CN108398550B - Composition, chip, preparation method of chip and detection device comprising chip - Google Patents
Composition, chip, preparation method of chip and detection device comprising chip Download PDFInfo
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- CN108398550B CN108398550B CN201810186409.5A CN201810186409A CN108398550B CN 108398550 B CN108398550 B CN 108398550B CN 201810186409 A CN201810186409 A CN 201810186409A CN 108398550 B CN108398550 B CN 108398550B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/34—Genitourinary disorders
- G01N2800/347—Renal failures; Glomerular diseases; Tubulointerstitial diseases, e.g. nephritic syndrome, glomerulonephritis; Renovascular diseases, e.g. renal artery occlusion, nephropathy
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention relates to the technical field of biological detection, in particular to a composition, a chip, a preparation method of the composition and the chip and a detection device comprising the chip. The detection kit has high sensitivity and specificity, can detect 12 antibodies at a time, has the advantages of higher accuracy, simple and convenient operation, antigen saving, cost reduction and the like, optimizes related reagent formulas and treatment methods, ensures that the stability of the kit is better (2-8 ℃ for refrigeration for 2 years, and can be stored at room temperature for 6 months), and is more convenient for storage and transportation.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a composition, a chip, a preparation method of the composition and the chip, and a detection device comprising the chip.
Background
Autoimmune-related nephritis is a disease in which an immune complex is deposited on the kidney due to the binding of an autoantigen and an autoantibody, resulting in damage to the kidney. Including Lupus Nephritis (LN), Primary Membranous Nephropathy (PMN), anti-neutrophil cytoplasmic antibody (ANCA) -associated renal damage, and the like. The incidence of immune nephritis in China is still high so far. The disease is a persistent or progressive development of underlying pathological changes, which makes the disease persistent and chronic, rather than simply a chronic stage of acute nephritis. Early diagnosis aids in the clinical management of autoimmune diseases. Research has found that specific autoantibodies, such as ANCA-associated vasculitis anti-Myeloperoxidase (MPO) antibody, anti-protease 3 antibody (PR3), anti-glomerular basement membrane antibody (GBM), anti-endothelial cell antibody, anti-Lactoferrin (LF) antibody, anti-human lysosomal associated membrane protein (LAMP-2) antibody, are present in the serum of most patients with autoimmune renal disease; the antibody is characterized by comprising an anti-C1 q antibody, an anti-Nucleosome (Nucleosome) antibody and an anti-double-chain DNA (dsDNA) antibody of Lupus Nephritis (LN), an anti-phospholipase A2 receptor (PLA2R) antibody and a thrombospondin type 1 (THSD7A) antibody of primary membranous nephropathy (IMN), and a fusion protein antibody prepared by the research of the company and fusing an anti-phospholipase A2 receptor (PLA2R) and a thrombospondin type 1 (THSD7A), wherein the specific autoantibodies PT are important components in the diagnostic standard of autoimmune nephropathy.
At present, a plurality of methods for detecting autoimmune-related nephritis antibodies comprise an indirect immunofluorescence assay, an enzyme-linked immunosorbent assay or an immunoblotting method, but have respective defects. The indirect immunofluorescence assay infers the class of autoantibodies through the formed fluorescent conjugates, lacks a specific objective diagnosis, and requires secondary confirmation by other techniques, such as immunoblotting, enzyme-linked immunization, and the like. The enzyme-linked immunosorbent assay can only detect a single antibody in one test, has low efficiency and higher cost, and has certain limitation in the aspect of diagnosis and application. The method has the problems of large sample amount, complex operation, overlong detection time, higher cost and the like. And the stable period of the kit is generally 2-8 ℃ for 1 year.
The ELISA-Array technology not only keeps the advantages of simple operation and low cost of the ELISA method, but also has the advantage of high flux of the protein chip technology; the technology fixes a plurality of markers in the pores of a microporous plate in a microarray form through a spotting instrument, and can detect a plurality of pathogens simultaneously. It can realize multiple detections of a sample in a micropore, and greatly reduces the amount of samples and reagents. However, at present, no research on ELISA-Array technology detection aiming at autoimmune nephropathy exists in China. Therefore, the detection kit for the autoimmune nephritis antibody, which is high in sensitivity, specificity and stability, has important practical significance.
