Localization of the Trypanosoma cruzi-specific Mr 25,000 antigen by immune electron microscopy using monoclonal antibodies

Two monoclonal antibodies reacted with the Trypanosoma cruzi-specific antigen of an apparent Mr 25,000 from all developmental forms (Tachibana et al. 1986). This T. cruzi-specific antigen was found at the plasma membrane by immunoperoxidase electron microscopy using the monoclonal antibodies TCF48 and TCF87. The TCF48 and TCF87-treated cells showed stain deposits at the plasma membrane clearly distinguishable from those in cells treated with a monoclonal antibody against a surface antigen. This suggests that the epitope(s) of the Mr 25,000 antigen is located on the inner surface or in the matrix of the plasma membrane. TCF48 and TCF87 also reacted with an antigen on the microtubules of the axoneme, but not with the subpellicular microtubules. These results suggest that the T. cruzi-specific Mr 25,000 antigen is common to both the plasma membrane and axoneme but it is not located at the subpellicular microtubules. Its identity and that of the surface antigen, Gp25 (Scharfstein et al. 1983) as well as its role in the pathogenicity of the parasite are discussed.     

A 40 kDa nuclear membrane protein changes its concentration and localization within the nuclear envelope of the yeast Saccharomyces cerevisiae in a cell-cycle dependent manner

In order to identify and characterize structural components in the nuclear membrane of Saccharomyces cerevisiae which show a cell-cycle dependent regulation, we have undertaken a combined biochemical/immunofluorescence microscopy approach. Antisera raised against nuclear membrane proteins from yeast lead to the identification of a 40 kDa membrane protein which cofractionated with nuclei upon cell fractionation. This 40 kDa membrane protein partitioned into the Triton X-114 phase and was not extracted from purified nuclei at alkaline pH. Using affinity-purified antibodies against this protein, the antigen was localized at the nuclear periphery suggesting that it is an integral constituent of the nuclear envelope. However, the 40 kDa antigen revealed a heterogenous distribution within the nuclear membrane: in indirect immunofluorescence microscopy, nuclei isolated from an asynchronously growing yeast culture showed either no immunodetectable antigen or contained it in a cap-, dot- or ring-like conformation. Using synchronized yeast cultures, we could demonstrate cell-cycle dependent changes of concentration and localization of the 40 kDa protein within the nuclear envelope.     

Monoclonal antibodies to antigens abnormally expressed in breast cancer

We report the production, screening, and characterization of ten murine monoclonal antibodies directed at antigens that are expressed abnormally in human breast tumors. Immunoperoxidase staining of frozen and fixed tissues shows the antigens to be present at low levels on the luminal membrane of normal breast cells and at high levels in the cytoplasm and surface membrane of breast tumor cells. The ten antibodies appear to recognize six different epitopes on the basis of their quantitative differences in reactivity against four antigen preparations, as measured by ELISA. Immunoblots show that eight of the ten antibodies recognize a 300,000 MW molecule from breast tumor preparations; six of these antibodies also react with a second molecule from the same tumor preparations of 280,000 MW. Seven antibodies react with an antigen from milk fat globule membrane of 330,000 MW. It therefore appears that the two molecules from tumor tissue and the one molecule from normal tissue share common epitopes. Selected antibodies were tested for reactivity against 25 primary breast tumors and 14 pairs of primary and metastatic breast tumors. Three antibodies have broad reactivity and stain more than 80% of primary tumors; the three other antibodies identify subsets of those tumors. Results of staining pairs of primary and metastatic lesions show that metastases continue to express antigens of the primary lesion in a high percentage of cells.     

