CN104614535B - TMA detection kit and its preparation method and application - Google Patents
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Classifications
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- 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
- G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
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- 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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
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- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- Hematology (AREA)
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- General Physics & Mathematics (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
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- General Health & Medical Sciences (AREA)
- Rheumatology (AREA)
- Rehabilitation Therapy (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention provides a kind of chemiluminescence immune detection reagent kit for being used to detect TMA and preparation method thereof, the kit includes being marked with the protein A antigens of trace labelling thing or is marked with the anti-human igg of trace labelling thing and anti-human IgM, and the attachment of the thyroid microsomal antigen and protein carrier of the thyroid microsomal antigen of coating magnetic ball or coating magnetic ball.Present invention also offers the detection method of TMA concentration.TMA concentration is detected using kit provided by the invention, is not related to enzymatic reaction, it is not necessary to put up a bridge by enzyme, therefore the reagent holding time is grown, it is stable；It can also be detected by Full-automatic chemiluminescence method, reduce the operating time, reduce manual operation error, while using the specificity of chemical tracing label, improve detection sensitivity and accuracy.
Description
Technical Field
The invention relates to a detection kit, in particular to a chemiluminescence immunoassay kit for detecting thyroid microsome antibodies. The invention also relates to a preparation method of the kit and a method for detecting the concentration of the thyroid microsome antibody by using the kit.
Background
Thyroid Microsome Antibody (TMA) is an antibody against Thyroid Microsomes (TM), and is one of autoantibodies caused by autoimmune thyroid diseases. TAM and anti-thyroglobulin antibodies (TGA) have been recognized as important markers in the course of thyroid autoimmune, the most representative antibodies. TAM and TGA are indispensable indicators in the diagnosis of autoimmune thyroid diseases, and are one of specific means for histologically diagnosing autoimmune thyroid diseases.
For various thyroid diseases, the detection rate of anti-TM positive is as follows: the hashimoto thyroiditis is 50-100%; hypothyroidism was 88.9%; thyroid tumor 13.1%; simple goiter is 8.6%; subacute thyroiditis 17.2% -25%; systemic Lupus Erythematosus (SLE) 15.4% -44.7%; other rheumatism was 30%. Normal persons also had 8.4% positive rate against TM.
Serum TMA levels are significantly higher for patients with autoimmune thyroiditis (i.e., Graves' disease) than for normal and other non-autoimmune thyroid disease patients. Therefore, the TMA level has important value for differential diagnosis of autoimmune thyroiditis, and the diagnosis accuracy rate of the combined application of the TMA level and the autoimmune thyroiditis can reach 98%.
Serum TMA level of patients with immune diseases such as hashimoto's thyroiditis, primary hypothyroidism and hyperthyroidism is also obviously higher than that of normal people, and particularly, the hashimoto's thyroiditis is more prominent, so the serum TMA level is a 'specific index' for diagnosing the diseases.
In particular, serum TMA levels are characterized as follows for some specific thyroid disorders. Firstly, hyperthyroidism: TMA shows strong positive, and the antibody level is higher than that of Hashimoto's thyroiditis; TMA can turn negative after part of patients are treated, but TMA of most clinically cured hyperthyroidism patients is determined to be weak positive for a long time; thyroid function should be reviewed regularly to prevent recurrence. ② hashimoto's thyroiditis, addison's disease: TMA shows strong positive; TMA of patients with the methylene inflammation is obviously higher than that of normal people and lower than that of patients with the hashimoto's thyroiditis. ③ Primary hypothyroidism: TMA was positive, but secondary hypothyroidism was TMA negative, which allowed secondary hypothyroidism to be identified. Thyroid cancer: the TMA increased significantly. Pregnancy autoimmune disease: TMA may be increased.
At present, the clinical methods for measuring TMA mainly include enzyme-linked immunosorbent assay, chemiluminescence immunoassay and the like. However, the enzyme-linked immunosorbent assay has many defects, the traditional enzyme-linked immunosorbent assay has long detection time, and meanwhile, the traditional enzyme-linked immunosorbent assay mainly depends on a series of complicated operations such as pure manual sample adding and the like, so that the efficiency is low, and the error of the experimental result is easy to cause; the enzymatic reaction is not thorough enough and is easily influenced by external interference factors such as temperature, time, material concentration and the like, so that the specificity is low, the sensitivity is poor and the detection range is narrow during detection. Chemiluminescence immunoassay is a technology for detecting and analyzing various antigens, haptens, antibodies, hormones, enzymes, fatty acids, vitamins, drugs and the like by combining a chemiluminescence assay technology with high sensitivity and high specificity immunoreaction, and is the most sensitive immunoassay technology so far. However, there are not many detection kits having excellent performance for detecting TMA by the chemiluminescence immunoassay. Therefore, there is still a need in the art for a detection kit for detecting TMA, which has high sensitivity and accuracy, is easy to operate, and can realize full automation of the detection process by means of an analyzer.
Disclosure of Invention
The invention aims to provide a chemiluminescence immunoassay kit for detecting TMA, so as to obtain high detection sensitivity and accuracy. The kit can also improve the stability of the detection reagent and prolong the storage time of the detection reagent.
The invention also provides a preparation method of the chemiluminescence immunoassay kit for detecting TMA.
