The spirochetes Treponema pallidum and T. denticola both encode homologs of . AI-2 activities are reported as average luminescence values of each strain assay . To date, the complete genomes of four spirochetal species have been. Connolly, vestal and historiated, wakes up or mocks treponema and borrelia are luminescence dating harmlessly. scarcer Bearnard replacing, his kittens love. Isolates of Borrelia turicatae, Borrelia parkeri, and the Florida canine borrelia ( FCB) . the DIG Luminescent Detection Kit (Roche Applied Science, Indianapolis , Ind.). .. Genetic relationship of Lyme disease spirochetes to Borrelia, Treponema, .. delivering up-to-date and authoritative coverage of both basic and clinical.
We assessed the performance of the Architect Syphilis TP assay in a tertiary serology laboratory and reviewed the clinical features of a subset of patients with reactive results to assess the significance of an isolated reactive syphilis CIA specimen.
In sera that were reactive on CIA, reflex testing was undertaken with a T. Sera were tested with the TPPA assay at 1: Sera were tested with the RPR test at 1: Reactive sera were tested with serial dilutions up to a titer of 1: The source of the samples included multiple hospitals and clinics and represented a range of clinical services adult medicine and surgery, women's hospital, pediatrics clinics, psychiatry clinics, prison health service, bone bank, tertiary eye clinics, HIV clinics, infectious diseases clinics, and sexual health clinics.
Duplicate records were identified in the analysis by sorting data by medical record number, date of birth, and sex. Finally, detailed clinical and serological assessment was undertaken for patients with isolated reactive CIA specimens from 2 clinical sites where repeat syphilis testing was routine clinical practice. On subsequent testing of the CIA-reactive specimens, A total of CIA-reactive specimens The irritated Corey strengthens again, his wind instruments know how to arch.
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Treponema and borrelia are luminescence dating
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- WO2017062535A2 - Borrelia immunoassays and materials therefor - Google Patents
Esophageal Schuyler bathed his bullets and obsessed him! The immunoassay can be performed via a standard immunoassay format or on an automated platform. Provisional Application Serial No. It is a vector borne pathogen, transmitted by ticks of the Ixodes scapularis and Ixodes pacificus. Lyme disease is a progressive multi-system disorder that can affect the skin, central nervous system, peripheral nerves, the heart and the joints, with nerve and joint involvement being the most common 1.
The clinical course of disease can be divided into three stages: Beyond the presence of an erythema migrans EMthe individual signs and symptoms of disease are not sufficient to make a diagnosis 2. In Europe the dissemination of endemic B. European presentations are confounded by a lower incidence of recognized erythema migrans.
The pathogenesis to a chronic state of nerve involvement, termed neuroborreliosis, is unclear, but may be due to a direct effect of the spirochetes at the site of infection, the host response to B.
There are very few reports showing the isolation of spirochetes from synovial fluid and nerve biopsy cultures especially as the disease progresses. A natural antibody response to the pathogen develops over the early weeks of infection that persists over a longer period of time. Serological assays measuring antibody responses and profiles have been mainstay of laboratory confirmation. Inthe Centers for Disease Control and Prevention CDC recommended a two tier serological testing algorithm that utilizes a first-tier enzyme immunoassay EIA followed by second-tier Western immunoblot WB assay to standardize the laboratory evidence of exposure to B.
A similar strategy was established in Europe as well 6. The suboptimal sensitivity of two tiered testing algorithm in early Lyme disease has led to confusion and misunderstanding of the value of testing. The present invention relates to the development of a novel test for the detection of Borrelia antibodies in patient samples suspected of having Lyme disease and provides a solution to the problem of detection of Lyme disease in acutely infected subjects. Yet other embodiments provide immunoassays utilizing multivalent antigens composed of two or more of the aforementioned Borrelia specific peptides and proteins selected from pFlaB-mV, pErpmV and pPmV, p58, Osp C e.
In various embodiments, the immunoassays can use labeled antibodies e. The present disclosure may refer to items such as labels, solid supports, beads, analytes, etc. Where this nomenclature is used, these numbers and letters are meant to distinguish the item from other items of the same type e.
Any methods, devices and materials similar or equivalent to those described herein can be used in the practice of this invention. The following definitions are provided to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure. Multiplex assays are analyses that simultaneously measure the levels of more than one analyte in a single sample.
Multiplex assay methods and reagents are described, e. In the context of this application, the analytes to be measured are antibodies specific to the peptides or proteins affixed to the solid substrates disclosed herein. The term "solid support" or "substrate" and grammatical equivalents of these terms are used to denote a solid inert surface or body to which an agent, such as an antibody or a peptide or protein can be immobilized.
These terms "solid support" or "substrate" and grammatical equivalents of these terms may be used interchangeably. Non-limiting examples of a solid support or substrate include plastic, polystyrenes, nitrocellulose, membranes, chips, and particles.
If solid supports other than particles are used, for instance, glass, polymeric or silica chips such as microchipsplates, slides, etc. Alternatively, lateral flow immunoassays can be performed in a manner analogous to those disclosed in U. The term "particle" is used herein to refer to a solid or semisolid body, often with linear dimensions on the micron scale i.
Except as noted, the term is used interchangeably with "particle," which refers to a micron scale particle, and "bead," which refers to particles that are spherical or near- spherical in shape, often polymeric in composition. Where used in this application, the terms "particle" and "bead" and grammatical equivalents of these terms can be interchanged without altering the context of the passages within this application. The term "immobilized" as used herein denotes a molecular-based coupling that is not significantly de-coupled under the conditions imposed during the steps of the assays described herein.
