WO2001020325A1 - Dosages immunologiques de membrane permettant de detecter plusieurs maladies transmises par des tiques - Google Patents

Dosages immunologiques de membrane permettant de detecter plusieurs maladies transmises par des tiques Download PDF

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Publication number
WO2001020325A1
WO2001020325A1 PCT/US1999/021814 US9921814W WO0120325A1 WO 2001020325 A1 WO2001020325 A1 WO 2001020325A1 US 9921814 W US9921814 W US 9921814W WO 0120325 A1 WO0120325 A1 WO 0120325A1
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Prior art keywords
membrane
set forth
analyte
tick
interest
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PCT/US1999/021814
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English (en)
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Andrew E. Levin
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Immunetics, Inc.
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Priority to AU60540/99A priority Critical patent/AU6054099A/en
Publication of WO2001020325A1 publication Critical patent/WO2001020325A1/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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • 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
    • G01N33/5302—Apparatus specially adapted for immunological test procedures

Definitions

  • the present invention relates generally to a membrane and diagnostic method for detecting multiple tick-borne and/or other vector-borne diseases, and more specifically, to a membrane immunoassay and method of using same for simultaneously detecting Lyme disease and additional tick-borne diseases.
  • Lyme disease is a progressive, systemic infection caused by the spirochete Borrelia burgdorferi. The disease is transmitted to man by the bite of the deer tick (Ixodes scapularis and other species). Diagnostic tests for Lyme disease rely mainly on the detection of human antibodies to spirochaetal antigens.
  • the principal test used for screening human sera for antibodies to the Lyme spirochete is enzyme-linked immunosorbent assay (ELISA). Due to the significant inaccuracies inherent in ELISA, sera which are ELISA-positive or indeterminate are often subjected to a confirmatory test. The confirmatory test now in most common use and officially recommended by the U.S. Centers for Disease Control (CDC) is the Western blot.
  • results of the Western blot test appear as a series of bands on a membrane strip.
  • the pattern of bands is compared with the band pattern of known positive sera to produce a diagnostic result. The exact position of bands, and the number of bands which correlate with positivity, differ depending on the pathogen.
  • U.S. Patent No. 5,187,065 discloses methods of detecting Lyme disease in mammals that otherwise show seronegativity due to the generation of immune complexes which "hide” the antibodies raised to the spirochete; disassociation of such complexes followed by immunological assay procedures such as ELISA are described.
  • U.S. Patent No. 5,470,712 provides bioassays which incorporate non-flagellar B. burgdorferi proteins, or antibodies raised to such proteins, to create an assay where such proteins or antibodies are bound to a surface and form complexes with certain components of the serum.
  • U.S. Patent No. 5,217,872 teaches a method of detecting B. burgdorferi antigens through an assay which utilizes vesicle proteins released from the spirochete, while U.S. Patent Nos. 5,494,797 and 5,324,630 teach the detection of the Lyme spirochete via oligonucleotide probes.
  • U.S. Patent No. 4,888,276 describes a reliable, noninvasive method for detecting antigens of B. burgdorferi from the urine of affected individuals and U.S. Patent No.
  • HGE Human granulocytic ehrlichiosis
  • HGE Human granulocytic ehrlichiosis
  • HGE Human granulocytic ehrlichiosis
  • Splenectomy, immunosuppression, and advanced age are significant negative prognostic indicators. Early symptoms may appear within one to several weeks post- infection, and typically include malaise, anorexia, and fatigue. In susceptible individuals, these progress quickly to more serious symptoms, including fever up to 40 °C, sweating, myalgia, nausea, vomiting, headache, shaking chills, emotional lability and depression, hemoglobinuria, hyperesthesia, and pulmonary edema.
  • Blood analyses may reveal anemia, thrombocytopenia, and low white blood cell count, while lactic dehydrogenase, bilirubin and transaminases may appear at elevated levels.
  • the wide range in symptoms makes the clinical diagnosis of babesiosis difficult, and additionally so, in view of the possibility of either confusion or coinfections with Lyme disease and/or ehrlichiosis.
  • Co-infected patients may be subject to more severe illness than caused by either pathogen alone. Significant frequencies of co-infection have been reported in areas endemic for babesiosis and Lyme disease. Mitchell, P.D. et al., J. Clin. Microbiol. 34:724-727 (1996). Both diseases appear to be rising in incidence, perhaps due to changes both in public awareness of tick-borne diseases and in the interactions of man and the surrounding natural environment. Because therapeutic treatment and prognosis differ for the various diseases, accurate diagnosis is essential for successful clinical management of the patient.
