WO2005124351A2

WO2005124351A2 - A sensitive antibody-based method for detecting cryptosporidium parvum oocysts in water

Info

Publication number: WO2005124351A2
Application number: PCT/US2005/018936
Authority: WO
Inventors: Mark C. Jenkins; Kalmia K. Kniel
Original assignee: The United States Of America, As Represented By The Secretary Of Agriculture
Priority date: 2004-06-09
Filing date: 2005-05-31
Publication date: 2005-12-29
Also published as: WO2005124351A3; WO2005124351A8

Abstract

A viral symbiont (CPV) of Cryptosporidium parvum and Cryptosporidium hominis sporozoites has been characterized and a CPV capsid protein, CPV40, has been identified as a target for sensitive detection of C. parvum. Recombinant CPV40 was produced in E. coli, purified by affinity chromatography, and used to prepare polyclonal rabbit sera specific for the viral capsid protein. Anti-rCPV40 recognized a 40 kDa and a 30 kDa protein in C. parvum oocysts and appeared to localize to the apical end of the parasite. Anti-rCPV40 serum was capable of detecting as few as one C. parvum or C. hominis oocyst in a dot blot assay, the sensitivity being at least 1000-fold greater than sera reactive with total native C. parvum protein or specific for the 41 kDa C. parvum oocyst surface antigen. Water samples were seeded with C parvum oocysts and incubated at 4, 20, or 25 °C for greater than 3 months to determine if CPV levels were correlated with oocyst infectivity and viral particle presence. While sporozoite infectivity declined by more than 75% after 1 month at 25 °C, the CPV signal was similar to that of control samples at 4 °C. By three months at 20 °C, the C. parvum oocysts were found to be non-infectious, but retained a high CPV signal. CPV is an excellent target for sensitive detection of C. parvum and C. hominis oocysts in water, but may persist for an indefinite time after oocysts become non-infectious.

Description

A SENSITIVE ANTIBODY-BASED METHOD FOR DETECTING Cryptosporidium parvum OOCYSTS IN WATER Background of the invention

Field of the Invention

Cryptosporidium parvum and Cryptosporidium hominis represent a threat to human health due to contamination of surface waters with feces from humans, domesticated and companion animals and wildlife. This invention relates to Cryptosporidium parvum virus (CPV), a viral symbiont of C. parvum and C. hominis sporozoites and to an antibody which specifically recognizes a 40 kDa and a 30 kDa protein of the viral capsid protein. The anti-rCPV40 antibody is capable of detecting as few as one C. parvum or C. hominis oocyst, thus CPV is an excellent target for sensitive detection of C. parvum and C. hominis oocysts in water.

Description of the Relevant Art

One survey has found that between 24% and 100% of surface waters in the U. S. contain C. parvum oocysts (Xiao et al. 2001a. Appl. Environ. Microbiol. 67: 1097-1101; Xiao et al. 2001b. Emerg. Infect. Dis. 7: 141-145). Because there are no drugs or disinfectants available to destroy the parasite, preventing Cryptosporidium oocysts from entering surface waters or filtering water during treatment to remove oocysts may be the only feasible approaches to preventing outbreaks. Any management practice that seeks to prevent surface water contamination must rely on sensitive and specific detection methods. Although many techniques have been described for detecting Cryptosporidium oocysts, most are neither specific nor sensitive enough for detecting extremely low contamination levels. The difficulty in detecting Cryptosporidium in large volumes of water is that, unlike most viruses and bacteria, the infectious dose of C. parvum for humans is very low (~ 150 oocysts; DuPont et al. 1995. New Engl. J. Med. 332: 855-859).

Traditionally, indicators and indicator organisms have provided a means to assess water quality. This is due in part to the fact that the indicator organisms are often more prevalent than the pathogens themselves, and historically the means of identification of pathogens may not have been well developed (Leclerc et al. 2000. J. Appl. Microbiol. 88: 5-21). The use of fecal coliforms as indicators for E. coli and bacteriophage as indicators for viruses provide two well-known examples (Leclerc et al., supra). This study describes the proposed use of an indicator, the symbiont, C. parvum virus (CPV), for detection of the protozoan parasite C. parvum and C hominis in water.

Double-stranded RNA (dsRNA) viruses belonging to the family Totiviridae, have been identified in several protozoan parasites, including Entamoeba, Trichomanas, Leishmania, Giardia, Babesia, Naegleria, and Eimeria (Wang and Wang. 1991. Parasitol. Today 7: 76- 80). Although C. parvum has been shown to harbor a dsRNA virus (CPV), CPV is dissimilar to viruses reported from other protozoa, in that it is classified as a member of the Partitiviridae family (Khramtsov and Upton. 2000. J. Virol. 1A: 5788-5795). Despite a modest amount of research, the function of these viruses remains unclear. Several authors have shown that viral symbionts may play a role in immunogenicity or host cell pathogenicity (Miller et al. 1988. Mol. Biochem. Parasitol. 28: 189-196; Ψ getal. 1987. J. Exp. Med. 166: 142-150; Weeks et al. 1992. J Virol. 66: 1389-1393). Other members of the family Partitiviridae, including the yeast viruses, often have little or no negative effect on the host (Khramtsov et al. 1997. Mol. Microbiol. 26: 289-300).

