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    • 专利摘要:
    Monoclonal antibody against N. ceranae and the hybridoma that produces it. Monoclonal antibody against a protein of the spore wall (exospore) of the microsporidium Nosema ceranae identified by the sequence SEQ ID NO: 1 produced by the hybridoma deposited in the ECACC with deposit number 19061101; hybridoma producing said monoclonal antibody; in vitro method of diagnosis of nosemosis caused by Nosema ceranae; kit to carry out the in vitro diagnosis of nosemosis caused by Nosema ceranae in bees, food products containing honey or products derived from it: use of said monoclonal antibody in the in vitro detection of Nosema ceranae spores and device for detecting said protein comprising said monoclonal antibody. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2803960A1
    申请号:ES201930700
    申请日:2019-07-29
    公开日:2021-02-01
    发明作者:De La Puente Carmen Aguila；Arias Fernando Izquierdo；Rodríguez Soledad Fenoy；Vadillo Carmen Fernández；Pascual Mariano Higes；Hernández Raquel Martín
    申请人:Instituto Regional De Investig Y Desarrollo Agroalimentario Y Forestal De Castilla La Mancha Iriaf；Fundacion Universitaria San Pablo CEU；
    IPC主号:
    专利说明:

    [0004] TECHNICAL SECTOR
    [0006] The invention belongs to the field of immunochemical techniques for the detection of antigen-antibody complexes. The invention relates to a monoclonal antibody produced by a hybridoma and to its use in in vitro methods of diagnosis of nosemosis caused by Nosema ceranae in bees.
    [0008] BACKGROUND OF THE INVENTION
    [0010] The economic importance of the beekeeping industry is indisputable since bees are pollinating insects that play a very important role in nature. The pollination process is critical in the production of food and, therefore, for the continuation of the human mode of subsistence. Furthermore, pollination is a process that directly links natural ecosystems with agricultural production systems. The role of these insects has meant that, at present, there is a high interest in the recent decrease in pollinators detected in different regions of the world.
    [0012] In recent years, high mortality has been observed in bees from different geographical areas and consequently a reduction in honey production. The analyzes carried out on these bees have shown an increase in the diagnosed cases of nosemosis. Nosemosis is a pathology described in honey-producing bees produced by intracellular organisms, specifically, microsporidia belonging to the genus Nosema. There are two species belonging to this genus related to this pathology: Nosema apis, a parasite of western and European bees ( Apis mellifera) and Nosema ceranae, a parasite of the Asian bee ( Apis cerana). In recent years, in the different beekeeping areas, both in Spain and in other countries, an increase in N. ceranae infections has been detected in western and European bees (A. mellifera). This fact, together with high bee mortality and, consequently, a reduction in honey production, makes it necessary to differentiate between N. ceranae and N. apis .
    [0014] The pathology produced by N. ceranae in A. mellifera is more aggressive than in its usual host A. cerana. Although N. apis nosemosis is acutely associated with diarrhea, in the case of that produced by N. ceranae, the picture is different, characterized by the absence of diarrhea symptoms (dry diarrhea) and the progressive weakening of the hives, causing their death in the most serious cases . This pathology is included within the possible causes of the Depopulation Syndrome of the Hives.
    [0016] In N. apis infections, the epithelial cells of A. mellifera in which the mature spores are found are typically surrounded by vegetative cells (merozoites) of the parasite. Infections by N. ceranae in their original host A. cerana, appear as isolated foci, of a single epithelial cell, in which the mature spores that have developed are also found. However, when N. ceranae infects A. mellifera, it presents a pattern similar to that of N. apis in this host, which rules out histology as a technique that allows the differential diagnosis of infection by N. ceranae and N. apis species. in A. mellifera.
    [0018] The spores of the species N. ceranae and N. apis are very similar and difficult to differentiate under the light microscope. The differentiation between the spores of N. ceranae and those of N. apis using the light microscope is quite complicated. In general, the spores of N. ceranae are slightly smaller (4.7 x 2.7 | jm) than those of N. apis (6 x 3 jm). However, if the size ranges that have been described for the spores of both species are compared, there is a small overlap. In this way, the smaller spores of N. apis could be confused with the larger ones of N. ceranae. Another characteristic that can be taken into account when differentiating these species by light microscopy is that the spores of N. ceranae, in some cases, are slightly bent, which gives them a less consistent appearance than those of N. apis . The greatest differences between the two species of Nosema appear when studying the ultrastructure of their spores. The polar tubule in N. apis is larger, with 26-32 turns, while that of N. ceranae only has 20-23 turns.
    [0020] Detection techniques for N. ceranae and N. apis have been developed based on the detection of molecular markers by polymerase chain reaction (PCR). The detection techniques based on PCR allow the detection of very low levels of infection produced by the parasite in all the stages of the cycle and the sequencing of its products allows the identification and differentiation of the two species of Nosema. One marker that has been used for the diagnosis of microsporidia is the 16S small subunit of ribosomal RNA (rRNA). In these parasites, the sequences of the genes of the 16S small subunit of rRNA are shorter and share little homologies with those of other eukaryotes.
    [0021] Furthermore, they are highly conserved sequences. Klee et al. developed a method for detecting N. ceranae based on restriction fragment length polymorphism analysis (RFLP-PCR) of the 16S small subunit of rRNA with which it is possible to distinguish between N. apis and N. ceranae (Klee et al., 2007).
    [0023] Martín-Hernández et al. designed a double PCR and multiple PCR technique that allows the simultaneous diagnosis of the two species in the same reaction (Martín-Hernández et al., 2007).
    [0025] Chen et al. designed a quantitative multiplex PCR in real time (Q-PCR) that allows the simultaneous detection of the two parasites and their quantification (Chen et al., 2009).
    [0027] These PCR-based detection techniques are associated with complications, as they are laborious, require expensive equipment and reagents, and are expensive.
    [0029] Various immunological techniques for the detection of N. ceranae spores have been developed based on the detection of spores by means of antibodies.
