WO1990003575A1

WO1990003575A1 - Process for producing campylobacter pylori-specific antigens

Info

Publication number: WO1990003575A1
Application number: PCT/GB1989/001144
Authority: WO
Inventors: Diane Georgine Elizabeth Newell
Original assignee: Public Health Laboratory Service Board
Priority date: 1988-09-29
Filing date: 1989-09-28
Publication date: 1990-04-05
Also published as: ZA897301B; GB8822875D0; AU4485789A; GB2223756A

Abstract

A process for producing $i(Campylobacter pylori)-specific antigens comprises separating the proteins into fractions by a chromatographic technique, preferably by FPLC, removing the fractions responsible for cross-reaction with antibody not directed against $i(Campylobacter) species other than $i(Campylobacter pylori), and optionally recombining the remaining fractions. In this way, material suitable for use in sero-diagnostic assay for gastritis/peptic ulceration may be produced.

Description

PROCESS FOR PRODUCING CAMPYLOBACTER PYLORI -SPECIFIC ANTIGENS

This invention relates to process for producing Campylobacter pylori-spec±f±c antigens.

Gastritis, non-ulcer dyspepsia and peptic ulcer disease are common chronic conditions. Their aetiology and pathogenesis are not completely understood. The term gastritis is often inadequate for cases of vague upper abdominal complaints after exclusion of reflux oesophagitis, ulcer disease, gallstone disease and pancreatic disease.

In 1983, much excitement was generated when Warren and Marshall found curved or spiral Campylobacter-like bacteria in endoscopic antral mucosal biopsies from patients with gastritis and peptic ulceration.

Initially clinicians doubted that these micro-organisms could be primary pathogens, able to colonize normal antral mucosa and induce active chronic gastritis. However, the association between these newly described micro-organisms and gastritis was rapidly confirmed in several countries.

In 1980 Warren (319) in Australia observed curved and S-shaped bacilli in 135 gastric biopsy specimens whenever there was active gastritis. On light microscopy these bacilli resembled Campylobacter jejuni .

However, studies of the chemical composition and ultrastruetural studies showed fundamental differences between these Campylobacter-l±ke organisms and other Campylobacte .

Subsequently, based on rules of taxonomy, the name C. pylori was designated (189) for these newly discovered organisms. It is now generally accepted that Campylobacter pylori is a causitive organism of the aforementioned gastric disorders and a need has arisen for an effective diagnostic technique for determining the presence of the organism in the gastric ucosa of patients.

Currently such techniques involve gastric endoscopic biopsy. Clearly invasive procedures are undesirable in view of their expense and inconvenience and the discomfort caused to patients.

Patients infected with C. pylori develop serum antibodies of the IgG class during infection and it would be highly desirable if a serum-diagnostic test for infection could be devised.

A number of techniques have been developed to detect anti-C. pylori antibodies in serum, including haemagglutination, bacterial agglutination and complement fixation. However, the most commonly used systems are those based on enzyme-linked immunosorbant assays (ELISA) . Such assays are quick, relatively simple and economic. Moreover, ELISA is easily adapted to detect the different antibody isotypes consistent with mucosal antibody responses.

The sensitivity and specificity of the F-LISA technique is large! dependant upon the C. pylori antigen coupled to the plastic support. Previously, whole cells or sonicated whole cell antigens were used.

They tend to have unacceptably high backgrounds, presumably due to the non-specific absorption of immunoglobulins, and a poor sensitivity due to the low proportion of specific immunogens. It is generally accepted that the serod agnosis of C. pylori using these crude antigens is inadequate. One approach to improving the sensitivity and specificity of the ELISA i to develop better antigens. The most suitable antigenic material should have a high proportion of those components of C. pylori which are immunogenic during human infection. The immunogens should be common to all C. pylori strains but not present in other bacteria, especially other Campylobacters . The antigenic material should bind effectively to the various plastics used in ELISA microplate manufacture and maintain antigenic stability for reasonable periods once coupled to that plastic.

Our recent studies have attempted to establish the value of various antigens with respect to these criteria. Among the antigens investigated are: a) an acid extractable material, which has been previously shown to be better than whole cell sonicates and b) a recently described crude urease prepared according to the method of Dent et al. using a French Press method of cell disruption.

The immunogenicity of C. pylori components during human infections has been determined, at the molecular level, by either a) Western blotting after SDS-PAGE or b) the immunoprecipitation and subsequent electrophoresiε of I-surface labelled, solubilised bacteria (radio- immunoprecipitation: RIPA) . These techniques are complementary and do not necessarily give the same antigenic profiles.