Disclosure of Invention
In view of the above, the present invention provides a composition, a chip, a method for preparing the same, and a detection device comprising the chip. The kit can detect 12 antibodies at one time, has the advantages of higher accuracy, simple and convenient operation, antigen saving, cost reduction and the like, optimizes related reagent formulas and treatment methods, ensures that the stability of the kit is better (2-8 ℃ for refrigeration for 2 years, and can be stored at room temperature for 6 months), and is more convenient in storage and transportation conditions.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a composition, which comprises one or a mixture of more than two of citric acid, mannitol, PVA2W, PEG1W, acacia gum and sodium p-hydroxybenzoate.
In some embodiments of the invention, the volume ratio of citric acid, mannitol and PVA2W in the composition is (1-1.5): (0.3-0.6): 0.8-1.5.
In other embodiments of the present invention, the following components are included in the composition:
in other embodiments of the invention, the composition further comprises a mixture of one or more of BSA, Proclin300, PBS, or disodium phosphate.
In other embodiments of the present invention, the BSA is 1 to 2.5% by weight in the composition;
the volume percentage content of the Proclin300 in the composition is 0.05%;
the concentration of PBS is 0.01M;
the concentration of the disodium hydrogen phosphate is 0.01M.
The invention also provides the application of the composition in the preparation of a chip and/or a detection device.
The invention also provides a chip coated with the composition.
In some embodiments of the invention, the chip is further coated with an autoimmune nephropathy-associated antigen.
In some embodiments of the invention, the autoimmune nephropathy associated antigen comprises at least one of GBM, PR3, MPO, LF, LAMP-2, PLA2R, THSD7A, PT protein, endothelial cells, C1q, dsDNA, nucleosomes.
In some embodiments of the invention, the chip further comprises a quality control point and/or a reference point; the quality control points comprise at least one positive quality control Point (PC), at least one negative quality control point (NC), at least one sample quality control point (SC) and/or at least one enzyme-labeled quality control point (EC); the reference points include reference curve points (S1-S3) of different concentrations and/or at least one chip position reference point (Loc).
In some embodiments of the invention, the positive control sites are coated with human IgG or coated with a BSA-DNP conjugate; the negative quality control point is coated with trace concentration of human IgG or other proteins irrelevant to the autoimmune nephropathy which is lower than the reaction signal value; the sample quality control point is coated with anti-human IgG; the enzyme-labeled quality control points are coated with human IgG, anti-rabbit antibodies or anti-sheep antibodies; the reference curve points are coated with human IgG at different concentrations; the chip location reference points were coated with a solution of human IgG of known concentration.
The invention provides a preparation method of the chip, which comprises the following steps:
step 1: diluting the autoimmune nephropathy associated antigen, the protein of the quality control point and/or the protein of the reference point by a coating buffer solution, and coating the diluted product on the substrate of the chip in a dot array form;
and 2, step: sealing the chip by a sealing stabilizer;
the blocking stabilizer comprises the composition.
In some embodiments of the invention, the blocking stabilizer comprises 1% to 2% BSA (g/v), preferably 1% BSA (g/v).
In some embodiments of the invention, the blocking stabilizer further comprises 1% to 1.5% citric acid (v/v), preferably 1% citric acid (v/v).
In some embodiments of the present invention, the blocking stabilizer further comprises 0.3% to 0.6% mannitol (v/v), preferably 0.5% mannitol (v/v).
In some embodiments of the invention, the blocking stabilizer further comprises 0.8% to 1.5% PVA2W (v/v), preferably 1% PVA2W (v/v).
In some embodiments of the invention, the blocking stabilizer further comprises 0.05% (v/v) of Proclin 300.
In some embodiments of the invention, the blocking stabilizer is a 0.01M disodium phosphate solution containing 1% to 2% BSA (g/v), 1% to 1.5% citric acid (v/v), 0.3% to 0.6% mannitol (v/v), 0.8% to 1.5% PVA2W (v/v) and 0.05% (v/v) Proclin 300.
In some embodiments of the invention, the blocking stabilizer is a 0.01M disodium phosphate solution containing 1% BSA (g/v), 1% citric acid (v/v), 0.5% mannitol (v/v), 1% PVA2W (v/v) and 0.05% (v/v) Proclin 300.
The invention also provides a chip prepared by the preparation method.
