Mouse Ascitic Fluid Research Articles

Anti-protein C monoclonal antibody induces thrombus in mice

Protein C (PC) is considered to be an important regulator of blood coagulation and fibrinolysis. During the production of monoclonal antibodies (MoAbs) against human PC in mouse ascitic fluid, one hybridoma was found to induce heavy thrombus in mice, resulting in severe hemorrhage. Intravenous infusion of the purified MoAb (PC01) from this hybridoma also caused thrombosis in mice. The crossreacting substance was then isolated from mouse plasma with PC01 immunoaffinity column, which was identified as mouse PC by several criteria. Mouse PC prolonged the activated partial thromboplastin time of mouse plasma, and PC01 neutralized this in vitro anti-coagulant activity. Therefore, heavy thrombosis observed in PC01-treated mice is likely to be ascribed to the defect of PC caused by PC01.

Anti-Protein C Monoclonal Antibody Induces Thrombus in Mice

Protein C (PC) is considered to be an important regulator of blood coagulation and fibrinolysis. During the production of monoclonal antibodies (MoAbs) against human PC in mouse ascitic fluid, one hybridoma was found to induce heavy thrombus in mice, resulting in severe hemorrhage. Intravenous infusion of the purified MoAb (PC01) from this hybridoma also caused thrombosis in mice. The crossreacting substance was then isolated from mouse plasma with PC01 immunoaffinity column, which was identified as mouse PC by several criteria. Mouse PC prolonged the activated partial thromboplastin time of mouse plasma, and PC01 neutralized this in vitro anti-coagulant activity. Therefore, heavy thrombosis observed in PC01-treated mice is likely to be ascribed to the defect of PC caused by PC01.

Anion-exchange and size-exclusion chromatography of proteins in mouse ascites fluid: Changes in the protein pattern during tumour growthIn vivo

During the transition of Ehrlich mouse ascites tumour cells from the proliferating to the resting state of growth a large loss of purine and pyrimidine compounds occurs. This decrease is accompanied by a change in the amount of protein in the supernate of ascites fluid, which is known from the study of the ATP-consumption during protein synthesis. The ascites fluid was investigated by anion-exchange and size-exclusion chromatography (SEC). SDS-PAGE (sodium, dodecyl sulfate-polyacrylamide gel electrophoresis) data were compared with SEC data. The total amount of protein increased by 50% between day 5 and 12 of growth. At least 5 new peaks are observed in the chromatograms of an ion-exchange separation of Ehrlich ascites fluid at day 12 postinoculation. The amounts of transferrin, albumin and IgG (immunoglobulin G) were increased to 132, 134, 157%, respectively duringin vivo growth.

Monoclonal Antibody in the Identification of Haemophilus Somnus

Electrophoretic comparisons of outer membrane proteins of Haemophilus somnus isolates revealed 2 major protein bands (46 and 14 kilodaltons [kD]) common to all isolates tested. A monoclonal antibody raised against H. somnus reacted to the 46-kD band. Coagglutination tests were performed using a monoclonal antibody coagglutination assay. The monoclonal reagent was produced by incubating Cowan strain Staphylococcus aureus suspension, used as a source of crude protein A, with mouse ascitic fluid monoclonal antibody or goat anti-H. somnus hyperimmune serum. Bacteria to be tested were suspended at a concentration of 4.5 x 10(9) cells/ml. The coagglutination test was performed by the addition of 50 microliters of the monoclonal reagent to 50 microliters of the bacterial suspension on a glass plate and manual rotation for 2-3 minutes. The coagglutination assay using Cowan strain Staphylococcus aureus protein A, coupled with the monoclonal antibody, agglutinated 10 different H. somnus isolates. The antibody reagent did not coagglutinate with Actinobacillus suis, A. equuli, Pasteurella haemolytica, P. multocida, or P. pneumotropica under similar test conditions.

Antigens and antibodies in the ascitic fluid of mice infected with Fasciola gigantica

Fluke antigens were detected in the ascitic fluid of mice infected with Fasciola gigantica. The precipitating antibodies reactive against the antigens from metacercariae were detected as early as 2 weeks after the infection, and those against the adult and immature worms appeared 3 and 4 weeks later.

