CN110907653B - Kit for detecting thyroid stimulating hormone, preparation method and application thereof - Google Patents
Kit for detecting thyroid stimulating hormone, preparation method and application thereof Download PDFInfo
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- CN110907653B CN110907653B CN201911244704.2A CN201911244704A CN110907653B CN 110907653 B CN110907653 B CN 110907653B CN 201911244704 A CN201911244704 A CN 201911244704A CN 110907653 B CN110907653 B CN 110907653B
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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
- G01N33/74—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
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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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/588—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with semiconductor nanocrystal label, e.g. quantum dots
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/575—Hormones
- G01N2333/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g. HCG; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
Abstract
The invention relates to the technical field of in-vitro reagent diagnosis, and discloses a kit for detecting thyroid stimulating hormone antibody, a preparation method and application thereof, wherein the kit comprises one or more than two components of a thyroid stimulating hormone antibody-quantum dot 1 cross-linked substance, a thyroid stimulating hormone antibody-quantum dot 2 cross-linked substance marked by alkaline phosphatase, a thyroid stimulating hormone antibody marked by alkaline phosphatase-quantum dot 3 cross-linked substance and a bovine serum albumin-quantum dot 4 cross-linked substance, which are coated by a solid phase carrier. The quantum dots are selected as the cross-linked luminescent material of the main component of the thyroid stimulating hormone kit, and the formed cross-linked substance can conveniently, quickly and accurately obtain the fluorescence state and intensity of the quantum dots by applying exciting light on the premise of not influencing the detection accuracy of the kit, so that the distribution state of a target substance in a reagent is indicated, the reason for abnormal results caused by abnormal samples is judged, and the kit can be applied to the preparation of a thyroid stimulating hormone detection product.
Description
Technical Field
The invention relates to the technical field of in-vitro reagent diagnosis, in particular to a kit for detecting thyroid stimulating hormone antibody and a preparation method and application thereof.
Background
Unlike small-scale production, the determination of the state of mixing is usually visually observed in the industry when large-scale reagent production is carried out. However, the actual homogeneous state may not be obtained by visual observation, and the determination of the blending time and blending manner that are actually required requires many tests or is obtained only by experience, has uncertainty, and is extremely wasteful of manpower and material resources. In the production process, if the product is split-charged in an inhomogeneous state, the quality of the product may be affected, and the prolonged stirring and blending time may reduce the production efficiency and even cause the risk of deterioration.
In vitro diagnostic reagents such as thyroid stimulating hormone usually comprise components such as a solid phase, a labeled conjugate, a diluent and the like, and the existing states of active materials and auxiliary materials in the components in the reagents cannot be generally obtained by visual observation. Since the distribution of a specific component in a reagent may be non-uniform due to storage conditions, environmental factors, or long-term storage, there is no simple and convenient way to observe the distribution of the specific component in the reagent.
Furthermore, interference is inevitable in immunodiagnostics due to the complexity of detecting specimens. Types of interference in immunodiagnostics include cross-reactivity with analyte analogs, interference with endogenous antibodies, interference with binding proteins, interference from pretreatment of the sample, and the like. In addition, matrix components other than the analyte in the sample may also nonspecifically bind to components in the reagent, affecting the immunoassay signal value. For the interference elimination in immunodiagnosis, whether the analysis pretreatment of the specimen has a problem or not is firstly eliminated, and then repeated tests are carried out on different manufacturers, different platforms or other instruments. The method is characterized in that the method comprises the following steps of processing a specimen by using techniques such as heterophilic antibody blocking tubes, PEG precipitation and the like, or verifying the interference reasons by means of gradient dilution, solvent extraction and the like. However, in general, the cause or mode of interference is estimated by confirming a specific interfering factor, by adding an excessively suspected interfering substance or the like to a reagent, or by adding a substance that specifically or non-specifically binds to a suspected interfering factor in a sample, and by detecting a change in a detection signal value after the sample is treated. However, this approach does not clearly "see" the interference factors and lacks an intuitive understanding of the interference situation.
Therefore, two main problems to be solved by the in vitro diagnostic reagent at present are how to observe the distribution state of specific components in the reagent quickly and conveniently and how to display the abnormal results intuitively.
Disclosure of Invention
In view of the above, the invention aims to provide a kit for detecting thyroid stimulating hormone, which can rapidly, conveniently and accurately observe the distribution state of each component of the kit in a reagent on the premise of not influencing the detection of the kit, and is beneficial to quality control;
another object of the present invention is to provide a kit for detecting thyroid stimulating hormone, which enables the kit to visually display the cause of abnormal results caused by abnormal specimens, and to provide applications of the kit in this respect;
the invention also aims to provide the application of the kit in analyzing the pollution condition of each component of the kit of the sample adding needle of the instrument;
in order to achieve the above purpose, the invention provides the following technical scheme:
a kit for detecting thyroid stimulating hormone comprises one or more than two components as follows:
the solid phase carrier is coated with a thyroid stimulating hormone antibody-quantum dot 1 cross-linked substance, an alkaline phosphatase-marked thyroid stimulating hormone antibody-quantum dot 2 cross-linked substance, an alkaline phosphatase-quantum dot 3 cross-linked substance-marked thyroid stimulating hormone antibody and a bovine serum albumin-quantum dot 4 cross-linked substance; the particle sizes of the quantum dots in the components are different.
According to the thyroid stimulating hormone detection kit, each main component of the thyroid stimulating hormone detection kit is crosslinked with the quantum dot, and the fluorescence intensity of the quantum dot is obtained by applying exciting light, so that the degree of association between the fluorescence intensity and a target object is established, the distribution state of each component in a related reagent is visually obtained according to the relative fluorescence intensity, whether a related pollutant exists in an instrument sample adding needle or not can be judged according to the relative fluorescence intensity, and the reason of an abnormal result caused by an abnormal sample is visually analyzed through the change of the fluorescence position, state and intensity.
The spectral characteristics of the quantum dots mainly depend on the radius of the semiconductor nano particles, the smaller the radius is, the wider the energy band is, the blue shift is caused by the absorption band, and the blue fluorescence emitted by the particles is generated; the larger the radius, the red shift of the absorption band is, the particles emit green, yellow, orange, red and other fluorescence, and the quantum dots with different particle sizes can be excited by light with the same wavelength to emit fluorescence with different colors.
