WO1996027799A1 - Procede pour diagnostiquer des infections - Google Patents

Procede pour diagnostiquer des infections Download PDF

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Publication number
WO1996027799A1
WO1996027799A1 PCT/EP1996/000979 EP9600979W WO9627799A1 WO 1996027799 A1 WO1996027799 A1 WO 1996027799A1 EP 9600979 W EP9600979 W EP 9600979W WO 9627799 A1 WO9627799 A1 WO 9627799A1
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WO
WIPO (PCT)
Prior art keywords
proteins
protein
vpl
igg
denatured
Prior art date
Application number
PCT/EP1996/000979
Other languages
English (en)
Inventor
Klaus Hedman
Maria Söderlund
Original Assignee
Klaus Hedman
Soederlund Maria
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 Klaus Hedman, Soederlund Maria filed Critical Klaus Hedman
Priority to EP96907383A priority Critical patent/EP0813683A1/fr
Priority to AU51033/96A priority patent/AU5103396A/en
Publication of WO1996027799A1 publication Critical patent/WO1996027799A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
    • 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
    • G01N33/56983—Viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14211—Erythrovirus, e.g. B19 virus
    • C12N2750/14222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/01—DNA viruses
    • G01N2333/015—Parvoviridae, e.g. feline panleukopenia virus, human Parvovirus

