EP3213076A1 - Protéines recombinantes de borrelia et utilisation de celles-ci - Google Patents

Protéines recombinantes de borrelia et utilisation de celles-ci

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Publication number
EP3213076A1
EP3213076A1 EP15782207.3A EP15782207A EP3213076A1 EP 3213076 A1 EP3213076 A1 EP 3213076A1 EP 15782207 A EP15782207 A EP 15782207A EP 3213076 A1 EP3213076 A1 EP 3213076A1
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EP
European Patent Office
Prior art keywords
borrelia
antigens
recombinant
proteins
antibodies
Prior art date
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EP15782207.3A
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German (de)
English (en)
Inventor
Benjamin J. Luft
John F. Bruno
Yun Xu
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Research Foundation of State University of New York
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Research Foundation of State University of New York
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Publication of EP3213076A1 publication Critical patent/EP3213076A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • C07K14/20—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Spirochaetales (O), e.g. Treponema, Leptospira
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/20—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Spirochaetales (O), e.g. Treponema, Leptospira
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/26—Infectious diseases, e.g. generalised sepsis

Definitions

  • Lyme disease is the most common vector-borne disease in North America and Europe, and its range and incidence are increasing. Human Lyme disease is caused by several members of a group of closely related spirochetes belonging to the Borrelia burgdorferi sensu lato species complex. The spirochete is transmitted to humans via ticks of the genus Ixodes (Steere, A. C, N. Engl. J.Med. 1989; 321 :586- 96). It is a progressive multisystem disorder characterized by an initial cutaneous infection that can spread early in infection to secondary sites that include the nervous system, heart and joints (Masuzawa, T. et al, Microbiol. Immunol.
  • the present invention encompasses novel recombinant Borrelia burgdorferi proteins and their use in highly sensitive and specific methods of diagnosing Lyme disease in a subject.
  • the present invention is drawn to recombinant Borrelia fusion protein constructs comprising at least three recombinant Borrelia burgdorferi protein antigens. These constructs are also referred to herein as Borrelia chimeric protein antigens, or Borrelia chimeras.
  • the fusion protein constructs are described in Tables 4, 5, 6 and 8 herein.
  • the present invention is also drawn to methods of using one, or more, or all, of these recombinant Borrelia fusion proteins in assays for assessing test samples for detecting the presence of antibodies to Borrelia burgdorferi in the test sample.
  • the Borrelia fusion proteins described herein e.g., Tables 4, 5, 6 and 8 can be used to prepare protein arrays, or microarrays, for use in the detection methods, as well as in ELISA, other immunological assays, or any assay method capable of detecting antibody-antigen binding or antibody-antigen binding complexes.
  • the microarray comprises the 25 recombinant Borrelia antigens and chimeric proteins of Table 6. In another embodiment, the microarray comprises the 11 recombinant Borrelia antigens and chimeric proteins of Table 8.
  • a subject such as a mammal, including a human
  • methods of diagnosing Lyme disease in a subject by assessing a test sample obtained from the subject for antibodies reactive with one, or more of the recombinant Borrelia burgdorferi fusion proteins of Tables 4, 5, 6 or 8, or, additionally one, or more, of the recombinant Borrelia antigens listed in Table 1 , wherein the detection of antibody-antigen reactions (such as antibody-antigen bound complexes, also referred to herein as antibody-antigen reaction products) are indicative of Lyme disease in the individual.
  • antibody-antigen reactions such as antibody-antigen bound complexes, also referred to herein as antibody-antigen reaction products
  • Such assays to detect antibody-antigen complexes are well-known to those of skill in the art.
  • microarrays are used in the detection/diagnostic assays, wherein the microarrays comprise the recombinant Borrelia chimeric proteins described herein as in Tables 4, 5, 6 and 8, and also recombinant Borrelia antigens described in Table 1.
  • the microarray comprises the 25 recombinant Borrelia antigens and chimeric proteins of Table 6.
  • the microarray comprises the 11 recombinant Borrelia antigens and chimeric proteins of Table 8.