  • Babesia infects and multiplies within the erythrocytes of the host and thus laboratory testing for babesiosis has traditionally been based on examination of Giemsa-stained blood smears. Telford, S.R. Ill et al., Topley and Wilson's Microbiology, in press; Diagnostic Medical Par asitology, Washington, D.C., p. 131-135 (Second Edition, 1993).
  • Babesia may be visualized in parasitized erythrocytes as pear-shaped piroplasms (hence the common name for babesiosis, "piroplasmosis") or rings, and infrequently as tetrads (maltese cross forms) which are considered as definitive evidence of infection.
  • Parasitemia in infected individuals may vary between l-20%>, while in splenectomized patients, it may attain 85%>. Nevertheless, low level parasitemia is common enough so that failure to observe the parasite in blood smears does not prove the absence of infection with Babesia.
  • Babesia may also be revealed by inoculation of patient blood samples into hamsters, which develop high levels of parasitemia; however, this technique may require up to 6 weeks to yield detectable results.
  • PCR polymerase chain reaction
  • Human ehrlichiosis a potentially life-threatening disease, on the other hand, is caused by infection with rickettsiae of the genus Ehrlichia. Bakken, J.S. et al., JAMA, 275:199-205 (1996). Ehrlichiae are obligate intracellular gram-negative bacteria. Two types of ehrlichiosis have been identified, monocytic, caused by E. chaffeensis, and granulocytic, caused by HGE. First identified as a distinct ehrlichial disease in 1994, Dumler, J.S. and J.S. Bakken, Clin. Infect. Dis., 20:1102-1110 (1995), HGE is caused by an as yet unidentified organism of the E. equi/E.
  • phagocytophila genogroup which primarily infect horses, dogs and cattle. In the United States, approximately 200 cases of HGE have been diagnosed since 1994. Chen, S.M. et al., J. Clin. Microbiol., 32:589-595 (1994). The geographic distribution of human infection closely parallels that of Lyme borreliosis, with greatest concentration in the northeast, upper midwest and northern California. Closely related or antigenically crossreactive Ehrlichiae have been reported to be associated with infections of humans and animals in Switzerland, Sweden, Norway, Germany and other countries.
  • the agent of HGE Upon infection of the host, the agent of HGE invades granulocytes in the blood and forms morulae, or optically dense intracellular inclusions. Goodman, J.L. et al., N. Engl. J. Med., 334:209-215 (1996). Various organs and tissues may be foci of infection, frequently liver, spleen, bone marrow and lymph nodes. The exact mechanism of pathogenesis remains unclear. The host immune defense systems appear to be impaired in susceptible individuals, many of whom contract lethal opportunistic infections with pathogens including Aspergillus, Cryptococcus, Candida, and Herpes. Chen, S.M. et al., J. Clin. Microbiol., 32:589-595 (1994).
  • HGE humans infected with HGE develop an immune response which can be detected by serologic assays including immunocytology, immunofluorescence and immunoblotting. Immunofluores-cence tests based on surrogate antigens, e.g., E. equi and E. phagocytophila, Walker, D.H. and Dumler, J.S., Emerging Infectious Diseases, 2(1): 18-29 (1996), have been developed based on the crossreactivity of these species with HGE.
  • surrogate antigens e.g., E. equi and E. phagocytophila, Walker, D.H. and Dumler, J.S., Emerging Infectious Diseases, 2(1): 18-29 (1996)
  • the present invention provides a membrane immunoassay and method of using same for the simultaneous detection of antibodies to multiple tick-borne agents of, for example, babesiosis, HGE, and Lyme disease.
  • the immunoassay in an embodiment, is based on recombinant protein antigens specific to certain tick-borne agents, which protein antigens have been applied to a nitrocellulose membrane, or other applicable membrane made from, for instance, nylon, polyvinyldifluoridene or a mixture thereof.
  • the protein antigens may be applied to the nitrocellulose membrane as individual stripes containing a B. microti-specific antigen, an HGE-specific antigen, or a B.
  • burgdorferi-specific antigen In the event there are several B. microti-specific antigens, several HGE-specific antigens or several B. burgdorferi-spccific antigens, a mixture of the species-specific antigens may be provided in one stripe.
