EP0233261A1 - Anticorps monoclonaux reactifs contre le campylobacter pyloridis - Google Patents

Anticorps monoclonaux reactifs contre le campylobacter pyloridis

Info

Publication number
EP0233261A1
EP0233261A1 EP19860905155 EP86905155A EP0233261A1 EP 0233261 A1 EP0233261 A1 EP 0233261A1 EP 19860905155 EP19860905155 EP 19860905155 EP 86905155 A EP86905155 A EP 86905155A EP 0233261 A1 EP0233261 A1 EP 0233261A1
Authority
EP
European Patent Office
Prior art keywords
membrane
mab
antigen
pclo
antibodies
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP19860905155
Other languages
German (de)
English (en)
Inventor
Gregory Murray Winn
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Individual
Original Assignee
Individual
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Individual filed Critical Individual
Publication of EP0233261A1 publication Critical patent/EP0233261A1/fr
Pending legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
    • C07K16/121—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Helicobacter (Campylobacter) (G)

Definitions

  • the present invention relates to monoclonal antibodies which are reactive against Campylobacter pyloridis.
  • Campylobacter pyloridis is implicated with gastric ulcers, duodenal ulcers and gastritis in humans.
  • the Campy- lobacter pyloridis orgaiism is known to occur as a strain identified as NCTC11639 but this is only one example of many possible strains.
  • Patients affected by antral gastritis generally had Campylobacter pyloridis in their gastric mucosa whereas Campylobacer pyloridis was not shown to be present on normal gastric mucosa (as reported in B.J. Marshall et.al., THE MEDICAL JOURNAL OF AUSTRALIA Vol 142 April 1985 p.439-444) .
  • Campylobacter pyloridis was originally isolated from patients with duodenal ulcers and was named in 1984
  • Campylobacter pyloridis Patients infected with Campylobacter pyloridis suffer from varying degrees of gastritis, gastric ulcer, duodenal ulcer, reflux oesophagitis and associated
  • the bacteria is sensitive to some antibiotics and some bismuth compounds and has been eradicated by treatment -with thesematerials.
  • Campylobacter pyloridis (as reported in Joseph Alper and B.J. Spalding, CHEMICAL WEEK, January 23, 1985 P 17-18) .
  • diagnosis is confirmed by gastric endoscopy, biopsy and subsequent culture.
  • NS-l-Ag 4/1 (a mouse myeloma cell line used in hybridoma studies) - NS-1
  • the present invention provides in another aspect a method for the detection of PCLO antigen or antibodies to PCLO which comprises contacting biological material, particularly from the human body, with Mabs which are reactive against Campylobacter pyloridis antigens.
  • the material from the body may be tissue, body secretions or blood.
  • the Mabs of the present invention may be produced by the use of lymphocyte hybridoma technology.
  • a mouse or other subject is injected with PCLO which leads to the production of antibodies b lymphocytes in various organs including the spleen.
  • the spleen may be removed from the animal and the lymphocytes harvested. These cells cannot survive in vitro indefinitely.
  • An NSI myeloma cell line has been developed which is able to live in continuous culture. These two cell types may be fused using established techniques to produce a hybridoma which can live indefinitely in vitro and can also produce antibodies.
  • the hybridoma culture may be subjected to limiting dilution cell isolation.
  • the object is to obtain a population of cells derived from a single hybrid cell. These cells secrete an antibody into the culture medium in which they are grown. This medium is removed and reacted with a range of different bacteria. This is to ascertain the range and specificity of the antibody. Typically most of the bacteria selected are related to PCLO although a wide range are tested. If the antibodies produced are negative to all bacteria tested except PCLO then there is a high probability that the antibodies are specific to PCLO. The limiting dilution technique is typically repeated a number of times to ensure that a monoclonal antibody is obtained and that the selected cell line will continue to produce antibodies indefinitely.
  • the lymphocyte hybridoma technique is described in S. Fazekas de St. Groth and D. Scheidegger, JOURNAL OF IMMUNOLOGICAL METHODS, Vol 35 1980, p. 1-21. BRIEF DESCRIPTION OF THE DRAWINGS In the accompanying drawings there is shown in:-
  • Figure 1 a schematic upper perspective view of a cassette used to detect chemicals reactive with Mabs; and Figure 2 is a schematic longitudinal section through the middle of the cassette of Figure 1. DESCRIPTION OF THE INVENTION
  • PCLO is grown on brain heart infusion horse blood agar (BHIA-3) in a 10% atmosphere of CO at 37°C.
  • PCLO samples are preferably taken from a number of different people in case there are serotypic variations.
  • the grown PCLO isolated from different people was then pooled and an antigen preparation made for the immunisation of BALB/c female mice.
  • the PCLO is subjected to sonication to break up the cells, expose more antigens and render the culture soluble.
