EP0830449A1 - Test de diagnostic precoce - Google Patents

Test de diagnostic precoce

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Publication number
EP0830449A1
EP0830449A1 EP96921518A EP96921518A EP0830449A1 EP 0830449 A1 EP0830449 A1 EP 0830449A1 EP 96921518 A EP96921518 A EP 96921518A EP 96921518 A EP96921518 A EP 96921518A EP 0830449 A1 EP0830449 A1 EP 0830449A1
Authority
EP
European Patent Office
Prior art keywords
burgdorferi
fluid
tissue
spp
binding
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP96921518A
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German (de)
English (en)
Inventor
Hallie M. Krider
Sandra Lee Bushmich
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Connecticut
Original Assignee
University of Connecticut
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Filing date
Publication date
Priority claimed from US08/659,369 external-priority patent/US5985595A/en
Application filed by University of Connecticut filed Critical University of Connecticut
Publication of EP0830449A1 publication Critical patent/EP0830449A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • 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
    • 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
    • G01N33/56966—Animal cells
    • G01N33/56972—White blood cells

Definitions

  • the present invention relates to diagnostic tests and more particularly to a method for the rapid and reliable detection of the presence of pathogens or other foreign substances in a human or animal.
  • Lyme borreliosis is the most prevalent tick- borne disease in the United States as well as one of the most important tick-borne infectious diseases worldwide. It is endemic in much of the northeastern United States, Minnesota, Wisconsin, and parts of the Pacific Northwest.
  • the bacterial spirochete Borrelia burgdorferi is the causative agent for Lyme disease. Infection with B . burgdorferi produces local and systemic manifestations. Local symptoms that appear early after infection include a skin lesion at the site of the tick bite, termed erythema chronicum migrans
  • ECM ECM
  • fever and flu-like illness Weeks to months after infection, systemic manifestations that include rheumatic, cardiac and neurological symptoms appear.
  • the early local phase of B . burgdorferi infection is easily treatable with antibiotics.
  • NK natural killer
  • antibiotic treatment of Lyme borreliosis should be administered early in the course of the disease, when spirochetemia and the polymorphonuclear leukocyte (PMN) phagocytic responses are prominent.
  • PMN polymorphonuclear leukocyte
  • Early, appropriate treatment aborts progression to more severe cardiac neurological and arthritic manifestations in the majority of cases.
  • early treatment is more cost effective than less successful treatments for later arthritic and neurological manifestations of Lyme disease, which may include extended periods of intravenous antibiotic therapy.
  • early diagnosis of Lyme disease is essential. Lyme disease is currently difficult to definitively diagnose. The early symptoms are often non-specific. Further, although B .
  • burgdorferi- infected Ixodid ticks are the predominant source of infection, some patients have no history of a tick bite and 25-30% of the patients do not develop ECM.
  • a screening test which reliably detects B . burgdorferi infection early in the course of the disease would allow early diagnosis and treatment, and eliminate the need for the physician to treat patients based on clinical suspicion alone.
  • B . burgdorferi spirochetes have been demonstrated in the blood, urine, cerebrospinal fluid (CSF) and synovial fluid of infected animals and humans, their numbers are too few, and their presence too variable, to allow reliable diagnosis by traditional dark field examination. Culture of these organisms from body fluids is difficult due to the low number of spirochetes in samples and the slow growth rate of wild strain 23. burgdorferi in culture; the process can take up to five weeks. Bacterial contamination of samples is another common difficulty, as the rich Barbour-Stoenner-Kelly (BSK) media needed to support 23. burgdorferi growth will also encourage growth of contaminants, and the few antibiotics that can be used to decrease contamination also slow B . burgdorferi growth considerably.
  • BSK rich Barbour-Stoenner-Kelly
  • syphilitic patients using current diagnostic tests, may test falsely positive for Lyme Disease, and Lyme Disease patients may test falsely positive for syphilis. This is problematic due to the different treatments involved for each of these diseases as well as the social stigma attached to syphilis as a sexually transmitted disease.
  • antibiotics has reduced the fatality rate of acute bacterial meningitis to less than 10% in cases recognized early, but when diagnosed late it is often fatal.
  • Current methods of diagnosis include a lumbar puncture followed by culturing and examination of the cerebrospinal fluid (CSF) for the presence of bacteria.
  • CSF cerebrospinal fluid
  • Such a test is time consuming, and lumbar puncture can cause neurological damage in the presence of a brain abscess or other mass lesion.