  • the Figure depicts the amino acid sequences of the chimeric antigens of Table 6 (SEQ ID NOS: 3-27) and variants of OC2/9 (SEQ ID NOS: 28-30). DETAILED DESCRIPTION OF THE INVENTION
  • this library of highly antigenic Borrelia proteins has been used to develop a set of recombinant Borrelia chimeric antigen proteins as shown in Tables 4, 5 and 6 and a highly sensitive and specific recombinant protein-based assay containing the 25 recombinant and chimeric antigens of Table 6, or the 11 recombinant antigens and recombinant chimeric antigens (Table 8) as described herein.
  • the overall sensitivity and specificity of the Lyme disease assay as described herein is shown in Tables 2, 3, 7, 9 and 10.
  • one or more recombinant cell envelope protein antigens, recombinant antigens and/or chimeric antigens are used.
  • a set of two or more antigens are used.
  • Other embodiments encompassed herein include three antigens, four antigens, five antigens and so forth, up to the 11 antigens of Table 8, the 16 antigens of Table 4, the 25 antigens of Table 6, the 96 antigens of Table 5 or the 48 antigens of Table 1, or all of the antigens from the afore-mentioned Tables.
  • Representative sets include the set of proteins shown in Tables 1, 4, 5, 6, and 8.
  • Such sets can further include homo logs and paralogs of the cell envelope proteins.
  • the set consists essentially of the antigens set forth in Table 6.
  • the set consists essentially of the antigens set forth in Table 8.
  • one or more other proteins as identified herein are used, other combinations of proteins are used (e.g., proteins selected from any of Tables 1, 4, 5, 6 and 8, in combination).
  • Such combinations include sets of two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or other groups of proteins (e.g., with one or more selected from Table 1, Table 5 and/or Table 6).
  • test sample from an individual is assessed for the presence of antibodies to one or more proteins (e.g., recombinant or chimeric antigens) of B. burgdorferi).
  • the "test sample” is a sample of blood, serum, cerebrospinal fluid, or other appropriate biological fluid from the individual.
  • the test sample is assessed for the presence of antibodies to one or more proteins using routine methods established in the art.
  • the assessment is performed using a microarray of Borrelia proteins.
  • an array eg.
  • a microarray as described below, or a protein or set of proteins as described herein is exposed to the test sample from the individual, and any resultant binding of antibodies (if present in the test sample) to the proteins is determined.
  • the presence of antibodies bound to (also referred to herein as "reactive with") one, or more, antigens is indicative of antibodies specific to those proteins of B. burgdorferi.
  • the presence of such antibodies is diagnostic for Lyme disease in the individual from whom the test sample was obtained.
  • the assay methods described herein encompass arrays of B. burgdorferi antigens.
  • the array comprises a subset of recombinant proteins of B. burgdorferi, such as the set the proteins set forth in Tables 1, 4, 5, 6 or 8.
  • other arrays include various subsets of proteins of B.
  • microarray consists essentially of all of the proteins set forth in Table 6, or consists of all of the proteins set forth in Table 6.
  • the microarray consists essentially of all of the proteins set forth in Table 8, or consists of all of the proteins of Table 8.
  • other microarrays include various subsets of proteins of B. burgdorferi, such as subsets of the proteins set forth in Table 1 or in Table 5 (e.g., sets of two or more, four or more, six or more, eight or more, or other groups of proteins as set forth in Table 6 or Table 8).
  • inventions of the present invention encompass methods for the evaluation/assessment of other or additional proteins of B. burgdorferi as potential candidates for development of a diagnostic test for Lyme disease, and also for assessment of proteins of B. burgdorferi as potential candidates for development of vaccines to protect against Lyme disease.
  • one or more proteins of B. burgdorferi such as described herein in Table 6 or Table 8 (e.g., in a microarray as described above), are exposed to sera from one or more individuals known to have Lyme disease, and the proteins to which antibodies from the sera bind are then determined.
