WO2019169793A1 - Composition, chip and preparation method for chip and detection device including chip - Google Patents

Abstract

Disclosed are a composition, a chip and a preparation method for the chip and a detection device including the chip. The detection device has high sensitivity and specificity, can detect 12 antibodies at a time, has the advantages of high accuracy, convenient operation, saving on antigens, cost reduction, etc., and also optimizes related reagent formulations and treatment methods, and makes the stability of a kit better and storage and transportation conditions more convenient.

Description

Composition, chip, preparation method thereof and detection device including the same

The present application claims priority to Chinese Patent Application No. 201810186409.5, entitled "A Composition, Chip, Preparation Method Therefor, and Detection Device Containing the Chip", filed on March 7, 2018, the Chinese Patent Application. The entire content of which is incorporated herein by reference.

Technical field

The present invention relates to the field of biodetection technology, and in particular, to a composition, a chip, a preparation method thereof and a detection device including the same.

Background technique

Autoimmune-associated nephritis is a type of disease in which kidney complexes are caused by the deposition of immune complexes in the kidneys due to the combination of autoantigens and autoantibodies. Including lupus nephritis (LN), primary membranous nephropathy (PMN), anti-neutrophil cytoplasmic antibody (ANCA)-related renal damage. The incidence of immunological nephritis in China is still high today. The disease is a persistent or progressive development of a potential pathological change, which delays the disease and becomes chronic, not simply a chronic stage of acute nephritis. Early diagnosis contributes to the clinical control of autoimmune diseases. Studies have found that most autoimmune nephropathy patients have specific autoantibodies in their serum, such as ANCA-associated vasculitis anti-myeloperoxidase (MPO) antibody, anti-protease 3 antibody (PR3), anti-glomerular substrate Anti-C1q antibody, anti-nucleosome (Nucleosome) antibody and anti-double-stranded DNA (dsDNA) antibody, primary membranous nephropathy (IMN) anti-phospholipase A2 receptor (PLA2R) antibody and type 1 thrombospondin 7A (THSD7A) antibody, and our company The fusion protein PT protein antibody fused to the phospholipase A2 receptor (PLA2R) and the type 1 thrombospondin 7A domain (THSD7A) was studied. These specific autoantibodies are important components in the diagnostic criteria for autoimmune nephropathy.

There are many methods for detecting autoimmune-associated glomerulonephritis antibodies, including indirect immunofluorescence assays, enzyme-linked immunosorbent assays or immunoblotting, but each has its own disadvantages. The indirect immunofluorescence assay is based on the formation of fluorescent conjugates to infer the class of autoantibodies. Lack of a specific objective diagnosis requires secondary confirmation using other techniques, such as immunoblotting and enzyme-linked immunosorbent assays. Enzyme-linked immunosorbent assay can only detect a single antibody in one test, which has low efficiency and high cost, and has certain limitations in diagnostic applications. The imprinting method requires a large amount of specimens, complicated operation, long detection time, and high cost. And the stability period of the kit is generally 2-8 ° C stable period of 1 year.

ELISA-Array technology not only maintains the advantages of easy operation and low cost of ELISA, but also has the advantage of high throughput of protein chip technology. This technology fixes various markers to microplates in the form of microarrays by spotting instrument. Multiple pathogens can be detected simultaneously in the plate well. It enables multiple assays of a single sample in a single well, greatly reducing the amount of specimens and reagents. However, there is currently no research on ELISA-Array technology for autoimmune nephropathy in China. Therefore, it is of great practical significance to provide a highly sensitive and specific detection kit for autoimmune-associated glomerulonephritis antibodies with higher stability.

Summary of the invention

In view of the above, the present invention provides a composition, a chip, a method of preparing the same, and a detecting device including the same. The kit can detect 12 kinds of antibodies at a time, and has the advantages of high accuracy, simple operation, saving antigen, reducing cost, etc., and optimizing the related reagent formula and processing method to make the stability of the kit better (2- Refrigerated at 8 ° C for 2 years, room temperature can be stored for 6 months), storage and transportation conditions are more convenient.

In order to achieve the above object, the present invention provides the following technical solutions:

The present invention provides a composition comprising a mixture of one or more of citric acid, mannitol, PVA2W, PEG1W, gum arabic, sodium p-hydroxybenzoate.