  • a nitrocellulose membrane is prepared with one stripe containing B. burgdorferi antigens, one stripe containing B. microti antigens, and one stripe containing HGE antigens.
  • the membrane in an embodiment, may be provided within an apparatus for use in a flow-through assay.
  • Such an apparatus includes an upper plate having a plurality of channels extending from a first surface to a second surface of the upper plate, a lower plate for receiving the upper plate, and a wicking member positioned between the upper and lower plate.
  • the membrane is designed to be placed between the upper plate and the wicking member, such that when fluid, such as serum samples and reagents, are added on to the membrane, the wicking member may act to distribute and absorb the liquid substantially uniformly through the membrane.
  • the apparatus in one embodiment, includes a vacuum port coupled to the lower plate, so that vacuum pressure may be used to facilitate the flow- through process.
  • the membrane produced in accordance with an embodiment of the invention may be used in a flow-through immunoassay, in which samples and reagents are incubated briefly on the membrane, then permitted to flow through the membrane. If subsequently there is an appearance of a colored stripe on the membrane surface, such a stripe is indicative of a positive test result for the corresponding antigen.
  • Fig. 1 illustrates an immunoassay membrane in accordance with an embodiment of the present invention.
  • Figs. 2A-C illustrate representative results from tests of serum samples, some of which are positive for various combinations of B. burgdorferi (Lyme), HGE and Babesia antigens.
  • Fig. 3 illustrates a cassette for use with one embodiment of an immunoassay of the present invention.
  • Fig. 4 illustrates an instrument for performing rapid membrane flow-through immunoassays.
  • Fig. 5 illustrates the specificity results for normal serum samples.
  • Fig. 6 illustrates the sensitivity of detection results for HGE and B. burgdorferi antibodies.
  • Fig. 7 illustrates the sensitivity of detection results for 5. microti antibodies.
  • a membrane panel for the simultaneous detection of antibodies to the agents of multiple tick-borne and/or other vector-borne diseases, and methodologies for detecting same are provided.
  • a membrane 10 for use in connection with a flow-through membrane immunoassay is shown.
  • the membrane 10 in a preferred embodiment, includes at least three recombinant protein antigens for the detection of antibodies to the agents of babesiosis, HGE, and Lyme disease respectively.
  • These recombinant protein antigens namely Babesia microti, HGE and Borrelia burgdorferi, can be immobilized as separate colorless stripes 12 directly on the membrane using methods known in the art (discussed below).
  • An additional stripe containing, for instance, an anti-human IgG antibody may be provided on the membrane as a control band (not shown).
  • the anti-IgG antibody in this control band when bound to antibody in the sample, serves as an indicator showing whether the sample contains IgG antibody in sufficient quantity to be detected.
  • the band of antibody may also serve as verification that the assay procedure functioned properly (e.g., that the reagents have been added and that they are active).
  • the band of antibody may further serve to verify that a sample has indeed been added to the membrane.
  • the stripes 12 and the control band are generally colorless prior to performing the immunoassay.
  • the antigens used herewith may be applied to the membrane in any applicable pattern, for example, a dot or a square.
  • the membrane 10 of the present invention may be a single strip of nitrocellulose paper for testing one serum sample, or a panel 14 having a plurality of strips 16 for testing multiple samples at once.
  • Other materials may be used for making the membrane 10, examples of which include but not limited to, nylon, polyvinyldifluoridene, or a mixture thereof.
  • the membrane may be provided with eight strips 16 to permit, for example, eight different samples to be tested at a time.
  • the membrane immunoassay in one embodiment, is similar in principle to conventional ELISA, with the exception that a membrane is substituted for the plastic microplate, and the antigen is applied to the membrane as, for example, a stripe.
  • the application of the antigen stripes to the membrane is known in the art.
  • the antigens may be solubilized in a Tris-HCl/SDS/Bromophenol Blue buffer and applied in stripes to a nitrocellulose membrane using a BioDotTM instrument, an Immunetics Miniblotter®, or manually with a calligraphy pen. Other methods are disclosed in Aeppli et al., J. Immunol.
  • the membrane immunoassay of the present invention generally comprises initially introducing a serum sample, which may contain antibody specific to the antigen of interest, directly to a membrane 10. The membrane 10 is then washed to remove unbound antibody. Antibody which is bound to the antigens on the membrane is detected by incubation with a second, anti-human immunoglobulin antibody. This second antibody is linked to an enzyme, such as alkaline phosphatase, which reacts with an applied substrate to yield a colored product from a colorless one, or by converting a nonfluorescent substrate into a fluorescent one. In using this immunoassay, the presence of a particular antibody is indicated by deposition of a colored, insoluble reaction product on the membrane corresponding to the position at which the antigen is placed.
