CN114689846A - Human vasculitis detection kit - Google Patents
Human vasculitis detection kit Download PDFInfo
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- CN114689846A CN114689846A CN202210308588.1A CN202210308588A CN114689846A CN 114689846 A CN114689846 A CN 114689846A CN 202210308588 A CN202210308588 A CN 202210308588A CN 114689846 A CN114689846 A CN 114689846A
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The application relates to the technical field of medical detection, and particularly discloses a human vasculitis detection kit. The kit comprises: a mixed solution of fluorescence-encoded magnetic microspheres respectively coated by PR3 antigen, MPO antigen and GBM antigen; using a secondary antibody solution labeled by fluorescein; a sample diluent; and (4) cleaning the microspheres. The kit provided by the application can simultaneously obtain qualitative and quantitative results of the anti-PR 3 antibody, the anti-MPO antibody and the anti-GBM antibody.
Description
Technical Field
The application relates to the technical field of medical detection, in particular to a human vasculitis detection kit.
Background
Systemic vasculitis (systemic vasculitis) is a group of diseases with vascular necrosis and inflammation as main pathological changes, and clinical manifestations are different due to different types, sizes, parts and pathological features of affected blood vessels. Common vasculitis include Wegener's granulomatosis, microscopic vasculitis, hemorrhagic pulmonary-nephritis syndrome, polyarteritis nodosa, and the like. It usually affects multiple systems throughout the body, and may cause dysfunction of multiple systems and organs, but may be limited to a certain organ. Because the clinical manifestations of vasculitis are complex, the disease condition is serious, the early diagnosis and treatment are difficult, the disability rate and the fatality rate are high, and the prognosis is poor, the early treatment of vasculitis is very important.
The characteristic anti-neutrophil cytoplasmic antibody (ANCA) is an autoantibody with a neutrophil and monocyte cytoplasmic component as a target antigen, and comprises a large amount of autoantibodies, wherein an anti-protease 3(PR3) antibody and an anti-Myeloperoxidase (MPO) antibody are the most important ANCA antibodies. Studies have shown that anti-PR 3 antibodies and anti-MPO antibodies are involved in the pathogenesis of vasculitis. The two antibodies can activate human neutrophils and cause degranulation reaction, so that the neutrophils release a large amount of harmful proteolytic enzyme and oxygen free radical, thereby causing damage to the blood vessel wall and causing vasculitis. In addition, anti-Glomerular Basement Membrane (GBM) antibodies can also be detected in patients with the goodpasture-nephritis syndrome. Therefore, the anti-PR 3 antibody, the anti-MPO antibody and the anti-GBM antibody are all detection indexes of vasculitis.
Currently, enzyme linked immunosorbent assay (ELISA), Radioimmunoassay (RIA), indirect immunofluorescence assay (IIF), and the like are commonly used as detection methods for the detection index. The ELISA method is complex to operate, samples need to be preprocessed, and the detection time is long; although the RIA method can directly detect the plasma as a sample, the RIA method not only pollutes the environment, but also has certain radioactive radiation to an operator, and simultaneously needs sophisticated equipment, so the cost is higher; the IIF method is relatively complicated in operation, requires an expensive fluorescence microscope, can perform only qualitative detection, cannot perform quantitative determination, and cannot distinguish non-specific identification according to the size of molecular weight in analysis results. Meanwhile, the detection method can only realize qualitative or quantitative detection of the single detection index.
Disclosure of Invention
In order to obtain qualitative and quantitative results of the anti-PR 3 antibody, the anti-MPO antibody and the anti-GBM antibody simultaneously, the application provides a human vasculitis detection kit.
The application provides a human vasculitis detect reagent box, adopts following technical scheme:
a human vasculitis detection kit, comprising:
mixed solution of fluorescence coding magnetic microspheres respectively coated by PR3 antigen, MPO antigen and GBM antigen;
using a fluorescein-labeled secondary antibody solution;
a sample diluent;
and (4) cleaning the microspheres.
The kit of the application mainly comprises a mixed solution and a secondary antibody solution. The mixed solution contains fluorescence coding magnetic microspheres respectively coated by PR3 antigen, MPO antigen and GBM antigen; in the process of quantitative combined detection of the anti-PR 3 antibody, the anti-MPO antibody and the anti-GBM antibody, a sample to be detected is firstly reacted with a mixed solution, and the PR3 antigen, the MPO antigen and the GBM antigen in the mixed solution are respectively identified and combined with the anti-PR 3 antibody, the anti-MPO antibody and the anti-GBM antibody to be detected in the sample to be detected, so that the anti-PR 3 antibody, the anti-MPO antibody and the anti-GBM antibody to be detected in the sample to be detected are indirectly combined with the fluorescence-encoded magnetic microspheres.
