CN114814203B - Antinuclear antibody joint detection kit - Google Patents
Antinuclear antibody joint detection kit Download PDFInfo
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Abstract
The application relates to the technical field of medical detection, and particularly discloses an anti-nuclear antibody combined detection kit. The kit comprises: se:Sup>A mixed solution of fluorescent coding microspheres respectively coated by dsDNA antigen, smD1 antigen, nucleosome antigen, histone antigen, SS-A/Ro 60 antigen, SS-A/Ro 52 antigen, SS-B/se:Sup>A antigen, CENP-B antigen, scl70 antigen, jo-1 antigen, PM-Scl antigen, U1-snRNP antigen, ribosomal P protein antigen, PCNA antigen and AMA-M2 antigen; using a fluorescein-labeled secondary antibody solution; a sample diluent; cleaning the microspheres; the LC-1 antigen comprises an antigen A and an antigen B; the dsDNA antigen is circular phage PM2 DNA. The kit provided by the application can perform qualitative and quantitative detection on 15 antinuclear antibodies at the same time, and has higher accuracy.
Description
Technical Field
The application relates to the technical field of medical detection, in particular to an anti-nuclear antibody combined detection kit.
Background
Antinuclear antibodies (antinuclear antibody, ANA), also known as anti-nucleic acid antigen antibodies, are a group of autoantibodies produced against DNA, RNA, proteins or molecular complexes of these substances within the nucleus of a cell. Depending on the properties of the individual molecules within the nucleus, the ANAs can be classified as anti-DNA antibodies, anti-histone antibodies, anti-non-histone antibodies, anti-nucleolar antibodies, and the like. Each major class is in turn subdivided into a number of classes due to different antigenic properties. Thus, ANA is a broad set of autoantibodies each of different clinical significance, more precisely the antinuclear antibody profile. ANA is mainly found in IgG, also in IgM, igA, even IgD and IgE.
Antibodies detected by anti-nuclear antibody profiling include:
(1) anti-dsDNA antibodies: anti-double stranded DNA (dsDNA) antibodies are specific antibodies to Systemic Lupus Erythematosus (SLE), and are closely related to the activity of the disease. In addition to anti-Sm antibodies, anti-dsDNA antibodies were used as a serological index for SLE, with a positive rate of 20-90% depending on the detection method and the degree of disease activity. At the same time, anti-dsDNA antibodies may also be found in other autoimmune and infectious diseases, rarely in healthy people.
(2) anti-SmD 1 antibody: anti-Smith (Sm) antibodies have high specificity for SLE, the positive detection rate of anti-Sm antibodies in patients is 5-40%, and anti-Sm antibodies are considered to be involved in the disease pathogenesis together with anti-dsDNA antibodies, however, the sensitivity of anti-Sm antibodies is low. Whereas peptide numbers 83-119 of SmD1 in the Sm core antigen had the best sensitivity and specificity for SLE.
In contrast, the sensitivity of anti-SmD 1 antibodies was 68% which is much higher than that of anti-Sm antibodies (32%). Meanwhile, the sensitivity of the anti-SmD 1 antibody combined with the anti-dsDNA antibody can reach 79 percent, which is higher than that of the anti-Sm antibody combined with the anti-dsDNA antibody (67 percent).
(3) Anti-nucleosome (Nucleosomes) antibodies: the anti-Nucleosomes antibody is also a specific index of SLE, the positive rate of the anti-Nucleosomes antibody in patients reaches 50-95%, and the detection rate of SLE can be improved by combining the anti-Nucleosomes antibody with the anti-dsDNA antibody.
(4) Anti-histone (Histones) antibodies: anti-Histones antibodies are found in a variety of autoimmune diseases, and anti-histone (Histones) antibodies are specific antibodies to drug-induced lupus. If the anti-Histone antibody is detected as negative, drug-induced lupus can be eliminated. However, if an anti-histonia antibody is detected as positive, it may be a variety of autoimmune diseases. The positive rate of the anti-Histone antibodies in the systemic lupus erythematosus patients is 30-70%, the positive rate of the anti-Histone antibodies in the rheumatoid arthritis patients is 5-50%, the positive rate of the anti-Histone antibodies in the autoimmune hepatitis patients and the systemic sclerosis patients is 35%, and the anti-Histone antibodies can also show positive in other autoimmune diseases.
(5) anti-SS-se:Sup>A/Ro 60 antibody: the positive rate of the anti-SS-A/Ro 60 antibody in the primary Sjogren syndrome patient is 70-90%; anti-SS-A/Ro 60 antibodies can also be found in systemic lupus erythematosus, where the positive rate of anti-SS-A/Ro 60 antibodies is 20-60%; the positive rate of anti-SS-se:Sup>A/Ro 60 antibodies in neonatal lupus erythematosus is 100%, and anti-SS-se:Sup>A/Ro 60 antibodies can be transferred to the fetus vise:Sup>A the placentse:Sup>A, thereby causing an inflammatory response of the fetus and congenital heart block in the neonate.
(6) anti-SS-se:Sup>A/Ro 52 antibody: anti-SS-se:Sup>A/Ro 52 antibodies are se:Sup>A non-specific indicator of autoimmune disease and can be positive in se:Sup>A variety of autoimmune diseases.
(7) anti-SS-B/La antibodies: the anti-SS-B/La antibody is a marker antibody of Sjogren syndrome, and the positive rate of the anti-SS-B/La antibody in the primary Sjogren syndrome is 40%; anti-SS-B/La antibodies also showed positive in patients with systemic lupus erythematosus. In Sjogren's syndrome, anti-SS-A and anti-SS-B antibodies are typically present simultaneously.
(8) Anti-centromere (CENP-B) antibodies: the anti-CENP-B antibody is a marker antibody for localized cutaneous sclerosis, the positive rate is 13.4-45.1%, and the appearance of the anti-CENP-B antibody indicates a good prognosis.
(9) Anti-topoisomerase i (Scl 70) antibody: the anti-Scl 70 antibody is a marker antibody for diffuse skin sclerosis, the positive rate is 18-59.5%, and the anti-Scl 70 antibody can be used as a biomarker for poor prognosis, and rapidly progressive skin thickening, renal crisis, gastric Antral Vasodilation (GAVE) and cancer appear.
(10) Antihistamine acyl tRNA synthetase (Jo-1) antibody: the anti-Jo-1 antibody is a myositis specific antibody, the anti-Jo-1 antibody is one of the anti-synthetase antibodies with the highest positive rate, and the positive rate is 22-30%.
(11) anti-PM-Scl antibody: the anti-PM-Scl antibody is a myositis-related autoantibody with a positive rate of 5-10%, and can also be positive in combined connective tissue disease and systemic sclerosis.
(12) anti-U1-snRNP antibody: high titers of anti-U1-snRNP antibodies were present in Mixed Connective Tissue Disease (MCTD) patients. The positive rate of anti-U1-snRNP antibodies in myositis patients is 10-15%, and can also be positive in combined connective tissue disease, systemic sclerosis and systemic lupus erythematosus.
(13) Anti-ribosomal P protein antibodies: the anti-ribosomal P protein antibody is related antibody of SLE, the positive rate is 6-20%, and the positive rate is related to the involvement of the nervous system, kidney and liver of SLE.
(14) Anti-proliferative nuclear antigen (PCNA) antibodies: anti-PCNA antibodies are also SLE related antibodies with a positive rate of 5-10%.
(15) Anti-mitochondrial M2 (AMA-M2) antibodies: high titers of anti-AMA-M2 antibodies are markers of primary cholangitis, with good sensitivity and specificity to primary cholangitis (PBC).
The antinuclear antibodies appear before clinical symptoms, part of the autoantibodies also participate in the progress of diseases, and a certain autoimmune disease can detect various antinuclear antibodies, and one antinuclear antibody can also relate to various related autoimmune diseases.
