WO1990015326A1 - Test de formation des rosettes spontanees pour le diagnostic d'une infection active - Google Patents

Test de formation des rosettes spontanees pour le diagnostic d'une infection active Download PDF

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Publication number
WO1990015326A1
WO1990015326A1 PCT/SE1990/000382 SE9000382W WO9015326A1 WO 1990015326 A1 WO1990015326 A1 WO 1990015326A1 SE 9000382 W SE9000382 W SE 9000382W WO 9015326 A1 WO9015326 A1 WO 9015326A1
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WO
WIPO (PCT)
Prior art keywords
microorganism
white blood
blood cells
antibodies
liquid medium
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Application number
PCT/SE1990/000382
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English (en)
Inventor
Olle RINGDÉN
Lola Markling
Anders Ingvarsson
Original Assignee
Karobio Aktiebolag
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.)
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Publication date
Application filed by Karobio Aktiebolag filed Critical Karobio Aktiebolag
Publication of WO1990015326A1 publication Critical patent/WO1990015326A1/fr

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    • 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

Definitions

  • the present invention pertains to a method of diagnosing in vitro an active infection caused by a microorganism, whereby the presence of said micro ⁇ organism, and/or phagocytized parts thereof, exposed on white blood cells is determined in a sample of white blood cells separated from whole blood. It further pertains to a diagnostic assay kit adapted to the method of the invention.
  • Background of the invention Early detection of an active infection in a patient has always been recognized by the clinician as one of the most important factors for a successful treat ⁇ ment. The importance of an early detection has been accentuated during the last years, owing to the fact that it now is possible to slow down the progress of and in some cases cure patients from i.a. viral infections, provided a rapid and correct diagnosis can be established. Traditional diagnosis has in prin ⁇ ciple been performed by using one of the following three methods:
  • Microorganisms can be recovered from several sites during an acute infection or reacti- vation of a latent infection by the cultivation of samples and the identification of a micro ⁇ organism.
  • Use of monoclonal antibodies The use of monoclonal antibodies specific to well characterized epitopes on a microorganism of interest makes it possible to detect the microorganism either directly from a patient sample or after prior amplification by cultivation.
  • DNA hybridization technology has recently become available for the detection of a number of infectious diseases. This technology can be applied both to direct samples and to cultivated microorganisms.
  • a common feature of the above mentioned techniques is that they do not distinguish between active and past infection. Moreover, they are often tedious and contain many working steps, and when cultivation methods are used, the time between sampling and an estabished diagnosis involving certain microorganisms (viruses, parasites, some bacteria) is very long.
  • the assay steps differ significantly from the ones used in the claimed invention, primarily due to the fact that enzyme-label ⁇ led anti-antibodies which require staining for detec- tion, are used.
  • the disclosed assay can, if necessary, be carried out within 3 hours.
  • the present invention provides a method of diagno ⁇ sing in vitro an active infection caused by a microorga ⁇ nism, which is rapid and easy to perform without the need of any sophisticated equipment. Moreover, the method is sensitive and allows detection at an early stage of an oncoming infection and provides a tool to follow the patient from onset to the end of a speci ⁇ fic microbial infection, since it reflects an active infection. Moreover, the effectiveness of the chosen treatment can be monitored by using the method of the invention. The time between sampling and answer is comparatively short, in one embodiment of the inven ⁇ tion approximately two hours, and in another embodi ⁇ ment of the invention approximately an hour and a half.
  • a method of diagnosing in vitro an active infection caused by a microorganism whereby the presence of said microorganism, and/or phagocytized parts thereof, exposed on white blood cells is determined in a sample of white blood cells separated from whole blood, said sample of white blood cells in a liquid medium being incubated with either a) an excess amount of antibodies directed against said microorganism, followed by incubating the mixture with an excess amount of anti- -antibodies coated on a carrier having a par ⁇ ticle size detectable in a light microscope, or b) an excess amount of antibodies directed against said microorganism and coated on a carrier having a particle size detectable in a light microscope, followed by placing a drop of the incubated mixture resulting from a) or b) on a microscope slide and, with the aid of a light microscope, determining the presence of said microorganism, and/or phagocytized parts thereof, exposed on white blood cells as so-called rose
