EP0859791A1 - Procede de detection d'ookystes de cryptosporidium - Google Patents

Procede de detection d'ookystes de cryptosporidium

Info

Publication number
EP0859791A1
EP0859791A1 EP96928273A EP96928273A EP0859791A1 EP 0859791 A1 EP0859791 A1 EP 0859791A1 EP 96928273 A EP96928273 A EP 96928273A EP 96928273 A EP96928273 A EP 96928273A EP 0859791 A1 EP0859791 A1 EP 0859791A1
Authority
EP
European Patent Office
Prior art keywords
oocysts
antibody
sample
excysted
cryptosporidium
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP96928273A
Other languages
German (de)
English (en)
Other versions
EP0859791A4 (fr
Inventor
Graham Vesey
Keith Williams
Duncan Veal
Alan Champion
Natalia Pererva
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Australian Water Technologies Pty Ltd
Access Macquarie Ltd
Original Assignee
Australian Water Technologies Pty Ltd
Macquarie Research Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Australian Water Technologies Pty Ltd, Macquarie Research Ltd filed Critical Australian Water Technologies Pty Ltd
Publication of EP0859791A1 publication Critical patent/EP0859791A1/fr
Publication of EP0859791A4 publication Critical patent/EP0859791A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/20—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans from protozoa
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56905—Protozoa