The invention also provides a detection device which comprises the chip.
In some embodiments of the invention, the detection device further comprises an enzyme-labeled dilution stabilizer; the enzyme-labeled diluent stabilizer comprises 0.01M PBS.
In some embodiments of the invention, the detection device further comprises an enzyme-labeled dilution stabilizer; the enzyme-labeled dilution stabilizer also comprises 0.05M citric acid.
In some embodiments of the invention, the detection device further comprises an enzyme-labeled dilution stabilizer; the enzyme-labeled diluent stabilizer also comprises 1.5-2.5% (g/v) BSA, preferably 2% (g/v) BSA.
In some embodiments of the invention, the detection device further comprises an enzyme-labeled dilution stabilizer; the enzyme-labeled diluent stabilizer also comprises 1.5-2% (v/v) of PEG1W, preferably 2% (v/v) of PEG 1W.
In some embodiments of the invention, the detection device further comprises an enzyme-labeled dilution stabilizer; the enzyme-labeled diluent stabilizer also comprises 0.1-0.2% (g/v) of gum arabic, and preferably 0.1% (g/v) of gum arabic.
In some embodiments of the invention, the detection device further comprises an enzyme-labeled dilution stabilizer; the enzyme-labeled diluent stabilizer also comprises 0.2 to 0.5 percent of sodium p-hydroxybenzoate (v/v), and preferably 0.3 percent of sodium p-hydroxybenzoate (v/v).
In some embodiments of the invention, the detection device further comprises an enzyme-labeled dilution stabilizer; the enzyme labeling dilution stabilizer also comprises 0.05% (v/v) Proclin 300.
In some embodiments of the invention, the detection device further comprises an enzyme-labeled dilution stabilizer; the enzyme labeling diluting stabilizer comprises 0.01M PBS, 0.05M citric acid, 1.5-2.5% (g/v) BSA, 1.5-2% (v/v) PEG1W, 0.1-0.2% (g/v) gum arabic, 0.2-0.5% sodium p-hydroxybenzoate (v/v) and 0.05% (v/v) Proclin 300.
In some embodiments of the invention, the detection device further comprises an enzyme-labeled dilution stabilizer;
the enzyme-labeled diluent stabilizer comprises 0.01M PBS, 0.05M citric acid, 2% (g/v) BSA, 2% (v/v) PEG1W, 0.1% (g/v) gum arabic, 0.3% sodium p-hydroxybenzoate (v/v) and 0.05% (v/v) Proclin 300.
In some embodiments of the invention, the detection device further comprises a mixture of one or more of a label, a sample diluent, a washing solution, and a developing solution.
In some embodiments of the invention, the label is an anti-human IgG antibody labeled with a label conjugate;
the label conjugate comprises an enzyme label, avidin or acridinium ester;
the enzyme label is horseradish peroxidase or alkaline phosphatase;
the anti-human IgG antibody is a rabbit anti-human IgG antibody or a goat anti-human IgG antibody;
the sample diluent is a 0.02M Tris solution of pH7.4 containing 0.15M NaCl, 0.05% Tween20, 0.01% casein;
the washing solution is 0.02M Tris solution with pH7.4 containing 0.15M NaCl and 0.05% Tween 20;
the color development liquid is ECL.
The invention also provides a detection method of the autoimmune nephropathy based on the detection device, which comprises the steps of taking a sample to be detected, diluting the sample with the sample diluent, coating the sample to be detected on the chip, adding the marker, adding the developing solution for developing color in a dark place, and obtaining a detection result of the fluorescence detection device to obtain a signal value of the antibody related to the autoimmune nephropathy in the sample to be detected.
The invention utilizes ELISA-Array technology to prepare a highly sensitive and specific detection kit for the autoimmune-related nephritis antibodies, can detect 12 antibodies at one time, has the advantages of higher accuracy, simple and convenient operation, antigen saving, cost reduction and the like, and optimizes related reagent formulas and treatment methods, so that the stability of the kit is better (2-8 ℃ for 2 years in refrigeration and can be stored at room temperature for 6 months), and the storage and transportation conditions are more convenient.
Detailed Description
The invention discloses a composition, a chip, a preparation method thereof and a detection device comprising the chip, and a person skilled in the art can realize the purpose by properly improving process parameters by taking the contents into consideration. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The protein chip technology adopted by the invention prepares the autoantibody spectrum protein chip kit related to the autoimmune nephropathy, which has the advantages of high throughput, multiple detection indexes, stable performance, good repeatability and high accuracy.