The secondary antibody solution labeled with fluorescein contained an anti-human IgG antibody. After the anti-PR 3 antibody, the anti-MPO antibody and the anti-GBM antibody to be detected in the sample to be detected are indirectly combined with the fluorescence-encoded magnetic microspheres, the fluorescein-labeled anti-human IgG antibody is used for identifying and combining with the anti-PR 3 antibody, the anti-MPO antibody and the anti-GBM antibody to be detected in the sample to be detected, so that the fluorescence labeling of the anti-PR 3 antibody, the anti-MPO antibody and the anti-GBM antibody to be detected in the sample to be detected is completed. And detecting the anti-PR 3 antibody, the anti-MPO antibody and the anti-GBM antibody to be detected in the sample to be detected by using a flow cytometer to obtain the fluorescence signal intensities corresponding to the anti-PR 3 antibody, the anti-MPO antibody and the anti-GBM antibody to be detected in the sample to be detected, and finding out the relative concentrations of the antibodies corresponding to the anti-PR 3 antibody, the anti-MPO antibody and the anti-GBM antibody to be detected in the sample to be detected based on the fluorescence signal intensities corresponding to the anti-PR 3 antibody, the anti-MPO antibody and the anti-GBM antibody to be detected in the sample to be detected according to the relation between the relative concentrations of the antibodies and the fluorescence signal intensities in a standard curve prepared by using a calibrator, so as to obtain the concentrations corresponding to the anti-PR 3 antibody, the anti-MPO antibody and the anti-GBM antibody to be detected in the sample to be detected, and completing the detection of the relative concentrations of the anti-PR 3 antibody, the anti-MPO antibody and the anti-GBM antibody to be detected in the sample to be detected.
The kit comprises a mixed solution and a secondary antibody solution, and also comprises a calibrator and a buffer solution. The calibrator is a standard concentration solution containing an anti-PR 3 antibody, an anti-MPO antibody and an anti-GBM antibody, and the kit is used for detecting the fluorescence signal intensity of the standard solutions with different concentrations, so as to obtain a standard curve of the anti-PR 3 antibody, the anti-MPO antibody and the anti-GBM antibody and the fluorescence signal intensity. Therefore, the kit of the application can be used for simultaneously detecting and obtaining the relative concentrations of the anti-PR 3 antibody, the anti-MPO antibody and the anti-GBM antibody in a sample to be detected.
The sample dilution was 1xPBS containing 1% BSA.
The microsphere cleaning solution is 1xPBS containing 1% of Tween-20.
Preferably, in the mixed solution, the coating ratio of the PR3 antigen to the fluorescent coding magnetic microsphere is as follows: 40-50. mu.g of antigen coating 1X 107Magnetic fluorescent encoding microspheres.
Preferably, in the mixed solution, the coating ratio of the MPO antigen to the fluorescence-encoded magnetic microspheres is as follows: 40-50. mu.g of antigen coating 1X 107Magnetic fluorescent encoding microspheres.
Preferably, in the mixed solution, the coating ratio of the GBM antigen to the fluorescent-coded magnetic microspheres is: 40-50. mu.g of antigen coating 1X 107Magnetic fluorescent encoding microspheres.
By adopting the technical scheme, through experimental analysis, when the coating proportion of the PR3 antigen, the MPO antigen and the GBM antigen to the fluorescent coding magnetic microspheres is controlled within the range, the fluorescent signal intensity obtained by detection under the same antigen relative concentration is basically consistent; when the coating proportion of the PR3 antigen, the MPO antigen and the GBM antigen to the fluorescent coding magnetic microspheres is smaller or larger than the range, the fluorescence signal intensity obtained by detecting the same antigen relative concentration is smaller than the fluorescence signal intensity when the coating proportion of the PR3 antigen, the MPO antigen and the GBM antigen is in the range. Therefore, when the coating proportion of the PR3 antigen, the MPO antigen and the GBM antigen to the magnetic fluorescent coding microspheres is controlled within the range, the coating proportion can be obtainedObtaining stable fluorescence signal intensity, so that the relative concentrations of the anti-PR 3 antibody, the anti-MPO antibody and the anti-GBM antibody in the finally obtained sample to be detected are accurate, and therefore, the coating proportion of the PR3 antigen, the MPO antigen, the GBM antigen and the fluorescence coding magnetic microsphere is controlled as follows: 40-50. mu.g antigen coating 1X 107Magnetic fluorescent encoding microspheres.
Preferably, the secondary antibody solution contains anti-human IgG antibody, and the concentration of the anti-human IgG antibody is 0.5-4 [ mu ] g/ml.
By adopting the technical scheme, in the process of preparing the secondary antibody solution through experimental analysis, the concentration of the fluorescein-labeled anti-human IgG antibody is controlled within the range, and the change gradients of the fluorescence signal intensity obtained by detection under the same antibody relative concentration are basically consistent; when the concentration of fluorescein-labeled anti-human IgG was 0.3. mu.g/ml, the intensity of the fluorescence signal obtained by detection was low at the high-point value of the relative concentration of the antibody, whereas when the concentration of fluorescein-labeled anti-human IgG was 6. mu.g/ml, the intensity of the fluorescence signal obtained by detection was too high at the 0-point value of the relative concentration of the antibody. Therefore, when the concentration of the fluorescein-labeled anti-human IgG antibody in the solution B is controlled within the range, stable fluorescence signal intensity can be obtained, and the finally obtained anti-PR 3 antibody, anti-MPO antibody and anti-GBM antibody in the sample to be detected have accurate relative concentrations. Therefore, the concentration of the fluorescein-labeled anti-human IgG antibody in the secondary antibody solution was controlled to be in the range of 0.5 to 4. mu.g/ml.
Preferably, the fluorescein comprises Phycoerythrin (PE), PE-Cy7, Fluorescein Isothiocyanate (FITC), peridinin-chlorophyll-protein complex (PerCP), Allophycocyanin (APC), Alexa Fluo 488.
By adopting the above scheme, the fluorescein used in the present application is as described above, and the anti-human IgG antibody can be fluorescently labeled using the above fluorescein.
