An ELISA-disc procedure for antibodies toPseudomonas pseudomallei: application for a serological study of melioidosis in an endemic area

The optimization and development of an ELISA-disc procedure for the detection of antibodies to whole cell surface antigens and purified exotoxin ofPseudomonas pseudomallei is described. Comparison of the serum agglutination test (SAT), the serum based enzyme-linked immunosorbent assay (ELISA) and the ELISA-disc procedures used on goat and human sera demonstrated a high correlation in their ability to detect antibodies specific forP. pseudomallei antigens. A serological survey using the ELISA-disc method was carried out on a normal human population in Sabah, Malaysia, an area known to be endemic for melioidosis. The prevalances of antibodies towards cell surface antigens and exotoxin ofP. pseudomallei were 28% and 8%, respectively. As a procedure, the ELISA-disc technique reported here is technically simple and provides savings in costs and is thus deemed suitable for seroepidemiological surveillance of melioidosis in remote areas of South-East Asia.     

Inhibition of Candida albicans attachment to extracellular matrix by antibodies which recognize hydrophobic cell wall proteins

Cell surface hydrophobicity influences the adhesive properties of the opportunistic fungal pathogen Candida albicans. Hydrophobic proteins are present in the C. albicans cell wall. These proteins were used to generate a polyclonal antiserum and monoclonal antibodies. We characterized three of these monoclonal antibodies (designated 6C5, 5F8 and 5D8) that recognize different hydrophobic cell wall proteins. Initial characterization of the three antigens, and assessment of their distribution among various Candida species was also carried out. Further, pretreatment of germ tube initials with the mAb inhibits binding of these cells to immobilized extracellular matrix. These results suggest that these hydrophobic proteins are involved in C. albicans adhesion events.     

Cortical thymocyte differentiation in thymomas: an immunohistologic analysis of the pathologic microenvironment

Four monoclonal antibodies (BH11, T2/30, AG3, and BC3) were produced against different epithelial components of the normal thymus. An immunohistologic study was performed on 13 thymomas by the use of these and other stromal and lymphocyte-specific reagents. The aim of this study was to find possible relationships between the proliferating thymoma epithelial cell type and the T cell composition of thymomas. Our results indicate that cortical T cell differentiation is present in thymomas, and that this differentiation is induced in the absence of detectable levels of MHC class II antigens on the epithelial component in most cases. The role of the MHC class II antigens cannot be excluded, however, because these antigens were always present on macrophages. Analysis of the selected group of thymomas, each of which contained epithelial cells homogeneously stained by at least one of the described monoclonal antibodies, showed that the cortical type T cell inducer capacity of thymomas is independent of the epithelial type predominant in the tumor.     

Characterization of antigens recognized by new monoclonal antibodies raised against culture filtrate proteins of Mycobacterium bovis bacillus Calmette-Guérin

Effective protection against Mycobacterium tuberculosis may be achieved in experimental animals by immunization with proteins secreted by tuberculous bacilli in the extracellular milieu during growth. In this study, monoclonal antibodies were raised against Mycobacterium bovis bacillus Calmette-Guérin (BCG) culture filtrate proteins or live BCG, in an attempt to identify novel mycobacterial secretion antigens: the localization of the antigens recognized by the monoclonal antibodies within the mycobacterial cell was studied and interspecies reactivity was also investigated. The monoclonal antibodies obtained recognized proteins of molecular mass ranging from 5 to 82 kDa, with a prevailing frequency in the 30 kDa region. Three of the monoclonal antibodies recognized proteins present only in culture filtrates, one reacted with a cytoplasmic antigen, while the remaining antibodies recognized components which were mainly associated with the cell wall and the cytoplasmic membrane. The chemical nature and possible identity of the antigens was checked. Three monoclonal antibodies are likely to react with novel mycobacterial antigens of 5, 42 and 82 kDa, respectively.     

Ultrastructural Localization of Monoclonal IgG Antibodies for Antigenic Sites of Eimeria tenella Oocysts,Sporocysts, and Sporozoites1

Monoclonal IgG antibodies against sporozoites of Eimeria tenella were obtained from the ascites fluid of BALB/c mice. Oocysts, sporocysts, and sporozoites were exposed to medium 199, normal ascites fluid, or monoclonal antibodies 1A, 9D, 3D3II, or 2G8f. Specimens were then incubated with ferritin-conjugated goat anti-mouse IgG antibody. Ferritin was uniformly distributed over the surface of sporozoites exposed to 1A, 9D, or 3D3II; ferritin was localized in patches on sporozoites exposed to 2G8f. A uniform layer of ferritin was present on the inner layer of oocyst walls and on the Stieda body and outer surface of sporocysts exposed to 1A, 9D or 3D3II. In specimens treated with 2G8f, ferritin was present on the inner layer of the oocyst wall and the Stieda body, but not on the sporocyst wall. No ferritin was found on specimens exposed to medium 199 or normal ascites fluid. Monoclonal antibodies 1A, 9D, and 3D3II, but not 2G8f, caused complement-mediated lysis of sporozoites. These findings indicate that oocysts, sporocysts, and sporozoites of E. tenella contain common antigens specific for each monoclonal antibody tested.     

