Immunoassay method using syphilis treponema pallidum antigen 47 kda (TPN47) as antigen

Abstract

Provided is an immunoassay for detecting a specific antibody against Treponema pallidum in serum by using syphilis Treponema pallidum antigen 47 kDa (TpN47) as an antigen.　Specifically provided are: a protein comprising a sequence having a structure such that a part of an amino acid sequence for C domain of TpN47 is deleted; and an immunoassay method using the protein as an antigen.

Description

Immunoassay method using Treponema pallidum antigen 47 kDa (TpN47) as an antigen

[0001] The present invention relates to an immunoassay method using Treponema pallidum antigen 47 kDa (TpN47) as an antigen, a reagent therefor, and a method using said reagent.

[0002] Treponema pallidum belongs to the spirochete bacterial family and is the causative agent of syphilis. Syphilis is primarily transmitted by sexual contact and can also be transmitted from mother to baby during pregnancy. The disease is characterized by distinct clinical stages and a long period of latent, asymptomatic infection. Many people do not notice symptoms and therefore remain unaware of syphilis infection for years.

[0003] Although effective treatments have been available since the introduction of penicillin in the mid-20th century, syphilis remains a significant global health problem. Identifying patients infected with Treponema pallidum is essential to support antibiotic therapy and prevent the spread of syphilis. Diagnostic tools such as immunoassays are used to detect antibodies to Treponema pallidum in serum.

[0004] After infection, serum antibodies first increase in antibody titers against cardiolipin (tests using non-treponemal antigens: VDRL, RPR card, RPR automated method), followed by an increase in specific antibody titers against Treponema pallidum (tests using Treponema pallidum antigens: FTA-ABS, TPHA, TPPA, TP antibody automated method). Anticardiolipin antibody titers decrease in response to treatment, so they are also used to assess treatment efficacy. However, because they are not specific antigens, biological false-positive reactions are possible. On the other hand, anti-Treponema pallidum antibody tests are characterized by high specificity, but even after treatment, antibody titers continue to be positive, although they gradually decrease, making it difficult to distinguish from previous syphilis infection.

[0005] Japanese Patent Application Publication No. 09-288110

[0006] V Sambri et al., Clin Diagn Lab Immunol. 2001 May; 8(3):534-9

[0007] The present invention provides an immunoassay for detecting Treponema pallidum-specific antibodies in serum using Treponema pallidum antigen 47 kDa (TpN47) as an antigen. TpN47 is a large protein consisting of 434 amino acid residues and is capable of detecting anti-Treponema pallidum antibodies, which are IgG and IgM. However, to facilitate differentiation from previous syphilis infections, it is believed necessary to further increase the sensitivity of the assay for IgM, which increases in the early stages of syphilis infection. Furthermore, increasing the sensitivity of the assay for IgM, which increases in the early stages of syphilis infection, could lead to early detection of syphilis infection. Therefore, the present invention provides an immunoassay for detecting Treponema pallidum-specific IgM antibodies in serum using Treponema pallidum antigen 47 kDa (TpN47) as an antigen.

[0008] As a result of extensive research, the present inventors discovered that the reaction with anti-Treponema pallidum IgM antibodies can be improved by deleting a portion of the C domain of the TpN47 antigen, and thus completed the present invention.

[0009] That is, the present invention is as follows: [1] A protein comprising a sequence in which a part of the amino acid sequence of the C domain of TpN47 is deleted. [2] The protein of [1], in which the part of the amino acid sequence of the C domain of TpN47 is deleted from 110 to 127 amino acids on the C-terminal side. [3] The protein of [1], which is deleted from the amino acids 225 to 351, 226 to 351, 227 to 351, 228 to 351, 229 to 351, 230 to 351, 231 to 351, 232 to 351, 233 to 351, 234 to 351, 235 to 351, 236 to 351, 237 to 351, 238 to 351, 239 to 351, 240 to 351, 241 to 351, or 242 to 351 of the amino acid sequence of full-length TpN47 shown in SEQ ID NO: 1. [4] The protein of [2] or [3], which consists of the amino acid sequence shown in SEQ ID NO: 4. [5] A TpN47 antigen comprising any one of the proteins [1] to [4]. [6] A method for measuring anti-Treponema pallidum antibodies by immunoassay using any one of the proteins [1] to [4] or the TpN47 antigen of [5] as an antigen. [7] The method of [6] for measuring anti-Treponema pallidum IgM antibodies with high sensitivity. [8] The method of [6] or [7], which is an agglutination method. [9] A kit for measuring anti-Treponema pallidum antibodies by immunoassay, which comprises any one of the proteins [1] to [4] or the TpN47 antigen of [5] as the measurement antigen.