  • the separation of a sample of white blood cells from whole blood can be effected by any method known in the art, e.g. by gradient centrifugation using a mixture of sodium metrizoate and Ficoll according to the method of B ⁇ 'yum, A. (Scand. J. Clin. Lab. Invest. 21, Suppl. 97 (1968)).
  • The- ' term "liquid medium” used in the specification and claims denotes any suitable liquid medium which does not lyse the white blood cells. Examples of such media are physiological saline solution or a culture medium, such as RPMI 1640 medium.
  • Antibodies directed against a microorganism of interest can either be selected from commercially available ones or be prepared according to standard procedures. (See e.g. Fazekas de St Groth, Scheidegger D., Production of monoclonal antibodies: Strategy and tactics. J. Immunol. Meth. 35:1, 1980).
  • Both monoclonal and polyclonal antibodies can be used in the method.
  • the carrier particles used in the method of the invention should have a particle size detectable in a light microscope, and in a preferred embodiment of the invention the carrier particles are microspheres having an average diameter of 3-5 ⁇ m.
  • the carrier material should be such that antibodies can be coated on the particles.
  • the incubations according to the method of the invention are performed at approximately 4°C.
  • washing of the first incubated mixture is performed with e.g. phosphate buffered saline, followed by separation of a mixture of free and antibody-bound white blood cells.
  • the separated mixture of white blood cells is then diluted with a liquid medium prior to incubation with anti- -antibodies coated on a carrier. This intermediate washing procedure which removes non-bound antibodies, facilitates the detection of rosettes on the microscope slide.
  • rosettes in the present invention infected white blood cells on which antigen-antibody bound carrier particles are attached
  • So-called rosettes are well known to the man skilled in the art (see e.g. Jondahl M. , Holm G., igzell H. , J. Exp. Med., 136; p. 207, 1972).
  • the method according to the invention can be applied to diagnosing in vitro an active infection caused by any specific microorganism, provided anti ⁇ bodies directed against said microorganism are, or can be made, available.
  • Particularly important micro ⁇ organisms causing active infections are Cytomegalovirus (CMV), Candida, Aspergillus, Human immunodeficiency virus (HIV), Hepatitis virus, Toxoplas a, Epstein- -Barr-virus (EBV), Pneumocytis carinii, and Herpes virus.
  • CMV Cytomegalovirus
  • Candida Aspergillus
  • HCV Human immunodeficiency virus
  • HCV Human immunodeficiency virus
  • Hepatitis virus Hepatitis virus
  • Toxoplas a Epstein- -Barr-virus
  • EBV Epstein- -Barr-virus
  • Pneumocytis carinii Herpes virus.
  • the microorganism is Cytomegalovirus
  • the method according to the invention it is possible to calculate the percentage of infected white blood cells in the starting sample of white blood cells by calibrating the starting sample of white blood cells to a predetermined number of cells per ml of liquid medium, and screening, after the last incubation, an appropriate number of cells for the number of rosettes.
  • the presence of at least two different microorga ⁇ nisms is determined simultaneously with the aid of at least two differently coloured carrier particles. This is possible due to the fact that the colour of the carrier particle is detectable in a light micro ⁇ scope.
  • carrier partic ⁇ les of different colours, e.g. blue, red, brown and white.
  • kits for the determination of an active infection caused by a microorganism.
  • the kit comprises in separate containers either the items a) a calibrated amount of antibodies directed against a selected microorganism in a liquid medium, and a calibrated amount of carrier particles pre- coated with anti-antibodies in a liquid medium,
  • a calibrated amount of carrier particles pre- coated with antibodies directed against a selected microorganism and optionally washing solutions, media for separation of white blood cells from whole blood, and reagents for testing the activity of the antibodies.
  • the kit is adapted for use in the method according to the invention.
  • the calibrated suspensions are in lyophilized form for reconstitution with a calculated amount of a liquid medium.
  • the liquid medium is optionally supplied in a separate container in the kit.
  • the assay kit may comprise at least two different items as defined in a) or b) of this aspect of the invention, each of said items comprising carrier particles which with respect to each other are differently coloured. Diagnostic assay procedure
  • a sample of whole blood from a patient is mixed
  • Lymphoprep is a trade mark of Nycomed AS Diagnostica, Norway, and contains sodium metrizoate 9.6% (w/v) and Ficoll 5.6% (w/v)).
