JP2016191581A - Detection method of trypanosome infection - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide means for quickly and easily diagnosing an African trypanosomiasis at high sensitivity.SOLUTION: Disclosed are: a diagnostic product of the African trypanosomiasis containing at least three kinds of antigens selected from a group which is composed of polypeptide (ISG64) including an amino acid sequence indicated in an array number 1, polypeptide (ISG65) including an amino acid sequence indicated in an array number 2, polypeptide (FCaBP) including an amino acid sequence indicated in an array number 3, and polypeptide (GM6) including an amino acid sequence indicated in an array number 4; a diagnostic kit of the African trypanosomiasis which includes a sold phase and the antigen, and in which the antigen is coupled to the sold phase; and a detection method of African trypanosomiasis which includes a process (1) for making a biological specimen derived from a subject come into contact with the antigen and producing a composite of an antibody and the antigen which withstands the antigen which is contained in the biological specimen, and a process (2) for detecting the antibody in the composite which is produced in the process (1).SELECTED DRAWING: None

Description

  The present invention relates to a method for detecting trypanosomiasis and relates to the field of serodiagnosis of human African trypanosomiasis.

  Human African trypanosomiasis (HAT), or African sleeping sickness, is an infection caused by Trypanosoma protozoa. Trypanosoma is a protozoan classified as a kinetoplast, and it is pathogenic to humans, Trypanosoma cruzi causing Chagas disease in the Americas, and Trypanosoma brucei gambiense (Tbg) causing African sleeping sickness in the African continent. Trypanosoma brucei rhodesiense (Tbr). African sleeping sickness is mediated by tsetse flies, but the pathogen trypanosoma circumvents human immune mechanisms by mutating its surface antigen (variant surface glycoprotein: VSG) and is present in the blood. VSG can include nearly infinite types of mutations, but infectious protozoa express only one of them. Although human immunity is established and protozoa are destroyed, a small amount of mutant appears before immunity is established, so that protozoa with new VSG can proliferate. On the other hand, in Chagas disease, trypanosomes are parasitized inside the cells, avoiding the immune mechanism.

  African trypanosomiasis is divided into an acute phase (first phase) such as fever after infection and a later phase (second phase) in which sleep becomes uncertain and eventually becomes a coma. In gambiense type trypanosoma, central nervous system symptoms appear several months to several years after the onset of acute disease. In rhodesiense type trypanosoma, invasion of the central nervous system occurs within a few weeks to several months after the onset of acute disease (Non-patent Document 1). ). Therefore, it is necessary to make a diagnosis as early as possible.

The following detection methods are used as a method for diagnosing African trypanosomiasis.
1) Protozoa detection method Diagnose early in the disease by microscopic observation of trypanosoma in live specimens or Giemsa-stained peripheral blood thin or thick smears, or in aspirate from swollen lymph nodes. In the advanced stage, trypanosomes may be detected only in centrifuge cerebrospinal fluid. This method is effective when a certain number of protozoa grow, and early detection is difficult. However, for the definitive diagnosis of African trypanosomiasis, it is common to detect trypanosoma protozoa parasitic in the blood or cerebrospinal fluid of a patient with a microscope.
2) Antigen detection method Antigens are detected using serological tests (immunofluorescence test, ELISA, Western blot, card agglutination, etc.). It cannot respond and detection sensitivity becomes low.
3) Gene detection method
The method of detecting the trypanosoma gene using the PCR method or the LAMP method can detect trypanosome DNA in the sample with high sensitivity. However, in addition to stable power supply and laboratory equipment, the detection method is expensive. New equipment, reagents and molecular biology experimental techniques are required. Therefore, it is difficult to spread as a field diagnostic method in the tropics.

As a method for detecting and diagnosing Trypanosoma cruzi infection that causes Chagas disease, a method using four recombinant polypeptides is known (Patent Document 1).
In addition, for the diagnosis of human African trypanosomiasis (HAT), several antigens have been identified in proteomics experiments using IgG obtained from HAT patients and Lysate of Trypanosoma brucei brucei (Tbb) (Non-patent Document 2). .

JP 2010-507100 A

Merck Manual 18th Section-Infectious Diseases, Chapter-Extra-Intestinal Protozoa African Trypanosomiasis Revised November 2005 PLOS Neglected Tropical Diseases 2013, Vol.7 (2) e2087

Trypanosoma brucei genome decoding was completed in 2005. A significant part of the gene is related to the variant surface glycoprotein (VSG), and it was found that VSG expressed during cell division is frequently replaced to avoid the host immune mechanism. Therefore, it is difficult to increase the detection sensitivity and specificity by a normal method.
An object of the present invention is to provide a means for diagnosing African trypanosomiasis quickly, conveniently and with high sensitivity. In particular, the object is to provide means suitable for point-of-care testing (POCT) of African trypanosomiasis.

  As a result of intensive studies, the inventors have found a plurality of antigens with few mutations unlike trypanosoma VSG, and combined an IgG detection system with an IgM detection system, so that African trypanosomiasis can be detected with high sensitivity and high specificity. It reached the point where it could be detected, and the present invention was completed.