[10] The kit of [9], which is an agglutination method. This specification incorporates the disclosures of Japanese Patent Application No. 2023-126558, from which the present application claims priority.

[0010] By measuring anti-Treponema pallidum antibodies using the TpN47 antigen of the present invention, anti-Treponema pallidum IgM antibodies can be measured with high sensitivity, and as a result, syphilis infection can be detected with high sensitivity at an early stage of infection.

[0011] The present invention will be described in detail below. In this specification, "%" means mass % (w / v %) unless otherwise specified.

[0012] The present invention relates to a protein in which a part of the C domain of the 47 kDa surface antigen (TpN47) of Treponema pallidum is deleted.

[0013] It is known that Treponema pallidum cell surfaces contain surface antigens of various molecular weights, the major ones being 47 kDa, 42 kDa, 17 kDa, and 15 kDa. These antigens are clearly separated by electrophoresis. Genes encoding not only the 47 kDa surface antigen but also the 17 kDa and 15 kDa surface antigens have already been cloned, and the antigens are being produced by genetic engineering. The amino acid sequences of these antigens have also been determined.

[0014] The amino acid sequence of full-length TpN47 (referred to as full-length TpN47) is shown in SEQ ID NO: 1, the amino acid sequence of TpN47 (referred to as ABCD domain) obtained by excluding the amino acid sequence of the signal peptide (amino acids 1 to 19) and the lipid-modified cysteine ​​residue (amino acid 20) from the amino acid sequence of full-length TpN47 is shown in SEQ ID NO: 2, the amino acid sequence of TpN47 from which the D domain has been deleted (referred to as ABC domain) is shown in SEQ ID NO: 3, the amino acid sequence of TpN47 from which part of the amino acid sequence of the C domain has been deleted (referred to as AB+D domain) is shown in SEQ ID NO: 4, and the amino acid sequence of TpN47 from which the ABC domain has been deleted (referred to as D domain) is shown in SEQ ID NO: 5.

[0015] The crystal structure and biological role of TpN47 have also been reported, and it is known to have a four-domain structure: A, B, C, and D. Counting from the N-terminus of the amino acid sequence of full-length TpN47 (SEQ ID NO: 1, SwissProt P29723), domain A consists of amino acids 26 to 62 (amino acid acids 6 to 42 in the amino acid sequence of SEQ ID NO: 2) (A1 domain) and 175 to 223 (amino acid acids 155 to 203 in the amino acid sequence of SEQ ID NO: 2) (A2 domain), domain B consists of amino acids 63 to 174 (amino acid acids 43 to 154 in the amino acid sequence of SEQ ID NO: 2), domain C consists of amino acids 224 to 351 (amino acid acids 204 to 331 in the amino acid sequence of SEQ ID NO: 2), and domain D consists of amino acids 352 to 434 (amino acid acids 332 to 414 in the amino acid sequence of SEQ ID NO: 2).

[0016] The TpN47 protein used as an antigen for measurement in the present invention does not contain the signal peptide amino acid sequence (amino acids 1 to 19) and lipid-modified cysteine ​​residue (amino acid 20) of the amino acid sequence of full-length TpN47 shown in SEQ ID NO: 1.

