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WO1997026007A1 - Antigenes mycobacteriens et leurs procedes de detection - Google Patents

Antigenes mycobacteriens et leurs procedes de detection Download PDF

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Publication number
WO1997026007A1
WO1997026007A1 PCT/US1997/000754 US9700754W WO9726007A1 WO 1997026007 A1 WO1997026007 A1 WO 1997026007A1 US 9700754 W US9700754 W US 9700754W WO 9726007 A1 WO9726007 A1 WO 9726007A1
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antigen
kda
antibodies
antigens
patients
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PCT/US1997/000754
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English (en)
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Matthew J. Fenton
John Bernardo
Jawahar L. Raina
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Trustees Of Boston University
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Priority to AU17501/97A priority Critical patent/AU1750197A/en
Publication of WO1997026007A1 publication Critical patent/WO1997026007A1/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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/04—Mycobacterium, e.g. Mycobacterium tuberculosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/35—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
    • C07K16/1289—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Mycobacteriaceae (F)

Definitions

  • This invention relates to two mycobactenal antigens, the significance of which is reported herein for the first time. Serum from patients with active tuberculosis has been found to contain antibodies specific for these antigens, but the titer of these antibodies changes in patients whose disease is no longer active. Therefore, this invention is directed to assays for detection of active tuberculosis, based on determination of antibodies specific for these antigens in patient samples.
  • Tuberculosis is responsible for more deaths worldwide each year than any other single infectious disease.
  • TB Tuberculosis
  • a critical component of any successful TB control program is the availability of a rapid and low cost method for identifying persons with active TB disease so that they may be treated and reduce future spread.
  • Diagnosis of TB is usually based on clinical suspicion and demonstration of Mycobacterium tuberculosis (Mtb) in sputum samples utilizing procedures that may take up to 8 weeks. Delays in establishing a diagnosis add to costs of care by necessitating isolation of patients and often further complicate management by delaying initiation of appropriate treatment and allowing further spread of disease.
  • Mtb Mycobacterium tuberculosis
  • the other conventional diagnostic test diagnostic skin testing using purified protein derivative (PPD, a crude extract derived from heat- killed Mtb), is indicative of previous infection, but cannot discriminate between infection and active disease.
  • PPD purified protein derivative
  • the skin test is also hampered by a lack of sensitivity and specificity, especially in immunocompromised individuals. In many cases, persons with skin reactivity to PPD and radiological evidence of pulmonary lesions are placed into isolation and treated with antimicrobial drugs while awaiting the demonstration of Mtb in sputum samples. Thus, there is a need for an inexpensive diagnostic test for active TB which is rapid and selective.
  • Mycobactenal antigens Although TB is predominantly controlled by cell-mediated immunity and delayed-type hypersensitivity responses, there is substantial activation of B lymphocytes by mycobacterial antigens (reviewed in Collins, 1994, Vet. Microbiol, 40:95). Initial identification of irnmunodominant antigens was based on serological studies using antisera from TB patients. There has been steady progress in identifying and characterizing several classes of molecules from Mtb that may function as key protective immunity-inducing antigens.
  • Mtb possesses a complex mixture of antigenic determinants which include of proteins, carbohydrates, lipids, and nucleic acids.
  • Much effort has been directed into the identification of Mtb protein antigens, which have been described primarily based upon their relative molecular weights. These include proteins of 65 kDa (Shinnick, 1987, J. Bacteriol., 169: 1080), 32 kDa (Borremans, et al., 1989, Infect andlmmun., 57: 3123), 38 kDa (Chandramuki, et al., 1989, J. Clin.
  • Microbiol., 27: 821 as well as proteins of 10 kDa, 16 kDa, 24 kDa, and 30 kDa (Torres, et al., 1994, Clin. Exp. Immunol., 96: 75).
  • the functions of most Mtb proteins have not been identified to date, although some have been shown to serve as heat shock proteins, based on their homology to eukaryotic heat shock proteins (Garcia, et al., 1989, Infect, andlmmun., 57: 204). Efforts to develop vaccines have focused on the identification of mycobacterial antigens recognized by T cells.
  • T cell antigens are predominantly proteins
  • B cell antigens can be proteins, carbohydrates, lipids, and nucleic acids.
  • antigenic epitopes recognized by T cells are typically chemical in nature
  • antigenic epitopes recognized by antibodies can be either chemical or conformational in nature.
  • carbohydrate antigens the epitopes are predominantly chemical in nature, although the presence of repeating sugar moieties usually enhances the antigenicity of the molecule.
  • Mtb possesses antigenic determinants that are shared by nontuberculous environmental mycobacteria, and exposure to these organisms can generate a false positive PPD skin test. Some of these shared antigens have been identified as mycobacterial stress proteins, including a variety of heat shock proteins. Evidence for the ability of heat shock proteins to serve as antigens was provided by the observation that a large number of T cell clones react with a 65 kDa mycobacterial heat shock protein (HSP-65). Approximately 20% of T cell clones generated after immunization of mice with Mtb were found to react with HSP-65 (Kaufmann, et al., 1987, Eur. J. Immunol., 17:351).
  • HSP-65 is not a major target of the early protective immune response to Mtb (Orme, et al., 1993, J. Infect. Dis., 167: 1481).
  • a mycobacterial product that is a potent B cell antigen is the cell wall glycolipid lipoarabinomannan (LAM).
  • LAM is a complex glycolipid containing multiple linear and branched arabinose and mannose groups linked to a phosphatidylinositol unit at its reducing end.
  • the phosphatidylinositol moiety of LAM has been proposed to play a role in "anchoring" LAM to the membrane (Hunter, et al., 1986, J. Biol. Chem., 261: 12345, Hunter, et al., 1990, J Biol. Chem., 265: 9272).
  • LAM has been shown to be secreted from macrophages infected with Mtb in vitro (Sturgill-Koszycki, et al., 1994, Science, 263:678), and the presence of anti-LAM antibodies in sera from patients with active TB suggests that LAM is also released from infected macrophages in vivo ( Sada, et al., 1990, J. Clin. Microbiol., 2:2587). In addition to its antigenicity, LAM has been shown to have multiple effects on the immune system, including suppression of T cell activation (Kaplan, et al., 1987, J. Immunol, 138: 3028), and induction of the release of tumor necrosis factor (Moreno, et al., 1989, Clin. and Exper. Immunol., 76: 240).
  • Serodiagnostic assays using cruder protein fractions as antigen such as the A-60 fraction of PPD (Daftary, et al., 1995, Indian J. Med. Sci., 48:39) and a 55-67 kDa phospholipid-associated protein fraction from Mtb (Kaushik, et al., 1993, Med Microbiol. Immunol. , 182:317), have been reported to give greater than 90% sensitivity. Sensitivities using a variety of purified mycobacterial protein antigens (MW 10 kDa, 16 kDa, 24 kDa, 30 kDa, 38 kDa, and 70 kDa) have demonstrated lower sensitivity, with values reported to range between 29-51%.
