Unlabeled Antibody Method Research Articles

Detection of estrogen receptor (ER) in the rat brain using rat anti-ER monoclonal IgG with the unlabeled antibody method

Application of Sternberger's unlabeled antibody enzyme method for detection of the estrogen receptor (ER) using a rat primary antibody with rat tissues has been discouraged, presumably because nonspecific staining of endogenous IgG was expected with the required anti-rat IgG bridging antibody. Because the blood-brain barrier greatly reduces immunoglobulin infiltration into the brain, we hypothesized that rat brain tissue could be specifically immunostained using rat IgG primary antibodies. A rat monoclonal anti-ER antibody (H222) specifically stained ERs in the brains of ovariectomized but not in ovariectomized estrogen-treated rats. In contrast, the uterus, a well-perfused target organ stained intensely in a nonspecific fashion. Dense populations of estrogen receptors were observed in the medial preoptic area, the bed nucleus of the stria terminalis, and the arcuate and ventromedial nuclei of the hypothalamus. A monoclonal rat IgG directed against alpha-tubulin labeled primarily cortical dendrites quite distinct from the neuronal nuclei that are the primary antigenic sites for the estrogen receptor antibody. These results confirm that the sensitive unlabeled antibody method can be applied to rat brain tissues, even when the primary antibody is rat IgG and that labeling of endogenous IgG may be used as a simple method to evaluate the integrity of the blood brain barrier.

The use of peroxidase anti-peroxidase (PAP) complexes in the detection of plant viruses by ELISA

An ELISA test system for detection of plant viruses in field samples is described, based on the unlabelled antibody method using a peroxidase-antiperoxidase (PAP) complex. Novel features of the system include the use of acid-treated naked bacteria as combined carrier-adjuvants for the production of rabbit antiviral antibodies, and the use of acid-treated chicken antibodies (IgY) for antigen trapping in the ELISA. Systems for detection of potato virus Y (PVY), potato leafroll virus (PLRV), grapevine fanleaf virus (GFV) and grapevine virus A (GVA) were developed and compared with conventional direct double antibody sandwich (DAS) systems in tests with both purified virus and field samples. The PAP systems offer improved sensitivity, no background problems in the outer rows of the microtitre plates and are much easier to visualize with the naked eye if no plate reader is available.

The substitution of glucose-antiglucose oxidase complex (GAG) for peroxidase-antiperoxidase (PAP) in immunohistochemical studies of skin.

The substitution of glucose-antiglucose oxidase complex (GAG) for peroxidase antiperoxidase (PAP) in the Sternberger three-layer unlabeled antibody method avoids the problems associated with the use of the chromogen 3,3,diaminobenzidine (DAB). The blue final reaction product obtained using the GAG complex is not obscured by melanin granules, which is often the case when using the PAP-DAB combination. The replacement of PAP with GAG complexes facilitates the detection of melanocytes in melanoma and blue nevi. The absence of endogenous glucose oxidase in mammals provides for greater image contrast, which is not possible using alternative chromogens and immunoperoxidase.

Immunocytochemical demonstration of pituitary cell types in the teleost Poecilia latipinna, by light and electron microscopy

Using the unlabelled antibody method at the light microscope level, and the immunogold method at the electron microscope level, the distribution of the different adenohypophysial cells was demonstrated in the teleost Poecilia latipinna, by means of antisera to both teleostean and mammalian pituitary hormones and their subunits. Anti-salmon prolactin, but not anti-rat or-ovine prolactin, gave a specific staining of the acidophils of the rostral pars distalis (RPD), while anti-trout growth hormone (GH), but not anti-rat GH, stained similar but always separate cells in the proximal pars distalis (PPD). Antisera to the whole molecules of mammalian glycoprotein hormones stained the entire population of basophils in the PPD, but separate populations of gonadotrophs and thyrotrophs could be discriminated using anti-salmon gonadotrophin and anti-human thyrotrophin β subunit. Antisera to ACTH (1–24) and (11–24) sequences, as well as β-endorphin and met-enkephalin, stained the lead haematoxylin-positive cells of the RPD and pars intermedia (PI), whereas anti-α-MSH stained only the PI cells. Ultrastructural examination showed that these immunoreactivities were present in the same secretory granules, and were always greater in pale granules rather than electron dense granules. In the RPD, blebs of ACTH-immunoreactive cytoplasm were found to protrude through the gaps in the basement membrane into the neurohypophysis. The second “PAS-positive” cell type of the PI showed a strong cross-reaction with anti-salmon gonadotrophin, suggesting that it may produce a glycoprotein chemically related to the gonadotrophin(s).

