Compositions and methods for the detection of mycoplasma

Abstract

Provided herein are compositions and methods for detecting and treating Mycoplasma haemofelis and Mycoplasma haemocanis.

Description

[Technical Field]

[0001] (Priority) This application claims the benefit of U.S. Patent Application No. 63 / 477,850, filed December 30, 2022, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Haemotropic mycoplasmas (hemoplasmas) are nonculturable, wall-less bacteria that bind to the surface of red blood cells in vertebrate hosts (previously known as species of Haemobartonella and Eperithrozoon). Haemotropic mycoplasmas are a clade within the genus Mycoplasma and are phylogenetically related to the pneumoniae group of Mycoplasmas.

[0003] Mycoplasma haemocanis is a hemotropic mycoplasmal blood pathogen that can cause acute disease in immunosuppressed or splenectomized dogs. Cats can be infected with Mycoplasma haemofelis, which causes asymptomatic infection and hemolytic anemia. Mycoplasma haemofelis and Mycoplasma haemocanis infections have traditionally been detected by cytological evaluation of blood smears to detect polymorphic bacteria on host red blood cells. Cytological detection of Mycoplasma haemofelis often provides false positives due to artifact and false negatives due to variable parasitemia levels in sick cats. PCR assays are difficult to implement in veterinary practice due to specialized equipment requirements and quality control issues. The art is in need of better and more accessible detection methods.

[0004] Anemia is one of the common hematologic abnormalities seen in infected cats. Mycoplasma haemofelis (Mhf), feline leukemia virus (FeLV), and feline immunodeficiency virus (FIV) are causative agents of feline infectious anemia and fever. It is important for clinicians to distinguish viral agents, such as FIV and FeLV, from bacterial agents, such as Mhf, for clear treatment.

[0005] Current diagnosis relies on PCR using whole blood samples. Because infections are largely cyclical, the presence of mycoplasma in the blood after infection may be less abundant or even undetectable. Furthermore, although RBCs are replenished with every cycle and the infection appears to be cleared, mycoplasma can recur.

[0006] The maximum copy number after M. haemofelis infection is approximately 14–15 days post infection (DPI) (blood 10 8.6 ~10 9.6 Although M. haemofelis copy numbers occur in the range of 0.1 to 0.2 copies / ml, there are large fluctuations in M. haemofelis copy numbers over time. These fluctuations in copy number can be as large and rapid as a 4 log difference over 2 or 3 days, or a 7 log difference over 12 days. [Brief explanation of the drawings]

[0007] [Figure 1] FIG. 1 shows an indirect ELISA assay for the detection of anti- M. haemofelis antibodies in serum samples using rTDX1362. [Figure 2-1] FIG. 2 shows a diagram of TDX1362 (SEQ ID NO: 9). [Figure 2-2] Same as above. [Figure 3] FIG. 3 shows testing of the B4 peptide, C11 peptide, and mature TDX1362 polypeptide with M. haemofelis negative serum samples. [Figure 4A] 4A-4C show testing of the B4 peptide, the C11 peptide, and the mature TDX1362 polypeptide with an M. haemofelis positive serum sample. [Figure 4B] Same as above. [Figure 4C] Same as above. [Figure 5A-1] 5A-5B show the results of an indirect ELISA antibody assay for point mutation alanine scanning. SESLRDLEKARRWCV is SEQ ID NO: 5, SFVAYTKDACTKPKK is SEQ ID NO: 79, SESLRDLEKARAWCV is SEQ ID NO: 67, and SFVAYTKDWCTKPKA is SEQ ID NO: 85. [Figure 5A-2] Same as above. [Figure 5A-3] Same as above. [Figure 5A-4] Same as above. [Figure 5B] Same as above. [Figure 6] FIG. 6 shows an indirect ELISA assay using recombinant TDX1452 used to test antibody responses in cats experimentally infected with M. haemofelis over time. [Figure 7] FIG. 7 shows the detection of M. haemofelis in experimentally infected cats using a labeled polyclonal antibody specific for TDX1452. [Figure 8-1] FIG. 8 shows epitope mapping by alanine scanning of the B5 and C12 peptides. [Figure 8-2] Same as above. [Figure 8-3] Same as above. [Figure 8-4] Same as above. [Figure 8-5] Same as above. [Figure 9] FIG. 9 shows a diagram of TDX1452 (SEQ ID NO: 142). [Figure 10-1]10 shows a series of 10 overlapping 15mer peptides of TDX1452 are used to elucidate the immunodominant region of the protein: DDAGAKALREHCKTK is SEQ ID NO: 143, DGITGNESDASKKLL is SEQ ID NO: 144, EITKVLNAYKVNSPT is SEQ ID NO: 145, EKCKELYEKPSDTPS is SEQ ID NO: 146, ESNDWKQKSQSHTSS is SEQ ID NO: 147, FSEDFQDTLEKVKLW is SEQ ID NO: 148, HCKTKLEVNSFSEDF is SEQ ID NO: 149, KALREHCKTKLEVNS is SEQ ID NO: 150, KEDLLKKLKK WCVIP is SEQ ID NO: 1, KKLKKWCVIPKTVN is SEQ ID NO: 151, KQKSQSHTSSSNNKF is SEQ ID NO: 152, KSPIFNKSKPTLRSE is SEQ ID NO: 153, KTVNQRLGDLNYTAL is SEQ ID NO: 154, LEVNSFSEDFQDTLE is SEQ ID NO: 155, LNAYKVNSPTVTLKF is SEQ ID NO: 156, LYEKPSDTPSKEDLL is SEQ ID NO: 157, NESDASKKLLEKCKE is SEQ ID NO: 15 8, NKSKPTLRSEVEGDK is SEQ ID NO: 159, NYTALSTNGPTQGTT is SEQ ID NO: 160, QDTLEKVKLWCSVTK is SEQ ID NO: 3, RLGDLNYTALSTNGP is SEQ ID NO: 161, SDTPSKEDLLKKLKK is SEQ ID NO: 162, SHTSSSNNKFTGISV is SEQ ID NO: 163, SKKLLEKCKELYEKP is SEQ ID NO: 164, SNNKFTGISVTGTGN is SEQ ID NO: 165, SRNTEEI TKVLNAYK is SEQ ID NO: 166, STNGPTQGTTESNDW is SEQ ID NO: 167, TGISVTGTGNDDAGA is SEQ ID NO: 168, TGTGNDDAGAKALRE is SEQ ID NO: 169, TLRSEVEGDKWVFLT is SEQ ID NO: 170, TQGTTESNDWKQKSQ is SEQ ID NO: 171, VEGDKWVFLTSRNTE is SEQ ID NO: 172, VNSPTVTLKFDGITG is SEQ ID NO: 173, VTLKFDGITGNESDA isSEQ ID NO: 174, WCVIPKTVNQRLGDL is SEQ ID NO: 175, and WVFLTSRNTEEITKV is SEQ ID NO: 65. [Figure 10-2] Same as above. [Figure 10-3] Same as above. [Figure 10-4] Same as above. Summary of the Invention

[0008] Provided herein are one or more polypeptides, e.g., purified polypeptides, comprising one or more of the following: (i) a polypeptide set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 37, 54, 74, 97, 111, 140, 141, or 151; (ii) a polypeptide comprising SEQ ID NO: 1, (a) W at position 11 is retained and one, two, or three other amino acids are substituted with conservative amino acid substitutions; (b) the W at position 11 is retained and conservative amino acid substitutions are at one, two, or three of the following positions: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, or 15; or (c) a polypeptide in which the W at position 11 is retained and conservative amino acid substitutions are made at one, two, or three of the following positions: 2, 3, 4, 6, 7, 8, or 14; (iii) a polypeptide comprising SEQ ID NO: 2, (a) W at position 11 is retained and one, two, or three other amino acids are replaced with conservative amino acid substitutions; (b) the W at position 11 is retained and conservative amino acid substitutions are at one, two, or three of the following positions: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, or 15; or (c) a polypeptide in which the W at position 11 is retained and conservative amino acid substitutions are made at one, two, or three of the following positions: 2, 3, 4, 6, 7, 8, or 14; (iv) a polypeptide comprising SEQ ID NO: 3, (a) W at position 10 is retained and one, two, or three other amino acids are replaced with conservative amino acid substitutions; (b) the W at position 10 is retained and conservative amino acid substitutions are at one, two, or three of the following positions: 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, or 15; or (c) a polypeptide in which the W at position 11 is retained and conservative amino acid substitutions are one, two, or three at the following positions: 1, 3, 5, 6, 7, 8, 14, or 15; (v) a polypeptide comprising SEQ ID NO: 4, (a) W at position 10 is retained and one, two, or three other amino acids are replaced with conservative amino acid substitutions; (b) the W at position 10 is retained and conservative amino acid substitutions are at one, two, or three of the following positions: 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, or 15; or (c) a polypeptide in which the W at position 11 is retained and conservative amino acid substitutions are one, two, or three at the following positions: 1, 3, 5, 6, 7, 8, 14, or 15; (vi) a polypeptide comprising SEQ ID NO: 5, (a) K at position 9 and W at position 13 are retained, and one, two, or three other amino acids are replaced with conservative amino acid substitutions; (b) the K at position 9 and the W at position 13 are retained, and conservative amino acid substitutions are at one, two, or three of the following positions: 1, 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 14, or 15; or (c) a polypeptide in which the K at position 9 and the W at position 13 are retained and conservative amino acid substitutions are made at one, two, or three of the following positions: 2, 3, 4, or 5; (vii) a polypeptide comprising SEQ ID NO: 6, (a) K at position 9 and W at position 13 are retained, and one, two, or three other amino acids are replaced with conservative amino acid substitutions; (b) the K at position 9 and the W at position 13 are retained, and conservative amino acid substitutions are at one, two, or three of the following positions: 1, 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 14, or 15; or (c) a polypeptide in which the K at position 9 and the W at position 13 are retained and conservative amino acid substitutions are made at one, two, or three of the following positions: 2, 3, 4, or 5; (viii) a polypeptide comprising SEQ ID NO: 7, (a) K at position 7, D at position 8, and W at position 9 are retained, and one, two, or three other amino acids are replaced with conservative amino acid substitutions; (b) the K at position 7, the D at position 8, and the W at position 9 are retained, and conservative amino acid substitutions are at one, two, or three of the following positions: 1, 2, 3, 4, 5, 6, 10, 11, 12, 13, 14, or 15; or (c) a polypeptide in which the K at position 7, the D at position 8, and the W at position 9 are retained and conservative amino acid substitutions are one, two, or three at the following positions: 3, 4, or 15; (ix) a polypeptide comprising SEQ ID NO: 8, (a) D at position 8 and W at position 9 are retained, and one, two, or three other amino acids are replaced with conservative amino acid substitutions; (b) the D at position 8 and the W at position 9 are retained, and conservative amino acid substitutions are at one, two, or three of the following positions: 1, 2, 3, 4, 5, 6, 7, 10, 11, 12, 13, 14, or 15; or (c) a polypeptide in which the D at position 8 and the W at position 9 are retained and conservative amino acid substitutions are made at one, two, or three of the following positions: 3, 4, 7, or 15; (x) a polypeptide comprising SEQ ID NO: 10; (xi) A polypeptide having 95% or more sequence identity to SEQ ID NO: 10, wherein amino acids 75 to 89, 170 to 184, or 75 to 89 and 170 to 184 are retained; (xii) a polypeptide comprising SEQ ID NO: 12; (xiii) A polypeptide having 95% or more sequence identity to SEQ ID NO: 12, wherein amino acids 82 to 96, 177 to 191, or 82 to 96 and 177 to 191 are retained; (xiv) a polypeptide comprising SEQ ID NO: 9 or SEQ ID NO: 11; (xv) a polypeptide comprising a polypeptide comprising SEQ ID NO: 141, (a) W at position 6 is retained and one, two, or three other amino acids are replaced with conservative amino acid substitutions; (b) the W at position 6 is retained and conservative amino acid substitutions are at one, two, or three of the following positions: 1, 2, 3, 4, 5, 7, 8, 9, or 10; or (c) a polypeptide in which the W at position 6 is retained and position 7 is substituted with A, and optionally the conservative amino acid substitutions are at one, two, or three of the following positions: 1, 2, 3, 4, 5, 8, 9, or 10; (xvi) A polypeptide comprising a polypeptide comprising SEQ ID NO: 151, (a) W at position 6 is retained and one, two, or three other amino acids are replaced with conservative amino acid substitutions; (b) the W at position 6 is retained and conservative amino acid substitutions are at one, two, or three of the following positions: 1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, or 14; or (c) a polypeptide in which the W at position 6 is retained and position 7 is substituted with A, and optionally conservative amino acid substitutions are at one, two, or three of the following positions: 1, 2, 3, 4, 5, 8, 9, 10, 11, 12, 13, or 14; (xvii) A polypeptide comprising a polypeptide comprising SEQ ID NO: 97, (a) W at position 11 is retained and one, two, or three other amino acids are substituted with conservative amino acid substitutions; (b) the W at position 11 is retained and conservative amino acid substitutions are at one, two, or three of the following positions: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, or 15; or (c) a polypeptide in which W at position 11 is retained and A is substituted at position 12, and optionally conservative amino acid substitutions are at one, two, or three of the following positions: 1, 2, 3, 4, 5, 8, 9, 10, 13, 14, or 15; (xviii) A polypeptide comprising SEQ ID NO: 74, (a) K at position 4 and W at position 8 are retained, and one, two, or three other amino acids are replaced with conservative amino acid substitutions; or (b) a polypeptide in which the K at position 4 and the W at position 8 are retained and conservative amino acid substitutions are made at one, two, or three of the following positions: 1, 2, 3, 5, 6, 7, or 10; (xix) A polypeptide comprising SEQ ID NO: 37, (a) K at position 4 and W at position 8 are retained, and one, two, or three other amino acids are replaced with conservative amino acid substitutions; or (b) a polypeptide in which the K at position 4 and the W at position 8 are retained and conservative amino acid substitutions are made at one, two, or three of the following positions: 1, 2, 3, 5, 6, 7, 11, 12, 13, 14, or 15; (xx) A polypeptide comprising SEQ ID NO: 54, (a) K at position 12, D at position 13, and W at position 14 are retained, and one, two, or three other amino acids are replaced with conservative amino acid substitutions; (b) the K at position 12, the D at position 13, and the W at position 14 are retained, and conservative amino acid substitutions are at one, two, or three of the following positions: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 15; or (c) a polypeptide in which the K at position 12, the D at position 13, and the W at position 14 are retained, and conservative amino acid substitutions are one, two, or three at the following positions: 7, or 8; (xxi) A polypeptide comprising SEQ ID NO: 140, (a) K at position 7, D at position 8, and W at position 9 are retained, and one, two, or three other amino acids are replaced with conservative amino acid substitutions; (b) the K at position 7, the D at position 8, and the W at position 9 are retained, and conservative amino acid substitutions are at one, two, or three of the following positions: 1, 2, 3, 4, 5, 6, or 10; or (c) a polypeptide in which the K at position 7, the D at position 8, and the W at position 9 are retained, and conservative amino acid substitutions are one, two, or three at the following positions: 3, or 4; (xxii) A polypeptide comprising SEQ ID NO: 111, (a) W at position 10 is retained and one, two, or three other amino acids are replaced with conservative amino acid substitutions; (b) the W at position 10 is retained and conservative amino acid substitutions are at one, two, or three of the following positions: 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, or 15; or (c) A polypeptide in which the W at position 11 is retained and conservative amino acid substitutions are at one, two, or three of the following positions: 1, 3, 5, 6, 7, 8, 14, or 15.

[0009] These polypeptides may be present in fusion proteins comprising two, three, four, five, six, seven, or more polypeptides having 90% or greater sequence identity to one or more of the polypeptides described herein.

