A monoclonal antibody that defines rostrocaudal gradients in the mammalian nervous system

Spinal cord axons display a rostrocaudal, positional bias in their innervation of sympathetic ganglia and intercostal skeletal muscles. In an effort to examine the molecular basis of this positional specificity, we used the cyclophosphamide immunosuppression method to produce monoclonal antibodies that bind preferentially to rostral ganglia. The staining distribution of one of these antibodies, ROCA1, has been analyzed using a novel histological method. A graded decline in binding is observed along the chain of adult rat sympathetic ganglia, as well as in the nerves innervating intercostal muscles. The antigen is identified on immunoblots as a 65 kd protein, whose distribution corresponds to the pattern found histologically. Surprisingly, ROCA1 appears to bind to glial cells, implying rostrocaudal, molecular differences in their surfaces.     

A monoclonal antibody that defines basal cells of stratified epithelia in various human and rabbit tissues

Summary Monoclonal antibodies were produced against monkey lung lavage fluid by using a mouuse hybridoma technique. One monoclonal antibody, KP8D4, specifically reacted with basal cells in human bronchial epithelia by immunohistological staining of acetone-fixed, frozen sections and it recognized a protein with an apparent molecular weight of 84000, as determined by gel immunoblotting. The distribution of this protein was immunohistochemically examined in various human tissues (lung, tongue, esophagus, stomach, intestine, liver, pancreas, salivary gland, spleen, thymus, heart, aorta, vena cava, prostate, breast, kidney, urinary bladder, thyroid, brain, skin, striated muscle) and various tissues of rats, rabbits and pigs. The results showed a specific affinity of KP8D4 to basal cells of stratified epithelia in the various human and rabbit tissues. This antibody may be a useful tool for studies of normal development and diverse pathological disorders.     

A monoclonal antibody that defines basal cells of stratified epithelia in various human and rabbit tissues

Monoclonal antibodies were produced against monkey lung lavage fluid by using a mouse hybridoma technique. One monoclonal antibody, KP8D4, specifically reacted with basal cells in human bronchial epithelia by immunohistological staining of acetone-fixed, frozen sections and it recognized a protein with an apparent molecular weight of 84000, as determined by gel immunoblotting. The distribution of this protein was immunohistochemically examined in various human tissues (lung, tongue, esophagus, stomach, intestine, liver, pancreas, salivary gland, spleen, thymus, heart, aorta, vena cava, prostate, breast, kidney, urinary bladder, thyroid, brain, skin, striated muscle) and various tissues of rats, rabbits and pigs. The results showed a specific affinity of KP8D4 to basal cells of stratified epithelia in the various human and rabbit tissues. This antibody may be a useful tool for studies of normal development and diverse pathological disorders.     

Characterization of a high-affinity monoclonal antibody to calcineurin whose epitope defines a new structural domain of calcineurin A

Monoclonal antibodies have been raised against native calcineurin using conventional in vivo immunization and hybridoma procedures. The relatively high affinity of nonimmune IgG for the two subunits of calcineurin resulted in large nonspecific binding values for immunoassays of native, dissociated and denatured calcineurin, which complicated the antibody screening. Monoclonal aCn5, a high-affinity IgG1 that exhibits specific binding, was characterized. Other calmodulin-binding proteins tested were not recognized by aCn5. Simple binding properties were exhibited in solid-phase experiments, Kd = 26 (+/- 4) pM, but the stoichiometry was low. The loss of immunoreactivity after denaturation of calcineurin indicated that the aCn5 epitope is of the assembled topographic, not segmental, type. The epitope was located to the A subunit and affinity was unaffected by the presence of calcineurin B. The epitope remained intact after proteolytic removal of the amino-terminal 20 residues of calcineurin A essential for phosphatase activity, and the carboxyl-terminal inhibitory and calmodulin-binding domains. The calmodulin-binding peptide derived from calcineurin, cA8, was not recognized by aCn5. Addition of Ca2+, Mn2+, Ni2+, chelators or dithiothreitol did not influence the affinity of aCn5 for the holoenzyme. Phosphatase activity of calcineurin, in the presence and absence of calmodulin and after removal of the inhibitory domain, was little affected by aCn5. Thus, the aCn5 epitope defines a previously unidentified structural domain of calcineurin A located in a region of the proteolytically resistant core that is topologically distinct from the catalytic, inhibitory, calmodulin-binding and calcineurin-B-binding domains, and not functionally connected with calcineurin B or the putative metal-binding domain(s).     

