CN108398550A - A kind of composition, chip and preparation method thereof and include the chip detection device - Google Patents
A kind of composition, chip and preparation method thereof and include the chip detection device Download PDFInfo
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- CN108398550A CN108398550A CN201810186409.5A CN201810186409A CN108398550A CN 108398550 A CN108398550 A CN 108398550A CN 201810186409 A CN201810186409 A CN 201810186409A CN 108398550 A CN108398550 A CN 108398550A
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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
- G01N33/531—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
- G01N33/558—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 present invention relates to technical field of biological, more particularly to a kind of composition, chip and preparation method thereof and include the chip detection device.The detection kit has High sensitivity and specificity, it can be with 12 kinds of antibody of one-time detection, have many advantages, such as higher accuracy, it is easy to operate, save antigen, reduce cost, optimize relevant agent prescription and processing method simultaneously, making the stability of kit, more preferable (2 8 DEG C refrigerate 2 years, room temperature can store 6 months), storage and traffic condition are more convenient.
Description
Technical field
The present invention relates to technical field of biological, more particularly to a kind of composition, chip and preparation method thereof and comprising
There is the detection device of the chip.
Background technology
Autoimmunity related glomerulonephritis is to form immune complex deposit since autoantigen and autoantibody are combined and exist
Kidney leads to a kind of disease of kidney injury.Including lupus nephritis (LN), primary membranous nephropathy (PMN), anti-neutrality grain is thin
Born of the same parents' endochylema antibody (ANCA) correlation renal damage etc..So far still very high in the incidence of Chinese immune nephritis.The disease is latent
In the duration or progress sexual development of pathological change, makes protracted inflammation, become chronic, the chronic rank without being merely acute nephritis
Section.Early diagnosis contributes to the clinic control of autoimmune disease.Research is it has been found that most autoimmune nephrosis is suffered from
There is specific autoantibodies, such as anti-myeloperoxidase (MPO) antibody, the anti-egg of ANCA relevant blood vessel inflammation in person's serum
It is white 3 antibody of enzyme (PR3), AGBM antibody (GBM), anti-endothelial cell antibodies, anti-lactoferrin (LF) antibody, anti-human
Lysosomal associated membrane albumen (LAMP-2) antibody；Anti-C1q antibodies, the anti-nucleosome (Nucleosome) of lupus nephritis (LN) are anti-
Body and anti-double-chain DNA (dsDNA) antibody, anti-phospholipase A2 receptor (PLA2R) antibody of primary membranous nephropathy (IMN) and 1 type
Anti- phospholipase A2 receptor (PLA2R) and 1 type blood prepared by the research of thrombospondin 7A (THSD7A) antibody, also our company
The fusion protein PT protein antibodies of the domains platelet reactive protein 7A (THSD7A) fusion, these specific autoantibodies are autoimmunity
Important component part in property diagnosis of nephropathy standard.
There are the method for many detection autoimmunity related glomerulonephritis antibody, including indirect immunofluorescence analysis method, enzyme at present
Join immunoabsorption or Western blot, but haves the shortcomings that respective.Indirect immunofluorescence analysis method is by formation
Fluorescence conjugate infers the classification of autoantibody, lacks a specific objectively diagnosis, needs to utilize other technologies progress two
Secondary confirmation, such as Western blot, enzyme-linked immunization.Once experiment can only detect monospecific antibody, efficiency to enzyme linked immunosorbent assay
Low, cost is higher, is had some limitations in terms of diagnostic application.Specimen amount needed for Diagnosis of Sghistosomiasis notation is big, cumbersome, inspection
Survey overlong time, the problems such as cost is higher.And the stationary phase of kit is typically all 2-8 DEG C of 1 year stationary phase.
ELISA-Array technologies not only remain the advantage easy to operate, at low cost of ELISA method, and have egg
The advantage of white chip technology high throughput；A variety of markers are fixed on microwell plate by the technology in the form of a microarray by point sample instrument
In plate hole, multiple pathogens can be detected simultaneously.It can realize a variety of detections of a sample in a micropore, greatly reduce
The amount of sample and reagent.But the current country there is no the research of the ELISA-Array technologies detection for autoimmune nephrosis.
Therefore it provides a kind of High sensitivity and special, the better autoimmunity related glomerulonephritis antibody of stability detection kit
It has important practical significance.