  • Detection of antibodies against the non-protein antigens have also been used as the basis for serodiagnostic assays.
  • Assays based on LAM or cord factor (trehalose-6,6'-dimycolate) have demonstrated a range of sensitivities from 70-95% (Sada, et al., 1990, J. Clin. Microbiol., 2: 2587; (Park, et al., 1993, Tubercule Lung Dis., 74:317; Maekura, et al., 1993, Amer. Rev. Resp. Dis., 148:997).
  • This invention provides a purified antigen having the characteristics of a high molecular weight antigen obtained from the aqueous extract of autoclaved cells oi Mycobacterium sp. by digestion of the extract with proteinase K and collection of an ultrafiltrate residue from the digested extract by ultrafiltration through an ultrafiltration membrane, the high molecular weight antigen being retained in the ultrafiltrate residue, the purified antigen being immunoreactive with antibodies from patients suffering from active disease caused by said Mycobacterium sp.
  • the ultrafiltration membrane is a 20 kDa cutoff membrane, more preferably a 30 kDa cutoff membrane.
  • this invention provides a method for obtaining an antigen reactive with antibodies present in patients suffering from active tuberculosis.
  • the method comprises the steps of obtaining an extract from cells of Mycobacterium sp., digesting the extract with proteinase K, and ultrafiltering the digested extract using a 30 kDa cutoff membrane, the antigen being retained in the ultrafiltration residue.
  • the Mycobacterium sp. isM avium o ⁇ M. bovis; more preferably, the Mycobacterium sp. is M. tuberculosis.
  • this invention provides a purified antigen having the characteristics of an antigen of 40 kDa apparent molecular weight measured by SDS-PAGE, obtained from an extract of autoclaved cells of Mycobacterium tuberculosis by digestion of the extract with proteinase K and collection of a residue from the digested extract by ultrafiltration using a 30 kDa cutoff membrane, the antigen of about 40 kDa apparent molecular weight being retained by the ultrafiltration membrane, the purified antigen being immunoreactive with antibodies from patients suffering from active tuberculosis.
  • this invention provides a purified antigen immunoreactive with antibodies from patients suffering from active tuberculosis, which antigen is released from autoclaved cells of Mycobacterium tuberculosis, is resistant to digestion by proteinase K, as determined by retention of the antigen by a 30 kDa cutoff ultrafiltration membrane, is resistant to alkanolysis by 0.1 N NaOH, as measured by antigenicity, has an apparent molecular weight of about 40 kDa by SDS PAGE, can be stained with periodate-Schiff reagent, but not ethidium bromide, and is not soluble in either phenol or chloroform.
  • this invention additionally provides a diagnostic method for determining the presence of active disease caused by mycobacteria in a patient comprising detecting, in a sample from the patient, antibodies specifically immunoreactive with an antigen having the characteristics of an antigen according to this invention.
  • This invention additionally provides a diagnostic method for determining the presence of active tuberculosis in a patient comprising detecting, in a sample from the patient, antibodies specifically immunoreactive with an antigen having the characteristics of an antigen according to these embodiments.
  • this invention provides a diagnostic kit for detecting active tuberculosis, comprising an antigen according to this invention and reagent means for detecting antibody binding to said antigen.
  • the antigen in the kit is immobilized on a solid support.
  • this invention provides a method of eliciting an immune response in a mammal, comprising injecting an antigen according to this invention into the mammal.
  • a serodiagnostic test has been developed which detects the presence of serum antibodies against a novel, protease-resistant cell- associated antigen with an apparent MW by SDS PAGE of about 40 kDa (hereinafter termed "the 40 kDa antigen"), which is expressed by Mtb.
  • the 40 kDa antigen The overwhelming majority of sera obtained from patients with active TB contained antibodies against this 40 kDa antigen and a co-purifying antigen with an apparent molecular weight by SDS PAGE of about 6 kDa (hereinafter termed "the 6 kDa antigen”).
  • Sera from healthy and BCG- vaccinated persons generally did not contain antibodies which recognized the 40 kDa or 6 kDa antigens.
  • a TB serodiagnostic kit based on the 40 kDa antigen can be used by untrained personnel to screen large numbers of samples outside of a hospital setting, because the chemical features of this antigen indicate that such a kit would not require special equipment or storage conditions.
  • a serodiagnostic test based on the 6 kDa antigen can provide a clinical marker of previous disease.
  • Figure 1 shows Western blots of Mtb antigen and purified LAM developed with a monoclonal antibody specific for LAM.
  • Figure 2 shows the effect of mild alkaline treatment on the antigenicity of LAM and Mtb antigen.
  • Figure 3 shows Western blots of Mtb antigen and LAM developed with TB-positive serum.
  • Figure 4 shows Western blots of Mtb antigen and LAM developed with serum from a TB patient post-treatment.
  • Figure 5 shows Western blots of Mtb antigen and LAM developed with serum from another post-treatment TB patient.
  • Figure 6 shows dot blot assays using normal and TB sera collected from various donors.
  • Figure 7 shows dot blot assays using secondary antibodies specific for human IgG and IgM.
  • TB skin tests involve intracutaneous injection of PPD to detect cell mediated immunity manifested by delayed-type hypersensitivity reactions.
  • Dermal sensitivity to PPD fails to discriminate between persons with active disease, healthy persons who have been previously infected with Mtb but do not have active disease, and many persons who have been vaccinated with BCG.
  • many TB patients, whether or not HIV-infected fail to generate a positive skin reaction to PPD.
  • T cells from healthy individuals living in the same household with TB patients show stronger recognition responses to mycobacterial antigenic peptides than do T cells from the patients, although the opposite trend characterizes their antibody and B cell responses to these antigens (Falla, et al., 1991, Infect. Immun., 59:2265).
  • the antigen or antigens in PPD responsible for recognition of TB infection is/are unknown.
  • living Mtb play an active role in modulating host immune responses, it is likely that long-term protective immunity is generated by secreted mycobacterial antigens produced by phagocytosed viable Mtb.
  • Selection of appropriate antigen(s) is the key to developing a sensitive and specific assay that is diagnostic for active TB disease based on detecting the presence of specific antibodies in serum.
  • Initial efforts in identifying novel mycobacterial antigens were based on the premise that TB patients can generate humoral responses against non-protein antigens.
  • the present invention is based on the discovery that antibodies present in the serum of patients with active TB (as opposed to TB infected persons with no disease or uriinfected persons) recognize a cell-associated, heat-stable, protease-resistant antigen present in Mtb, having an apparent MW of 40 kDa. Studies show that over 90% of sera from TB patients tested contained either IgG and/or IgM antibodies against this antigen. Serum from patients infected with M.
  • avium (MAI serum), a mycobacterial pathogen often seen in patients with AIDS, also contained antibodies which recognized the 40 kDa antigen. Concurrent infection with HIV did not necessarily eliminate the ability of either TB or MAI patients to raise a humoral response against the antigen, although the sensitivity of diagnostic tests based on this antigen appeared lower in HIV-infected individuals. Sera from normal (PPD negative) and BCG-vaccinated persons did not contain antibodies which recognized this antigen.