A monoclonal antibody detects a difference in the cellular composition of developing and regenerating limbs of newts

We have previously described a monoclonal antibody (called 22/18) that reacts with the early blastemal cells of the regenerating limb of the newt (Notophthalmus viridescens). In embryos of two newt species the antibody reacts with the epidermis, glial cells in the neural tube, the lens and cells in a restricted region of the aorta. In the developing limb bud less than 1% of the mesenchymal cells were reactive with 22/18, although most cells stained brightly with an antibody to another cytoskeletal component. When limbs were amputated prior to the arrival of nerves (axons and Schwann cells) at the amputation plane there was no extra reactivity with 22/18 as compared to the contralateral unamputated control, even though the amputated buds regenerated satisfactorily. Limbs amputated after nerves are present at the plane of amputation respond by forming a 22/18-positive blastema. The appearance of the 22/18 responses is a function of the stage of limb development as shown by amputation of forelimb and hindlimb buds at a larval stage where development of the forelimb is greatly advanced relative to the hindlimb. The distribution of the 22/18-positive cells in larval blastemas showed them to be closely associated with axons as detected by double staining with an antiserum to a neurofilament subunit. The clear antigenic difference between development and regeneration may be related to the relationship between embryonic regulation and epimorphic regeneration, and also to the acquisition of nerve-dependent proliferation of blastemal cells.     

A monoclonal antiidiotypic antibody with rheumatoid factor activity defines a cross-reactive idiotope on murine anticollagen antibodies.

A syngeneic antiidiotypic mAb, C1C3, was characterized as to its binding to monoclonal anti-collagen II (-CII) auto-antibodies reactive with different epitopes of the native CII molecule. Both by direct binding and by inhibition ELISA studies, the anti-idiotypic antibody was shown to react with a cross-reactive idiotope present on Fab fragments of most, but not all, tested anti-CII mAb, whereas the binding to Fab fragments from normal mouse IgG was low. As previously described, C1C3 bound to isolated Fc fragments from normal mouse IgG. The binding to intact normal mouse IgG was, however, weak, and only isolated Fc-gamma fragments, not intact IgG, competed efficiently with Fab fragments of anti-CII antibodies for binding to the antiidiotypic antibody. The antibody was shown to self-associate, i.e., to behave similarly to certain IgG rheumatoid factors obtained from patients with rheumatoid arthritis. The presented data indicate that the described anti-anti-CII mAb may be representative of antibodies involved in the physiologic regulation of autoimmunity to CII and, consequently, may be used as a tool for further studies on idiotypic regulation in CII-induced arthritis.     

A monoclonal antibody against a neuron-specific 65-kDa protein with laminar expression in the developing cerebral cortex

The extracellular matrix component chondroitin sulfate supports the survival of neocortical neurons and influences their differentiation in culture. During development of the rat forebrain expression of chondroitin sulfate peaks at around birth. To elucidate functional partners of this important player of neurogenesis, a monoclonal antibody, termed MAb-9, was generated after immunization with chondroitin sulfate-binding proteins from neonatal rat brain. In western blots of neonatal tissue, MAb-9 recognized a major brain-specific 65-kDa protein band. While most of the 65-kDa protein was present in the soluble compartment, a significant fraction was membrane-associated. Prolonged extraction of brain membranes with CHAPS revealed three additional minor protein bands of approximately 48, 50, and 58 kDa. Of these, the 50-kDa protein specifically bound to chondroitin sulfate C-Sepharose. Immunocytochemical studies and western blot analyses of cultures of neocortical neurons and astrocytes demonstrated that MAb-9 recognizes a neuron-specific cytosolic protein. In the developing cerebral cortex the protein was detectable by immunohistochemistry in the preplate and ventricular zones as early as embryonic day 15. On embryonic day 18, the protein was found in the marginal zone and the subplate, but it was not present in the emerging cortical plate. At the neonatal stage the immunoreactivity was distributed throughout the cerebral cortex with prominent staining of the marginal zone. A similar pattern was observed in the adult animal. Notably, the laminar distribution of MAb-9 immunoreactivity in the cerebral cortex during the prenatal period closely resembled the expression pattern reported for chondroitin sulfate. Thus, MAb-9 recognizes a neuronal cytosolic protein which might participate in neurotrophic signaling events triggered by chondroitin sulfate.     

