CN110095592A - A kind of c reactive protein monofilm detection dry plate - Google Patents
A kind of c reactive protein monofilm detection dry plate Download PDFInfo
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- CN110095592A CN110095592A CN201910367557.1A CN201910367557A CN110095592A CN 110095592 A CN110095592 A CN 110095592A CN 201910367557 A CN201910367557 A CN 201910367557A CN 110095592 A CN110095592 A CN 110095592A
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- dry plate
- supporting layer
- diffusion agent
- crp
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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
-
- 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
-
- 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
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
- G01N33/537—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody
-
- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
- G01N33/6857—Antibody fragments
Abstract
The invention discloses a kind of c reactive protein (CRP) monofilms to detect dry plate comprising upper supporting layer, lower supporting layer, diffusion agent layer and photic zone, and upper supporting layer and lower supporting layer are mutually glued.And diffusion agent layer is arranged on the photic zone between upper supporting layer and lower supporting layer, it is made of water-soluble material, the reagent of participation chemical reaction and spherical white particle etc., comprising: solidification phosphocholine, the anti-CRP antibody of the mouse of peroxidase labelling, calcium chloride, 2- (3,5- dimethoxy-4 's-hydroxyphenyl) -4,5- is to (4- dimethyl amido phenyl) imidazoles, adhesive, surfactant, protein, white spherical particles etc..It is linear higher, reproducible, easy to operate, voluntarily easy to detect, specific good that c reactive protein (CRP) monofilm of the present invention detection dry plate has many advantages, such as, and the drugs such as brufen, etamcylate, theophylline on the measurement of dry plate without influence.
Description
Technical field
The invention belongs to technical field of in-vitro clinical diagnostic reagent, and in particular to a kind of monofilm for biochemical analysis is dry
Piece-c reactive protein monofilm detection dry plate.
Background technique
Biofluid such as serum, blood plasma, whole blood, urine, cerebrospinal fluid are very important in medical diagnosis on disease.It is existing
Biochemical analyzer and matched reagent have many products, such as 5600 Full-automatic biochemical immunity analysis instrument of VITROS, Beckman
SYNCHRONization-LX20 series automatic clinical chemistry analyzer, Hitachi 7180 serial automatic clinical chemistry analyzer, more magnificent YS-
The various semiautomatic biochemistry analysis series such as 2000 III type semi-automatic biochemical analyzers.But not due to the liquid reagent retention time
Can be too long and not readily transportable, so Dry-type biochemical analysis also has gradually developed, as a kind of means solved the problems, such as, such as blood
Sugared instrument is exactly Typical Representative, is not necessarily to additional agents.
Dry chemical, which refers to, is in advance fixed on reagent some or all of needed for reaction in the carrier with special construction, surveys
Biological fluid is applied directly to secure on the carrier of reagent by timing, using the moisture in sample as solvent, causes determinand
Occur to chemically react and generate reaction signal with the reagent in carrier, be shown in finally by the detection of certain instrument anti-on carrier
Induction signal variation, to measure the concentration of determinand in sample.Dry analysis technology recent decades achieve it is very big into
Step, can apply in the field that wet-chemical can not touch, such as battlefield, field or cosmic space clinical examination, apply also for
The fields such as food safety, environmental monitoring.
Dry chemical product is substantially the product of offshore company in the market, probably there is three classes.The first kind is with Johnson & Johnson, U.S. public affairs
Department is the dry chemical technology of the Multilayer film dry plate of representative；Second class is the dry chemical technology of kyoto, Japan company；Third class is auspicious
The dry chemical product of scholar Roche company.Dry chemical technology using more and more extensive, improves dry plate to sample in present biochemical analysis
The diffusion of product and the influence factor in inhibition chemical reaction are vital in the overall performance for improving dry plate.
C reactive protein (C-reaction protein, CRP) is found in nineteen thirty by Tillet and Francis.Initially he
Observe that the serum of some acute patients can react with the pod membrane C- polysaccharide of streptococcus pneumonia, then confirm can be more with C-
The substance of sugar reaction is a kind of protein, thus this protein is named as C reactive protein.CRP is that body non-specificity is exempted from
A part of epidemic disease mechanism, it combines C- polysaccharide, in Ca2+In the presence of in combination with phosphocholine on cell membrane.The detection of CRP is in clinic
Using quite extensive, the diagnosis and differential diagnosis including acute infectious diseases, the monitoring of post-operative infection；Antibiotic curative effect
Observation；Course of disease detection and Index for diagnosis etc..The existing test kit technology in China is linear poor equal to lack there are inconvenient to carry
Point.China's dry plate relies primarily on import, and key technology is the preparation of dry chemistry reagent dry plate, the dry plate of existing market, generally
All there is the deficiencies of preparation is complicated, at high cost.