The present invention provides the following.
[1] Polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 (ISG64), polypeptide containing the amino acid sequence shown in SEQ ID NO: 2 (ISG65), polypeptide containing the amino acid sequence shown in SEQ ID NO: 3 (FCaBP) and SEQ ID NO: A diagnostic agent for African trypanosomiasis comprising at least three antigens selected from the group consisting of a polypeptide (GM6) comprising the amino acid sequence shown in FIG.
[2] The diagnostic agent according to [1], wherein the antigen is ISG64, ISG65 and FCaBP (3 types), or ISG64, ISG65, FCaBP and GM6 (4 types).
[3] The diagnostic agent according to [1] or [2], further comprising at least one antibody selected from the group consisting of an anti-human IgG antibody and an anti-human IgM antibody.
[4] The diagnostic agent according to any one of [1] to [3], wherein the antigen is bound to a solid phase.
[5] The diagnostic agent according to [3], wherein the antigen and the antibody are bound to a solid phase.
[6] A solid phase, a polypeptide (ISG64) containing the amino acid sequence shown in SEQ ID NO: 1, a polypeptide (ISG65) containing the amino acid sequence shown in SEQ ID NO: 2, and a polypeptide (FCaBP containing the amino acid sequence shown in SEQ ID NO: 3) And at least three kinds of antigens selected from the group consisting of a polypeptide (GM6) comprising the amino acid sequence shown in SEQ ID NO: 4, and the antigens bound to the solid phase, a kit for diagnosis of African trypanosomiasis .
[7] The kit according to [6], wherein the antigen is ISG64, ISG65 and FCaBP (3 types) or ISG64, ISG65, FCaBP and GM6 (4 types).
[8] The kit according to [6] or [7], further comprising at least one antibody selected from the group consisting of an anti-human IgG antibody and an anti-human IgM antibody.
[9] The kit according to any one of [6] to [8], further comprising a reagent for detecting an anti-human IgG antibody and / or an anti-human IgM antibody.
[10] The kit according to [9], wherein the detection reagent is bound to the anti-human IgG antibody and / or anti-human IgM antibody.
[11] The following steps:
(1) a biological sample derived from a subject;
A polypeptide containing the amino acid sequence shown in SEQ ID NO: 1 (ISG64), a polypeptide containing the amino acid sequence shown in SEQ ID NO: 2 (ISG65), a polypeptide containing the amino acid sequence shown in SEQ ID NO: 3 (FCaBP), and SEQ ID NO: 4 Contacting with at least three antigens selected from the group consisting of a polypeptide (GM6) containing an amino acid sequence to form a complex of an antibody against the antigen and the antigen contained in a biological sample; and (2) Detecting the antibody in the complex formed in step (1);
A method for detecting African trypanosomiasis, comprising:
[12] The method according to [11], wherein the step (2) detects an antibody against the antigen in the complex using at least one antibody selected from the group consisting of an anti-human IgG antibody and an anti-human IgM antibody. Detection method.
[13] The detection method according to [11], wherein the step (2) detects an antibody against the antigen in the complex using two types of antibodies, an anti-human IgG antibody and an anti-human IgM antibody.

According to the present invention, a rapid and simple determination means for African trypanosomiasis is provided, and a point-of-care test (POCT) for African trypanosomiasis is possible.
According to the diagnostic agent and diagnostic kit of the present invention, the presence or absence of trypanosoma infection and the presence or absence of African trypanosomiasis can be determined with high sensitivity, specificity and simplicity. According to the detection method of the present invention, the presence or absence of infection with African trypanosomiasis can be appropriately determined alone or in combination with other diagnostic methods.

FIG. 2 is a boxplot for T.b.g. antigen and L. donovani antigen. Box whisker represents the fluorescent signal for the antigen. A-H: T.b.g. antigen, I-K: L. donovani antigen. The box shows the boundary between the 25th and 75th percentile values and the median value in the box. The whiskers show the 10th and 90th percentile values. The sera used are as follows. Cg: healthy control for T.b.g. patient, Pg1: T.b.g. patient stage 1, Pg2: T.b.g. patient stage 2, Cr: healthy control for T.b.r. patient, Pr: T.b.r. patient and Ld: visceral leishmania patient. It is a heat map which shows the reactivity which measured T.b.g. antigen with the IgM detection system. Cg: healthy control for T.b.g. patient, Pg1: T.b.g. patient stage 1, Pg2: T.b.g. patient stage 2 and VL: visceral leishmania patient. ×: Negative, ●: Positive, ○: Strongly positive. It is a heat map which shows the reactivity which measured T.b.r. antigen with the IgM detection system. Cr: healthy control for T.b.r. patient, Pr1: T.b.r. patient stage 1, Pr2: T.b.r. patient stage 2 and VL: visceral leishmania patient. ×: Negative, ●: Positive, ○: Strongly positive. It is a heat map which shows the reactivity which measured L. donovani antigen with the IgM detection system. Cg: Healthy control for T.b.g. patients, Cr: Healthy control for T.b.r. patients and VL: Visceral Leishmania patients. ×: Negative, ●: Positive, ○: Strongly positive. It is the graph which measured the non-trypanosoma antigen with the IgM detection system. Cg: healthy controls for T.b.g. patients and Pg: T.b.g. patients (including stage 1 and stage 2). It is a conceptual diagram of the immunochromatography method.

  In this specification, the abbreviations of amino acids, (poly) peptides, (poly) nucleotides, etc. are defined by IUPAC-IUB [IUPAC-IUB Communication on Biological Nomenclature, Eur. J. Biochem., 138: 9 (1984). ], “Guidelines for the preparation of specifications including base sequences or amino acid sequences” (edited by the Japan Patent Office), and conventional symbols in the field.

Definitions In the present invention, trypanosoma refers to protists classified as kinetoplasts. It is Trypanosoma cruzi that causes Chagas disease in the Americas, and Trypanosoma brucei gambiense (Tbg) and Trypanosoma brucei rhodesiense (Tbr) that cause African sleeping sickness in the African continent. In the present invention, Trypanosoma brucei gambiense (Tbg) and Trypanosoma brucei rhodesiense (Tbr) are targeted.

  In the present invention, human African trypanosomiasis (also referred to as HAT or African sleeping sickness) refers to an infectious disease caused by Tsetse fly-mediated Trypanosoma brucei gambiense (T.b.g.) or Trypanosoma brucei rhodesiense (T.b.r.). In the present invention, “human African trypanosomiasis” and “African trypanosomiasis” are used synonymously. Trypanosoma avoids human immune mechanisms by mutating its surface antigen (VSG) and is present in the blood. VSG can include nearly infinite types of mutations, but infectious protozoa express only one of them. Although human immunity is established and protozoa are destroyed, a small amount of mutant appears before immunity is established, so that protozoa with new VSG can proliferate. African trypanosomiasis can also be transmitted through blood transfusions.