  • no bacteria are found in the culture it is difficult to determine whether viral meningitis or bacterial meningitis is the diagnosis, and therefore how to treat the patient. Due to the rapid progress of the disease, when bacterial meningitis is seriously suspected, administration of antibiotics must begin before the results of such diagnostic tests.
  • septicemia invasion of the circulation by pathogenic bacteria and their toxins
  • septic shock characterized by acute circulatory failure and multiple organ failure including the kidneys, lungs and heart.
  • Mortality ranges from 25-90%, and is higher when treatment is not begun soon enough. Therefore, administration of antibiotics should begin before the results of blood cultures are known. This means treatment must be selected based on the physician's educated guess, and unnecessary agents may be administered. Further, culture results may be negative, especially in patients who have had prior antibiotic therapy, but a negative culture does not exclude septicemia.
  • Gonorrhea a sexually transmitted infection caused by the bacterium Neisseria gonorrhoeae, can lead to complications such as bactere ia and gonococcal arthritis if left untreated. However, it is a disease for which there is no rapid nonculture diagnostic test.
  • a urethral Gram stain allows identification of the causative organism in about 90% of men, but a cervical Gram stain is only about 60% sensitive in women. Nor is there a reliable ⁇ erologic diagnostic test for gonorrhea currently available.
  • Syphilis is caused by the spirochete bacterium Treponema pallidum .
  • the screening tests are easy to perform and inexpensive, but have a high rate of false positives and do not become positive until three to six weeks after the initial infection.
  • the treponemal tests are more accurate, but do not become positive until three to four weeks after infection.
  • An immediate diagnosis of syphilis can be made by demonstrating T . pallidum in fluid from lesions by darkfield microscopy, but skill is needed to collect and correctly identify the organism.
  • the present invention provides a method for detecting exposure to a foreign substance in a human or animal, comprising the steps of obtaining a tissue or fluid sample from the animal or human which contains polymorphonuclear leukocytes.
  • the sample is contacted with the foreign substance of interest.
  • the fluid or tissue is labelled with one or more labels which allow detection of the white blood cells and of the foreign substance (e.g. a DNA fluorochrome) either before, during or after contacting the sample with the foreign substance.
  • An appropriate method e.g., fluorescence microscopy
  • the degree of binding can indicate exposure to, or the presence of, the foreign substance, especially when compared to a control.
  • the leukocytes are neutrophils.
  • the neutrophils can be from any fluid or tissue from an animal or human.
  • the foreign substance can be any pathogen, e.g. bacteria, viruses, yeasts, fungi, protozoans, parasitic animals, or biological molecules including a protein, lipoprotein (e.g., in a particularly preferred embodiment, OspA or OspC of 23. burgdorferi ) , a lipopolysaccharide or a glycoprotein.
  • the foreign substance is a bacteria selected from the group consisting of Borrelia spp . , E . coli, Bordetella pertussis, Neisseria spp . , Staphylococcus spp. , Leptospira spp . , Treponema spp . , Lister ia spp . and Mycoplasma spp .
  • the foreign substance is a virus selected from the group consisting of parvoviruses, herpesviruses, canine distemper virus, papovavirus and parainfluenza viruses.
  • the fluid or tissue obtained from the human or animal is selected from the group consisting of blood, respiratory secretions, urine, cerebrospinal fluid, exudate from skin lesions or abscesses, exudate from the lacrimal sac, and synovial fluid.
  • the method thus provides a reliable way to ascertain whether an animal or human has been exposed to the foreign substance, for instance whether the animal or human has been infected by, inoculated with or otherwise exposed to an antigen, immunogen or pathogen. Thus, one can reliably ascertain whether an animal or human has been infected, inoculated or otherwise exposed to the foreign substance.