  • a patient is an animal infected with or suspected of infection with a mycobacterium, particularly an animal suffering from mycobacterial disease.
  • a human patient suffering from a mycobacterial disease will exhibit clinical symptoms associated with that disease.
  • This active disease state may be differentiated from mere infection by mycobacteria, which may or may not result in clinically recognizable disease state, for example as discussed above concerning PPD-positive individuals who have never exhibited active TB.
  • Patients may include human adults or children, either hospitalized and unhospitalized individuals, including infants.
  • Animal patients include mammals, such as goats, pigs, sheep, horses, cats, dogs, alpacas, non-human primates, rabbits, and especially cattle, elk, deer, bison, camels or llamas.
  • Antigens described herein may be obtained from the crude soluble fraction of autoclaved Mtb cultures. Autoclaved cultures are centrifuged to remove insoluble debris, and the supernatant is digested with Proteinase K. The crude antigen preparation may be further fractionated by SDS-PAGE, ion exchange chromatography, and/or fractional precipitation. Protease treatment is effective at eliminating most of the mycobacterial proteins, as judged by SDS-PAGE and staining with Coomassie Blue or silver nitrate. The antigen preparation was determined not to contain DNA or RNA based on the absence of an absorbance peak at 260 nm and the inability to stain with ethidium bromide, but the antigens gave positive results in various carbohydrate assays.
  • Extracts of autoclaved Mtb were prepared from two Mtb strains, H37Ra (an avirulent strain in humans) and H37Rv (a human virulent strain), and analyzed by SDS-PAGE. Identical SDS-PAGE gels were prepared, and one gel was stained for protein with Coomassie blue, while the other gel was electrophoretically transferred onto nitrocellulose membranes. The SDS-PAGE protein profiles generated from each Mtb extract were virtually identical.
  • the 40 kDa antigenic determinant does not represent a major fraction ofthe Coomassie blue-stainable material in extracts prepared from autoclaved Mtb cultures.
  • Crude Mtb extracts were digested with Proteinase K (1 U/ml) for 15-60 min at 55 °C, concentrated 10-20 fold by ultrafiltration using a 30 kDa cut-off membrane, and then analyzed by immunoblotting.
  • Low molecular weight Proteinase K-digested contaminants were removed by repeated dilution and ultrafiltration/concentration steps, whereas high molecular weight contaminants were insignificant as determined by SDS- PAGE analysis of the antigen concentrates.
  • Immunoblot analysis of the Proteinase K-digested antigen preparations demonstrated that antibodies in the sera from TB patients still recognized a molecule with an apparent molecular size of 40 kDa, as judged by SDS-PAGE.
  • the 40 kDa antigen does not appear to contain protein material susceptible to proteinase K.
  • non-protein antigens such as carbohydrates as the basis for a serodiagnostic assay.
  • One advantage is that immunoreactivity of carbohydrates is defined by a linear arrangement of sugar groups, whereas the immunoreactivity of many protein epitopes is determined by the conformational arrangement of amino acids which are in proximity only due to folding of the polypeptide chain. While the conformational epitopes on protein antigens are destroyed by heat denaturation, the antigenicity of non-protein molecules is not heat labile.
  • autoclaved cultures of virulent Mtb bacilli could be safely and easily used as a crude source of non-protein antigens. Demonstration that the antigenicity of the 40 kDa antigen survives autoclaving and proteinase K digestion, suggests that this antigen may offer the stability desirable in serodiagnostic assays.
  • carbohydrate antigens are generally more potent B cell stimuli compared with protein antigens. This is most likely due to the repeating chemical structure of carbohydrates which can effectively crosslink Ig molecules on the B cell surface, thus generating a stronger activation signal than that which would be generated by proteins which would be unable to crosslink multiple surface Ig molecules.
  • Supematants of autoclaved Mtb cultures digested with Proteinase K (I U/ml) in the presence of 0.1% SDS at 55 C° for 18 hr produce a crude fraction which can be concentrated by ultrafiltration in an Amicon positive pressure concentrator using 30 kDa-cutoff membranes.
  • the lysate retained by the 30 kDa-cutoff membrane can be dialyzed in phosphate buffered saline (PBS) using 30 kDa-cutoff membranes and stored at 4 C°.
  • PBS phosphate buffered saline
  • This ultrafiltration method retains antigenic material that appears at about 6 kDa as well as 40 kDa on Western blots developed with TB-positive serum. Attempts to separate the material in these two bands by other size-based separations were not successful. Both antigens are retained by 30 kDa and 50 kDa cut-off dialysis membranes, with or without the addition of denaturants. On a molecular sizing HPLC column in non- denaturing buffer, the antigens traveled with the void peak having over 100 kDa apparent molecular weight. Together the results from Western Blots and HPLC columns suggest that in the absence of a denaturant the 40 kDa and 6 kDa antigens are complexed into large macromolecular aggregates.
  • the apparent molecular weight of the 6 kDa antigen may simply reflect the aberrant mobility of carbohydrates on SDS-PAGE, and the true molecular size ofthis antigen may be greater.
  • Crude antigen preparations (not fractionated on DEAE-Sephadex) contain contaminating protein (especially at approximately 65 and 10 kDa, as judged by silver stained SDS gels) and nucleic acid (as judged by absorbance at 260 nm).
  • DEAE-purified antigen preparations are free of contaminating protein (as judged by silver-stained SDS-PAGE gels and absorbance at 280 nm) and nucleic acids (as judged by ethidium bromide-stained gels and absorbance at 260 nm). Furthermore, the antigenicity of the both 40 kDa and 6 kDa antigens is unaffected by RNAse A or DNAse I treatment.
  • a sugar-specific staining procedure was used to determine if the 40 kDa antigen contained, or consisted entirely of, carbohydrate.
  • Purified antigen prepared by crystallization as described below
  • Gels stained by this method revealed a major band at 40 kDa, indicating that the antigen is, or contains, carbohydrate.
  • the Western blotting showed a broad band of immune reactivity in the region of 30-40 kDa, suggesting the presence of multiple, closely-spaced bands; such a pattem is frequently observed with carbohydrate antigens.
  • the 40 kDa and 6 kDa antigens both are bound by the lectin concanavalin A (Con A), like arabinomannan and LAM, but unlike arabinogalactan.
  • Con A concanavalin A
  • the antigenicity of the 40 kDa and 6 kDa antigens is destroyed by periodic acid treatment. Both antigens are lysozyme and mannosidase resistant.
  • the 40 kDa and 6 kDa antigens are freely soluble in aqueous buffers, unlike cord factor and acylated trehaloses. Cord factor and acylated trehaloses do not bind to diethylaminoethyl groups (DEAE), in contrast to the antigens of this invention.