Definitions

  • the present invention relates to a method for diagnosing infections caused by microbes whereby antibody reactivities towards at least two proteins or antigenic determinants derived from the microbes are compared.
  • Human parvovirus B19 is structurally the simplest known human-pathogenic virus. Its icosahedral capsid is made up of two structural proteins, VPl (83 JD) and VP2 (58 kD) . The smaller protein VP2 is the major capsid constituent, while VPl comprises only ⁇ 10% of the capsid. The amino acid sequence of VP2 overlaps with that of VPl, so that a portion of only 227 N-terminal amino acids is unique to VPl (Ozawa, K., et al., J. Virol. 61 (1987) , 2395-2406) .
  • Parvovirus causes erythema infectiosum (fifth disease) , a common childhood exanthem (Anderson M.J., et al., J. Hyg. Camb. 93 (1984) , 85-93; Plummer F.A., et al., N. Engl. M. Med. 313 (1985) , 74-79) which, especially in adults, is often coupled to transient polyarthritis or arthralgia affecting typically knees and small joints (Reid D.M. , et al., Lancet 1 (1985) , 422-425; White D.G., et al., Lancet 1 (1985) , 419-421) .
  • the pathogen can be transmitted from mother to fetus causing hydrops fetalis and fetal death (Anand A. , et al., N. Engl. J. Med. 316 (1987) , 183-186; Brown T. , et al. , Lancet 2 (1984) , 1033-1034; Hall S.M., et al., BMJ 300 (1990) , 1166-1170) .
  • patients which shortened red cell survival compensated by accelerated erythropoiesis parvovirus may cause aplastic anemia (Pattison J.R., et al., Lancet 1 (1981) , 664-665) .
  • the technical problem underlying the present invention is to provide a novel assay and a kit for the diagnosis of infections caused by microbes, especially for determining the stage of an infection.
  • the present invention relates to a method for the diagnosis of infections caused by microbes which comprises comparing antibody reactivities towards antigenic determinants of at least two proteins derived from the microbes.
  • microbe includes microorganisms, e.g. bacteria or fungi, as well as viruses.
  • Figure 1 shows the results of an indirect IgG-EIA with human parvovirus capsid proteins. Capsids composed of VPl and VP2 (VPl/2) or VP2 alone (VP2) on polystyrene were treated with serum pools, collected on the indicated days, and bound IgG was measured.
  • Figure 2 shows the results of an IgG-EIA with chemically modified proteins: A, biotin-VP2 capsids on streptavidin; and B, VP2 capsids, C, VP1/2 capsids or D, ⁇ VPl protein on polystyrene.
  • the serum pools used are as in figure 1. The protein was treated prior to IgG binding.
  • Figure 3 shows immunoblots wherein the following proteins were used: A, ⁇ VPl protein; B VP1/2 capsids; C, VP2 capsids. Lanes 1 to 6 contain the same serum pools as in the previous figures, and lane 7 the control pools with no B19 antibodies. Numbers refer to molecular weights (kD) of marker proteins (M) . Arrows indicate migration positions of ⁇ VPl (140 kD) , VPl (83 kD) and VP2 (58 kD) .
  • Figure 4 shows an evaluation of the new diagnostic assays by serum pools.
  • Figure 5 shows an evaluation of the new diagnostic assay by individual follow-up samples.
  • the 2 circles indicate a serum pair with exceptionally high relative binding to linear-VP2 determinants in the first sample, leveling off in 14 days (sample interval) .
  • Past imm denotes past immunity.
  • the present invention relates to a method wherein one of the at least two proteins originates from a modification in the primary structure of the other(s) .
  • the present invention relates to a method wherein the at least two proteins have the same primary structure but different secondary or tertiary structure.
  • microbes are viruses.
  • proteins are capsid proteins in isolated form or in assembled capsids.
  • the two proteins are the denatured VP2 and VPl proteins of the human parvovirus B19 whereby the VPl protein can be the entire protein or the portion unique to the VPl protein.
  • the method of the present invention is based on an immunoassay which employs proteins derived from microbes and antibodies contained in sera or obtained according to conventional methods (see Ex. 1) .
  • immunoassays are well-known in the art (see, for example, "Immunoassay: A Practical guide", D.W. Chan and M.T. Perlstein eds. 1987) and include, for example, radioimmunoassay (RIA) , enzyme immunoadsorbent assay (EIA, ELISA) , Immunoblotting, complement fixation, immunodiffusion, immunoelectrophoretic or immunofluorescent assay and the like.
  • RIA radioimmunoassay
  • EIA enzyme immunoadsorbent assay
  • Immunoblotting complement fixation, immunodiffusion, immunoelectrophoretic or immunofluorescent assay and the like.
  • Suitable assays include both solid phase (heterogeneous) and non-solid phase (homogeneous) protocols.
  • the assays may be run using competitive or non-competitive formats, and using a wide variety of labels, such as radioisotopes, enzymes, fluorescers, chemiluminescers, spin labels, and the like.
  • a majority of suitable assays rely on heterogeneous protocols where the ligand is immobilized on a variety of solid phases, such as dipsticks, particulates, microspheres, magnetic particles, test tubes, microtiter wells, and the like.
  • the immunoassay is EIA or Immunoblotting.
  • the antigens used in the assays i.e. the capsids of parvovirus B19, consisting either of VPl and VP2 together in the approximate ratio 1:11 (VPl/2 capsids) , or of VP2 alone (VP2 capsids)
  • the capsids of parvovirus B19 consisting either of VPl and VP2 together in the approximate ratio 1:11 (VPl/2 capsids) , or of VP2 alone (VP2 capsids)
  • VP2 capsids can, for example, be produced in insect cells by the baculovirus expression system as described in Brown, C.S., et al., Virus Res. 15 (1990) , 197-212 or Brown C.S., et al., J. Virol. 65(5) (1991), 2702-2706.
  • human IgGs to parvovirus capsids consisting either of protein VPl and VP2 in the native proportion (VP1/2) or of VP2 alone (VP2) were directly coated onto plastic, e.g. polystyrene, and the responses were assessed using pools of sera.
  • the two proteins are the native and the denatured VP2 protein. Contrary to the directly immobilized antigen, the streptavidin-attached biotinylated VP2 capsids in native form showed very high immunoreactivity at all time points after infection ( Figure 2A) . In order to assess whether the difference in immunoreactivity was due to protein conformation, we exposed these capsids to protein denaturants followed by reducing and alkylating agents.
  • the VP2:VP2 absorbance ratios stayed above the threshold of 3.6 for 36/38 patients, whereas lower ratios (2.6 and 2.2) were obtained for 2 patients of 10 with acute EBV infection.
  • the mean ratio of the 38 acutely infected control patients was 11.7, with an SD of 5.4.
  • Another object of the present invention is to provide kits for the diagnosis of an infection caused by microbes by comparing antibody reactivity towards antigenic determinants of at least two proteins derived from the microbes.
  • the kit contains denatured VP2 and VPl proteins of the human parvovirus B19.
  • the kit contains the native and the denatured VP2 protein of the human parvovirus B19.
  • the VP2 protein is biotinylated.
  • the minor capsid protein VPl stabilizes the virus particle and, in general, is an important immunogen for neutralizing B19 immunity (Kurtzman G.J., et al., J. Clin. Invest. 84 (1989) , 1114-1123; Bansal G.P., et al. , J. Inf. Dis. 167 (1993) , 1034-1044) .
  • urea destroyed the conformational VP2 determinants irreversibly was unexpected , since its effect on protein antigenicity is usually reversible (Hedman K. , et al., J. Clin. Immunol. 8 (1988) , 214-221; Hedman K. , et al.
  • Sera Acute-phase serum samples were obtained from 61 symptomatic patients with B19 infection fulfilling strict diagnostic criteria: seroconversion or -4-fold rise in titer of B19-IgG and detectable B19 IgM antibodies (Soderlund M. , et al., J. Clin. Microbiol. 30(2) (1992) , 305-311) . All the infections were primary as verified by low avidity of IgG in the acute phase, with subsequent avidity maturation (Soderlund M. , et al., J. Inf. Dis. 171 (1995) , in press) . Characteristic symptoms were rash or arthralgia/arthritis, usually with a self-limiting course.
  • Pools of sera were created by combining equal volumes of 7-17 of the samples above at the following time points after onset: 0-7 days (a), 13-15 days (b) , 40- 100 days (c) , 150-250 days (d) and 300-400 days (e) .
  • Pool (f) comprised the past-immunity sera, whereas pool (g) was made up of the samples of 12 non-immune controls.
  • EIAs with chemically modified antigens The VPl/2 capsids or the ⁇ VPl antigen in PBS were applied onto polystyrene microtiter wells (Biohit, Helsinki, Finland) overnight, and the biotin-VP2 capsid on streptavidin-coated plates, for 30 minutes at 22°C.
  • the immobilized antigens underwent one of the following 30- minutes treatments at 37°C: 1) 0,5 M Tris-HCl pH 8,0
  • TB urea-TB
  • DTT dithiothreitol
  • IAA iodacetamide
  • the VP1/2 capsids (30 ng/well) or the VP2 capsids (20 ng/well) in PBS were separately adsorbed onto polystyrene microwell plates (Labsystems, Helsinki, Finland) overnight at 22°C.
  • the sensitized wells were washed 2 times, 5 min each, with 8 M urea in PBS and then 3 times 10 min each with PBST.
  • the serum samples (1:100 in PBST) were kept on the immobilized antigens for one hour at 37°C.
  • the PBST-washed wells were treated with alkaline phosphatase-conjugated anti-human IgG and substrate, 30 min. each.
  • the ratio of the VP1/2 and VP2 absorbances was plotted against time after onset of symptoms.
  • Example 4 Native versus denatured VP2 EIA
  • Biotin-VP2 capsids were first treated with 8 M urea in PBS for 2 hours at 4°C, followed by dialysis
  • biotin-VP2 capsids were suspended directly in PBST. Both antigens were separately mixed with sera (1:200) and applied onto streptavidin-coated microwells in a shaking incubator for 30 min at 22°C.
  • the antigens in reducing sample buffer (2% SDS, 5% ⁇ - mercaptoethanol, 10% glycerol, 62.5 mM Tris-HCl, pH 6.8) were heated 5 min at 95-100°C and run in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred electrophoretically to nitrocellulose filters (Schleicher and Schuell, Germany) according to standard techniques.
  • the filters were blocked with TEN-Tween (5 mM EDTA, 150 mM NaCl, 0.05% Tween-20 in 50 mM Tris-HCl, pH 7.4) for 45 min, and sera (1:100 in TEN-Tween) were applied for 1 hour.
  • Peroxidase-conjugated anti-human IgG was allowed to react 1 hour followed by the substrates (dia inobenzidine plus H 2 0 2 ) 5 min. (Soderlund M., et al., J. Clin. Microbiol. 30(2) (1992) , 305-311) .