  • Figure 1 is a graphical representation of the percentage of neutrophils out of total white blood cells in blood from Balb/C mice injected with 23. burgdorferi ; Balb/C mice injected with Freund's adjuvant; and a control group of Balb/C mice injected only with BSK medium;
  • Figure 2 is a graphical representation of the percentage of white blood cells binding 23. burgdorferi spirochetes out of total white blood cells in blood from Balb/C mice injected with B . burgdorferi ; Balb/C mice injected with Freund's adjuvant," and a control group of Balb/C mice injected only with BSK medium;
  • Figure 3 is a graphical representation of the percentage of white blood cells from mice injected with one of strains N-40, 2684 or 25550 of B . burgdorferi binding 23. burgdorferi spirochetes of strain N-40;
  • Figure 4 is a graphical representation of the percentage of white blood cells from mice injected with one of strains N-40, 2684 or 25550 of B .burgdorferi binding B . burgdorferi spirochetes of strain 2684;
  • Figure 5 is a graphical representation of the percentage of white blood cells from mice injected with strain N-40 of 23. burgdorferi binding B . burgdorferi spirochetes of strain 25550; the percentage of white blood cells from mice injected with strain 2684 of B . burgdorferi binding 23. burgdorferi spirochetes of strain 25550; and the percentage of white blood cells from mice injected with strain 25550 of 23. burgdorferi binding 23. burgdorferi spirochetes of strain 25550;
  • Figure 6 is a graphical representation of the percentage of neutrophils out of total white blood cells in the blood of mice infected with 23. burgdorferi versus the percentage of neutrophils out of total white blood cells in the blood of mice injected with BSK medium;
  • Figure 7 is a graphical representation of the percentage of white blood cells from mice injected with
  • mice injected with Staphylococcus spp . binding B. burgdorferi spirochetes of strain N-40 the percentage of white blood cells from mice injected with
  • Figures 8a and 8b are graphical representations of the percentage of white blood cells from mice injected with distemper, herpes, parvo and influenza pathogens binding B . burgdorferi spirochetes of strain N-40;
  • Figure 9 is a graphical representation of the comparison of Leptospira and B. burgdorferi binding in white blood cells of Leptospira infected mice;
  • Figure 10 is a graphical representation of the percentage of cells bound to B. burgdorferi spirochetes of strain N-40 in male mice inoculated with the N-40 strain
  • Figure 11a is a graphical representation of the percentage of neutrophils bound to B. burgdorferi spirochetes of strain N-40 in mice intradermally inoculated with B. burgdorferi ;
  • Figure lib shows an immunoblot analysis of mice infected intradermally with B. burgdorferi spirochetes of strain N-40;
  • Figures 12a and 12b are graphical representations of the percentage neutrophils bound in mice serially bled by retroorbital bleeding (Fig. 12a) or serially killed by guillitine bleeding (Fig. 12b) in mice infected with B. burgdorferi spirochetes of strain N-40; and
  • Figure 13 is a graphical representation of the percentage of cells bound in human volunteers naturally and not (controls) exposed to B. burgdorferi .
  • the present invention is concerned with a method for the rapid and reliable detection of the presence of pathogens or other foreign substances in a human or animal.
  • Neutrophils are a type of polymorphnuclear leukocyte (PMN) , whose primary known functions are phagocytosis (the ingestion and destruction of particulate material) and the release of chemicals involved in inflammation. They comprise approximately 50-70% of total leukocytes (white blood cells) in normal human blood. It has been found that when whole blood of seropositive animals is mixed with cultured B. burgdorferi , neutrophils bind large numbers of the spirochetes. This response has been observed in canine, equine, bovine, caprine and murine blood.
  • PMN polymorphnuclear leukocyte
  • the blood smears can be .stained, using stains such as DAPI, Hoechst (e.g., Hoechst 33258) , acridines (e.g., Acridine Orange) and other fluorescent DNA stains to visualize the binding.
  • Antibodies linked to fluorochromes or colorigenic agents such as fluorescein isothiocyanate (FITC) , rhodamines, Texas Reds, and the like, are also suitable.
  • FITC fluorescein isothiocyanate
  • rhodamines rhodamines
  • Texas Reds and the like.
  • the method of the claimed invention is not limited to the use of whole blood but can involve the examination of smears of any body fluid or tissue where white blood cells are likely to be found, e.g. urine, respiratory secretions, CSF, the exudate from skin lesions or abscesses, exudate from the lacrimal sac, synovial fluid, and the like
  • the origin of the specificity of the binding response, and the origin of the cells that bind, is not yet known.
  • the increase in the number of neutrophils which bind spirochetes is matched by only a slight increase in the total number or fraction of the cells which are neutrophils. That is, neutrophilia is not a necessary component of the syndrome associated with a host response to early Lyme disease.
  • the cells could be recruiting recognition and/or binding factors from some other source.
  • the rise in the number of competent cells may not result from new cells produced in response to infection.
  • the material is spread on slides, air dried, fixed, stained and examined using a microscope. The presence and degree of cell binding is then observed by indirect techniques.
