So-called antireceptor sera

Experiments in delayed type hypersensitivity transfer were carried out with the aim of studying the ability of rabbit antisera against peritoneal exudate cells of rats sensitized with bovine gamma globulin or rabbit kidney tissue antigen to block peritoneal exudate cells of guinea pigs. In the serological test the antisera prepared against the cells of sensitized rats and tentatively named "receptor antisera", reacted not only with the cells of these rats, respectively, but also with guinea pig cells. In hypersensitivity transfer experiments in guinea pigs receptor antisera showed a blocking effect on the transferred cells, making them incapable of transferring hypersensitivity, i. e. rabbit antisera against rat peritoneal exudate cells reacted with guinea pig cells. This interaction was specific: the blocking effect was manifested only when guinea pigs whose cells were used in the transfer were sensitized with the same antigen as the rats against whose cells the receptor antisera had been prepared. The control antisera, taken for the treatment of the transferred cells in the same doses as the receptor antisera, had no blocking effect on the cells.     

Multiple hormone storage by cells of the human pituitary

While immunostaining serial semi-thin sections of acrylic resin-embedded normal human pituitary using antisera to human pituitary hormones, it became clear that several cells were stained by more than one antiserum. The tissue had been surgically excised from a patient with a prolactinoma. The tumor, which was immunoreactive only with antiprolactin antiserum, was distinctly different from the pieces of tissue under study which had normal pituitary architecture and demonstrated immunoreactivity with antisera against all six of the common pituitary hormones. A major immunoelectron microscopic investigation, using immunocolloidal gold and immunoperoxidase methods, revealed cells in which follicle-stimulating hormone (FSH), luteinizing hormone (LH), and prolactin (PRL) were co-localized to the same electron-dense granules. Some similar cells also possessed electron-lucent granules immunoreactive only for anti-PRL antiserum. Adrenocorticotrophic hormone (ACTH) and PRL were also found in the same cell but were very largely localized to separate, morphologically different populations of electron-dense and -lucent storage granules. By employing double immunolabeling, a few granules in the ACTH/PRL cells were shown to be immunoreactive to both anti-ACTH and anti-PRL antisera. The possibility that the multipotential stem cells is discussed.     

A modification of the unlabeled antibody enzyme method using heterologous antisera for the light microscopic and ultrastructural localization of insulin, glucagon and growth hormone.

The requirement of using homologous antisera (primary antiserum and peroxidase-antiperoxidase (PAP) complex raised in the same species) in the unlabeled antibody enzyme method has been investigated at the light and electron microscopic level using the localization of insulin, glucagon and growth hormone as model systems. Optimum immunocytochemical staining for all three antigens was observed when sheep or goat antirabbit gamma-globulin (S-ARgammaG or G-ARgammaG) were used to couple rabbit peroxidase-antiperoxidase complex with either guinea pig antisera to insulin (GP-AIS) or glucagon (GP-AGS), or monkey antisera to rat growth hormone (M-ARGH). The cross-reactivity between S-ARgammaG or G-ARgammaG and immunoglobulins in these primary antisera were substantiated by immunoelectrophoresis and radioimmunoassay. S-ARgammaG was shown to produce precipitation arcs with GP-AIS and M-ARGH that were similar to those seen when the latter were reacted with rabbit antiguinea pig gamma-globulin antiserum and goat antimonkey gamma-globulin antiserum, respectively. Radioimmunoassay results revealed that immunoprecipitation of 6-10% as compared to homologous antisera controls yielded excellent staining localization when S-ARgammaG was used for immunocytochemistry. Thus, heterologous antisera (primary antiserum and PAP complex raised in different species) may be used in the unlabeled antibody enzyme method as long as the coupling antiserum shows cross-reactivity with immunoglobulins of the primary antiserum and the PAP complex.     