Definitions

  • the present invention relates to antibodies reactive to recently excysted oocysts of Cr ⁇ tospo ⁇ dium and methods of detecting viable Cryptosporidium oocysts using the antibodies.
  • Cryptosporidium is amongst the most common pathogens responsible for diarrhoeal disease in humans (Current 1986). 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 (Smith and Rose 1990. Badenoch 1990). Surveys have shown that many surface water supplies are contaminated with Ci ⁇ ptospo dium oocysts (LeChevallier et al 1991. Rose 1988).
  • Ci ⁇ ptospo ⁇ dium oocysts in water relies on the concentration of particulate matter including oocysts from large volumes of water prior to staining with fluorescently labelled antibodies.
  • detection and identification of fluorescently labelled oocysts required examination of the sample using epifluorescence microscopy.
  • the development of flow cytometric detection methods has alleviated many of these problems and enabled the routine monitoring of water for the presence of Ci ⁇ ptospo ⁇ dium oocysts (Vesey et al 1994).
  • a major limitation of this methodology is the lack of oocyst viability measurements.
  • a further problem with the currently employed method for flow cytometric analysis of water samples for Cryptosporidium is the requirement of a flow cytometer that can sort particles for subsequent microscopical examination. Sorting flow cytometers are expensive and sophisticated instruments that require a highly skilled operator.
  • the flow cytometric method involves staining of samples with a fluorescently labelled monoclonal antibody specific to the surface of Ci ⁇ ptospo ⁇ dium 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 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 bind the oocyst-specific antibody.
  • Analysis flow cytometers are available which are simple to operate and relatively inexpensive. These cytometers, however, are unable to perform sorting. To enable the detection of Cryptosporidium oocysts using one of these analysis cytometers 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 (Vesey et al 1994B) if the microorganism is labelled with two different antibodies.
  • the present invention consists in a method of detecting the presence of viable Cryptosporidium oocysts in a sample containing Cryptosporidium oocysts, the method comprising the steps of: a) treating the sample so as to cause any viable oocysts of
  • Ci ⁇ ptospo ⁇ dium in the sample to excyst b) exposing the treated sample to an antibody that binds specifically to recently excysted Cryptospo ⁇ dium oocysts such that the antibody binds to recently excysted C ptospo ⁇ dium oocysts in the sample; and c) detecting the presence of oocyst-bound antibody in the sample.
  • an antibody that binds specifically to recently excysted Cryptospo ⁇ dium oocysts such that the antibody binds to recently excysted C ptospo ⁇ dium oocysts in the sample
  • c) detecting the presence of oocyst-bound antibody in the sample oocyst-bound antibody in the sample. It will be appreciated that any treatment of the sample that causes oocysts to undergo excystation would be suitable. It is.
  • the oocysts are caused to excyst by incubating the sample at about 37°C under acidic conditions, followed by incubating the sample under neutral to alkaline conditions at about 37°C. More preferably, the oocysts are caused to excyst by incubating the sample at 37°C at pH 2 to 4 for 10 to 60 minutes, followed by incubating the sample at 37 ⁇ C at pH 7 to 9 for 10 to 60 minutes. The sample can be washed between the steps to facilitate the removal of the buffers and replacement with fresh buffers at the required pH.
  • excysted Cryptosporidium oocysts are defined as oocysts that have excysted within several hours from treatment.
  • the oocysts are exposed to antibody within one hour or less from being excysted to ensure optimal binding. It has been found that the antigen or antigens present on recently excysted oocysts to which antibodies can be made do not remain intact over prolonged periods. It will be appreciated that this period can be increased if the treated sample or oocysts are preserved in some manner. For example, freezing of the sample has been found to preserve the antigenicity of the excysted oocyst past this several hour period.
  • the present inventors have found that short-lived antigens are present on recently excysted C ptospo ⁇ dium oocysts and that specific antibodies can be raised against these antigens. These antibodies can be used to detect viable
  • Ci ⁇ ptospo ⁇ dium oocysts in samples. It will be appreciated that by following the teaching of the present invention useful antibodies can be produced against recently excysted Ci ⁇ ptospo ⁇ dium oocysts.
  • the sample is analysed by flow cytometry or microscopy to detect the oocyst- bound antibody.
  • the binding of the antibody to the recently excysted oocysts can be measured indirectly by further treating the sample with a fluorescently- labelled ligand that binds specifically to the antibody and measuring the binding of the labelled ligand to the oocyte-bound antibody.
  • the antibody can be fluorescently labelled prior to use and the binding of the antibody to the recently excysted oocysts can be detected by measuring directly the fluorescence of the bound antibody.
  • the antibody is a monoclonal antibody, more preferably the monoclonal antibody is Cry4, Cry5 or Cry6. and most preferably Cry4.
  • the treated oocysts are exposed to a first antibody that binds specifically to recently excysted oocysts and a second antibody that binds specifically to the surface of Ci ⁇ ptospo ⁇ dium oocysts.
  • the first and second antibodies are labelled with different fluorescent markers such that antibody binding can be detected by measuring the respective fluorescence of each fluorescent marker with a simple analysis-only flow cytometer.
  • the second antibody preferably binds to both viable and non-viable oocysts and the detection of the binding of one or more of the antibodies to the oocysts can be used to indicate the presence Ci ⁇ ptospo ⁇ dium oocysts in the sample.
  • the first antibody is Cry4, Cry5 or Cry6, preferably
  • the method of the present invention is particularly suitable for the detection of viable Cn ⁇ tospo ⁇ dium parvum oocysts.
  • the present invention consists in an antibody that binds specifically to recently excysted oocysts of Ci ⁇ ptospo ⁇ dium.
  • the antibody is a monoclonal antibody and more preferably the monoclonal antibody is Cry4. Cry5 or Cry6.
  • the present invention consists in an hybridoma cell producing a monoclonal antibody that binds specifically to recently excysted oocysts of Cryptosporidium. More preferably the hybridoma cell produces the monoclonal antibody Cry4, Cry5 or Cry6. In a fourth aspect, the present invention consists in a ligand or ligands of recently excysted oocysts of Cryptosporidium that is specifically bound by the monoclonal antibody Cry4, Cry5 or Cry6.
