Immunoassay method using treponema pallidum antigen 17 kda (TPN17) as antigen

Abstract

To provide an immunoassay for detecting a specific antibody against Treponema pallidum in serum by using Treponema pallidum antigen 17 kDa (TpN17) as an antigen.　The present invention provides a protein that contains a sequence in which part of the amino acid sequence of TpN17 has been deleted and an immunoassay method that uses the protein as an antigen.

Description

Immunoassay method using Treponema pallidum antigen 17 kDa (TpN17) as an antigen

[0001] The present invention relates to an immunoassay method using Treponema pallidum antigen 17 kDa (TpN17) 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 T. 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 aims to provide an immunoassay that uses Treponema pallidum antigen 17 kDa (TpN17) as an antigen to detect Treponema pallidum-specific antibodies in serum. TpN17 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 measurement sensitivity for IgM, which increases in the early stages of syphilis infection. Furthermore, increasing the measurement sensitivity for IgM, which increases in the early stages of syphilis infection, could lead to early detection of syphilis infection. Therefore, the present invention aims to provide an immunoassay that uses Treponema pallidum antigen 17 kDa (TpN17) as an antigen to detect Treponema pallidum-specific IgM antibodies in serum.

[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 amino acid sequence of the TpN17 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 portion of the amino acid sequence of TpN17 has been deleted. [2] The protein of [1], wherein the deletion of the portion of the amino acid sequence of TpN17 is a deletion of 1 to 20 amino acids at the N-terminus of the amino acid sequence shown in SEQ ID NO: 1. [3] The protein of [2], wherein the deletion of the portion of the amino acid sequence of TpN17 is a deletion of 11 amino acids at the N-terminus, and the protein has the amino acid sequence shown in SEQ ID NO: 3. [4] A TpN17 antigen comprising any of the proteins of [1] to [3]. [5] A method for measuring anti-Treponema pallidum antibodies by immunoassay using any of the proteins of [1] to [3] or the TpN17 antigen of [4] as an antigen. [6] The method of [5], which measures anti-Treponema pallidum IgM antibodies with high sensitivity. [7] The method of [5] or [6], which is an agglutination method. [8] A kit for measuring anti-Treponema pallidum antibodies by immunoassay, comprising any one of the proteins of [1] to [3] or the TpN17 antigen of [4] as an antigen for measurement. [9] The kit of [8], which is an agglutination method. This specification incorporates the disclosure of Japanese Patent Application No. 2023-126467, from which the present application claims priority.

[0010] By measuring anti-Treponema pallidum antibodies using the TpN17 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 amino acid sequence of the 17 kDa surface antigen (TpN17) 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 protein of the present invention has enhanced reactivity with anti-Treponema pallidum antibody IgM by deleting a portion of the amino acid sequence of TpN17. Furthermore, deleting a portion of the amino acid sequence of TpN17 increases the measured value, enabling highly sensitive measurement of anti-Treponema pallidum antibody. Specifically, by using a protein in which a portion of the amino acid sequence of the 17 kDa surface antigen (TpN17) of Treponema pallidum has been deleted, anti-Treponema pallidum IgM antibody can be measured with high sensitivity.

[0015] The amino acid sequence of full-length TpN17 excluding the amino acid sequence of the signal peptide and lipid-modified cysteine ​​residues is shown in SEQ ID NO: 1, the amino acid sequence of full-length TpN17 is shown in SEQ ID NO: 2 (SwissProt P29722), and the amino acid sequence of ΔTpN17, in which 11 amino acids have been deleted from the N-terminus of the amino acid sequence of TpN17 in SEQ ID NO: 1, is shown in SEQ ID NO: 3.