Preferably, the present invention selects carboxylated quantum dots, more preferably CdSe quantum dots; in the embodiment of the invention, four CdSe quantum dots with particle sizes of 2.2nm, 3.1nm, 4.5nm and 7.7nm are selected, the emitted light of the CdSe quantum dots under the excitation of a 400nm ultraviolet lamp is blue light, green light, orange light and red light respectively, the emitted light spectrums are basically not overlapped, the same excitation light source can be used for multi-channel synchronous detection, and the fluorescence of different colors can help to distinguish the objects to be observed.
Preferably, the solid phase carrier is a magnetic particle.
In the verification test of the invention, the thyroid stimulating hormone antibody-quantum dot 2 cross-linked substance marked by alkaline phosphatase is taken as a test object, green fluorescent protein with the same function is selected as a control, and the influence of different fluorescent luminescent substances on the actual detection result is verified (whether alkaline phosphatase is influenced or not and the luminescent value of the alkaline phosphatase is related to the detection result is mainly considered), and the result shows that, compared with a negative control, the cross-linked substance does not influence the normal detection of the thyroid stimulating hormone, the luminescent value of the alkaline phosphatase is not influenced, but the luminescent value of the alkaline phosphatase of the cross-linked substance of the green fluorescent protein is reduced by nearly 70 percent and is obviously lower than that of the negative control, which indicates that for a thyroid stimulating hormone detection kit, only proper fluorescent luminescent substances can meet the effect required by the invention.
The components of the kit are main substances for detecting the thyroid stimulating hormone, but in the reagent of the kit, the kit often needs to be combined with some auxiliary materials to form the reagent, and the auxiliary materials are mainly used for preserving, buffering, providing pigments, maintaining the stability of protein and the like, and in the specific implementation mode of the invention, the auxiliary materials are as follows: each 1000 liters of the auxiliary material solution contains PBS pH7.5, bovine serum albumin 3%, gentamicin 10mg and carmine 20 mg. The distribution state of the main substances in the reagent can be observed quickly, conveniently and accurately, and the quality of the product can be controlled; therefore, the invention provides a method for detecting the distribution state of the components of the kit in the reagent, wherein exciting light is applied to the reagent containing the components of the kit, the relative fluorescence intensity results of the reagent are counted at different time points, and the distribution state of the components of the kit in the reagent is judged through the change of the relative fluorescence intensity. The method can detect the distribution state of the target object in the production process or the storage state of the reagent, thereby determining whether to perform the blending operation according to the distribution state and counting the time required by the blending operation.
In the specific embodiment of the invention, the invention provides a method for detecting the distribution state of each component of the kit in a reagent in the production process, the components of the kit are put into a reagent auxiliary material to be stirred, an excitation light source and a fluorescence detection device are arranged at a position far away from the feeding position, and excitation light and fluorescence detection quantity are externally applied at different time points. In the initial feeding stage, the components are not uniformly distributed in the reagent, the detected fluorescence quantity increases along with the increase of time, and then the fluorescence quantity reaches a stable state at a certain time point, so that the target is judged to be in a uniform distribution state, and the time point corresponding to the stable fluorescence state is the time required for the components to reach the uniform state.
Meanwhile, the invention also provides a method for detecting the distribution state of each component of the kit in a reagent in a storage state, wherein the reagent containing each component of the kit is transferred to a room temperature environment from the storage state, quantum dot fluorescence is excited by exciting light, and a photographing and fluorescence detection device is used for observation and recording. And (3) uniformly mixing the reagents, exciting the fluorescence of the quantum dots by exciting light at different time points, and observing and recording by using a photographing and fluorescence detection device. And judging whether the reagent needs to be uniformly mixed or not and the time for uniformly mixing according to the result of the fluorescence intensity. Wherein the storage state includes, but is not limited to, repeated freezing and thawing at-20- -30 deg.C and 2-8 deg.C for multiple times, long-term storage at 37-40 deg.C, and long-term storage at 2-8 deg.C.
When the reagent sampling needle of the detection instrument is a fixed needle, the reagent sampling needle is usually cleaned by washing with purified water, and the cleaning mode often has a poor effect after long-time use or when the instrument is not maintained sufficiently, and the condition is called carrying pollution of the reagent needle. Generally, the judgment of the carrying pollution of the reagent needle needs to be inferred through blank experiments of the carrying pollution kit, and the method cannot judge specific carried objects and carrying quantity and has certain limitation. Whether the reagent needle is carried or not and the carrying amount of the reagent needle can be quickly, simply and conveniently identified through all the components of the quantum dot tracing kit.
The invention also provides a method for detecting the pollution condition of the sample adding needle of the instrument, firstly establishing a standard curve of the concentration and the relative fluorescence intensity of the components in the kit; after the kit is used for detecting the thyroid stimulating hormone, the sample adding needle is washed by water, then exciting light is applied to count the relative fluorescence intensity and the fluorescence color of the sample adding needle, and the relative fluorescence intensity and the fluorescence color are compared with those of a negative control sample adding needle, so that the type and the pollution amount of pollutants are judged.
