A multiplicity of erythrocyte glycolipids of the neolacto series revealed by immuno-thin-layer chromatography with monoclonal anti-I and anti-i antibodies

The thin-layer-chromatography immunostaining procedure was applied to human erythrocyte glycolipids using monoclonal anti-i and anti-I antibodies which are directed against epitopes on linear and branched carbohydrate chains of the neolacto (poly-N-acetyl-lactosamine) series. An examination of native and mild-acid-treated glycolipids from normal adult (I_{adul t} antigen type), neonatal (i_cord) , and I-antigen-deficient adult (i_adult) erythrocytes enabled certain structural inferences to be made as follows: (a) cells of both I and i phenotypes contain a multiplicity of glycolipids of the neolacto series whose backbones consist of 8 or more sugar residues; (b) the octasaccharide backbones are predominantly linear in cells of i phenotype and branched in those of I type; and (c) more complex glycolipids having decasaccharide and larger backbones with both linear and branched sequences occur in erythrocytes of both phenotypes, 0     

Differences in carbohydrate moieties of high molecular weight glycoproteins of human lymphocytes of T and B origins revealed by monoclonal autoantibodies with anti-I and anti-i specificities

NADPH reduced rabbit liver microsomal enzymes catalyzed anaerobic dehalogenation of halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) to produce CF₂CHCl and CF₃CH₂Cl. Anaerobic dehalogenation was optimal at pH7.4 and was blocked by either oxygen or carbon monoxide. The degree of inhibition of anaerobic dehalogenation by carbon monoxide was closely correlated to the proportion of carbon monoxide complex of cytochrome P450. Anaerobic dehalogenation was enhanced by pretreatment of the animals with phenobarbital but not with methylcholanthrene.     

Endo-beta-D-galactosidases of Bacteroides fragilis and Escherichia freundii hydrolyze linear but not branched oligosaccharide domains of glycolipids of the neolacto series

The specificities of the endo-beta-galactosidases of Bacteroides fragilis and Escherichia freundii towards linear and branched oligosaccharides of the lacto-N-glycosyl series were investigated using as substrates glycolipids containing (a) linear neolactotetra - or hexaosyl sequences, (b) branched biantennary neolactooctaosyl sequences, and (c) triantennary neolactononaor dodecaglycosyl sequences. Glycolipid and oligosaccharide hydrolysis products were identified by tlc and/or paper chromatography. The rate of hydrolysis was assessed in time course experiments in which the oligosaccharides released were quantified as 3H-labeled alditols. The salient observations were as follows. (i) With the substrates thus far tested in the present and a previous study ( Scudder , P., Uemura , K., Dolby , J., Fukuda, M.N., and Feizi , T. (1983) Biochem. J. 213, 485-494), the endo-beta-galactosidases from B. fragilis and E. freudii have indistinguishable specificities. (ii) The beta-galactosidic linkage of the branch point sequence (Formula: see text) is completely resistant to hydrolysis by these enzymes, although the unbranched sequence GlcNAc beta 1-3Gal beta 1-4GlcNAc/Glc is readily cleaved. (iii) At an optimal concentration of detergent, the endo-beta-galactosidase susceptibility of the GlcNAc beta 1-3Gal beta 1-4Glc sequence near the ceramide moiety of branched glycolipids is similar to that of the corresponding sequence in linear glycolipids.     

A new membrane antigen revealed by monoclonal antibodies is associated with motoneuron axonal pathways

