Monoclonal antibody against interferon-gamma (ifn-gamma) inducing factor (ifig, il-18)

a monoclonal antibody and interferon-gamma technology, which is applied in the field of monoclonal antibodies against interferon-gamma (ifngamma) inducing factors (ifig, il18), can solve the problems of not being able to establish monoclonal antibodies specific to the protein, and not being able to obtain a desired amount of preparations with a constant,

Inactive Publication Date: 2014-11-13
HAYASHIBARA BIOCHEMICAL LAB INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides a novel protein that induces the production of interferon-γ (IFN-γ) by immunocompetent cells. The protein has specific physicochemical properties, including a molecular weight of 19,000±5,000 daltons on gel filtration and sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), an isoelectric point (pI) of4.8±1.0 on chromatofocusing, partial amino acid sequences in SEQ ID NOs:1 and 2, and biological activity of inducing the production of IFN-γ by immunocompetent cells. The invention also provides a DNA that encodes the protein, a replicable recombinant DNA containing the DNA and a self-replicable vector, a transformant obtained by introducing the recombinant DNA into an appropriate host, a process for preparing the protein, a monoclonal antibody specific to the protein, a method for detecting the protein, and a method for purifying the protein.

Problems solved by technology

These mitogens, however, have problems of their molecular and quality varying dependently on their origins and purification methods, as well as the difficulty of obtaining a desired amount of preparations with a constant IFN-γ inducibility.
In addition, most of these mitogens induce unfavorable side effects when administered to living bodies, and some of them may cause toxicity, so that it is substantially difficult to induce the IFN-γ production by the direct administration to living bodies.
The material most suitable for the purification and assay is a monoclonal antibody, but such a monoclonal antibody specific to the protein is not established.

Method used

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  • Monoclonal antibody against interferon-gamma (ifn-gamma) inducing factor (ifig, il-18)
  • Monoclonal antibody against interferon-gamma (ifn-gamma) inducing factor (ifig, il-18)
  • Monoclonal antibody against interferon-gamma (ifn-gamma) inducing factor (ifig, il-18)

Examples

Experimental program
Comparison scheme
Effect test

experiment 1

Preparation of Purified Protein

[0099]To 600 8-week-old female CD-1 mice was intraperitonealy injected one mg / mouse of dead Corynebacterium parvum (ATCC 11827) which had been obtained by preheating at 60° C. for one hour, and the mice were fed in usual manner for 7 days and intravenously injected with one μg / mouse of a purified lipopolysaccharide derived from Escherichia coli. On 1-2 hours after the intravenous injection, the mice were sacrificed by dislocating their cervical vertebrae to collect their blood from hearts, followed by removing their livers, disrupting them by a homogenizer in 8-fold volumes of 50 mM phosphate buffer (pH 7.3), and extracting the resultant. The resultant extract was centrifuged at about 8,000 rpm for 20 min, and an about 9 L of the resultant supernatant was admixed with a saturated ammonium sulfate in 50 mM phosphate buffer (pH 7.3) to give a saturation degree of 45 w / v %. The resultant solution was allowed to stand at 4° C. for 18 hours and centrifuged ...

experiment 2

Physicochemical Property of Protein

Experiment 2-1

Molecular Weight

[0102]In accordance with the method reported by U. K. Laemmli in Nature, Vol. 227, pp. 680-685 (1970), the purified protein prepared by the method in Experiment 1 was electrophoresed in a sodium dodecylsulfate (SDS) polyacrylamide gel free of reducing agent to mainly show a single protein band with an IFN-γ inducing activity at a position corresponding to about 19,000±5,000 daltons. The marker proteins used in this experiment were calf serum albumin (MW=67,000 daltons), ovalbumin (MW=45,000 daltons), soybean trypsin inhibitor (MW=20,100 daltons), and α-lactalbumin (MW=14,400 daltons).

experiment 2-2

Isoelectric Point

[0103]The purified protein in Experiment 1 was chromatofocused to give an isoelectric point of about 4.8±1.0.

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Abstract

A protein which induces the IFN-γ production by immunocompetent cells and has a molecular weight of 19,000±5,000 daltons on SDS-PAGE or gel filtration method and a pI of 4.8±1.0 on chromatofocusing. The protein is isolated from mouse liver and can be purified by a monoclonal antibody specific to it. The monoclonal antibody can be also used for assaying the protein.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a continuation application of copending parent application Ser. No. 09 / 050,249, filed Mar. 30, 1998, which is a continuation of application Ser. No. 08 / 502,535, filed Jul. 14, 1995, now issued as U.S. Pat. No. 5,912,324.BACKGROUND OF THE INVENTION[0002]1. Field of the Invention[0003]The present invention relates to a protein which induces the interferon-γ (hereinafter abbreviated as “IFN-γ”) production by immunocompetent cells, and a monoclonal antibody specific to the protein.[0004]2. Description of the Prior Art[0005]It is said that IFN-γ is a protein which has antiviral-, antioncotic- and immunoregulatory-activities and which is produced by immunocompetent cells stimulated with antigens and / or mitogens. Because of these biological activities, IFN-γ is expected to be used as an antitumor agent since the discovery, and energetically studied on clinical trials as a therapeutic agent for malignant tumors in general incl...

Claims

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Application Information

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Patent Type & AuthorityApplications(United States)
IPC IPC(8): C07K16/24G01N33/68C07K14/52C12N1/21C12N5/20C12N15/19C12N15/24
CPCC07K16/244C07K16/24G01N33/6869C07K2316/96A61K2039/505C07K14/52G01N33/6863G01N33/6866G01N33/6872C07K2317/76
InventorOKAMURA, HARUKITANIMOTO, TADAOTORIGOE, KAKUJIKUNIKATA, TOSHITANIGUCHI, MUTSUKOKOHNO, KEIZOKURIMOTO, MASASHI
OwnerHAYASHIBARA BIOCHEMICAL LAB INC