CN109444429A - Utilize the kit of Chemiluminescence Immunoassay measurement laminin - Google Patents
Utilize the kit of Chemiluminescence Immunoassay measurement laminin Download PDFInfo
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- CN109444429A CN109444429A CN201811521732.XA CN201811521732A CN109444429A CN 109444429 A CN109444429 A CN 109444429A CN 201811521732 A CN201811521732 A CN 201811521732A CN 109444429 A CN109444429 A CN 109444429A
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- laminin
- magnetic particle
- kit
- chemiluminescence immunoassay
- sample
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/08—Hepato-biliairy disorders other than hepatitis
- G01N2800/085—Liver diseases, e.g. portal hypertension, fibrosis, cirrhosis, bilirubin
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- Proteomics, Peptides & Aminoacids (AREA)
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- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention discloses a kind of kit using Chemiluminescence Immunoassay measurement laminin, the magnetic particle suspension including combining laminin monoclonal antibody；The laminin monoclonal antibody of horseradish peroxidase-labeled；Laminin series of calibration product are added with releasing agent in the magnetic particle suspension and enzyme conjugates.The advantage of the invention is that the LN molecule polymerizeing in sample can be dissociated quickly and effectively, will not change with the storage of sample；It is fast to detect speed, process stabilization, "dead" pollution, in the presence of no magnetic field, magnetic particle floats on a liquid, so that antigen-antibody reaction is similar to homogeneous reaction；Magnetic particle is convenient to separate under the action of externally-applied magnetic field；The partial size of magnetic particle reaches nanoscale, so that coating solid phase, close to liquid phase state, opposite micro-pore plate type reduces coating, multiple operating procedures for influencing accuracy such as closing, and the accuracy and stability of detection are greatly improved.
Description
Technical field
The present invention relates to immunoassay medicine is dual-purpose, more particularly, to a kind of viscous using Chemiluminescence Immunoassay measurement layer
The even kit of albumen.
Background technique
Laminin (LN) is a kind of macromolecular glycoprotein, is the heterotrimer being made of 3 polypeptide chains of α, β, γ, point
Son amount is greater than 800kD.The seldom of LN content is distributed mainly on the positions such as vascular wall, bile duct wall and lymph tube wall in normal liver tissue.
When liver fibrosis, the permeability for being largely deposited on sinusoidal endothelial cell gap reduction endothelial cell makes its capillarisa tion, and makes
Portal vein pressure increases, therefore can reflect liver tissue inflammation grade and portal area fibrosis.Meanwhile the cause of disease that LN and liver fibrosis are formed is close
Cut phase is closed: studies have shown that LN level is apparently higher than viral and biliary cirrhosis in alcoholic cirrhosis, and can be reflected
Alcoholic liver disease whether there is portal hypertension and portal hypertension degree.
Have at present to the measuring method of laminin: 1, Elisa measuring method: this method is generally used for sxemiquantitative
Measurement, accuracy of measurement, sensitivity, precision are poor, and operation is comparatively laborious, is unfavorable for being widely applied in clinic.2,
Radioimmunoassay method: this method is because reagent has the defects of radioactive pollution and reagent validity period of short duration (half-life period) to exist
Gradually it is eliminated.
Chemiluminescence immune assay (Chemiluminescence Immunoassay, CLIA) is nearly ten years in the world
The very fast on-radiation immuno analytical method of development, is after radiommunoassay (RIA) and EIA enzyme immunoassay in range
(EIA) the microdetermination technology of a kind of hypersensitivity to grow up after, with highly sensitive, detection range is wide, operation
It is easy quickly, the advantages that marker stability is good, pollution-free, instrument simple economy.It is radioimmunoassay and normal enzyme
The substituent of immunoassay is that current immune quantitative analyzes optimal method.
Since the LN molecule in serum is in polymeric form, only moiety site exposure can be identified by antibody, but this shape
Testing result under state is relatively low.After serum is pulled out in vitro, with increasing for resting period, the LN molecule meeting of polymeric form
It gradually dissociates, at this moment testing result can gradually rise, and under 2-8 DEG C of storage condition, 5-7 days LN molecules of blood sampling have dissociated substantially
Entirely, testing result reaches plateau, and under -20 DEG C of storage condition, then need the several months that can just will be completely dissociated.But for doctor
For institute, the same day will go out examining report, therefore the LN molecule in sample cannot be waited voluntarily to dissociate, and without dissociation
Pattern detection Lower result will cause the erroneous judgement of doctor.
Summary of the invention
The purpose of the present invention is to provide one kind can quickly dissociate the LN molecule polymerizeing in sample, thus Accurate Determining layer
The kit of Fibronectin.
To achieve the above object, the present invention can take following technical proposals:
Kit of the present invention using Chemiluminescence Immunoassay measurement laminin, including combine a layer adhesion egg
The magnetic particle suspension of white monoclonal antibody；The laminin monoclonal antibody of horseradish peroxidase-labeled；Layer adhesion egg
White series of calibration product are added with releasing agent in the magnetic particle suspension and enzyme conjugates.
