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Detection kit of anti-double-stranded DNA (Deoxyribonucleic Acid) antibody IgG and detection method of detection kit

A technology for detection kits and reagents, which is applied in biological testing, measuring devices, material inspection products, etc., can solve the problems of difficult standardization, low sensitivity, inability to distinguish non-specific recognition by molecular weight, shorten reaction time, improve clinical sensitivity and The effect of the linear range

Inactive Publication Date: 2018-07-06
SUZHOU HAOOUBO BIOPHARML
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Among them, radioimmunoassay is considered to be the gold standard for detecting anti-double-stranded DNA antibodies. Although the repeatability is good, it can only detect high-affinity antibodies, cannot distinguish antibody subtypes, and requires specific equipment, and isotope contamination is relatively high. serious
Indirect immunofluorescence can be used as a semi-quantitative assay, but it has subjective judgment errors and difficult standardization; and it is impossible to distinguish non-specific recognition based on molecular weight when analyzing results
Although the dot immunogold filtration method is simple and fast, it has low specificity and cannot be used for quantitative detection
Enzyme-linked immunosorbent assay can be used for high-throughput sample detection. The method is simple and easy to standardize. It can detect different types of antibodies. High and low affinity antibodies can be measured at the same time. It is widely recognized, but this method still has certain limitations: (1) The detection reagent is open in the detection process, which is easy to cause cross-contamination between various reagents and affect the detection results; (2) The detection range of ELISA is narrow and the sensitivity is low; (3) ELISA The detection time of immunosorbent method is long, and the total time required to complete a test is generally more than 2 hours, which cannot fully meet the needs of rapid clinical diagnosis; Hysteresis
[0007] For example, China Patent Application No. 201610249121.9, the invention patent of the patent name "a magnetic particle chemiluminescence quantitative assay kit for anti-double-stranded DNA antibody IgG and its preparation and detection method", which applies the magnetic particle chemiluminescence quantitative detection technology to anti-double-stranded DNA antibody IgG In the detection of stranded DNA antibodies, the sensitivity and linear range have been improved, but it does not disclose the source of double-stranded DNA antigens and the composition of chemiluminescent substrates

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  • Detection kit of anti-double-stranded DNA (Deoxyribonucleic Acid) antibody IgG and detection method of detection kit
  • Detection kit of anti-double-stranded DNA (Deoxyribonucleic Acid) antibody IgG and detection method of detection kit
  • Detection kit of anti-double-stranded DNA (Deoxyribonucleic Acid) antibody IgG and detection method of detection kit

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Embodiment 1

[0037] The preparation of the first reagent of embodiment one

[0038] 1. Materials and instruments:

[0039] Materials: Double-stranded DNA antigen from Escherichia coli, double-stranded DNA antigen from calf thymus, double-stranded DNA antigen from herring sperm, photobiotin, tris buffer, glycerol , phosphate buffer saline;

[0040] Instrument: Reagent cryogenic storage box.

[0041] 2. Preparation steps:

[0042] Step 1: Mix 0.8 mg double-stranded DNA antigen from Escherichia coli with 0.05 mg photobiotin, and let stand at 25°C for 30 minutes;

[0043] Step 2: Add 12uL of tris buffer solution with a substance concentration of 0.05mol / L, and let stand at 25°C for 15min;

[0044] Step 3: Add 350uL of glycerol to obtain biotinylated double-stranded DNA antigen from Escherichia coli, and store it at -20°C for later use;

[0045] Step 4: Mix 0.8 mg double-stranded DNA antigen from calf thymus with 0.05 mg photobiotin, and let stand at 25°C for 30 minutes;

[0046] Step 5: ...

Embodiment 2

[0052] The preparation of the second reagent of embodiment two

[0053] 1. Materials and instruments:

[0054] Materials: anti-human IgG antibody, 2-iminosulfane hydrochloride coupling agent, glycine, alkaline phosphatase, tris buffer;

[0055] Instruments: Reagent cryogenic storage box, G-25 gel column, Supperdex200 gel purification column.

[0056] 2. Preparation steps:

[0057] Step 1: Add 3mg of anti-human IgG antibody to 40mL of 2-iminosulfane hydrochloride coupling agent with a concentration of 10mg / mL, and let stand at 20°C for 20min;

[0058] Step 2: Add 2mL of 0.08mol / L glycine solution, let it stand at 20°C for 4min, use G-25 gel column to desalt, collect the activated anti-human IgG antibody, and store it at 5°C for later use;

[0059] Step 3: Add 3 mg alkaline phosphatase solution to 4 mg / mL 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester solution at 25°C Let it stand for 30 minutes, use G-25 gel column to desalt, collect the activated alka...

Embodiment 3

[0062] The preparation of embodiment three calibrator

[0063] 1. Materials and instruments:

[0064] Materials: anti-double-stranded DNA antibody, phosphate buffer, standard;

[0065] 2. Preparation steps:

[0066] Select an anti-double-stranded DNA antibody, dilute it in a certain ratio with a phosphate buffer solution with a pH of 7-7.5 and a substance concentration of 0.01mol / L, and prepare a concentration of 10RU / mL and 400RU / mL with reference to the standard. calibrators.

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Abstract

The invention discloses a detection kit of an anti-double-stranded DNA (Deoxyribonucleic Acid) antibody IgG. The detection kit comprises a first reagent, a second reagent, a magnetic micro-particle separation reagent, a chemical light emission substrate, a calibration standard, a quality control product and a cleaning fluid, wherein the first reagent is a solution with a mixed double-stranded DNAantigen coupled with biotin; the mixed double-stranded DNA antigen consists of a double-stranded DNA antigen extracted from escherichia coli, a double-stranded DNA antigen extracted from calf thymus,and a double-stranded DNA antigen extracted from herring sperms; and the second reagent is a solution with an anti-human IgG antibody coupled with alkaline phosphatase. The antigen in the antigen reagent of the detection kit of the anti-double-stranded DNA antibody IgG is the mixed double-stranded DNA antigen coupled with biotin, and due to complementariness of multi-source antigens, the clinicalsensitivity can be greatly improved, and the linear range can be greatly increased. The standard time that all procedures of the detection kit are completed and results are obtained is 45 minutes, andthe reaction time of the detection kit is greatly shortened when being compared with that of an enzyme linked immunosorbent assay method.

Description

technical field [0001] The invention belongs to the technical field of in vitro diagnosis, and in particular relates to a detection kit for anti-double-stranded DNA antibody IgG and a detection method for the detection kit. Background technique [0002] Systemic lupus erythematosus (SLE) is an autoimmune disease that invades the connective tissue of the whole body and most organs. It mostly occurs in young women of childbearing age. , is a medical disease that urgently needs to be studied and solved, and is also one of the hot spots of research. [0003] Clinical studies have found that there are a variety of autoantibodies in the blood of SLE patients, especially antinuclear antibodies, which are often accompanied by elevated autoantibody titers during the active stage of the disease. Among them, anti-double-stranded DNA antibody is the most common autoimmune antinuclear antibody in SLE patients, and its positive rate is about 50%-80%. Therefore, detecting the content of ...

Claims

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Application Information

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IPC IPC(8): G01N33/68
CPCG01N33/6854G01N33/6893G01N2800/104
Inventor 李庆春崔利歌柳乐赵婷黎静雯杨苏清徐乐
Owner SUZHOU HAOOUBO BIOPHARML