Invention content
In view of this, the present invention provides a kind of composition, chip and preparation method thereof and includes the detection dress of the chip
It sets.The kit can with 12 kinds of antibody of one-time detection, have higher accuracy, it is easy to operate, save antigen, reduce cost etc.
Advantage, while optimizing relevant agent prescription and processing method, making the stability of kit, more preferable (2-8 DEG C refrigerates 2 years, room
Temperature can store 6 months), storage and traffic condition are more convenient.
In order to achieve the above-mentioned object of the invention, the present invention provides following technical scheme：
The present invention provides a kind of composition, including citric acid, mannitol, PVA2W, PEG1W, gum arabic, to hydroxyl
Mixture more than one or both of yl benzoic acid sodium.
In some specific embodiments of the present invention, the volume ratio of citric acid, mannitol, PVA2W in the composition
For (1~1.5):(0.3~0.6):(0.8~1.5).
In other specific embodiments of the present invention, the composition includes following component：
In other specific embodiments of the present invention, the composition further includes BSA, Proclin300, PBS or phosphorus
Mixture more than one or both of sour disodium hydrogen.
In other specific embodiments of the present invention, the quality volume basis of the BSA in the composition contains
Amount is 1~2.5%；
The volumn concentrations of the Proclin300 in the composition are 0.05%；
A concentration of 0.01M of the PBS；
A concentration of 0.01M of the disodium hydrogen phosphate.
The present invention also provides application of the composition in preparing chip and/or detection device.
The present invention also provides a kind of chip, it is coated with the composition.
In some specific embodiments of the present invention, the chip is also coated with autoimmune nephrosis related antigen.
The present invention some specific embodiments in, the autoimmune nephrosis related antigen include GBM, PR3,
At least one of MPO, LF, LAMP-2, PLA2R, THSD7A, PT albumen, endothelial cell, C1q, dsDNA, nucleosome.
In some specific embodiments of the present invention, the chip also includes Quality Control point and/or reference point；The Quality Control
Point comprising 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 mark Quality Control point (EC)；The reference point includes the reference curve point (S1-S3) and/or at least one of various concentration
A chip position reference point (Loc).
In some specific embodiments of the present invention, the positive quality control point coating human IgG or coating BSA-DNP couplings
Object；Human IgG or others and autoimmune nephrosis of the feminine gender Quality Control point coating less than the micro-concentrations of reaction signal value
Unrelated albumen；The sample Quality Control point is coating anti-human igg；The antibody or anti-of the enzyme mark Quality Control point coating human IgG, anti-rabbit
The antibody of sheep；The human IgG of the reference curve point coating various concentration；The people of the chip position reference point coating known concentration
IgG solution.
The present invention provides the preparation methods of the chip, include the following steps：
Step 1：By the autoimmune nephrosis related antigen, the albumen of Quality Control point and/or the reference point
After the coated buffer solution dilution of albumen, the matrix of the chip is coated in the form of lattice array；
Step 2：The chip is closed through closed stablity agent；
The closed stablity agent includes the composition.
In some specific embodiments of the present invention, 1%~2%BSA (g/v) is contained in the closed stablity agent, it is excellent
Select 1%BSA (g/v).
In some specific embodiments of the present invention, also containing 1%~1.5% citric acid in the closed stablity agent
(v/v), preferably 1% citric acid (v/v).
In some specific embodiments of the present invention, also containing 0.3%~0.6% sweet dew in the closed stablity agent
Alcohol (v/v), preferably 0.5% mannitol (v/v).
In some specific embodiments of the present invention, also containing 0.8%~1.5% in the closed stablity agent
PVA2W (v/v), preferably 1% PVA2W (v/v).
Also contain 0.05% (v/v's) in some specific embodiments of the present invention, in the closed stablity agent
Proclin300。
In some specific embodiments of the present invention, the closed stablity agent is 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
The disodium phosphate soln of the 0.01M of the Proclin300 of 0.05% (v/v).
In some specific embodiments of the present invention, the closed stablity agent is to contain 1%BSA (g/v), 1% lemon
Lemon acid (v/v), 0.5% mannitol (v/v), the 0.01M of the Proclin300 of 1% PVA2W (v/v) and 0.05% (v/v)
Disodium phosphate soln.