  • an enzyme such as alkaline phosphatase
  • the results appear as a colored stripe (Figs. 2A-C), where antigen is applied on to the membrane as a stripe.
  • immunoassays are typically based on unfractionated antigens and may not provide the means to distinguish between antibody reactions with antigen fractions, which are either more or less specific to a given pathogen
  • the membrane immunoassay of the present invention is sufficiently accurate, as will be discussed hereinafter in detail, to be used in the screening and detection of multiple tick-born diseases.
  • the analysis of if ⁇ besz ⁇ -positive, HGE- positive, and B are examples of the membrane immunoassay of the present invention.
  • burgdorferi-positive samples to obtain sensitivities of the present immunoassay, as compared to traditional Western Blot, provides sensitivity of detection results of 100% for HGE, 96%> for Babesia, and 88% for B. burgdorferi.
  • the agents of babesiosis, HGE, and Lyme disease may be transmitted by vectors other than ticks, for instance, mosquitos, the present invention is also designed for detection of such agents.
  • the immunoassay is performed by placing an eight-strip membrane panel 10 into a cassette 20 having eight channels 22 corresponding to the eight strips on the membrane panel 10.
  • the cassette 20 in an embodiment, includes an upper plate 24, a lower plate 26 capable of receiving the upper plate 24, and a sheet of wicking paper 28 positioned between the upper and lower parts.
  • the membrane 10 is placed over the wicking paper 28 and the cassette upper plate 24 placed within recess 29 of the lower plate 26.
  • the assembled cassette 20 is wetted thoroughly by dipping into a tray containing distilled water or a wash buffer, such as phosphate-buffered saline with 0.05%> Tween-20 detergent.
  • the cassette 20 may be transferred to between panels 32 and 33 of instrument 30 (Fig. 4) for performing rapid membrane flow-through immunoassay.
  • instrument 30 One such instrument is the CodaXcelTM from Immunetics, Inc. in Cambridge, Massachusetts.
  • the instrument 30 can either include a mechanical system or a vacuum locking system, which can be activated to exert a downward pressure against the cassette 20.
  • the pressure exerted by the instrument 30 on the cassette 20 effectively causes the cassette 20 to form a liquid-tight seal between the cassette channels 22.
  • a rocking platform 34 on the instrument 30 may also be activated at this time, if desired, to rock the cassette 20. Although a rocking motion can be used, the present immunoassay may be performed without the activation of the rocking platform 34.
  • Samples of serum, plasma, or other fluids are then added to each channel 22 with a pipet.
  • serum or plasma is diluted 1 : 100 in a buffer containing phosphate-buffered saline and 0.05%> Tween-20, but dilutions can vary between 1:1 and 1 :10,000 depending on the type of sample and the relative concentration of antibodies in the test sample.
  • Cerebrospinal fluid for instance, may be tested at a dilution of 1 : 1 , since antibody concentrations are generally much lower than those of serum, while a sample of purified polyclonal or monoclonal antibody may require dilution in excess of 1:10,000 to yield a concentration appropriate for the assay.
  • test sample The volume of test sample, and of all other solutions and reagents used in the following steps, is approximately 0.5 ml per cassette channel. Smaller or larger volumes may also be used, with moderate effects upon the assay performance.
  • the test samples e.g., serum or plasma
  • the test samples are next incubated in the cassette for about 4 minutes, after which they may be permitted to flow through the membrane. It should be noted that the time period disclosed hereinafter may be varied in all steps. However, such variance may affect the assay sensitivity.
  • the use of wicking member 28 can permit substantially uniform distribution of liquid through the membrane 20. Specifically, the wicking member 28, which may be a filter paper or other absorbent material, can act to pull liquid substantially uniformly along the surface of the membrane 20 and through the membrane 20.
  • the sample may be aspirated through the membrane 10 by the use of a vacuum.
  • the lower plate 26 is designed to permit coupling of the cassette 20 to a vacuum source.
  • the lower plate 26 may include a plurality of channels (not shown) similar to channels 22 of the upper plate 24, or a vacuum port (not shown) to permit engagement of the lower part to a vacuum source.
