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WO2008021862A2 - Procédés et COMPOSITIONS pour la DéTECTION OF MYCOPLASMA PNEUMONIAE - Google Patents

Procédés et COMPOSITIONS pour la DéTECTION OF MYCOPLASMA PNEUMONIAE Download PDF

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Publication number
WO2008021862A2
WO2008021862A2 PCT/US2007/075441 US2007075441W WO2008021862A2 WO 2008021862 A2 WO2008021862 A2 WO 2008021862A2 US 2007075441 W US2007075441 W US 2007075441W WO 2008021862 A2 WO2008021862 A2 WO 2008021862A2
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Prior art keywords
pneumoniae
sample
antibody
protein
antibodies
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WO2008021862A3 (fr
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Alan H. Davis
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Abbott Diagnostics Scarborough Inc
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Binax Inc
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/56933—Mycoplasma
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
    • C07K16/1289—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Mycobacteriaceae (F)

Definitions

  • Mycoplasma pneumoniae is an extracellular pathogen that attaches to and destroys ciliated epithelial cells of the respiratory tract mucosa.
  • M. pneumoniae is believed to be responsible for approximately 20% to 30% of community acquired pneumonia infections and has been implicated in asthma and chronic obstructive pulmonary disease.
  • M. pneumoniae infections can lead to gastrointestinal, hematologic, neurologic, dermatologic, musculoskeletal, joint and cardiovascular pathologies, in part manifested as arthritis, pericarditis, and central nervous system disorders.
  • the pathogenic potential of M. pneumoniae is substantial, and it is becoming clear that M. pneumoniae-associatcd diseases are greatly underreported because of the wide diversity of clinical manifestations linked to M.
  • M. pneumoniae infections difficulties in cultivating these pathogens directly from clinical specimens and inadequate diagnostic tools.
  • Diagnosis of the M. pneumoniae pathogen responsible in these cases is difficult and is usually based on comparison of serum antibody titers between acute and convalescent phases. A fourfold or greater rise in antibody titer in the latter compared to the former indicates the patient was infected with the organism. Improved methods of detecting M. pneumoniae are thus needed.
  • compositions, methods and devices for detecting M. pneumoniae We have discovered novel epitopes of M. pneumoniae that may be used to generate antibodies and other agents capable of specifically binding the epitopes. These antibodies and other compounds may be incorporated into a variety of methods for the standardized, sensitive and/or specific detection of M. pneumoniae. [0004] The methods and compositions may be further incorporated into any test format or device suitable for the practice of the methods. Also provided are kits, reagents, etc. for the practice of the methods.
  • FIGURE 1 depicts the sequence of recombinant CARDS ("rCARDS”) protein (SEQ ID NO: 1
  • Epitopes recognized by polyclonal anti-rCARDS rabbit antisera are in italics.
  • the red S/I represents one of the known amino acid changes in different isolates.
  • FIGURE 2 depicts peptides encompassing the identified rCARDS epitopes were synthesized (FIGURE 2).
  • the shaded regions in FIGURE 2 represent amino acids not included in M. pneumoniae rCARDS and are added to enable solubility in aqueous media and to allow attachment of the peptides to solid phases through the sulfur atom contained in cysteine residues.
  • FIGURE 3 depicts rCARDS epitope sequences (SEQ ID NOS 2 through 6) that were synthesized as well as their location in the rCARDS protein.
  • FIGURE 4 depicts the results of all possible combinations of antigen capture and detection assays using the starting IgG pool and affinity purified anti-peptide antibodies.
  • FIGURE 5 shows that antigen capture assays using the starting IgG pool, a combination of anti peptide (3, 4, and 6) antibodies and anti-peptide 4 antibodies were all equally efficacious. Anti-peptide 2 conjugate was used for detection. The figure legend is as indicated next to the graph.
  • FIGURE 6 depicts results of an antigen capture assay challenged using a second recombinant protein, Pl.
  • Antigen capture was effected using the starting IgG pool; a combination of anti-peptides 3, 4, and 5; or anti-peptide 4 antibodies.
  • Anti-peptide 2 or the IgG pool conjugate were used for detection.
  • the figure legend is as indicated next to the graph.
  • FIGURE 7 depicts the sequence of a fragment of recombinant Pl (“rPl”) protein
  • FIGURE 8 represents an isometric view of an exemplary assay device that may be used to implement the methods described herein.
  • FIGURE 9 represents a cross-sectional side elevation of the device shown in
  • FIGURE 10 shows an enlarged view of the sample collector, macroporous body and test strip in the device illustrated in FIGURES 8 and 9.
  • FIGURE 11 depicts the results of rCARDS detection using an anti-rCARDS immuno chromatographic device, as described in Example 4.
  • FIGURE 12 depicts a Western blot of various extractions of ⁇ -irradiated M pneumoniae using Zwittergen.
  • FIGURE 13 depicts the results of an antigen capture EIA used to determine the efficacy of Zwittergen extraction from cultured M. pneumoniae cells.
  • FIGURE 14 depicts the results of an antigen capture EIA used to determine the efficacy of Zwittergen extraction of CARDS from Mycoplasma cells spiked into buffers with or without detergent.
  • the buffers were pre-conditioned by addition of throat and oral swabs made from an individual.
  • amino acid is intended to embrace all molecules, whether natural or synthetic, which include both an amino functionality and an acid functionality and capable of being included in a polymer of naturally-occurring amino acids.
  • exemplary amino acids include naturally-occurring amino acids; analogs, derivatives and congeners thereof; amino acid analogs having variant side chains; and all stereoisomers of any of any of the foregoing.