  • the bacterial antigen preparations are preferably further purified by the use of one or both of the following methods. a) Preparation of Lipopolysaccharide (LPS) as described in 0. Westphal and K. Jann METHODS OF CARBOHYDRATE CHEMISTRY Vol. 5, 1965 p. 83-7, or E. Staub, METHODS OF IMMUNOLOGY AND IMMUNOCHEMISTRY, Vol. 1, 1967, p. 28-34; and b) Preparation of Pol saccharide as described in
  • mice were immunised with the sonicated PCLO preparation via the intra peritoneal route.
  • the blood serum of the mice was tested to determine the production of antibodies to PCLO. This can be demonstrated by an ELISA technique.
  • a positive result w ⁇ s obtained the mice were given a further immunising dose of the antigen preparation.
  • the spleen cells were removed from the mice by sterile surgical technique and then fused with the mouse myeloma cell line NS-l-Ag 4/1 by the cell fusion technique.
  • the fusion was carried out in the presence of 50% v/v polyethylene glycol 1500, 5% v/v DMSO in 45% v/v RPMI-1640 culture media.
  • the product of the fusion was then distributed in multiwell tissue culture plates in a medium of RPMI-1640, 10% Foetal Calf Serum (heat inactivated) , 0 -1 mM hypoxanthine, Q.016mM thymidine, 0.4 pM. aminopterin, 0.2mM glutamine, 10 IU/mL penicillin and 10 IU/mL streptomycin.
  • the trays were incubated at 37°C in an atmosphere of 7% CO . After 5-7 days, visible hybridoma colonies were evident.
  • PCLO specific Mabs may be detected by an ELISA using crude and purified antigens.
  • the antigen preparations used included PCLO,. other campylobacters and other bacteria.
  • the antigen preparations were bound to multi-well ELISA trays.
  • Supernatants from wells containing hybridomas were added to the trays and incubated.
  • Specifically bound Mab was detected using either an alkaline phosphatase or peroxidase labelled anti-mouse immunoglobulin antiserum and a suitable substrate colour reaction system. Further, it was found that the presence of PCLO specific Mabs could be detected by heat fixing suspensions of PCLO on microscope slides and incubating this with hybridoma supernatants.
  • the PCLO specific Mab may be similarly detected.
  • the culture is preferably subjected to limiting dilution on more than one occasion and prefer ⁇ ably on 3 occasions to ensure monoclonality. Further, the ELISA test is done after each limiting dilution.
  • the isolated clones capable of producing the PCLO specific Mabs of the present invention are stored in liquid nitrogen until required for Mab production or for further testing of antigens.
  • the Mabs of the present invention can be used to identify PCLO antigens present in gastric biopsies, sera and body secretions. Further, the Mabs of the present invention can be used to detect serum antibodies to PCLO.
  • specific antigen is prepared using a solid phase Mab immuno affinity technique.
  • this Mab was covalen ⁇ y attached to cyanogen bromide activated agarose beads via primary amino groups.
  • the gel so produced was then loaded into a chromatography column and crude PCLO antigen extract was applied to the column.
  • the Mab binds to the specific antigen that distinguishes PCLO from other bacteria.
  • the remainder of the extract was removed by washing with phosphate buffer at pH8.0.
  • Elution of the specific antigen was achieved by using a chaotropic ion such as thiocyanate buffer at pH 8.0.
  • the eluted antigen is perma ⁇ iently bound to a membrane, such as a nitrocellulose membrane.
  • the membrane was then loaded into a cassette as illustrated in the accompanying drawings.
  • the cassette comprises a plastic body 10 containing a layer of an absorbent wick 12 on which is mounted the membrane 14.
  • a well 16 is formed in the plastic and a paper cover 18 is mounted over the upper end of the cassette.
  • the cassette is then ready for use in detection of serum antibodies produced by PCLO infection. This may be done by the technique now to be described but it is to be understood that reagent volumes and dilutions may be varied to suit particular requirements.
  • the colour reagent is typically 3mg/mL 4-chloro-l-naphthol in methanol ' diluted 1:5 just prior to use with saline containing - 0.1% hydrogen peroxide.
  • the colour reagent reacts with the peroxidase labelled anti-human immunoglobulin which has adhered to the antibody antigen complex which is specific to the Mab and produces a purple colouration of the membrane in a short period of time such as about 15 minutes.
  • the colouration indicates the presence of PCLO antibodies in the serum.
  • An alternative procedure is to detect PCLO antigen. This may be done by using a cassette similar to that shown in Figures 1 and 2, except that a removable pre- filter is placed over the membrane 14.
  • IOOJUL of the suspected PCLO source such as serum, gastric juice or faeces
  • Mab labelled with horse-radish peroxidase was then applied to the membrane and incubated for 15 minutes.
  • the membrane was washed with 2mL of saline and a substrate colour reagent applied.
  • the presence of PCLO antigen is indicated by a purple colour on the membrane which appears in a short period of time such as 15 " minutes.
  • the labelled Mab adheres to the antigen and provides a basis for the selective colouration of the membrane. Modifications and variations such as would be apparent to a skilled addressee are deemed within the scope of the present invention.