Currently, the detection of such autoantibodies is mostly single autoantibody detection alone. However, since the pathogenesis of autoimmune diseases is closely related to immune function, and there are many kinds of diseases, there may be many diseases at the same time. Therefore, the detection result of only a single autoantibody cannot accurately locate autoimmune disease. Since the detection of the autoantibodies is performed individually, it takes a long period of time to obtain the detection results of the various autoantibodies.
Disclosure of Invention
The application provides an anti-nuclear antibody combined detection kit, which can carry out qualitative and quantitative detection on 15 anti-nuclear antibodies simultaneously and has higher accuracy.
The application provides an antinuclear antibody joint detection kit, which adopts the following technical scheme:
an antinuclear antibody joint detection kit, said kit comprising:
se:Sup>A mixed solution of fluorescent coding microspheres respectively coated by dsDNA antigen, smD1 antigen, nucleosome antigen, histone antigen, SS-A/Ro 60 antigen, SS-A/Ro 52 antigen, SS-B/se:Sup>A antigen, CENP-B antigen, scl70 antigen, jo-1 antigen, PM-Scl antigen, U1-snRNP antigen, ribosomal P protein antigen, PCNA antigen and AMA-M2 antigen;
Using a fluorescein-labeled secondary antibody solution; a sample diluent; cleaning the microspheres;
the dsDNA antigen is phage PM2DNA;
the nucleotide sequence of the phage PM2DNA is shown as SEQ ID NO: 1.
According to the application, phage PM2DNA is selected as the mixed solution in the dsDNA antigen preparation kit, so that the accuracy of negative and positive detection results can be effectively improved. The selection of phage PM2DNA as dsDNA antigen has a higher positive and negative compliance than when dsDNA antigen was selected from DIARECT, HUMAN-LIA, HUMAN-ELISA and Sigma. Therefore, phage PM2DNA is selected as dsDNA antigen, and the accuracy of the detection result can be effectively improved.
The kit mainly comprises a mixed solution and a secondary antibody solution. The mixed solution contains fluorescent coding magnetic microspheres respectively coated by dsDNA antigen, smD1 antigen, nucleosome antigen, histone antigen, SS-A/Ro 60 antigen, SS-A/Ro 52 antigen, SS-B/se:Sup>A antigen, CENP-B antigen, scl70 antigen, jo-1 antigen, PM-Scl antigen, U1-snRNP antigen, ribosomal P protein antigen, PCNA antigen and AMA-M2 antigen; in the process of carrying out quantitative combined detection on an anti-dsDNA antibody, an anti-SmD 1 antibody, an anti-Nucleosome antibody, an anti-Histone antibody, an anti-SS-A/Ro 60 antibody, an anti-SS-A/Ro 52 antibody, an anti-SS-B/se:Sup>A antibody, an anti-CENP-B antibody, an anti-Scl 70 antibody, an anti-Jo-1 antibody, an anti-PM-Scl antibody, an anti-U1-snRNP antibody, an anti-ribosomal P protein antibody, an anti-PCNA antibody and an anti-AMA-M2 antibody, firstly, reacting se:Sup>A sample to be detected with se:Sup>A mixed solution, the dsDNA antigen, smD1 antigen, nucleosome antigen, histone antigen, SS-A/Ro 60 antigen, SS-A/Ro 52 antigen, SS-B/se:Sup>A antigen, CENP-B antigen, scl70 antigen, jo-1 antigen, PM-Scl antigen, U1-snRNP antigen, ribosomal P protein antigen, PCNA antigen, AMA-M2 antigen in the mixed solution are respectively combined with the anti-dsDNA antibody, anti-SmD 1 antibody, anti-Nucleosome antibody, anti-Histone antibody, anti-SS-A/Ro 60 antibody, anti-SS-A/Ro 52 antibody, anti-SS-B/se:Sup>A antibody, anti-CENP-B antibody, anti-Scl 70 antibody, anti-Jo-1 antibody, anti-PM-Scl antibody, anti-U1-snRNP antibody, anti-ribosomal P protein antibody, anti-PCNA antibody, anti-AMA-M2 antibody in the sample, and the fluorescent coded magnetic microsphere.
The secondary antibody solution labeled with fluorescein contains an anti-human IgG antibody. After the anti-dsDNA antibody, the anti-SmD 1 antibody, the anti-Nucleosome antibody, the anti-Histone antibody, the anti-SS-A/Ro 60 antibody, the anti-SS-A/Ro 52 antibody, the anti-SS-B/se:Sup>A antibody, the anti-CENP-B antibody, the anti-Scl 70 antibody, the anti-Jo-1 antibody, the anti-PM-Scl antibody, the anti-U1-snRNP antibody, the anti-ribosomal P protein antibody, the anti-PCNA antibody and the anti-AMA-M2 antibody are indirectly combined with the fluorescent coding magnetic microsphere in the sample to be tested, and then the fluorescein is used for labeling the anti-human IgG antibody and recognizing and combining the anti-dsDNA antibody, the anti-SmD 1 antibody, the anti-Nucleome antibody, the anti-Histone antibody, the anti-SS-A/Ro 60 antibody, the anti-SS-A/Ro 52 antibody, the anti-SS-B/se:Sup>A antibody, the anti-CENP-B antibody, the anti-Scl 70 antibody, the anti-Jo-1 antibody, the anti-PM-Scl antibody, the anti-U1-snRNP antibody, the anti-ribosomal P protein antibody, the anti-PCNA antibody and the anti-AMA-M2 antibody in the sample to be tested, thereby completing the recognition and combination of the anti-dsDNA antibody, the anti-SmD 1 antibody, the anti-Nucleome antibody, the anti-Histones antibody, the anti-SS-A/Ro 60 antibody, the anti-SS-A/Ro 52 antibody, the anti-SS-B/se:Sup>A antibody, the anti-CENP-B antibody, the anti-Scl 70 antibody, the anti-Jo-1 antibody, the anti-PM-Scl antibody, the anti-U1-snP antibody, the anti-RNP protein, the anti-RNA protein and the anti-PCNA 2 antibody in the sample to be tested. Detecting the anti-dsDNA antibody, the SmD1 antibody, the Nucleome antibody, the anti-Histone antibody, the anti-SS-A/Ro 60 antibody, the anti-SS-A/Ro 52 antibody, the anti-SS-B/se:Sup>A antibody, the anti-CENP-B antibody, the anti-Scl 70 antibody, the anti-Jo-1 antibody, the anti-PM-Scl antibody, the anti-U1-snRNP antibody, the anti-ribosomal P protein antibody, the anti-PCNA antibody and the anti-AMA-M2 antibody in se:Sup>A sample to be detected by using se:Sup>A flow cytometer to obtain the corresponding signal intensities of the anti-dsDNA antibody, the anti-SmD 1 antibody, the anti-Nucleome antibody, the anti-Histone antibody, the anti-SS-A/Ro 60 antibody, the anti-SS-A/Ro 52 antibody, the anti-SS-B/se:Sup>A antibody, the anti-CENP-B antibody, the anti-Scl 70 antibody, the anti-Jo-1 antibody, the anti-Scl antibody, the anti-U1-snP antibody, the anti-PCNA protein antibody and the anti-AMA-M2 antibody in the sample to be detected, and then based on the relation between the relative concentration of the antibodies and the fluorescence signal intensity in the standard curve prepared by using the calibrator, the relative concentrations of the antibodies to be detected in the sample to be detected, namely, the anti-dsDNA antibody, the anti-SmD 1 antibody, the anti-Nucleome antibody, the anti-Histone antibody, the anti-SS-A/Ro 60 antibody, the anti-SS-A/Ro 52 antibody, the anti-SS-B/se:Sup>A antibody, the anti-CENP-B antibody, the anti-Scl 70 antibody, the anti-Jo-1 antibody, the anti-PM-Scl antibody, the anti-U1-snRNP antibody, the anti-ribosomal P protein antibody, the anti-PCNA antibody and the anti-AMA-M2 antibody, are detected, so as to obtain the relative concentrations of the antibodies to be detected in the sample to be detected, the relative concentrations of the anti-dsDNA antibody, the anti-SmD 1 antibody, the anti-Nucleosome antibody, the anti-SS-A/Ro 60 antibody, the anti-SS-A/Ro 52 antibody, the anti-SS-B/se:Sup>A antibody, the anti-CENP-B antibody, the anti-Scl 70 antibody, the anti-Jo-1 antibody, the anti-PM-Scl antibody, the anti-U1-snRNP antibody, the anti-ribosomal P protein antibody, the anti-PCNA antibody, and the anti-AMA-M2 antibody in the sample to be tested were determined by the relative concentrations of the anti-dsDNA antibody, the anti-SmD 1 antibody, the anti-Nucleosome antibody, the anti-Histone antibody, the anti-SS-A/Ro 60 antibody, the anti-SS-A/Ro 52 antibody, the anti-SS-B/se:Sup>A antibody, the anti-CENP-B antibody, the anti-Scl 70 antibody, the anti-Jo-1 antibody, the anti-PM-Scl antibody, the anti-U1-snRNP antibody, the anti-ribosomal P protein antibody, the anti-PCNA and the anti-AMA-M2 antibody.
