Kit for trypanosoma kirschisi antibody detection

Abstract

The invention provides a kit for detecting a trypanosoma kirschneri antibody, and relates to the technical field of biological detection. The kit for detecting the trypanosoma kirschneri antibody comprises a component A, a component B, a component C, a preserving fluid of the component A, a diluent of the component B and a diluent of the component C, the component A is a magnetic bead coated with a trypanosoma kirschneri antigen; the component B is avidin marked with a tracing marker; the component C is a trypanosoma kirschneri antigen marked with biotin; the preservation solution of the component A comprises a buffer solution containing bovine serum albumin; the diluent of the component B comprises a casein-containing buffer solution; the diluent of the component C comprises a buffer solution containing a surfactant. The kit is high in detection specificity, and can accurately detect the trypanosoma kirschisi antibody. The kit can also realize full-automatic chemiluminescence detection by virtue of a chemiluminescence immunoassay analyzer, so that the detection efficiency is improved, and manual operation errors are reduced.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, and in particular to a kit for detecting Trypanosoma cruzi antibodies. Background Technology

[0002] Chagas disease, also known as American trypanosomiasis or Chagas disease, is a zoonotic parasitic protozoan disease caused by Trypanosoma cruzi. It belongs to the extraintestinal protozoan class of infectious diseases. Trypanosoma cruzi is a single-celled protozoan that can grow in various forms in the blood and cells of mammals (including humans) and in the digestive tract of kissing bugs (also known as triangular bugs, "kissing" bugs, or "bedbugs").

[0003] Currently, the main methods for detecting Chagas antibodies on the market are enzyme-linked immunosorbent assay (ELISA) and chemiluminescence immunoassay (CLI). ELISA uses a double-antibody sandwich to capture the target analyte, which is then detected by colorimetric reaction with horseradish peroxidase (HRP) or alkaline phosphatase (ALP). While ELISA is used to detect Chagas antibodies in serum, this method suffers from a significant matrix effect, affecting detection accuracy and limiting its further application in clinical Chagas testing.

[0004] Chemiluminescence immunoassays employ a double-antigen sandwich method to capture the target analyte, and then use a luminescent label to generate a chemiluminescent signal for detection. However, in chemiluminescence methods for detecting Chagas using the double-antigen sandwich method, most of the antigens that can pair up with each other have poor specificity and are prone to non-specific binding, thus limiting the detection sensitivity.

[0005] In view of this, the present invention is hereby proposed. Summary of the Invention

[0006] The purpose of this invention is to provide a kit for detecting Trypanosoma cruzi antibodies to solve the above-mentioned technical problems.

[0007] To achieve the above objectives, the following technical solution is adopted:

[0008] In a first aspect, the present invention provides a kit for detecting Trypanosoma cruzi antibodies, comprising component A, component B, a preservation solution of component A, component C, a diluent for component B, and a diluent for component C.

[0009] Component A is a magnetic bead coated with Trypanosoma cruzi antigen;

[0010] Component B is an avidin labeled with a tracer marker;

[0011] Component C is biotin-labeled Trypanosoma cruzi antigen;

[0012] The preservation solution of component A includes a buffer solution containing 1 wt% to 3 wt% bovine serum albumin;

[0013] The diluent for component B includes a buffer solution containing 0.3 wt% to 0.5 wt% casein;

[0014] The diluent for component C includes a buffer solution containing 0.5 wt% to 1 wt% surfactant.

[0015] As a further embodiment, the magnetic beads include carboxyl magnetic beads.

[0016] As a further embodiment, the pH of the carboxylated magnetic beads coated with Trypanosoma cruzi antigen is 7.

[0017] As a further embodiment, the tracer markers include horseradish peroxidase, alkaline phosphatase, acridine ester, isoluminol and its derivatives, and ruthenium terpyridine.

[0018] As a further embodiment, the buffer solution in the dilution of component B includes PBS buffer.

[0019] Preferably, the diluent of component B further includes CaCl2 and a preservative.

[0020] As a further embodiment, the surfactant includes SDS, CHAPS, CTAB, TW20, and Triton X-100, preferably Triton X-100.

[0021] As a further embodiment, the buffer solution in the dilution of component C includes Tris buffer;

[0022] Preferably, the diluted solution of component C further includes a sealing agent and a preservative.