In some embodiments of the invention, the volume ratio of citric acid, mannitol, PVA2W in the composition is (1 to 1.5): (0.3 to 0.6): (0.8 to 1.5).

In other specific embodiments of the invention, the composition comprises the following components:

In other specific embodiments of the invention, the composition further comprises a mixture of one or more of BSA, Proclin 300, PBS or disodium hydrogen phosphate.

In other specific embodiments of the present invention, the mass percentage of the BSA in the composition is from 1 to 2.5%;

The volume percentage of the Proclin 300 in the composition is 0.05%;

The concentration of the PBS is 0.01M;

The concentration of the disodium hydrogen phosphate was 0.01 M.

The invention also provides for the use of the compositions described in the preparation of chips and/or detection devices.

The invention also provides a chip coated with the composition.

In some embodiments of the invention, the chip is further coated with an autoimmune kidney disease associated antigen.

In some specific embodiments of the invention, the autoimmune nephropathy-associated antigen comprises GBM, PR3, MPO, LF, LAMP-2, PLA2R, THSD7A, PT protein, endothelial cells, C1q, dsDNA, nucleosomes At least one.

In some embodiments of the invention, the chip further includes a quality control point and/or a reference point; the quality control point includes at least one positive property control point (PC), at least one negative nature control point (NC), At least one sample control point (SC) and/or at least one enzyme standard control point (EC); the reference point comprising different concentration reference curve points (S1-S3) and/or at least one chip position reference point (Loc) ).

In some embodiments of the invention, the positive property control is coated with a human IgG or a BSA-DNP conjugate; the negative property control is coated with a trace concentration of human IgG or a lower than the response signal value Other proteins not related to autoimmune nephropathy; the sample control point is coated with anti-human IgG; the enzyme-labeled control point is coated with human IgG, anti-rabbit antibody or anti-sheep antibody; the reference curve The dots were coated with different concentrations of human IgG; the chip position reference points were coated with a known concentration of human IgG solution.

The invention provides a preparation method of the chip, comprising the following steps:

Step 1: the autoimmune nephropathy-associated antigen, the protein of the control point and/or the protein of the reference point are diluted with a coating buffer, and coated in a matrix of the chip in a dot array;

Step 2: blocking the chip by blocking the stabilizer;

The blocking stabilizer comprises the composition.

In some embodiments of the invention, the blocking stabilizer contains from 1% to 2% BSA (g/v), preferably 1% BSA (g/v).

In some embodiments of the invention, the blocking stabilizer further comprises from 1% to 1.5% citric acid (v/v), preferably 1% citric acid (v/v).

In some embodiments of the invention, the blocking stabilizer further comprises from 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 from 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) Proclin 300.

In some embodiments of the invention, the blocking stabilizer is 1% to 2% BSA (g/v), 1% to 1.5% citric acid (v/v), 0.3% to 0.6% nectar Alcohol (v/v), 0.8% to 1.5% PVA2W (v/v) and 0.05% (v/v) Proclin 300 in 0.01 M sodium hydrogen phosphate solution.

In some embodiments of the invention, the blocking stabilizer is 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 in 0.01 M sodium disodium hydrogen phosphate solution.

The present invention also provides a chip produced by the preparation method.

The invention also provides a detection device comprising the chip described above.

In some embodiments of the invention, the detection device further comprises an enzyme standard dilution stabilizer; the enzyme standard dilution stabilizer comprises 0.01 M PBS.

In some embodiments of the invention, the detection device further comprises an enzyme standard dilution stabilizer; the enzyme standard dilution stabilizer further comprises 0.05 M citric acid.

In some embodiments of the present invention, the detecting device further comprises an enzyme standard dilution stabilizer; the enzyme standard dilution stabilizer further comprises 1.5% to 2.5% (g/v) BSA, preferably 2% (g/ v) BSA.

In some embodiments of the invention, the detection device further comprises an enzyme standard dilution stabilizer; the enzyme standard dilution stabilizer further comprises 1.5% to 2% (v/v) PEG1W, preferably 2% (v/ v) PEG1W.

In some embodiments of the present invention, the detecting device further comprises an enzyme standard dilution stabilizer; the enzyme standard dilution stabilizer further comprises 0.1% to 0.2% (g/v) gum arabic, preferably 0.1% (g) /v) gum arabic.