  • antibody refers to an immunoglobulin, derivatives thereof which maintain specific binding ability, and proteins having a binding domain which is homologous or largely homologous to an immunoglobulin binding domain. These proteins may be derived from natural sources, or partly or wholly synthetically produced. An antibody may be monoclonal or polyclonal.
  • the antibody may be a member of any immunoglobulin class, including, for example, any of the classes: IgG, IgM, IgA, IgD, and IgE.
  • immunoglobulin class including, for example, any of the classes: IgG, IgM, IgA, IgD, and IgE.
  • antibodies used with the methods and compositions described herein are derivatives of the IgG class.
  • antibody fragment refers to any derivative of an antibody which is less than full-length. In exemplary embodiments, the antibody fragment retains at least a significant portion of the full-length antibody's specific binding ability. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, scFv, Fv, dsFv diabody, and Fd fragments.
  • the antibody fragment may be produced by any means. For instance, the antibody fragment may be enzymatically or chemically produced by fragmentation of an intact antibody, it may be recombinantly produced from a gene encoding the partial antibody sequence, or it may be wholly or partially synthetically produced. The antibody fragment may optionally be a single chain antibody fragment.
  • the fragment may comprise multiple chains which are linked together, for instance, by disulfide linkages.
  • the fragment may also optionally be a multimolecular complex.
  • a functional antibody fragment will typically comprise at least about 50 amino acids and more typically will comprise at least about 200 amino acids.
  • purified refers to an object species that is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition).
  • a “purified fraction” is a composition wherein the object species comprises at least about 50 percent (on a molar basis) of all species present.
  • the solvent or matrix in which the species is dissolved or dispersed is usually not included in such determination; instead, only the species (including the one of interest) dissolved or dispersed are taken into account.
  • a purified composition will have one species that comprises more than about 80 percent of all species present in the composition, more than about 85%, 90%, 95%, 99% or more of all species present.
  • the object species may be purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single species.
  • a skilled artisan may purify a polypeptide of the invention using standard techniques for protein purification in light of the teachings herein. Purity of a polypeptide may be determined by a number of methods known to those of skill in the art, including for example, amino -terminal amino acid sequence analysis, gel electrophoresis and mass- spectrometry analysis.
  • Recombinant protein refers to a polypeptide which is produced by recombinant DNA techniques, wherein generally, DNA encoding the polypeptide is inserted into a suitable expression vector which is in turn used to transform a host cell to produce the heterologous protein. That is, the polypeptide is expressed from a heterologous nucleic acid.
  • sample refers to any sample potentially containing Mycoplasma pneumoniae bacteria.
  • a sample may be a bodily fluid such as blood, urine, mucous or saliva, or a respiratory sample, such as a nasopharyngeal wash or aspirate, nasal swab, nasopharyngeal swab, nasal wash, throat swab, transtracheal aspirate, broncho alveolar lavage, elution buffer used to wash a respiratory sample, etc.
  • a respiratory sample such as a nasopharyngeal wash or aspirate, nasal swab, nasopharyngeal swab, nasal wash, throat swab, transtracheal aspirate, broncho alveolar lavage, elution buffer used to wash a respiratory sample, etc.
  • isolated and/or purified polypeptides comprising epitopes of proteins from M. pneumoniae. These polypeptides are referred to herein collectively as "polypeptide epitopes of M. pneumoniae.”
  • isolated and/or purified polypeptides comprising epitopes of rCARDS protein (SEQ ID NO: 1) and rPl protein (SEQ ID NO: 7) from M. pneumoniae.
  • polypeptide sequences comprising epitopes of rCARDS protein are SEQ ID NOs: 2 through 6
  • polypeptide sequences comprising epitopes of rPl protein are SEQ ID NOs: 8 through 10.
  • Additional epitopes may be identified using the procedures outlined in the Exemplification for other M. pneumoniae proteins, as well as using methods for epitope identification as known to those of skill in the art. Further, other isolates or variants of the CARDS and Pl proteins may be used in such epitope identification methods. Such isolates or variants retain the same naturally-occurring activity of CARDS or Pl, but may have, amino acid substitutions, deletions, or additions, which substitutions may consist in whole or part by conservative amino acid substitutions.
  • polypeptides comprising epitopes may be produced using standard polypeptide synthesis methods as will be known to one of skill in the art. Alternatively, such polypeptides may be produced using recombinant techniques. Chemical synthesis of the polypeptides may be carried out using a variety of art recognized methods, including stepwise solid phase synthesis, semi- synthesis through the conformationally-assisted re- ligation of peptide fragments, enzymatic ligation of cloned or synthetic peptide segments, and chemical ligation. Native chemical ligation employs a chemoselective reaction of two unprotected peptide segments to produce a transient thioester-linked intermediate.
  • the transient thioester-linked intermediate then spontaneously undergoes a rearrangement to provide the full length ligation product having a native peptide bond at the ligation site.
  • Full length ligation products are chemically identical to proteins produced by cell free synthesis. Full length ligation products may be refolded and/or oxidized, as allowed, to form native disulfide-containing protein molecules, (see e.g., U.S. Patent Nos. 6,184,344 and 6,174,530; and T. W. Muir et al, Curr. Opin. Biotech. (1993): vol. 4, p 420; M. Miller, et al., Science (1989): vol. 246, p 1149; A.
  • modified polypeptide epitope of M. pneumoniae for such purposes as enhancing therapeutic or prophylactic efficacy, or stability (e.g., ex vivo shelf life, resistance to proteolytic degradation in vivo, etc.).
  • modified polypeptides when designed to retain at least one activity of the naturally-occurring form of the protein, that is, as an epitope recognized by an anti-M pneumoniae protein antibody, are considered "functional equivalents" of the polypeptides described in more detail herein.