The kit comprises a mixed solution and a secondary antibody solution, and also comprises a calibrator and a buffer solution. The calibrator is se:Sup>A standard concentration solution containing anti-dsDNA antibody, anti-SmD 1 antibody, anti-Nucleosome antibody, anti-Histone antibody, anti-SS-A/Ro 60 antibody, anti-SS-A/Ro 52 antibody, anti-SS-B/se:Sup>A antibody, anti-CENP-B antibody, anti-Scl 70 antibody, anti-Jo-1 antibody, anti-PM-Scl antibody, anti-U1-snRNP antibody, anti-ribosomal P protein antibody, anti-PCNA antibody, anti-AMA-M2 antibody, and fluorescent signal intensities of the standard solutions of different concentrations are detected by the kit, thereby obtaining se:Sup>A standard curve of anti-dsDNA antibody, anti-SmD 1 antibody, anti-Nucleosome antibody, anti-Histone antibody, anti-SS-A/Ro 60 antibody, anti-SS-A/Ro 52 antibody, anti-SS-B/se:Sup>A antibody, anti-CENP-B antibody, anti-Scl 70 antibody, anti-Jo-1 antibody, anti-PM-Scl antibody, anti-U1-snP antibody, anti-RNA protein, and NA-RNA-2 antibody. Therefore, the relative concentrations of the anti-dsDNA antibody, the anti-SmD 1 antibody, the anti-Nucleome antibody, the anti-Histone antibody, the anti-SS-A/Ro 60 antibody, the anti-SS-A/Ro 52 antibody, the anti-SS-B/se:Sup>A antibody, the anti-CENP-B antibody, the anti-Scl 70 antibody, the anti-Jo-1 antibody, the anti-PM-Scl antibody, the anti-U1-snRNP antibody, the anti-ribosomal P protein antibody, the anti-PCNA antibody and the anti-AMA-M2 antibody in se:Sup>A sample to be detected can be detected and obtained simultaneously by using the kit.
The sample dilution was 1xPBS containing 1% BSA.
The microsphere cleaning liquid is 1xPBS containing 1% of Tween-20.
Preferably, the dsDNA antigen is circular phage PM2DNA.
Phage PM2DNA has phage PM2 linear DNA and phage PM2 circular DNA, and the positive coincidence rate when dsDNA antigen is circular phage PM2DNA is higher than that when dsDNA antigen is linear phage PM2DNA. Thus, the dsDNA antigen is further preferably phage PM2 circular DNA.
U1-snRNP68/70KD is a compound of U1-snRNP68 KD and U1-snRNP 70 KD.
Preferably, the weight ratio of RNP A, U1-snRNP C and U1-snRNP68/70KD in the U1-snRNP antigen is (1-30): (1-30): (1-30).
Preferably, the weight ratio of RNP A, U1-snRNP C and U1-snRNP68/70KD in the U1-snRNP antigen is (5-20): (10-30): (10-30).
In a specific embodiment, the weight ratio of RNP A, U1-snRNP C, U1-snRNP68/70KD in the U1-snRNP antigen may be 5: (10-30): (10-30), 5: (20-30): (10-30), 5:20: (20-30), 5:20:20.
in a specific embodiment, the weight ratio of RNP A, U1-snRNP C, U1-snRNP68/70KD in the U1-snRNP antigen may be 30:30: 30. 10:20: 30. 10:20: 20. 10:30:20.
In a specific embodiment, the weight ratio of RNP A, U1-snRNP C, U1-snRNP68/70KD in the U1-snRNP antigen may be 5:10: 10. 5:20: 30. 5:20: 20. 5:30:20.
as is evident from experimental analysis, when the addition ratio of RNP A, U1-snRNP C or U1-snRNP68/70KD is (5-20): (10-30): (10-30), and the positive coincidence rate in the detection result is more than 83.33%. In particular, when RNP A, U1-snRNP C or U1-snRNP68/70KD is added in a ratio of 5:20: at 20, the positive coincidence rate in the detection result can reach 100%. Therefore, when RNP A, U1-snRNP C or U1-snRNP68/70KD is mixed as the U1-snRNP antigen, the positive coincidence rate in the detection result can be effectively improved by reducing the addition amount of RNP A, so that the accuracy of the detection result is improved.
Preferably, the secondary antibody solution contains anti-human IgG antibody, and the concentration of the anti-human IgG antibody is 10-50 mug/ml.
In a specific embodiment, the concentration of anti-human IgG antibodies in the secondary antibody solution is from 10 to 30 μg/ml.
Through 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 variation gradient of the fluorescence signal intensity obtained by detection under the same relative concentration of the antibody is basically consistent; when the concentration of the fluorescein-labeled anti-human IgG was 5. Mu.g/ml, the intensity of the fluorescence signal obtained by detection of the relative concentration of the antibody at the high spot value was low, whereas when the concentration of the fluorescein-labeled anti-human IgG was 60. Mu.g/ml, the intensity of the fluorescence signal obtained by detection of the relative concentration of the antibody at the 0 spot value was too high. As is clear from the above, the concentration of the fluorescein-labeled anti-human IgG antibody in the solution B was controlled within the above range, and thus se:Sup>A relatively stable fluorescence signal intensity could be obtained, so that the relative concentrations of the finally obtained anti-dsDNA antibody, anti-SmD 1 antibody, anti-Nucleome antibody, anti-Histone antibody, anti-SS-A/Ro 60 antibody, anti-SS-A/Ro 52 antibody, anti-SS-B/se:Sup>A antibody, anti-CENP-B antibody, anti-Scl 70 antibody, anti-Jo-1 antibody, anti-PM-Scl antibody, anti-U1-snRNP antibody, anti-ribosomal P protein antibody, anti-PCNA antibody, and anti-AMA-M2 antibody were relatively accurate. Thus, the concentration of the fluorescein-labeled anti-human IgG antibody in the secondary antibody solution was controlled to be in the range of 10-50. Mu.g/ml.
Preferably, the fluorescent coding microsphere is a magnetic fluorescent coding microsphere.
The application uses the magnetic fluorescent coding microsphere to match with full automation without manual operation, thereby reducing the workload of the testers.
Preferably, the luciferin comprises Phycoerythrin (PE), PE-Cy7, fluorescein Isothiocyanate (FITC), polymethine-chlorophyll-protein complex (PerCP), allophycocyanin (APC), alexa Fluo488.
