CZ2001709A3 - Murine lymphocyte hybridoma PED 3C8/2H6 - Google Patents

Abstract

The present invention relates to a murine lymphocytic hybridoma PED 3C8/2H6 being deposited in the Collection of Zoopathogenic Microorganism of the Research Institute of Veterinary Medicine in Brno under designation CAPM-TK-8/01-PED 3C8/2H6, which hybridoma produces a monoclonal antibody reacting specifically with dominant antigens of pig epizootic diarrhea (PED) virus. The monoclonal antibody reacts at immunoperoxidase and ELISA assay specifically with PED virus. At Western blot analysis, the monoclonal antibody reacts with a membrane viral protein M with the size of 22 to 28 kDa. The prepared monoclonal antibody is usable for both further research and careful and specific proof of PED virus in infected cells and in faeces of individuals suffering from diarrhea disease.

Description

Technical field

The subject of the invention is the murine lymphocyte hybridoma PED 3C8 / 2H6 useful in veterinary medicine for highly specific and sensitive diagnosis of porcine epizootic diarrhea.

BACKGROUND OF THE INVENTION

The agent of porcine epizootic diarrhea (PED) is a single stranded RNA coronavirus with a virion size of 80 to 160 nm. Virions are covered with protuberances of 20 nm length forming a characteristic hemming that gives the impression of a corona. In general, the following major structural proteins are shown for coronaviruses: glycoprotein S of 150 to 200 kDa, membrane protein M of 20 to 30 kDa, and nucleocapsid phosphoprotein of 45 to 57 kDa.

coronavirus PED is characterized by a high affinity for the enterocytes forming the villi cover of the small intestine, especially jejunum and ileum. Together with rotaviruses and coronavirus VGP / TGE (transmissible gastroenteritis), PED is one of the most important agents of porcine gastroenteritis of viral origin. PED coronavirus and VGP / TGE coronavirus are morphologically identical, but their antigenic composition is quite different. In all viral gastroenteritis, the disintegration of infected enterocytes leads to desquamation of the intestinal epithelium and damage to the intestinal barrier. As a result of this damage, extensive losses of proteins, salts and significant changes in the ionic balance of the organism occur. Damaged intestinal barrier allows penetration of toxins, bacteria, fungi and other harmful substances into the body. Clinical signs of these changes are severe diarrhea, often leading to death, depending on the age of the animal. In surviving individuals, secondary complications (bacterial and fungal infections, etc.) lead to chronic disturbances, stunting, and decreased increments.

The severity of viral gastroenteritis (together with gastroenteritis of bacterial origin) lies in high losses (deaths of newborn piglets and stunts and reduction in growth in surviving animals), which significantly affect farmers' economic performance. The introduction of preventive and prophylactic measures requires rapid and specific diagnosis of the causative agent. The specific diagnosis of pathogens of viral gastroenteritis has not been performed routinely yet, but due to extraordinary professional, financial and time demands only in specially indicated cases at research institutes. In general, the following methods can be used to diagnose viral gastroenteritis:

(a) virological methods involving the isolation and multiplication of virus from a faeces sample of a diarrhea individual on cell cultures and subsequent immunocytochemical detection of the virus by specific polyclonal or monoclonal antibodies. These methods are very time, methodically and financially demanding. Examination of larger numbers of samples is unrealistic.

b) detection of virus in faeces samples by electron microscopy and immunoelectron /

microscopic examination. The method requires demanding instrumentation as well as methodological and professional knowledge. Lets you examine smaller sample files.

(c) immunohistochemical methods allowing specific detection of virus in histological sections of small intestine specimens collected post mortem. The method allows screening of smaller sample sets and requires the use of high-quality specific antibodies and conjugates (fluorescent and enzymatic).