Membrane antigens of human bronchial epithelial cells identified by monoclonal antibodies

Summary Subpopulations of normal bronchial epithelial cells were identified using a series of murine monoclonal antibodies. These antibodies were used to stain intact bronchial epithelial cells in culture by indirect immunofluorescence. LAM 2 reacted with 80%, LAM 6 with 75%, LAM 7 with 60%, and LAM 8 with 5% of these cells. Sections of human bronchial epithelium were also stained with these antibodies by immunoperoxidase methods. LAM 2 was found to bind with 80%, LAM 6 with 65%, LAM 7 with 50%, and LAM 8 with less than 1% of bronchial epithelial cells. LAM 2 stained both columnar epithelial cells and basal cells; LAM 6 stained mainly basal cells and only a small proportion of columnar cells; LAM 7 showed specificity for basal cells; LAM 8 distinctly stained single cells in the basal cell layer. These antibodies were previously shown to react with the surface membrane of human lung carcinomas, ranging from the broad reactivity of LAM 2 with small cell and non-small cell lung cancers to the highly restricted reactivity of LAM 8 with small cell carcinomas of the lung. Thus, membrane antigens have been identified in bronchial epithelial cells by monoclonal antibodies which exhibit a similar range of cellular reactivity in vitro as in vivo. Inasmuch as these antibodies recognize subsets of cells which could not be easily distinguished by morphologic characteristics, these reagents may be useful in classifying bronchial epithelial cells.     

Brugia pahangi: stage-specific antigens on microfilariae detected by serum from infected jirds or by monoclonal antibodies

Experiments were carried out to determine whether there are stage-specific antigens on microfilariae of Brugia pahangi, using sera from Mongolian jirds infected with B. pahangi and monoclonal antibodies against microfilariae of B. pahangi. These studies showed that microfilariae have both stage-specific and nonspecific antigens. The nonspecific antigens were also present on adult worms and on infective larvae. Among monoclonal antibodies, 6 out of 14 clones produced antibodies against the microfilarial stage-specific antigens, and 8 clones produced antibodies against nonspecific antigens. These monoclonal antibodies could not distinguish between adults, microfilariae, or infective larvae of B. malayi and B. pahangi.     

Monoclonal antibodies to cell surface components of zoospores and cysts of the fungusPythium aphanidermatum reveal species-specific antigens

A panel of monoclonal antibodies (MAbs) designated PA1 to PA8 has been raised against cell surface components of zoospores and cysts of the pathogenic fungusPythium aphanidermatum. The antibodies were selected on the basis of binding assays using indirect immunofluorescence. Four binding patterns were observed: PA1 labeled the entire zoospore surface including both flagella, PA2 binding was restricted to the anterior flagellum, PA3–PA6 bound to the adhesive cell coat secreted by zoospores during encystment, and PA7 and PA8 labeled zoospores and the cyst cell wall. Electron microscopic immunogold labeling of zoospores showed that PA2 bound to the mastigonemes on the anterior flagellum. The MAbs were tested for binding to zoospores and cysts of several isolates ofP. aphanidermatum, and to zoospores and cysts of several species ofPythium, Phystophthora, Aphanomyces, andSaprolegnia. The results showed that the antigens recognized by MAbs PA1–PA6 were restricted toP. aphanidermatum, whereas those recognized by PA7 and PA8 occurred on all species tested.     

Cell surface antigens during submerged development of Myxococcus xanthus examined with monoclonal antibodies

Eighteen monoclonal antibodies directed against cell surface antigens of Myxococcus xanthus were followed by enzyme-linked immunosorbent assay. Three of the monoclonal antibodies were specifically directed against antigens present only on cells undergoing fruiting body development. These cell surface antigens became detectable by the early preaggregation stage (2 to 4 h) of development and increased until early aggregation (9 to 10 h), after which the concentrations of two of the cell surface antigens remained constant and the concentration of the third decreased. The remaining 15 monoclonal antibodies recognized cell surface antigens that were shared by vegetative and developing cells. Based on their relative concentrations during development, these shared antigens can be grouped into three classes. In the first class antigen concentration remained constant, in the second it decreased, and in the third it increased. Western blots of cell surface antigens were probed with monoclonal antibodies. Five monoclonal antibodies reacted with material in distinct bands, five monoclonal antibodies reacted with multiple, diffuse bands, and eight monoclonal antibodies were not reactive in Western blots.     