[0023] As a further measure, quality control materials for Trypanosoma cruzi antibodies are also included.

[0024] As a further embodiment, the steps for detecting Trypanosoma cruzi antibodies in the kit include:

[0025] Component A is mixed with the preservation solution of component A to obtain a suspension of component A;

[0026] Component B is mixed with a diluted solution of component B to obtain a solution of component B;

[0027] A solution of component C is obtained by mixing component C with a diluted solution of component C.

[0028] The suspension of component A, the solution of component C, and the sample to be tested were mixed and incubated for the first time. After solid-liquid separation, the solution of component B was added to the precipitate for the second incubation. After solid-liquid separation, the concentration of Trypanosoma cruzi antibody in the sample to be tested was detected based on the signal intensity of the tracer marker or the excitation signal intensity in the precipitate.

[0029] As a further embodiment, in the suspension of component A, the concentration of magnetic beads is 0.1–2 mg / mL, and the concentration of Trypanosoma cruzi antigen is 50–5000 ng / mL.

[0030] In the solution of component B, the concentration of avidin labeled with the tracer is 5–5000 ng / mL;

[0031] In the solution of component C, the concentration of biotin-labeled Trypanosoma cruzi antigen is 5–5000 ng / mL.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] The kit for detecting Trypanosoma cruzi antibodies provided by this invention reduces matrix effects by using a buffer containing casein; improves detection stability by using a buffer containing surfactants; and enhances positive and negative contrast and improves concordance rate through an avidin-biotin system. The kit provides high detection specificity and can accurately detect Trypanosoma cruzi antibodies. This kit can also be used with a chemiluminescence immunoassay analyzer to achieve fully automated chemiluminescence detection, thereby improving detection efficiency and reducing human error. Detailed Implementation

[0034] The embodiments and examples of the present invention will be described in detail below. However, those skilled in the art will understand that the following embodiments and examples are for illustrative purposes only and should not be considered as limiting the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0035] In a first aspect, the present invention provides a kit for detecting Trypanosoma cruzi antibodies, comprising component A, component B, a preservation solution of component A, component C, a diluent for component B, and a diluent for component C.

[0036] Component A is a magnetic bead coated with Trypanosoma cruzi antigen;

[0037] Component B is an avidin labeled with a tracer marker;

[0038] Component C is biotin-labeled Trypanosoma cruzi antigen;

[0039] The preservation solution of component A shown includes a buffer containing 1 wt% to 3 wt% bovine serum albumin, wherein the concentration of bovine serum albumin can be, for example, but not limited to, 1 wt%, 2 wt% or 3 wt%.

[0040] The diluent for component B includes a buffer containing 0.3 wt% to 0.5 wt% casein, wherein the concentration of casein can be, for example, but is not limited to, 0.3 wt%, 0.4 wt%, or 0.5 wt%.

[0041] The diluent for component C includes a buffer solution containing 0.5 wt% to 1 wt% of a surfactant, wherein the concentration of the surfactant may be, for example, but is not limited to, 0.5 wt%, 0.8 wt%, or 1 wt%.

[0042] In some alternative embodiments, the magnetic beads are a composite of Fe2O3 or Fe3O4 magnetic nanoparticles and organic polymer materials, and the magnetic beads are optionally surface-modified to have one or more active functional groups.

[0043] In some alternative embodiments, the magnetic beads have a particle size of 0.1-5 μm.

[0044] In some alternative embodiments, the magnetic beads are carboxyl magnetic beads.

[0045] In some alternative embodiments, the Trypanosoma cruzi antigen is selected from natural antigens or commercially available Chagas recombinant antigens and chimeric antigens, such as the Chagas antigen (T. cruziAg) from Zhengzhou Yimeno Biotechnology Co., Ltd.

[0046] In some optional embodiments, the method for preparing magnetic beads coated with Trypanosoma cruzi antigen includes:

[0047] Magnetic beads and activator were added to the non-magnetically stirred coating buffer for activation. After activation, Trypanosoma cruzi antigen was added for coating. After coating, magnetic bead preservation solution was added and stored at 2-8℃.

