WO2016127301A1 - rT3化学发光免疫检测试剂盒及其检测方法和应用 - Google Patents

rT3化学发光免疫检测试剂盒及其检测方法和应用 Download PDF

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WO2016127301A1
WO2016127301A1 PCT/CN2015/072616 CN2015072616W WO2016127301A1 WO 2016127301 A1 WO2016127301 A1 WO 2016127301A1 CN 2015072616 W CN2015072616 W CN 2015072616W WO 2016127301 A1 WO2016127301 A1 WO 2016127301A1
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triiodothyronine
antibody
labeled
antigen
magnetic
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PCT/CN2015/072616
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English (en)
French (fr)
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饶微
徐红
梁咏秋
罗凯
杨雅丽
李婷华
袁锦云
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深圳市新产业生物医学工程股份有限公司
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G01N33/78—Thyroid gland hormones, e.g. T3, T4, TBH, TBG or their receptors

Definitions

  • the invention relates to the technical field of in vitro diagnostic detection, in particular to a rT3 (anti-triiodothyronine) chemiluminescence immunoassay kit and a detection method and application thereof.
  • rT3 anti-triiodothyronine
  • Anti-triiodothyronine also known as 3,3',5'-triiodothyronine, is a thyroid hormone present in normal human serum, which is T4 (thyroxine) in the liver and kidney.
  • T4 thyroxine
  • 5-iodide is used to remove an iodine from the tyrosine ring.
  • rT3 degrades faster in the body and its half-life is 30-60 minutes. It has a very low content in the blood and a low biological activity, but there are significant differences in serum levels under different physiological and pathological conditions. Therefore, determination of serum rT3 levels is still clinically significant.
  • cord blood and amniotic fluid have higher levels of rT3, which can reflect fetal thyroid function.
  • rT3 can reflect fetal thyroid function.
  • serum rT3 concentration there is no difference in serum rT3 concentration between men and women. From children to 40 years old, it tends to decline with age, and after 40 years old, it increases with age.
  • rT3 hyperthyroidism or hypothyroidism: rT3 is elevated in hyperthyroidism, and is more sensitive than T3, T4. When treated with antithyroid drugs, T3 decreased faster, while rT3 decreased slowly. When both T3 and rT3 were normal, it indicated that the drug was appropriate; if rT3 and T4 were lower than normal, it indicated overdose. The reduction of rT3 is very important for the judgment of mild or subclinical hypothyroidism.
  • T3 decline shows a decrease in the T3 / rT3 ratio.
  • High rT3 phenomenon caused by drugs The use of drugs such as amiodarone, glucocorticoids, propylthiouracil, and dextroamine can cause high rT3 phenomenon, so rT3 can also be used to monitor the use of the above drugs.
  • the methods for determining serum anti-triiodothyronine (rT3) in clinical practice include radioactive immunoassay and enzyme-linked immunosorbent assay, while radioimmunoassay and enzyme-linked immunosorbent assay have many shortcomings.
  • the traditional radioactive immunoassay or enzyme-linked immunosorbent assay has a long detection time, and mainly relies on a series of cumbersome operations such as manual manual loading, which is inefficient and easily leads to large errors in the experimental results; since the enzymatic reaction is not thorough enough and is susceptible to external
  • the influence of interference factors, such as temperature, time and material concentration is therefore low in specificity, poor in sensitivity, and narrow in detection range.
  • the radioactive immunization method also has radioactive contamination, short half-life of the marker, and radioactive damage to the operator.
  • the object of the present invention is to overcome the defects of the prior art and provide an rT3 chemiluminescence immunoassay kit, which is a chemiluminescence assay with high sensitivity and a highly specific immune response. Combined, it has the advantages of high sensitivity, good specificity and wide detection range.
  • the present invention adopts the following technical solutions:
  • An rT3 chemiluminescence immunoassay kit comprising the following components:
  • a marker system comprising an anti-triiodothyronine antigen or an anti-triiodothyronine antibody directly or indirectly linked to a labeled tracer;
  • Magnetic microsphere system including the corresponding direct or indirect linkage of anti-triiodothyronine Magnetic microspheres of antibodies or anti-triiodothyronine antigens.
  • the magnetic microsphere system comprises magnetic microspheres directly or indirectly linked to the anti-triiodothyronine antibody
  • the magnetic microsphere system comprises magnetic microspheres that are directly or indirectly linked to the anti-triiodothyronine antigen.
  • direct linkage is a direct binding linkage
  • indirect linkage is a bridge capable of binding to each other by biotin and streptavidin, or fluorescein isothiocyanate and anti-isothiocyanate antibody. Connected in an indirect combination.
  • the invention provides an rT3 chemiluminescence immunoassay kit, which utilizes a chemiluminescence immunoassay method for detecting rT3, has the advantages of high sensitivity, good specificity and wide detection range, and the kit in the invention is magnetic micro As a solid phase system in the detection, the ball can be uniformly suspended in the reaction system when reacting with the analyte, and can be better combined with the analyte, and can be easily separated by applying a magnetic field during separation. Easy to operate features. More importantly, the test kit can be used in conjunction with a conventional chemiluminescence analyzer to achieve fully automated detection, avoiding experimental errors that may be caused by human factors, and improving analysis efficiency.
  • the magnetic microspheres indirectly linked to an anti-triiodothyronine antibody are biotin-labeled anti-anti-triiodothyronine antibodies, and The streptavidin-coated magnetic microspheres; or the magnetic microspheres indirectly linked to the anti-triiodothyronine antibody are anti-anti-triiodothyronine antibodies labeled with fluorescein isothiocyanate, And magnetic anti-isothiocyanate antibody coated magnetic microspheres.
  • the magnetic microspheres indirectly linked to the anti-triiodothyronine antigen are fluorescein isothiocyanate-labeled anti-triiodothyronine antigen , And magnetic anti-isothiocyanate antibody-coated magnetic microspheres; or the magnetic microspheres indirectly linked to the anti-triiodothyronine antigen by biotin-labeled anti-triiodothyronine antigen, and a chain Mycelium-coated magnetic microspheres.
  • the indirect linked marker tracer anti-triiodothyronine antigen is biotinylated anti-triiodothyronine antigen, and streptavidin An avidin-labeled marker tracer composition; or the indirect linked marker tracer anti-triiodothyronine antigen is fluorescein isothiocyanate-labeled anti-triiodothyronine antigen, and is resistant to The fluorescein thiocyanate antibody-labeled marker tracer composition.
  • the indirect linked marker tracer anti-triiodothyronine antibody is labeled with biotin-labeled anti-triiodothyronine antibody, and a streptavidin-labeled labeled tracer composition; or the indirect linked labeled tracer anti-triiodothyronine antibody is labeled with a counter-triiodothyronine antigen labeled with fluorescein isothiocyanate, And a marker tracer composed of an anti-isothiocyanate antibody labeled.
  • the above anti-isothiocyanate antibody may be a monoclonal antibody or a polyclonal antibody.
  • the above scheme enriches the marker system and the magnetic microsphere system used for the detection, and can be flexibly selected according to different needs.
  • the anti-triiodothyronine antigen or anti-triiodothyronine antibody
  • the anti-triiodothyronine ammonia can also be connected by the indirect bridging method described above.
  • Acid antigen or anti-triiodothyronine antibody
  • magnetic microspheres can be directly linked to the magnetic microspheres in the magnetic microsphere system.
  • the labeled tracer can be directly linked to the anti-triiodothyronine antibody (or anti-triiodothyronine antigen) in the marker system, and the labeled tracer can also be linked by the indirect bridging method described above.
  • the connection manner (direct connection or indirect connection) adopted in the above magnetic microsphere system and the marker system does not affect or limit each other, and the direct connection method can be simultaneously used in the magnetic microsphere system and the marker system.