In addition, the invention also provides an application of the TMA chemiluminescence immunoassay kit provided by the invention. Particularly, the kit is adopted, the TMA detection method is carried out by a full-automatic chemiluminescence method, the operation time can be reduced, the manual operation error can be reduced, and the detection sensitivity can be improved by utilizing the specificity of the chemical tracer markers.
According to the present invention, there is provided a chemiluminescent immunoassay kit for detecting thyroid microsome antibodies, the kit comprising protein a labeled with a tracer marker or anti-human IgG and anti-human IgM labeled with a tracer marker, and a magnetic bead-coated thyroid microsome antigen or a conjugate of a magnetic bead-coated thyroid microsome antigen and a protein carrier. Wherein the tracer label directly or indirectly labels protein A, or directly or indirectly labels anti-human IgG and anti-human IgM; the thyroid microsome antigen or the connector of the thyroid microsome antigen and the protein carrier directly or indirectly coats the magnetic ball.
Protein a is a s.aureus cell wall protein that specifically binds to the Fc region of human and mammalian antibodies (primarily IgG). Thus, protein a is bound to sepharose in a manner that produces an affinity filler for antibody purification. The Fab fragment (antigen-binding fragment) of anti-IgG can bind to the Fc fragment (crystalline fragment) of an IgG antibody. The Fab fragment (antigen-binding fragment) of anti-IgM is capable of binding to the Fc fragment (crystalline fragment) of IgM antibody.
The protein carrier to which the present invention is applicable may be selected from at least one of protein carriers commonly used in the art. For example, the protein carrier is selected from at least one of Bovine Serum Albumin (BSA), Human Serum Albumin (HSA), Rabbit Serum Albumin (RSA), hemocyanin (KLH), bovine IgG, human IgG, Ovalbumin (OVA), myoglobin, and thyroglobulin.
According to the invention, the tracer label may be chosen from among the tracer labels commonly used in the art for labeling antigens or antibodies, for example from at least one of adamantane, luminol and derivatives thereof, isoluminol and derivatives thereof, acridinium esters, alkaline phosphatase and horseradish peroxidase, preferably N- (4-aminobutyl) -N-ethyl isoluminol (ABEI).
According to the invention, the tracer marker preferably indirectly labels protein a or indirectly labels anti-human IgG and anti-human IgM through a Fluorescein Isothiocyanate (FITC) and anti-FITC antibody system or a Streptavidin (SA) and Biotin (Biotin) system.
The direct labeling means that the tracer marker is directly connected with the antigen for labeling; the "indirect labeling" refers to labeling protein A or anti-human IgG and anti-human IgM with tracer labels through an intermediate medium linking system, including but not limited to FITC and anti-FITC antibody system or SA and Biotin system. The inventor finds that indirect labeling is beneficial to weakening space effect, is beneficial to amplifying signals and enables detection to be more sensitive.
According to the invention, the thyroid microsomal antigen (or a conjugate thereof to a protein carrier) is preferably coated indirectly on the magnetic spheres via a fluorescein isothiocyanate and anti-fluorescein isothiocyanate antibody system or a streptavidin and biotin system.
The "direct coating" refers to coating the magnetic spheres directly by using a TM antigen (or a connector of the TM antigen and a protein carrier); the "indirect coating" refers to coating the magnetic ball by TM antigen (or its conjugate with protein carrier) through an intermediate medium linking system, including but not limited to FITC and anti-FITC antibody system or SA and Biotin system. Also, indirect coating has the advantages of reducing the spatial effect, amplifying the signal and making the detection more sensitive.
In some embodiments of the invention, the kit comprises protein a labeled with a tracer marker or anti-human IgG and anti-human IgM labeled with a tracer marker, and one component selected from the group consisting of: a) thyroid microsomal antigen directly coating magnetic spheres (or its linker to a protein carrier); b) biotinylated thyroid microsomal antigen (or its conjugate to a protein carrier) and streptavidin directly coating the magnetic beads; c) thyroid microsome antigen directly labeled with fluorescein isothiocyanate (or its conjugate with protein carrier) and goat anti-fluorescein isothiocyanate antibody directly coating magnetic ball.
Kits provided according to the invention may also comprise a low-point calibrator and a high-point calibrator for TMA, and optionally a buffer. The low-point calibrator and the high-point calibrator are relative, wherein the low-point calibrator is a calibrator obtained by diluting TMA (TMA) with a 50% bovine serum product to a concentration of 0-30 IU/ml; the "high-point calibrator" refers to a calibrator obtained by diluting TMA antigen with 50% bovine serum preparation to a concentration of 500-.
Magnetic spheres, also referred to as magnetic beads, suitable for use in the present invention may be magnetic microspheres commonly used in the art. Preferably, the magnetic spheres used in the present invention are those of nanoscale Fe2O3Or Fe3O4The magnetic particles and the organic polymer material are compounded to form micron-sized solid phase microspheres with superparamagnetism and extremely large protein adsorption capacity, and the micron-sized solid phase microspheres have the properties that the micron-sized solid phase microspheres can be quickly magnetized under the action of an external magnetic field and have zero residual magnetism after the magnetic field is removed. The kind of the organic polymer material is not particularly limited, and may be selected as needed.
The magnetic microspheres used in the invention can meet the requirement that the diameter is 0.1-5 mu m, and the magnetic microspheres can also be modified on the surface to carry various active functional groups, including but not limited to-OH, -COOH and-NH2。
In one embodiment, the magnetic sphere is Fe2O3Or Fe3O4A complex of magnetic nanoparticles and an organic polymer material, and having a particle diameter of 0.1 to 5 μm.