Phycoerythrin is a fluorescent protein separated and purified from red algae, is a novel fluorescent labeling dye, and has wide application in the fluorescent labeling and living body imaging of antibodies such as immunofluorescence, immunohistochemistry, flow cytometry detection and the like because the phycoerythrin has strong fluorescence, good light absorption performance, high quantum yield and wide excitation and emission ranges in a visible spectrum region. PE-Cy7 is a phycoerythrin-based conjugated fluorescent dye.
Fluorescein isothiocyanate is a biochemical reagent and also a medical diagnosis medicine, is mainly used for fluorescent dye in a fluorescent antibody technology, can be combined with various antibody proteins, ensures that the combined antibody does not lose the specificity of combining with a certain antigen, has strong green fluorescence in an alkaline solution, is used for various aspects of medicine, agriculture, livestock husbandry and the like, and can be used for quickly diagnosing diseases caused by bacteria, viruses, parasites and the like. Alexa Fluo488 is a small molecule fluorescent dye, the excitation and emission wavelength is similar to that of fluorescein isothiocyanate, but the light stability is stronger.
The polydatin-chlorophyll-protein complex is isolated from the phylum violencia and has a very high quenching coefficient, high quantum efficiency and a large stokes shift. PerCP belongs to a living fluorescent imaging protein marking dye, can generate fluorescence with higher and better light resistance, and is an excellent substitute for the blue-green pigment dye with shorter wavelength. PerCP is generally used for fluorescent immunolabeling, and in addition, PerCP is also used for flow cytometry analysis of multicolor cells, so that PerCP can be used in multicolor analysis together with FITC, PE and other fluorescent substances.
The detection method of the kit provided by the application is as follows:
(1) diluting a sample to be detected by using a sample diluent to obtain a diluted sample;
(2) mixing the diluted sample obtained in the step (1) with the mixed solution, and reacting at 37 ℃ for 20min to obtain an antibody-antigen-microsphere compound;
(3) cleaning the antibody-antigen-microsphere composite obtained in the step (2) by using a microsphere cleaning solution, and removing the microsphere cleaning solution;
(4) uniformly mixing the antibody-antigen-microsphere complex cleaned in the step (3) with a secondary antibody solution, and reacting for 20min at 37 ℃ to obtain a labeled secondary antibody-antigen-microsphere complex;
(5) cleaning the secondary antibody-antigen-microsphere compound obtained in the step (4) by using a microsphere cleaning solution, and removing the microsphere cleaning solution;
(6) and (4) resuspending the secondary antibody-antigen-microsphere complex cleaned in the step (5) by using a microsphere cleaning solution, carrying out sample loading detection, and analyzing a detection result.
The kit and the detection method are utilized to detect a sample to be detected, specifically comprising the reaction of the sample to be detected and a mixed solution, and the anti-PR 3 antibody, the anti-MPO antibody and the anti-GBM antibody in the sample to be detected are combined with the PR3 antigen, the MPO antigen and the GBM antigen on the fluorescence coding magnetic microsphere to obtain an antibody-antigen-microsphere compound, so that the positioning of the anti-PR 3 antibody, the anti-MPO antibody and the anti-GBM antibody to be detected in the sample to be detected is realized; then reacting the antibody-antigen-microsphere compound with a secondary antibody solution, and performing antigen-antibody binding reaction by using a fluorescein-labeled anti-human IgG antibody in the secondary antibody solution to react with an anti-PR 3 antibody, an anti-MPO antibody and an anti-GBM antibody to be detected positioned on the fluorescence-encoded magnetic microsphere, so as to complete the fluorescence labeling of the anti-PR 3 antibody, the anti-MPO antibody and the anti-GBM antibody to be detected in a sample to be detected, and obtain a secondary antibody-antigen-microsphere compound; and finally, performing constant volume on the secondary antibody-antigen-microsphere compound to obtain a detection solution, detecting the detection solution by using a flow cytometer to obtain fluorescence signal intensities corresponding to the anti-PR 3 antibody, the anti-MPO antibody and the anti-GBM antibody to be detected in the sample to be detected, and finding out the corresponding antigen-antibody relative concentrations according to a standard curve of the relation between the antigen-antibody relative concentration and the fluorescence signal intensity obtained by using a detection calibrator, on the basis of the fluorescence signal intensities corresponding to the anti-PR 3 antibody, the anti-MPO antibody and the anti-GBM antibody to be detected in the sample to be detected, so as to obtain the corresponding concentrations of the anti-PR 3 antibody, the anti-MPO antibody and the anti-GBM antibody to be detected in the sample to be detected, and complete the detection of the relative concentrations of the anti-PR 3 antibody, the anti-MPO antibody and the anti-GBM antibody in the sample to be detected.
The reaction time of the sample to be tested and the mixed solution can be 10-30 min. Through experimental analysis, when the reaction time of the sample to be detected and the mixed solution is controlled within the range, the fluorescence signal intensity of the anti-PR 3 antibody, the anti-MPO antibody and the anti-GBM antibody which are obtained through detection shows a continuously increasing trend; when the reaction time of the sample to be detected and the mixed solution is less than the range, the fluorescence signal intensity of the anti-PR 3 antibody, the anti-MPO antibody and the anti-GBM antibody obtained by detection is less than the fluorescence signal intensity of the anti-PR 3 antibody, the anti-MPO antibody and the anti-GBM antibody obtained by detection when the reaction time is in the range; when the reaction time of the sample to be detected and the mixed solution is longer than the range, the increase trend of the fluorescence signal intensity of the anti-PR 3 antibody, the anti-MPO antibody and the anti-GBM antibody obtained by detection is slowed down and is basically unchanged. Therefore, in order to obtain more accurate information of the anti-PR 3 antibody, the anti-MPO antibody and the anti-GBM antibody in the sample to be tested, the reaction time of the sample to be tested and the mixed solution is selected and controlled within the range of 10-30 min.