[0017] The protein of the present invention enhances the reactivity of anti-Treponema pallidum antibodies with IgM by deleting a portion of the amino acid sequence of the C domain of the TpN47 antigen. Furthermore, even when a portion of the amino acid sequence of the C domain of TpN47 is deleted, the measured values ​​are equivalent to those obtained when TpN47, which is derived from the full-length TpN47 amino acid sequence omitting the signal peptide amino acid sequence (amino acids 1 to 19) and the lipid-modified cysteine ​​residue (amino acid 20), is used, enabling highly sensitive measurement of anti-Treponema pallidum antibodies. Specifically, the use of a protein in which a portion of the amino acid sequence of the C domain of the 47 kDa surface antigen (TpN47) of Treponema pallidum is deleted enables highly sensitive measurement of anti-Treponema pallidum IgM antibodies. The partial deletion of the amino acid sequence of the C domain of the TpN47 antigen is 110 to 127, preferably 115 to 125, more preferably 118 to 122, and particularly preferably 120, amino acids from the C-terminal side of the amino acid sequence of the C domain of the TpN47 antigen.The deletions are at amino acids 225 to 351, 226 to 351, 227 to 351, 228 to 351, 229 to 351, 230 to 351, 231 to 351, 232 to 351, 233 to 351, 234 to 351, 235 to 351, 236 to 351, 237 to 351, 238 to 351, 239 to 351, 240 to 351, 241 to 351, or 242 to 351 of the amino acid sequence of full-length TpN47 shown in SEQ ID NO: 1. The amino acids are deleted (amino acids at positions 205 to 331, amino acids at positions 206 to 331, amino acids at positions 207 to 331, amino acids at positions 208 to 331, amino acids at positions 209 to 331, amino acids at positions 210 to 331, amino acids at positions 211 to 331, amino acids at positions 212 to 331, amino acids at positions 213 to 331, amino acids at positions 214 to 331, amino acids at positions 215 to 331, amino acids at positions 216 to 331, amino acids at positions 217 to 331, amino acids at positions 218 to 331, amino acids at positions 219 to 331, amino acids at positions 220 to 331, amino acids at positions 221 to 331, or amino acids at positions 222 to 331 of the amino acid sequence shown in SEQ ID NO: 2).

[0018] The amino acid sequences of SEQ ID NOs: 1 to 5 are shown below: SEQ ID NO: 1 MKVKYALLSAGALQLLVVGCGSSHHETHYGYATLSYADYWAGELGQSRDVLLAGNAEADRAGDLDAGMFDAVSRATHGHGAFRQQFQYAVEVLGEKVLSKQETEDSRGRKKWEYETDPSVTKMVRASASFQDLGEDGEIKFEAVEGAVALADRASSFMVDSEEYKITNVKVHGMKFVPVAVPHELKGIAKEKFHFVEDSRVTENTNGLKTMLTEDSF SARKVSSMESPHDLVVDTVGTGYHSRFGSDAEASVMLKRADGSELSHREFIDYVMNFNTVRYDYYGDDASYTNLMASYGTKHSADSWWKTGRVPRISCGINYGFDRFK GSGPGYYRLTLIANGYRDVVADVRFLPKYEGNIDIGLKGKVLTIGGADAETLMDAAVDVFADGQPKLVSDQAVSLGQNVLSADFTPGTEYTVEVRFKEFGSVRAKVVAQ SEQ ID NO: 2 GSSHHETHYGYATLSYADYWAGELGQSRDVLLAGNAEADRAGDLDAGMFDAVSRATHGHGAFRQQFQYAVEVLGEKVLSKQETEDSRGRKKWEYETDPSVTKMVRASASFQDLGEDGEIKFEAVEGAVALADRASSFMVDSEEYKITNVKVHGMKFVPVAVPHELKGIAKEKFHFVEDSRVTENTNGLKTMLTEDSFSARKVSSMESPHDLVVDTVGTGYHSRFGSDAEASVMLKRADGSELSHREFIDYVMNFNTVRYDYYGDDASYTNLMASYGTKHSADSWWKTGRVPRISCGINYGFDRFKGSGPGYYRLTLIANGYRDVVADVRFLPKYEGNIDIGLKGKVLTIGGADAETLMDAAVDVFADGQPKLVSDQAVSLGQNVLSADFTPGTEYTVEVRFKEFGSVRAKVVAQ SEQ ID NO: 3GSSHHETHYGYATLSYADYWAGELGQSRDVLLAGNAEADRAGDLDAGMFDAVSRATHGHGAFRQQFQYAVEVLGEKVLSKQETEDSRGRKKWEYETDPSVTKMVRASASFQDLGEDGEIKFEAVEGAVALADRASSFMVDSEEYKITNVKVHGMKFVPVAVPHELKGIAKEKFHFVEDSRVTENTNGLKTMLTEDSFSARKVSSMESPHDLVVDTVGTGYHSRFGSDAEASVMLKRADGSELSHREFIDYVMNFNTVRYDYYGDDASYTNLMASYGTKHSADSWWKTGRVPRISCGINYGFDRFKGSGPGYYRLTLIANGYRDVVADVRFL SEQ ID NO: 4 GSSHHETHYGYATLSYADYWAGELGQSRDVLLAGNAEADRAGDLDAGMFDAVSRATHGHGAFRQQFQYAVEVLGEKVLSKQETEDSRGRKKWEYETDPSVTKMVRASASFQDLGEDGEIKFEAVEGAVALADRASSFMVDSEEYKITNVKVHGMKFVPVAVPHELKGIAKEKFHFVEDSRVTENTNGLKTMLTEDSFSARKVSSMESPHDLPKYEGNIDIGLKGKVLTIGGADAETLMDAAVDVFADGQPKLVSDQAVSLGQNVLSADFTPGTEYTVEVRFKEFGSVRAKVVAQ SEQ ID NO: 5 PKYEGNIDIGLKGKVLTIGGADAETLMDAAVDVFADGQPKLVSDQAVSLGQNVLSADFTPGTEYTVEVRFKEFGSVRAKVVAQ