Chick embryonic motoneurons selectively grow out from the spinal cord as the first step of their selective axonal growth. In order to detect the molecules responsible for motoneuron outgrowth from the cord, we produced and immunohistochemically screened many monoclonal antibodies (MAbs) against cord and somite. We found that two of them, called M7412 and M7902, selectively bound to the cell surface of the anterior half of the sclerotome, where motoneurons selectively extend their axons. Immunohistochemistry and immunoblot results were identical for these antibodies and the antigen was called M7412 antigen. Although neural crest cells also migrate into the anterior half of the sclerotome, the expression of M7412 antigen by sclerotome cells was independent of the neural crest, because neural crest removal did not affect the appearance of the antigen. Furthermore, MAb M7412 bound to the mesenchymal cells along presumptive major nerve trunks in the limb and to the structures surrounding myotubes in muscles during the formation of intramuscular nerve branches. These results suggest that M7412 antigen might be a substrate for general, but not specific, growth of motoneuron axons. If this is the case, we must also infer that some molecule inhibitory for motoneuron growth is localized in the posterior half of sclerotome, because at upper cervical levels the M7412 antigen was also expressed intensely in the posterior sclerotome, whereas motoneurons still grew only into the anterior half. The M7412 antigen was transiently expressed in such various tissues as somite; muscles; blood vessels; spinal cord cells, especially motoneurons innervating the limb; and dorsal root and other peripheral ganglion cells. The M7412 antigenic molecule was extractable with NP40 from a membrane fraction of whole chick embryos and its molecular weight was estimated to be 70 kDa from immunoblot analysis. Thus, our monoclonal antibodies have revealed a new membrane-associated molecule which is likely to play a role in cell-cell interactions during development of motoneurons.     

Unusual structure of human splenic sinusoids revealed by monoclonal antibodies

The sinusoid of the human spleen is an unusual vascular structure which is involved in the removal of damaged or aged erythrocytes. The sinusoid also permits the migration of leukocytes from the cords of Billroth into the circulation. The sinusoids were studied with a variety of monoclonal antibodies in an effort to further delineate their structure. Immunohistochemical studies of sinusoids with antibodies to the intermediate filament protein vimentin and the T cell antigen Leu 2 revealed unusual rod-shaped and banded structures. These structures are distinct from the annular fibers detected by histochemical methods. They are not detected in other types of endothelium. The sinusoids are also distinct in that they fail to react with antibody Leu M3 which stains other types of endothelium. The sinusoids are similar to other types of endothelium in their immunoreactivity with antibodies to Factor VIII and HLA-DR antigens.     

Domain structure of neurofilament subunits as revealed by monoclonal antibodies

Monoclonal antibodies have been prepared against purified neurofilament (NF) subunits (NF68, NF150, and NF200). From 25 fusions, several hundred strongly positive antibodies have been obtained. Among them are antibodies against the specific subunits as well as antibodies recognizing common antigenic determinants. These have all been characterized according to the following properties: ELISA (enzyme-linked immunosorbant assay) testing against each subunit, immunoblots against enriched neurofilament preparation, immunoblots of cyanogen bromide or chymotrypsin-treated neurofilaments, immunofluorescence with PC12 cells, and immunohistochemistry of cerebellum. Whereas the antibodies against the NF68 and NF150 appear to react with single cyanogen bromide fragments, the antibodies against the NF200 react with multiple cyanogen bromide fragments. These data are consistent with the hypothesis that the NF200 is partially composed of several repeated structural determinants. Furthermore, all of the antibodies that react with the NF200 recognize the solubilized "sidearm" domain from limited chymotryptic digestions. The locations of the common and variable domains of the three subunits are discussed in light of these results.     

Unusual structure of human splenic sinusoids revealed by monoclonal antibodies

Summary The sinusoid of the human spleen is an unusual vascular structure which is involved in the removal of damaged or aged erythrocytes. The sinusoid also permits the migration of leukocytes from the cords of Billroth into the circulation. The sinusoids were studied with a variety of monoclonal antibodies in an effort to further delineate their structure. Immunohistochemical studies of sinusoids with antibodies to the intermediate filament protein vimentin and the T cell antigen Leu 2 revealed unusual rod-shaped and banded structures. These structures are distinct from the annular fibers detected by histochemical methods. They are not detected in other types of endothelium. The sinusoids are also distinct in that they fail to react with antibody Leu M3 which stains other types of endothelium. The sinusoids are similar to other types of endothelium in their immunoreactivity with antibodies to Factor VIII and HLADR antigens.     