[0048] In some embodiments, the activator includes EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide), with the mass ratio of EDC and NHS to the magnetic beads being 1:0.2; the coating buffer is 0.01M PBS buffer at pH 7.35.

[0049] In some alternative embodiments, the pH of the carboxylated magnetic beads coated with Trypanosoma cruzi antigen is 7.0.

[0050] The inventors discovered that coating magnetic beads within the aforementioned pH range yielded the greatest contrast between positive and negative pH readings.

[0051] In some alternative embodiments, the Trypanosoma cruzi antigen is directly or indirectly coated with magnetic beads;

[0052] The indirect coating method for magnetic beads includes indirect coating via a system of fluorescein isothiocyanate and anti-fluorescein isothiocyanate antibody.

[0053] In some alternative embodiments, the tracer markers include, but are not limited to, horseradish peroxidase, alkaline phosphatase, acridine ester, isoluminol and its derivatives, and ruthenium terpyridine.

[0054] This invention does not impose any particular limitation on the specific type of tracer used; any type commonly used in the art is acceptable. Those skilled in the art can select the appropriate tracer based on the luminescence principle being detected. For example, when the luminescence mode is enzymatic chemiluminescence, horseradish peroxidase or alkaline phosphatase can be used as the tracer, and luminol and hydrogen peroxide or adamantane and its derivatives as the luminescent substrate. When the luminescence mode is direct chemiluminescence, non-enzymatic small-molecule organic compounds can be used as the tracer, such as acridinium ester, isoluminol ABEI, and its derivatives. When the luminescence mode is electrochemiluminescence, ruthenium tripyridine can be selected as the tracer, which undergoes an electrochemical reaction under the influence of tripropylamine and voltage, emitting visible light.

[0055] In some optional embodiments, the buffer solution in the dilution of component B includes, but is not limited to, PBS buffer.

[0056] Preferably, the diluent of component B further includes CaCl2 and a preservative, wherein CaCl2 is used to provide divalent cations, which can help maintain the spatial conformational stability of the protein by binding to the negatively charged sites (such as carboxyl groups, hydroxyl groups, etc.) in the protein molecule, and its concentration can be, for example, 0.01 wt%; the preservative can be selected from gentamicin sulfate or P300, and its concentration can be, for example, 0.1 wt%.

[0057] In some alternative embodiments, the surfactant includes, but is not limited to, SDS, CHAPS, CTAB, TW20 and Triton X-100, preferably Triton X-100.

[0058] The inventors discovered that using Triton X-100 to prepare the dilution of component C resulted in better stability of the kit.

[0059] In some alternative embodiments, the buffer solution in the dilution of component C includes, but is not limited to, Tris buffer.

[0060] Preferably, the diluent of component C further includes a blocking agent and a preservative. In some specific embodiments, the blocking agent includes, but is not limited to, bovine serum albumin (BSA), casein, calf serum, etc., preferably 1 wt% bovine serum albumin (BSA); the preservative can be gentamicin sulfate or P300, and its concentration can be, for example, 0.1 wt%.

[0061] In some optional implementations, a quality control of Trypanosoma cruzi antibodies is also included.

[0062] In some alternative implementations, the quality control samples include low-point quality control samples and high-point quality control samples.

[0063] In some optional implementations, the step of detecting Trypanosoma cruzi antibodies using the kit includes:

[0064] Component A is mixed with the preservation solution of component A to obtain a suspension of component A;

[0065] Component B is mixed with a diluted solution of component B to obtain a solution of component B;

[0066] A solution of component C is obtained by mixing component C with a diluted solution of component C.

[0067] The suspension of component A, the solution of component C, and the sample to be tested were mixed and incubated for the first time. After solid-liquid separation, the solution of component B was added to the precipitate for the second incubation. After solid-liquid separation, the concentration of Trypanosoma cruzi antibody in the sample to be tested was detected based on the signal intensity of the tracer marker or the excitation signal intensity in the precipitate.

[0068] In some optional embodiments, the concentration of magnetic beads in the suspension of component A may be, for example, but not limited to, 0.1 mg / mL, 1 mg / mL or 2 mg / mL, and the concentration of Trypanosoma cruzi antigen may be, for example, but not limited to, 50 ng / mL, 100 ng / mL, 1000 ng / mL or 5000 ng / mL.