  • the indirect connection method is adopted, and the magnetic microsphere system can be directly connected, the marker system adopts indirect connection, or the magnetic microsphere system uses indirect connection, and the marker system adopts direct connection. It can be understood that the above indirect bridging connection manner can also be indirectly connected by using other indirect labeling systems.
  • the above-mentioned labeled tracers include the following: 1. Markers capable of direct luminescence used in chemiluminescence immunoassay, such as luminol and its derivatives, isoluminol or its derivatives, acridinium ester, etc.; A chemiluminescent enzyme immunoassay uses a label that can illuminate with a corresponding substrate, such as alkaline phosphatase or peroxidase.
  • the labeled tracer is a luminescent label selected from the group consisting of: adamantane, luminol and derivatives thereof, isoluminol and its derivatives, acridinium ester.
  • adamantane luminol and derivatives thereof
  • isoluminol and its derivatives acridinium ester.
  • the substrate complexed with the above luminescent label comprises H 2 O 2 -microperoxidase, H 2 O 2 - catalase, H 2 O 2 - lactoperoxidase, H 2 O 2 - views hemin, H 2 O 2 - hemin, hypochlorite -CoCl 2, persulfate, potassium At least one of sodium periodate, H 2 O 2 -K 3 Fe(CN) 6 , xanthine-hypoxanthine oxidase
  • the above luminescent label refers to a compound that participates in energy transfer in the luminescence reaction and finally releases energy in the form of emitted photons, which can be catalyzed by a catalyst and oxidized by an oxidant to form an excited state intermediate when the excited state When the intermediate returns to a stable ground state, photons (hM) are simultaneously emitted.
  • the labeled tracer is a chemiluminescent catalyst selected from the group consisting of: alkaline phosphatase, peroxidase.
  • the luminescence is quantitatively detected in conjunction with a corresponding chemiluminescent substrate, including NaOH and H 2 O 2 , also including adamantane, luminol and its derivatives, isoluminol or its At least one of the derivatives is preferably N-(4-aminobutyl)-N-ethylisoluminol (ABEI).
  • the magnetic microspheres are directly or indirectly linked to one or more anti-triiodothyronine antibodies. And, understandably, when the magnetic microspheres are directly connected or indirectly When a plurality of anti-anti-triiodothyronine antibodies are linked, the binding sites of the plurality of (ie, at least two) anti-triiodothyronine antibodies and the anti-triiodothyronine antigen are different from each other.
  • the anti-triiodothyronine antibody is a monoclonal antibody and/or a polyclonal antibody.
  • Magnetic microspheres suitable for use in the present invention are also referred to as magnetic beads or magnetic spheres and may be magnetic microspheres commonly used in the art.
  • the magnetic ball used in the present invention is a composite of nano-scale Fe 2 O 3 or Fe 3 O 4 magnetic particles and an organic polymer material to form a micron-scale having superparamagnetic and extremely large protein adsorption capacity.
  • the solid phase microspheres have the property of being magnetized rapidly under the action of an external magnetic field and having zero remanence after the magnetic field is withdrawn.
  • the type of the organic polymer material is not particularly limited and may be selected as needed.
  • the magnetic microspheres used in the present invention should be capable of satisfying a diameter of 0.1 to 5 ⁇ m, and the magnetic microspheres may also have various active functional groups by surface modification, including but not limited to -OH, -COOH, -NH 2 .
  • the magnetic microspheres are a composite of Fe 2 O 3 or Fe 3 O 4 magnetic nanoparticles and an organic polymer material, and have a particle diameter of 0.1 to 5 ⁇ m, and the magnetic microspheres Optionally carrying one or more reactive functional groups by surface modification.
  • test kit further comprises the following components:
  • Calibrator solution a calibrator solution containing an anti-triiodothyronine antigen concentration of 0.2-6 ng/mL and a calibrator solution having a concentration of 8-20 ng/mL. After investigating a large number of real sample anti-triiodothyronine antigen concentration ranges, the inventors combined the characteristics of the test kit to set the concentration of the calibrator within the above range, which has a better detection effect.
  • the invention also discloses a preparation method of the above rT3 chemiluminescence immunoassay kit, comprising the following steps:
  • Labeling of antibody or antigen Put the antigen or antibody into a dialysis bag, place it in dialysate for dialysis, and then add the label to the dialyzed solution, and then purify after the reaction;
  • Coating of magnetic microspheres magnetic microspheres are suspended in a buffer, and then a coating is added to form a reaction system, which is then purified after the reaction.
  • the antigen is an anti-triiodothyronine antigen
  • the antibody is an anti-triiodothyronine antibody.
  • the above antibody may be either a monoclonal antibody or a polyclonal antibody.
  • 1-cyclohexyl-2-morpholineethylcarbodiimide p-toluenesulfonate (CMC) may also be coated and attached at a concentration of preferably 10 mg/mL. Can increase the coating effect.
  • Purification of the labeled antibody or antigen is preferably carried out using a G-25 gel column.
  • the activated ester of the labeled tracer may be ABEI-hemisuccinimide N-Hydroxysuccinimide or the like, and only needs to be capable of binding to an antigen or an antibody.
  • the label is: an activated ester of a labeled tracer, fluorescein isothiocyanate or biotin;
  • the coating is: an anti-triiodothyronine antibody, anti-three Iodine thyronine antigen, anti-isothiocyanate fluorescein antibody or streptomycin avidin.
  • the label in the label of the antibody or antigen, is added in an amount of 200-400 ⁇ g of label, preferably 300 ⁇ g of label per 100 ⁇ g of antigen; 200-400 ⁇ g of label is added per 1 mg of antibody, preferably 300 ⁇ g of label; reaction temperature of 35-40 ° C, preferably 37 ° C, reaction time of 1-3 hours, preferably 2 hours; in the coating of the magnetic microspheres, the amount of the coating is added per 20 mg of magnetic
  • the microspheres are added with 100-140 ⁇ g of a coating, preferably 120 ⁇ g of the coating, at a reaction temperature of 35-40 ° C, preferably 37 ° C, for a reaction time of 12-36 hours, preferably 24 hours. With the above parameter ratio, it is possible to have an optimum labeling rate and luminous efficiency.
  • the purified labeled antigen or antibody in the label of the antibody or antigen, is diluted 150-250 times, preferably 200-fold, to be used; in the label of the antibody or antigen, After purification, the coated magnetic microspheres are diluted to 0.5-1.5 mg/mL, preferably 1 mg/mL, to be used; the volume ratio of the above labeled antigen or antibody solution to the coated magnetic microsphere solution is 60- 100:20, preferably 80:20. When used in combination with the above ratio, a better detection effect can be obtained.
  • the dialysate in the label of the antibody or antigen, is preferably 0.1 mol/L of pH 9.5 carbonate buffer; in the coating of magnetic microspheres, the buffer is preferably pH 3.6 acetic acid. Buffer.
  • the dialysate and buffer described above are capable of promoting better labeling and coating reactions.
  • the invention also discloses an rT3 chemiluminescence immunoassay method, which adopts the above rT3 chemiluminescence immunoassay kit, comprising the following steps:
  • reaction mixing the sample to be tested with the marker system and the magnetic microsphere system, and incubating to obtain a reaction product;
  • the detection method of the invention adopts the chemiluminescence immunocompetition method for detection, and has the advantages of high sensitivity, good specificity and wide detection range.
  • the above rT3 chemiluminescence immunoassay method uses a chemiluminescence analyzer to detect anti-triiodothyronine. With operating specifications, no one has the advantage of introducing errors and full automation.