Renin–containing cells in kidney transplants into the anterior eye chamber

As a part of experiments aimed at the study of the effects of denervation on the development of the renin—containing juxtaglomerular epithelioid granular cells (JEG-cells), pieces of fetal rat kidneys were transplanted to the anterior eye chamber of adult animals. These allografts survived and organized an organotypic tissue, with the exception that the renin—containing cells significantly increased in number. The implantations were performed on 15 adult female Wistar (Ivanovas GMBH, BRD) or ZUR/Siv (Zuchthygiene, Zurich, Switzerland) rats anesthetized with 0.1 mliter Nembutal and 0.1 muter Ketalar i.p. and pretreated with a drop of 1% Atropin— sulfate on the cornea [1]. The kidney tissue was derived from 17 to 19 day old fetuses, sacrificed in utero by an overdose of Nembutal to the mother. The kidney was removed with heat—sterilized instruments and cut in small pieces in RPMI1640 cell culture medium supplemented with 10% fetal calf serum. The tissue pieces (kidney cortex or medulla alone and cortex and medulla together; approximately 3 x I x 1 mm) were aspirated in the tip of a Pasteur—pipette and inserted on the iris of the host eye through an incision of the cornea. The animals recovered rapidly and did not show any impairment due to the intervention or the implant. After various survival times (14 days to eight months) the rats were anesthetized again and perfused through the left ventricle with Bouin—fixative. The whole eye with the implanted kidney was dissected out, dehydrated, embedded in paraplast and cut with a microtome. Adjacent, 5 jm thick sections through the kidney implant were stained with hematoxylin—eosin (HE) or incubated with antibodies against rat—kidney renin and angiotensin 11. The immunohistochemistry was performed according to the unlabeled antibody method as described earlier [21. Approximately 80% of the implants survived. They were visible by naked eye as white spots on the anterior surface of the red iris of the host animal. The size of the implanted piece of tissue increased only slightly from the original one. In a few cases, the implant adhered to the cornea without provoking particular reactions. In HE-stained sections, the implants displayed morphologies always reminiscent of a normal kidney, but mostly fairly disorganized and immature (Fig. IA, 3). The kidney implant was supplied by vessels of the iris and was Fig. JA. HE-stained longitudinal section through the eye of an adult female rat in which a piece of fetal rat kidney cortex was implanted for /4 days. The implant grew on the iris (IR) and adhered to the cornea (C). The blood vessels to the implant are derived from iris capillaries (arrow). x 76. B. Consecutive section to IA incubated with antisera against rat—kidney renin. A dilated longitudinally cut (arrow) and two cross—cut (arrowheads) blood vessels contain renin—immunoreactive cells in their walls. In the former the immunoreactivity can be followed over an unusually long distance. x 76. C. Enlargement of Fig. I B. The abundant renin immunoreactive cells (arrowheads) belonging to a vessel contacting the glomerulus (G) is overwhelming. x 300.

The unlabeled antibody method: comparison of peroxidase-antiperoxidase with avidin-biotin complex by a new method of quantification.

Upon plotting of areas against optical densities in immunocytochemically stained tissue sections, hyperbolic curves were obtained which could be reduced to two straight lines, one representing variations in stained structures, and the other variations in background. The slopes of the stained structure lines reflected staining intensity independently of total area of stained structure in a section. The ratio of slopes of the stained structure and background lines reflected immunocytochemical sensitivity. A comparison of the peroxidase-antiperoxidase (PAP) method with the avidin-biotin complex (ABC) method showed that at usual antibody dilutions the PAP method was much more sensitive than the ABC method, while at impractically high antibody dilutions it was moderately more sensitive. Once sufficient dilutions of antibodies were reached, staining intensities dropped sharply with the PAP method. On the other hand, the dilution curves were flat with the ABC method. The ABC method consequently appeared unsuitable for estimating variations in concentration of antigen or for distinguishing high or low concentrations of antigen. The ABC method provided a stain for myelin even in the absence of any antibodies.

