New Panel Of Monoclonal Antibodies Research Articles

P.253 - Evaluation of a panel of new monoclonal antibodies to α913-DG

P.253 - Evaluation of a panel of new monoclonal antibodies to α913-DG

Monoclonal antibodies against Vibrio vulnificus RtxA1 elicit protective immunity through distinct mechanisms.

Vibrio vulnificus causes rapidly progressing septicemia with an extremely high mortality rate (≥50%), even with aggressive antibiotic treatment. The bacteria secrete multifunctional autoprocessing repeats-in-toxin (MARTX) toxins, which are involved in the pathogenesis of Gram-negative Vibrio species. Recently, we reported that immunization with the C-terminal region of V. vulnificus RtxA1/MARTXVv, RtxA1-C, elicits a protective immune response against V. vulnificus through a poorly defined mechanism. In this study, we generated a panel of new monoclonal antibodies (MAbs) against V. vulnificus RtxA1-C and investigated their protective efficacies and mechanisms in a mouse model of infection. Prophylactic administration of seven MAbs strongly protected mice against lethal V. vulnificus infection (more than 90% survival). Moreover, three of these MAbs (21RA, 24RA, and 47RA) demonstrated marked efficacy as postexposure therapy. Notably, 21RA was therapeutically effective against lethal V. vulnificus infection by a variety of routes. Using Fab fragments and a neutropenic mouse model, we showed that 21RA and 24RA mediate protection from V. vulnificus infection through an Fc-independent and/or neutrophil-independent pathway. In contrast, 47RA-mediated protection was dependent on its Fc region and was reduced to 50% in neutropenic mice compared with 21RA-mediated and 24RA-mediated protection. Bacteriological study indicated that 21RA appears to enhance the clearance of V. vulnificus from the blood. Overall, these studies suggest that humoral immunity controls V. vulnificus infection through at least two different mechanisms. Furthermore, our panel of MAbs could provide attractive candidates for the further development of immunoprophylaxis/therapeutics and other therapies against V. vulnificus that target the MARTX toxin.

Detection of the dystroglycanopathy protein, fukutin, using a new panel of site-specific monoclonal antibodies

Mutations in the gene encoding fukutin protein cause Fukuyama muscular dystrophy, a severe congenital disorder that occurs mainly in Japan. A major consequence of the mutation is reduced glycosylation of alpha-dystroglycan, which is also a feature of other forms of congenital and limb-girdle muscular dystrophy. Immunodetection of endogenous fukutin in cells and tissues has been difficult and this has hampered progress in understanding fukutin function and disease pathogenesis. Using a new panel of monoclonal antibodies which bind to different defined sites on the fukutin molecule, we now show that fukutin has the predicted size for a protein without extensive glycosylation and is present at the Golgi apparatus at very low levels. These antibodies should enable more rapid future progress in understanding the molecular function of fukutin.

Flow cytometry analysis of leukocytes in induced sputum from asthmatic patients

Inflammatory cell counts in induced sputum from asthmatic patients partially correlate with respiratory physiology data. To identify and quantify these inflammatory components, microscopy has been useful but it is not without its limitations. Flow cytometry could be an alternative but still has underlying methodological difficulties. While passing airways, leukocytes undergo morphologic cellular changes that alter their conventional phenotype. To demonstrate the usefulness of cytometry in accurately identifying cellular profiles in induced sputum of asthmatic and chronic cough patients, we introduced a new panel of monoclonal antibodies against specific subset markers. To identify neutrophils, sputum cells were stained with CD45 and CD66b. To identify eosinophils, sputum cells were stained with anti-CD45 and anti-CD125. We co-stained CD45, CD14 and CD66b to identify macrophages as CD45+CD14+CD66b− cells. Comparable results of trypan blue exclusion and annexin V-FITC suggested that cytometry manipulation did not decrease cellular viability. Range values were similar in microscopy neutrophils (median 19.9%, range 1.7–90.1%) and CD45+CD66b+ neutrophils (median 31% range 0.9–89%). After gating out CD45− non-leukocyte events, CD45+ and SSC dot-plots defined three patterns of leukocyte distribution. The eosinophil range in microscopic examination was 0–71.3% (median 2.85%) whereas CD45+CD125+ cell range in cytometry was 0–29% (median 3.7%). Since no exclusive markers were found on airways macrophages, we co-stained CD45, CD14 and CD66b to identify macrophages as CD45+CD14+CD66b− cells. Microscopy showed that macrophage and CD45+CD14+CD66b− cell counts were comparable (median 52.3 and range 6.7–94.8 vs median 61 and range 10.5–97.7 respectively). Correlations between neutrophils, eosinophils and macrophages in microscopic examination and flow cytometry were strong (R=0.725, 0.747 and 0.532, respectively p<0.001). This study validates effectiveness of combining specific antibodies and cytometry to quantify inflammatory leukocytes in induced sputum. Multiple markers at a single cell level will deepen our knowledge concerning the phenotype of airway leukocytes.