  • the invention also discloses an application of the above rT3 chemiluminescence immunoassay kit in a chemiluminescence analyzer.
  • the kit is applied to a chemiluminescence platform with operational specifications, and no one has the advantage of introducing errors and fully automating.
  • the present invention has the following beneficial effects:
  • the rT3 chemiluminescence immunoassay kit of the invention combines the high sensitivity chemiluminescence measurement technology with the high specific immunological reaction, and has high sensitivity, specificity and detection range.
  • the kit of the present invention uses magnetic microspheres as a solid phase system in the detection, which can be uniformly suspended in the reaction system when reacting with the analyte, and can be better combined with the analyte and can be When the magnetic field is separated, the magnetic field can be easily separated, and the operation is convenient.
  • the test kit can be used in conjunction with a conventional chemiluminescence analyzer to achieve fully automated detection, avoiding experimental errors that may be caused by human factors, and improving analysis efficiency.
  • the detection kit combines the possible concentration of the analyte in the real sample, and the characteristics of the label system and the magnetic microsphere system in the kit, and the components are optimized, and the optimal detection range is obtained.
  • the rT3 chemiluminescence immunoassay method of the invention has the advantages of high sensitivity, good specificity and wide detection range. Moreover, the detection kit can be used together with the chemiluminescence analyzer in the conventional technology to realize fully automated detection, avoiding experimental errors that may be caused by human factors, and improving the analysis efficiency.
  • Example 1 is a comparison diagram of detection results of a detection kit prepared in Example 1 and a reference radioimmunoassay kit in an experimental example of the present invention
  • Figure 3 is a comparison diagram of detection results of the detection kits prepared in Examples 1 and 3 in the experimental examples of the present invention.
  • Fig. 4 is a graph showing the comparison of the detection results of the test kits prepared in Examples 1 and 4 in the experimental examples of the present invention.
  • Anti-triiodothyronine antigen source: purchased from Sigma, article number: T0281.
  • Anti-anti-triiodothyronine antibody source: purchased from Biogenesis, clone number: 9160-7004.
  • ABEI Produced for Shenzhen New Industry Biomedical Co., Ltd.
  • Magnetic microspheres source: Shenzhen New Industry Biomedical Engineering Co., Ltd. produces 80% particle size distribution of 1-5 ⁇ m, magnetization intensity of 4000 Gauss when the precipitation time is 10-15 seconds, BSA is 30mg when the protein adsorption concentration is 0.8 mg - 1.2 mg.
  • Maglumi2000 chemiluminescence analyzer source: Shenzhen New Industry Biomedical Engineering Co., Ltd.
  • An rT3 chemiluminescence immunoassay kit comprising the following components:
  • Marker system A solution comprising a counter-triiodothyronine antigen (rT3 antigen) labeled with a luminescent marker (ABEI).
  • the rT3 antigen labeled with ABEI was diluted at a ratio of 1:200 with a 0.5 M phosphate buffer dilution of pH 7.4.
  • Magnetic microsphere system A solution comprising magnetic microspheres linked to an anti-triiodothyronine antibody (anti-rT3 antibody).
  • the anti-rT3 antibody-coated magnetic microspheres were diluted to 1 mg/mL with a pH of 8.0 Tris-HCl buffer.
  • calibrator solution low point school including anti-triiodothyronine antigen concentration of 0.326 ng / mL The standard solution and the high point calibrator solution at a concentration of 8.132 ng/mL.
  • the preparation method of the rT3 chemiluminescence immunoassay kit of the present embodiment is as follows:
  • step 3) purified ligation product can be diluted 1:2 with a D2 solution.
  • Preparation buffer (A solution): Weigh 2.55 g of sodium acetate trihydrate and dissolve it with 4,500 ml of purified water, then add 14 ml of acetic acid, and then dilute to 5000 ml to obtain an acetic acid buffer having a pH of 3.6.
  • BSA was dissolved in 0.05 M PBS of pH 7.4 to make the concentration of BSA 0.5 g/ml, which is a magnetic bead cleaning solution.
  • the magnetic bead suspension is added, wherein the magnetic bead suspension is prepared by dissolving BSA and methyl cellulose (MC) in 0.05 M pH 7.4 PBS to make the concentration of BSA 0.5 g/
  • the concentration of ml and MC was 0.4 g/ml, and the suspension concentration was 20 mg/ml, that is, a magnetic microsphere solution coated with anti-rT3 antibody was obtained.
  • the rT3 antigen was diluted with 50% bovine serum preparation into a low point calibrator solution with a concentration of 0.326 ng/mL and a high point calibrator solution with a concentration of 8.132 ng/mL.
  • the method for performing rT3 chemiluminescence immunoassay using the detection kit of the present embodiment comprises the following steps:
  • the sample to be tested may be directly obtained serum, plasma and whole blood, or may be obtained by extracting a human blood sample.
  • Reaction Mix the above-mentioned sample to be tested with the marker system and the magnetic microsphere system, and incubate to make the anti-triiodothyronine in the sample to be tested compete with the ABEI-labeled anti-triiodothyronine antigen.
  • the magnetic microspheres coated with anti-triiodothyronine antibody were combined to obtain a reaction product.
  • the above detection method can also be combined with a chemiluminescence analyzer, and the specific steps are as follows:
  • the sample to be tested is serum or plasma.
  • An rT3 chemiluminescence immunoassay kit is basically the same as the test kit of Example 1, except that:
  • Magnetic microsphere system magnetic microspheres linked to anti-triiodothyronine antibody (anti-rT3 antibody) fluorescein isothiocyanate labeled with anti-triiodothyronine antibody (anti-rT3 antibody)
  • the FITC fluorescein isothiocyanate labeled with anti-triiodothyronine antibody (anti-rT3 antibody)
  • the FITC fluorescein polyclonal antibody coated magnetic microsphere solution.
  • anti-rT3 polyclonal antibody-labeled FITC was diluted 1:200 in 0.5M phosphate buffer phosphate buffer
  • goat anti-FITC polyclonal antibody-coated magnetic microspheres were diluted to 1 mg/mL in C solution. .
  • the preparation method of the rT3 chemiluminescence immunoassay kit of the present embodiment is basically the same as the preparation method of the first embodiment, except that:
  • Anti-rT3 antibody-labeled FITC was prepared by the following method:
  • Sheep anti-FITC polyclonal antibody-coated magnetic microspheres were prepared by the following method:
  • the method for performing rT3 chemiluminescence immunoassay using the detection kit of the present embodiment is basically the same as the method of Example 1, except that:
  • An rT3 chemiluminescence immunoassay kit is basically the same as the test kit of Example 1, except that:
  • Marker system A solution of an anti-triiodothyronine antibody (anti-rT3 antibody) comprising a marker-labeled tracer (ABEI).
  • the anti-rT3 antibody labeled with ABEI was diluted at a ratio of 1:200 with a 0.5 M phosphate buffer phosphate buffer.
  • Magnetic microsphere system A solution comprising magnetic microspheres linked to an anti-triiodothyronine antigen (rT3 antigen).
  • the rT3 antigen-coated magnetic microspheres were diluted to 1 mg/mL with a pH of 8.0 Tris-HCl buffer.
  • the preparation method of the rT3 chemiluminescence immunoassay kit of the present embodiment is basically the same as the preparation method of the first embodiment, except that:
  • the method for performing rT3 chemiluminescence immunoassay using the detection kit of the present embodiment is basically the same as the method of Example 1, except that:
  • An rT3 chemiluminescence immunoassay kit is basically the same as the test kit of Example 3, except that:
  • Magnetic microsphere system a magnetic microsphere to which an anti-triiodothyronine antigen (rT3 antigen) is attached, a solution of fluorescein isothiocyanate (FITC) labeled with an anti-triiodothyronine antigen (rT3 antigen), And a goat anti-isothiocyanate fluorescein polyclonal antibody coated magnetic microsphere solution.