The present invention also provides chips made from the preparation method.
The present invention also provides a kind of detection devices, including above-mentioned chip.
In some specific embodiments of the present invention, the detection device further includes enzyme mark dilution stabilizer；The enzyme
Mark dilution stabilizer includes 0.01M PBS.
In some specific embodiments of the present invention, the detection device further includes enzyme mark dilution stabilizer；The enzyme
Mark dilution stabilizer further includes the citric acid of 0.05M.
In some specific embodiments of the present invention, the detection device further includes enzyme mark dilution stabilizer；The enzyme
Mark dilution stabilizer further includes the BSA, the BSA of preferably 2% (g/v) of 1.5%~2.5% (g/v).
In some specific embodiments of the present invention, the detection device further includes enzyme mark dilution stabilizer；The enzyme
Mark dilution stabilizer further includes the PEG1W, the PEG1W of preferably 2% (v/v) of 1.5%~2% (v/v).
In some specific embodiments of the present invention, the detection device further includes enzyme mark dilution stabilizer；The enzyme
Mark dilution stabilizer further includes the gum arabic of 0.1%~0.2% (g/v), the gum arabic of preferably 0.1% (g/v).
In some specific embodiments of the present invention, the detection device further includes enzyme mark dilution stabilizer；The enzyme
Mark dilution stabilizer further includes 0.2%~0.5% P-hydroxybenzoic acid sodium (v/v), preferably 0.3% P-hydroxybenzoic acid sodium
(v/v)。
In some specific embodiments of the present invention, the detection device further includes enzyme mark dilution stabilizer；The enzyme
Mark dilution stabilizer further includes the Proclin300 of 0.05% (v/v).
In some specific embodiments of the present invention, the detection device further includes enzyme mark dilution stabilizer；The enzyme
Mark dilution stabilizer includes 0.01M PBS, the citric acid of 0.05M, the BSA of 1.5%~2.5% (g/v), 1.5%~2% (v/
V) PEG1W, the gum arabic of 0.1%~0.2% (g/v), 0.2%~0.5% P-hydroxybenzoic acid sodium (v/v) and
The Proclin300 of 0.05% (v/v).
In some specific embodiments of the present invention, the detection device further includes enzyme mark dilution stabilizer；
The enzyme mark dilution stabilizer includes 0.01M PBS, the citric acid of 0.05M, the BSA of 2% (g/v), 2% (v/v)
PEG1W, the gum arabic of 0.1% (g/v), 0.3% P-hydroxybenzoic acid sodium (v/v) and 0.05% (v/v's)
Proclin300。
In some specific embodiments of the present invention, the detection device further includes marker, sample diluting liquid, washing
Mixture more than one or both of liquid, developing solution.
In some specific embodiments of the present invention, the marker is the anti-human IgG antibodies of production label；
The production includes enzyme marker, Avidin or acridinium ester；
The enzyme marker is horseradish peroxidase or alkaline phosphatase；
The anti-human IgG antibodies are rabbit anti-human igg's antibody or goat anti-human igg antibody；
The sample diluting liquid is the pH7.4 comprising 0.15M NaCl, 0.05%Tween20,0.01% casein
0.02M Tris solution；
The cleaning solution is the 0.02M Tris solution of the pH7.4 comprising 0.15M NaCl, 0.05%Tween20；
The developing solution is ECL.
The present invention also provides a kind of detection methods of the autoimmune nephrosis based on the detection device, take to be measured
After sample is diluted with the sample diluting liquid in coating to the chip, the marker is added, the developing solution is added and is protected from light
Colour developing, fluorescence detection device testing result obtain the associated antibodies signal value of the autoimmune nephrosis in sample to be tested.
The present invention utilizes ELISA-Array technologies, is prepared for a kind of High sensitivity and special autoimmunity correlation kidney
The detection kit of scorching antibody, it can with 12 kinds of antibody of one-time detection, have higher accuracy, it is easy to operate, save antigen,
The advantages that reducing cost, while relevant agent prescription and processing method are optimized, keep the stability of kit (2-8 DEG C more preferable
Refrigeration 2 years, room temperature can store 6 months), storage and traffic condition are more convenient.