Tissue distribution of Diablo/Smac revealed by monoclonal antibodies

Diablo/Smac is a mammalian pro-apoptotic protein that can antagonize the inhibitor of apoptosis proteins (IAPs). We have produced monoclonal antibodies specific for Diablo and have used these to examine its tissue distribution and subcellular localization in healthy and apoptotic cells. Diablo could be detected in a wide range of mouse tissues including liver, kidney, lung, intestine, pancreas and testes by Western blot analysis. Immunohistochemical analysis found Diablo to be most abundant in the germinal cells of the testes, the parenchymal cells of the liver and the tubule cells of the kidney. In support of previous subcellular localization analysis, Diablo was present within the mitochondria of healthy cells, but released into the cytosol following the induction of apoptosis by UV.     

Interactions of calmodulin with metal ions and with its target proteins revealed by conformation-sensitive monoclonal antibodies

Two monoclonal antibodies (mAbs) raised against bovine calmodulin (CaM), CAM1 and CAM4, enable one to monitor conformational changes that occur in the molecule. The interaction of CAM1 with CaM depends on the Ca2+ occupancy of its Ca(2+)-binding sites. CAM4, in contrast, interacts with CaM in a Ca(2+)-independent manner, interacting with both holoCaM and EGTA-treated CaM to a similar extent. Their interaction with various CaMs, CaM tryptic fragments and chemically modified CaM, as well as molecular graphics, led to identification of the CAM1 and CAM4 epitopes on the C- and N-terminal lobes of CAM respectively. The two mAbs were used as macromolecular probes to detect conformational changes occurring in the CaM molecule upon binding of metal ions and target proteins and peptides. MAb CAM1 successfully detected changes associated with Al3+ binding even in the presence of Ca2+, indicating that Al3+ and Ca2+ ions may bind to the protein simultaneously, leading to a new conformation of the molecule. MAbs CAM1 and CAM4 were used to follow the interactions of CaM with its target peptides and proteins. Complexes with melittin, mastoparan, calcineurin and phosphodiesterase showed different immunological properties on an immuno-enzyme electrode, indicating unique structural properties for each complex.     

Heterogeneity of mouse Thy 1.2 antigen expression revealed by monoclonal antibodies

A different sensitivity of T cells from C57B1/6 and DBA/2 mice to treatment with the monoclonal anti-Thy 1.2 F7D5 serum as compared with a conventional alloantiserum is reported. Depletion of T helper cells, Con A-, PHA-, MLC-, and GVH-reactive cells from a DBA/2 or C57B1/6 spleen cell population was readily achieved with the conventional alloserum. In contrast, the F7D5 antiserum abolished all T functions studied in C57B1/6 spleen cells whereas it was totally or partially ineffective on DBA/2 spleen cells when T helper, MLC, or GVH reactivity were assayed. It did however eliminate the capacity of DBA/2 spleen cells to respond to stimulation with Con A or PHA. Analysis in an Ortho-Cytofluorograf of thymocytes and sIg^? lymphocytes labeled with either GAMB-F or F7D5 + RAM Ig-F showed no difference at the level of the thymocytes: Thy 1.2 antigen as revealed by either GAMB or F7D5 is similarly expressed in the two mouse strains. The fluorescence profiles of splenic T lymphocytes indicated a reduced representation per unit cell basis of the Thy 1.2 antigenic determinant recognized by F7D5 in DBA/2 mice. Moreover, this same determinant is expressed in only 70% of all Thy 1.2-positive cells detected in DBA/2 sIg^? population. This implies that, in DBA/2 mice, maturation of T cells is accompanied by a complete or partial loss of the F7D5 Thy 1.2 determinant and that T helper functions and MLC and GVH reactivity are mediated by T cells which express little or none of this F7D5 Thy 1.2 determinant.     

Adsorption equilibrium in immuno-affinity chromatography with polyclonal and monoclonal antibodies

The effects of pH, ionic strength, anion species, and antibody concentration on the adsorption equilibrium between immobilized antibodies and antigens were studied by use of anti-BSA, anti-HSA, anti-BlgG, and monoclonal anti-HSA coupled to Sepharose 4B. The polyclonal antibodies possessed average binding affinities of the order of 10(8)M(-1), and the heterogeneity was accounted for by assuming a normal distribution of the free energy of antibody-antigen combination. The monoclonal antibody, on the other hand, showed a homogeneous affinity of the Langmuir type. Bound antigens could be eluted by lowering pH or adding a chaotropic anion, and their purity was very high. The antibody ligand was sufficiently stable for repeated use.     

