EP0233261A1

EP0233261A1 - Monoclonal antibodies reactive against campylobacter pyloridis

Info

Publication number: EP0233261A1
Application number: EP19860905155
Authority: EP
Inventors: Gregory Murray Winn
Original assignee: Individual
Current assignee: Individual
Priority date: 1985-08-16
Filing date: 1986-08-18
Publication date: 1987-08-26
Also published as: WO1987001119A1

Abstract

Anticorps monoclonaux caractérisés en ce qu'ils sont spécifiquement réactifs contre les antigènes du Campylobacter pyloridis (PCLO).Monoclonal antibodies characterized in that they are specifically reactive against the antigens of Campylobacter pyloridis (PCLO).

Description

TITLE

MONOCLONAL ANTIBODIES REACTIVE AGAINST CAMPYLOBACTER PYLORIDIS

DESCRIPTION

The present invention relates to monoclonal antibodies which are reactive against Campylobacter pyloridis.

FIELD OF THE INVENTION

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. Work has been done to show that the association between Campylobacter pyloridis and antral gastritis exceeds 80%. 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) .

Further- a study has been undertaken to directly link Campylobacter pyloridis with gastritis as the primary cause thereof (as reported in B.J. Marshall et.al. , THE MEDICAL JOURNAL OF AUSTRALIA Vol 142, April 1985 p.436- 439) .

Still further, it has been noted that Campylobacter pyloridis is responsive to pharmaceutical compounds (as /

2. reported in B.J. Marshall et. al., THE MEDICAL JOURNAL OF AUSTRALIA Vol. 142 April 1985 P439-444) . Campylobacter pyloridis was originally isolated from patients with duodenal ulcers and was named in 1984

5 (as reported in B.J. Marshall et. al. , THE LANCET, June 16, 1984, P 1313-1314) .

Patients infected with Campylobacter pyloridis suffer from varying degrees of gastritis, gastric ulcer, duodenal ulcer, reflux oesophagitis and associated

10 symptoms. The bacteria is sensitive to some antibiotics and some bismuth compounds and has been eradicated by treatment -with thesematerials.

It is estimated that 10-20% of the Australian population will develop gastritis or gastric/duodental ulcers and

15 about 75% of these people will be infected by Campylobacter pyloridis (as reported in Joseph Alper and B.J. Spalding, CHEMICAL WEEK, January 23, 1985 P 17-18) . At the present time diagnosis is confirmed by gastric endoscopy, biopsy and subsequent culture. There is a

20 need for a means of conducting a preliminary test of patients to ascertain whether they are likely to be infected by Campylobacter pyloridis before conducting gastric endoscopy and biopsy. SUMMARY OF THE INVENTION

In accordance with one aspect of the present invention there is provided monoclonal antibodies which are specifically reactive against Campylobacter pyloridis antigens. In this specification the following abbreviations will henceforth be used: -

Campylobacter pyloridis ' - PCLO

Monoclonal Antibody - Mab

NS-l-Ag 4/1 (a mouse myeloma cell line used in hybridoma studies) - NS-1

A strain of inbred white mice - BALB/c

Enzyme Linked Immonosorbent

Assay - ELISA Further, 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. In this technique 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. To ensure onoclonality 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

The following description is by way of example only and it is to be understood that equivalent techniques to those described can be used. 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.

Preferably, 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

T.Y. Liu, JOURNAL OF BIOLOGICAL CHEMISTRY, Vol. 246, 1971, p. 2849. BALB/c 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. When a positive result wεs obtained the mice were given a further immunising dose of the antigen preparation. After a suitable period of time such as four days, 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. In this case 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. Those cells with hybridoma colonies were screened for antibody production specifically reactive to PCLO by techniques described in detail hereinafter, to ehsure that positive hybridomas were detected. The presence of 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.

When a hybridoma capable of producing PCLO specific Mab has been isolated 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.

After the conclusion of the limiting dilution procedures and the ELISA tests 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.

Firstly, specific antigen is prepared using a solid phase Mab immuno affinity technique. In this Mab was covalenϋy 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 permaϊiently bound to a membrane, such as a nitrocellulose membrane. Any vacant binding sites on the membrane are blocked such as by means of bovine serum albumin. 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. Further, all incubations were carried out at room temperature, about 20°C« About 100 μL of serum was diluted 1:100 v/v in 0.015 M •Saline and the diluted serum was applied to the membrane 14. The cassette was incubated for 15 minutes. 2mL of saline was then added to the membrane to wash away non-specific serum components. Then IOOJUL of horse- radish-peroxidase labelled anti-human immunoglobulin diluted 1:500 v/v was applied to the membrane and the s unit incubated for 15 minutes. The membrane was again .washed. lOO_juL of substrate colour reagent was then applied to the membrane. 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. The membrane wa's coated with Mab and blocked with albumin. IOOJUL of the suspected PCLO source such as serum, gastric juice or faeces, was applied to the pre-filter and allowed to soak through the pre-filter to the membrane. The cassette was then incubated for 15 minutes and the pre-filter discarded.

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. In this case 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.

Claims

1. Monoclonal antibodies characterised in that they are selectively reactive against Campylobacter Pyloridis (PCLO) antigens.

2. Monoclonal antibodies according to claim 1, characterised in that antibodies are produced by injecting PCLO into an animal subject to cause lymphocytes to produce antibodies, the antibody producing lymphocytes are removed from the animal and fused with a continuous culture cell line to produce a lymphocyte 0 hybridoma which is capable of producing antibodies.

3. Monoclonal antibodies according to claim 2 , characterised in that the lymphocyte hybridoma cells are cultured and subjected to repeated limiting dilution with intervening ELISA after each limiting 5 dilution procedure to isolate antibodies specific to PCLO.

4. A method of producing a membrane having attached to it antigen which is specific to the Mab of any one of claims 1 to 3, characterised in that it comprises Q covalently binding the Mab of any one of claims 1 to 3 to a solidphase, applying PCLO antigen extract to the bound Mab such that any Mab specific antigen attaches to the bound Mab, then removing the remainder of the extract from the column, then eluting the specific 5 antigen from the bound Mab and applying the eluted antigen to the membrane.

5. A membrane characterised in that it has attached to it antigen which is specific to the Mab of any one of claims 1 to 3.

6. A method for the detection of PCLO antibodies in body material characterised in that it comprises contacting the material with a membrane according to claim 5, then contacting the membrane with an enzyme labelled anti-human immunoglobulin and then adding a colour reagent which reacts with the enzyme label to produce a colouration when the enzyme label is present through adherence of antibody to the antigen attached' to the membrane.

7. A method for the detection of PCLO antigen in body material characterised in that it comprises contacting the membrane with Mab according to any one of claims 1 to 3, then contacting the material with a membrane according according to claim 5, then contacting the membrane with enzyme labelled Mab according to any one of claims 1 to 3 , and then contacting the membrane with a colour reagent to produce a colouration when the enzyme label is present through adherence of the antigen to the first applied Mab.