By adopting the above scheme, the fluorescein used in the application can be used for fluorescent labeling of anti-human IgG antibodies as described above.
Phycoerythrin is a fluorescent protein separated and purified from red algae, and is a novel fluorescent marking dye, and has strong fluorescence, good light absorption performance and high quantum yield, and has wide excitation and emission range in the visible spectrum region, so that the phycoerythrin has wide application in fluorescent marking and living body imaging of antibodies such as immunofluorescence, immunohistochemistry, flow cytometry detection and the like. PE-Cy7 is a conjugated fluorescent dye based on phycoerythrin.
Fluorescein isothiocyanate is a biochemical reagent and also a medical diagnostic medicine, is mainly used for fluorescent dye in fluorescent antibody technology, can be combined with various antibody proteins, does not lose the specificity of combining with certain antigen, has strong green fluorescence in alkaline solution, is used for various aspects such as medicine, agriculture, animal husbandry and the like, and can be used for rapidly diagnosing diseases caused by bacterial viruses, parasites and the like. Alexa Fluo488 is a small-molecule fluorescent dye, and the excitation and emission wavelength is similar to that of fluorescein isothiocyanate, but the light stability is stronger.
The polymorphin-chlorophyll-protein complex is isolated from the phylum of the class Phaeoda and has a very high quenching coefficient, high quantum energy efficiency and a large Stokes shift. PerCP belongs to an in-vivo fluorescence imaging protein labeling dye, can generate fluorescence with higher light resistance, and is a substitute for blue Lan Suran dye with shorter wavelength. PerCP is commonly used for fluorescent immunolabeling, and in addition, is also used for flow cytometric analysis of multi-color cells, so that PerCP can be used in multi-color analysis along with FITC, PE, and other fluorescent substances.
The detection method of the kit provided by the application comprises the following steps:
(1) Diluting a sample to be tested by using a sample diluent to obtain a diluted sample;
(2) Mixing the diluted sample obtained in the step (1) with the mixed solution A, and reacting for 20min at 37 ℃ to obtain an antibody-antigen-microsphere compound;
(3) Cleaning the antibody-antigen-microsphere compound obtained in the step (2) by utilizing 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 anti-antibody-antigen-microsphere compound obtained in the step (4) by utilizing microsphere cleaning liquid, and removing the microsphere cleaning liquid;
(6) And (3) re-suspending the secondary anti-antibody-antigen-microsphere compound cleaned in the step (5) by utilizing microsphere cleaning liquid, loading and detecting, and analyzing the detection result.
The reagent kit and the detection method thereof are utilized to detect the sample to be detected, which specifically comprises the reaction of the sample to be detected and mixed liquid, and the anti-dsDNA antibody, the anti-SmD 1 antibody, the anti-Nucleosome antibody, the anti-Histone antibody, the anti-SS-A/Ro 60 antibody, the anti-SS-A/Ro 52 antibody, the anti-SS-B/se:Sup>A antibody, the anti-CENP-B antibody, the anti-Scl 70 antibody, the anti-Jo-1 antibody, the anti-PM-Scl antibody, the anti-U1-snRNP antibody, the anti-ribosomal P protein antibody, the anti-PCNA antibody, the anti-AMA-M2 antibody, the dsDNA antigen on fluorescent coded magnetic microspheres, the SmD1 antigen, the Nucleosome antigen, the Histones antigen, the SS-A/Ro 60 antigen, the SS-A/Ro 52 antigen, the SS-B/se:Sup>A antigen, the CENP-B antigen, the Scl70 antigen, the Jo-1 antigen, the PM-Scl antigen, the U1-snP antigen, the PCNA antigen and the RNA 2 antigen, obtaining an antibody-antigen-microsphere complex, thereby realizing the localization of an anti-dsDNA antibody, an anti-SmD 1 antibody, an anti-Nucleosome antibody, an anti-Histone antibody, an anti-SS-A/Ro 60 antibody, an anti-SS-A/Ro 52 antibody, an anti-SS-B/se:Sup>A antibody, an anti-CENP-B antibody, an anti-Scl 70 antibody, an anti-Jo-1 antibody, an anti-PM-Scl antibody, an anti-U1-snRNP antibody, an anti-ribosomal P protein antibody, an anti-PCNA antibody, and an anti-AMA-M2 antibody to be detected in se:Sup>A sample to be detected; then reacting the antibody-antigen-microsphere complex with se:Sup>A secondary anti-solution, and carrying out antigen-antibody binding reaction by utilizing an anti-human IgG antibody marked by fluorescein in the secondary anti-solution and an anti-dsDNA antibody, an anti-SmD 1 antibody, an anti-Nucleosome antibody, an anti-Histone antibody, an anti-SS-A/Ro 60 antibody, an anti-SS-A/Ro 52 antibody, an anti-SS-B/se:Sup>A antibody, an anti-CENP-B antibody, an anti-Scl 70 antibody, an anti-Jo-1 antibody, an anti-PM-Scl antibody, an anti-U1-snRNP antibody, an anti-ribosomal P protein antibody, an anti-PCNA antibody and an anti-AMA-M2 antibody to be detected on the fluorescence-encoded magnetic microsphere, thereby completing the detection of the anti-dsDNA antibody, the anti-SmD 1 antibody, the anti-Nucleosome antibody, the anti-Histones antibody, the anti-SS-A/Ro 60 antibody, the anti-SS-A/Ro 52 antibody, the anti-SS-B/se:Sup>A antibody, the anti-CENP-B antibody, the anti-Scl 70 antibody, the anti-Scl 1 antibody, the anti-PCNA antibody, the anti-RNA-M2 antibody, and the anti-RNA-protein-antibody; finally, the secondary anti-antibody-antigen-microsphere complex is subjected to constant volume to obtain detection liquid, the detection liquid is detected by se:Sup>A flow cytometry to obtain fluorescent signal intensities corresponding to anti-dsDNA antibodies, anti-SmD 1 antibodies, anti-Nucleome antibodies, anti-Histone antibodies, anti-SS-A/Ro 60 antibodies, anti-SS-A/Ro 52 antibodies, anti-SS-B/se:Sup>A antibodies, anti-CENP-B antibodies, anti-Scl 70 antibodies, anti-Jo-1 antibodies, anti-PM-Scl antibodies, anti-U1-snRNP antibodies, anti-ribosomal P protein antibodies, anti-PCNA antibodies and anti-AMA-M2 antibodies in se:Sup>A sample to be detected, and then according to se:Sup>A standard curve of the relation between the relative concentration of the antigen antibodies and the fluorescent signal intensities obtained by using se:Sup>A detection calibrator, the relative concentrations of the antigen and antibody corresponding to the anti-dsDNA antibody, the anti-SmD 1 antibody, the anti-Nucleome antibody, the anti-Histone antibody, the anti-SS-A/Ro 60 antibody, the anti-SS-A/Ro 52 antibody, the anti-SS-B/se:Sup>A antibody, the anti-CENP-B antibody, the anti-Scl 70 antibody, the anti-Jo-1 antibody, the anti-PM-Scl antibody, the anti-U1-snRNP antibody, the anti-ribosomal P protein antibody, the anti-PCNA antibody and the anti-AMA-M2 antibody are detected based on the fluorescence signal intensities corresponding to the anti-dsDNA antibody, the anti-SmD 1 antibody, the anti-Nucleome antibody, the anti-Histones antibody, the anti-SS-A/Ro 60 antibody, the anti-SS-A/Ro 52 antibody, the anti-SS-B/se:Sup>A antibody, the anti-CENP-B antibody, the anti-Scl 70 antibody, the anti-Jo-1 antibody and the anti-PM-Scl antibody in the sample, the relative concentrations of the anti-KM-1 antibody, the anti-C-1 antibody, the anti-SA antibody, the anti-AMA-M2 antibody, the anti-Sp 100 antibody, the anti-Gp 210 antibody, the anti-ASMA antibody, the anti-dsDNA antibody, the anti-SmD 1 antibody, the anti-Nucleosome antibody, the anti-Histone antibody, the anti-SS-A/Ro 60 antibody, the anti-SS-A/Ro 52 antibody, the anti-SS-B/se:Sup>A antibody, the anti-CENP-B antibody, the anti-Scl 70 antibody, the anti-Jo-1 antibody, the anti-PM-Scl antibody, the anti-U1-snRNP antibody, the anti-ribosomal P protein antibody, the anti-PCNA antibody and the anti-AMA-M2 antibody in the sample to be tested are detected.