The dsRNA virus of interest in the present study was found in all C. parvum and C. hominis isolates, but not in other Cryptosporidium species (Khramtsov et al. 2000. J Parasitol. 86: 275-282). Each viral particle contains two RNA segments contained within a capsid composed of one dominant protein. The viral particles are isometric with a diameter of 31 run (Kharamtsov and Upton, supra). It has been hypothesized that the virus is conserved and passed during cell division and gamete fusion (Kharamtsov and Upton, supra); however, the route of transmission, importance to infection, and localization are unknown.

The importance and necessity of immediate detection and public notification during outbreaks of cryptosporidiosis became clear during the recent problems in Kansas where oocyst contamination most likely originated in a public swimming pool and led to over 100 cases of cryptosporidiosis (Painter, S. 2003. The Wichita Eagle, Friday, September 19, 2003, Wichita, Kansas; Rothschild, S. 2003. Lawrence Journal World, Saturday, September 20, 2003, Topeka, Kansas). Thus, there is a 5 need for an agent useful for detection of C. parvum and C. hominis in environmental samples. The goal of this work was to utilize molecular and antigenic features of the viral symbiont as targets in a novel assay for detecting C. parvum and C. hominis oocysts/sporozoites in water.

Summary of the Invention 10 We have characterized a viral symbiont (CPV) of C. parvum and C. hominis sporozoites and have determined that CPV can be used as an indicator to detect presence of the protozoan parasite C. parvum and C. hominis in water. We have produced an antibody which specifically recognizes a 40 kDa and a 30 kDa protein of the viral capsid protein. The anti-rCPV40 antibody is capable of detecting as few as one C. parvum or C. hominis oocyst. 15 In accordance with this discovery, it is an object of the invention to provide an antibody that specifically binds to CPV, in particular, to native CPV 40 kDA protein (CPV40), to recombinant CPV 40 kDA protein (rCPV40), and to native 30 kDa protein (CPV30).

_0 It is a further object of the invention to provide an assay that is a specific and sensitive assay for the detection of C. parvum and C. hominis oocysts, particularly for the detection of C. parvum and C hominis oocysts in water.

In particular, this invention comprises a method to identify and enumerate the presence of C. _5 parvum and C. hominis in water. This invention comprises a method of identifying the presence of C. parvum and C. hominis in water, comprising: contacting a sample from the water source, or a concentrate of a sample from the water source, with an anti-rCPV antibody; detecting any selective binding of the antibody to any antigenic CPV-specific peptide present in the water; and determining that such binding is an indication of C. parvum or C. hominis infection. In particular, it is an additional object of the invention to contact a sample or concentrated sample with an anti-rCPV40 antibody.

It is further part of this invention to identify C. parvum and C. hominis in sea water and in water rinses of fruits and vegetables.

Also part of this invention is a Cryptosporidium detection kit, comprising anti-CPV-specific or anti- rCPV-specific antibodies, particularly anti-rCPV40 antibody; and instructions for the use of the kit.

Other objects and advantages of this invention will become readily apparent from the ensuing description.

Brief Description of the Drawings Figure 1 shows the SDS-PAGE/immunoblotting analysis of E. co/.-expressed recombinant CPV40 protein. Coomassie blue staining of NiNTA-purified rCPV40 protein, Lane A; Immunostaining of purified rCPV40 protein, Lane B or native C. parvum virus 40 kDa protein (nCPV40), Lane C by serum specific for rCPV40. Molecular size markers, in kilodaltons, Lane M.

Figure 2 shows a comparison of immuno-dot blotting signal of serial dilutions of C. parvum oocysts in deionized water using anti-C. parvum oocyst surface protein pre-immunization serum (1), anti-recombinant CPV40 pre-immunization serum (2), anti-rCPV40 post-immunization serum (3), anti-C. parvum oocyst 41 kDa surface protein post-immunization serum (4), or anti-C. parvum oocyst total protein post-immunization serum (5).

Figure 3 shows the immunofluorescence staining of excysted and methanol-fixed C. parvum sporozoites with anti-recombinant CPV40 serum.

Detailed Description of the Invention The use of the C. parvum viral symbiont (CPV) as a target for sensitive detection of C. parvum oocysts in water has several advantages. It is always present in C. parvum and C. hominis oocysts and appears to be specific for oocysts ( C parvum and C. hominis) that are infectious to humans (Xiao et al., supra). Perhaps the greatest advantage of the CPV is the ease of detection of CPV due to its abundance in each oocyst, for approximately 2000 CPV particles are present in each oocyst. The CPV capsid protein appears to be a good target for detecting C. parvum and C hominis oocysts in water samples. As few as one oocyst was detectable in deionized water using antiserum specific for rCPV40 antigen. The assay is therefore at least 1000-fold more sensitive than other assays that use antibodies reactive with the parasite itself.

While our data indicate that antibodies directed to CPV are capable of detecting low numbers of C. parvum oocysts, this assay, similar to immunofluorescence antibody (IFA)-based techniques, does not appear to be useful for differentiating infectious from non-infectious oocysts. It is probable that, similar to most viruses in the Partitiviridae family, CPV is stable for long periods of time. Our data also suggest that CPV capsid protein does not readily break down after C. parvum oocysts become non-infectious. The CPV assay should provide water quality laboratories an excellent alternative to the immunofluorescence assay for detecting C. parvum and C. hominis in drinking water. The primary concern of water utilities is whether C. parvum or C. hominis is present in water that has undergone treatment. Although non-infectious oocysts are not a concern to water utilities, the presence of oocysts indicates a problem in one to several points in the treatment process. In spite of years of intense investigation, no sensitive molecular-based assay that can detect extremely low numbers of C. parvum and C. hominis oocysts is being used by water quality laboratories. Use of an immunoblot method for detecting CPV represents a significant advance in this effort because it is amenable to a dipstick-type assay and does not rely on expensive equipment, unlike PCR, for analyzing water samples. Furthermore, while immunofluorescence requires a skilled microscopist for examining the samples under epifluorescence, this assay does not.