Summary of the invention
The purpose of the present invention is overcoming defect of the existing technology, a kind of C reactive protein (CRP) monofilm detection is provided
Dry plate.
Realizing the technical solution of the object of the invention is: a kind of C reactive protein (CRP) monofilm detection dry plate, including upper support
Layer, lower supporting layer, diffusion agent layer and photic zone；The upper supporting layer and lower supporting layer are mutually glued, the diffusion
Reagent layer is arranged on the photic zone between upper supporting layer and lower supporting layer；The diffusion agent layer includes water-soluble material and ginseng
With the reagent of chemical reaction, and spherical white particle.
The upper supporting layer and lower supporting layer are transparent poly terephthalic acid class material, polyethylene, polypropylene, polyvinyl chloride
Or glassware, light transmittance is 80% or more.
The diffusion agent layer includes:
Reagent: solidification phosphocholine 0.03-0.1mg/cm2,
The anti-CRP antibody 0.0002-0.0008U/cm of the mouse of horseradish peroxidase-labeled2,
0.04-0.1mg/cm of calcium chloride2
2- (3,5- dimethoxy-4 '-hydroxyphenyl) -4,5- is to (4- dimethyl amido phenyl) imidazoles 0.01-0.07mg/cm2
Adhesive 0.1-1.5 μ L/cm2
Surfactant 0.5-2 μ L/cm2
Protein 1-10 μ g/cm2
Spherical white particle 0.05-0.5mg/cm2。
C reactive protein (CRP) monofilm detection dry plate of the present invention is preferred are as follows:
Reagent: solidification phosphocholine 0.05-0.08mg/cm2；
The anti-CRP antibody 0.0004-0.0007U/cm of the mouse of horseradish peroxidase-labeled2；
Calcium chloride 0.05-0.09mg/cm2；
2- (3,5- dimethoxy-4 '-hydroxyphenyl) -4,5- is to (4- dimethyl amido phenyl) imidazoles 0.02-0.05mg/
cm2；
Adhesive 0.3-1 μ L/cm2；
Surfactant 0.8-1.5 μ L/cm2；
Protein 2-8 μ g/cm2；
Polymeric beads 0.15-0.25mg/cm2。
Raw material selection gist is in C reactive protein (CRP) monofilm detection dry plate of the present invention:
Solidify phosphocholine: in conjunction with CRP, simulating cellular environment
The anti-CRP antibody of the mouse of horseradish peroxidase-labeled: oxidation hydrogen peroxide, with CRP antigen binding
Calcium chloride: Ca needed for reaction is provided2+
2- (3,5- dimethoxy-4 '-hydroxyphenyl) -4,5- is to (4- dimethyl amido phenyl) imidazoles: conducive to blue pigment
Quickly formed
Adhesive: by its viscosity, spherical particles are connected
Surfactant: the surface energy of water is reduced, Serodiagnosis is conducive to
Protein: providing environment required for reacting, and simulates cellular environment
Spherical white particle: as carrier, reacting environment is provided.
The photic zone is formed using visible-range light transmittance in 80% or more transparent plastic sheet or glass preparation, thick
Degree is 0.5-2mm.
The photic zone can be by various transparent plastics (such as polythene PE, polyethylene terephtalate, poly- third
Alkene PP, polycarbonate etc.), glass etc. be prepared, especially with PET.It is euphotic with a thickness of 0.5-2mm, be with 1-1.5mm
Most preferably.80% or more visible-range light transmittance, 95% the above are best.Light source penetrates photic zone from instrument connection incidence.
Realizing the organization plan of the object of the invention is: C reactive protein (CRP) monofilm test dry plate of the invention includes upper
Supporting layer, lower supporting layer, diffusion agent layer and photic zone.The upper supporting layer and lower supporting layer are mutually glued, upper
The test section of supporting layer is provided with well, and the corresponding position of lower supporting layer is provided with instrument connection, and diffusion agent layer passes through coating process
Coated between the upper supporting layer and lower supporting layer on photic zone, being set at well and instrument connection.
Above-mentioned Multilayer film dry plate, main sample to be used is serum, blood plasma and urine, if needing to shift to an earlier date with whole blood
To whole blood processing, serum is obtained.
The sample can be added directly into diffusion agent layer by well.The effect of diffusion agent layer is uniform
By sample diffusion and the place of reaction is provided, and reflected light can be played the role of.In addition, diffusion layer, which also functions to removal detection, to be influenced
The effect of the factor.
The diffusion agent layer is the perforated membrane of one layer of uniform each Xiang Junxiang, be barium sulfate, titanium dioxide, barium titanate,
The ball-types white particle such as polystyrene, particle size range is between 0.1um-5.0um, and wet-film thickness is 100-300 μm, thickness of dry film
It is 50-200 μm.