The reaction time of the sample to be tested and the mixed solution can be 10-30min. Through experimental analysis, when the reaction time of se:Sup>A sample to be tested and the mixed solution is controlled within the above range, the fluorescent signal intensity of the obtained anti-KM-1 antibody, anti-C-1 antibody, anti-SA antibody, anti-AMA-M2 antibody, anti-Sp 100 antibody, anti-Gp 210 antibody and anti-ASMA antibody, anti-SmD 1 antibody, anti-Nucleosome antibody, anti-Histone antibody, anti-SS-A/Ro 60 antibody, anti-SS-A/Ro 52 antibody, anti-SS-B/se:Sup>A antibody, anti-CENP-B antibody, anti-Scl 70 antibody, anti-Jo-1 antibody, anti-PM-Scl antibody, anti-U1-snRNP antibody, anti-ribosomal P protein antibody, anti-PCNA antibody and anti-AMA-M2 antibody shows se:Sup>A continuous increase trend; when the reaction time of the sample to be tested and the mixed solution A is smaller than the above range, detecting that the fluorescence signal intensity of the obtained anti-dsDNA antibody, the anti-SmD 1 antibody, the anti-Nucleosome antibody, the anti-Histone antibody, the anti-SS-A/Ro 60 antibody, the anti-SS-A/Ro 52 antibody, the anti-SS-B/se:Sup>A antibody, the anti-CENP-B antibody, the anti-Scl 70 antibody, the anti-Jo-1 antibody, the anti-PM-Scl antibody, the anti-U1-snRNP antibody, the anti-ribosomal P protein antibody, the anti-PCNA antibody, the anti-AMA-M2 antibody is smaller than the fluorescence signal intensity of the obtained anti-dsDNA antibody, the anti-SmD 1 antibody, the anti-Nucleome antibody, the anti-Histone antibody, the anti-SS-A/Ro 60 antibody, the anti-SS-A/Ro 52 antibody, the anti-SS-B/se:Sup>A antibody, the anti-CENP-B antibody, the anti-Jo-1 antibody, the anti-Scl 1 antibody, the anti-Scl 1-sP antibody, the anti-RNA protein antibody and the anti-AMM 2 antibody is in the above range; when the reaction time of the sample to be tested and the mixed solution is longer than the above range, the fluorescent signal intensity increasing trend of the obtained anti-dsDNA antibody, anti-SmD 1 antibody, anti-Nucleosome antibody, anti-Histone antibody, anti-SS-A/Ro 60 antibody, anti-SS-A/Ro 52 antibody, anti-SS-B/se:Sup>A antibody, anti-CENP-B antibody, anti-Scl 70 antibody, anti-Jo-1 antibody, anti-PM-Scl antibody, anti-U1-snRNP antibody, anti-ribosomal P protein antibody, anti-PCNA antibody and anti-AMA-M2 antibody is slowed down and is basically unchanged. Therefore, in order to obtain more accurate information of the anti-dsDNA antibody, the anti-SmD 1 antibody, the anti-Nucleosome antibody, the anti-Histone antibody, the anti-SS-A/Ro 60 antibody, the anti-SS-A/Ro 52 antibody, the anti-SS-B/se:Sup>A antibody, the anti-CENP-B antibody, the anti-Scl 70 antibody, the anti-Jo-1 antibody, the anti-PM-Scl antibody, the anti-U1-snRNP antibody, the anti-ribosomal P protein antibody, the anti-PCNA antibody and the anti-AMA-M2 antibody in the sample to be tested, the reaction time of the sample and the mixed solution is controlled within se:Sup>A range of 10-30min.
Preferably, the reaction time of the sample to be tested and the mixed solution is 20min.
The reaction time of the antibody-antigen-microsphere complex with the secondary antibody solution may be 10-30min. Through experimental analysis, when the reaction time of the antibody-antigen-microsphere complex and the secondary antibody solution is controlled within the above range, the fluorescence signal intensity of the obtained anti-dsDNA antibody, anti-SmD 1 antibody, anti-Nucleosome antibody, anti-Histone antibody, anti-SS-A/Ro 60 antibody, anti-SS-A/Ro 52 antibody, anti-SS-B/se:Sup>A antibody, anti-CENP-B antibody, anti-Scl 70 antibody, anti-Jo-1 antibody, anti-PM-Scl antibody, anti-U1-snRNP antibody, anti-ribosomal P protein antibody, anti-PCNA antibody and anti-AMA-M2 antibody is continuously increased; when the reaction time of the antibody-antigen-microsphere complex with the secondary antibody solution is less than the above range, the fluorescent signal intensity of the anti-dsDNA antibody, anti-SmD 1 antibody, anti-Nucleosome antibody, anti-Histone antibody, anti-SS-A/Ro 60 antibody, anti-SS-A/Ro 52 antibody, anti-SS-B/se:Sup>A antibody, anti-CENP-B antibody, anti-Scl 70 antibody, anti-Jo-1 antibody, anti-PM-Scl antibody, anti-U1-snRNP antibody, anti-ribosomal P protein antibody, anti-PCNA antibody, anti-AMA-M2 antibody obtained by the detection is less than the fluorescent signal intensity of the anti-dsDNA antibody, anti-SmD 1 antibody, anti-Nucleosome antibody, anti-Histone antibody, anti-SS-A/Ro 60 antibody, anti-SS-A/Ro 52 antibody, anti-SS-B/se:Sup>A antibody, anti-CENP-B antibody, anti-Scl 70 antibody, anti-Jo-1 antibody, anti-PM-Scl, anti-RNP 1, anti-RNP antibody, anti-RNA antibody obtained by the detection is less than 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 trend of increasing the fluorescence signal intensity of the obtained anti-dsDNA antibody, anti-SmD 1 antibody, anti-Nucleosome antibody, anti-Histone antibody, anti-SS-A/Ro 60 antibody, anti-SS-A/Ro 52 antibody, anti-SS-B/se:Sup>A antibody, anti-CENP-B antibody, anti-Scl 70 antibody, anti-Jo-1 antibody, anti-PM-Scl antibody, anti-U1-snRNP antibody, anti-ribosomal P protein antibody, anti-PCNA antibody, anti-AMA-M2 antibody has been slowed down and is substantially unchanged. Therefore, in order to obtain more accurate information of the anti-dsDNA antibody, the anti-SmD 1 antibody, the anti-Nucleosome antibody, the anti-Histone antibody, the anti-SS-A/Ro 60 antibody, the anti-SS-A/Ro 52 antibody, the anti-SS-B/se:Sup>A antibody, the anti-CENP-B antibody, the anti-Scl 70 antibody, the anti-Jo-1 antibody, the anti-PM-Scl antibody, the anti-U1-snRNP antibody, the anti-ribosomal P protein antibody, the anti-PCNA antibody and the anti-AMA-M2 antibody in the sample to be tested, the reaction time of the antibody-antigen-microsphere complex and the secondary antibody solution is controlled within the range of 10-30min.
Preferably, the reaction time of the antibody-antigen-microsphere complex with the secondary antibody solution is 20min.
Preferably, the reaction temperature of the mixed solution and the sample to be tested, and the reaction temperature of the antibody-antigen-microsphere complex and the secondary antibody solution are 37+/-1 ℃.