    [0031] Aronstein et al. developed a polyclonal antibody against a N. ceranae spore wall protein that has specificity for N. ceranae against N. apis (Aronstein et al., 2011).
    [0033] Over polyclonal antibodies, monoclonal antibodies have advantages, such as monospecificity, known biological activity, and constant specificity.
    [0035] Document ES2548425A1 describes a monoclonal antibody that recognizes the spore of microsporidia of the species Nosema apis and describes its use in the diagnosis, by immunochemical techniques, of nosemosis of the bee caused by Nosema apis.
    [0037] Document ES2548424A1 describes a monoclonal antibody that recognizes the microsporidia spore of the species Nosema ceranae and describes its use in the diagnosis, by immunochemical techniques, of nosemosis of the bee caused by Nosema ceranae. This document does not describe the antigen against which the monoclonal antibody is directed. In ES2548424A1 it is described that it is not ruled out that the antibody recognizes an antigen of the endospore or of some structure of the sporoplasm of the spore.
    [0038] The monoclonal antibody of the invention specifically recognizes a protein of the spore wall of N. ceranae identified by the sequence SEQ ID NO: 1 with access number A0A0F9YW02 in the UniProtKB database.
    [0040] Cornman et al. and Pelin et al. described sequences that code for proteins, obtained by prediction from data of N. ceranae DNA sequences obtained by DNA sequencing techniques, and described the amino acid sequence SEQ
    [0041] ID NO: 1 (Cornman et al., 2009; Pelin et al., 2015).
    [0043] The biological function of this cell wall protein of the N. ceranae spore identified by the sequence SEQ ID NO: 1 is unknown. Li et al. they compared the sequence
    [0044] of this protein of N. ceranae with the sequence of the protein SWP5, of the wall of the spore
    [0045] of Nosema bombycis, microsporidium that infects the silkworm, determining that
    [0046] there is 25% identity between both sequences (Li et al., 2012).
    [0048] No monoclonal antibody directed against the disease has been described in the state of the art.
    [0049] cell wall protein of the N. ceranae spore identified by the sequence SEQ ID
    [0050] NO: 1.
    [0052] DESCRIPTION OF THE INVENTION
    [0054] The NosCer74 monoclonal antibody differs from the state of the art in that it specifically recognizes a protein from the wall of the N. ceranae spore identified by the sequence SEQ ID NO: 1.
    [0056] Specific recognition of this protein is associated with an improvement in diagnosis
    [0057] in bees from the nosemosis caused by N. ceranae.
    [0059] The specific recognition of the peptides identified by the sequences SEQ ID NO: 2 3, belonging to the protein
    [0060] ceranae and a complete absence of cross-reactivity against N. apis spores. Said specific recognition is also associated with an improved reproducibility in the diagnosis in bees of the nosemosis caused by N. ceranae.
    [0062] The specific recognition of the peptides identified by the sequences SEQ ID NO: 2 3, belonging to the protein
    [0063] and it is also associated with a lack of cross-reactivity against spores of other microsporidia, such as Nosema locustae, a microsporidium that infects other arthropods.
    [0065] The use of immunochemical techniques based on the monoclonal antibody NosCer74 allows a faster, cheaper, less laborious and more reliable diagnosis of the nosemosis caused by N. ceranae with differentiation at the species level in bees than the diagnosis by PCR.
    [0067] The technical problem to be solved would therefore consist in providing a monoclonal antibody associated with an improved diagnosis in bees of nosemosis caused by N. ceranae, in which a high specificity is obtained against N. ceranae spores, a total absence of cross reactivity against spores of N. apis and a higher reproducibility.
    [0069] The present invention, defined by the object of the claims, provides a solution to said technical problem.
    [0071] The present invention refers to a monoclonal antibody against a protein of the spore wall (exospore) of microsporidium Nosema ceranae identified by the sequence SEQ ID NO: 1, in which said monoclonal antibody is produced by the hybridoma deposited in the ECACC with deposit number 19061101 (also identified as NosCer74 herein).
    [0073] In a preferred embodiment, said monoclonal antibody specifically recognizes the epitopes identified by the sequences SEQ ID NO: 2 and 3.
    [0075] In another preferred embodiment, said monoclonal antibody is conjugated with a labeling probe.
    [0077] In a more preferred embodiment of said monoclonal antibody, said labeling probe is selected from the group consisting of a fluorophore, a chromophore, a bioluminescent probe, an enzyme, and colloidal gold.
    [0079] In another preferred embodiment, said monoclonal antibody is radioactively labeled.
    [0080] Said radioactively labeled monoclonal antibody comprises spontaneously disintegrating radioactive atoms, nuclei or isotopes. These radioactive atoms comprise unstable nuclei that remain in an excited state and reach their ground state by losing energy. This loss of energy is in the form of electromagnetic emissions or particle emissions.
    [0081] In one embodiment, the present invention relates to a composition comprising said monoclonal antibody.
    [0083] Said composition can comprise aqueous solvents. Examples of aqueous solvents are: water, saline, and buffers. Examples of buffers are: phosphate buffer, saline phosphate buffer, acetate buffer, borate-chloride buffer, carbonate buffer, glycine buffer, and Tris buffer. Said composition may comprise diluents, cosolvents, preservatives, stabilizers, antioxidants and other additives, such as, for example, antimicrobial agents, chelating agents, thickening agents, emulsifying agents, dispersing agents, etc. Said composition can be a liquid solution, liquid suspension, emulsion, solid form suitable to be dissolved or placed in suspension and composition based on water.
    [0085] In another embodiment, the present invention relates to a hybridoma deposited with ECACC with deposit number 19061101 (also identified as NosCer74 hybridoma herein).
    [0087] Said monoclonal antibody can be obtained by culturing said hybridoma in a commercially available cell culture medium and isolating said monoclonal antibody, secreted by said hybridoma, from said cell culture medium and optionally purifying said monoclonal antibody. In such a production method, cells, cell debris, lipids and coagulated material can be removed, typically by centrifugation followed by filtration. The concentration of said monoclonal antibody can also be increased by ultrafiltration or dialysis. Most of the impurities are usually anions that can be removed by ion exchange chromatography. Impurities can also be removed by size exclusion chromatography or affinity chromatography.
