RESUMO
The advantages offered by a mouse IgG1 monoclonal antibody to human alpha-foetoprotein (AFP) for the preparation of [125I]antibody for use in an immunoradiometric assay (IRMA) have been investigated. The antibody was isolated from ascites fluid by sodium sulphate precipitation followed by gel filtration on Sephadex G-200. The freeze-dried powder and solutions thereof were stable and were used for iodination to 1 atom 125I/molecule antibody by the chloramine-T procedure. At high antigen concentrations 70-80% of the added [125I]Ab was present in the sandwich. Linear response curves in the range 1-100 micrograms antigen/l incubate were obtained when [125I]Ab was in slight excess. In this region an Ag : Ab ratio of 1.9 : 1 was obtained which is consistent with the saturation of a bifunctional antibody. Although non-specific binding (in the absence of antigen) was consistently less than 0.1% of added [125I]Ab, this was the main factor in determining assay detection limits. The serum AFP levels from both non-pregnant and pregnant subjects as measured by the IRMA using the [125I]monoclonal Ab and by radioimmunoassay (RIA) using a sheep antiserum to AFP were in excellent agreement. The IRMA was manipulatively simple, employed a shorter incubation time (2 h), required shorter counting times than the RIA and gave a much wider working range. The provision of a monoclonal antibody for labelling removes the one major practicability barrier which otherwise limits the development and use of the potentially superior IRMA system.
Assuntos
Anticorpos Monoclonais/imunologia , alfa-Fetoproteínas/análise , Animais , Líquido Ascítico/análise , Feminino , Humanos , Imunoensaio/métodos , Masculino , Camundongos , Radioimunoensaio , OvinosRESUMO
Serum and plasma from human and domestic animals contain variable amounts of non-specific material(s) which may be mistaken for hormone in assays for human LH and FSH, based upon antisera of high sensitivity and hormonal monospecificity. The non-specific response curves are generally, but not invariably, less steep than those of the hormone standards and endogenous homologous hormones. The levels of non-specific intrusion can be of sufficient magnitude to obscure specific estimations seriously, particularly at low hormone levels, unless the assays are designed to minimize this effect. The non-specific effects could be minimized (but not abolished) by careful optimization of the assays which involved making the response curve as sensitive as possible and incorporating the serum at a final dilution of 1 : 2, since further dilution increased the relative contribution of the non-specific substance(s). The optimized assays require only 48 h of total incubation and show a sevenfold increase in the mean concentration of LH between sera from prepubertal children and adults accompanied by a mean threefold difference in the concentration of FSH.
Assuntos
Hormônio Foliculoestimulante/sangue , Hormônio Luteinizante/sangue , Adulto , Animais , Criança , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Radioimunoensaio/métodos , Radioimunoensaio/normas , Temperatura , Fatores de TempoRESUMO
Serotonin (5-HT) plays a role in mediating the oestradiol-induced surge of luteinising hormone (LH), but so far the 5-HT receptor subtype involved has not been identified. Our previous in-situ hybridization and pharmacological studies suggest that the action of 5-HT involves the 5-HT2A receptor. The aim of the present study was to investigate this possibility by the direct approach of determining whether 5-HT2A receptor antagonists block the oestradiol-induced surge of luteinising hormone releasing hormone (LHRH). Adult female Wistar rats, which had shown at least two consecutive 4-day oestrous cycles, were ovariectomised under halothane anaesthesia in the morning of dioestrus and injected with vehicle (arachis oil) alone or oestradiol benzoate (OB). At 12.00 h of the next day, presumptive pro-oestrus, the animals were injected intraperitoneally with one of three 5-HT2A antagonists, a selective 5-HT reuptake inhibitor (fluoxetine), or the appropriate vehicles; hypophysial portal blood was then collected under alphaxalone anaesthesia between 15.00 and 19.00 h. The amount of LHRH released into hypophysial portal blood during consecutive 30-min periods was determined by radioimmunoassay. As expected, oestradiol, but not oil, triggered a surge of LHRH in hypophysial portal blood with a peak at about 16.00 h of presumptive pro-oestrus. This oestradiol-induced surge of LHRH was blocked by ketanserin, ritanserin and the highly selective 5-HT2A receptor antagonist, RP62203, but not by fluoxetine. These results provide the first direct evidence that the 5-HT2A receptor plays an important role in the oestradiol-induced surge of LHRH.