The kit provided by the invention comprises a protein chip, 12 antigens of GBM, PR3, MPO, LF, LAMP-2, PLA2R, THSD7A, PT protein, endothelial cells, C1q, dsDNA and nucleosomes and required reference point protein are coated on a chip substrate in a dot array form of 4 x 5 by a high-precision spotting instrument, and corresponding related antibodies in a sample are captured by the immunological principle of antigen-antibody specific reaction.
Reference points in embodiments of the present invention include: at least one negative control point (NC) and one positive control Point (PC); at least one sample quality control point (SC) and one enzyme-labeled quality control point (EC); at least 3 standard curve points (S1-S3) and a chip-self-coated location reference point (Loc).
In the embodiment of the invention, the positive quality control point can be human IgG, and the used enzyme-linked immunosorbent assay marker is the marker of the anti-human IgG. In other embodiments, the spot may also be coated with BSA-conjugated DNP, and the enzyme-linked immuno label used is a mixture of anti-human IgG and anti-DNP labels.
In the embodiment of the present invention, the negative control point can be a trace concentration of human IgG lower than the reaction signal value, and can also be other unrelated proteins in other embodiments.
In the embodiment of the invention, the sample quality control point can be goat anti-human IgG, and in other embodiments, other anti-human IgG can be used, such as rabbit anti-human IgG, mouse anti-human IgG, and the like.
In the embodiment of the present invention, the enzyme-labeled quality control point may be human IgG (enzyme-labeled anti-human IgG), and in other embodiments, other enzyme-labeled quality control points may be used: such as anti-rabbit antibodies (enzyme-labeled with rabbit anti-human IgG), or anti-sheep antibodies (enzyme-labeled with sheep anti-human IgG).
In the embodiment of the invention, the standard curve points are coated with human IgG with three concentrations, namely low, medium and high, and are used for internal calibration and comparison and interpretation of results.
In the embodiment of the invention, the position reference point of the chip is coated with 2 mu g/ml human IgG solution, which is mainly used for positioning when the array is subjected to value taking.
In the embodiment of the invention, the chip substrate is a 96-hole enzyme labeling reaction plate, and in other embodiments, the substrate can also be a carrier suitable for protein adhesion, such as a glass slide, various chemical films, porous silica gel and the like.
The antigen provided by the invention comprises several or more than 12 antigens, and is uniformly coated on a chip substrate by using different antigen coating buffers. The antigen coating buffer solution is a CB buffer solution with pH9.6, a Tris buffer solution with pH8.5 and a PBS buffer solution with pH7.4, wherein trehalose, glycerol, PEG or PVP and Proclin300 preservative are added, and additives such as water-soluble cyclodextrin and the like are added, so that the coating effect is better, more stable and uniform, and the coated points are more regular, mellow and smaller in CV. The PEG is PEG-400, and the water-soluble cyclodextrin can be 0.02% of 2-hydroxy-beta-cyclodextrin, and can also be Captisol, carboxymethyl-beta-cyclodextrin and the like.
The chip is coated under the conditions of 2-8 ℃ and overnight coating for 24-30 hours.
The chip is blocked when the coating is finished, and the blocking stabilizer used in the invention is 0.01M disodium hydrogen phosphate solution containing 1% BSA, 1% citric acid, 0.5% mannitol, 1% PVA2W and 0.05% Proclin 300.
The enzyme label used by the kit is rabbit anti-human IgG labeled by HRP, the kit comprises an enzyme label working solution and an enzyme label diluent, wherein the enzyme label working solution is an enzyme label with a final concentration of HPR labeled rabbit anti-human IgG diluted by an enzyme label diluent stabilizer, and the enzyme label diluent stabilizer is 0.01M PBS, 0.05M citric acid, 2% BSA (g/v), 2% (v/v) PEG1W, 0.1% (g/v) Arabic gum, 0.3% (v/v) sodium p-hydroxybenzoate and 0.05% (v/v) Proclin 300. In other embodiments, other enzyme-labeled anti-human IgG, such as goat anti-human IgG; in other embodiments, other label conjugates can be used, such as AP, avidin, acridinium esters, and the like.
