WO2002059605A1 - Methode et necessaire de detection d'une reponse immunitaire a mediation cellulaire - Google Patents

Methode et necessaire de detection d'une reponse immunitaire a mediation cellulaire Download PDF

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Publication number
WO2002059605A1
WO2002059605A1 PCT/DK2002/000013 DK0200013W WO02059605A1 WO 2002059605 A1 WO2002059605 A1 WO 2002059605A1 DK 0200013 W DK0200013 W DK 0200013W WO 02059605 A1 WO02059605 A1 WO 02059605A1
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WIPO (PCT)
Prior art keywords
lymphocytes
cell
immune response
whole blood
antigen
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PCT/DK2002/000013
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English (en)
Inventor
Martin Munk
Peter Andersen
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Statens Serum Institut
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Publication of WO2002059605A1 publication Critical patent/WO2002059605A1/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
    • G01N33/56911—Bacteria
    • G01N33/5695—Mycobacteria
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system

Definitions

  • This invention relates to an in vitro diagnostic method of detecting a cell- mediated immune response to one or more specific antigens in a human or animal.
  • the invention relates to a method for detecting gamma in- terferon released by sensitised lymphocytes originating from a whole blood sample from the human or animal.
  • the major characteristic of the immune system is to maintain the host free of infective agents. Both the humoral immunity dependent on B lymphocytes, and cell-mediated immunity dependent on T lymphocytes, are crucial for this pur- pose. IntraceUular bacteria are relatively protected from humoral immunity and consequently, processed and presented proteins from these microbes can promote the activation of T-lymphocytes. Accordingly, acquired resistance against intraceUular bacterial pathogens, such as Mycobacterium tuberculosis, depend on T-lymphocytes and interferon- ⁇ (IFN ⁇ ) which in synergy with other cyto- kines are crucial for resistance against infections caused by intraceUular bacterial pathogens. Therefore, IFN ⁇ production by stimulated T-lymphocytes are a major effector function of ceU-mediated responses to antigens from intraceUular bacterial pathogens and is useful for diagnostic purposes.
  • IFN ⁇ interferon- ⁇
  • a rapid laboratory confirmation of infection prior to the onset of cUnical symptoms is crucial for effective health measures against outbreaks of bacteria infections.
  • the lack of sensitivity or specificity, as well as the length of the time period required for current methods impairs identification of M. tuberculosis infected individuals.
  • Methods for rapid identification of e.g. M. tuberculosis in- fection in different clinical specimens can include microscopy, bacilli culture, PCR, nested-PCR, or DNA-RNA hybridisation.
  • sensitivity, specificity and positive prediction values vary between these methods and should be interpreted cautiously together with the clinical picture.
  • Most mycobacteria related PCR systems in clinical laboratories amplify a portion of the ⁇ S6110 insert
  • PPD Tuberculin Purified Protein Derivative
  • PPD is not able to distinguish between active disease, BCG vacci- nated donors, previous contact with bacilli, cross reactive antigens found in other mycobacteria species or positivity due to boosting phenomenon (1).
  • application of PPD to the skin may lead to adverse skin reactions, such as ulceration or necrosis at the site of skin test, as weU as systemic effects Uke febrile or hypersensitivity reaction.
  • IFN ⁇ assay a simple and rapid whole blood IFN ⁇ assay was adapted for detection of PPD reactivity in humans (3).
  • the assay requires no ceU separation procedures and the two step simultaneous enzyme immunoassay (EIA) can be standardized in a serology laboratory.
  • EIA enzyme immunoassay
  • the present invention relates to an in vitro diagnostic method of detecting a cell-mediated immune response to one or more specific antigens in a human or animal.