Western blots of total protein profiles of complex antigens, like C. pylori acid extract, demonstrates a remarkable complexity and diversity in the immune response. Each patient appears to produce an apparently unique pattern of antibodies. RIPAs also suggest that the pattern of radiolabelled antigens precipitated by human sera is variable. A number of major protein antigens react with sera using these techniques. All of these antigens are present, to some extent, in the whole cell sonicates, the acid extract and the crude urease preparation. Human sera also react variably in Western blots with all of the major proteins of a purified urease preparation.

Although quantitative differences occur in the antibody patterns the diversity is qualitatively the same in both Western blots and RIPAs, whether homologous or heterologous strains are used as the antigen. The response, therefore, appears to be unrelated to antigenic variations in the bacterial strains or at least in the surface protein antigens. Such diversity is more likely to be a reflection of the variability in the host response to the infection and perhaps to the extent of the damage to the gastric epithelium and thereby systemic antigen exposure.

The considerable variation in antibody responses mentioned above means that only a few potentially universal antigens have been identified. On the basis of Western blotting, one antigen, a 120,000 molecular weight protein, appeared to be immunolabelled by most human sera, but even this protein is not present in all C. pylori isolates. This røeuld suggest that th uεc cf a purified unicomponent protein antigen would be unlikely to detect the immune response of all patients.

The most commonly used methods for preparation of material for use in this type of assay are whole-cell sonication and acid extraction. In both cases unbroken cells and cell debris are removed by centrifugation and the resulting supernatant is used in the assay with little or no further treatment. This provides antigenic material that reacts with human antibodies directed against Campylobacter pylori but parts of this material also cross-react with antibodies directed against other Campylobacter species. This cross reactivity leads to a lower specificity of the serodiagnostic assay for gastritis/peptic ulceration. For example people who have had a recent infection with Campylobacter jejuni (C. jejuni) would be positive in tests using the whole cell sonicate or acid extract material.

In summary, the assays currently available suffer from high backgrounds due to non-specific binding, and poor specifity due to antigenic crossreactivity between the Campylobacter species.

It is therefore desirable to provide a process for purifying Campylobacter pylori proteins that removes those parts of the material which bind, specifically or non-specifically, to antibodies against bacteria other than Campylobacter pylori (C. pylori ) .

The problem to which the present invention relates is thus to devise a commercial method for producing antigens which are specific to antibodies against C. pylori , but do not cross-react with antibodies against other Campylobacter species, especially C. jejuni .

It has now been unexpectedly found that such C. pylori-specific antigens can be economically produced by subjecting a crude protein- containing extract of C. pylori to a chromatographic separation technique.

Chromatography is a well known separation technique whereby individual chemical compounds in a mixture are resolved from each other by the selective process of distribution between two phases. The distribution of chemical species to be separated occurs in a dynamic process between a mobile phase and a stationary phase. According to one aspect of the invention there is provided a process for producing Campylobacter pylori-specific antigens suitable for use in sero-diagnostic assay for gastritis/peptic ulceration, comprising subjecting proteins obtained from a C. pylori culture to chromatographical separation into a number of fractions, removing fractions responsible for cross-reaction with antibody directed against Campylobacter species other than Campylobacter pylori , and recovering one or more fractions containing Campylobacter pylori-specific- antigens.

The chromatographic separation used in the method of the invention preferably is one capable of separating proteins on the basis of size. Such techniques include for example so-called exclusion or permeation chromatography, otherwise termed "gel filtration". Gels which are commonly used include cross-linked dextrans (Sephadex) agarose (Sepharose, Bio-Gel A, Sagavac) , polyacrylamide (Bio-Gel P) , and polystyrenes (Bio-Beads S) .

The use of gel filtration avoids drawbacks of other protein separation techniques Fo example mpare to SDS-PAGE, no protein- denaturing agents need to be included in the medium and compared to ion exchange chromatographic methods, the separation does not depend on charge differences between individual protein species.

Separation is preferably effected using a FPLC technique, which can enable a single step separation to be carried out rapidly and with good reproducibility. The use of FPLC in protein purification has been well documented. Very broadly, fast protein liquid chromatqgraphy processes use a gel-filtration column packed with a gel such as Superose-6 matrix to achieve the separation.

It has been found that by using a chromatographic separation technique, it is possible to remove those parts of the material responsible for cross-reaction with antibodies directed against non-C. pylori proteins. This has the dual effect of reducing antigen/antibody cross-reaction with antibody raised to other Campylobacter species that may be present in the test material, and reducing the non-specific background, i.e. reactions that lower the sensitivity of the assay. The resulting purified material has been found to be particularly well suited for use as the basis of a sero-diagnostic assay for gastritis/peptic ulceration.