[0019] The protein of the present invention can also be obtained by incorporating DNA encoding a protein in which a portion of the amino acid sequence of the C domain of TpN47 has been deleted into a plasmid vector, which is then introduced into a host cell for expression.

[0020] The host cell is not particularly limited as long as it is capable of producing the protein of the present invention, and may be, for example, an insect cell (e.g., Sf9, Hi5 cells, etc.), Escherichia coli, yeast (e.g., Saccharomyces cerevisiae, Saccharomyces pombe, Pichia pastori, etc.), mammalian cell (e.g., CHO, HEK), or any other cell.

[0021] The protein of the present invention can be produced using a plant expression system, or can be produced using a genetically modified plant. Plant transient expression systems can be carried out using known methods. Examples of such methods include, but are not limited to, the agroinfiltration method, the plant virus vector method, the magnICON® system, and the particle gun method.

[0022] When the agroinfiltration method is used, Agrobacterium is transformed with the above vector, and the Agrobacterium is then infected into a plant, whereby a plant that expresses the above protein can be obtained.

[0023] In the present invention, the term "TpN47 antigen" also refers to a protein in which a portion of the C domain of the 47 kDa surface antigen (TpN47) of Treponema pallidum has been deleted. That is, the present invention encompasses TpN47 antigens consisting of a protein in which a portion of the C domain of the 47 kDa surface antigen (TpN47) of Treponema pallidum has been deleted.

[0024] Furthermore, the present invention includes a method for measuring and immunoassaying Treponema pallidum TpN47 using a protein obtained by deleting a portion of the C domain of the 47 kDa surface antigen (TpN47) of Treponema pallidum as an antigen. By measuring TpN47, the presence or absence of Treponema pallidum in a test sample collected from a subject can be detected. By detecting the presence or absence of Treponema pallidum in a test sample, it is possible to detect whether a subject is infected with Treponema pallidum, i.e., whether the subject has syphilis, or to obtain auxiliary data for diagnosing the same. By using a protein obtained by deleting a portion of the C domain of the 47 kDa surface antigen (TpN47) of Treponema pallidum as an antigen, it is possible to detect anti-Treponema pallidum TpN47 IgM antibodies, which appear at an early stage of infection, with high sensitivity, thereby enabling syphilis to be detected at an early stage of infection.