  African trypanosomiasis is divided into an acute phase (first phase) such as fever after infection and a later phase (second phase) in which sleep becomes uncertain and eventually becomes a coma. In gambiense type trypanosoma, central nervous system symptoms appear several months to several years after the onset of acute disease, whereas in rhodesiense type trypanosoma, central nervous system invasion occurs within a few weeks to several months after the onset of acute disease. Therefore, it is necessary to make a diagnosis as early as possible.

In the present invention, the determination of the presence or absence of African trypanosomiasis includes the evaluation and examination according to a certain standard, and the present invention alone or in combination with another examination To include.
Current testing methods for African trypanosomiasis include protozoa detection; antigen detection using serological tests (immunofluorescence, ELISA, Western blot, card agglutination, etc.); PCR or LAMP A gene detection method for detecting a trypanosoma gene by using a protein is used. It is desirable to use the present invention as a tool for initial diagnosis or primary screening of African trypanosomiasis and use these detection methods for definitive diagnosis.

1. Diagnostic Agent for African Trypanosomiasis The antigen contained as an active ingredient in the diagnostic agent of the present invention is selected from the polypeptides shown below.

ISG64: polypeptide NAKLTKDGALALCKLTDVADLVATKKADKIRKDTEGFADELKLWLDRLEHWLQTLETRAHSNNGYSKLSDADTKKVKEIYEKAKGKVSEQLPKAKEFGEEAGKRHQEVTEAAKRARGWGLDDEGQNSSGLHQLLEWYCGTKEDNANNQKCDGVKVKEHYLGRERNPIDCKGTGSTVPFYLDVTSGTMKEALENWERKKPKSDGEPVNNNWKADYDSATNKLKELEESHEKGKKTVNDVSGFYNAAYALHSGLSAGKPLSEVLVEAKEASKRGAKITNPGGAAPEATQRGVGTSTGEGGATETTGGTSTTISTGTGTGTTSGTEPGVVGADADFGDLLETSDR comprising the amino acid sequence shown in SEQ ID NO: 1 (SEQ ID NO: 1)
The ISG64 antigen used in the Examples is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 and a polypeptide in which a tag is fused to the N-terminus and C-terminus thereof (SEQ ID NO: 5).
maswshpqfekgalevlfqgpgyqdpNAKLTKDGALALCKLTDVADLVATKKADKIRKDTEGFADELKLWLDRLEHWLQTLETRAHSNNGYSKLSDADTKKVKEIYEKAKGKVSEQLPKAKEFGEEAGKRHQEVTEAAKRARGWGLDDEGQNSSGLHQLLEWYCGTKEDNANNQKCDGVKVKEHYLGRERNPIDCKGTGSTVPFYLDVTSGTMKEALENWERKKPKSDGEPVNNNWKADYDSATNKLKELEESHEKGKKTVNDVSGFYNAAYALHSGLSAGKPLSEVLVEAKEASKRGAKITNPGGAAPEATQRGVGTSTGEGGATETTGGTSTTISTGTGTGTTSGTEPGVVGADADFGDLLETSDRvdaaaelalvprgssahhhhhhhhhh (SEQ ID NO: 5)

ISG65: polypeptide ATNGGDKHVKANKKLDQEGANLLCKMKHLADKVANKGGEDLKKKTKGFEDDVLLEVERVNNWLEKLGDRKQYNDGYAKLSDSDIEKVKEIFTKAKDGITKQLPEAKKAGENAEKLYDEVKKAAEHARGQKLDDDRDKSTGLYRILDWYCFKEEENAGKSDNCDGVKFSEHHGTHRRRNVIDCGDEKANKYGDASSKTLEDTLKQWESKKPQAAGASGGNDVCKATASSESHPCTMTEEWQTPYKETVKKLKELEGAHERGKKAHDAMLGYANTAHATNRKVEQGKPLTEVIAAAKEAGKKGAKIIIPAAAPSTPTNSTKNEDSAPTEHVDRGIATNETQVEVGIDADFDGLLEATEAAEVKSRHQRT comprising the amino acid sequence shown in SEQ ID NO: 2 (SEQ ID NO: 2)
The ISG65 antigen used in the Examples is a polypeptide having the amino acid sequence shown in SEQ ID NO: 2 and a polypeptide in which a tag is fused to the N-terminus and C-terminus thereof (SEQ ID NO: 6).
maswshpqfekgalevlfqgpgyqdpATNGGDKHVKANKKLDQEGANLLCKMKHLADKVANKGGEDLKKKTKGFEDDVLLEVERVNNWLEKLGDRKQYNDGYAKLSDSDIEKVKEIFTKAKDGITKQLPEAKKAGENAEKLYDEVKKAAEHARGQKLDDDRDKSTGLYRILDWYCFKEEENAGKSDNCDGVKFSEHHGTHRRRNVIDCGDEKANKYGDASSKTLEDTLKQWESKKPQAAGASGGNDVCKATASSESHPCTMTEEWQTPYKETVKKLKELEGAHERGKKAHDAMLGYANTAHATNRKVEQGKPLTEVIAAAKEAGKKGAKIIIPAAAPSTPTNSTKNEDSAPTEHVDRGIATNETQVEVGIDADFDGLLEATEAAEVKSRHQRTvdaaaelalvprgssahhhhhhhhhh (SEQ ID NO: 6)

FCaBP: polypeptide comprising the amino acid sequence shown in SEQ ID NO: 3
MGCSGSKNASNPKDGAASKGGKDGKTTADRKVAWERIRCAIPRDKDAESKSRRIELFKQFDTNGTGKLGFREVLDGCYGILKLDEFTTHLPDIVQRAFDKAKDLGNKVKGVGEEDLVEFLEFRLMLCYIYDIFELTVMFDTMDKDGSLLLELQEGKEPDVDEVDGDGPDGTDVT
The FCaBP antigen used in the Examples is a polypeptide having the amino acid sequence shown in SEQ ID NO: 3 and a polypeptide in which a tag is fused to the N-terminus and C-terminus (SEQ ID NO: 7).
maswshpqfekgalevlfqgpgyqMGCSGSKNASNPKDGAASKGGKDGKTTADRKVAWERIRCAIPRDKDAESKSRRIELFKQFDTNGTGKLGFREVLDGCYGILKLDEFTTHLPDIVQRAFDKAKDLGNKVKGVGEEDLVEFLEFRLMLCYIYDIFELTVMFDTMDKDGSLLLELQEFKEALPKLKEWGVDITDATTVFNEIDTNGSGVVTFDEFSCWAVTKKLQVCGDPDDEENGANEGDGANAGDGVPAAEelalvprgssahhhhhhhhhh (SEQ ID NO: 7)