  • the protocol for a simple hematocrit test is altered to include the mixing of reagents prior to centrifuging the blood. Enzymatic or fluorescent tags would show if there is cell binding, and would allow a quantitative evaluation. In another prefered embodiment, enzymes may be attached in order to generate a colorimetric reaction that could be read in the serum, or a precipitated product that would appear at the layer of the cells. A sophisticated reader of this type exists as the QBC instrument marketed by Becton Dickinson. Flow cytometry, e.g., fluorescent cell sorting utilizing a fluorescence-activated cell sorter (FACS) machine is useful with the claimed method. Film readers and other approaches may also be used with the claimed method.
  • FACS fluorescence-activated cell sorter
  • fluroescent or enzyme- linked tags to provide the qualitative and quantitative markers for the test has the advantage of allowing a positive internal control and standard to be incorporated into the test. That is, antibodies which will bind other cellular components to ensure that the reagents are still active, the blood is properly preserved, mixing was complete, etc. , can be added.
  • mice were innoculated with B. burgdorferi , Freund's adjuvant or control BSK media. The mice were bled three times weekly for two weeks following infection. The whole blood was mixed with cultured B. burgdorferi spirochetes. Thin blood smears were prepared and stained with a DNA fluorochro e to visualize white blood cell nuclei and the B. burgdorferi spirochetes.
  • the results, shown in Figure 2 showed an increase in B. burgdorferi binding to neutrophils in the blood from B. burgdorferi-infected mice compared to mice innoculated with Freund's adjuvant and the control mice. The binding response was detectable as early as day 2-4 post infection (preceding measurable antibody response in mice by approximately one week) . These results suggested that the binding response was specific, and occurred very early.
  • mice were infected with one of three isolates of B. burdorferi (N-40, 2684, or 25550). A control group was innoculated with BSK media. The mice were bled twice weekly for one month. Aliquots of each whole blood sample were incubated with one of the three strains of B. burgdorferi . Blood smears were prepared, and the binding properties of the white blood cells for the cultured B. burgdorferi spirochetes was quantitated. The binding response was also compared with Western blot analysis. The percentage of neutrophils among the white blood cells in the infected and control mice are shown in Figure 6. The percentage of white blood cells of mice innoculated with each of the three strains of B.
  • Double-blind experiments (to avoid observer bias) have been conducted which show that other organisms do not produce cross reactions with B. burgdorferi . None of the injected agents induced the binding of Lyme spirochetes above that of the background. It can thus be asserted that there is no evidence that specificity is compromised by secondary or concomitant infections by other organisms.
  • the method of the claimed invention is not limited to the detection of Borrelia infection.
  • Responsive cells have been shown to recognize organisms other than Borrelia .
  • active binding of E. coli bacteria by cells from mice that were injected with E . coli has been observed.
  • the cell recognition reaction has been induced with isolated proteins.
  • mice were injected with the lipoprotein OspA which had been cloned and expressed by E. coli .
  • OspA is the most abundant outer surface protein of B. burgdorf ri , which has been the focus of much research as a protective antigen for use in a Lyme disease vaccine, as described in published international patent application number WO 92/14488. Blood drawn from the mice was mixed with living B.
  • the method of the present invention can be used to rapidly and accurately diagnose human and veterinary diseases in their early stages.
  • diseases include bacterial infections, e.g., septicemia; bacterial meningitis; gonorrhea, syphilis and other sexually transmitted diseases; streptococcal infections, including those caused by S . pneumoniae, S . pyogenes, and S . mutans ; staphylococcal infections, including those caused by S . aureus and S . epidermidis ; listeriosis in humans and circling disease in cattle (both caused by Listeria SPP * / primarily L. monocytogenes) ; Hemophili ⁇ spp. infections, including those caused by H .
  • bacterial infections e.g., septicemia; bacterial meningitis; gonorrhea, syphilis and other sexually transmitted diseases
  • streptococcal infections including those caused by S . pneumoniae, S . pyogene
  • influenzae H. parainfluenzae, H . aphrophilis, H . aegyptius, H . ducreyi and H. ⁇ omnus ; relapsing fever; treponematoses, including bejel, yaws and pinta; leptospirosis; mycoplasmal infections, including those caused by Mycoplasma pneumoniae ; and chlamydia infections, including those caused by Chlamydia psittaci and C. trachomatis .
  • Such diseases also include yeast or fungal infections, e.g. candidiasis, blastomycosis and histoplasmosi ⁇ , as well as viral infections, e.g. those caused by herpesviruses or papovaviruses such as papilloma.
  • yeast or fungal infections e.g. candidiasis, blastomycosis and histoplasmosi ⁇
  • viral infections e.g. those caused by herpesviruses or papovaviruses such as papilloma.
