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Recombinant human bivalent diabody against rabies virus and uses thereof

A rabies virus and double-chain antibody technology, applied in the field of immunology, can solve complex biological processes, expensive and other problems

Inactive Publication Date: 2014-02-19
INDIAN IMMUNOLOGICALS LIMITED
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The use of monoclonal antibodies for the evaluation of rabies virus glycoproteins involves the generation of antibodies from hybridoma cell lines, which is expensive and requires complex biological processes

Method used

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  • Recombinant human bivalent diabody against rabies virus and uses thereof
  • Recombinant human bivalent diabody against rabies virus and uses thereof
  • Recombinant human bivalent diabody against rabies virus and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Human X mouse heterohybridoma producing human anti-rabies virus monoclonal antibody

[0065] The rabies virus strain used in the present invention is a Pasteur virus (PV) strain described in the literature.

[0066] Human X mouse heterohybridomas were generated by fusing human immune B cells with human X mouse heteromyeloma (Champion et al. (2000); The development of monoclonal human rabies virus-neutralizing antibodies as a substitute for pooled human immune globulin in the prophylactic treatment of rabies virus exposure, Journal of Immunological Methods 235(1-2):81-90). Heterohybridomas were developed by fusing naive peripheral blood B cells with the heteromyeloma cell line K6H6 / B5 (Carroll et al. (1986); Mouse x human heterohybridomas as fusion partners with human B cell tumors. Journal of Immunological Methods 89( 1):61-72)). The K6H6 / B5 cell line was chosen because it has been successfully used to clone human antiviral antibodies (Siemoneit et al. (1994). Isolati...

Embodiment 2

[0070] Screening of 8 novel human IgG antibody (huMab) clones

[0071] Clones were screened for specific human Mab secretion by indirect ELISA using purified inactivated rabies virus antigen (PV strain).

[0072] Antibody purification

[0073] Human monoclonal antibodies (Mabs) secreted by heterohybridoma clones have been affinity purified on protein A sepharose columns. Check the purified antibody on a gel ( Figure 7 ). Lane 1 shows BSA as a standard with a molecular weight of 66Kda. Lanes 2-5 show purified R16E5 human monoclonal antibody with a molecular weight of 160 KDa. The main criterion for selecting 4 of the 8 clones was the rabies virus neutralization profile as determined by RFFIT.

[0074] Assay to confirm production of human anti-rabies virus monoclonal antibody by heterohybridoma cells

[0075] Several assays such as indirect ELISA, cellular ELISA, RFFIT and MNT were performed to confirm the rabies virus specificity of huMabs secreted by heterohybridoma...

Embodiment 3

[0085] Construction of diabody fragments

[0086] RNA extraction and amplification of antibody variable domain sequences

[0087] Total RNA was isolated from rabies resistant heterohybridoma (R16E5) and DNA was synthesized by RT-PCR. The nucleotide sequence of the amplified c-DNA is shown in SEQ ID NO:25. The cDNA amplified by RT-PCR was used as a template to amplify variable domains of antibodies using universal primers having the nucleotide sequences shown in SEQ ID NO:1 to SEQ ID NO:20. The amplified variable domains were assembled by splicing using overlap extension PCR to form diabodies and cloned into TOPO vectors for sequence verification ( Figure 1A , Figure 1B with Figure 1C ).

[0088] Primers used to amplify the variable domains of the heavy and light chains were as follows:

[0089] Forward primer for human variable heavy chain

[0090] HuVH1a: SEQ ID NO: 1

[0091] GGCGGCGGCGGCTCCGGTGGTGGTCAGGTGCAGCTGGTGCAGTCTGG

[0092] HuVH2a:SEQ ID NO:2

[0093...

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Abstract

The present invention provides recombinant human bivalent diabody against rabies virus capable of recognizing rabies virus glycoprotein and neutralizing rabies viruses and a method for production thereof. The present invention further provides polynucleotide encoding the recombinant bivalent diabody. The bivalent diabody disclosed in the present invention is also useful for quantitation of the rabies virus glycoprotein for evaluating the vaccine quality and predicting the vaccine potency.

Description

field of invention [0001] The present invention relates to the field of immunology, in particular to the production of recombinant antibody fragments. The invention specifically relates to the production of recombinant human bivalent diabody proteins against rabies virus. Background of the invention [0002] Rabies is an necessarily fatal viral infection of the nervous system of warm-blooded animals, including humans. It is transmitted through the bite of an infected animal, usually from a dog (Jackson, A.C., (2003). Rabies virus infection: an update. Journal for Neurovirology, 9; 253-258). An estimated 2.5 to 3 million people in India require post-exposure vaccination each year (Hemachudha T, Phuapradit P. (1997). Rabies. Current Opinion in Neurology. 10; 260-267). Potency assays for rabies vaccines have been described involving the use of in vivo tests in mice (WHO, 1992) and in vitro methods based on the evaluation of rabies virus glycoproteins (Perrin et al., (1990). I...

Claims

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Application Information

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IPC IPC(8): C07K16/10A61K39/395C07K14/145A61P31/14
CPCC12N2760/20111C07K2317/626C07K2317/21G01N33/56983A61K2039/505C07K2317/76C07K16/10A61P31/14
Inventor 文卡塔·尼迈格答·斯里戴维赛卢麦尼·那咖拉简戴乌·常德兰阿尔沃·维路帕努尔·斯里尼瓦桑
Owner INDIAN IMMUNOLOGICALS LIMITED
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