[0069] In the solution of component B, the concentration of avidin labeled with the tracer can be, for example, but not limited to, 5 ng / mL, 100 ng / mL, 1000 ng / mL or 5000 ng / mL;

[0070] In the solution of component C, the concentration of biotin-labeled Trypanosoma cruzi antigen can be, for example, but not limited to, 5 ng / mL, 50 ng / mL, 100 ng / mL, 1000 ng / mL or 5000 ng / mL.

[0071] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0072] In the following examples, the Trypanosoma cruzi antigen is the chagas antigen (T. cruzi Ag) from Zhengzhou Yimeno Biotechnology Co., Ltd.; the tracer is horseradish peroxidase, with luminol and hydrogen peroxide as luminescent substrates; and the magnetic beads are carboxyl magnetic beads.

[0073] Example 1

[0074] Experimental group 1:

[0075] A kit comprising component A, a preservation solution for component A, component B, and a diluent for component B;

[0076] Component A consists of magnetic beads coated with Trypanosoma cruzi antigen;

[0077] The preservation solution for component A is PBS buffer;

[0078] Component B is Trypanosoma cruzi antigen labeled with horseradish peroxidase;

[0079] The diluent for component B was a PBS buffer containing 0.01 wt% CaCl2 and 0.1 wt% preservatives (gentamicin sulfate and P300).

[0080] Experimental group 2:

[0081] A kit comprising component A, a preservation solution for component A, component B, and a diluent for component B;

[0082] Component A consists of magnetic beads coated with Trypanosoma cruzi antigen;

[0083] The preservation solution for component A is PBS buffer;

[0084] Component B is an enzyme-labeled secondary antibody containing horseradish peroxidase;

[0085] The diluent for component B was a PBS buffer containing 0.01 wt% CaCl2 and 0.1 wt% preservatives (gentamicin sulfate and P300).

[0086] The above-mentioned kits were used to test the samples (P1-P44 were samples that tested positive by Roche Diagnostics GmbH, Abbott Ireland Diagnostics Division, and Wiener Laboratorios S.AIC; clinical samples 3-4 were 200 clinically false-positive samples tested using the indirect method). The testing steps for experimental groups 1 and 2 are as follows:

[0087] The magnetic beads coated with Trypanosoma cruzi antigen were diluted 1:10 with the component A preservation solution to obtain a suspension of component A.

[0088] Component B is mixed with a diluted solution of component B at a ratio of 1:3000 to obtain a solution of component B.

[0089] The suspension of component A was mixed with the sample to be tested for the first incubation. After magnetic separation, the solution of component B was added to the precipitate for the second incubation. After magnetic separation again, the excitation substrate was added to the precipitate, and the light signal intensity was detected (using an AutoLumo A2000 Plus fully automated chemiluminescence analyzer (Antu Experimental Instruments (Zhengzhou) Co., Ltd.)). The light signal intensity of the low-point calibrator and high-point calibrator of total Trypanosoma cruzi antibody was measured using the same method, and a standard curve between the concentration of total Trypanosoma cruzi antibody and the light intensity was obtained. The light signal intensity of the sample to be tested was compared with the standard curve to obtain the concentration of total Trypanosoma cruzi antibody in the sample to be tested.

[0090] The test results are shown in Table 1.

[0091] Table 1

[0092]

[0093]

[0094] The results above show that the sandwich method (experimental group 1) has better specificity than the indirect method when using the conventional indirect method (experimental group 2).

[0095] Example 2

[0096] Based on experimental group 1, the inventors optimized the pH of the antigen-coated magnetic beads:

[0097] Experimental Group 3: Magnetic beads and cross-linking agent were added to the non-magnetically stirred coating buffer for activation. After activation, Trypanosoma cruzi antigen was added for coating. PBS coating buffer with pH 5.0 was used as the coating buffer. After coating, blocking solution was added for blocking. After blocking, magnetic microparticle preservation solution was added and stored at 2-8℃.

[0098] Experimental Group 4: The magnetic bead coating process was the same as that of Experimental Group 3, but the coating buffer used was PBS coating buffer with pH 7.0.