Assuntos
Estradiol/farmacologia , Hormônio Liberador de Gonadotropina/metabolismo , Receptores de Serotonina/fisiologia , Anestesia , Anestésicos , Animais , Óxidos S-Cíclicos/farmacologia , Diestro , Feminino , Fluoxetina/farmacologia , Hormônio Liberador de Gonadotropina/sangue , Ketanserina/farmacologia , Naftalenos/farmacologia , Ovariectomia , Hipófise/irrigação sanguínea , Pregnanodionas , Proestro , Ratos , Ratos Wistar , Receptor 5-HT2A de Serotonina , Ritanserina/farmacologia , Antagonistas da Serotonina/farmacologia , Inibidores Seletivos de Recaptação de Serotonina/farmacologiaRESUMO
Neuropeptides, including substance P (SP), calcitonin gene-related peptide (CGRP) and somatostatin (SS) in dorsal root ganglia (DRG) may play a role in neurogenic inflammation and pain transmission. Adrenal corticosteroids regulate neuropeptide synthesis in some areas of the CNS and may modulate neurogenic inflammation and sensory perception. We have investigated the effects of adrenalectomy and dexamethasone (0.2 mg/kg/day) treatment on neuropeptide content of rat cervical DRG using specific and sensitive radioimmunoassays. In control animals, a differential distribution of neuropeptide was found; SP and CGRP content increased from C4 to C7 in contrast to SS content, which decreased from C4 to C7. Ten days following adrenalectomy, the mean SS content of cervical DRG decreased significantly to 79.6 +/- 4.5% of sham-operated controls. In contrast, SP and CGRP content increased significantly 10 days after adrenalectomy to 134.6 +/- 6.9% and 132.0 +/- 11.6% of sham-operated controls, respectively. The effects of adrenalectomy on CGRP and SS were reversed by administration of dexamethasone. These results suggest that glucocorticoids affect the neuropeptide content of DRG in the adult rat.
Assuntos
Adrenalectomia , Dexametasona/farmacologia , Gânglios Espinais/metabolismo , Neuropeptídeos/metabolismo , Hormônio Adrenocorticotrópico/sangue , Animais , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Corticosterona/sangue , Gânglios Espinais/efeitos dos fármacos , Masculino , RNA Mensageiro/metabolismo , Radioimunoensaio , Ratos , Ratos Endogâmicos , Receptores de Glucocorticoides/biossíntese , Receptores de Glucocorticoides/efeitos dos fármacos , Somatostatina/metabolismo , Substância P/metabolismoRESUMO
Neurotensin immunoreactivity and choline acetyltransferase (ChAT) activity were measured in post-mortem brain from 10 cases of Down's syndrome (7 aged 53-63 years, one aged 27 years, one aged 16 months and one aged 10 months), 6 cases of Alzheimer-type dementia (ATD) and 19 control subjects (13 aged 40-88 years and 6 aged 9-18 months). Neurotensin concentrations in anterior and basal hypothalamus, amygdala, septal area, caudate nucleus and temporal cortex were unaltered in ATD. The concentrations of neurotensin were significantly increased in the caudate nucleus, temporal cortex and frontal cortex in the cases of Down's syndrome aged 53-63 years with the neuropathological features of ATD, and were also increased in the cerebral cortex of the 27-year-old, which did not have the neuropathological features of ATD, and in two infant Down's cases. ChAT activity was reduced in the ATD and the 53-63-year-old cases of Down's syndrome, but not in the 27-year or 10-month-old Down's cases. The increased neurotensin concentrations appear to be a feature of Down's syndrome not related to the presence of plaques and neurofibrillary tangles or to a deficit in ChAT activity.