[0016] The amino acid sequences of SEQ ID NOs: 1 to 3 are shown below. SEQ ID NO: 1 VSCTTVCPHAGKAKAEKVECALKGGIFRGTLPAADCPGIDTTVTFNADGTAQKVELALEKKSAPSPLTYRGTWMVREDGIVELSLVSSEQSKAPHEKELYELIDSNSVRYMGAPGAGKPSKEMAPFYVLKKTKK SEQ ID NO: 2 MKGSVRALCAFLGVGALGSALCVSCTTVCPHAGKAKAEKVECALKGGIFRGTLPAADCPGIDTTVTFNADGTAQKVELALEKKSAPSPLTYRGTWMVREDGIVELSLVSSEQSKAPHEKELYELIDSNSVRYMGAPGAGKPSKEMAPFYVLKKTKK SEQ ID NO: 3 KAKAEKVECALKGGIFRGTLPAADCPGIDTTVTFNADGTAQKVELALEKKSAPSPLTYRGTWMVREDGIVELSLVSSEQSKAPHEKELYELIDSNSVRYMGAPGAGKPSKEMAPFYVLKKTKK

[0017] The TpN17 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 TpN17 shown in SEQ ID NO: 2.

[0018] The partial deletion of the amino acid sequence of TpN17 is 1 to 20, preferably 5 to 15, more preferably 7 to 14, even more preferably 8 to 13, even more preferably 9 to 12, and particularly preferably 11, deletions from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1. That is, deletions of amino acids at positions 1 to 42, 1 to 41, 1 to 40, 1 to 39, 1 to 38, 1 to 37, 1 to 36, 1 to 35, 1 to 34, 1 to 33, 1 to 32, 1 to 31, 1 to 30, 1 to 29, 1 to 28, 1 to 27, 1 to 26, 1 to 25, 1 to 24, or 1 to 23 of the amino acid sequence of full-length TpN17 shown in SEQ ID NO: 2. Among these, deletions of amino acids at positions 1 to 33 are preferred.

[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 TpN17 has been deleted into a plasmid vector, and then introducing this 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, a protein in which a portion of the amino acid sequence of the 17 kDa surface antigen (TpN17) of Treponema pallidum has been deleted is referred to as the "TpN17 antigen." That is, the present invention encompasses TpN17 antigens consisting of a protein in which a portion of the amino acid sequence of the 17 kDa surface antigen (TpN17) of Treponema pallidum has been deleted.

[0024] Furthermore, the present invention includes a method for measuring and immunoassaying Treponema pallidum 17 kDa surface antigen (TpN17) using a protein in which a portion of the amino acid sequence of the TpN17 has been deleted as an antigen. Measuring TpN17 allows the presence or absence of Treponema pallidum in a test sample collected from a subject to be detected. Detecting the presence or absence of Treponema pallidum in a test sample allows for the detection of whether a subject is infected with Treponema pallidum, i.e., suffering from syphilis, or for obtaining auxiliary data for diagnosing the same. Using a protein in which a portion of the amino acid sequence of the TpN17 17 kDa surface antigen of Treponema pallidum has been deleted as an antigen allows for the highly sensitive detection of anti-Treponema pallidum TpN17 IgM antibodies, which appear at an early stage of infection, and thus allows for the detection of syphilis 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 TpN17 has been 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 TpN17 TpN17 was prepared using a plant transient expression system. TpN17 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, TpN17 (sequence number 1), which lacks the signal peptide amino acid sequence and lipid-modified cysteine ​​residues from the amino acid sequence of full-length TpN17, and ΔTpN17 (sequence number 3), which lacks a portion of the amino acid sequence, were expressed and produced.

[0039] TpN15 and TpN47 were also prepared in a similar manner. The amino acid sequences of TpN15 and TpN47 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 method was prepared using TpN17, ΔTpN17, TpN15, and TpN47 prepared in (1) as follows: Sensitized particles prepared by supporting 3.9 mg of TpN17, ΔTpN17, TpN15, and TpN47 per mL of polystyrene latex suspension were suspended in a buffer solution (Bis-Tris, pH 6.0) to a concentration of 0.12%, to prepare a second reagent.

[0042] (4) Measurement by Automated Analyser Automatic measurements were performed by the endpoint method using a Hitachi Automated Analyser 7180 (Hitachi High-Tech Corporation).