Glycoconjugates are stage- and position-specific cell surface molecules in the developing olfactory system, 1: The CC1 immunoreactive glycolipid defines a rostrocaudal gradient in the rat vomeronasal system

Primary sensory neurons in the vomeronasal organ (VNO) project axons to the glomeruli of the accessory olfactory bulb (AOB) where they form connections with mitral cell dendrites. We demonstrate here that monoclonal antibodies to specific carbohydrate antigens define stage- and position-specific events during the development of the vomeronasal system (VN). CC1 monoclonal antibodies react with specific N-acetyl galactosamine containing glycolipids. In the embryo, CC1 antigens are expressed throughout the VNO and on vomeronasal nerves. Beginning approximately at birth and continuing into adults, CC1 expression is spatially restricted in the VNO to centrally located cell bodies. In the postnatal AOB, CC1 is expressed in the nerve layer and glomeruli, but only in the rostral half of the AOB. These data suggest that CC1 antigens may participate in the targeting of axons from centrally located VNO neurons to rostral glomeruli in the AOB. In contrast, CC2 monoclonal antibodies, which recognize complex α-galactosyl and α-fucosyl glycoproteins and glycolipids, react with all VNO cell bodies and VN nerves from embryonic (E) day 15 to adults. CC2 antibodies do not distinguish rostral from caudal regions of the AOB, nor are the CC2 glycoconjugates developmentally regulated. P-Path monoclonal antibodies, which recognize 9-O-acetyl sialic acid, react with cell bodies in the VNO and nerve fibers from E13 to postnatal (P) day 2. P-Path immunoreactivity disappears from the VNO system almost completely by P14, when only a few P-Path reactive nerve fibers can be seen. These studies suggest that specific cell surface glycoconjugates may participate in spatially and temporally selective cell–cell interactions during development and maintenance of vomeronasal connections.     

A monoclonal antibody reactive with a 15-kDa cytoplasmic granule-associated protein defines a subpopulation of CD8+ T lymphocytes

We have recently described a novel method for the production and characterization of mAb reactive with T cell-restricted intracellular antigens. From a panel of antibodies that react specifically with permeabilized T lymphocytes but not with permeabilized B lymphocytes or native T cells, we have selected one, designated TIA-1, that reacts with 20 to 36% of digitonin permeabilized peripheral blood T lymphocytes. Flow cytometric analysis of purified CD4+ and CD8+ subsets showed TIA-1 to recognize a subpopulation of 49 to 64% of CD8+ lymphocytes. Little or no reactivity with CD4+ resting T lymphocytes was observed. TIA-1 did not react with any of a panel of T cell lines, B cell lines, or monocytoid cell lines. TIA-1 reacted strongly with NK cell clones and CD8+ cytolytic T cell clones, and less strongly with CD4+-activated T cell clones, suggesting a preferential expression in cells possessing cytolytic potential. Cell fractionation experiments showed TIA-1 to be membrane associated. Furthermore, Percoll gradient fractionation of a cytolytic T cell clone (T4T8C1) showed the majority of TIA-1 to be contained in a low density membrane fraction that also contained serine protease activity. Immunoelectron microscopy showed TIA-1 to decorate the membranes of electron lucent and electron dense cytoplasmic granules in this same cytolytic T cell clone. Biochemical analysis showed TIA-1 to be a 15-kDa protein in unstimulated T cells. Upon activation with Con A or anti-CD3 antibodies. TIA-1 was induced to form disulfide linked dimers, trimers, and tetramers of the basic 15-kDa unit. Taken together, our data suggest that TIA-1 is a cytolytic granule associated protein that may define a subpopulation of resting CD8+ T lymphocytes possessing cytolytic potential.     