  • Cy5 intensity/Cy3 intensity ratio of fluorescence as described in the Examples, can be used.
  • any proteins greater than the mean ratio of the reactivity of the Lyme sera to a negative control plus three times the standard deviation indicates significant interactions between antibodies present in the Lyme sera and the B. burgdorferi protein. It is reasonable to believe that such proteins are useful for vaccine compositions to elicit an immune response in a subject, as described below.
  • Such proteins are also proteins which can be used in diagnostic tests for Lyme disease (e.g., in the methods described above), as well as in microarrays as described herein, and additionally can be used as potential vaccine candidates.
  • the present invention is also drawn to pharmaceutical compositions which can be used to vaccinate and/or treat Borrelia infection in an animal or human.
  • the pharmaceutical composition comprises one, or more, Borrelia burgdorferi antigen, such as one shown in Tables 5, 6 or 8, or another Borrelia burgdorferi antigen, such as one shown in Table 1 , or a protein derived from a cell envelope protein or other protein (e.g., a protein having modifications such as insertions, deletions, or other alterations, or a protein that forms part of a chimeric protein, such as those described in U.S.
  • the pharmaceutical composition can also be administered together with a physiologically-acceptable carrier, an excipient and/or an adjuvant.
  • Suitable adjuvants are well known in the art (see for example PCT Publication WO 2011/00110060600A1100A1100A1100A1100A1100A1100A1100A1100A1100A1100A1100A1100A1100A1100A1100A1100A1100A, a pharmaceutically-acceptable carrier, an excipient and/or an adjuvant.
  • Suitable adjuvants are well known in the art (see for example PCT Publication WO
  • the pharmaceutical compositions used to vaccinate and/or treat Borrelia infection can be prepared using methods for preparing vaccines which are well known in the art.
  • the proteins described herein can be isolated and/or purified by known techniques, such as by size exclusion chromatography, affinity chromatography, ion exchange chromatography, preparative electrophoresis, selective precipitation or combinations thereof.
  • the prepared proteins can be mixed with suitable other reagents as described herein, such that the protein is at a suitable concentration.
  • the dosage of the protein will vary and depends upon the age, weight and/or physical condition of the animal, e.g., mammal, human, to be treated.
  • the optimal dosage can be determined by routine optimization techniques, using suitable animal models.
  • Administration of the pharmaceutical composition to be used as a vaccine can be by any suitable technique.
  • suitable techniques for administration of the pharmaceutical composition include, but are not limited to, injection, e.g., subcutaneous injection, intramuscular injection, intravenous injection, intra peritoneal injection; mucosal administration, e.g., exposing nasal mucosa to nose drops containing the proteins of the present invention; oral administration; and DNA immunization.
  • proteins identified herein as reacting with sera of individuals with Lyme disease can be used as prognostic markers enabling one skilled in the art to tailor treatment for disease by targeting those specific proteins. For example, should an individual's serum demonstrate reactivity with a particular subset of proteins, therapy can be initiated to reduce and/or eliminate the presence of those proteins in the individual, as shown by reducing and/or eliminating reactivity of the individual's serum with those proteins.
  • the present invention is also drawn to diagnostic and/or prognostic kits which comprise the proteins described herein (e.g., in a microarray as described above).
  • the kit also includes reagents for detecting antibody-antigen complexes that are formed between the protein and antibodies that are present in a sample, e.g., a user-supplied host sample.
  • Protein arrays A total of 416 genes were identified in the B. burgdorferi genome comparison. Of these, 350 (84%) produced a product that was the correct size when PCR was performed, and all 350 were successfully cloned into the T7 expression vector pET28b. Sequence confirmed plasmids were expressed using the overnight autoexpression system; expressed proteins were purified using IDA resin and printed onto nitrocellulose coated FAST slides (see for example, US Serial No. 12/784,584 and US Serial No. 12/989,003, the teaching of which are incorporated herein). In addition, representatives of several different OspC types were amplified from human isolates.
  • the OspC types included in this study were types A, B, C, D, E, F, G, H, I, J, K and U.