    [0088] In yet another embodiment, the present invention relates to an in vitro method of diagnosis of nosemosis caused by Nosema ceranae, wherein said method comprises:
    [0089] - bringing said monoclonal antibody or a composition comprising it into contact with a preparation obtained from an isolated sample of one or more arthropods, thus forming an antigen-antibody complex, if said antigen is present in said preparation,
    [0090] - detect said antigen-antibody complex and
    [0091] - determine that said preparation contains spores of Nosema ceranae in case of positive detection of said complex, thus determining that said arthropod or arthropods are affected by nosemosis caused by Nosema ceranae.
    [0092] In said diagnostic method, said arthropod can be an arthropod that acts as a mechanical vector that favors the transmission and dissemination of Nosema ceranae spores.
    [0094] In a preferred embodiment of said diagnostic method, said arthropod is a bee. In an even more preferred embodiment, said bee is of the Apis mellifera or Apis cerana species
    [0096] In said diagnostic method, the detection of said antigen-antibody complex can be detected directly when said monoclonal antibody is conjugated with a labeling probe (fluorophore, chromophore, bioluminescent probe, enzyme or colloidal gold) or is radioactively labeled by means of fluorescence detection techniques, light absorption, etc.
    [0098] In such a method, detection can be carried out indirectly using, for example, a secondary antibody that specifically recognizes said monoclonal antibody. Said secondary antibody can be conjugated to a labeling probe (fluorophore, chromophore, bioluminescent probe, enzyme or colloidal gold) or radioactively labeled.
    [0100] In a preferred embodiment of said diagnostic method, the detection of said antigen-antibody complex is carried out by means of a technique selected from the group consisting of immunofluorescence, immunohistochemistry, immunochromatography, immunoblotting ( Western Blot), ELISA and microarrays.
    [0102] In a more preferred embodiment of said diagnostic method, said immunofluorescence is indirect or direct immunofluorescence.
    [0104] In an even more preferred embodiment of said diagnostic method, said preparation is a homogenate of bees from a hive, food products comprising honey or products derived from honey and in case of positive detection of said complex it is determined that said hive of The one from which the homogenate was obtained, or from which the honey was obtained from the products that comprise it or are derived from it, is affected by nosemosis caused by Nosema ceranae.
    [0106] In yet another embodiment, the present invention relates to a kit for carrying out the in vitro diagnosis of nosemosis caused by Nosema ceranae in arthropods, wherein said kit comprises:
    [0107] - said monoclonal antibody or the composition comprising it and
    [0108] - Buffer solutions to carry out the immunological reaction to form the antigen-antibody complex and to carry out the detection of said complex.
    [0110] Examples of buffer solutions are: phosphate buffer, saline phosphate buffer, acetate buffer, borate-chloride buffer, carbonate buffer, glycine buffer, and Tris buffer.
    [0112] In a preferred embodiment of said kit, said arthropods are bees.
    [0114] In a preferred embodiment, said kit is selected from the group consisting of a fluorescence kit, an immunoenzymatic kit, and an immunochromatographic kit.
    [0116] In a preferred embodiment, said kit further comprises reagents to carry out the immunological reaction to form the antigen-antibody complex and to carry out the detection of said complex.
    [0118] In a more preferred embodiment, said kit further comprises a secondary antibody that specifically recognizes said monoclonal antibody.
    [0120] In an even more preferred embodiment of said kit, said secondary antibody is an antibody that specifically recognizes mouse antibodies.
    [0122] In yet another embodiment, the present invention relates to the use of said monoclonal antibody or the composition comprising it in the in vitro detection of Nosema ceranae spores in a sample.
    [0124] In this use, the in vitro detection of Nosema ceranae spores can be carried out directly when said monoclonal antibody is conjugated with a labeling probe or is radioactively labeled by means of fluorescence detection, light absorption, enzymatic techniques, etc. .
    [0126] In such use, detection can be performed indirectly using, for example, a secondary antibody that specifically recognizes said monoclonal antibody. Said secondary antibody can be conjugated to a labeling or radiolabelled probe.
    [0128] In a preferred embodiment of said use, said detection comprises contacting said antibody with said sample to form an antigen-antibody complex, detecting said complex, and determining that said sample contains Nosema ceranae spores.
    [0129] In a more preferred embodiment of said use, the detection of said antigen-antibody complex is carried out by means of a technique selected from the group consisting of immunofluorescence, immunohistochemistry, immunochromatography, immunoblotting ( Western Blot), ELISA and microarrays.
    [0131] In a preferred embodiment of said use, said immunofluorescence is indirect or direct immunofluorescence.
    [0133] In an even more preferred embodiment of said use, said sample is a biological sample from bees, or from honey, or from products derived from honey.
    [0135] In yet another embodiment, the present invention relates to a device or kit for detecting the microsporidium Nosema ceranae spore wall protein identified by the sequence SEQ ID NO: 1, wherein said device or kit comprises said antibody monoclonal or the composition comprising said monoclonal antibody.
    [0137] In a preferred embodiment, said device or said kit for detecting the microsporidium Nosema ceranae spore wall protein identified by the sequence SEQ ID NO: 1, wherein said device or kit comprises said monoclonal antibody and wherein said monoclonal antibody recognizes said microsporidium Nosema ceranae spore wall protein identified by the sequence SEQ ID NO: 1.
    [0139] In a preferred embodiment of said device or said kit, said monoclonal antibody is immobilized on a surface of said device or said kit.
    [0141] In a more preferred embodiment, said device is selected from the group consisting of a biosensor, an immunochromatographic device and a microarray.
    [0143] In another embodiment, the present invention relates to said monoclonal antibody or a composition comprising it for use in the in vitro diagnosis of nosemosis in bees and / or in hives.
    [0145] In a further embodiment, the present invention relates to said monoclonal antibody for use in the manufacture of a composition, device, biosensor or kit, for the diagnosis of nosemosis in bees and / or in hives.