Separation of luminal and abluminal membrane enriched domains from cultured bovine aortic endothelial cells: monoclonal antibodies specific for endothelial cell plasma membranes

Two kinds of membrane (luminal and abluminal membrane domains) fractions have been isolated from bovine aortic endothelial cells by fractionation of whole cell homogenate on discontinuous sucrose density gradients. The luminal membrane domain was enriched 12-16-fold for angiotensin-converting enzyme activity and 8-10-fold in alkaline phosphatase activity. The abluminal membrane domain displayed an enrichment of 8-fold in (Na+ + K+)-ATPase activity. Both of the membrane domains were minimally contaminated with mitochondria, microsomes and Golgi bodies, as assessed by their corresponding marker enzyme activities. 125I-labeling of endothelial cell monolayers by the Enzymo-Bead lactoperoxidase-catalyzed iodination procedure, followed by isolation of membranes, revealed that the radioactivity was predominantly associated with membranes enriched in angiotensin-converting enzyme activity, corresponding to the luminal membrane domain. However, when cells were radioiodinated in suspension culture, radioactivity was found equally associated in both the luminal and abluminal membrane fractions. Electron microscopy of freeze-fractured and sectioned material showed both luminal and abluminal membrane domains to be in the form of vesicles varying in size from 100 to 400 nm in diameter. To characterize the separation of endothelial cell membrane domains, we have attempted to prepare monoclonal antibodies specific for endothelial cells. Several clones were obtained, producing antibodies which bound to endothelial cells of arterial, venous and capillary origin. Two antibodies of these clones, XIVC6 and XVD2, were studied in more detail. In the ELISA assay, these antibodies reacted with bovine vascular endothelial cells, but not with human umbilical cord endothelial cells, nor with bovine corneal endothelial cells, smooth muscle cells or fibroblasts. Both of these antibodies are directed against an antigen of approximately 130 kDa, under reducing and non-reducing conditions, as assayed by the immunoprecipitation method. Western blot analysis of luminal and abluminal membrane fractions revealed that only MAb XVD2 reacted with an antigen, indicating that the antibody XIVC6 is directed against an epitope which is denatured by SDS. Moreover, MAb XVD2 preferentially reacted with the luminal membrane compared to the abluminal membrane domain of the endothelial cell. These monoclonal antibodies do not react with platelet membrane proteins, indicating that this 130 kDa membrane antigen is not common to both endothelial cells and platelets.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ultrastructural localization of human kidney antigens using monoclonal antibodies.

A highly sensitive method of ultrastructural-immunoperoxidase staining was developed for use with monoclonal antibodies which have been raised in this laboratory to a variety of antigens of the human kidney. Because of the susceptibility of the antigens to fixation and processing, a four layer, pre-embedding method of staining was used. Results confirmed and clarified previously reported light microscopy results, indicating that an antigen recognized by the PHM5 antibody was found on the podocyte cell membrane within the glomerulus and was not present within the glomerular basement membrane. The antigen was also present on the extraglomerular endothelial cell membrane. The study also demonstrated the presence of an antigen specific to endothelial cells throughout the renal cortex, and gave further insight into the precise localization of glomerular basement membrane components including fibronectin. The method of staining is now being used together with detailed ultrastructural studies to identify the cells produced from isolated glomeruli in tissue culture.     

Detection of cytoskeletal proteins in small cell lung carcinoma

Small cell lung carcinoma (SCLC) is the most aggressive of lung tumors, metastasize widely and are virtually incurable by surgical means. Therefore, the classification of lung cancer into SCLC and non-small cell lung carcinoma is essential for disease prognosis and treatment. For this purpose we have compared the immunohistochemical distribution of different cytoskeletal proteins as tumor markers. Analysis was performed by using of monoclonal antibodies directed against cytokeratins, neurofilaments, betaIII-tubulin, epithelial membrane antigen and neuron-specific enolase. Our results indicate that keratin and epithelial membrane antigen are reliable epithelial markers for SCLC. In addition, the positive staining with monoclonal antibodies TU-20 against betaIII-tubulin and neuron-specific enolase was found in some cases of SCLC. We suggest, that these antibodies could be a useful tool for complex immunohistochemical diagnosis of SCLC.     