  • rT3 antigen an anti-triiodothyronine antigen
  • FITC fluorescein isothiocyanate
  • rT3 antigen-labeled FITC was diluted 1:200 in 0.5M phosphate buffer phosphate buffer
  • goat anti-FITC polyclonal antibody-coated magnetic microspheres were pH 8.0 in Tris-HCl buffer. Dilute to 1 mg/mL.
  • the preparation method of the rT3 chemiluminescence immunoassay kit of the present embodiment is basically the same as the preparation method of the third embodiment, and the difference is that:
  • the rT3 antigen-labeled FITC was prepared by the following method:
  • Sheep anti-FITC polyclonal antibody-coated magnetic microspheres were prepared as in Example 2.
  • the method for performing rT3 chemiluminescence immunodetection using the detection kit of the present embodiment is basically the same as the method of Example 3, except that:
  • the rT3 chemiluminescence immunoassay kit and the detection method of the above embodiments are combined with a chemiluminescence analyzer for clinical test comparison, and the reference kit is a corresponding mainstream commercial radioimmunoassay kit (abbreviated as a radioactive reagent). Box), the sample was collected from the Nuclear Medicine Department of Shenzhen Peking University Hospital, a total of 140 cases, the specific results are shown in the table below.
  • the specimens 1, 2, 11, 18, and 30 were diagnosed with hypothyroidism, and the specimens 75, 82, 114, and 138 were confirmed by hypothyroidism.
  • the test kit prepared in Example 1 was in good agreement with the reference radioimmunoassay kit.
  • kits of the third embodiment and the fourth embodiment are used for the above sample detection, and the results obtained in the third embodiment and the fourth embodiment are compared with the first embodiment, and the correlation coefficients are all above 0.99, as shown in the figure. 3. As shown in Figure 4, it has good consistency.
  • the measured value of the detection kit of the present invention is more consistent with the actual value, and the clinical coincidence rate is higher, indicating that the detection capability of the detection kit of the present invention is stronger.

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Abstract

提供一种rT3化学发光免疫检测试剂盒及其应用。该检测试剂盒包括以下组分：1）标记物体系：包括直接或间接连接标记示踪物的反三碘甲状腺原氨酸抗原或抗反三碘甲状腺原氨酸抗体；2）磁性微球体系：包括相应的直接或间接连接的抗反三碘甲状腺原氨酸抗体或反三碘甲状腺原氨酸抗原。该检测试剂盒可用于反三碘甲状腺原氨酸的化学发光免疫检测。

Description

rT3化学发光免疫检测试剂盒及其检测方法和应用 技术领域
本发明涉及体外诊断检测技术领域，特别是涉及一种rT3(反三碘甲状腺原氨酸)化学发光免疫检测试剂盒及其检测方法和应用。
背景技术
反三碘甲状腺原氨酸(rT3)又称3，3′，5′-三碘甲腺原氨酸，是正常人血清中存在的一种甲状腺激素，是T4(甲状腺素)在肝、肾、垂体及心肌等组织中，经5-脱碘酶脱去酪氨酸环上的一个碘而生成的。rT3在体内降解速度较快，其半寿期为30-60分钟。它在血液中含量甚微，生物活性很低，但在不同的生理及病理状况下，血清含量有显著区别，因此，测定血清中rT3水平在临床上仍有一定意义。例如：妊娠时，脐血及羊水rT3含量较高，能反映胎儿甲状腺功能状态。总体来说，血清中rT3浓度男女间无差异，从儿童到40岁之间随年龄增长呈下降趋势，而40岁以后又随年龄增长呈上升趋势。
具体来说，当rT3增高或降低，有助于以下几种情况的判断：
A，甲状腺功能亢进或减退症：甲亢时rT3升高，而且比T3，T4灵敏。当用抗甲状腺药物治疗时，T3降低较快，而rT3下降缓慢，当T3和rT3都正常，表示用药适当；若rT3和T4均低于正常，则表示用药过量。rT3的降低对于轻型或亚临床型甲状腺功能减退的判断有非常重要的意义。
B，甲状腺疾病：急性心肌梗塞，肝硬变、糖尿病、尿素症、脑血管病、胃癌等类疾病还可出现T3下降，故显示出T3/rT3比值降低。
C.替代治疗的观察：甲状腺机能减退症服用甲状腺素替代治疗后，rT3、T3均升高提示用量过大，而甲状腺功能减退症在使用抗甲亢药物后出现rT3下降，则提示药物用量过大，故rT3测定可用于监测用药。
