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Title:  Antibodies to cryptosporidium

United States Patent:  6,777,222

Issued:  August 17, 2004

Inventors:  Vesey; Graham (New South Wales, AU); Weir; Christopher (New South Wales, AU); Williams; Keith Leslie (New South Wales, AU); Slade; Martin Basil (New South Wales, AU); Veal; Duncan (New South Wales, AU)

Assignee:  Macquarie Research Ltd. (Sydney, AU); Sydney Water Corporation (Sydney, AU)

Appl. No.:  424048

Filed:  February 24, 2000

PCT Filed:  May 19, 1998

PCT NO:  PCT/AU98/00368

PCT PUB.NO.:  WO98/52974

PCT PUB. Date:  November 26, 1998

Abstract

A method of producing IgG1 subclass antibodies reactive to the surface of Crystosporidium oocysts, the method comprising: (a) separating at least a protion of the Cryptosporidium oocyst wall from the internal sporozoites to form an oocyst-wall preparation; (b) treating the separated oocyst-wall preparation so as to obtain an oocyst antigen preparation capable of eliciting a detectable IgG1 immune response in an animal to the surface of the oocyst; (c) immunizing an animal with the oocyst antigen preparation so as to elicit an IgG1 immune response in the animal; and (d) obtaining from the animal IgG1 antibodies reactive to the surface of Cryptosporidium oocysts. IgG1 antibodies reactive to the surface of Cryptosporidium oocysts.

BACKGROUND PRIOR ART

The protozoan parasite Cryptosporidium is amongst the most common pathogens responsible for diarrhoeal disease in humans. Infection occurs when Cryptosporidium oocysts shed in the faeces of infected individuals are ingested by new hosts. Recently, several large outbreaks of cryptosporidiosis have occurred in which drinking water has been identified as the source of infection. Surveys have shown that many surface water supplies are contaminated with Cryptosporidium oocysts.

Laboratory methods used to detect Cryptosporidium often involve the use of antibodies to this organism. Typical methods used to analyse water samples for the presence of this organism include microscopy and cytometry or a combination of these techniques. Flow cytometric methods involve staining of samples with a fluorescently labelled monoclonal antibody specific to the surface of Cryptosporidium oocysts and then analysis with a sorter flow cytometer. Particles with the fluorescence and light scatter characteristics of Cryptosporidium oocysts are sorted onto a microscope slide and examined manually using epifluorescence microscopy to confirm their identity as oocysts. This confirmation step is necessary because with a single antibody the cytometer is unable to distinguish oocysts from all other particles present in water samples. The particles that the cytometer can mistake as oocysts are autofluorescent particles such as algae or particles that non-specifically bind the oocyst-specific antibody.

Analysis-only flow cytometers are available which are simple to operate and relatively inexpensive. These cytometers are unable to perform sorting. To enable the detection of Cryptosporidium oocysts using an analysis-only cytometer the discrimination achieved by the cytometer must be improved so that non-oocyst particles are not mistaken as oocysts. The present inventors have shown previously that it is possible to detect a single specific microorganism in turbid water samples with an analysis cytometer if the microorganism is labelled with two different antibodies.

Unfortunately, the antibodies for Cryptosporidium presently available are not ideal due their stickiness and there is a need for more specific and reactive antibodies to the surface of Cryptosporidium oocysts. Monoclonal antibodies (mAbs) that are specific to the surface of Cryptosporidium oocysts are used for detecting Cryptosporidium in clinical and environmental samples. All available mAbs that bind to the surface of Cryptosporidium oocysts are of the immunoglobulin M (IgM) or IgG3 subclass. Monoclonal antibodies of the IgG1 subclass would be preferable because they usually show less non-specific binding. Such mAbs would be more suitable in methods currently used for the detection and identification of Cryptosporidium. Unfortunately, past attempts by workers in the field to produce IgG1 monoclonal antibodies to Cryptosporidium have been unsuccessful or not substantiated (Smith, 1994; MacDonald et al., 1991). It is generally considered that due to the antigenic characteristics of this organism, this class of antibody is not produced by infected or immunised animals (Smith, 1994).

In WO 97/08204 filed by the present inventors, monoclonal antibodies to a range of Cryptosporidium oocyst antigens were developed. Whole or excysted oocysts that were exposed to various treatments were used as antigens. From a total of eight fusions that included screening several thousand hybridomas only one hybridoma was identified that was specific to the surface of Cryptosporidium oocysts. This monoclonal antibody was of the IgM immunological subclass.

The present inventors have now developed a new method that allows the production of IgG1 antibodies to the surface of Cryptosporidium oocysts.

DISCLOSURE OF INVENTION

In a first aspect, the present invention consists in a method of producing IgG1 subclass antibodies reactive to the surface of Cryptosporidium oocysts comprising: (a) pretreating Cryptosporidium oocysts with a reagent so as to remove the surface layer of the oocysts to form an oocyst antigen preparation; (b) separating the oocysts from the antigen preparation so as to obtain a separated oocyst antigen preparation capable of eliciting a detectable IgG1 immune response in an animal to the surface of the oocyst; (c) immunising an animal with the separated oocyst antigen preparation so as to elicit an IgG1 immune response in the animal; and (d) obtaining from the animal IgG1 antibodies reactive to the surface of Cryptosporidium oocysts.

It will be appreciated that once a suitable immune response has been stimulated in an animal, for example in a laboratory mouse, monoclonal antibodies of IgG1 subclass may be generated by standard techniques from that animal.