In some embodiments of the present invention, the detecting device further comprises an enzyme standard dilution stabilizer; the enzyme standard dilution stabilizer further comprises 0.2% to 0.5% sodium p-hydroxybenzoate (v/v), preferably 0.3%. Sodium p-hydroxybenzoate (v/v).

In some embodiments of the invention, the detection device further comprises an enzyme standard dilution stabilizer; the enzyme standard dilution stabilizer further comprises 0.05% (v/v) Proclin 300.

In some embodiments of the present invention, the detecting device further comprises an enzyme standard dilution stabilizer; the enzyme standard dilution stabilizer comprises 0.01 M PBS, 0.05 M citric acid, 1.5% to 2.5% (g/v) BSA, 1.5% to 2% (v/v) PEG1W, 0.1% to 0.2% (g/v) gum arabic, 0.2% to 0.5% sodium p-hydroxybenzoate (v/v) and 0.05% (v /v) Proclin300.

In some embodiments of the present invention, the detecting device further comprises an enzyme standard dilution stabilizer;

The enzyme standard dilution stabilizer comprises 0.01 M PBS, 0.05 M 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 marker, a sample diluent, a wash solution, a color developing solution.

In some specific embodiments of the invention, the marker is a labeled conjugate labeled anti-human IgG antibody;

The labeled conjugate comprises an enzyme label, an avidin or an 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 dilution is a 0.02 M Tris solution of pH 7.4 comprising 0.15 M NaCl, 0.05% Tween 20, 0.01% casein;

The washing liquid is a 0.02 M Tris solution of pH 7.4 containing 0.15 M NaCl, 0.05% Tween 20;

The color developing solution is ECL.

The invention also provides a method for detecting autoimmune nephropathy based on the detecting device, wherein the sample to be tested is diluted with the sample diluent and coated onto the chip, and the label is added and added to the sample. The color liquid is protected from light and the color is detected by a fluorescence detecting device, and the antibody signal value of the autoimmune kidney disease in the sample to be tested is obtained.

The invention uses the ELISA-Array technology to prepare a highly sensitive and specific autoimmune-associated glomerulone antibody detection kit, which can detect 12 kinds of antibodies at a time, has high accuracy, simple operation, saves antigen and reduces Cost and other advantages, while optimizing the relevant reagent formulations and treatment methods, the stability of the kit is better (2-8 ° C refrigeration for 2 years, room temperature can be stored for 6 months), storage and transportation conditions are more convenient.

Detailed ways

The invention discloses a composition, a chip and a preparation method thereof and a detecting device comprising the same, and those skilled in the art can learn from the contents of the paper and appropriately improve the process parameters. It is to be understood that all such alternatives and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The method and the application of the present invention have been described by the preferred embodiments, and it is obvious that the method and application described herein may be modified or appropriately modified and combined without departing from the scope of the present invention. The technique of the present invention is applied.

The protein chip technology used in the invention prepares an autoimmune nephropathy-related autoantibody protein chip kit with high flux, multiple detection indexes, stable performance, good repeatability and high accuracy.

The kit provided by the invention comprises a protein chip, and GBM, PR3, MPO, LF, LAMP-2, PLA2R, THSD7A, PT protein, endothelial cells, C1q, dsDNA, nucleosomes are obtained by a high-precision spotting instrument. These 12 antigens and the required reference point proteins are coated on the chip substrate in a 4*5 dot array form, and the corresponding related antibodies in the sample are captured by the immunological principle of antigen-antibody specific reaction.

Reference points in embodiments of the invention include: at least one negative nature control point (NC) and one positive nature control point (PC); at least one sample control point (SC) and one enzyme standard control point (EC); Three standard curve points (S1-S3) and a position reference point (Loc) coated by the chip itself.

In the examples of the present invention, the positive control point may be human IgG, and the enzyme-linked immunolabel used is a marker against human IgG. In other embodiments, the point may also be coated with BSA-conjugated DNP, and the enzyme-linked immunolabel used is a mixture of a marker against human IgG and a marker against DNP.

In embodiments of the invention, the negative property control point may be a minor concentration of human IgG below the response signal value, and in other embodiments may be other unrelated proteins.