Specific implementation mode
The invention discloses a kind of composition, chip and preparation method thereof and include the chip detection device, ability
Field technique personnel can use for reference present disclosure, be suitably modified technological parameter realization.In particular, it should be pointed out that all similar replaces
Change and change apparent to those skilled in the art, they are considered as being included in the present invention.The side of the present invention
Method and application be described by preferred embodiment, related personnel obviously can not depart from the content of present invention, spirit and
Method described herein and application are modified or are suitably changed and combined in range, to realize and apply the technology of the present invention.
The protein biochip technology that the present invention uses, it is stable, reproducible to be prepared for high-throughput, more Testing index, performance,
The high autoimmune nephrosis related auto-antibodies spectrin chip agent box of accuracy.
Kit provided by the invention includes a kind of protein chip, by the point sample instrument of high precision by GBM, PR3, MPO,
This 12 kinds of antigens of LF, LAMP-2, PLA2R, THSD7A, PT albumen, endothelial cell, C1q, dsDNA, nucleosome and required
Reference point albumen is coated in the form of the lattice array of 4*5 in chip matrix, and the immunology reacted by antigen and antibody specific is former
It manages to capture corresponding associated antibodies in sample.
Reference point includes in the embodiment of the present invention：At least one feminine gender Quality Control point (NC) and a positive quality control point
(PC)；At least one sample Quality Control point (SC) and an enzyme mark Quality Control point (EC)；At least three standard curve point (S1-S3) and
One coated position reference point of chip (Loc) itself.
Positive quality control point can be human IgG in the embodiment of the present invention, and the enzyme linked immunological marker used is exactly anti-human igg
Marker.The point can also be coated with the DNP of BSA couplings in other embodiments, and the enzyme linked immunological marker used is exactly anti-
The mixed liquor of the marker of the marker of human IgG and anti-DNP.
In the embodiment of the present invention, negative Quality Control point can be less than the human IgG of the micro-concentrations of reaction signal value, at it
Can also be other unrelated proteins in his embodiment.
In the embodiment of the present invention, sample Quality Control point can be the IgG of goat-anti people, can also use it in other embodiments
His anti-human igg, such as rabbit anti-human igg, mouse anti-human igg etc..
In the embodiment of the present invention, enzyme mark Quality Control point can be human IgG (enzyme mark is the enzyme mark of anti-human igg), in other realities
Others can also be used by applying in example：Such as antibody (the enzyme mark of the antibody (enzyme mark is rabbit anti-human igg) of anti-rabbit, or anti-sheep
With regard to the IgG with goat-anti people).
In the embodiment of the present invention, it is the human IgG of basic, normal, high three kinds of concentration that standard curve point is coated, for internal fixed
Mark, comparison interpretation as a result.
In the embodiment of the present invention, it is the human IgG solution of 2 μ g/ml that the position reference point of chip itself is coated, mainly
Positioning action when to array value.
The embodiment of the present invention chips matrix is 96 hole enzyme reaction plates, and in other examples, which may be used also
To be the carrier of the suitable protein attachments such as slide, various chemical films, Bio-sil.
Antigen provided by the invention includes several or a variety of in above-mentioned 12 kinds of antigen, by using different antigen coats
Buffer solution is uniformly coated in chip matrix.The antigen coat buffer solution is the Tris of the CB buffer solutions of PH9.6, PH8.5
The PBS buffer solution of buffer solution and PH7.4, wherein be added to trehalose, glycerine, PEG or PVP and Proclin300 anti-corrosions
Agent, while being added to the additives such as water soluble Beta-cyclodextrin so that coating effect is more preferable, and more stable, uniform, coated point is more advised
Then, mellow and full, CV smallers.The PEG is PEG-400, and the water soluble Beta-cyclodextrin can be 0.02% 2- hydroxy-betas-ring paste
Essence can also be Captisol, carboxymethyl-beta-cyclodextrin etc..
The coated condition of chip is 2-8 DEG C, and 24-30 hour is coated with overnight.
Coating terminates to need to close chip, and the closed stablity agent used in the present invention is 1% lemon containing 1%BSA
In addition lemon acid, 0.5% mannitol also added the phosphoric acid hydrogen of the 0.01M of 1% PVA2W and 0.05% Proclin300
Two sodium solutions.