    [0146] Unless otherwise defined, all technical and scientific terms have the same meaning as those commonly understood by a person skilled in the field of the invention. Methods and materials similar and equivalent to those described herein can be used in the practice of the present invention.
    [0148] Throughout the description and claims, the term "comprises", "comprising" and their variants are not limiting in nature and therefore are not intended to exclude other technical characteristics. The term "comprises", "comprising" and their variants, throughout the description and claims, specifically includes the term "consists of", "consisting of" and their variants.
    [0150] Definitions
    [0152] In the present specification, the term "labeling probe" refers to a probe or molecule conjugated with the monoclonal antibody NosCer74 that allows the detection of said monoclonal antibody by means of fluorescence detection, light absorption, enzymatic techniques, etc.
    [0154] In this specification, the term "fluorophore" (also called fluorochrome) refers to a molecule that absorbs energy of a specific wavelength and emits it, with lower energy, in a determined one of higher wavelength. Examples of fluorophores are: fluorescein isothiocyanate (ITFC), ethidium bromide, propidium iodide, phycoerythrin, cyanine, phycocyanin, allophycocyanin, 6-FAM, Cy5, Cy3, TAMRA, JOE, MAX, TET, Cy5.5, ROX, TYE 563, Ykima Yellow, HEX, TEX 615, TYE 665, TYE 705, Alexa Fluorine (350, 405, 488, 532, 546, 555, 568, 594, 647, 660, 680, 750), LI-COR IR ( 700, 800, 800CW), ATTO (488, 532, 550, 565, Rho101, 590, 633, 647N), Rhodamines (Green-X, Red-X, 5-TAMRA), WellRED (D4, D3, D2), Texas Red, Texas Red-X, Lightcycler 640, Dt 750, GFP, Oregon Green, Pacific Blue, Pacific Orange, Pacific Green, Coumarin, Tetramethylrhodamine (TRITC), BODIPY FL, Super Bright (436, 600, 645, 702), DAPI, SYTOX Green, SYTO 9, TO-PRO-3, Qdot (525, 565, 605, 655, 705, 800).
    [0156] As used herein, the term "chromophore" refers to a molecule or group that has electrons capable of absorbing energy and becoming excited, at different wavelengths. Chromophores have unsaturated groups responsible for the absorption of light.
    [0157] In the present specification, the term "bioluminescent probe" refers to a probe or molecule that participates in a biochemical reaction through which some living organisms produce light. A well-known example of a bioluminescent reaction is that involving the enzyme luciferase. and the luciferin protein.
    [0159] As used herein, the term "immunofluorescence" refers to an immunostaining technique that uses antibodies chemically bound to a fluorescent molecule to detect the presence of an antigen. In this technique, the antigen can be detected in live cells or in tissue samples. .
    [0161] Herein, the term "indirect immunofluorescence" (also known as IFI) refers to an immunofluorescence technique that makes use of two antibodies; the primary antibody is the one that recognizes and binds to the target molecule, while the secondary which is the one that is marked with the fluorophore, recognizes the primary and binds to it.
    [0163] Herein, the term "immunohistochemistry" refers to an assay in which an antigen is detected and made visible. This assay enables the location of an antigen at the tissue or cellular level in a tissue sample to be identified. It is based on the use of antibodies that specifically bind to an antigen to be identified.
    [0165] In the present specification, the term "immunochromatography" refers to an immunodiagnostic technique whose main advantages are simplicity and speed. There are many applications of this technique, both in the field of tests, because it is not necessary reagents or instrumentation additionally, such as in the health field. A well-known example is the pregnancy test commercially distributed in pharmacies. The technique can be performed using a device (in this specification, this device will be referred to as an "immunochromatographic device") or kit developed to detect the presence (or absence) of a target compound in the sample (the matrix). Said device can be presented in a strip format, in which the test sample flows along a solid substrate (eg nitrocellulose membrane) by means of capillary action or lateral flow.
    [0166] As used herein, the term "ELISA" refers to an enzyme-linked immunosorbent assay (peroxidase, alkaline phosphatase, etc.), which is an immunoassay technique in which an immobilized antigen is detected by an antibody bound to a enzyme capable of generating a detectable product (OPD, TMB, etc.) from a substrate, by means of a color change or some other type of change caused by the enzymatic action on said substrate. In this technique there may be a primary antibody that recognizes antigen and which in turn is recognized by a secondary antibody linked to said enzyme. Antigen can be indirectly detected in the sample by spectrophotometrically measured color changes.
    [0168] As used herein, the term "immunoblotting" refers to a technique for identifying specific proteins in a complex mixture of proteins, such as cell or tissue extracts. The assay is typically performed in three steps: separation by size and charge (electrophoresis), transfer to a solid support (membrane), and finally visualization using an antibody or antibodies. The Western Blot assay is an example of an immunoblot assay.
    [0170] As used herein, the term "microarray" ( microarray or lab-on-a-chip) refers to a two-dimensional matrix on a solid substrate (generally a glass sheet or a cell with a thin silicon film) that analyzes large quantities of biological material using miniaturized, multiplexed and parallel high-throughput detection and processing methods. An example of microarrays are antigen microarrays and antibody microarrays.
    [0172] In the present specification, the term "biosensor" refers to a device for the measurement of biological or chemical parameters. It usually combines a component of a biological nature and another of a physical-chemical nature. It consists of a biological sensor, for example an antibody; a transducer, which couples the other two elements and translates the signal emitted by the sensor; and a detector, for example, optical, piezoelectric, thermal, magnetic, etc. A well-known example of a biosensor is one that measures glucose in the blood.
    [0174] BRIEF DESCRIPTION OF THE DRAWINGS
    [0176] Figure 1. Immunization protocol of mice of the BALB / c strain immunized by intraperitoneal administration of Nosema ceranae spores from hives with infected bees.
    [0178] Figure 2. Reactivity of the supernatant (undiluted) of the monoclonal antibody NosCer74 against the spores of N. ceranae by IFI.