According to the present invention, the antibody may be a monoclonal antibody or a polyclonal antibody.
The present invention also provides a method for preparing a kit as described above, comprising the steps of: i) directly or indirectly labeling protein A or directly or indirectly labeling anti-human IgG and anti-human IgM by using a tracer label; and ii) coating the thyroid microsomal antigen (or its conjugate to a protein carrier) directly or indirectly with a magnetic bead; wherein, the indirect labeling comprises labeling a tracer marker with protein A or anti-human IgG and anti-human IgM through a fluorescein isothiocyanate and anti-fluorescein isothiocyanate antibody system or a streptavidin and biotin system; the indirect coating comprises indirectly coating the thyroid microsome antigen (or the conjugate thereof with a protein carrier) with magnetic spheres through a fluorescein isothiocyanate and anti-fluorescein isothiocyanate antibody system or a streptavidin and biotin system.
The preparation method of the kit according to the invention can also comprise the preparation of the low-point calibrator and the high-point calibrator, and can further comprise the assembly of the kit.
According to the present invention, there is also provided a method for detecting TMA concentration, which comprises detecting TMA concentration in a sample to be tested by a chemiluminescence immunoassay using the kit as described above.
Specifically, the method for detecting the concentration of TMA provided by the invention comprises the steps of mixing a sample to be detected, protein A directly or indirectly marked with a tracer marker or anti-human IgG and anti-human IgM marked with a tracer marker, and TMA directly or indirectly coated with a magnetic sphere or a connector of TMA and a protein carrier, enabling the TMA, the protein A or the anti-human IgG to be detected, the anti-human IgM and the TMA coated with the magnetic sphere or the connector of TMA and the protein carrier to form a sandwich-shaped immune complex, adding an excitation substrate, measuring the luminous intensity, and calculating the TMA concentration of the sample to be detected by referring to a standard curve of the concentration of a TMA calibrator and the luminous intensity.
In one embodiment, the method for detecting TMA comprises detecting TMA concentration by a chemiluminescent immunoassay analyzer using a kit as described above provided according to the present invention. In a preferred embodiment of the invention, the process is carried out fully automatically. According to the present invention, the chemiluminescence immunoassay analyzer is preferably a Maglumi series chemiluminescence immunoassay analyzer (produced by Shenzhen New Industrial biomedical engineering, Inc.).
The invention has the beneficial effects that:
1. high specificity, good sensitivity, high accuracy and wider detection range.
2. The operation is simpler and more convenient, and meanwhile, the kit can be matched with a chemiluminescence immunoassay analyzer (especially a Magummi series chemiluminescence immunoassay analyzer) for use, so that full automation is realized in the sample determination process, the detection of the TMA concentration can be simply, conveniently, quickly and massively carried out, and the detection system error is ensured to be smaller.
3. The invention detects TMA by chemiluminescence immunoassay, which avoids the defects of long detection time, low efficiency, easy cause of large error of experimental result, easy influence of external interference factors, low specificity, poor sensitivity and the like of the conventional enzyme-linked immunoassay; and the reagent does not need to be bridged by enzyme, so the reagent has long storage time and is stable.
Drawings
FIG. 1 is a graph comparing experimental results data of example 1 and example 2;
FIG. 2 is a graph comparing the data of the experimental results of example 1 and example 3;
FIG. 3 is a graph comparing the data of the experimental results of example 1 and example 4.
Detailed Description
The invention will be further explained and illustrated with reference to the following non-limiting examples. It should be noted, however, that the following detailed description of the present invention is not intended to limit the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.
In the following examples:
protein A is purchased from Shenzhen Yazhi Biotech GmbH;
anti-human IgG and anti-human IgM labeled with tracer markers were purchased from Medix
TM antigens were purchased from Meridian, USA;
goat anti-FITC polyclonal antibody was purchased from Jackson, usa;
the magnetic microspheres and the ABEI are both produced by Shenzhen New Industrial biomedical corporation Limited;
FITC was purchased from Sigma, USA;
biotin and streptavidin were both available from Biosources, USA;
TMA calibrants were purchased from Biodesign corporation, usa;
maglumi 2000 chemiluminescence immunoassay analyzer: shenzhen new industry biomedical engineering shares, Ltd.
A sample to be detected: 120 clinical serum samples from Hospital of Shenzhen, north Dazhen, at least 7 of which were serum samples from patients diagnosed with thyroid function disease.
Example 1
(1) Labelling of protein A
Preparation of dialysate (solution F): adding Na into 5000ml beaker2CO314.31g，NaHCO326.46g, adding purified water to dilute to 4500 ml. The prepared F solution is placed on a magnetic stirrer for standby.
Selecting a dialysis bag with a proper interception amount (14000 is commonly used), measuring the dialysis bag with a proper size, tightening one end after wetting, and testing leakage of purified water for 3 times (without leakage).
Mu.g of protein A was adjusted to 1ml with 0.1mol/L of a carbonic acid buffer solution (solution F) having a pH of 9.5. The resulting solution was added to a dialyzate, and dialyzed at room temperature for 2 hours with stirring, and 300. mu.g of an ABEI activated ester was added to the dialyzed solution to react at 37 ℃ for 2 hours.
The ligation products of protein A and ABEI were purified by G-25 gel column.