C reactive protein (CRP) monofilm test dry plate of the invention measures C reactive protein by following methods: using calcium
As capturing agent, on phosphocholine is covalently bound on particle surface, it is integrated to the list that horseradish peroxidase is immune marker
On monoclonal CRP antibody.One drop clinical samples drop is equably spread on dry plate, and through diffusion layer.CRP and phosphorus in sample
The capture particle of sour choline interlinkage is formed a kind of undissolved sandwich in conjunction with the anti crp antibody of horseradish peroxidase-labeled
Structural compounds.Then immune washing lotion, unbonded substance are added on dry plate, while also providing for the colourless of enzyme medium
Hydrogen peroxide needed for stain oxidation reaction.
Measuring method using C reactive protein of the present invention (CRP) monofilm test dry plate is as follows: sample is added drop-wise to test
At hole, after 30-38 DEG C of reaction 4-6min, immune washing lotion is added drop-wise at instrument connection, 30-38 DEG C of reaction 1-3min.After reacting
Dry plate immediately under 670nm light carry out dyestuff reading.Reference standard curve graph can check in the concentration of CRP according to signal value.
Wherein, washing lotion dosage 10-15 μ L is immunized in amount of samples 10-15 μ L.
The present invention has the effect of positive: it is linear higher, it is reproducible；Due to easy to operate, patient can voluntarily be detected, special
It is anisotropic more preferable, as brufen, etamcylate, theophylline etc. have biggish clinic suitable for automatic clinical chemistry analyzer without influence to this
Value.
Detailed description of the invention
Fig. 1 is that C reactive protein of the present invention (CRP) monofilm tests dry plate structural profile illustration.
Fig. 2 is the canonical plotting that C reactive protein of the present invention (CRP) monofilm tests dry plate.
Fig. 3 is the canonical plotting for comparing C reactive protein (CRP) monofilm test dry plate.
Specific embodiment
In order that the present invention can be more clearly and readily understood, right below according to specific embodiment and in conjunction with attached drawing
The present invention is described in further detail.
To C reactive protein of the present invention (CRP) monofilm test dry plate and dry plate is compared below with reference to Fig. 1, Fig. 2 and table
Performance is compared.Abscissa is serum-concentration in Fig. 2, Fig. 3, and ordinate is signal value.
Table 1 is that dry plate of the present invention and comparison dry plate measure linear equation comparison.
Table 2 is present invention test dry plate and the comparison of contrast test dry plate accuracy measurement result.
Embodiment:
Solidify phosphocholine 0.06mg/cm2,
The anti-CRP antibody 0.0006U/cm of the mouse of horseradish peroxidase-labeled2,
Calcium chloride 0.07mg/cm2
2- (3,5- dimethoxy-4 '-hydroxyphenyl) -4,5- is to (4- dimethyl amido phenyl) imidazoles 0.02mg/cm2
0.3 μ L/cm of adhesive2
1.2 μ L/cm of surfactant2
5 μ g/cm of protein2
Spherical white particle 0.15mg/cm2
Comparative example:
Solidify phosphocholine 0.09mg/cm2,
The anti-CRP antibody 0.0008U/cm of the mouse of horseradish peroxidase-labeled2,
Calcium chloride 0.05mg/cm2
2- (3,5- dimethoxy-4 '-hydroxyphenyl) -4,5- is to (4- dimethyl amido phenyl) imidazoles 0.06mg/cm2
0.25 μ L/cm of adhesive2
0.5 μ L/cm of surfactant2
1 μ g/cm of protein2
TiO2 0.1mg/cm2
The performance test of C reactive protein (CRP) monofilm test dry plate of the present invention is as follows:
Test 1: linear equation
Prepare 64.500,49.000,32.250,25.800,21.500,18.429,16.125,14.333,8.063,
4.031mg/L C reactive protein standard solution, it is dry with C reactive protein of the present invention (CRP) monofilm test dry plate and comparison respectively
Piece measures its variance, the results are shown in Table 1.
The dry plate of the present invention of table 1 and comparison dry plate measurement linear equation comparison
C reactive protein (CRP) monofilm test dry plate and comparison C reactive protein (CRP) monofilm of the present invention test dry plate
Test method is as follows: sample being added drop-wise at instrument connection, after 30-38 DEG C of reaction 4-6min, immune washing lotion is added drop-wise to instrument connection
Place, 30-38 DEG C of reaction 1-3min.Dry plate after reaction is carried out under 670nm light to the reading of dyestuff immediately.Reference standard curve
Figure, can check in the concentration of CRP according to signal value.Wherein, washing lotion dosage 10-15 μ L is immunized in amount of samples 10-15 μ L.
For dry plate of the present invention compared with comparing dry plate, variance is bigger, linear more preferable.
