GB2290866A - Helicobactor pylori carbonic anhydrase antibody detection - Google Patents

Description

-l- 2290866 HELICOBACTER PYLORI CARBONIC ANHYDRASE-LIKE PROTEIN The

present invention relates to Helicobacter pylori Mpylori) carbonic anhydrase-like (CAL) protein and, in particular, to the use of such protein in the diagnosis of H.nylori infection.

H.pylori (formerly Campylobacter pyloridis or C.pylori) is a spiralshaped Gram negative human gastric pathogen. H.pylori is well documented in its association with gastric and duodenal ulcer disease as well as gastric cancer. The microorganism has been described as the most common chronic infectious agent of man. Despite its incidence, much of the pathogenic mechanism involving H.pylori in gastroduodenal disease is unknown.

The action of autoantibodies directed towards specific tissue targets is known to have aetiological ramifications in organ-specific autoimmune disease. Known examples include: the acetylcholine receptor in myasthenia gravis; streptococcal antigens in rheumatic heart disease; glutamate decarboxylase in insulin-dependant diabetes mellitus (IDDM); Lyme disease; and Goodpasture's syndrome.

The production of antibodies that cross-react with gastric autoantigens has been proposed as a pathological mechanism which links H.pYlori and gastritis (Negrini et al, Gastroenterology, 1991, 101, 437-445), although to date, no such gastric autoantigen has been identified. Indeed, the converse has also been suggested since H.pYlori-associated gastritis is rare in AIDS patients, possibly as a result of immunodeficiency (Marano et al, Am.J. Gastroenterol., 1993, 88, 887-90).

Carbonic anhydrase (CA) is a ubiquitous gastric metalloenzyme which has three isoforms in human tissue, CAI and CAII which are present in most tissues including the stomach and CAIII which is only present in skeletal muscle. The tertiary structures of all three CAs and the amino acid sequences involved in zinc binding are conserved. Carbonic anhydrase has also been implicated in the formation and secretion of gastric and pancreatic juices.

We have now surprisingly found that H.pvlori has carbonic anhydrase-like proteins. We have shown that patients with high titre antibodies to H. Pvlori showed an antibody response to carbonic anhydrase (CA).

Prior art in the diagnosis of H.pylori mediated gastric diseases include Campylobacter DNA probes capable of hybridizing to rRNA of H.pvlori (EP-A0350205); H.nylori oligonucleotides specific for the H.Pvlori urease gene sequences (WO 91/09049); serological detection and diagnosis of H.Pvlori infection by serological immunoassay; and detection of H.pvlori.antigens and antigenic fragments (WO 89/08843, WO 89/09497 and EP-A-0329570).

It would, however, be highly desirable to have a reliable and rapid means for diagnosing H.Pylori infection from a clinical sample from a patient, as a means of early diagnosis of gastric or peptic ulceration, or of gastric cancer.

Accordingly, the present invention provides a method for the diagnosis of H.Pylori infection by detecting the presence of antibodies to carbonic anhydrase protein or a fragment thereof comprising contacting a sample to be tested with carbonic anhydrase protein or a fragment thereof and detecting the presence of antigen-antibody complex.

The present invention further provides a method for the diagnosis of H. Pylori infection by detecting the presence of antibodies to carbonic anhydrase or a fragment thereof comprising contacting a sample to be tested with carbonic anhydrase protein or a fragment thereof, then further contacting the sample reacted with the carbonic anhydrase protein or fragment thereof with a second anti-human antibody and detecting either the presence of antigen-second antibody complex or detecting bound second antibody by reaction with a labelled third antibody.

It will be understood that the term "carbonic anhydrase protein" refers to carbonic anhydrase protein or carbonic anhydrase-like protein. The term "fragment" will be understood to include any carbonic anhydrase or carbonic anhydrase-like protein which is capable of eliciting an immunogenic response in the host.

The detection of the antigen-antibody complex may be by any procedure known in the art. Such procedures include identification by a label attached to the carbonic anhydrase protein or fragment thereof or by immunoprecipitation. Labels may include radioactive, fluorescent, chemiluminescent, dye molecules or enzyme labels. The detection of the antigen-antibody complex may include an amplification system such as those utilizing biotin and avidin.

The second anti-human antibody may be a mixture of rabbit anti-human antibodies or a mixture of goat anti-human antibodies. Alternatively, the second anti-human antibody may be anti-human IgA, anti-human IgG or antihuman IgM.

The third antibody which may be used to detect bound second antibody is selected so that it will react with the second antibody. For example, if the second antibody is rabbit anti-human antibody the third antibody may be horse anti-rabbit antibody or goat anti-rabbit antibody.

The sample to be tested may comprise gastric mucosa, dental plaque, saliva, gastric juice, a gastric biopsy sample or faeces. Preferably the carbonic anhydrase protein or a fragment thereof is H.Dylori carbonic anhydrase-like protein although it may be human CAI or CAII, or other carbonic anhydrase proteins or fragments thereof.