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
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  • Virology (AREA)
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  • Proteomics, Peptides & Aminoacids (AREA)
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Abstract

L'invention concerne deux nouveaux tests permettant d'effectuer des diagnostics sur des patients. Avec le premier, on compare les réactivités d'IgG vis-à-vis d'épitopes de conformation et d'épitopes linéaires de VP2. Avec le second, on compare les réactivités de l'IgG vis-à-vis des antigènes dénaturés VP2 et VP1. Les deux tests conviennent à la vérification de l'état d'infection d'un humain par un parvovirus.
PCT/EP1996/000979 1995-03-08 1996-03-07 Procede pour diagnostiquer des infections WO1996027799A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP96907383A EP0813683A1 (fr) 1995-03-08 1996-03-07 Procede pour diagnostiquer des infections
AU51033/96A AU5103396A (en) 1995-03-08 1996-03-07 Method for the diagnosis of injections

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP95103356.2 1995-03-08
EP95103356 1995-03-08

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WO1996027799A1 true WO1996027799A1 (fr) 1996-09-12

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7094541B2 (en) 2001-08-31 2006-08-22 Gen-Probe Incorporated Assay for detection of human parvovirus B19 nucleic acid
WO2010099378A2 (fr) 2009-02-26 2010-09-02 Gen-Probe Incorporated Dosage de détection de l'acide nucléique du parvovirus humain
WO2013012708A1 (fr) 2011-07-15 2013-01-24 Gen-Probe Incorporated Compositions et procédés de détection de l'acide nucléique du parvovirus humain et de détection des acides nucléiques du virus de l'hépatite a dans des dosages monoplexes ou multiplexes