Test 2: accuracy
It can be obtained by table 1, the standard curve that C reactive protein (CRP) monofilm test dry plate of the present invention obtains are as follows: y=-
0.00207x+0.40386, the standard curve that comparison C reactive protein (CRP) monofilm test dry plate obtains are as follows: y=-
0.000322x+0.08938, respectively with C reactive protein of the present invention (CRP) monofilm test dry plate and comparison C reactive protein
(CRP) blood serum sample of monofilm test dry plate measurement various concentration, obtains signal value after test, by checking in serum on standard curve
Concentration value.It the results are shown in Table 2.
2 present invention test dry plate of table and the comparison of contrast test dry plate accuracy measurement result
As can be seen that the value ratio that present invention test dry plate measures compares dry plate closer to theoretical value from table 2, accuracy is more
Height, it is more preferable that the present invention tests dry plate.
Particular embodiments described above has carried out further in detail the purpose of the present invention, technical scheme and beneficial effects
It describes in detail bright, it is to be understood that the above is only a specific embodiment of the present invention, is not intended to restrict the invention, all
Any modification, equivalent substitution, improvement and etc. done within the spirit and principles in the present invention should be included in protection of the invention
Within the scope of.
Claims (8)
1. a kind of c reactive protein monofilm detects dry plate, it is characterised in that including upper supporting layer, lower supporting layer, diffusion agent layer and
Photic zone；The upper supporting layer and lower supporting layer are mutually glued, and the diffusion agent layer setting is in upper supporting layer under
On photic zone between supporting layer；The diffusion agent layer includes water-soluble material and the reagent for participating in chemical reaction, Yi Jiqiu
Shape white particle.
2. dry plate according to claim 1, which is characterized in that the upper supporting layer and lower supporting layer gather to be transparent to benzene two
First acrylic materials, polyethylene, polypropylene, polyvinyl chloride or glassware, light transmittance is 80% or more.
3. dry plate according to claim 1, which is characterized in that the diffusion agent layer includes:
Reagent: solidification phosphocholine 0.03-0.1mg/cm2,
The anti-CRP antibody 0.0002-0.0008U/cm of the mouse of horseradish peroxidase-labeled2,
Calcium chloride 0.04-0.1mg/cm2
2- (3,5- dimethoxy-4 '-hydroxyphenyl)-4,5- is to (4- dimethyl amido phenyl) 0.01-0.07mg/cm of imidazoles2
Adhesive 0.1-1.5 μ L/cm2
Surfactant 0.5-2 μ L/cm2
Protein 1-10 μ g/cm2
Spherical white particle 0.05-0.5mg/cm2。
4. dry plate according to claim 1, which is characterized in that the diffusion agent layer includes:
Reagent: solidification phosphocholine 0.05-0.08mg/cm2,
The anti-CRP antibody 0.0004-0.0007U/cm of the mouse of horseradish peroxidase-labeled2,
Calcium chloride 0.05-0.09mg/cm2
2- (3,5- dimethoxy-4 '-hydroxyphenyl) -4,5- is to (4- dimethyl amido phenyl) imidazoles 0.02-0.05mg/cm2
Adhesive 0.3-1 μ L/cm2
Surfactant 0.8-1.5 μ L/cm2
Protein 2-8 μ g/cm2
Spherical white particle 0.15-0.25mg/cm2。
5. dry plate according to claim 1, which is characterized in that the photic zone is using visible-range light transmittance 80%
Above transparent plastic sheet or glass preparation forms, with a thickness of 0.5-2mm.
6. dry plate according to claim 1, which is characterized in that the euphotic material is selected from polyethylene, is poly- to benzene two
Formic acid glycol ester, polypropylene or polycarbonate, it is seen that 95% or more optical range light transmittance, with a thickness of 1-1.5mm.
7. dry plate according to claim 1, which is characterized in that the test section of the upper supporting layer is provided with well, described
The corresponding position of lower supporting layer is provided with instrument connection, and the diffusion agent layer is coated on photic zone by coating process.
8. dry plate according to claim 1, which is characterized in that the diffusion agent layer is one layer of uniform each Xiang Junxiang's
Perforated membrane, the ball-type white particle are barium sulfate, titanium dioxide, barium titanate, polystyrene, and particle size range is in 0.1um-
Between 5.0um, wet-film thickness is 100-300 μm, and thickness of dry film is 50-200 μm.
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Cited By (2)
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| CN110794146A (en) * | 2019-10-31 | 2020-02-14 | 无锡锦帛诚医疗器械科技有限公司 | Total protein quantitative detection dry tablet for in vitro diagnosis |
| CN110791547A (en) * | 2019-10-31 | 2020-02-14 | 无锡锦帛诚医疗器械科技有限公司 | Creatinine quantitative detection dry sheet for in vitro diagnosis |
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Application publication date: 20190806 |
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