Generally, when abnormal results of tests are encountered, a clinical laboratory or a manufacturer needs to examine the interference reasons step by step and further process to estimate the interference reasons, but the interference factors cannot be clearly seen, and the specific interference factors can be locked under a fluorescence microscope by tracing the components of the kit, mixing the interference samples with the components and processing the mixture so as to more accurately understand and judge the combination mode and degree of the interference factors. Therefore, the invention also provides a method for analyzing the abnormal result cause caused by the thyroid stimulating hormone abnormal specimen, which comprises the following steps:
step 1, screening abnormal samples from the detected samples, centrifuging the abnormal samples, and taking supernatant for later use;
step 3, mixing the components in the kit with the abnormal sample and the normal sample respectively, and incubating and reacting;
when the interference of an anti-alkaline phosphatase antibody occurs, a thyroid stimulating hormone antibody-thyroid stimulating hormone-alkaline phosphatase labeled thyroid stimulating hormone antibody complex exists on a solid phase carrier, and compared with a sample without the interference, the position, the state and the fluorescence intensity of fluorescence emitted by a thyroid stimulating hormone antibody-quantum dot 1 cross-linked product coated by the solid phase carrier, a thyroid stimulating hormone antibody-quantum dot 2 cross-linked product labeled by alkaline phosphatase and a bovine serum albumin-quantum dot 4 cross-linked product in the kit are not influenced, and the position, the state and the fluorescence intensity of fluorescence emitted by a thyroid stimulating hormone antibody labeled by alkaline phosphatase-quantum dot 3 cross-linked product are obviously changed;
when the interference of endogenous antibodies occurs, the two situations are divided, wherein the situation is that the endogenous antibodies are combined with a thyroid stimulating hormone antibody coated by a solid phase carrier and a thyroid stimulating hormone antibody marked by alkaline phosphatase to form a compound, so that a false positive result is caused; in this case, the thyroid stimulating hormone antibody complex 1 coated with the thyroid stimulating hormone antibody-thyroid stimulating hormone-alkaline phosphatase label and the thyroid stimulating hormone antibody complex 2 coated with the thyroid stimulating hormone antibody-endogenous antibody-alkaline phosphatase label are present on the solid phase carrier at the same time, and compared with a sample without such interference, the position, state and fluorescence intensity of the fluorescence emitted by the thyroid stimulating hormone antibody-quantum dot 1 cross-linked product and the bovine serum albumin-quantum dot 4 cross-linked product coated by the solid phase carrier are not affected, the position and state of fluorescence emitted by the thyroid stimulating hormone antibody marked by alkaline phosphatase-quantum dot 2 cross-linked substance and the thyroid stimulating hormone antibody marked by alkaline phosphatase-quantum dot 3 cross-linked substance are changed, the fluorescence quantity is obviously increased, and correspondingly, the fluorescence quantity is reduced in a liquid phase;
the situation is that the endogenous antibody is respectively combined with a thyroid stimulating hormone antibody coated by a solid phase carrier and a thyroid stimulating hormone antibody marked by alkaline phosphatase, so that a false negative result is caused; in this case, thyroid stimulating hormone antibody complex 1 and thyroid stimulating hormone antibody-endogenous antibody complex 2 both coated with thyroid stimulating hormone antibody-thyroid stimulating hormone-alkaline phosphatase label are present on the solid phase carrier, and with respect to the sample without such interference, the position, state and fluorescence intensity of the fluorescence emitted by the thyroid stimulating hormone antibody-quantum dot 1 cross-linked product and bovine serum albumin-quantum dot 4 cross-linked product coated with the solid phase carrier are not affected, while the position, state and fluorescence amount of the fluorescence emitted by the thyroid stimulating hormone antibody-quantum dot 2 cross-linked product and the thyroid stimulating hormone antibody labeled with alkaline phosphatase-quantum dot 3 cross-linked product are changed, and the fluorescence amount is significantly reduced, and correspondingly, the fluorescence amount thereof is increased in the liquid phase.
Wherein, the abnormal result caused by the abnormal sample is that the test result is not in accordance with clinical symptoms or patterns, or the detection result is not in accordance with a high-level reference method.
In a specific embodiment of the invention, the quantum dot in the thyroid stimulating hormone antibody-quantum dot 1 cross-linked product coated by the solid phase carrier is CdSe, the particle size is 2.2nm, and the emitted light under the excitation of an ultraviolet lamp with the wavelength of 400nm is blue light;
the quantum dots in the thyroid stimulating hormone antibody-quantum dot 2 cross-linked substance marked by alkaline phosphatase are CdSe, the particle size is 3.1nm, and the emitted light under the excitation of an ultraviolet lamp with the wavelength of 400nm is green light;
the quantum dots in the thyroid stimulating hormone antibody marked by the alkaline phosphatase-quantum dot 3 cross-linked substance are CdSe, the particle size is 4.5nm, and the emitted light under the excitation of an ultraviolet lamp with the wavelength of 400nm is orange light;
the quantum dots in the bovine serum albumin-quantum dot 4 cross-linked material are CdSe, the particle size is 7.7nm, and the emitted light under the excitation of a 400nm ultraviolet lamp is red light;
according to the examples of the quantum dots with different particle diameters, the situations of abnormal result reasons are visually presented through a graph 1, wherein A represents a double-antibody sandwich compound formed by a normal sample and a thyroid stimulating hormone antibody coated by a solid phase carrier in a reagent and a thyroid stimulating hormone antibody marked by alkaline phosphatase; b represents the interference of the anti-alkaline phosphatase antibody in the abnormal sample; c and D represent two cases of endogenous antibody interference in the abnormal sample; e represents a solid phase carrier, F represents the solid phase carrier coated with thyroid stimulating hormone antibody cross-linked 2.2nm CdSe, and the emitted light under the excitation of a 400nm ultraviolet lamp is blue fluorescence; g represents thyroid stimulating hormone in the sample; h represents alkaline phosphatase-labeled thyroid stimulating hormone antibody crosslinked 3.1nm CdSe, and emitted light under 400nm ultraviolet lamp excitation is green fluorescence; i represents thyroid stimulating hormone antibody labeled alkaline phosphatase crosslinked 4.5nm CdSe, and emitted light under 400nm ultraviolet lamp excitation light is orange fluorescence; j represents an anti-alkaline phosphatase antibody; k represents an endogenous antibody.
Based on the description of the technical effects and principles, the invention provides the application of one or more of the following aspects of the kit:
the application in the preparation of products for detecting thyroid stimulating hormone;
the application of the detection instrument in the detection of the contamination condition of a sample application needle, wherein the contamination is each component of a kit;
the application of the distribution state of each component of the detection kit in a reagent;
the application of analyzing abnormal result reasons caused by thyroid stimulating hormone abnormal samples.
In addition, the invention also provides a preparation method of the kit, EDC and NHS are used as cross-linking agents, quantum dots with different particle sizes are selected to respectively carry out chemical cross-linking on the thyroid stimulating hormone antibody, alkaline phosphatase and bovine serum albumin, and a thyroid stimulating hormone antibody-quantum dot 1 cross-linked substance, a thyroid stimulating hormone antibody-quantum dot 2 cross-linked substance, an alkaline phosphatase-quantum dot 3 cross-linked substance and a bovine serum albumin-quantum dot 4 cross-linked substance are obtained;
coating the solid phase carrier with a thyroid stimulating hormone antibody-quantum dot 1 cross-linked substance to obtain the solid phase carrier coated with the thyroid stimulating hormone antibody-quantum dot 1 cross-linked substance, marking the thyroid stimulating hormone antibody-quantum dot 2 cross-linked substance with alkaline phosphatase to obtain the thyroid stimulating hormone antibody-quantum dot 2 cross-linked substance marked with phosphatase, and marking the thyroid stimulating hormone antibody with the alkaline phosphatase-quantum dot 3 cross-linked substance to obtain the thyroid stimulating hormone antibody marked with the alkaline phosphatase-quantum dot 3 cross-linked substance.
