Monoclonal antibodies against spore antigens of Bacillus anthracis

A murine monoclonal antibody produced against heat inactivated spores of Bacillus anthracis Ames, reacted with live or inactivated spores of several anthrax strains in indirect immunofluorescence (IF) tests. The reactive anthrax strain gave only a moderate degree of reaction. No staining of anthrax vegetative cells was observed. The monoclonal did not react with spores of non-anthrax Bacillus strains that gave cross reactions with mouse hyperimmune antiserum raised against Ames spores. The staining of individual spores in B. anthracis preparations was more heterogeneous with the monoclonal antibody than with the hyperimmune serum. Evidence is produced that the epitope for this monoclonal is not stable during long-term storage of inactivated spore preparations, and is not fully available for reaction with antibody until late in spore maturation. The monoclonal did not react by immunoblotting (Western blotting) of spore extracts. A monoclonal antibody produced against Ames spore extracts reacted with about 1% of Ames spores in IF tests, but not reproducible reactions with other anthrax strains were recorded. This monoclonal interacted with three bands in Western blots of anthrax spore extracts.     

Monoclonal antibodies against spore antigens of Bacillus anthracis

Abstract A murine monoclonal antibody produced against heat inactivated spores of Bacillus anthracis Ames, reacted with live or inactivated spores of several anthrax strains in indirect immunofluorescence (IF) tests. The reactive anthrax strain gave only a moderate degree of reaction. No staining of anthrax vegetative cells was observed. The monoclonal did not react with spores of non-anthrax Bacillus strains that gave cross reactions with mouse hyperimmune antiserum raised against Ames spores. The staining of individual spores in B. anthracis preparations was more heterogeneous with the monoclonal antibody than with the hyperimmune serum. Evidence is produced that the epitope for this monoclonal is not stable during long-term storage of inactivated spore preparations, and is not fully available for reaction with antibody until late in spore maturation. The monoclonal did not react by immunoblotting (Western blotting) of spore extracts.     

Characterization and epitope mapping of neutralizing monoclonal antibodies produced by immunization with oligomeric simian immunodeficiency virus envelope protein

In an attempt to generate broadly cross-reactive, neutralizing monoclonal antibodies (MAbs) to simian immunodeficiency virus (SIV), we compared two immunization protocols using different preparations of oligomeric SIV envelope (Env) glycoproteins. In the first protocol, mice were immunized with soluble gp140 (sgp140) from CP-MAC, a laboratory-adapted variant of SIVmacBK28. Hybridomas were screened by enzyme-linked immunosorbent assay, and a panel of 65 MAbs that recognized epitopes throughout the Env protein was generated. In general, these MAbs detected Env by Western blotting, were at least weakly positive in fluorescence-activated cell sorting (FACS) analysis of Env-expressing cells, and preferentially recognized monomeric Env protein. A subset of these antibodies directed toward the V1/V2 loop, the V3 loop, or nonlinear epitopes were capable of neutralizing CP-MAC, a closely related isolate (SIVmac1A11), and/or two more divergent strains (SIVsmDeltaB670 CL3 and SIVsm543-3E). In the second protocol, mice were immunized with unfixed CP-MAC-infected cells and MAbs were screened for the ability to inhibit cell-cell fusion. In contrast to MAbs generated against sgp140, the seven MAbs produced using this protocol did not react with Env by Western blotting and were strongly positive by FACS analysis, and several reacted preferentially with oligomeric Env. All seven MAbs potently neutralized SIVmac1A11, and several neutralized SIVsmDeltaB670 CL3 and/or SIVsm543-3E. MAbs that inhibited gp120 binding to CD4, CCR5, or both were identified in both groups. MAbs to the V3 loop and one MAb reactive with the V1/V2 loop interfered with CCR5 binding, indicating that these regions of Env play similar roles for SIV and human immunodeficiency virus. Remarkably, several of the MAbs generated against infected cells blocked CCR5 binding in a V3-independent manner, suggesting that they may recognize a region analogous to the conserved coreceptor binding site in gp120. Finally, all neutralizing MAbs blocked infection through the alternate coreceptor STRL33 much more efficiently than infection through CCR5, a finding that has important implications for SIV neutralization assays using CCR5-negative human T-cell lines.     