D.药物引起的高rT3现象：使用乙胺碘呋酮、糖皮质激素、丙基硫氧嘧啶、心得胺等药物可出现高rT3现象，故rT3还可用于监测上述药物的使用。
目前，临床上测定血清反三碘甲状腺原氨酸(rT3)的方法主要有放射性免疫法、酶联免疫法等，而放射性免疫法及酶联免疫法存在诸多不足。如：传统的放射性免疫法或酶联免疫法方法检测时间长，同时主要依靠纯手工加样等系列繁琐操作，效率低，容易导致实验结果误差大；由于酶促反应不够彻底，且易受外部干扰因素影响，如温度、时间及材料浓度影响，因此检测时特异性低，灵敏度差，检测范围窄。并且，放射性免疫法还存在放射性污染、标记物半衰期短、对操作者具有放射性损伤等缺陷。
发明内容
基于此，本发明的目的在于克服现有技术的缺陷，提供一种rT3化学发光免疫检测试剂盒，该化学发光免疫检测试剂盒是将具有高灵敏度的化学发光测定技术与高特异性的免疫反应相结合，具有灵敏度高，特异性好和检测范围宽的优点。
为实现上述目的，本发明采取以下技术方案：
一种rT3化学发光免疫检测试剂盒，包括以下组分：
1)标记物体系：包括直接连接或间接连接标记示踪物的反三碘甲状腺原氨酸抗原或抗反三碘甲状腺原氨酸抗体；
2)磁性微球体系：包括相应的直接连接或间接连接抗反三碘甲状腺原氨酸 抗体或反三碘甲状腺原氨酸抗原的磁性微球。
并且，当标记物体系包括直接连接或间接连接标记示踪物的反三碘甲状腺原氨酸抗原，则磁性微球体系包括直接连接或间接连接抗反三碘甲状腺原氨酸抗体的磁性微球；当标记物体系包括直接连接或间接连接标记示踪物的抗反三碘甲状腺原氨酸抗体，则磁性微球体系包括直接连接或间接连接反三碘甲状腺原氨酸抗原的磁性微球。
上述“直接连接”为直接结合的连接，上述“间接连接”为通过生物素和链霉亲和素，或异硫氰酸荧光素和抗异硫氰酸荧光素抗体等能够相互结合的搭桥物以间接结合的方式连接。
本发明提供了一种rT3化学发光免疫检测试剂盒，利用化学发光免疫的方法来检测rT3，具有灵敏度高，特异性好和检测范围宽的优点，并且，本发明中的试剂盒，以磁性微球作为检测中的固相体系，既能在与待测物反应时均匀的悬浮于反应体系中，与待测物更好的结合，又能在分离时外加磁场即可方便的实现分离，具有操作方便的特点。更为重要的是，该检测试剂盒能与常规技术中的化学发光分析仪配合使用，实现全自动化检测，避免了人为因素可能导致的实验误差，并提高了分析效率。
在其中一个实施例中，所述2)磁性微球体系中，所述间接连接抗反三碘甲状腺原氨酸抗体的磁性微球由生物素标记的抗反三碘甲状腺原氨酸抗体，和链霉亲和素包被的磁性微球组成；或所述间接连接抗反三碘甲状腺原氨酸抗体的磁性微球由异硫氰酸荧光素标记的抗反三碘甲状腺原氨酸抗体，和抗异硫氰酸荧光素抗体包被的磁性微球组成。
在其中一个实施例中，所述2)磁性微球体系中，所述间接连接反三碘甲状腺原氨酸抗原的磁性微球由异硫氰酸荧光素标记的反三碘甲状腺原氨酸抗原， 和抗异硫氰酸荧光素抗体包被的磁性微球组成；或所述间接连接反三碘甲状腺原氨酸抗原的磁性微球由生物素标记的反三碘甲状腺原氨酸抗原，和链霉亲和素包被的磁性微球组成。
在其中一个实施例中，所述1)标记物体系中，所述间接连接标记示踪物的反三碘甲状腺原氨酸抗原由生物素标记的反三碘甲状腺原氨酸抗原，和链霉亲和素标记的标记示踪物组成；或所述间接连接标记示踪物的反三碘甲状腺原氨酸抗原由异硫氰酸荧光素标记的反三碘甲状腺原氨酸抗原，和抗异硫氰酸荧光素抗体标记的标记示踪物组成。
在其中一个实施例中，所述1)标记物体系中，所述间接连接标记示踪物的抗反三碘甲状腺原氨酸抗体由生物素标记的抗反三碘甲状腺原氨酸抗体，和链霉亲和素标记的标记示踪物组成；或所述间接连接标记示踪物的抗反三碘甲状腺原氨酸抗体由异硫氰酸荧光素标记的反三碘甲状腺原氨酸抗原，和抗异硫氰酸荧光素抗体标记的标记示踪物组成。
上述抗异硫氰酸荧光素抗体既可以为单克隆抗体，也可以为多克隆抗体。
上述方案丰富了检测所使用的标记物体系和磁性微球体系，可根据不同需求灵活选择。例如可以在磁性微球体系中直接将反三碘甲状腺原氨酸抗原(或抗反三碘甲状腺原氨酸抗体)连接磁性微球，也可以通过上述的间接搭桥方式连接反三碘甲状腺原氨酸抗原(或抗反三碘甲状腺原氨酸抗体)和磁性微球。同样，可以在标记物体系中将标记示踪物直接连接抗反三碘甲状腺原氨酸抗体(或反三碘甲状腺原氨酸抗原)，也可以通过上述的间接搭桥方式连接标记示踪物和抗反三碘甲状腺原氨酸抗体(或反三碘甲状腺原氨酸抗原)。并且，上述磁性微球体系和标记物体系中采用何种连接方式(直接连接或间接连接)并无相互影响或限定，既可在磁性微球体系和标记物体系中同时采用直接连接方式或 同时采用间接连接方式，也可磁性微球体系选用直接连接，标记物体系采用间接连接，或者磁性微球体系选用间接连接，标记物体系采用直接连接。可以理解的，上述间接搭桥连接方式还可采用其它间接标记体系进行间接连接。
上述标记示踪物包括以下几种：1、化学发光免疫分析使用的能够直接发光的标记物，如鲁米诺及其衍生物、异鲁米诺或其衍生物、吖啶酯等；2、化学发光酶免疫分析使用的配合相应底物可以发光的标记物，如碱性磷酸酶或过氧化物酶等。
在其中一个实施例中，所述标记示踪物为发光标记物，选自：金刚烷、鲁米诺及其衍生物、异鲁米诺及其衍生物、吖啶酯。优选N-(4-氨基丁基)-N-乙基异鲁米诺(ABEI)，与上述发光标记物配合的底物包括H2O2-微过氧化物酶、H2O2-过氧化氢酶、H2O2-乳过氧化物酶、H2O2-次氯血红素、H2O2-氯化血红素、次氯酸盐-CoCl2、过硫酸盐、过氧化钾、高碘酸钠、H2O2-K3Fe(CN)6、黄嘌呤-次黄嘌呤氧化酶、叔丁醇钾中的至少一种。
上述发光标记物是指在发光反应中参与能量转移并最终以发射光子的形式释放能量的化合物，该化合物能够经催化剂的催化和氧化剂的氧化，形成一个激发态的中间体，当这种激发态中间体回到稳定的基态时，同时发射出光子(hM)。
在其中一个实施例中，所述标记示踪物为化学发光催化剂，选自：碱性磷酸酶、过氧化物酶。使用时，配合相应的化学发光底物即可发光被定量检测，所述化学发光底物包括NaOH和H2O2，还包括金刚烷、鲁米诺及其衍生物、异鲁米诺或其衍生物中的至少一种，优选N-(4-氨丁基)-N-乙基异鲁米诺(ABEI)。
在其中一个实施例中，所述磁性微球上直接连接或间接连接一种或多种抗反三碘甲状腺原氨酸抗体。并且，可以理解的，当磁性微球上直接连接或间接 连接多种抗反三碘甲状腺原氨酸抗体时，该多种(即至少两种)抗反三碘甲状腺原氨酸抗体与反三碘甲状腺原氨酸抗原的结合位点互不相同。
其中一个实施例中，所述抗反三碘甲状腺原氨酸抗体为单克隆抗体和/或多克隆抗体。
适用于本发明的磁性微球也称为磁珠或磁球，可以是本领域中常用的磁性微球。优选的是，本发明使用的磁球，是将纳米级的Fe2O3或Fe3O4磁性粒子和有机高分子材料进行复合，形成具有超顺磁性和极大量蛋白吸附容量的微米级的固相微球，具有在外加磁场作用下可迅速被磁化，在撤走磁场后剩磁为零的属性。其中，所述有机高分子材料的种类没有特别限制，可根据需要进行选择。
本发明所使用的磁性微球应能满足直径为0.1-5μm，磁性微球还可以通过表面改性而带有多种活性功能基团，包括但不限于-OH、-COOH、-NH2。
在其中一个实施例中，所述磁性微球为Fe2O3或Fe3O4磁性纳米粒子与有机高分子材料的复合体，并具有0.1-5μm的粒径，并且，所述磁性微球任选地通过表面改性而带有一种或多种活性功能基团。