  • Wash buffer such as phosphate-buffered saline with 0.05%> Tween-20, or similar solution
  • the buffer is thereafter aspirated immediately through the membrane 10, and replaced with fresh buffer twice.
  • a detection reagent is added to the cassette channels 22.
  • the detection reagent may be, for example, horseradish peroxidase-conjugated goat anti-human IgG, alkaline phosphatase-conjugated goat-anti-human IgG, horseradish peroxidase-conjugated Protein A or Protein G, or similar enzyme conjugates using various enzymes and specific for different immunoglobulin types.
  • the detection reagent may also be a colloidal gold-antibody or other colloidal conjugate, a radioactively labeled antibody, or a wide variety of other reagents.
  • alkaline phosphatase-conjugated goat anti-human IgG is added and incubated for about 4 minutes, then aspirated through the membrane. Following this step, wash buffer is added once and aspirated immediately, followed by addition of distilled water. After the distilled water has been aspirated, an enzyme substrate is added.
  • the enzyme substrate that is added may be 5- bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium.
  • the enzyme substrate that is added may be tetramethylbenzidene or 4-chloro-l- napthol, both of which are available commercially as stabilized solutions.
  • the substrate is incubated for about 2 minutes, then aspirated through the membrane. Following a final wash with distilled water and aspiration, the cassette 20 is removed from the instrument 30 and opened to release the membrane 10. The membrane 10 is removed and allowed to dry at room temperature.
  • a complete immunoassay can be performed in approximately 15 minutes with this method, instead of several hours, as seen with conventional Western Blot methodology.
  • the immunoassay may be performed without the use of the instrument 30 for performing rapid flow through immunoassay.
  • the following protocol provides an example of such a method. Place a single strip of membrane into each channel of the cassette and exerting pressure on the cassette to effectively cause the cassette to form a liquid-tight seal between the cassette channels. Dispense 0.5-1.0 ml of wash buffer into each channel. Thereafter, place the cassette on a rocking platform and incubate for about 1 minute to thoroughly wet the strips. All incubations on the rocking platform are performed at the speed of 10-15 cycles/min. Aspirate the liquid completely from individual channels using a disposable pipet tip connected to a vacuum system (a water aspirator with a trap is sufficient).
  • Tilting the cassette to empty channels should be minimized, as this could cause liquid to cross over and mix with adjacent samples during the subsequent steps.
  • Load samples to be tested immediately, in order to avoid drying of the strips. Include Positive and Negative Controls in each assay run. Pipet about 0.5-1.0 ml of diluted IgG Negative Control Serum and about 0.5- 1.0 ml of diluted IgG Positive Control Serum, including antibodies reactive with B. burgdorferi, HGE, and B. microti antigens present on the membrane into separate channels. Pipet about 0.5-1.0 ml of diluted serum sample into the appropriate numbered channel corresponding to the sample sequence in the protocol. Incubate on the rocking platform for about 30 minutes. Aspirate liquid from each channel by vacuum as described above.
  • Reactivity of antibodies in the test samples with specific antigens is indicated by the appearance of colored bands over the position of the corresponding antigen stripes.
  • a sample can be tested simultaneously for the presence of antibodies to Borrelia burgdorferi , HGE and Babesia microti using this assay.
  • a detectable band is interpreted as a positive assay result.
  • an intensity indicator stripe may be provided on the membrane, so as to permit determination of a threshold level for positive results of each pathogen.
  • Band results may be read manually, for instance, by eye, or be read by an automated device. For example, the bands on the membrane may be scanned by a CCD camera or other device for digital image acquisition and interpretation by software algorithms.
  • Test results in conjunction with other clinical data, may thus be used to aid in the diagnosis of Lyme disease, HGE, babesiosis, or combinations of these diseases in a human or animal patient.
  • kits comprising a membrane of the present invention.
  • the kits may also include reagents, as well as apparatus for performing the methods described herein. Examplif ⁇ cation
  • Recombinant antigens derived from Babesia microti and human granulocytic Ehrlichiae were provided by Corixa, Inc.
  • Babesia microti recombinant antigens represented immunodominant sequences of unknown relationship to native B. microti proteins.
  • the recombinant HGE antigen was a 44 kDa protein.
  • the strategy employed to isolate these novel immunodominant antigens relied on creation of genomic expression libraries from pathogen-infected cells (Corixa, Inc.). Sheared DNA from infected HL60 (HGE) or hamster red blood cells (Babesia) was incorporated into the Lambda Zap ⁇ /EcoRl/CIAP vector.