The chromogenic substrate used in the present invention is an enhanced chemiluminescent substrate (ECL) and the reading of the reaction results is performed by a fluorescent detection device or instrument. Other chromogenic substrates, such as p-NPP, TMB, etc., may be used in other embodiments.
The invention utilizes ELISA-Array technology to prepare a highly sensitive and specific autoimmune nephropathy antibody spectrum detection kit, the technology is low in price after being commercialized, and the stability of the kit is better than that of related products (generally 2-8 ℃ for 1 year) on the market.
The composition, the chip, the preparation method thereof and the raw materials and reagents used in the detection device containing the chip can be purchased from the market.
The invention is further illustrated by the following examples:
examples 1 to 3
1. Coating of the chip:
1) the chip array design and the specific distribution of the antigens and reference points are shown in tables 1 and 2:
TABLE 1
| · | · | · | · | · |
| · | · | · | · | · |
| · | · | · | · | · |
| · | · | · | · | · |
TABLE 2
| PC | NC | GBM | LAMP-2 | Endothelial cells |
| SC | S1 | PR3 | PLA2R | Nucleosome |
| EC | S2 | MPO | THSD7A | dsDNA |
| Loc | S3 | LF | PT proteins | C1q |
2) The specific coating process is as follows:
first, the antibodies and related proteins were diluted as follows:
the PC, NC, S1, S2, S3, and EC spots in the array were coated with 2. mu.g/ml, 0.01. mu.g/ml, 0.5. mu.g/ml, 2. mu.g/ml, 4. mu.g/ml, and 2. mu.g/ml human IgG, respectively, and the dilution buffer was CB buffer pH9.6 (containing 2.5% PEG4000, 5% trehalose, 0.05% Proclin300, and 15% glycerol).
SC dots were coated with 2. mu.g/ml goat anti-human IgG antibody and the dilution buffer was CB buffer pH9.6 (supra).
The Loc dots were coated with 2. mu.g/ml human IgG and the dilution buffer was CB buffer pH 9.6.
Dilution of coating antigen:
the dsDNA, nucleosomes, C1q were diluted to final concentrations of 40. mu.g/ml, 12. mu.g/ml, 15. mu.g/ml, respectively, with 0.01M PBS buffer (0.5% PVP, 5% trehalose, 0.05% Proclin300, 0.02% 2-hydroxy-beta-cyclodextrin) at pH7.4-pH 7.6.
PLA2R, THSD7A, LF, MPO, PR3, GBM were diluted with CB buffer (3% PEG4000, 2.5% trehalose, 0.02% 2-hydroxy-beta-cyclodextrin, 0.05% Proclin300, and 15% glycerol) at pH9.6 to concentrations of 8. mu.g/ml, 15. mu.g/ml, 11. mu.g/ml, 12. mu.g/ml, 20. mu.g/ml, respectively.
LAMP-2, PT proteins and endothelial cells were diluted with 0.02M Tris buffer (2.5% PEG4000, 0.05% Proclin300, 3% trehalose, and 0.01% 2-hydroxy-. beta. -cyclodextrin and 15% glycerol) at pH8.5 to final concentrations, respectively: 20. mu.g/ml, 40. mu.g/ml and 30. mu.g/ml.
All the diluted antibodies or antigens as described above were filtered through 0.22um filters, and then coated on the array as required by a Biodot precise spotting machine in a volume of 10nl per spot. After all protein coating is completed, the chip is covered with a membrane and is placed at 2-8 ℃ for 24-30h overnight for coating.
2. Sealing of
The coated chip is taken out, washed 3 times by PBST washing solution with PH7.4, then 150ul of blocking solution (1-2% BSA (g/v), 1-1.5% citric acid (v/v), 0.3-0.6% mannitol (v/v) is added into each hole, 0.8-1.5% PVA2W (v/v) and 0.05% (v/v) 0.01M disodium hydrogen phosphate solution of Proclin300 are additionally added, the rest condition is shown in the table 3, the chip is kept stand and sealed for 35min at room temperature, then the chip is patted dry, is placed and dried for 4h at the humidity of less than 15 percent at RT, and is sealed and stored at the temperature of 2-8 ℃.