Presence of a receptor for the active component of iscador in ascites fluid of tumour bearing mice

Tumour bearing mice exhibit a specific “receptor” in the ascites fluid which binds with the active component isolated from Iscador. This “receptor” was found to be a protein which inhibited the cytotoxicity of Iscador and its isolated active component at low concentration. The receptor protein was also found in the sonicates of tumour cells which are susceptible to the action of Iscador but not in lymphocytes which were not susceptible to Iscador or its isolated active component. The receptor was separated on a Sephadex G-50 column. Activity was lost upon heat denaturation and dialysis.

Production of an equine monoclonal antibody specific for the H7 hemagglutinin of equine influenza virus

Peripheral blood leucocytes from a pony previously exposed to equine influenza virus (H3, N8) and vaccinated with killed virus (H3, N8 and H7, N7 subtypes) were cultured in vitro with live A/equine/Prague/56 (H7, N7). On the sixth day of culture, cells were harvested and fused with mouse myeloma cells (X63-Ag8.653). From this fusion, one hemagglutinin specific, equine IgG monoclonal antibody secreting hybridoma was identified and cloned twice by limiting dilution. The antibody inhibited hemagglutination by nine H7 equine influenza virus isolates obtained over a 21-year period, but did not inhibit A/equine/Miami/63 (H3, N8), or A/PR/8/34 (H1, N1). The neutralizing titer of hybridoma induced, nude mouse ascitic fluid was 10 −4.5 when tested in eggs against 100 egg infective doses (EID 50) A/equine/Prague/1/56. The hybridoma continued to synthesize antibody during more than 4 months in continuous culture.

Molecular Characterization of a Neutralizing Domain of the Japanese Encephalitis Virus Structural Glycoprotein

Expression of antigenic fragments of the Japanese encephalitis virus envelope protein (E) in Escherichia coli has been used to define the boundaries of an antigenic domain that contains the binding sites for 10 anti-E monoclonal antibodies (MAbs). All of these antibodies neutralized the virus in vitro and some of them passively protected mice from a fatal virus challenge. We have shown previously that nine of these antibodies react with the antigenic determinants encoded by a 405 bp fragment of viral cDNA. To determine the amino acid sequences of specific determinants, truncated polypeptides were expressed as fusion proteins in E. coli following progressive Bal 31 exonuclease digestion of the 5' and 3' ends of the cDNA fragment. Examination of the immunoreactivity of these polypeptides revealed that the region from methionine 303 to tryptophan 396 was the shortest sequence capable of reacting with any of the 10 MAbs or with a polyclonal, antiviral hyperimmune mouse ascitic fluid. Biochemical tests showed that an intramolecular disulphide cross-linkage between cysteine 304 and cysteine 335 of the E protein sequence was required for presentation of the binding site(s) for these MAbs. Although this 95 amino acid antigenic domain appeared to be capable of forming several conformational neutralizing epitopes, it was not an effective immunogen for inducing neutralizing or protective antibodies in mice.

Bispecific antibody-producing hybrid hybridoma and its use in one-step immunoassays for human lymphotoxin.

A hybrid hybridoma cell line secreting a bispecific monoclonal antibody (MAb) was constructed by fusing horseradish peroxidase (HRPO)-immunized mouse splenocytes with previously established mouse hybridomas secreting anti-human lymphotoxin (hLT). This cell line was grown in ascitic fluid in mice to obtain large quantities of hybrid MAbs and a bispecific antibody, reacting with both HRPO and hLT, was separated from the monospecific antibody or other inactive immunoglobulin populations by hydroxylapatite chromatography. Sodium dodecylsulfate-polyacrylamide gel electrophoresis demonstrated that the bispecific antibody molecule contained two different types of heavy and light chains of both anti-HRPO and anti-hLT origin. The bispecific antibody was used to establish one-step enzyme immunoassays (EIAs) employing competitive and sandwich systems. The simple sandwich EIA was able to detect 1-100 U/ml of hLT and there was good correlation (r = 0.96) between hLT concentrations measured by the one-step EIA and a bioassay using L929 cell-lysis.