  • Each library was screened using either human Babesia-positive serum or HGE-infected mouse serum. Phagemid was obtained from positive plaques and sequenced using forward, reverse, and internal primers. Twenty (20) Babesia clones and eighteen (18) HGE clones were obtained and expressed in pET 17b constructs and purified by affinity chromatography using nickel affinity columns. Purified protein was used in ELISA and Western Blot format to determine immunological reactivity with Babesia- and HGE-positive sera. Three Babesia-sp ⁇ cific recombinant antigens, BMNI-2, BMNI-17, and MN-10, and two HGE-specific recombinant antigens, HGE-1 and HGE-3 were evaluated.
  • a recombinant 44-kDa major outer membrane protein of HGE was obtained from Dr. Yasuko Rikihisa at Ohio State University. This protein has been shown to be the major antigen recognized by sera of HGE-infected humans.
  • purified HGE genomic DNA was digested and ligated into the Lambda ZAPII vector.
  • the gene library was constructed by infecting E. coli Xll-Blue MRF with recombinant phage. Clones expressing Ehrlichia proteins were identified with equine anti-HGE serum. Positive recombinant phagemids were used to transform E. coli SOLR cells, and positive clones were analyzed by Western Blotting with the equine anti-HGE serum.
  • p44 44-kDa protein
  • OspC and p41(internal fragment), recombinant antigens were obtained from Mikrogen GmbH (Munich, Germany). Both OspC and p41 (flagellin) are well-studied antigens used in various commercial and non-commercial diagnostic immunoassays.
  • Recombinant p35-VlsE was obtained from Dr. Steven Norris at Texas A&M University. This antigen appears to be expressed in vivo but not in spirochetes grown in vitro, and according to recent reports may be a promising candidate antigen for serodiagnosis of Lyme disease.
  • Native HGE antigen was obtamed from Dr. J. Stephen Dumler.
  • the antigen was prepared by culturing a human isolate of HGE in HL60 cells followed by renograffm gradient purification of the Ehrlichiae. The resulting material was solubilized in SDS sample buffer for electrophoresis and Western Blotting.
  • B. burgdorferi antigen, strain B31 (low passage) was provided from Immunetics, Inc.'s stock. Spirochetes grown in BSK-II medium were harvested, washed in phosphate-buffered saline, and sonicated. The resulting suspension was solubilized in SDS sample buffer for SDS-PAGE and Western Blotting.
  • nitrocellulose membranes e.g., 0.2 nitrocellulose membranes obtained from Schleicher & Schuell.
  • the membranes were formatted with a size of approximately 5 X 10 cm to fit within a cassette for use with a CodaXcelTM instrument.
  • the CodaXcelTM is an instrument designed for performing rapid membrane flow-through immunoassays, and can be obtained from Immunetics, Inc. in Cambridge, Massachusetts.
  • the nitrocellulose membranes were prepared with one stripe containing B. burgdorferi antigens, one stripe containing an HGE antigen, and one stripe containing B. microti antigens.
  • Stripes were applied to the membranes using, for example, a BioDotTM instrument, an Immunetics Miniblotter® , or manually with a calligraphy pen. The membranes were dried at room temperature and stored for later use.
  • the antibody will bind to the antigen. Free antibody is washed away, the blot is treated with a second antibody which is capable of binding to a site on the first antibody, and the blot is rinsed again to remove excess antibody.
  • the second antibody may carry a radiolabel or may be linked to an enzyme as in an ELISA technique. The enzyme linked to the antibody may then in turn react with a substrate applied to the blot which, for example, generates a colored product.
  • the bands may be visualized through the technique of autoradiography, where the radioactive blot is exposed to photographic film for a time sufficient to visualize the protein band or bands of interest. The presence of very small quantities of antigen may thus be detected due to the highly sensitive nature of the Western blotting technique, and hence its value as a confirmatory test.
  • Serum samples of known immunoreactivity to Babesia, HGE, and B. burgdorferi- specific antigens were used to optimize the immunoassay.
  • Optimal concentrations of Babesia, HGE, and B. burgdorferi antigens, as well as serum and enzyme conjugates were determined by varying these parameters independently in test runs on positive and negative serum standards.
  • Antibody detection was effected using a mixture of goat anti -human IgG and goat anti-human IgM, both conjugated to alkaline phosphatase.