Assuntos
Doença de Alzheimer/metabolismo , Química Encefálica , Síndrome de Down/metabolismo , Neurotensina/análise , Adulto , Idoso , Tonsila do Cerebelo/análise , Núcleo Caudado/análise , Lobo Frontal/análise , Humanos , Hipotálamo/análise , Pessoa de Meia-Idade , Radioimunoensaio , Septo Pelúcido/análise , Lobo Temporal/análiseRESUMO
An immunoradiometric assay (IRMA) for h-LH based upon an 125I-labelled mouse monoclonal antibody (MAb) to h-LH with an effective equilibrium constant of 5.8 X 10(9) l/mol is described. A total incubation time of 3 h at room temperature was required, separation by means of the sucrose layering procedure took a further 1 h and counting times were 1 min/tube. Using the first IRP for h-LH (prep. 68/40) as standard, the detection limit was 0.1 U/l serum and the within-assay CV for duplicate determinations was less than 10% over the range 1-280 U/l and less than 3% at 10-100 U/l. The epitope, with which the MAb reacted, shared structures, on the alpha- and beta-subunits of LH since the assay responded to the intact hormone, but not to either of the subunits. Specificity was greater than 100 000:1 for h-LH vs. h-FSH and greater than 10 000:1 for h-LH vs. h-TSH. h-CG and h-LH were approximately equipotent. The results on 604 unselected samples were generally very similar to those found by RIA except at levels below 2 U/l for which the IRMA regularly gave lower results suggesting relative freedom from non-specific serum effects. The new assay, based upon potentially limitless supplies of a very stable reagent offers advantages of speed, sensitivity, range, and precision over conventional RIA. The specificity appears to be excellent. Although there are marginally more steps the total staff involvement is less than with conventional methods employing centrifugation.
Assuntos
Hormônio Luteinizante/sangue , Animais , Anticorpos Monoclonais , Gonadotropina Coriônica/imunologia , Humanos , Concentração de Íons de Hidrogênio , Radioisótopos do Iodo , Radioimunoensaio/métodosRESUMO
The stability of the International Reference Preparation of human TSH for immunoassay (previously distributed in ampoules coded 68/38) and of Research Standard A for HTSH has been examined by radioimmunoassay of accelerated degradation samples (stored as the dry ampouled preparations). Potency estimates on degradation samples suggest a high order of stability of immunopotency recognisable by two antisera (MRC 72/356 and Hunter 3A). For the International Reference Preparation the loss of immunopotency during storage at -20 degrees C is predicted to have been 0.56% per year.
Assuntos
Radioimunoensaio , Tireotropina/análise , Estabilidade de Medicamentos , Armazenamento de Medicamentos , Humanos , Soros Imunes , Temperatura , Tireotropina/imunologia , Tireotropina/normasRESUMO
The hypothesis that ECT produces selective effects on hypothalamic-pituitary activity was investigated by determining the effect of ECT on pituitary hormone release in nine depressed patients. After ECT there were massive and rapid increases in the plasma concentrations of nicotine- and oestrogen-stimulated neurophysin (NSN and ESN), prolactin (PRL) and adrenocorticotropin (ACTH), smaller increases in plasma luteinizing hormone (LH) and cortisol, a significant decrease in plasma growth hormone (GH) concentration but no change in plasma thyrotropin (TSH). There was significant attenuation of PRL responses with repeated ECT. The hormonal responses to ECT cannot simply be attributed to stress, since a similar pattern of increases in plasma hormone concentrations did not occur in psychologically normal patients in whom plasma hormone concentrations were measured during induction of anaesthesia and abdominal incision for cholecystectomy. Analysis of these hormonal responses in terms of the knowledge available on the neurotransmitter control of pituitary hormone release suggests that some of these hormonal responses to ECT may be mediated by the activation of serotonergic neurones, while others are probably due to direct stimulation of the neuroendocrine neurones themselves.