A novel rat monoclonal antibody directed against a population of activated mouse peritoneal macrophages

A hybridoma clone secreting rat monoclonal antibody (MAB) designated as 3F3.5F and which reacted with a population of activated tumoricidal mouse peritoneal macrophage (M phi) was produced by the fusion of mouse myeloma cells with rat spleen cells immunized against adherent BCG-activated mouse peritoneal exudate cells (adherent BCG-PEC). The antibody was cytotoxic and of the rat IgM class. The specific reactivity of the antibody with mouse primary cells and cell lines was examined by complement-dependent cytotoxicity and indirect immunofluorescence flow cytometry analysis. The antibody was found to bind to about 40% of the adherent BCG-PEC activated in vivo and elicited peritoneal macrophages activated in vitro by lymphokine and lipopolysaccharide (LPS), to about 35% of polymorphonuclear neutrophils (PMN) 15 hr after intraperitoneal injection of BCG, to about 30% of bone marrow cells from BCG-infected mice, to about 10% of P815 mastocytoma cells and to thioglycollate-induced PEC to some degree. It did not bind to other cells tested including BCG-induced peritoneal lymphocytes, non-tumoricidal PEC, thymocytes, spleen cells, resting bone marrow cells from normal mice, lymphomas, myelomas, fibroblasts, or macrophage-cell lines. Pretreatment of adherent BCG-PEC with MAB 3F3.5F and rabbit complement caused a considerable decrease in tumor cytotoxicity toward P815 cells, but the same pretreatment of non-adherent BCG-PEC had no inhibitory effect on natural killer activity for YAC-1 cells.     

Immunocytochemical analysis of embryonic compartmentation with a monoclonal antibody against a cytokeratin-related antigen

Summary Mab 113F4, a monoclonal antibody recognizing an antigen in the outer synaptic layer of the chick neural retina, also recognizes an antigen appearing in all three germ layers of the gastrulating chick embryo. However, as neurulation proceeds, the antigen is down-regulated in three distinct patterns. First, the antigen is lost specifically from those trunk ectodermal cells destined to form the neural plate and, later, the neural tube. It remains absent from any neural derivative until day 13 when it appears in the outer synaptic layer of the neural retina, coincident with synaptogenesis in this region. Second, the entirety of the head ectoderm loses this antigen as the head lifts off the blastoderm. This down-regulation is followed later by a similar loss of antigen expression in the trunk ectoderm. Third, expression in the mesoderm becomes limited to the lateral plate and extraembryonic epithelia. Endodermal derivatives continue to express the antigen throughout development. Antigen 113F4 is localized within the cytoplasm and is organized in a fibrillar pattern. The intracellular localization of this antigen and its characteristic spatio-temporal tissue distribution are consistent with the antigen being a cytokeratin or cytokeratin-related antigen. The changes in tissue distribution suggest a possible role in tissue modelling in response to inductive interactions during development.     

Immunocytochemical identification of bovine Langerhans cells by use of a monoclonal antibody directed against class II MHC antigens

We undertook a study to develop a reliable light microscopic technique for identifying Langerhans cells (LC) in bovine epidermis. Monoclonal antibodies (MCA) detecting bovine class II MHC antigens were used in conjunction with an avidin-biotin-peroxidase complex (ABC) immunocytochemical staining method. The specificity of the MCA for LC was confirmed ultrastructurally by use of gold-labeled second antibody. Epidermal sheets and epidermal single-cell suspensions examined by light microscopy confirm that bovine epidermal LC express class II antigens. Anti-bovine class II MCA is a dependable reagent for identification of LC in normal bovine epidermis.     