Precisely tuned antibodies nab HIV

Newly discovered neutralizing antibodies that target sites on the envelope proteins of HIV-1 provide a window on how some of the most powerful of these antibodies acquire their potency and breadth of activity. See Letter p.466 Antibodies that can neutralize many different strains of a virus — known as broadly neutralizing antibodies — are the focus of the search for a universal flu vaccine. Dennis Burton and colleagues report the isolation of a new panel of monoclonal antibodies with broad neutralizing activity against HIV that could be promising as immunotherapeutics. The antibodies recognize novel glycosylated epitopes on the viral spike, and some are ten times more potent than previously reported antibodies.

Genomic and antigenic characterization of viruses from the 1993 Italian foot-and-mouth disease outbreak

The origin and evolution of the type O foot-and-mouth disease viruses (FMDV) that caused the outbreak occurrence in Italy in 1993, the first episode of the disease in the EU after adoption of a non-vaccination policy in 1991, have been studied by the analysis of sequences encoding three main antigenic sites on the viral capsid proteins. The phylogenetic tree derived from sequences spanning the carboxyterminal end of VP1 showed that these Italian viruses were grouped in the ME-SA topotype, closely related to viruses that circulated previously in the Middle East. The analysis of the nucleotide sequences in VP1, VP2 and VP3 showed a co-circulation during the epizootic of genetic variants, including viruses with amino acid replacements in VP3. For some of the isolates analyzed, values of fixation of nucleotide substitutions per year were observed in the three regions analyzed, ranging from 1.5 to 5.1 x 10(-2). The use of a panel of new monoclonal antibodies raised against an isolate from this outbreak, as well as monoclonal antibodies to FMDV O1-Switzerland 1965, showed differences in the reactivity pattern among some of the Italian isolates analyzed, which were consistent with the co-circulation of antigenic variants. These results support the potential for FMDV diversification in a limited period of time and under epidemiological conditions in which no vaccination campaigns were being implemented.

Characterisation of the transcription factor, SIX5, using a new panel of monoclonal antibodies

SIX5 is a member of the human SIX family of transcription factors, many of which are involved in eye development. However, SIX5 transcripts are known to be present at very low levels in cells and no study has yet convincingly demonstrated detection of endogenous SIX5 protein by Western blotting or immunolocalisation. We have produced a new panel of 18 monoclonal antibodies (mAbs) that recognise at least four different epitopes in order to identify authentic human SIX5 protein in cells and tissues. Phage-displayed peptide libraries were used to identify individual amino-acids important for antibody binding within each epitope. Endogenous SIX5 migrated in SDS-PAGE with an apparent M(r) of 100 kDa and was present at similar levels in all foetal tissues and cell lines tested. In HeLa cells, it was located in the nucleoplasm with a granular distribution. An mRNA for a shorter splicing isoform of SIX5, with an altered carboxy-terminus, has been described, but further mAbs specific for this isoform did not detect any endogenous protein. We conclude that the full-length isoform is the major functional protein in vivo while the putative shorter protein is undetectable and may not be expressed at all.

A lamin A/C beta-strand containing the site of lipodystrophy mutations is a major surface epitope for a new panel of monoclonal antibodies.

Using a phage-displayed peptide library, we have identified the epitope recognized by a new panel of five monoclonal antibodies (mAbs) raised against full-length recombinant human lamin A. The mAbs were found to recognize both lamin A and C by Western blotting and immunolocalization at the nuclear rim. A nine-amino acid consensus sequence PLLTYRFPP in the common immunoglobulin-like (Ig-like) domain of lamin A/C contains the binding site for all five mAbs. Three-dimensional structure of the Ig-like domain of lamin A/C shows this sequence is a complete beta-strand. This sequence includes arginine-482 (R482) which is mutated in most cases of Dunnigan-type familial partial lipodystrophy (FPLD). R482 may be part of an interaction site on the surface of lamin A/C for lamin-binding proteins associated with lipodystrophy.