The present invention further provides a kit for the diagnosis of H. Pylori infection by the detection of antibodies to carbonic anhydrase comprising carbonic anhydrase protein or a fragment thereof. Kits according to the present invention may include appropriately labelled reagents and additional materials such as buffer solution, other H.Pylori proteins such as urease, H.pYlori cytotoxins and flagelli, means for detecting results of the diagnostic method and assay instructions. The kit components may be packaged in a suitable kit-container.

The present invention will now be described in more detail with reference to the following examples:

Example 1

Growth of H.Pylori strain and extraction of proteins H.Pylori strain NCTC 11638 was grown in liquid culture by adding one harvested plate of culture to 100m1 of Brucella broth (Difco) containing 2% p-cyclodextrin (Signa) and 0.2m1 of reconstituted H.pvlori selective supplement (Oxoid) in a 500 ml conical flask. The flask was incubated with gentle shaking (100 r.p.m.) for 3 days under microaerophilic conditions (Campypak, BBL) at 37 0 C. The H.pylori cells were collected by centrifugation and the proteins in the supernatant precipitated as previously described in Milton D.L., Norqvist, A., and Wolf Watz, H., (1992), J. Bacteriol., 174:7235-7244. Cellular proteins were extracted by resuspending two plates of growth in 1.5 ml of protein-extraction buffer (lomM Tris pH 7.5, 1 lmM MgCl 21 0.15mM EDTA, lmM DTT, 1 lmM PMSF, 2mgmL pepstating A(Sigma), 0.5 2mgmL leupeptin (Sigma), adding 200mgml of 10% SDS and boiling for 5 minutes. The suspension was then cooled on ice for 5 minutes and the supernatant collected by centrifugation at 12500 r.p.m. for five minutes. The protein concentration was determined according to Bradford, M., (1976), Anal Biochem, 72, 248-252, (1976).

Identification of carbonic anhydrase-like proteins in H.pylori protein extracts.

40pg of soluble total (HpT) and extracellular (HpE) H.pYlori protein extracts were analysed on vertical minigels (Hoeffer Scientific), comprising a 5% acrylamide stacking gel and a 13% resolving gel, according to the procedure of Laemmli, U.K., 1970, Nature, 227, 680-685.

Electrophoresis was performed at 20mA. Proteins were transferred to a nitrocellulose membrane by semi-dry blotting using transfer buffer (48mM Tris pH 8, 39mM glycine, 20% methanol, 1.3mM SDS) and an ATTO AE-6675 Horizblot transfer unit (Genetic Research International) according to the manufacturer's instructions. The nitrocellulose membrane was then dried and placed in blocking solution (1% bovine serum albumin in wash buffer (1OmM Tris pH7.5, 10OmM NaCl, 0.1% Tween 20) for 1 hour at room temperature with constant rocking. Primary antibody (either rabbit antihuman CAI or rabbit anti-human CAII prepared as described below) were added to the blocking buffer at a concentration of 1:1000 and incubation was continued for 1 hour. The membrane was then briefly washed twice with wash buffer, once for 15 min and once for 5 min with rocking. The HRP-labelled antibody (goat anti-rabbit) was added to the membrane at a concentration of 1:1000 in wash buffer and incubated for 1 hour as above. The membrane was then washed once for 15 min, and four times for 5 min with wash buffer as above. The membrane was then developed using ECL substrate reagents (Amersham) and exposed to Fuji RX X-ray film according to the manufacturerfs instructions. Re-probing of blots was performed after stripping antibodies bound to the blotted proteins by incubating in 20 mM glycine pH2.5, 0.055% Tween 20 overnight at room temperature with continuous shaking.

The result of the Western blots of the two SDS PAGE separations of total (HpT) and extracellular (HpE) proteins of H.pvlori., are shown in Fig. 1. Bovine carbonic anhydrase (CA) was clearly detected in the molecular weight marker (MWM) track.

The H.Pylori total protein extracts (HpT) had three prominent proteins which cross-reacted with both anti-CAI and anti-CAII antibodies. The approximate molecular weight of these H.Dylori CAL proteins were 57 (the most prominent), 42. and 34 kDa (arrowed with molecular weights in Figure 1). only the 57 kDa protein was prominent in extracellular H.Pylori protein extracts (HpE). Total H.pylori cellular proteins were fractionated on an AH Sepharose affinity column conjugated with p- aminomethylbenzene sulphonamide hydrogen chloride. When subjected to SDS PAGE and Western blot analysis, only the potassium thiocyanate and urea fractions reacted with the anti-CAI antibodies (not shown) indicating that these proteins bound strongly to the sulphonamide via a CA active site.

H.pvlori CAL proteins were not shown to be essential for growth since nine culture collection and seven clinical isolates were resistant to three sulphonamide CA inhibitors as well as the antimicrobial sulphanilamide (MICs>64 mg/1).