  • modified polypeptides may be produced, for instance, by amino acid substitution, deletion, or addition, which substitutions may consist in whole or part by conservative amino acid substitutions.
  • the polypeptide epitopes of M. pneumoniae may be modified so as to increase their immunogenicity.
  • a polypeptide such as an antigenically or immunologically equivalent derivative, may be associated, for example by conjugation, with an immunogenic carrier protein for example bovine serum albumin (BSA) or keyhole limpet haemocyanin (KLH).
  • BSA bovine serum albumin
  • KLH keyhole limpet haemocyanin
  • a multiple antigenic peptide comprising multiple copies of the protein or polypeptide, or an antigenically or immunologically equivalent polypeptide thereof may be sufficiently antigenic to improve immunogenicity so as to obviate the use of a carrier.
  • Truncated polypeptides may be prepared. Truncated polypeptides have from 1 to 20 or more amino acid residues removed from either or both the N- and C-termini. Such truncated polypeptides may prove more amenable to expression, purification or characterization than the full-length polypeptide.
  • agents that bind epitopes of M. pneumoniae proteins are provided also.
  • agents that bind epitopes of M. pneumoniae proteins are anti-M pneumoniae protein antibodies or antigen-binding fragments thereof, including polyclonal and monoclonal antibodies, prepared according to conventional methodology.
  • Antibodies and antigen-binding fragments thereof that bind epitopes of M. pneumoniae proteins are useful for determining M. pneumoniae presence or levels.
  • Antibodies and antigen-binding fragments thereof that bind epitopes of M. pneumoniae proteins and are useful for determining M. pneumoniae presence or levels include but are not limited to: antibodies or antigen-binding fragments thereof that bind specifically to M. pneumoniae proteins or fragments or analogs thereof. In certain embodiments, the antibodies or antigen-binding fragments there bind specifically to the epitopes comprising SEQ ID NOs: 2 through 6 and 8 through 10. [0039]
  • the paratrope is involved in the binding of the antibody to its epitope (see, in general, Clark, W. R.
  • the pFc' and Fc regions are effectors of the complement cascade but are not involved in antigen binding.
  • an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region designated an Fab fragment
  • Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain denoted Fd.
  • the Fd fragments are the major determinant of antibody specificity (a single Fd fragment may be associated with up to ten different light chains without altering antibody specificity) and Fd fragments retain epitope-binding ability in isolation.
  • CDRs complementarity determining regions
  • Frs framework regions
  • non-CDR regions of a mammalian antibody may be replaced with similar regions of conspecific or heterospecific antibodies while retaining the epitopic specificity of the original antibody.
  • This is most clearly manifested in the development and use of "humanized" antibodies in which non- human CDRs are covalent Iy joined to human FR and/or Fc/pFc' regions to produce a functional antibody. See, e.g., U.S. Pat. Nos. 4,816,567, 5,225,539, 5,585,089, 5,693,762 and 5,859,205.
  • Fully human monoclonal antibodies also can be prepared by immunizing mice transgenic for large portions of human immunoglobulin heavy and light chain loci. Following immunization of these mice (e.g., XenoMouse (Abgenix), HuMAb mice (Medarex/GenPharm)), monoclonal antibodies can be prepared according to standard hybridoma technology. These monoclonal antibodies will have human immunoglobulin amino acid sequences and therefore will not provoke human anti-mouse antibody (HAMA) responses when administered to humans.
  • HAMA human anti-mouse antibody
  • the present invention also provides for F(ab')2, Fab, Fv and Fd fragments; chimeric antibodies in which the Fc and/or FR and/or CDRl and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric F(ab')2 fragment antibodies in which the FR and/or CDRl and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric Fab fragment antibodies in which the FR and/or CDRl and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; and chimeric Fd fragment antibodies in which the FR and/or CDRl and/or CDR2 regions have been replaced by homologous human or non-human sequences.
  • the present invention also includes so-called single chain antibodies.
  • the invention involves polypeptides of numerous size and type that bind specifically to epitopes of M. pneumoniae proteins. These polypeptides may be derived also from sources other than antibody technology.
  • polypeptide binding agents can be provided by degenerate peptide libraries which can be readily prepared in solution, in immobilized form or as phage display libraries.
  • Combinatorial libraries also can be synthesized of peptides containing one or more amino acids. Libraries further can be synthesized of peptoids and non-peptide synthetic moieties.
  • Phage display can be particularly effective in identifying binding peptides useful according to the invention. Briefly, one prepares a phage library (using e.g. ml3, fd, or lambda phage), displaying inserts from 4 to about 80 amino acid residues using conventional procedures. The inserts may represent, for example, a completely degenerate or biased array. One then can select phage-bearing inserts which bind to M. pneumoniae proteins. This process can be repeated through several cycles of reselection of phage that bind to the M. pneumoniae proteins. Repeated rounds lead to enrichment of phage bearing particular sequences. DNA sequences analysis can be conducted to identify the sequences of the expressed polypeptides.
  • the minimal linear portion of the sequence that binds to the M. pneumoniae proteins can be determined.
  • Yeast two-hybrid screening methods also may be used to identify polypeptides that bind to the M. pneumoniae proteins.
  • M. pneumoniae proteins can be used to screen peptide libraries, including phage display libraries, to identify and select peptide binding partners of the M. pneumoniae proteins.
  • the antibodies, antibody fragments and other agents may be coupled to specific diagnostic labeling agents for imaging of the protein or fragment thereof.
  • exemplary labels include, but are not limited to, fluorescent labels, radiolabels, colloidal labels such as carbon or gold, enzymatic labels and particulate labels such as latex particles, dyes, or sols.