In the embodiment of the present invention, the sample control point may be a goat anti-human IgG, and in other embodiments, other anti-human IgG, such as rabbit anti-human IgG, mouse anti-human IgG or the like may also be used.

In the embodiment of the present invention, the enzyme labeling control point may be human IgG (the enzyme label is an enzyme label of anti-human IgG), and other embodiments may also be used in other embodiments: such as an anti-rabbit antibody (the enzyme label is Rabbit anti-human IgG), or anti-goat antibody (the enzyme is labeled with goat anti-human IgG).

In the embodiment of the present invention, the standard curve points are coated with low, medium and high concentrations of human IgG for internal calibration, and the results are aligned.

In the embodiment of the present invention, the position reference point of the chip itself is coated with a 2 μg/ml human IgG solution, which is mainly a positioning effect when the array is valued.

In the embodiment of the present invention, the chip substrate is a 96-well enzyme-labeled reaction plate. In other embodiments, the substrate may also be a carrier suitable for protein attachment such as a slide, various chemical films, and porous silica gel.

The antigen provided by the present invention includes several or more of the above 12 antigens, and is uniformly coated on the chip substrate by using different antigen coating buffers. The antigen coating buffer is pH 9.6 CB buffer, pH 8.5 Tris buffer and pH 7.4 PBS buffer, which is supplemented with trehalose, glycerin, PEG or PVP, and Proclin 300 preservative. Additions such as water-soluble cyclodextrin are added to make the coating effect better, more stable and uniform, and the coating points are more regular, rounded, and CV smaller. The PEG is PEG-400, and the water-soluble cyclodextrin may be 0.02% 2-hydroxy-β-cyclodextrin, or may be Captisol, carboxymethyl-β-cyclodextrin or the like.

The chip was coated at 2-8 ° C for 24-30 hours overnight coating.

The end of the coating requires the closure of the chip. The blocking stabilizer used in the present invention contains 1% BSA, 1% citric acid, 0.5% mannitol, and 1% PVA2W and 0.05% Proclin 300 0.01 M. A solution of disodium hydrogen phosphate.

The enzyme used in the kit of the present invention is labeled as HRP-labeled rabbit anti-human IgG, and the kit contains an enzyme standard working solution for diluting HPR-labeled rabbit anti-human IgG with an enzyme-labeled dilution stabilizer to an enzyme label using a final concentration. The enzyme standard dilution stabilizer is 0.01 M PBS, 0.05 M citric acid, 2% BSA (g/v), 2% (v/v) PEG1W, 0.1% (g/v) gum arabic, 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, may be used; in other embodiments, other labeled conjugates such as AP, avidin, acridinium ester, etc. may also be used. .

The chromogenic substrate used in the present invention is an enhanced chemiluminescent substrate (ECL) which is read by a fluorescence detecting device or apparatus. Other chromogenic substrates such as p-NPP, TMB, and the like can also 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 productization, and the stability of the kit is higher than that of the current market (general 2- 8 ° C 1 year) should be good.

The composition, the chip, the preparation method thereof and the raw materials and reagents used in the detection device comprising the chip are commercially available.

The present invention is further illustrated below in conjunction with the embodiments:

Examples 1-3

1, the package of the chip:

1) The chip array design and the specific distribution of antigen and reference points are shown in Table 1 and Table 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 protein	C1q

2) Specific coating process:

First, dilute antibodies and related proteins as follows:

The PC, NC, S1, S2, S3, and EC points in the array were coated with 2 μg/ml, 0.01 μg/ml, 0.5 μg/ml, 2 μg/ml, 4 μg/ml, and 2 μg/ml of human IgG, respectively. The buffer was pH 9.6 CB buffer (containing 2.5% PEG 4000, 5% trehalose, 0.05% Proclin 300, and 15% glycerol).

The SC spot was coated with 2 μg/ml of goat anti-human IgG antibody, and the dilution buffer was pH 9.6 CB buffer (ibid.).

The Loc spot was coated with 2 μg/ml of human IgG, and the dilution buffer was pH 9.6 CB buffer.

Dilution of coated antigen:

dsDNA, nucleosome, and C1q were respectively used in 0.01 M PBS buffer of pH 7.4-pH 7.6 (containing 0.5% PVP, 5% trehalose, 0.05% Proclin 300, 0.02% 2-hydroxy-β). - cyclodextrin) was diluted to a final concentration of 40 μg/ml, 12 μg/ml, and 15 μg/ml, respectively.