Development of a diagnostic system for Burkholderia pseudomallei infections

Monoclonal antibodies were generated against whole cell lysate of Burkholderia pseudomallei. Two out of 6 monoclonal antibodies were found specific and exhibited high affinity against B. pseudomallei, one of which, was utilized to develop sandwich ELISA for detection of specific B. pseudomallei antigen. Immunoassays were found to be specific as no reaction was observed with closely related Burkholderia and Pseudomonas species. Blood samples from experimentally infected mice were found positive for isolation till 4 days post infection (DPI) and ELISA till 10 DPI. One out of 40 sick animal serum samples tested in Thailand was found positive by sandwich ELISA that was earlier confirmed by isolation of B. pseudomallei. The results indicate the potentiality of the assay for its applicability in specific diagnosis of septicaemic melioidosis.     

Distribution of H type 1-4 chains of the ABO(H) system in different cell types of human respiratory epithelium.

We used three anti-H monoclonal antibodies (MAbs) specific for H Type 1, H Type 2, and H Type 3/4 antigens to investigate the distribution of H Type 1-H Type 4 chains of the ABO(H) histo-blood group in the human respiratory system. Strong staining of H Type 1 chain and weak staining of H Type 2 chain were observed in mucous cells of submucosal glands of bronchial epithelium, which were dependent on the secretor status. No H Type 3/4 chains were detected in mucous cells. Serous cells of submucosal glands of respiratory system showed no staining by three anti-H antibodies. H Type 1 and H Type 3/4 antigens were detected heterogeneously in apical surfaces of bronchial epithelium from secretors but not from nonsecretors. In contrast, basal cells of bronchial epithelium expressed H Type 2 irrespective of the secretor status, probably regulated by the H gene. Some alveolar Type II cells contained only H Types 3/4, which were dependent on the secretor status, whereas alveolar Type I cells had no H antigens. Our results indicated that different cell types in respiratory epithelium expressed different types of carbohydrate chains of histo-blood group antigens under the control of the H or the Se gene.     

Detection of leishmania antigen in kala azar patients using monoclonal antibodies.

Twenty-one monoclonal antibodies were produced against promastigote antigens of Leishmania donovani. Five monoclonal antibodies (Hyb.17, 6, 5, 4 and 2) identifying molecules associated with various L. donovani antigenic determinants ranging from 42-116 kDa were selected as 'capture antibodies' and compared with specific anti-leishmania antisera for detection of circulating leishmania antigens in kala azar patients' sera in a competitive-enzyme-linked immunosorbent assay system (ELISA). The anti-leishmania antisera could detect circulating antigen in 30% of kala azar cases while out of the five monoclonals, Hyb.17 could effectively detect circulating leishmania antigen in 85.4%. The efficacy of Hyb.6 was however low (31.7%). The antigens recognized by these monoclonal antibodies in the western blot assay could possibly represent the ones circulating in sera of patients suffering from kala azar. A cocktail of these monoclonal antibodies may be more useful than the conventional polyclonal antisera in detection of circulating antigen for clinical diagnosis of kala azar.     

Monoclonal antibodies to adhesive cell coat glycoproteins secreted by zoospores of the green alga Enteromorpha

Zoospores of Enteromorpha compressa (L.) Grev. secrete an adhesive cell coat which is involved in their attachment to various substrata. Two monoclonal antibodies (mAbs), designated Ent 1 and Ent 6, were raised against settled zoospores displaying secreted adhesive. Both antibodies labelled specifically the anterior region of the cell containing putative adhesive vesicles. During settlement the antigens recognised by both mAbs were secreted but whereas Ent 6 recognised a fibrillar material released within a few minutes of settlement, Ent 1 recognised components which were associated predominantly with the developing cell wall at later time points. Both mAbs also labelled a Golgi-rich region of settled spores, suggesting that these antigens are also synthesised after settlement. Both mAbs labelled the cell walls of vegetative tissue. Competitive enzyme-linked immunosorbent assay indicated that the two antibodies recognise separate, but overlapping epitopes. In spore settlement assays the Ent 6 immunoglobulin strongly reduced initial adhesion at low concentration whereas the inhibitory effects of Ent 1 occurred at later time points. On analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, (SDS-PAGE) both MAbs recognised a major buffer- and SDS-soluble, polydisperse 110-kDa antigen. The 110-kDa component was present in extracts of zoospores and sporulating tissue, but absent, in soluble form, from vegetative tissue. Deglycosylation of zoospore extract with anhydrous HF and peptide N-glycosidase digestion, showed that the major 110-kDa antigen is an N-linked glycan, and that the epitope is borne by the protein component. Time-course experiments showed that the Ent 6 antigen became progressively insoluble after zoospore attachment. Taken together, the data indicate that the two antibodies recognise separate but closely related antigens which have distinctive roles in adhesion and cell wall development. Received: 8 February 1999 / Accepted: 26 July 1999     

