Immune Ascites Research Articles

Cross-Reactive Antibodies to the NS1 Protein of Omsk Hemorrhagic Fever Virus Are Absent in the Sera of Patients with Tick-Borne Encephalitis.

Omsk hemorrhagic fever virus (OHFV) is a member of the tick-borne encephalitis virus (TBEV) complex of the Flaviviridae family. Currently, there are no data on the cross-reactivity of antibodies to the NS1 proteins of OHFV and TBEV. Such data are of major interest for monitoring viral encephalitis of unknown etiology due to the increasing geographical distribution of OHFV. In this study, a recombinant OHFV NS1 protein was produced using the Escherichia coli expression system and purified. The recombinant OHFV NS1 protein was recognized by specific mice immune ascetic fluids to the native OHFV NS1 protein. A Western blot analysis and ELISA of the recombinant NS1 proteins of OHFV and TBEV were used to study the cross-reactivity of antibodies from immune ascites fluid obtained from OHFV-infected mice and mAbs against TBEV NS1. Anti-TBEV NS1 mouse monoclonal antibodies (mAbs) have been shown to not be cross-reactive to the OHFV NS1 protein. Sera from patients with confirmed tick-borne encephalitis (TBE) were examined by ELISA using recombinant OHFV NS1 and TBEV NS1 proteins as antigens. It was shown for the first time that cross-reactive antibodies to the OHFV NS1 protein were not detected in the sera of TBE patients, whereas the sera contained antibodies to the TBEV NS1 protein.

Isolation of two strains of West Nile virus during an outbreak in southern Russia, 1999.

From July to September 1999, a widespread outbreak of meningoencephalitis associated with West Nile virus (Flavivirus, Flaviviridae) occurred in southern Russia, with hundreds of cases and dozens of deaths. Two strains of West Nile virus isolated from patient serum and brain-tissue samples reacted in hemagglutination-inhibition and neutralization tests with patients' convalescent-phase sera and immune ascites fluid from other strains of West Nile virus.

Purification and partial characterization of an early pregnancy factor-induced suppressor factor (EPF-S1).

The immunomodulatory properties of early pregnancy factor (EPF) are mediated through induction of at least two lymphokines, designated EPF-S1 and EPF-S2 (previously estimated M(r) 15,000 and 55,000 respectively). The activity of the former is MHC-restricted while the latter is restricted to a locus (or loci) outside the MHC. The present study established further criteria by which EPF-S1 and EPF-S2 might be distinguished from each other and compared with other suppressor factors. In addition, techniques have been developed to purify EPF-S1 to homogeneity. Congenic mouse strains were used to map the genetic restriction of EPF-S2 in the rosette inhibition test and high performance gel permeation chromatography was used to demonstrate that EPF-S1 induces EPF-S2 but not vice versa. Further studies then focused on isolation of this first component of the cascade, EPF-S1, from immune ascites (from growth in athymic mice of the anti-EPF-S1 producing rat-mouse hybridoma R2T gamma, in which EPF-S1 is complexed to antibody). Techniques used were acidification followed by application to Sep-pak C18 cartridges, high performance cation-exchange chromatography and two reversed-phased HPLC steps on a C3 column. Purified material was analyzed by SDS-PAGE and Edman degradation. Approximately 10 micrograms EPF-S1 were isolated fom 60 ml ascitic fluid. Homogeneity of the purified material was demonstrated by SDS-PAGE, where it ran as a single band of approximate M(r) 12,000 coincident with biological activity. Attempts at Edman degradation indicate that the molecule is N-blocked. Definitive primary characterization of EPF-S1 must await the preparation and isolation of proteolytic fragments of the molecule, but the present studies establish conditions which make such structural analysis possible.

Methods of measuring antibodies in sera/plasma, ascites, and tissue-culture media.

One of the easiest and most sensitive methods to screen for, to quantify, and to determine the quality of antibodies in the serum of mice, the spleens of which may be used for fusions, is by solid-phase enzyme-linked immunoassays (EIA) (,). In these procedures, the wells of 96-well plates or strip plates are coated with antigen. Antibody that binds to the antigen is detected by its reaction with an anti-immunoglobulin that has been conjugated to a reporter molecule, usually horseradish peroxidase (HRP). Quantification of antibody is accomplished through diluting sera to “extinction” levels of detection. The quality of antibody binding to antigen is determined by using conjugated antibodies, specific for isotypes rather than an anti-immunoglobulin. The procedures work equally well for detecting monoclonal antibodies (MAbs) present in tissue-culture medium from primary cultures of hybridomas, cloned hybridomas, and antibodies in immune ascites. In the latter situations, the option exists to use polyclonal or isotype-specific anti-immunoglobulin reagents conjugated to enzyme. The choice of second step reagent used depends on whether the detection system is intended to detect antibodies of a single or all immunoglobulin isotypes. A 96-well plate configuration for EIA is presented here as an example.