Characterization of neutralization sites on the circulating variant of swine vesicular disease virus (SVDV): a new site is shared by SVDV and the related coxsackie B5 virus

Using a panel of new monoclonal antibodies (mAbs), five neutralizing, conformation-dependent sites have been identified on the antigenic variant of swine vesicular disease virus (SVDV) circulating currently. In studies on the antigenic conservation of these sites, the four antigenic/genetic groups of SVDV described showed distinguishable patterns, confirming this classification. By sequencing mAb-resistant mutants, the five sites have been mapped precisely and localized on a three-dimensional model of the SVDV capsid. All were found to be orientated, to a different extent, towards the external surface of the capsid. Three of the five sites, located in VP1, VP2 and VP3, correspond to epitopes identified previously in historic isolates as sites 1, 2a and 3b, respectively. Another site, site IV, which maps to position 258 of VP1, corresponds to an epitope reported recently and is described in this study to be specific for isolates of the most recent antigenic group of SVDV. A fifth site is described for the first time and corresponds to the unique neutralizing site that is common to both SVDV and coxsackie B5 virus; it maps to positions 95 and 98 of VP1, but may also include positions nearby that belong to site 1 on the BC-loop of VP1, suggesting the classification of site Ia. These results may have useful diagnostic and epidemiological applications, since mAbs to the new conserved site Ia provide universal reagents for SVDV detection systems, while the specificity of mAbs to site IV make them unique markers for the most recent strains of SVDV.

Study of the distribution pattern of Schistosoma haematobium egg antigens recognised by six different monoclonal antibodies in the parasite and the host.

Recently a new panel of monoclonal antibodies was developed against soluble egg antigens in the hatching fluid of Schistosoma mansoni. These antibodies have been used to develop an improved ELISA for the detection of circulating soluble egg antigens in serum and urine that would have a higher sensitivity in the immunodiagnosis of S. mansoni infections. Although these antibodies showed no improvement in the immunodiagnosis of S. mansoni infections compared with egg antigen-based ELISAs already described (Nourel Din et al., 1994a), they may have a potential role in the identification of S. haematobium infections. This study has looked into the immunolocalisation of S. haematobium egg antigens in both the parasite and the host as recognised by four newly developed monoclonal antibodies (290-2D9-A, 290-2E6-A, 290-2H12-A and 290-4A8-A) and two already described antibodies (114-5B1-A and 114-4D12-A). The antibodies 114-5B1-A and 114-4D12-A appeared to have in S. haematobium eggs a similar staining pattern when compared to S. mansoni eggs. The antibodies prepared against the hatching fluid showed a characteristic signal, especially 290-2E6-A. These antibodies recognised a component originating from the lateral glands of the miracidium. In the host a similar immunohistochemical tissue localisation pattern (mainly phagocytising reticulo-endothelial cells) was seen as previously described for S. mansoni infected hamsters.

Antigenic characterization of the nucleocapsid protein of Newcastle disease virus by means of a new panel of monoclonal antibodies

Nine monoclonal antibodies (MAB) against nucleocapsid protein (NP) of Newcastle disease virus (NDV) have been prepared and characterized. All the MABs were classified into three groups by means of the competitive binding assay. At least three antigenic sites were delineated on the NP. The 1st site includes two closely located epitopes; the 2nd site includes two related and two distinct epitopes; the 3rd site includes two closely related and one distinct epitopes.

Localization of rabbit huntingtin using a new panel of monoclonal antibodies

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by the expansion of a CAG repeat which is expressed as a polyglutamine tract near the N-terminus of the gene product, huntingtin. N-terminal huntingtin fragments form intranuclear aggregates in HD patients and these may be involved in the pathogenesis. Monoclonal antibodies (mAbs) against three different regions of huntingtin (amino acids 997–1276, 1844–2131 and 2703–2911) have been produced and two of the epitopes have been identified using phage displayed peptide libraries. All mAbs reacted with 350 kDa huntingtin on Western blots and one mAb from each region was selected for further study by strong immunoreactivity with neurons in different regions of rabbit brain and by ability to immunoprecipitate native huntingtin. Subcellular fractionation and sucrose density centrifugation of rabbit brain extract showed that most of the huntingtin exists as a high molecular weight complex in the cytoplasm. Two outstanding problems have been addressed; the location of huntingtin in tissues outside the central nervous system and whether huntingtin is present in the nucleus of normal cells. We conclude that huntingtin is present at low levels in most non-neuronal cells though we have identified an interstitial cell type in skin with very high immunoreactivity. Using both immunolocalization and nuclear purification methods, we were unable to exclude the possibility that a small proportion of full-length huntingtin is present in the nucleus.

Localization of myotonic dystrophy protein kinase in human and rabbit tissues using a new panel of monoclonal antibodies.