Preferably, the method for crosslinking the quantum dots by each component comprises the following steps:
adding the carboxylated quantum dots into a reaction container, and respectively adding the components into the reaction container to slowly vibrate and uniformly mix; weighing EDC and NHS, adding into a reaction vessel, shaking at room temperature, mixing uniformly, filtering with a filter membrane after the reaction is finished, and purifying the cross-linked substance.
In a specific embodiment of the invention, the method for crosslinking the quantum dots by the components comprises the following steps:
adding 1uM of carboxylated quantum dots into a reaction container, and respectively adding 0.5mg of each component into the reaction container to slowly shake and uniformly mix for 30 min; weighing 1mg of EDC and 1mg of NHS, adding into a reaction vessel, shaking and mixing uniformly for 2h at room temperature, and filtering with a 0.22um filter membrane after the reaction is finished; the crosslinked product is further purified by passage through an ultrafiltration tube or a chromatography column.
Preferably, the cross-linked product further comprises a step of obtaining a thyroid stimulating hormone antibody-quantum dot 2 cross-linked product and then labeling with alkaline phosphatase to obtain an alkaline phosphatase-thyroid stimulating hormone antibody-quantum dot 2 cross-linked product; wherein, the alkaline phosphatase is marked on the thyroid stimulating hormone antibody.
Preferably, the labeling of alkaline phosphatase is performed by:
weighing alkaline phosphatase, dissolving the alkaline phosphatase in a glutaraldehyde solution, and standing overnight at room temperature; after the reaction, the solution passes through a chromatographic column, elution is carried out, and brown effluent liquid is collected; diluting the thyroid stimulating hormone antibody-quantum dot 2 cross-linked substance with PBS, slowly adding alkaline phosphatase solution (collected brown effluent) under stirring, adding carbonic acid buffer solution, and reacting at room temperature; then NaBH is added4And (3) uniformly mixing the solution, reacting at low temperature, dialyzing the solution overnight after reaction, and filtering by using a filter membrane, wherein the filtrate is the alkaline phosphatase-thyroid stimulating hormone antibody-quantum dot 2 cross-linked substance.
In a specific embodiment of the present invention, the labeling of alkaline phosphatase as described above is performed by:
weighing 50mg of alkaline phosphatase, dissolving in 1% glutaraldehyde solution, and standing at room temperature overnight; eluting the solution after the reaction by a Sephadex G-25 chromatographic column, and collecting brown effluent; diluting 10ul of thyroid stimulating hormone antibody-quantum dot 2 cross-linked substance to 3ml with PBS, slowly adding into alkaline phosphatase solution under stirring, and maintaining for 1 hr; adding 0.3ml of carbonic acid buffer solution with the pH value of 9.5, and reacting for 2 hours at room temperature; 0.1ml of freshly prepared 4mg/ml NaBH was added4Mixing the solution, and reacting at 2-8 deg.C for 2 hr; putting the solution after reaction into a dialysis bag, and dialyzing with PBS (phosphate buffer solution) with pH7.4 overnight; use 0.22And filtering the solution through a uM filter membrane, wherein the filtrate is alkaline phosphatase-thyroid stimulating hormone antibody-quantum dot 2 cross-linked substance.
Preferably, the method for coating the thyroid stimulating hormone antibody-quantum dot 1 cross-linked substance by the solid phase carrier comprises the following steps:
activating the solid phase carrier by EDC and NHS, adding the thyroid stimulating hormone antibody-quantum dot 1 cross-linked substance into the activated solid phase carrier, uniformly mixing by shaking, extracting supernatant, adding a protective solution containing protein, uniformly mixing by shaking, and finally fixing the volume by using the protective solution.
In a specific embodiment of the present invention, the method for coating the thyroid stimulating hormone antibody-quantum dot 1 cross-linked product with the solid phase carrier comprises the following steps:
collecting 500uL stock solution of carboxyl magnetic beads, washing with PBS buffer solution with pH7.5 for 5-10 times, adding 20mg/ml EDC and 20mg/ml NHS to activate for 1h, and washing with PBS buffer solution with pH7.5 for 5-10 times; adding the thyroid stimulating hormone antibody-quantum dot 1 cross-linked substance into the activated magnetic beads, and uniformly mixing for 2h by shaking; and (3) extracting the supernatant, adding a protective solution containing protein, shaking and uniformly mixing for 1h, repeating for 4 times, and fixing the volume to 30ml by using the protective solution.
Preferably, the method for labeling the thyroid stimulating hormone antibody with the alkaline phosphatase-quantum dot 3 cross-linked substance is as follows:
weighing the alkaline phosphatase-quantum dot 3 cross-linked substance, dissolving in glutaraldehyde solution, and standing overnight at room temperature; after the reaction, the solution passes through a chromatographic column, elution is carried out, and brown effluent liquid is collected; diluting thyroid stimulating hormone antibody with PBS, slowly adding alkaline phosphatase-quantum dot 3 cross-linked substance solution (collected brown effluent) under stirring, adding carbonic acid buffer solution, and reacting at room temperature; then NaBH is added4And (3) uniformly mixing the solution, then placing the mixture at a low temperature for reaction, dialyzing the solution overnight after the reaction, and filtering the solution by using a filter membrane, wherein the filtrate is alkaline phosphatase-quantum dot 3 cross-linked substance labeled thyroid stimulating hormone antibody.
In a specific embodiment of the present invention, the method for labeling a thyroid stimulating hormone antibody with the alkaline phosphatase-quantum dot 3 cross-linked substance is as follows:
weighing 50mg of alkaline phosphatase-quantum dot 3 cross-linked substance, dissolving in 1% glutaraldehyde solution, and standing at room temperatureStaying overnight; eluting the solution after the reaction by a Sephadex G-25 chromatographic column, and collecting brown effluent; diluting 10ul of thyroid stimulating hormone antibody to 3ml by PBS, slowly adding the thyroid stimulating hormone antibody into the alkaline phosphatase-quantum dot 3 cross-linked substance solution under the condition of stirring, and keeping the solution for 1 hour; adding 0.3ml of carbonic acid buffer solution with the pH value of 9.5, and reacting for 2 hours at room temperature; 0.1ml of freshly prepared 4mg/ml NaBH was added4Mixing the solution, and reacting at 2-8 deg.C for 2 hr; putting the solution after reaction into a dialysis bag, and dialyzing with PBS (phosphate buffer solution) with pH7.4 overnight; the filtration was performed using a 0.22uM filter and the filtrate was labeled with thyrotropin antibody using alkaline phosphatase-quantum dot 3 cross-linker.