  • DEAE diethylaminoethyl groups
  • the antigens of this invention are released from a DEAE-Sephadex column at > 500 mM NaCl, unlike LAM or deacylated LAM (chemically similar to arabinomannan) which eluted at ⁇ 100 mM NaCl.
  • LAM deacylated LAM
  • the antigens are acidic polysaccharides, unlike LAM or arabinomannan which are neutral polysaccharides.
  • mycobacterial carbohydrate antigens, such as LAM also contain lipids.
  • the 40 kDa and 6 kDa antigens are not extractable in phenol or chloroform, unlike acylated trehaloses and cord factor, although the 40 kDa antigen can be selectively precipitated by 2 volumes of isopropanol.
  • the antigenicity and mobility of the 40 kDa antigen and 6 kDa antigen on SDS-PAGE are essentially unaffected by mild alkalinolysis, unlike LAM, cord factor, or acylated trehaloses.
  • Limited NaOH treatment 0.1 N NaOH, 2 hr, 37° C
  • This treatment failed to alter either the antigenicity or mobility of the antigen on SDS-PAGE and suggests that the 40 kDa antigen is not an ester-linked glycolipid.
  • LAM was sensitive to NaOH treatment (Hunter, et al., 1986, J. Biol. Chem., 261 : 12345), thus further distmguishing the 40 kDa antigen from LAM.
  • the 40 kDa and 6 kDa antigens are recognized by both serum IgG and IgM in serum from patients with active TB. In patients who previously had TB and do not have active disease, serum antibodies can be found against the 6 kDa antigen, but not the 40 kDa antigen. An additional antigen is observed at 15-20 kDa using sera from some patients or cows, or when using Protein G is used instead of anti-IgG and -IgM reagents to detect bound serum antibodies.
  • This 20 kDa antigen like the 40 kDa antigen is both eluted form DEAE Sephadex at > 500 mM NaCl and precipitated by 2 vol. isopropanol. However, the 20 kDa antigen does not bind well to Concanavalin A.
  • the inventors have found that a monoclonal antibody against LAM (CS-35) did not recognize either the 40 kDa or 6 kDa Mtb antigens, although sera from active TB patients recognized both LAM and the 40 kDa antigen. These data demonstrate that the 40 kDa antigen is antigenically distinct from LAM. Using immobilized 40 kDa Mtb antigen, the isotype of antigen- bound serum antibodies was determined. Enzyme-conjugated secondary antibodies which were specific for either human IgM or IgG were used to determine which isotypes were responsible for the reactivity in human sera.
  • Example 6 describes the results of dot blots using dilutions of the 40 kDa Mtb antigen applied to a membrane.
  • Membranes were incubated with either TB patient (TB) or normal (N) sera, and bound human serum antibodies were detected using enzyme-conjugated secondary antibodies (2nd Ab) specific for human IgM (M) or IgG (G).
  • 2nd Ab enzyme-conjugated secondary antibodies
  • M human IgM
  • IgG IgG
  • the 40 kDa antigen was recognized by both IgM and IgG antibodies present in the sera of TB patients.
  • sera contained only IgM antibodies which recognized the antigen (see Figure 9). Because early humoral responses are dominated by an IgM response, this observation may reflect the history of TB in a patient. For example, primary TB may preferentially elicit an IgM response whereas reactivation TB may elicit both and IgM and an IgG response.
  • Antigen maybe extracted from cells of Mycobacteria sp., such as M. avium, M. bovis, M. chelonei, M. fortuitum, M. gastris, M. intracellulare, M. kansasii, M. leprae, M. nonchromogenicum, M. phei, M. simiae, M. smegmatis, M. tuberculosis, and M. ulcerans. While antigen extracted from Mtb is described herein as representative ofthis invention, using other mycobacteria may be preferable, because Mtb cultures should be maintained in a biosafety level 3 (P-3) laboratory facility.
  • P-3 biosafety level 3
  • Mtb or other mycobacteria can be grown according to standard procedures for mycobacteria.
  • Mycobacteria cultures for preparation of the antigen of this invention may be harvested at any stage of growth, but late log phase or stationary phase is preferred for recovery of larger amounts of cellular material.
  • the antigen disclosed herein is generally is not released into the culture medium by logarithmically growing cells, but rather must be extracted from the cellular mass. Extraction is generally into an aqueous buffer, and may be facilitated by freeze-thaw cycling, sonication, refluxing in alcohol, or other known cell lytic procedures.
  • the antigen may be prepared from autoclaved Mtb cells. Usually culture media containing Mtb cells is autoclaved without separation.
  • This source is preferred to attempting extraction of the antigen from viable Mtb; the use of autoclaved material minimizes the health risk during preparation. Late-log phase Mtb cultures can autoclaved, and the bacilli removed by centrifugation. The desired antigenic activity was not found in medium recovered from viable bacterial cultures, but 40 kDa antigen is released into the culture medium following autoclaving. Additional antigenic material was obtained by reextraction of the cellular pellet, suggesting that the antigen is cell wall associated.
  • antigen prepared by extraction from autoclaved M. tuberculosis cells will be recognized by sera from TB patients, further purification ofthe antigen is prefened to increase specificity.
  • the proteinase is proteinase K, which may be added at lOO ⁇ g/ml to provide an excess, and digestion is preferably carried out in the presence of 0.1% SDS.
  • Bulk protein may altematively be removed from the lysate by digestion with one or more other proteases, so long as substantial protein degradation occurs. Modification of digestion conditions to accommodate protease substitutions is within the skill of the art. Components resistant to digestion can be size-selected by dialysis and concentrated by ultrafiltration. Degraded peptide fragments can be removed by ultrafiltration; the antigen will be retained by a 30 kDa cutoff membrane after digestion with proteinase K. Concentrated preparation ofthe 40 kDa and 6 kDa antigens can then be filtered and stored at 4°C.
  • the concentrated antigen may be further purified using ion exchange chromatography.
  • antigen preparation may be applied to a DEAE column equilibrated in 10 mM Tris-HCl (pH 7.4), 0.5% Triton X-100, 1 mM EDTA. The column will then be washed extensively and eluted using a 0-500 mM linear NaCl gradient. The eluted fractions should be collected, tested for immunoreactivity (for example using a dot blot assay as described herein), and pooled. Pooled samples may be dialyzed and concentrated. The antigens may be quantitated based on carbohydrate content of the fractions using the phenol-sulfuric acid colorimetric assay. Additional purification may be achieved by reverse-phase HPLC using, for example, a C-18 HPLC column.
  • the concentrated material from the Proteinase K digestion may be dialyzed into buffer containing a denaturant, such as 4M urea or l%Triton-X100 and applied to an anion exchange column, such as a DEAE column.
  • a denaturant such as 4M urea or l%Triton-X100
  • the concentrated proteinase K digest typically contains lipid material which may interfere with column flow. This contaminating lipid may be removed by extraction with organic solvents or saponification by NaOH followed by extraction of the unsaponified material into chloroform before applying the antigenic concentrate to the column. Contaminating protein can be removed by washing the column with buffer plus denaturant, the wash preferably having moderate ionic strength, such as about 300 mM NaCl.