Induction of an immune response through the idiotypic network with monoclonal anti-idiotype antibodies in the carcinoembryonic antigen system.

Anti-idiotype antibodies can mimic the conformational epitopes of the original antigen and act as antigen substitutes for vaccination and/or serological purposes. To investigate this possibility concerning the tumor marker carcinoembryonic antigen (CEA), BALB/c mice were immunized with the previously described anti-CEA monoclonal antibody (MAb) 5.D11 (AB1). After cell fusion, 15 stable cloned cell lines secreting anti-Ids (AB2) were obtained. Selected MAbs gave various degrees of inhibition (up to 100%) of the binding of 125I-labeled CEA to MAb 5.D11. Absence of reactivity of anti-Id MAbs with normal mouse IgG was first demonstrated by the fact that anti-Id MAbs were not absorbed by passage through a mouse IgG column, and second because they bound specifically to non-reduced MAb 5.D11 on Western blots. Anti-5.D11 MAbs did not inhibit binding to CEA of MAb 10.B9, another anti-CEA antibody obtained in the same fusion as 5.D11, or that of several anti-CEA MAbs reported in an international workshop, with the exception of two other anti-CEA MAbs, both directed against the GOLD IV epitope. When applied to an Id-anti-Id competitive radioimmunoassay, a sensitivity of 2 ng/ml of CEA was obtained, which is sufficient for monitoring circulating CEA in carcinoma patients. To verify that the anti-Id MAbs have the potential to be used as CEA vaccines, syngeneic BALB/c mice were immunized with these MAbs (AB2). Sera from immunized mice were demonstrated to contain AB3 antibodies recognizing the original antigen, CEA, both in enzyme immunoassay and by immunoperoxidase staining of human colon carcinoma. These results open the perspective of vaccination against colorectal carcinoma through the use of anti-idiotype antibodies as antigen substitutes.     

Production and characterization of a novel monoclonal antibody against Vibrio parahaemolyticus F0F1 ATP synthase's delta subunit and its application for rapid identification of the pathogen

We raised monoclonal antibodies (MAbs) against Vibrio parahaemolyticus cell extracts. One of the MAbs, designated MAb-VP34, reacted in enzyme-linked immunosorbent assays (ELISAs) with 140 V. parahaemolyticus strains, regardless of serotype or origin. MAb-VP34 did not detectably react with 96 strains belonging to 27 other Vibrio species (except for Vibrio natriegens) or with 29 non-Vibrio species. These results show that MAb-VP34 is highly specific for V. parahaemolyticus. Western blotting and mass spectrometry analyses revealed that MAb-VP34 recognized V. parahaemolyticus F₀F₁ ATP synthase's delta subunit.Using MAb-VP34, a rapid and simple immunodot blotting assay (VP-Dot) was developed to determine whether bacterial colonies growing on selective agar, represented V. parahaemolyticus. To evaluate VP-Dot, 20 V. parahaemolyticus strains and 19 non-related strains were tested. The results indicated that VP-Dot is a reliable tool for identification of V. parahaemolyticus colonies. The simple VP-Dot procedure took 40 min, indicating that the MAb-VP34 based immunological method will greatly reduce labor, time, and costs required to verify V. parahaemolyticus colonies as compared with the conventional biochemical test.     