The term "peptide" as used herein refers to a molecular chain of amino acids with a biological activity {e.g., capable of binding antibody specific for CPV), and does not refer to a specific length of the product. Thus, inter alia, proteins, oligopeptides, polypeptides and fusion proteins as well as fusion peptides are included. Further, CPV40 and rCPV40 are interchangeable as reagents for generating CPV-specific antibodies. Thus, inter alia, reference to CPV40 encompasses rCPV40, and reference to rCPV40 encompasses CPV40.

The term "antibody," as used herein, includes, but is not limited to a polypeptide substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof which specifically bind and recognize an analyte (antigen). Examples include polyclonal, monoclonal, chimeric, humanized, CDR-grafted, and single chain antibodies, and the like. Fragments of immunoglobulins, including Fab fragments and fragments produced by an expression library, including phage display. See, e.g., Paul, Fundamental Immunology, Third Ed., 1993, Raven Press, New York, for antibody structure and terminology.

The phrases "specifically binds to", "specifically immunoreactive with", or "specific for" when referring to an antibody or other binding moiety refers to a binding reaction which is determinative of the presence of the target analyte in the presence of a heterogeneous population of proteins and other biologies. Thus, under designated assay conditions, the specified binding moieties bind preferentially to a particular target analyte and do not bind in a significant amount to other components present in a test sample. Specific binding to a target analyte under such conditions may require a binding moiety that is selected for its specificity for a particular target analyte. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with an analyte. See Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, for a description of immunoassay formats and conditions that can be used to determine specific immuno-reactivity. Typically a specific or selective reaction will be at least twice background signal to noise and more typically more than 10 to 100 times background.

For RT-PCR, mRNA is transcribed into cDNA using a gene specific primer (or oligo dT if the gene sequence is unknown) and reverse transcriptase. After the first strand cDNA is produced (the RT reaction), the second DNA strand is generated using an upstream gene specific primer. This second reaction, i.e., the PCR part, with downstream and upstream primers is repeated 25-35 times to produce a DNA fragment originating from the mRNA (See Example 6).

5 The proteins, CPV40 and rCPV40 can be used as immunogens to generate monoclonal and polyclonal antibodies that are selectively specific for CPV.

To prepare antibodies, a host animal is immunized using the CPV40 or rCPV40 protein as the immunogen. The host serum or plasma is collected following an appropriate time interval to provide 0 a composition comprising antibodies reactive with the immunogen. Methods of antibody (polyclonal and monoclonal) production and isolation are well known in the art. See, for example, Harlow et al. 1988, supra. Purification methods may include salt precipitation (for example, with ammonium sulfate), ion exchange chromatography (for example, on a cationic or anionic exchange column run at neutral pH and eluted with step gradients of increasing ionic strength), gel filtration

15 chromatography (including gel filtration HPLC), and chromatography on affinity resins such as protein A, protein G, hydroxyapatite, and anti-antibody.

In another embodiment, the monoclonal antibody of the invention is a chimeric monoclonal antibody or a humanized monoclonal antibody, produced by techniques well-known in the art.

_0 When a haptenic peptide of protein CPV40 is used, (i.e., a peptide which reacts with anti-CPV40- specific antibodies, but cannot itself elicit an immune response), it can be conjugated to an immunogenic carrier molecule. For example, an oligopeptide containing one or more epitopes of CPV40 protein may be haptenic. Conjugation to an immunogenic carrier can render the oligopeptide

_5 immunogenic. Preferred carrier proteins for the haptenic peptides of CPV40 are tetanus toxin or toxoid, diphtheria toxin or toxoid and any mutant forms of these proteins such as CRMι₉₇. Others include exotoxin A of Pseudomonas, heat labile toxin of E. coli and rotaviral particles (including rotavirus and VP6 particles). Alternatively, a fragment or epitope of the carrier protein or other immunogenic protein can be used. For example, the hapten can be coupled to a T cell epitope of a bacterial toxin. See U.S. Patents 5,785,973 and 5,601,831, the teachings of which are incorporated herein. In addition, immunogenicity of CPV40 could be increased by conjugation of a carrier molecule, for example, dipalmityl lysine. (See Hopp, 1984. Mol. Immunol. 21: 13-16, incorporated herein by reference.)

Antibodies of the present invention can also be labeled by conjugation to any detectable group, such as fluorescent labels, enzyme labels, and radionuclides. Suitable detectable labels may be selected from among those known in the art, including, but not limited to, radionuclides, enzymes, specific binding pair components, colloidal dye substances, fluorochromes, reducing substances, latexes, digoxigenin, metals, particulates, dansyl lysine, antibodies, protein A, protein G, electron dense materials, chromophores, and the like. Effectively, any suitable label, whether directly or indirectly detectable, may be employed. One skilled in the art will clearly recognize that these labels set forth above are merely illustrative of the different labels that could be utilized in this invention.