In addition to enabling removal in a single-step of material that reduces the specificity and sensitivity of the sero-diagnostic assay, the use of chromatographic separation (especially gel filtration, e.g. FPLC) has other advantages over existing methods. The first of these is reproducibility and the second is ease of scale-up. Both are properties inherent in the technology end are equally important in the context of providing sizeable quantities of material with no batch-to-batch variation.

Various procedures may be used in order to distinguish Campylobacter pylori-specific antigens (hereinafter CPSA) from proteins responsible for cross-reacting with antibody against Campylobacter species other than Campylobacter pylori - (hereinafter CRA) .

One procedure involves the use of antibodies against Campylobacter jejuni antigens. Using this procedure, a given fraction resulting from the chromatographic separation may be tested for reaction with one or more such antibodies and rejected or isolated on the basis of the test result.

Although both polyclonal and monoclonal antibodies may be used in order to distinguish CPSA-containing fractions from CRA-containing ones, it is preferable to use monoclonal antibodies. Particularly suitable ^•available monoclonal antibodies are as follows:

Antibody Characteristic

CF5 - (a common epitope of C. jejivni flagella)

CP12 - reacts with the 31KD polypeptide associated with the urease activity in C. jejuni (but not C. pylori)

Hybridomas producing the above antibodies are deposited in the European Collection of Animal Cells under Accession Nos. 85052401 (24th May 1985) and 890925OI (25th September 1989) .

In another procedure for distinguishing fractions, the fractions removed and recovered may be selected on the basis of molecular weight. Thus tests ha^e >« <«ie that Campylobacter pylori-specific antigens are present in fractions corresponding to molecular weights in the range 115,000 to 500,000 Daltons.

Alternatively fractions may be selected on the basis of specific fraction numbers in gel-filtration chromatograpy proceures carried out under standard conditions.

In a preferred aspect the present invention provides a process for preparing Campylobacter pylori antigens using a fast protein liquid chromatography technique which includes the following steps: (a) subjecting a cell suspension of C. pylori to ultrasonicatio .

(b) separating a supernatant fraction from cell debris.

(c) subjecting the supernatant fraction to gel filtration, and

(d) recovering one or more fractions containing Campylobacter pylori-specific antigens.

A more specific method according to the invention involves the following steps:

(a) growing the Campylobacter pylori cells on a solid medium in a defined atmosphere,

(b) removing the cells and resuspending them in sterile phosphate buffered saline solution,

(c) subjecting the cell suspension to ultra-sonication.

(d) centrifuging the resulting material and retaining the supernatant material,

(e) filtering said supernatant material through a low protein bindino filter unit.

(f) preparing a gel filtration column by packing it with a Superose-6 matrix an equilibrating said column with sterile phosphate buffered saline solution,

(g) loading with filtrate recovered from step (e) into said column, (h) eluting the sample with sterile phosphate buffered saline solution, (i) analysing the samples recovered and (j) recombining the relevant fractions and recovering the

Campylobacter pylori antigen product. The invention also includes within its scope CPSA-type Campylobacter pylori proteins or antigens recovered by the process of the invention. Such material can be used in sero-diagnostic assays as discussed above and accordingly test kits for sero-diagnostic assay including such proteins or antigens as reagents form a further aspect of the invention.

In the first step of a preferred process of the instant invention Campylobacter pylori cells are grown on conventional solid medium in a defined atmosphere (Oxoid Campylobacter gas pack) . The cells are removed and resuspended in sterile phosphate buffered saline solution. In the next step of the process the cell suspension is subjected to ultrasonication. The resulting material is centrifuged for the appropriate period of time. The pelletal material is discarded and the supernatant is retained for treatment in the next step.

In the next step of the process the supernatant is filtered through a sterile low-protein binding Millipore filter. A Pharmacia Serial No. gel filtration column (a glass column packed with a Superose-6 matrix) is used for the subseαuent gel filtration. Prior to the loading of the material, the column is pre-equilibrated with sterile phosphate buffered saline solution. The filtrate is then loaded into an inoculating loop and loaded onto the gel filtration column. The sample is eluted from the column with sterile phosphate buffered saline at a low flow rate. The fractions are collected and retained for analysis and a sample trace is provided. CPSA an CRA-containing fractions are identified in the last step of the process the relevant fractions are recombined to provide the antigenic material for sero-diagnostic assay. The invention is further illustrated by the following specific but non-limiting example:

EXAMPLE 1

Campylobacter pylori organisms were grown on conventional solid •medium (5% defibrinated horse blood agar) in a defined atmosphere (Oxoid Campylobacter gas pack) . The cells from five petri-dishes were removed and resuspended in approximately 1 ml of sterile phosphate buffered saline solution.