  • the method comprises the steps of adding an erythrocyte lysing buffer to a whole blood sample from the human or animal, centrifuging the sample, resuspending the peUet in a ceU culture medium to a volume less than the original whole blood volume, incubating the resuspension with the specific antigen, and detecting the presence of gamma interferon (IFN ⁇ ) released by the sensitized lymphocytes in said whole blood sample to indicate a ceU-mediated immune response of said specific antigen.
  • IFN ⁇ gamma interferon
  • the cell lysis is preferably carried out by adding an erythrocyte lysing buffer which in a preferred embodiment, contains NH + and KHCO 3 .
  • an erythrocyte lysing buffer which in a preferred embodiment, contains NH + and KHCO 3 .
  • Other methods of lysis may in principle be used. The method should be selected to avoid damage to the lymphocytes which will preventthem exhibiting a ceU mediated im-
  • SUBSTITUTE SHEET mune response in subsequent stages Methods which may not be used include exposure to lysing antibodies and the use of hypertonic medium.
  • the lymphocytes are enriched, i.e. the concentration of lymphocytes in said sample is increased between the steps of lysing the erythrocytes and incubating the lymphocytes with antigen.
  • said lymphocytes may be separated from the sample, suitably by centrifugation and are resuspended in ceU culture me- dium to produce a resuspension volume less than the volume of said whole blood sample.
  • said resuspension volume is less than that of the whole blood sample by a factor of at least 1.5, more preferably from 1.5 to 3, e.g. about 2. Going to higher concentrations of the lymphocytes may reduce cytokine production.
  • the detection of the immune response is preferably carried out by detecting the presence of gamma interferon (IFN- ⁇ ) released by sensitised lymphocytes from said whole blood sample to indicate a ceU-mediated immune response to said antigen.
  • IFN- ⁇ gamma interferon
  • T-ceU proliferation assays involving the uptake of labelled substance such as radioactive thymidine or 5-bromo-2'- deoxyuridine may be used as well as flow cytometry ceU dilution analysis.
  • the production of cytokines other than gamma interferon may be detected by known assay procedures. These include assays for IL2, IL4, IL5, IL10, IL12, IL18. TNF or TGF ⁇ .
  • the gamma interferon released from the sensitised lymphocytes can be detected in an immunoassay, such as an enzyme-Unked immunosorbent assay.
  • the specific antigen or antigens may be coated onto latex beads prior to contact with the lymphocytes.
  • the specific antigen or antigens are an antigen or antigens of M. tuberculosis, M. paratuberculosis or M bovis or an antigen immunologically cross-reactive therewith.
  • the invention also provides a diagnostic kit suitable for use in a method according to the present invention.
  • an in vitro diagnostic method of detecting a ceU-mediated immune response to one or more specific antigens in a human or animal comprises the steps of: (i) adding erythrocyte lysing buffer to a whole blood sample from the human or animal, (ii) centrifuging the sample from step (i), (iii) resuspend- ing the peUet from step (ii) in a cell culture medium to a volume less than the original whole blood volume, (iv) incubating the resuspension with the specific antigen, and (v) detecting the presence of gamma interferon (IFN ⁇ ) released by the sensitised lymphocytes in said whole blood sample to indicate a cell- mediated immune response of said specific antigen.
  • IFN ⁇ gamma interferon
  • antigens may be tested singly in separate resuspensions prepared from the same whole blood sample. However, the presence of reactivity to any one of a number of antigens may be tested for simultaneously by ex- posing T-ceUs to a mixture of such antigens.
  • Lymphocytes definitely needs physical contact with antigen presenting ceUs in order to react (respond) to a specific antigen.
  • High amount of erythrocytes can impair such contact and subsequent lymphocyte activation.
  • most erythrocytes in vitro die after 24 hours. Consequently, the concentration of different intraceUular components released into ceU-culture medium from dead ceUs impair the assay or lyse many of the stimulated ceUs responsible for the production of cytokines to be detected.
  • the lysis may be carried out by adding a buffered erythro-
  • the buffered erythrocyte lysing solution contains preferably NH C1 and KHCO 3 which efficiently buffers suspensions of lysed ceUs.