  • the desired antigens are eluted from the washed column by increasing the ionic strength to greater than 500 mM NaCl, preferably at least about 800 mM.
  • the antigen may be purified by fractionating the cellular extract by ion exchange chromatography before Proteinase K digestion.
  • the antigen may be purified by immuno-affinity methods using antibodies specific for the antigen. Preferably, irnmuno-affinity methods, such as affinity chromatography, will use purified polyclonal or monoclonal antibodies prepared as described herein.
  • the mixture of 40 kDa and 6 kDa antigens can be fractionated by selectively precipitating the 40 kDa antigen with a polar organic solvent, such as isopropanol.
  • the 40 kDa antigen may also be separated from the 6 kDa antigen by treating the proteinase K digest with a polar solvent, such as ethanol, isopropyl alcohol, butyl alcohol, acetone, etc. Purification of the 40 kDa antigen by precipitation with a polar solvent is generally less satisfactory if attempted on crude extracts before the bulk protein is removed by Proteinase K digestion and/or column chromatography.
  • the antigenic material which survives proteinase K digestion is surprisingly stable. Little or no loss of antigenic activity is observed at 37 °C for 1 day, and this extrapolates to approximately 3 months of stability when stored at 4°C.
  • Prediluted (ready-to-use form) secondary antibody conjugates are stable for at least one year at 4°C.
  • Serum samples obtained from patients with active TB (either HIV+ or HIV-), and serum samples from patients infected with M. avium, which were tested to determine if they contained antibodies which recognized the 40 kDa antigen.
  • Samples from healthy PPD negative, healthy PPD-positive, and BCG-vaccinated persons were also tested. Representative examples ofthe results obtained using the dot blot assay and normal and TB sera collected from various donors are shown in Example 6. The results indicate that a serodiagnostic assay using the 40 kDa antigen is highly sensitive and specific for active mycobacterial disease state. The sensitivity of this assay appears to be reduced by co-infection with HIV.
  • the 6 kDa antigen described herein is also associated with the TB disease state, but not limited to sera from patients with active disease. Rather, reactivity of a patient's serum with the 6 kDa antigen reflects a history of active TB, with antibodies to the 6 kDa antigen being found in sera from both active and recovered TB patients. On the other hand, sera from PPD-positive individuals with no history of active TB (such as BCG- vaccinated individuals) do not react with the 6 kDa antigen. Thus, the 6 kDa antigen provides a clinical marker of previous disease.
  • Comparison of serum reactivity with the 40 kDa antigen to reactivity with the 6 kDa antigen can be useful diagnostically in ruling out active TB in an individual with TB-like symptoms, including abnormal chest X-ray, lung scarring, etc.
  • Detection of antibodies immunoreactive with the antigens of this invention may be accomplished using techniques that are well known to those skilled in the art. These antibodies can detected by a variety of immunometric assay techniques.
  • the assays of the present invention can be directly used to detect antibodies which are specifically immuno-reactive with the 40 kDa and/or the 6 kDa antigens ofthis invention.
  • a particularly useful stain employs peroxidase, hydrogen peroxide and a chromogenic substance such as aminoethyl carbazole.
  • the peroxidase (a well known enzyme available from many sources) can be directly coupled to an anti-human IgG or IgM or complexed via one or more antibodies to an antibody which specifically binds the 40 kDa antigen.
  • a goat anti-peroxidase antibody and a goat anti-human antibody can be complexed via an anti-goat IgG.
  • Other chromogenic substances and enzymes may also be used. Radiolabeling of antibodies may also be used to detect antibody binding. Labeled antibodies may be immunoreactive with anti-human IgG or IgM. Again, such techniques are well known.
  • Solution assay methods including calorimetric, chemiluminescent or fluorescent immunoassays such as ELISA, sandwich and competitive immunoassays, immuno-diffusion, radio immunoassay, immunoassay, Western blot and other techniques, may be used to detect and quantitate the antibodies from a patient by assaying any sample from the patient that contains antibodies.
  • Antibodies may be quantitated in a biological fluid, such as whole blood, serum, plasma, effusions, ascites, urine, cerebrospinal fluid, and bronchoalveolar lavage fluid using any immunologic detection means known in the art.
  • Preferred methods employ immunological detection in samples which include serum.
  • Prefened assay techniques include: radioimmunoassay, enzyme linked immunoadsorbent assay, complement fixation, nephelometric assay, immunodiffusion or immunoelectrophoretic assay and the like.
  • Cellular elements and lipid may be removed from fluids, e.g., by centrifugation.
  • dilute fluids such as urine or bronchoalveolar lavage fluid, antibodies may be concentrated, e.g., by ultra-filtration or salting-out.
  • Preferred assay formats are described below.
  • partially-purified or completely purified 40 kDa and/or 6 kDa Mtb antigens are applied in five serial 2-fold dilutions to strips of nylon membranes (0.7 cm x 5 cm) using high precision ceramic micropumps which are capable of delivering 0. l-1.0 ⁇ l volume per stroke with a maximum of 5% variability.
  • Purified human IgG 1 ⁇ g/dot is applied on the membrane strip as a positive control for the validity of the test reagents.
  • the membrane dot blot strips are air-dried, blocked with a blocking buffer of PBST (PBS with 0.05% Tween-20) and 5% normal rabbit serum, and then dried at 40 °C for 1 hr.
  • PBST PBS with 0.05% Tween-20
  • control or TB- positive sera (1 : 25 dilution) is applied to the strips, incubated for 10 minutes at room temperature, and washed free of non-specifically bound antibodies.
  • a detection antibody e.g., an alkaline phosphatase-linked goat polyclonal antibody recognizing human IgG and/or IgM
  • the appropriate substrate e.g. BCIP/NBT; Kierkegaard & Perry
  • dot blots are scored visually.
  • these antigens may be separated as described herein, and individually applied to membrane strips. Western blotting.
  • the partially-purified Mtb antigen mixture are separated on SDS-PAGE under standard conditions and blotted to nylon membranes.
  • purified LAM preparations may also separated on gels for comparative purposes. Membranes are cut into several identical strips, blocked with PBST containing 5% rabbit serum, and processed with TB patient sera analogous to the method used for dot blots.
  • the wells of 96 well microtiter plates are coated with partially purified 40 kDa and 6 kDa antigens diluted in 100 ⁇ l of PBS, incubated at room temperature for 60 minutes, and washed with PBST to remove unbound antigen. Wells are then blocked with solution of PBST with 1% BSA for 1 hr at room temperature, washed again, and dried for 1 hr at 40 °C. Wells not receiving antigen are used as a negative control, and wells receiving human IgG will serve as positive controls. For each assay, normal human or TB-positive serum (1 :25 dilution) are added, incubated for 60 minutes at room temperature, then washed to remove unbound antibodies.
  • Samples are processed in duplicate.