Search for the optimal fetal cell antibody: results of immunophenotyping studies using flow cytometry

Fetal nucleated cells circulating in maternal peripheral blood are a noninvasive source of fetal DNA for prenatal genetic diagnoses. The successful isolation of fetal cells from maternal blood depends upon identification of differences between fetal and maternal cell surface antigen expression. To our best knowledge, a monoclonal antibody that binds only fetal blood cells has not yet been identified. We studied antigens recognized by six different monoclonal antibodies for their biologic expression on fetal blood cells as a function of gestational age, and compared their ability to bind fetal but not maternal cells. The results suggest a relationship between gestational age and nucleated cell surface antigen expression. The monoclonal antibodies FB3-2, H3-3, CD71 and 2-6B/6 are suitable reagents for first or early second trimester fetal cell isolation, although FB3-2 and H3-3 are more specific for fetal cells due to significantly lower expression of these antigens on maternal mononuclear cells. The observation that samples from fetuses with chromosome abnormalities or multiple structural anomalies express higher levels of these antigens indicates that these reagents will potentiate the detection of abnormal fetal cells in maternal blood samples. Received: 23 November 1996 / Accepted: 13 February 1997     

An immunogold-silver staining method for detection of cell-surface antigens in light microscopy

An immunogold-silver staining technique for detection of cell-surface antigens in cell suspensions was developed. Leukocyte cell suspensions were first incubated with monoclonal antibodies directed against cell-surface antigens and then with colloidal gold-labeled goat anti-mouse antibodies. Cytocentrifuge preparations of the cell suspensions were immersed in a physical developer containing silver lactate and hydroquinone as reducing substance. The preparations were then counterstained and mounted. In light microscopy, cells reacting with the monoclonal antibodies showed dark granules on their surface membrane. An optimal morphology, as revealed by a May-Grünwald-Giemsa counterstain, permitted accurate cell identification. The labeling was influenced by the gold particle diameter and the concentration of the gold reagents, by the duration of incubation in the physical developer, and by the composition and temperature of this medium. The T-cell subsets enumerated with this method in the peripheral blood of normal adults were identical to those found with other methods. The sensitivity of the technique was comparable with that of immunofluorescence microscopy. This immunogold-silver staining procedure proved to be a reliable tool for detection of cell-surface antigens in light microscopy.     

Monoclonal autoantibodies to different epithelial structures of the thymus gland obtained by the immunization with streptococcal group A antigens

By the indirect immunofluorescence method it was shown to which epithelial thymus structures monoclonal antibodies (mAT) reacting with the different epidermal structures are directed. These mAT related to the autoantibodies were obtained earlier, as a result of lymphoid cells polyclonal activation, by the immunization of BALB/c mice with streptococcal group A nonspecific protein antigens of the cell wall. It was shown that mAT A6/1, reacting with the basal layer of the skin epithelium are directed to the epithelium of the cortical and medullar thymus zones, which is regarded as the so called endocrinal epithelium. These mAT, by the study with immunoblotting method, react with the protein of mV SOkD, B5/1 mAT to the skin epithelium, on the thymus sections react with the single cells around the Hassel bodies.     