  • fluorescent labels are available from and/or extensively described in the Handbook of Fluorescent Probes and Research Products 8 th Ed. (2001), available from Molecular Probes, Eugene, OR., as well as many other manufacturers.
  • the antibody, antibody fragment or other binding agent is fused to a molecule that is readily detectable either by its presence or activity, including, but not limited to, luciferase, fluorescent protein (e.g., green fluorescent protein), chloramphenicol acetyl transferase, ⁇ -galactosidase, secreted placental alkaline phosphatase, ⁇ -lactamase, human growth hormone, and other secreted enzyme reporters.
  • fluorescent protein e.g., green fluorescent protein
  • chloramphenicol acetyl transferase e.g., chloramphenicol acetyl transferase
  • ⁇ -galactosidase e.g., secreted placental alkaline phosphatase
  • ⁇ -lactamase ⁇ -lactamase
  • human growth hormone e.g., human growth hormone
  • kits comprising the use of one or more antibodies, antibody fragments, or other agents specific for a polypeptide epitope of an M. pneumoniae to detect M. pneumoniae bacteria, e.g., that may have infected a subject.
  • the antibodies, antibody fragments, or other agents may bind M. pneumoniae proteins in a sample prepared from lysed bacteria, or M. pneumoniae proteins present on M. pneumoniae bacteria.
  • a method for detection of M. pneumoniae bacteria comprises the steps of providing a sample, preferably a respiratory sample, suspected of containing M. pneumoniae bacteria and/or proteins.
  • a sample can be tested by any known methods in order to confirm the presence of M. pneumoniae bacteria and/or proteins in the sample.
  • a sample suspected of containing M. pneumoniae bacteria and/or proteins can be tested by performing an immunoassay, sandwich immunoassay, competitive immunoassay, simultaneous immunoassay, nucleic acid detection, hybridization PCR, gPCR, enzyme assay, or a combination thereof on said sample.
  • a general immuno chromatographic test device that is able to detect M.
  • the sample may be a respiratory sample, such as a swab.
  • the swab may be directly used in an assay method or device, or washed with an elution buffer and the eluate used in the assay method or device.
  • the sample is a throat swab, or is prepared therefrom.
  • a sample may be prepared from a throat swab as follows.
  • a throat swab is taken from a patient presenting with respiratory symptoms consistent with M. pneumoniae infection.
  • the throat swab may be then placed in a buffer, for example, comprising a detergent such as Brij 58, Zwittergen 3-14 or cholate.
  • a detergent such as Brij 58, Zwittergen 3-14 or cholate.
  • Zwittergen 3-14 is used.
  • insoluble components may be removed from the sample, and the supernatant used as the test sample.
  • the sample may comprise M. pneumoniae proteins extracted from M. pneumoniae bacteria. Methods for bacterial cell lysis and subsequent protein extraction are well-known in the art.
  • the methods may include providing an antibody, antibody fragment or other agent specific for a polypeptide epitope of M. pneumoniae, wherein the antibody, antibody fragment or other agent binds to the epitope as present on the M. pneumoniae protein from which the polypeptide epitope was derived, if present in the sample being tested, to form complexes. In the event that the sample does not contain the epitope, no complex is formed.
  • the complexes may be removed from the sample by any removal method suitable for removing all or substantially all of the complexes from the sample mixture.
  • the removal methods include removal by way of filtration, centrifugation, size exclusion chromatography, gravimetric, phase, reverse phase, polyethylene glycol precipitation, magnetic, or a combination thereof.
  • the methods may further comprise contacting a sample potentially containing complexes with a second antibody, antibody fragment or other agent specific for a polypeptide epitope of M. pneumoniae.
  • the second antibody, antibody fragment or other agent may be the same or different as the first antibody, antibody fragment or other agent specific for a polypeptide epitope of M. pneumoniae.
  • the second antibody or antibody fragment or agent may be labeled for ease of detection, and the label can be any suitable label that is able to be detected.
  • the label may be a radiolabel, fluorescent label, colloidal label, enzymatic label, particulate label or molecule that is readily detectable either by its presence or activity.
  • the methods may further comprise detecting the presence or absence of the first antibody-M pneumoniae protein or bacteria-second antibody complexes, wherein the presence of such complexes indicates the presence of M. pneumoniae.
  • the sample mixture can be tested by any known methods able to detect the complexes.
  • the detection methods may include performing an immunoassay, sandwich immunoassay, competitive immunoassay, simultaneous immunoassay, nucleic acid detection, hybridization, PCR, gPCR, enzyme assay, or a combination thereof on the sample.
  • the sample can be tested by a general immuno chromatographic test device.
  • a method comprises: (a) contacting a sample with a first antibody, antibody fragment or agent specific for a polypeptide epitope of M. pneumoniae to form, if M. pneumoniae protein comprising the epitope or M. pneumoniae bacterium is present in the sample, a first complex comprising the first antibody, antibody fragment or other binding agent and M. pneumoniae protein comprising the epitope or M.
  • EIA direct enzyme immunoassay
  • Western blot Western blot
  • lateral flow detection of a M pneumoniae protein is accomplished by one of the three following specific methods: (1) direct enzyme immunoassay ("EIA"); (2) Western blot; and (3) lateral flow.
  • EIA plates may be coated with a first antibody, antibody fragment or other agent specific for a polypeptide epitope of M. pneumoniae and washed, then blocked and washed. A patient sample may be added to the plate, which is then incubated and washed. A second antibody, antibody fragment or other agent specific for a polypeptide epitope of M. pneumoniae that is conjugated to a reporter system may be then added, and the plate incubated and washed.
  • Substrate for the reporter system may then be added, and substrate development allowed. The development may then be stopped and the degree of substrate development read on a plate reader or other suitable device. The presence of the polypeptide epitope of M. pneumoniae is indicated by signal development higher than reaction produced in the absence of the organism.