TABLE 3
| Group of | Example 1 | Example 2 | Example 3 |
| BSA(g/v) | 1％ | 2％ | 1.5％ |
| Citric acid (v/v) | 1％ | 1.5％ | 1.3％ |
| Mannitol (v/v) | 0.5％ | 0.3％ | 0.6％ |
| PVA2W(v/v) | 1％ | 0.8％ | 1.5％ |
| Proclin300(v/v) | 0.05％ | 0.05％ | 0.05％ |
| Disodium hydrogen phosphate solution | 0.01M | 0.01M | 0.01M |
3. Preparation of enzyme-labeled reagent
HRP-labeled rabbit anti-human IgG was diluted 4K-fold (4000-fold) with an enzyme-labeled stabilization buffer (0.01M PBS, 0.05M citric acid, 1.5% -2.5% BSA (g/v), 1.5% -2% PEG1W (v/v), 0.1% -0.2% acacia (g/v), 0.2% -0.5% sodium p-hydroxybenzoate (v/v), and 0.05% Proclin300(v/v) at pH7.4, and mixed well with the HRP-labeled rabbit anti-human IgG added as detailed in Table 4.
TABLE 4
| Group of | Example 1 | Example 2 | Example 3 |
| Citric acid | 1％ | 1.5％ | 1.3％ |
| BSA(g/v) | 2％ | 1.5％ | 2.5％ |
| PEG1W(v/v) | 2％ | 1.5％ | 1.8％ |
| Arabic gum (g/v) | 0.1％ | 0.2％ | 0.15％ |
| P-hydroxybenzoic acid sodium salt (v/v) | 0.3％ | 0.5％ | 0.2％ |
| Proclin300(v/v) | 0.05％ | 0.05％ | 0.05％ |
| PBS solution | 0.01M | 0.01M | 0.01M |
4. Reaction system of kit
1) Taking out the chip reagent, and balancing to room temperature;
2) sample adding: negative and positive control sera, and 101-fold diluted test sample with sample diluent (0.02M Tris, 0.15M NaCl, 0.05% Tween20, 0.01% casein, pH7.4) were added to test chip wells at 100uL per well for reaction.
3) Incubation: the reaction was carried out at room temperature for 30 min. Add 300uL of washing solution (0.02M Tris, 0.15M NaCl, 0.05% Tween20, pH7.4) and wash 3 times for 1min each.
4) Adding an enzyme labeling reagent: 100ul of enzyme label (rabbit anti-human HRP) was added to each well;
5) and (3) incubation: the reaction was carried out at room temperature for 30 min. Add 300uL of washing solution, wash 3 times for 1min each time.
6) Color development: adding 50uL ECL color developing agent into each hole, reacting for 30min at room temperature in a dark place, and then sucking to dry.
7) And (3) detection: and within 30min, reading and analyzing the signal value of each reaction hole corresponding to the detection index through a Berlotte chemiluminescence chip analyzer, and calculating and judging the positive and negative of the result and the intensity through a standard curve calibrated by reference points S1-S3.
Example 4 evaluation of accuracy
1. anti-GBM IgG positive sera and 10 anti-GBM IgG negative sera were determined from 10 cases screened with ELISA kit of Abnova corporation and tested with the chip kit prepared in examples 1 to 3, and the results are shown in the following Table 5(+ positive-negative).
TABLE 5 anti-GBM IgG serum test results for the chips prepared in examples 1-3
2. anti-PR 3IgG positive sera and 10 anti-PR 3IgG negative sera were determined from 10 cases screened with ELISA kit of EUROIMMUN corporation, and tested on the chips prepared in examples 1-3, and the results are shown in the following Table 6(+ positive-negative).
TABLE 6 anti-torch-IgG type antibody profiling chip prepared in examples 1-3 test anti-PR 3IgG serum results
3. The results of the tests on the chips prepared in examples 1 to 3 were as shown in Table 7 below (+ indicates positive, and-indicates negative) using 10 cases of anti-MPO IgG positive serum and 10 cases of anti-MPO IgG negative serum screened by ELISA kit of Invitrogen.
TABLE 7 results of testing anti-MPO IgG serum on chips prepared in examples 1-3
4. anti-LF IgG positive sera and 10 anti-LF IgG negative sera were determined using 10 cases of screening with ELISA kit of cusbio corporation and tested using the chips prepared in examples 1-3, and the results are shown in Table 8 below (+ positive, -negative).
