Demonstration of immunochemical identity between the synaptic vesicle proteins synaptin and synaptophysin/p38

The synaptic vesicle proteins synaptin and synaptophysin/p38 were shown to be immunochemically identical. Western immunoblot analysis of Triton X-100 extracts from rat brain showed that polyclonal polyspecific anti-synaptin antibodies and monoclonal antibody SY38 against synaptophysin both reacted with a band of 38 kDa. In two-dimensional immunoblots of chromaffin granule membranes from bovine adrenal medulla anti-synaptin and anti-synaptophysin antibodies also recognized the same component. Finally, in a Western immunoblotting experiment SY38 reacted with an immuno-isolated synaptin antigen.     

Comparative immunohistochemistry and histochemistry of dipeptidyl peptidase IV in rat organs during development

Summary The occurrence of dipeptidyl peptidase (DPP) IV during development in Wistar rat organs was studied on day 10, 16 and 21 of gestation and on day 1, 4, 8, 13, 21, 26 and 60 after birth comparing immunohistochemistry and activity histochemistry. A polyclonal antibody, as well as monoclonal antibodies recognizing four different epitopes (A-D) of the DPP IV molecule, were employed for the immunohistochemical studies. In all investigated tissues, immunoreactivity with the polyclonal antibody appeared earlier than DPP IV activity and was already present on day 10 of gestation in the plasma membranes of embryonic and extraembryonic (decidual) cells. At these and other sites, e.g. brain capillary endothelium and tracheal or bronchial epithelium, immunoreactivity with the polyclonal antibody decreased or disappeared after birth and enzyme activity never developed. Immunoreactivity with the monoclonal antibodies appeared later than that with the polyclonal antibody, and mostly in those structures where DPP IV activity was subsequently found. The monoclonal antibody against epitope D showed a high reactivity in the epididymal duct, renal collecting ducts and in all domains of the hepatocyte plasma membrane, where neither DPP IV activity nor immunoreactivity with the other antibodies were observed. Our results also suggest that DPP IV might be present as a molecule before it becomes catalytically active and that immunoreactivity occurs at more sites than DPP IV activity. However, it cannot be excluded that the polyclonal antibody and the monoclonal antibody against the epitope D cross-react with as yet uncharacterized proteins, which express common epitopes during embryonic development, but are not present in the tissues of adult Wistar rats.     

Monoclonal antibodies to rabbit and pig zonae pellucidae distinguish species-specific and shared antigenic determinants

Monoclonal antibodies against rabbit or porcine zonae pellucidae (ZP) demonstrate species-specific and shared antigenic determinants. In addition, these antibodies are used to characterize the biochemical nature of these determinants. All of six monoclonal antibodies developed against porcine ZP react with porcine but not with rabbit ZP. Only one of seven monoclonal antibodies developed against rabbit ZP cross-reacts with porcine ZP. None of these antibodies recognized antigens associated with other tissues tested. High-resolution, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) followed by immunoblotting was used to demonstrate that the cross-reactive antibody recognizes an antigenic determinant which is associated with the major low molecular weight glycoprotein of both the pig and rabbit ZP. Since this antibody recognizes all charge species of this glycoprotein, it is apparent that the antigenic determinant recognized by this antibody involves protein. Further studies demonstrate that proteolytic digestion of ZP will destroy the antigenic determinant while glycosidic digestion of ZP has no effect on antibody binding. Although polyclonal antibodies to this glycoprotein inhibit sperm from binding to the zona pellucida, this monoclonal antibody does not affect sperm binding. None of the species-specific antibodies recognize ZP glycoproteins following 2D-PAGE. This is a property typical of antibodies directed against conformational antigenic determinants. The presence of common as well as unique zona antigenic determinants could explain why ZP proteins induce heteroantibodies which result in infertility while alloimmunization has no effect on fertility.     

Preparation of mycoplasma immunogens from plants and a comparison of polyclonal and monoclonal antibodies made against primula yellows MLO-associated antigens

A method is described for obtaining from plants partially purified preparations of mycoplasma-like organisms (MLO) which are suitable for use as immunogens for polyclonal or monoclonal antibody production, and as antigens for directly coating ELISA plates. Using this method a mouse monoclonal antibody to primula yellows MLO was prepared, and its characteristics compared with those of primula yellows polyclonal antibodies from rabbits and also against polyclonal antibodies made to similar preparations of European aster yellows MLO. No serological distinction was obtained between any of the homologous or heterologous combinations of antibody and MLO preparation using ELISA, fluorescence microscopy with FITC-labelled antibodies, or immunoprobes of western blots of partially purified MLO preparations. By contrast, there were no cross-reactions between the primula or aster yellows antibodies or MLO preparations and preparations of clover phyllody or tomato big bud MLOs or their respective polyclonal antibodies. The primula yellows MLO monoclonal and polyclonal antibodies, and also the European aster yellows MLO polyclonal antibodies, all appeared to recognize only a single major antigen of approximate M, = 22 400 daltons. Some possible explanations for the apparent specificity of the polyclinic antisera for a single antigen, and the relevance to MLO preparation procedures are discussed.     