[0010] Yet another embodiment provides a polypeptide having fewer than 190 total amino acids and comprising 90% or more sequence identity to the polypeptide set forth in SEQ ID NO: 10 or 12. In some aspects, one or more polypeptides described herein are not naturally occurring. One or more polypeptides may be lyophilized, dehydrated, or dried. One or more polypeptides may further comprise one or more labels or tags. One or more polypeptides may be immobilized on a support. The polypeptide may be present in an immunocomplex with one or more antibodies that specifically bind to the polypeptide set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 37, 54, 74, 97, 111, 140, 141, or 151. The one or more polypeptides may comprise one or more secretory signal sequences, one or more epitope tags, or one or more secretory signal sequences and one or more epitope tags.

[0011] Another embodiment provides a method for detecting anti-Mycoplasma haemofelis or anti-Mycoplasma haemocanis antibodies, or specific binding fragments thereof. The method includes (a) contacting a test sample with one or more polypeptides described herein and (b) detecting complexes between the anti-Mycoplasma haemofelis or anti-Mycoplasma haemocanis antibodies, or specific binding fragments thereof, and the one or more polypeptides. The one or more polypeptides may be immobilized on a support. The complexes may be detected using one or more secondary antibodies, or specific binding fragments thereof, that specifically bind to the anti-Mycoplasma haemofelis or anti-Mycoplasma haemocanis antibodies, or specific binding fragments thereof. The secondary antibodies, or specific binding fragments thereof, may contain one or more tags or labels. The complexes may be detected using one or more detector polypeptides, where the one or more detector polypeptides are one or more polypeptides described herein (e.g., the polypeptides described in claim 1 or 3). The one or more detector polypeptides may contain labels or tags.

[0012] Another embodiment provides a method for diagnosing a disease caused by Mycoplasma haemofelis or Mycoplasma haemocanis antibodies in a subject by (a) contacting a test sample with one or more polypeptides described herein (e.g., a polypeptide described in claim 1 or claim 3) and (b) detecting a complex between an anti-Mycoplasma haemofelis or anti-Mycoplasma haemocanis antibody, or a specific-binding fragment thereof, and one or more polypeptides. The subject may have been infected with Mycoplasma haemofelis or Mycoplasma haemocanis for less than 15 days. The method may further include comparing the amount of the complex in the sample with a control sample or control standard, where an elevated level of the complex compared to the control sample or control standard is indicative of a disease caused by Mycoplasma haemofelis or Mycoplasma haemocanis. The method may further include administering a treatment for the disease caused by Mycoplasma haemofelis or Mycoplasma haemocanis for which the complex is detected. The method may further include determining the amount of anti-Mycoplasma haemofelis or anti-Mycoplasma haemocanis antibody, or a specific binding fragment thereof, in the sample. The subject may be a human or a non-human animal. The test sample may be blood, plasma, serum, or lymph.

[0013] In some embodiments, methods provide for detecting anti-Mycoplasma haemofelis or anti-Mycoplasma haemocanis antibodies, or specific binding fragments thereof, by competitive immunoassay, sandwich immunoassay, enzyme-linked immunoassay (ELISA), immunohistochemistry, immunoturbidimetry, particle-enhanced immunoturbidimetry, radioimmunoassay (RIA), fluorescence immunoassay (FIA), multiplex immunoassay, protein / peptide array immunoassay, solid-phase radioimmunoassay (SPRIA), indirect immunofluorescence assay (IIF), chemiluminescence immunoassay (CIA), particle-based multiplex assay (PMAT), dot blot assay, Western blot assay, surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), microscale thermophoresis (MST), biolayer interferometry, or grating-binding interferometry. Multiplex assays may further test for feline leukemia virus (FeLV), feline immunodeficiency virus (FIV), or both FeLV and FIV.

[0014] Yet another embodiment provides a kit for diagnosing a disease caused by Mycoplasma haemofelis or Mycoplasma haemocanis, the kit comprising: (a) one or more polypeptides described herein (e.g., a polypeptide of claim 1 or claim 3), wherein the polypeptide is not naturally occurring; and (b) one or more reagents that promote binding of one or more polypeptides to anti-Mycoplasma haemofelis antibodies, or anti-Mycoplasma haemocanis antibodies, or specific binding fragments thereof, present in the test sample.

[0015] One embodiment provides a method of treating a disease caused by Mycoplasma haemofelis or Mycoplasma haemocanis in a subject by (a) contacting a test sample with one or more polypeptides described herein, (b) detecting complexes between an anti-Mycoplasma haemofelis antibody or an anti-Mycoplasma haemocanis antibody, or a specific binding fragment thereof, and the one or more polypeptides, and (c) treating the subject with one or more antibiotics if complexes between an anti-Mycoplasma haemofelis antibody or an anti-Mycoplasma haemocanis antibody, or a specific binding fragment thereof, and the one or more polypeptides are detected. The subject may have been infected with Mycoplasma haemofelis or Mycoplasma haemocanis for less than 15 days (e.g., less than 15, 14, 13, 12, 11, 10, 9, 8, or 7 days). The amount of the complex in the sample relative to the amount in a control sample or control standard can be determined, and an increase in the level of the complex compared to the control sample or control standard is indicative of a disease caused by Mycoplasma haemofelis or Mycoplasma haemocanis. An amount of anti-Mycoplasma haemofelis antibody or anti-Mycoplasma haemocanis antibody, or a specific binding fragment thereof, can be determined in the sample. The subject can be a human or a non-human animal. The test sample can be blood, plasma, serum, or lymph. Anti-Mycoplasma haemofelis antibodies or anti-Mycoplasma haemocanis antibodies, or specific binding fragments thereof, may be detected by competitive immunoassay, sandwich immunoassay, enzyme-linked immunoassay (ELISA), immunohistochemistry assay, immunoturbidimetry, particle-enhanced immunoturbidimetry, radioimmunoassay (RIA), fluorescence immunoassay (FIA), multiplex immunoassay, protein / peptide array immunoassay, solid-phase radioimmunoassay (SPRIA), indirect immunofluorescence assay (IIF), chemiluminescence immunoassay (CIA), particle-based multiplex test (PMAT), dot blot assay, Western blot assay, surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), microscale thermophoresis (MST), biolayer interferometry, or grating-coupled interferometry.

[0016] Another aspect provides polynucleotides that encode one or more of the polypeptides or fusion proteins disclosed herein. DETAILED DESCRIPTION OF THE INVENTION

[0017] Provided herein are compositions and methods for detecting and treating Mycoplasma haemofelis and Mycoplasma haemocanis.

[0018] Polypeptides A polypeptide is a polymer in which amide bonds covalently link three or more amino acids. Polypeptides may be post-translationally modified. A purified polypeptide is a polypeptide preparation that is substantially free of cellular material, other types of polypeptides, chemical precursors, chemicals used to synthesize the polypeptide, or combinations thereof. A polypeptide preparation that is substantially free of cellular material, culture medium, chemical precursors, and chemicals used to synthesize the polypeptide has no more than about 30%, 20%, 10%, 5%, or 1% of other polypeptides, culture medium, chemical precursors, and / or other chemicals used in synthesis. Thus, a purified polypeptide is about 70%, 80%, 90%, 95%, 99%, or more pure.

[0019] The term "polypeptide" can refer to one or more types of polypeptides, or a set of polypeptides. "Polypeptide" can also refer to a mixture of two or more different types of polypeptides, including, but not limited to, full-length proteins, truncated polypeptides, or polypeptide fragments. The term "polypeptide," whether in the plural or singular, can each mean "one or more polypeptides."

[0020] In some embodiments, the polypeptide comprises the feline TDX1452 B5 epitope: KEDLLKKLKKWCVIP (SEQ ID NO: 1). In some embodiments, the TDX1452 polypeptide comprises the canine TDX1452 B5 epitope: KDIFLRQIKKWCVVP (SEQ ID NO: 2). In some embodiments, the TDX1452 polypeptide comprises the feline TDX1452 C12 epitope: QDTLEKVKLWCSVTK (SEQ ID NO: 3). In some embodiments, the TDX1452 polypeptide comprises the canine TDX1452 C12 epitope: DDALRASTLWCSVST (SEQ ID NO: 4). The feline and canine epitopes are aligned below.

[0021] The TDX1452 epitope is aligned: B5 cat KEDLLKKLKKWCVIP (SEQ ID NO: 1) B5 dog KDIFLRQIKKWCVVP (SEQ ID NO: 2) C12 cat QDTLEKVKLWCSVTK (SEQ ID NO: 3) C12 Dog DDALRASTLWCSVST (SEQ ID NO: 4)

[0022] In particular, the experiments in Examples 3-5 demonstrate that specific binding can be retained when amino acid substitutions are made, and therefore antibodies specific for Mycoplasma haemofelis and Mycoplasma haemocanis are expected to retain specific binding to polypeptides with conservative amino acid substitutions.

[0023] In some embodiments, the polypeptide comprises the TDX1452 feline B5 epitope comprising SEQ ID NO: 1, wherein the W at position 11 is retained and one, two, three, four, five, or more other amino acids are substituted with conservative amino acid substitutions. In one embodiment, the W at position 11 is retained and conservative amino acid substitutions are made at one, two, three, four, five, or more of the following positions: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, or 15. In one embodiment, the W at position 11 is retained and conservative amino acid substitutions are made at one, two, three, four, five, or more of the following positions: 2, 3, 4, 6, 7, 8, or 14.

[0024] In some embodiments, the polypeptide comprises the TDX1452 canine B5 epitope comprising SEQ ID NO:2, wherein the W at position 11 is retained and one, two, three, four, five, or more other amino acids are substituted with conservative amino acid substitutions. In one embodiment, the W at position 11 is retained and conservative amino acid substitutions are made at one, two, three, four, five, or more of the following positions: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, or 15. In one embodiment, the W at position 11 is retained and conservative amino acid substitutions are made at one, two, three, four, five, or more of the following positions: 2, 3, 4, 6, 7, 8, or 14.

[0025] In some embodiments, the polypeptide comprises the TDX1452 feline C12 epitope comprising SEQ ID NO: 3, wherein the W at position 10 is retained and one, two, three, four, five, or more other amino acids are substituted with conservative amino acid substitutions. In one embodiment, the W at position 10 is retained and conservative amino acid substitutions are made at one, two, three, four, five, or more of the following positions: 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, or 15. In one embodiment, the W at position 11 is retained and conservative amino acid substitutions are made at one, two, three, four, five, or more of the following positions: 1, 3, 5, 6, 7, 8, 14, or 15.

[0026] In some embodiments, the polypeptide comprises the TDX1452 canine C12 epitope comprising SEQ ID NO: 4, wherein the W at position 10 is retained and one, two, three, four, five, or more other amino acids are substituted with conservative amino acid substitutions. In one embodiment, the W at position 10 is retained and conservative amino acid substitutions are made at one, two, three, four, five, or more of the following positions: 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, or 15. In one embodiment, the W at position 11 is retained and conservative amino acid substitutions are made at one, two, three, four, five, or more of the following positions: 1, 3, 5, 6, 7, 8, 14, or 15.

[0027] In some embodiments, the polypeptide comprises the feline TDX1362 B4 epitope: SESLRDLEKARRWCV (SEQ ID NO: 5). In some embodiments, the TDX1362 polypeptide comprises the canine TDX1362 B4 epitope: SNKAEDLEKARRWCV (SEQ ID NO: 6). In some embodiments, the polypeptide comprises the feline TDX1362 C11 epitope: SFVAYTKDWCTKPKK (SEQ ID NO: 7). In some embodiments, the polypeptide comprises the canine TDX1362 C11 epitope: SFLSYTRDWCTKPKN (SEQ ID NO: 8). The feline and canine epitopes are aligned below: B4 cat SESLRDLEKARRWCV (SEQ ID NO: 5) B4 dog SNKAEDLEKARRWCV (SEQ ID NO: 6) C11 cat SFVAYTKDWCTKPKK (SEQ ID NO: 7) C11 Dog SFLSYTRDWCTKPKN (SEQ ID NO: 8)

[0028] In some embodiments, the polypeptide comprises the TDX1362 feline B4 epitope comprising SEQ ID NO: 5, wherein K at position 9 and W at position 13 are retained and one, two, three, four, five, or more other amino acids are substituted with conservative amino acid substitutions. In one embodiment, K at position 9 and W at position 13 are retained and conservative amino acid substitutions are made at one, two, three, four, five, or more of the following positions: 1, 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 14, or 15. In one embodiment, K at position 9 and W at position 13 are retained and conservative amino acid substitutions are made at one, two, three, four, five, or more of the following positions: 2, 3, 4, or 5.

[0029] In some embodiments, the polypeptide comprises the TDX1362 canine B4 epitope comprising SEQ ID NO:6, wherein the K at position 9 and the W at position 13 are retained and one, two, three, four, five, or more other amino acids are substituted with conservative amino acid substitutions. In one embodiment, the K at position 9 and the W at position 13 are retained and conservative amino acid substitutions are made at one, two, three, four, five, or more of the following positions: 1, 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 14, or 15. In one embodiment, the K at position 9 and the W at position 13 are retained and conservative amino acid substitutions are made at one, two, three, four, five, or more of the following positions: 2, 3, 4, or 5.

[0030] In some embodiments, the polypeptide comprises the TDX1362 feline C11 epitope comprising SEQ ID NO: 7, wherein K at position 7, D at position 8, and W at position 9 are retained, and one, two, three, four, five, or more other amino acids are substituted with conservative amino acid substitutions. In one embodiment, K at position 7, D at position 8, and W at position 9 are retained, and conservative amino acid substitutions are made at one, two, three, four, five, or more of the following positions: 1, 2, 3, 4, 5, 6, 10, 11, 12, 13, 14, or 15. In one embodiment, K at position 7, D at position 8, and W at position 9 are retained, and conservative amino acid substitutions are made at one, two, three, four, five, or more of the following positions: 3, 4, or 15.

[0031] In some embodiments, the polypeptide comprises the TDX1362 canine C11 epitope comprising SEQ ID NO: 8, wherein the D at position 8 and the W at position 9 are retained and one, two, three, four, five, or more other amino acids are substituted with conservative amino acid substitutions. In one embodiment, the D at position 8 and the W at position 9 are retained and conservative amino acid substitutions are made at one, two, three, four, five, or more of the following positions: 1, 2, 3, 4, 5, 6, 7, 10, 11, 12, 13, 14, or 15. In one embodiment, the D at position 8 and the W at position 9 are retained and conservative amino acid substitutions are made at one, two, three, four, five, or more of the following positions: 3, 4, 7, or 15.

[0032] In some embodiments, the polypeptide comprises a TDX1362 polypeptide: MSIPLKFLAGAAGVGTVSTGAYFAISHDKGTNIKDRLEASGYSILNLDKGDSEKWEKIKE AYGKEDDEALRFNGVNKNDASTIVGIKTACSSLLASKSESLRDLEKARRWCVVPVTVSSR IGDNLALLSSQDSQDTSLWNAKLEEHKKEASKFTKIPNVWDDKANSDELKLTAFKKKCEE MSKLNTFDKDFESFVAYTKDWCTKPKKG (SEQ ID NO: 9).

[0033] In some embodiments, the polypeptide comprises a TDX1362 polypeptide without a signal sequence: AISHDKGTNIKDRLEASGYSILNLDKGDSEKWEKIKE AYGKEDDEALRFNGVNKNDASTIVGIKTACSSLLASKSESLRDLEKARRWCVVPVTVSSR IGDNLALLSSQDSQDTSLWNAKLEEHKKEASKFTKIPNVWDDKANSDELKLTAFKKKCEE MSKLNTFDKDFESFVAYTKDWCTKPKKG (SEQ ID NO: 10).

[0034] In one embodiment, variant TDX1362 polypeptides are provided. Variants can include polypeptides having 80, 85, 90, 93, 95, 96, 97, 98, 99% or more sequence identity to SEQ ID NO: 10, wherein amino acids 75-89, 170-184, or 75-89 and 170-184 (bolded above) are retained, i.e., not substituted or deleted.