Select host cell proteins coelute with monoclonal antibodies in protein A chromatography

The most significant factor contributing to the presence of host cell protein (HCP) impurities in Protein A chromatography eluates is their association with the product monoclonal antibodies (mAbs) has been reported previously, and it has been suggested that more efficacious column washes may be developed by targeting the disruption of the mAbs-HCP interaction. However, characterization of this interaction is not straight forward as it is likely to involve multiple proteins and/or types of interaction. This work is an attempt to begin to understand the contribution of HCP subpopulations and/or mAb interaction propensity to the variability in HCP levels in the Protein A eluate. We performed a flowthrough (FT) recycling study with product respiking using two antibody molecules of apparently different HCP interaction propensities. In each case, the ELISA assay showed depletion of select subpopulations of HCP in Protein A eluates in subsequent column runs, while the feedstock HCP in the FTs remained unchanged from its native harvested cell culture fluid (HCCF) levels. In a separate study, the final FT from each molecule's recycling study was cross-spiked with various mAbs. In this case, Protein A eluate levels remained low for all but two molecules which were known as having high apparent HCP interaction propensity. The results of these studies suggest that mAbs may preferentially bind to select subsets of HCPs, and the degree of interaction and/or identity of the associated HCPs may vary depending on the mAb.     

Immunohistochemical heterogeneity of alpha-tubulin in human epithelia revealed with monoclonal antibodies

Four monoclonal antibodies raised to alpha subunit of pig brain tubulin (TU-01, TU-02, TU-03, TU-04) were used to study immunohistochemical heterogeneity of alpha tubulin in human epithelia. Selective reactivity was detected in the skin and trachea/bronchi, whereas all other epithelia investigated reacted uniformly with all four monoclonal antibodies. In the skin TU-01 reacted very strongly with all layers except the basal layer; TU-02 reacted strongly with granular layer and was unreactive or only weakly reactive with others; TU-03 reacted very strongly with basal layer and weakly to moderately with superficial layers; TU-04 reacted strongly with the granular layer of epidermis and was unreactive with other layers. In the trachea and major bronchi TU-01 reacted with the entire epithelial layer; TU-02 reacted only with superficial layer; TU-03 reacted with superficial and basal layer; TU-04 reacted only with superficial layer. Different staining patterns obtained with these four monoclonal antibodies indicate that there is immunohistochemical heterogeneity of alpha tubulin in some but not all normal human epithelia.     

Immunohistochemical heterogeneity of alpha-tubulin in human epithelia revealed with monoclonal antibodies

Summary Four monoclonal antibodies raised to alpha subunit of pig brain tubulin (TU-01, TU-02, TU-03, TU-04) were used to study immunohistochemical heterogeneity of alpha tubulin in human epithelia. Selective reactivity was detected in the skin and trachea/bronchi, whereas all other epithelia investigated reacted uniformly with all four monoclonal antibodies. In the skin TU-01 reacted very strongly with all layers except the basal layer; TU-02 reacted strongly with granular layer and was unreactive or only weakly reactive with others; TU-03 reacted very strongly with basal layer and weakly to moderately with superficial layers; TU-04 reacted strongly with the granular layer of epidermis and was unreactive with other layers. In the trachea and major bronchi TU-01 reacted with the entire epithelial layer; TU-02 reacted only with superficial layer; TU-03 reacted with superficial and basal layer; TU-04 reacted only with superficial layer. Different staining patterns obtained with these four monoclonal antibodies indicate that there is immunohistochemical heterogeneity of alpha tubulin in some but not all normal human epithelia.     