D2Preparing a solution: 200ml of 0.5M phosphate buffer (P001 solution), 20g of BSA, 8g of NaN were added3、2gMgCl2·6H2O, 600ml of glycerin, adding purified water to reach the constant volume of 2000ml, and filtering.
The purified ligation product was used as D2The solution was diluted to a concentration of 0.025. mu.g/ml based on protein A.
(2) Coating of TM antigens
Preparation of solution A: 2.55g of sodium acetate trihydrate are weighed, dissolved in 4500ml of purified water, then 14ml of acetic acid is added and mixed evenly, and the volume is adjusted to 5000ml (pH is 3.6).
Adding the solution A with the volume 5 times of the coating volume into a small white bottle, putting the bottle into an ultrasonic instrument, stirring and cleaning the bottle for 2 to 3 minutes while carrying out ultrasonic treatment, then placing the bottle on a magnet, and pouring out the supernatant after the supernatant is clear. This step was repeated three times.
Adding the magnetic spheres into an acetic acid buffer solution with pH3.6 equal to the coating volume to ensure that the suspension concentration of the magnetic spheres is 20mg/ml, adding 1-cyclohexyl-2-morpholine ethyl carbodiimide p-toluenesulfonate (CMC) to ensure that the concentration is 10mg/ml, and adding purified TM antigen according to a certain ratio.
The mixed solution of the magnetic ball and the TM antigen is put into a constant-temperature shaking water bath box to react for 24 hours at 37 ℃ (shaking speed of a shaking water bath: 260 rpm).
Preparing Phosphate Buffer Solution (PBS) with the pH value of 7.4, adding 2.5g of BSA for dissolving, and uniformly mixing to obtain the magnetic ball cleaning solution.
Pouring the mixed solution of the magnetic ball and the TM antigen which are well bathed in the warm water into a beaker, then placing the beaker on a magnet for precipitation, pouring out the supernatant, adding 5 times of magnetic ball cleaning solution for stirring and cleaning, then placing the beaker on the magnet, pouring out the supernatant after the supernatant is clear, and repeating the cleaning step for four times.
C, preparing a solution: 160g of Methylcellulose (MC) is weighed and poured into a 5000ml beaker, purified water is added to 4000ml, and the mixture is heated and stirred for dissolving for 2 hours in a water bath cabinet at the temperature of 90 ℃. Another 4000ml of 0.5M phosphate buffer was added with 80g of NaN3(analytically pure), 80ml of T-20 (analytically pure), mixing, and filtering. The two solutions were mixed well and 200g BSA was added and water was added to 40000 ml.
And (3) suspending the cleaned magnetic spheres in the solution C, wherein the suspension concentration is 20mg/ml, so as to obtain the magnetic spheres coated with the TM antigen, and the volume of the suspension is the coating volume of the embodiment.
The suspension of magnetic beads coated with TM antigen was further diluted to 0.5mg/ml for use.
(3) TMA low-point calibrator and high-point calibrator
TMA standard was diluted with calibrator dilutions (50% bovine serum preparation) to two high and low calibrator calibration points with concentrations of 591.58IU/ml and 25.36IU/ml, respectively.
(4) Reaction: and mixing the sample to be detected and the high-low point calibrator with a marker system and a magnetic sphere system respectively, and incubating to combine the thyroid microsome antibody in the sample to be detected with the ABEI-labeled protein A to obtain a reaction product.
The concrete sample adding steps are as follows: mu.l of the sample to be tested or the high and low spot calibrator was added, and then 20. mu.l of the magnetic sphere solution coated with the TM antigen (prepared in step (2) above) was added, followed by washing after 10 minutes of reaction, and 100. mu.l of the ABEI-labeled protein A solution (prepared in step (1) above) was added to form a complex.
(5) And (3) detection: the reaction product is precipitated by an external magnetic fieldPrecipitating, removing supernatant, washing with buffer solution, adding chemiluminescence excitant (NaOH and H)2O2) And detecting the emitted relative light intensity by using a Maglumi 2000 chemiluminescence immunoassay analyzer (the specific operation steps can refer to the specification of the Maglumi 2000 chemiluminescence immunoassay analyzer), and calculating to obtain the TMA content of the sample to be detected. The results are shown in Table 1.
Example 2
(1) Protein A biotinylation comprises the following specific steps:
preparation of dialysate (solution F): adding Na into 5000ml beaker2CO314.31g，NaHCO326.46g, adding purified water to dilute to 4500 ml. The prepared F solution is placed on a magnetic stirrer for standby.
Mu.g of biotin and 1mg of protein A were taken and adjusted to 1ml with 0.1mol/L of a carbonic acid buffer (solution F) having a pH of 9.5. The resulting mixture was added to a dialysate, and dialyzed at room temperature for 2 hours with stirring, followed by reaction at 37 ℃ for 2 hours.
Purifying by G-25 gel column.
A solution of D2 was prepared according to the method of example 1, and the purified ligation product was purified using D2The solution was diluted to a concentration of 0.025. mu.g/ml.
(2) Labeling of SA
100. mu.g of SA was adjusted to 1ml with 0.1mol/L of pH9.5 carbonate buffer (solution F). The resulting solution was added to a dialyzate, and dialyzed at room temperature for 2 hours with stirring, and 300. mu.g of an ABEI activated ester was added to the dialyzed solution to react at 37 ℃ for 2 hours.
The ligation products of SA and ABEI were purified using a G-25 gel column.
Purified ligation product D2The solution was diluted to a concentration of 10. mu.g/ml based on SA and was ready for use.