The cell suspension was then subjected to ultrasonication for 4 x 30 s (30 s. intervals) at an amplitude of 10 microns. The resultant material was centrifuged at 12,000 g for minutes. The pelletal material was discarded and the supernatant retained for further treatment.

A 500 μl aliquot of the supernatant was filtered through a sterile 0.2 μm low-protein binding Millipore filter unit. The filtrate was then loaded onto a gel-filtration column packed with a Superose-6 matrix. Prior to loading of-^: the material the column was pre-equilibrated with sterile phosphate buffered saline solution.

The sample was eluted from the column with 50 ml of sterile phosphate buffered saline at a flow rate of 0.2 ml/min. One ml fractions were collected and retained for analysis. A sample trace is shown in Figure 1.

The relevant fractions containing antigenic material which binds to antibodies to Campylobacter pylori (CPSA-fractions) , were then recombined to provide antigenic material suitable for use in a sero-diagnostic assay for gastritis/peptic ulceration. EXAMPLE 2

The results of a further characteristic separation of Campylobacter pylori-specific antigens in a further experiment are shown in Figure 2. In this Figure, the fractions responsible for urease activity (cross-hatched) are shown.

EXAMPLE 3

Antigen-containing fractions separated in accordance with the invention were tested against serum samples from a panel of patients known (as a result of analysis of biopsy samples) to be Campylobacter pylori positive or negative. The fractions were used as ELISA antigens against the serum samples. The results of these tests are shown in Figure 3. from which it is readily apparent that fractions between 14 and 21 contain antigens which are highly specific to Campylobacter pylori .

In Example 1, the gel filtration column was a standard pre-packed column obtained from Pharmacia designated "Superose 6", Serial No. 17/0537/01.

The phosphate buffered saline had the following composition:

Obviously many modifications and variations of the invention may be made without departing from the essence and the scope thereof and only such limitations should be applied as indicated in the appended claims.

Claims

1. A process for producing Campylobacter pylori-specific antigens (CPSA) suitable for use in serodiagnostic assay for gastritis/peptic ulceration, comprising subjecting proteins obtained from a C. pylori culture to chromatographic separation into a number of fractions, removing fractions responsible for cross-reaction with antibody directed against Campylobacter species other than Campylobacter pylori ,(CRA) , and recovering one or more fractions containing Campylobacter pylori-specific antigens (CPSA) .

2. A process according to Claim 1 wherein a plurality of CPSA- containing fractions are recombined.

3. A process according to Claim 1 or Claim 2 wherein CPSA-containing fractions and CRA-containing fractions are separated on the basis of molecular size.

4. A process according to Claim 1 or Claim 2 wherein CPSA-containing fractions and CRA-containing fractions are separated on the basis of molecular weight.

5. A process according to any preceding claim where said chromatographic separation comprises a gel filtration procedure. U

6. A process according to any preceding claim where said chromatographic separation comprises a fast protein liquid chromatography procedure.

7. A process according to Claim 6, wherein the separation is achieved using a fast protein liquid chromatography technique in a single step.

8. A process according to any preceding claim, wherein the . reconstruction of the mixture of Campylobacter pylori proteins into defined mixtures results in the production of an antigenic material which will specifically and selectively bind the antibody in serum from patients with gastritis/peptic ulceration.

9. A process according to any preceding claim comprising the following steps:

(a) subjecting a cell suspension of C. pylori to ultrasonication.

(b) separating a supernatant fraction from cell debris.

(c. sub.iectinc the supernatant fraction to gel filtration, and (d) recovering one or more fractions containing Campylobacter p lori-specific antigens.

10. A process for preparing Campylobacter pylori antigens using a fast protein liquid chromatography technique which includes the following steps:

(a) growing the Campylobacter pylori cells on a solid medium in a defined atmosphere, (b) removing the cells and resuspending them in sterile phosphate buffered saline solution,

(c) subjecting the cell suspension to ultra-sonication.

(d) centrifuging the resulting material and retaining the supernatant material,

(e) filtering said supernatant material through a low protein binding filter unit,

Campylobacter pylori antigen product.

13 A -orocess according to any preceding claim wherein prior to said removing and recovering steps, CPSA-containing fractions and CRA- containing fractions are identified on the basis of reaction with one or more antibodies.

12. A process according to Claim 11 wherein said antibodies comprise monoclonal antibodies.

13. A process according to Claim 11 wherein said monoclonal antibodies are the antibodies designated CF5 and CP12. 14. A process for producing Campylobacter pylori proteins substantially as herein described.

15- Campylobacter pylori protein or antigen purified by the process of any one of the preceding claims.

16. A test kit for use in the sero-diagnosis of C. pylori infection comprising a protein or antigen as claimed in Claim 1 .