Preferably, the reaction time of the sample to be detected and the mixed solution is 20 min.
The reaction time of the antibody-antigen-microsphere complex with the secondary antibody solution may be 10-30 min. Through experimental analysis, when the reaction time of the antibody-antigen-microsphere complex and the secondary antibody solution is controlled within the range, the fluorescence signal intensity of the anti-PR 3 antibody, the anti-MPO antibody and the anti-GBM antibody obtained through detection shows a continuously increasing trend; when the reaction time of the antibody-antigen-microsphere complex with the secondary antibody solution is less than the above range, the fluorescence signal intensities of the anti-PR 3 antibody, the anti-MPO antibody and the anti-GBM antibody detected to be obtained are less than the fluorescence signal intensities of the anti-PR 3 antibody, the anti-MPO antibody and the anti-GBM antibody detected to be obtained when the reaction time is within the above range; when the reaction time of the antibody-antigen-microsphere complex with the secondary antibody solution is longer than the above range, the increase tendency of the fluorescence signal intensity of the anti-PR 3 antibody, the anti-MPO antibody and the anti-GBM antibody obtained by detection is reduced and is basically unchanged. Therefore, in order to obtain more accurate information of the anti-PR 3 antibody, the anti-MPO antibody and the anti-GBM antibody in the sample to be tested, the reaction time of the antibody-antigen-microsphere complex and the secondary antibody solution is selected and controlled within the range of 10-30 min.
Preferably, the reaction time of the antibody-antigen-microsphere complex and the secondary antibody solution is 20 min.
Preferably, the reaction temperature of the sample to be detected and the mixed solution, the antibody-antigen-microsphere complex and the secondary antibody solution is 37 +/-1 ℃.
Through experimental analysis, when the reaction temperature is 37 ℃, the fluorescence signal intensities of the anti-PR 3 antibody, the anti-MPO antibody and the anti-GBM antibody obtained through detection are all larger than the fluorescence signal intensities of the anti-PR 3 antibody, the anti-MPO antibody and the anti-GBM antibody obtained through detection when the incubation temperature is room temperature (18-25 ℃). Because, in order to obtain more accurate information of the anti-PR 3 antibody, the anti-MPO antibody and the anti-GBM antibody in the sample to be tested, the incubation temperature is selected and controlled within the range of 37 +/-1 ℃.
Preferably, the sample to be tested is a whole blood sample.
Preferably, the sample to be tested is a serum or plasma sample.
Preferably, the adding proportion of the sample to be detected and the mixed solution is as follows: and correspondingly adding 50-500 fluorescent coding magnetic microspheres prepared by antigens into each 1 mu L of sample to be detected.
Preferably, the addition amount of the sample to be tested is 10-100. mu.L. Preferably 50. mu.L.
By adopting the technical scheme, through experimental analysis, the adding proportion of the sample to be detected and the mixed solution is controlled within the range, and the final detection can be ensured to obtain more accurate fluorescence signal intensity, so that more accurate relative concentration of the antibody can be obtained. In a similar way, when the addition amount of the sample to be detected is controlled within the range, the final detection can be ensured to obtain more accurate fluorescence signal intensity, so that more accurate relative concentration of the antigen and the antibody can be obtained.
In summary, the present application has the following beneficial effects:
the kit provided by the application can be used for simultaneously completing qualitative and quantitative detection of the anti-PR 3 antibody, the anti-MPO antibody and the anti-GBM antibody in a sample to be detected in a single time. Namely, the detection results of 3 detection indexes are obtained through one detection. Compared with the prior art that the linear immunoblotting method can only provide qualitative/semi-quantitative detection results, the enzyme-linked immunosorbent assay method can only perform quantitative detection, and the kit provided by the application can simultaneously realize qualitative and quantitative detection. Meanwhile, the detection by using the kit reduces the dependence on manpower and reduces the burden of testers.
Detailed Description
The application provides a human vasculitis detection kit, the kit includes: mixed solution of fluorescence coding magnetic microspheres respectively coated by PR3 antigen, MPO antigen and GBM antigen; using a fluorescein-labeled secondary antibody solution; a sample diluent; and (4) cleaning the microspheres.
In the mixed solution, the coating proportion of the PR3 antigen to the fluorescent coding magnetic microsphere is as follows: 40-50. mu.g of antigen coating 1X 107Magnetic fluorescent encoding microspheres. In the mixed solution, the coating proportion of MPO antigen and the fluorescence coding magnetic microspheres is as follows: 40-50. mu.g of antigen coating 1X 107Magnetic fluorescent encoding microspheres. In the mixed solution, the coating proportion of the GBM antigen to the fluorescence coding magnetic microspheres is as follows: 40-50. mu.g of antigen coating 1X 107Magnetic fluorescent encoding microspheres.
The secondary antibody solution labeled by fluorescein contains anti-human IgG antibody, and the concentration of the anti-human IgG antibody is 0.5-4 mug/ml. The preparation method of the secondary antibody solution comprises the following steps:
1. preparing a PBS-TBP solution, wherein the formula is as follows:
na is mixed with2HPO4·12H2O 2.9g、KH2PO4 0.2g、NaCl 8g、KCl 0.2g、DI H2Mixing O0.9L, and adding T-200.2 ml, PC-3000.5 ml and BSA 1 g; the pH was adjusted to 7.4 and then made constant to 1L with sterile water.