Further study of the problem of specific cellular receptors in delayed hypersensitivity]

The work presents the materials obtained as a result of the further study of specific T lymphocyte receptors with the use of so-called receptor antisera, i. e. antisera against the lymphoid cells of mice sensitized with one of the two antigens (Macobacterium tuberculosis or bovine gamma globulin); thus these differed from control antisera against the lymphoid cells of intact mice. These mouse antisera reacted with the lymphoid cells of guinea pigs in experiments of delayed-type hypersensitivity transfer. The cells of sensitized guinea pigs lost their ability to transfer hypersensitivity if, prior to their injection into the recipient guinea pigs, these cells were treated with the above-mentioned mouse antisera, i. e. antisera against the lymphoid cells of mice had a blocking effect on the lymphoid cells of guinea pigs. The blocking action of the antisera proved to be specific: antisera against the lymphoid cells of mice sensitized to bovine gamma gloublin blocked the cells of guinea pigs, also sensitized to bovine gamma globulins, but did not block the cells sensitized to Mycobacterium tuberculosis. The control antisera, taken in the same doses as the factor antisera, did not show a blocking effect on the specific activity of lymphoid cells.     

GnRH相关肽在大鼠垂体前叶的细胞学定位

本研究应用特异性抗GnRH相关肽(GAP)N端11个氨基酸的抗血清和六种垂体前叶激素的抗血清,通过免疫组织化学双重染色技术观察GAP在大鼠垂体前叶细胞的定位。结果发现,GAP样免疫反应性物质存在于LH细胞和FSH细胞,而未见于GH、PRL、TSH和ACTH细胞。本文首次证明GAP存在于正常大鼠垂体促性腺激素细胞,为GAP调节LH和FSH的分泌提供了形态学证据;也支持GAP的功能序列在其分子的N端,或GAP进一步裂解出N端片段而发挥作用。     

Immunological relatedness of human and porcine growth hormones.

The immunological relatedness of human and porcine growth hormones is examined by means of labelled human growth hormone and guinea pig antiserum. 1) Labelled human growth hormone is found in the precipitate after reaction with antiserum against porcine growth hormone. Parallel dilution curves are obtained with antisera against human and porcine growth hormones. 2) After addition of antiserum against porcine growth hormone, all the radioactivity is eluted from Sephadex G-100 with the void volume. 3) The addition of an excess of porcine hormone displaces labelled human growth hormone from antibodies against human growth hormone to the same extent as an excess of non-labelled human growth hormone does. 4) The standard radioimmunoprecipitation curves for porcine and human growth hormones obtained in the assay system for the human hormone are parallel in slope, provided that the human hormone and our preparation of the porcine hormone are introduced at a proportion of 1 to 560. 5) In a double diffusion test in agarose gel layers, with human and porcine growth hormones diffusing against guinea pig anti-porcine serum, cross reaction is observed. The conclusion is drawn that with guinea pig antisera, human and porcine growth hormones behave immunologically in a similar fashion. Labelled human growth hormone seems to have only such immunodeterminants as are also found in porcine growth hormone.     

Immunohistochemical Identification of the Adenohypophysial Cells in the Indian Paddy Field Frog,Rana limnocharis

Cells of the pituitary pars distalis (PD) and pars intermedia (PI) in the frog Rana limnocharis have been identified by an unlabelled antibody enzyme method using antisera developed in rabbit against mammalian hypophysial hormones. On the basis of their immunoreactivity, six types of cells, viz. thyrotropic (TSH), gonadotropic (GTH), prolactin (PRL), growth hormone (GH), corticotropic (ACTH) and melanotropic (MSH), cells have been recognized. GTH and PRL cells are distributed throughout the PD. GH cells usually occur in the anterodorsal and central region of the gland. Immunoreactive TSH cells are fewer in number and are localized in the ventromedian region of the PD. Cells showing immunoreactivity to ACTH 1–24 antiserum are encountered in the rostroventral part of the PD. Cells of the PI also show immunoreactivity to ACTH 1–24 antiserum. PI cells cross-react with α-MSH antiserum at all dilutions up to 1: 50 000. However, when the same antiserum was used at dilutions up to 1: 20 000, the ACTH cells of the PD also showed cross-reactivity.     

Isolation and characterization of growth hormone from blue fox pituitary glands

Growth hormone has been purified to homogeneity from blue fox pituitary glands. It has 191 amino acids with two disulfide bridges and a single tryptophan residue. The somatotropin activity is only 8% when compared with the bovine hormone in the receptor-binding assay. From radioimmunoassay data using baboon antisera to porcine or bovine growth hormone, the fox hormone has 14-17% immunoreactivity of bovine or porcine hormone.     