Production and characterization of monoclonal antibodies against human glycoalbumin

Hybridomas secreting monoclonal antibodies specific for nonenzymatically glycated albumin were produced by fusion of SP2 0 myeloma cells with spleen cells from BALB/c mice immunized with unreduced nonenzymatically glycated albumin prepared from human plasma. Wells containing hybridomas secreting antibodies against glycoalbumin were identified by binding, in an enzyme-linked immunosorbent assay, to glycoalbumin isolated from human plasma or to albumin that had been glycated in vitro. The colony designated A717, which secreted antibodies discriminating between glycated versus unglycated albumin, was cloned four times by limiting dilution and used for further study, performed with monoclonal antibody purified from mouse ascites fluid. Specificity of A717 was demonstrated by immunoblotting and by ELISA, wherein the monoclonal antibody reacted preferentially with glycated albumin but insignificantly with unglycated albumin. Immunoblotting of human plasma with A717 on nitrocellulose yielded a single band, the electrophoretic mobility of which corresponded with that of authentic glycated albumin, indicating site specificity for glycated epitopes residing in albumin but not in other nonenzymatically glycated serum proteins. A717 differs from other antibodies raised against glycated albumin and other proteins, which recognize glycated residues only after reductive conversion to glucitollysine and which do not discriminate between different glycated proteins. Thus, this report describes the establishment of the first hybridoma secreting monoclonal antibody raised against unreduced glycated albumin, which is the physiologic form occuring in vivo, and for the epitope when it resides in albumin but not other proteins.

Gamma globulin-derived standards for the determination of molecular weights, transfer, and immunodetection efficiencies in protein blotting procedures

Molecular weight markers which are detectable using labeled antispecies antibodies or labeled Protein A have been prepared for use as standards on protein blots. The standards were prepared by the controlled reduction followed by subsequent alkylation of gamma globulin. Separate sets of standards were prepared using gamma globulins derived from human, mouse, rabbit, and sheep species. Standards were also prepared using monoclonal-derived gamma globulins from human myeloma fluid and mouse ascites fluid. Standards produced from monoclonal-derived gamma globulins produced very sharp bands on sodium dodecyl sulfate-polyacrylamide gels and proved to be excellent standards for this technique alone. However, the markers were uniquely suitable for use as standards in protein blotting procedures because their detection was achieved by the procedure used to detect the transferred antigen(s). The detection of immunoglobulin G (IgG)-derived standards on protein blots from all the species listed above was demonstrated using appropriate horseradish peroxidase (HRP)-conjugated antispecies antibodies. The use of other detection systems (biotin-labeled antibody and subsequent detection with HRP-steptavidin, HRP-Protein A) was also validated with human IgG-derived standards. Furthermore, the standards were shown to be suitable for use on both nitrocellulose and cationized nylon-based supports and could be used when adjacent samples were run under reducing conditions. Hence the gamma globulin-derived standards serve as both a control to check the adequacy of transfer and immunodetection systems and as markers which enable the molecular weights of detected antigens to be calculated.

Antigen-induced bronchopulmonary alterations in the guinea pig: a new model of passive sensitization mediated by mouse IgE antibodies.

A selective model to study the IgE-mediated anaphylactic bronchoconstriction (BC) in the guinea pig is needed, since human asthma involves mainly this class of antibody. However, most procedures presently available for passive homologous or active sensitization lead to responses which are mediated both by IgE and IgG antibodies. In this study, we developed an anaphylactic model in which guinea pigs are passively sensitized with mouse ascitic fluid containing dinitrophenol (DNP)-specific IgE antibodies. Challenge of sensitized animals with DNP coupled to bovine serum albumin evokes a bronchoconstrictor response that is maximal 5 h after sensitization. The resulting anaphylactic BC is not blocked by the H1 histamine antagonist mepyramine, by the peptido-leukotriene antagonist FLP 55712 nor by the platelet-activating factor antagonist BN 52021 alone. However, when the sensitized animals are pretreated with the three drugs in combination, significantly reduced BC was observed upon challenge with the antigen. This latter result indicates that IgE-dependent BC involves the participation of different mediators, a characteristic shared in common with allergic asthma in human.

32] Immunoprecipitation of ribonucleoproteins using autoantibodies

This chapter describes immunoprecipitation procedures developed to identify and characterize the components of small ribonucleoproteins (RNPs) using patient polyclonal or mouse monoclonal autoantibodies. Monoclonal antibodies, either in the form of mouse ascites fluid or cell culture supernatants, can be used for immunoprecipitation of small RNPs. Tissue culture cells are the preferred source of extracts to immunoprecipitate small RNPs. Tissues can be used, but as tissues are bathed in serum before removal from the animal, ribonuclease levels are usually high and can result in RNA degradation during the immunoprecipitation procedure. If phosphoproteins are to be analyzed, RNAs contained in the immunoprecipitate must first be destroyed or the RNAs will produce an intolerably high background in the Laemmli gel. Immunoprecipitation will detect all proteins in a RNP complex, whether the proteins are antigenic or not. The antigenic protein(s) themselves can be detected by transfer from the gel to nitrocellulose and by Western blotting.