[0035] In certain embodiments, the polypeptide comprises a TDX1452 polypeptide linked to the following MGS His6 tag / thrombin cleavage site: MGSSHHHHHHSSGLVPRGSHMKSPIFNKSKPTLRSEVEGDKWVFLTSRNTEEITKVLNAY KVNSPTVTLKFDGITGNESDASKKLLEKCKELYEKPSDTPSKEDLLKKLKKWCVIPKTVN QRLGDLNYTALSTNGPTQGTTESNDWKQKSQSHTSSSNNKFTGISVTGTGNDDAGAKALR EHCKTKLEVNSFSEDFQDTLEKVKLWCSVTK (SEQ ID NO: 11).

[0036] The MGS His6 tag / thrombin cleavage site is MGSSHHHHHHSSGLVPRGSH (SEQ ID NO: 13).

[0037] The TDX1452 polypeptide without the MGS His6 tag / thrombin cleavage site is shown below. MKSPIFNKSKPTLRSEVEGDKWVFLTSRNTEEITKVLNAY KVNSPTVTLKFDGITGNESDASKKLLEKCKELYEKPSDTPSKEDLLKKLKKWCVIPKTVN QRLGDLNYTALSTNGPTQGTTESNDWKQKSQSHTSSSNNKFTGISVTGTGNDDAGAKALR EHCKTKLEVNSFSEDFQDTLEKVKLWCSVTK (SEQ ID NO: 12).

[0038] In one embodiment, variant TDX1452 polypeptides are provided. Variants can include polypeptides having 80, 85, 90, 93, 95, 96, 97, 98, 99% or more sequence identity to SEQ ID NO: 12, wherein amino acids 82-96, 177-191, or 82-96 and 177-191 (bolded above) are retained, i.e., not substituted or deleted.

[0039] In one embodiment, a polypeptide comprises two or more (e.g., 2, 3, 4, 5, 6, 7 or more) of SEQ ID NOs: 1-12 fused or linked together. In one embodiment, a polypeptide may be multimeric, i.e., a polypeptide may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, or more copies of one of SEQ ID NOs: 1-12 fused or linked together. In one embodiment, a polypeptide comprises one or more of a label, tag, signal sequence, cleavage site (e.g., a thrombin cleavage site or an enterokinase cleavage site), solid support (e.g., beads, or membrane).

[0040] Any of these polypeptides can be immobilized on a support, such as a barcoded magnetic bead (BMB), and used in a detection assay.

[0041] Any one of the above-described polypeptides or variant polypeptides can be non-naturally occurring. In some embodiments, substitution, deletion, or addition of one or more amino acids can change any of SEQ ID NOs: 1-12 into a non-naturally occurring polypeptide.

[0042] A secretory signal sequence is a peptide sequence (or a polynucleotide encoding a peptide sequence) that is present at the N-terminus (or in some cases, the C-terminus) of a peptide sequence. Secretory signal sequences can be variously referred to, including, but not limited to, a signal sequence, a targeting signal, a localization signal, a localization sequence, a transit peptide, a leader sequence, a leader peptide, a prepro sequence, a pre sequence, or a secretory signal peptide. Secretory signal sequences can be about 10 to 110 amino acids in length (e.g., about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, or 110 amino acids in length). Secretory signal sequences that are components of larger polypeptides can be useful for targeting and directing the larger polypeptide through the secretory pathway of the cell in which it is synthesized. In some embodiments, the larger polypeptide is cleaved to remove the secretory signal sequence during transit through the secretory pathway. The secretory signal sequence can be endogenous or can be genetically engineered.

[0043] Secretory signal sequences can be synthesized, for example, according to the rules established by von Heinje (Eur. J. Biochem. 133: 17-21, 1983; J. Mol. Biol. 184: 99-105, 1985; Nuc. Acids. Res. 14: 4683-3690, 1986). Examples of secretory signal sequences are shown in Table 1. Any secretory signal sequence known in the art or known to one of ordinary skill in the art can be used in the polypeptides and methods described herein. [Table 1] JPEG2026501555000003.jpg102166

[0044] The polypeptide can have about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to one or more polypeptides (e.g., 1, 2, 3, 4, 5, 6, 7, or more) set forth in SEQ ID NOs: 1-12, and can further include one or more (e.g., 1, 2, 3, 4, 5, or more) secretory signal sequences, one or more (e.g., 1, 2, 3, 4, 5, or more) epitope tags, or one or more secretory signal sequences and one or more epitope tags. These elements can be present as a fusion protein with one or more linkers between the individual proteins or sequences that make up the fusion protein. Alternatively, there can be no linkers between the individual proteins or sequences that make up the fusion protein. The elements of the fusion protein (i.e., the polypeptides set forth in SEQ ID NOs: 1-12, the secretory signal sequences, and the epitope tags) can occur in any order within the fusion protein. Thus, these polypeptides are non-naturally occurring and have different properties than naturally occurring polypeptides, including, for example, a reduced tendency to form secondary structures and the ability to be conjugated to a solid phase for testing.

[0045] Certain embodiments provide purified polypeptides comprising SEQ ID NOs: 1-12, or fragments thereof. The polypeptide fragment can be less than about 50, 40, 30, 20, 15, 14, 13, or 12 total contiguous amino acids.

[0046] In certain aspects, the polypeptide fragment is more than about 5, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 95, 100, 150, 200, or 210 total contiguous amino acid sequence of SEQ ID NOs: 1-12. In another embodiment, the polypeptide has less than about 95, 75, 50, 25, or 10 total amino acids and comprises 70%, 80%, 90%, 95%, or more sequence identity to a polypeptide set forth in SEQ ID NOs: 1-12. In another embodiment, the polypeptide has less than about 200, 190, 180, 170, 160, 150, 100, 95, 90, 80, 70, 60, 50, 40, 35, 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, or 5 total amino acids and has 70%, 80%, 90%, 95% or more sequence identity to a polypeptide set forth in SEQ ID NOs: 1-12.

[0047] For example, SEQ ID NO:9 is 208 amino acids long. In one embodiment, the polypeptide can include additional amino acids to extend the polypeptide, for example, to 250 amino acids in length. The extra or additional amino acids can be, for example, a label, a tag, additional Mycoplasma haemofelis amino acids, amino acids unrelated to Mycoplasma haemofelis, amino acids that can be used for purification, amino acids that can be used to increase the solubility of the polypeptide, amino acids that improve other characteristics of the polypeptide, or other amino acids. In one embodiment, the additional amino acids are not Mycoplasma haemofelis amino acids. In this example, the 250 amino acid long polypeptide has about 70%, 80%, 90%, 95%, or more sequence identity to the polypeptide set forth in SEQ ID NO:9 over the 208 consecutive amino acids of SEQ ID NO:9, while the remaining 42 amino acids of the 208 amino acid polypeptide may not have sequence identity to, for example, SEQ ID NO:9.

[0048] The fact that the polypeptides (e.g., SEQ ID NOS: 1-8) are smaller than full-length Mycoplasma haemofelis or Mycoplasma haemocanis polypeptides can be important because the smaller polypeptides may have greater specificity and / or sensitivity than the full-length polypeptides in detection or diagnostic assays. Furthermore, these smaller polypeptides may be less expensive to produce and may be obtained with greater purity than the full-length polypeptides.

[0049] In one embodiment, the polypeptide, or fragment thereof, is non-naturally occurring. That is, the polypeptide or fragment comprises 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 75, or more non-naturally occurring amino acids. In one embodiment, the non-naturally occurring amino acids may provide beneficial properties, such as increasing the solubility of the polypeptide, or increasing the sensitivity or specificity of the polypeptide in an assay.

[0050] The terms "sequence identity" and "percent identity" are used interchangeably herein. To determine the percent identity of two polypeptide molecules or two polynucleotide sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced into a first polypeptide or polynucleotide sequence for optimal alignment with a second polypeptide or polynucleotide sequence). The amino acids or nucleotides at corresponding amino acid or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions (i.e., overlapping positions) × 100). In some embodiments, the length of a reference sequence (e.g., SEQ ID NOS: 1-12) aligned for comparison purposes is at least 50, 60, 70, or 80% of the length of the comparison sequence, and in some embodiments, at least 90% or 100%. In one embodiment, the two sequences are the same length.

[0051] The desired range of sequence identity is approximately 80% to 100%, and integer values ​​therebetween. The percent identity between the disclosed sequence and the claimed sequence can be at least 80%, at least 83%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or at least 99.9%. Generally, a perfect match indicates 100% identity over the length of the reference sequence (e.g., SEQ ID NOs: 1-12).

[0052] Polypeptides that are sufficiently similar to the polypeptides described herein can be used herein. Polypeptides that are about 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5% or more identical to the polypeptides described herein can also be used herein.

[0053] Polypeptide variants differ by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or more amino acid residues (e.g., amino acid additions, substitutions, or deletions) from a peptide set forth in SEQ ID NOs: 1-12, or a fragment thereof. Where this comparison requires alignment, the sequences are aligned for maximum sequence identity. The site of mutation can occur anywhere in the polypeptide. In one embodiment, a variant has about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the original polypeptide.

[0054] Variant polypeptides can generally be identified by modifying one of the polypeptide sequences described herein and evaluating the properties of the modified polypeptide to determine whether it is biologically equivalent. A variant is biologically equivalent if it reacts substantially identically to a polypeptide described herein in an assay such as an immunohistochemical assay, enzyme-linked immunosorbent assay (ELISA), immunoturbidimetry, particle-enhanced immunoturbidimetry, particle-enhanced immunoturbidimetry, radioimmunoassay (RIA), enzyme-linked immunosorbent assay, Western blot assay, or other suitable assay. In other words, a variant is biologically equivalent if it possesses 90-110% of the activity of the original polypeptide. In one embodiment, the assay is a competitive assay, in which a biologically equivalent polypeptide has the ability to reduce binding of a polypeptide described herein to a corresponding reactive antigen or antibody by about 80%, 95%, 99%, or 100%. Antibodies that specifically bind to the corresponding polypeptide also specifically bind to the variant polypeptide.

[0055] Variant polypeptides have one or more conservative amino acid mutations or other minor modifications and retain biological activity, i.e., are biologically functional equivalents of SEQ ID NOS: 1-12, or fragments thereof. Variant polypeptides may have labels, tags, additional Mycoplasma haemofelis or Mycoplasma haemocanis amino acids, amino acids unrelated to Mycoplasma haemofelis, or Mycoplasma haemocanis amino acids that can be used for purification, amino acids that can be used to increase the solubility of the polypeptide, amino acids to improve other characteristics of the polypeptide, or other amino acids. In one embodiment, the additional amino acids are not Mycoplasma haemofelis or Mycoplasma haemocanis amino acids.

[0056] Methods for introducing mutations into amino acid sequences are well known to those skilled in the art. See, for example, Ausubel (ed.), Current Protocols in Molecular Biology, John Wiley and Sons, Inc. (1994); Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor laboratory, Cold Spring Harbor, NY (1989). Mutations can also be introduced using commercially available kits, such as the QuikChange™ Site-Directed Mutagenesis Kit (Stratagene). Generating functionally active variant polypeptides by substituting amino acids that do not affect the function of the polypeptide can be achieved by those skilled in the art. Variant polypeptides can also be chemically synthesized.

[0057] A variant polypeptide may have conservative amino acid substitutions at one or more predicted non-essential amino acid residues. A conservative substitution is one in which an amino acid is substituted for another amino acid with similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic index of the polypeptide to remain substantially unchanged. Generally, the following groups of amino acids represent conservative changes: (1) alanine, proline, glycine, glutamic acid, aspartic acid, glutamine, asparagine, serine, threonine; (2) cysteine, serine, tyrosine, threonine; (3) valine, isoleucine, leucine, methionine, alanine, phenylalanine; (4) lysine, arginine, histidine; and (5) phenylalanine, tyrosine, tryptophan, histidine. In one embodiment, a polypeptide has no more than about 1, 2, 3, 4, 5, 10, or 20 conservative amino acid substitutions.

[0058] The polypeptides can be fusion proteins that can contain amino acid linkers, amino acid spacers, signal sequences, TMR stop signaling sequences, transmembrane domains, and other amino acid sequences such as ligands useful for protein purification (glutathione-S-transferase, histidine tags (e.g., about 6, 7, 8, 9, 10, or more His residues), Staphylococcal protein A, or combinations thereof. In one embodiment, the polypeptides can be fusion proteins containing, for example, FLAG (e.g., DYKDDDDK; SEQ ID NO: 14), HA (YPYDVPDYAC; SEQ ID NO: 15), or VEGF (e.g., VEGF; SEQ ID NO: 16). SEQ ID NO: 15), myc (EQKLISEEDLC; SEQ ID NO: 16), V5 (GKPIPNPLLGLDST; SEQ ID NO: 17), E-tag (GAPVPYPDPLEPR; SEQ ID NO: 18), VSV-g (YTDIEMNRLGK; SEQ ID NO: 19), 6xHis (HHHHHHH; SEQ ID NO: 20), or HSV (QPELAPEDPEDC; SEQ ID NO: 21). Antibodies, such as monoclonal antibodies, can specifically bind to epitope tags and can be used to purify polypeptides comprising epitope tags.

[0059] Fusion proteins can comprise two or more different amino acid sequences operably linked to each other. Fusion protein constructs can be chemically synthesized using organic synthesis techniques by linking individual polypeptide fragments together in a fixed sequence. Fusion proteins can also be chemically synthesized. Fusion protein constructs can also be expressed by recombinant host cells (e.g., E. coli) cultured in vitro, which harbor an expression vector carrying a specific recombinant DNA sequence encoding amino acid residues in the appropriate sequence. Heterologous polypeptides can be fused, for example, to the N-terminus or C-terminus of the polypeptide. Multiple polypeptides can be present in the fusion protein. Fragments of polypeptides can be present in the fusion protein. Fusion proteins can comprise, for example, one, two, three, four, five, six, seven, or more of SEQ ID NOs: 1-12, fragments thereof, or combinations thereof. Polypeptides can be in multimeric form. In other words, a polypeptide can comprise two or more copies (e.g., two, three, four, five, six, seven, or more) of SEQ ID NOs: 1-12, fragments thereof, or combinations thereof. A polypeptide can include, for example, a fusion protein of two, three, four, five, six, seven, or more polypeptides having about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NOs: 1-12, or a fusion protein of at least two polypeptides having about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NOs: 1-12. A polypeptide can be a fusion protein, which can include one or more linkers between the individual proteins (i.e., SEQ ID NOs: 1-12) that make up the fusion protein. Alternatively, there may be no linker between the individual proteins that make up the fusion protein.The fusion polypeptide may contain other amino acid sequences such as amino acid linkers, amino acid spacers, signal sequences, TMR stop signaling sequences, transmembrane domains, and ligands useful for protein purification (such as glutathione-S-transferase, histidine tags, epitope tags, and Staphylococcal protein A), or combinations thereof.

[0060] Yet another component of the fusion protein may be a secretory (signal) sequence. These sequences may enable secretion of the fusion protein from the host cell during expression. The secretory (signal) sequence may be the sequence of the heterologous protein being produced if it has such a sequence, or may be derived from another secreted protein (e.g., t-PA), or synthesized de novo. The polynucleotide sequence encoding the secretory (signal) sequence may be operably linked to the fusion protein DNA sequence, i.e., the two sequences are joined in the correct reading frame and positioned to direct the newly synthesized polypeptide into the secretory pathway of the host cell. The polynucleotide sequence encoding the secretory (signal) sequence is generally positioned 5' to the DNA sequence encoding the polypeptide of interest, although certain signal sequences may be positioned elsewhere in the DNA sequence of interest (see, e.g., Welch et al., U.S. Pat. No. 5,037,743; Holland et al., U.S. Pat. No. 5,143,830).