GM6: polypeptide comprising the amino acid sequence shown in SEQ ID NO: 4
RRKLIAEDREGNATRIAELEVAMNEHSHELAKLKASDSRSFLDPMPEGVPLSELGLDKDEKFSTMEEERRKLIAEDREGNATRIAELEVAMNEHSHELAKLKASDSRSFLDPMPEGVPLSELGLDKDEKFSTMEEERRKLIAEDREGNATRIAELEVAMNEHSHELAKLKASDSRSFLDPMPEGVSTSELGLDKDEF
The GM6 antigen used in the Examples is a polypeptide having the amino acid sequence shown in SEQ ID NO: 4 and a polypeptide in which a tag is fused to the N-terminus and C-terminus thereof (SEQ ID NO: 8).
maswshpqfekgalevlfqgpgyqdpRRKLIAEDREGNATRIAELEVAMNEHSHELAKLKASDSRSFLDPMPEGVPLSELGLDKDEKFSTMEEERRKLIAEDREGNATRIAELEVAMNEHSHELAKLKASDSRSFLDPMPEGVPLSELGLDKDEKFSTMEEERRKLIAEDREGNALKDHPEGASH

The polypeptide in the present invention is not limited to the amino acid sequence represented by the above SEQ ID No., but in the amino acid sequence represented by any one of SEQ ID Nos. Individual, more preferably 1 or 2 amino acid residues are substituted, deleted, or added, so long as it has the same antigenicity as the polypeptide before mutation. Included in the polypeptide of the invention.
Here, “having equivalent antigenicity” means having a high homology with a part of the protein derived from Tbg or Tbr and a low homology with a protein derived from the human subject. Equivalent antigenicity can be confirmed by Blast search of the amino acid sequence after mutation.

  Examples of “amino acid residue substitution” include conservative amino acid substitution. Conservative amino acid substitution refers to substitution of a specific amino acid with an amino acid having a side chain having the same properties as the side chain of the amino acid. Specifically, in a conservative amino acid substitution, a particular amino acid is replaced with another amino acid belonging to the same group as the amino acid. Groups of amino acids having side chains of similar nature are known in the art. For example, such amino acid groups include amino acids having basic side chains (eg, lysine, arginine, histidine), amino acids having acidic side chains (eg, aspartic acid, glutamic acid), amino acids having neutral side chains. (For example, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan). In addition, the amino acid having a neutral side chain further includes an amino acid having a polar side chain (for example, glycine, asparagine, glutamine, serine, threonine, tyrosine), and an amino acid having a nonpolar side chain (for example, alanine, valine, Leucine, isoleucine, proline, phenylalanine, methionine, tryptophan). In addition, as other groups, for example, amino acids having aromatic side chains (for example, phenylalanine, tryptophan, tyrosine), amino acids having side chains including hydroxyl groups (alcoholic hydroxyl groups, phenolic hydroxyl groups) (for example, serine, threonine, Tyrosine). Also included are substitution of histidine and methionine, substitution of glutamic acid and serine, and the like.

  Examples of the “deletion of amino acid residue” include selecting and deleting an arbitrary amino acid residue from the amino acid sequence represented by each SEQ ID NO. Preferably, it is a deletion of 1 to 5 amino acid residues continuously from the N-terminal or C-terminal of the amino acid sequence represented by each SEQ ID NO.

  Examples of “addition of amino acid residues” include adding 1 to 5 amino acid residues to the N-terminal or C-terminal side of the amino acid sequence represented by each SEQ ID NO. Preferably, the linkage between the polypeptide and the solid phase or the addition of an amino acid residue for imparting water solubility to the polypeptide. Alternatively, when a nucleic acid encoding the polypeptide of the present invention is incorporated into an expression vector to prepare a polypeptide which is an expression product, any amino acid residue derived from the expression vector is present at the N-terminus and / or C-terminus of the polypeptide. Groups may be added.

  The polypeptide of the present invention can be prepared by a method known per se. For example, it may be prepared by a peptide synthesis method using a normal peptide synthesizer, or may be prepared as a recombinant protein by a gene recombination technique. The recombinant protein may be prepared in either a cell system or a cell-free system. Polypeptides can be prepared by a gene recombination technique by a conventional method using a commercially available expression vector. In this case, in order to facilitate the purification of the polypeptide, it is preferable to express the polypeptide so that a tag is added to the N-terminus and / or C-terminus of the polypeptide. Examples of expression vectors used for this purpose include pET52b (Novagen), pET32 (Novagen), and pET41 (Novagen).

The polypeptide of the present invention may be a polypeptide consisting of the amino acid sequence shown in each SEQ ID No. or a polypeptide consisting of a variant amino acid sequence of the amino acid sequence shown in each SEQ ID No., and the amino acid sequence shown in each SEQ ID No. or a variation thereof It may be a fusion protein comprising an amino acid sequence and further having a tag added to the N-terminus and / or C-terminus of those amino acid sequences.
Examples of the tag include His tag, GST tag, FLAG tag, Trx tag, S tag, StrepTagII (IBA), Twin-Strp-tag (IBA), and the like.

Antigens contained as an active ingredient in the diagnostic agent of the present invention are at least three antigens selected from the group consisting of ISG64, ISG65, FCaBP and GM6,
ISG64, ISG65 and FCaBP
ISG64, ISG65 and GM6
ISG64, FCaBP and GM6
ISG65, FCaBP and GM6
ISG64, ISG65, FCaBP and GM6
There are five ways. From the viewpoint of sensitivity and specificity, three types of ISG64, ISG65 and FCaBP, and four types of ISG64, ISG65, FCaBP and GM6 are preferable.