[0099] Experimental Group 5: The magnetic bead coating process was the same as that of Experimental Group 3, but the coating buffer used was PBS coating buffer with pH 9.0.

[0100] The detection results of magnetic beads with different pH coatings are shown in Table 2.

[0101] Table 2

[0102] experimental group Experimental group 3 Experimental group 4 Experimental group 5 CHAGAS-P2 14.35 15.51 12.05 CHAGAS-P4 2.97 3.07 3.02 CHAGAS-P5 14.4 15.19 14.32 CHAGAS-P6 18.5 17.76 16.38 CHAGAS-P8 14.94 16.13 16.41 Clinical 8 0.35 0.17 0.37 Clinical 9 0.37 0.27 1.36 Clinical 10 0.37 0.23 0.84 Clinical 11 0.46 0.26 0.27 Clinical 12 0.67 0.14 1.47 P / N 29.35 63.23 14.43

[0103] The results above show that the contrast between positive and negative values ​​was greatest when the pH of the 4-coated experimental group was around 7.

[0104] Example 3

[0105] Based on the above results, the inventors further adopted the avidin and biotin system, setting component B as avidin labeled with horseradish peroxidase; and component C as Trypanosoma cruzi antigen labeled with biotin.

[0106] Experimental group 6:

[0107] A kit comprising component A, a preservation solution for component A, component B, and a diluent for component B;

[0108] Component A consists of magnetic beads coated with Trypanosoma cruzi antigen;

[0109] The preservation solution for component A is PBS buffer;

[0110] Component B is Trypanosoma cruzi antigen labeled with horseradish peroxidase;

[0111] The diluent for component B was a PBS buffer containing 0.01 wt% CaCl2 and 0.1 wt% preservatives (gentamicin sulfate and P300).

[0112] Experimental group 7:

[0113] A kit includes component A, a preservation solution for component A, component B, a diluent for component B, component C, and a diluent for component C.

[0114] Component A consists of magnetic beads coated with Trypanosoma cruzi antigen;

[0115] The preservation solution for component A is PBS buffer;

[0116] Component B is an avidin enzyme labeled with horseradish peroxidase;

[0117] The diluent for component B was a PBS buffer containing 0.01 wt% CaCl2 and 0.1 wt% preservatives (gentamicin sulfate and P300).

[0118] Component C is biotin-labeled Trypanosoma cruzi antigen.

[0119] The diluent for component C was Tris buffer containing 1 wt% bovine serum albumin (BSA) and 0.1 wt% preservatives (gentamicin sulfate and P300).

[0120] The above-mentioned kit was used to test the samples (P1-P44 were samples that tested positive by Roche Diagnostics GmbH, Abbott Ireland Diagnostics Division, and Wiener Laboratorios S.AIC; clinical samples 3-4 were 200 false positive samples detected by indirect method). The testing steps are as follows:

[0121] The magnetic beads coated with Trypanosoma cruzi antigen were diluted to a final volume using the component A preservation solution to obtain a suspension of component A (the concentration of the magnetic beads in the solution was 0.5 mg / mL, and the concentration of Trypanosoma cruzi antigen was 3000 ng / mL).

[0122] Component B is mixed with a dilution of component B to obtain a solution of component B (the concentration of component B in the solution is 2000 ng / mL);

[0123] Component C was mixed with a dilution of component C to obtain a solution of component C (the concentration of component C in the solution was 2000 ng / mL);

[0124] The suspension of component A, the solution of component C, and the sample to be tested were mixed and incubated for the first time. After magnetic separation, the solution of component B was added to the precipitate for the second incubation. After magnetic separation again, the excitation substrate was added to the precipitate, and the light signal intensity was detected (using an AutoLumo A2000 Plus fully automated chemiluminescence analyzer (Antu Experimental Instruments (Zhengzhou) Co., Ltd.)). The light signal intensity of the low-point calibrator and high-point calibrator of total Trypanosoma cruzi antibody was measured using the same method, and a standard curve between the concentration of total Trypanosoma cruzi antibody and the light intensity was obtained. The light signal intensity of the sample to be tested was compared with the standard curve to obtain the concentration of total Trypanosoma cruzi antibody in the sample to be tested.

[0125] The test results are shown in Table 3.