Enzyme used in kit of the present invention is designated as the rabbit anti-human igg of HRP labels, and the enzyme mark working solution that the kit includes is
The HPR rabbit anti-human iggs marked are diluted to the enzyme marker using final concentration with enzyme mark dilution stabilizer, the enzyme mark dilution is steady
Determine agent be the citric acid of 0.01M PBS, 0.05M, 2% BSA (g/v), the PEG1W of 2% (v/v), 0.1% (g/v) I
The Proclin300 of uncle's natural gum, the P-hydroxybenzoic acid sodium of 0.3% (v/v) and 0.05% (v/v).In other embodiments,
Other enzyme mark anti-human igg, such as goat anti-human igg can also be used；In other embodiments, other labels can also be used even
Join object, such as AP, Avidin, acridinium ester etc..
The chromogenic substrate that the present invention uses is enhanced chemical luminous substrate (ECL), passes through fluorescence detection device or instrument
Carry out the reading of reaction result.Other chromogenic substrates, such as p-NPP, TMB etc. can also be used in other embodiments.
The present invention utilizes ELISA-Array technologies, is prepared for a kind of High sensitivity and special autoimmune nephrosis is anti-
It is cheap after the body spectrum detection kit technical products, and the stability of kit is than the Related product (one of present market
As 2-8 DEG C 1 year) will get well.
Composition provided by the invention, chip and preparation method thereof and include the chip detection device in it is raw materials used
And reagent is available on the market.
With reference to embodiment, the present invention is further explained：
Examples 1 to 3
1, the coating of chip：
1) the specific distribution of chip array design and antigen and reference point is as shown in table 1, table 2：
Table 1
| · | · | · | · | · |
| · | · | · | · | · |
| · | · | · | · | · |
| · | · | · | · | · |
Table 2
| PC | NC | GBM | LAMP-2 | Endothelial cell |
| SC | S1 | PR3 | PLA2R | Nucleosome |
| EC | S2 | MPO | THSD7A | dsDNA |
| Loc | S3 | LF | PT albumen | C1q |
2) specifically it is coated with process：
First, by dilution antibody as described below and GAP-associated protein GAP：
PC, NC, S1, S2, S3, EC point in array coated respectively is 2 μ g/ml, 0.01 μ g/ml, 0.5 μ g/ml, 2 μ g/
The human IgG of ml, 4 μ g/ml, 2 μ g/ml, the CB buffer solutions that dilution buffer is PH9.6 are (wherein containing 2.5% PEG4000,5%
Trehalose, 0.05% Proclin300 and 15% glycerine).
It is the goat anti-human igg antibody of 2 μ g/ml that SC points are coated, and dilution buffer is the CB buffer solutions (being same as above) of PH9.6.
It is the human IgG of 2 μ g/ml that Loc points are coated, and dilution buffer is the CB buffer solutions of PH9.6.
The dilution of envelope antigen：
DsDNA, nucleosome, C1q respectively use PH7.4-PH7.6 0.01M PBS buffer solution (wherein contain 0.5% PVP,
5% trehalose, 0.05% Proclin300,0.02% 2- hydroxy-beta-cyclodextrins) be diluted, final concentration is respectively
40μg/ml、12μg/ml、15μg/ml。
PLA2R, THSD7A, LF, MPO, PR3, GBM use CB buffer solutions (3% PEG4000,2.5% of PH9.6 respectively
The glycerine of trehalose, 0.02% 2- hydroxy-beta-cyclodextrins, 0.05% Proclin300 and 15%) it is diluted to 8 μ of concentration
g/ml、15μg/ml、15μg/ml、11μg/ml、12μg/ml、20μg/ml。
LAMP-2, PT albumen and endothelial cell use the Tris buffer solutions of the 0.02M of PH8.5 (wherein to contain 2.5% respectively
PEG4000,0.05% Proclin300,3% trehalose and 0.01% 2- hydroxy-beta-cyclodextrins and 15% it is sweet
Oil) it is diluted, final concentration is respectively：20 μ g/ml, 40 μ g/ml and 30 μ g/ml.
All antibody or antigen good according to above-mentioned dilution use the membrane filtration of 0.22um respectively, then pass through BioDot essences
Close point sample instrument carries out the coating of array as requested, and coating volume is each point 10nl.After whole albumen coatings are completed, by core
Piece is lived with membrane cover, is placed in 2-8 DEG C, is coated with 24-30h overnight.