(3) TM antigen coated magnetic ball
Solution A was prepared as in example 1.
Adding the solution A with the volume 5 times of the coating volume into a small white bottle, putting the bottle into an ultrasonic instrument, stirring and cleaning the bottle for 2 to 3 minutes while carrying out ultrasonic treatment, then placing the bottle on a magnet, and pouring out the supernatant after the supernatant is clear. This step was repeated three times.
The magnetic beads were added to the same coating volume of pH3.6 acetate buffer to give a magnetic bead suspension concentration of 20mg/ml, CMC was added to give a concentration of 10mg/ml, and purified TM antigen was added.
The magnetic ball suspension is put into a constant-temperature shaking water bath box to react for 24 hours at 37 ℃ (shaking speed of a shaking water bath: 260 rpm).
500ml of pH7.4PBS buffer solution is prepared, 2.5g of BSA is added and mixed and dissolved, thus obtaining the magnetic ball cleaning solution.
Pouring the magnetic ball suspension liquid after warm bath into a beaker, then placing the beaker on a magnet for precipitation, pouring out the supernatant, adding 5 times of magnetic ball cleaning liquid for stirring and cleaning, then placing the beaker on the magnet, pouring out the supernatant after the supernatant is clear, and repeating the cleaning step for four times.
Suspending the washed magnetic spheres coated with the TM antigen in the solution C prepared according to the example 1, wherein the suspension concentration is 20mg/ml, and obtaining the magnetic sphere suspension coated with the TM antigen, and the volume of the suspension is the coating volume in the step.
The magnetic sphere suspension was further diluted to 0.5mg/ml for use.
(4) TMA low-point calibrator and high-point calibrator
TMA antigen was diluted with 50% bovine serum preparation at different ratios to two high and low calibrator points at concentrations of 591.58IU/ml and 15.298 IU/ml.
(5) Reaction: and mixing the sample to be detected or the high-low point calibrator with the marker system and the magnetic microsphere system respectively, and incubating to combine the thyroid microsome antibody in the sample to be detected with the biotinylated protein A to obtain a reaction product. The concrete sample adding steps are as follows: after 10 minutes of reaction, 50. mu.l of SA solution (prepared in step (2) above) and 50. mu.l of biotinylated protein A solution (prepared in step (1) above) were added to the reaction mixture, and a complex was formed.
(6) And (3) detection: precipitating the reaction product with an external magnetic field, removing supernatant, washing with buffer solution, and adding chemiluminescence excitant (NaOH and H)2O2) And detecting the emitted relative light intensity by using a Maglumi 2000 chemiluminescence immunoassay analyzer, and calculating to obtain the TMA content in the sample to be detected. The results are shown in Table 1.
Example 3
(1) Labelling of protein A
Preparation of dialysate (solution F): adding Na into 5000ml beaker2CO314.31g，NaHCO326.46g, adding purified water to dilute to 4500 ml. The prepared F solution is placed on a magnetic stirrer for standby.
Selecting a dialysis bag with a proper interception amount (14000 is commonly used), measuring the dialysis bag with a proper size, tightening one end after wetting, and testing leakage of purified water for 3 times (without leakage).
Mu.g of protein A was adjusted to 1ml with 0.1mol/L of a carbonic acid buffer solution (solution F) having a pH of 9.5. The resulting solution was added to a dialyzate, and dialyzed at room temperature for 2 hours with stirring, and 300. mu.g of an ABEI activated ester was added to the dialyzed solution to react at 37 ℃ for 2 hours.
The ligation products of protein A and ABEI were purified by G-25 gel column.
D2Preparing a solution: 200ml of 0.5M phosphate buffer (P001 solution), 20g of BSA, 8g of NaN were added3、2gMgCl2·6H2O, 600ml of glycerin, adding purified water to reach the constant volume of 2000ml, and filtering.
The purified ligation product was used as D2The solution was diluted to a concentration of 0.025. mu.g/ml based on protein A.
(2) FITC labeling of TM antigens
100. mu.g of the TM antigen was adjusted to 1ml with 0.1mol/L of a carbonic acid buffer solution (solution F) having a pH of 9.5. The resulting mixture was added to a dialyzate, dialyzed at room temperature for 2 hours with stirring, and added to 300. mu.g of FITC-activated ester to react at 37 ℃ for 2 hours.
The ligation product of TM antigen and FITC was purified by G-25 gel column.
The purified ligation product was used as D2The solution was diluted to a concentration of 0.1. mu.g/ml based on the TM antigen.
(3) Goat anti-FITC polyclonal antibody coated magnetic ball
2.55g of sodium acetate trihydrate are weighed, dissolved in 4500ml of purified water, then added with, for example, 14ml of acetic acid, mixed evenly, and then added with purified water to reach 5000ml (pH 3.6).
Adding the solution A with the volume 5 times of the coating volume into a small white bottle, putting the bottle into an ultrasonic instrument, stirring and cleaning the bottle for 2 to 3 minutes while carrying out ultrasonic treatment, then placing the bottle on a magnet, and pouring out the supernatant after the supernatant is clear. This step was repeated three times.
Adding the magnetic spheres into an equal coating volume of pH3.6 acetic acid buffer solution to ensure that the suspension concentration of the magnetic spheres is 20mg/ml, adding CMC to ensure that the concentration is 10mg/ml, and adding the purified goat anti-FITC polyclonal antibody according to a certain ratio.