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991004330A1 (fr) * 1989-09-14 1991-04-04 Rijksuniversiteit Te Leiden Proteines du parvovirus b19 humain et particules d'aspect viral, leur production et leur utilisation dans les analyses diagnostiques et les vaccins
WO1991012269A1 (fr) * 1990-02-08 1991-08-22 Mikrogen Molekularbiologische Entwicklungs-Gmbh Peptides ou polypeptides immunologiquement actifs du parvovirus b19
EP0620231A1 (fr) * 1993-04-06 1994-10-19 Shin-Etsu Chemical Co., Ltd. Peptide se rapportant à un épitope de parvovirus B19 humain

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991004330A1 (fr) * 1989-09-14 1991-04-04 Rijksuniversiteit Te Leiden Proteines du parvovirus b19 humain et particules d'aspect viral, leur production et leur utilisation dans les analyses diagnostiques et les vaccins
WO1991012269A1 (fr) * 1990-02-08 1991-08-22 Mikrogen Molekularbiologische Entwicklungs-Gmbh Peptides ou polypeptides immunologiquement actifs du parvovirus b19
EP0620231A1 (fr) * 1993-04-06 1994-10-19 Shin-Etsu Chemical Co., Ltd. Peptide se rapportant à un épitope de parvovirus B19 humain

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
F. MORINET ET AL.: "Development of an IgM antibody capture test using labelled fusion protein as antigen for diagnosis of B19 human parvovirus infections", BEHRING INSTITUTE MITTEILUNGEN, vol. 85, August 1990 (1990-08-01), MARBURG DE, pages 28 - 34, XP000196154 *
G.J. KURTZMAN ET AL.: "Immune response to B19 parvovirus and antibody defect in persistent viral infection", JOURNAL OF CLINICAL INVESTIGATION, vol. 84, no. 4, October 1989 (1989-10-01), NEW YORK US, pages 1114 - 1123, XP000196152 *
K. YASHIMOTO ET AL.: "A second neutralizing epitope of B19 parvovirus implicates the spike region in the immune response", JOURNAL OF VIROLOGY, vol. 65, no. 12, December 1991 (1991-12-01), BALTIMORE US, pages 7056 - 7060, XP000196156 *
M. SÖDERLUND ET AL.: "Epitope type-specific IgG responses to capsid proteins VP1 and VP2 of human parvovirus B19", THE JOURNAL OF INFECTIOUS DISEASES, vol. 172, December 1995 (1995-12-01), CHICAGO US, pages 1431 - 1436, XP000196150 *
M. SÖDERLUND ET AL.: "Prokaryotic expression of a VP1 polypeptide antigen for diagnosis by a human parvovirus B19 antibody enzyme immunoassay", JOURNAL OF CLINICAL MICROBIOLOGY, vol. 30, no. 2, February 1992 (1992-02-01), WASHINGTON US, pages 305 - 311, XP000196091 *
M.M. SALIMANS ET AL.: "Recombinant parvovirus B19 capsids as a new substrate for detection of B19-specific IgG and IgM antibodies by an enzyme-linked immunosorbent assay", JOURNAL OF VIROLOGICAL METHODS, vol. 39, no. 3, September 1992 (1992-09-01), AMSTERDAM NL, pages 247 - 258, XP000196153 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7094541B2 (en) 2001-08-31 2006-08-22 Gen-Probe Incorporated Assay for detection of human parvovirus B19 nucleic acid
WO2010099378A2 (fr) 2009-02-26 2010-09-02 Gen-Probe Incorporated Dosage de détection de l'acide nucléique du parvovirus humain
EP3118208A1 (fr) 2009-02-26 2017-01-18 Gen-Probe Incorporated Dosage pour la détection de l'acide nucléique parvovirus humain
EP3257859A1 (fr) 2009-02-26 2017-12-20 Gen-Probe Incorporated Dosage pour la détection de l'acide nucléique de parvovirus humain
EP3705486A1 (fr) 2009-02-26 2020-09-09 Gen-Probe Incorporated Dosage pour la détection de l'acide nucléique de parvovirus humain
WO2013012708A1 (fr) 2011-07-15 2013-01-24 Gen-Probe Incorporated Compositions et procédés de détection de l'acide nucléique du parvovirus humain et de détection des acides nucléiques du virus de l'hépatite a dans des dosages monoplexes ou multiplexes
EP3009522A1 (fr) 2011-07-15 2016-04-20 Gen-Probe Incorporated Compositions et procédé pour détecter de l'acide nucléique du parvovirus humain et des acides nucléiques du virus de l'hépatite a dans des dosages multiplexes ou uniques
US11268159B2 (en) 2011-07-15 2022-03-08 Gen-Probe Incorporated Compositions and method for detecting hepatitis a virus nucleic acids in single-plex or multiplex assays

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AU5103396A (en) 1996-09-23

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