Glycoconjugates are stage- and position-specific cell surface molecules in the developing olfactory system, 1: The CC1 immunoreactive glycolipid defines a rostrocaudal gradient in the rat vomeronasal system.

Primary sensory neurons in the vomeronasal organ (VNO) project axons to the glomeruli of the accessory olfactory bulb (AOB) where they form connections with mitral cell dendrites. We demonstrate here that monoclonal antibodies to specific carbohydrate antigens define stage- and position-specific events during the development of the vomeronasal system (VN). CC1 monoclonal antibodies react with specific N-acetyl galactosamine containing glycolipids. In the embryo, CC1 antigens are expressed throughout the VNO and on vomeronasal nerves. Beginning approximately at birth and continuing into adults, CC1 expression is spatially restricted in the VNO to centrally located cell bodies. In the postnatal AOB, CC1 is expressed in the nerve layer and glomeruli, but only in the rostral half of the AOB. These data suggest that CC1 antigens may participate in the targeting of axons from centrally located VNO neurons to rostral glomeruli in the AOB. In contrast, CC2 monoclonal antibodies, which recognize complex alpha-galactosyl and alpha-fucosyl glycoproteins and glycolipids, react with all VNO cell bodies and VN nerves from embryonic (E) day 15 to adults. CC2 antibodies do not distinguish rostral from caudal regions of the AOB, nor are the CC2 glycoconjugates developmentally regulated. P-Path monoclonal antibodies, which recognize 9-O-acetyl sialic acid, react with cell bodies in the VNO and nerve fibers from E13 to postnatal (P) day 2. P-Path immunoreactivity disappears from the VNO system almost completely by P14, when only a few P-Path reactive nerve fibers can be seen. These studies suggest that specific cell surface glycoconjugates may participate in spatially and temporally selective cell-cell interactions during development and maintenance of vomeronasal connections.     

Characterization of an anti-Ly-6 monoclonal antibody which defines and activates cytolytic T lymphocytes

HK1.4 mAb was identified based on its ability to stimulate proliferation of cloned murine CTL. Within the lymphoid lineage, mAb HK1.4 bound exclusively to CTL, regardless of the expression of Lyt-2 or MHC restriction. HK1.4 mAb also bound to 40% of bone marrow cells and less than 5% of thymocytes from all mouse strains tested. Based on the tissue distribution of the determinant with which it reacted and the ability to cross-block binding of the anti-Ly-6 mAb H9/25, mAb HK1.4 appeared to react with a product of the Ly-6 locus. However, significant differences were observed between the properties of mAb HK1.4 and other, previously described anti-Ly-6 mAb. Cell proliferation and lymphokine release by cloned CTL were stimulated by culture with mAb HK1.4 alone or in the presence of non-stimulatory levels of IL-2. This proliferation and lymphokine release were not blocked by the addition of soluble anti-Lyt-2 or anti-IL-2R mAb. Activation induced by HK1.4 mAb proceeds in the absence of accessory cells, of cross-linking of the TCR, or the addition of mitogens or PMA. Stimulation of cells by anti-TCR mAb was not blocked by the addition of soluble HK1.4 mAb, and the stimulatory effects of HK1.4 and anti-TCR mAb were not additive. However, IL-2-driven proliferation of CTL clones was dramatically inhibited by the addition of HK1.4 mAb.HK1.4 mAb had no effect on Ag-specific or lectin-facilitated cytolysis. Taken together, these data indicate that mAb HK1.4 operates via an IL-2-independent pathway of activation that is also independent of the TCR.     

A monoclonal antibody identifying a T-cell marker in the horse

A cell surface molecule of equine T lymphocytes was identified and characterized using a mouse monoclonal antibody, HT23A. The molecule was detected on all T cells but not on other cells in peripheral blood, with the possible exception of a small subpopulation (about 5%) of B cells, as assessed by indirect immunofluorescence and flow cytometry. HT23A labelled T cell areas of horse lymph nodes and spleen when used in an indirect immunoperoxidase assay on frozen sections. Macrophages and neutrophils were not labelled by the antibody nor were frozen sections of horse liver, kidney, or brain. HT23A precipitated a molecule of approximately 69 kDa from 125Iodine labelled horse lymphocytes.     