According to the technical scheme, the quantum dots are selected as the cross-linked luminescent material of the main component of the thyroid stimulating hormone kit, and the formed cross-linked material can conveniently, quickly and accurately obtain the fluorescence state and intensity of the quantum dots by applying exciting light on the premise of not influencing the detection accuracy of the kit, so that the distribution state of a target object in a reagent is indicated, the reason for abnormal results caused by abnormal samples is judged, and the kit can be applied to the preparation of thyroid stimulating hormone detection products.
Drawings
FIG. 1 is a schematic diagram showing the interference principle of a thyroid stimulating hormone normal sample, an anti-alkaline phosphatase antibody abnormal sample and an endogenous antibody abnormal sample; wherein A represents a double-antibody sandwich compound formed by a normal sample and a thyroid stimulating hormone antibody coated by a solid phase carrier in a reagent and a thyroid stimulating hormone antibody marked by alkaline phosphatase; b represents the interference of the anti-alkaline phosphatase antibody in the abnormal sample; c and D represent two cases of endogenous antibody interference in the abnormal sample; e represents a solid phase carrier, F represents the solid phase carrier coated with thyroid stimulating hormone antibody cross-linked 2.2nm CdSe, and the emitted light under the excitation of a 400nm ultraviolet lamp is blue fluorescence; g represents thyroid stimulating hormone in the sample; h represents alkaline phosphatase-labeled thyroid stimulating hormone antibody crosslinked 3.1nm CdSe, and emitted light under 400nm ultraviolet lamp excitation is green fluorescence; i represents thyroid stimulating hormone antibody labeled alkaline phosphatase crosslinked 4.5nm CdSe, and emitted light under 400nm ultraviolet lamp excitation light is orange fluorescence; j represents an anti-alkaline phosphatase antibody; k represents an endogenous antibody;
FIG. 2 is a standard curve of the concentration and fluorescence intensity of the alkaline phosphatase-labeled thyroid stimulating hormone antibody-quantum dot 2 cross-linked substance; wherein A represents an alkaline phosphatase-labeled thyroid stimulating hormone antibody-green fluorescent protein cross-linked substance, B represents an alkaline phosphatase-labeled thyroid stimulating hormone antibody-quantum dot 2 cross-linked substance, and C represents an alkaline phosphatase-labeled thyroid stimulating hormone antibody;
FIG. 3 shows the contamination status of a thyroxine antibody detection instrument labeled by quantum dot tracing promoted alkaline phosphatase; wherein A represents an alkaline phosphatase-labeled thyroid stimulating hormone antibody-quantum dot 2 cross-linked product, B represents an alkaline phosphatase-labeled thyroid stimulating hormone antibody-green fluorescent protein cross-linked product, and C represents an alkaline phosphatase-labeled thyroid stimulating hormone antibody.
Detailed Description
The invention discloses a kit for detecting thyroid stimulating hormone antibody, a preparation method and application thereof, and a person skilled in the art can realize the detection by properly improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the kit of the present invention and its preparation and use have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that the techniques of the present invention may be practiced and used with modification, or appropriate modification and combination, of the kit and its preparation and use described herein without departing from the spirit, scope, and spirit of the invention.
The kit for detecting thyroid stimulating hormone antibody provided by the invention, the preparation method and the application thereof are further explained below.
Example 1: thyroid stimulating hormone antibody and CdSe quantum dot crosslinking method
The general procedure for carboxyl-terminal activation and amino-terminal cross-linking using EDC and NHS is well known in the art, i.e.
Adding 10uM of carboxylated quantum dot CdSe into a reaction vessel; adding 5mg thyroid stimulating hormone antibody protein into a reaction container, slowly shaking and uniformly mixing for 30 min; weighing 10mg/ml EDC and 10mg/ml NHS, adding into a reaction vessel, and shaking and mixing uniformly for 2h at room temperature; after the reaction is finished, filtering by using a 0.22um filter membrane; the crosslinked product is further purified by passage through an ultrafiltration tube or a chromatography column.
Chemical crosslinking of other reagents and components with CdSe quantum dots is described above with reference to the above methods.
Example 2: preparation of Components of the kit
1. Magnetic particle coated thyroid stimulating hormone antibody cross-linked CdSe
The chemical crosslinking method of the thyroid stimulating hormone antibody and the quantum dots in the example 1 is the same as that of the CdSe quantum dots with the size of 2.2nm for crosslinking.
The 500uL stock solution of the carboxyl magnetic beads was washed 5 to 10 times with PBS buffer at pH7.5, activated for 1 hour by adding 20mg/ml EDC and 20mg/ml NHS, and washed 5 to 10 times with PBS buffer at pH 7.5.
Adding the thyroid stimulating hormone antibody marked with the CdSe quantum dots into the activated magnetic beads, and uniformly mixing for 2 hours by shaking.
And (3) extracting the supernatant, adding a protective solution containing protein, shaking and uniformly mixing for 1h, repeating for 4 times, and fixing the volume to 30ml by using the protective solution.
2. Alkaline phosphatase-labeled thyroid-stimulating hormone antibody cross-linked CdSe
The CdSe quantum dots with the particle size of 3.1nm are selected for crosslinking, and the marking method is the same as the chemical crosslinking method of the thyroid stimulating hormone antibody and the quantum dots in the embodiment 1.
50mg of alkaline phosphatase was weighed out and dissolved in 1% glutaraldehyde solution, and left to stand at room temperature overnight.
And (4) eluting the solution after the reaction through a Sephadex G-25 chromatographic column, and collecting brown effluent.
10ul of quantum dot crosslinked antibody was diluted to 3ml with PBS and slowly added to the alkaline phosphatase solution with stirring for 1 hour.
0.3ml of a carbonic acid buffer solution having a pH of 9.5 was added thereto, and the reaction was carried out at room temperature for 2 hours.
0.1ml of newly prepared 4mg/ml NaBH4 solution is added, mixed evenly and placed at 2-8 ℃ for reaction for 2 hours.
The solution was put into a dialysis bag and dialyzed overnight against PBS buffer having a pH of 7.4.
The filter is carried out by using a 0.22uM filter membrane, and the filtrate is alkaline phosphatase-labeled thyroid stimulating hormone antibody crosslinked CdSe 3.1 uM.