TABLE 8 anti-LFIgG serum test results for chips prepared in examples 1-3
5. The results of the tests using the chips prepared in examples 1 to 3, which were obtained by using 10 cases of the anti-LAMP-2 IgG positive serum and 10 cases of the anti-LAMP-2 IgG negative serum screened by the ELISA kit of biorbyt corporation, are shown in the following Table 9(+ indicates positive, and-indicates negative).
TABLE 9 results of the chip prepared in examples 1 to 3 for testing the LAMP-2 IgG-resistant serum
6. Infection with anti-PLA 2R IgG positive serum and 10 anti-PLA 2R IgG negative serum were determined using 10 ELISA kits from EUROIMMUN, and the results of the tests using the chips prepared in examples 1-3 are shown in Table 10 below (+ indicates positive and-indicates negative).
TABLE 10 anti-PLA 2R IgG serum test results for chips prepared in examples 1-3
7. anti-THSD 7A IgG-positive sera and 10 anti-THSD 7A IgG-negative sera were determined by 10 screening samples using the EUROIMMUN kit (indirect immunofluorescence) and tested using the chips prepared in examples 1 to 3, and the results are shown in the following Table 11(+ positive and negative).
TABLE 11 results of testing anti-THSD 7A IgG serum on chips prepared in examples 1-3
8. Meanwhile, anti-PT protein IgG positive serum and 10 anti-PT protein IgG negative serum were determined by using 10 cases of ELISA kit of EUROIMMUN company and EUROIMMUN department kit (indirect immunofluorescence method), and the results were as shown in Table 12 below (the results indicate positive and negative). (the PT protein antibody is a fusion protein PT protein antibody which is prepared by the research of the company and is fused with the phospholipase A2 receptor (PLA2R) and the thrombospondin type 1 7A domain (THSD 7A)).
TABLE 12 anti-PT protein IgG serum test results for chips prepared in examples 1-3
9. Anti-endothelial cell antibody IgG positive serum and anti-endothelial cell antibody IgG negative serum were determined for 10 cases screened with ELISA kit of cusabio corporation, and the test was performed using the chips prepared in examples 1 to 3, and the results are shown in the following Table 13(+ represents positive, and-represents negative).
TABLE 13 results of IgG serum test for anti-endothelial cell antibody on the chips prepared in examples 1 to 3
10. anti-C1 q IgG-positive sera and 10 anti-C1 q IgG-negative sera were determined from 10 cases screened by ELISA kit of CD company, and tested on the chip prepared in examples 1-3, the results are shown in the following Table 14(+ positive-negative).
TABLE 14 anti-C1 q IgG serum test results for chips prepared in examples 1-3
11. The anti-dsDNA IgG positive serum and 10 anti-dsDNA IgG negative serum were determined by 10 screening using ELISA kit of Abcam company, and the results of the test using the chips prepared in examples 1 to 3 were as shown in the following Table 15(+ means positive, and-means negative).
TABLE 15 anti-dsDNA IgG serum test results for chips prepared in examples 1-3
12. Anti-nucleosome IgG positive serum and 10 anti-nucleosome IgG negative serum were determined by 10 screening samples using ELISA kit of NOVATEINBIO company, and the results of the test using the chips prepared in examples 1 to 3 were as shown in the following Table 16(+ indicates positive, and-indicates negative).
TABLE 16 results of anti-nucleosome IgG serum test on the chips prepared in examples 1-3
In conclusion, the chip prepared by the invention meets the requirement on the accuracy of testing various autoimmune nephropathy-associated antigen IgG, and has high accuracy.
Example 5 results of sensitivity and specificity tests
The patient samples were 85 positive serum samples from patients with autoimmune disease and 102 negative serum samples from normal healthy persons, which were diagnosed by renal puncture and clinical manifestation.
TABLE 17
The data show that the sensitivity (positive coincidence rate) of the chip kit disclosed by the invention on the detection of a clinical self-immune nephropathy patient (a self-immune nephropathy patient diagnosed by renal puncture and clinical symptoms together) can reach 98.8%, and the specificity (negative correctness rate) reaches 100%.
Example 6 stability test results of the kit
The stability experiment result of the self-immune kidney disease related antibody spectrum chip kit is as follows: watch 18
The stabilization period at the temperature of 2-8 ℃ can reach 2 years.