Immunoreactivity of vasopressin and a novel pituitary protein ‘7B2’ in long-evans and brattleboro rat hypothalamus and hypophysis

The unlabeled antibody (peroxidase-anti-peroxidase) method was used to simultaneously localize vasopressin and a novel pituitary protein designated ‘7B2’ in rat hypothalamus and pituitary. Results showed the common localization of both substances within magnocellular neurons of supraoptic, supraoptic retrochiasmic and paraventricular nuclei. The distribution was also similar in the inner zone of the median eminence and in the posterior lobe of the pituitary gland. Only 7B2 antiserum labeled the external zone of the median eminence and the intermediate and anterior lobes of the pituitary. In the Brattleboro rat the anterior and intermediate lobes were strongly labeled with 7B2-IR and there was some 7B2-staining in the hypothalamus and the posterior lobe, but the intensity of the reaction was diminished.

Improved immunocytochemical staining through the use of Fab fragments of primary antibody, Fab-specific second antibody, and Fab-horseradish peroxidase.

A modification of the unlabeled antibody method of immunocytochemistry is described here that offers increased immunoreagent penetration and greatly reduced background staining. The method involves the following alterations to the conventional technique; the use of Fab fragments of primary antibody, rather than whole immunoglobulin G (IgG) or serum; the use of a second, or link, anti-rabbit IgG serum that is Fab fragment-specific, rather than directed against the whole rabbit IgG molecule; the use of the Fab--horseradish peroxidase complex described by JR Slemmon, PM Salvaterra, and K Saito (J Histochem Cytochem 28:10, 1980), rather than peroxidase--antiperoxidase (PAP). Steps 2 and 3 alone brought about a significant reduction in background staining, but did not increase the depth of immunostaining, as compared to the PAP technique. When all three steps were combined, however, background staining was further reduced, and there was a five- to tenfold increase in the depth of immunostaining. These readily made changes should be useful in preembedding immunocytochemistry whenever enhanced reagent penetration is required.

Species-specific second antibodies reduce spurious staining in immunocytochemistry.

Spurious staining related to the second (linking) antibodies was observed in immunocytochemical specimens processed with an unlabeled antibody method. Some of this staining was suspected to result from species cross-reactivity of the second antibodies with endogenous immunoglobulin Gs in the tissue. Therefore, species-specific second antibodies were obtained, and the staining patterns of tissue processed with such antibodies were compared with those of tissue processed with standard (nonspecies-specific) second antibodies. In these studies, a monoclonal antibody to choline acetyltransferase (ChAT) was utilized as the primary antibody, and a similarly prepared monoclonal antibody that did not react with ChAT served as a control antibody. Spurious staining that included staining of discrete tissue and cellular components as well as amorphous background staining was present in both control and experimental tissue processed with standard second antibodies. Such staining was virtually eliminated in tissue processed with species-specific second antibodies. In specimens from the central nervous system, for example, species-specific second antibodies greatly reduced dark staining within the area postrema, in the pia-arachnoid membranes, and around blood vessels as well as the staining of small dot-like structures within some large neurons. In addition, the general level of background staining was reduced in both adult and developing tissues, thus permitting clearer visualization of many positively stained structures. In peripheral tissues such as skeletal muscle, spurious staining of connective tissue elements was eliminated, allowing the observation of previously occluded ChAT-positive structures such as nerve fibers and motor end-plates. Thus, species-specific second antibodies appear to be very useful for immunocytochemistry, particularly when the primary antibody and the tissue to be studied are from closely related species.