[0025] The present invention can be applied to any immunological measurement method utilizing an antigen-antibody reaction, such as immunodiffusion, immunoturbidimetry, immunonephelometry, hemagglutination, latex, enzyme immunoassay (EIA), radioimmunoassay (RIA), immunochromatography, etc. Of these, the present invention is particularly effective in immunoturbidimetry, immunonephelometry, and agglutination methods such as hemagglutination and latex agglutination.

[0026] The agglutination method is an immunoassay that uses an insoluble carrier such as latex particles, bentonite, collodion, kaolin, fixed sheep red blood cells, etc. As the insoluble carrier, latex is preferred, and examples of latex particles that can be used include polystyrene latex particles, styrene-butadiene copolymer latex particles, and polyvinyl toluene latex particles. In an immunoassay using the agglutination method, the insoluble carrier is sensitized with an antigen, i.e., the antigen is bound to the surface of the carrier.

[0027] The immunoassay of the present invention is preferably performed using an automated analyzer. The automated analyzer is an automated analyzer for biochemical testing applicable to immunoassays, employing a mechanism for dispensing reagents into reaction vessels via tubes using a continuous dispensing method. That is, the automated analyzer is equipped with a means for placing reagents inside or outside the apparatus, and continuously dispenses reagents into reaction vessels provided in the apparatus through tubes. Dispensing of reagents through tubes is performed using a combination of a pump, such as a peristaltic pump, and a switching valve. Various commercially available analyzers of this type are available, including the Hitachi Automated Analyzer 7180, Hitachi Automated Analyzer 7250, Hitachi Automated Analyzer 7450, and Hitachi Automated Analyzer 7350, all manufactured by Hitachi High-Tech Corporation.

[0028] In a method using an automated analyzer, antibodies are measured by optically capturing agglutinates formed by binding of antigen-sensitized carriers to target antibodies in a reaction vessel. Specifically, the agglutinates are irradiated with light ranging from visible light to near-infrared light, and the degree of carrier particle agglutination is measured by detecting changes in absorbance or scattered light intensity. Measurement methods include the one-point method, two-point method, three-point method, and rate method, and the method of the present invention can be applied to any of these methods. A test sample is dispensed into a reaction vessel in advance, and then a first reagent and a second reagent are dispensed therein to allow a reaction, and the degree of agglutination is measured. The degree of agglutination can be used as an index to quantify the target antigen or antibody in the test sample. The order of addition of the first and second reagents is not limited, but preferably the first reagent is dispensed before the second reagent, or the first and second reagents are dispensed simultaneously. In this case, by creating a standard curve relating the concentration of the analyte to the degree of agglutination in advance using a plurality of samples with known concentrations of the analyte, the concentration of the analyte in the test sample can be calculated based on the standard curve.

[0029] The first reagent may include a buffer solution, which may contain other stabilizing reagents such as surfactants, protective reagents, etc. The second reagent may include the above-mentioned particles sensitized with an antigen that specifically binds to the antibody to be measured, which may contain other stabilizing reagents such as surfactants, protective reagents, etc.

[0030] The method for sensitizing the antigen to the carrier is not particularly limited and may be any known method. For example, the antigen may be physically adsorbed to the carrier or chemically bound to the carrier. More specifically, the antigen can be sensitized to the carrier by mixing the antigen and the carrier and then warming and shaking at 30-37°C for 1-2 hours. The amount of antigen sensitized to the carrier can be appropriately determined depending on the particle size of the carrier used. After sensitizing the antigen to the carrier, it is preferable to block the unsensitized portion on the carrier surface with bovine serum albumin, human serum albumin, rabbit serum albumin, ovalbumin, or the like. The antigen-sensitized carrier is preferably kept as a dispersion in a medium until it is reacted with the test sample. In this case, for example, a phosphate buffer solution, a glycine buffer solution, or the like can be used as the medium. Bovine serum albumin, gelatin, gum arabic, or the like may be added to the medium as needed. The antigen-sensitized carrier thus prepared is reacted with a test sample, and the reactivity between the sensitized antigen and the antibody in the test sample can be determined based on the presence or absence of agglutination or the degree of agglutination, thereby detecting the antibody in the test sample.