The mixed solution of the magnetic ball and the goat anti-FITC polyclonal antibody is put into a constant-temperature shaking water bath box to react for 24 hours at 37 ℃ (shaking speed of a shaking water bath: 260 rpm).
And (3) preparing pH7.4PBS buffer solution, adding 2.5g of BSA for dissolving, and uniformly mixing to obtain the magnetic ball cleaning solution.
Pouring the mixed solution of the magnetic balls and the goat anti-FITC polyclonal antibody which are well bathed in the warm water into a beaker, then placing the beaker on a magnet for precipitation, pouring off the supernatant, adding 5 times of magnetic ball cleaning solution for stirring and cleaning, then placing the beaker on the magnet, pouring off the supernatant after the supernatant is clear, and repeating the cleaning step for four times.
C, preparing a solution: 160g of Methylcellulose (MC) is weighed and poured into a 5000ml beaker, purified water is added to 4000ml, and the mixture is heated and stirred for dissolving for 2 hours in a water bath cabinet at the temperature of 90 ℃. Another 4000ml of 0.5M phosphate buffer was added with 80g of NaN3(analytically pure), 80ml of T-20 (analytically pure), mixing, and filtering. The two solutions were mixed well and 200g BSA was added and water was added to 40000 ml.
Suspending the magnetic spheres coated by the goat anti-FITC polyclonal antibody in the solution C prepared in the step (3) at a suspension concentration of 20mg/ml to obtain a goat anti-FITC polyclonal antibody coated magnetic sphere suspension, wherein the volume of the suspension is the coating volume in the step.
The suspension of goat anti-FITC polyclonal antibody coated magnetic beads was further diluted to 1mg/ml for use.
(4) TMA low-point calibrator and high-point calibrator
The TMA standard is diluted by a calibrator diluent and a 50% bovine serum product according to different proportions to form two high and low calibrator calibration points with the concentrations of 591.58IU/ml and 25.36IU/ml respectively.
(5) Reaction: and mixing the sample to be detected or the high-low point calibrator with a marker system and a magnetic microsphere system respectively, and incubating to combine the thyroid microsome antibody in the sample to be detected with the marked protein A to obtain a reaction product. The concrete sample adding steps are as follows: adding 10 mu l of sample to be detected or high-low point calibrator, adding 40 mu l of FITC solution for marking TM antigen (prepared in the step (2) above), adding 20 mu l of magnetic sphere solution coated by goat anti-FITC polyclonal antibody (prepared in the step (3) above), reacting for 10 minutes, washing and adding ABEI marked protein A solution (prepared in the step (1) above), and forming the complex.
(6) And (3) detection: adding magnetic field to precipitate the reaction product, removing supernatant, washing with buffer solution, and adding chemiluminescenceElicitor (NaOH and H)2O2) And detecting the emitted relative light intensity by using a Maglumi 2000 chemiluminescence immunoassay analyzer, and calculating to obtain the TMA content in the sample to be detected. The results are shown in Table 1.
Example 4
(1) Labeling of anti-human IgG and anti-human IgM
(1.1) labeling of anti-human IgG
Preparation of dialysate (solution F): adding Na into 5000ml beaker2CO314.31g，NaHCO326.46g, adding purified water to dilute to 4500 ml. The prepared F solution is placed on a magnetic stirrer for standby.
Selecting a dialysis bag with a proper interception amount (14000 is commonly used), measuring the dialysis bag with a proper size, tightening one end after wetting, and testing leakage of purified water for 3 times (without leakage).
Mu.g of anti-human IgG was adjusted to 1ml with 0.1mol/L of pH9.5 carbonate buffer (solution F). The resulting solution was added to a dialyzate, and dialyzed at room temperature for 2 hours with stirring, and 300. mu.g of an ABEI activated ester was added to the dialyzed solution to react at 37 ℃ for 2 hours.
The ligation products of anti-human IgG and ABEI were purified by G-25 gel column.
D2Preparing a solution: 200ml of 0.5M phosphate buffer (P001 solution), 20g of BSA, 8g of NaN were added3、2gMgCl2·6H2O, 600ml of glycerin, adding purified water to reach the constant volume of 2000ml, and filtering. The purified ligation product was used as D2The solution was diluted so that the concentration thereof was 0.025. mu.g/ml as anti-human IgG.
(1.2) labeling of anti-human IgM
Preparation of dialysate (solution F): adding Na into 5000ml beaker2CO314.31g，NaHCO326.46g, adding purified water to dilute to 4500 ml. The prepared F solution is placed on a magnetic stirrer for standby.
Selecting a dialysis bag with a proper interception amount (14000 is commonly used), measuring the dialysis bag with a proper size, tightening one end after wetting, and testing leakage of purified water for 3 times (without leakage).
Mu.g of anti-human IgM was adjusted to 1ml with 0.1mol/L of a carbonic acid buffer solution (solution F) having a pH of 9.5. The resulting solution was added to a dialyzate, and dialyzed at room temperature for 2 hours with stirring, and 300. mu.g of an ABEI activated ester was added to the dialyzed solution to react at 37 ℃ for 2 hours.
The ligation products of anti-human IgM with ABEI were purified by G-25 gel column.
D2Preparing a solution: 200ml of 0.5M phosphate buffer (P001 solution), 20g of BSA, 8g of NaN were added3、2gMgCl2·6H2O, 600ml of glycerin, adding purified water to reach the constant volume of 2000ml, and filtering.