The Use of Antiavidin Antibody and Avidin-Biotin-Peroxidase Complex in Immunoperoxidase Technics

Avidin has an extraordinary affinity for the small-molecule vitamin biotin. Covalently coupling biotin or avidin to peroxidase molecules does not interfere with their normal biochemical functions. The avidin or biotin molecules, either peroxidase conjugated or unconjugated, can be brought to the antigen sites by means of an antiavidin antibody. Several immunohistochemical staining technics based on this principal have been described. The method utilizing an avidin-biotin-peroxidase complex was found to be more sensitive than the unlabeled antibody (PAP) method. This method involved four sequential staining procedures: (1) primary antibody (goat anti-human antigen); (2) secondary antibody (rabbit antigoat IgG) added in relative excess; (3) goat antiavidin antibody; (4) avidin-biotin-peroxidase complex. The applications of this technic are discussed.

Identification of erythrocytes in intervillous thrombi: A study using immunoperoxidase identification of hemoglobins

The origin of the erythrocytes in intervillous thrombi has never been definitively established. Fetal hemoglobin-containing erythrocytes were identified in histologic sections of intervillous thrombi using the peroxidase-antiperoxidase unlabeled antibody method. In 85 per cent of intervillous thrombi containing well-preserved erythrocytes, fetal hemoglobin-containing cells were found in quantities well in excess of the number seen in maternal blood. This definitely demonstrates the presence of erythrocytes of fetal origin in intervillous thrombi. Possible pathogenic mechanisms of intervillous thrombosis and fetal-maternal hemorrhage are discussed.

An unlabeled antibody method using glucose oxidase-antiglucose oxidase complexes (GAG): a sensitive alternative to immunoperoxidase for the detection of tissue antigens.

Immunoenzyme staining for tissue antigens using glucose oxidase as marker enzyme was compared with immunoperoxidase techniques. Both the indirect, conjugated antibody method and the unlabeled antibody procedure employing preformed complexes of glucose oxidase-antiglucose oxidase (GAG) and peroxidase-antiperoxidase (PAP) were used to stain carcinoembryonic antigen (CEA) in paraffin tissue sections. The localization of CEA within a tissue specimen was the same in all cases, and background staining was minimal. The percentage of positive specimens detected with GAG and PAP was similar but was slightly greater with glucose oxidase compared to peroxidase conjugates. The glucose oxidase conjugates and GAG were similar to the comparable immunoperoxidase reagents in enzyme-antibody molar ratio, retention of antibody and enzyme activity, immunohistochemical staining dilutions, and stability. Immunoglucose oxidase and immunoperoxidase were combined to localize CEA and colon-specific antigen-p simultaneously. Excellent contrast and staining separation were shown between the enzymatic reaction products of the two systems. Since immunoglucose oxidase methods are as sensitive as the comparable immunoperoxidase techniques, they should be considered as a reliable alternative to the latter, especially when endogenous peroxidase activity may be a problem. Furthermore, the two methods can be conveniently combined for the simultaneous detection of two antigens.

Protein A, avidin, and biotin in immunohistochemistry.

Conjugated and unlabeled peroxidase antibody methods have proven to be quite satisfactory in localizing sites of antigen-antibody reaction. The use of avidin-biotin-peroxidase complex (ABC), as well as protein A, can contribute significantly to the field of immunohistochemistry. The sensitivity and specificity of several immunohistochemical methods is compared. In general, the ABC method produced the most intense staining and the least background staining of any method tested. The unlabeled antibody (peroxidase-antiperoxidase: PAP) method also yielded satisfactory results, but it was less intense than the ABC method. In comparison to the PAP method, the indirect conjugated method presented slightly inferior staining intensities and significantly higher background staining. Protein A techniques produced a range of staining sensitivities similar to or inferior to the PAP technique. The main disadvantage in using protein A is that it reacts with intrinsic immunoglobulin (Ig) G, thus producing an intense background. Therefore, its use is not recommended on tissues that have either abundant immunoglobulins in their interstitium or numerous IgG-containing plasma cells.

Determination of brain enolase isozymes with an enzyme immunoassay at the level of single neurons.