2. The prepared PBS-TBP solution, the secondary antibody labeled with fluorescein are mixed with 1: 2500-1: 20000 to make the final concentration 0.5-4 ug/ml. Preferably, the secondary antibody labeled with fluorescein is labeled with 1: 10000 dilution to a final concentration of 1 ug/ml.
The fluorescein used comprises Phycoerythrin (PE), Fluorescein Isothiocyanate (FITC), fucoxanthin-chlorophyll-protein complex (PerCP), and Allophycocyanin (APC). The sample to be detected of the kit is a whole blood sample or a serum or plasma sample. The reaction time of the mixed liquid and the sample to be detected is 10-30 min. The reaction time of the substance after the mixed solution reacts with the sample to be detected and the secondary antibody solution is 10-30 min. The adding proportion of the sample to be detected and the mixed solution is as follows: and correspondingly adding 50-500 fluorescent coding magnetic microspheres prepared by antigens into each 1 mu L of sample to be detected.
The present application is described in further detail below with reference to examples 1 to 18, comparative examples 1 to 18, and the test.
Preparation example
Preparation example 1
The preparation example provides a mixed solution of fluorescent coding magnetic microspheres respectively coated by PR3 antigen, MPO antigen and GBM antigen. The preparation method of the mixed solution specifically comprises the following steps:
(1) pretreatment:
obtaining microspheres: 100 μ L of fluorescent-coded magnetic microsphere suspension (about 1X 10)7Microspheres) are placed in 3 centrifuge tubes, the 3 centrifuge tubes are placed in a magnetic frame for magnetic adsorption, and supernatant is removed after separation;
washing and resuspending microspheres: respectively adding 200 mu L of microsphere cleaning solution into 3 centrifuge tubes, carrying out vortex for 10s, placing the centrifuge tubes on a magnetic frame, carrying out magnetic adsorption, separating and removing supernatant; washing is continuously repeated for two times; and respectively adding 100 mu L of microsphere washing solution into 3 centrifuge tubes to resuspend the microspheres, and vortexing for 10s to uniformly mix the microspheres to obtain 3 microsphere resuspension solutions A.
(2) And (3) activation:
activating the microspheres: respectively adding 10 mu L of activating agent solution into the 3 microsphere resuspension A, carrying out vortex for 10s, then carrying out light-shielding reaction for 20-30min at room temperature, placing 3 centrifuge tubes on a magnetic frame, carrying out magnetic adsorption, separating and removing supernatant;
washing and resuspending the microspheres after activation: respectively adding 200 mu L of microsphere cleaning solution into 3 centrifuge tubes, performing vortex for 10s, placing the 3 centrifuge tubes on a magnetic frame, performing magnetic adsorption, separating and removing supernatant; washing is continuously repeated for two times; and respectively adding 100 mu L of microsphere washing solution into 3 centrifuge tubes to resuspend the microspheres, and vortexing for 10s to uniformly mix the microspheres to obtain 3 microsphere resuspension solutions B.
(3) Coating:
coating microspheres: adding PR3 antigen, MPO antigen and GBM antigen into the 3 microsphere resuspension B respectively; after vortex mixing, reacting for 24 hours at room temperature in a dark place; placing the 3 reacted centrifuge tubes in a magnetic frame, performing magnetic adsorption, separating and removing supernatant;
sealing after coating: respectively adding sealing liquid with the same volume into 3 centrifuge tubes, carrying out sealing reaction, sealing at room temperature in a dark place for 4h, placing the 3 centrifuge tubes after reaction in a magnetic frame, carrying out magnetic adsorption, separating and removing supernatant;
washing and resuspending the coated microspheres: respectively adding 200 mu L of microsphere preservation buffer solution into 3 centrifuge tubes, carrying out vortex for 10s, placing the 3 centrifuge tubes in a magnetic frame, carrying out magnetic adsorption, separating and removing supernatant; washing is continuously repeated for two times; and respectively adding 1ml of microsphere preservation buffer solution into 3 centrifuge tubes to resuspend the microspheres, and vortexing for 10 seconds to uniformly mix the microspheres to obtain 3 fluorescent coding magnetic microsphere resuspension solutions.
(4) And (3) storage: placing 3 kinds of fluorescent coding magnetic microsphere heavy suspension liquid at 2-8 ℃ and keeping away from light; and (3) mixing the fluorescent coding magnetic microsphere resuspension according to the weight ratio of 1: 1: 1, namely obtaining mixed liquid.
Preparation examples 2 to 13
Preparation examples 2 to 13 each provide a mixture of fluorescent-encoded magnetic microspheres coated with PR3 antigen, MPO antigen, and GBM antigen.
Preparation examples 2 to 13 differ from preparation example 1 in the amounts of PR3 antigen, MPO antigen, GBM antigen added in the coating step of the preparation method. Specifically, the results are shown in Table 1.
TABLE 1 addition amounts of PR3 antigen, MPO antigen, GBM antigen in preparation examples 1-13
Preparation examples 14 to 28
Preparation examples 14 to 28 each provide a mixture of fluorescently encoded magnetic microspheres coated with PR3 antigen, MPO antigen or GBM antigen. Preparation examples 14 to 28 differ from preparation example 1 in the kind of antigen added in the coating step of the preparation method and the amount thereof added. Specifically, as shown in table 2.
Preparation examples 14 to 18 each provided a mixture of fluorescent-encoded magnetic microspheres coated with PR3 antigen.
Preparation examples 19 to 23 each provide a mixture of fluorescence-encoded magnetic microspheres coated with MPO antigen.