[0031] Detection using the agglutination method using sensitized particles involves adding several tens to hundreds of microliters of physiological saline or an appropriate buffer solution to several microliters to several tens of microliters of a test sample in a suitable reaction vessel, mixing, and incubating for several minutes. Then, several tens to hundreds of microliters of antigen-bound sensitized particles are added and incubated for several minutes. The turbidity caused by the agglutination of sensitized particles resulting from the antigen-antibody reaction can be measured by measuring the absorbance at an appropriate wavelength (e.g., 570 nm). Alternatively, several tens to hundreds of microliters of a sensitized particle solution suspended in a buffer solution can be added to several microliters of the reaction sample. Suitable buffer solutions with a pH of 5.0 to 10, such as phosphate buffer, borate buffer, and Tris buffer, can be used.

[0032] The test sample to be subjected to the immunoassay method of the present invention is not particularly limited, but is preferably a body fluid such as blood, serum, plasma, urine, stool, saliva, tissue fluid, cerebrospinal fluid, swab, or a dilution thereof, and more preferably blood, serum, plasma, or a dilution thereof.

[0033] In the agglutination method, the first reagent and the second reagent containing latex particles are mixed together.

[0034] Furthermore, the present invention also encompasses an immunoassay kit comprising the assay reagent. The immunoassay kit comprises, as an assay antigen, a protein comprising a sequence in which a portion of the amino acid sequence of the C domain of TpN47 is deleted. If the buffer composition does not contain antigen-sensitized particles, the immunoassay kit may also contain antigen-sensitized particles separately, and may further include a positive control, a negative control, instructions, etc.

[0035] The present invention will be specifically explained by the following examples, but the present invention is not limited to these examples.

[0036] (1) Preparation of TpN47 TpN47 was prepared using a plant transient expression system. TpN47 was cloned into a tobacco mosaic virus (TMV) vector (Icon Genetics). This vector was introduced into Agrobacterium, which was transformed and cultured. The resulting culture mixture was then infiltrated into Nicotiana benthamiana leaves and harvested approximately one week later. The harvested leaves (infected leaves) were frozen.

[0037] 200 g of frozen infected leaves were weighed and crushed in a cutter mixer. 200 mL of extraction solution (100 mM Tris, 250 mM NaCl, 5 mM EDTA, 40 mM sodium ascorbate) was added to the crushed material, and the infected leaves were crushed repeatedly. The mixture was centrifuged and the supernatant was collected. The collected solution was filtered through a 0.22 μm filter. The filtrate was subjected to affinity column chromatography to remove impurities.

[0038] Using the above method, the following TpN47 variants were expressed and prepared: the ABCD domain (SEQ ID NO: 2), the ABC domain with the D domain deleted (SEQ ID NO: 3), the AB+D domain with a partial deletion of the C domain (SEQ ID NO: 4), and the D domain with the ABC domain deleted (SEQ ID NO: 5).

[0039] TpN15 and TpN17 were also prepared in a similar manner. The amino acid sequences of TpN15 and TpN17 and the DNA sequences encoding them are known.

[0040] (2) Preparation of First Reagent A surfactant and bovine serum albumin were added to a buffer solution (Tris, pH 8.0) to prepare a first reagent.

[0041] (3) Preparation of Second Reagent A measurement reagent for the immunoagglutination assay was prepared using the ABCD domain, ABC domain, AB+D domain, and D domain of TpN47 prepared in (1) and TpN15 and TpN17 as follows: Sensitized particles prepared by supporting 3.9 mg of the ABCD domain, ABC domain, AB+D domain, and D domain of TpN47 prepared in (1) and TpN15 and TpN17 per mL of polystyrene latex suspension were suspended in a buffer solution (Bis-Tris, pH 7.0) to a concentration of 0.12%, to prepare a second reagent.