Methods for labeling antibodies have been described, for example, by Hunter et al. ( 1962. Nature 194:495-496) and by David et al. (1974. Biochem. 13: 1014). Additional methods for labeling antibodies have been described in U.S. Pat. Nos. 3,940,475 and 3,645,090. The label may be radioactive, i.e. , contain a radionuclide. Some examples of useful radionuclides include ³²P, ¹²⁵1, ^13II, ^{1 π}In, and ³H. Use of radionuclides have been described in U.K. patent document No.2,034,323, U.S. Pat. Nos. 4,358,535, and 4,302,204.

Some examples of non-radioactive labels include enzymes, chromophores, atoms and molecules detectable by electron microscopy, and metal ions detectable by their magnetic properties. Some useful enzymatic labels include enzymes that cause a detectable change in a substrate. Some useful enzymes and their substrates include, for example, horseradish peroxidase (pyrogallol and o- phenylenediamine), beta-galactosidase (fluorescein beta-D-galactopyranoside), and alkaline phosphatase (5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium). The use of enzymatic labels have been described in U.K. 2,019,404, EP 63,879, and by Rotman (1961. Proc. Natl. Acad. Sci. USA 47: 1981-1991). Useful chromophores include, for example, fluorescent, chemiluminescent, and bioluminescent molecules, as well as dyes. Some specific chromophores useful in the present invention include, for example, fluorescein, rhodamine, Texas red, phycoerythrin, umbelliferone, luminol.

The labels may be conjugated to the antibody probe by methods that are well known in the art. The labels may be directly attached through a functional group on the probe. The probe either contains or can be caused to contain such a functional group. Some examples of suitable functional groups include, for example, amino, carboxyl, sulfhydryl, maleimide, isocyanate, isothiocyanate. Alternatively, labels such as enzymes and chromophoric molecules may be conjugated to the antibodies by means of coupling agents, such as dialdehydes, carbodumides, dimaleimides, and the like.

The label may also be conjugated to the antibody probe by means of a ligand attached to the probe by a method described above and a receptor for that ligand attached to the label. Any of the known ligand-receptor combinations is suitable. Some suitable ligand-receptor pairs include, for example, biotin-avidin or -streptavidin, and antibody-antigen. The biotin-avidin combination is preferred. Thus, the anti-CPV antibodies of the invention can be derivatized by conjugation to biotin, and used, upon addition of species of avidins that have been rendered detectable by conjugation to fluorescent labels, enzyme labels, radionuclides, electron dense labels, substrates, etc., in a multiplicity of immunochemical and immunohistological applications.

The antibodies of the invention may also be attached or bound to substrate materials according to methods known to those skilled in the art. Such materials are generally substantially solid and relatively insoluble, imparting stability to physical and chemical disruption of the antibodies, and permitting the antibodies to be arranged in specific spatial distributions. Among substrate materials, materials may be chosen according to the artisan's desired ends, and include materials such as membranes (e.g. , Immobilon), gels, hydrogels, resins, beads, nitrocellulose, nylon filters, microtiter plates, culture flasks, polymeric materials, and the like, without limitation. In a preferred embodiment, the detection method employs an antibody that has been detectably labeled with a marker moiety. In other embodiments, the method may employ an antibody of the invention that has been bound to a substrate material. In the method, the composition may also include other reagents such as other antibodies that differentially detect other CPV antigens.

The CPV- binding method of the invention includes methods known in the art that employ antibodies to bind target substances specifically. Preferred methods include methods of detecting the presence C. parvum and C. hominis. Also provided herein is a method of detecting the presence of C. parvum and C. hominis in water samples. The detection of the antibody-polypeptide complex may be conducted by any method known in the art. This includes solid phase, double antibody, sandwich double antibody, and triple antibody assays, and the like, including radioimmunoassay, enzyme-linked immunosorbent assay, fluorescent assay, including flow cytometry, chemiluminescent assay, competitive immunoassay, membrane-based immunoassay, immunomagnetic separation, precipitation, agglutination, antigen capture, or the like.

Assays for detecting the presence of proteins with antibodies have been previously described, and follow known formats, such as standard blot and ELISA formats. These formats are normally based on incubating an antibody with a sample suspected of containing the protein and detecting the presence of a complex between the antibody and the protein. The antibody is labeled either before, during, or after the incubation step. The protein is preferably immobilized prior to detection. Immobilization may be accomplished by directly binding the protein to a solid surface, such as a membrane or microtiter well, or by binding the protein to immobilized antibodies.

For immunoblotting assays, serial dilutions of C. parvum oocysts (standards) are prepared in deionized water. Oocysts are either untreated or treated by three cycles of freeze-thawing in liquid nitrogen and then vacuum-blotted onto Immobilon membrane, a polyvinylidine fluoride (PVDF) microporous membrane (Millipore) in 100 μl at the oocyst numbers using a dot blot apparatus (Bio- Rad, Hercules, CA) (See Example 3 for details). Briefly, water samples are applied to and drawn through the membrane by vacuum, thus immobilizing the oocysts (and viral capsid protein). The blots are tested with a dilution of pre-immunization serum or antiserum which specifically binds to rCPV40, total C. parvum oocyst/sporozoite protein, or rCP41 (oocyst wall protein). Oocysts are detected by probing the membranes with biotinylated goat-anti-rabbit IgG followed by incubation with avidin-alkaline phosphatase and final incubation with phosphatase substrate, to visualize antibody binding. Other detection moieties can be used.