    [0179] DESCRIPTION OF REALIZATION MODES
    [0181] Example 1. Obtaining the monoclonal antibody NosCer74
    [0183] 10 7 week old female mice of the BALB / c strain (Charles Rives Laboratories International, Inc.) were immunized by intraperitoneal administration of 5x107 or 2.5x107 purified spores of Nosema ceranae from hives with infected bees diluted in a final volume of 0.2 ml of phosphate buffer saline (PBS) per mouse.
    [0185] Immunization and bleeding was carried out in the Animal Facility of the Faculty of Pharmacy of the San Pablo-CEU University (registration number in the CAM: EX-029-UC and European registration ES280220000015) and following the rules established by the Royal Decree 233/1988 of March 14 that transposes the European Directive 86/609 / EEC.
    [0187] The mice were immunized for a total of 182 days ( Figure 1 ) which included the days of inoculation (up to day 152 inclusive) and those of bleeding (up to day 182 inclusive). The pattern of the inocula is shown below:
    [0189] Inocula:
    [0191] - Day 1: 5x107 purified spores of Nosema ceranae were inoculated intraperitoneally. in 0.2 ml of PBS per mouse.
    [0192] - Day 12: 5x107 purified spores of Nosema ceranae were inoculated intraperitoneally in 0.2 ml of PBS per mouse.
    [0193] - Day 97: 5x107 purified spores of Nosema ceranae were inoculated intraperitoneally. in 0.2 ml of PBS per mouse.
    [0194] - Day 121: 2.5x107 purified spores of Nosema ceranae were inoculated intraperitoneally in 0.2 ml of PBS per mouse.
    [0195] - Day 152: 2.5x107 purified spores of Nosema ceranae were inoculated intraperitoneally. in 0.2 ml of PBS per mouse.
    [0197] During this time, the evolution and rate of antibodies produced by mice after immunization against Nosema ceranae spores were studied. For this, said sera were obtained by means of the extraction of blood by the saphenous route (Microvette R Cat. 16440, CB 300, Sarstedt), obtaining a volume of 0.1 to 0.4 ml of blood per mouse. The polyclonal sera obtained were titrated by indirect immunofluorescence (IFI) in order to know the level of antibodies produced against N. ceranae. For IFI, microscope slides were prepared with a 107 spore / ml suspension of N. ceranae. The slides were air-dried, fixed in methanol-acetone (1: 1) for 10 minutes and then incubated with the polyclonal sera obtained. After incubation in a humid chamber for 30 minutes at 37 ° C, the slides were washed three times in distilled water. Fluorescein isothiocyanate conjugated whole molecule or secondary anti-mouse IgG antibody (catalog number F2012; Sigma, St. Louis, Mo) was diluted 1: 300 in PBS buffer with 0.2% Evans Blue and added to the slides for incubation at 37 ° C for 30 min. Subsequently, the slides were washed, the coverslips were placed with PVA-DABCO mounting medium, and the samples were examined under UV light microscopy (excitation wavelength 490 nm and observation wavelength 514 nm).
    [0199] The bleeding pattern is shown below, as well as the serum titer obtained in each of them:
    [0201] Indents:
    [0203] - Day 0: the blood was extracted via the saphenous route, obtaining a volume of 150 jl of blood per mouse. The polyclonal serum obtained presented a serum titer of 0, with absence of humoral response against N. ceranae.
    [0205] - Day 49: the blood was extracted via the saphenous route, obtaining a volume of 150 µl of blood per mouse. The polyclonal serum obtained presented a serum titer of 400 against N. ceranae .
    [0207] - Day 112: the blood was extracted via the saphenous route, obtaining a volume of 370 jl of blood per mouse. The polyclonal serum obtained presented a serum titer of 1,600 against N. ceranae.
    [0209] - Day 131: blood was extracted via the saphenous route, obtaining a volume of 200 jl of blood per mouse. The polyclonal serum obtained presented a serum titer of 1,600 against N. ceranae.
    [0211] - Day 182: blood was extracted via the saphenous route, obtaining a volume of 200 jl of blood per mouse. The polyclonal serum obtained presented a serum titer of 3,200 against N. ceranae .
    [0213] Once the immunization protocol was completed, 3 or 4 days before cell fusion, a booster dose was administered intravenously to stimulate splenocytes. Splenocytes were harvested and fused with P3X63-Ag8.653 myeloma cells (CEU San Pablo University, Madrid, Spain) using polyethylene glycol (PEG) as agent. membrane fusing chemical. Six fusions were carried out, in which the hybridomas were seeded in 96-well plates previously lined with thymocytes and RPMI-1640 culture medium containing 18% heat-inactivated bovine serum (Linus Cat. No. S01805) and supplemented with hypoxanthine-aminopterin-thymidine (HAT).
    [0215] The BALB / c strain of mice used is characterized by high histocompatibility with the myeloma or plasmacytoma line (P3X63-Ag.8.653) used in the fusion, since it is derived from the same animal strain, favoring the stability of the hybrids obtained in cell fusions.
    [0217] During the immunization period, hyperimmune sera from immunized mice were pooled and tested against their homologous species, N. ceranae and their hererologous species, N. apis, by IFI ( Table 1 ). Titres against N. ceranae increased with each inoculation, reaching a maximum titer of 3200 in the last bleeding. A strong reaction to N. apis (serum titer 3200) appeared on the third bleed.
    [0219] Table 1. Reactivity of polyclonal antisera from mice against spores of N. ceranae and N. apis
    [0221]
    [0224] From the six cell fusions that were carried out, 3050 hybridomas were obtained that secreted antibodies against N. ceranae. The NosCer74 hybridoma, which produced a stronger reaction, was selected and cloned at least three times by limiting dilution ensuring that all cells came from the same progenitor cell. For this, the Hybridoma NosCer74 was diluted and dispensed at concentrations of 5, 1 and 0.5 cells per well, in 96-well plates previously lined with thymocytes. Several clones (at least three) of each selected hybrid were carried out until guaranteeing its "monoclonality" with a positivity of 100% in the plates of 1 and 0.5 cells / well, using the IFI technique.