Solid-phase adsorption method for removing undesired antibodies from polyclonal antiserum

We have developed a novel and simple method, requiring only a small amount of antigen, for removal of undesired antibodies from antiserum. The method was established using a well-characterized antiserum against rat luteinizing hormone (anti-rLH). Wells of polystyrene tissue culture plates were coated with rat LH (rLH). Anti-rLH diluted 1:3000 was added to rLH-coated wells and shaken to remove LH antibodies. Control anti-rLH was treated in a similar manner in non-rLH-coated wells. Both antisera were tested by immunocytochemistry on rat pituitaries. Antiserum from rLH-coated wells stained no cells, whereas the control serum stained cells that were morphologically typical of LH cells. The effectiveness of this antibody removal was also confirmed in a modified ELISA. In another experiment, anti-rLH and anti-hTSH beta sera were mixed. The final dilution of both antisera was 1:10,000. Anti-rLH was removed by the purification method described. Completeness of antibody removal was confirmed by a double-immunohistochemical staining of rat pituitary in which sections were first stained by the PAP method and then stained with an immunofluorescence procedure after elution of the first antigen-antibody complex. The mixed antiserum incubated in rLH-coated wells did not stain LH cells. There was no co-localization between the LH immunopositivity demonstrated by an anti-rLH serum using immunofluorescence and cells immunostained with the purified antiserum using the PAP method. As indicated in ELISA, the titer of the TSH beta antiserum was not decreased compared to that of the untreated, mixed control antiserum, and the LH antibodies were eliminated by the treatment. This new purification method has four distinct advantages: (a) antiserum is not treated chemically; (b) it requires only a small amount of antigen compared with the amount required for affinity chromatography; (c) neither the undesired antigen-antibody complex(es) nor an excess amount of antigen is present in the purified antiserum; and (d) removal of undesired antibodies can be monitored by ELISA.     

Evaluation of Fluorescein Isothiocyanate-labeled Whole Antiserum in the Immunofluorescent Identification of Microorganisms

Portions of a whole antiserum to Histoplasma capsulatum were reacted with amounts of fluorescein isothiocyanate (FITC) that ranged from 50 to 400 mug/mg of protein. Portions of the globulin from the same antiserum were reacted with amounts of FITC that ranged from 12.5 to 50 mug of FITC per mg of protein. The globulin conjugates (postlabeled globulins), the whole serum conjugates, and the globulins from the whole serum conjugates (prelabeled globulins) were compared with respect to their fluorescein-protein (F:P) ratios and fluorescent-antibody (FA) activities. The whole serum sample treated with 50 mug of FITC per mg of protein was least reactive in FA tests, and its globulin had the lowest F:P. All other conjugates had globulins with F:P ratios that were considered to be adequate for high FA activity. It was found, however, that the prelabeled globulins were considerably less reactive than the postlabeled globulins or the whole serum conjugates. A larger amount of brightly staining reagent per milliliter of original serum could be obtained from labeled whole serum than from postlabeled globulin. Lissamine-rhodamine conjugated to bovine serum albumin (LRBSA) was evaluated as a counterstain to be used in conjunction with FITC-labeled whole antisera. The counterstain was effective in masking nonspecific FITC fluorescence in Formalin-fixed tissues and in culture smears of fungi. Masking was incomplete in culture smears of a bacterium and in blood smears containing a protozoan.     

Cross-reactivity between human and fish pituitary hormones as demonstrated by immunocytochemistry

Summary In this communication we describe the immunocytochemical cross-reactivity between antisera to various human pituitary hormones and specific hormone producing cell types in the pituitary gland of sexually mature male platyfish (Xiphophorus maculatus). Antisera to human pituitary hormones cross-reacted either with cells known to produce corresponding hormones (or hormone subunits) in the platyfish (e.g., ACTH, prolactin, TSH , LH , FSH , TSH ) or with no pituitary cells at all (e.g., LH , FSH ). The one exception was antiserum to human growth hormone which cross-reacted with MSH and ACTH producing cells. The platyfish pituitary is proposed as a test system for immunocytochemically screening antisera for purity and specificity in order to determine their applicability in particular studies.     