High-resolution immunogold localization of Giardia cyst wall antigens using field emission SEM with secondary and backscatter electron imaging

We describe here the ultrastructural localization of Giardia cyst antigens in the filaments associated with the outer portion of intact cysts and on developing cyst wall filaments in encysting trophozoites. Post-embedding immunogold labeling of thin sections of intact Giardia cysts with polyclonal and monoclonal antibodies specific for cyst wall antigens (major protein bands of approximately 29, 75, 88, and 102 KD on Western blots) showed strong labeling of the filamentous cyst wall, whereas no labeling was seen on the membranous portion. High-resolution field emission scanning electron microscopy (FESEM) of Giardia cysts revealed that the cyst wall-specific polyclonal rabbit antisera and monoclonal mouse antibody produced gold labeling of 20-nm filaments in the cyst wall as detected with secondary electron imaging (SEI) and backscatter electron imaging (BEI) at 10 kV, despite coating of the cells with platinum by ion sputtering. FESEM studies of encysting Giardia trophozoites demonstrated that immunostaining with antibodies to cyst wall antigens produced colloidal gold labeling of developing cyst wall filaments on the cell surface; however, the intervening membrane domains were unlabeled. Substitution of normal serum for cyst wall-specific antibodies, or preabsorption of specific antibodies with Giardia cysts, eliminated immunolabeling of the filaments.     

Expression of epithelial antigens in primary cultures of normal human breast analysed with monoclonal antibodies

Abstract. Primary cultures of normal human breast were stained with monoclonal antibodies to see if antigens characteristic of luminal epithelial cells are retained in culture. Three monoclonal antibodies were used, LICR-LON-M8, LICR-LON-M18, and LICR-LON-M24, all specific for the cell surface of luminal epithelial as opposed to myoepithelial or stromal cells in the breast, and each staining a different subset of the epithelial cells in the intact tissue. Cultures were prepared from reduction mammoplasty samples by digestion with collagenase. The surface layer of cells was stained by immunofluorescence without fixation. (Cells underneath the surface layer were not accessible to this mode of staining). The antibodies stained patches of cells resembling flattened epithelium. These patches of cells cannot be distinguished by phase contrast microscopy without reference to the staining, in fact the boundaries of the cells are not usually resolved by phase contrast microscopy. Electron microscopy of sections through these cells show they are very flattened. They lie on top of the polygonal and elongated cells that dominate the phase contrast image. Two of the antibodies, M8 and M24, stain subsets of these epithelial-like cells at all stages of culture. The third antibody, Ml8, stains such cells initially, but after the first few days staining is predominantly found on the polygonal and elongated cells, then this also gradually disappears. It is possible that the cells stained by antibody Ml8 are converting from the epithelial-like morphology to the cuboidal and elongated morphology. Many cells are not stained by any of the antibodies, so appear either to by myoepithelial in origin or to have lost their luminal epithelial surface antigens at an early stage. This analysis draws attention to the variety of cell types in these cultures and the limitations of phase contrast microscopy as a means of analysing them.     

HLA antigen expression at the single cell level on a K562 X B cell hybrid: an analysis with monoclonal antibodies using bacterial binding assays

The influence of different genetic environments on the expression of HLA complex-controlled antigens has been investigated using cell lines with various defects in the synthesis of these molecules and a somatic cell hybrid derived from them. A very sensitive bacterial binding assay allowing simultaneous evaluation of the morphology of a given cell and the quantity of a surface molecule has been developed for these studies. The fetal erythroid cell line K562, the Burkitt's lymphoma-derived cell line DAUDI, and their hybrid DUTKO1 have been employed. K562 and the hybrid, but not DAUDI, expressed HLA-A,B,C heavy chains as detected by the monoclonal antibody W6/32.HL, while two monoclonal antibodies (TU48 and 2BC4) against the supertypic specificities HLA-Bw4 and Bw6 showed no reactivity. The presence of human Ia-like antigens on the cell surfaces was investigated with a panel of eight monoclonal antibodies. K562 cells were completely unreactive, and DAUDI cells gave the expected positive reaction, but about 1% or less of the cells in the DUTKO1 population appeared to express these antigens as well. We discuss possible reasons for the failure to detect HLA-B antigens with monoclonal antibodies and the lack of complete "dominance" of the K562 genome in the hybrid cell line.     

Monoclonal antibodies to the antigens of human HeLa tumor cells

A panel of 6 hybridomas "XEJIMA" producing monoclonal antibodies specific to HeLa cells is prepared. Monoclonal antibodies do not bind to antigens of human diploid fibroblasts, human continuous B- and T-lymphocytes and animal cell lines. The specificity of monoclonal antibodies to cellular antigens of 5 HeLa-like cell lines and 6 human tumour cells lines, not contaminated with HeLa cells, is determined. Antibody containing ascitic fluid and culture media of hybridomas XEJIMA-3, -12, -13, and -22 significantly decrease the attachment of HeLa cells to the surface of culture flasks. Monoclonal antibodies XEJIMA-11, -12 and -13 block the multiplication of HeLa cells. The effect depends on serum concentration in the nutrient medium.