PLA2R, THSD7A, LF, MPO, PR3, and GBM were respectively used in CB buffer of PH9.6 (3% PEG4000, 2.5% trehalose, 0.02% 2-hydroxy-β-cyclodextrin, 0.05% Proclin300, And 15% glycerol) was diluted to a concentration of 8 μg/ml, 15 μg/ml, 15 μg/ml, 11 μg/ml, 12 μg/ml, and 20 μg/ml.

LAMP-2, PT protein and endothelial cells were respectively 0.02 M Tris buffer (containing 2.5% PEG4000, 0.05% Proclin300, 3% trehalose, and 0.01% 2-hydroxy-β-) at pH 8.5. The cyclodextrin and 15% glycerol were diluted to a final concentration of 20 μg/ml, 40 μg/ml and 30 μg/ml, respectively.

All of the diluted antibodies or antigens were filtered through a 0.22 um filter, and then coated by the BioDot precision spotter as required, with a volume of 10 nl per spot. After all protein coating was completed, the chip was covered with a membrane, placed at 2-8 ° C, and overnight coated for 24-30 h.

2, closed

Remove the coated chips, wash them 3 times with PBST washing solution of pH 7.4, then add 150 ul of blocking solution per well (1%-2% BSA (g/v), 1%-1.5% citric acid (v /v), 0.3%-0.6% mannitol (v/v), plus 0.8%-1.5% PVA2W (v/v) and 0.05% (v/v) Proclin300 0.01M hydrogen phosphate The disodium solution was added as shown in Table 3, and it was allowed to stand at room temperature for 35 min, then patted dry, and the humidity was <15%. The RT was left to dry for 4 h, then sealed and stored at 2-8 °C.

table 3

Group	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 standard reagent

Stabilizing buffer with pH 7.4 (0.01 M PBS, 0.05 M citric acid, 1.5%-2.5% BSA (g/v), 1.5%-2% PEG1W (v/v), 0.1% -0.2% gum arabic (g/v), 0.2%-0.5% sodium p-hydroxybenzoate (v/v) and 0.05% Proclin 300 (v/v), as shown in Table 4) HRP-labeled rabbit anti- Human IgG was diluted to 4K times (4000 times) and mixed.

Table 4

Group	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%

Gum arabic (g/v)	0.1%	0.2%	0.15%
Sodium p-hydroxybenzoate (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, the reaction system of the kit

1) remove the chip reagent and equilibrate to room temperature;

2) Loading: Negative and positive control serum, and sample dilutions (0.02 M Tris, 0.15 M NaCl, 0.05% Tween 20, 0.01% casein, pH 7.4) were diluted 101 times for each sample. 100 uL was added to the well of the chip to be tested for reaction.

3) Incubation: react at room temperature for 30 min. 300 uL of washing solution (0.02 M Tris, 0.15 M NaCl, 0.05% Tween 20, pH 7.4) was added and washed 3 times for 1 min each time.

4) Add enzyme labeling reagent: add 100 ul of enzyme label (rabbit anti-human HRP) per well;

5) Incubation: react at room temperature for 30 min. Add 300 uL of washing solution and wash 3 times for 1 min each time.

6) Color development: 50 μL of ECL developer was added to each well, and the reaction was allowed to stand at room temperature for 30 minutes in the dark, followed by blotting.

7) Detection: Within 30 minutes, the signal value of each reaction well corresponding to the detection index is read and analyzed by the Schlott chemiluminescence chip analyzer, and the standard curve of the S1-S3 reference point calibration is used to calculate and judge the negative result of the result and strength.

Example 4 accuracy evaluation

1. Ten anti-GBM IgG positive sera and 10 anti-GBM IgG negative sera were screened by Abnova ELISA kit, and tested with the chip kits prepared in Examples 1-3. The results are shown in Table 5 below (+ indicates positive, - indicates negative).

Table 5 The chips prepared in Examples 1-3 were tested for anti-GBM IgG serum results.

2. Ten anti-PR3 IgG-positive sera and 10 anti-PR3 IgG-negative sera were screened using the EUROIMMUN ELISA kit and tested with the chips prepared in Examples 1-3. The results are shown in Table 6 below (+ indicates positive, - indicates negative).