There is considerable confusion in the literature about the size of the myotonic dystrophy protein kinase (DMPK) and its localization within tissues. We have used a new panel of monoclonal antibodies (mAbs) to begin to resolve these issues, which are important for understanding the possible role of DMPK in myotonic dystrophy. Antisera raised against the catalytic and coil domains of DMPK recognized a major 55 kDa protein and a minor 72-80 kDa doublet on western blots of human skeletal muscle. Ten mAbs, five against the catalytic domain and five against the coil region, recognized only the 72-80 kDa doublet. The 72 kDa protein was present in all tissues tested, whereas the 80 kDa component was variably expressed, mainly in skeletal and cardiac muscles. The 72 kDa protein was absent in a DMPK knockout mouse and was greatly increased in a transgenic mouse overexpressing human DMPK, confirming its identity as authentic DMPK. Two mAbs against the catalytic domain recognized only the more abundant 55 kDa protein, which was found only in skeletal muscle. Nine out of 10 mAbs located DMPK to intercalated discs in human heart, an affected tissue in myotonic dystrophy. However, co-localization of DMPK with acetylcholine receptors at neuromuscular junctions was not observed with any of the mAbs. Subcellular fractionation and sedimentation analysis suggest that a major proportion of the DMPK in skeletal muscle and brain is cytosolic.

Functional topography of myelin‐associated glycoprotein. II. Mapping of domains on molecular fragments

The myelin-associated glycoprotein (MAG), an adhesion molecule of the immunoglobulin (Ig) superfamily with five Ig-like domains, was investigated with regard to its binding site(s) for the neuronal cell surface, collagen I, and heparin, using a panel of new monoclonal antibodies and cyanogen bromide cleavage fragments of MAG. All antibodies generated competed with each other for binding to MAG, indicating that they reacted with identical or closely related epitopes. Mapping of the reactive epitopes on recombinant deletion fragments of MAG expressed by Chinese hamster ovary (CHO) fibroblasts showed reactivity of monoclonal antibody 513 with domains I, II, and III, comprising the amino-terminal end of the extracellular domain. Monoclonal antibody 15 recognized domain III only. Binding of MAG-containing liposomes to neurons was blocked by antibodies 15 and 513. Cyanogen bromide (CNBr) fragments of domains I, II, and III bound to collagen type I under isotonic buffer conditions. CNBr fragments containing domains I and II were involved in binding to heparin. These observations suggest that domain III may be important for binding to the neuronal cell surface receptor for MAG, while domains I, II, and III interact with collagen type I and domains II and III with heparin.

Monoclonal antibodies to surfactant protein D: evaluation of immunoreactivity in normal rat lung and in a radiation-induced fibrosis model.

This report describes the development of a new panel of monoclonal antibodies established after immunization of mice with purified surfactant protein D of the rat. To enhance the detection of SP-D in formalin- or Schaffer-fixed samples, immunohistochemistry was performed by using microwave pretreatment of paraffin sections. Using these new antibodies that bind to type II epithelial cells, Clara cells, and alveolar macrophages, the responses of lung parenchymal cells were examined in a radiation-induced fibrosis model. Increased accumulation of extracellular SP-D in the alveolar space was found. Double staining with anti-surfactant protein A antibodies revealed different Clara cell populations containing one or both types of surfactant proteins.

Characterization and epitope mapping of a new panel of monoclonal antibodies to estradiol receptor

A new panel of monoclonal antibodies to the calf uterus estrogen receptor was prepared. Thirteen antibodies were characterized for their isotype and for the affinity for the antigen. These antibodies recognize the human receptor and can be used in Western blot analysis. The location of the epitopes was mapped on the antigen structure using synthetic fragments of estrogen receptor, and it was possible to group the antibodies in five groups. Many antibodies were useful for the purification of estrogen receptor from tissue extracts by immunoaffinity chromatography. The reciprocal inhibition of the antibodies for the antigen binding was measured with an immunoadsorption assay. This was maximal and symmetrical for antibody pairs within the same group, but was incomplete and, in some instances, asymmetrical between pairs of antibodies from different groups. One antibody was able to inhibit the estrogen receptor-DNA interaction, whereas two others were unable to recognize the receptor-DNA complexes. This new panel of antibodies is a useful addition to the existing tools for studying structure and function of the estrogen receptor. (Steroids 58:4–12, 1993)

Characterization of microglia and macrophages in the central nervous system of rats: definition of the differential expression of molecules using standard and novel monoclonal antibodies in normal CNS and in four models of parenchymal reaction.