Ultrasensitive enzyme immunoassay systems for the assay of rat brain enolase isozymes (alpha alpha, alpha gamma, and gamma gamma forms) were prepared by use of beta-D-galactosidase from Escherichia coli as label and the purified rabbit antibodies to alpha alpha and gamma gamma enolases. The antibodies were purified from the immunoglobulin G (IgG) fractions of antisera by immunoaffinity chromatography with a column of the corresponding antigen-coupled Sepharose. Sandwich-type immunoassay systems with the galactosidase-labeled antibody Fab' fragments and the antibody F(ab')2-immobilized polystyrene beads could determine amounts as small as 1 amol (10(-18) mol) of each isozyme. Purkinje cell bodies picked up from the bulk-separated fraction by means of a nylon loop were subjected to the assay at the level of single cells. In contrast to previous report, this neuron contained not only the gamma gamma but also the alpha gamma and alpha alpha enolases at a level of amol per cell body, although the concentration of gamma gamma was the highest. Immunohistochemical experiments on te cerebellum with the peroxidase-labeled antirabbit IgG antibody and the unlabeled antibody method confirmed the above results, and indicated that both alpha and gamma subunits of hte enolase were stained intensely in axons.

Immunocytochemical localization of GABAergic neurones at the electron microscopical level.

Antibodies prepared to purified brain glutamic acid decarboxylase (GAD), the synthesizing enzyme for the neurotrasmitter, γ-aminobutyric, acid (GABA), have been utilized with an unlabelled antibody method to localize GABAergic neurones in both light and electron microscopic preparations. A modification of Sternberger's peroxidase-antiperoxidase (PAP) complex is used to localize the site of anti-GAD binding, and the PAP complex is visualized with diaminobenzidine and H2O2. The reaction product is visible in both the light and electron microscopes. The ability to localize and identify labelled profiles in the electron microscope provides more functional information than light microscopical preparations. For example, the GAD-positive reaction product occurs mostly in association with synaptic vesicles within axon terminats, and this localization indicates the importance of GAD for the packaging and storage of GABA. The somata and dendrites of neurones giving rise to these terminals are visualized in colchicine-injected material. The GABAergic neurones form axo-somatic, axo-dendritic, axo-axonal and dendro-dendritic synapses in various regions of the rat central nervous system. Pretreatments of animals with anterograde degeneration have shown the significance of some of the GABAergic terminals that form axo-axonal synapses in the spinal cord. An many brain regions, such as the cerebral cortex, hippocampus and olfactory bulb, virtually all of the GABAergic synapses are derived from local circuit neurones. In other regions such as the cerebellum and neostriatum, the GABAergic terminals are derived from both local circuit neurones and the local axon collaterals of projection neurones that have their somata within these regions. A third type of configuration of GABAergic terminals occurs in the globus pallidus and substantia nigra where these terminals are derived from distant brain regions, axon collaterals of projection neurones and from local circuit neurones. Together, these results indicate the complex organization of the GABAergic system of the brain that has been vividly revealed with electron in croscopical immunocytochemistry.

Localization of lipophosphonoglycan in membranes of Acanthamoeba by using specific antibodies.

Antibodies against two electrophoretically distinct forms of lipophosphonoglycan (LPG) were produced in rabbits. Antibody specificity was demonstrated by the coupled antibody 125I-protein A assay (Adair et al., J. Cell Biol. 79:281-285, 1978). Indirect immunofluorescent labeling of intact Acanthamoeba showed that antibodies to both LPG components had the same uniform distribution on the cell surface. Both antibodies also bound to the cytoplasmic surface of isolated phagosomes. The location of LPG in other membranes of the amoeba was demonstrated on sections by the unlabeled antibody method. Although LPG was absent from the nuclear membrane, virtually all of the internal vacuole membranes were labeled, including the contractile vacuole. Antibodies directed against LPG were utilized to label lipophosphonoglycan in the plasma membrane of living amoebae. Labeled membrane was internalized and then localized by immunofluorescence in cytoplasmic vacuoles within 10 min of incubation. Although these results are evidence for exchange between plasma and cytoplasmic vacuolar membranes, the contractile vacuole remained unlabeled and can be considered, therefore, a separate membrane compartment. Concanavalin A also was bound and internalized by the amoeba, but electron microscopy showed that this label caused pronounced membrane perturbation, limiting its usefulness as a membrane marker in this system.