  • Peroxidase- conjugated goat anti-human Ig (recognizing IgG and/or IgM) is used to detect the bound immune complexes, and an appropriate substrate (e.g., OPD) is added for color development.
  • ELISA plates are read at 450 nm. ELISA assay tests are scored as positive where absorbance reading is greater than a calculated cutoff value (e.g. 0. 10 absorbance units above the mean of several analyzed normal human sera). For subsequent analysis of reactivity with individual 40 kDa and 6 kDa antigens, these antigens may be separated as described herein, and individually appUed to the microliter plate wells.
  • immunoassays of this invention measure binding of antisera to antigens purified by anion exchange chromatyraphy, or to antigenic material of comparable purity.
  • Specific binding moieties which, as used herein, refer to molecules capable of binding to the 40 kDa antigen with high specificity, as for example an antibody specific for the 40 kDa antigen.
  • Specific binding moieties may include whole immunoglobulin G (IgG) antibodies made up of four immunoglobulin peptide chains, two heavy chains and two light chains and immunoglobulin M (IgM), as well as immunoglobulin fragments, which are protein molecules related to antibodies and which retain the epitopic binding specificity of the original antibody, such as Fab, F(ab)' 2 , Fv, etc.
  • IgG immunoglobulin G
  • IgM immunoglobulin M
  • Specific binding moieties for the 40 kDa antigen also include single chain antibodies and recombinant peptides constructed to retain the paratope configuration of antibodies specific for the 40 kDa antigen, as well as other molecules constructed to specifically bind the antigen.
  • Antibodies which are specifically reactive with the antigen of this invention may be obtained in a number of ways which will be readily apparent to those skilled in the art.
  • the antigen obtained as described above can be injected into an animal as an immunogen to elicit polyclonal antibody production. Purification ofthe antibodies can be accomplished by selective binding of antibodies from the serum ofthe immunized animal to the 40 kDa antigen purified as described herein.
  • antibodies which specifically bind to this antigen can be isolated (e.g., from serum of humans with active TB) by binding to immobilized 40 kDa antigen and subsequently eluted from the immobilized antigen.
  • This invention also contemplates monoclonal antibodies specifically immunoreactive with the 40 kDa antigen, which may be prepared according to well known methods (See, e.g., Kohler and Milstein, 1976, Eur. J. Immunol, 6:611), using the antigen of this invention as an immunogen, using it for selection or using it for both functions. These and other methods for preparing antibodies that are specifically immunoreactive with the 40 kDa antigen are easily within the skill of the ordinary worker in the art. Recombinantly produced antibodies, including single chain antibodies of equivalent specificity may be prepared by recombinant DNA methods, including conventional molecular biology, microbiology, and recombinant DNA techniques within the skill ofthe art.
  • Suitable recombinant antibodies may be obtained using phage display technology, as described in a very comprehensive and well-written article by Burton and Barbas, 1994, Adv. Immunol., 57: 191, and also discussed in Winter et al., 1994, Ann. Rev. Immunol, 12:433, both of which are incorporated herein by reference.
  • the vectors described by Burton and Barbas may be used to make a library from which the sequences of the antibodies ofthis invention may be selected.
  • DNA segments or oligonucleotides having specific sequences can be synthesized chemically or isolated by one of several approaches.
  • the basic strategies for identifying, amplifying and isolating desired DNA sequences as well as assembling them into larger DNA molecules containing the desired sequence domains in the desired order, are well known to those of ordinary skill in the art. See, e.g., Sambrook, et al., (1989); B. Perbal, (1984).
  • DNA segments conesponding to immunoglobulin variable regions may be isolated using the polymerase chain reaction (M.A. Innis, et al., "PCR Protocols: A Guide To Methods and Applications," Academic Press, 1990).
  • Suitable primers may be constructed using sequences selected from the constant region flanking the variable regions, as shown in Kabat, et al. (1991), "Sequences of Proteins of Immunological Interest," Fifth Edition, U.S. Dept. of Health & Human Services, Washington D.C.
  • a complete sequence may be assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See, e.g., Edge (1981) Nature, 292:756; Nambair, et al. (1984) Science, 223: 1299; Jay, et al. (1984) J. Biol. Chem., 259:6311.
  • the assembled sequence can be cloned into any suitable vector or replicon and maintained there in a composition which is substantially free of vectors that do not contain the assembled sequence.
  • This provides a reservoir of the assembled sequence, and segments or the entire sequence can be extracted from the reservoir by excising from DNA in the reservoir material with restriction enzymes or by PCR amplification.
  • Numerous cloning vectors are known to those of skill in the art, and the selection of an appropriate cloning vector is a matter of choice (see, e.g., Sambrook, et al., incorporated herein by reference).
  • the construction of vectors containing desired DNA segments linked by appropriate DNA sequences is accomplished by techniques similar to those used to construct the segments.
  • vectors may be constructed to contain additional DNA segments, such as bacterial origins of replication to make shuttle vectors (for shuttling between prokaryotic hosts and mammalian hosts), etc.
  • Antigens purified as described above may be used in standard binding studies to select suitable clones encoding recombinant antibodies specific for the antigens.
  • Antibodies specific for the antigens of this invention may be used to identify the antigens of this invention.
  • a preparation of affinity- purified antibodies is useful as a standard for the diagnostic assay.
  • the antibodies ofthis invention may also be used in competitive immunoassays, sandwich immunoassays, etc., for detection of serum antibodies specific for the 40 kDa and 6 kDa antigens in samples of patient serum.
  • Antibodies e.g., monoclonal antibodies, affinity purified antibodies, antibody fragments or recombinant antibodies
  • of equivalent specificity may also be injected as immunogens to elicit anti-idiotype antibodies.
  • anti-idiotype antibodies will bind to serum antibodies specific for the antigens of this invention, and therefore anti-idiotype antibodies may be used in immunoassays, such as those described herein for diagnosis of active TB, in place of the 40 kDa or 6 kDa antigens. Anti-idiotype antibodies may likewise be substituted for the antigens in other applications, such as injection to elicit an immune response.
  • Example 1 Preparation and purification of the 40 kDa antigen.
  • Mtb cultures (in 1 liter batches) were grown at 37 °C in Middlebrook 7H9 medium supplemented with Tween 80 and ADC (Sigma). Mtb cultures were maintained in a biosafety level 3 (P-3) laboratory facility. Late-log phase Mtb cultures were autoclaved, and the bacterial debris removed by centrifugation. The 40 kDa antigen and 6 kDa antigen were released into the culture medium following autoclaving. Furthermore, this antigenic activity was not found in medium recovered from viable bacterial cultures.
  • the autoclave extract was digested with proteinase K, as the antigen is resistant to such digestion, while nearly all antigenic proteins are degraded by this procedure.
  • Supernatant was recovered after autoclaving and digested with Proteinase K (lOO ⁇ g/ml) in the presence of 0.1% SDS at 55 °C for 18 hr.
  • Degraded peptide fragments were removed by ultrafiltration; both the 40 kDa and the 6 kDa were retained by a 30 kDa cutoff membrane after digestion with proteinase K.