Multiple forms of ram and bull sperm hyaluronidase revealed by using monoclonal antibodies

Two monoclonal anti-sperm hyaluronidase-producing cell lines were isolated following inoculation of mice with ram sperm hyaluronidase monomer. Both lines produced antibodies of the IgG1 class; these bound to ram hyaluronidase after 'Western blotting' but did not recognize the native enzyme. Whereas the 1A4 antibody was specific for ram hyaluronidase, and did not react with 'blotted' bull, boar or rabbit hyaluronidase, the 1D6 antibody recognized bull as well as ram hyaluronidase. The antibodies could be used for immunocytochemical localization of hyaluronidase in fixed spermatozoa. However, although some form of denaturation was required to unmask or form the epitopes with which the antibodies reacted, the degree and type of fixation required was critical, for the epitopes were readily destroyed; in particular, they were very sensitive to chemical modification such as glutaraldehyde treatment. It could be demonstrated that, like ram, bull spermatozoa contained an extended oligomeric family of hyaluronidase forms, apparently the result of intermolecular disulphide cross-linking of monomers. In spermatozoa of both species, the enzyme was confined to the anterior acrosomal region of the head.     

Diversity of antigenic determinants of porcine zona pellucida revealed by monoclonal antibodies

Monoclonal antibodies derived from ten hybrid cell clones, generated against porcine zona pellucida gave strong immunofluorescence with zona but the pattern varied from patchy, thin rim to heavy precipitation type of rim. Five of the 6 monoclonals studied prevented the binding of the porcine epididymal sperm to homologous oocytesin vitro, whereas the sixth one was partially effective. All of the 6 monoclonale of this batch inhibited the lysis of zonae by proteolytic enzymes even at dilutions up to 1 × 10⁻³. Three of the four monoclonals prepared in a subsequent batch gave strong immunofluorescent reactions and had high titres as determined by enzyme immunoassay. These monoclonals did not, however, protect the zonae against lysis by proteolytic enzymes. These properties are suggestive of the heterogeneity of the antigenic determinants in zona and emphasize the employment of appropriate bioassays for screening and selection of bioeffective antibodies.     

Dissection of the human erythrocyte spectrin molecule using monoclonal antibodies.

One IgM and three IgG monoclonal antibodies specific to band 1 of human erythrocyte spectrin have been characterised. The antigenic sites of the IgG antibodies have been identified and mapped by radioimmune labelling of tryptic fragments of spectrin fractionated by SDS slab gel electrophoresis and blotted onto nitrocellulose filters. The binding site of one of these antibodies has also been directly visualised in the electron microscope after low-angle shadowing of the antibody-spectrin dimer complex, and lies at that end of the dimer which is responsible for tetramer formation.     

Tumor-specific idiotopes on suppressor factors and suppressor cells revealed by monoclonal anti-idiotope antibodies

Two monoclonal anti-idiotope antibodies, previously found to induce tumor-specific cell-mediated immunity in mice, were examined for their relationship to tumor-associated suppressor factors (SF), produced in culture by spleen cells from tumor-bearing mice or present in sera from such mice. A leukocyte adherence inhibition assay was used to detect cellular immunoreactivity to tumor antigens and its inhibition by SF, using peritoneal cells from mice bearing tumor or sensitized with anti-idiotope antibody. The SF were specifically absorbed by the corresponding anti-idiotope antibodies coupled to a solid phase and were recovered by elution. They were also specifically neutralized by the addition of the respective antibodies to the assay system. Anti-idiotope antibody, used with complement to pretreat spleen cells from tumor-bearing mice, prevented these cells from producing SF in culture. Tumor antigen-reactive effector cells, suppressor cells, and SF thus share similar idiotopes, permitting their respective functions to be modulated by appropriate anti-idiotopes.     

Purification of the bacterium-like organism associated with greening disease of citrus by immunoaffinity chromatography and monoclonal antibodies

An immunoaffinity chromatographic procedure with monoclonal antibodies (MA) has been developed for purification of the uncultivable, bacterium-like organism associated with greening disease of citrus. The greening organism (GO) was partially purified from leaf midribs of infected periwinkle plants by differential centrifugation. The GO present in such preparations was retained on an affinity matrix consisting of CNBr-activated Sepharose 4B on which GO-specific MA had been covalently linked. The unbound plant material was washed from the matrix, and the GOs were eluted with 0.1M glycine (pH 11.5). Purified GOs were compared with organisms observed in the initial plant preparation by both immunofluorescence and electron microscopic techniques. The morphology and serological characteristics of the GO were retained following purification procedures.