA degradation by aminooxyacetic acid (100 mg/kg, i.p.). GABA accumulation was linear in all areas for 90 min (p < 0.01), and GABA turnover was estimated as the slope of the line formed by increased GABA concentration versus time, determined by linear regression. There was considerable regional variation both in the initial steady-state concentrations of GABA and in the rates of GABA turnover. Of 10 discrete brain structures, GABA turnover was highest in the medial preoptic nucleus and lowest in the caudate nucleus. Turnover times in the terminal fields of known GABAergic projection neurons ranged sevenfold, from 2.6 h in the substantia nigra to 0.4 h in the lateral vestibular nucleus. The effect of castration on GABA turnover in 13 microdissected brain regions was investigated by measuring regional GABA concentrations before and 30 min after injection of aminooxyacetic acid in intact rats or 2 or 6 days postcastration. Following castration, steady-state GABA concentrations were increased, and GABA turnover decreased in the diagonal band of Broca, the medial preoptic area, and the median eminence. GABA turnover increased in the medial septal nucleus and was unaffected in the cortex, striatum, and hindbrain. These results are consistent with the hypothesis that testosterone negative-feedback control of luteinizing hormone-releasing hormone involves steroid-sensitive GABAergic neurons in the rostral and medial basal hypothalamus.

Aminooxyacetic Acid↗

PubMed1993

Differential effects of adrenalectomy on the prolactin-induced suppression of LH and FSH secretion after castration in male rats.

Hyperprolactinaemia inhibits gonadotrophin secretion in males and females of many species. The aim of this study was to determine the role of the adrenal gland in mediating the inhibitory effects of prolactin by contrasting the effects of acute hyperprolactinaemia on LH and FSH secretion in adrenal-intact and adrenalectomized rats with and without physiological corticosterone replacement. Adult male rats were administered purified ovine prolactin every 12 h (2.4 mg per injection s.c.) beginning at the time of castration. Blood samples were collected every 3 h for 36 h, then every 12 h until 10 days after castration. Ovine prolactin significantly reduced LH secretion in all groups from approximately 15 to 48 h after castration. In contrast, plasma FSH concentrations were reduced by ovine prolactin from 21 to 48 h only in the adrenal-intact rats and not in the adrenalectomized or adrenalectomized plus corticosterone groups. In all groups, ovine prolactin inhibited endogenous prolactin secretion in rats by short-loop autofeedback as soon as 3 h after the first ovine prolactin injection and throughout the 10 days of the study. Adrenalectomy per se, with or without corticosterone replacement, also had a differential effect on LH and FSH secretion after castration, causing only a transient delay in the rise in LH after castration, but inducing a significant and long-lasting inhibition of FSH secretion. The results demonstrate that ovine prolactin-induced suppression of LH secretion after castration occurs with or without the adrenal glands. Suppression of FSH secretion after castration by ovine prolactin, however, may involve an adrenal component.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Glands↗

PubMed1993

Effect of alcohol, neurohypophysectomy, and vasopressin antagonists on hemorrhage-induced bradycardia in the rat.

During initial stages of hemorrhage in the rat, cardiovascular compensation leads to a tachycardia (mean +/- SE, 5.2 +/- 0.7%; n = 23) that helps prevent a large fall in blood pressure. This compensatory phase is followed by a decompensatory phase in which mean arterial pressure and heart rate fall. A rise in arginine vasopressin (AVP) levels has been postulated as the cause of this hemorrhage-induced bradycardia (HIB). The object of the present study was to determine whether interference with AVP release by alcohol anesthesia or neurohypophysectomy or by blockade of AVP receptors in the plasma or cerebral spinal fluid could attenuate HIB. Male Wistar rats were anesthetized with pentobarbital, surgically prepared, and bled to maintain a blood pressure of 40-50 mm Hg. After hemorrhage, heart rate decreased 15 +/- 2% (n = 6) with alcohol anesthesia compared with 32 +/- 3% (n = 7) with pentobarbital. After neurohypophysectomy, however, HIB remained unchanged (-15 +/- 2%; n = 5) compared with sham-operated controls (-19 +/- 3%; n = 6). Peripheral administration of two nonselective V1/V2 antagonists and one V2 antagonist had no effect on HIB, whereas a V1 antagonist significantly attenuated the heart rate decrease (-15 +/- 4%; n = 6) compared with controls (-32 +/- 3%; n = 7). None of the AVP antagonists tested at one tenth the peripheral dose had any effect on HIB when administered into the lateral ventricle of the brain, although a mixed serotonin, dopamine, and catecholamine antagonist, spiperone, potentiated the response. It was concluded that although peripheral release of AVP may be partially involved in the heart rate response to hemorrhage, central AVP release and central AVP receptors were not involved in HIB.