Preparation examples 24 to 28 each provide a mixture of fluorescent-encoded magnetic microspheres coated with GBM antigen.
TABLE 2 addition amounts of PR3 antigen, MPO antigen or GBM antigen in preparation examples 14 to 28
Preparation example 29
This preparation provides a secondary antibody solution labeled with fluorescein. The preparation method of the secondary antibody solution marked by the fluorescein specifically comprises the following steps:
1. preparing a PBS-TBP solution, wherein the formula is as follows:
mixing Na2HPO4·12H2O 2.9g、KH2PO4 0.2g、NaCl 8g、KCl 0.2g、DI H2Mixing O0.9L, and adding T-200.2 ml, PC-3000.5 ml and BSA 1 g; the pH was adjusted to 7.4 and then made constant to 1L with sterile water.
2. The prepared PBS-TBP solution and the secondary antibody solution labeled with fluorescein are mixed in a proportion of 1: 10000 dilution to a final concentration of 1 ug/ml.
Preparation examples 30 to 31
Preparation examples 30 to 31 each provide a secondary antibody solution labeled with fluorescein. Preparations 30 to 31 differed from preparation 29 in that: the dilution ratio of the secondary antibody solution labeled with fluorescein was used. Specifically, the results are shown in Table 3.
TABLE 3 dilution ratio of secondary antibody solutions labeled with fluorescein in preparation examples 29 to 31
| Preparation example | Dilution ratio |
| 29 | 1:10000 |
| 30 | 1:2500 |
| 31 | 1:20000 |
Examples
Examples 1 to 15
Examples 1-15 provide a human vasculitis assay kit, respectively. Examples 1 to 15 differ in the mixture and the secondary antibody solution labeled with fluorescein. The details are shown in Table 4.
The components of the human vasculitis detection kit provided by the invention are as follows:
mixed solution of fluorescence coding magnetic microspheres respectively coated by PR3 antigen, MPO antigen and GBM antigen; using a fluorescein-labeled secondary antibody solution; a sample diluent; and (4) cleaning the microspheres.
TABLE 4 kinds of mixed solutions and secondary antibody solutions in examples 1 to 15
Example 16
The embodiment provides a human vasculitis detection kit. This embodiment is different from embodiment 11 in that: the secondary antibody solution labeled with fluorescein was stored at 4 ℃ for one year.
Example 17
The embodiment provides a human vasculitis detection kit. The present embodiment is different from embodiment 14 in that: the secondary antibody solution labeled with fluorescein was stored at 4 ℃ for one year.
Example 18
The embodiment provides a human vasculitis detection kit. This embodiment is different from embodiment 15 in that: the secondary antibody solution labeled with fluorescein was stored at 4 ℃ for one year.
Comparative example
Comparative examples 1 to 15
Comparative examples 1-15 provide a human vasculitis detection kit, respectively. Comparative examples 1 to 15 differ in the mixture of the components. Specifically, the results are shown in Table 5.
TABLE 5 kinds of the mixed solution and the secondary antibody solution in comparative examples 1 to 15
Test for detection
The human vasculitis detection kits provided in the above examples 1-18 and comparative examples 1-15 were used to detect samples to be detected, respectively.
The types of the samples to be detected are respectively 1-PR3 positive, 2-MPO positive, 3-GBM positive and 4-negative.
The 4 samples to be tested were tested using the human vasculitis test kits provided in examples 1-18, respectively.
The human vasculitis detection kit provided in the comparative examples 1-5 is used for respectively detecting the 1-PR3 positive and 4-negative 2 samples to be detected.
And (3) respectively detecting the 2-MPO positive and 4-negative samples to be detected by using the human vasculitis detection kit provided by the comparative examples 6-10.
The human vasculitis detection kits provided in comparative examples 11-15 are used to detect the 2 samples to be detected, namely 3-GBM positive samples and 4-negative samples respectively.
The detection method of the human vasculitis detection kit comprises the following steps:
(1) diluting a sample to be detected by using a sample diluent to obtain a diluted sample;
(2) mixing 100 mu l of the diluted sample obtained in the step (1) with the mixed solution, and reacting for 20min at 37 ℃ to obtain an antibody-antigen-microsphere compound;
(3) cleaning the antibody-antigen-microsphere composite obtained in the step (2) by using a microsphere cleaning solution, and removing the microsphere cleaning solution;
(4) uniformly mixing the antibody-antigen-microsphere complex cleaned in the step (3) with a second antibody solution, and reacting at 37 ℃ for 20min to obtain a marked second antibody-antigen-microsphere complex;
(5) cleaning the secondary antibody-antigen-microsphere compound obtained in the step (4) by using a microsphere cleaning solution, and removing the microsphere cleaning solution;
(6) and (4) resuspending the secondary antibody-antigen-microsphere complex cleaned in the step (5) by using a microsphere cleaning solution, carrying out sample loading detection, and analyzing a detection result.
The results of the measurements are shown in tables 6 and 7.
TABLE 6 test results using the human vasculitis kit provided in examples 1-18
With reference to table 6, it is understood from the results of the tests of comparative examples 1 to 5 that the accuracy of the test results for three antibodies can be improved when the amount of addition of the PR3 antigen is controlled within the range of 10 to 50 μ g, as can be seen from the results of the tests of the four samples, by adjusting the amount of addition of the PR3 antigen while keeping the amounts of addition of the MPO antigen and the GBM antigen constant. Particularly, when the amount of addition of the PR3 antigen is controlled within the range of 40-50. mu.g, the accuracy of the detection results of the three antibodies can be further improved.