Monoclonal antibodies demonstrate limited structural homology between myosin isozymes from Acanthamoeba

We used a library of 31 monoclonal and six polyclonal antibodies to compare the structures of the two classes of cytoplasmic myosin isozymes isolated from Acanthamoeba: myosin-I, a 150,000-mol-wt, globular molecule; and myosin-II, a 400,000-mol-wt molecule with two heads and a 90-nm tail. This analysis confirms that myosin-I and -II are unique gene products and provides the first evidence that these isozymes have at least one structurally homologous region functionally important for myosin's role in contractility. Characterization of the 23 myosin-II monoclonal antibody binding sites by antibody staining of one-dimensional peptide maps and solid phase, competitive binding assays demonstrate that they bind to at least 15 unique sites on the myosin-II heavy chain. The antibodies can be grouped into six families, whose members bind close to one another. None of the monoclonal antibodies bind to myosin-II light chains and polyclonal antibodies against myosin-II light or heavy chain bind only to myosin-II light or heavy chains, respectively: no antibody binds both heavy and light chains. Six of eight monoclonal antibodies and one of two polyclonal sera that react with the myosin-I heavy chain also bind to determinants on the myosin-II heavy chain. The cross-reactive monoclonal antibodies bind to the region of myosin-II recognized by the largest family of myosin-II monoclonal antibodies. In the two papers that immediately follow, we show that this family of monoclonal antibodies to myosin-II binds to the myosin-II tail near the junction with the heads and inhibits both the actin-activated ATPase of myosin-II and contraction of gelled cytoplasmic extracts of Acanthamoeba cytoplasm. Further, this structurally homologous region may play a key role in energy transduction by cytoplasmic myosins.     

Monoclonal antibody as a probe for structure and function of an Escherichia coli outer membrane protein

Eight independently derived monoclonal antibodies directed against the LamB protein were produced and characterized. By using these antibodies as probes, we identified four distinct topological and functional regions in the LamB molecule. Four monoclonal antibodies recognize antigenic determinants of the protein exposed on the outer side of the membrane. Two of these have their binding sites located in a region involved in maltose transport. One monoclonal antibody presumably binds to a determinant which is normally hidden in the membrane and three monoclonal antibodies recognize determinants facing the periplasmic space.     

Immunological study of a neurofilament protein variant (S150) with polyclonal and monoclonal antibodies

Ten polyclonal neurofilament antibodies were tested for domain specificity with immunoblots of chymotrypsin digests of a neurofilament protein of 150 kDa (NF 150K). In contrast to most monoclonal antibodies previously reported, the five polyclonal antibodies which showed domain specificity reacted with the 40 kDa α-helical rod domain of the molecule. (With one exception, monoclonal antibodies reacted with the 200 kDa carboxy-terminal peripheral domain.) Of these ten polyclonal antibodies only two reacted with an isoelectric variant of NK 150 K (S150) isolated by Liem and collaborators (Wong, J., Hutchison, S.B. and Liem, R.K.H. (1984) J. Biol. Chem. 259, 10867–10874) from bovine brain. 13 monoclonal antibodies were also tested for reactivity with S150 protein. With one exception, none of these antibodies reacted with this variant, not even a monoclonal antibody which we have previously shown to react with a non-phosphorylated epitope located in the rod domain of NF 150K. We suggest that either there are modifications other than dephosphorylation in the S150 isoelectric variant or, alternatively, that it is not derived from NF 150K.     