3. Alkaline phosphatase crosslinked CdSe labeled thyroid stimulating hormone antibody
And (3) selecting a CdSe quantum dot with the wavelength of 4.5nm to perform crosslinking on the alkaline phosphatase to obtain an alkaline phosphatase crosslinked CdSe 4.5um quantum dot compound, wherein the crosslinking method is the same as the chemical crosslinking method of the thyroid stimulating hormone antibody and the quantum dot in the example 1.
Weighing 50mg of the alkaline phosphatase crosslinked CdSe 4.5um quantum dot complex, dissolving in 1% glutaraldehyde solution, and standing overnight at room temperature.
And (4) eluting the solution after the reaction through a Sephadex G-25 chromatographic column, and collecting brown effluent.
10ul of thyroid stimulating hormone antibody was added and diluted to 3ml with PBS, and slowly added to the alkaline phosphatase solution with stirring for 1 hour.
0.3ml of a carbonic acid buffer solution having a pH of 9.5 was added thereto, and the reaction was carried out at room temperature for 2 hours.
0.1ml of newly prepared 4mg/ml NaBH4 solution is added, mixed evenly and placed at 2-8 ℃ for reaction for 2 hours.
The solution was put into a dialysis bag and dialyzed overnight against PBS buffer having a pH of 7.4.
Filtration was performed using a 0.22uM filter and the filtrate was alkaline phosphatase-crosslinked CdSe-labeled thyroid stimulating hormone antibody.
4. Bovine serum albumin crosslinked CdSe
Adding 10uM of carboxylated quantum dot CdSe with the particle size of 7.7nm into a reaction container;
adding 5mg bovine serum albumin into a reaction vessel, and slowly shaking and uniformly mixing for 30 min;
weighing 10mg/ml EDC and 10mg/ml NHS, adding into a reaction vessel, and shaking and mixing uniformly for 2h at room temperature;
after the reaction is finished, filtering by using a 0.22um filter membrane;
the crosslinked product is further purified by passage through an ultrafiltration tube or a chromatography column.
Example 3: standard curve of concentration and fluorescence intensity of alkaline phosphatase-labeled thyroid stimulating hormone antibody-quantum dot cross-linked substance
Preparing 0.05M PBS pH7.40 buffer solution;
alkaline phosphatase-thyroid stimulating hormone antibody (for negative control), alkaline phosphatase-labeled thyroid stimulating hormone antibody-CdSe 3.1um conjugate, and alkaline phosphatase-labeled thyroid stimulating hormone antibody-green fluorescent protein conjugate (the conjugate was prepared according to the quantum dot conjugate method in example 1, using EDC and NHS as the conjugate) were prepared in the above-mentioned buffers at 0.5, 1, 2, 4, 8, and 16mg/ml, respectively.
The solution with each concentration is excited by ultraviolet with the wavelength of 360nm to generate fluorescence, a photographing and fluorescence detection device is used for observation and recording, the data are shown in a table 1, and the corresponding standard curve is shown in a table 2.
TABLE 1
The results in table 1 and fig. 2 show that the relative fluorescence intensity of the cross-linked quantum dots and the cross-linked substance of the green fluorescent protein can be changed with the change of the concentration, i.e., the cross-linked product can indicate the concentration by using the fluorescence intensity.
Example 3: detection accuracy verification of alkaline phosphatase-labeled thyroid stimulating hormone antibody-quantum dot cross-linked substance
Preparing an enzyme conjugate diluent containing 0.05M PBS pH7.40 buffer solution, 1% bovine serum albumin, 0.5% sodium chloride and 0.1% proclin-300;
the enzyme conjugate diluent is used for respectively preparing 1mg/ml alkaline phosphatase-thyroid stimulating hormone antibody (negative control), alkaline phosphatase labeled thyroid stimulating hormone antibody-CdSe 3.1um cross-linked substance and alkaline phosphatase labeled thyroid stimulating hormone antibody-green fluorescent protein cross-linked substance as enzyme conjugate.
Forty clinical samples were tested with the magnetic particle suspension containing thyroid stimulating hormone antibody as the solid phase component and the above three solutions as the enzyme conjugate, and the change in signal value was recorded and analyzed, the data are shown in table 2.
TABLE 2
As is evident from the data in the column of "alkaline phosphatase-thyroid stimulating hormone antibody-quantum dot cross-linked material/negative control" in the data in table 2, the thyroid stimulating hormone antibody labeled with alkaline phosphatase for detection has little influence on the luminescence value of alkaline phosphatase after cross-linking the quantum dot, while the luminescence value of alkaline phosphatase in the alkaline phosphatase-thyroid stimulating hormone antibody-green fluorescent protein cross-linked material is reduced by nearly 70%, which obviously influences the accuracy of actual detection; although the results of example 2 show that the fluorescent substance can be used to indicate the concentration of the components of the kit, the results of this example show that only suitable substances do not affect the emission value of alkaline phosphatase.
Example 4: detecting contamination condition of sample adding needle of full-automatic instrument
Preparing 0.05M PBS pH7.40 buffer solution;
alkaline phosphatase-thyroid stimulating hormone antibody (negative control), alkaline phosphatase-thyroid stimulating hormone antibody-CdSe 3.1um conjugate, and alkaline phosphatase-thyroid stimulating hormone antibody-green fluorescent protein conjugate were prepared in the above-mentioned buffers at concentrations of 0, 5, 10, 50, 100, and 200mg/ml, respectively.
The solutions with the concentrations of the reagents are loaded on a full-automatic instrument by using the reagent components, and a program reagent needle is set to be washed twice by purified water after the reagent needle is loaded.
After washing, the reagent needle is irradiated by ultraviolet with the wavelength of 360nm, a photographing and fluorescence detection device is used for observation and recording, the data is shown in a table 3, and the data is shown in a figure 3 corresponding to the attached figure.
TABLE 3
As can be seen from the results of Table 3 and FIG. 3, when the extreme high concentrations of 50mg/ml alkaline phosphatase-thyroid stimulating hormone antibody-CdSe 3.1um cross-linker, alkaline phosphatase-thyroid stimulating hormone antibody-green fluorescent protein cross-linker were added to the reagent needle, the reagent needle was contaminated with carryover, and the degree of carryover increased with the increase in the concentration of the cross-linker in the reagent.