[0061] In one embodiment, the polypeptides described herein are present in an immune complex with one or more antibodies, or specific binding fragments thereof, that specifically bind to the polypeptides set forth in SEQ ID NOs: 1-12. The one or more antibodies, or specific binding fragments thereof, can be anti-Mycoplasma haemofelis antibodies, anti-Mycoplasma haemocanis antibodies, or specific binding fragments thereof.

[0062] Polypeptides (e.g., SEQ ID NOS: 1-12) may be lyophilized, dehydrated, or dried, e.g., freeze-dried. Lyophilized polypeptides can be obtained by placing a polypeptide preparation at a low temperature to remove water from the sample. Dehydrated polypeptide compositions can be obtained by drying a polypeptide preparation by removing water. A dried polypeptide preparation can refer to an air-dried (e.g., lyophilized) polypeptide preparation.

[0063] Polynucleotides A polynucleotide contains less than the entire microbial genome and can be a single-stranded or double-stranded nucleic acid. A polynucleotide can be RNA, mRNA, DNA, cDNA, genomic DNA, chemically synthesized RNA or DNA, or a combination thereof. A polynucleotide can include, for example, a gene, an open reading frame, a non-coding region, or a regulatory element.

[0064] A gene is any polynucleotide molecule that encodes a polypeptide, protein, or fragment thereof, and optionally includes one or more regulatory elements before (5' non-coding sequences) and after (3' non-coding sequences) the coding sequence. In one embodiment, a gene does not include regulatory elements before or after the coding sequence. A native or wild-type gene refers to a gene found in nature, optionally having its own regulatory elements before and after the coding sequence. A chimeric or recombinant gene refers to any gene that is not a native or wild-type gene, and optionally includes regulatory elements before and after the coding sequence, where all or some of the coding sequence and / or regulatory elements are not found together in nature. Thus, a chimeric or recombinant gene includes regulatory elements and coding sequences from different sources, or regulatory elements and coding sequences from the same source but arranged differently than found in nature. A gene can include a full-length gene sequence (e.g., a gene sequence found in nature and / or encoding a full-length polypeptide or protein) and can also include a partial gene sequence (e.g., a fragment of a gene sequence found in nature and / or a gene sequence encoding a protein, or a fragment of a polypeptide or protein). A gene can also include modified gene sequences (e.g., modified compared to the sequence found in nature). Thus, a gene is not limited to the native or full-length gene sequence found in nature.

[0065] A polynucleotide may be purified to be free from other components, such as proteins, lipids, and other polynucleotides. For example, a polynucleotide may be 50%, 75%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% purified. A polynucleotide present among hundreds to millions of other polynucleotide molecules, for example, in a cDNA or genomic library or a gel slice containing a genomic DNA restriction digest, is not considered a purified polynucleotide. A polynucleotide may encode a polypeptide described herein (e.g., SEQ ID NOs: 1-12).

[0066] A polynucleotide may include additional heterologous nucleotides that do not naturally occur contiguous with the polynucleotide. As used herein, the term "heterologous" refers to a combination of elements that are not naturally occurring or that are obtained from different sources.

[0067] A polynucleotide may be isolated. An isolated polynucleotide is a naturally occurring polynucleotide that is not immediately contiguous with one or both of the 5' and 3' flanking genomic sequences with which it is naturally associated. An isolated polynucleotide may be, for example, a recombinant DNA molecule of any length, provided that the naturally occurring nucleic acid sequences immediately adjacent to the recombinant DNA molecule in the natural genome are removed or absent. Isolated polynucleotides also include non-naturally occurring nucleic acid molecules. A polynucleotide may encode a full-length polypeptide, a polypeptide fragment, and a variant or fusion polypeptide.

[0068] Degenerate polynucleotide sequences encoding the polypeptides described herein, as well as homologous nucleotide sequences that are at least about 80%, or about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polynucleotides described herein and their complements, are also polynucleotides. Degenerate nucleotide sequences are polynucleotides that encode the polypeptides described herein, or fragments thereof, but differ in nucleic acid sequence from the wild-type polynucleotide sequence due to the degeneracy of the genetic code. Complementary DNA (cDNA) molecules, species homologs, and variants of polynucleotides that encode biologically functional polypeptides are also contemplated herein.

[0069] Polynucleotides can be derived from nucleic acid sequences present in, for example, mycoplasma species. Polynucleotides can also be synthesized in the laboratory, for example, using an automated synthesizer. Amplification methods such as PCR can be used to amplify polynucleotides from either genomic DNA or cDNA encoding the polypeptide.

[0070] The polynucleotide may include non-coding sequences or coding sequences for a naturally occurring polypeptide, and may also encode modified sequences that do not occur in nature.

[0071] Unless otherwise indicated, the term polynucleotide, or gene, includes reference to the designated sequence and its complementary sequence.

[0072] The expression product of a gene, or polynucleotide, is often a protein or polypeptide, although for non-protein-coding genes, such as rRNA genes or tRNA genes, the product is functional RNA. The process of gene expression is used by all known forms of life, i.e., eukaryotes (including multicellular organisms), prokaryotes (including bacteria and archaea), and viruses, to generate the macromolecular machinery for survival. Several steps in the gene expression process can be regulated, including protein transcription, up-regulation, RNA splicing, translation, and post-translational modification.

[0073] sign One or more of the polypeptides described herein (including detector polypeptides) can be conjugated to one or more labels, or tags, which can be attached directly or indirectly to a desired component of the assay (e.g., an antibody, polypeptide, or support).

[0074] In one embodiment, the polypeptides are comprised of one or more scaffolds, carriers and / or linkers such as streptavidin (SA) and derivatives thereof, biotin, immunoglobulins, antibodies (monoclonal, polyclonal, and recombinant), antibody fragments, and derivatives thereof, the leucine zipper domains of AP-1 (jun and fos), hexa-his (metal chelating moiety), hexa-hat Further included are lectins that mediate binding to a variety of compounds, including GST (glutathione S-transferase) glutathione affinity, calmodulin-binding peptide (CBP), strep-tag, cellulose-binding domain, maltose-binding protein, S-peptide tag, chitin-binding tag, immunoreactive epitope, epitope tag, E2Tag, HA epitope tag, Myc epitope, FLAG epitope, AU1 and AU5 epitope, Glu-Glu epitope, KT3 epitope, IRS epitope, Btag epitope, protein kinase-C epitope, VSV epitope, carbohydrates, lipids, and proteins (e.g., Con A (Canavaria ensiformis), or WGA (wheat germ agglutinin)), and tetranectin, or protein A or G (antibody affinity).

[0075] Multimers can include multiple identical or different polypeptides linked by binding moieties.

[0076] A polypeptide can include more than one label, such as two or more labels, wherein the two or more labels are the same or different.

[0077] The polypeptides can include one or more fluorophore labels, such as, for example, fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, fluorescamine, 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid, sodium salt, 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid, pyrene-1-butanolic acid, AlexaFluor 350 (7-amino-6-sulfonic acid-4-methylcoumarin-3-acetic acid, AMCA (7-amino-4-methylcoumarin-3-acetic acid), 7-hydroxy-4-methylcoumarin-3-acetic acid, Marina Blue (6,8-difluoro-7-hydroxy-4-methylcoumarin-3-acetic acid), 7-dimethylamino-coumarin-4-acetic acid, fluorescamine-N-butylamine adduct, 7-hydroxy-coumarin-3-carboxylic acid, Cascade Blue (pyrene-trisulfonic acid acetyl azide), Cascade Yellow, Pacific Blue (6,8-difluoro-7-hydroxycoumarin-3-carboxylic acid, 7-diethylamino-coumarin-3-carboxylic acid, N-(((4-azidobenzoyl)amino)ethyl)-4-amino-3,6-disulfo-1,8-naphthalimide, dipotassium salt), Alexa Fluor 430, 3-perylenedodecanoic acid, 8-hydroxypyrene-1,3,6-trisulfonic acid, trisodium salt, 12-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoic acid, N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethyl-enediamine, Oregon Green 488 (difluorocarboxyfluorescein), 5-iodoacetamidofluorescein, propidium iodide-DNA adduct, carboxyfluorescein, or a combination thereof.

[0078] In one aspect, the polypeptide can include a fluorescent label, such as a simple fluorescent label, e.g., Fluor dys, Pacific Blue®, Pacific Orange®, Cascade yellow®, AlexaFluor®, AF405, AF488, AF500, AF514, AF532, AF546, AF555, AF568, AF594, AF610, AF633, AF635, AF647, AF680, AF700, AF710, AF750, AF800, Quantum Dot-based dyes, QDot® Nanocrystls (Invitrogen, Molecular Probes), Qdot® 525, Qdot® 565, Qdot® 585, Qdot® 605, Qdot® 655, Qdot® 705, Qdot® 800, DyLight® dyes (Pierce) (DL), DL549, DL649, DL680, DL800, fluorescein (Flu) or any derivative thereof, e.g., FITC, Cy dyes, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, fluorescent proteins, RPE , PerCp, APC, green fluorescent protein, GFP and GFP-derived mutant proteins, BFP, CFP, YFP, DsRed, T1, Dimer2, mRFP1, MBana, mOrange, dTomato, tdTomato, mTangerine, mStrawberry, mCherry, tandem dyes, RPE-Cy5, RPE-Cy5.5, RPE-Cy7, RPE-AlexaFluor® tandem conjugates, RPE-Alexa610, RPE-TxRed, APC-Aleca600, APC-Alexa610, APC-Alexa750, APC-Cy5, APC-Cy5.5. Multi-fluorescent dye assemblies, including multiple fluorescent dyes attached to polymer molecules, e.g., peptides / proteins, dextrans, polysaccharides, any combination of fluorescent dyes involved in the generation of FRET (fluorescence resonance energy transfer)-based techniques, ionophores, ion-chelating fluorescent probes (props), probes that change wavelength when bound to specific ions, probes that change intensity when bound to specific ions such as calcium, or combinations thereof.

[0079] In one embodiment, the polypeptide may include one or more labels capable of absorbing light, such as a chromophore or dye.In one embodiment, the polypeptide can include one or more labels capable of emitting light upon excitation, for example, one or more fluorescent dyes, such as those of the AlexaFluor® (AF) family, including AF® 350, AF405, AF430, AF488, AF500, AF514, AF532, AF546, AF555, AF568, AF594, AF610, AF633, AF635, AF647, AF680, AF700, AF710, AF750, and AF800; Quantum Dot (Qdot®) dye family (Qdot525®, Qdot®565, Qdot®585, Qdot®605, Qdot®655, Qdot®705, Qdot®800), DyLight® dye (DL) family (e.g., DL549, DL649, DL680, DL800), small fluorescent dyes (e.g., FITC, Pacific Blue®, Pacific Orange®, Cascade yellow®, Marina Blue®, DSred, Dsred-2, 7-AAD, TO-Pro-3, the Cy dye family (e.g., Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7), phycobiliproteins (R-phycoerythrin (RPE), PerCP, allophycocyanin (APC), B-phycoerythrin, C-phycocyanin), fluorescent proteins (e.g., (E)GFP and GFP (enhanced) green fluorescent protein, etc.) derived mutant proteins, BFP, CFP, YFP, DsRed, T1, Dimer2, mRFP1, MB Anana, mOrange, dTomato, tdTomato, mTangerine, tandem dyes with RPE (e.g., RPE-Cy5, RPE-Cy5.5, RPE-Cy7, RPE-AlexaFluor® tandem conjugates, RPE-Alexa610, RPE-TxRed), tandem dyes with APC (e.g., APC-Aleca600, APC-Alexa610, APC-Alexa750, APC-Cy5, APC-Cy5.5), and calcium dyes (e.g., Indo-1-Ca). 2+ , Indo-2-Ca2+ ) is available.

[0080] In one embodiment, the polypeptide may include one or more labels capable of reflecting light, such as gold, plastic, glass, polystyrene, and / or pollen.

[0081] In one embodiment, the polypeptide can include one or more chemiluminescent labels, such as luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt, or oxalate ester.

[0082] In one embodiment, the polypeptide may comprise a bioluminescent label (eg, luciferin, luciferase, or aequorin).

[0083] In one embodiment, the polypeptide can include a radiolabel (eg, such a radionuclide, isotope, light-emitting label, p-emitting label, or gamma-emitting label).

[0084] In one embodiment, the polypeptide comprises an enzymatic polypeptide (e.g., an enzyme that can catalyze a reaction between chemicals within the region of the labeled molecule, an enzyme that can produce a light signal (chemiluminescence), where the enzyme catalyzes a reaction between chemicals within the near environment of the labeled molecule, resulting in the precipitation of a chromophore dye, or where the enzyme catalyzes a reaction between chemicals within the near environment of the labeled molecule, resulting in a precipitate that can be detected by an additional layer of detection molecule.

[0085] In one embodiment, the polypeptide can include an enzymatic label, such as peroxidase, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, kasalase, glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholinesterase, horseradish peroxidase, alkaline phosphatase.

[0086] In one embodiment, the polypeptide can comprise an ionophore or chelating chemical compound, such as a lanthanide (e.g., a fluorescent, phosphorescent, or paramagnetic lanthanide), a DNA fluorescent stain (e.g., propidium iodide, Hoechst stain, DAPI, AMC, DraQ5™, and Acridine orange), a nucleic acid label (e.g., a DNA label, an RNA label, or an artificial nucleic acid label), and / or a nucleic acid comprising one or more of: a) a barcode region, b) a 5' first primer region (forward), c) a 3' second primer region (reverse), d) a random nucleotide region, e) a connector molecule, f) a stability-increasing component, g) a short nucleotide linker between any of the above components, h) an adapter for sequencing, or i) an annealing region.

[0087] Detection of labels, or tags, can be carried out using many different methods. For example, radioactive labels can be detected using a scintillation counter, photographic film in autoradiography, or storage phosphor imaging. If the label is fluorescent, the label can be detected by exciting the fluorescent dye with light of the appropriate wavelength and detecting the resulting fluorescence. Fluorescence can be detected visually, by photographic film, by the use of electronic detectors such as charge-coupled devices (CCDs) or photomultipliers. Similarly, enzymatic labels can be detected by providing the enzyme with an appropriate substrate and detecting the resulting reaction product. Simple colorimetric labels can be detected by observing the color associated with the label. Pairs of fluorophores can have distinct emission patterns (wavelengths) so that they can be easily distinguished when used in assays.

[0088] While the components described herein (e.g., polypeptides, antibodies, specific binding fragments thereof) can be provided with labels, labels are not required for detection of polypeptide / antibody complexes, as many options exist for label-free detection, including, for example, surface plasmon resonance, biolayer interferometry, and grating-binding interferometry detection assays.

[0089] support In one embodiment, assay components (e.g., polypeptides, or antibodies, or specific binding fragments thereof) can be immobilized on a support. A support is any material suitable, or capable of being modified to be suitable, for attachment of one or more polypeptides, antibodies, or specific binding fragments described herein. Examples of supports include glass and modified or functionalized glass, plastics (acrylic, polystyrene, methylstyrene, polyurethane, Teflon®, etc.), paramagnetic materials, triazoles, carbon graphite, titanium oxide, cross-linked dextrans such as latex or Sepharose, cellulose polysaccharides, nylon or nitrocellulose, ceramics, resins, silicon or silicon-based materials, including silicon and modified silicon, carbon metals, inorganic glass, fiber optic bundles, and various other polymers. In one embodiment, the support can be located within a microtiter well plate (e.g., a 96-well, 384-well, or 1536-well plate). In one embodiment, the support can be located within a flow cell or flow cell device (e.g., a flow cell on a protein chip). The support can be a solid support.