As is known in the art, other types of immunoassays may involve one step or two steps. In a one-step assay, the target molecule, if it is present, is immobilized and incubated with a labeled antibody. The labeled antibody binds to the immobilized target molecule. After washing to remove unbound molecules, the sample is assayed for the presence of the label. Alternatively, the immobilized target molecule can be incubated with an antibody and then detected indirectly by labeled antibodies or known immunodetection methods described above.

In a two-step assay, immobilized target molecule is incubated with an unlabeled first antibody. The target molecule-antibody complex, if present, is then bound to a second, labeled antibody that is specific for the unlabeled antibody. The sample is washed and assayed for the presence of the label, as described above.

The immunometric assays described above include simultaneous sandwich, forward sandwich, and reverse sandwich immunoassays. These terms are well known to those skilled in the art.

The specific concentrations of labeled and immobilized antibodies, the temperature and time of incubation, as well as other such assay conditions, can be varied, depending upon various factors including the concentration of antigen in the sample, the nature of the sample and the like. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation.

Many solid phase immunoabsorbents are known and can be used in the present invention. Well- known i munoabsorbents include beads formed from glass, polystyrene, polypropylene, dextran, nylon, and other material; and tubes formed from or coated with such materials, and the like. The immobilized antibodies may be covalently or physically bound to the solid phase immunoabsorbent, by techniques such as covalent bonding via an amide or ester linkage or by absorption.

The invention further provides diagnostic and experimental kits that include anti-CPV specific antibody, and enable the detection of CPV in a specific and reproducible manner. In these kits, the antibodies may be provided with means for binding to detectable marker moieties or substrate surfaces. Alternatively, the kits may include the antibodies already bound to marker moieties or substrates. The kits may further include positive and/or negative control reagents as well as other reagents for adapting the use of the antibodies of the invention to particular experimental and/or diagnostic techniques as desired. The kits may be prepared for in vitro use, and may be particularly adapted for performance of any of the methods of the invention, such as ELISA. For example, kits containing antibody bound to multi-well microtiter plates can be manufactured.

Also provided herein is another C. parvum and C. hominis detection kit, that comprises anti-CPV antibodies having specificity for rCPV40; and instructions for use of the kit. Thus, kit may be utilized for the detection of CPV peptides, a sign that there is parasite present in the water. In addition to the above, the kits may also comprise a control, anti-antibodies, protein A/G, and the like, suitable for conducting the different assays referred to above.

EXAMPLES Having now generally described this invention, the same will be better understood by reference to certain specific examples, which are included herein only to further illustrate the invention and are not intended to limit the scope of the invention as defined by the claims.

EXAMPLE 1 Preparation of Parasites

Cryptosporidium parvum (Beltsville-1 strain) oocysts were obtained by infecting a 1 day old calf with 10⁶ oocysts. The calf was obtained at birth from the dairy herd at the Beltsville Agricultural Research Center. Feces were collected from days 3-10 post-infection, pooled, and passed through a series of sieves of increasingly finer mesh, ending with a 45μm sieve. Sieved fecal material was mixed with 2M sucrose and subjected to centrifugation (60 min, 9000 X g). The supernatant containing oocysts was diluted in water and then subjected to continuous flow centrifugation. The pellet was suspended in water, then layered onto a discontinuous cesium chloride gradient of 1.05, 1.1, and 1.4 g/cm³ and then centrifuged for 60 min at 9000 X g. The oocysts were aspirated from the interface and suspended in distilled water, stored at 4°C, and used within 6 months after collection.

EXAMPLE 2 Production of recombinant viral capsid protein and immunization of rabbits for production of antisera against recombinant protein

Expression of recombinant CPV was carried out using the pET28 vector (Novagen, Madison, WI). The recombinant plasmid was introduced into Escherichia coli BL21(DE3) cells (Novagen) by standard transformation procedures (Hanahan, D. 1983. J. Mol. Biol. 166: 557-580). A time course study to identify the time of IPTG induction leading to peak expression of recombinant protein indicated that a 4 h induction was optimal. Recombinant CPV protein, rCPV40, was isolated by lysing PTG-induced E. coli with denaturing column binding buffer (0.2 M NaPO₄, pH 7.8, 0.5M NaCl, 8 M Urea; Invitrogen, Carlsbad, CA) and purified by passage over NiNTA resin followed by elution with denaturing column elution buffer (0.2 M NaPO , pH 4.0, 0.5M NaCl_, 8 M Urea, Invitrogen). Protein samples were analyzed by SDS-PAGE to confirm the purity of eluted recombinant protein. Two New Zealand White rabbits (Covance, Denver, PA) were each immunized twice over a 6 week period by intramuscular injection with recombinant CPV 40 kDa protein (rCPV40) in phosphate buffered saline (PBS) containing LnmunoMax SR adjuvant (Zonagen, The Woodlands, TX). The rabbits were bled for serum by central articular artery puncture 2 weeks after the last booster immunization. EXAMPLE 3 SDS-PAGE and immunob lotting of recombinant viral capsid protein and C parvum protein.