Clinical radioimmunolocalization with a rat monoclonal antibody directed against c-erbB-2

Lymph node status is still the single most important prognostic factor in breast cancer and surgery remains the only reliable means of providing this information. This study evaluates using a highly specific radiolabeled monoclonal antibody to provide equivalent information. The optimum labeling conditions for radiolabeling a monoclonal antibody against the gene product of the protooncogene c-erbB-2 with Tc99m were established. This immunoconjugate was next evaluated in a mouse model system and averaged 20% localization of the total injected dose per gram of tumor at 24h. Ten patients have had this immunoconjugate, with planar and tomographic reconstructed images being obtained at 24 h. The resulting images were compared to histopathological examination of the surgical specimens. Three patients acted as normal controls, two patients were selected on the basis of inappropriate sampling of adjacent ductal carcinomain situ, three patients demonstrated only moderate antigen expression, and two patients demonstrated excellent tumor localization in both breast primary and regional node metastases. The high specificity of this antibody, ease of labeling, and excellent localization performance with a good antigen target encourage the development of this system as a method of localization and a potential means of antibody-guided therapy.     

Immunocytochemical analysis of embryonic compartmentation with a monoclonal antibody against a cytokeratin-related antigen.

Mab 113F4, a monoclonal antibody recognizing an antigen in the outer synaptic layer of the chick neural retina, also recognizes an antigen appearing in all three germ layers of the gastrulating chick embryo. However, as neurulation proceeds, the antigen is down-regulated in three distinct patterns. First, the antigen is lost specifically from those trunk ectodermal cells destined to form the neural plate and, later, the neural tube. It remains absent from any neural derivative until day 13 when it appears in the outer synaptic layer of the neural retina, coincident with synaptogenesis in this region. Second, the entirety of the head ectoderm loses this antigen as the head lifts off the blastoderm. This down-regulation is followed later by a similar loss of antigen expression in the trunk ectoderm. Third, expression in the mesoderm becomes limited to the lateral plate and extraembryonic epithelia. Endodermal derivatives continue to express the antigen throughout development. Antigen 113F4 is localized within the cytoplasm and is organized in a fibrillar pattern. The intracellular localization of this antigen and its characteristic spatio-temporal tissue distribution are consistent with the antigen being a cytokeratin or cytokeratin-related antigen. The changes in tissue distribution suggest a possible role in tissue modelling in response to inductive interactions during development.     

Immunocytochemical staining of ovarian cyst aspirates with monoclonal antibody against inhibin

mccluggage w. g., patterson a., white j. and anderson n. h. (1998) Cytopathology 9, 336–342
Immunocytochemical staining of ovarian cyst aspirates with monoclonal antibody against inhibin
Inhibin is a peptide hormone which is produced by ovarian granulosa cells during normal follicular development. It is important that granulosa cells are recognized in fine needle aspirates (FNAs) of ovarian cystic lesions, as this allows definite recognition of a functional cyst and exclusion of a potentially neoplastic epithelial lined cyst. Occasionally the distinction between granulosa and epithelial cells may be difficult, especially when aspirates from functional cysts are unusually cellular. In the present study, FNAs from 33 ovarian cystic lesions were immunostained with a monoclonal antibody against inhibin. Nine cases of peritoneal fluid containing malignant cells in patients subsequently confirmed to have ovarian adenocarcinoma were also stained. Where possible the cytological and immunocytochemical findings were correlated with subsequent biopsy. In most cases in which cytology suggested a functional cyst there was a strong positive staining with anti-inhibin, although occasional cases were negative. One case originally thought to contain epithelial cells stained strongly positive with anti-inhibin and on review was felt to represent a cellular functional cyst. In all other cases where cells were considered to be epithelial there was no staining with anti-inhibin. The study shows that immunocytochemical staining with anti-inhibin may be of value in confirming the presence of granulosa cells, thus establishing a diagnosis of functional cyst. Although negative staining does not exclude a functional cyst, positive staining with anti-inhibin allows exclusion of an epithelial lined cyst and may avoid unnecessary surgical intervention.     