在其中一个实施例中，该检测试剂盒还包括以下组分：
3)校准品溶液：包括反三碘甲状腺原氨酸抗原浓度为0.2-6ng/mL的校准品溶液和浓度为8-20ng/mL的校准品溶液。本发明人考察了大量的真实样本反三碘甲状腺原氨酸抗原浓度范围后，结合该检测试剂盒的特点，将校准品浓度设置于上述范围内，具有较佳的检测效果。
本发明还公开了一种上述的rT3化学发光免疫检测试剂盒的制备方法，包括以下步骤：
抗体或抗原的标记：将抗原或抗体放入透析袋中，置于透析液中进行透析，随后往透析好的溶液中加入标记物，反应后经纯化，即得；
磁性微球的包被：以缓冲液悬浮磁性微球，再加入包被物，组成反应体系，经反应后再进行纯化，即得。
所述抗原为反三碘甲状腺原氨酸抗原；
所述抗体为抗反三碘甲状腺原氨酸抗体。
上述抗体既可以为单抗，也可以为多抗。上述包被磁性微球的反应体系中，还可加入1-环已基-2-吗啉乙基碳二亚胺对甲苯磺酸盐(CMC)进行包被连接，其浓度优选10mg/mL，可以增加包被效果。经标记的抗体或抗原的纯化优选采用G-25凝胶柱进行纯化。所述标记示踪物的活化酯可以为ABEI-半琥珀酰胺酸N-羟基琥珀酰亚胺酯(ABEI-hemisuccinimide N-Hydroxysuccinimide)等，仅需满足能与抗原或抗体结合即可。
在其中一个实施例中，所述标记物为：标记示踪物的活化酯，异硫氰酸荧光素或生物素；所述包被物为：抗反三碘甲状腺原氨酸抗体，反三碘甲状腺原氨酸抗原，抗异硫氰酸荧光素抗体或链霉素亲和素。上述方案丰富了检测所使用的标记物体系和磁性微球体系，可根据不同需求灵活选择。
在其中一个实施例中，所述抗体或抗原的标记中，所述标记物的加入量为每100μg抗原加入200-400μg标记物，优选300μg标记物；每1mg抗体加入200-400μg标记物，优选300μg标记物；反应温度为35-40℃，优选37℃，反应时间为1-3小时，优选2小时；所述磁性微球的包被中，所述包被物的加入量为每20mg磁性微球加入100-140μg包被物，优选120μg包被物，反应温度为35-40℃，优选37℃，反应时间为12-36小时，优选24小时。采用上述参数配比，能够具有最佳的标记率及发光效率。
在其中一个实施例中，所述抗体或抗原的标记中，将纯化后经标记的抗原或抗体稀释150-250倍，优选稀释200倍，待用；所述抗体或抗原的标记中，将 纯化后经包被的磁性微球稀释至0.5-1.5mg/mL，优选1mg/mL，待用；上述经标记的抗原或抗体溶液与经包被的磁性微球溶液的使用体积比为60-100：20，优选80：20。以上述比例配合使用，能获得较佳的检测效果。
在其中一个实施例中，所述抗体或抗原的标记中，所述透析液优选0.1mol/L的pH9.5碳酸缓冲液；磁性微球的包被中，所述缓冲液优选pH3.6醋酸缓冲液。上述透析液和缓冲液能够促使标记和包被反应进行得更好。
本发明还公开了一种rT3化学发光免疫检测方法，采用上述的rT3化学发光免疫检测试剂盒，包括以下步骤：
1)反应：将待测样本与标记物体系和磁性微球体系混合，温育，得到反应产物；
2)检测：外加磁场将上述反应产物沉淀，去除上清液，清洗后，加入化学发光激发物，检测发出的相对光强度，计算得到反三碘甲状腺原氨酸的含量。
本发明的检测方法，采用了化学发光免疫竞争法进行检测，具有灵敏度高，特异性好和检测范围宽的优点。
在其中一个实施例中，上述rT3化学发光免疫检测方法，是利用化学发光分析仪进行反三碘甲状腺原氨酸的检测。具有操作规范，无人为引入误差及全自动化的优点。
本发明还公开了一种上述的rT3化学发光免疫检测试剂盒在化学发光分析仪中的应用。将该试剂盒应用于化学发光平台，具有操作规范，无人为引入误差及全自动化的优点。
与现有技术相比，本发明具有以下有益效果：
本发明的一种rT3化学发光免疫检测试剂盒，将具有高灵敏度的化学发光测定技术与高特异性的免疫反应相结合，具有灵敏度高，特异性好和检测范围 宽的优点。并且，本发明中的试剂盒，以磁性微球作为检测中的固相体系，既能在与待测物反应时均匀的悬浮于反应体系中，与待测物更好的结合，又能在分离时外加磁场即可方便的实现分离，具有操作方便的特点。更为重要的是，该检测试剂盒能与常规技术中的化学发光分析仪配合使用，实现全自动化检测，避免了人为因素可能导致的实验误差，并提高了分析效率。
该检测试剂盒结合真实样本中待测物可能的浓度，以及该试剂盒中标记物体系和磁性微球体系的特点，对其中各组分进行了优选，得到了最佳的检测用量范围。
本发明的rT3化学发光免疫检测方法，具有灵敏度高，特异性好和检测范围宽的优点。并且，还可将该检测试剂盒与常规技术中的化学发光分析仪配合使用，实现全自动化检测，避免了人为因素可能导致的实验误差，并提高了分析效率。
附图说明
图1为本发明实验例中实施例1制备的检测试剂盒与参比的放免试剂盒的检测结果对比图；
图2为本发明实验例中实施例1和2制备的检测试剂盒的检测结果对比图；
图3为本发明实验例中实施例1和3制备的检测试剂盒的检测结果对比图；
图4为本发明实验例中实施例1和4制备的检测试剂盒的检测结果对比图。
具体实施方式
以下结合实施例对本发明做进一步的说明，但并不对本发明造成任何限制。
以下实施例中：
反三碘甲状腺原氨酸抗原，来源：从Sigma公司采购，货号：T0281。
抗反三碘甲状腺原氨酸抗体，来源：从Biogenesis公司采购，克隆号：9160-7004。
羊抗FITC多克隆抗体，来源：美国Jackson公司。
FITC：购自美国Sigma公司。
ABEI：为深圳新产业生物医学股份有限公司生产。
磁性微球，来源：深圳市新产业生物医学工程股份有限公司生产，80％粒径分布为1-5μm，磁化强度为4000高斯时沉淀时间为10-15秒，BSA为30mg时蛋白吸附浓度为0.8mg-1.2mg。
Maglumi2000化学发光分析仪，来源：深圳市新产业生物医学工程股份有限公司。
实施例1
一种rT3化学发光免疫检测试剂盒，包括以下组分：
1)标记物体系：包括标记发光标记物(ABEI)的反三碘甲状腺原氨酸抗原(rT3抗原)的溶液。
其中，标记ABEI的rT3抗原以pH7.4的0.5M磷酸盐缓冲稀释液按照1：200的比例稀释。
2)磁性微球体系：包括连接抗反三碘甲状腺原氨酸抗体(抗rT3抗体)的磁性微球的溶液。
其中，抗rT3抗体包被的磁性微球以pH值为8.0的Tris-HCl缓冲液稀释至1mg/mL。
3)校准品溶液：包括反三碘甲状腺原氨酸抗原浓度为0.326ng/mL的低点校 准品溶液和浓度为8.132ng/mL的高点校准品溶液。
本实施例的rT3化学发光免疫检测试剂盒的制备方法如下：
一、标记物体系的制备。
1)透析液的配制：在5000ml烧杯中加入Na2CO314.31g，NaHCO326.46g，加水定容至4500ml，即得0.1mol/L pH9.5的碳酸缓冲液。上述配制好的透析液置于磁力搅拌器上备用。
2)选用截留量为14000的透析袋，量取合适的尺寸，取100μg的rT3抗原用透析液调整到1ml，放入透析液中，室温搅拌透析2小时，将透析好的溶液加入300μg ABEI-半琥珀酰胺酸N-羟基琥珀酰亚胺酯(ABEI-hemisuccinimide N-Hydroxysuccinimide)，37℃反应2小时。
3)以G-25凝胶柱纯化上述反应得到的标记ABEI的rT3抗原。