Table 6 Anti-torch-IgG type antibody profile chips prepared in Examples 1 to 3 were tested for anti-PR3 IgG serum results.

3. Ten anti-MPO IgG positive sera and 10 anti-MPO IgG negative sera were screened by Invitrogen ELISA kit, and the chips prepared in Examples 1-3 were tested. The results are shown in Table 7 below (+ indicates positive, - indicates negative). ).

Table 7 Chips prepared in Examples 1-3 were tested for anti-MPO IgG serum results.

4. Ten anti-LF IgG-positive sera and 10 anti-LF IgG-negative sera were screened by cusbio ELISA kit, and tested with the chips prepared in Examples 1-3. The results are shown in Table 8 below (+ indicates positive, - indicates negative). ).

Table 8 Chips prepared in Examples 1-3 were tested for anti-LFIgG serum results.

5. Ten anti-LAMP-2 IgG positive sera and 10 anti-LAMP-2 IgG negative sera were screened by biorbyt ELISA kit, and the chips prepared in Examples 1-3 were tested. The results are shown in Table 9 below (+ indicates positive , - means negative).

Table 9 Chips prepared in Examples 1-3 were tested for anti-LAMP-2 IgG serum results.

6. 10 cases of anti-PLA2R IgG positive serum and 10 anti-PLA2R IgG negative serum were screened by EUROIMMUN ELISA kit, and the chips prepared in Examples 1 to 3 were tested. The results are shown in Table 10 below (+ indicates positive, - Indicates negative).

Table 10 Chips prepared in Examples 1-3 were tested for anti-PLA2R IgG serum results.

7. Ten anti-THSD7A IgG-positive sera and 10 anti-THSD7A IgG-negative sera were screened using EUROIMMUN kit (indirect immunofluorescence) and tested with the chips prepared in Examples 1-3. The results are shown in Table 11 below (+ Positive, - negative.

Table 11 Chips prepared in Examples 1-3 were tested for anti-THSD7A IgG serum results.

8. At the same time, 10 anti-PT protein IgG positive serum and 10 anti-PT protein IgG negative serum were selected by using EUROIMMUN ELISA kit and EUROIMMUN kit (indirect immunofluorescence), and prepared by using Examples 1-3. The chip was tested and the results are shown in Table 12 below (+ indicates positive, - indicates negative). (The PT protein antibody is a fusion protein PT protein antibody fused by the anti-phospholipase A2 receptor (PLA2R) prepared by our company and the type 1 thrombospondin 7A domain (THSD7A)).

Table 12 Chips prepared in Examples 1-3 were tested for anti-PT protein IgG serum results.

9. Ten anti-endothelial cell antibody IgG-positive sera and 10 anti-endothelial cell antibody IgG-negative sera were screened by cusabio ELISA kit, and the chips prepared in Examples 1 to 3 were tested. The results are shown in Table 13 below (+ indicates Positive, - indicates negative).

Table 13 Chips prepared in Examples 1-3 were tested for anti-endothelial cell antibody IgG serum results.

10. Ten anti-C1q IgG positive sera and 10 anti-C1q IgG negative sera were screened by CD company ELISA kit, and tested with the chips prepared in Examples 1-3. The results are shown in Table 14 below (+ indicates positive, - indicates negative).

Table 14 Chips prepared in Examples 1-3 were tested for anti-C1q IgG serum results.

11. Ten anti-dsDNA IgG-positive sera and 10 anti-dsDNA IgG-negative sera were screened by Abcam ELISA kit, and tested with the chips prepared in Examples 1-3. The results are shown in Table 15 below (+ indicates positive, - indicates negative).

Table 15 Chips prepared in Examples 1-3 were tested for anti-dsDNA IgG serum results.

12. Ten anti-nuclear IgG-positive sera and 10 anti-nucleosome IgG-negative sera were screened by NOVATEINBIO ELISA kit, and tested with the chips prepared in Examples 1-3. The results are shown in Table 16 below (+ indicates Positive, - indicates negative).

Table 16 Chips prepared in Examples 1-3 were tested for anti-nucleosome IgG serum results.

In summary, the chip prepared by the invention tests the accuracy of each autoimmune nephropathy-associated antigen IgG according to requirements and has high accuracy.