Characterization of a tubular basement membrane component reactive with autoantibodies associated with tubulointerstitial nephritis

A kidney tubular basement membrane (TBM) component that is bound by antibodies from individuals with anti-TBM antibody-associated tubulointerstitial nephritis (TIN) was purified and characterized (TIN antigen). TIN antigen was prepared from rabbit TBM by extraction with guanidine and purified by ion-exchange, gel filtration, and reversed-phase chromatography. Based upon yields of protein and antibody reactivity, TIN antigen accounts for about 9% of the mass of TBM and thus is a major component of this basement membrane. A predominant 58-kDa form comprises about 90% of purified TIN antigen, and a 50-kDa form accounts for the remainder. The two forms share the amino-terminal sequence Ser-Ile-Phe-Gln-Gly-Gln-Tyr-X-Arg-Ser-Phe-Gly- and give similar tryptic peptide maps, indicating that they are structurally related. Their amino acid compositions overall are similar to laminin and entactin/nidogen. The absence of hydroxyproline and hydroxylysine and the low levels of glycine in TIN antigen indicate that it is noncollagenous. No similarities were found between other known proteins and sequences of tryptic peptides and the amino terminus of TIN antigen, suggesting that it is distinct from other characterized basement membrane components. A goat polyclonal antibody toward rabbit TIN antigen showed the same kidney distribution as human antibodies and was completely inhibited in enzyme-linked immunosorbent assay by purified TIN antigen. These data further support the idea that TIN antigen is the primary target for anti-TBM antibodies associated with TIN. This research presents methods to prepare TIN antigen for biochemical studies and investigations of its role in anti-TBM autoimmune TIN.     

Immuno-isolation of vesicles using antigenic sites either located on the cytoplasmic or the exoplasmic domain of an implanted viral protein. A quantitative analysis

In this study, we present a new general approach for immuno-isolation: a foreign integral membrane protein, the G-protein of vesicular stomatitis virus (VSV), is implanted into the plasma membrane for subsequent immuno-isolation. A quantitative analysis was accomplished using the erythrocyte plasma membrane as a model system. The virus was artificially bound to the membrane via a lectin and subsequently fused at low pH. Vesicles of two opposite orientations were prepared from erythrocytes with fused G-protein. Right-side-out and inside-out vesicles expose the exoplasmic and the cytoplasmic domains of the G-protein on their surfaces respectively. In immuno-isolation experiments antibodies against each of the domains of the G-protein were used. Vesicles were presented to an immunoadsorbent (ImAd) consisting of a solid support with appropriate antibodies bound to its surface. Two commonly used immunoadsorbents prepared from either polyacrylamide beads or fixed Staphylococcus aureus cells were compared and found to have identical immuno-isolation efficiencies. It was possible to control and quantitate the amount of implanted antigen. Therefore, we were able to show that the critical antigen density required for immuno-isolation is 50 G molecules/micron2 plasma membrane surface area for both types of vesicle/antibody couples. This analysis showed that vesicles presenting either the cytoplasmic or the exoplasmic domain of the G-protein are immuno-isolated with the same efficiency.     

Human beta-adrenergic receptors. Simultaneous purification of beta 1- and beta 2-adrenergic-receptor peptides.

Monoclonal antibodies to insulin secretory granule membranes were obtained following immunization of mice with granule membranes purified from a rat transplantable insulinoma. The specificities of the antibodies were investigated by using binding assays with different insulinoma subcellular fractions, by indirect immunofluorescence studies with intact and permeabilized cells, and by immunoblotting of granule membrane proteins fractionated by SDS/polyacrylamide-gel electrophoresis. Fifty-six antibodies were characterized initially, and 21 representative cell lines were cloned. The antibodies fell into four categories: (1) binding preferentially to secretory granules, and reacting with a component of approx. 80,000 Da on immunoblots (antigen designated SGM 80); (2) binding preferentially to secretory granules, and reacting with components of approx. 110,000 and 50,000 Da on immunoblots (antigen designated SGM 110); (3) binding preferentially to secretory granules but unreactive on immunoblots; (4) binding to membrane antigen(s) with a widespread intracellular distribution which included granules and plasma membranes. The antigens SGM 80 and SGM 110 were studied in more detail and both were shown to be integral membrane glycoproteins with antigenic determinants located on the internal face of the secretory granule membrane. These antigens were also present in normal rat islets of Langerhans and similar components were detected by immunoblotting in secretory granules from anterior pituitary and adrenal medulla. Proteins which were immunologically related to SGM 80 and SGM 110, but distinct in molecular size, were also identified in liver. It is concluded that secretory granules contain specific components which are restricted in subcellular location but widespread in tissue distribution. The antibodies obtained will be valuable reagents in the further investigation of the biogenesis and turnover of insulin secretory granules.     