Analysis of epitope specificity of monoclonal antibodies by counterflow isotachophoresis on nitrocellulose membranes

When working with monoclonal antibodies (MCAB) to an individual antigen (AG) it is necessary to determine that epitope specificity, i.e., their targeting toward the same or different determinants. Competitive analysis of MCAB is usually used for this purpose, by solidphase radioimmunoassay [4], for which purified AG and radioactive labeling of each of the MCAB to be compared are required. In the investigation described below it is shown that epitope specificity of MCAB can be analyzed by counterflow isotachophoresis (ITP) on a nitrocellulose membrane (NCM) [I] on a model of MCAB to human (HAFP) and mouse (MAFP) a-fetoprotein (AFP). The method does not require purified AG or MCAB, or their radioactive labeling, it canbe performed automatically, and requires only the simplest electrophoretic equipment. Rat MCAB to MAFP and mouse MCAB to HAFP were used. Rat MCAB were obtained as described previously [6]. Culture media (CM) of three hybridomas (D6, G~, Ha), both initial and concentrated tenfold by precipitation with (NH~)2SO~ (0.33 of saturation), were used for analysis. MCAB to HAFP were obtained by fusing the splenocytes of a mouse immunized with a purified HAFP preparation and myeloma X63-AgS.653 cells. MCAB of three cell lines (D1o, De, Es) in mouse ascites fluid were used. Rabbit antiserum of MAFP (anti-MAFP) was obtained as described previously [2] and neonatal mouse serum (NMS) was used as the MAFP preparation. A commercial immunodiagnostic serum for primary carcinoma of the liver (N. F. Gamaleya Institute of Epidemiology and Microbiology, Academy of Medical Sciences of the USSR), including rabbit anti-HAFP and AG, containing 50 ~g HAFP in i ml and with a large excess of serum albumin, was used as the system for determining HAFP. Mouse MCAB to rat L~ IgG (rat anti-L K containing 3 mg of antibodies in i ml [5] and rat IgG (i0 mg/ml), generously provided by A. Yu. Rudenskii (AllUnion Research Institute of Genetics, Main Administration of the Microbiological Industry of the USSR), were used. Fab'-fragments of donkey antiserum to rabbit IgG, conjugated with horseradish peroxidase (P) (Fab'-P), were obtained by V. S. Poltorandina by the method in [8]. To abolish cross reactions between reagents used in the analysis, embryonic calf serum (ECS) was added to the anti-MAFP, and rat IgG and ECS were added to the Fab'-P in concentrations chosen beforehand.

A Monoclonal Antibody to Exfoliated Surface Vesicles That Recognizes a Membrane-Associated Erythroid Burst-Promoting Activity

A monoclonal antibody (MoAb) recognizing a membrane-associated erythroid burst-promoting factor was prepared by immunizing BALB/c mice with plasma membrane-derived vesicles exfoliated from lymphocytes under serum-free conditions. Hybrids secreting antibody reactive with lymphocyte plasma membranes were formally cloned and IgG was purified from monoclonal supernatants or from BALB/c mouse ascites fluid. Two clones (D3-E4 and D3-G9) were found to suppress burst forming unit-erythroid (BFU-E) proliferation when added directly to serum-free human marrow culture. Inhibition to a level of 100% was observed in a dose-dependent fashion over a wide range of antibody concentrations (0-200 micrograms/mL). Neither antibody altered the proliferation of colony forming unit-granulocyte macrophage (CFU-GM) or colony forming unit-granulocyte-erythroid-monocyte-megakaryocyte (CFU-GEMM) progenitor cells in human bone marrow culture. The D3-E4 clone was found to produce an IgG1 antibody which adsorbs an erythroid burst-promoting activity (BPA) from supernatants of, and octylglucoside extracts of shed vesicles present in, serum-free, lymphocyte conditioned medium (LCM), and which recognizes a vesicular protein of Mr approximate 30,000 on immunoblots of membrane proteins electrophoresed on sodium dodecyl sulfate (SDS)/polyacrylamide and transferred to nitrocellulose. In contrast, the D3-G9 clone was found to produce an IgG1 cytotoxic antibody. These antibodies will be important to the study of cell-cell and growth factor-cell interactions in vitro.