Through experimental analysis, the fluorescent signal intensity of the obtained anti-dsDNA antibody, the anti-SmD 1 antibody, the anti-Nucleome antibody, the anti-Histone antibody, the anti-SS-A/Ro 60 antibody, the anti-SS-A/Ro 52 antibody, the anti-SS-B/se:Sup>A antibody, the anti-CENP-B antibody, the anti-Scl 70 antibody, the anti-Jo-1 antibody, the anti-PM-Scl antibody, the anti-U1-snRNP antibody, the anti-ribosomal P protein antibody, the anti-PCNA antibody and the anti-AMA-M2 antibody is larger than that of the anti-dsDNA antibody, the anti-SmD 1 antibody, the anti-Nucleome antibody, the anti-Histone antibody, the anti-SS-A/Ro 60 antibody, the anti-SS-A/Ro 52 antibody, the anti-SS-B/se:Sup>A antibody, the anti-Scl 70 antibody, the anti-Jo-1 antibody, the anti-Scl antibody, the anti-U1-snRNP antibody, the anti-RNA protein antibody and the anti-RNA protein antibody obtained through detection at room temperature (18-25 ℃) is higher than that of the fluorescent signal intensity of the anti-RNA antibody. Therefore, in order to obtain more accurate information of the anti-dsDNA antibody, the anti-SmD 1 antibody, the anti-Nucleosome antibody, the anti-Histone antibody, the anti-SS-A/Ro 60 antibody, the anti-SS-A/Ro 52 antibody, the anti-SS-B/se:Sup>A antibody, the anti-CENP-B antibody, the anti-Scl 70 antibody, the anti-Jo-1 antibody, the anti-PM-Scl antibody, the anti-U1-snRNP antibody, the anti-ribosomal P protein antibody, the anti-PCNA antibody and the anti-AMA-M2 antibody in the sample to be tested, the incubation temperature is 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 addition ratio of the sample to be measured and the mixed solution is as follows: every 1 mu L of sample to be detected is correspondingly added with 50-500 fluorescent coding magnetic microspheres coated with antigens.
Preferably, the amount of the sample to be measured is 10-100. Mu.L. Preferably 50. Mu.L.
By adopting the technical scheme, through experimental analysis, the addition proportion of the sample to be detected and the mixed solution is controlled within the range, so that the final detection can be ensured to obtain more accurate fluorescent signal intensity, and more accurate relative concentration of the antibody can be obtained. Similarly, 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 antigen-antibody relative concentration can be obtained.
In summary, the application has the following beneficial effects:
the kit provided by the application can simultaneously complete qualitative and quantitative detection of an anti-dsDNA antibody, an anti-SmD 1 antibody, an anti-Nucleome antibody, an anti-Histone antibody, an anti-SS-A/Ro 60 antibody, an anti-SS-A/Ro 52 antibody, an anti-SS-B/se:Sup>A antibody, an anti-CENP-B antibody, an anti-Scl 70 antibody, an anti-Jo-1 antibody, an anti-PM-Scl antibody, an anti-U1-snRNP antibody, an anti-ribosomal P protein antibody, an anti-PCNA antibody and an anti-AMA-M2 antibody in se:Sup>A sample to be detected. Namely, through one-time detection, detection results of 15 detection indexes are obtained. Compared with the linear immunoblotting method in the related art, the method can only provide qualitative/semi-quantitative detection results, and the ELISA method can only perform quantitative detection.
Detailed Description
The application provides an antinuclear antibody combined detection kit, which comprises the following components: se:Sup>A mixed solution of fluorescent coding microspheres respectively coated by dsDNA antigen, smD1 antigen, nucleosome antigen, histone antigen, SS-A/Ro 60 antigen, SS-A/Ro 52 antigen, SS-B/se:Sup>A antigen, CENP-B antigen, scl70 antigen, jo-1 antigen, PM-Scl antigen, U1-snRNP antigen, ribosomal P protein antigen, PCNA antigen and AMA-M2 antigen; using a fluorescein-labeled secondary antibody solution; a sample diluent; and (5) cleaning the microspheres. Wherein the dsDNA antigen is phage PM2DNA. The nucleotide sequence of phage PM2DNA is shown in SEQ ID NO: 1. Further, the dsDNA antigen is circular phage PM2DNA.
Wherein the SmD1 antigen is SmD1 core 83-119 peptide fragment. The SmD1 antigen has an amino acid sequence shown in SEQ ID NO: 2.
The weight ratio of RNP A, U1-snRNP C and U1-snRNP 68/70KD in the U1-snRNP antigen is (1-30): (1-30): (1-30). Further, the weight ratio of RNP A, U1-snRNP C and U1-snRNP 68/70KD in the U1-snRNP antigen is (5-20): (10-30): (10-30).
The fluorescent coding microsphere is a magnetic fluorescent coding microsphere. The secondary antibody solution contains anti-human IgG antibody, and the concentration of the anti-human IgG antibody is 10-50 mug/ml. The luciferin comprises Phycoerythrin (PE), fluorescein Isothiocyanate (FITC), fucoxanthin-chlorophyll-protein complex (PerCP), and Allophycocyanin (APC). The sample to be tested of the kit is a whole blood sample or a serum or plasma sample. The reaction time of the mixed solution and the sample to be tested is 10-30min. The reaction time of the substance reacted by the mixed solution and the sample to be detected and the secondary antibody solution is 10-30min. The adding proportion of the sample to be tested and the mixed solution is as follows: every 1 mu L of sample to be detected is correspondingly added with 50-500 fluorescent coding magnetic microspheres coated with antigens. The reaction temperature of the mixed solution and the sample to be tested, and the reaction temperature of the antibody-antigen-microsphere complex and the secondary antibody solution are 37+/-1 ℃.
Wherein, the preparation method of dsDNA antigen is as follows:
extracting phage PM2DNA by using a phage DNA extraction kit; then cloning the selected gene fragment by PCR, wherein the gene fragment cloned by PCR is shown as SEQ ID NO:1, namely phage PM2 linear DNA;
constructing phage PM2 linear DNA into pCDNA plasmid to obtain phage PM2 circular DNA.
The present application will be described in further detail with reference to preparation examples 1 to 25, examples 1 to 20, comparative examples 1 to 4 and detection tests.
Preparation example
Preparation example 1
The preparation example provides se:Sup>A mixed solution of fluorescent coding microspheres respectively coated by dsDNA antigen, smD1 antigen, nucleosome antigen, histone antigen, SS-A/Ro 60 antigen, SS-A/Ro 52 antigen, SS-B/se:Sup>A antigen, CENP-B antigen, scl70 antigen, jo-1 antigen, PM-Scl antigen, U1-snRNP antigen, ribosomal P protein antigen, PCNA antigen and AMA-M2 antigen.
In this preparation, the dsDNA antigen is of the type phage PM2 linear DNA.
The preparation method of the mixed solution specifically comprises the following steps:
(1) Pretreatment:
obtaining microspheres: 100. Mu.L of each suspension (about 1X 10) 7 Microspheres) are placed in 15 centrifuge tubes, the 15 centrifuge tubes are placed in a magnetic rack for magnetic adsorption, and the supernatant is separated and discarded;
Washing and resuspension of the microspheres: adding 200 mu L of microsphere washing liquid into 15 centrifuge tubes respectively, swirling for 10s, placing the centrifuge tubes in a magnetic rack, magnetically adsorbing, and separating and discarding supernatant; repeating the washing twice; 100 mu L of microsphere washing liquid is added into 15 centrifuge tubes to resuspend the microspheres, and the microspheres are mixed by vortexing for 10s, so as to obtain 15 microsphere heavy suspension A.