Animals↗

PubMed1991

Posterior pituitary lobectomy chronically attenuates the nocturnal surge of prolactin in early pregnancy.

The posterior pituitary gland contains a potent PRL-releasing factor (PRF). The aim of this study was to determine whether this PRF was involved in the luteotropic PRL surges of early pregnancy. The posterior pituitary was removed from a group of rats at least 4 weeks before the experiment (LOBEX). LOBEX rats were tested for diabetes insipidus and checked postmortem for the absence of posterior lobes. Plasma samples were taken from these chronically LOBEX rats as well as from sham-operated and unoperated controls beginning at noon on day 3 of pregnancy and continuing for 3 days at times most likely to demonstrate the twice daily surges of PRL secretion. PRL in all groups was basal (less than 12 ng/ml) at noon and rose to a diurnal peak at 1800 h. While the unoperated and sham-operated controls then displayed a clear nocturnal surge of PRL, peaking at 300-400 ng/ml at 0300 h, LOBEX rats had a significantly attenuated peak of 102.7 +/- 17.7 ng/ml (P less than 0.001). On subsequent nights the nocturnal peaks were similarly attenuated in LOBEX rats, averaging 80.3 and 42.1 ng/ml on days 5 and 6, respectively. LOBEX rats all had normal sized litters, but lactation yields were depressed. These data support the hypothesis that the posterior pituitary secretes a potent PRL-releasing factor because its removal partially blocked the mating-induced nocturnal surge of PRL.

Animals↗

PubMed1991

Role of the placenta in the control of the ante-partum surge of prolactin in the rat.

A nocturnal surge of prolactin secretion occurs in the dark period preceding parturition in the rat. The aim of this study was to examine the role of the placenta in the control of this prolactin surge. Plasma prolactin and progesterone were measured by radioimmunoassay in serial blood samples collected after surgical removal of conceptuses during late pregnancy, and after intracerebroventricular (i.c.v.) injection of placental lactogen (PL) before the prolactin surge. In intact control animals, prolactin secretion remained low until a nocturnal surge of secretion occurred in the dark period preceding parturition, peaking at 269 +/- 51 (S.E.M.) micrograms/l at 03.00 h on day 21. Progesterone levels fell from greater than 200 nmol/l on day 19 to less than 40 nmol/l by 12.00 h on day 20 of pregnancy. PL levels during late pregnancy were modified by partial or complete removal of conceptuses at 10.00 h on day 19 of pregnancy. Removal of all but one or two conceptuses did not change the normal pattern of prolactin or progesterone secretion. Removal of all conceptuses, however, induced a large nocturnal surge of prolactin secretion, peaking at 211.7 +/- 78 micrograms/l at 03.00 h on day 20, 24 h earlier than the surge in intact animals. Progesterone levels after removal of all conceptuses fell to less than 40 nmol/l by 23.00 h on day 19, approximately 12 h before the decline in intact animals. Maintenance of increased progesterone levels after conceptus removal using silicone tubing implants significantly (P less than 0.05) reduced the peak of the premature prolactin surge to 79.7 +/- 18 micrograms/l at 05.00 h on day 20.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Intrahypothalamic pituitary grafts elevate prolactin in the cerebrospinal fluid and attenuate prolactin release following ether stress.

Anterior pituitary (AP) tissue grafted into the hypothalamus of female rats inhibits the luteotrophic prolactin (PRL) secretion which normally follows mating. Dopamine blockade has been shown to overcome this inhibition, suggesting that the grafts suppress PRL release from the in situ pituitary by the action of graft PRL increasing dopamine activity in the hypothalamus. To examine whether PRL levels in the cerebrospinal fluid (CSF) were elevated by the AP grafts, CSF samples were taken from 5 control rats and 10 rats bearing intrahypothalamic AP grafts. Mean PRL concentrations in the CSF of the control rats were 3.0 +/- 0.8 ng/ml. The grafted rats had significantly higher concentrations of PRL in their CSF, averaging 23.2 +/- 4.2 ng/ml (P less than 0.005). Plasma PRL concentrations were similar in the control and grafted rats. PRL release in response to 5 min of ether stress was examined in 8 control and 11 grafted rats. In control animals, PRL rose from 4.2 +/- 1.5 to 44.7 +/- 9.0 ng/ml following exposure to ether, but the response was significantly attenuated in the grafted rats, peaking at 9.3 +/- 1.4 ng/ml (P less than 0.001). This inhibition of response due to the grafts was evident within 1 week of graft placement. The results confirm that the presence of intrahypothalamic AP grafts led to the accumulation of supranormal PRL concentrations in the CSF. This elevated PRL suppressed pituitary PRL release in response to ether stress, probably by an autoregulatory feedback activation of the inhibitory tuberoinfundibular dopaminergic neurons in the hypothalamus.