[0126] Table 3

[0127]

[0128]

[0129] The results above show that the avidin sandwich method enhances the contrast between positive and negative results and improves the positive concordance rate.

[0130] Example 4

[0131] The inventors optimized the concentration of the blocking agent added to the preservation solution of component A, adding 1 wt%, 2 wt%, and 3 wt% bovine serum albumin (BSA) respectively. The experimental conditions were set as follows:

[0132] Experimental group 8:

[0133] A kit includes component A, a preservation solution for component A, component B, a diluent for component B, component C, and a diluent for component C; the difference from experimental group 7 is that the preservation solution for component A also contains 1 wt% bovine serum albumin (BSA).

[0134] Experimental group 9:

[0135] A kit comprising component A, a preservation solution for component A, component B, a diluent for component B, component C, and a diluent for component C; the difference from experimental group 7 is that the preservation solution for component A also contains 2 wt% bovine serum albumin (BSA).

[0136] Experimental group 10:

[0137] A kit includes component A, a preservation solution for component A, component B, a diluent for component B, component C, and a diluent for component C; the difference from experimental group 7 is that the preservation solution for component A also contains 3 wt% bovine serum albumin (BSA).

[0138] The reagent kits under different experimental conditions were tested according to the detection method in Example 3, and the experimental results are as follows:

[0139] Table 4

[0140] experimental group Experimental group 8 Experimental group 9 Experimental group 10 CHAGAS-P2 401.92 378.39 122.44 CHAGAS-P4 3.71 2.99 3.09 CHAGAS-P5 421.73 410.40 116.76 CHAGAS-P6 192.38 186.40 70.37 CHAGAS-P8 98.75 91.62 36.59 Clinical 8 0.98 0.35 0.37 Clinical 9 0.27 0.38 1.39 Clinical 10 0.38 0.38 0.87 Clinical 11 0.26 0.47 0.28 Clinical 12 0.35 0.68 1.47 P / N 626.74 589.14 102.58

[0141] The results above show that adding bovine serum albumin (BSA) to the preservation solution of component A improves the sample to a certain extent, with the largest contrast between positive and negative samples when 1 wt% BSA is added.

[0142] Example 5

[0143] During the real-time testing of Example 3, it was found that after 6 months of testing with the kit in Experimental Group 1, the values ​​of negative samples increased, showing a significant matrix effect. To address this issue, the inventors further optimized the diluent for component B based on Experimental Group 8, by adding 0.3 wt% bovine serum albumin (BSA), casein, or fetal bovine serum to the diluent for component B. The experimental conditions were set as follows:

[0144] Experimental group 11:

[0145] A kit includes component A, a preservation solution for component A, component B, a diluent for component B, component C, and a diluent for component C; the only difference from kit 8 in experimental group is that the diluent for component B also contains 0.3 wt% bovine serum albumin (BSA).

[0146] Experimental group 12:

[0147] A kit includes component A, a preservation solution for component A, component B, a diluent for component B, component C, and a diluent for component C; the only difference from kit 8 of experimental group is that the diluent for component B also contains 0.3 wt% casein.

[0148] Experimental group 13:

[0149] A kit includes component A, a preservation solution for component A, component B, a diluent for component B, component C, and a diluent for component C; the only difference from kit 8 in experimental group is that the diluent for component B also contains 0.3 wt% fetal bovine serum.

[0150] After 6 months, the reagent kits for experimental groups 11-13 were tested according to the detection method in Example 3. The test results are shown in Table 5.

[0151] Table 5

[0152]

[0153]

[0154] The results above show that the reagent kit in experimental group 12 can solve the above problems.

[0155] Example 5

[0156] Further optimization of casein concentration was achieved by setting up different experimental groups:

[0157] Experimental group 14: The reagent kit is the same as that for experimental group 12.

[0158] Experimental Group 15: A kit comprising component A, a preservation solution for component A, component B, a diluent for component B, component C, and a diluent for component C; the only difference from the kit in Experimental Group 12 is that the diluent for component B contains 0.4 wt% casein.

[0159] Experimental Group 16: A kit comprising component A, a preservation solution for component A, component B, a diluent for component B, component C, and a diluent for component C; the only difference from the kit in Experimental Group 12 is that the diluent for component B contains 0.5 wt% casein.