  • IgG- and IgM-specific conjugates were mixed to maximize the likelihood of detecting an antibody-positive sample irrespective of isotype. Isotype specificity could be determined, if necessary, by re-testing a positive specimen on an isotype-specific immunoblot .
  • Membranes were examined visually for optimal signal to background ratio. Sensitivities and specificities were determined by testing a limited number of sera against each individual protein antigen. Combinations of species-specific antigens in a single stripe were then tested to increase sensitivity in some cases. Results of sensitivity and specificity values based on combinations of species-specific antigens for detection of antibodies to B. burgdorferi and Babesia, and a single antigen for HGE were obtained. The assay conditions selected were as follows:
  • OspC 100 g/ml p35-VlsE 5 g/ml ifabesz ⁇ -specific proteins were combined into one stripe, as were B. burgdorferi-specific proteins.
  • the membranes of the present invention were used to test representative serum samples from (i) Krause/Ryan Serum Samples, (ii) NYB Serum Samples, and (iii) Dumler Serum Samples, for the presence of antibodies to the agents of babesiosis (Babesia microti), HGE, and Lyme disease (Borrelia burgdorferi). To detect the antibodies, one membrane was inserted into an eight-channel cassette and placed on to an instrument for performing rapid membrane flow-through immunoassay, and the serum samples added, as described above.
  • Figs. 2A-C Results from several runs of these serum samples, including some which were positive for various combinations of the three pathogens are shown in Figs. 2A-C.
  • Visible bands in each channel indicate a positive reaction of the antibody with one or more of the antigens. The absence of any band denotes a negative result overall. Positive immunoreaction was verified by re-testing that sample on a conventional strip Western Blot, as shown. In other words, if the sample was a positive for Lyme (sample 2 in Fig. 2 A), a Western Blot was performed to verify the presence of antibody to B. burgdorferi in the sample. Similarly, if the sample was positive for Lyme, HGE and Babesia (sample 3 in Fig.
  • Immunoassays were performed on the Dumler Serum Samples using the membranes of the present invention and the protocol of Example 1 to detect the presence of antibodies to the agents of babesiosis, HGE, and Lyme disease.
  • the results of the assays obtained with the membrane of the present invention are illustrated in Fig. 6 under the heading "Tick Screen”.
  • Western Blots were also performed on the Dumler Serum Samples for confirmation of both positive and negative results to HGE and Lyme antibodies.
  • Immunoassays were performed on the NYB Serum Samples using the membranes of the present invention and the protocol of Example 1 to detect the presence of antibodies to the agents of Lyme disease, HGE and babesiosis.
  • the results of the assays obtained with the membrane of the present invention are illustrated in Fig. 6 under the heading "Tick Screen”.
  • Immunoassays were performed on the Krause/Ryan Serum Samples using the membranes of the present invention and the protocol of Example 1 to detect the presence of antibodies to the agents of babesiosis, Lyme disease and HGE.
  • the results of the assays obtained with the membrane of the present invention are illustrated in Fig. 7 under the heading "Tick Screen”.
  • Western Blots were also performed on the Krause/Ryan Serum Samples for confirmation of both positive and negative results to the B. microti antibodies obtained by the membrane immunoassay of the present invention.
  • the membrane when used in connection with a flow-through immunoassay, as described above, provides a test that is as substantially accurate as the Western Blot, and thus may be used effectively in the serodiagnosis of at least Lyme disease, HGE and babesiosis, the three principal tick-borne pathogens.
  • the membrane can also provide a basis for an immunoassay which can quickly and accurately test multiple serum samples for the presence of multiple tick-borne and/or other vector-borne pathogens.
  • pathogens similar to the one disclosed above are transmitted by vectors other than ticks, for example, by mosquitos
  • the membrane can also be used to detect for the presence of such pathogens.
  • the immunoassay when performed in accordance with an embodiment of the invention, can be completed in about 15 minutes.
  • the membrane and method of the present invention have been described in detail in connection with the tick-borne pathogens B. microti, HGE and B. burgdorferi, it will be appreciated that it is within the scope of the present invention that the membrane and method may be modified to detect antibodies to other pathogens, including Ehrlichia chaff eensis, the agent of Human Monocytic Ehrlichiosis, and Rickettsia rickettsia, the agent of Rocky Mountain Spotted Fever.
  • pathogens including Ehrlichia chaff eensis, the agent of Human Monocytic Ehrlichiosis, and Rickettsia rickettsia, the agent of Rocky Mountain Spotted Fever.