Production of a monoclonal antibody directed against rat liver glutathione-insulin transhydrogenase

A hybridoma cell line secreting monoclonal antibody specific for glutathione-insulin transhydrogenase has been produced by fusing mouse myeloma cells with spleen cells from mice immunized to purified rat liver glutathione-insulin transhydrogenase. The secreted antibody isotypes were found to be: Ig gamma 1 heavy chains and kappa light chains. This monoclonal antibody has been used to screen glutathione-insulin transhydrogenase in various rat tissue extracts (liver, fat, heart, testis, spleen, lung and kidney) following separation on NaDodSO4/urea polyacrylamide disc-gel electrophoresis and electrophoretic transfer to nitrocellulose. Screening with the monoclonal antibody showed the presence of one immunoreactive protein band equal in molecular weight to that of purified rat liver GIT (Mr 53,000) in extracts of all tissues studied and a second immunoreactive protein band of lower molecular weight (Mr 49,000) in spleen and lung tissue extracts. Separation of these two proteins by HPLC using a TSK-DEAE column demonstrated that both proteins exhibit insulin degrading activity. These data indicate that GIT may occur in multiple forms in some tissues.     

Preliminary crystallographic study of the Fab fragment of a monoclonal antibody directed against a cobra cardiotoxin

We report on the preparation, crystallization and preliminary X-ray crystallographic study of the Fab fragments from a murine monoclonal anti-cardiotoxin antibody M gamma 2-3 directed against a cobra cardiotoxin. The Fab fragment has been crystallized from polyethylene glycol 8000 solutions in a form suitable for high-resolution, X-ray crystallographic studies. The crystals are monoclinic, space group C2, with a = 161.2 A, b = 40.4 A, c = 96.5 A, beta = 118.3 degrees.     

A monoclonal antibody directed against the head region of vimentin

The production and identification of a monoclonal antibody directed against an epitope in the aminoterminal head region of vimentin is described. Enzyme-linked immunosorbent assay, protein blotting and indirect immunofluorescence were used. The wide range of cross-reactivity within cytoskeletal proteins observed for this antibody gives evidence for a determinant in an evolutionarily conserved region. Computer comparison of aminoacid sequences of the immunoreactive proteins and biochemical cleavage of vimentin provide possible clues to some antigenic determinants.     

An immunoprotective monoclonal antibody directed against Leptospira interrogans serovar copenhageni

A monoclonal antibody (mAb) was prepared by hybridoma technology in BALB/c mice immunized to Leptospira interrogans serovar copenhageni. This mAb agglutinated serovars copenhageni and icterohaemorrhagiae to high titres and protected hamsters, dogs and monkeys against challenge with a virulent strain of serovar copenhageni. The mAb gave protection to hamsters at dilutions up to 1 in 1000; at a 1 in 10 dilution the protective effect lasted for at least two weeks. Biochemical analysis by SDS-PAGE and Western blotting indicated that this mAb reacted with an epitope of a carbohydrate nature.     

Two monoclonal antibody lines directed against subunit IV of cytochrome oxidase: a study of opposite effects

Two monoclonal lines of antibodies were isolated with specificities against the amino half of Subunit IV of beef heart cytochrome oxidase. The lines had nonoverlapping epitopes. Both bound to the matrix face of membranous oxidase, neither bound to the cytoplasmic face. One line (QA4/C4) stimulated electron transfer in soluble or membranous oxidase, while the other (QA4) inhibited that activity by both oxidase preparations. These effects on electron transfer activity were not altered by the inclusion or omission of detergent. ATP depressed the binding of either antibody to either soluble or membranous oxidase. In the absence of ATP, QA4/C4 stimulated electron transfer only in the high affinity phase of cytochrome c oxidation (with decreased KM and increased Vmax), causing slight inhibition in the low affinity phase (with decreased KM). In the presence of ATP, QA4/C4 abolished the high affinity phase, but did not alter the ATP influence on the low affinity phase. In the absence of ATP, antibodies of line QA4 abolished the low affinity phase, leaving a high affinity phase similar to that induced by ATP. In the presence of ATP, QA4 abolished the high affinity phase, leaving a low affinity phase similar to that seen with ATP alone. This behavior is consistent with the dissection of two catalytic sites for cytochrome c and more than one ATP affector site.     