C. parvum oocyst protein (equivalent to 10⁷ oocysts/lane) or recombinant protein eluates were mixed with sample buffer containing 2-mercaptoethanol (Laemmeli, U.K. 1970. Nature 227: 680-685), heated for 3 min in a boiling water bath, and applied to 12.5%₀ SDS-polyacrylamide gel electrophoresis. The gels were transblotted to Immobilon (Millipore, Bedford, MA) membrane. The antigen-impregnated membranes were rinsed briefly with PBS, then immersed in PBS containing 2% non-fat dry milk (NFDM) to block non-specific antibody binding in subsequent steps. After blocking, the membranes were incubated for 1 hr with a 1 : 100 dilution of rabbit antiserum specific for the recombinant viral capsid protein, for native C. parvum oocysts, or for recombinant CP41 C. parvum oocyst wall protein in PBS containing 0.05% Tween 20 (PBS-Tw20). The membranes were then probed for 1 hr with biotinylated goat-anti-rabbit IgG (heavy and light chain specific, Vector Laboratories, Burlingame, CA) followed by a 1 hr incubation with avidin-alkaline phosphatase (Sigma Chemical Co., St. Louis, MO), and final incubation in phosphatase substrate (0.38 mM 5- Bromo-4-chloro-3'-indolylphosphate p-toluidine salt (BCIP), 2.45 mM nitro-blue tetrazolium chloride (NBT) in alkaline phosphatase buffer 0.1 M Tris, 0.1 M NaCl, 5 mM MgCl₂, pH 9.5) to visualize antibody binding. Membranes were washed with PBS-Tw20 three times for five min between each incubation step, and an additional final wash with PBS before substrate addition.

For testing assay sensitivity, serial dilutions (10⁶ - 10 ) of C. parvum oocysts were prepared in deionized water. Oocysts were either untreated or treated by three cycles of freeze-thawing in liquid nitrogen and then vacuum-blotted onto Immobilon membrane (Millipore) in 100 μl at the oocyst numbers using a dot blot apparatus (Bio-Rad, Hercules, CA). The blots were tested with a 1 :2500 dilution of pre-immunization serum or antiserum which specifically binds to rCPV40, total C. parvum oocyst/sporozoite protein, or rCP41 (oocyst wall protein).

Analysis of affinity-purified rCPV40 by SDS-PAGE and Coomassie blue staining revealed a -40 kDa protein that was absent in preparations derived from E. coli harboring non-recombinant pET28 vector (Figure 1, Lane A). Immunoblotting analysis using anti-serum specific for rCPV40 recognized both the 40 kDa recombinant protein (Figure 1 , Lane B), and a 40 kDa native C. parvum protein (Figure 1 , Lane C). A minor 30 kDa native C. parvum oocyst protein was also recognized by anti-rCPV40 serum. The relationship of this protein to the 40 kDa antigen is unknown (Figure 1, Lane C).

By immunoblotting, anti-rCPV serum detected as few as one C. parvum oocyst that had been suspended in deionized water and applied to the Immobilon membrane (Figure 2). The sensitivity of anti-rCPV serum was at least 1000-fold greater than that observed with antiserum to oocyst protein extract or to a 41 kDa oocyst surface protein (Figure 2). The signal intensity observed with anti- rCPV serum was greater with C. parvum oocysts that had been disrupted by repeated freezing and thawing compared to intact oocysts (data not shown). In a control study, (10¹ - 10⁶ ) Trichomonas vaginalis ( a cocktail of three strains ATCC 30236, 30238, 30001) or (10¹ - 10⁶ ) Giardia duodenalis cysts drawn onto membrane did not show any measurable reactivity with anti-rCPV40 serum (data not shown).

EXAMPLE 4 Immunofluorescence antibody (IF A) staining

C. parvum oocysts were first excysted in vitro using standard methods (Gut and Nelson. 1999. J. Eukaryotic Microbiol. 46: 56S-57S). The excysted sporozoite/oocyst mixture was pipetted onto multi-well glass slides, allowed to air dry, and then treated with cold methanol for 1 min. After fixation, the slides were rinsed with PBS, allowed to air dry, and then immunostained with a 1 : 100 dilution of serum directed to rCPV40, total C. parvum oocyst protein (positive control), or pre- immune serum (negative control) for 2 hr at room temperature (RT). The slides were rinsed three times for 10 s/wash with PBS, air-dried, and incubated with a 1:50 dilution of FITC-labeled sheep anti-rabbit IgG (H+ L chain specific, Sigma) for 1 h at RT. The slides were rinsed three times for 10 s/wash with PBS, air-dried, and overlaid with VectaStain mounting medium (Vector Laboratories) and a coverslip, and examined by epifluorescence microscopy.

IFA staining of excysted sporozoites revealed the CPV protein to be concentrated in the apical portion of C. parvum sporozoites (Figure 3). Negligible staining of C parvum sporozoites was observed with control pre-immunization serum (data not shown). EXAMPLE 5 C. parvum infectivity assays

Oocysts were stored in a water bath at temperatures of 25, 20, or 4°C. For mouse infection, 9-day- old BALB/c mice were inoculated with 10⁴ C. parvum oocysts by gastric intubation. The mice were killed by CO₂ exposure 5 days post-infection, and the ileum was placed in DNA extraction buffer (0.2 M Tris, pH 8.0, 0.1 M EDTA, 0.4 M NaCl) plus proteinase K. Intestinal tissue was always harvested from control (non-infected) mice first. DNA samples were extracted for DNA using the Qiagen Dneasy Tissue Kit (Qiagen, Valencia, CA). C. parvum development was assessed by polymerase chain reaction (PCR) on intestinal DNA samples as described using primers specific for C. parvum (Jenkins et al. 1998. J. Parasitol. 84: 182-186).