4)D2溶液的配制：加入200ml 0.5M磷酸盐缓冲液、20g牛血清白蛋白(BSA)、8g NaN3(防腐剂)、2g MgCl2·6H2O、600ml甘油，定容到2000mL，过滤，即得。
5)将上述步骤3)纯化好的连接产物用D2溶液按1：2稀释，即可。
二、磁性微球体系的制备。
1)配制缓冲液(A溶液)：称取2.55g三水合乙酸钠用4500ml纯化水溶解后再加如14ml乙酸混匀后，定容至5000ml，即得pH为3.6的醋酸缓冲液。
2)向装有磁性微球的瓶中加入上述pH3.6醋酸缓冲液悬浮磁性微球浓度为20mg/mL，再加入1-环已基-2-吗啉乙基碳二亚胺对甲苯磺酸盐(CMC)(浓度为10mg/ml)，加入100μg纯化的抗rT3抗体，组成反应体系。
3)将上述反应体系放入恒温震荡水浴箱中37℃反应24小时(震荡水浴锅振摇速度为260rpm)。
4)磁珠清洗液的配制：在0.05M pH7.4的PBS中溶入BSA，使BSA的浓度为0.5g/ml，即为磁珠清洗液。
5)将反应好的反应体系倒入烧杯中，然后置于磁铁上沉淀后，倒掉上清，加入5倍体积的磁珠清洗液搅拌清洗，然后放置在磁铁上，待上清液清亮后倒掉上清液。
6)清洗完毕后，加入磁珠悬浮液，其中磁珠悬浮液的制备方法为：在0.05MpH7.4的PBS中溶入BSA和甲基纤维素(MC)，使BSA的浓度为0.5g/ml，MC的浓度为0.4g/ml，悬浮浓度为20mg/ml，即得到抗rT3抗体包被的磁性微球溶液。
三、校准品溶液的制备。
将rT3抗原用50％牛血清制品按不同比例稀释成浓度为0.326ng/mL的低点校准品溶液和浓度为8.132ng/mL的高点校准品溶液。
采用本实施例的检测试剂盒进行rT3化学发光免疫检测的方法，包括以下步骤：
1)获取待测样本：待测样本可为直接得到的血清、血浆及全血，也可以是通过抽取人体血样进行分离得到。
2)反应：将上述待测样本与标记物体系和磁性微球体系混合，温育，使待测样本中的反三碘甲状腺原氨酸与经ABEI标记的反三碘甲状腺原氨酸抗原竞争结合抗反三碘甲状腺原氨酸抗体包被的磁性微球，得到反应产物。
3)检测：外加磁场将上述反应产物沉淀，去除上清液，并以缓冲液清洗后，加入化学发光激发物(NaOH和H2O2)，检测发出的相对光强度，通过计算得到反三碘甲状腺原氨酸的含量。
上述检测方法还可与化学发光分析仪配合，具体步骤如下：
1、在样本架上依次加载校准品或待测样本，待测样本为血清或者血浆。
2、将样本架插入Maglumi2000化学发光分析仪样本仓，编辑标本号开始运行试验，具体加样步骤为：校准品或待测样本加50μl，然后加标记物体系(标记ABEI的rT3抗原)溶液80μl，加0.1g/ml 8-苯胺基萘磺酸镁(ANS)的稀释液80μl，加入磁性微球体系溶液20μl，混匀，37℃温育15分钟，仪器自动清洗两遍后直接进入测量室得到各个标本的光强度信号，通过十点曲线及两点定标自动拟合出待测样本的rT3浓度值。
实施例2
一种rT3化学发光免疫检测试剂盒，与实施例1的检测试剂盒基本相同，不同之处在于：
2)磁性微球体系：连接抗反三碘甲状腺原氨酸抗体(抗rT3抗体)的磁性微球由抗反三碘甲状腺原氨酸抗体(抗rT3抗体)标记的异硫氰酸荧光素(FITC)溶液，和羊抗异硫氰酸荧光素多克隆抗体包被的磁性微球溶液组成。
其中：抗rT3多克隆抗体标记的FITC以0.5M的pH7.4磷酸盐缓冲稀释液按照1：200的比例稀释，羊抗FITC多克隆抗体包被的磁性微球以C溶液稀释至1mg/mL。
本实施例的rT3化学发光免疫检测试剂盒的制备方法与实施例1的制备方法基本相同，不同之处在于：
二、磁性微球体系中：
抗rT3抗体标记的FITC通过以下方法制备：
3)取1mg抗rT3抗体用透析液调整到1ml。放入透析液中，室温搅拌透析2小时，将透析好的溶液加入300μg FITC，室温边摇边反应24h。
羊抗FITC多克隆抗体包被的磁性微球通过以下方法制备：
3)加入上述pH3.6醋酸缓冲液悬浮磁珠浓度20mg/mL，再加入CMC(浓度为10mg/ml)，加入纯化的羊抗FITC抗体，组成反应体系。
采用本实施例的检测试剂盒进行rT3化学发光免疫检测的方法与实施例1的方法基本相同，不同之处在于：
2)反应：先将抗反三碘甲状腺原氨酸抗体(抗rT3抗体)标记的异硫氰酸荧光素(FITC)溶液，和羊抗异硫氰酸荧光素多克隆抗体包被的磁性微球溶液混合，得到连接抗反三碘甲状腺原氨酸抗体(抗rT3抗体)的磁性微球，再将此磁性微球体系与待测样本与标记物体系混合。
实施例3
一种rT3化学发光免疫检测试剂盒，与实施例1的检测试剂盒基本相同，不同之处在于：
1)标记物体系：包括标记标记示踪物(ABEI)的抗反三碘甲状腺原氨酸抗体(抗rT3抗体)的溶液。
其中，标记ABEI的抗rT3抗体以0.5M的pH7.4磷酸盐缓冲稀释液按照1：200的比例稀释。
2)磁性微球体系：包括连接反三碘甲状腺原氨酸抗原(rT3抗原)的磁性微球的溶液。
其中，rT3抗原包被的磁性微球以pH值为8.0的Tris-HCl缓冲液稀释至1mg/mL。
本实施例的rT3化学发光免疫检测试剂盒的制备方法与实施例1的制备方法基本相同，不同之处在于：
一、标记物体系的制备。
3)取1mg的抗rT3抗体用透析液调整到1ml，放入透析液中，室温搅拌透析2小时，将透析好的溶液加入300μg ABEI活化酯4-(氨丁基)-N-乙基异鲁米诺，37℃反应2小时。
二、磁性微球体系的制备。
3)加入pH3.6醋酸缓冲液悬浮磁性微球浓度为20mg/mL，再加入CMC(浓度为10mg/ml)，加入100μg纯化的rT3抗原，组成反应体系。
采用本实施例的检测试剂盒进行rT3化学发光免疫检测的方法与实施例1的方法基本相同，不同之处在于：
2)反应：采用本实施例中的标记物体系和磁性微球体系。
实施例4
一种rT3化学发光免疫检测试剂盒，与实施例3的检测试剂盒基本相同，不同之处在于：
2)磁性微球体系：连接反三碘甲状腺原氨酸抗原(rT3抗原)的磁性微球由反三碘甲状腺原氨酸抗原(rT3抗原)标记的异硫氰酸荧光素(FITC)溶液，和羊抗异硫氰酸荧光素多克隆抗体包被的磁性微球溶液组成。
其中：rT3抗原标记的FITC以0.5M的pH7.4磷酸盐缓冲稀释液按照1：200的比例稀释，羊抗FITC多克隆抗体包被的磁性微球以pH值为8.0的Tris-HCl缓冲液稀释至1mg/mL。
本实施例的rT3化学发光免疫检测试剂盒的制备方法与实施例3的制备方法基本相同，不同之处在于：
二、磁性微球体系中：
rT3抗原标记的FITC通过以下方法制备：
3)取100μg rT3抗原用透析液调整到1ml。放入透析液中，室温搅拌透析2小时，将透析好的溶液加入300μg FITC，室温边摇边反应24h。
羊抗FITC多克隆抗体包被的磁性微球按照实施例2中的方法制备。
采用本实施例的检测试剂盒进行rT3化学发光免疫检测的方法与实施例3的方法基本相同，不同之处在于：
2)反应：先将反三碘甲状腺原氨酸抗原(rT3抗原)标记的异硫氰酸荧光素(FITC)溶液，和羊抗异硫氰酸荧光素多克隆抗体包被的磁性微球溶液混合，得到连接反三碘甲状腺原氨酸抗原(rT3抗原)的磁性微球，再将此磁性微球体系与待测样本与标记物体系混合。
实验例