[0042] (4) Measurement by Automated Analyzer: Automated measurements were performed using the Hitachi Automated Analyzer 7180 (Hitachi High-Tech Corporation) using the endpoint method. Serum samples were measured using the first reagent described above and a second reagent containing sensitized particles of the ABCD domain, ABC domain, AB+D domain, and D domain of TpN47. 100 μL of the first reagent was added to 8.5 μL of sample solution, and the mixture was stirred and mixed at 37°C. After leaving the mixture for 5 minutes, 50 μL of the second reagent was added, followed by further stirring and mixing at 37°C. The agglutination reaction over approximately 5 minutes was measured as the change in absorbance, and the anti-Treponema pallidum antibody concentration of each sample was calculated from a calibration curve prepared using a standard solution (HEPES buffer, bovine serum albumin, sodium chloride, anti-Treponema pallidum antibody, pH 7.4).

[0043] (5) Comparison of TpN47 Reactivity Measurement values ​​of the reagents prepared in (1) using the ABCD domain, ABC domain, AB+D domain, and D domain of TpN47 were compared with those of standard samples. Serum samples (two samples) collected from syphilis patients were used as samples, and a dilution series of a standard solution (HEPES buffer, bovine serum albumin, sodium chloride, anti-Treponema pallidum antibody, pH 7.4) was prepared. Each diluted sample was measured by the method in (4) using the first reagent prepared in (2) and the second reagent prepared in (3) using the ABCD domain, ABC domain, AB+D domain, and D domain of TpN47. The results are shown in Table 1.

[0044]

[0045] The results in Table 1 show that the reagent using TpN47, which consists of only the D domain with the ABC domain deleted, had lower reactivity to the standard solution than the reagent using TpN47, which consists of the AB+D domain with the ABCD domain and a portion of the C domain deleted.

[0046] Furthermore, using rabbit immune serum against TpN47 as a sample, measurements were performed by the method of (4) using the first reagent prepared in (2) and the second reagent prepared in (3) using the ABCD domain, ABC domain, AB+D domain, and D domain of TpN47. The results are shown in Table 2.

[0047]

[0048] The results in Table 2 show that, compared to a reagent using TpN47 consisting of an AB+D domain in which the ABCD and C domains have been partially deleted, a reagent using TpN47 consisting of only the D domain in which the ABC domain has been deleted had lower reactivity with rabbit immune serum against TpN47.

[0049] (6) Comparison of TpN47 serum sample measurements The anti-TP antibody titer values ​​of positive serum samples were compared for the reagents using each TpN47 prepared in (1). Two serum samples (serum 1 and 2) were measured by the method in (4) using the first reagent prepared in (2) and the second reagent prepared in (3) using the ABCD domain, ABC domain, AB+D domain, and D domain of TpN47. The results are shown in Table 3.

[0050]

[0051] From the results in Table 3, compared to the reagent using TpN47 consisting of an ABC domain with the ABCD domain and D domain deleted, the reagent using TpN47 consisting of an AB+D domain with a partial deletion of the C domain had higher measurement values ​​for positive serum samples and higher sensitivity.

[0052] Additionally, anti-Treponema pallidum antibodies in human serum were detected using recomLine Treponema IgG (trade name, commercially available from MIKROGEN) and recomLine Treponema IgM (trade name, commercially available from MIKROGEN) to detect IgG and IgM anti-TpN15, anti-TpN17, and anti-TpN47 antibodies contained in serum samples 1 and 2. The presence or absence of antibodies against each Treponema pallidum antigen (TpN17, TpN47, and TpN15) and the intensity of the detected bands were compared with the cutoff bands of each test strip according to the criteria in Table 4.

[0053]

[0054] The results are shown in Table 5.

[0055]

[0056] The results in Table 5 show that serum samples 1 and 2 were samples in which anti-TpN47 IgM antibodies were detected.

[0057] These results suggest that the protein of the present invention containing a sequence in which a portion of the C domain of TpN47 has been deleted reacts with high sensitivity to IgM antibodies.