The diagnostic agent of the present invention preferably further contains at least one antibody selected from the group consisting of an anti-human IgG antibody and an anti-human IgM antibody.
That is, it is preferable to use an anti-human IgG antibody, an anti-human IgM antibody, or both an anti-human IgG antibody and an anti-human IgM antibody in order to detect a complex of the anti-trypanosoma antibody and the antigen in the sample.

The anti-human IgG antibody or anti-human IgM antibody is not particularly limited as long as it is an antibody capable of detecting human IgG or human IgM, and is a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a single chain antibody, or, for example, F ( ab) Fragments such as ₂ , Fab ', Fab, Fv (variable fragment of antibody), scFv (single chain Fv), dsFv (disulphide stabilized Fv), sdAb (single domain antibody), and Fab expression libraries And a part of the antibody having antigen binding property. From the viewpoint of specificity, a monoclonal antibody is preferable.

  The production of said antibodies is well known in the art. In the present invention, a commercially available anti-human IgG antibody or anti-human IgM antibody can be preferably used.

The diagnostic agent of the present invention may consist of only the above antigen, only the above antigen and antibody, or may contain a pharmaceutically acceptable carrier. As a pharmaceutically acceptable carrier, when preparing the diagnostic agent of the present invention as a detection reagent and a liquid, various carriers conventionally used as pharmaceutical materials, such as diluents, solvents, solubilizers, suspending agents, It may contain an isotonic agent, a buffering agent and the like. Furthermore, in order to prevent protein aggregation, it is preferable to add a surfactant such as Tween 20 (registered trademark) or Tween 80 (registered trademark).
Diluents include water, physiological saline and the like.
Examples of the solvent include water, physiological saline, ethanol and the like.
Examples of the solubilizer include cyclodextrins.
Examples of the suspending agent include gum arabic and carmellose.
Examples of the isotonic agent include inorganic salts such as sodium chloride and potassium chloride, and carbohydrates such as glycerin, mannitol and sorbitol.
Examples of the buffer include phosphate buffer, acetate buffer, borate buffer, carbonate buffer, citrate buffer, Tris buffer, and the like.
Those skilled in the art can appropriately determine the mixing ratio of these carriers.

  In the diagnostic agent of the present invention, the antigen may be bound to a solid phase for the convenience of examination. Alternatively, the antigen and the antibody may be bound to a solid phase. In that case, it can be used in the same manner as the diagnostic kit of the present invention described later. The binding between the solid phase and the antigen and / or antibody will be explained in the diagnostic kit.

  By using the diagnostic agent of the present invention to examine a biological sample of a subject by the detection method of the present invention, which will be described later, it is possible to more easily determine the presence or absence of African trypanosomiasis.

2. Diagnostic Kit for African Trypanosomiasis The antigen contained in the kit of the present invention is the same as the antigen described in “1. Diagnostic agent for African trypanosomiasis”.
The anti-human IgG antibody and / or anti-human IgM antibody contained in the kit of the present invention is the same as the antibody described in “1. Diagnostic agent for African trypanosomiasis”.

In the kit of the present invention, the antigen is bound to a solid phase in advance.
The solid phase that can be used in the kit of the present invention is not particularly limited, and is a plate (eg, microwell plate), tube, bead (eg, plastic beads such as polystyrene, magnetic beads), chromatography carrier (eg, Examples thereof include filter papers, water-absorbing substrates such as nitrocellulose membranes, Sepharose), membranes (eg, nitrocellulose membranes, PVDF membranes), gels (eg, polyacrylamide gels), metal membranes (eg, gold membranes), and the like. Of these, plates, beads, chromatographic carriers and membranes are preferably used. More preferred are MagPlex (registered trademark) beads (Luminex) used for the Luminex method, filter paper or nitrocellulose membrane for immunochromatography, and microwell plate used for the ELISA method.
In the kit of the present invention, in addition to the antigen, the antibody may be bound to the solid phase.

  The bond is not particularly limited, and examples thereof include electrostatic interaction, hydrophobic interaction, hydrogen bond, covalent bond, ionic bond, and physical adsorption. The solid phase may be directly bonded to the solid phase or indirectly bonded to the solid phase using a substance known per se.

  The kit of the present invention may contain other antigens or reagents in addition to the antigens and antibodies, and the other antigens or reagents may be previously combined with the antigens and antibodies. , May be stored in separate containers. Other antigens or reagents include other antigens derived from trypanosoma, substrate agents, labeling substances, solid phases, reaction vessels, treatment solutions, antigens, buffers for diluting antibodies, positive controls (eg, Sera from trypanosoma-infected animals), negative controls (eg, sera from non-trypanosoma-infected animals), instructions describing the protocol, and the like. These elements can be mixed in advance if necessary.

The antibody may be directly or indirectly labeled with a labeling substance. Examples of labeling substances include fluorescent substances (eg, FITC, rhodamine), radioactive substances (eg, ³² P, ³⁵ S, ¹⁴ C, ³ H), enzymes (eg, alkaline phosphatase, peroxidase), colored particles (eg, metal ( Gold) colloidal particles, colored latex), biotin and the like.

  The form of the kit of the present invention is not particularly limited, but for the purpose of simple diagnosis, it can be an integrated kit in which the components of the kit of the present invention are integrated. Examples of the form of the integrated kit include a cassette type using an immunochromatography method.

  The use of the kit of the present invention makes it possible to more easily determine the presence or absence of African trypanosomiasis.

3. Method for detecting African trypanosomiasis The method for detecting African trypanosomiasis according to the present invention comprises the following steps:
(1) a biological sample derived from a subject;
A step of contacting at least three antigens selected from the group consisting of ISG64, ISG65, FCaBP and GM6 to form a complex of an antibody against the antigen contained in a biological sample and the antigen; and (2) step ( The method comprises a step of detecting an antibody in the complex formed in 1) (hereinafter also referred to as the detection method of the present invention).

In the detection method of the present invention, after the step (2),
(3) It is preferable to include a step of determining whether or not the subject is suffering from African trypanosomiasis based on the detection result of step (2).