Production of a monoclonal antibody directed against the recombinant SEL1L protein

SEL1L, highly similar to the C elegans sel-1 gene, is a recently cloned human gene whose function is under investigation. SEL1L is differentially expressed in tumors and normal tissues and seems to play a role in tumor growth and aggressiveness. We used the recombinant N-terminus of the SEL1L protein to immunize a Balb/c mouse and produce a monoclonal antibody. A hybridoma secreting an antibody specifically reacting on the SEL1L recombinant fragment was selected. This monoclonal antibody, named MSel1, recognizes the SEL1L protein by Western blotting, immunofluorescence and immunohistochemistry on normal and tumor cells. MSel1 is able to recognize SEL1L even on archival tumor specimens and is therefore particularly appropriate to study SEL1L involvement in tumor progression.     

Structural analysis of the epitope recognized by a monoclonal antibody directed against tricyclic antidepressants

After somatic cell fusion between splenocytes of immunized BALB/c mice and NS-1 myeloma cells, a clone was obtained that secreted an anti-nortriptyline antibody of the IgG1 kappa isotype. The association constant of this antibody for pharmacologically active tricyclic antidepressant drugs ranged from 0.6 X 10(7) to 3 X 10(7) M-1. From thermodynamic and binding studies as well as tridimensional structures of tested compounds, the epitope recognized by the monoclonal antibody appeared to include both a hydrophobic tricycle in which the two phenyl rings form an angle of 120 to 130 degrees, and a side chain in which the amino group is separated from the two lateral rings of the tricyclic structure by a distance of approximately 5.9 A and 7.5 A, respectively. This conformation seems to be the one interacting with muscarinic acetylcholine brain receptors.     

Characterization of a monoclonal antibody directed against the murine B lymphocyte receptor for IgE

A rat hybridoma producing a high-affinity IgG2a monoclonal antibody (B3B4) directed against against the murine lymphocyte IgE receptor (Fc epsilon R) was established by using purified Fc epsilon R from Fc epsilon R+ murine hybridoma B cells as immunogen. The monoclonal and polyclonal anti-Fc epsilon R inhibited the binding of IgE to the murine lymphocyte Fc epsilon R and were also used to isolate the Fc epsilon R. B3B4 specifically recognized only the 49-Kd Fc epsilon R on murine B lymphocyte as determined by immunoprecipitation and SDS-PAGE analysis. In addition to its reaction with intact Fc epsilon R, B3B4 also recognized Fc epsilon R fragments that were present in the culture media of Fc epsilon R+ hybridoma cells. The predominant fragments isolated were 38 Kd and 28 Kd by SDS-PAGE analysis. When tested for reactivity with other cell types, B3B4 was highly specific for murine B lineage cells in that it did not significantly react with Fc epsilon R on macrophages and T cells and, in addition, did not react with the high affinity mast cell Fc epsilon R. B3B4 completely blocked IgE rosetting, and a reciprocal inhibition of binding was seen in a dose-dependent fashion between IgE and B3B4, indicating a close proximity of the IgE and B3B4 binding sites. Saturation binding analysis indicated that the Fab' fragment of B3B4 bound to twice as many sites/cell as IgE, suggesting that there are two identical B3B4 determinants per 49-Kd Fc epsilon R or that the IgE binding site is formed by the association of at least two 49-Kd Fc epsilon R. However, unlike IgE, neither B3B4 nor F(ab')2-B3B4 nor Fab'-B3B4 were very effective in causing Fc epsilon R upregulation on murine hybridoma B cells; in fact, B3B4 prevented this upregulation when added in combination with IgE. These results suggest that a site-specific interaction provided only by IgE may be essential for ligand-specific upregulation. Both polyclonal and monoclonal antibodies will be useful in further studies concerning the functional relationship between the membrane Fc epsilon R and the soluble Fc epsilon R fragments.     