Localization of lipophosphonoglycan in membranes of Acanthamoeba by using specific antibodies.

Antibodies against two electrophoretically distinct forms of lipophosphonoglycan (LPG) were produced in rabbits. Antibody specificity was demonstrated by the coupled antibody 125I-protein A assay (Adair et al., J. Cell Biol. 79:281-285, 1978). Indirect immunofluorescent labeling of intact Acanthamoeba showed that antibodies to both LPG components had the same uniform distribution on the cell surface. Both antibodies also bound to the cytoplasmic surface of isolated phagosomes. The location of LPG in other membranes of the amoeba was demonstrated on sections by the unlabeled antibody method. Although LPG was absent from the nuclear membrane, virtually all of the internal vacuole membranes were labeled, including the contractile vacuole. Antibodies directed against LPG were utilized to label lipophosphonoglycan in the plasma membrane of living amoebae. Labeled membrane was internalized and then localized by immunofluorescence in cytoplasmic vacuoles within 10 min of incubation. Although these results are evidence for exchange between plasma and cytoplasmic vacuolar membranes, the contractile vacuole remained unlabeled and can be considered, therefore, a separate membrane compartment. Concanavalin A also was bound and internalized by the amoeba, but electron microscopy showed that this label caused pronounced membrane perturbation, limiting its usefulness as a membrane marker in this system.

Immunohistochemical localization of dipeptidyl aminopeptidase (DAP) IV in the rat endocrine organs.

Immunohistochemical localization of dipeptidyl aminopeptidase (DAP) IV in the rat endocrine organs was studied using the unlabeled antibody method. Fresh frozen, chloroform-acetone treated sections of the endocrine organs (pineal, pituitary, thyroid, parathyroid, adrenal gland and pancreas) were investigated for the present study.A fairly intense immunoreaction of DAP IV was detected in the capillary endothelium of the thyroid gland and adrenal cortex. The capillary endothelium of the pineal, pituitary, parathyroid gland and pancreatic islets showed faint immunoreaction. No immunoreaction could be seen in the parenchymal cells of these endocrine organs except for the follicular epithelial cells of the thyroid gland.These results indicated that DAP IV in the endocrine organs was restricted to the capillary endothelium, and showed organ-dependent differences. Especially, it seemed that DAP IV in the capillary endothelium might be of importance in the thyroid and adrenal glands.

Immunohistochemical demonstration of viral antigens in paraffin embedded autopsy specimens of virally infected central nervous system.

Viral antigens are preserved in routinely processed and paraffin embedded CNS tissue to an extent that they can be traced by the highly sensitive and specific unlabeled antibody method. Using this method, it was possible to visualize antigens of measles, influenza A, polio, varicella-zoster, herpes simplex, cytomegalo, parainfluenza I, lymphocytic choriomeningitis, Moloney, and Friend virus.

Light and electron microscopic observation on the appearance of immunoreactive LHRH in perinatal rat hypothalamus.

The appearance and localization of LHRH were studied in the developing hypothalamus of perinatal rats using the unlabelled antibody method. By light microscopy, immunoreactive LHRH was first detected as brown dots on day 18.5 of gestation in the OVLT and on day 19.5 in the median eminence, respectively. When the median eminence was examined by the preembedding immunohistochemistry technique for electron microscopy, the occurrence of immunoreactive LHRH fibers could be demonstrated on day 18.5. These fibers were thin and very occasionally encountered near the surface of the lateral regions of the median eminence. The axoplasm contained a few immunopositive secretory granules and also extragranular immunoreactive products. With development, a gradual increase was noted both in number and size of nerve fibers with a concomitant accumulation of secretory granules within the axoplasm. A possible physiological significance of LHRH is discussed in relation to the onset of hypothalamo-hypophysial system in fetal life.