  • the method of the present invention is also useful for detecting the presence of, or exposure to, protozoal infections, e.g., those caused by tricho onads and opalinids, as well as infections by parasitic worms such as ascarides, cestodes, Filarioidea , amblystomes and roundworms.
  • protozoal infections e.g., those caused by tricho onads and opalinids
  • parasitic worms such as ascarides, cestodes, Filarioidea , amblystomes and roundworms.
  • the method of the claimed invention includes the use of any label, and any suitable imaging method, which allows the binding of the foreign substance to the neutrophils to be observed.
  • the cells are stained with a fluorescent DNA stain, such as Hoechst 33258 or DAPI and viewed under a microscope with an ultraviolet or near-ultraviolet light source.
  • a fluorescent DNA stain such as Hoechst 33258 or DAPI
  • phase contrast microscopy Nomarski differential contrast microscopy, Hoffman contrast microscopy, darkfield microscopy, interference microscopy or low power scanning or transmission electron microscopy.
  • phase contrast microscopy Nomarski differential contrast microscopy
  • Hoffman contrast microscopy Hoffman contrast microscopy
  • darkfield microscopy darkfield microscopy
  • interference microscopy or low power scanning or transmission electron microscopy.
  • the method of the present invention may be automated.
  • an automated digital imaging apparatus could be used with the method of the present invention.
  • the samples are prepared on glass slides which are mounted on the motorized stage of a
  • the image of the microscopic field is collected by a video camera after automatic focusing.
  • the scanning process proceeds as follows: the camera image is digitized by an analog to digital converter and stored in the memory of the computer.
  • the computer program analyzes the image by searching the characteristic image components. The position of the microscope stage and focusing are saved if any positive elements are found. The computer adjusts the stage and/or focusing for the next field. A complete scan of all of the mounted sample is made by repeating the above process.
  • Strains 2684, N-40 and 25550 were inoculation groups: Strains 2684, N-40 and 25550, and a BSK media control. Cultures of the three strains were started from frozen cultures. Cultures were counted on a Petroff-Hauser Chamber the morning of inoculation. Strain N-40 was passage 3, 90% motile, and at a concentration of 7.1 x 10 7 spirochetes/ml. Strain 2684 was passage 3, 90% motile, and at a concentration of 6.Ox IO 7 spirochetes/ml. Strain 25550 was passage 4, 70% motile, and at a concentration of 3.0 x IO 7 spirochetes/ml. B. burgdorferi cultures were grown in BSK media at 33-34°C.
  • mice Prior to inoculation, the mice were anesthetized with methoxyflurane (Pitman-Moore) and the lower left quadrant of the abdomen was shaved and sterilized with Betadine followed by 95% ethanol. The mice were then inoculated intraperitoneally using a 27 gauge tuberculin syringe with 0.1 to 0.3 ml inoculum under sterile conditions. The mice were carefully observed for several hours following inoculation for after effects of the anesthesia and inoculation.
  • methoxyflurane Pane-Moore
  • mice Each group consisted of 28 mice; 3 mice were euthanized on each of days 0, 3, 7, 10, 14, 17, 21, 24 and 28. The remaining mouse was euthanized on day 36. Sampling of the mice was done on the day of inoculation and every three to four days following the inoculation. At each sampling date, three mice were euthanized for each treatment group. Whole blood from two of these mice was used for the test, and serum was saved from the third for Western blots (Example 2) . The mouse was deeply anesthetized with methoxyflurane, decapitated using surgical scissors, and the blood collected using a funnel into a small beaker to which a drop of heparinized saline had been added.
  • the blood was aliquotted into 1.0 ml round bottomed Nunc Cryotubules (USA Scientific, Ocala, FL) . Cultures were counted immediately prior to incubation. Incubations were done with each of the three strains. Spirochete concentrations of strain N- 40 ranged from approximately 2 to 6 x IO 7 spirochetes/ml, and were all passage 3. Strain 2684 had spirochete concentrations ranging from 2.5 to 4.5 x IO 7 spirochetes/ml. All cultures were passage 4. Strain 25550 had spirochete concentrations ranging from 1.0 to 6.1 x IO 7 spirochetes/ml, and were either passage 4 or 5.
  • mice inoculated with BSK provided a baseline for non-specific binding
  • the values for these mice were averaged over the course of each experiment, and one standard error was calculated.
  • standard errors indicated that data for each of the three treatment groups (i.e., mice incoulated with strains 2684, N-40 and 25550) were similar enough to justify the combining of this data, and calculating one standard error for each sampling date. This was done for the N-40 ( Figure 3) and 2684 ( Figure 4) incubations, but not for the 25550 incubation ( Figure 5) .
  • SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis
  • Laemmli Laemmli SE 600 vertical slab gel electrophoresis unit, Hoefer Scientific, San Francisco, CA
  • Spirochetes were prepared by washing 150 ml of whole cells from log phase cultures of B . burgdorferi three times by centrifugation at 10,000 x g for 15 minutes in a 0.1% solution of merthiolate and IX PBS. The cells were resuspended and the protein concentration was determined using the methods of Bradford (Bradford, 1976) .
  • Electrophoresis was done at a constant current of 100mA until the dye-front reached 1 cm from the bottom of the gel. Proteins were pre-equilibrated in transfer buffer (25mM Tris base, 38mM glycine, 20% methanol, pH 8.3) , for 30 minutes, then transferred onto 0.45 ⁇ m nitrocellulose membranes as previously described by Towbin et al. (Towbin et al., 1979) .
  • nitrocellulose membranes were stained in Ponceau's stain (Sigma, St. Louis, MO) for approximately 10 minutes to determine if proteins were properly transferred.
  • the membranes were cut into strips, placed in a small tray and blocked with 2% Bovine Serum Albumin Fraction IV (BSA) and 1% horse serum in Tris buffered saline for one hour at room temperature with rocking. strips were rinsed (three 10 minute washes) in was buffer (150 mM NaCl, lOmM Tris, 0.05% TweenTM 20), overlaid with experimental calf sera (diluted 1:100) and incubated as before for two hours.
  • BSA Bovine Serum Albumin Fraction IV
  • Serum was removed, strips were washed as described and goat anti-mouse heavy and light chain IgG phosphatase labeled conjugate (Kirkegaard & Perry, Gaithersburg, MD) (diluted 1:500) was added and incubated for one hour. The strips were rinsed as before and reacted with BCIP/NBT (phosphatase substrate) (Kirkegaard & Perry, Gaithersburg, MD) until optimal protein band development was achieved (1-10 minutes) . The reaction was stopped by the removal of the substrate and rinsing the strips several times with distilled water.
  • BCIP/NBT phosphatase substrate
  • Canine distemper MLV (Solvay, Lot # 81000A, 0.1 ml, 1/10 dog dose) 4.
  • Coagulase negative Staphylococcus spp. (cow skin isolate) (0.3 ml of 1.5 x 10 8
  • Treponema bryantii (8 x 10 7 spirochetes/mouse; culture 95% dead) 12.
  • T bryantii Counts were done of cultures the morning of inoculation. Syringes for treatments 1-9 were loaded 30 to 120 minutes prior to inoculation. Syringes for treatments 10 and 11 were loaded immediately prior to inoculation. The mice were anesthetized and inoculated as described in Example 1.
  • mice There were 15 mice for each treatment group. Three mice were euthanized by guillotine for each of days 0, 3, 7, 10 and 14, and blood collected, as described in Example 1. Incubations were done with each of the three strains of B. burgdorferi (N-40, 2684 and 25550) . Cultures were counted immediately prior to incubation. Spirochete concentrations of strain N-40 ranged from 1.5 to 3.5 x IO 7 spirochetes/ml and were passages 4 to 7. Strain 2684 had 1 to 3 x IO 7 spirochetes/ml and were passages 3 to 9. Strain 25550 had 0.75 to 2.5 x 10 7 spirochetes/ml, and were passages 4 to 6. For incubating, 150 ⁇ l of blood was mixed with 150 ⁇ l of culture. Smears were made and slides were fixed as described in Example 1.
  • incubations v/ere also done on selected mice with E . coli and Staphylococcus . These incubations were all done on blood from day 10 mice.
  • E . coli strain K-12 was incubated with blood from mice inoculated with BSK, N-40 and E . coli . Seventy-five ⁇ l E . coli (concentration 5 x IO 8 CFU/ml) was incubated with 150 ⁇ l blood.
  • Staphylococcus was incubated with blood from mice inoculated with Staphylococcus . Fifteen ⁇ l of culture (concentration 1 x IO 8 CFU/ml) were incubated with 150 ⁇ l blood.
  • mice in three groups were inoculated according to the method described in Example 1.
  • the first group was inoculated with OspA (Connaught)
  • the second group with Staphylococcus spp .
  • the third group was inoculated with OspA (Connaught)
  • the third group was inoculated with Staphylococcus spp .
  • mice were euthanized, and blood collected, at days 0, 3, 7, 10 and 14 as described in Example 1.
  • OspA represents disagreement between the samples, so that the values for those points are not established.