A monoclonal antibody specific to a song system nuclear antigen in estrildine finches.

This paper describes a monoclonal antibody that recognizes a molecule whose expression is mostly restricted to some of the forebrain areas that control singing behavior in adult estrildine species studied, including the zebra, Bengalese, and spice finches. When the song system displays extreme sexual dimorphism, as in these species, antibody staining occurs only in the male's song nuclei. However, protein expression is identical in both sexes of estrildine finches, in which females also have a well-developed song system. Canaries appear to lack the protein, but it can be induced in female zebra finches by early estrogen treatment. Antibody staining patterns in the zebra finch show that the protein's expression is developmentally regulated to coincide with the abrupt increase in the volume and cell size of the male's or the estrogen-treated female's song system.     

Identification of two variants of troponin T in the developing chicken heart using a monoclonal antibody

Troponin T (TNT) expressed in the developing chicken cardiac muscle was examined by immunoblotting combined with two-dimensional electrophoresis (2-D PAGE) and peptide mapping. When the whole lysate of the neonatal heart was examined by 2-D PAGE, two TNT variants were detected on the gel by monoclonal antibody to TNT. Expression of the two variants was developmentally regulated: one isoform (type I) was expressed from embryonic through neonatal stages, and the other (type II) from the late embryonic stage through adulthood during cardiac muscle development. The type-I isoform, but not type-II isoform, was also expressed transiently in chicken skeletal muscle at embryonic stages. As judged from the peptide maps, the two isoforms differed in the N-terminal region but not in the C-terminal region.     

A monoclonal antibody defining a lymphoma-associated antigen in man

A monoclonal antibody (Ab 89) was developed against a lymphoma-associated antigen on the tumor cells of a patient (N.B.) with a B cell, poorly differentiated lymphocytic lymphoma (D-PDL). By indirect immunofluorescence, Ab 89 was shown to react only with N.B. lymphoma cells and was unreactive with normal N.B. lymphoid cells, normal fractionated peripheral blood cells, other normal lymphoid tissues, fetal tissues, and non-lymphoid malignant cells. In addition, Ab 89 was unreactive with conventional immunoglobulins, private and public HLA antigens, or Ia-like antigens. More importantly, Ab 89 was reactive with some B cell lymphoid malignancies. Of the 57 B cell lymphomas tested, it was found that Ab 89 reacted with approximately 10% of B cell D-PDL and B cell chronic lymphocytic leukemia (CLL). Of interest was the finding that N.B. serum contained a circulating antigen which could specifically block the reactivity of Ab 89. The data obtained suggests that Ab 89 defines a tumor-associated antigen shared by a unique subgroup of B cell lymphomas. The group of patients reactive with Ab 89 did not fall into any histopathologic classification system. These data support the notion that there is greater heterogeneity of B cell lymphomas than may have been previously recognized.     

Studies of the developing chick retina using monoclonal antibody 8A2 that recognizes a novel set of gangliosides

A monoclonal antibody, Mab 8A2, that recognizes a novel set of gangliosides was produced by immunizing a mouse with Embryonic Day 14 chick optic nerve. Immunohistochemical studies of the developing chick retina revealed a complex pattern of Mab 8A2 immunoreactivity. Initially, staining is concentrated in the optic fiber layer in the central retina. Later in development, the most intense staining is seen at the periphery of the retina and 8A2 immunoreactivity appears in other retina layers. In the adult retina, 8A2 immunoreactivity is lost from the optic fiber layer but persists in the inner plexiform layer, inner nuclear layer, and outer plexiform layer. Cell culture experiments showed intense staining of neurites from retinal ganglion cells but no staining of Muller cells. Biochemical characterization of the epitope recognized by Mab 8A2 suggests that it includes a 9-O-acetyl group that is present on five different gangliosides. The 8A2 immunoreactive gangliosides are distinct from and have slower mobilities on thin-layer chromatographs than those recognized by Mab D1.1 which recognizes 9-O-acetyl GD3.     