[0058] (7) Comparison with other companies' products The first reagent prepared in (2) and the second reagent (the present invention) prepared in (3) using the AB+D domain TpN47 with a partial deletion of the C domain prepared in (1) were used to compare the measured values ​​of anti-TP antibody titers of three other companies' products (other companies' products 1 to 3). The reagent of the present invention was measured by the method of (4).

[0059] The specimens used were the Syphilis Seroconversion Panel (trade name, commercially available from SeraCare), a set of plasma samples collected over time from syphilis patients. The plasma samples collected over time were also used to detect anti-Treponema pallidum antibodies in human serum using recomLine Treponema IgG (trade name, commercially available from MIKROGEN) and recomLine Treponema IgM (trade name, commercially available from MIKROGEN). The presence or absence of antibodies against each Treponema pallidum antigen and the intensity of the detected bands were compared with the cutoff bands of each test strip according to the criteria in Table 4. The results are shown in Table 6.

[0060]

[0061] The results in Table 6 show that the reagent using the AB+D domain TpN47, in which the C domain was partially deleted, was able to detect anti-Treponema pallidum antibodies from plasma samples of syphilis patients on day 31. On the other hand, the competitor's product was able to detect anti-Treponema pallidum antibodies from plasma samples of syphilis patients on day 45 at the earliest. In other words, the reagent using the AB+D domain TpN47, in which the C domain was partially deleted, was able to detect anti-Treponema pallidum antibodies earlier than the competitor's product.

[0062] Furthermore, the results of detecting various anti-Treponema pallidum antibodies in plasma samples confirmed the presence of IgM antibodies against TpN47 on the 31st day.

[0063] From the above results, it was found that the protein of the present invention containing a sequence in which part of the C domain of TpN47 was deleted reacted with high sensitivity to the anti-TpN47 IgM antibody that appeared first in patient plasma, and was able to measure the antibody in syphilis patients at an earlier stage than other companies' products.

[0064] The method of the present invention makes it possible to detect syphilis infection at an early stage of infection.

[0065] SEQ ID NOs: 2-5 Synthesis All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

1. A protein containing a sequence in which a portion of the amino acid sequence of the C domain of TpN47 is deleted.

2. The protein according to claim 1, wherein the partial deletion of the amino acid sequence of the C domain of TpN47 is a deletion of 110 to 127 amino acids from the C-terminus.

3. The protein according to claim 1, which is deleted from the amino acids 225 to 351, 226 to 351, 227 to 351, 228 to 351, 229 to 351, 230 to 351, 231 to 351, 232 to 351, 233 to 351, 234 to 351, 235 to 351, 236 to 351, 237 to 351, 238 to 351, 239 to 351, 240 to 351, 241 to 351, or 242 to 351 of the amino acid sequence of full-length TpN47 shown in SEQ ID NO:

1.

4. The protein according to claim 2, which consists of the amino acid sequence shown in SEQ ID NO:

4.

5. The protein according to claim 3, which consists of the amino acid sequence shown in SEQ ID NO:

4.

6. A TpN47 antigen comprising a protein according to any one of claims 1 to 5.

7. A method for measuring anti-Treponema pallidum antibodies by immunoassay using the protein according to any one of claims 1 to 5 as an antigen.

8. A method for measuring anti-Treponema pallidum antibodies by immunoassay using the TpN47 antigen according to claim 6 as an antigen.

9. The method according to claim 7, which measures anti-Treponema pallidum IgM antibodies with high sensitivity.

10. The method according to claim 8, which measures anti-Treponema pallidum IgM antibodies with high sensitivity.

11. The method of claim 7, which is an agglomeration method.

12. The method of claim 8, which is an agglomeration method.

13. The method of claim 9, which is an agglomeration method.

14. The method of claim 10, which is an agglomeration method.

15. A kit for measuring anti-Treponema pallidum antibodies by immunoassay, comprising the protein according to any one of claims 1 to 5 as an antigen for measurement.

16. A kit for measuring anti-Treponema pallidum antibodies by immunoassay, comprising the TpN47 antigen according to claim 6 as an antigen for measurement.

17. The kit according to claim 15, which is an agglutination method.

18. The kit according to claim 16, which is an agglutination method.

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