  • the cell suspen- sion is centrifuged once at low speed without brakes on and the peUet is resus- pended in cell-culture medium supplemented with human serum in a smaller volume than the original whole blood volume, e.g. for producing a 2-fold concentrated leukocyte cell suspension. Consequently, an elevated frequency of antigen responding ceUs per ml are present and therefore stimulated in each weU of the assay, resulting in a higher IFN ⁇ production than the conventional whole blood assay.
  • the cell culture in the improved method according to the invention can be maintained for 4 days, as compared to the elevated ceU lysis in whole blood assay after 2 days culture. This favours the stimulation of specific cells, compared to 24 hs cultures.
  • the detection of the presence of IFN ⁇ may be performed by any suitable means, for example, by means of an immunoassay such as an enzyme-linked immunoassay (ELISA) using monoclonal antibodies specific for IFN ⁇ .
  • an immunoassay such as an enzyme-linked immunoassay (ELISA) using monoclonal antibodies specific for IFN ⁇ .
  • ELISA enzyme-linked immunoassay
  • detection systems may be simply qualitative, or they may be quantitative of the amount of IFN ⁇ produced. Similar comments apply to the detection of other cy- tokines.
  • Values of IFN ⁇ production in vitro in the present invention is higher than detected in other whole blood assays. Consequently, sensitivity is significantly higher by this method compared to other whole blood assays.
  • the number of leukocytes per ml obtained by this procedure is compared with that in whole blood.
  • Peripheral blood cells from each of several donors was either left untreated (fresh whole blood) or enriched by the improved whole blood method of Example 1 (improved whole blood).
  • the absence of interfering erythrocytes increased ceU contact and performance of IFN ⁇ production by enriched cells.
  • SUBSTITUTE SHEET are removed and added into another 96 well plate either to perform serial fold dilutions or stored at -20°C until further use.
  • IFN ⁇ secreted into cell-culture supernatants can be analysed by an enzyme-linked immunoassay (ELISA) using commercially available monoclonal antibodies specific for human IFN ⁇ (11, 12). Briefly, 96- weU plates (NUNC) are incubated with a catching anti-human IFN ⁇ monoclonal antibody (Endogen, USA) diluted in PBS at 4°C for 18 h. After washing and blocking of the weUs, ceU culture supernatants are added and the wells are in- cubated for 2h. The weUs are washed and biotin-labeled polyclonal goat-anti- human IFN ⁇ antibodies (Endogen) are added for 30 min.
  • ELISA enzyme-linked immunoassay
  • the reaction is visualized by adding alkaUne phosphatase-conjugated streptavidin (Jackson Laboratories, USA) for 30 min foUowed by p-nitrophenyl phosphate (Sigma) dUuted in the appropriate buffer.
  • Concentrations of IFN ⁇ in the samples are calculated using the standard curve generated from recombinant human IFN ⁇ (Endogen) and results are expressed in pg/ml. The minimum assay sensitivity is 10 pg/ml. Results are given in Table 2.
  • EXAMPLE 2A COMPARISON OF CELL MEDIATED RESPONSES OF IMPROVED WHOLE BLOOD ASSAY
  • Peripheral blood cells from each of several donors were either left untreated (fresh whole blood) or enriched by the improved whole blood method (improved w.b.) of Example 1.
  • improved w.b. ceUs were incubated together with antigen (PPD or CFP7) either at 1 ml/weU (as for fresh whole blood) or at 0,2 ml/weU (as described in this invention).
  • Table 2a shows the IFN ⁇ production induced by ceU-mediated responses to PPD or CFP7 by the method described in this invention and cytokine production analysed by ELISA (as per Example 2).
  • Results are expressed as IFN ⁇ titers (pg/ml) of ceU culture supernatants.
  • EXAMPLE 3A IMPROVED WHOLE BLOOD ASSAY FOR TB DIAGNOSIS
  • Table 3a and 3b shows the production of IFN ⁇ induced by cell-mediated responses to M. tuberculosis specific antigens (ESAT6 or Hybrid A2 that is composed of ESAT6 and CFP10 (6) proteins) by fresh whole blood (Table 3a) or the improved whole blood method (Table 3b) described in this invention and analysed by ELISA (as per Example 2).
  • ESAT6 or Hybrid A2 that is composed of ESAT6 and CFP10 (6) proteins