For cell culture infection, human ileocecal adenocarcinoma cells (HCT-8 cells; ATCC CCL-244, American Type Culture Collection, Manassas, VA) were maintained in RPMI 1640 medium (Mediatech Cellgro) supplemented with L-glutamine (300 mg/1; Mediatech Cellgro) and HEPES (25 mM; Mediatech Cellgro). For normal cell maintenance, the medium was supplemented with 5% fetal bovine serum (FBS; Biofluids, Rockville, MD). Growth medium with 10% FBS was used for parasite infection. Stock HCT-8 cells were maintained in 75 ml tissue culture flasks in a 5% CO₂ atmosphere at 37°C and 100% humidity, and passaged every 3-5 days. Cells were lifted from the surface after incubation for 10 min at 37 °C with a solution of 0.25% (W/V) trypsin and 0.53 mM EDTA in PBS (Mediatech Cellgro). Cell viability was assessed with trypan blue exclusion (0.02% in PBS) and cells were counted using a hemocytometer. Collected cells were seeded on sterile 22 mm² glass coverslips in six-well cluster plates (Corning-Corning, NY) at 1 X 10⁶ cells/well and grown to ~ 95% confluency in maintenance medium (48 h). For infection of monolayers and before inoculation with oocysts, the maintenance medium was removed and 2-3 ml of growth medium added to each well in a 6 well cluster plate. Cells were then incubated with oocysts at numbers of 10⁶ /ml for 90-120 min. After this time, each inoculated well was washed twice with HBSS to remove unexcysted oocysts, oocyst walls, and other materials that may have been liberated from the excysted oocysts. Cells in cluster plates were then incubated for 48 h with 3-4 ml maintenance medium/well.

Parasite infection was assessed 48 h post-infection using an immunohistochemistry stain as described previously (Phelps et al. 2001. J. Eukaryot. Microbiol. 48: 40-41). Briefly, coverslips in cluster plates were fixed with 100% methanol for 20-30 min and washed twice for 5 min with PBS. Coverslips were then treated first with a rabbit anti-C. parvum primary antibody (courtesy of C. Dykstra, Auburn University), followed by a biotinylated anti-rabbit secondary antibody (Vectastain ABC Kit, Vector Laboratories) and an avidin-biotin complex (ABC reagent; Vectastain ABC Kit, Vector Laboratories). Developmental stages were visualized with an immunoperoxidase stain using hydrogen peroxide (Sigma) and diaminobenzidine tetrahydrochloride (DAB, Sigma), with hematoxylin (Fisher Scientific) used as a counter stain. With this system, Cryptosporidium life cycle stages appear brown against a blue and purple background. Oocysts that were frozen in liquid nitrogen for 2 h before host cell inoculation served as a negative control. Positive infection was based on scoring the presence or absence of living stages (sexual gamonts and asexual meronts) in 50-100 sequential and non-overlapping fields visualized at a 400X magnification using phase contrast microscopy. Fields containing one or more C. parvum life stages were scored as positive, while fields containing none were scored as negative.

For PCR, at 48 h post-infection, infected cell monolayers were incubated in DNA extraction buffer (0.2 M Tris, pH 8.0, 0.1 M EDTA, 0.4 M NaCl) plus 0.1 mg/ml proteinase K and Tween 20 at 37 °C for 4-8 h and then overnight at 4°C. DNA was prepared by phenol-chloroform and chloroform extractions, precipitated with ethanol, and then analyzed by PCR as described above.

EXAMPLE 6 RT-PCR for CPV

C parvum oocysts that were stored in water baths at 20, 25, or 4°C were pelleted by centrifugation and resuspended in 100 μl PBS. RNAse inhibitor (20 U/pellet, Applied Biosystems, Foster City, CA) was added to the pellets, which were freeze-thawed three times and then applied to a Qiagen viral RNA kit (Qiagen). RT-PCR was performed using Superscript One-Step RT-PCR with Platinum Taq kit (Invitrogen) with 5 μl of RNA using described conditions and primers (Kozwich et al. 2000. Appl. Environ. Microbiol. 66: 2711-2717).

C. parvum oocysts showed reduced infectivity over time in water incubated for 1-3 months at 20 °C or 25 °C (Table 1). After 2 months at 25 °C, the oocysts were no longer infective to mice as measured by PCR or to cell culture as indicated by immunohistochemistry; however, at this time infectivity was found to occur in cell culture by PCR analysis. C. parvum oocysts stored at 20 °C for 3 months were found to be non-infectious by both mouse infectivity and cell culture assays. Again infection of cell culture as determined by PCR was variable, with a weak positive signal observed. Infectivity remained high for oocysts stored at 4°C at all time points. Although C. parvum oocyst infectivity decreased rapidly at 20 °C and 25 °C, CPV was detected by RT-PCR after 3 months incubation at all three temperatures.

Table 1. Comparison of RT-PCR for CPV with infectivity of C. parvum oocysts in mice and cell cultures to determine viability of C. parvum stored in sterile water at three different temperatures. Time point Mouse infection Cell Culture Infection CPV

PCR PCR Histochemistrv RT-PCR

1 Month 4°C + + + + 20°C + + + + 25 °C + + + +

2 Months 4°C + + + + 20°C + + +

25°C + +

3 Months

4°C + + + + 20°C +/- + 25°C +/- +

All publications and patents mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.

It is understood that the foregoing detailed description is given merely by way of illustration and that modifications and variations may be made therein without departing from the spirit and scope of the invention.