    [0226] The NosCer74 hybridoma was injected intraperitoneally into BALB / c mice for the generation of ascites fluid. Monoclonal antibody (mAb) NosCer74 was purified from ascites fluid by precipitation with ammonium sulfate and dialysis against PBS buffer (24 hours at 4 ° C).
    [0228] Example 2. Characterization of the monoclonal antibody NosCer74
    [0230] The isotype determination of the mAb NosCer74 was carried out using the Pierce® Rapid Isotyping Kit with Kappa and Lambda-Mouse from Thermo Scientific Cat n ° 26179, following the protocol described by the manufacturer. NosCer74 mAb was determined to be an IgM immunoglobulin.
    [0232] The supernatant and ascites fluid containing the mAb NosCer74 were tested against N. ceranae by IFI. IFI was performed as described in Example 1, but in this case, the slides were incubated with the hybridoma culture supernatants diluted 1/25 to 1/800, or ascites fluid diluted 1/25 to 1 / 51200.
    [0234] The mAb NosCer74 strongly reacted with the exospore ( Figure 2 ). Regarding the supernatants ( Table 2 ), the NosCer74 antibody reached a titer of 200 against N. ceranae. On the other hand, given its higher concentration of mAb, the ascites fluid titers increased with respect to those of the supernatants ( Table 3 ). The ascites fluid titer against N. ceranae reached a titer of 3200.
    [0236] Table 2. Titers of the supernatant saturated with the mAb NosCer74 against N. ceranae
    [0239] Table 3. Ascites fluid titers with the mAb NosCer74 against N. ceranae
    [0241]
    [0244] The mAb NosCer74 was tested against several heterologous microsporidia: E. intestinalis, E. cuniculi, E. hellem, E. bieneusi, V. corneae, A. algerae and N. apis, the latter being the heterologous species that infects hives, using IFI. The IFI was performed as described in Example 1, but, in this case, the slides were prepared with 107 spores / ml of different microsporidia: N. apis, E. intestinalis, E. cuniculi, E. hellem, E. bieneusi , V. corneae or A. algerae. It is noteworthy that, in addition to its high specificity against its homologous antigen, there is a total absence of cross-reactivity of the antibody against N. apis spores ( Table 4 ).
    [0246] Table 4. Evaluation of cross-reactions of the mAb NosCer74 against different species of microsporidia
    [0248]
    [0250] These results support the use of the mAb NosCer74 for the differential diagnosis of the two Nosema species by IFI in samples from hives.
    [0251] Example 3. Proteomic characterization of the epitope recognized by the monoclonal antibody NosCer74
    [0253] A proteomic characterization was carried out using the replica gel methodology using two 12% SDS-PAGE gels with soluble Nosema ceranae spore antigen for subsequent staining and identification of the antigenic profile, and in parallel, a Western Blot was performed using the NosCer74 mAb . The proteomic characterization was carried out in collaboration with the Proteomics Service of the Molecular Biology Center "Severo Ochoa" (CBM-CSIC).
    [0254] Two antigenic bands recognized by the mAb (called Bands 1 and 2) were identified for subsequent tryptic digestion of the replica gel for analysis by liquid chromatography-mass spectrometry / mass spectrometry (LC-MS / MS). The results obtained revealed the high presence of cytosolic peptides (sporoplasm) in both bands except for two non-cytosolic peptides present in Bands 1 and 2, identified by the sequences SEQ ID NO: 2 and 3.
    [0256] The UniProt-Nosema ceranae database was searched using the Sequest search engine, using the Proteome Discoverer 2.2 software and the PEAKS 8.5 software. The search identified a protein from the spore wall of N. ceranae that contains the two peptides identified by the sequences SEQ ID NO: 2 and 3, present in Bands 1 and 2. The accession number of said protein in the UniProtKB database is A0A0F9YW02. The sequence of said protein is SEQ ID NO: 1.
    [0258] The mAb shows positive reactivity, appearing the exospore (wall) of the fluorescent microsporidium in the images obtained by IFI. This fact shows that the peptides identified by the sequences SEQ ID NO: 2 and 3 are the epitope or antigenic target of the mAb NosCer74.
    [0260] Example 4. Comparative test. Diagnostic test for spores of N. cerenae by IFI with the monoclonal antibody NosCer74 Vs. assays by PCR and light microscopy
    [0261] A blind study was conducted on a total of 180 samples from the Regional Beekeeping Center (CAR, Junta de Communities de Castilla-La Mancha, Guadalajara). Each sample consisted of a homogenate of bees from the corresponding hive. Hives infected with N. ceranae, with N. apis, co-infected with the two species simultaneously and free of infection, were used, which served as negative control of the study. In all samples the presence or absence of N. ceranae had been previously studied and N. apis by phase contrast optical microscopy (MO) and by molecular techniques (multiplex PCR).
    [0263] The results obtained from the 180 hives by means of the IFI technique with the mAb NosCer74 against N. ceranae were compared with the results obtained by optical microscopy with phase contrast and multiplex PCR, taken as the reference technique for this example.
    [0265] The results corresponding to the detection by the IFI technique with the mAb NosCer74 showed the presence of N. ceranae spores in 98 of the 180 hives, while in the remaining 82, these were not detected. This is reflected in Table 5 with 54.4% positive and 45.6% negative. The results obtained with the PCR for the diagnosis of this species revealed 52.2% of hives infected by N. ceranae ( Table 5 ). On the other hand, light microscopy showed results similar to those of the IFI, detecting 90 positive hives (50%) for the presence of N. ceranae and 90 negative (50%) ( Table 5 ). However, with respect to the differential diagnosis of both species (since light microscopy identifies both), the number of false diagnoses increased due to the cases in which this technique confused both species ( Table 6 ).