  • a Western blot to detect a polypeptide epitope of M. pneumoniae may be accomplished as follows. A patient sample is mixed with SDS gel sample buffer. The control may comprise a protein comprising the polypeptide epitope of M. pneumoniae to indicated electrophoretic migration. Electrophoresis may then be performed. The resolved components are transferred to nitrocellulose or other suitable membrane, which is then blocked and washed.
  • An antibody, antibody fragment or other agent specific for a polypeptide epitope of M. pneumoniae is added to the membrane, and the membrane and antibody mixture incubated and washed.
  • An antibody that is coupled to a reporter system and that is directed against the animal species of the antibody, antibody fragment or other agent specific for a polypeptide epitope of M. pneumoniae originally added to the membrane is added to the membrane, and the membrane and antibody mixture incubated and washed.
  • a substrate suitable for Western blot development (such as a visible or chemiluminescent product) is added, and the membrane incubated. The development may then be stopped and the degree of substrate development determined.
  • kits and devices for the practice of the above-described methods are also provided.
  • Devices for practice of the methods include lateral flow devices (wherein the reagents employed in the reaction may be dried or immobilized onto a chromatographic support contained within the device), a test strip, or other support for practice of the methods.
  • a method incorporating lateral flow may be practiced as follows. A patient sample may be added to the sample pad of a lateral flow device. The sample pad or other component may contain an extractant such as the detergent Zwittergen 3-14.
  • the sample moves into the conjugate pad where an antibody, antibody fragment or other agent specific for a polypeptide epitope of M. pneumoniae reacts with any protein comprising the polypeptide epitope of M. pneumoniae in the patient sample.
  • the antibody, antibody fragment or other agent is conjugated to a reporter (for example, gold, latex, or other microparticle, or any other type of reporter system) in a dried format.
  • a reporter for example, gold, latex, or other microparticle, or any other type of reporter system
  • the patient sample moves through the device. If the epitope is present in the sample, the polypeptide comprising it will be captured by the antibody, antibody fragment or other agent specific for a polypeptide epitope of M. pneumoniae localized on the membrane. Signal may be generated by the reporter on the conjugate antibody, antibody fragment or other agent, which indicates the presence of M.
  • kits and devices may include a support, reagents and wash and incubation buffers.
  • kits and devices can contain any number or combination of reagents or components.
  • the kits can comprise one or more of the above components in any number of separate containers, tubes, vials and the like or such components can be combined in various combinations in such containers.
  • Kit components may be packaged for either manual or partially or wholly automated practice of the foregoing methods. Further, instructions for the use of a device or kit may be included with the device or kit.
  • Such kits and devices may have a variety of uses, including, for example, diagnosis, therapy, and other applications.
  • the immunoassay can also include a distal sink at the end opposite to the sample application pad to absorb any excess liquid after testing has run to completion.
  • the sample application pad is a porous pad able to absorb the sample to be tested and transfer the absorbed sample to the conjugate pad by capillary action.
  • the conjugate pad includes one or more dried labeled molecules or reagents, such as antibodies, capable of specifically binding to the one or more analytes of interest forming a analyte-labeled reagent complex.
  • the conjugate pad may also include one or more stabilizing compounds that are able to induce thermal stability and also stability as to conditions imposed by humidity and temperature.
  • the conjugate pad is a porous pad able to absorb the transferred sample from the sample application pad and transfer the sample to the nitrocellulose strip by capillary action.
  • the nitrocellulose strip is able to absorb the sample from the conjugate pad and transfer the sample by capillary action downstream to the test result zone and the control zone.
  • the test result zone of the immunoassay device includes one or more immobilized molecules or reagents, such as antibodies or antibody fragments, capable of specifically binding to the one or more analytes of interest or any portion of the analyte- labeled reagent complex.
  • the control zone of the immunoassay device may include one or more immobilized molecules or reagents, such as antibodies or antibody fragments, capable of specifically binding to the one or more labeled reagent.
  • the immunoassay device may also comprise agents to prevent non-specific binding, as well as liquid reagents to aid in sample flow, pH control, control of non-specific binding, etc.
  • the sample travels through the sample application pad, the conjugate pad, and nitrocellulose strip by capillary action.
  • the sample solublizes the dried labeled molecule or reagent, and if the analyte of interest is present in the sample, the solublized labeled molecule or reagent binds the analyte of interest forming an analyte-labeled reagent complex, otherwise, if the analyte of interest is not present in the sample, no complex is formed.
  • the labeled reagent binds to the immobilized reagent of the control zone forming a detectable line indicating that the test has run to completion. Any excess liquid sample, after the testing has run to completion, can be absorbed the distal sink of the device.
  • the analyte may be a protein or other component from M. pneumoniae or M. pneumoniae bacterium.
  • a device may comprise a carrier upon which is disposed (a) a sample receiving zone comprising mobilizable labeled first antibodies, antibody fragments or agents specific for a polypeptide epitope of M. pneumoniae; and (b) a capture zone comprising immobilized second antibodies, antibody fragments or agents specific for a polypeptide epitope of M. pneumoniae.
  • the first and second antibody, antibody fragment or agent specific for a polypeptide epitope of M. pneumoniae may be the same, or different.
  • a device may comprise a carrier defining a flow path extending at least from a sample receiving zone to a capture zone, mobilizable labeled first antibodies, antibody fragments or agents specific for a polypeptide epitope of M. pneumoniae, and second antibodies, antibody fragments or agents specific for a polypeptide epitope of M. pneumoniae disposed along the flow path; wherein: a liquid sample received by the receiving zone migrates along the flow path mobilizing the labeled antibodies, antibody fragments or agents, and in the presence of M. pneumoniae bacteria or protein, the labeled and second antibodies, antibody fragments or agents cooperate to capture the antibodies, antibody fragments or agents in the capture zone.