  • the mixture is centrifugated for 20 minutes at 2000 rpm.
  • the interphase band containing white blood cells is sucked off using a pasteur pipette and is transferred to another centri ⁇ fuge tube.
  • the cells are washed three times with physio ⁇ logical NaCl for 6 minutes at 1500 rpm.
  • the cells are counted in a Btirker chamber and are diluted in a RPMI 1640 medium (manufacturer Gibco BRL) to a pre ⁇ determined number of cells per ml of liquid medium, e.g. 10 x 10 cells per ml.
  • RPMI 1640 medium manufactured by RPMI 1640 cells.
  • test tube is incubated on ice for 30 minutes (i.e. at 0°-4°C). The test tube is gently shaken from time to time during the incubation, to keep the anti ⁇ bodies suspended in the liquid medium. 4. The contents of the test tube are washed twice with 1 ml portions of phosphate buffered saline (PBS), pH 7.4, comprising 2.0% fetal calf serum, and centri ⁇ fugated at 1500 rpm.
  • PBS phosphate buffered saline
  • the resulting pellet is suspended in 0.9 ml RPMI 1640 medium, and 50 ⁇ l of a suspension comprising 4 x 10 microspheres coated with anti-antibodies per ml of PBS, is added while working in a refrigerated room.
  • test tube is rotated and incubated for 30 minutes in a refrigerated room (at 4°C). 7.
  • the contents of the test tube is mixed using a Pasteur pipette, to suspend the microspheres and cells to an even distribution.
  • One drop of the suspended mixture is placed on a microscope slide which is then covered with a cover glass, followed by visual inspec- tion in an ordinary light microscope.
  • An appropriate number of cells e.g. 200 cells, are screened for the number of rosettes with the aid of an appropriate magnification objective, e.g. 40x magnification.
  • an appropriate magnification objective e.g. 40x magnification.
  • the percentage of infected white blood cells in the starting sample of white blood cells is calculated (with the aid of the number of rosettes) .
  • test tube is rotated and incubated for 30 minutes in a refrigerated room (at 4°C).
  • test tube 3 The contents of the test tube are mixed using a Pasteur pipette to suspend the microspheres and cells to an even distribution.
  • One drop of the suspended mixture is placed on a microscope slide which is then covered with a cover glass, followed by visual inspec ⁇ tion in an ordinary light microscope.
  • EXAMPLE 1 The method according to alternative a) of the invention was used for the determination of the per ⁇ centage of Cytomegalovirus-infected white blood cells in blood samples taken from 9 patients who had received transplants at Huddinge University Hospital, Sweden.
  • the microspheres carrying anti-antibodies were sheep anti-mouse IgG 1 (FC) coated on activated micro- spheres, Dynabeds, from Dynal AS, Norway.
  • MAS 127 monoclonal antibody against Cytomegalovirus with specificity for human Cytomegalovirus early antigen, a product of Sera-lab, Great Britain, purchased from AB Kemila Preparat, Sollentuna, Sweden.
  • Cytomegalovirus with specificity for DNA binding protein purchased from Dr. Lenore Pereira, the University of California,
  • the patient 1. was a pancreas transplant patient; the patients 2., 3., 4. and 9. were kidney transplant patients; and the patients 5., 6., 7. and 8. were bone marrow transplant patients.
  • M denotes male and F denotes female.
  • Serology had been performed by the Swiss County Council Central Microbiological Laboratory, Sweden, + denotes raise in IgM or IgG titres; - de ⁇ notes unchanged IgM or IgG titres.
  • Cultivation in fibroblasts and virus detection had been performed by the Swedish County Council Central Microbiological Laboratory, Sweden, and B denotes blood, U denotes urine and Bal denotes bronco- 5 alveolar lavage.
  • microspheres carrying anti-an ⁇ tibodies were the same as the ones used in Example 1; and the antibodies directed against Cytomegalovirus were M 127 as in Example 1.
  • Cytomegalovirus matrix protein (64-68 kD) from Biolnvent International, Lund, Sweden.
  • the antibodies were coated on red carboxylated microspheres (Polyscience) using the carbodiimide kit from Polyscience, Inc. USA, in accordance with the manufacturer's instructions. The results are given in Table II.
  • the method according to alternative a) of the invention was used for the determination of the percen ⁇ tage of Candida-infected white blood cells in blood samples taken from 8 patients who had received trans ⁇ plants at Huddinge University Hospital, Sweden.
  • microspheres carrying anti-antibodies were the same as the ones used in Example 1.
  • the antibodies directed against Candida were MAB 806, mouse anti-Candida albicans monoclonal antibody having no cross reactivity with other yeast, Lactobacillus Streptococcus/- Pediococcus, from Chemicon International, USA. The results are given in Table III.
  • the patients 1., 2., 4., 5., and 7. were kidney transplant patients; the patient 3. was a liver transplant patient; and the patients 6. and 8. were kidney + pancreas transplant patients.
  • M denotes male and F denotes female.