Cytochrome-C localizes in secretory granules in pancreas and anterior pituitary

We used quantitative immunogold electron microscopy to evaluate the subcellular distribution of cytochrome-c in normal rat tissues, employing a wide variety of monoclonal and polyclonal antibodies against mammalian cytochrome-c. Immunogold labeling of tissues embedded in the acrylic resin LR Gold shows highly specific labeling of mitochondria in all tissues examined, including adrenal gland, cerebellum, cerebral cortex, heart, kidney, liver, pituitary, pancreas, skeletal muscle, spleen and thyroid. In pancreatic acinar cells and anterior pituitary, however, there was also strong cytochrome-c reactivity in zymogen granules and growth hormone granules, respectively. In the pancreas, strong immunoreactivity is also detected in condensing vacuoles and in the acinar lumen. Immunocytochemical controls included (i) use of monoclonal antibodies to horse cytochrome-c which recognize an epitope not present in rat cytochrome-c, (ii) preadsorption of antibodies with purified cytochrome-c, and (iii) omission of the primary antibody. The indicated presence of cytochrome-c outside mitochondria in certain tissues under normal physiological conditions raises interesting questions concerning translocation mechanisms and the cellular functions of cytochrome-c.     

Recognition of covalent insulin-receptor complexes on viable adipocytes by anti-insulin antibodies

Isolated rat adipocytes were photo-affinity-labelled with B2-(4-azido-2-nitrophenylacetyl)-des-PheB1-insulin or B29-(4-azido-2-nitrophenylacetyl)insulin. Four anti-insulin antibodies (3 monoclonal, 1 polyclonal) were tested for their ability to inhibit the persistent stimulation of lipogenesis caused by the covalently bound insulin [Brandenburg et al. (1980) Nature (London) 286, 821-822]. The polyclonal and 2 monoclonal antibodies, directed against the C-terminus of the B-chain, gave a significant depression, while one antibody, directed against the region A(8-10), was without effect. Under reversible conditions, without irradiation, all antibodies completely inhibited lipogenesis. For the polyclonal antibody this is shown in a dose-dependent way. It is concluded that the effective antibodies can recognize their epitope because it is accessible on the surface of the complex and does not represent part of the receptor-binding surface of insulin. This binding leads to interference with the generation and/or transmittance of the biological signal.     

MAPPING OF HIV-1 GAG EPITOPES RECOGNIZED BY POLYCLONAL ANTIBODIES USING GENE-FRAGMENT PHAGE DISPLAY SYSTEM

Phage display has emerged as a powerful technique for mapping epitopes recognised by monoclonal and polyclonal antibodies. We have recently developed a simple gene-fragment phage display system and have shown its utility in mapping epitope recognised by a monoclonal antibody. In the present study, we have employed this system in mapping epitopes recognised by polyclonal antibodies raised against HIV-1 capsid protein, p24 which is derived from proteolytic cleavage of Gag polyprotein. HIV-1 gag DNA was fragmented by DNase I and the fragments (50–250 bp) were cloned into gene-fragment phage display vector to construct a library of phages displaying peptides. This phage library was used for affinity selection of phages displaying epitopes recognised by rabbit anti-p24 polyclonal antibodies. Selected phages contained sequences from two discrete regions of p24, demonstrating the presence of two antigenic regions.

The DNA sequences encoding these regions were also cloned and expressed as GST fusion proteins. The immunoreactivity of these epitopes as GST fusion proteins, or as phage-displayed peptides, was comparable in ELISA system using same anti-p24 polyclonal antibodies. The results indicate that the gene-fragment based phage display system can be used efficiently to identify epitopes recognised by polyclonal antibodies, and phage displayed epitopes can be directly employed in ELISA to detect antibodies.     

Mapping of hiv-1 Gag epitopes recognized by polyclonal antibodies using gene-fragment phage display system.

Phage display has emerged as a powerful technique for mapping epitopes recognised by monoclonal and polyclonal antibodies. We have recently developed a simple gene-fragment phage display system and have shown its utility in mapping epitope recognised by a monoclonal antibody. In the present study, we have employed this system in mapping epitopes recognised by polyclonal antibodies raised against HIV-1 capsid protein, p24 which is derived from proteolytic cleavage of Gag polyprotein. HIV-1 gag DNA was fragmented by DNase I and the fragments (50-250 bp) were cloned into gene-fragment phage display vector to construct a library of phages displaying peptides. This phage library was used for affinity selection of phages displaying epitopes recognised by rabbit anti-p24 polyclonal antibodies. Selected phages contained sequences from two discrete regions of p24, demonstrating the presence of two antigenic regions. The DNA sequences encoding these regions were also cloned and expressed as GST fusion proteins. The immunoreactivity of these epitopes as GST fusion proteins, or as phage-displayed peptides, was comparable in ELISA system using same anti-p24 polyclonal antibodies. The results indicate that the gene-fragment based phage display system can be used efficiently to identify epitopes recognised by polyclonal antibodies, and phage displayed epitopes can be directly employed in ELISA to detect antibodies.     