  • the second antibodies, antibody fragments or agents may be disposed in the capture zone when the device is in the unused state.
  • the device of FIGURES 8 through 10 may be used to implement the methods described herein.
  • the device comprises a housing or casing 100 of elongate rectangular form having at one end 101 a portion 102 of reduced cross-sectional area.
  • a cap 103 can be fitted onto portion 102 and can abut against the shoulder 104 at end 101 of the housing.
  • Cap 103 is shown separated from housing 100.
  • Extending beyond end 105 of portion 102 is a porous sample collector 106.
  • Upper face 107 of housing 100 incorporates two apertures 108 and 109.
  • the housing is constructed of an upper half 110 and a lower half 111.
  • housing 100 is of hollow construction.
  • Porous sample collector 106 extends into housing 100.
  • the inner end 112 of sample collector 106 is recessed to accommodate a macroporous body 113 of plastics material.
  • Aqueous liquid sample applied to collector 106 can pass freely into macroporous body 113, rapidly saturating it.
  • macroporous body 113 is in liquid permeable contact with a strip of porous carrier material 114.
  • the housing is constructed of an upper half 110 and a lower half 111 and strip 114 overlap to ensure that there is adequate contact between these two components and that a liquid sample applied to sample collector 106 can permeate via macroporous body 113 and into strip 114.
  • Strip 114 extends further into housing 100.
  • a gap 115 can be left in the housing 100 by arranging for the strip 114 to overlap macroporous body 113 only partially.
  • Strip 114 is "backed" by a supporting strip 116 formed of transparent moisture-impermeable plastics material. Strip 114 extends beyond apertures 108 and 109. Means are provided within housing 100 by webs 117 and 118 to hold strip 114 firmly in place. In this respect, the internal constructional details of the housing are not a significant aspect of the invention as long as the strip is held firmly in place within the housing, sample collector 106 is firmly retained in the housing, and adequate fluid permeable contact is maintained between sample collector 106, macroporous body 113 and strip 114.
  • the transparent backing strip 116 lies between strip 114 and apertures 108 and 109 and can act as a seal against ingress of moisture from outside the housing 100 via these apertures.
  • the residual space 119 within the housing can contain moisture-absorbant material, such as silica gel, to help maintain the strip 114 in the dry state during storage.
  • the reagent-containing detection zone in strip 114 is not depicted in FIGURE 9, but the zone containing the immobilised unlabelled reagent will lie in the region exposed through aperture 108 in order that when the device has been used in an assay, the result can be observed through aperture 108.
  • Aperture 109 provides means through which a control zone containing further reagents which may enable the adequate permeation of sample through the strip to be observed.
  • the collector 106 need not be recessed to accommodate the macroporous body 113.
  • the device is an opposable-element, detachable-element or other multiple component chromatographic test device such as those described in U.S.
  • polypeptides comprising epitope sequences and antibodies, antibody fragments, or other agents specific for polypeptide epitopes of M. pneumoniae proteins may be used in conformational analysis of M. pneumoniae proteins and rational drug design.
  • a number of techniques can be used to screen, identify, select and design chemical entities capable of associating with M. pneumoniae proteins, structurally homologous molecules (for example, those found in plants), and other molecules.
  • Knowledge of the structure for a M. pneumoniae proteins permits the design and/or identification of molecules and/or other modulators which have a shape complementary to the conformation of a M pneumoniae protein, or more particularly, a druggable region thereof.
  • Detection of the toxin during infection might be an approach for diagnosis of the pathogen. This may be especially relevant since M. pneumoniae infection has been associated with asthma (for ex. Reviewed in: Johnston and Martin 2005 Am. J. Resp. Crit. Care. Med. 172: 1078-89).
  • SEQ ID NO: 1 The sequence (SEQ ID NO: 1) of recombinant CARDS ("rCARDS") from M. pneumoniae is depicted in FIGURE 1.
  • Rabbits were immunized and boosted with rCARDS protein using standard protocols. An array of overlapping peptides encompassing the rCARDS sequence was purchased. Each peptide contained 17 amino acids of the rCARDS sequence and overlapped each of its neighbor sequences by 10 amino acids.
  • Each peptide was synthesized with a cysteine residue at its C-terminus which is used to covalently couple the peptide to commercially available microtiter plates. Peptides were bound to the microtiter plates in solvent. Unreacted sites were rendered inert using a cysteine solution, and plastic, protein binding sites were blocked with a solution of bovine serum albumen. Microtiter plates were washed using standard conditions. [0081] Polyclonal anti-rCARDS rabbit antisera were subjected to a serial dilution series. A control antiserum raised against a second M. pneumoniae protein was included in the experiment. Each dilution was applied to the immobilized peptide and allowed to react. Plates were washed.
  • Example 1 The work in Example 1 identified epitopes in recombinant CARDS (rCARDS) using polyclonal anti-rCARDS rabbit antisera, which are potential sites for targeting antigen capture and detection of M. pneumoniae CARDS toxin.
  • rCARDS recombinant CARDS
  • polyclonal anti-rCARDS rabbit antisera which are potential sites for targeting antigen capture and detection of M. pneumoniae CARDS toxin.
  • Peptides encompassing the identified rCARDS epitopes were synthesized (FIGURE 2).
  • the shaded regions in FIGURE 2 represent amino acids not included in M. pneumoniae rCARDS and are added to enable solubility in aqueous media and to allow attachment of the peptides to solid phases through the sulfur atom contained in cysteine residues.