Example 5: analysis method for abnormal results caused by thyroid stimulating hormone abnormal sample
Screening abnormal samples from the test specimens, taking the abnormal samples out of the blood collection tube, centrifuging at 14000rpm for 10min, and taking the supernatant for later use.
10 normal samples of known undisturbed samples with consistent analyte concentration were screened from the same batch of test samples as the abnormal samples, removed from the blood collection tube, centrifuged at 14000rpm for 10min, and the supernatant was taken for use.
The magnetic particles in the kit are coated with thyroid stimulating hormone antibody crosslinked CdSe cross-linked by 20ul, and 50ul of other reagents are respectively mixed with 100ul of the normal and abnormal samples uniformly and incubated at 37 ℃ for 30 min.
The liquid phase and solid phase (magnetic particles) of the incubated mixture were separated by magnetic force, and 20ul of each was placed on a slide.
The wavelength of the fluorescence microscope excitation light is adjusted to 400nm, four fluorescence is observed in four channels, and the fluorescence position and the fluorescence quantity represent the state of the target object combined with the fluorescence. The following lists the state changes due to the presence of abnormal factors in several specimens (FIG. 1).
When the interference of an anti-alkaline phosphatase antibody occurs, a thyroid stimulating hormone antibody-thyroid stimulating hormone-alkaline phosphatase labeled thyroid stimulating hormone antibody complex exists on the solid phase carrier, and compared with a sample without the interference, the position, the state and the fluorescence intensity of the fluorescence emitted by the thyroid stimulating hormone antibody-quantum dot cross-linked product coated by the solid phase carrier, the thyroid stimulating hormone antibody-quantum dot cross-linked product labeled by alkaline phosphatase and the bovine serum albumin-quantum dot cross-linked product in the kit are not influenced, and the position, the state and the fluorescence intensity of the fluorescence emitted by the thyroid stimulating hormone antibody labeled by alkaline phosphatase-quantum dot cross-linked product are obviously changed;
when the interference of endogenous antibodies occurs, the two situations are divided, wherein the situation is that the endogenous antibodies are combined with a thyroid stimulating hormone antibody coated by a solid phase carrier and a thyroid stimulating hormone antibody marked by alkaline phosphatase to form a compound, so that a false positive result is caused; in this case, a thyroid stimulating hormone antibody complex 1 coated with a thyroid stimulating hormone antibody-thyroid stimulating hormone-alkaline phosphatase label and a thyroid stimulating hormone antibody complex 2 coated with a thyroid stimulating hormone antibody-endogenous antibody-alkaline phosphatase label are present on the solid phase carrier at the same time, and the position, state and fluorescence intensity of fluorescence emitted by the thyroid stimulating hormone antibody-quantum dot cross-linked product and the bovine serum albumin-quantum dot cross-linked product coated with the solid phase carrier are not affected relative to a sample without such interference, the position and state of fluorescence emitted by the thyroid stimulating hormone antibody marked by alkaline phosphatase and the thyroid stimulating hormone antibody marked by the alkaline phosphatase-quantum dot cross-linking substance are changed, the fluorescence quantity is obviously increased, and correspondingly, the fluorescence quantity is reduced in a liquid phase;
the situation is that the endogenous antibody is respectively combined with a thyroid stimulating hormone antibody coated by a solid phase carrier and a thyroid stimulating hormone antibody marked by alkaline phosphatase, so that a false negative result is caused; in this case, a thyroid stimulating hormone antibody complex 1 and a thyroid stimulating hormone antibody-endogenous antibody complex 2 both coated with a thyroid stimulating hormone antibody-thyroid stimulating hormone-alkaline phosphatase label are present on the solid phase carrier, and with respect to a sample without such interference, the position, state and fluorescence intensity of the fluorescence emitted by the thyroid stimulating hormone antibody-quantum dot cross-linked product and the bovine serum albumin-quantum dot cross-linked product coated with the solid phase carrier are not affected, while the position and state of the fluorescence emitted by the thyroid stimulating hormone antibody-quantum dot cross-linked product coated with the alkaline phosphatase label and the thyroid stimulating hormone antibody labeled with the alkaline phosphatase-quantum dot cross-linked product are changed, the fluorescence amount is significantly reduced, and the fluorescence amount is increased in the liquid phase.
Wherein, the abnormal result caused by the abnormal sample is that the test result is not in accordance with clinical symptoms or patterns, or the detection result is not in accordance with a high-level reference method.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A kit for detecting thyroid stimulating hormone is characterized by comprising the following components:
the solid phase carrier is coated with a thyroid stimulating hormone antibody-quantum dot 1 cross-linked substance, an alkaline phosphatase-marked thyroid stimulating hormone antibody-quantum dot 2 cross-linked substance, an alkaline phosphatase-quantum dot 3 cross-linked substance-marked thyroid stimulating hormone antibody and a bovine serum albumin-quantum dot 4 cross-linked substance;
the particle sizes of the quantum dots in the components are different;
the preparation method comprises the following steps:
EDC and NHS are adopted as cross-linking agents, quantum dots with different particle sizes are selected to respectively carry out chemical cross-linking on a thyroid stimulating hormone antibody, alkaline phosphatase and bovine serum albumin, and a thyroid stimulating hormone antibody-quantum dot 1 cross-linked substance, a thyroid stimulating hormone antibody-quantum dot 2 cross-linked substance, an alkaline phosphatase-quantum dot 3 cross-linked substance and a bovine serum albumin-quantum dot 4 cross-linked substance are obtained;
coating the solid phase carrier with a thyroid stimulating hormone antibody-quantum dot 1 cross-linked substance to obtain the solid phase carrier coated with the thyroid stimulating hormone antibody-quantum dot 1 cross-linked substance, marking the thyroid stimulating hormone antibody-quantum dot 2 cross-linked substance with alkaline phosphatase to obtain the thyroid stimulating hormone antibody-quantum dot 2 cross-linked substance marked with phosphatase, and marking the thyroid stimulating hormone antibody with the alkaline phosphatase-quantum dot 3 cross-linked substance to obtain the thyroid stimulating hormone antibody marked with the alkaline phosphatase-quantum dot 3 cross-linked substance.
2. The kit of claim 1, wherein the quantum dot is a carboxylated quantum dot.
3. The kit of claim 2, wherein the carboxylated quantum dot is CdSe.
4. The kit of claim 1, wherein the solid support is a magnetic microparticle.
5. Use of a kit according to any one of claims 1 to 4 in any one or more of:
the application in preparing products for detecting thyroid stimulating hormone;
the application of the detection instrument in the detection of the contamination condition of a sample application needle, wherein the contamination is each component of a kit;
the application of the distribution state of each component of the detection kit in a reagent;
the application of analyzing abnormal result reasons caused by thyroid stimulating hormone abnormal samples.