Localization of H-2Kk in developing mouse teeth using monoclonal antibody

The in situ distribution of H-2 antigens during mouse tooth morphogenesis was investigated using monoclonal antibodies to H-2Kk and indirect immunofluorescent techniques. H-2 antigens were detected in the basement membrane region of fetal molars; they were absent from both the epithelial and dental mesenchyme. H-2 antigens were not found in newborn and 4-day-old mouse molars.     

Immunohistochemical localization of WE-14 in the developing porcine sympathoadrenal cell lineage

Immunohistochemical investigation of the post-translational processing of chromogranin A (CgA) to generate WE-14 in the sympathoadrenal cell lineage of the developing porcine fetus (F) detected intense CgA and weak WE-14 immunoreactivity in migrating neuroblast cells of the diffuse sympathetic ganglia adjacent to the dorsal aorta and projecting toward the cortical mass at F24-27. F37-42; WE-14 immunoreactivity was detected in chromaffinoblasts at the periphery of the developing cortex and at F54-56 days gestation WE-14 immunoreactivity was detected in a large population of central medullary cells. From F74 to F76 days and thereafter the number of cells exhibiting intense WE-14 immunostaining decreased, and the majority of chromaffin cells exhibited uniform weak WE-14 immunostaining. At postnatal day 1 (P1) intense WE-14 immunoreactivity was primarily confined to clusters of chromaffin cells with weak immunostaining in the general population. The transitory neuroblasts, chromaffinoblasts, and maturing chromaffin cell population exhibited uniform intense CgA immunostaining through gestation and after birth. Additional observations detected intense CgA and WE-14 immunostaining in extrachromaffin tissue at P1 and in neuronal-like cells in vessels of the aortic arch at F37. This study has demonstrated that CgA is post-translationally processed to generate WE-14 during early fetal development in the migrating progenitor cells of the porcine sympathoadrenal lineage.     

A monoclonal antibody driven biodiagnostic system for the quantitative screening of breast cancer

A prospective clinical parametric study comprising women afflicted by breast cancer and otherwise healthy participants was undertaken. The mean plasmatic concentration of putative leucine amino peptidase and nucleoside diphosphate phosphotransferase enzymatic complex in breast cancer cases was significantly elevated [43.9 +/- 2.8 microg ml(-1) (n = 9)] when compared to those found in otherwise healthy women [8.07 +/- 0.14 microg ml(-1) (n = 8)]. Women without images compatible with any tumours (n = 13) had a mean concentration of 10.77 +/- 1.49 microg ml(-1). The mean value obtained in women with fibroadenomas was 10.15 +/- 0.81 microg ml(-1) (n = 6) and with cystic fibrosis mastopathy 8.75 +/- 0.28 microg ml(-1) (n = 7). The efficacy of a tandem quantitative biodiagnostic system as a parametric screening tool for the early detection of breast cancer is underlined, raising the possibility of increasing the cost effectiveness of current imaging non-parametric technologies.     

Embryonic myosin heavy chain as a differentiation marker of developing human skeletal muscle and rhabdomyosarcoma. A monoclonal antibody study

Hybridoma cell lines were obtained from the fusion of NS-O myeloma cells with spleen cells of mice immunized with bovine fetal skeletal myosin. A stable hybridoma clone, BF-G6, produced immunoglobulin G1 k antibodies reacting specifically with embryonic-type myosin heavy chains present in fetal but not in neonatal or adult human skeletal muscle, as determined by enzyme immunoassay and immunoblot analysis. Fetal but not adult skeletal muscle fibers were stained by this monoclonal antibody in indirect immunofluorescence assays; smooth muscle cells and cardiac muscle cells, as well as non-muscle cells were also unreactive. Solid tumors of infants and children were tested for reactivity with BF-G6 by immunofluorescence and immunoperoxidase staining. Embryonic myosin heavy chain was expressed in rhabdomyosarcomas but not in other types of tumor, except for Wilms' tumor. Rhabdomyosarcoma cells isolated from a bone marrow metastasis and grown in vitro for several months were also labelled by BF-G6. Embryonic myosin heavy chain can thus be used as a specific differentiation marker of normal and neoplastic skeletal muscle tissue.