  • This study also included the highly antigenic B31 cell envelope proteins that were identified in an earlier protein array study (Xu Yet al, 2008). In total, the arrays contained approximately 400 samples including expressed Borrelia proteins, negative controls and blanks. Arrays were probed with sera from Lyme disease patients and antibodies were visualized with Cy5 -conjugated goat antihuman lgG or Cy5 -conjugated goat anti-human lgM to determine antibody isotype (Xu Y et al, 2008).
  • CDC Centers for Disease Control
  • samples were obtained from Dr. Mark Soloski, Johns Hopkins School of Medicine, Baltimore MD.
  • a total of 46 samples per group were analyzed.
  • sera were obtained from patients with diseases associated with serological responses that are known to produce cross- reactivity in Lyme disease tests. These included patients with syphilis, lupus, and rheumatoid arthritis. Normal control sera were obtained from healthy donors.
  • EXAMPLE 2 Protein Arrays using Recombinant Chimeric Borrelia Proteins. Overview. A library of chimeric proteins using 48 highly antigenic Borrelia proteins was designed for use in developing a recombinant chimera based diagnostic assay for Lyme disease. Construction of recombinant chimeras containing genes from several genospecies allows one to generate one protein that confers antigenicity to multiple strains, revealing the great potential and adaptability of this technique. In addition, the use of pure protein preparations as antigens offers greater flexibility in adapting the test to different assay formats. It is reasonable to believe that an assay of recombinant chimeric proteins will present superior sensitivity in detecting antibodies against B. burgdorferi for the early stages of the disease, and equivalent sensitivity for the late stages of the disease, when compared to the best whole-cell assays.
  • BB_0312 purine-binding chemotaxis protein (BB_0744,Bbu297_W44,BB_A74)
  • BB_0546 conserved hypothetical protein BB_0744/Bbu297_W44/BB_A74
  • BbuJDl_S13 conserved hypothetical protein BbuJDl_S13/BB_H18/BbuN40_X36
  • BB_0858 hypothetical protein BB_0067a/BB_A16/BB_0024
  • BB_A14 Borrelia orf-D family BB_A16/BB_0024/BB_0067a
  • BB_A16 outer surface protein B (OspB) BB_A16/BB_0067a/BB_0024
  • BB_ 22 conserved hypothetical protein BbuJDl_F31/Bbu297_F32/BB_0337
  • BbuJDl_F31 vmp-like sequence (Vis) protein BB_S42/BB_0364/BB_A66
  • burgdorferi antigens were amplified using a unique gene-specific primer pair that we specifically designed for chimeric protein construction.
  • the primers were designed from the genomic sequence of B. burgdorferi strains and each contains a novel DNA cassettes coding for the recognition sequence for three restriction
  • the 5' primer (5 * -AATTGGTACCCCAGGATCCCATATG + 15MER ORF specific sequence) (SEQ ID NO: l) contains Kpnl (underlined), BamHI (bold) and Ndel (italics) recognition sequence.
  • the 3 * primer (5'GCGGGATCCGGTACCGTCGAC +15mer ORF specific sequence) (SEQ ID NO:2) contains BamHI, Kpnl and Sail (dashed underline) recognition sequences. For amplification, ten ng of genomic DNA were used as template in a 50- ⁇ PCR reaction containing two ORF-specific primer pairs.
  • primer sets were designed to amplify coding regions without a membrane anchoring signal sequence (Dunn et al., 1990).
  • PCR amplification was performed under stringent conditions with Platinum Taq DNA polymerase High Fidelity (Invitrogen) using conditions we have previously described (Xu et al, 2003).
  • the PCR products were visualized by agarose gel electrophoresis.
  • the products were purified (PCR purification kit, Qiagen) and quantified by UV absorbance.
  • amplified products were cleaved with Ndel/BamHI (for VI position fragment), or BamHI/Kpnl (for 2nd position fragment) or Kpnl/Sall (for 3rd position fragment).
  • the 1st position fragments were directionally cloned into the unique Ndel and BamHI sites of the T7-based expression vector pET-28 (Novagen).