2, it closes
The chip being coated with is taken out, is cleaned 3 times with the PBST cleaning solutions of PH7.4, the confining liquid of 150ul is then added per hole
(citric acid (v/v) of 1%-2%BSA (g/v), 1%-1.5%, the mannitol (v/v) of 0.3%-0.6%, in addition also added
The disodium phosphate soln of the 0.01M of the Proclin300 of the PVA2W (v/v) and 0.05% (v/v) of 0.8%-1.5%, addition
Not busy feelings are shown in as shown in table 3), it is stored at room temperature closing 35min, is then patted dry, in humidity<15%, RT place dry 4h, seal afterwards,
2-8 DEG C of preservation.
Table 3
| Group | Embodiment 1 | Embodiment 2 | Embodiment 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 phosphate soln | 0.01M | 0.01M | 0.01M |
3, enzyme marking reagent is prepared
Enzyme mark stabilizing buffer (citric acid of 0.01M PBS, 0.05M, the BSA of 1.5%-2.5% for being 7.4 with pH value
(g/v), the PEG1W (v/v) of 1.5%-2%, the gum arabic (g/v) of 0.1%-0.2%, 0.2%-0.5% to hydroxyl
Sodium benzoate (v/v) and 0.05% Proclin300 (v/v), to be shown in Table the rabbit anti-human igg for 4) marking HRP dilute for addition details
It releases to 4K times (4000 times), mixing.
Table 4
| Group | Embodiment 1 | Embodiment 2 | Embodiment 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% |
| P-hydroxybenzoic acid sodium (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 kit
1) chip reagent, balance to room temperature are taken out；
2) it is loaded：By negative and positive control serum and with sample diluting liquid (0.02M Tris, 0.15M NaCl,
0.05%Tween20,0.01% casein, PH7.4) 101 times of sample to be tested is diluted, chip to be measured is added per hole 100uL
It is reacted in hole.
3) it incubates：React at room temperature 30min.Add 300uL cleaning solutions (0.02M Tris, 0.15M NaCl, 0.05%
Tween20, PH7.4), it washs 3 times, each 1min.
4) add enzyme marking reagent：The enzyme marker (rabbit-anti people HRP) of 100ul is added per hole；
5) it incubates：React at room temperature 30min.Add 300uL cleaning solutions, washs 3 times, each 1min.
6) it develops the color：ECL color developing agent 50uL are added per hole, room temperature is protected from light 30min, blots later.
7) it detects：In 30min, is read by butcher bird spy's chemical luminous chip analyzer and analyze each reacting hole correspondence inspection
Survey the signal value of index, the standard curve calibrated by S1-S3 reference points, to calculate the yin and yang attribute and intensity of simultaneously judging result.
4 evaluation of the accuracy of embodiment
1, with the anti-GBM IgG positive serums of 10 determinations and 10 anti-GBM of the ELISA kit screening of Abnova companies
IgG negative serums, the chip agent box prepared with Examples 1 to 3 are tested, as a result as (+expression is positive ,-expression for the following table 5
It is negative).
The anti-GBM IgG serum results of chip testing prepared by 5 Examples 1 to 3 of table
2, anti-with the anti-PR3IgG positive serums of 10 determinations and 10 of EUROIMMUN companies ELISA kit screening
PR3IgG negative serums, the chip testing prepared with Examples 1 to 3, as a result such as the following table 6 (+expression is positive, and-expression is negative).
The anti-anti- PR3IgG serum result of torch-IgG types antibody repertoire chip testing prepared by 6 Examples 1 to 3 of table
3, anti-with the anti-MPO IgG positive serums of 10 determinations and 10 of Invitrogen companies ELISA kit screening
MPO IgG negative serums, chip prepared by Examples 1 to 3 are tested, as a result as (+expression is positive, and-expression is cloudy for the following table 7
Property).
The anti-MPO IgG serum results of chip testing prepared by 7 Examples 1 to 3 of table
4, with the anti-LF IgG positive serums of 10 determinations and 10 anti-LF IgG of the ELISA kit screening of cusbio companies
Negative serum, the chip testing prepared with Examples 1 to 3, as a result such as the following table 8 (+expression is positive, and-expression is negative).
