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Anti-campylobacter jejuni antibodies and uses therefor

A technology of Campylobacter jejuni and antibodies, which can be applied in the direction of antibodies, antibacterial drugs, antibacterial immunoglobulins, etc., can solve the problems of unrealistic cost and feasibility.

Inactive Publication Date: 2015-08-26
NAT RES COUNCIL OF CANADA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies in chickens also suggest that active immunization can reduce the level of enteric infection by C. jejuni, but the time window to achieve an adequate immune response before early sacrifice in chickens, as well as cost and feasibility, make this approach unrealistic

Method used

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  • Anti-campylobacter jejuni antibodies and uses therefor
  • Anti-campylobacter jejuni antibodies and uses therefor
  • Anti-campylobacter jejuni antibodies and uses therefor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0154] [Example 1: Preparation of Antigen]

[0155] Flagella were prepared for use as antigens in subsequent examples.

[0156] C. jejuni (strain 81-176) flagella were isolated as previously described (Power et al., 2003). Briefly, to prepare flagella, Campylobacter jejuni (C. jejuni) was cultured overnight, and cells were scraped into Muller-Hinton broth and incubated overnight. Cells were then harvested by centrifugation and resuspended in 100 mL of Tris-buffered saline. Flagella were sheared from the cells using a Waring mixer on ice. Cell debris was pelleted by centrifugation, and the supernatant was transferred to an ultracentrifuge tube. Flagella were pelleted by centrifugation at 45,000 rpm for 1 hour. Further purification was performed by resuspension in 2% SDS and centrifugation of samples. Resuspend the pellet in 200-500 μL of dH 2 O.

Embodiment 2

[0157] [Example 2: Alpaca immunity and serum response]

[0158] To isolate the V targeting the flagella of Campylobacter jejuni (C. jejuni) H H, alpacas were immunized with the flagellar antigen obtained in Example 1.

[0159] Male alpacas (Lama glama) were immunized subcutaneously with C. jejuni flagella (Example 1). A total of 7 injections were performed and, for each injection, 100 μg of antigen in a total volume of 0.5 ml was mixed with an equivalent volume of complete (day 1) or incomplete (day 21,35,49,63) Freund's Agent (Sigma) mixed. The last 2 injections (days 76 and 90) were performed with 100 μg of antigen without adjuvant. Pre-immune blood (15-20 ml) was collected before the first injection and on days 21, 49, 76 and 90. Specific immune responses were analyzed by ELISA using total preimmune and immune sera. Alpaca serum from day 90 was fractionated according to Hamers-Casterman et al. (1993). Protein G and A columns (GE HEALTHCARE) were used for serum fractio...

Embodiment 3

[0161] [Example 3: Flagella-binding V H Library construction and selection for H]

[0162] Construction of hyperimmune alpaca V based on RNA isolated from serum collected in Example 2 H H library.

[0163] Phage display libraries were constructed as previously described (Arbabi Ghahroudi et al., 2009). Briefly, approximately 1 × 10 7 Lymphocytes, total RNA was isolated using the QIAamp RNA blood mini kit (Qiagen, Mississauga, Ontario, Canada). 1-strand cDNA was synthesized with oligo(dT) primers using 5 μg of total RNA as template according to the manufacturer's recommendations (GE HEALTHCARE) (Arbabi Ghahroudi et al., 2009). By using oligonucleotide MJ1-3 (sense) and two kinds of CH2 domain antisense primers CH2 and CH2b3 (primer sequences see Arbabi Ghahroudi et al., 2009) to amplify the variable and part of the constant region DNA, and the heavy chain fragment (550-650 bp in length) was gel-purified by using the QIA Quick Gel Extraction Kit (Qiagene). The variable reg...

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Abstract

Campylobacter jejuni is a leading cause of bacterial food-borne diseases in humans, ranging from acute diarrheal disease to neurological disorders. An isolated or purified antibody or fragment thereof specific to C. jejuni is described. The antibody or fragment thereof binds to a flagellar protein and reduces motility of C. jejuni. The antibody or fragment thereof is derived from a heavy chain IgG variable domain fragment (VHH) of a camelid animal immunized with C. jejuni flagellar protein. A multivalent form, as well as a phage format, of the antibody or fragment thereof is described. Methods of reducing presence of C. jejuni in an animal or an animal environment, methods and formulations for treating C. jejuni infection, and method of detecting C. jejuni are also described.

Description

【Technical field】 [0001] The present disclosure generally relates to antibodies, fragments thereof and derivatives and uses related to the proteins. The described antibodies and fragments are directed against the flagellin of Campylobacter jejuni (C. jejuni). 【Background technique】 [0002] The genus Campylobacter includes a large morphologically diverse group of bacteria (spiral, curved or rod-shaped), with more than 35 species and subspecies reported, 20 of which were found to be pathogenic to humans, Causes intestinal and extraintestinal disease, or colonizes at a diverse number of sites in humans. (Man, 2011). [0003] Campylobacter jejuni, a Gram-negative spiral bacterium, is by far one of the most prevalent foodborne pathogens and the leading cause of bacterial gastroenteritis in the human world. In North America, Campylobacteriosis Outnumbers Diseases Caused by Salmonella, Shigella, Listeria, and E.coli Combinations This is the case reported (Stern & Robach, 2003;...

Claims

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Application Information

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IPC IPC(8): C07K16/12A61K39/40A61K47/48A61P31/04C07K19/00C12N15/13G01N33/569
CPCC07K2317/92C07K2317/569C07K2317/35C07K2317/22A61K2039/505C07K16/121G01N2333/205G01N33/56922A61P31/04Y02A50/30A61K39/40A61K45/06C07K2317/565G01N2469/10
Inventor M·阿巴比格赫劳迪A·瑞尔兹C·M·西曼斯基G·哈萨克J·坦纳R·麦肯兹
Owner NAT RES COUNCIL OF CANADA
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