    [0267] Table 5. Percentage of positive and negative results in the detection of N. ceranae in 180 hives by indirect immunofluorescence (IFI) using the monoclonal antibody NosCer74, by PCR and by light microscopy (OM)
    [0269]
    [0272] Table 6. Summary of false positives and negatives obtained by indirect immunofluorescence (IFI) using the NosCer74 monoclonal antibody as well as by light microscopy (MO)
    [0274]
    [0277] To determine the significance of the differences in the results obtained by the different techniques, a statistical analysis was carried out. Using the EPIDAT 3.1 program (Vidal et al., 2004) a Diagnostic Test was carried out to be able to compare the sensitivity and Specificity of the IFI technique with the NosCer74 antibody over the other two techniques.
    [0279] When studying the results obtained in the Diagnostic Test, it can be observed how the IFI using the mAb NosCer74 presented a higher sensitivity than the OM for the detection of N. ceranae, and yet the specificity was higher in the case of OM ( Table 7 ) .
    [0281] Table 7. Percentages of sensitivity and specificity (Confidence Interval) of the Diagnostic Test comparing CRP versus OM and IFI using NosCer74 versus N. ceranae (95% confidence level)
    [0283]
    [0286] As a complement to the Diagnostic Test and to give greater robustness to the statistical study, a concordance analysis of the results obtained by the different techniques was performed in parallel, using Cohen's Kappa Statistical Method, which was interpreted according to the scale developed by Altman (Altman , 1991). Likewise, a comparison of proportions was carried out using the Chi square method. Both analyzes were carried out with the PASW 18 statistical program (IBM SPSS).
    [0288] The statistical comparison of the results of the multiplex PCR and the IFI with the mAb NosCer74 obtained a Chi square value of 121.723 (p <0.05) and a very good Agreement Index ( k = 0.822) according to the Altman scale ( Table 8 ). On the other hand, when comparing OM with CRP, a significant correlation was obtained with a Chi square value of 115.428 (p <0.05) and a Concordance Index k = 0.8, which in the Altman scale was related to good agreement ( Table 8 ).
    [0290] Table 8. Concordance of light microscopy (OM) and indirect immunofluorescence (IFI) using the NosCer74 monoclonal antibody, compared with multiplex PCR (gold standard) in Chi-square and Cohen's Kappa analysis
    [0292]
    [0293] The differences between sensitivity and specificity between IFI, OM and CRP could only be explained by false diagnoses. In the case of IFI, it is necessary to highlight that these differences could be due to the fact that PCR cannot detect extruded spores (Martínez Fernández, 1995), while IFI with mAb NosCer74 is based on its reactivity against the wall of the spore. This is an advantage of NosCer74-based immunochemical detection over PCR detection.
    [0295] REFERENCE TO THE DEPOSITED BIOLOGICAL MATERIAL
    [0297] Hybridoma NosCer74 was deposited on June 11, 2019, under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure, with the International Depository Authority (IDA) European Collection of Authenticated Cell Cultures (ECACC), with address at Culture Collections, Public Health England, Porton Down, Salisbury, SP4 0JG, United Kingdom, by the depositor Fundación Universitaria San Pablo CEU, with address at Universidad CEU San Pablo, C / Issac Peral 58. 28040 Madrid , Spain. The depositor identified the hybridoma with the reference "Hybridoma NosCer74". After successfully completing the quality control tests of the hybridoma, the IDA certified the acceptance of the hybridoma, confirmed the date on which the IDA received the deposit, the date indicated above, and assigned the deposit number 19061101 to the hybridoma.
    [0299] The NosCer74 hybridoma was obtained by fusion of splenocytes from BALB / c mice (Charles Rives Laboratories International, Inc.) with P3X63-Ag8.653 myeloma cells (Universidad CEU San Pablo, Madrid, Spain), using polyethylene glycol (PEG) as agent. membrane fusing chemical. The splenocytes had previously been immunized with spores of the microsporidium Nosema ceranae.
    [0301] The NosCer74 hybridoma produces monoclonal antibodies (IgM) against a wall protein of the exospore of Nosema ceranae. The accession number of this protein in the UniProtKB database is A0A0F9YW02. The sequence of said protein is SEQ ID NO: 1.
    [0303] The NosCer74 hybridoma grows in suspension. A suitable culture medium for growth is RPMI 1640 culture medium supplemented with 18% fetal bovine serum and 2 mM L-glutamine. Both the culture medium and supplementary ingredients are commercially available. It is advisable to grow said hybridoma at a temperature of 37 ° C in the presence of 5% CO 2 and maintain the culture at a concentration between 3-5x105 cells / ml.
    [0304] FREE TEXT OF THE SEQUENCE LIST
    [0306] Table 9. Free text of the sequence list
    [0308]
    [0311] LIST OF BIBLIOGRAPHIC REFERENCES
    [0313] Altman, D. G. (1991). Practical statistics for medical research. Chapman and Hall (New York).
    [0314] Aronstein, KA et al. (2011). Evaluation of Nosema ceranae spore-specific polyclonal antibodies, Journal of Apicultural Research, 50: 2, 145-151.
    [0316] Chen et al. (2009). Asymmetrical coexistence of Nosema ceranae and Nosema apis in honey bees. J Invertebr Pathol 101, 204-209.
    [0318] Cornman RS et al. (2009). Genomic Analyzes of the Microsporidian Nosema ceranae, an Emergent Pathogen of Honey Bees. PLoS Pathog 5 (6): e1000466.
    [0320] Klee, J. et al. (2007). Dispersal widespread of the microsporidian Nosema ceranae, an emergent pathogen of the western honey bee, Apis mellifera. J Invertebr Pathol 96, 1-10.
    [0321] Li, Z. et al. (2012). SWP5, a spore wall protein, interacts with polar tube proteins in the parasitic microsporidian Nosema bombycis. Eukaryot Cell. Feb; 11 (2): 229-37.
    [0323] Martín-Hernández, R. et al. (2007). Outcome of colonization of Apis mellifera by Nosema ceranae. Appl Environ Microbiol 73, 6331-6338.
    [0325] Martínez Fernández, AR (1995). Microsporidia: Parasitic Protists New Human Pathogens. In, RAdFId Spain, ed. (Madrid).