[0160] The above-mentioned reagent kit was tested according to the detection method of Example 3, and the results are shown in Table 6.

[0161] Table 6

[0162]

[0163]

[0164] The results showed that a casein concentration of 0.3 wt% was suitable.

[0165] Example 6

[0166] Furthermore, during the real-time testing, it was found that the positive sample values ​​increased after 6 months of use, which is an abnormal phenomenon. The inventors discovered that adding a surfactant to the diluent of component C can solve this problem. Different experimental groups were set up by adding 1 wt% of different types of surfactants to the diluent of component C:

[0167] Experimental group 17:

[0168] A kit includes component A, a preservation solution of component A, component B, a diluent for component B, component C, and a diluent for component C; the only difference from the kit in experimental group 14 is that the diluent for component C also contains 1 wt% of the anionic surfactant SDS.

[0169] Experimental group 18:

[0170] A kit includes component A, a preservation solution of component A, component B, a diluent for component B, component C, and a diluent for component C; the only difference from the kit in experimental group 14 is that the diluent for component C also contains 1 wt% of the amphoteric surfactant CHAPS.

[0171] Experimental group 19:

[0172] A kit includes component A, a preservation solution of component A, component B, a diluent for component B, component C, and a diluent for component C; the only difference from the kit in experimental group 14 is that the diluent for component C also contains 1 wt% of the amphoteric surfactant CTAB.

[0173] Experimental group 20:

[0174] A kit includes component A, a preservation solution of component A, component B, a diluent for component B, component C, and a diluent for component C; the only difference from the kit in experimental group 14 is that the diluent for component C also contains 1 wt% of the cationic surfactant TW20.

[0175] Experimental group 21:

[0176] A kit includes component A, a preservation solution of component A, component B, a diluent for component B, component C, and a diluent for component C; the only difference from kit 14 in experimental group is that the diluent for component C also contains 1 wt% of the nonionic surfactant Triton X-100.

[0177] The detection results of the above reagent kit according to the detection method of Example 3 are shown in Table 7.

[0178] Table 7

[0179]

[0180]

[0181] The results above show that the price reduction of reagent kit 21 in experimental group 21 met the requirements after 6 months of real-time testing.

[0182] Example 7

[0183] The concentration of the surfactant used in experimental group 21 was optimized, and the experimental group was set up as follows:

[0184] Experimental group 22:

[0185] A kit includes component A, a preservation solution of component A, component B, a diluent for component B, component C, and a diluent for component C; the only difference from the kit in experimental group 21 is that the concentration of Triton X-100 in the diluent for component C is 0.5 wt%.

[0186] Experimental group 23:

[0187] A kit includes component A, a preservation solution for component A, component B, a diluent for component B, component C, and a diluent for component C; the only difference from the kit in experimental group 21 is that the diluent for component C has a Triton X-100 concentration of 0.8 wt%.

[0188] Experimental group 24: The reagent kit is the same as that for experimental group 21.

[0189] The results of the above reagent kits tested according to the detection method in Example 3 are shown in Table 8.

[0190] Table 8

[0191]

[0192]

[0193] In summary, based on the above research results, a kit according to an embodiment of the present invention is proposed, comprising component A, a preservation solution for component A, component B, component C, a diluent for component B, and a diluent for component C;

[0194] Component A consists of magnetic beads coated with Trypanosoma cruzi antigen;

[0195] Component B is an avidin labeled with horseradish peroxidase;

[0196] Component C is biotin-labeled Trypanosoma cruzi antigen;

[0197] The preservation solution for component A is PBS buffer containing 1 wt% bovine serum albumin.

[0198] The diluent for component B was a PBS buffer containing 0.01 wt% CaCl2, 0.1 wt% preservatives (gentamicin sulfate and P300), and 0.3 wt% casein.

[0199] The diluent for component C was Tris buffer containing 1 wt% bovine serum albumin (BSA) as a blocking agent, 1 wt% Triton X-100, and 0.1 wt% preservatives (gentamicin sulfate and P300).

[0200] The Trypanosoma cruzi antigen was coated onto magnetic beads at pH 7.