  The subject in the detection method of the present invention is a human suspected of having trypanosoma infection.

  The biological sample to be measured by the detection method of the present invention is not particularly limited as long as it can be collected from humans, and body fluid samples such as blood, cerebrospinal fluid, lymph fluid, saliva, urine, lymph nodes, etc. Biopsy of the tissue. A blood sample is preferable because the burden on the subject is small, and examples of the blood sample include whole blood, plasma, and serum.

  Step (1) is a step of forming a complex of an antibody against the antigen contained in the biological sample and the antigen.

  The antigen can be used in a soluble state to which nothing else is bound, but may be bound to a solid phase. When using an antigen bound to a solid phase, the kit of the present invention can be preferably used.

  Depending on the type and mode of the solid phase, a phosphate buffer solution such as bovine serum albumin (BSA) or bovine milk protein is brought into contact with the solid phase in order to suppress non-specific adsorption or non-specific reaction. Generally, the solid phase surface portion that has not been coated is blocked with the BSA, bovine milk protein, or the like.

  As long as the antigen and the biological sample derived from the subject are in contact with each other as long as the antigen and the antibody in the biological sample can interact with each other, the aspect, order, and specific method are not particularly limited. The contact can be made, for example, by adding a biological sample to a plate on which the antigen is immobilized. For example, the contact can be made by mixing a biological sample and beads on which an antigen is immobilized in a container. In the case of immunochromatography, contact can also be made by dropping a specimen on the sample dropping part of the solid phase and leaving it for a certain period of time.

  The time for maintaining such contact is not particularly limited as long as it is sufficient for the antigen and the antibody contained in the subject-derived biological sample to bind to form a complex, but usually several seconds to It is a dozen hours. Moreover, as temperature conditions which perform a contact, it is 4 to 50 degreeC normally, 4 to 37 degreeC is preferable and the room temperature of about 15 to 30 degreeC is the most preferable. Furthermore, the pH condition for carrying out the reaction is preferably 5.0 to 9.0, particularly preferably the neutral range of 6.0 to 8.0.

  Step (2) is a step of detecting the antibody in the complex formed in the step (1).

  For the detection, any method capable of detecting an antibody can be used. In the present invention, in order to increase sensitivity and specificity, it is desirable to use a detection method utilizing an anti-human IgG antibody and / or an anti-human IgM antibody. The anti-human IgG antibody and anti-human IgM antibody can easily detect the complex by a detection method corresponding to the labeling substance by using an antibody labeled with the labeling substance. For example, the reaction can be performed by adding an anti-human IgG antibody and / or anti-human IgM antibody to the reaction system in which the complex is formed, and allowing to stand for a predetermined time. For example, in the case of immunochromatography, an anti-human IgG antibody is preliminarily contained in the sample dropping portion of the solid phase, and the antibody in the sample and the anti-human IgG antibody move on the solid phase while forming an immune complex. Detection can be done by trapping the immune complex on an antigen immobilized on a phase.

  In order to increase detection sensitivity and specificity, it is preferable to detect using both anti-human IgG antibody and anti-human IgM antibody. In this case, the same sample may be performed simultaneously or separately, and the order of detection is not limited in separate cases. In general, when a living body is infected with a pathogen, it is usual that IgM is detected transiently in a large amount at the beginning of infection, and then IgG is detected. In the case of trypanosomes, the protozoan VSG repeats mutations, so that there are infinite number of mutants, so every time the living body's immune system recognizes the mutant, it recognizes that it is an initial infection and IgM is produced continuously. . Therefore, IgM is difficult to detect IgM against a specific antigen because it is low in specificity and easy to aggregate. However, in order to increase the detection rate of trypanosoma-infected patients, ie, sensitivity, anti-human IgM It is preferred to use antibodies. Moreover, the specificity can achieve a specificity higher than a reference value by using the at least three kinds of antigens.

  When the complex is detected in step (2), it can be determined that the subject has a high probability of suffering from African trypanosomiasis. If the complex is not detected or below the detection limit, the subject can be determined to have a low probability of not suffering from African trypanosomiasis.

  Suitable specific examples of the detection include an enzyme immunoassay (EIA method), a fluorescence immunoassay (FIA), an immunochromatography method, a radioimmunoassay and the like, and these analysis methods are well known to those skilled in the art.

  When the EIA method is selected as the detection method in step (2), an enzyme-labeled antibody is used. Examples of the enzyme used for labeling include peroxidase, alkaline phosphatase, glucose oxidase, β-galactosidase and the like. As the substrate agent used for the detection of the enzyme, an appropriate one is selected according to the selected labeling enzyme. For example, when peroxidase is selected as an enzyme, o-phenylenediamine (OPD), tetramethylbenzidine (TMB) or the like is used. When alkaline phosphatase is selected, p-nitrophenyl phosphate (PNPP) or the like is used. used. As the reaction stop solution and substrate solution, conventionally known ones can be appropriately used without particular limitation depending on the selected enzyme.

When the FIA method is selected as the detection method in step (2), the complex can be detected by substituting the enzyme used as the label in the EIA method with a fluorescent substance.
As the fluorescent substance, chemical substances such as APC, PE, Cy2, Cy3, Cy5, ECD, FITC, PerCP, Alexa (registered trademark) Fluor, fluorescein, and rhodamine can be preferably used. Labeling with these chemical substances can be carried out by a method known per se.
The fluorescence can be detected using a commercially available measuring instrument, a fluorescence microscope, or the like. When using the Luminex method, by using a flowmetry analyzer such as the Luminex200 device (manufactured by Luminex) and recommended analysis software, multiple items can be measured from a small amount of sample in 30 minutes to 1 hour. preferable.

  As a detection method in the step (2), an immunochromatography method is preferably selected in a situation where a measuring device is not used (for example, field diagnosis in a tropical region). In the case of detection by an immunochromatography method, for example, the antigen solid-phased on a water-absorbing substrate such as filter paper or a nitrocellulose membrane, a biological sample developed on the membrane, and a labeled anti-human IgG antibody and A complex is formed between the three anti-human IgM antibodies. The labeled anti-human IgG antibody and anti-human IgM antibody may be pre-mixed with the biological sample, or supplied onto the water-absorbing substrate so that the biological sample comes into contact with the biological sample before contacting the antigen. It may be left. The formation of the complex can be detected by a technique corresponding to the label. For example, when colloidal gold particles are used as a label, a region where the labeled antibody is accumulated exhibits a red color, and is thus detected. A conceptual diagram of the immunochromatography method is shown in FIG.