[0090] In one embodiment, the support can be a magnetic bead, such as a magnetic barcoded bead, a microsphere, a particle, a membrane, a chip, a slide, a well, or a test tube. Beads include microspheres or particles, which can be small, discrete, non-planar particles of micrometer or nanometer dimensions. Beads can be spherical or irregular. Beads can be porous. In one embodiment, the support can have a patterned surface suitable for immobilizing polypeptides in an ordered pattern (e.g., a protein chip).

[0091] In one embodiment, one or more polypeptides described herein can be immobilized to a support via a linker molecule. One or more polypeptides can be conjugated to a support using any suitable methodology. In one embodiment, one or more polypeptides are coupled to a support using a conjugation reagent, including covalent and non-covalent conjugation reagents. Covalent conjugation reagents can include any chemical or biological reagent that can be used to covalently immobilize a polypeptide on a surface. Covalent conjugation reagents include, for example, carboxyl-amine reactive groups such as carbodiimides such as EDC or DCC, amine reactive groups such as N-hydroxysuccinimide (NHS) esters or imidoesters, sulfhydryl reactive crosslinkers such as maleimides, haloacetyls, or pyridyl disulfides, carbonyl reactive crosslinkers such as hydrazides or alkoxyamines, photoreactive crosslinkers such as aryl azides or didilines, or chemoselective ligation groups such as Staudinger reaction pairs. Non-covalent immobilization reagents may include any chemical or biological reagent that can be used to non-covalently immobilize a polypeptide on a surface, such as an affinity tag such as biotin, or a capture reagent such as streptavidin, or an anti-tag antibody such as an anti-His6 antibody or an anti-Myc antibody.

[0092] Methods of detection and diagnosis In one embodiment, the assay method can include contacting a test sample with one or more polypeptides under conditions suitable for the formation of a complex between the polypeptide and an antibody or its specific binding fragment. The one or more polypeptides can include, for example, about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NOs: 1-12, fragments, or variants of these polypeptides described herein. The complex of the polypeptide and the antibody or its specific binding fragment can then be detected. In one embodiment, the complex of the polypeptide and the antibody or specific binding fragment can also comprise one or more labels or tags. If a complex is detected, then the sample contains an anti-Mycoplasma haemofelis antibody or an anti-Mycoplasma haemocanis antibody.

[0093] In one embodiment, a method includes diagnosing a disease caused by Mycoplasma haemofelis or Mycoplasma haemocanis in a subject. A test sample can be contacted with one or more polypeptides that share about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NOs: 1-12, fragments, or variants described herein. Complexes of anti-Mycoplasma haemofelis or anti-Mycoplasma haemocanis antibodies, or specific binding fragments thereof, and one or more polypeptides present in the sample are then detected. The subject may have been infected with Mycoplasma haemofelis or anti-Mycoplasma haemocanis for less than about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 28, or 35 days. That is, detection (using any method described herein) may occur at about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 28, or 35 days after infection. The amount of anti-Mycoplasma haemofelis or anti-Mycoplasma haemocanis antibody, or a specific binding fragment thereof, in the sample may be determined. The amount of the complex in the sample can be compared to a control sample or a control standard, and an elevated level of the complex compared to the control sample or the control standard is indicative of a disease caused by Mycoplasma haemofelis or Mycoplasma haemocanis. If the complex is detected, the subject can be administered a treatment. The subject can be a human or a non-human mammal, such as a dog, horse, cow, or cat. The test sample can be, for example, blood, plasma, serum, lymph, or any other bodily fluid sample. In some embodiments, the test sample is removed from the subject or patient, and the detection and diagnosis methods are completed ex vivo or in vitro.

[0094] In one embodiment, specific binding between one or more polypeptides described herein and one or more anti-Mycoplasma haemofelis antibodies or anti-Mycoplasma haemocanis antibodies, or specific binding fragments thereof, is detected using a secondary antibody or specific binding fragment thereof. In one example, specific binding may be detected using a secondary antibody. For example, a secondary antibody or specific binding fragment thereof may bind an anti-Mycoplasma haemofelis antibody, an anti-Mycoplasma haemocanis antibody, or a specific binding fragment thereof. In one embodiment, the secondary antibody is an anti-species antibody, such as a rabbit anti-dog antibody or a rabbit anti-human antibody. In one embodiment, the secondary antibody is covalently or non-covalently bound to a label or tag that can be used to detect the polypeptide / anti-Mycoplasma antibody / secondary antibody complex. In one example, the secondary antibody may be conjugated to biotin or another label or tag. A streptavidin conjugate (if biotin-labeled), such as a streptavidin fluorescent dye conjugate, may be used to detect the polypeptide / anti-mycoplasma antibody / secondary antibody complex.

[0095] In one embodiment, specific binding between one or more polypeptides described herein and one or more anti-Mycoplasma haemofelis antibodies, anti-Mycoplasma haemocanis antibodies, or specific-binding fragments thereof is detected using one or more detector polypeptides that specifically bind to the anti-Mycoplasma haemofelis antibodies, anti-Mycoplasma haemocanis antibodies, or specific-binding fragments thereof. In one example, the one or more detector polypeptides comprise about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a polypeptide, fragment, or variant thereof set forth in SEQ ID NOS: 1-12 described herein. In one embodiment, the one or more detector polypeptides are covalently or noncovalently linked to a label or tag that can be used to detect the polypeptide / anti-Mycoplasma antibody / detector polypeptide complex. In one example, the detector polypeptide can be conjugated to a label or tag, such as biotin. A streptavidin conjugate (in the case of biotin labeling), such as a streptavidin fluorescent dye conjugate, may be used to detect the polypeptide / anti-mycoplasma antibody / detector polypeptide complex.

[0096] In one embodiment, one or more anti-Mycoplasma haemofelis antibodies, anti-Mycoplasma haemocanis antibodies, or specific binding fragments thereof can be immobilized on a support. In one aspect, these antibodies are polyclonal antibodies. One or more of the polypeptides described herein can specifically bind to the immobilized antibodies. A sample can be added to the assay, and any anti-Mycoplasma haemofelis antibodies, anti-Mycoplasma haemocanis antibodies, or specific binding fragments thereof will specifically bind to one or more polypeptides. Antibodies from the sample can be detected with a labeled secondary antibody, such as an anti-cat or anti-dog secondary antibody.

[0097] Detection of specific binding between one or more polypeptides described herein and one or more antibodies or specific binding fragments thereof can be accomplished using any suitable method, such as a competitive immunoassay, sandwich immunoassay, enzyme-linked immunoassay (ELISA), immunohistochemistry, immunoturbidimetry, particle-enhanced immunoturbidimetry, radioimmunoassay (RIA), fluorescence immunoassay (FIA), multiplex immunoassay, protein / peptide array immunoassay, solid-phase radioimmunoassay (SPRIA), indirect immunofluorescence assay (IIF), chemiluminescence immunoassay (CIA), particle-based multiplex assay (PMAT), dot blot assay, or Western blot assay. Other assay methods that can be used to detect antibodies and / or antibody / polypeptide complexes include, for example, surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), microscale thermophoresis (MST), biolayer interferometry, or grating-binding interferometry.

[0098] The antibodies described herein can be polyclonal antibodies, monoclonal antibodies, single-chain antibodies (scFv), or specific binding fragments of antibodies. A specific binding fragment of an antibody is one or more portions of a complete antibody that contain the antigen-binding site or variable region of the complete antibody, lacking the constant heavy chain domain of the Fc region of the antibody. Examples of antibody fragments that bind to an antigen include Fab, Fab', Fab'-SH, F(ab')2, and Fv fragments. The antibodies described herein can be of any class, including, for example, IgG, IgM, IgA, IgD, and IgE. In one embodiment, polyclonal antibodies can be generated using one or more of the polypeptides described herein in rabbits or any other mammal. In one technique, a polypeptide of the present disclosure is introduced into a host animal, such as, for example, a rabbit, mouse, rat, guinea pig, goat, pig, cow, sheep, donkey, dog, cat, chicken, or horse. Although an enhanced immune response can be elicited in a host animal by associating the polypeptide with a carrier and / or exposing the host to an adjuvant, it should be understood that the present disclosure does not require that the polypeptide be associated with a carrier or that the host be exposed to an adjuvant. Exemplary carriers that can be used for this purpose include bovine serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Exemplary adjuvants include Freund's complete or incomplete adjuvant and MDL-TDM adjuvant. Regardless of whether the polypeptide is associated with such a carrier or whether the host is exposed to an adjuvant, booster immunizations can optionally be performed with the host animal subsequently bled one or more times. Polyclonal antibodies that specifically bind to the polypeptide can then be purified from the bleeding or antisera obtained from the bleeding. Such purification can be achieved, for example, by using affinity chromatography techniques involving associating the polypeptide with a solid support. Such affinity chromatography techniques are well known to those skilled in the art.

[0099] These polypeptides may be used in the assays described herein. Polyclonal antibody populations may be prepared by administering an antigen (e.g., one or more polypeptides described herein), or antigenic fragments thereof, to a mammal (e.g., rabbit) and purifying a population of polyclonal antibodies from the serum or plasma of the mammal. These polyclonal antibodies may specifically bind to one or more of the polypeptides described herein.

[0100] "Specifically binds" or "specific" means that a first antigen, e.g., a polypeptide as set forth in SEQ ID NOS: 1-12 described herein, a fragment thereof, or a variant thereof, recognizes an anti-Mycoplasma haemofelis antibody, an anti-Mycoplasma haemocanis antibody, or a specific-binding fragment thereof, and binds to the antibody or specific-binding fragment with greater affinity than other non-specific molecules. A non-specific molecule is an antigen that does not share a common epitope with the first antigen. In one embodiment, the non-specific molecule is not a Mycoplasma haemofelis polypeptide and is not related to Mycoplasma haemocanis. In one embodiment, the non-specific molecule is not a Mycoplasma haemocanis polypeptide and is not related to Mycoplasma haemocanis. In one embodiment, the non-specific molecule is not derived from a Mycoplasma organism. For example, an antibody raised against a first antigen (e.g., Mycoplasma haemofelis) that binds more efficiently than a non-specific antigen can be described as specifically binding to the first antigen. A polypeptide can be 10 -6 Binding affinity (K D ) specifically binds to an anti-Mycoplasma haemofelis antibody, anti-Mycoplasma haemocanis antibody, or specific binding fragment. In one embodiment, the polypeptide specifically binds to an anti-Mycoplasma haemofelis antibody, anti-Mycoplasma haemocanis antibody, or specific binding fragment when bound at 2 x 10 -6 Affinity (K D ) specifically binds to an anti-Mycoplasma haemofelis antibody, an anti-Mycoplasma haemocanis antibody, or a specific binding fragment thereof. In one embodiment, the polypeptide has a binding affinity of at least 10 -6 M, 10 -7 M, 10 -8M, 10 -9 Affinity (K D ) specifically binds to an anti-Mycoplasma haemofelis antibody, an anti-Mycoplasma haemocanis antibody, or a specific binding fragment thereof. In certain embodiments, affinity is measured by surface plasmon resonance or a KinExA assay. In one embodiment, the method specifically detects Mycoplasma haemofelis and not other species of Mycoplasma. In one embodiment, the method specifically detects Mycoplasma haemocanis and not other species of Mycoplasma. In one embodiment, the method specifically detects Mycoplasma haemofelis and Mycoplasma haemocanis.

[0101] kit The kit may include one or more of the polypeptides described herein. The kit may include one or more anti-Mycoplasma haemofelis antibodies, anti-Mycoplasma haemocanis antibodies, or specific binding fragments thereof. The kit may include a secondary anti-species antibody, such as an anti-feline species antibody or an anti-canine species antibody. Any of these components may be labeled and / or immobilized on a support.

[0102] For example, the label of the kit may comprise a fluorophore, an enzyme, a chemiluminescent moiety, a radioactive moiety, an organic dye, a small molecule, a polypeptide, or a functional fragment thereof. In some embodiments, the label of the kit comprises phycoerythrin (PE). In some embodiments, the label of the kit comprises fluorescein isothiocyanate. In some embodiments, the label may be conjugated to a secondary antibody or a detection polypeptide.

[0103] The kit may include a positive control or standard. In some embodiments, the positive control or standard may be a sample containing a detectable amount of anti-Mycoplasma haemofelis and / or anti-Mycoplasma haemocanis antibodies. In one embodiment, the positive control or standard may be obtained from a diseased subject with anti-Mycoplasma haemofelis or anti-Mycoplasma haemocanis antibodies. In one embodiment, the positive control or standard may include in vitro synthesized or otherwise obtained anti-Mycoplasma haemofelis or anti-Mycoplasma haemocanis antibodies. In one embodiment, the kit may include a negative control or standard. The negative control or standard may be a sample that does not contain a detectable amount of anti-Mycoplasma haemofelis or anti-Mycoplasma haemocanis antibodies. In some embodiments, the negative control or standard may be obtained from a healthy control individual or may be synthesized in vitro. For example, the negative control may include water or a buffer solution.

[0104] In some embodiments, the kit may include a standard curve for determining the amount of anti-Mycoplasma haemofelis antibody, Mycoplasma haemofelis polypeptide, anti-Mycoplasma haemocanis antibody, or Mycoplasma haemocanis polypeptide in a sample. The anti-Mycoplasma haemofelis antibody, Mycoplasma haemofelis polypeptide, anti-Mycoplasma haemocanis antibody, or Mycoplasma haemocanis polypeptide may be used to generate a standard curve for the assay. The standard curve may be used to determine the concentration of anti-Mycoplasma haemofelis antibody, Mycoplasma haemofelis polypeptide, anti-Mycoplasma haemocanis antibody, or Mycoplasma haemocanis polypeptide in a sample. The standard curve is obtained by relating the measured amount to the concentration of anti-Mycoplasma haemofelis antibody, Mycoplasma haemofelis polypeptide, anti-Mycoplasma haemocanis antibody, or Mycoplasma haemocanis polypeptide in "known" samples, i.e., standards of known concentration. These standards provide a reference for determining unknown concentrations of anti-Mycoplasma haemofelis antibodies, Mycoplasma haemofelis polypeptides, anti-Mycoplasma haemocanis antibodies, or Mycoplasma haemocanis polypeptides in samples. The amounts of the standards can span the range of concentrations expected to be found in the "unknown" or "test" sample concentrations.

[0105] The kit may further include one or more assay reagents that promote binding of one or more polypeptides to anti-Mycoplasma haemofelis antibodies, anti-Mycoplasma haemocanis antibodies, or specific binding fragments thereof. Assay reagents may be substances, mixtures, materials, or components useful for carrying out the intended purpose of the kit. Reagents may include, for example, conjugate reagents, buffers, standards, positive controls, labels, sample collection devices, instructions, etc.

[0106] The kit may include one or more buffers, such as a wash buffer. Wash buffers may include, for example, tris(hydroxymethyl)aminomethane (Tris)-based buffers, such as Tris-buffered saline (TBS), or phosphate buffers, such as phosphate-buffered saline (PBS). The wash buffer may comprise a detergent, such as an ionic or non-ionic detergent. In some embodiments, the wash buffer may be a PBS buffer containing about 0.001, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08% or more Tween® 20 (polysorbate), at a pH of about 7.0, 7.2, 7.4, 7.6, or 7.8.

[0107] The kit may include a dilution buffer, which may include, for example, a carrier protein such as bovine serum albumin (BSA) and a detergent such as Tween® 20 (polysorbate).

[0108] The kit may include a detection or assay buffer. The detection or assay buffer may be, for example, a colorimetric detection or assay buffer, a fluorescent detection or assay buffer, or a chemiluminescent detection or assay buffer. Colorimetric detection or assay buffers include, for example, PNPP (p-nitrophenyl phosphate), ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)), or OPD (o-phenylenediamine). Fluorescent detection or assay buffers include QuantaBlu® or QuantaRed® (Thermo Scientific, Waltham, Massachusetts). Chemiluminescent detection or assay buffers may include luminol or luciferin. The detection or assay buffer may also include a trigger, such as H2O2, and a tracer, such as an isoluminol conjugate.