Preparation of Primary Antibodies Rabbit Anti-human CAI and Rabbit Antihurnan CAII 500m1 of human red blood cells were chloroform extracted at 40C and separated by centrifugation. The red cells were then purified on a AH Sepharose affinity column conjugated with p-aminomethylbenzene sulphoramide hydrogen chloride. The potassium iodide fraction yielded the CAI protein and the azide fraction yielded the CAII protein. The fractions were dialysed against 1OmM Tris, pH 8.3, and purified on a FPLC Mono Q column (Pharmacia) according to the manufacturer's instructions. The CAI protein was eluted from the potassium iodide fraction with 0.1M NaCl and the CAII protein was eluted from the azide fraction with 0.1M NaCl. The extracts were freeze dried and the protein content assayed using the Pierce BCA protein assay reagent according to the manufacturer's instructions (Pierce(UK) Ltd.).

Preparations comprising 300pg of the CAI or CAII proteins blended in 1. 5m1 water and 1.5m1 complete Freund's adjuvant were prepared and lml injected subcutaneously into rabbits. After 1 month the rabbits were injected intramuscularly with 300pg of the CAI or CAII protein absorbed on alum and the procedure repeated after 6 months.

The rabbits were then bled and the antibodies collected according to standard procedures known in the art.

Example 2

Correlation of CAL proteins in H.Pylori protein extracts with serum from infected patients To correlate the CAL proteins of H.Pylori with the antibody response in patients infected with this microorganism, the protein separations from Example 1 were stripped overnight at room temperature and re-probed with pooled sera from seven patients (absorbed with Escherichia coli), sero- positive or sero-negative (by ELISA) for H.Pylori (as shown in Fig. 2). Sera from patients infected with H.Pylori (detected with anti-human Ig HRP antibody conjugate at 1:1000 dilution) strongly identified the 57 and 34 kDa, CAL and HAP proteins respectively, amongst others of H.pylori, as well as the carbonic anhydrase molecular weight marker (30 kDa). Sera from sero-negative patients failed to react with the CA marker or any proteins in the H.pylori tracks (not shown). This suggests that patients infected with H.pvlori produce a significant antibody response to both H. Pvlori metalloenzymes (CAL and HAP) and, since CA is a prominant native gastric enzyme, this would result in antibody-mediated gastroduodenal inflammation.

Example 3

Blocking of rabbit anti-CAI antibodies by serum from infected patients infected with H.pYlori.

To confirm that the CA epitopes were similar in the polyclonal human and rabbit antibodies, a Western blot of total H.pylori proteins as described in Example 2 was first blocked with sera from patients with high or low titre antibodies against H.1pylori.

Three identical separations of H.pylori total cellular proteins (HpT) were pre-blocked with either sera from patients with high or low titre antibodies against H.pYlori in blocking solution (at 1:250 dilution), or blocking solution alone. The CA molecular weight marker (MWM) served as a positive control.

After washing in the manner as described for the CAI antibody in Example 1, the separation was then probed and detected using the rabbit anti-CAI as in Figure 1 (except that the sera were absorbed with both E.coli and normal human serum). Figure"3 shows a reduction in overall signal in the track blocked with serum from infected patients compared with that of the control (no blocking) or the sera from patients with low antibody titres against H.Pylori. More importantly, the signal from the prominant 57 kDa CAL protein of H.pylori (arrowed) is significantly reduced in the seropositive tack, but not in the control or sero-negative tracks. This confirms that patients infected with H.Pylori produce antibodies to the same epitopes as the rabbit anti-human CAI polyclonal antibody.

Claims (9)

CLAIMS:

1. A method for the diagnosis of H.Pylori infection by detecting the presence of antibodies to carbonic anhydrase protein or a fragment thereof comprising contacting a sample to be tested with carbonic anhydrase protein or a fragment thereof and detecting the presence of antigen-antibody complex.

2. A method for the diagnosis of H.pvlori infection by detecting the presence of antibodies to carbonic anhydrase or a fragment thereof comprising contacting a sample to be tested with carbonic anhydrase protein or a fragment thereof and then further contacting the sample reacted with the carbonic anhydrase protein or fragment thereof with a second anti-human antibody and detecting either the presence of antigensecond antibody complex or detecting bound second antibody by reaction with a labelled third antibody.

3. A method as claimed in claim 2 wherein the second anti-human antibody is labelled.

4. A method as claimed in claim 2 or claim 3 wherein the second antihuman antibody is a mixture of rabbit anti-human antibodies.

5. A method as claimed in claim 2 or claim 3 wherein the second antihuman anti-body is anti-human IgA, anti-human IgG or anti-human IgM.

6. A method as claimed in any one of claims 1 to 5 wherein the sample to be tested comprises gastric mucosa, dental plaque, saliva, gastric juice, a gastric biopsy sample or faeces.

7. A process as claimed in any one of claims 1 to 6 wherein the carbonic anhydrase protein or fragment thereof is H.pylori derived.

8. A kit for the diagnosis of H.nvlori infection comprising carbonic anhydrase protein or a fragment thereof.

9. A kit as claimed in claim 8 further comprising means for detecting an antigen-antibody complex.