From the results of the comparative examples 3 and 6 to 9, it is understood that the accuracy of the results of the detection of the three antibodies can be improved by adjusting the addition amount of the MPO antigen under the condition that the addition amounts of the PR3 antigen and the GBM antigen are kept constant, and from the results of the detection of the four samples, when the addition amount of the MPO antigen is controlled within the range of 10 to 50. mu.g. Particularly, when the addition amount of the MPO antigen is controlled within the range of 40 to 50. mu.g, the accuracy of the results of the detection of the three antibodies can be further improved.
From the results of the comparative examples 8 and 10 to 13, it was found that the accuracy of the results of the detection of the three antibodies can be improved by adjusting the amount of the GBM antigen to be added while keeping the amounts of the PR3 antigen and the MPO antigen to be added constant, and by controlling the amount of the GBM antigen to be added within the range of 10 to 50. mu.g from the results of the detection of the four samples. Particularly, when the amount of the GBM antigen to be added is controlled within the range of 40 to 50. mu.g, the accuracy of the results of detecting three antibodies can be further improved. Particularly, when the addition amount of the PR3 antigen in the mixed solution is 40 mu g, the addition amount of the MPO antigen is 50 mu g, and the addition amount of the GBM antigen is 40 mu g, the detection results of the three antibodies in the sample to be detected have the best accuracy.
From the results of the tests of comparative examples 11 and 14 to 15, it was found that when the amount of the PR3 antigen added to the mixed solution was 40 μ g, the amount of the MPO antigen added to the mixed solution was 50 μ g, and the amount of the GBM antigen added to the mixed solution was 40 μ g, the ratio of the amount of the secondary antibody solution labeled with fluorescein to the total amount of the mixed solution was 1: 10000 is diluted according to the proportion, and when the final concentration is 1ug/ml, the accuracy of the detection result of the three antibodies in the sample to be detected is optimal.
From the results of comparing examples 11, 14 to 15 and examples 16 to 18, it can be seen that when a secondary antibody solution labeled with fluorescein was used, the ratio of 1: 10000, and when the final concentration is 1ug/ml, the detection result of the three antibodies in the sample to be detected still has better accuracy after the secondary antibody solution marked by the fluorescein is stored for one year at 4 ℃.
TABLE 7 test results Using the human vasculitis kit provided in comparative examples 1-15
With reference to Table 7, it is understood from the results of the comparative examples 1 to 5 that when the amount of PR3 antibody added is controlled to be in the range of 15 to 25. mu.g when PR3 antigen is added alone, the accuracy of the result of PR3 antibody detection can be improved. Particularly, when the addition amount of the PR3 antigen is controlled within the range of 15-20 mug, the accuracy of the detection result of the PR3 antibody can be further improved.
As is clear from the results of the comparative examples 6 to 10, when the MPO antibody alone is detected, the accuracy of the result of the detection of the MPO antibody can be improved when the amount of addition of the MPO antigen is controlled within the range of 15 to 25. mu.g. Particularly, when the amount of addition of the MPO antigen is controlled within the range of 15 to 20. mu.g, the accuracy of the result of MPO antibody detection can be further improved.
As is clear from the results of comparative examples 11 to 15, when the GBM antibody is detected alone, the accuracy of the result of detection of the GBM antibody can be improved when the amount of the GBM antigen to be added is controlled within the range of 15 to 30. mu.g. Particularly, when the amount of the GBM antigen to be added is controlled within the range of 20 to 30. mu.g, the accuracy of the detection result of the GBM antibody can be further improved.
Further, from the results of the tests of comparative examples 1 to 13 and comparative examples 1 to 15, it was found that when the PR3 antibody, the MPO antibody, and the GBM antibody were tested in combination, the amounts of the PR3 antigen, the MPO antigen, and the GBM antigen added to the mixed solution were completely different from the amounts of the three antigens added to the mixed solution when the three antibodies were tested individually.
Test 2
The human vasculitis detection kits provided in the above example 11 were used to detect samples to be detected, respectively.
The detection method differs from the "detection test one" in the conditions of the two reactions. Specifically, the results are shown in Table 8.
TABLE 8 conditions for the two reactions in test two
The type of the sample to be detected is the same as the detection test I. The results are shown in Table 9.
TABLE 9 results of measurements under different reaction conditions
With reference to table 9, it can be seen from the comparison between the detection results of nos. 1-2 and 3-5 that the accuracy of the detection results of the three antibodies in the sample to be detected can be improved by controlling the reaction temperature of the diluted sample of the sample to be detected and the mixed solution and the reaction temperature of the antibody-antigen-microsphere complex and the second antibody solution at 37 ℃ compared with the room temperature.
According to the detection results of the comparison numbers 3-5, the reaction temperature of the diluted sample of the sample to be detected and the mixed solution is 37 ℃ and the reaction time is 20min, and meanwhile, the accuracy of the detection results of the three antibodies in the sample to be detected can be further improved when the reaction temperature of the antibody-antigen-microsphere compound and the second antibody solution is 37 ℃ and the reaction time is 20 min.
Comparative example 16
The comparative example provides a method for detecting a sample to be detected by using an enzyme-linked immunosorbent assay.