  • This vector provides an N-terminal poly (His) affinity tag fused to the expressed proteins to aid in purification on nickel-Sepharose columns.
  • Plasmids containing fragment 1 now served as vectors for all subsequent cloning.
  • vectors were digested with BamHI/XhoIl, and restriction digested amplicons of fragment two and three were ligated simultaneously into the digested vector. Following transformation, plasmids were purified and sequenced.
  • Serum samples Serum samples. The antigenicity of the chimeric proteins was tested using CDC sera samples C (12 samples) and E (11 samples), and Baltimore sera samples VI (28 samples) and V2 (23 samples), described above. In addition, 32 more sera samples were obtained from the CDC (CDC-R1). These included sera from 12 Lyme patients. 12 patients with diseases associated with cross-reactivity in Lyme disease tests and 8 normal controls. Moreover, 35 sera from patients with diseases associated with serological responses that are known to produce crossreactivity in Lyme disease tests were obtained from Bioreclamation LLC, Westbury, NY. These included patients with syphilis, lupus, and rheumatoid arthritis. Seven normal control sera were also obtained from healthy donors.
  • Microarray For protein microarray, recombinant proteins and each of the individual 48 Borrelia antigens were printed onto nitrocellulose-coated FAST glass slides. In addition, recombinant proteins OspB-OspC-Flagellin (B-C-Fia), OspA- p39-p93 (A-39-93) and an OspC dimer comprised of OspC Type Band Type H (OC2/9) from an early study were also printed (Gomes Solecki et. al, 2000). Each slide in the arrays also contained 10 immobilized BSA spots for background determination. Proteome chips were probed with serum from patients with untreated early Lyme disease and sick non-Lyme patients using the Fast Pak protein array kit.
  • slides were first blocked overnight at 4°C in protein array-blocking buffer before incubation in primary antibody (human sera and mouse anti His-Tag for quantitation) for 2 h.
  • Antibodies were visualized with Cy5 -conjugated goat anti- human lgG or Cy5 -conjugated goat anti-human lgM (to detect bound human antibodies) and Cy 3 -conjugated goat anti mouse lgG (to quantify the amount of recombinant protein in each spot).
  • the slides are' stringently washed and then scanned with an Axon GenePix 4200 A microarray scanner. The raw data was captured and analyzed with Gene Pix Pro image analysis software.
  • the PMT gain was adjusted to equall .O in all the arrays with power setting at 50%.
  • a global background subtraction method was used to subtract the background from each spot using the average mean intensity value of BSA from each slide (Xu et al., 2008).
  • the spot was considered positive and included for further ratio analysis if the median fluorescence intensity of a spot is more than 1000 and the SNR (signal- noise-ratio) of a spot is more than 4.
  • SNR signal- noise-ratio
  • a serum panel composed of samples from patients with early Lyme disease was used to screen arrays of chimeric proteins to determine their sensitivity to detect anti-5. burgdorferi antibodies. Although a considerable amount of heterogeneity in reactivity of individual serum samples to the arrayed proteins was observed, there was remarkable consistency in the reactivity of individual samples to the 6 combinations of each recombinant protein. In fact, among the 288 arrayed proteins, the majority of them delectably elicited an antibody response in humans with natural infections using both secondary antibodies. Moreover, the chimeric constructs did not vary significantly in their immunoreactivities when compared to individual proteins. Although there were sample-specific responses, there was a subset of proteins recognized in common by a majority of the sera.
  • variants were made of chimeric antigen OC2/9 (SEQ ID NO: 27) and are shown as SEQ ID NOS: 28-30 of the Figure. These variants, as well as variants of the other chimeric antigens described herein, can be made and tested for efficacy using the procedures as described herein.
  • the overall sensitivity and specificity of the Lyme assay for this set of sera specimens are shown below in Table 7.
  • 5 protein arrays containing 25 recombinant Borrelia proteins out performed 2 tier testing using the Lyme disease serum panel described above.