[0109] The kit may include a stop solution, which can terminate or delay further development of the detection reagent and the corresponding assay signal. Examples of the stop solution include a low pH buffer (e.g., glycine buffer, pH 2.0), a chaotropic agent (e.g., guanidinium chloride, sodium dodecyl sulfate (SDS)), a reducing agent (e.g., dithiothreitol, β-mercaptoethanol), etc.

[0110] The provided kits may include devices for collecting biological samples, such as collection tubes, columns, swabs, syringes, needles, etc. The kits may include instructions for using the components of the kit. The instructions may provide details regarding protocols and analytical techniques.

[0111] The components of the kit can be in any physical state, for example, one or more of the components can be lyophilized, in aqueous solution, or frozen.

[0112] Kits can be designed for specific assay techniques. For example, the kits can be immunoassay kits, competitive immunoassay kits, sandwich immunoassay kits, enzyme-linked immunoassay (ELISA) kits, immunohistochemistry assay kits, immunoturbidimetric assay kits, particle-enhanced immunoturbidimetric assay kits, radioimmunoassay (RIA) kits, fluorescent immunoassay (FIA) kits, multiplex immunoassay kits, protein / peptide array immunoassay kits, solid-phase radioimmunoassay (SPRIA) kits, indirect immunofluorescence assay (IIF) kits, chemiluminescence immunoassay (CIA) kits, particle-based multiplex test (PMAT) kits, dot blot assay kits, Western blot assay kits, surface plasmon resonance (SPR) kits, isothermal titration calorimetry (ITC) kits, microscale thermophoresis (MST) kits, biolayer interferometry kits, or grating-binding interferometry kits. In one embodiment, the ELISA kits can include, for example, a wash buffer, a sample diluent, a secondary antibody, a secondary antibody-enzyme conjugate, a detection polypeptide, a labeled detection polypeptide, a detection reagent, and a stop solution. In one embodiment, a dot blot kit may include, for example, a wash buffer, a sample diluent, a secondary antibody-enzyme conjugate, a detection reagent, and a stop solution. In some embodiments, a chemiluminescent immunoassay kit may include, for example, a wash buffer, a sample diluent, a tracer (e.g., an isoluminol conjugate), and a trigger (e.g., HO). In one embodiment, a multiplex kit may include, for example, a wash buffer, a sample diluent, and a secondary antibody-enzyme conjugate.

[0113] Treatment method Once detected or diagnosed, Mycoplasma haemofelis or Mycoplasma haemocanis can be treated with, for example, antibiotic agents, protein or peptide agents, nucleic acid-based agents, anti-inflammatory agents, other agents, immunomodulatory therapies, alternative therapies, or combinations thereof.

[0114] For example, Mycoplasma haemofelis or Mycoplasma haemocanis can be treated with a number of antibiotics, such as tetracycline, fluoroquinolones (e.g., enrofloxacin), doxycycline, furosemide, robenidine, or marbofloxacin. Antibiotic treatment can be administered at approximately 5-10 mg / kg for up to 28 days. In one embodiment, doxycycline can be used at 5-10 mg / kg for up to 28 days. If bacteremia persists or recurs, antibiotic treatment can be switched to marbofloxacin (2 mg / kg once daily for 14 days). Other agents, such as robenidine and those listed in U.S. Patent No. 10,370,341 (incorporated herein by reference), can be used for treatment. Because the primary mechanism of injury is immune-mediated targeting of infected host cells, glucocorticoids can also be used to reduce erythrophagocytosis in cases of severe hemolysis. Supportive care such as oxygen and blood transfusions may be used in treatment.

[0115] Methods for treating Mycoplasma haemofelis or Mycoplasma haemocanis can include contacting a test sample with one or more polypeptides described herein. Complexes between anti-Mycoplasma haemofelis or anti-Mycoplasma haemocanis, or specific binding fragments thereof, and one or more polypeptides can be detected as described herein. The amount of complexes can be compared to a positive control or standard and / or a negative control or standard. If the amount of complexes indicates infection, the subject can be treated with one or more antibiotics or other treatments described herein and / or known to those of skill in the art.

[0116] A control or standard may be used for comparison. The control or standard value may be determined prior to performing assays simultaneously in parallel in a multiplex assay or other assay format. The control or standard may be a first level determined by assaying a first sample obtained from a patient. The control or standard may be, for example, a level or range detected in a population of healthy subjects, Mycoplasma haemofelis or Mycoplasma haemocanis patients, or severe Mycoplasma haemofelis or Mycoplasma haemocanis patients.

[0117] The present composition and method will be described in more detail below, and the examples described herein are intended to be merely illustrative, since many modifications and variations will be apparent to those skilled in the art.Terms used herein generally have their ordinary meanings in the art within the context of the compositions and methods described herein, and in the specific context in which each term is used.Some terms are more specifically defined herein to provide practitioners with additional guidance regarding the description of the present composition and method.

[0118] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items. As used in this description and throughout the following claims, the meaning of "a," "an," and "the" includes plural as well as singular reference unless the context clearly indicates otherwise. The term "about" in connection with a numerical value means that the value varies above and below 5%. For example, a value of about 100 means 95 to 105 (or any value between 95 and 105).

[0119] All patents, patent applications, and other scientific or technical literature mentioned anywhere in this specification are incorporated herein by reference in their entirety. The embodiments illustratively described herein may suitably be practiced in the absence of any element(s), limitation(ies), specifically or non-specifically disclosed herein. Thus, for example, in each instance herein, any of the terms "comprising," "consisting essentially of," and "consisting of" may be replaced with either of the other two terms while retaining their ordinary meaning. The terms and expressions used are used as terms of description, not of limitation, and there is no intention in the use of such terms and expressions to exclude any equivalents of the shown and described features or portions thereof, recognizing that various modifications are possible within the scope of the claims. Thus, while the present methods and compositions have been specifically disclosed by embodiments and optional features, it will be understood that modifications and variations of the concepts disclosed herein may occur to those skilled in the art, and that such modifications and variations are deemed to be within the scope of the compositions and methods as defined by the description and the appended claims.

[0120] Any single term, element, phrase, group of terms, phrases, or elements described herein may each be specifically excluded from the claims.

[0121] Whenever a range is given herein, such as a temperature range, time range, composition, or concentration range, all intermediate ranges and subranges, as well as all individual values ​​included in the given range, are intended to be included in the disclosure. It is understood that any subrange or individual value within a range or subrange included in the description herein can be excluded from the embodiments herein. It is understood that any element or step included in the description herein can be excluded from the claimed composition or method.

[0122] Additionally, when features or aspects of compositions and methods are described in terms of a Markush group or other grouping of alternatives, one of skill in the art will recognize that the compositions and methods are also described in terms of any individual member or subgroup of members of the Markush group or other group.

[0123] The following are provided for illustrative purposes only and are not intended to limit the scope of the embodiments broadly described above. [Example]

[0124] Discovery of marker TDX1362 The Mycoplasma haemofelis genome contains approximately 80% hypothetical proteins, i.e., proteins with no predicted function. Potentially useful markers were selected through sequence analysis and integrative analysis. The M. haemofelis genome was compared with the nonpathogenic M. haemominutum genome. Thirteen distinct proteins unique to M. haemofelis were selected for screening with infected samples. The 13 promising markers were cloned into expression vectors. The expressed proteins were subjected to immunoblot screening. Several expressed proteins were purified and subjected to ELISA analysis for binding to Mycoplasma haemofelis-specific antibodies. The polynucleotide encoding TDX1362 was sequenced (see SEQ ID NO: 9). The full-length TDX1362 was cloned into pET28a, allowing the expressed protein to contain a pET28a 6xHis tag and a thrombin cleavage site (see SEQ ID NO: 13). To express the recombinant protein, the signal sequence was removed, and only the gene sequence encoding the mature protein was cloned into the pET28a vector. The N-terminal region was tagged with a His tag and a thrombin site (MGSSHHHHHHSSGLVPRGSH; SEQ ID NO: 13). The recombinant protein TDX1362 was expressed in E. coli BL21 using IPTG induction. The expressed recombinant protein migrated according to the predicted molecular weight of 21 kDa. The mature protein is shown in SEQ ID NO: 10.

[0125] The recombinant TDX1362 protein was purified using an IMAC column. Briefly, cells were suspended in PBS pH 7.2 and lysed with a cell disruptant. The lysate was centrifuged at 10,000 g for 30 minutes. The supernatant was carefully removed and subjected to purification on an IMAC column. The purified recombinant TDX1362 was checked by SDS-PAGE to confirm its purity. The total yield of recombinant protein was estimated by OD280. Approximately 30 mg / liter was recovered. The immunoreactivity of the TDX1362 protein was confirmed by immunoblot screening and negative screening against sera from M. haemofelis-infected cats and naturally infected cats.

[0126] An indirect ELISA assay was designed in which TDX1362 was immobilized on Immulon IV plates. Briefly, plates were coated with recombinant TDX1362 overnight at 4°C, washed with PBST, and blocked with 2% BSA for 2-3 hours. Plates were washed again and stored at 4°C for future use. Plates were thawed at room temperature for 3-4 hours before use. Serum samples were diluted in PRRS diluent, added to the plates, and incubated at room temperature for 1 hour. Plates were washed again with PBST, and a secondary antibody, goat anti-IgG HRP, diluted in enzyme conjugate diluent, was added and incubated at room temperature for 30 minutes. Plates were washed with PBST, substrate was added, and the plate was read at 450 nm. Sera from experimentally infected M. haemofelis cats, sera from samples PCR-positive for M. haemofelis, samples PCR-negative for M. haemofelis, and samples from the general cat population were added to the assay plate. Labeled anti-cat IgG was added to the assay plate, and the label was then detected.

[0127] An indirect ELISA assay was used to test antibody responses in cats experimentally infected with M. haemofelis over time. Recombinant proteins were coated onto Immulon IV plates overnight at 4°C, and the plates were blocked with blocking solution (1% BSA in 100 mM Tris pH 7.5). The plates were washed with 5X PBST, and serum samples from experimentally infected cats were diluted with PRRS diluent, added to the plates, and incubated at room temperature for 1 hour. The plates were washed again with 5X PBST, and secondary antibody (anti-cat IgG H+L) was added to the plates and incubated for 30 minutes. The plates were washed, substrate was added, and the plates were read at 450 nm. The indirect ELISA assay was able to detect anti-M. haemofelis antibodies in serum samples as early as approximately 1.5 weeks. See Figure 1. [Example]

[0128] TDX1362 was subjected to epitope mapping. 15-mer peptides covering TDX1362 with 10 amino acid overlaps were tested as follows: 01 LCBiot-MAISHDKGTNIKDRL-amide 15 A1 SEQ ID NO: 22 02 LCBiot-DKGTNIKDRLEASGY-amide 15 A2 SEQ ID NO: 23 03 LCBiot-IKDRLEASGYSILNL-amide 15 A3 SEQ ID NO: 24 04 LCBiot-EASGYSILNLDKGDS-amide 15 A4 SEQ ID NO: 25 05 LCBiot-SILNLDKGDSEKWEK-amide 15 A5 SEQ ID NO: 26 06 LCBiot-DKGDSEKWEKIKEAY-amide 15 A6 SEQ ID NO: 27 07 LCBiot-EKWEKIKEAYGKEDD-amide 15 A7 SEQ ID NO: 28 08 LCBiot-IKEAYGKEDDEALRF-amide 15 A8 SEQ ID NO: 29 09 LCBiot-GKEDDEALRFNGVNK-amide 15 A9 SEQ ID NO: 30 10 LCBiot-EALRFNGVNKNDAST-amide 15 A10 SEQ ID NO: 31 11 LCBiot-NGVNKNDASTIVGIK-amide 15 A11 SEQ ID NO: 32 12 LCBiot-NDASTIVGIKTACSS-amide 15 A12 SEQ ID NO: 33 13 LCBiot-IVGIKTACSSLLASK-amide 15 B1 SEQ ID NO: 34 14 LCBiot-TACSSLLASKSESLR-amide 15 B2 SEQ ID NO: 35 15 LCBiot-LLASKSESLRDLEKA-amide 15 B3 SEQ ID NO: 36 16 LCBiot-SESLRDLEKARRWCV-amide 15 B4 SEQ ID NO: 5 17 LCBiot-DLEKARRWCVVPVTV-amide 15 B5 SEQ ID NO: 37 18 LCBiot-RRWCVVPVTVSSRIG-amide 15 B6 SEQ ID NO: 38 19 LCBiot-VPVTVSSRIGDNLAL-amide 15 B7 SEQ ID NO: 39 20 LCBiot-SSRIGDNLALLSSQD-amide 15 B8 SEQ ID NO: 40 21 LCBiot-DNLALLSSQDSQDTS-amide 15 B9 SEQ ID NO: 41 22 LCBiot-LSSQDSQDTSLWNAK-amide 15 B10 SEQ ID NO: 42 23 LCBiot-SQDTSLWNAKLEEHK-amide 15 B11 SEQ ID NO: 43 24 LCBiot-LWNAKLEEHKKEASK-amide 15 B12 SEQ ID NO: 44 25 LCBiot-LEEHKKEASKFTKIP-amide 15 C1 SEQ ID NO: 45 26 LCBiot-KEASKFTKIPNVWDD-amide 15 C2 SEQ ID NO: 46 27 LCBiot-FTKIPNVWDDKANSD-amide 15 C3 SEQ ID NO: 47 28 LCBiot-NVWDDKANSDELKLT-amide 15 C4 SEQ ID NO: 48 29 LCBiot-KANSDELKLTAFKKK-amide 15 C5 SEQ ID NO: 49 30 LCBiot-ELKLTAFKKKCEEMS-amide 15 C6 SEQ ID NO: 50 31 LCBiot-AFKKKCEEMSKLNTF-amide 15 C7 SEQ ID NO: 51 32 LCBiot-CEEMSKLNTFDKDFE-amide 15 C8 SEQ ID NO: 52 33 LCBiot-KLNTFDKDFESFVAY-amide 15 C9 SEQ ID NO: 53 34 LCBiot-DKDFESFVAYTKDWC-amide 15 C10 SEQ ID NO: 54 35 LCBiot-SFVAYTKDWCTKPKK-amide 15 C11 SEQ ID NO: 7 36 LCBiot-TKDWCTKPKKG-amide 11 C12 SEQ ID NO: 55 B4: LCBiot-SESLRDLEKARRWCV-amide 15 (SEQ ID NO: 5) B5: LCBiot-DLEKARRWCVVPVTV-amide 15 (SEQ ID NO: 37) C10:LCBiot-DKDFESFVAYTKDWC-amide 15 (SEQ ID NO: 54) C11:LCBiot-SFVAYTKDWCTKPKK-amide 15 (SEQ ID NO: 7) The sequence overlap between B4 and B5 indicates that antibodies from M. haemofelis infected cats bind within a polypeptide sequence shared by B4 and B5. B4 / B5: DLEKARRWCV (SEQ ID NO: 74) The sequence overlap between C10 and C11 indicates that antibodies from M. haemofelis infected cats bind within a polypeptide sequence shared by C10 and C11. C10 / C11:SFVAYTKDWC (SEQ ID NO: 140)

[0129] Thirty-six peptides (15mers) with 10 amino acid overlaps covering the entire full-length protein were synthesized and labeled with biotin at the N-terminus.