采用上述实施例的rT3化学发光免疫检测试剂盒及检测方法，与化学发光分析仪配合，进行临床测试比对，参比试剂盒为相应的市面上主流商品化放射免疫检测试剂盒(简称放免试剂盒)，样本为深圳北大医院核医学科收集样本，共140例，具体结果如下表所示。
表1 临床样本测定对比结果(单位：ng/ml)
Figure PCTCN2015072616-appb-000001
Figure PCTCN2015072616-appb-000002
Figure PCTCN2015072616-appb-000003
上述140例临床样本，其中1、2、11、18、30号标本为甲减确诊患者，75、82、114、138号标本为甲减确诊患者。通过上述对比实验结果可以证明，除去确诊的9例标本，实施例1与参比试剂盒两种方法得到的结果直线方程为y＝1.050x，相关系数R＝0.920，如图1所示，可见实施例1制备的检测试剂盒与参比的放免试剂盒有较好的一致性。
而将实施例2与实施例1得到的结果相对比，二者的直线方程为y＝1.000x， 相关系数R＝0.999，如图2所示，可见本方法制备的检测试剂盒，无论是采用直接连接方式，或是间接连接方式，其检测结果具有较好的一致性，检测结果稳定。而对于9例甲减、甲亢确诊患者，放免试剂盒未检出但是有偏低或偏高的趋势，但用本实施例1、2的试剂盒和检测方法均能全部检出。
并且，本发明中还将实施例3和实施例4的试剂盒用于上述样本检测，实施例3和实施例4得到的结果与实施例1相比，其相关系数均在0.99以上，如图3、图4所示，具有较好的一致性。
综上所述，本发明的检测试剂盒的测量值与实际值符合程度更好，临床符合率更高，说明本发明检测试剂盒的检测能力更强。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (14)

  1. 一种rT3化学发光免疫检测试剂盒，其特征在于，包括以下组分：
    1)标记物体系：包括直接连接或间接连接标记示踪物的反三碘甲状腺原氨酸抗原或抗反三碘甲状腺原氨酸抗体；
    2)磁性微球体系：包括相应的直接连接或间接连接抗反三碘甲状腺原氨酸抗体或反三碘甲状腺原氨酸抗原的磁性微球。
  2. 根据权利要求1所述的rT3化学发光免疫检测试剂盒，其特征在于，所述2)磁性微球体系中，所述间接连接抗反三碘甲状腺原氨酸抗体的磁性微球由生物素标记的抗反三碘甲状腺原氨酸抗体，和链霉亲和素包被的磁性微球组成；或所述间接连接抗反三碘甲状腺原氨酸抗体的磁性微球由异硫氰酸荧光素标记的抗反三碘甲状腺原氨酸抗体，和抗异硫氰酸荧光素抗体包被的磁性微球组成。
  3. 根据权利要求1所述的rT3化学发光免疫检测试剂盒，其特征在于，所述2)磁性微球体系中，所述间接连接反三碘甲状腺原氨酸抗原的磁性微球由异硫氰酸荧光素标记的反三碘甲状腺原氨酸抗原，和抗异硫氰酸荧光素抗体包被的磁性微球组成；或所述间接连接反三碘甲状腺原氨酸抗原的磁性微球由生物素标记的反三碘甲状腺原氨酸抗原，和链霉亲和素包被的磁性微球组成。
  4. 根据权利要求1所述的rT3化学发光免疫检测试剂盒，其特征在于，所述1)标记物体系中，所述间接连接标记示踪物的反三碘甲状腺原氨酸抗原由生物素标记的反三碘甲状腺原氨酸抗原，和链霉亲和素标记的标记示踪物组成；或所述间接连接标记示踪物的反三碘甲状腺原氨酸抗原由异硫氰酸荧光素标记的反三碘甲状腺原氨酸抗原，和抗异硫氰酸荧光素抗体标记的标记示踪物组成。
  5. 根据权利要求1所述的rT3化学发光免疫检测试剂盒，其特征在于，所述1)标记物体系中，所述间接连接标记示踪物的抗反三碘甲状腺原氨酸抗体由 生物素标记的抗反三碘甲状腺原氨酸抗体，和链霉亲和素标记的标记示踪物组成；或所述间接连接标记示踪物的抗反三碘甲状腺原氨酸抗体由异硫氰酸荧光素标记的抗反三碘甲状腺原氨酸抗体，和抗异硫氰酸荧光素抗体标记的标记示踪物组成。
  6. 根据权利要求1-5任一项所述的rT3化学发光免疫检测试剂盒，其特征在于，所述标记示踪物为发光标记物，选自：金刚烷、鲁米诺及其衍生物、异鲁米诺及其衍生物、吖啶酯。
  7. 根据权利要求1-5任一项所述的rT3化学发光免疫检测试剂盒，其特征在于，所述标记示踪物为化学发光催化剂，选自：碱性磷酸酶、过氧化物酶。
  8. 根据权利要求1-5任一项所述的rT3化学发光免疫检测试剂盒，其特征在于，所述磁性微球上直接连接或间接连接一种或多种抗反三碘甲状腺原氨酸抗体。
  9. 根据权利要求1-5任一项所述的rT3化学发光免疫检测试剂盒，其特征在于，所述抗反三碘甲状腺原氨酸抗体为单克隆抗体和/或多克隆抗体。
  10. 根据权利要求1-5任一项所述的rT3化学发光免疫检测试剂盒，其特征在于，所述磁性微球为Fe2O3或Fe3O4磁性纳米粒子与有机高分子材料的复合体，并具有0.1-5μm的粒径，并且，所述磁性微球任选地通过表面改性而带有一种或多种活性功能基团。
  11. 根据权利要求1-5任一项所述的rT3化学发光免疫检测试剂盒，其特征在于，还包括以下组分：
    3)校准品溶液：包括反三碘甲状腺原氨酸抗原浓度为0.2-6ng/mL的校准品溶液和浓度为8-20ng/mL的校准品溶液。
  12. 一种rT3化学发光免疫检测方法，其特征在于，采用权利要求1-11任 一项所述的rT3化学发光免疫检测试剂盒，包括以下步骤：
    1)反应：将待测样本与标记物体系和磁性微球体系混合，温育，得到反应产物；
    2)检测：外加磁场将上述反应产物沉淀，去除上清液，清洗后，加入化学发光激发物，检测发出的相对光强度，计算得到反三碘甲状腺原氨酸的含量。
  13. 根据权利要求12所述的rT3化学发光免疫检测方法，其特征在于，利用化学发光分析仪进行反三碘甲状腺原氨酸的检测。
  14. 权利要求1-11任一项所述的rT3化学发光免疫检测试剂盒在化学发光分析仪中的应用。
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CN111505273A (zh) * 2020-04-10 2020-08-07 蓝怡科技集团股份有限公司 一种地高辛的非竞争型化学发光免疫检测试剂盒及其应用
CN112557660A (zh) * 2020-12-09 2021-03-26 江苏泽成生物技术有限公司 一种磁微粒化学发光法her2检测试剂盒及制备方法
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CN113702638A (zh) * 2021-08-24 2021-11-26 山东大学 一种基于CuInS2@ZnS纳米晶定量检测CEA抗原的化学发光免疫试剂盒
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CN113493514A (zh) * 2020-03-20 2021-10-12 郑州达诺生物技术有限公司 抗三碘甲状腺原氨酸单抗的酶结合物、总三碘甲状腺原氨酸定量检测试剂盒及其使用方法
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CN115043898A (zh) * 2021-03-08 2022-09-13 迈克生物股份有限公司 生物素化抗原衍生物及相关试剂盒和用途
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