The anti-LFIgG serum result of chip testing prepared by 8 Examples 1 to 3 of table
5, anti-with the anti-LAMP-2IgG positive serums of 10 determinations and 10 of biorbyt companies ELISA kit screening
LAMP-2IgG negative serums, chip prepared by Examples 1 to 3 are tested, as a result as (+expression is positive, and-expression is cloudy for the following table 9
Property).
The anti-LAMP-2IgG serum result of chip testing prepared by 9 Examples 1 to 3 of table
6, it is determined with 10 of EUROIMMUN companies ELISA kit screening and infects anti-PLA2R IgG positive serums and 10
The anti-PLA2R IgG negative serums of example, the chip prepared with Examples 1 to 3 are tested, as a result as the following table 10 (+indicate positive ,-
Indicate negative).
The anti-PLA2R IgG serum results of chip testing prepared by 10 Examples 1 to 3 of table
7, with 10 anti-THSD7A IgG positive bloods of determination of EUROIMMUN companies kit (glimmering method is immunized indirectly) screening
Cleer and peaceful 10 anti-THSD7A IgG negative serums, the chip prepared with Examples 1 to 3 are tested, as a result such as the following table 11 (+table
Show the positive ,-indicate negative).
The anti-THSD7A IgG serum results of chip testing prepared by 11 Examples 1 to 3 of table
8 while with two kinds of EUROIMMUN companies ELISA kit and EUROIMMUN departments kit (immune glimmering method indirectly)
The anti-PT protein Is gG positive serums of 10 determinations and 10 anti-PT protein Is gG negative serums of screening are prepared with Examples 1 to 3
Chip is tested, as a result such as the following table 12 (+expression is positive, and-expression is negative).(PT protein antibodies are prepared by our company's research
The fusion protein PT albumen that anti-phospholipase A2 receptor (PLA2R) is merged with 1 domains type thrombospondin 7A (THSD7A) is anti-
Body).
The anti-PT protein Is gG serum results of chip testing prepared by 12 Examples 1 to 3 of table
9, with 10 determining anti-endothelial cell antibodies IgG positive serums and 10 of cusabio companies ELISA kit screening
Example anti-endothelial cell antibodies IgG negative serums, the chip prepared with Examples 1 to 3 are tested, as a result such as the following table 13 (+expression
The positive ,-indicate negative).
Chip testing anti-endothelial cell antibodies IgG serum results prepared by 13 Examples 1 to 3 of table
10, with the anti-C1q IgG positive serums of 10 determinations and 10 anti-C1q IgG of the ELISA kit screening of CD companies
Negative serum, the chip prepared with Examples 1 to 3 are tested, as a result such as the following table 14 (+expression is positive, and-expression is negative).
The anti-C1q IgG serum results of chip testing prepared by 14 Examples 1 to 3 of table
11, anti-with the 10 determining anti-dsDNA IgG positive serums and 10 of Abcam companies ELISA kit screening
DsDNA IgG negative serums, the chip prepared with Examples 1 to 3 are tested, as a result as (+expression is positive ,-expression for the following table 15
It is negative).
Chip testing anti-dsDNA IgG serum results prepared by 15 Examples 1 to 3 of table
12, with the anti-nucleosome IgG positive serums of 10 determinations and 10 of NOVATEINBIO companies ELISA kit screening
The anti-nucleosome IgG negative serums of example, the chip prepared with Examples 1 to 3 are tested, as a result as the following table 16 (+indicate positive ,-
Indicate negative).
The anti-nucleosome IgG serum results of chip testing prepared by 16 Examples 1 to 3 of table
In conclusion the accuracy symbol of chip testing items autoimmune nephrosis related antigen IgG prepared by the present invention
It closes and requires, accuracy is high.
5 sensibility and specificity test result of embodiment
Clinical samples are diagnosed as jointly from the positive serum sample 85 for exempting from nephrotic by renal fibroblast and clinical manifestation
Example, 102, the negative serum sample of normal healthy people.
Table 17
Data above can be seen that the chip agent box of the present invention to clinically from exempt from nephrotic (by renal fibroblast and
What clinical symptoms were made a definite diagnosis jointly exempts from nephrotic certainly) up to 98.8%, specificity is (negative for the susceptibility (positive coincidence rate) of detection
Accuracy) up to 100%, illustrate that the kit susceptibility of the present invention is high, specificity is good, and the diagnosis to exempting from nephrosis certainly can provide more
It accurately completely refers to and instructs.