  • antigens unrelated to tick-borne pathogens for instance, autoimmune, rheumatoid, viral and other infections, may also be incorporated as appropriate for the differential diagnosis of conditions for which distinction between tick- borne and other etiologies is desired.
  • a combination of antigens for detecting various diseases may be further chosen based on similarity of resulting disease symptoms, known antigenic cross-reactivity or other criteria.

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Abstract

L'invention concerne un dosage immunologique de membranes à renouvellement continu, et des techniques utilisant ledit dosage immunologique qui permettent de détecter simultanément plusieurs maladies transmises par les tiques en un seul dosage. Pour détecter des infections et/ou des co-infections dues à plusieurs agents transmis par des tiques, la membrane comprend au moins trois antigènes différents, chacun étant capable de détecter des anticorps contre l'agent d'une maladie considérée transmise par des tiques, par exemple, la babésiose, une infection HGE, ou la borréliose.
PCT/US1999/021814 1999-09-16 1999-09-20 Dosages immunologiques de membrane permettant de detecter plusieurs maladies transmises par des tiques WO2001020325A1 (fr)

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AU60540/99A AU6054099A (en) 1999-09-16 1999-09-20 Membrane immunoassays for detection of multiple tick-borne diseases

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US09/398,162 1999-09-16

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003046561A1 (fr) * 2001-11-26 2003-06-05 Bortech Oy Methode de diagnostic de la borreliose de lyme aux stades precoce et avance
FR2874093A1 (fr) * 2004-08-03 2006-02-10 Inodiag Sa Methode et trousse de determination du statut vaccinal de personnes
WO2006093651A2 (fr) * 2005-02-25 2006-09-08 Immunosciences Lab, Inc. Procedes et ensemble pour diagnostiquer les maladies transmises par les tiques
EP1844331A1 (fr) * 2005-02-02 2007-10-17 Standard Diagnostics, Inc. Dispositif d'immunodosage non continu et méthode d'immunodosage doté dudit dispositif
CN103091482A (zh) * 2013-01-11 2013-05-08 苏州浩欧博生物医药有限公司 一种酶联免疫法中用于放置检测条的容器
WO2021142294A1 (fr) * 2020-01-10 2021-07-15 Quidel Corporation Bandelette réactive à substrat structuré permettant une analyse sur membrane multiplexe destinée à un diagnostic de maladie

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WO1991002073A1 (fr) * 1989-07-28 1991-02-21 Pierce Chemical Company Enceinte a vide pour immunoanalyse possedant une plaque microtitre
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WO1999000655A2 (fr) * 1997-06-27 1999-01-07 Immunetics, Inc. Appareil et procede d'essai de liaison rapide en ecoulement continu
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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003046561A1 (fr) * 2001-11-26 2003-06-05 Bortech Oy Methode de diagnostic de la borreliose de lyme aux stades precoce et avance
FR2874093A1 (fr) * 2004-08-03 2006-02-10 Inodiag Sa Methode et trousse de determination du statut vaccinal de personnes
WO2006024761A1 (fr) * 2004-08-03 2006-03-09 Inodiag Methode et trousse de determination du statut vaccinal de personnes
EP1844331A1 (fr) * 2005-02-02 2007-10-17 Standard Diagnostics, Inc. Dispositif d'immunodosage non continu et méthode d'immunodosage doté dudit dispositif
EP1844331A4 (fr) * 2005-02-02 2008-10-01 Standard Diagnostics Inc Dispositif d'immunodosage non continu et méthode d'immunodosage doté dudit dispositif
WO2006093651A2 (fr) * 2005-02-25 2006-09-08 Immunosciences Lab, Inc. Procedes et ensemble pour diagnostiquer les maladies transmises par les tiques
WO2006093651A3 (fr) * 2005-02-25 2007-02-01 Immunosciences Lab Inc Procedes et ensemble pour diagnostiquer les maladies transmises par les tiques
US7390626B2 (en) 2005-02-25 2008-06-24 Immunosciences Lab., Inc. Methods and kit for diagnosing tick borne illnesses
CN103091482A (zh) * 2013-01-11 2013-05-08 苏州浩欧博生物医药有限公司 一种酶联免疫法中用于放置检测条的容器
WO2021142294A1 (fr) * 2020-01-10 2021-07-15 Quidel Corporation Bandelette réactive à substrat structuré permettant une analyse sur membrane multiplexe destinée à un diagnostic de maladie

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