Characterization of an antiserum to guinea pig antithrombin III (AT III). I. Reactivity with guinea pig T lymphocytes

In the course of studies investigating the effects of antisera prepared against a variety of guinea pig proteins on lymphocyte function, a goat antiserum prepared against a guinea pig gamma-globulin preparation was found to react with guinea pig T lymphocytes. This antiserum, serum 592, contained a significant titer of antibodies that were cytotoxic for a subpopulation of lymph node cells and thymocytes, and mitogenic for lymph node T cells. Immunoelectrophoretic analysis and selective absorptions of the antiserum demonstrated that the antigen recognized on thymocytes was also present on an alpha 2 globulin in guinea pig plasma, which, on the basis of physiochemical characteristics and heparin-binding affinity, appeared to be guinea pig antithrombin III (AT III). Although the antiserum was shown to contain antibodies to both protein and carbohydrate determinants on the AT III molecule, studies comparing the effects of 7 M guanidine and periodate oxidation on the antigenicity of the AT III determinant also recognized on the thymocytes indicated that this shared antigenic determinant was carbohydrate in nature. The thymocyte membrane molecule bearing this determinant was also isolated and was found to be a 210,000-dalton macromolecule that was very sensitive to proteolytic and/or autolytic degradation. These data raise the interesting possibility that guinea pig lymphoid cells may have a membrane-associated protease inhibitor related to plasma AT III.     

Polyclonal Antisera to the Individual Neurofilament Triplet Protdins: A Characterization Using ELISA and Immunoblotting

In this article, the preparation and characterization of polyclonal rabbit antisera against the individual polypeptides of bovine neurofilament (68, 150, and 200 kilodaltons) is described. Selected antisera against the 68- and 150-kilodalton neurofilament polypeptides were specific for the corresponding antigen in homogenates of bovine, rat, and human brain as judged by immunoblots. The antisera against the 200-kilodalton neurofilament polypeptide cross-reacted to some extent with the 150-kilodalton neurofilament polypeptide, especially with the human antigen. The most specific antisera were used to develop an enzyme-linked immunosorbent assay (ELISA), and the cross-reactivities between the antisera and the different bovine and rat neurofilament polypeptides were determined. Contrary to the results in the immunoblots, the antiserum against the 200-kilodalton neurofilament polypeptide was subunit-specific, as was the 150-kilodalton antiserum. The 68-kilodalton antiserum displayed a minute cross-reactivity against bovine 150- and 200-kilodalton neurofilaments, but it cross-reacted somewhat more with the rat 150- and 200-kilodalton antigens. Even so, the subunit specificity of the antisera is high enough to enable the development of a quantitative ELISA for determination of the individual bovine or rat neurofilament polypeptides in a mixture. This study is the necessary preparation for such an assay.     

Absorption of antisera for studies on specific enzyme turnover.

Antisera were raised to acetyl-CoA carboxylase and 6-phosphogluconate dehydrogenase from mammary glands of lactating rabbits, and cytochrome oxidase from rat liver. The enzymes were all highly purified but gave rise to multispecific antisera when tested against tissue extracts. Absorption procedures were devised to free the antisera of contaminating antibodies. Antisera to acetyl-CoA carboxylase and cytochrome oxidase were absorbed with fractions discarded during enzyme purification. The antiserum to 6-phospho-gluconate dehydrogenase was absorbed with a tissue extract from an early stage in mammary-gland differentiation. Monospecific antisera are essential for enzyme turnover studies and therefore antisera should be extensively tested and absorbed before use. A general procedure for the absorption of antisera to purified enzymes has been devised on the basis of accepted principles of antisera absorption.     

Immunoelectron microscopic demonstration of pancreatic polypeptide in midgut epithelium of hematophagous dipterans

Midguts of mosquitoes, Aedes aegypti and Anopheles stephensi, and of the tsetse fly, Glossina morsitans morsitans, as well as guinea pig pancreas, were prepared for electron microscopy by using low-temperature embedding in Lowicryl K4M. Rabbit antiserum to bovine pancreatic polypeptide (PP) crossreacted with secretory granules of pancreatic PP-producing cells and of the clear cells in mosquito gut. Rabbit antiserum to human somatostatin crossreacted with the control tissue, guinea pig pancreas D-cells, but not with the mosquito clear cells. None of the antisera used showed a distinct reaction with the endocrine-like cells of tsetse fly midgut. Positive reactions were revealed by gold as electron-dense marker. The gold particles were coated with protein A-gold or goat antibodies to rabbit immunoglobulin.     