[0043] Serum samples were assayed using the first reagent and a second reagent containing TpN17- or ΔTpN17-sensitized particles. 100 μL of the first reagent was added to 8.5 μL of sample solution, and the mixture was stirred at 37°C. After leaving the mixture for 5 minutes, 50 μL of the second reagent was added, and the mixture was further stirred 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).

[0044] (5) Comparison of the reactivity of TpN17 The measured values ​​for the samples of the reagents using TpN17 (SEQ ID NO: 1), which was prepared by excluding the amino acid sequence of the signal peptide and the lipid-modified cysteine ​​residues from the amino acid sequence of the full-length TpN17 prepared in (1), and ΔTpN17 (SEQ ID NO: 3), which was prepared by deleting a portion of the amino acid sequence, were compared.

[0045] Serum samples (two samples) collected from syphilis patients were used. The samples were measured by the method described in (4) using the first reagent prepared in (2) and the second reagent prepared in (3) using TpN17 or ΔTpN17.

[0046] In addition, anti-Treponema pallidum antibodies in human serum collected from syphilis patients were detected 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 to 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 1.

[0047]

[0048] The results are shown in Table 2.

[0049]

[0050] The results in Table 2 show that the reagent using ΔTpN17, in which part of the amino acid sequence was deleted, gave higher measurement values ​​and was able to detect more antibodies than the reagent using TpN17, in which the amino acid sequence of the signal peptide and the lipid-modified cysteine ​​residue were removed from the amino acid sequence of the full-length TpN17.

[0051] Furthermore, the results of detecting various anti-Treponema pallidum antibodies in the serum samples indicated that sample 1 contained IgM and IgG, and sample 2 contained IgM.

[0052] The difference in measurement values ​​between the reagent using ΔTpN17, which has a portion of the amino acid sequence deleted, and the reagent using TpN17, which has the signal peptide amino acid sequence and lipid-modified cysteine ​​residues removed from the amino acid sequence of full-length TpN17, was greater for sample 2 than for sample 1. This indicates that the protein of the present invention, in which a portion of the amino acid sequence of TpN17 has been deleted, reacts sensitively to anti-TpN17 IgM antibodies, which appear early in patient serum, and makes it possible to measure antibodies in syphilis patients at an early stage.

[0053] The method of the present invention makes it possible to detect syphilis infection at an early stage of infection.

[0054] SEQ ID NO: 3 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 TpN17 has been deleted.

2. The protein according to claim 1, wherein the partial deletion of the amino acid sequence of TpN17 is a deletion of 1 to 20 amino acids from the N-terminus of the amino acid sequence shown in SEQ ID NO:

1.

3. The protein according to claim 2, which comprises the amino acid sequence shown in SEQ ID NO:3, in which the partial deletion of the amino acid sequence of TpN17 is the deletion of 11 amino acids at the N-terminus.

4. A TpN17 antigen comprising the protein according to any one of claims 1 to 3.

5. A method for measuring anti-Treponema pallidum antibodies by immunoassay using the protein according to any one of claims 1 to 3 as an antigen.

6. A method for measuring anti-Treponema pallidum antibodies by immunoassay using the TpN17 antigen according to claim 4 as an antigen.

7. The method according to claim 5, which measures anti-Treponema pallidum IgM antibody with high sensitivity.

8. The method according to claim 6, which measures anti-Treponema pallidum IgM antibody with high sensitivity.

9. The method of claim 5, which is an agglomeration method.

10. The method of claim 6, which is an agglomeration method.

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

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

13. A kit for measuring anti-Treponema pallidum antibodies by immunoassay, comprising the protein according to any one of claims 1 to 3 as an antigen for measurement.

14. A kit for measuring anti-Treponema pallidum antibodies by immunoassay, comprising the TpN17 antigen according to claim 4 as an antigen for measurement.

15. The kit according to claim 13, which is an agglutination method.

16. The kit according to claim 14, which is an agglutination method.

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