[0130] Neutravidin plates were coated with 100 μl of peptide diluted in PBS. The plates were incubated at room temperature for 1 hour with shaking, then washed three times with PBS-T. Feline serum samples from cats infected with M. haemofelis were diluted in 1X PBS pH 7.2 + 2% BSA. The plates were incubated at room temperature for 1 hour with shaking, then washed three times with PBS-T. 100 μl of secondary antibody (anti-cat IgG H+L) was added to each well at a dilution of 1:5000. The plates were incubated at room temperature for 1 hour with shaking, then washed five times with PBS-T. The plates were developed with 100 μl of TMB substrate. Negative samples (i.e., serum from cats not infected with M. haemofelis) showed no significant reactivity to the peptide. Peptides B4 (SEQ ID NO: 5), B5 (SEQ ID NO: 37), C10 (SEQ ID NO: 54), and C11 (SEQ ID NO: 7) showed reactivity with sera from M. haemofelis-infected cats in this assay, thus identifying the immunodominant region.

[0131] B4, B5, C10, and C11 were selected for immunogenicity testing in rabbits immunized with recombinant TDX1362 protein. Briefly, 100 μl of peptide diluted in PBS was coated onto a neutravidin plate. The plate was incubated at room temperature for 1 hour with shaking. The plate was washed three times with PBS-T. Serum samples from rabbits immunized against TDX1362 were diluted in 1XPBS pH 7.2 + 2% BSA. The plate was incubated at room temperature for 1 hour with shaking. The plate was washed three times with PBS-T. 100 μl of secondary antibody (anti-rabbit IgG H+L) was added to each well at a dilution of 1:5000. The plate was incubated at room temperature for 1 hour with shaking, then washed five times with PBS-T. The plate was developed with 100 μl of TMB substrate. Negative samples showed no significant reactivity to the peptides. B4 and B5 weakly detected antibodies in the serum of rabbits immunized with recombinant TDX1362 protein, whereas C10 and C11 did not detect antibodies in the serum of rabbits immunized with recombinant TDX1362 protein.

[0132] B4, C11, and mature TDX1362 polypeptides were tested with M. haemofelis-negative serum samples (see Figure 3). In the ELISA assay, TDX1362 had a specificity of 21 / 29, C11 had a specificity of 26 / 29, and B4 had a specificity of 25 / 29.

[0133] B4, C11, and mature TDX1362 polypeptides were tested in M. haemofelis-positive serum samples (see Figures 4A-C). In the ELISA assay, TDX1362 had a sensitivity of 29 / 41, C11 had a sensitivity of 22 / 41, and B4 had a sensitivity of 25 / 41.

[0134] Polyclonal antibodies against recombinant TDX1362 protein were generated in rabbits using standard protocols (SDIX, LLC, Newark, Delaware, USA). Briefly, preimmune serum was collected prior to rabbit immunization. Animals were subcutaneously immunized with 200 μg of immunogen using Freund's complete adjuvant, followed by booster doses on days 21, 35, and 50. Blood was collected on days 45, 60, and 70. Serum samples derived from the blood were evaluated using an ELISA assay with recombinant antigen-coated plates to determine antibody titers against the immunogen. An antigen capture assay was designed using biotinylated rabbit polyclonal anti-M. haemofelis antibody. The biotinylated rabbit polyclonal anti-M. haemofelis antibody was immobilized on a neutravidin plate. Serum samples were added to the plate so that any M. haemofelis antigen present in the sample could specifically bind to the biotinylated rabbit polyclonal anti-M. haemofelis antibody. Assays were also performed using test samples of M. haemofelis antigen. Binding reactions were detected using horseradish peroxidase-labeled rabbit polyclonal anti-M. haemofelis antibody.

[0135] Evaluation of the antigen capture assay was performed using a positive control of recombinant mature TDX1362 at concentrations of 1 μg / ml, 2 μg / ml, or 4 μg / ml, and a negative control of recombinant control TDX926 at concentrations of 1 μg / ml, 2 μg / ml, or 4 μg / ml. The concentrations of M. haemofelis antigen were 1, 0.5, 0.25, 0.125, 0.065, 0.0313, 0.0156, 0.0078, 0.0039, 0.002, 0.0001, and 0.0005 μg / ml. Briefly, purified rabbit IgG against rTDX1362 and rTDX926 (negative control) was coated onto plates (5 μg / ml). Recombinant protein TDX1362 was serially diluted to concentrations ranging from 1 μg / ml to 0.005 μg / ml and incubated on the IgG-coated plate for 30 minutes. The plate was washed three times with PBST to remove nonspecific binding. Purified pAb HRP conjugates against rTDX1362 and rTDX926 were added and incubated for 1 hour. The plate was washed again, substrate was added, and the plate was read at 450 nm. The results are shown in Table 2 below. 2 μg / ml of conjugate was determined to be the optimal concentration based on these assays. [Table 2]

[0136] Samples from PCR-positive and PCR-negative sera from cats were also tested, and the results are shown in Table 3 below. [Table 3]

[0137] The results show that 2 μg / ml of the conjugate is suitable for use in the detection assay. [Example]

[0138] Alanine scanning analysis of polypeptides B4 and C11 was completed.

[0139] B4: LCBiot-SESLRDLEKARRWCV-amide 15 (SEQ ID NO: 5)

[0140] C11:LCBiot-SFVAYTKDWCTKPKK-amide 15 (SEQ ID NO: 7)

[0141] Alanine substitution mutants of B4 are shown below. [Table 4]

[0142] In the above, number 1 (B4 M1) is SEQ ID NO: 56, number 2 (B4 M2) is SEQ ID NO: 57, number 3 (B4 M3) is SEQ ID NO: 58, number 4 (B4 M4) is SEQ ID NO: 59, number 5 (B4 M5) is SEQ ID NO: 60, number 6 (B4 M6) is SEQ ID NO: 61, number 7 (B4 M7) is SEQ ID NO: 62, number 8 (B4 M8) is SEQ ID NO: 63, number 9 (B4 M9) is SEQ ID NO: 64, number 10 (B4 M10) is SEQ ID NO: 5, number 11 (B4 M11) is SEQ ID NO: 66, number 12 (B4 M12) is SEQ ID NO: 67, number 13 (B4 M13) is SEQ ID NO: 68, number 14 (B4 M14) is SEQ ID NO: 69, and number 15 (B4 M15) is SEQ ID NO: 70.

[0143] Alanine substitution mutants of C11 are shown below. [Table 5]

[0144] The above number 1 (C11 M1) is SEQ ID NO: 71, number 2 (C11 M2) is SEQ ID NO: 72, number 3 (C11 M3) is SEQ ID NO: 73, number 4 (C11 M4) is SEQ ID NO: 7, number 5 (C11 M5) is SEQ ID NO: 75, number 6 (C11 M6) is SEQ ID NO: 76, number 7 (C11 M7) is SEQ ID NO: 77, number 8 (C11 M8) is SEQ ID NO: 78, number 9 (C11 M9) is SEQ ID NO: 79, number 10 (C11 M10) is SEQ ID NO: 80, number 11 (C11 M11) is SEQ ID NO: 81, number 12 (C11 M12) is SEQ ID NO: 82, number 13 (C11 M13) is SEQ ID NO: 83, number 14 (C11 M14) is SEQ ID NO: 84, and number 15 (C11 M15) is SEQ ID NO: 85.

[0145] The peptides were tested in an indirect ELISA antibody assay for alanine scanning. Briefly, peptides were coated onto neutravidin plates overnight (3x plate washer washes). Samples (field negatives and experimentally induced negatives) were plated at 1:200, 1:400, and 1:800 at room temperature for 1 hour (3x plate washer washes). Secondary antibody (goat anti-cat HRP) 1:5000 was coated for 1 hour at room temperature and 5x washes. Substrate was added, and after 5 minutes, stop solution was added and read at 450°C. The results are shown in Figures 5A and 5B.

[0146] The B4M12 peptide, SESLRDLEKARAWCV (SEQ ID NO: 67), showed consistently high OD values, indicating increased affinity for the feline antibody. The B4M9 peptide, SESLRDLEAARRWCV (SEQ ID NO: 64), showed consistently low OD values, indicating low affinity for the feline antibody.

[0147] Both B4M9 and C11M9 carry lysine to alanine substitutions that destroy binding activity, therefore these lysine residues should be retained to retain binding activity.

[0148] Both B4M12 and C11M15 possess favorable substitutions that increase the observed OD values, suggesting enhanced antibody interaction. The change from a positively charged side chain to a nonpolar, hydrophobic side chain in B4M9 results in a decrease in affinity for the feline antibody, suggesting that this is important for antibody-peptide interaction. In the case of B4M12, the change from a negatively charged side chain to a nonpolar, hydrophobic side chain results in an increase in affinity for the feline antibody.

[0149] TDX1362 and TDX1452 were tested in a multiplex ELISA assay. The feline serum samples tested are shown in Table 4. [Table 6] [Table 7]

[0150] TDX-1362 and TDX1452 were tested in a multiplex assay together with FIV and FeLV assays. See Table 5. Briefly, recombinant TDX1452 and TDX1362 were conjugated to barcoded magnetic beads. Cat serum was added to the assay plate. Biotin-labeled recombinant TDX1452 and TDX1362 were added to the assay plate. The labeled peptide was detected using streptavidin-PE. The results are shown in Table 6. The data indicate that serum can be tested for Mhf in multiplex with FIV and FELV testing. [Table 8] [Table 9]

[0151] The marker showed a 4-6% positivity rate across samples from the United States and Brazil. Male cats showed higher positivity rates, consistent with previous findings (Table 7). There was an increase in Mhf PCR positivity rates in anemic, FIV, or FeLV-positive, and male cats (Tables 8-10). [Table 10] [Table 11] [Table 12] Approximately 2 / 5 of FIV-positive cats were Mhf-positive. Total FeLV-positive - 46 FeLV / Mhf concordance - 17 / 46 (39%) [Table 13] [Example]

[0152] M. haemofelis PCR-positive and -negative samples were screened by LCMS. TDX 1452 was identified as a candidate marker. Recombinant TDX1452 was expressed in E. coli and purified using an IMAC column.

[0153] An indirect ELISA assay was designed using TDX1452 immobilized on Immulon IV plates. Briefly, plates were coated with recombinant TDX1452 overnight at 4°C, washed with PBST, and blocked with 2% BSA for 2–3 hours. Plates were washed again and stored at 4°C for future use. Plates were thawed at room temperature for 3–4 hours before use. Serum samples were diluted in PRRS diluent, added to the plates, and incubated at room temperature for 1 hour. Plates were washed again with PBST, and a secondary antibody, goat anti-IgG HRP, diluted in enzyme conjugate diluent, was added and incubated at room temperature for 30 minutes. Plates were washed with PBST, substrate was added, and the assay was read at 450 nm. Sera from experimentally infected M. haemofelis cats, sera from samples PCR-positive for M. haemofelis, samples PCR-negative for M. haemofelis, and samples from the general cat population were added to the assay plate. Labeled anti-cat IgG was added to the assay plate, and the label was then detected.

[0154] An indirect ELISA assay was used to test antibody responses in cats experimentally infected with M. haemofelis over time. Recombinant proteins were coated onto Immulon IV plates overnight at 4°C, and the plates were blocked with blocking solution (1% BSA in 100 mM Tris pH 7.5). The plates were washed with 5X PBST, and serum samples from experimentally infected cats were diluted with PRRS diluent, added to the plates, and incubated at room temperature for 1 hour. The plates were washed again with 5X PBST, and a secondary antibody (anti-cat IgG H+L) was added to the plates and incubated for 30 minutes. The plates were washed, substrate was added, and the plates were read at 450 nm. The indirect ELISA assay was able to detect anti-M. haemofelis in serum samples as early as approximately 4.5 weeks post-infection. See Figure 6.

[0155] In another assay, streptavidin plates were coated with biotinylated anti-TDX1452 recombinant antigen polyclonal antibodies. Polyclonal antibodies against recombinant TDX1452 protein were generated in rabbits using standard protocols (SDIX, LLC, Newark, Delaware, USA). Briefly, preimmune serum was collected before rabbit immunization. Animals were subcutaneously immunized with 200 μg of immunogen using Freund's complete adjuvant, followed by booster doses on days 21, 35, and 50. Blood samples were collected on days 45, 60, and 70. Serum samples derived from the blood were evaluated using an ELISA assay with recombinant antigen-coated plates to determine antibody titers against the immunogen. Serially diluted anti-TDX1452 recombinant antigen was used as a control in the plate assay. 50 mL of undiluted serum / plasma sample was added to the plate and incubated at room temperature for 30 minutes. 50 mL of HRP-conjugated anti-TDX1452 pAb was used for detection. The plates were incubated at room temperature for 30 minutes. 50 mL of TMB was added to each well and the plates were incubated at room temperature for 10 minutes. 50 mL of stop solution was added and the plates were read at A650 nm. The test results for experimentally infected cats are shown in Figure 7.

[0156] A series of 15-mer peptides overlapping by 10 amino acids of TDX1452 (FIG. 9) were generated to elucidate the immunodominant regions of the protein. FIG.

[0157] Peptides B5, B6, and C12 showed high affinity for the feline antibody. B5 KEDLLKKLKKWCVIP (SEQ ID NO: 1) B6 KKLKKWCVIPKTVN (SEQ ID NO: 151) C12 QDTLEKVKLWCSVTK (SEQ ID NO: 3)

[0158] The sequence overlap between B5 and B6 indicates that antibodies from M. haemofelis infected cats bind within a polypeptide sequence shared by B5 and B6. B5 / B6: KKLKKWCVIP (SEQ ID NO: 141)

[0159] Peptides were coated onto neutravidin plates overnight (washed 3x with a plate washer). Serum samples (cat field Mhf positive, cat field Mhf negative, rabbit, cat transiently Mhf infected; and SPF negative) were diluted 1:200 and incubated for 1 hour at room temperature (3x with a plate washer). Secondary antibodies 1:5000 were coated for 1 hour at room temperature. The plates were washed and substrate was added. After 5 minutes, stop solution was added. The plates were read at 450 nm. B5, B6, and C12 showed strong immunological responses. [Example]

[0160] Alanine substitutions were made for each amino acid position in the 15-mer peptide. Immunodominant regions B5 and C12 were synthesized with these substitutions.

[0161] The B5 alanine substitution is shown below. [Table 14]

[0162] The C12 alanine substitution is shown below. [Table 15]

[0163] Glycine was used in positions where alanine was already present. Critical amino acids can be detected when a large decrease in OD is observed, suggesting that the substituted amino acid is important for antibody binding. This experiment also provides an opportunity to suggest favorable substitutions when the OD value is high or increased compared to the original peptide.

[0164] An indirect assay was used for epitope mapping of the alanine-substituted B5 and C12 peptides. The peptides were biotinylated at the N-terminus, coated onto neutravidin plates at 1 μg / mL, and incubated overnight at 4°C. The plates were washed three times with a plate washer. Samples were incubated for 1 hour at room temperature (3x plate washer). Secondary antibody was coated onto the plates for 1 hour at room temperature and washed. Substrate was added, followed by stop solution 5 minutes later. The plates were read at 450 nm.

[0165] The results are shown in FIG. 8 and Table 12. [Table 16]

[0166] The B5M12 peptide, KEDLLKKLKKWAVIP (SEQ ID NO: 97), showed a higher OD value than the wild-type B5M0 peptide, KEDLLKKLKKWCVIP (SEQ ID NO: 1), indicating that the B5M12(C>A) mutation increased binding to the feline anti-Mhf antibody. The B5M11 peptide, KEDLLKKLKKACVIP (SEQ ID NO: 96), showed a lower OD value than the wild-type B5M0 peptide, indicating that the substituted amino acid residue (tryptophan) is important for antibody-peptide interaction and should be retained to maintain binding activity.

[0167] The C12M11 peptide, QDTLEKVKLWASVTK (SEQ ID NO: 111), showed a higher OD value than the wild-type C12M0 peptide, QDTLEKVKLWCSVTK (SEQ ID NO: 3), indicating that the C12M11 (C>A) mutation increased binding to the feline anti-Mhf antibody. The C12M10 peptide, QDTLEKVKLACSVTK (SEQ ID NO: 110), showed a lower OD value than the wild-type C12M0 peptide, indicating that the substituted amino acid residue (tryptophan) is important for antibody-peptide interaction and should be retained to maintain binding activity.