Biochemical characterization and epididymal localization of the maturation-dependent ram sperm surface antigen ESA152

We examine here the biochemical properties and epididymal localization of a maturation dependent ram sperm surface antigen. A monoclonal antibody, ESA152, identifies an antigen that is present on the surface of ejaculated sperm, but is absent from testicular sperm. Crosslinking of the ESA152 antigen with bivalent antibodies induces the acrosome reaction, redistributing the antigen into the anterior region of the sperm head where it associates with the fusion product of the plasma membrane and the outer acrosomal membrane. The ESA152 antigen appears as a polypeptide of 18 kDa on immunoblots of SDS-polyacrylamide gels. The ESA152 epitope includes the sialic acid termini of N-linked oligosaccharides, as shown by its sensitivity to neuraminidase and endoglycosidase F. The ESA152 antigen is a highly hydrophobic integral membrane protein that resists aqueous extraction, partitions into the detergent phase of Triton-X-114, and solubilizes in chloroform-methanol mixtures. The anchoring of ESA152 is unaffected by phosphtidylinositol specific phospholipase C. The antigen is absent from extracts of caput and corpus epididymidis but appears abruptly in the first segment of the cauda. Immunofluorescence reveals that the ESA152 epitope first appears in clusters of cells in the luminal epithelium of the proximal cauda, prior to or concurrent with its appearance on sperm. © 1993 Wiley-Liss, Inc.     

Human glomerular basement membrane. Antigenic determinants in human urine

Antisera against particulate human glomerular basement membrane prepared from cadaver kidneys were raised in rabbits. It was shown that both normal individuals and patients with glomerular and tubular diseases excrete in their urine several antigens reactive with these antibodies. One antigen crossreacted immunologically with an antigen from human glomerular basement membrane while several others did not. One of the urinary antigens and the antigen crossreacting with the basement membrane were separated from the others by ion exchange chromatography and gel filtration, respectively.The pattern of antigen excretion differed depending on the underlying renal disease but the multitude of different antigens detected complicates the interpretation of the patterns of excretion in different diseases.     

A Rapid Method to Characterize Mouse IgG Antibodies and Isolate Native Antigen Binding IgG B Cell Hybridomas

B cell hybridomas are an important source of monoclonal antibodies. In this paper, we developed a high-throughput method to characterize mouse IgG antibodies using surface plasmon resonance technology. This assay rapidly determines their sub-isotypes, whether they bind native antigen and their approximate affinities for the antigen using only 50 μl of hybridoma cell culture supernatant. Moreover, we found that mouse hybridomas secreting IgG antibodies also have membrane form IgG expression without Igα. Based on this surface IgG, we used flow cytometry to isolate rare γ2a isotype switched variants from a γ2b antibody secreting hybridoma cell line. Also, we used fluorescent antigen to single cell sort antigen binding hybridoma cells from bulk mixture of fused hybridoma cells instead of the traditional multi-microwell plate screening and limiting dilution sub-cloning thus saving time and labor. The IgG monoclonal antibodies specific for the native antigen identified with these methods are suitable for in vivo therapeutic uses, but also for sandwich ELISA assays, histology, flow cytometry, immune precipitation and x-ray crystallography.     

Prevalence of a Vibrio cholerae 18-kDa antigen in vibrios

Abstract An 18-kDa protein that occurs in Vibrio cholerae has been described as an in vivo and low-iron regulated outer membrane antigen. Monoclonal antibodies which recognized this antigen were protective as passive vaccines in the infant rabbit model of cholera disease. In this study, those monoclonal antibodies were used in three immunological assays for surveillance of various bacteria for the 18-kDa antigen. ELISA, and Western blot assays gave variable results with bacteria or outer membrane preparations. The biodot assay was the most sensitive test, detecting the 18-kDa antigen in 29 of 29 V. cholerae strains, independent of biotype or serotype. A few other Gram-negative bacteria and V. parahaemolyicus reacted weakly with our antibodies and antiserum.     

Nonlabeled secondary antibodies augment/maintain the binding of primary, specific antibodies to cell membrane antigens.