(2) Activating:
activating microspheres: adding 10 mu L of activator solution into 15 microsphere heavy suspensions A respectively, swirling for 10s, performing light-proof reaction for 20-30min at room temperature, placing 15 centrifuge tubes in a magnetic rack, performing magnetic adsorption, separating and discarding supernatant;
washing and resuspension of the microspheres after activation: adding 200 mu L of microsphere washing liquid into 15 centrifuge tubes respectively, swirling for 10s, placing the 15 centrifuge tubes in a magnetic rack, magnetically adsorbing, separating and discarding supernatant; repeating the washing twice; 100. Mu.L of microsphere washing liquid was added to each of 15 centrifuge tubes to resuspend the microspheres, and the microspheres were vortexed for 10s to mix the microspheres evenly to obtain 715 microsphere resuspension B.
(3) Coating:
and (3) coating microspheres: adding dsDNA antigen, smD1 antigen, nucleosome antigen, histone antigen, SS-A/Ro 60 antigen, SS-A/Ro 52 antigen, SS-B/se:Sup>A antigen, CENP-B antigen, scl70 antigen, jo-1 antigen, PM-Scl antigen, ribosomal P protein antigen, PCNA antigen and AMA-M2 antigen into 15 microsphere heavy suspension B respectively, wherein the addition amount is 30 mug, and continuing adding U1-snRNP antigen; after vortex mixing, carrying out light-shielding reaction for 24 hours at room temperature; placing the 15 centrifuge tubes after the reaction in a magnetic rack, magnetically adsorbing, separating and discarding supernatant; wherein, the addition amount of RNP A, U1-snRNP C and U1-snRNP 68/70KD in the U1-snRNP antigen is 30 mug, 0 mug and 0 mug respectively.
Sealing after coating: adding an equal volume of sealing liquid into 15 centrifuge tubes respectively, performing sealing reaction, sealing for 4 hours at room temperature in a dark place, placing the 15 centrifuge tubes after the reaction in a magnetic rack, performing magnetic adsorption, and separating and discarding supernatant.
Washing and resuspension of coated microspheres: adding 200 mu L of microsphere preservation buffer solution into 15 centrifuge tubes respectively, swirling for 10s, placing the 15 centrifuge tubes in a magnetic rack, magnetically adsorbing, separating and discarding supernatant; repeating the washing twice; 1ml of microsphere preservation buffer solution is added into 15 centrifuge tubes respectively to resuspend the microspheres, and the microspheres are mixed evenly by vortexing for 10s, so as to obtain 15 fluorescent coding magnetic microsphere heavy suspensions.
(4) And (3) preserving: respectively placing 15 fluorescent coding magnetic microsphere heavy suspensions at 2-8deg.C and keeping away from light; mixing 15 fluorescent coding magnetic microsphere heavy suspensions according to equal weight to obtain a mixed solution.
PREPARATION EXAMPLES 2 to 14
Preparation examples 2 to 14 provide se:Sup>A mixed solution of fluorescent-encoded microspheres coated with dsDNA antigen, smD1 antigen, nucleosome antigen, histone antigen, SS-A/Ro 60 antigen, SS-A/Ro 52 antigen, SS-B/se:Sup>A antigen, CENP-B antigen, scl70 antigen, jo-1 antigen, PM-Scl antigen, U1-snRNP antigen, ribosomal P protein antigen, PCNA antigen, AMA-M2 antigen, respectively.
The above preparation example is different in that: the addition amount of RNP A, U1-snRNP C and U1-snRNP68/70KD in the U1-snRNP antigen.
The amounts of the three antigen materials added to the mixed solution U1-snRNP antigen prepared in the above preparation example are shown in Table 1.
TABLE 1 addition amount of three antigen raw materials in the mixed solution U1-snRNP antigen of preparation examples 1-14
Preparation examples 15 to 19
Preparation examples 15-19 provide se:Sup>A mixture of fluorescent-encoded microspheres coated with dsDNA antigen, smD1 antigen, nucleosome antigen, histone antigen, SS-A/Ro 60 antigen, SS-A/Ro 52 antigen, SS-B/se:Sup>A antigen, CENP-B antigen, scl70 antigen, jo-1 antigen, PM-Scl antigen, U1-snRNP antigen, ribosomal P protein antigen, PCNA antigen, AMA-M2 antigen, respectively.
Preparation examples 15 to 19 differ from preparation example 1 in that: a selected species of dsDNA antigen. As shown in table 2.
TABLE 2 kinds of dsDNA antigens in preparation examples 15-20
| Preparation example | Class of dsDNA antigens | dsDNA source |
| 11 | Phage PM 2-loop | Phage DNA |
| 15 | Phage PM 2-line | Phage DNA |
| 16 | DIARECT | Derived from bacteria |
| 17 | HUMAN-LIA | Derived from bacteria |
| 18 | HUMAN-ELISA | Synthetic DNA (pUC 18) |
| 19 | Sigma | Derived from calf thymus |
Preparation example 20
The preparation example provides a secondary antibody solution marked by fluorescein. The preparation method of the secondary antibody solution marked by fluorescein specifically comprises the following steps:
1. PBS-TNP solution is prepared according to the following formula:
na is mixed with 2 HPO 4 ·12H 2 O 2.9g、KH 2 PO 4 0.2g、NaCl 8g、KCl 0.2g、DI H 2 O0.9L was mixed and added with T-20.2 ml, PC-300.5 ml, BSA 1g; the pH was adjusted to 7.4 and then fixed to a volume of 1L with sterile water.
2. The prepared PBS-TNP solution was mixed with a secondary antibody solution labeled with fluorescein at a ratio of 1:10000 to give a final concentration of 5ug/ml.
Preparation examples 21 to 24
Preparation examples 21 to 24 provide secondary antibody solutions labeled with fluorescein, respectively. Preparation examples 21 to 24 differ from preparation example 20 in that: final concentration of the fluorescein-labeled secondary antibody solution was used. Specifically, the results are shown in Table 3.
TABLE 3 final concentration of secondary antibody solutions labeled with fluorescein in PREPARATIVE EXAMPLES 20-24
| Preparation example | Final concentration (ug/ml) |
| 20 | 5 |
| 21 | 10 |
| 22 | 20 |
| 23 | 30 |
| 24 | 50 |
Examples
Examples 1 to 19
Examples 1-19 provide an antinuclear antibody combination detection kit, respectively. Examples 1 to 19 differ in the mixture and the secondary antibody solution labeled with fluorescein. Specifically, the results are shown in Table 4.
The human autoimmune disease detection kit comprises the following components:
se:Sup>A mixed solution of fluorescent coding microspheres respectively coated by dsDNA antigen, smD1 antigen, nucleosome antigen, histone antigen, SS-A/Ro 60 antigen, SS-A/Ro 52 antigen, SS-B/se:Sup>A antigen, CENP-B antigen, scl70 antigen, jo-1 antigen, PM-Scl antigen, U1-snRNP antigen, ribosomal P protein antigen, PCNA antigen and AMA-M2 antigen; using a fluorescein-labeled secondary antibody solution; a sample diluent; and (5) cleaning the microspheres.
TABLE 4 kinds of mixed solution and secondary antibody solution in examples 1 to 19
Comparative example
Comparative examples 1 to 4
Comparative examples 1-4 each provide an antinuclear antibody combination detection kit. Comparative examples 1 to 4 differ from example 1 in the mixture of the components. Specifically, the results are shown in Table 5.
TABLE 5 kinds of mixed solution and secondary antibody solution in comparative examples 1 to 4
Test 1
And (3) detecting the sample to be detected by using the anti-nuclear antibody combined detection kit provided in the above examples 1-14 and 16-19 respectively. The types and numbers of samples to be tested are shown in table 6, respectively.