  • the sensitivity of the protein array described herein was compared with two-tier testing using 29 of the early Lyme disease patients.
  • the sensitivity of the instant protein array with lgM or lgG secondary antibody was 10 100% at initial presentation (Samples VI) and 87% during convalescence, 3 weeks later (Samples V2). In comparison, the percentages were 48% and 71%, respectively, for standard 2-tiered testing.
  • Table 7 Sensitivity and specificity of protein arrays using IgM and IgG secondary antibodies.
  • V2 three week 20/28 19/23 18/23 20/23 follow-up (71) (83) (78) (87)
  • Serum samples Serum samples. Sera from 70 Lyme disease patients were used to the sensitivity of protein arrays of the 25 recombinant proteins. This included 28 early Lyme patients from the CDC, Baltimore samples VI and V2 described above, and 8 Lyme arthritis, 6 neurologic Lyme and 2 cardiac Lyme patients also from the CDC. In addition, for specificity studies, sera were obtained from 83 patients (sick non-Lyme) with diseases associated with serological responses that are known to produce cross- reactivity in Lyme disease test including syphilis, fibromyalgia, and rheumatoid arthritis. Normal control sera were obtained from 16 healthy donors from an area of endemicity and 36 healthy donors from non-endemic areas.
  • Microarray For protein microarray, the 25 recombinant proteins of Table 6 were printed onto nitrocellulose-coated FAST glass slides. Proteome chips were probed with serum and analyzed as described above.
  • the overall sensitivity of our array of 11 proteins is shown in Table 9.
  • the sensitivity of the protein array with lgM or lgG secondary antibody was 71% for sera from the CDC and for the Baltimore samples 88% at initial presentation (Samples VI) and 88%> during convalescence, 3 weeks later (Samples V2). In comparison, the sensitivity was 44%> for standard 2-tiered testing for these same early Lyme patients.
  • the sensitivity of the array was 100% for Lyme arthritis, neurologic Lyme and cardiac Lyme sera (Table 9).
  • Table 9 Serologic Responses in Well-Characterized Lyme disease Patients: Sensitivity.
  • the specificity of the 11 antigen array assay was evaluated by testing sera from normal controls and from patient with diseases associated with serological responses that are known to produce cross-reactivity (Sick-non-Lyme) in currently used tests. Among the 52 healthy controls from areas in which Lyme disease is endemic, none had positive results with the assay. For the potential cross reactive patients 11 of 83 samples had positive IgM or IgG antibody responses in our assay. Thus, the overall specificity for the array assay was 100% for normal controls and 87% in Sick-non-Lyme patients.
  • microarray assays described herein offer superior sensitivity, specificity and precision in detecting antibodies against B. burgdorferi for not only early stages of the disease, but shows equivalent sensitivity for the late stages of the disease as well.
  • a standardized sensitive and specific single-tier Lyme disease assay of recombinant chimeric Borrelia proteins that will potentially have a wide range of coverage and specificity against Lyme disease is now available.
  • these assays will have significant commercial potential for the development of a next-generation rapid, single-tier point of care assay.

Abstract

La présente invention concerne des protéines de Borrelia burgdoferi recombinantes et des procédés d'analyse d'un échantillon en vue de détecter la présence d'anticorps dirigés contre certaines protéines de Borrelia burgdorferi, ainsi que des méthodes de diagnostic de la maladie de Lyme. L'invention concerne également des microréseaux de protéines de Borrelia burgdorferi.
EP15782207.3A 2014-10-07 2015-10-06 Protéines recombinantes de borrelia et utilisation de celles-ci Withdrawn EP3213076A1 (fr)

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WO2018017998A1 (fr) 2016-07-22 2018-01-25 The Research Foundation For The State University Of New York Protéines recombinantes de borrelia et leurs procédés d'utilisation
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CN113302493A (zh) * 2018-10-01 2021-08-24 小利兰·斯坦福大学托管委员会 同时解析多种抗体同种型的高特异性和灵敏度免疫吸附诊断测定

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