Immunohistochemical localization of heparan sulfate proteoglycan in human gastrointestinal tract

Heparan sulfate proteoglycans (HS-PGs) are associated with important cell functions, for example, cell motility, cell adhesion, and oncogenesis. We examined the localization of HS-PGs in normal and carcinoma tissues of the gastrointestinal tract to help elucidate their roles in these organs. Fresh surgical materials from 134 patients with carcinoma of the stomach or large intestine and 26 patients with various diseases of the small intestine were immunostained after fixation with 10E4 (an antibody against the HS of HS-PG) as a primary antibody. Immunoelectron microscopy (immunogold method) was also performed. The basolateral surfaces of normal tissues of the large and small intestines were strongly stained with antibody confirmed by electron microscopy. In the stomach, lesions with intestinal metaplasia showed the same staining as the intestines, although normal gastric tissue showed staining only in some parts of the basal layer of fundic and pyloric glands. Carcinoma tissues in all cases examined showed staining with antibody. Better results were obtained after fixation in acetic alcohol or zinc-containing solutions than in ordinary formalin. These characteristic localizations of HS-PG in intestines and stomachs suggest that this kind of HS-PG staining could be a hallmark characteristic of the intestine.     

Production and properties of monoclonal antibodies to Solanum nodiflorum mottle sobemovirus

Black raspberry necrosis virus (BRNV) reaches only very low concentrations in herbaceous plants and is difficult to maintain in culture. However, in a mixed culture with an unrelated virus, Solanum nodiflorum mottle (SNMV), in the genus Sobemovirus, the concentration of BRNV particles increases about 1000‐fold. In attempts to produce monoclonal antibodies (MAbs) to BRNV for diagnostic use, purified virus particles from the mixed virus culture were used as immunogen and the resultant antibodies screened against cultures of SNMV alone, BRNV+SNMV and healthy plant extracts. None of the virus‐specific MAbs obtained in this way was specific to BRNV but six were specific to SNMV. Although the original objective was not achieved, the SNMV MAbs were characterised and used to study serological properties of SNMV and other Sobemoviruses. Characterisation of the six SNMV MAbs showed that four were IgG3, one IgG1 and the other IgG2b. SNMV was detected by all six MAbs in ELISA, by five in Western blotting, by three in agarose gel double diffusion tests, but only one was suitable for trapping virus particles in immuno‐electron microscopy (IEM). In Western blotting using virus in sap extracts of Nicotiana clevelandii, each of the five MAbs detected a single major band of Mc. 31 000 in sap containing SNMV, and additional bands of lower mass attributed to degradation of coat protein. In various serological tests, no cross‐reactions were detected between SNMV and seven other viruses from the genus Sobemovirus. However, in IEM but not in Western blotting, significant cross‐reactions were observed between SNMV and Velvet tobacco mottle virus, another species from the genus Sobemovirus. The significance of these different findings is discussed.     

人小肠三叶因子在大肠杆菌中的表达

利用PCR技术将人小肠三叶因子(hITF)基因重组入表达载体pGEX-4T-1,构建了融合蛋白GST-hITF的重组表达质粒pGTF,在大肠杆菌中诱导表达。表达的融合蛋白经亲和层析、凝血酶切和凝胶过滤层析得到纯化的hITF蛋白。测定了重组蛋白的氨基酸组成、分子量及其对酸和蛋白酶的抗性。Western印迹表明重组蛋白具有hITF的抗原性,并对大鼠胃溃疡具有明显的预防和保护作用。     

Reactivity of an anti-(human gastric carcinoma) monoclonal antibody with core-related peptides of gastrointestinal mucin

Summary A murine anti-(human gastric carcinoma) monoclonal antibody, GL-013 (IgG1), which reacts with a high-molecular-mass glycoprotein from colorectal tumour tissue [Yang and Price (1989) Anticancer Res 9: 1707], was examined for reactivity against a panel of purified and partially purified antigens associated with tumours of the gastrointestinal tract. These included carcinoembryonic antigen (CEA), normal cross-reacting antigen, Y-hapten glycoproteins, and perchloric acid extracts and glycolipid preparations from colorectal tumours. While the GL-013 antibody failed to bind to these antigens, it was found to react strongly with synthetic peptides with sequences based upon that reported for the protein core of a human gastrointestinal mucin [Barnd et al. (1989) Proc Natl Acad Sci USA 86: 7159; Gum et al. (1989) J Biol Chem 264: 6480]. In control tests, a series of other anti-(colorectal tumour) antibodies (IgG1 and IgG3), with broad reactivity towards gastrointestinal carcinomas, as well as an anti-CEA antibody, (IgG1) failed to react with the synthetic peptides. It is concluded that the anti-(gastric carcinoma) monoclonal antibody GL-013 binds to a threonine-rich peptide epitope expressed within the protein core of gastrointestinal mucins. Present address: Cancer Research Institute, China Medical University, Shenyang, Liaoning, People's Republic of China     