BACKGROUND: In studies on surface membrane antigen expression using immunofluorescence techniques, it is commonly observed that direct staining gives weaker signals than the signals following indirect staining with fluorochrome-conjugated secondary antibodies. This is most marked when cells have also been permeabilized in order to stain intracellular protein. The commonly accepted explanation for this observation is that fluorochrome-conjugated secondary antibodies bind to a higher number of binding sites on the primary antibody, as compared to the binding of conjugated primary antibodies to the membrane antigens. Another hypothesis might be that the antibody/antibody complexes formed on the membranes when using the indirect technique may have an augmented ability to bind the membrane epitopes. The present study was performed in order to check this hypothesis. MATERIALS AND METHODS: Peripheral blood mononuclear cells were stained with fluorochrome-conjugated anti-CD antibodies directly without or with a second-step application of nonconjugated goat anti-mouse IgG antibodies, followed by different fixation and permeabilization methods. The cells were analyzed by flow cytometry. RESULTS: A second-step application of nonconjugated goat anti-mouse IgG antibodies following direct staining with fluorochrome-conjugated anti-CD antibodies gave a significant increase in membrane antigen expression on permeabilized cells as compared to direct staining alone. The secondary antibody must be bivalent, since whole IgG or F(ab')(2) fragments of the goat anti-mouse antibodies showed effects, while Fab fragments did not. CONCLUSIONS: Nonlabeled secondary antibodies are able to influence the binding of primary, specific antibodies to cell membrane antigens on cells treated with permeabilizing agents necessary for staining intracellular proteins. The improved membrane antigen expression seems to be due to the formation of a network of primary and secondary antibodies on the cell surface, with increased ability for maintaining binding to CD antigens.     

Characterization of rat hepatocyte plasma membrane domains by monoclonal antibodies

The hepatocyte plasma membrane consists of three morphologically and functionally distinct domains, the sinusoidal, the lateral and the canalicular. To study the distribution of antigenic determinants among these domains, we prepared monoclonal antibodies by immunizing mice with a crude, plasma membrane-enriched liver fraction. Four monoclonal antibodies were obtained that recognized various parts of the rat hepatocyte plasma membrane when tested by indirect immunofluorescence and immunoperoxidase assay performed on formaldehyde-fixed liver tissue. Each antibody gave a different staining pattern when analyzed by light and electron microscopy. A59 exclusively labelled the part of the sinusoidal membrane facing the sinusoids. A39 mainly labelled the sinusoidal membrane. B1 mainly labelled the lateral membrane, while the labelling by B10 was almost completely limited to the canalicular membrane. Immunoblotting showed that the antibody B1 recognized an antigen of approximately 100 kilodaltons and that B10 recognized an antigen of approximately 125 to 130 kilodaltons. These antibodies allow us to distinguish the three domains of the hepatocyte plasma membrane.     

Human glomerular basement membrane. Antigenic determinants in human urine.

Antisera against particulate human glomerular basement membrane prepared from cadaver kidneys were raised in rabbits. It was shown that both normal individuals and patients with glomerular and tubular diseases excrete in their urine several antigens reactive with these antibodies. One antigen crossreacted immunologically with an antigen from human glomerular basement membrane while several others did not. One of the urinary antigens and the antigen crossreacting with the basement membrane were separated from the others by ion exchange chromatography and gel filtration, respectively. The pattern of anttigen excretion differed depending on the underlying renal disease but the multitude of different antigens detected complicates the interpretation of the patterns of excretion in different diseases.     

Structural characterization of developmentally expressed antigenic markers on chicken erythrocytes using monoclonal antibodies