The releasing agent is citric acid, gluconic acid, triethanolamine, 8- aniline -1-naphthalene sulfonic aicd (ANS), ethylenediamine tetrem
Sour (EDTA), bis- (the 2- amino-ethyl ether) tetraacethyls (EGTA) of ethylene glycol, polyethylene glycol, in polyvinylpyrrolidone (PVP)
It is a kind of.
The partial size of the magnetic particle is nanoscale.
The advantage of the invention is that the LN molecule polymerizeing in sample can be dissociated quickly and effectively, blood sampling same day testing result and
The testing result of 2-8 DEG C of storage 7 days is consistent after blood sampling, will not change with the storage of sample；It is fast to detect speed, 40 minutes
Result can be gone out；Process stabilization, "dead" pollution etc., in the presence of no magnetic field, magnetic particle is suspended in liquid
In, so that antigen-antibody reaction is similar to homogeneous reaction；Magnetic particle is convenient to separate under the action of externally-applied magnetic field, washing
Quickly；The partial size of magnetic particle reaches nanoscale, so that coating solid phase, close to liquid phase state, opposite micro-pore plate type reduces
Coating, multiple operating procedures for influencing accuracy such as closing, the accuracy and stability of detection are greatly improved.The present invention
Accuracy is within 6% in the analysis of kit, between day within accuracy 10%, between batch within accuracy 12%；Kit of the present invention
High sensitivity, sensitivity for analysis are not higher than 5ng/mL, are excused from an examination agent and to put agent of being excused from an examination better than enzyme；Stabilization of kit of the present invention is good,
Storage 1 year can at least be stablized at 2-8 DEG C；Kit specificity of the present invention is good and hyaluronic acid (HA), III type of homologous series
Precollagen N end peptide (III NP of P), type Ⅳ collagen (Col IV) are without intersection.
Detailed description of the invention
Fig. 1 is the calibration curve figure of kit fitting of the present invention.
Specific embodiment
More clear explanation is done to the present invention below by specific embodiment, in order to the reason of those skilled in the art
Solution.Unless otherwise specified, reagent used in the embodiment of the present invention and detecting instrument are commercial product, and method therefor is ability
Domain conventional method.
The kit (Magnetism particulate immuno chemistry luminescence method) of the preparation measurement laminin of embodiment 1
1, magnetic particle suspension is prepared
The carboxylated magnetic particle stoste that partial size is 1nm is mixed well, appropriate progress Magneto separate is taken out, removes supernatant, be added
The acetate buffer solution of 0.05M, PH5.5 carry out resuspension cleaning, are repeated 2 times, and crosslinking agent EDC 10mg is added to magnetic particle surface carboxylic
Base is activated, and native purified or recombination laminin antigen 3uL is added and is coupled, room temperature concussion reaction 2h, magnetic point
From removal supernatant, with the 0.05M for being added to 2% isinglass and 1% ANS, the phosphate buffer of PH7.0 is protected liquid as envelope and carried out
2-8 DEG C of preservation is placed in closing.
2, enzyme conjugates is prepared
The BSA and 1% ANS that 0.5% is added in 0.02M, the phosphate of PH7.0 are configured to dilution 1L, add horseradish peroxidating
The laminin monoclonal antibody 5uL of object enzyme label, is uniformly mixed, and places 2-8 DEG C of preservation.
3, calibration object is prepared
The BSA that 0.5% is added in 0.02M, the phosphate of PH7.0 is configured to dilution 1L, is divided into 5 equal portions, and addition is natural respectively
Purifying or recombination III procollagen type N-terminal peptide antigen be configured to 50ng/mL, 100ng/mL, 200ng/mL, 400ng/mL,
The series of concentrations of 800ng/mL places 2-8 DEG C of preservation.
The application method of the kit of the present invention of embodiment 2
Coagulate pipe using no added common tube/separation sebific duct/rush and acquire whole blood 5mL, in 37 DEG C of standings 30min, afterwards with 4000 turns/
The revolving speed of min is centrifuged 10min, and the serum after taking centrifugation is as detection sample.To may not exceed 8 small being placed at room temperature for after sample collection
When, if sample need to be placed in 2 ~ 8 DEG C of refrigerator by detecting not in 8 hours, if need 48 hours or more save or transport,
It should freeze in -20 DEG C hereinafter, avoiding multigelation.It is restored to room temperature before use, gently shakes mixing.
Testing procedure
1) calibration object (for calibrating) or sample are separately added into reaction vessel (hereinafter referred to as " hole "), sample-adding amount is 50 μ
The hole l/.
2) every hole is separately added into 20 μ l of magnetic particle suspension.
3) 50 μ l of enzyme conjugates is added in every hole.
4) it is incubated 34 minutes for 37 DEG C after mixing.
5) cleaning solution washs 5 times.