6. The preparation method of the kit according to claim 1, characterized in that EDC and NHS are used as cross-linking agents, quantum dots with different particle sizes are selected to respectively carry out chemical cross-linking on thyroid stimulating hormone antibody, alkaline phosphatase and bovine serum albumin, and thyroid stimulating hormone antibody-quantum dot 1 cross-linked substance, thyroid stimulating hormone antibody-quantum dot 2 cross-linked substance, alkaline phosphatase-quantum dot 3 cross-linked substance and bovine serum albumin-quantum dot 4 cross-linked substance are obtained;
coating the solid phase carrier with a thyroid stimulating hormone antibody-quantum dot 1 cross-linked substance to obtain the solid phase carrier coated with the thyroid stimulating hormone antibody-quantum dot 1 cross-linked substance, marking the thyroid stimulating hormone antibody-quantum dot 2 cross-linked substance with alkaline phosphatase to obtain the thyroid stimulating hormone antibody-quantum dot 2 cross-linked substance marked with phosphatase, and marking the thyroid stimulating hormone antibody with the alkaline phosphatase-quantum dot 3 cross-linked substance to obtain the thyroid stimulating hormone antibody marked with the alkaline phosphatase-quantum dot 3 cross-linked substance.
7. A method for detecting the distribution state of the components of the kit according to any one of claims 1 to 4 in a reagent, characterized in that excitation light is applied to the reagent containing the components of the kit according to any one of claims 1 to 4, the results of the relative fluorescence intensities of the reagents are counted at different time points, and the distribution state of the components of the kit in the reagent is judged by the change of the relative fluorescence intensities.
8. A method for detecting the contamination of a sample application needle of an instrument, characterized by establishing a standard curve of the concentration of the components in the kit according to any one of claims 1 to 4 against the relative fluorescence intensity;
after thyroid stimulating hormone is detected by using the kit according to any one of claims 1 to 4, the sample application needle is washed with water, and then excitation light is applied to count the relative fluorescence intensity and fluorescence color of the sample application needle, and the type and amount of the contamination are judged by comparing with the sample application needle of a negative control.
9. A method for analyzing causes of abnormal results caused by thyroid stimulating hormone abnormal specimens, comprising:
step 1, screening abnormal samples from the detected samples, centrifuging the abnormal samples, and taking supernatant for later use;
step 2, screening multiple normal samples with consistent thyroid stimulating hormone concentration from the detection samples in the same batch as the step 1, and centrifuging to take the supernatant for later use;
step 3, mixing the components in the kit of any one of claims 1 to 4 with the abnormal sample and the normal sample respectively, and incubating and reacting;
step 4, separating the solid phase carrier part and the liquid phase part in the reaction solution in the step 3, placing the solid phase carrier part and the liquid phase part under a fluorescence microscope for observation, and visually analyzing the cause of the abnormal result through the fluorescence position and the fluorescence quantity;
when the interference of an anti-alkaline phosphatase antibody occurs, a thyroid stimulating hormone antibody-thyroid stimulating hormone-alkaline phosphatase labeled thyroid stimulating hormone antibody complex exists on the solid phase carrier, and compared with a sample without the interference, the position, the state and the fluorescence intensity of fluorescence emitted by the thyroid stimulating hormone antibody-quantum dot cross-linked product coated by the solid phase carrier, the thyroid stimulating hormone antibody-quantum dot cross-linked product labeled by the alkaline phosphatase and the bovine serum albumin-quantum dot cross-linked product are not influenced, and the position, the state and the fluorescence intensity of fluorescence emitted by the thyroid stimulating hormone antibody labeled by the alkaline phosphatase-quantum dot cross-linked product are obviously changed;
when the interference of endogenous antibodies occurs, the two situations are divided, wherein the situation is that the endogenous antibodies are combined with a thyroid stimulating hormone antibody coated by a solid phase carrier and a thyroid stimulating hormone antibody marked by alkaline phosphatase to form a compound, so that a false positive result is caused; in this case, a thyroid stimulating hormone antibody complex 1 coated with a thyroid stimulating hormone antibody-thyroid stimulating hormone-alkaline phosphatase label and a thyroid stimulating hormone antibody complex 2 coated with a thyroid stimulating hormone antibody-endogenous antibody-alkaline phosphatase label are present on the solid phase carrier at the same time, and the position, state and fluorescence intensity of fluorescence emitted by the thyroid stimulating hormone antibody-quantum dot cross-linked product and the bovine serum albumin-quantum dot cross-linked product coated with the solid phase carrier are not affected relative to a sample without such interference, the position and state of fluorescence emitted by the thyroid stimulating hormone antibody marked by alkaline phosphatase and the thyroid stimulating hormone antibody marked by the alkaline phosphatase-quantum dot cross-linking substance are changed, the fluorescence quantity is obviously increased, and correspondingly, the fluorescence quantity is reduced in a liquid phase;
the situation is that the endogenous antibody is respectively combined with a thyroid stimulating hormone antibody coated by a solid phase carrier and a thyroid stimulating hormone antibody marked by alkaline phosphatase, so that a false negative result is caused; in this case, a thyroid stimulating hormone antibody complex 1 and a thyroid stimulating hormone antibody-endogenous antibody complex 2 both coated with a thyroid stimulating hormone antibody-thyroid stimulating hormone-alkaline phosphatase label are present on the solid phase carrier, and with respect to a sample without such interference, the position, state and fluorescence intensity of the fluorescence emitted by the thyroid stimulating hormone antibody-quantum dot cross-linked product and the bovine serum albumin-quantum dot cross-linked product coated with the solid phase carrier are not affected, while the position and state of the fluorescence emitted by the thyroid stimulating hormone antibody-quantum dot cross-linked product coated with the alkaline phosphatase label and the thyroid stimulating hormone antibody labeled with the alkaline phosphatase-quantum dot cross-linked product are changed, the fluorescence amount is significantly reduced, and the fluorescence amount is increased in the liquid phase.
10. The method of claim 9, wherein the abnormal specimen causes an abnormal result in which the test result does not correspond to a clinical symptom or pattern, or the test result does not correspond to a high-level reference method.
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