d) immunochemical methods allowing direct detection of the virus in the faeces of the diarrhea individual (ELISA, DOT-ELISA, etc.) using virus-specific polyclonal or monoclonal antibodies and enzyme conjugated antibodies. Polyclonal antiviral sera prepared by immunization of experimental animals kept under normal conditions are unsuitable for these needs (the presence of antibodies against other viruses and bacteria and the possibility of cross-reactions of these sera does not guarantee sufficient specificity). Specific antiviral sera are therefore difficult and costly to obtain by immunizing animals reared under sterile (germ free) conditions or under controlled colonization of the gastrointestinal tract by apatogenic E. coli, etc. (SPF - specific pathogen free rearing). The quality and specificity of the diagnostic methods listed under a) to d) above (immunocytochemical, immunoelectron microscopic, immunohistochemical, ELISA etc. virus detection) also depend on the quality of these sera and antibodies. Assuring these conditions, immunochemical methods (ELISA, DOT-ELISA) allow rapid and sufficiently sensitive examination of large sets of field samples.

The immunoassays under these conditions allow large numbers of field samples to be immunostained

SUMMARY OF THE INVENTION

5histochemical

Problems with the preparation of specific polyclonal antiviral sera can be overcome by the use of a monoclonal antibody reacting specifically only with PED dominant antigens. The mouse lymphocyte hybridoma PED 3C8 / 2HO deposited in the Collection of Zoopathogenic Microorganisms of the Research Institute of Veterinary Medicine in Brno under the designation CAPM-TK-8/01 -PED 3C8 / 2HO, which produces a monoclonal antibody against membrane protein M of 22-28 kDa obtained by a method known in the literature (KOHLER, G., MILSTEIN, C.: Continuous Cultures of Fused Cells Secreting Antibody to Predefined Specificity, Nature 256, 495-4-97 (1975); Monoclonal Antibodies, Principles and Practice, JW Goding ed. (Acad. Press, 315 (1986)) by selecting and cloning producing hybridoma cells resulting from the fusion of mouse myeloma line Sp 2/0 and lymphocyte cells obtained from the spleen of BALB / c mice immunized with purified PED virus.

The advantage of the murine lymphocyte hybridoma PED 3C8 / 2H6 is that it produces a monoclonal antibody that is immunochemically and biologically homogeneous and reacts specifically with viral antigens only.

The high specificity of the monoclonal antibody produced by the PED 3C8 / 2H6 hybridoma was confirmed by examination of the monoclonal antibody by Western blot analysis, ELISA and specific detection of PED virus in infected cell lines and intestinal enterocytes of experimentally infected piglets by immunoperoxidase assay.

Producing hybridoma cells are non-immortal ”(stored indefinitely in a liquid nitrogen environment), allowing monoclonal antibodies of consistent quality to be produced in any amount, thereby performing standard, highly specific, and sensitive diagnostics.

Compared to the prior art based on the use of polyclonal antibodies, the use of monoclonal antibodies represents a qualitatively higher degree in the diagnosis of viral gastroenteritis and in the field of further studies of these diseases.

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DETAILED DESCRIPTION OF THE INVENTION

To obtain more monoclonal antibodies, the hybridoma-producing hybridoma is administered intraperitoneally to BALB / c mice that have been sensitized 7-30 days prior to intraperitoneal administration of Pristan (Sigma) or paraffin oil. The ascites fluid of these mice contains high concentrations of monoclonal antibodies. Their quality, concentration, immunoglobulin class, specificity and other parameters are verified by ELISA by determination of antibody titers, Western blot analysis, agar-gel immunodiffusion using class-specific antisera against mouse immunoglobulins, etc. Monoclonal antibodies of purified zasititic fluid>

(salting-out, ion-exchange, or affinity chromatography) are used either directly or in the form of a peroxidase conjugate in immunochemical and immunocytochemical assays. The monoclonal antibody is useful for purification of viral antigens by affinity chromatography, analysis of structural proteins of field virus isolates and in particular for rapid and specific diagnosis of PED-induced gastroenteritis of piglets (immunoelectron microscopic virus detection, immunocytochemical virus detection in infected cells) piglets, etc.).

Claims (1)

PATENT CLAIMS Mice lymphocytic hybridoma PED 308 / 2H6 producing a monoclonal antibody against the membrane protein M of 22 to 28 kDa of porcine epizootic diarrhea virus, deposited in the Collection of Zoopathogenic Microorganisms of the Research Institute of Veterinary Medicine in Brno under the designation CAPM-TK-8/01-PED 3C8 / .