The detection method specifically comprises the following steps:
(1) sucking 100ul of diluted sample, standard and quality control blood and sterilizing (without dilution) and putting the sample, the standard and the quality control blood and sterilizing into each reaction hole, and using a dilution buffer solution to replace a blood drop to make a blank; and sealing the opening by using a self-adhesive cover plate. Incubating for 1h at room temperature;
(2) wash wells 3 times with wash solution (300 μ L per well);
(3) removing the washing buffer, and blotting on filter paper or cloth; 100ul of enzyme label was added to each well. Incubation at room temperature for 30 minutes;
(4) wash wells 3 times with wash solution (300 μ L per well);
(5) removing washing liquid, and blotting on filter paper or cloth; 100ul of TMB substrate was added per well. Incubating at room temperature in dark for 10 min; the incubation time required above 25 ℃ can be shortened, but should not be less than 5 min;
(6) 100ul of stop solution is added into each hole; detection is carried out within 10min at 450 nm.
Comparative example 17
The comparative example provides a method for detecting a sample to be detected by using a linear immunoblotting method.
The detection method specifically comprises the following steps:
(1) sample dilution buffer 1: 101, diluting a sample to be detected;
(2) putting the membrane strip into a sample adding groove, adding 1mL of washing buffer solution into each groove, wetting the membrane strip, and incubating at room temperature for 1 min; removing the washing buffer;
(3) adding 1mL of diluted sample into each groove, and incubating for 30min at room temperature; removing liquid, and washing for 3 times;
(4) adding 1mL of enzyme conjugate into each groove, and incubating for 30min at room temperature; removing liquid, and washing for 3 times;
(5) adding 1mL of substrate solution into each tank, and incubating for 10min at room temperature;
(6) adding 1mL of purified water into each tank, and incubating for 1min at room temperature;
(7) removing liquid, adding 1mL of stop solution into each tank, and incubating at room temperature for 5 min;
(8) the liquid was removed and the strips were thoroughly dried.
Test III
The human vasculitis kit provided in example 11 and the detection method thereof, the enzyme-linked immunosorbent assay provided in comparative example 16, and the linear immunoblotting method provided in comparative example 17 were used to detect the sample to be detected, respectively. The results are shown in tables 10 to 11.
The type of the sample to be detected is the same as the detection test I.
The test results obtained by using the kit and the test method thereof provided in example 11 of the present application are shown in table 6.
The results of the detection using the enzyme-linked immunosorbent assay provided in comparative example 16 are shown in Table 10.
TABLE 10 comparative example 16 detection results of enzyme-linked immunosorbent assay
As can be seen from table 10, although the elisa can obtain various concentrations of antibodies, the elisa can only obtain one concentration of antibody at a time, and if a plurality of antibodies are to be detected, a plurality of detection tests are required. Compared with the table 6, the kit provided by the application can obtain detection results of various antibodies only by detecting once, is simple, convenient and quick, and improves the detection efficiency of the antibodies.
The results of the assays obtained using the linear immunoblotting method provided in comparative example 17 are shown in Table 11.
TABLE 11 test results of comparative example 17
As can be seen from table 11, the linear immunoblotting method can only determine whether the sample to be tested is positive, and cannot obtain a quantitative result. Compared with the table 6, the kit provided by the application can not only qualitatively determine whether the sample to be detected is positive, but also obtain a quantitative result, and the detection efficiency is higher.
The specific embodiments are only for explaining the present application and are not limiting to the present application, and those skilled in the art can make modifications to the embodiments without inventive contribution as required after reading the present specification, but all the embodiments are protected by patent law within the scope of the claims of the present application.
Claims (10)
1. A human vasculitis detection kit, comprising:
mixed solution of fluorescence coding magnetic microspheres respectively coated by PR3 antigen, MPO antigen and GBM antigen;
using a fluorescein-labeled secondary antibody solution;
a sample diluent;
and (4) cleaning the microspheres.
2. The human vasculitis detection kit of claim 1, wherein in the mixed solution, the coating ratio of the PR3 antigen to the fluorescent-encoded magnetic microspheres is as follows: 40-50. mu.g of antigen coating 1X 107Magnetic fluorescent encoding microspheres.
3. The human vasculitis detection kit of claim 1, wherein in the mixed solution, the coating ratio of the MPO antigen to the fluorescence-encoded magnetic microspheres is as follows: 40-50. mu.g of antigen coating 1X 107Magnetic fluorescent encoding microspheres.
4. The human vasculitis detection kit of claim 1, wherein the coating ratio of the GBM antigen to the fluorescent-coded magnetic microspheres in the mixed solution is as follows: 40-50. mu.g of antigen coating 1X 107Magnetic fluorescent encoding microspheres.
5. The human vasculitis detection kit of claim 1, wherein the secondary antibody solution comprises anti-human IgG antibodies, and the concentration of the anti-human IgG antibodies is 0.5-4 μ g/ml.
6. The human vasculitis detection kit of claim 1, wherein the fluorescein comprises Phycoerythrin (PE), Fluorescein Isothiocyanate (FITC), polymethacrylflavin-chlorophyll-protein complex (PerCP), Allophycocyanin (APC).
7. The human vasculitis detection kit of claim 1, wherein the sample to be detected of the kit is a whole blood sample or a serum or plasma sample.
8. The human vasculitis detection kit of claim 1, wherein the reaction time of the mixed solution and the sample to be detected is 10-30 min.
9. The human vasculitis detection kit of claim 1, wherein a reaction time of a substance of the mixture solution reacted with the sample to be detected and the secondary antibody solution is 10-30 min.
10. The human vasculitis detection kit according to claim 1, wherein the ratio of the sample to be detected to the mixed solution is as follows: and correspondingly adding 50-500 fluorescent coding magnetic microspheres prepared by antigens into each 1 mu L of sample to be detected.
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