Characterization of a monoclonal antibody directed against the epidermal growth factor receptor binding site

Summary In this work a new monoclonal antibody (mAb), designated MGR1, which recognizes the epidermal growth factor receptor (EGF-R) binding site, is described. The main characteristic of this mAb is its ability to discriminate between cells that express normal levels of EGF-R from cells with overexpression, the detectability threshold by immunocytochemical tests being 5 × 10⁴ receptors/cell of 10 µm diameter. MGR1 was found to inhibit EGF binding on the relevant target cells, and vice versa its binding was inhibited by EGF, which indicated that MGR1 recognizes the EGF receptor binding site. MGR1 exerted an inhibitory effect on both the in vitro and in vivo growth of cells with EGF-R overexpression, but had no effect on cells with a normal expression of the receptor. Tumour growth inhibition in athymic mice was also obtained on already implanted tumours. MGR1 therefore seems to be an adequate reagent for the development of immunotherapeutical approaches suitable for the treatment of tumours with EGF-R overexpression.     

Production of a monoclonal antibody directed against the high-affinity nerve growth factor receptor.

The high-affinity nerve growth factor receptor corresponds to the tyrosine protein kinase encoded by the proto-oncogene trkA. Different findings suggest that nerve growth factor (NGF) can be operative in the growth modulation of tumor cell lines possessing high-affinity binding sites for this molecule. Using as immunizing material the SKNBE neuroblastoma cell line transfected with proto-trkA we produced a monoclonal antibody (MAb) able to recognize the high-affinity nerve growth factor receptor. The selected MAb, designated MGR12, is directed against an epitope present on the extracellular domain of the receptor since it showed reactivity on living trkA-expressing cells and was able to immunoprecipitate the proto-trkA molecule. The MGR12 MAb is directed against a non-functional epitope since it neither inhibited NGF binding nor induced receptor internalization. This new reagent appears to be an appropriate tool for analyzing the expression of high-affinity nerve growth factor receptor in tumors of different origin and for elucidating its involvement in tumor progression.     

Cellular proteins reactive with monoclonal antibodies directed against simian virus 40 T-antigen.

Several recently isolated monoclonal antibodies which reacted with simian virus 40 T antigens also reacted with proteins found in uninfected and untransformed cells. The proteins were different from each other, PAb419 reacting with a 35,000-molecular-weight protein, PAb427 reacting with a 75,000-molecular-weight phosphoprotein, PAb405 reacting with a 150,000-molecular-weight phosphoprotein, and PAb204 reacting with a 68,000-molecular-weight protein. It is suggested that although some of these cross-reactions may be fortuitous, they may, as an alternative, reflect similarities of shape and perhaps function between domains of the viral T antigen and the relevant host proteins.     

Immunocytochemical staining of breast carcinoma with the monoclonal antibody NCRC 11: a new prognostic indicator.

The staining of breast cancer with a new monoclonal antibody, NCRC 11, was studied in a series of 126 women with primary breast carcinoma. Tumour samples embedded in paraffin were tested, and the minimum duration of follow up was five years or to death. Altogether 119 tumours stained positively. There was a strong relation between the intensity of staining, divided on a four point scale, and patient survival. Patients whose tumours exhibited intense staining had an improved survival compared with those with less intensely staining tumours (p less than 0.0001). Staining related weakly to histological grade but not significantly to oestrogen receptor state or the pathological stage of lymph node disease. Mathematical analysis showed the relation to survival to be independent of the other known prognostic factors. Inclusion of intensity of staining with other factors in a prognostic index might permit a more accurate estimation of prognosis in patients with breast cancer.     

A new monoclonal antibody (3D3) generated with human respiratory mucins and directed against Lewis determinants

We have prepared a monoclonal antibody (MAb), 3D3, raised againstpurified human respiratory mucins. This antibody recognizedmucins and proteolytically derived glycopeptides. The epitoperecognized by the antibody was destroyed by -L-fucosidase, indicatingthat it was present on the carbohydrate moieties. Structuralspecificity was determined by adsorption on a variety of synthetic,insolubilized oligosaccharides. Several lines of evidence indicatethat the 3D3 MAb reacted strongly with the Lewis (Le^b) antigen,but also recognized Le^a and Le^y determinants. This antibodymight be useful to study mucin secretion. human bronchial mucins Lewis b