  • Figure 1 shows flow cytometric analysis of pure excysted oocysts labelled with monoclonal antibodies
  • Figure 2 shows flow cytometric analysis of pure non-excysted oocysts reacted with monoclonal antibodies
  • Figure 3 shows flow cytometric analysis of pure excysted oocysts stained with Cry4
  • Figure 4 shows flow cytometric analysis of environmental samples seeded with excysted oocysts and treated with two antibodies Cry26 and Cry4; Modes for Carrying Out the Invention
  • Cryptosporidium oocysts Cryptosporidium parvum oocysts cultured in lambs and purified by density gradient centrifugation were purchased from the Moredun Animal Research Institute, Edinburgh. Monoclonal antibodies. Two female balbC mice were injected with oocyst preparations as presented in Table 1.. Mice were sacrificed, spleen cells dissected and fused with NSl mouse myeloma cells and the resulting hybridomas cloned. Clones were screened for anti-oocyst antibody production by screening against fresh and excysted oocyst preparations with flow cytometry.
  • Excysted oocysts were prepared by excysting oocysts and then washing in saline solution.
  • Excysted SDS treated oocysts were prepared by excysting oocysts, treating with 1% (w/v) sodium deoxycholate at 21°C for 10 minutes and then washing in saline solution.
  • I.P. intraperitoneal I.V. - intravenous
  • Sonicated oocysts were prepared by sonicating a heat killed (80°C for 10 minutes) oocysts suspension until no intact oocysts were visible using light microscopy.
  • mice 1 and 2 represent flow cytometric analysis of oocysts and excysted oocysts reacted against a selection of the antibodies.
  • Oocysts 10 5 (existed or fresh) were aliquotted into each well of a 96 well plate, and 100 ⁇ l hybridoma super supernatant added plus 10 ⁇ l of FITC- coupled sheep anti-mouse antibody (1/40 dilution) Silenus. Samples were incubated at 37°C for 30 minutes, then mixed with 200 ⁇ l of phosphate buffered saline and analysed by flow cytometry (Vesey, et al, 1994B). The flow cytometer was calibrated with dilutions of a commercially available anti- Cr ⁇ ptospo ⁇ dium antibody so that positive and negative controls were defined. Analysis of all 41 clones by ELISA, indirect immuno-fluorescence and
  • Antibodies Cry26 and Cry4 were purified using EZ-Sep (Amrad Phamacia Biotech, Boronia. Australia) according to the manufacturer's instructions. Purified Cry26 antibody was conjugated with CY3 (Biological Detection Systems, PA, USA) according to the manufacturer's instructions. Table 2. Characteristics of monoclonal antibodies generated against Ci ⁇ ptospo ⁇ dium parvum oocysts
  • Oocysts (1 x IO 8 ) were surfaced sterilised by suspending in 1 ml of 70% (v/v) ethanol for 5 min and then washing by centrifuging at 13000g for 2 min, discarding the supernatant and resuspending in phosphate buffered saline (PBS), pH 7.4. Excystation was then performed by suspending in 1 ml of acidified PBS. pH 2.75.
  • PBS phosphate buffered saline
  • bovine serum albumin fraction V was then added to a final concentration of 2% (w/v) before the addition of 20 ⁇ l of CY3 conjugated Cry26 antibody (approximately 0.055 mg/ml). The sample was incubated at 37° for 10 min and then analysed by flow cytometry.
  • the two non-fluorescent populations 1 and 4 were observed to be full oocysts and empty oocysts.
  • the higher light scatter of population 4 indicates that this population represents the full oocysts. Therefore population 1 must represent the empty oocysts.
  • the lack of populations 2 and 3 in the sample that was not excysted indicates that the antigen recognised by Cry4 is only present in recently excysted oocysts.
  • Fig. 3 gives a pattern that is characteristic of antibodies that react specifically with recently excysted Cryptosporidium oocysts. It will be appreciated that the flow cytometric analysis as described can be used to determine whether any particular antibody reacts to recently excysted
  • Results of the analysis of environmental water samples that had been seeded with oocysts stained with both the Cry26 and Cry4 antibodies are presented in Figure 4.
  • the first graph represents side scatter versus red fluorescence (ie. the fluorescence due to binding of CY3 labelled Cry26 and the second graph represents side scatter versus green fluorescence (ie, the fluorescence due to binding of FITC labelled Cry4).
  • a box was drawn on the first graph around an area containing the stained oocysts. This box was then used to gate graph 2 (ie. the only particles that appear on graph 2 are those that appeared in the box).
  • Ci ⁇ ptospo ⁇ dium oocysts The antigen is not accessible in oocysts that have not excysted nor is it present in oocysts that have excysted prior to the excystation treatment. Furthermore, the antigen recognised by Cry4 is removed if oocysts are excysted in the presence of bacteria. This would indicate that the antigen is destroyed by bacterial enzymes.
  • oocyst viability has been determined on pure samples of oocysts by performing excystation and then manually counting the number of full and empty oocysts. This method is tedious and labour intensive.
  • the development of the antibody Cry4 or other antibodies that react specifically with recently excysted oocysts will enable immunofluorescence assays employing flow cytometry or other automated technologies to replace this manual methodology.
  • Ci ⁇ ptospo ⁇ dium oocysts based on the uptake or exclusion of the fluorogenic vital dyes propridium iodide (PI) and 4'6-diamidino-2-phenylindole (DAPI) have been reported (Campbell et al 1992). The authors report that dead oocysts take up PI and fluoresce red, whereas live oocysts exclude PI but are permeable to DAPI resulting in the sporozoites within the oocysts fluorescing blue. The method has proven useful for viability studies on pure oocysts (Robertson et al 1992).
  • Cry4 antibody to stain samples after they have been excysted enable the routine detection of viable Ci ⁇ ptospo ⁇ dium oocysts in environmental samples.
  • the present inventors have found that the antigen recognised by Cry4 rapidly degrades in samples containing bacterial activity. Therefore, oocysts which have excysted in the environment prior to sample collection will no longer contain the antigen recognised by Cry4.
  • Ci ⁇ ptospo ⁇ dium oocysts with more than one antibody because all commercially available antibodies recognise the same epitope on the surface of the oocyst wall (Moore et al 1995).
  • an antibody which reacts specifically to recently excysted oocysts and a surface antibody according to on method of the present invention it is now possible to dual label oocysts and detect them using a simple analysis-only flow cytometer.