Expression of catalase and glutathione peroxidase in renal insufficiency

Chronic renal failure (CRF) is associated with oxidative stress, the precise mechanism of which is yet to be elucidated. The present study was undertaken to investigate in renal insufficiency the expression of catalase and glutathione peroxidase, which play a critical role in antioxidant defense system by catalyzing detoxification of hydrogen peroxide (H2O2) and organic hydroperoxides. Rats were randomly assigned to the CRF (5/6 nephrectomized) and sham-operated control groups and observed for 6 weeks. Renal and thoracic aortic catalase and glutathione peroxidase protein abundance was measured by Western blotting. The enzyme activities in the renal and aortic extracts, hepatic glutathione levels, blood pressure and urinary nitric oxide metabolites (NO(x)) excretion were also measured. Blood pressure and urinary nitric oxide metabolite (NO(x)) excretion were also measured. The CRF group showed a significant down-regulation of both immunodetectable catalase and glutathione peroxidase proteins in the remnant kidney. Catalase activity was also significantly decreased in the remnant kidney whereas glutathione peroxidase activity was not significantly affected. Furthermore, the protein abundance of catalase was unchanged whereas the enzyme activity was significantly decreased in the thoracic aorta of CRF animals compared to the sham-operated controls. By contrast, both the protein abundance and the enzyme activity of glutathione peroxidase were not significantly affected in the aorta of CRF animals compared to the sham-operated controls. This was coupled with marked arterial hypertension, significant reduction of hepatic glutathione levels and urinary NO(x) excretion pointing to increased inactivation and sequestration of NO by superoxide. These events point to the role of impaired antioxidant defense system in the pathogenesis of oxidative stress in CRF.     

幽门螺杆菌感染促进胃炎儿童胃粘膜细胞的增殖

本研究旨在探讨幽门螺杆菌感染对小儿慢性胃炎患者细胞增殖的影响,使用内镜检查消化不良患者的上消化道症状,使用改良的Giemsa染色检测胃粘膜活组织中幽门螺杆菌,用苏木精/曙红和改良的吉姆萨染色活组织,并通过光学显微镜研究染色后胃粘膜样品组织病理学变化,RT-PCR检测各组胃粘膜细胞中调控细胞凋亡的Bcl-2、Bcl-xl、Bax和PCNA的mRNA表达,提取胃粘膜细胞蛋白质,利用蛋白质免疫印迹分析蛋白质浓度。组织化学染色结果表明,与对照相比,患有胃炎和幽门杆菌感染后的胃炎患者胃粘膜细胞明显增加,且幽门螺杆菌感染后细胞增殖更显著(p<0.05);幽门螺杆菌感染后Bcl-2和Bcl-xl,PCNA在患者体内表达显著上调(p<0.05),而细胞促凋亡因子Bax基因在胃炎患者感染幽门螺杆菌后被显著下调(p<0.05),蛋白免疫印迹分析Bcl-2,Bcl-xl,Bax和PCNA蛋白表达趋势与基因表达一致,说明结果可靠。幽门螺杆菌感染会显著提高慢性胃炎儿童患者胃粘膜细胞的增殖。     

Production and some characteristics of monoclonal antibodies against beet necrotic yellow vein virus