The stability experiment result of 6 kit of embodiment
From the stability experiment result for exempting from nephrosis associated antibodies spectrum chip kit：Table 18
2-8 DEG C of stationary phase was up to 2 years.
Table 19
18-28 DEG C of 9 months stationary phase.
Embodiment 7
Contrast agent box：(the ingredient of general confining liquid and enzyme mark dilution：0.01M PBS (PH7.4)+10%BSA).
Table 20
Table 21
Above-mentioned experiment is using kit of the invention and the kit for having used general confining liquid and enzyme mark dilution
(remaining condition is the same) is Experimental comparison, the stablizing effect of kit as a result of the invention be better than used general confining liquid and
The general reagent box of enzyme mark dilution.
Data show that the stability of kit of the invention test can store 2 years at 2-8 DEG C, and (18-28 DEG C can put room temperature
Set 9 months), it was demonstrated that its stability is preferable.It is compared simultaneously with the confining liquid and enzyme mark dilution used in the prior art, and
The stabilization of kit of the present invention is more excellent.
Embodiment 8
By embodiment 1-3 reagent preparations box compared with preparing conventional ELISA kit antigen dosage：
Table 22
In conclusion showing that kit provided by the invention can effectively reduce the usage amount of related antigen, ensureing to try
While the sensitivity and specificity of agent box, relevant antigen use cost is reduced.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of composition, which is characterized in that including citric acid, mannitol, PVA2W, PEG1W, gum arabic, para hydroxybenzene
Mixture more than one or both of sodium formate；
Citric acid described in the composition, mannitol, PVA2W volume ratio be (1~1.5):(0.3~0.6):(0.8~
1.5) or
The composition includes following component：
2. composition according to claim 1, which is characterized in that further include BSA, Proclin300, PBS or phosphoric acid hydrogen two
Mixture more than one or both of sodium.
3. application of the composition according to claim 1 or 2 in preparing chip and/or detection device.
4. a kind of chip, which is characterized in that it is coated with just like composition described in any one of claim 1 to 5.
5. chip according to claim 4, which is characterized in that be also coated with autoimmune nephrosis related antigen.
6. chip according to claim 4 or 5, which is characterized in that the chip also includes Quality Control point and/or reference point；
The Quality Control point includes 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 mark Quality Control point (EC)；The reference point includes the reference curve point (S1-S3) of various concentration
And/or at least one chip position reference point (Loc).
7. the preparation method of claim 4 to 6 any one of them chip, includes the following steps：
Step 1：By the autoimmune nephrosis related antigen, the albumen of the albumen of Quality Control point and/or the reference point
After coated buffer solution dilution, the matrix of the chip is coated in the form of lattice array；
Step 2：The chip is closed through closed stablity agent；
The closed stablity agent includes such as composition described in any one of claim 1 to 5.
8. chip made from preparation method according to claim 7.
9. a kind of detection device, which is characterized in that including such as 4 to 6 any one of them chips or core as claimed in claim 8
Piece.
10. a kind of detection side based on such as autoimmune nephrosis of claim 16 to 20 any one of them detection device
Method, which is characterized in that after taking sample to be tested to be diluted with the sample diluting liquid in coating to the chip, the label is added
Object is added the developing solution and is protected from light colour developing, and fluorescence detection device testing result obtains the autoimmune nephrosis in sample to be tested
Associated antibodies signal value.
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| CN201810186409.5A CN108398550B (en) | 2018-03-07 | 2018-03-07 | Composition, chip, preparation method of chip and detection device comprising chip |
| PCT/CN2018/092770 WO2019169793A1 (en) | 2018-03-07 | 2018-06-26 | Composition, chip and preparation method for chip and detection device including chip |
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| CN112748242A (en) * | 2020-12-21 | 2021-05-04 | 珠海碳云智能科技有限公司 | Secondary antibody buffer confining liquid for polypeptide chip technology platform detection, kit comprising same and application thereof |
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| CN115015538A (en) * | 2022-05-17 | 2022-09-06 | 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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Also Published As
| Publication number | Publication date |
|---|---|
| WO2019169793A1 (en) | 2019-09-12 |
| CN113238039B (en) | 2023-03-31 |
| CN113238039A (en) | 2021-08-10 |
| CN108398550B (en) | 2022-07-26 |
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