Monoclonal antibodies selected for embryonic and adult erythrocyte specificity have been used to characterize developmentally expressed markers on the surfaces of mature circulating erythrocytes of young and adult chickens. The data presented demonstrate that the antigenic changes which occur on the avian erythrocyte membrane with organismic maturation can be accounted for, at least in part, by changes in the expression of structurally different, but possibly related polypeptides. Monoclonal antibodies selected for specific reactivity with the erythrocytes of newly hatched chicks recognize a glycoprotein of 48,000 daltons apparent molecular weight. On two-dimensional isoelectric focusing gels, this antigen, which appears identical in all strains studied, displays microheterogeneity; consisting of eight to nine closely spaced spots with an isoelectric midpoint of approximately 5.5. This antigen is not expressed on the circulating erythrocytes of mature birds; however, an antigen with similar, but perhaps not completely identical structure, can be detected within the adult bone marrow. The monoclonal antibodies which show preferential binding to the circulating erythrocytes of adult birds also immune precipitate an antigen of 48,000 daltons apparent molecular weight, but this antigen has a more basic isoelectric point. The adult antigen is polymorphic. Slightly different patterns were obtained on two-dimensional gels with erythrocytes from inbred birds having different major histocompatibility genotypes. It has a major component near pH 7.0 and additional focusing spots usually occurring at a slightly lower molecular weight near pH 6.8 or 6.6 depending upon strain. Competitive radiobinding assays with B-system-specific alloantisers suggest that these antigens may in fact be antigens of the polymorphic BG locus of the chicken major histocompatibility complex. One-dimensional peptide mapping of the immune precipitated embryonic and adult erythrocyte polypeptides demonstrate that the antigens are borne on distinct but possibly related polypeptides. Both common and unique peptide fragments are found in the digestion products. Selective solubilization of the chicken erythrocyte membrane suggests that the antigens are integral membrane proteins extractable with nonionic detergent but not with reagents which remove peripheral proteins.     

Tissue distribution and biochemical and functional properties of Tp55 (CD27), a novel T cell differentiation antigen

Two monoclonal antibodies (CLB-CD 27/1 and CLB-CD 27/2) were raised against a novel determinant on human T lymphocytes. One of these antibodies, CLB-CD 27/1 (clone 9F4), was grouped by the Third International Workshop and Conference on Human Leucocyte Differentiation Antigens together with three other monoclonal antibodies (VIT 14, OKT 18A, and S152) in the new cluster CD27. In this paper we show that antibodies belonging to this cluster recognize an antigen present on a large subset of peripheral T lymphocytes and most medullary thymocytes. At least two different nonoverlapping epitopes were identified with directly labeled monoclonal antibodies. Immunoprecipitation studies indicate that the target antigen of CD27 antibodies is a polypeptide of 55 kDa, which appears in the form of a disulfide-linked homodimer on the T lymphocyte membrane (Tp55). Stimulation of T cells via the T3/T cell antigen-receptor complex, with either phytohemagglutinin or CD3 monoclonal antibodies, resulted in a fivefold increase in the membrane expression of Tp55, whereas activation by phorbol myristate acetate caused a marked down-regulation. Moreover, an additional molecule of 32 kDa was precipitated from the membrane of activated but not of resting T cells. Addition of CD27 antibodies to cultures stimulated with either phytohemagglutinin or CD3 monoclonal antibody led to enhanced proliferation, whereas no effect was observed in phorbol myristate acetate or interleukin 2-stimulated cultures. The possible role of the Tp55 antigen in T cell activation is discussed.     

HIV-1 antibodies and vaccine antigen selectively interact with lipid domains

The rare, broadly neutralizing antibodies, 4E10 and 2F5, that target the HIV-1 membrane proximal external region also associate with HIV-1 membrane lipids as part of a required first-step in HIV-1 neutralization. HIV-1 virions have high concentration of cholesterol and sphingomyelin, which are able to organize into liquid-ordered domains (i.e., lipid rafts), and could influence the interaction of neutralizing antibodies with epitopes proximal to the membrane. The objective of this research is to understand how these lipid domains contribute to 2F5/4E10 membrane interactions and to antigen presentation in liposomal form of HIV-1 vaccines. To this end we have engineered biomimetic supported lipid bilayers and are able to use atomic force microscopy to visualize membrane domains, antigen clustering, and antibody–membrane interactions. Our results demonstrate that 2F5/4E10 do not interact with highly ordered gel and liquid-ordered domains and exclusively bind to a liquid-disordered lipid phase. This suggests that vaccine liposomes that contain key viral membrane components, such as high cholesterol content, may not be advantageous for 2F5/4E10 vaccine strategies. Rather, vaccine liposomes that primarily contain a liquid-disordered phase may be more likely to elicit production of lipid reactive, 2F5- and 4E10-like antibodies.