    [0326] Pelin A. et al. (2015). Genome analyzes suggest the presence of polyploidy and recent humandriven expansions in eight global populations of the honeybee pathogen Nosema ceranae. Environ Microbiol., 17 (11): 4443-58.
    [0328] Vidal, X. H. et al. (2004). EPIDAT 3.0 Program for epidemiological analysis of tabulated data. Rev Esp Public Health 78, 277-280.
    权利要求:
    Claims (29)
    [1]
    1. A monoclonal antibody against a protein of the spore wall of microsporidium Nosema ceranae identified by the sequence SEQ ID NO: 1, in which said monoclonal antibody is produced by the hybridoma deposited in the ECACC with deposit number 19061101.
    [2]
    2. The monoclonal antibody according to claim 1, which specifically recognizes the epitopes identified by the sequences SEQ ID NO: 2 and 3.
    [3]
    The monoclonal antibody according to claim 1 or 2, wherein said monoclonal antibody is conjugated to a label probe.
    [4]
    The monoclonal antibody according to claim 3, wherein said labeling probe is selected from the group consisting of a fluorophore, a chromophore, a bioluminescent probe, an enzyme, and colloidal gold.
    [5]
    The monoclonal antibody according to claim 1 or 2, wherein said monoclonal antibody is radioactively labeled.
    [6]
    6. A composition comprising the monoclonal antibody according to any of claims 1-5.
    [7]
    7. A hybridoma deposited with the ECACC with deposit number 19061101.
    [8]
    8. An in vitro method of diagnosis of nosemosis caused by Nosema ceranae, wherein said method comprises:
    - Contacting the monoclonal antibody according to any of claims 1-5 or a composition comprising it, according to claim 6, with a preparation obtained from an isolated sample of one or more arthropods, thus forming an antigen-antibody complex , if said antigen is present in said preparation,
    - detect said antigen-antibody complex and
    - determine that said preparation contains spores of Nosema ceranae in case of positive detection of said complex, thus determining that said arthropod or arthropods are affected by nosemosis caused by Nosema ceranae.
    [9]
    The method according to claim 8, wherein said arthropod is a bee.
    [10]
    The method according to claim 9, wherein said bee is of the Apis mellifera or Apis cerana species.
    [11]
    The method according to any of claims 8-10, wherein the detection of said antigen-antibody complex is carried out by means of a technique selected from the group consisting of immunofluorescence, immunohistochemistry, immunochromatography, immunoblotting, ELISA and microarrays .
    [12]
    The method according to claim 11, wherein said immunofluorescence is indirect or direct immunofluorescence.
    [13]
    The method according to any of claims 8-12, wherein said preparation is a homogenate of bees from a hive, food products comprising honey or products derived from honey and in case of positive detection of said complex it is determined that said hive from which the homogenate was obtained, or from which the honey was obtained from the products that comprise it or that are derived from it, is affected by nosemosis caused by Nosema ceranae.
    [14]
    14. A kit for carrying out the in vitro diagnosis of nosemosis caused by Nosema ceranae in arthropods, wherein said kit comprises:
    - the monoclonal antibody according to any of claims 1-5 or the composition comprising it according to claim 6 and
    - Buffer solutions to carry out the immunological reaction to form the antigen-antibody complex and to carry out the detection of said complex.
    [15]
    The kit according to claim 14, wherein said arthropods are bees.
    [16]
    16. The kit according to claim 14 or 15, wherein said kit is selected from the group consisting of a fluorescence kit, an immunoenzyme kit and an immunochromatographic kit.
    [17]
    17. The kit according to any of claims 14-16, further comprising reagents for carrying out the immunological reaction to form the antigen-antibody complex and for carrying out the detection of said complex.
    [18]
    18. The kit according to any of claims 14-17, further comprising a secondary antibody that specifically recognizes said monoclonal antibody.
    [19]
    19. The kit according to claim 18, wherein said secondary antibody is an antibody that specifically recognizes mouse antibodies.
    [20]
    20. Use of the monoclonal antibody according to any of claims 1-5 or of the composition comprising it according to claim 6 in the in vitro detection of Nosema ceranae spores in a sample.
    [21]
    21. The use according to claim 20, wherein said detection comprises contacting said antibody or said composition comprising it with said sample to form an antigen-antibody complex, detecting said complex and determining that said sample contains spores of Nosema ceranae .
    [22]
    22. The use according to claim 21, wherein the detection of said antigen-antibody complex is carried out by means of a technique selected from the group consisting of immunofluorescence, immunohistochemistry, immunochromatography, immunoblotting, ELISA and microarrays.
    [23]
    23. The use according to claim 22, wherein said immunofluorescence is indirect or direct immunofluorescence.
    [24]
    24. The use according to any of claims 21-23, wherein said sample is a biological sample derived from bees, or from honey, or from products derived from honey.
    [25]
    25. A device or kit for detecting the microsporidium Nosema ceranae spore wall protein identified by the sequence SEQ ID NO: 1, wherein said device or kit comprises the monoclonal antibody according to any of claims 1-5 , or the composition comprising said monoclonal antibody, according to claim 6.
    [26]
    26. The device or kit according to claim 25, wherein said monoclonal antibody is immobilized on a surface of said device or said kit.
    [27]
    27. The device according to claim 25 or 26, wherein said device is selected from the group consisting of a biosensor, an immunochromatographic device, and a microarray.
    [28]
    28. The monoclonal antibody according to any of claims 1-5 or of a composition comprising it according to claim 6 for use in the in vitro diagnosis of nosemosis in bees and / or hives.
    [29]
    29. The monoclonal antibody according to any of claims 1-5 for use in the manufacture of a composition, device, biosensor or kit, for the diagnosis of nosemosis in bees and / or in hives.
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    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    ES2548424A1|2014-04-15|2015-10-16|Fundación Universitaria San Pablo - Ceu|7d2 monoclonal antibody specific anti-exospora nosema ceranae for the diagnosis by immunochemical techniques of the nosemosis of the bee |
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