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Abstract

La présente invention concerne une méthode de diagnostic in vitro qui permet de détecter chez l'homme ou l'animal une réponse immunitaire à médiation cellulaire dans un petit échantillon de sang entier frais relativement à un ou plusieurs antigènes spécifiques. La méthode consiste à lyser des érythrocytes à l'aide d'un tampon contenant NH4+ et KHCO¿3?; à centrifuger l'échantillon et à resuspendre la granule cellulaire dans un milieu de culture cellulaire. Des cellules en 0.2 ml sont incubées dans une plaquettes 96 cupules avec l'antigène spécifique pendant 4 à 5 jours, et une production de cytokine - de préférence de l'interféron gamma (IFNΩ) libéré, est détectée par la méthode ELISA ou toute autre méthode pouvant indiquer une réponse immunitaire à médiation cellulaire audit antigène spécifique. La méthode de l'invention permet de réduire le temps et le coût des tests de diagnostic de la tuberculose à médiation cellulaire, et offre une libération de l'interféron gamma considérablement supérieure, avec un nombre inférieur de cellules, par comparaison avec une autre méthode utilisant des échantillons de sang frais. La méthode de l'invention est donc fiable et convient pour une réponse aux lymphocytes T spécifiques.
PCT/DK2002/000013 2001-01-25 2002-01-08 Methode et necessaire de detection d'une reponse immunitaire a mediation cellulaire WO2002059605A1 (fr)

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GB0101973A GB0101973D0 (en) 2001-01-25 2001-01-25 Improved in vitro diagnostic method of detecting a cell-mediated immune response
GB0101973.6 2001-01-25

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005080990A2 (fr) 2004-02-19 2005-09-01 Istituto Nazionale Delle Malattie Infettive 'lazzaro Spallanzani' Essai de diagnostic immunitaire pour diagnostiquer et pour surveiller une infection de tuberculose
CN102062774A (zh) * 2009-11-13 2011-05-18 程小星 检测结核病治疗效果的试剂盒
CN108548917A (zh) * 2018-03-01 2018-09-18 广州市雷德生物科技有限公司 一种记忆性免疫细胞的检测体系及其应用

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EP0302435A1 (fr) * 1987-07-31 1989-02-08 Neorx Corporation Méthode d'augmentation de la cytotoxicité cellulaire dépendants d'anticorps
US5543300A (en) * 1991-10-26 1996-08-06 Torf Establishment Method and composition for determining the immunological activity of bioactive substances
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INT J. TUBERC. LUNG DIS, vol. 2, no. 6, June 1998 (1998-06-01), pages 443 - 450 *
ULRICHS T ET AL: "DIFFERENTIAL T CELL RESPONSES TO MYCOBACTERIUM TUBERCULOSIS ESAT6 IN TUBERCULOSIS PATIENTS AND HEALTHY DONORS", EUROPEAN JOURNAL OF IMMUNOLOGY, WEINHEIM, DE, vol. 28, no. 12, December 1998 (1998-12-01), pages 3949 - 3958, XP000891644, ISSN: 0014-2980 *
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005080990A2 (fr) 2004-02-19 2005-09-01 Istituto Nazionale Delle Malattie Infettive 'lazzaro Spallanzani' Essai de diagnostic immunitaire pour diagnostiquer et pour surveiller une infection de tuberculose
CN102062774A (zh) * 2009-11-13 2011-05-18 程小星 检测结核病治疗效果的试剂盒
CN108548917A (zh) * 2018-03-01 2018-09-18 广州市雷德生物科技有限公司 一种记忆性免疫细胞的检测体系及其应用

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