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Abstract

L'invention porte sur un procédé de détection d'ookystes de Cryptosporidium dans un échantillon contenant des ookystes de Cryptosporidium. Le procédé consiste à: a) traiter l'échantillon de manière à dékyster tous les ookystes viables de Cryptosporidium présents dans l'échantillon, b) exposer l'échantillon traité à un anticorps qui se fixe spécifiquement aux ookystes de Cryptosporidium venant d'être dékystés, de telle sorte que l'anticorps se fixe aux ookystes de Cryptosporidium venant d'être dékystés dans l'échantillon, et c) détecter la présence d'un anticorps lié aux ookystes dans l'échantillon.
EP96928273A 1995-08-30 1996-08-30 Procede de detection d'ookystes de cryptosporidium Withdrawn EP0859791A4 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
AUPN5146/95 1995-08-30
AUPN5146A AUPN514695A0 (en) 1995-08-30 1995-08-30 Method for detection of cryptosporidium oocysts
PCT/AU1996/000543 WO1997008204A1 (fr) 1995-08-30 1996-08-30 Procede de detection d'ookystes de cryptospodidium

Publications (2)

Publication Number Publication Date
EP0859791A1 true EP0859791A1 (fr) 1998-08-26
EP0859791A4 EP0859791A4 (fr) 2000-01-05

Family

ID=3789474

Family Applications (1)

Application Number Title Priority Date Filing Date
EP96928273A Withdrawn EP0859791A4 (fr) 1995-08-30 1996-08-30 Procede de detection d'ookystes de cryptosporidium

Country Status (4)

Country Link
EP (1) EP0859791A4 (fr)
AU (1) AUPN514695A0 (fr)
NZ (1) NZ315977A (fr)
WO (1) WO1997008204A1 (fr)

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2201490T3 (es) * 1997-05-19 2004-03-16 Macquarie Research Ltd. Anticuerpos igg1 reactivos con la superficie de oocistos de cryptosporidium.
US6475747B2 (en) 1997-10-28 2002-11-05 The United States Of America As Represented By The Department Of Health And Human Services Method for detecting Cryptosporidium parvum oocysts
AUPQ196199A0 (en) * 1999-08-02 1999-08-26 Btf Pty Ltd Internal quality control
US7098042B2 (en) 1999-09-02 2006-08-29 Btf Pty Ltd. Internal quality control
AUPQ629100A0 (en) * 2000-03-16 2000-04-15 Btf Pty Ltd Process for preparing controlled samples of particles such as microorganisms and cells
CA2419154A1 (fr) 2000-09-12 2002-03-21 Gen-Probe Incorporated Compositions, procedes et trousses pour determiner la presence d'organismes du genre cryptosporidium dans un echantillon a analyser
CN116143875A (zh) * 2022-07-09 2023-05-23 吉林大学 隐孢子虫cgd2_3080卵囊壁外壁标记蛋白质及其应用

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993024649A1 (fr) * 1992-05-29 1993-12-09 The Regents Of The University Of California Polypeptides, acides nucleiques, vecteurs de cryptosporidium et procedes d'utilisation

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CRABTREE K D ET AL: "The detection of cryptosporidium oocysts and Giardia cysts in cistern water in the U.S. Virgin Islands" WATER RESEARCH,NL,ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM, vol. 30, no. 1, page 208-216 XP004035105 ISSN: 0043-1354 *
FERRARI B C ET AL: "Comparison of Cryptosporidium-specific and Giardia-specific monoclonal antibodies for monitoring water samples" WATER RESEARCH,NL,ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM, vol. 33, no. 7, page 1611-1617 XP004159530 ISSN: 0043-1354 *
See also references of WO9708204A1 *

Also Published As

Publication number Publication date
NZ315977A (en) 1999-11-29
AUPN514695A0 (en) 1995-09-21
WO1997008204A1 (fr) 1997-03-06
EP0859791A4 (fr) 2000-01-05

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