  • the antigen-containing fraction was then size-selected from the crude digest by dialysis in phosphate buffered saline (PBS) using 30 kDa-cutoff membranes and concentrated 20- 50 fold by ultrafiltration using 30 kDa-cutoff membranes (Centricon-30). Concentrated preparation of the antigens in PBS was then filtered through 0.2 ⁇ m filters and stored at 4°C.
  • PBS phosphate buffered saline
  • the antigens which survive proteinase K digestion are surprisingly stable. Little or no loss of antigenic activity was observed in the concentrated preparation in PBS at 37 °C for 1 day, and this extrapolates to approximately 3 months of stability when stored at 4°C. Prediluted (ready- to-use form) secondary antibody conjugates are stable for at least one year at 4°C.
  • the resolublized antigen preparation was determined not to contain DNA or RNA based on the absence of an absorbance peak at 260 nm and the inability to bind ethidium bromide.
  • the preparation was diluted 1 : 10 in PBS, and the ultraviolet (UV) abso ⁇ tion of this solution was determined in a Beckman DU-65 spectrophotometer.
  • the UV spectrum showed a peak at 280 nm, suggestive of residual protein contamination. DNA contamination may also be expected, if the extraction procedure is particularly severe (such as reextraction ofthe pellet).
  • Example 2 Mycobacterial Antigen Preparation
  • Mycobacteria were cultured in 1 liter of Middlebrook 7H9 medium containing ADC and Tween 80 until the culture reached late-log growth. Then the culture was autoclaved for 30 min., and the autoclaved material was centrifuged to pellet insoluble debris (20,000 rpm, 30 min). The supernatant was transfened to a fresh flask, and the pellet was resuspended in 250 ml Buffer A (10 mM NaCl, 10 mM Tris-HCl (pH 7.4), and 1 mM EDTA) and recentrifuged. The supematants were pooled and SDS was added to 0.1 % final concentration.
  • Buffer A (10 mM NaCl, 10 mM Tris-HCl (pH 7.4), and 1 mM EDTA
  • Proteinase K was added to the pooled supematants to 100 ⁇ g/ml final concentration and allowed to digest the mycobacterial lysate at 55 °C for 12-18 hr.
  • the resulting clarified lysate was concentrated 20-fold using an Amicon concentrator unit with a 30 kDa- cutoff membrane, and the concentrate was dialyzed against Buffer A using a 12-14 kDa cutoff membrane.
  • the concentrated lysate was then dialyzed at room temperature against Buffer B (Buffer A containing 4 M urea).
  • the resultant lysate was loaded onto a DEAE-Sephadex column pre-equilibrated in Buffer B, and the column was washed with 5 column volumes of Buffer B to remove unbound material. Proteins were eluted from the column using 10 column volumes of Buffer B containing 300 mM NaCl. Antigens were eluted from the column using 5 column volumes of Buffer B containing 800 mM NaCl. The eluate containing antigens was dialyzed against Buffer A and concentrated.
  • 40 kDa antigen was isolated from either crude or DEAE- purified material by addition of 2 volumes of isopropanol, followed by incubating for 1 hr at room temperature, and centrifuging to pellet the precipitate which contains the 40 kDa antigen. The pellet was resuspended in Buffer A at 37 °C and dialyzed against Buffer A at room temperature.
  • Example 3 Comparison of LAM and Mtb Antigens.
  • the crude Mtb antigens prepared by the procedure of Example 1 were compared with LAM by several criteria. SDS gel fractionated antigens were electrophoretically transfened onto nylon membranes. Purified LAM was fractionated in adjacent lanes for comparison. Initially, a binding to the lectin concanavalin A (Con A) was used to detect the presence of carbohydrates.
  • Con A concanavalin A
  • Reactivity with the anti-LAM monoclonal antibody CS-35 identified a specific band of approximately 40 kDa in the LAM preparation, which was not observed in the crude Mtb antigen preparation ( Figure 1).
  • Detection of LAM was accomplished by incubating the membranes with the anti-LAM monoclonal antibody CS35, followed by goat-anti mouse-HRP.
  • Figure 1 shows SDS gels developed with CS35. Mtb antigen concentrate was apphed to the gel in lane 1, a 1:5 dilution in lane 2, and a 1 :25 dilution in lane 3.
  • Purified LAM was placed in lane 4, and a 1 :5 dilution of purified LAM in lane 5.
  • lane 1 contains purified Mtb antigens incubated with 0.10 N NaOH at 37 degrees for 2h; lane 2: LAM incubated with 0.10 N NaOH at 37 degrees for 2h; lane 3: Mtb Antigens incubated without 0.10 N NaOH at 37 degrees for 2h; lane 4: Mtb antigens incubated without 0.1 N NaOH at 37 degrees for 2h, lane 5: untreated Mtb antigen, and Lane 6: untreated LAM.
  • the absence of a band in lane 2 shows that immunoreactivity of LAM, but not Mtb antigens, was abolished by mild base treatment. Treatment with alpha-mannosidase abolished the ability of
  • Mannosidase-sensitive NO YES Example 4. Reactivity of serum antibodies against crude Mtb antigens and LAM.
  • Western blots of antigens extracted from various mycobacterial species were developed with antisera from a normal (TB-free) individual, two different TB-positive sera and antisera from normal and M. bovis- infected cows.
  • the antigens used in the Western blots developed with human sera were extracted by tiie procedure in Example 1 from M. bovis, M. kansasii, and M. avium, and the resulting material compared to Mtb antigen prepared by the procedures in Example 1 or Example 2.
  • the antigens used the experiments with cow serum were extracted by the procedure in Example 1 from bovis BCG or a clinical isolate of M. bovis.
  • Example 6 Dot blot- and ELISA-based serodiagnostics.
  • Dot blot format Three dilutions (1 : 10, 1 :25, 1 :50) of the concentrated 40 kDa antigen stock solution from Example 1 are deposited onto a nitrocellulose membrane (0.7 cm x 5 cm) using high precision ceramic micropumps which are capable of delivering 0.1-1.0 ⁇ l volume per stroke with a maximum of 5% variability.
  • Purified human IgG (1 ⁇ g/dot) is apphed on the membrane strip as a positive control for the validity ofthe test reagents.
  • the membrane dot blot strips are air-dried, blocked with a blocking buffer containing 2% BSA, washed with PBS containing 0.05% Tween-20, and then dried at 40 °C for 1 hr. Dried dot blots are packed in static-free, air-tight pouches, heat-sealed, and stored at room temperature.
  • ELISA format One hundred ⁇ l of a 1 :500 dilution (in PBS) of concentrated 40 kDa antigen stock solution prepared as described in Example 1 is dispensed automatically into the wells of 96 well ELISA plates.