A monoclonal antibody to exfoliated surface vesicles that recognizes a membrane-associated erythroid burst-promoting activity

A monoclonal antibody (MoAb) recognizing a membrane-associated erythroid burst-promoting factor was prepared by immunizing BALB/c mice with plasma membrane-derived vesicles exfoliated from lymphocytes under serum-free conditions. Hybrids secreting antibody reactive with lymphocyte plasma membranes were formally cloned and IgG was purified from monoclonal supernatants or from BALB/c mouse ascites fluid. Two clones (D3-E4 and D3-G9) were found to suppress burst forming unit- erythroid (BFU-E) proliferation when added directly to serum-free human marrow culture. Inhibition to a level of 100% was observed in a dose- dependent fashion over a wide range of antibody concentrations (0–200 micrograms/mL). Neither antibody altered the proliferation of colony forming unit-granulocyte macrophage (CFU-GM) or colony forming unit- granulocyte-erythroid-monocyte-megakaryocyte (CFU-GEMM) progenitor cells in human bone marrow culture. The D3-E4 clone was found to produce an IgG1 antibody which adsorbs an erythroid burst-promoting activity (BPA) from supernatants of, and octylglucoside extracts of shed vesicles present in, serum-free, lymphocyte conditioned medium (LCM), and which recognizes a vesicular protein of Mr approximate 30,000 on immunoblots of membrane proteins electrophoresed on sodium dodecyl sulfate (SDS)/polyacrylamide and transferred to nitrocellulose. In contrast, the D3-G9 clone was found to produce an IgG1 cytotoxic antibody. These antibodies will be important to the study of cell-cell and growth factor-cell interactions in vitro.

Isolation of monoclonal antibodies to phencyclidine from ascites fluid by preparative isoelectric focusing in the Rotofor

A monoclonal antibody to phencyclidine was developed, produced in mouse ascites fluid, and purified. The purification used only preparative-scale isoelectric focusing in the Rotofor and dialysis. In 4 h, 25% (4 mg) of the antibody from 10 ml of ascites fluid was purified to homogeneity while 63% of the total antibody was recovered.

High Performance Liquid Chromatography of Mouse Monoclonal Antibodies on Spherical Hydroxyapatite Beads

Abstract High performance liquid chromatography (HPLC) on newly developed spherical beads of hydroxyapatite was applied for the simple purification of monoclonal antibodies (mAbs) secreted into mouse ascitic fluid. Sixteen mAbs including all four subclasses of IgG and IgM were separated successfully from serum albumin a major contaminant in the crude mAb preparation by a 30 min-linear gradient of phosphate ion concentration from 0.01M-0.3M at pH 7.2. Not only IgG mAbs but also IgM mAbs were quantitatively eluted from the column. Each antibody had a different retention time (apparent capacity factor of 2.24–4.14) in the chromatography and no relation was found between the retention time and the type of immunoglobulin (class or subclass). A monomeric form of IgM was also resolved successfully from IgM (pentamer) after its reduction with dithiothreitol; the monomer form of IgM was eluted from the column by a lower concentration of phosphate ion than was the pentamer. These results indicate that HPLC on the h...

Plasma nucleic acid adsorption on charcoal sorbents in patients with leukemia.

The study employing labelled precursors of nucleic acids with their double-strand structure was identified by bromide ethidium. In addition, the application of hemocarboperfusion revealed active and irreversible adsorption of nucleic acids and nucleoproteids from ascitic fluid of mice with NKLy leukemia and from plasma of leukemia patients.

Purification of monoclonal antibodies against the low-density lipoprotein receptor by preparative isotachophoresis

A preparative free-flow isotachophoretic method for the purification of monoclonal antibodies from mouse ascites fluid and tissue culture media is described. This high-resolution method allows the direct separation of monoclonal antibodies from antibody-containing tissue culture media or ascites fluid and gives a better separation from the major contaminant protein fractions and a higher recovery of the monoclonal antibodies than anion-exchange chromatography. The purification can run continuously and without any time-consuming regeneration procedures; the monoclonal antibody is obtained under mild conditions in a small electrolyte volume.