This report describes the development of a new panel of monoclonal antibodies produced following immunization of mice with cultured rat microglial cells. Using these new reagents and previously defined antibodies that bind to microglia or macrophages, the responses of parenchymal microglia, perivascular "microglial" cells, and infiltrating macrophage/monocytes were examined in 4 divergent models of central nervous system reaction. These were brain abscess, experimental allergic encephalomyelitis, Wallerian degeneration, and stab wound. No single new antibody was specific only for microglia; all antibodies positively staining microglial cells also labeled various subsets of macrophage/monocytic cells in normal tissues of the immune system. Moreover, the results indicate that microglia are capable of different levels and a variety of types of response, as defined by the molecules they elaborate. These findings suggest that these CNS resident cells belong to the extended monocyte/macrophage/dendritic cell family and that they do not respond in a stereotypic manner to all forms of CNS insult.

Alveolar macrophages in idiopathic pulmonary fibrosis display a more monocyte-like immunophenotype and an increased release of free oxygen radicals

Bronchoalveolar lavage (BAL) was performed in 13 patients with idiopathic pulmonary fibrosis (IPF) and in 10 control subjects. Free oxygen radical (FOR) production of alveolar macrophages (AM) and of blood monocytes was measured by luminol-dependent chemiluminescence (LDCL) without and after stimulation with zymosan A. We confirmed earlier studies that alveolar macrophages of IPF patients display a significantly elevated release of FOR under basic and under stimulated conditions. In contrast to alveolar macrophages, blood monocytes did not reveal altered LDCL in IPF. This indicates that the functional properties for augmented FOR release by IPF alveolar macrophages are acquired in the lungs and not in the peripheral circulation. To elucidate the possible mechanisms leading to the augmented LDCL response, immunophenotyping of alveolar macrophages was carried out. A panel of new monoclonal antibodies of the Ki-M-series, discriminating differentiation stages of monocyte/macrophage subpopulations, served for immunocytochemical staining. In IPF patients distribution of Ki-M2, Ki-M3, Ki-M6 and Ki-M8 positive AM demonstrated an increased proportion of alveolar macrophages expressing a more monocyte-like immunophenotype. Normally, monocytes as precursors of AM reveal a markedly stronger LDCL than alveolar macrophages themselves. Therefore, it seems likely that the increased LDCL of alveolar macrophages in IPF is due to the higher proportion of more immature monocyte-like cells in the alveoli of these patients.

Human embryonal carcinoma and yolk sac carcinoma in vitro: cell lineage relationships and possible paracrine growth regulatory interactions.

In this chapter, we will review some aspects of work in our laboratory on the control of growth and differentiation in human teratocarcinoma stem cells. We begin by describing the three cell types which we have isolated in vitro from human teratomas: embryonal carcinoma, yolk sac carcinoma resembling visceral endoderm (solid yolk sac carcinoma), and yolk sac carcinoma resembling parietal endoderm (endodermal sinus tumour). Next, we discuss the development of a new panel of monoclonal antibodies for the study of cell differentiation lineage in human teratomas. We then describe properties of a spontaneously differentiating multipotent clone of human embryonal carcinoma, and we present evidence of possible paracrine growth interaction between teratocarcinoma stem cells and yolk sac carcinoma cells.

Epitope clusters of Qa-2 antigens defined by a panel of new monoclonal antibodies.

A recently derived intra-MHC recombinant mouse strain, the C3H.KBR was found to produce a surprisingly high titer of anti-Qa antibodies when immunized with C3H.SW lymphocytes. By using this immunization combination, a panel of 10 mAb with specificity for determinants encoded by the Q region was produced. These reagents were analyzed for strain distribution by microcytotoxicity, immunofluorescence, and flow cytometry assays. Competitive inhibition analyses, performed by using fluorescein-labeled antibodies and normal spleen cells, defined at least three epitope clusters, or groups of spatially related determinants, detected by this panel. One epitope cluster was unique to this new series of antibodies in that it was not detected with seven previously described anti-Qa mAb. These antibodies also have been analyzed for reactivity with products of isolated Q-region genes by using transfected cell lines. The data indicate that the Q6d, Q7d, and Q10d genes encode determinants reactive with one or more mAb and that two of the three epitope clusters defined with normal cells map to the N and/or C2 domains of these molecules. The third epitope cluster is presumed to map to the C2 domain. These reagents should be useful in determining the number of Q-region genes expressed and in analyses of Q gene expression in subpopulations of normal cells, in transfected cell lines, and during differentiation and ontogeny.