  • Antigen capture and detection was performed in plastic microtiter plates. However, solid phase antigen capture and detection may be accomplished using any suitable solid phase.
  • Purified anti-rCARDS IgG or affinity purified anti-peptide antibodies were absorbed to the solid phase using standard methods.
  • a combination of affinity purified antibodies was immobilized on the solid phase.
  • the solid phase was washed with a buffer containing a mild detergent (PBST) to remove loosely adherent antibody. Unreacted sites on the solid phase were blocked by incubation in the presence of an irrelevant protein.
  • PBST mild detergent
  • rCARDS protein was added to the solid phase in PBST containing irrelevant protein.
  • a dilution series of rCARDS was simultaneously made to permit definition of the lowest detection limit of rC ARDS.
  • similar dilution series of a second recombinant M Similar dilution series of a second recombinant M.
  • pneumoniae protein (rPl) was used to challenge the specificity of the assay. Incubation allowed antigen capture by immobilized antibody. Washing was employed to remove unreacted antigen. Affinity purified anti-peptide antibodies or starting IgG pool coupled to HRP were added in individual reactions. The conjugated antibodies were allowed to bind to captured rCARDS by incubation. Washing was used to remove excess conjugate. Conjugate binding was determined with a suitable reporter substrate. The reactions were terminated by addition of inorganic acid. Antigen capture and detection were quantitated using a microtiter plate reader. The data were analyzed and plots were made. In some cases, the background reactions resulting when antigen is not included were subtracted for ease of comparison.
  • FIGURE 4 shows the results of all possible combinations of antigen capture and detection using the starting IgG pool and affinity purified anti-peptide antibodies. All combinations were successful. The strongest reactions are observed using the IgG pool as both capture and detection reagents. Anti-peptide 2-HRP conjugate antibodies for detection is generally as effective as use of the IgG pool conjugate. In most cases, use of the same affinity purified antibody as capture and detection reagent is not as efficacious as use of heterologous reagents. [0092] FIGURE 5 shows that antigen capture using the starting IgG pool, a combination of anti peptide (3, 4, and 6) antibodies and anti-peptide 4 antibodies were all equally efficacious. Anti-peptide 2 conjugate was used for detection.
  • Contaminating bacterial proteins produced during production of recombinant molecules are usually of concern.
  • the assay was challenged using a second recombinant protein, rPl (FIGURE 6).
  • Antigen capture is effected using the starting IgG pool; a combination of anti-peptides 3, 4, and 5; or anti-peptide 4 antibodies.
  • Anti-peptide 2 or the IgG pool conjugate were used for detection. All possible combinations showed utility.
  • Significant reaction with rPl was not observed. The latter observation suggests assay specificity and indicates that antibody directed against contaminating bacterial proteins are not of concern.
  • Example 3 Identification of Epitopes in Recombinant Pl
  • M. pneumoniae Pl adhesion protein has been described in the literature. Diagnosis of M. pneumoniae by detection of Pl protein may be a viable alternative to existing assays. Anti-recombinant Pl polyclonal antisera and antibodies may be useful reagents for this approach.
  • the sequence (SEQ ID NO: 7) of recombinant Pl (“rPl") fromM pneumoniae is depicted in FIGURE 7. Rabbits were immunized and boosted with rPl protein using standard protocols. Samples of mouse, monoclonal anti-authentic Pl protein were obtained. An array of overlapping peptides encompassing the rPl sequence was purchased.
  • Each peptide contained 14 amino acids of the rPl sequence and overlapped each of its neighbor sequences by 10 amino acids.
  • Each peptide was synthesized with a C (cysteine, cys) residue at its C-terminus which is used to covalently couple the peptide to commercially available microtiter plates. Peptides were bound to the microtiter plates in solvent. Unreacted sites were rendered inert using a cysteine solution, and plastic, protein binding sites were blocked with a solution of bovine serum albumen. Microtiter plates were washed using standard conditions.
  • Polyclonal anti-rPl rabbit antisera were subjected to a serial dilution series. A control antiserum raised against a second M. pneumoniae protein was included in the experiment. Samples of the monoclonal antibodies were also subjected to serial dilutions; a control was an irrelevant monoclonal antibody. Each dilution was applied to the immobilized peptide and allowed to react. Plates were washed. Appropriate, commercially available, secondary antibody (anti-rabbit or anti-mouse IgG) each coupled to a reporter system, horseradish peroxidase, was added in a fixed concentration and allowed to react. Plates were washed.
  • SEQ ID NO: 8 MAFRGSWVNRLGRVESVWDLKGVWAD [0099]
  • SEQ ID NO: 9 EHPNALAFQ VS VVE [00100]
  • SEQ ID NO: 10 STNSSPYLHLVKPKKVTQSDKLDDDLKNLLDPNQ
  • Immuno chromatography may be more easily used in physician office laboratories than microtiter plate formats. Experiments were designed to examine the efficacy of this approach.
  • the anti-rCARDS IgG pool was applied to two types of nitrocellulose differing in pore size at 3 concentrations.
  • the nitrocellulose was incorporated into dipstick type devices consisting of a porous sample pad, nitrocellulose, and an absorbent pad at the distal end.
  • Conjugates used in this work were colloidal gold coupled to the IgG pool or to antibodies that had affinity purified on SEQ ID NO: 2. In this experiment, the conjugates were maintained in a liquid state; conjugate solutions included a detergent and exogenous proteins to help prevent non-specific binding.