(3) A step of determining whether or not the subject is suffering from African trypanosomiasis based on the detection result of step (2). Detecting in step (2) using the result obtained in detection step (2) The presence or absence of African trypanosomiasis can be determined from the level of the antibody obtained. If the detection in step (2) is obtained as a qualitative result, it is determined that a positive result is likely to have African trypanosomiasis, and a negative result (below the detection limit) is likely to have African trypanosomiasis Can be determined to be low. When the detection of step (2) is obtained as a quantitative result by the Luminex method or the like, it is possible to determine the presence or absence of African trypanosomiasis in a subject by setting a cutoff value. The same subject can also be compared to previously measured levels.

  The reference level (cutoff value) is an appropriate reference set according to the purpose. The cut-off value is a value that can satisfy both high diagnostic sensitivity (prevalence of correct diagnosis) and high diagnostic specificity (prevalence of non-diagnostic diagnosis) when a disease is determined based on that value. Usually, if it is below the detection limit, it can be determined that the patient is not suffering from African trypanosomiasis.

  When a simple test such as immunochromatography is performed, the presence or absence of an antibody in a biological sample can be determined based on the presence or absence of a visually observed signal (coloring or the like).

  If the antibody level in the biological sample of the subject exceeds the cut-off value, it is determined that the subject is likely to be “afflicted with African trypanosomiasis”. If the cut-off value or less, the subject is determined to be less likely to be “afflicted with African trypanosomiasis”.

  The method of the present invention can also be used to evaluate the effect of treatment on patients undergoing treatment for African trypanosomiasis. A biological sample is collected from such a patient before treatment, during treatment and / or after treatment, and the effect of treatment can be known by examining changes in the amount of antibody. For example, if the concentration of the antibody in the later biological sample is lower than that in the previous biological sample, the treatment can be evaluated as effective.

  When a simple test such as immunochromatography is performed, if the signal (color development, etc.) is confirmed by visual observation, the subject is determined to have a high probability of being “afflicted with African trypanosomiasis,” and other diagnostic methods are used. In combination, the presence or absence of African trypanosomiasis can be determined.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further, this invention is not limited to these in any meaning.

Example 1 Selection of antigen for diagnosis of African trypanosomiasis <Selection of Tbg antigen>
If there are few mutations from the antigens identified in the proteomics experiment (Sullivan et al. PLOS Neglected Tropical Diseases 2013, Vol.7 (2) e2087) using IgG and Trypanosoma brucei brucei (Tbb) solution obtained from HAT patients Five types of antigens that were expected and expected to be specific were selected from protein homology searches by Protein Blast (ISG64, ISG65, Hypothetical 3020, Hypothetical 2120, Flagellar calcium-binding protein (FCaBP)). FCaBP is also one of the test antigens for Chagas disease, which has been preceded by serological diagnostic research. ISG64 and ISG65 are discussed in detail in the paper by Sullivan et al. Hypothetical 3020 and Hypothetical 2120 were only identified by mass spectrometry and have not been examined as test antigens.
In addition, tandem repeat antigens GM6, MARP1 (Muller et al. Parasitology, 1992, vol. 104, 111-120) and Tbg6 (Goto et al. Parasitology International, 2011, vol. 60) identified in Tbb and animal models. 538-540).
A comparative study was conducted on the above eight antigens.

<Selection of L. donovani antigen>
The L. donovani antigen was selected with reference to the following paper.
KRP42 (Takagi et al. Am. J. Trop. Med. Hyg. 76 (5), 2007)
K26 (Sundar et al. Trop Med Int Health, 12, 2007)
KMP11 (Souda, PLoS One, 8, e66110, 2013)
Here, KRP42 is an antigen very similar to K39, which is most widely used for serodiagnosis of L. donovani.

<Method>
All trypanosoma antigens were derived from Tbg and Leishmania was derived from L. donovani. CDNA was prepared by cloning by PCR or artificial gene synthesis, expressed in E. coli BL21 (DE3) by pET52b vector, and affinity purification by fusion tag was performed. The purified antigen is coupled to Luminex MagPlex® beads, all antigen beads are mixed and reacted with serum, and then anti-human IgG or IgM fluorescent antibody (R-phycoerythrin: R-PE) and The reaction was performed and measured with a Luminex 200 apparatus (Luminex Corporation). The Luminex method using Luminex200 is capable of measuring multiple antigens at once by coupling antigens to fluorescent beads and detecting them by flowmetry, but the basic principle is that of a general ELISA method. It is the same, and it is considered that the same result can be obtained by ELISA method.

Standard serum from WHO
Cg: Healthy control for Tbg patients
Pg1: Tbg patient stage 1
Pg2: Tbg patient stage 2
Cr: Healthy control for Tbr patients
Pr1: Tbr patient stage 1
Pr2: Tbr patient stage 2

  Visceral Leishmania patient serum: Ld (Bangladesh)

  Table 1 shows the structure of the recombinant antigen expressed in the pET52b vector.

<Experimental result>
The results of measurement using an anti-human IgG antibody are shown as a box plot (FIG. 1). Of the 8 Tbg antigens, 4 of ISG64, ISG65, FCaBP and GM6 showed reactivity in Tbg patient specimens. For all four antigens, stage 1 and stage 2 could not be detected specifically. ISG64, ISG65 and FCaBP are trypanosoma specific because they are negative in Leishmania patient specimens. The reaction of the three antigens ISG64, ISG65 and FCaBP with Tbr patient sera was weaker than Tbg patients. GM6 was also positive in Leishmania patient sera. GM6 showed the highest reactivity among the Tbg antigens examined in Tbr patient sera. This result is satisfactory because GM6 cross-reacts with Leishmania samples.
All three Leishmania antigens (KRP42, K26, KMP11) are Leishmania-specific because they were all negative for trypanosoma patient sera.