Four rat monoclonal antibodies (MAbs) specific for beet necrotic yellow vein virus (BNYVV) were produced. In indirect ELISA, all four MAbs reacted strongly with BNYVV infected plant leaf extracts (19 isolates from eight countries) but they did not react with beet soil-borne virus (BSBV), an unnamed rod-shaped soil-borne beet virus isolate (86 - 109) from Sweden or barley stripe mosaic virus (BSMV). However, two of the MAbs, MAFF 6 and MAFF 7 did not detect BNYVV in ELISA of infected sugar beet roots whereas MAbs MAFF 8 and MAFF 9 did detect virus in root extracts. In electro-blot immunoassay (EBIA), MAFF 6 and MAFF 7 readily detected BNYVV coat protein from leaf extracts whereas MAFF 8 and MAFF 9 reacted only weakly. None of the MAbs reacted with BSBV, 86 - 109, BSMV or plum pox virus in EBIA. MAFF 6 coated BNYVV particles which were trapped from infected leaf or root sap on to electron microscope grids by polyclonal antibodies. MAFF 6 was partially purified from tissue culture supernatant fluid by cation exchange chromatography and the preparation used to coat microtitre plates and successfully trap BNYVV in ELISA of leaf sap extracts.     

Establishment of monoclonal antibodies against carbohydrate moiety of gastric mucins distributed in the different sites and layers of rat gastric mucosa

Eight monoclonal antibodies (MAbs), designated RGM21 RGM42, were generated against mucin purified from the rat gastric mucosa. By applying ELISA, all of these MAbs were proved to react not only with the purified mucin, but also with the oligosaccharide mixture obtained from the antigenic mucin by alkaline borohydride treatment. Treatment of the mucin-attached ELISA well with trypsin, sodium periodate or galactose oxidase prior to the addition of the MAb was applied to characterize these MAbs. Histochemical observation indicated that all these MAbs were able to stain the formalin fixed-paraffin embedded sections of the rat gastroduodenal mucosa. Although each of these MAbs reacted with distinct mucus-producing cells localized in particular regions of the gastroduodenal mucosa, their staining specificity could generally be classified into four groups. These MAbs might be useful for estimating the physiological and pathological changes of mucins in the gastric mucosa.     

Production of monoclonal antibodies against grouper nervous necrosis virus (GNNV) and development of an antigen capture ELISA

Five (2 IgG, 3 IgM) monoclonal antibodies (MAbs) against the G9508KS strain of grouper nervous necrosis virus (GNNV) were produced and characterized. All 5 MAbs showed positive signals in the retina of GNNV-infected grouper larvae and in the cytoplasm of GNNV-infected GF-1 cells using immunohistochemistry staining. Two MAbs reacted with the denatured capsid protein derived from GNNV-infected GF-1 cells in Western blot analysis, but did not react with the GNNV recombinant capsid protein expressed by E. coli in an indirect immnunosorbent assay (ELISA). All 5 MAbs were able to neutralize GNNV, tiger puffer NNV (TPNNV) and barfin flounder NNV (BFNNV), while only 2 of the MAbs neutralized striped jack NNV (SJNNV). A capture ELISA system based on the use of MAbs for capture and a rabbit polyclonal antibody for detection was developed. When absorbance values higher than 0.5 were judged to be positive, the sensitivity of the capture ELISA system was 2.5 ng per well of purified GNNV protein or 6.5 x 10(4) TCID50 per well of GNNV supernatant from culture cells. This capture ELISA system could become a more specific and sensitive tool for NNV diagnosis in the field and in routine laboratories.     