  • the strips were used in assays as follows. rCARDS was serially diluted in
  • Example 5 Extraction of CARDS from cultured M. pneumoniae cells
  • Suitable methods of extraction were examined by adjusting aliquots of ⁇ - irradiated M. pneumoniae to desired concentrations of the potential extraction reagent. The aliquots were subjected to micro centrifugation at 10,000-15,000 xg to pellet material not solubilized by the extraction. The supernatant from each extraction was made suitable for electrophoresis under denaturing conditions. The pellet was dispersed in PBS using the same volume as the original aliquot and was then adjusted for denaturing gel electrophoresis. A control consisted of fractionation in the absence of extraction reagent (not-treated). The supernatants and pellets were subjected to electrophoresis and were transferred to nitrocellulose via Western blotting (FIGURE 12).
  • Nitrocellulose membranes were reacted with rabbit polyclonal-anti-rCARDS antisera followed by goat-anti rabbit IgG-horse radish conjugate. Filters were then developed with a precipitating substrate for horse radish peroxidase. An increase in intensity of the band observed in the supernatant lane compared to its paired pellet and to the untreated supernatant indicated which extraction reagent was a good candidate.
  • the detergents Brij 58, Zwittergen 3-14 and cholate showed efficacy. A dose response with each of these 3 reagents was observed. Zwittergen 3-14 showed almost complete solubilization using 0.1 % or more detergent.
  • Antigen capture EIA was also used to determine the efficacy of Zwittergen extraction (FIGURE 13).
  • An IgG preparation of an-rCARDS antisera was bound to plastic microtiter plate wells, ⁇ - irradiated M. pneumoniae cells were adjusted to 1% Zwittergen followed by micro centrifugation. The supernatant was subjected to a five fold serial dilution series in a buffer containing 1% carrier protein (BSA), 1% Zwittergen 3-14 in PBS on the microtiter plate. The pellet after extraction was dispersed in the same buffer using the starting volume and was also serially diluted. Comparison of the curves indicates that the supernatant contained at least 25 fold more CARDS than the pellet. Additional plots reflect rCARDS spiked into various buffers serving as controls for the buffer formulations.
  • Example 6 Detection of M. pneumoniae in spiked specimens
  • a positive control employed rCARDS spiked into swab-conditioned 1% Zwittergen 3-14, PBS. Higher signal from M. pneumoniae is produced in the presence of detergent compared to swab conditioned PBS, confirming the efficacy of Zwittergen extraction (FIGURE 14). The lack of signal for M. salivarium indicates assay specificity.
  • Example 7 Detection of CARDS in Respiratory Specimens [00111] Twenty-seven patient respiratory specimens were examined. These included transport media after elution of pharyngeal swabs, nasal aspirates, sputa and broncho alveolar lavage (BAL) specimens. Nine specimens were purported to have been taken from patients with M.

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Abstract

La présente invention concerne des compositions, des procédés et des dispositifs de détection de M. pneumoniae dans un échantillon.
PCT/US2007/075441 2006-08-08 2007-08-08 Procédés et COMPOSITIONS pour la DéTECTION OF MYCOPLASMA PNEUMONIAE Ceased WO2008021862A2 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9052315B2 (en) 2012-05-09 2015-06-09 Advanced Animal Diagnostics, Inc. Rapid detection of analytes in liquid samples
WO2017132578A1 (fr) * 2016-01-29 2017-08-03 Advanced Animal Diagnostics, Inc. Méthodes et compositions destinées à détecter l'exposition à mycoplasma
US9797893B2 (en) 2013-05-09 2017-10-24 Advanced Animal Diagnostics, Inc. Rapid detection of analytes in liquid samples
US10359614B2 (en) 2012-07-03 2019-07-23 Advanced Animal Diagnostics, Inc. Diagnostic apparatus
CN112433050A (zh) * 2020-11-04 2021-03-02 上海赛罕生物技术有限公司 一种肺炎衣原体IgM抗体检测试剂盒及其检测方法
US12474322B2 (en) 2016-03-22 2025-11-18 Advanced Animal Diagnostics, Inc. Methods and compositions for reducing antibiotic administration to farm animals

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US5026636A (en) * 1987-11-10 1991-06-25 The University Of Texas Board Of Regents Methods and compositions for production of mycoplasmal adhesins
US5369005A (en) * 1987-01-09 1994-11-29 Board Of Regents, The University Of Texas System Method for mycoplasma detection in a biological sample
EP1678199B1 (fr) * 2003-10-03 2013-03-20 The Board Of Regents, The University Of Texas System Procedes et compositions pour la lutte contre les exotoxines produites par mycoplasma pneumoniae

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9052315B2 (en) 2012-05-09 2015-06-09 Advanced Animal Diagnostics, Inc. Rapid detection of analytes in liquid samples
US9482670B2 (en) 2012-05-09 2016-11-01 Advanced Animal Diagnostic, Inc. Rapid detection of analytes in liquid samples
US10359614B2 (en) 2012-07-03 2019-07-23 Advanced Animal Diagnostics, Inc. Diagnostic apparatus
US9797893B2 (en) 2013-05-09 2017-10-24 Advanced Animal Diagnostics, Inc. Rapid detection of analytes in liquid samples
WO2017132578A1 (fr) * 2016-01-29 2017-08-03 Advanced Animal Diagnostics, Inc. Méthodes et compositions destinées à détecter l'exposition à mycoplasma
US10222383B2 (en) 2016-01-29 2019-03-05 Advanced Animal Diagnostics, Inc. Methods and compositions for detecting mycoplasma exposure
US12474322B2 (en) 2016-03-22 2025-11-18 Advanced Animal Diagnostics, Inc. Methods and compositions for reducing antibiotic administration to farm animals
CN112433050A (zh) * 2020-11-04 2021-03-02 上海赛罕生物技术有限公司 一种肺炎衣原体IgM抗体检测试剂盒及其检测方法

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