Watch 19
The stabilization period is 9 months at 18-28 ℃.
Example 7
A comparison kit: (typical composition of blocking solution and enzyme-labeled diluent: 0.01M PBS (pH7.4) + 10% BSA).
Watch 20
TABLE 21
The above test uses the test comparison between the reagent box of the invention and the reagent box using common sealing liquid and enzyme-labeled diluent (the other conditions are the same), and the result shows that the stability effect of the reagent box of the invention is better than that of the common reagent box using common sealing liquid and enzyme-labeled diluent.
The data show that the stability of the test kit can be stored for 2 years at 2-8 ℃, and the stability is better at room temperature (the test kit can be stored for 9 months at 18-28 ℃). Compared with a confining liquid and an enzyme-labeled diluent used in the prior art, the kit provided by the invention has better stability.
Example 8
The preparation of the kits according to examples 1-3 compared to the preparation of conventional ELISA kits for antigen use:
TABLE 22
In conclusion, the kit provided by the invention is shown to be capable of effectively reducing the usage amount of the related antigens, and reducing the usage cost of the related antigens while ensuring the sensitivity and specificity of the kit.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (1)
1. A chip, wherein it is coated with a composition;
the composition comprises the following components:
also coated with autoimmune nephropathy associated antigen;
the autoimmune nephropathy-associated antigen comprises at least one of GBM, PR3, MPO, LF, LAMP-2, PLA2R, THSD7A, PT protein, endothelial cells, C1q, dsDNA and nucleosome;
the chip also comprises a quality control point and/or a reference point; the quality control points comprise at least one positive quality control Point (PC), at least one negative quality control point (NC), at least one sample quality control point (SC) and/or at least one enzyme-labeled quality control point (EC); the reference points comprise reference curve points (S1-S3) with different concentrations and/or at least one chip position reference point (Loc);
the positive quality control points are coated with human IgG or BSA-DNP conjugate; the negative quality control point is coated with trace concentration of human IgG or other proteins irrelevant to the autoimmune nephropathy which are lower than the reaction signal value; the sample quality control point is coated anti-human IgG; the enzyme-labeled quality control points are coated with human IgG, anti-rabbit antibodies or anti-sheep antibodies; the reference curve points are coated with human IgG at different concentrations; the chip position reference point is coated with a human IgG solution with known concentration;
the preparation method of the chip comprises the following steps:
step 1: diluting the autoimmune nephropathy associated antigen, the protein of the quality control point and/or the protein of the reference point by a coating buffer solution, and coating the diluted autoimmune nephropathy associated antigen, the protein of the quality control point and/or the protein of the reference point on a substrate of the chip in a dot array form;
and 2, step: sealing the chip by a sealing stabilizer;
the blocking stabilizer is a 0.01M disodium hydrogen phosphate solution containing 1-2% BSA (g/v), 1-1.5% citric acid (v/v), 0.3-0.6% mannitol (v/v), 0.8-1.5% PVA2W (v/v) and 0.05% (v/v) Proclin 300.
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| CN110672856A (en) * | 2019-09-27 | 2020-01-10 | 广州博厚健康科技有限公司 | Food intolerance specificity IgG detection kit based on multiple microarrays |
| CN113125762A (en) * | 2019-12-31 | 2021-07-16 | 瑞博奥(广州)生物科技股份有限公司 | Detection chip for detecting female malignant tumor marker and preparation method and application thereof |
| CN111257553B (en) * | 2020-02-21 | 2023-03-28 | 深圳市伯劳特生物制品有限公司 | Stabilizer of protein quality control chip and method for preparing protein quality control chip |
| CN111239417B (en) * | 2020-02-21 | 2023-03-31 | 深圳市伯劳特生物制品有限公司 | Coating buffer solution, kit and application |
| CN112748242B (en) * | 2020-12-21 | 2023-07-21 | 珠海碳云智能科技有限公司 | Secondary antibody buffer sealing solution for polypeptide chip technology platform detection, kit comprising secondary antibody buffer sealing solution and application of secondary antibody buffer sealing solution |
| CN115015538B (en) * | 2022-05-17 | 2023-11-03 | 重庆国际免疫研究院 | Anti-phospholipid antibody detection kit and application thereof in detection of systemic lupus erythematosus nephritis |
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