  • Dynal Dynabeads were prepared by washing three sets of beads from 250ul aliquots of product (e.g. 10 8 beads) in several washes of PBS in a 1.5ml tube. The large magnet was used to remove the fines and solutions and the beads were suspended in 750ul of PBS. These beads met the following conditions: (1) the beads could be coated with approximately 5ug protein/IO 7 beads; (2) the concentration was approximately 10 8 beads/ml; and (3)the approximate concentration for intended use was 4 beads/cell or more for the reaction. Three groups of beads were prepared in this manner. Anti-Borrelia antibody was prepared from a serum pool of goats immunized to whole cells of B.
  • burgdorferi (Kirkegaard & Perry Laboratories, 2 Cessna Court Gaithersburg, MD, catalog no. 01-97-91) was suspended in a 50% glycerol solution at lmg/ml. A 50ul aliquot was added to the dynabeads, and the tube was gently agitated at room temperature for 20 minutes. BSA was added to a final concentration of 0.1%, and the tube was gently agitated overnight at 37°C. The BSA is recommended to properly "position" the antibody complex on the dynabeads. The beads were washed with PBS with 0.1% BSA using a magnet to remove the antibody solution, and the beads were suspended in a Tris-HCl buffer, and incubated for 24 hours at 37°C.
  • the last step serves to block any remaining groups on the beads, and washes off proteins which are not covalently bound to the beads. Failure to effectively block the beads results in non-specific interactions with other groups on cells with which they can form bonds, such as any free amine, thereby effectively reducing the specificity of the beads to zero.
  • the second and third group of beads were coated with either OspA-L (lipidated) or OspA-NL (non- lipidated) .
  • the OspA-NL was used at a initial concentration of 700ug/ml, and a total volume of 70ul was added to the beads;
  • OspA-L was used at an initial concentration of 1210ug/ml, and a total volume of 50ul was added to the beads. Both solutions were gently agitated at 37°C for 24 hours without the addition of BSA.
  • Washing was performed several times in order to ensure that samples contained no unbound (or only bound) cellular material.
  • cytobuckets These are three well assemblies which allow one to centrifuge the entire contents of a microfuge tube onto a 22mm 2 surface.
  • the experimental and control samples were prepared on the same slide, so that variations in staining that can occur between slides could be controlled.
  • the buckets were spun at very low rates, and the resulting surface had cells evenly spread over it, wherein the bound beads are visible, and the nuclei could be seen in phase contrast.
  • the slides were air dried and fixed in methanol for storage. This method is compatible with the use of aqueous Wright's stain, and it also removes the petroleum sealant used in the cytobucket assembly.
  • Antibody linked beads detected cells in infected animals which were not neutrophils, and may be useful in monitoring the entire seroconversion cycle. This experiment was repeated using blood taken from animals (mice, as in the previous examples) sacrificed at seven days post inoculation by the guillotine method. When spirochetes were added to the cells with gentle agitation, followed by the addition of Anti-Borrelia coated beads, a cell population of neutrophils was isolated which contained virtually none of these cell types in the controls. Other cell types were found in these preparations when fluorescence was used to evaluate the slides. Co-addition of beads and bacteria to the blood created a more severe background. It is likely that the use of BSK, which contains BSA, is a complicating factor in these experiments, and in all of the reactions which follow. Apparently, as the immune response rises, cells which recognize the BSA used in the blocking reactions become available.
  • PspA Pneumococcal surface protein A
  • the animals were sacrificed on the seventh day following inoculation.
  • WBC were analyzed by the method outlined in Example 1.
  • Dynabeads were coated with each of the aforementioned antigens and mixed with subject blood. Large cells were plentiful in the blood of the experimental animals, but absent or virtually absent from all control samples. That is, antigen-coated beads bound white blood cells from animals inoculated with the same antigen. Binding was virtually absent from blood of control animals.
  • Tetanus toxoid provided a positive response, but it was not easily differentiated from the control samples. This may indicate that the protein is less effective in invoking a cell binding response, that the protein was not well bound to the beads, or that the protein was largely degraded. Unlabeled PspA bound cells from both control and experimental animals, and thus, its specificity was marginal.
  • mice were inoculated intraperitoneally with one of:
  • mice (1.0xlO pfu/mouse), Treponema (9.0X10 6 pfu/mouse), Leptospira (5xl0 6 pfu/mouse) , Influenza virus (10 6 organisms/mouse), Parvovirus (10 6 organisms/mouse), Herpesvirus (IO 6 organisms/mouse) , Paramyxovirus (Canine distemper; IO 6 organisms/mouse) and BSK (control) .