Active immunization with EPF suppresses the formation of immune ascites in BALB/c mice

The production of early pregnancy factor (EPF) is not confined to pregnancy--EPF has been detected as a product of tumour and transformed cells in vivo and in vitro. In this study, EPF (or an EPF-like substance) was detected also in the serum of BALB/c mice, 7 days after intraperitoneal (i.p.) administration of the mineral oil pristane. Furthermore, EPF was present in serum from mineral oil-induced plasmacytoma-susceptible mice throughout the latent period potentially leading to tumour development, peaking around the time neoplastic cells were identified in ascites. Since mineral oil-induced granuloma is essential to development of immune ascites, involvement of EPF in this process was investigated. Active immunization of BALB/c mice with EPF was shown to suppress the production of immune ascites, induced by multiple i.p. injections of antigen in complete Freund's adjuvant. Of the mice immunized with EPF (n = 19), only 47% produced ascites, compared with 94% of mice receiving saline or human chorionic gonadotrophin and 100% of mice receiving keyhole limpet haemocyanin. Further investigations revealed that ascites was only produced in mice that maintained free-circulating EPF. These mice displayed the classic mineral oil-induced granuloma covering the tissues of the peritoneum. In contrast, the serum of mice that did not produce ascites tested negative for EPF and the peritonea of these mice were devoid of the oil granuloma. These studies suggest that EPF is involved in the initiation and maintenance of the inflammatory response of the peritoneum to mineral oil.

The blocking effect of antibodies against the products of the H-2 gene complex on lymphocyte complement (C3d) receptors. complement dependence and specificity.

Antisera against the products of the major histocompatibility system (MHS), produced by immunization between congenic mouse strains differing only at that segment of the 17th chromosome rosettes which bears the H-2gene complex, exert a reproducible blocking effect on formation of with EACm (C3d rosettes). In many instances this effect is probably due to specific antibodies reactive with the lymphocytes, as indicated by immunochemical and adsorption experiments. Purified antibodies from the C3H anti-C3H.B10 immune ascites failed to exert this blocking effect. The blocking capacity can be restored, however, by addition of fresh normal mouse, rabbit or fetal calf serum, but not by heat inactivated serum. These experiments show that the presently defined H-2K, H-2D and H-2L antigens as well as some Ia antigens are themselves not the C3d receptors. The blocking effects observed in the presence of complement are possibly due to the proximity of H-2 or Ia molecules and C3d receptors, or to a rearrangement of membrane components after reaction with anti-MHC anti-MHC antibodies and complement.

Immune Ascites in the Guinea Pig: Specificity of Cells and Antibody in an Induced Peritoneal Exudate

Strain 2 guinea pigs, immunized with Lys10-Lys(Dnp) in CFA and repeatedly injected intraperitoneally with adjuvant, developed ascites. The fluid was harvested over 8 months in total amounts up to 2 liters per animal and contained substantial amounts of cells and antibody which reacted with the immunizing antigen and related peptides. The antibody was of the IgG and IgA classes and showed restricted heterogeneity. Among synthetic Dnp-oligopeptides, both the cells, studied by antigen-stimulated thymidine incorporation, and the purified antibody, studied by fluorescence quenching, demonstrated the same specificity for the immunizing antigen as has previously been noted in lymph node cells and in serum antibody. The technique offers a means for studying more cells and more antibody than has previously been possible from individual guinea pigs.

Stimulation of antibody formation against influenza virus by immunization with the use of ascites tumor

To obtain immune ascites fluids, rats were immunized three times with living influenza virus and vaccinated with an ascites strain of ovarian tumor (OYa). Considerable stimulation of formation of antiviral antibodies was observed compared with animals not vaccinated with the tumor strain. Immunofluorescence tests revealed an increase in titer of antibodies against both structural antigens of the virus and antigens induced by the virus in the membranes of the infected tumor cells. The phenomenon observed is evidently based on the local accumulation of virus antigens on a considerable scale in cells of the strain OYa.

A Serological Approach to the Study of the Male Isoantigen in Mice

Earlier tests for the H-Y (male) antigen in mice were limited to male graft rejection by syngeneic females. The experiments described in this report attest to the specificity of a new serological test for this antigen. Anti-H-Y sera and immune ascites fluid with statistically significant cytotoxic indices were produced in C57BL/6Ha females; a cytotoxicity test of sufficient sensitivity to detect and titrate cytotoxic activity, or, conversely, to detect the presence of the male antigen on C57BL/6Ha male lymphoid cells in vitro, was devised.

Tumor cell-induced mouse ascites fluid as a source of viral antibodies.

Mice immunized by multiple injections of either influenza A or Newcastle disease virus produced antibody levels in the Sarcoma 180 cell-induced ascites comparable to levels found in blood plasma as measured by hemagglutination-inhibition test. The infectivity and interfering capacity of influenza viruses could also be neutralized by the specific immune ascites. It was possible to collect at least 10 times more ascites than heart blood.