The detection method of the anti-nuclear antibody combined detection kit comprises the following steps:
(1) Diluting a sample to be tested by using a sample diluent to obtain a diluted sample;
(2) Mixing the diluted sample obtained in the step (1) with the mixed solution A, and reacting for 20min at 37 ℃ to obtain an antibody-antigen-microsphere compound;
(3) Cleaning the antibody-antigen-microsphere compound obtained in the step (2) by utilizing 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 anti-antibody-antigen-microsphere compound obtained in the step (4) by utilizing microsphere cleaning liquid, and removing the microsphere cleaning liquid;
(6) And (3) re-suspending the secondary anti-antibody-antigen-microsphere compound cleaned in the step (5) by utilizing microsphere cleaning liquid, loading and detecting, and analyzing the detection result.
The results of the measurements are shown in tables 6 and 7.
TABLE 6 detection results of U1-snRNP Using the kits provided in examples 1-14, examples 16-19
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TABLE 7 analysis of the detection results of the kits provided in examples 1-14, examples 16-19
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As can be seen from a combination of tables 6 and 7, the low positive compliance of examples 1 to 3, particularly those of examples 1 and 3, indicates that the use of RNP A, U1-snRNP C or U1-snRNP 68/70KD alone as the U1-snRNP antigen significantly resulted in inaccurate detection results. In particular, when U1-snRNP C alone is used as the U1-snRNP antigen, significant false negatives are also detected.
As can be seen from the results of examples 4 to 6, when the addition ratio of RNP A, U1-snRNP C or U1-snRNP 68/70KD is 30:30:30, the positive compliance rate in the test results was 83.33%, and when RNP A, U1-snRNP C or U1-snRNP 68/70KD was added in a ratio of 20:20:20 or 10:10: and when the test is carried out at 10, the positive coincidence rate in the test result can only reach 66.67% or 50.00%, and the positive coincidence rate is low. Therefore, when the amounts of RNP A, U1-snRNP C or U1-snRNP 68/70KD are required to be uniform, the preferable amounts of all the three are 30. Mu.g.
Further, as is evident from the results of the test in examples 7 to 14, when RNP A, U1-snRNP C or U1-snRNP68/70KD is added in a ratio of (5-20): (10-30): (10-30), and the positive coincidence rate in the detection result is more than 83.33%. In particular, when RNP A, U1-snRNP C or U1-snRNP68/70KD is added in a ratio of 5:20: at 20, the positive coincidence rate in the detection result can reach 100%. Therefore, when RNP A, U1-snRNP C or U1-snRNP68/70KD is mixed as the U1-snRNP antigen, the positive coincidence rate in the detection result can be effectively improved by reducing the addition amount of RNP A, so that the accuracy of the detection result is improved.
As can be seen from the detection results of examples 11 and examples 16 to 19, the concentration of the secondary antibody solution has a large influence on the test results, and by comparing the positive coincidence rate in the detection results, it can be seen that the positive coincidence rate reaches 100% when the concentration of the secondary antibody solution is controlled to be 10-30ug/ml, and that the positive coincidence rate is less than 100% when the concentration of the secondary antibody solution is less than 10ug/ml or greater than 30 ug/ml. Therefore, the concentration of the secondary antibody solution was selected to be controlled within the range of 10-30 ug/ml.
Detection test II
The test samples were tested using the kits provided in examples 11, 15 and comparative examples 1-4, respectively. The detection method is shown as "detection test one". The types, the numbers and the detection results of the samples to be detected are shown in tables 8 and 9.
TABLE 8 detection results using the kits provided in example 11, example 15 and comparative examples 1-4
TABLE 9 analysis of the detection results of the kits provided in example 11, example 15, and comparative examples 1-4
In combination with tables 8 and 9, it is understood from the results of the test of comparative examples 11 and 15 that the positive coincidence rate is higher when the dsDNA antigen is the circular phage PM2DNA than when the dsDNA antigen is the linear phage PM2 DNA. Therefore, it is further preferred that the dsDNA antigen is circular phage PM2.
As can be seen from the results of comparative examples 11, 15 and 1-4, the positive and negative compliance rates were both greater when the dsDNA antigen was phage PM2DNA than when the dsDNA antigen was DIARECT, HUMAN-LIA, HUMAN-ELISA and Sigma. Thus, phage PM2DNA was selected as dsDNA antigen.
The present embodiment is only for explanation of the present application and is not to be construed as limiting the present application, and modifications to the present embodiment, which may not creatively contribute to the present application as required by those skilled in the art after reading the present specification, are all protected by patent laws within the scope of claims of the present application.
Sequence listing
<110> Beijing Hu Manzhi made technology Co., ltd
Nanjing Chen light medical science and technology Co Ltd
<120> an antinuclear antibody combined detection kit
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<170> SIPOSequenceListing 1.0
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<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 1
atgccagcag caaaaaagca aattgaagaa aagccagaag ttgagcaaga tttgggcgcg 60
ccagatttta gcgacttact cgacgatgat gaaaaaacgc taattgattc ggttgtaaat 120
gacgatgatg aaagcgacga attaaccgac gatgcaatag gtatggccgt tggtgagttg 180
gtcgggatgg gtgtcatgtt tttaactgat tacttggccg agcgacgcgg ggagcattgg 240
aacgtaagca ctaaagagtt aaagcaactt gcaaaggcgg ttgatggctc tgtaccagat 300
acagagctat cgccagcgtg ggcgcttgtt gctgtcagtg tgggcatgtt tgccccgcgt 360
gttgttgttg atattcaatt aaataaaaga aaggtgattg aagttgagaa cgacgacaaa 420
aaagcagatt ga 432
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<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 2
Met Leu Leu Val Ala Pro Leu Met Leu Leu Ser His Gly Thr Val Thr
1 5 10 15
Ile Gly
Claims (7)
1. An antinuclear antibody joint detection kit, characterized in that said kit comprises:
se:Sup>A mixed solution of fluorescent coding microspheres respectively coated by dsDNA antigen, smD1 antigen, nucleosome antigen, histone antigen, SS-A/Ro 60 antigen, SS-A/Ro52 antigen, SS-B/se:Sup>A antigen, CENP-B antigen, scl70 antigen, jo-1 antigen, PM-Scl antigen, U1-snRNP antigen, ribosomal P protein antigen, PCNA antigen and AMA-M2 antigen;
using a fluorescein-labeled secondary antibody solution; a sample diluent; cleaning the microspheres;
the dsDNA antigen is phage PM2 DNA;
the nucleotide sequence of the phage PM2 DNA is shown as SEQ ID NO:1 is shown in the specification;
the dsDNA antigen is circular phage PM2 DNA;
the U1-snRNP antigen comprises RNP A, U1-snRNP C and U1-snRNP 68/70KD in the weight ratio of (5-20): (10-30): (10-30), and the weight ratio of the three is not 1:1:1.
2. The antinuclear antibody joint detection kit according to claim 1, wherein said fluorescent-encoded microspheres are magnetic fluorescent-encoded microspheres.
3. The antinuclear antibody combined detection kit according to claim 1, wherein the secondary antibody solution contains an anti-human IgG antibody, and the concentration of the anti-human IgG antibody is 10 to 50 μg/ml.
4. The antinuclear antibody joint detection kit according to claim 1, wherein said fluorescein comprises phycoerythrin PE, fluorescein isothiocyanate FITC, polymethine-chlorophyll-protein complex PerCP, allophycocyanin APC.
5. The antinuclear antibody combined detection kit according to claim 1, wherein the reaction time between the mixed solution and the sample to be detected is 10-30min.
6. The kit for the combined detection of antinuclear antibodies according to claim 1, wherein the reaction time between the substance after the reaction of the mixed solution and the sample to be detected and the secondary antibody solution is 10-30min.
7. The kit for the combined detection of antinuclear antibodies according to claim 1, wherein the reaction temperature of the mixed solution with the sample to be detected and the antibody-antigen-microsphere complex with the secondary antibody solution is 37.+ -. 1 ℃.
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