Claims

Claims We claim:

1. An isolated antibody or an antigen-binding fragment thereof which specifically and selectively binds one or more epitopes of the recombinant Cryptosporidium parvum/ Cryptosporidium hominis viral capsid protein rCPV40, native CPV40, and or native CPV30.

2. The antigen-binding fragment according to Claim 1, wherein the antigen-binding fragment is a Fab, F(ab)₂, or Fv fragment.

3. The antibody of Claim 1, wherein the antibody is a monoclonal antibody or a polyclonal antibody.

4. The antibody according to Claim 1, wherein the antibody is detectably labeled by conjugation to a detectable moiety.

5. The antibody according to Claim 1, wherein the antibody is bound in a complex comprising a detectable moiety.

6. The antibody according to Claim 4 or to Claim 5, wherein the detectable moiety is selected from the group consisting of radionuclides, enzymes, specific binding pair components, colloidal dye substances , fluorochromes, reducing substances, latexes, digoxigenin, metals, particulates, dansyl lysine, antibodies, protein A, protein G, electron dense materials, and chromophores.

7. The antibody according to Claim 1, wherein the antibody is attached to a substrate.

8. The antibody according to Claim 7, wherein the substrate includes a component selected from the group consisting of membranes, gels, hydrogels, resins, beads, nitrocellulose, nylon filters, microtiter plates, culture flasks, and polymeric materials.

9. A composition for binding C. parvum and C. hominis virus, comprising an isolated antibody or an antigen-binding fragment thereof which specifically and selectively binds one or more epitopes of the recombinant Cryptosporidium parvum/ Cryptosporidium hominis viral capsid protein rCPV40, native CPV40, and native CPV30.

10. The composition according to Claim 9, wherein the antibody or fragment thereof is detectably labeled by conjugation to a detectable moiety.

11. The composition according to Claim 9, wherein the antibody or fragment thereof is bound in a complex comprising a detectable moiety.

12. The composition according to Claim 10 or to Claim 11, wherein the detectable moiety is selected from the group consisting of radionuclides, enzymes, specific binding pair components, colloidal dye substances, fluorochromes, reducing substances, latexes, digoxigenin, metals, particulates, dansyl lysine, antibodies, protein A, protein G, electron dense materials, and chromophores.

13. A composition according to Claim 9, wherein the antibody or fragment thereof is attached to a substrate.

14. A composition according to Claim 13, wherein the substrate includes a component selected from the group consisting of gels, hydrogels, resins, beads, nitrocellulose, nylon filters, microtiter plates, culture flasks, and polymeric materials.

15. A method of detecting and identifying Cryptosporidium parvum and Cryptosporidium hominis in a sample, wherein CPV40 protein, specific for CPV, is detected by an isolated antibody or an antigen-binding fragment which specifically and selectively binds to recombinant Cryptosporidium parvum viral capsid protein rCPV40, native CPV40, and/or native CPV30, comprising: contacting the sample with a composition comprising an isolated antibody or an antigen-binding fragment thereof which specifically and selectively binds one or more epitopes of the recombinant C. parvum/C. hominis viral capsid protein, rCPV40,

detecting the presence or absence of a protein-antibody complex, wherein the specific binding is indicative of said CPV40, native CPV40 or native CPV30 in the sample.

16. The method of Claim 15 wherein the antibodies are monoclonal or polyclonal.

17. The method according to Claim 15, wherein the sample is a water sample.

18. The method according to Claim 15, wherein the water sample is sea water or water rinses of fruits and vegetables.

19. The method according to Claim 15, wherein the method is immuno-blotting.

20. The method according to Claim 15, wherein the method is selected from the group consisting of enzyme-linked immunosorbent assay, agglutination, precipitation, immunodiffusion, immunoelectrophoresis, immunofluorescence, chemiluminescence, radioimmunoassay, and immunohistochemistry.

21. The method according to Claim 15, wherein the antibody or fragment thereof is detectably labeled by conjugation to a detectable moiety.

22. The method according to Claim 15, wherein the antibody or fragment thereof is bound in a complex comprising a detectable moiety.

23. The method according to Claim 21 or to Claim 22, wherein the detectable moiety is selected from the group consisting of radionuclides, enzymes, specific binding pair components, colloidal dye substances, fluorochromes, reducing substances, latexes, digoxigenin, metals, particulates, dansyl lysine, antibodies, protein A, protein G, electron dense materials, and chromophores.

24. The method according to Claim 15, wherein the antibody or fragment thereof is attached to a substrate.

25. The method according to Claim 24, wherein the substrate includes a component selected from the group consisting of membranes, gels, hydrogels, resins, beads, nitrocellulose, nylon filters, microtiter plates, culture flasks, and polymeric materials.

26. The method according to Claim 15, wherein the fragment thereof is a Fab, F(ab)₂, or Fv fragment.

27. A method of detecting and identifying Cryptosporidium parvum and Cryptosporidium hominis in a sample, wherein a nucleic acid encoding CPV40 protein, specific for CPV, is detected by RT-PCR.

28. A kit for detection oi Cryptosporidium parvum and Cryptosporidium hominis in a sample, wherein the kit comprises: a composition comprising the antibody or antigen-binding fragment thereof according to Claim 1; and

a container housing the composition.

29. The kit according to Claim 28, wherein the antibody or fragment thereof is detectably labeled by conjugation to a detectable moiety.

30. The kit according to Claim 28, wherein the antibody or fragment thereof is attached to a substrate.