  • Plates are then covered with an adhesive film and incubated at room temperature for 1 hr in order to allow antigen binding to the plate. Any unbound antigen in the wells is removed by washing with PBS containing
  • Dot blot and ELISA plates are used for serologjcal testing with panels of clinically confirmed TB-positive and normal human sera (1 :25 dilution), and primary antibody binding to the 40 kDa antigen is determined using secondary antibody conjugates (alkaline phosphatase for dot blots and peroxidase for ELISA) with appropriate enzyme substrates for detection.
  • Dot blots are scored visually, and ELISA plates are read at 450 nm. Total time for completion of the dot blot assay is approximately 30 min and less than 2 hr for the ELISA assay.
  • ELISA assay tests are scored as positive where absorbance reading are greater than a calculated cutoff value. Cutoff values are dete ⁇ riined by measuring the absorbance reading obtained using normal human sera (typically 0.1-0.3 OD 450 ) plus a conection factor to reflect the variability observed between normal serum donors.
  • Dot blot assays were performed using normal and TB sera collected from various donors. Dilutions of Mtb antigen prepared according to Example 1 were applied to membranes, and the membranes were first incubated with either TB patient or normal sera as indicated. Some TB serum donors were also HTV positive (lanes 1-3). Some normal sera were from healthy PPD positive donors (lanes 9 and 10). Bound human serum antibodies were detected using enzyme-conjugated secondary antibodies specific for both human IgG and IgM. A mixture of human IgG and IgM (hulg) is included as a positive control.
  • Figure 7 shows the results of dot blots using dilutions of similarly prepared Mtb antigen applied to a membrane.
  • Membranes were incubated with either TB patient (TB) or normal (N) sera, and bound human serum antibodies were detected using enzyme-conjugated secondary- antibodies (2nd Ab) specific for human IgM (M) or IgG (G).
  • 2nd Ab enzyme-conjugated secondary- antibodies specific for human IgM
  • M enzyme-conjugated secondary- antibodies
  • IgG IgG
  • the antigen was recognized by both IgM and IgG antibodies present in the sera of TB patients.
  • sera contained only IgM antibodies which recognized the antigen (see Figure 7).
  • the results Figures 6 and 7 indicate that a serodiagnostic assay using the Mtb antigen of this invention is highly sensitive and specific for active mycobacterial disease state.
  • Example 7 Analysis of serum antibody reactivity against the Mtb antigens.
  • the clinical status of sera was determined by sputum culture. 216 serum samples were tested by ELISA, and 455 serum samples were tested by dot blot analysis, as described in Example 6. Table 2 summarizes the results of these tests.
  • the ELISA assay showed a sensitivity of 99% and a specificity of 100% for the samples evaluated.
  • the dot blot produced a similarly high estimate of sensitivity (97%) and a somewhat lower estimate of specificity (81%).
  • ELISA Dot Blot: Sample Test pos./ Test negJ Test pos./ Test neg./ Source Sputum pos. Sputum nee. Sputum pos. Sputum neg.
  • Example 8 Blinded Study Correlating Antigen with Disease State
  • Example 6 A blinded study was performed on 196 serum samples obtained from subjects at Tartu University Lung Hospital in Estonia and Ben Taub General Hospital in Houston, Texas, using the dot blot assay as described in Example 6, except that Mtb antigens prepared as described in Example 1 were applied to each strip in serial 2-fold dilutions. Highest concentration of crude antigen is located at the bottom of the strip. A single application of human IgG at the top of each strip served as a positive control. Four technicians carrying out the testing found the test to be technically straight forward and relatively simple. Table 3 shows the conelation between the TB clinical status and the results of testing using the dot blot test-system.
  • Example 10 Testing of a dot blot serological test system.
  • Tests to determine the anti-TB antibodies in human serum or plasma specimens were performed utilizing the dot blot test-system of Example 8 using proteinase K-digested antigen concentrate. Antibodies were determined in plasma specimens of 108 persons with active TB, 42 persons with unspecified lung diseases and 50 healthy donors. There were the following results among patients infected with the Mycobacterium species complex: 54 with infiltration TB, 23 with focal TB, 9 with fibrous- cavemous TB, 2 with cavemous TB, 5 with disseminated TB, 3 with tubercular TB, 9 with intrathoracic lymph node TB, 3 with TB pleurisy.
  • sensitivity of the test-system for the screening of individuals with TB is 65.7%; specificity is 92% (100 - 8%). Furthermore, the test-system was found to be simple to conduct, no special equipment being necessary for the conducting of tests, and the test could be rapidly completed.
  • the dot blot test-system for the early diagnosis of Tuberculosis is especially useful for "field” conditions (primarily in the group of patients with a higher risk of tuberculosis).
  • test-system for screening patients with TB is 88.7%, with a number of false positive reactions of 11.3%.
  • Sensitivity ofthe test-system is 70.5%.
  • a positive feature in the use ofthe test system was reported to be the simplicity of test conducting; in that no special equipment was necessary, and test completion was rapid (30 specimens of the test having been completed in 1.5 hours).
  • the Russian Scientific Research Institute Pulmonology and Tuberculosis MZMP PF tested 44 specimens of sera from patients with pulmonary TB (adult persons: 30, and children: 14) as well as 51 specimens of sera from healthy donors using the dot blot test-system of Example 8 using proteinase K-digested antigen concentrate.

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Abstract

Le sérum prélevé chez des patients atteints de tuberculose évolutive s'est avéré contenir des anticorps spécifiques de deux antigènes mycobactériens dont l'importance est mise en évidence pour la première fois dans cette invention. Le titre de ces anticorps se modifie chez des patients dont la maladie n'est plus évolutive. Cette invention concerne en outre des tests de détection de la tuberculose évolutive, basés sur la détermination d'anticorps spécifiques de ces antigènes dans des échantillons prélevés sur des patients.
PCT/US1997/000754 1996-01-17 1997-01-17 Antigenes mycobacteriens et leurs procedes de detection WO1997026007A1 (fr)

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Cited By (18)

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Publication number Priority date Publication date Assignee Title
WO1997045742A1 (fr) * 1996-05-29 1997-12-04 Gerlach Gerald F Methode de diagnostic pour depister la paratuberculose subclinique chez des mammiferes
US7105170B2 (en) 2001-01-08 2006-09-12 The United States Of America As Represented By The Department Of Health And Human Services Latent human tuberculosis model, diagnostic antigens, and methods of use
WO2002054073A2 (fr) * 2001-01-08 2002-07-11 The Governement Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services, Centers For Disease Control And Prevention Modele de tuberculose humaine latente, antigenes diagnostiques et methodes d'utilisation associees
WO2002054073A3 (fr) * 2001-01-08 2003-08-28 Us Gov Health & Human Serv Modele de tuberculose humaine latente, antigenes diagnostiques et methodes d'utilisation associees
US8057797B2 (en) 2004-07-20 2011-11-15 Chemogen, Inc. Method of preparing enriched antibodies for detecting mycobacterial infection
US7615222B2 (en) 2004-07-20 2009-11-10 Chemogen, Inc. Enriched antibody for detecting mycobacterial infection, methods of use and diagnostic test employing same
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