<ROC (Receiver Operating Characteristics) analysis>
ROC analysis was performed on the trypanosome results in FIG. The results are shown in Table 2.

  In the ROC analysis, the sensitivity and specificity are obtained as a cutoff value at a value that maximizes sensitivity + specificity. As a result, as shown in Table 2, ISG64 showed the highest AUC value in T.b.g. patients. Among T.b.r. patients, ISG646 showed the highest AUC value, which was 0.615, inferior to that of T.b.g. patients. In addition, it was found that the IgG detection system using the above-mentioned one kind of antigen does not reach the standard with the sensitivity and specificity (0.80 or more and 0.95 or more, respectively).

  Table 3 shows the sensitivity and specificity when the positive definition is single, multiple, 3 antigens or more. When multiple antigens are defined as positive, inclusion of GM6 in the antigen increases sensitivity. In multiple assays including Leishmania, cross-reaction with Leishmania can be detected separately, so false positives can be avoided.

  Similarly, ROC analysis was conducted for Leishmania. The negative specimen was a trypanosoma control (Cg, Cr) (Tables 4 and 5). Leishmania is excellent in sensitivity and specificity even when the definition of positive is single, plural, or more than three antigens.

<Experimental result>
The results of measurement using the above four Tbg antigens and using anti-human IgM antibody instead of anti-human IgG antibody are shown in FIG. 2-4.
The measurement system using an anti-human IgM antibody has a higher sensitivity than the measurement system using an anti-human IgG antibody, but the specificity tends to decrease. Antigens that were elevated in the IgM detection system were also elevated in purified IgM derived from human myeloma cells (ie, non-immune) (data not shown), and therefore the specificity of the IgM detection system was judged to be low. In order to clarify this, trypanosoma was detected by the Multiplex method using a non-trypanosoma antigen and an anti-human IgM antibody. The results are shown in FIG.
Based on the above results, it is possible to test by combining a specific IgG detection system with an antigen that non-specifically detects hyper-IgM blood (not necessarily a Tbg antigen, but may be a Tbg antigen). Improvement in accuracy can be expected.

According to the present invention, a rapid and simple determination means for African trypanosomiasis is provided, and a point-of-care test (POCT) for African trypanosomiasis is possible.
According to the diagnostic agent and diagnostic kit of the present invention, the presence or absence of trypanosoma infection and the presence or absence of African trypanosomiasis can be determined with high sensitivity, specificity and simplicity. According to the detection method of the present invention, the presence or absence of infection with African trypanosomiasis can be appropriately determined alone or in combination with other diagnostic methods.

  Although several specific embodiments of the present invention have been described in detail, those skilled in the art will recognize that various modifications may be made to the specific embodiments shown without departing from the teachings and advantages of the invention. It is possible to make changes. Accordingly, all such modifications and changes are intended to be included within the spirit and scope of the invention as claimed in the following claims.

Claims (13)

  A polypeptide containing the amino acid sequence shown in SEQ ID NO: 1 (ISG64), a polypeptide containing the amino acid sequence shown in SEQ ID NO: 2 (ISG65), a polypeptide containing the amino acid sequence shown in SEQ ID NO: 3 (FCaBP), and SEQ ID NO: 4 A diagnostic agent for African trypanosomiasis comprising at least three antigens selected from the group consisting of a polypeptide (GM6) comprising an amino acid sequence.   The diagnostic agent according to claim 1, wherein the antigen is three kinds of ISG64, ISG65 and FCaBP, or four kinds of ISG64, ISG65, FCaBP and GM6.   The diagnostic agent according to claim 1 or 2, further comprising at least one antibody selected from the group consisting of an anti-human IgG antibody and an anti-human IgM antibody.   The diagnostic agent according to any one of claims 1 to 3, wherein the antigen is bound to a solid phase.   The diagnostic agent according to claim 3, wherein the antigen and the antibody are bound to a solid phase.   Solid phase, polypeptide containing the amino acid sequence shown in SEQ ID NO: 1 (ISG64), polypeptide containing the amino acid sequence shown in SEQ ID NO: 2 (ISG65), polypeptide containing the amino acid sequence shown in SEQ ID NO: 3 (FCaBP) and sequence A kit for diagnosis of African trypanosomiasis, comprising at least three antigens selected from the group consisting of a polypeptide (GM6) comprising the amino acid sequence shown in No. 4 and bound to the solid phase.   The kit according to claim 6, wherein the antigen is three kinds of ISG64, ISG65 and FCaBP, or four kinds of ISG64, ISG65, FCaBP and GM6.   The kit according to claim 6 or 7, further comprising at least one antibody selected from the group consisting of an anti-human IgG antibody and an anti-human IgM antibody.   The kit according to any one of claims 6 to 8, further comprising a reagent for detecting an anti-human IgG antibody and / or an anti-human IgM antibody.   The kit according to claim 9, wherein the detection reagent is bound to the anti-human IgG antibody and / or anti-human IgM antibody. The following steps:
(1) a biological sample derived from a subject;
A polypeptide containing the amino acid sequence shown in SEQ ID NO: 1 (ISG64), a polypeptide containing the amino acid sequence shown in SEQ ID NO: 2 (ISG65), a polypeptide containing the amino acid sequence shown in SEQ ID NO: 3 (FCaBP), and SEQ ID NO: 4 Contacting with at least three antigens selected from the group consisting of a polypeptide (GM6) containing an amino acid sequence to form a complex of an antibody against the antigen and the antigen contained in a biological sample; and (2) Detecting the antibody in the complex formed in step (1);
A method for detecting African trypanosomiasis, comprising:   The detection method according to claim 11, wherein step (2) detects an antibody against the antigen in the complex using at least one antibody selected from the group consisting of an anti-human IgG antibody and an anti-human IgM antibody.   The detection method according to claim 11, wherein the step (2) detects an antibody against the antigen in the complex using two kinds of antibodies, an anti-human IgG antibody and an anti-human IgM antibody.