The purified ligation product was used as D2The solution was diluted so that the concentration thereof was 0.025. mu.g/ml in terms of anti-human IgM.
(2) Coating of TM antigens
Preparation of solution A: 2.55g of sodium acetate trihydrate are weighed, dissolved in 4500ml of purified water, then 14ml of acetic acid is added and mixed evenly, and the volume is adjusted to 5000ml (pH is 3.6).
Adding the solution A with the volume 5 times of the coating volume into a small white bottle, putting the bottle into an ultrasonic instrument, stirring and cleaning the bottle for 2 to 3 minutes while carrying out ultrasonic treatment, then placing the bottle on a magnet, and pouring out the supernatant after the supernatant is clear. This step was repeated three times.
Adding the magnetic spheres into an acetic acid buffer solution with pH3.6 equal to the coating volume to ensure that the suspension concentration of the magnetic spheres is 20mg/ml, adding 1-cyclohexyl-2-morpholine ethyl carbodiimide p-toluenesulfonate (CMC) to ensure that the concentration is 10mg/ml, and adding purified TM antigen according to a certain ratio.
The mixed solution of the magnetic ball and the TM antigen is put into a constant-temperature shaking water bath box to react for 24 hours at 37 ℃ (shaking speed of a shaking water bath: 260 rpm).
Preparing Phosphate Buffer Solution (PBS) with the pH value of 7.4, adding 2.5g of BSA for dissolving, and uniformly mixing to obtain the magnetic ball cleaning solution.
Pouring the mixed solution of the magnetic ball and the TM antigen which are well bathed in the warm water into a beaker, then placing the beaker on a magnet for precipitation, pouring out the supernatant, adding 5 times of magnetic ball cleaning solution for stirring and cleaning, then placing the beaker on the magnet, pouring out the supernatant after the supernatant is clear, and repeating the cleaning step for four times.
C, preparing a solution: 160g of Methylcellulose (MC) is weighed and poured into a 5000ml beaker, purified water is added to 4000ml, and the mixture is heated and stirred for dissolving for 2 hours in a water bath cabinet at the temperature of 90 ℃. Another 4000ml of 0.5M phosphate buffer was added with 80g of NaN3(analytically pure), 80ml of T-20 (analytically pure), mixing, and filtering. The two solutions were mixed well and 200g BSA was added and water was added to 40000 ml.
And (3) suspending the cleaned magnetic spheres in the solution C, wherein the suspension concentration is 20mg/ml, so as to obtain the magnetic spheres coated with the TM antigen, and the volume of the suspension is the coating volume of the embodiment.
The suspension of magnetic beads coated with TM antigen was further diluted to 0.5mg/ml for use.
(3) TMA low-point calibrator and high-point calibrator
TMA standard was diluted with calibrator dilutions (50% bovine serum preparation) to two high and low calibrator calibration points with concentrations of 591.58IU/ml and 25.36IU/ml, respectively.
(4) Reaction: and mixing the sample to be detected and the high-low point calibrator with a marker system and a magnetic sphere system respectively, and incubating to combine the thyroid microsome antibody in the sample to be detected with the ABEI-labeled anti-human IgG and anti-human IgM to obtain a reaction product.
The concrete sample adding steps are as follows: mu.l of the sample to be tested or the high and low point calibrator was added, 20. mu.l of the magnetic sphere solution coated with the TM antigen (prepared in step (2) above) was added, and after reacting for 10 minutes, washing was performed, and 50. mu.l each of the ABEI solutions labeled with anti-human IgG and anti-human IgM (prepared in step (1) above) was added to form a complex.
(5) And (3) detection: precipitating the reaction product with an external magnetic field, removing supernatant, washing with buffer solution, and adding chemiluminescence excitant (NaOH and H)2O2) And detecting the emitted relative light intensity by using a Maglumi 2000 chemiluminescence immunoassay analyzer (the specific operation steps can refer to the specification of the Maglumi 2000 chemiluminescence immunoassay analyzer), and calculating to obtain the TMA content of the sample to be detected. The results are shown in Table 1.
Comparative example 1
The 120 samples to be detected are detected by adopting the existing mainstream commercial enzyme linked immunosorbent assay kit on the market, and compared with the detection result of the kit provided by the invention. The results are shown in Table 1 and FIGS. 1 to 3.
TABLE 1
*: through statistical analysis, the judgment threshold value is as follows: normal people is less than 10IU/ml, and the positive is more than or equal to 10 IU/ml; "+" indicates positive, and "-" indicates negative (normal).
From the above table and fig. 1 to 3, 120 clinical samples selected in the clinical comparative test at this time, of which at least sample nos. 3, 14, 28, 39, 50, 62, and 118 are patients with thyroid function diseases for confirmed diagnosis, can be obtained. For other samples, the detection results of the kit of the invention and the enzyme immunoassay kit are consistent. However, for the seven diagnosed patient samples, the TMA in the enzyme immunoassay test is negative, and the TMA in the kit and the chemiluminescence test method provided by the present invention is positive, which means that the technical solution provided by the present invention can greatly avoid the omission of TMA in the enzyme immunoassay platform. Furthermore, as can be seen from FIG. 1, the data in examples 1 and 4 are relatively consistent, indicating that anti-human IgG and anti-human IgM labeled with labeled ABEI were consistently effective with protein A labeled with ABEI. As can be seen from FIGS. 1 to 3, the results of the measurement using the kits of examples 1 to 4 have good consistency. Therefore, the TMA detection kit and the detection method provided by the invention have the advantages of more accurate, sensitive and specific detection results, and can reflect the real clinical situation, thereby having higher application value.