Claims

1. One or more polypeptides, including one or more of the following: (i) a polypeptide set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 37, 54, 74, 97, 111, 140, 141, or 151; (ii) a polypeptide comprising SEQ ID NO: 1, (a) W at position 11 is retained and one, two, or three other amino acids are substituted with conservative amino acid substitutions; (b) W at position 11 is retained and conservative amino acid substitutions are at one, two, or three of the following positions: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, or 15; (c) W at position 11 is retained and conservative amino acid substitutions are at one, two, or three of the following positions: 2, 3, 4, 6, 7, 8, or 14; or (d) a polypeptide in which W at position 11 is retained and position 12 is substituted with A, and optionally conservative amino acid substitutions are at one, two, or three of the following positions: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 13, 14, or 15; (iii) a polypeptide comprising SEQ ID NO: 2, (a) W at position 11 is retained and one, two, or three other amino acids are replaced with conservative amino acid substitutions; (b) the W at position 11 is retained and conservative amino acid substitutions are at one, two, or three of the following positions: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, or 15; or (c) a polypeptide in which the W at position 11 is retained and conservative amino acid substitutions are one, two, or three at the following positions: 2, 3, 4, 6, 7, 8, or 14; (iv) a polypeptide comprising SEQ ID NO: 3, (a) W at position 10 is retained and one, two, or three other amino acids are replaced with conservative amino acid substitutions; (b) the W at position 10 is retained and conservative amino acid substitutions are at one, two, or three of the following positions: 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, or 15; or (c) a polypeptide in which the W at position 11 is retained and conservative amino acid substitutions are one, two, or three at the following positions: 1, 3, 5, 6, 7, 8, 14, or 15; (v) a polypeptide comprising SEQ ID NO: 4, (a) W at position 10 is retained and one, two, or three other amino acids are replaced with conservative amino acid substitutions; (b) the W at position 10 is retained and conservative amino acid substitutions are at one, two, or three of the following positions: 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, or 15; or (c) a polypeptide in which the W at position 11 is retained and conservative amino acid substitutions are one, two, or three at the following positions: 1, 3, 5, 6, 7, 8, 14, or 15; (vi) a polypeptide comprising SEQ ID NO: 5, (a) K at position 9 and W at position 13 are retained, and one, two, or three other amino acids are replaced with conservative amino acid substitutions; (b) the K at position 9 and the W at position 13 are retained, and conservative amino acid substitutions are at one, two, or three of the following positions: 1, 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 14, or 15; or (c) a polypeptide in which the K at position 9 and the W at position 13 are retained and the conservative amino acid substitutions are one, two, or three at the following positions: 2, 3, 4, or 5; (vii) a polypeptide comprising SEQ ID NO: 6, (a) K at position 9 and W at position 13 are retained, and one, two, or three other amino acids are replaced with conservative amino acid substitutions; (b) the K at position 9 and the W at position 13 are retained, and conservative amino acid substitutions are at one, two, or three of the following positions: 1, 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 14, or 15; or (c) a polypeptide in which the K at position 9 and the W at position 13 are retained and the conservative amino acid substitutions are one, two, or three at the following positions: 2, 3, 4, or 5; (viii) a polypeptide comprising SEQ ID NO: 7, (a) K at position 7, D at position 8, and W at position 9 are retained, and one, two, or three other amino acids are replaced with conservative amino acid substitutions; (b) the K at position 7, the D at position 8, and the W at position 9 are retained, and conservative amino acid substitutions are at one, two, or three of the following positions: 1, 2, 3, 4, 5, 6, 10, 11, 12, 13, 14, or 15; or (c) a polypeptide in which K at position 7, D at position 8, and W at position 9 are retained and conservative amino acid substitutions are one, two, or three at the following positions: 3, 4, or 15; (ix) a polypeptide comprising SEQ ID NO: 8, (a) D at position 8 and W at position 9 are retained, and one, two, or three other amino acids are replaced with conservative amino acid substitutions; (b) the D at position 8 and the W at position 9 are retained, and conservative amino acid substitutions are at one, two, or three of the following positions: 1, 2, 3, 4, 5, 6, 7, 10, 11, 12, 13, 14, or 15; or (c) a polypeptide in which the D at position 8 and the W at position 9 are retained and conservative amino acid substitutions are one, two, or three at the following positions: 3, 4, 7, or 15; (x) a polypeptide comprising SEQ ID NO: 10; (xi) a polypeptide having 95% or greater sequence identity to SEQ ID NO: 10, wherein amino acids 75-89, 170-184, or 75-89 and 170-184 are retained; (xii) a polypeptide comprising SEQ ID NO: 12; (xiii) a polypeptide having 95% or greater sequence identity to SEQ ID NO: 12, wherein amino acids 82-96, 177-191, or 82-96 and 177-191 are retained; (xiv) a polypeptide comprising SEQ ID NO: 9, or SEQ ID NO: 11; (xv) a polypeptide comprising a polypeptide comprising SEQ ID NO: 141, (a) W at position 6 is retained and one, two, or three other amino acids are substituted with conservative amino acid substitutions; (b) the W at position 6 is retained and conservative amino acid substitutions are at one, two, or three of the following positions: 1, 2, 3, 4, 5, 7, 8, 9, or 10; or (c) a polypeptide in which W at position 6 is retained and position 7 is substituted with A, and optionally conservative amino acid substitutions are at one, two, or three of the following positions: 1, 2, 3, 4, 5, 8, 9, or 10; (xvi) a polypeptide comprising a polypeptide comprising SEQ ID NO: 151, (a) W at position 6 is retained and one, two, or three other amino acids are substituted with conservative amino acid substitutions; (b) the W at position 6 is retained and conservative amino acid substitutions are at one, two, or three of the following positions: 1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, or 14; or (c) a polypeptide in which W at position 6 is retained and position 7 is substituted with A, and optionally conservative amino acid substitutions are at one, two, or three of the following positions: 1, 2, 3, 4, 5, 8, 9, 10, 11, 12, 13, or 14; (xvii) a polypeptide comprising a polypeptide comprising SEQ ID NO: 97, (a) W at position 11 is retained and one, two, or three other amino acids are substituted with conservative amino acid substitutions; (b) the W at position 11 is retained and conservative amino acid substitutions are at one, two, or three of the following positions: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, or 15; or (c) a polypeptide in which W at position 11 is retained and position 12 is substituted with A, and optionally conservative amino acid substitutions are at one, two, or three of the following positions: 1, 2, 3, 4, 5, 8, 9, 10, 13, 14, or 15; (xviii) a polypeptide comprising SEQ ID NO: 74, (a) K at position 4 and W at position 8 are retained, and one, two, or three other amino acids are replaced with conservative amino acid substitutions; or (b) a polypeptide in which the K at position 4 and the W at position 8 are retained and conservative amino acid substitutions are one, two, or three at the following positions: 1, 2, 3, 5, 6, 7, or 10; (xix) A polypeptide comprising SEQ ID NO: 37, (a) K at position 4 and W at position 8 are retained, and one, two, or three other amino acids are replaced with conservative amino acid substitutions; or (b) a polypeptide in which the K at position 4 and the W at position 8 are retained and conservative amino acid substitutions are one, two, or three at the following positions: 1, 2, 3, 5, 6, 7, 11, 12, 13, 14, or 15; (xx) a polypeptide comprising SEQ ID NO: 54, (a) K at position 12, D at position 13, and W at position 14 are retained, and one, two, or three other amino acids are replaced with conservative amino acid substitutions; (b) the K at position 12, the D at position 13, and the W at position 14 are retained, and conservative amino acid substitutions are at one, two, or three of the following positions: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 15; or (C) A polypeptide in which K at position 12, D at position 13, and W at position 14 are retained, and conservative amino acid substitutions are one, two, or three at the following positions: 7, or 8. (xxi) a polypeptide comprising SEQ ID NO: 140, (a) K at position 7, D at position 8, and W at position 9 are retained, and one, two, or three other amino acids are replaced with conservative amino acid substitutions; (b) K at position 7, D at position 8, and W at position 9 are retained, and conservative amino acid substitutions are at one, two, or three of the following positions: 1, 2, 3, 4, 5, 6, or 10; or (c) a polypeptide in which K at position 7, D at position 8, and W at position 9 are retained, and conservative amino acid substitutions are one, two, or three at the following positions: 3, or 4; (xxii) a polypeptide comprising SEQ ID NO: 111, (a) W at position 10 is retained and one, two, or three other amino acids are replaced with conservative amino acid substitutions; (b) the W at position 10 is retained and conservative amino acid substitutions are at one, two, or three of the following positions: 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, or 15; or (c) A polypeptide in which the W at position 11 is retained and conservative amino acid substitutions are at one, two, or three of the following positions: 1, 3, 5, 6, 7, 8, 14, or 15.

2. A fusion protein comprising two, three, four, five, six, or seven polypeptides having 90% or more sequence identity with one or more of the polypeptides of claim 1.

3. A polypeptide having a total of less than 190 amino acids that has 90% or more sequence identity with the polypeptide set forth in SEQ ID NO: 10 or 12.

4. 10. The one or more polypeptides of claim 1, wherein the one or more polypeptides are not naturally occurring.

5. 10. The one or more polypeptides of claim 1, wherein the one or more polypeptides are lyophilized, dehydrated, or dried.

6. 10. The one or more polypeptides of claim 1, wherein the one or more polypeptides further comprise one or more labels or tags.

7. 10. The one or more polypeptides of claim 1, wherein the one or more polypeptides are immobilized on a support.

8. The one or more polypeptides of claim 1, wherein the one or more polypeptides are present in an immune complex with one or more antibodies that specifically bind to a polypeptide set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 37, 54, 74, 97, 111, 140, 141, or 151.

9. 10. The one or more polypeptides of claim 1, wherein the one or more polypeptides comprise one or more secretory signal sequences, one or more epitope tags, or one or more secretory signal sequences and one or more epitope tags.

10. 1. A method for detecting an anti-Mycoplasma haemofelis antibody or an anti-Mycoplasma haemocanis antibody, or a specific binding fragment thereof, comprising: (a) contacting a test sample with one or more polypeptides of claim 1; and (b) detecting a complex between the anti-Mycoplasma haemofelis antibody or the anti-Mycoplasma haemocanis antibody, or a specific binding fragment thereof, and the one or more polypeptides.

11. The method of claim 10 , wherein the one or more polypeptides are immobilized on a support.

12. 11. The method of claim 10, wherein the complex is detected using one or more secondary antibodies, or specific binding fragments thereof, that specifically bind to the anti-Mycoplasma haemofelis antibody or the anti-Mycoplasma haemocanis antibody, or specific binding fragments thereof.

13. The method of claim 12 , wherein the secondary antibody, or specific binding fragment thereof, comprises one or more tags or labels.

14. The method of claim 10, wherein the complex is detected using one or more detector polypeptides, and the one or more detector polypeptides are one or more polypeptides described in claim 1.

15. The method of claim 14 , wherein the one or more detector polypeptides comprise a label or tag.

16. A method for diagnosing a disease caused by Mycoplasma haemofelis antibodies or Mycoplasma haemocanis antibodies in a subject, the method comprising: (a) contacting a test sample with one or more polypeptides described in claim 1; and (b) detecting a complex between an anti-Mycoplasma haemofelis or anti-Mycoplasma haemocanis antibody, or a specific binding fragment thereof, and the one or more polypeptides.

17. 17. The method of claim 16, wherein the subject has been infected with Mycoplasma haemofelis or Mycoplasma haemocanis for less than 15 days.

18. 17. The method of claim 16, further comprising comparing the amount of the complex in the sample with a control sample or a control standard, wherein an elevated level of the complex compared to the control sample or control standard is indicative of a disease caused by Mycoplasma haemofelis or Mycoplasma haemocanis.

19. 17. The method of claim 16, further comprising administering a treatment for a disease caused by Mycoplasma haemofelis or Mycoplasma haemocanis if the complex is detected.

20. 17. The method of claim 16, further comprising determining the amount of the anti-Mycoplasma haemofelis antibody or anti-Mycoplasma haemocanis antibody, or specific binding fragment thereof, in the sample.

21. 17. The method of claim 16, wherein the subject is a non-human animal.

22. 17. The method of claim 16, wherein the test sample is blood, plasma, serum, or lymph.

23. 17. The method of claim 10 or claim 16, wherein the anti-Mycoplasma haemofelis antibody or anti-Mycoplasma haemocanis antibody, or a specific binding fragment thereof, is detected by competitive immunoassay, sandwich immunoassay, enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, immunoturbidimetry, particle-enhanced turbidimetric immunoassay, radioimmunoassay (RIA), fluorescence immunoassay (FIA), multiplex immunoassay, protein / peptide array immunoassay, solid-phase radioimmunoassay (SPRIA), indirect immunofluorescence (IIF), chemiluminescence immunoassay (CIA), particle-based multicomponent assay (PMAT), dot blotting, Western blotting, surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), microscale thermophoresis (MST), biolayer interferometry, or grating-coupled interferometry.

24. 24. The method of claim 23, wherein the multiplex immunoassay additionally tests for feline leukemia virus (FeLV), feline immunodeficiency virus (FIV), or both FeLV and FIV.

25. A kit for diagnosing a disease caused by Mycoplasma haemofelis or Mycoplasma haemocanis, the kit comprising: (a) one or more polypeptides of claim 1, wherein said polypeptides are not naturally occurring; (b) one or more reagents that promote binding of one or more polypeptides to anti-Mycoplasma haemofelis antibodies, or anti-Mycoplasma haemocanis antibodies, or specific binding fragments thereof, present in the test sample.

26. A method for treating a disease caused by Mycoplasma haemofelis antibodies or Mycoplasma haemocanis antibodies in a subject, comprising: (a) contacting a test sample with one or more polypeptides described in claim 1; (b) detecting a complex between anti-Mycoplasma haemofelis or anti-Mycoplasma haemophenis, or a specific binding fragment thereof, and the one or more polypeptides; and (c) treating the subject with one or more antibiotics if a complex between anti-Mycoplasma haemofelis or anti-Mycoplasma haemophenis antibodies, or a specific binding fragment thereof, and the one or more polypeptides is detected.

27. 27. The method of claim 26, wherein the subject has been infected with Mycoplasma haemofelis or Mycoplasma haemocanis for less than 15 days.

28. 27. The method of claim 26, further comprising comparing the amount of the complex in the sample with a control sample or control standard, wherein an elevated level of the complex compared to the control sample or control standard is indicative of a disease caused by Mycoplasma haemofelis or Mycoplasma haemocanis.

29. 27. The method of claim 26, further comprising determining the amount of the anti-Mycoplasma haemofelis antibody, or anti-Mycoplasma haemocanis antibody, or specific binding fragment thereof, in the sample.

30. 27. The method of claim 26, wherein the subject is a non-human animal.

31. 27. The method of claim 26, wherein the test sample is blood, plasma, serum, or lymph.

32. 27. The method of claim 26, wherein the anti-Mycoplasma haemofelis antibody or anti-Mycoplasma haemocanis antibody, or a specific binding fragment thereof, is detected by competitive immunoassay, sandwich immunoassay, enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, immunoturbidimetry, particle-enhanced turbidimetric immunoassay, radioimmunoassay (RIA), fluorescence immunoassay (FIA), multiplex immunoassay, protein / peptide array immunoassay, solid-phase radioimmunoassay (SPRIA), indirect immunofluorescence (IIF), chemiluminescence immunoassay (CIA), particle-based multicomponent assay (PMAT), dot blotting, Western blotting, surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), microscale thermophoresis (MST), biolayer interferometry, or grating-coupled interferometry.

33. A polynucleotide encoding one or more of the polypeptides of claim 1, the fusion protein of claim 2, or the polypeptide of claim 3.

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