An immunohistochemical method for the localization of N-acetylserotonin (NAS) in the central nervous system. Description, validation, and application of the technique

Antisera to N-acetylserotonin (NAS) were raised in rabbits by coupling NAS to bovine serum albumin (BSA) through a p-carboxybenzyl (PCB) bridge at the indole N. The specificity and applicability of these antisera in immunohistochemistry is reported. The anti-NAS antiserum and a fluorescein-labeled immunoglobulin were employed to investigate the topographic distribution of immunoreactive NAS (INAS) in the hindbrain (mesencephalon, cerebellum, pons, and medulla oblongata). Positive identification of INAS was confirmed in the granular layer of the cerebellum, the tractus spinalis nervi trigemini and the reticular formation. INAS was also identified in Purkinje cells, cerebellar nuclei, nucleus principalis nervi trigemini, nucleus tractus mesencephali, cochlear and vestibular nuclei, the locus coeruleus, and other brain stem regions. The pattern of INAS distribution is independent of serotonin (5-HT) and norepinephrine (NE), although certain loci could contain both INAS and serotonin or INAS and norepinephrine.     

Immunohistochemical characterization of pituitary stellate cells in rats

Pituitary stellate cells from the normal adult male rats were immunohistochemically investigated at the light microscopical level by the use of rat TSH-beta, porcine ACTH1-39, porcine ACTH17-39, rat FSH and ovine FSH antisera. They were characterized by the stellate shape and a mimic engulfment of acidophils. In the present study, they were identified to be the ACTH cells but some were TSH cells. Although most of the corticotrophs showed a peripheral fringe immunostained with the porcine ACTH17-39 antiserum, some others were stained diffusively throughout the cytoplasm. The latter cells coincided, in shape and in homogenous stainability of the cytoplasm, with the stellate TSH cells. Both cells did not correspond but were independent in distribution at the same site of the gland on the adjacent two sections. The stellate type of FSH cells could react with the ovine FSH antiserum, but not with the rat FSH antiserum. Absorption tests of the ovine FSH, procine ACTH1-39 and procine ACTH17-39 antisera were carried out by an application of procine ACTH. In consequence, the porcine ACTH)-39 and porcine ACTH17-39 antisera were absorbed efficaciously by the ACTH antigen at the dose of 10 micrograms/ml, but the ovine FSH antiserum was not enough absorbed by ACTH in the doses of less than 1 mg/ml. It was not finally concluded whether or not the single stellate cells produced ACTH and FSH.     

Effect of active and passive immunization with TRH on plasma TSH response to propylthiouracil.

The role of thyrotropin-releasing hormone (TRH) in the secretion of TSH from the anterior pituitary was investigated in rats by active and passive immunization with TRH. The plasma TSH response to propylthiouracil (PTU) in TRH-bovine serum albumin (BSA)-immunized rats was significantly lower than that of BSA-immunized or non-immunized rats. Similarly, the increased plasma TSH level following PTU treatment was significantly suppressed after iv injection of antiserum to TRH. However, the decline in plasma TSH levels was not complete. The results of the present study indicate, at least in part, the physiological significance of endogenous TRH in the regulation of pituitary TSH secretion.     

Immunohistochemical identification of cells in the pars distalis of the pituitary of the lizard Anolis carolinensis

Immunocharacteristics of the pars distalis cells of the pituitary of the male lizard A. carolinensis are determined by employing the immunoperoxidase technique with antisera to mammalian pituitary hormones. On the basis of their immunoreactivity, 5 different cell types with characteristic anatomical distribution are recognized. ACTH cells are found in the rostral half of the pars distalis, and PRL cells in the rostral two thirds of the pars distalis. GH and TSH cells are located in the caudal half of the pars distalis. GTH cells are distributed throughout the gland. When consecutive sections are stained with antiserum to ovine FSH or its beta-subunit and to ovine LH, the same cells show immunoreactivity to all the three antisera. None of the GTH cells show positive immunoreactivity to ovine beta-LH antiserum. The results suggest the existence of one gonadotropic cell type in the pituitary of this lizard.