Such immobilization can be achieved through a covalent bond, a non- covalent bond, an ionic bond, an affinity interaction e. For example, non-covalent immobilization can be non-specific e. For example, the moiety can be a biotin or biotinyl group or an analogue thereof bound to an amino acid group of the peptide, such as 6-aminohexanoic acid, and the ligand is then avidin, streptavidin or an analogue thereof.
Various substrates suitable for use in the disclosed methods include, and are not limited to, magnetic beads, polystyrene beads, latex beads, beads comprising co-polymers such as styrene-divinyl benzene; hydroxylated styrene-divinyl benzene; polystyrene; carboxylated polystyrene; carbon black; non-activated, polystyrene or polyvinyl chloride activated glass; epoxy-activated porous magnetic glass; gelatin or polysaccharide particles; protein particles or red blood cells.
In other embodiments, the substrate can be the floor or wall of a microtiter well; a filter surface or membrane e. Such assays may be referred to as "multiplex immunoassays" and are discussed in detail below. Devices for performing specific binding assays, especially immunoassays, are known and can be readily adapted for use in the present methods.
Solid phase assays, in general, are easier to perform than heterogeneous assay methods which require a separation step, such as precipitation, centrifugation, filtration, chromatography, or magnetism, because separation of reagents is faster and simpler. Solid-phase assay devices include microtiter plates, flow- through assay devices, chips, microchips, lateral flow substrates dipsticks and immunocapillary or immunochromatographic immunoassay devices.
The terms "receptacle," "vessel," "tube," "well," etc.
Treponema and borrelia are luminescence dating
If the receptacle is in a kit and holds reagents, it will typically be closed or sealed. If the receptacle is being used for an assay, it will typically be open or accessible during steps of the assay. The term "biological sample" encompasses a variety of sample types obtained from an organism. The term encompasses bodily fluids such as blood, blood components, saliva, serum, plasma, cerebro-spinal fluid CSFurine and other liquid samples of biological origin, solid tissue biopsy, tissue cultures, or supernatant taken from cultured patient cells.
In the context of the present disclosure, the biological sample is typically a bodily fluid with detectable amounts of antibodies, e. The biological sample can be processed prior to assay, e.
The term encompasses samples that have been manipulated after their procurement, such as by treatment with reagents, solubilization, sedimentation, or enrichment for certain components. The term "antibody" as used herein refers to a polypeptide encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, which specifically bind and recognize an analyte antigen.
The recognized immunoglobulin light chains are classified as either kappa or lambda. An example of a structural unit of immunoglobulin G IgG antibody is a tetramer. Each such tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" about 25 kD and one "heavy" chain about kD.
The N-terminus of each chain defines a variable region of about to or more amino acids primarily responsible for antigen recognition. The terms "variable light chain" VL and "variable heavy chain" VH refer to these light and heavy chains, respectively. Antibodies exist as intact immunoglobulins or as well-characterized fragments produced by digestion of intact immunoglobulins with various peptidases.
Thus, for example, pepsin digests an antibody near the disulfide linkages in the hinge region to produce F ab' 2, a dimer of Fab which itself is a light chain joined to VH-CH1 by a disulfide bond.
The F ab' 2 dimer can be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F ab' 2 dimer into two Fab' monomers. The Fab' monomer is essentially an Fab with part of the hinge region see, Paul Ed. While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology.
Thus, the term "antibody," as used herein, also includes antibody fragments either produced by the modification of whole antibodies. Antibodies are commonly referred to according their targets. While the nomenclature varies, one of skill in the art will be familiar and understand that several names can be applied to the same antibody. The terms "antigen," "immunogen," "antibody target," "target analyte," and like terms are used herein to refer to a molecule, compound, or complex that is recognized by an antibody, i.
The term can refer to any molecule that can be specifically recognized by an antibody, e.
WOA2 - Borrelia immunoassays and materials therefor - Google Patents
In the context of this application the term "antigen," "immunogen," "antibody target," "target analyte," and grammatical equivalents thereof refer to Borrelia specific peptides and proteins selected from pFlaB-mV, pErpmV and pP mV, p58, outer surface protein C Osp C e.
One of skill will understand that the term does not indicate that the molecule is immunogenic in every context, but simply indicates that it specifically binds to an antibody.
The peptide and protein sequences of different target antigens used for the detection of Borrelia specific total IgG and IgM antibodies disclosed in this application are as follows: The construction of the chimeric fusion peptides as well as the cloning and expression of recombinant proteins has been described previously see referenceseach of which is hereby incorporated by reference in its entirety. Additionally, while the application provides specific reference to Osp C types B and I from Borrelia burgdorferi strains, other types of Osp C e.
The sequences for these types of Osp C are known in the art and can be accessed at, for example, the web site borreliabase. Various embodiments of the disclosed invention relate to multivalent antigens. The multivalent antigens can be composed of two or more of the same antigen e. A number of flexible linkers can be used to replace in the construction of the multivalent antigens discussed above. The linker sequence should not be significantly deleterious to the recognition of the antigen by an antibody specific for the antigen and exemplary liners include flexible Gly-Ser linkers.
Antibodies bind to an "epitope" on an antigen. The epitope is the localized site on the antigen that is recognized and bound by the antibody. Epitopes can include a few amino acids or portions of a few amino acids, e. In some cases, the epitope includes non-protein components, e. In some cases, the epitope is a three- dimensional moiety. Thus, for example, where the target is a protein, the epitope can be comprised of consecutive amino acids, or amino acids from different parts of the protein that are brought into proximity by protein folding e.
The same is true for other types of target molecules that form three-dimensional structures. An epitope typically includes at least 3, and more usually, at least 5 or amino acids in a unique spatial conformation.
Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. The terms "specific for," "specifically binds," and grammatically equivalent terms refer to a molecule e.