Example 5 Sensitivity and Specificity Test Results

The patient samples were positively diagnosed as positive serum samples from patients with self-improvement nephropathy by renal puncture and clinical manifestations, and negative serum samples from normal healthy individuals were 102.

Table 17

As can be seen from the above data, the sensitivity of the chip kit of the present invention for clinically self-improving nephropathy patients (self-protected nephropathy patients diagnosed by renal puncture and clinical symptoms) can reach 98.8%, specificity. (negative correct rate) of 100%, indicating that the kit of the present invention has high sensitivity and specificity, and provides a more accurate and comprehensive reference and guidance for the diagnosis of self-improvement of kidney disease.

Example 6 Stability Test Results of the Kit

Stability test results of self-improvement kidney disease related antibody spectrum chip kit:

Table 18

The stable period of 2-8 °C can reach 2 years.

Table 19

The stability period of 18-28 ° C is 9 months.

Example 7

Comparison kit: (General composition of blocking solution and enzyme standard dilution: 0.01 M PBS (pH 7.4) + 10% BSA).

Table 20

Table 21

In the above test, the kit of the present invention was compared with a kit using a general blocking solution and an enzyme standard dilution solution (the rest of the conditions), and as a result, the stability of the kit of the present invention is better than that of the general closure. A common kit for liquid and enzyme dilutions.

The data shows that the stability of the kit test of the present invention can be stored at 2-8 ° C for 2 years, and at room temperature (18-28 ° C can be placed for 9 months), which proves that the stability is good. At the same time, compared with the blocking solution and the enzyme standard diluent used in the prior art, the kit of the present invention is also more stable.

Example 8

Comparing the preparation of the kit according to Examples 1-3 with the preparation of a conventional ELISA kit for antigen usage:

Table 22

In summary, it is shown that the kit provided by the invention can effectively reduce the use amount of the relevant antigen, and reduce the related antigen use cost while ensuring the sensitivity and specificity of the kit.

The composition, the chip, the preparation method thereof and the detection device including the same provided by the present invention are described in detail above. The principles and embodiments of the present invention have been described with reference to specific examples, and the description of the above embodiments is only to assist in understanding the method of the present invention and its core idea. It should be noted that those skilled in the art can make various modifications and changes to the present invention without departing from the spirit and scope of the invention.

Claims (10)

1. a composition comprising a mixture of one or more of citric acid, mannitol, PVA2W, PEG1W, gum arabic, sodium p-hydroxybenzoate;

   The volume ratio of the citric acid, mannitol, and PVA2W in the composition is (1 to 1.5): (0.3 to 0.6): (0.8 to 1.5) or

   The composition includes the following components:

   
2. The composition according to claim 1, further comprising a mixture of one or more of BSA, Proclin 300, PBS or disodium hydrogen phosphate.
3. Use of a composition according to claim 1 or 2 in the preparation of a chip and/or detection device.
4. A chip characterized in that it is coated with the composition according to any one of claims 1 to 5.
5. The chip according to claim 4, further comprising an autoimmune nephropathy-associated antigen.
6. The chip according to claim 4 or 5, wherein the chip further comprises a quality control point and/or a reference point; the quality control point comprises at least one positive property control point (PC), at least one negative property control Point (NC), at least one sample control point (SC), and/or at least one enzyme control point (EC); the reference point comprising different concentration reference curve points (S1-S3) and/or at least one chip Location reference point (Loc).
7. The method for preparing a chip according to any one of claims 4 to 6, comprising the steps of:

   Step 1: diluting the autoimmune nephropathy-associated antigen, the protein of the control point, and/or the protein of the reference point by coating buffer, and coating the matrix of the chip in a dot array form;

   Step 2: blocking the chip by blocking the stabilizer;

   The blocking stabilizer comprises the composition of any one of claims 1 to 5.
8. A chip produced by the production method according to claim 7.
9. A detecting device comprising the chip according to any one of 4 to 6 or the chip according to claim 8.
10. A method for detecting autoimmune nephropathy based on the detecting device according to any one of claims 16 to 20, wherein the sample to be tested is diluted with the sample diluent and coated onto the chip. The label is added, the color developing solution is added to protect from light, and the fluorescence detecting device detects the result, and the antibody signal value of the autoimmune nephropathy in the sample to be tested is obtained.