  • Mice two of each group) were guillotine bled at 0, 3, 7, 10 and 14 days post inoculation, and the blood was subjected to the Lyme test (ELT) described in Example 1. The results are presented in Figures 8a and 8b. No significant binding of B. burgdorferi could be seen when ELT was performed on blood from mice infected with these pathogens.
  • Leptospira infected mice (inoculated as in Example 7) were guillotine bled 10 days post inoculation, and the blood was monitored for its reaction with exogenous Leptospira and B. burgdorferi .
  • the results are presented in Figure 9, in which the data is presented as a bar graph of the total percentage WBC bound/total WBC (%Cb) , and % neutrophils binding>3 Bb/total WBC (%Nb>3). It was found that binding occurs between the white blood cells and exogenous Leptospira, but not between white blood cells and exogenous B. burgdorferi . These results indicated that the ELT may be useful for other spirochetes.
  • ELT was performed on a total of twelve 7 month old male mice inoculated intraperitoneally with B. burgdorferi or BSK (control) (six mice each), and were guillotine bled, two mice per day (one infected, one control) at 0, 3, 7, 14, 21 and 28 days post inoculation.
  • B. burgdorferi or BSK (control) (six mice each)
  • guillotine bled two mice per day (one infected, one control) at 0, 3, 7, 14, 21 and 28 days post inoculation.
  • the results are presented in Figure 10. It was found that the ELT of male mice infected with B. burgdorferi was similar to the response found in females.
  • mice (as in the previous examples) were inoculated intradermally with B. burgdorferi (10 3 organisms) and bled, two mice per day (infected) at: 0, 3, 7, 10, 14, 17 and 21 days post inoculation, and the response in ELT and immunoblot was determined. The results are presented in Figures 11a and lib. ELT binding response was strong and serological response
  • ELT smears Close examination of ELT smears revealed excessive masses of platelets with bound B. burgdorferi , and occasional neutrophils in the group infected with B . burgdorferi and bled retroorbitally. Mice infected with B. burgdorferi but bled by decapitation showed a low level of platelet clumping. Control mice (media inoculated) showed essentially no platelet clumping whether retroorbitally or guillotine bled. WBC differential and reticulocyte numbers were similar among experimental and control groups. A slight increase in platelet clumping was observed in B. burgdorferi infected, retroorbitally bled mice. B.
  • B. burgdorferi infection plus repetitive vascular trauma may activate platelets, which then bind the exogenous B. burgdorferi added to whole blood in the ELT. B. burgdorferi bound to platelets are prevented from binding to neutrophils, resulting in inhibition of the WBC binding response.
  • DH Lyme disease symptoms for approximately 1 month, treated for 10 days with amoxicillin, symptoms returned after cessation of antibiotic; western blot: 41kDa response only; ELT: moderate binding response, consisting of only NB ⁇ 3;
  • TM no history of lyme disease, works in laboratory with B . burgdorferi ; ELT: minimal binding response;
  • Seronegative Lyme Disease Dissociation of Specific T- and B-Lymphocyte Responses to Borrelia burgdorferi . N. Engl. J. Med. 319:1441-1446.

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Abstract

Le procédé décrit permet de dépister de manière précoce la maladie de Lyme et d'autres maladies infectieuses au cours de l'évolution de la maladie. On a découvert une différence frappante dans le degré de liaison superficielle d'agents infectieux cultivés à des leucocytes polynucléaires naturels (par exemple, des neutrophiles) chez des animaux infectés et non infectés. Cette différence peut être présente tôt dans l'évolution de la maladie, avant toute réponse mesurable des anticorps. Cette réaction de liaison peut être détectée et quantifiée rapidement et avec précision au moyen de diverses techniques de détection. Selon ce procédé, on visualise la liaison superficielle des agents infectieux cultivés aux leucocytes polynucléaires naturels afin de détecter l'infection.
EP96921518A 1995-06-07 1996-06-07 Test de diagnostic precoce Withdrawn EP0830449A1 (fr)

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US08/659,369 US5985595A (en) 1995-06-07 1996-06-06 Early detection of Borrelia infection
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US7682619B2 (en) 2006-04-06 2010-03-23 Cornell Research Foundation, Inc. Canine influenza virus
US9395365B2 (en) * 2009-04-02 2016-07-19 Abbott Point Of Care, Inc. Detection of infectious disease in a human or animal by measuring specific phagocytosis in a thin film sample of their anticoagulated blood
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