Although the present invention has been described in detail, modifications within the spirit and scope of the invention will be apparent to those skilled in the art. Further, it should be understood that the various aspects recited herein, portions of different embodiments, and various features recited may be combined or interchanged either in whole or in part. In the various embodiments described above, those embodiments that refer to another embodiment may be combined with other embodiments as appropriate, as will be appreciated by those skilled in the art. Furthermore, those skilled in the art will appreciate that the foregoing description is by way of example only, and is not intended to limit the invention.
Claims (8)
1. A chemiluminescence immunoassay kit for detecting thyroid microsome antibodies comprises protein A marked with a tracer marker, and thyroid microsome antigen coated with magnetosphere or a connector of the thyroid microsome antigen coated with magnetosphere and a protein carrier,
the tracer marker is selected from at least one of luminol and derivatives thereof, isoluminol and derivatives thereof and acridinium ester;
the magnetic ball is Fe2O3Or Fe3O4Magnetic nanoparticles and nanoparticles ofA composite of organic polymer materials.
2. The kit of claim 1, wherein the protein carrier is selected from at least one of bovine serum albumin, human serum albumin, rabbit serum albumin, hemocyanin, bovine IgG, human IgG, ovalbumin, myoglobin, and thyroglobulin.
3. The kit of claim 1, wherein the tracer label is N- (4-aminobutyl) -N-ethyl isoluminol.
4. The kit according to claim 1,
the tracer marker directly or indirectly marks the protein A, wherein the indirect marking comprises indirect marking through a fluorescein isothiocyanate and anti-fluorescein isothiocyanate antibody system or a streptavidin and biotin system;
the thyroid microsome antigen or the connector of the thyroid microsome antigen and the protein carrier directly or indirectly coats the magnetic spheres, wherein the indirect coating comprises indirect coating through a fluorescein isothiocyanate and anti-fluorescein isothiocyanate antibody system or streptavidin and a biotin system.
5. The kit according to claim 1, wherein the magnetic spheres have a particle size of 0.1-5 μm.
6. The kit of claim 4, comprising a protein A antigen labeled with a tracer marker and one component selected from the group consisting of:
a) thyroid microsome antigen directly coating magnetic sphere or connector of thyroid microsome antigen directly coating magnetic sphere and protein carrier;
b) biotinylated thyroid microsomal antigen or a conjugate of thyroid microsomal antigen and a protein carrier, and streptavidin directly coating a magnetic sphere;
c) thyroid microsome antigen directly labeled with fluorescein isothiocyanate or a conjugate of the thyroid microsome antigen and a protein carrier, and goat anti-fluorescein isothiocyanate antibody directly coating a magnetic ball.
7. The kit of any one of claims 1 to 6, wherein the kit further comprises a low point calibrator and a high point calibrator for thyroid microsomal antibodies, and optionally a buffer.
8. A method for preparing a kit according to any one of claims 1 to 7, comprising the steps of:
i) directly or indirectly labeling the protein A by using a tracer label; and
ii) coating the magnetic beads directly or indirectly with thyroid microsomal antigens or with a conjugate of thyroid microsomal antigens and a protein carrier;
wherein,
the indirect labeling comprises that the tracing label is used for labeling protein A through a fluorescein isothiocyanate and anti-fluorescein isothiocyanate antibody system or a streptavidin and biotin system,
the indirect coating comprises indirectly coating the thyroid microsome antigen or the connector of the thyroid microsome antigen and a protein carrier with a magnetic sphere through a fluorescein isothiocyanate and anti-fluorescein isothiocyanate antibody system or a streptavidin and biotin system.
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| CN201510068327.7A CN104614535B (en) | 2015-02-10 | 2015-02-10 | TMA detection kit and its preparation method and application |
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| CN100389327C (en) * | 2004-06-22 | 2008-05-21 | 北京倍爱康生物技术股份有限公司 | Magnetic separation enzyme-linked immune detecting method for thyroid gland peroxidase antibody |
| CN1928560A (en) * | 2006-05-26 | 2007-03-14 | 深圳市新产业生物医学工程有限公司 | Immune analysis reagent for magnetic separation alkali phosphatase enzyme mark and analytical detection method for same |
| JP5805190B2 (en) * | 2010-08-19 | 2015-11-04 | エフ．ホフマン−ラ ロシュ アーゲーＦ． Ｈｏｆｆｍａｎｎ−Ｌａ Ｒｏｃｈｅ Ａｋｔｉｅｎｇｅｓｅｌｌｓｃｈａｆｔ | Assays for the measurement of antibodies that bind to therapeutic monoclonal antibodies |
| CN102072957B (en) * | 2011-01-18 | 2013-08-07 | 威海威高生物科技有限公司 | Hepatitis C virus antibody diagnostic kit and preparation method thereof |
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| CN103336115A (en) * | 2013-07-17 | 2013-10-02 | 江阴泽成生物技术有限公司 | Magnetic particle chemiluminescence immune assay kit for TPOAb and detecting method thereof |
| CN104237513A (en) * | 2014-09-30 | 2014-12-24 | 博奥赛斯（天津）生物科技有限公司 | Thyroid peroxidase antibody magnetic-particle chemiluminescence immune quantitative testing kit |
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