Animals↗

PubMed1990

Effect of ovarian steroids on a nocturnal surge of prolactin secretion that precedes parturition in the rat.

PRL secretion in several physiological and experimental conditions, including early pregnancy, is linked to the daily photoperiod. The aim of this study was to examine the antepartum increase in PRL secretion for evidence of a circadian pattern of release, as seen during early pregnancy. During the last 3 days of pregnancy blood samples were taken six times daily by means of previously implanted jugular cannulae. Plasma PRL concentrations were then measured by RIA. PRL levels were less than 10 ng/ml in all animals on day 19 of pregnancy, but during the light period of day 20 there was an increase to an average of 30 +/- 10 ng/ml, with no evidence of a peak related to the time of day. However, in the dark period between days 20 and 21 there was a large surge of PRL secretion which reached peak levels of 356 +/- 39 ng/ml at 0500 h on day 21, then returned to 48 +/- 20 ng/ml at 1200 h, around the time of parturition. The peak always occurred at 0500 h and was not related to the time of parturition which ranged from 1000-2200 h on day 21. Bilateral ovariectomy (OVX) on day 19 advanced the time of delivery by approximately 12 h. In seven of nine animals, no surge of PRL secretion was observed during the dark period preceding parturition. Estradiol treatment after OVX on day 19 (OVX+E) advanced the time of delivery by approx 18 h. An antepartum PRL surge was present and was advanced by 24 h in all OVX+E animals, peaking at 0300 h on day 20. Progesterone treatment from day 18 to 21 in intact pregnant animals delayed parturition by approximately 18 h and prevented PRL secretion during the period of treatment. After progesterone treatment was stopped, a nocturnal surge of PRL secretion occurred, peaking at 0500 h on day 22, 24 h after the surge in normal animals. The results suggest that the increased PRL secretion during late pregnancy is linked to the daily photoperiod and is characterized by a nocturnal surge in the dark period preceding parturition. This surge is inhibited by progesterone, and it can be advanced 24 h by estradiol treatment in the absence of the ovaries.

Animals↗

PubMed1989

Failure of an indirect competitive enzyme-linked immunosorbent assay to measure prolactin in rat plasma samples.

An indirect competitive enzyme-linked immunosorbent assay (ELISA) for rat prolactin was applied to the measurement of prolactin (PRL) in plasma. The assay involved PRL adsorbed to a 96-well plate competing with soluble PRL in the sample for rabbit anti-rat PRL antibody-binding sites. Antibody bound by the PRL on the well was detected by a goat anti-rabbit immunoglobulin antibody conjugated to the enzyme horseradish peroxidase. Dilutions of PRL in plasma-free assay buffer produced accurate and reliable standard curves over a range of 1-500 ng/ml. Plasma or serum samples, however, gave very low absorbance values, suggesting falsely high levels of PRL in the samples. For example, hypophysectomized rat plasma and fetal calf serum gave readings equivalent to over 250 ng/ml rat PRL by ELISA, whereas radioimmunoassay confirmed the expected PRL values of less than 5 ng/ml. This result indicated considerable nonspecific interference by the plasma. Inclusion of 20% or 50% hypophysectomized rat plasma in the assay significantly lowered the absorbance values and the slope of the standard curve (P less than 0.05). Dilution of plasma samples 1:1 and 1:4 with assay buffer caused nonlinear changes in absorbance and resulted in inaccurate ELISA estimates of plasma PRL concentrations compared with radioimmunoassay. Several treatments were attempted to remove the interference from plasma. Preexposure of plasma to 56 degrees C for 30 min, dialysis for 24 hr, fractional precipitation with 5% polyethylene glycol, increased ionic strength of the buffer, preadsorption of plasma with rabbit immunoglobulins bound to a solid phase support, and covalent attachment of the PRL to the wells with 2% glutaraldehyde all failed to remove the interference from the plasma samples in the ELISA. Thus, while the assay worked effectively for plasma-free applications, it was unsuitable for measurement of PRL in plasma or serum samples.

Animals↗