Identification of tubulin in Dictyostelium discoideum: characterization of some unique properties

We used three antitubulin antibodies to localize Dictyostelium tubulin subunits on two-dimensional polyacrylamide gels by Western blotting. All three antibodies, a polyclonal antibody against sea urchin alpha- and beta-tubulin and two monoclonal antibodies against yeast alpha- tubulin, recognize the same set of polypeptides with a molecular weight of 55,000 while focusing at a pH far more basic than all other tubulins. Each antibody specifically stains the microtubule system of slime mold amoebae by indirect immunofluorescence. The microtubule system can be isolated as a major component of the amoeba cytoskeleton, and these preparations are greatly enriched for the presumptive tubulin subunits. The microtubules of these cytoskeletons are resistant to being depolymerized by millimolar concentrations of calcium, while they retain their cold sensitivity. Comparison of peptide maps of slime mold and brain alpha-tubulins indicates that the proteins are related but not identical. Possible explanations for these unusual characteristics are discussed.     

Epitopes of an influenza viral peptide recognized by antibody at single amino acid resolution

Antibodies raised against the synthetic peptide corresponding to the carboxy-terminal 24 amino acids (305-328) of the heavy chain of the hemagglutinin molecule of influenza virus A/X-31 (H3) bind this peptide at three antigenic sites. These sites were identified by assaying binding of polyclonal BALB/c mouse antipeptide sera to the complete set of all possible di-, tri, tetra-, penta-, hexa-, hepta-, and octapeptides homologous with the 24-residue sequence. Individual epitopes were defined and essential residues identified by testing the binding of monoclonal antibodies to sets of peptide analogues in which every one of the homologous residues was replaced in turn by each of the 19 alternative genetically coded amino acids. The immunodominant epitope was shown to be a linear sequence of five amino acids, 314LKLAT318. Replacement of any one of these residues with any other amino acid resulted in loss of antibody binding, indicating that all five are essential to the interaction and that they are probably contact residues. Another antigenic site contains at least two overlapping epitopes: polyclonal sera recognize predominantly an epitope or epitopes encompassed by the linear sequence 320MRNVPEKQT328, whereas the epitope defined by a particular monoclonal antibody comprises the seven amino acids 322NVPEKQT328, of which N322, E325, and Q327 were implicated as contact residues.     

Quantitation and intracellular localization of the 85K heat shock protein by using monoclonal and polyclonal antibodies.

Two monoclonal antibodies have been produced against the human 85,000-molecular-weight heat shock protein (hsp85). One of these, 16F1, cross-reacts with the murine homolog and is shown by peptide map immunoblots to be directed against an epitope different from that recognized by the other monoclonal antibody, 9D2. Both monoclonal antibodies recognize only a single Mr-85,000 species in two-dimensional immunoblots. Immunoprecipitation did not reveal an association of this heat shock protein with any other protein in HeLa cells. Immunoperoxidase staining showed a purely cytosolic distribution at both light and electron microscopic levels and no association with membranes, mitochondria, or other organelles. The 9D2 monoclonal and a polyclonal antimurine hsp85 antibody were used to identify the antigens and to quantitate their levels in a variety of normal tissues by immunoautoradiography. Relative abundance in the various tissues as determined by Coomassie blue staining correlates reasonably well with the immunoreactivity. Testis and brain, for example, have high hsp85 levels, whereas heart and skeletal muscle have little or none. The Mr-85,000 sodium dodecyl sulfate-polyacrylamide gel band in testis and brain lysates was further confirmed to be hsp85 by one-dimensional partial proteolytic peptide mapping. Based on these data and our previous observations showing that synthesis and levels of the protein are altered by depriving cultured cells of glucose, we speculate that intracellular hsp85 levels depend on differences in the intermediary metabolism of glucose in the various tissues. Furthermore, it appears that high basal levels of this heat shock protein may not necessarily protect cells against heat shock, since testis is one of the most heat-sensitive tissues and has the highest hsp85 level.     

Absence of pituitary prolactin epitopes in immunoreactive prolactin of rat brain.

Immunoreactive prolactin (ir-PRL) in rat brain has been consistently documented. However, the identity of this ir-PRL is controversial. Ir-PRL is defined by its ability to bind to PRL antibodies. All previous studies of brain ir-PRL have used polyclonal antibodies, at least one of which apparently crossreacts with a portion of the proopiomelanocortin molecule. To begin to define the epitopes comprising ir-PRL in the brain, we utilized two monoclonal antibodies (MAb) that recognize pituitary PRL in a variety of species, including rat. Immunocytochemistry was performed on rat brains and pituitary glands using two monoclonal and one polyclonal PRL antibody. Although both MAb immunostained lactotrophs of the rat pituitary gland, neither antibody immunostained cell bodies or neuronal processes in the brain. However, the polyclonal antiserum immunostained lactotrophs and a system of neuronal cell bodies and processes in the brain. Thus, epitopes found in pituitary PRL from several species are not found in ir-PRL in rat brain.     