[0201] The method for detecting the sample using this kit includes the following steps:

[0202] The magnetic beads coated with Trypanosoma cruzi antigen were diluted to a final volume using the component A preservation solution to obtain a suspension of component A (the concentration of the magnetic beads in the solution was 0.5 mg / mL, and the concentration of Trypanosoma cruzi antigen was 3000 ng / mL).

[0203] Component B is mixed with a dilution of component B to obtain a solution of component B (the concentration of component B in the solution is 2000 ng / mL);

[0204] Component C was mixed with a dilution of component C to obtain a solution of component C (the concentration of component C in the solution was 2000 ng / mL);

[0205] The suspension of component A, the solution of component C, and the sample to be tested were mixed and incubated for the first time. After magnetic separation, the solution of component B was added to the precipitate for the second incubation. After magnetic separation again, the excitation substrate was added to the precipitate, and the light signal intensity was detected (using an AutoLumo A2000 Plus fully automated chemiluminescence analyzer (Antu Experimental Instruments (Zhengzhou) Co., Ltd.)). The light signal intensity of the low-point calibrator and high-point calibrator of total Trypanosoma cruzi antibody was measured using the same method, and a standard curve between the concentration of total Trypanosoma cruzi antibody and the light intensity was obtained. The light signal intensity of the sample to be tested was compared with the standard curve to obtain the concentration of total Trypanosoma cruzi antibody in the sample to be tested.

[0206] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A kit for detecting Trypanosoma cruzi antibodies, characterized in that, Includes component A, component B, component C, a preservation solution for component A, a diluent for component B, and a diluent for component C; Component A is a magnetic bead coated with Trypanosoma cruzi antigen; Component B is an avidin labeled with a tracer marker; Component C is biotin-labeled Trypanosoma cruzi antigen; The preservation solution of component A includes a buffer solution containing 1 wt% to 3 wt% bovine serum albumin; The diluent for component B includes a buffer solution containing 0.3 wt% to 0.5 wt% casein; The diluent for component C includes a buffer solution containing 0.5 wt% to 1 wt% surfactant.

2. The reagent kit according to claim 1, characterized in that, The magnetic beads include carboxyl magnetic beads.

3. The reagent kit according to claim 2, characterized in that, The pH of the carboxylated magnetic beads coated with Trypanosoma cruzi antigen is 7.

0.

4. The reagent kit according to claim 1, characterized in that, The tracer markers include horseradish peroxidase, alkaline phosphatase, acridine ester, isoluminol and its derivatives, and ruthenium terpyridine.

5. The reagent kit according to claim 1, characterized in that, The dilution solution of component B includes PBS buffer. Preferably, the diluent of component B further includes CaCl2 and a preservative.

6. The reagent kit according to claim 1, characterized in that, The surfactants include SDS, CHAPS, CTAB, TW20 and Triton X-100, preferably Triton X-100.

7. The kit according to claim 1, characterized in that, The dilution solution of component C includes a Tris buffer solution. Preferably, the diluted solution of component C further includes a sealing agent and a preservative.

8. The reagent kit according to claim 1, characterized in that, It also includes quality control products for Trypanosoma cruzi antibodies.

9. The reagent kit according to any one of claims 1-8, characterized in that, The steps for detecting Trypanosoma cruzi antibodies using the kit include: Component A is mixed with the preservation solution of component A to obtain a suspension of component A; Component B is mixed with a diluted solution of component B to obtain a solution of component B; A solution of component C is obtained by mixing component C with a diluted solution of component C. The suspension of component A, the solution of component C, and the sample to be tested were mixed and incubated for the first time. After solid-liquid separation, the solution of component B was added to the precipitate for the second incubation. After solid-liquid separation, the concentration of Trypanosoma cruzi antibody in the sample to be tested was detected based on the signal intensity of the tracer marker or the excitation signal intensity in the precipitate.

10. The reagent kit according to claim 9, characterized in that, In the suspension of component A, the concentration of magnetic beads is 0.1–2 mg / mL, and the concentration of Trypanosoma cruzi antigen is 50–5000 ng / mL. In the solution of component B, the concentration of avidin labeled with the tracer is 5–5000 ng / mL; In the solution of component C, the concentration of biotin-labeled Trypanosoma cruzi antigen is 5–5000 ng / mL.

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