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  • Health & Medical Sciences (AREA)
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  • Immunology (AREA)
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Abstract

L'invention concerne un procédé de diagnostic in vitro d'une infection active provoquée par un microorganisme, consistant à déterminer la présence dudit microorganisme, et/ou de parties phagocytées de celui-ci, sur des globules blancs dans un échantillon de globules blancs prélevés sur du sang total. Selon le procédé, on utilise soit des anticorps contre ledit microorganisme + des anti-anticorps sur des particules porteuses, soit des anticorps contre ledit microorganisme sur des particules porteuses. On procède à la détection desdites rosettes dans un microscope classique. L'invention concerne également un kit d'analyse de diagnostic adapté au procédé.
PCT/SE1990/000382 1989-06-01 1990-06-01 Test de formation des rosettes spontanees pour le diagnostic d'une infection active WO1990015326A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
SE8901988A SE466623B (sv) 1989-06-01 1989-06-01 Saett och testsats foer diagnosticering av en aktiv infektion
SE8901988-9 1989-06-01

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WO1990015326A1 true WO1990015326A1 (fr) 1990-12-13

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2034464A (en) * 1978-10-19 1980-06-04 Harvard College Assay of infected cells and kit therefor
DE3232955A1 (de) * 1981-09-07 1983-03-17 Fuji Photo Film Co., Ltd., Minami-Ashigara, Kanagawa Mit antikoerper sensibilisierte mikrokapseln und ein verfahren zur messung von lymphozyten unter verwendung derselben auf der grundlage zellvermittelter immunitaet
GB2122345A (en) * 1982-06-18 1984-01-11 Bio Rad Laboratories Simultaneous assay for t and b lymphocytes
EP0294216A2 (fr) * 1987-06-05 1988-12-07 Interferon Sciences, Inc. Essai fonctionnel pour des t-lymphocytes

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2034464A (en) * 1978-10-19 1980-06-04 Harvard College Assay of infected cells and kit therefor
DE3232955A1 (de) * 1981-09-07 1983-03-17 Fuji Photo Film Co., Ltd., Minami-Ashigara, Kanagawa Mit antikoerper sensibilisierte mikrokapseln und ein verfahren zur messung von lymphozyten unter verwendung derselben auf der grundlage zellvermittelter immunitaet
GB2122345A (en) * 1982-06-18 1984-01-11 Bio Rad Laboratories Simultaneous assay for t and b lymphocytes
EP0294216A2 (fr) * 1987-06-05 1988-12-07 Interferon Sciences, Inc. Essai fonctionnel pour des t-lymphocytes

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
CHEMICAL ABSTRACTS, Vol. 103, No. 11, 16 September 1985, (Columbus, Ohio, US), KOBAYASHI MASAKI et al., "Determination of lymphocyte subpopulations using antibody-containing gelatin particles", see page 493, abstract 86412r; & JP,A,60 057 256 (85 57,256), 3 April 1985. *
DERWENT'S ABSTRACT, No. 73-514 960/36; & SU,A,365137, publ. week 7336. *
DIALOG INFORMATION SYSTEMS, Database Medline, File 154, Dialog Accession no. 06765530, RANA M T et al., "Paramagnetic microspheres as immunological markers for light and electron microscopy", & J IMMUNOL METHODS (Netherlands), 9 Dec 1988, 115(2) p. 209-17. *
METHODS IN ENZYMOLOGY, Vol. 108, 1984, B E ELIOTT et al., "Rosetting Techniques to Detect Cell Surface Markers on Mouse and Human Lymphoreticular Cells", see page 49 - page 64, see particularly pages 49-51 and 56-60. *
NATIONAL LIBRARY OF MEDICIN (NLM) DATABASE MEDLINE, File Med 89, NLM accession no. 88045333, MINAEVA V M et al., "Use of the specific rosette formation test in tick-borne encephalitis for the purpose of laboratory diagnosis", & ZH MIKROBIOL. EPIDEMIOL IMMUNOBIOL 1987 Jul;(7):41-4. *

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Publication number Publication date
SE466623B (sv) 1992-03-09
SE8901988L (sv) 1990-12-02
SE8901988D0 (sv) 1989-06-01
AU5858290A (en) 1991-01-07

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