Characterization and localization of dynein and myosins V and VI in the ovaries of queen bees

The presence of myosin and dynein in the ovaries of both Apis mellifera and Scaptotrigona postica was investigated in extracts and in histological sections. In the ovary extracts, motor proteins, myosins V, VI and dynein were detected by Western blot. In histological sections, they were detected by immunocytochemistry, using a mouse monoclonal antibody against the intermediary chain of dynein and a rabbit polyclonal antibody against the myosin V head domain. The myosin VI tail domain was recognized by a pig polyclonal antibody. The results show that these molecular motors are expressed in the ovaries of both bee species with few differences in location and intensity, in regions where movement of substances is expected during oogenesis. The fact that antibodies against vertebrate proteins recognize proteins of bee species indicates that the specific epitopes are evolutionarily well preserved.     

Study of the structural and functional properties of fibronectin--a high molecular weight plasma glycoproteins--by using monoclonal antibodies

Functional and structural properties of fibronectin--high molecular weight glyco-protein from human plasma--were studied by monoclonal antibodies against fibronectin. It was shown that monoclonal antibodies against human plasma fibronectin exhibit a certain species specificity. Antigenic determinant for our monoclonal antibody is located in the central part of the protein polypeptide chain--in the structural domain. The monoclonal antibodies studied do not inhibit any tested functions of fibronectin. In contrast, polyclonal antibodies are not species specific and inhibit all fibronectin functions.     

Separation of histones from Physarum polycephalum by ion-paired, reverse-phase high-performance liquid chromatography

A rapid, one-step method for the efficient purification of murine monoclonal antibodies from tissue culture supernatants is described. This process is based on affinity chromatography on protein A-Sepharose columns. It was found that murine monoclonal antibodies raised against tick-borne encephalitis virus frequently eluted at more than one pH value and these pH values did not always correspond to those of antibodies of the same subclass from polyclonal mouse sera. The two populations of antibody molecule eluting at different pH values showed no variation in molecular weight, isoelectric profiles, specific enzyme-linked immunosorbent assay titer, or antibody subclass.     

Monoclonal antibodies against the membrane-bound, flavin-linked D-lactate dehydrogenase of Escherichia coli: preparation, characterization, and use in immunoaffinity chromatography

Three mouse hybridoma cell lines are described that produce monoclonal antibodies directed against the membrane-bound, flavin adenine dinucleotide linked D-lactate dehydrogenase of Escherichia coli. In contrast to polyclonal antibodies produced in rabbits, none of the monoclonal antibodies inhibits enzyme activity. Immunoblots of D-lactate dehydrogenase proteolytic fragments indicate that each antibody is directed against a different region of the molecule. One monoclonal antibody, 1B2a, reacts with native, undigested D-lactate dehydrogenase only and is used to purify the enzyme in a single step. The protocol involves chromatography of a Triton X-100 extract of membrane vesicles containing D-lactate dehydrogenase on a column made with the monoclonal antibody coupled to a solid support. After the column is washed free of unadsorbed protein, elution at high pH in the presence of guanidine hydrochloride yields a fraction containing highly purified, catalytically active D-lactate dehydrogenase.     

Monoclonal antibodies specific for glial fibrillary acidic (GFA) protein and for each of the neurofilament triplet polypeptides

A panel of 10 mouse monoclonal antibodies specific for glial fibrillary acidic protein (GFA) has been isolated using porcine GFA as antigen. Although all antibodies recognize GFA purified from porcine spinal cord in the western blot technique, they can be subdivided into at least three groups on the basis of their reactivity against defined fragments of the molecule. Immunofluorescence staining patterns with the monoclonal antibodies performed on tissues and cell lines resemble those reported with conventional polyclonal antibodies directed against GFA. In particular astrocytes and Bergmann glia are strongly stained. In addition mouse monoclonal antibodies specific for either the 200 kd, or the 160 kd, or the 68 kd neurofilament triplet protein have been isolated and characterized. These antibodies are specific for neuronal cells and support conclusions made with similar antigen affinity-purified polyclonal antibodies. The combined set of monoclonal antibodies seems a valuable tool to characterize the different cell types of the nervous system.