Landscapes

  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Food Science & Technology (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Anticorps monoclonaux caractérisés en ce qu'ils sont spécifiquement réactifs contre les antigènes du Campylobacter pyloridis (PCLO).
EP19860905155 1985-08-16 1986-08-18 Anticorps monoclonaux reactifs contre le campylobacter pyloridis Pending EP0233261A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AU1998/85 1985-08-16
AU199885 1985-08-16

Publications (1)

Publication Number Publication Date
EP0233261A1 true EP0233261A1 (fr) 1987-08-26

Family

ID=3692494

Family Applications (1)

Application Number Title Priority Date Filing Date
EP19860905155 Pending EP0233261A1 (fr) 1985-08-16 1986-08-18 Anticorps monoclonaux reactifs contre le campylobacter pyloridis

Country Status (2)

Country Link
EP (1) EP0233261A1 (fr)
WO (1) WO1987001119A1 (fr)

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5459041A (en) * 1988-02-18 1995-10-17 Enteric Research Laboratories, Inc. Campylobacter pylori antigens and uses thereof for detection of Campylobacter pylori infection
US4882271A (en) * 1988-03-10 1989-11-21 Baylor College Of Medicine Process for preparation of high molecular weight cell-associated protein of campylobacter pylori and use for serological detection of campylobacter pylori infection
WO1989009407A1 (fr) * 1988-03-23 1989-10-05 Julie Claire Dent Detection d'anticorps contre l'urease campylobacter pylori et leurs reactifs
GB2223756A (en) * 1988-09-29 1990-04-18 Health Lab Service Board Protein purification process
FR2637612B1 (fr) * 1988-10-06 1993-09-10 Pasteur Institut Sequences de nucleotides codant pour une proteine a activite ureasique
USRE34101E (en) * 1991-03-19 1992-10-13 Baylor College Of Medicine Process for preparation of high molecular weight cell-associated protein of Campylobacter pylori and use for serological detection of Campylobacter pylori infection

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB8422651D0 (en) * 1984-09-07 1984-10-10 Technology Licence Co Ltd Monoclonal antibodies

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO8701119A1 *

Also Published As

Publication number Publication date
WO1987001119A1 (fr) 1987-02-26

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