In a preferred embodiment of the first aspect of the present invention, the reagent used to prepare the antigen preparation is a detergent, preferably the detergent is sodium dodecyl sulphate (SDS). One suitable pretreatment involves boiling the oocysts in the presence of SDS for a sufficient time to generate a suitable antigen preparation. When a concentration of 0.5% (w/v) SDS is used, boiling for 1 hour has been found to be particularly suitable.

Other suitable reagents include urea, detergents such as Triton X-100 or nonident, enzymes such chitinase, oxidising agents such as sodium hypochlorite, sodium periodate, ozone and reducing agents such as mercaptol ethanol and 1,1,1-trichloro-2,2-bis[4-chlorophenyl]ethane (DDT).

The pretreatment removes antigens from the surface of the oocyst in a form that will allow the generation of IgG1 antibodies when injected into an animal.

The animal may be immunised by any technique suitable for eliciting an immune response in an animal. Adjuvants may also be included with the antigen preparation prior to immunising the animal to promote a strong immune response in the animal.

In a further preferred embodiment, the antigen preparation also enhances the production of IgM antibodies when placed in an animal.

In a second aspect, the present invention consists in a method of producing IgG1 subclass antibodies reactive to the surface of Cryptosporidium oocysts, the method comprising:

(a) separating at least a portion of the Cryptosporidium oocyst wall from the internal sporozoites to form an oocyst-wall preparation:

(b) treating the separated oocyst-wall preparation so as to obtain an oocyst antigen preparation capable of eliciting a detectable IgG1 immune response in an animal to the surface of the oocyst;

(c) immunising an animal with the oocyst antigen preparation so as to elicit an IgG1 immune response in the animal; and

(d) obtaining from the animal IgG1 antibodies reactive to the surface of Cryptosporidium oocysts.

The present inventors have found that in order to obtain a suitable oocyst wall antigen preparation, the oocyst wall should be separated from internal sporozoite components. It appears that the internal sporozoite antigens are more immunodominant than oocyst wall antigens and their presence in an antigen preparation may mask the oocyst wall antigens. A mixed antigen preparation will usually result in raising antibodies to the sporozoite antigens.

The separation of the oocyst wall from the internal sporozoite (step (a)) can be achieved by any means. The present inventors have found that causing the oocyst to excyst followed by immuno-separation of the wall components is particularly suitable. Separation can also be achieved by surface labelling of whole oocysts with a ligand, such as biotin, allowing separation of cell wall fragments from internal components by reaction with a reagent reactive to the ligand, such as avidin, on an insoluble matrix or beads. It will be appreciated, however, that other separation methods known to the art would also be suitable. Examples include centrifugation, flow cytometry, density gradient separation, precipitation, immuno-labelling, ligand-binding, biotin-labelling and separation by avidin, and chromatographic separation.

It is not necessary to cause the oocyst to excyst by normal procedures. The oocysts can be freeze-thawed for example to promote initial separation of the wall from the internal sporozoites. Furthermore, the oocyst may be physically broken up by crushing, sonication, or grinding followed by separation.

The treating step (b) can be carried out by any means suitable. In particular, the present inventors have found that physically breaking up the cell wall can produce a good antigen preparation. This can be done by any means but the use of a bead beater is quite suitable.

The treatment removes antigens from the surface of the oocyst wall in a form that will allow the generation of IgG1 antibodies when injected into an animal.

It will be appreciated that once a suitable immune response has been stimulated in an animal, for example in a laboratory mouse, monoclonal antibodies of IgG1 subclass may be generated by standard techniques from that animal.

The animal may be immunised by any technique suitable for eliciting an immune response in an animal. Adjuvants may also be included with the antigen preparation prior to immunising the animal to promote a strong immune response in the animal.

In a further preferred embodiment, the antigen preparation also enhances the production of IgM antibodies when placed in an animal.

As the present inventors have determined methods that allow the production of useful IgG1 antibodies reactive to the surface of Cryptosporidium oocysts. it will be appreciated that similar antibodies to those produced by the present inventors may now be produced from the information and teaching provided herein.

In a third aspect, the present invention consists in substantially isolated IgG1 antibodies reactive to the surface of Cryptosporidium oocysts produced by the method according to the first or second aspects of the present invention.

Preferably, the antibodies are monoclonal antibodies.

In a fourth aspect, the present invention consists in a substantially isolated IgG1 antibody reactive to the surface of Cryptosporidium oocysts, the antibody having the oocyst binding and affinity characteristics of antibody CRY104.

Preferably, the antibody is a monoclonal antibody.

More preferably, the IgG1 monoclonal antibody is produced by clone CRY104.

In a fifth aspect, the present invention consists in the hybridoma clone CRY104.

The production of antibody CRY104 is defined in the specification such that persons skilled in the art will be able to produce other antibodies with Cryptosporidium oocyst binding and affinity characteristics similar to, or the same as, CRY104. CRY104 is recited in the specification as one example of a suitable IgG1 antibody reactive to the surface of Cryptosporidium oocysts that can be obtained from the teaching of the present invention. Accordingly, a sample of CRY104 is not required for a person to carry out the present invention.

Claim 1 of 36 Claims

What is claimed is:

1. A method of producing isolated IgG1 subclass antibodies reactive to the surface of Cryptosporidium oocysts, the method comprising:

(a) pretreating Cryptosporidium oocysts with a reagent so as to remove the surface layer of the oocysts to form an oocysts antigen preparation;

(b) separating the oocysts from the oocyst antigen preparation so as to obtain a separated oocyst antigen preparation free from sporozoite antigens and capable of eliciting a detectable IgG1 immune response in an animal to the surface of the oocyst;

(c) immunizing an animal with the separated oocyst antigen preparation so as to elicit an IgG1 response in the animal; and

(d) obtaining from the animal IgG1 antibodies reactive to the surface of Cryptosporidium oocysts.




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