Monoclonal antibodies against calcitonin. Characterization and application in light and electron microscopy immunocytochemistry

Twenty-seven monoclonal antibodies (MAbs) to synthetic human calcitonin (CT) were characterized for their reactivities with human CT peptide fragments by dot-blot analysis on nitrocellulose paper. Most of the antibodies bound to the C-terminus and fewer to the mid-region of CT. We have studied thyroid tissue specimens from several animal species after fixation in paraformaldehyde-, glutaraldehyde- or picric acid-containing mixtures and cryostat sectioning or embedment in paraffin or plastic (Epon 812 or Lowicryl 4KM) using this panel of MAbs. The site of antigen-antibody reaction was revealed either by immunoperoxidase, immunoalkaline phosphatase or by silver-enhanced immunogold staining methods. All MAbs were able to localize CT in human, rat and mouse thyroid C cells. Nineteen MAbs recognizing synthetic salmon CT and synthetic [Asu1,7]-eel CT by dot-blot, reacted with chicken ultimobranchial body C cells. One MAb recognizing native porcine CT by dot-blot, stained C cells in hog thyroid. Immunopositivity was confined to the cytoplasm and ultrastructural immunogold labelling demonstrated that cytoplasmic secretory granules were stained. Surgical specimens from human medullary thyroid carcinoma were also analysed for the presence of CT and a variable number of positive cells was found. Furthermore, Congo red-positive areas were shown to react with the MAbs. All conventional staining and immunoabsorption controls were negative. Hence, these MAbs may be suitable for use in routine immunopathological diagnosis of CT-producing tumors and for immunocytochemical localization of the three major CT variants in different animal species.     

Histochemical distribution of four types of enzymes and mucous cells in the gastrointestinal tract of reared half‐smooth tongue sole Cynoglossus semilaevis

The histochemical distribution of acid phosphatase (ACP), alkaline phosphatase (ALP), non‐specific esterase (NSE), peroxidase (POD) and mucous‐cell types was evaluated in the gastrointestinal tract of the half‐smooth tongue sole Cynoglossus semilaevis. The enzymes were detected in the entire stretch of the gastrointestinal tract. ACP activity was found in the supranuclear region of enterocytes and the lamina propria of the intestine, as well as the cytoplasm of epithelial cells of the stomach. The staining intensity of ACP in the anterior and posterior intestines was stronger than in the stomach. ALP activity was detected in the striated border of enterocytes and muscularis of the whole intestine, lamina propria and supranuclear cytoplasm of the enterocytes in the anterior intestine, as well as in the blood vessels of the stomach. The staining intensity for ALP in the anterior intestine was stronger than in the posterior segment and the latter was stronger than in the stomach. NSE activity was detected in the cytoplasm of the epithelial cells in the entire gastrointestinal tract, with the anterior intestine showing stronger intensity than the stomach. POD activity was located in the blood cells of the lamina propria of the gastrointestinal tract and the levels in the stomach were similar to the anterior and posterior intestines. Alcian blue (pH 2·5) periodic acid Schiff (AB‐PAS) histochemical results revealed three types of mucous cells in the gastrointestinal tract. Type I cells (PAS+AB‐) were observed among the gastric mucosa columnar cells in the stomach and enterocytes in the basal region of the villi and in the middle and top regions of the intestinal villi. Type II cells (PAS‐AB+) and type III cells (PAS+AB+) were not detected in the stomach but were distributed ubiquitously among enterocytes in the middle and top regions of the intestinal villi.     

Monoclonal antibodies that distinguish between active and inactive forms of human postheparin plasma hepatic triglyceride lipase

Hepatic triglyceride lipase (H-TGL) was purified from human postheparin plasma. Specific monoclonal antibodies (MAbs) were produced that discriminate between active (native) and inactive (denatured) forms of the enzyme. Mice immunized with native H-TGL resulted in MAbs that recognized only the native protein. The antibodies did not react with H-TGL treated with 1% sodium dodecyl sulfate or heated at 60 degrees C. The loss of immunoreactivity with heating correlated directly with the loss of enzyme activity and there was a corresponding increase in immunoreactivity with the MAbs prepared against the denatured enzyme. Western blot analysis of postheparin plasma with the MAbs against denatured H-TGL gave a single protein band of 65 kD; preheparin plasma showed no detectable immunoreactivity with either MAb. These immunochemical studies suggest that there are no circulating active or inactive forms of H-TGL in man. Furthermore, the MAbs provide the necessary reagents for development of immunoassays for H-TGL.