6) each 50 μ l of luminous substrate A and luminous substrate B is added in every hole.
7) 1 ~ 5 minute detection luminous intensity after mixing.
8) result calculates:
Four parameters (lin-log) fit approach is used first, using calibration object concentration value as X-axis, with calibration object luminous intensity values
Log value is that Y-axis establishes calibration curve, and the curve of fitting is as shown in Figure 1.
Corresponding concentration value is back-calculated according to the luminous intensity values of sample to be tested.Instrumental operating system can pass through the calibration of storage
The signal value that curve and test sample obtain calculates sample test result automatically.
The performance detection of the kit of the present invention of embodiment 3
1, sensitivity for analysis
Product are controlled as sensitivity with 0 value calibration product, 10 hole measurements is carried out, calculates the average (M) and standard deviation of its luminous value
(SD), the concentration value of M+2SD is back-calculated according to dose-response curve, acquired results are sensitivity for analysis.Testing result see the table below
1。
Table 1
As can be seen from Table 1, the sensitivity for analysis of this kit is far below 5ng/mL.
2, accuracy
High and low two kinds of Internal Quality Control product (Q1, Q2) are detected respectively with the same lot number kit, it is desirable that replication 4 days, daily
At least 5 repetitions calculate measurement result according to four parameters (lin-log) fit standard curve, then calculate at least 20 times measurements knot
The average value M and standard deviation SD of fruit, obtain coefficient of variation CV according to formula CV=SD/M*100%.Testing result see the table below 2.
Table 2
As can be seen from Table 2: accuracy is within 10% between the day of this kit.
3, specific
With calibration object dilution, compound concentration is 1000ng/mL hyaluronic acid (HA), III procollagen type N-terminal peptide of 100ng/mL respectively
(III NP of P), 1000ng/mL type Ⅳ collagen (C IV) is as specific quality-control product.With a batch kit, multiple holes measurement is specifically
Property quality-control product, by luminous value substitution dose-response curve be back-calculated concentration value, calculate the average value of concentration value.Testing result is shown in Table
3。
Table 3
As can be seen from Table 3: this kit and the hyaluronic acid (HA) of homologous series, III procollagen type N-terminal peptide (III NP of P),
Type Ⅳ collagen (Col IV) is without intersection.
4, new and old differences between samples
5 samples according to normal process acquire after respectively acquire complete when, 2-8 DEG C storage 2 days, 4 days, 7 days when use respectively
Kit and conventional kit of the present invention are detected, and compare two kinds of LN Concentration Testing results with the variation of resting period.
Kit detection LN concentration results (ng/mL) of the present invention is shown in Table 4 with the variation of resting period.
Table 4
Conventional kit detects LN concentration results (ng/mL) and is shown in Table 5 with the variation of resting period.
Table 5
The data from table 4, table 5 are not as it can be seen that using conventional kit (adding releasing agent), and as time increases, detection LN is dense
Result is spent in lasting rising, highest point was raised to by 7 days, and uses kit of the present invention (being added to releasing agent), and just acquisition is completed
Pattern detection result and house 7 days pattern detection result it is almost the same, subsequent result will not be with the storage of sample
Time increases and occurs significantly changing.Therefore the releasing agent added in kit of the present invention be it is effective, LN can be made
Detection the more conducively clinical application as a result, substantially reduce detection time can be detected correctly at the beginning of sample collection.
Note: full-automatic magnetic microparticle chemiluminescence instrument (AutoLumo A2000Plus) use herein is by Zhengzhou Antu
Bioengineering limited liability company provides.
Claims (3)
1. a kind of kit using Chemiluminescence Immunoassay measurement laminin, including combine laminin Dan Ke
The magnetic particle suspension of grand antibody；The laminin monoclonal antibody of horseradish peroxidase-labeled；Laminin series
Calibration object, it is characterised in that: releasing agent is added in the magnetic particle suspension and enzyme conjugates.
2. the kit according to claim 1 using Chemiluminescence Immunoassay measurement laminin, feature exist
In: the releasing agent is citric acid, gluconic acid, triethanolamine, 8- aniline -1-naphthalene sulfonic aicd, ethylenediamine tetra-acetic acid, ethylene glycol pair
One of (2- amino-ethyl ether) tetraacethyl, polyethylene glycol, polyvinylpyrrolidone.
3. the kit according to claim 1 using Chemiluminescence Immunoassay measurement laminin, feature exist
In: the partial size of the magnetic particle is nanoscale.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811521732.XA CN109444429A (en) | 2018-12-13 | 2018-12-13 | Utilize the kit of Chemiluminescence Immunoassay measurement laminin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811521732.XA CN109444429A (en) | 2018-12-13 | 2018-12-13 | Utilize the kit of Chemiluminescence Immunoassay measurement laminin |
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| CN109444429A true CN109444429A (en) | 2019-03-08 |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103364568A (en) * | 2013-07-18 | 2013-10-23 | 博奥赛斯（天津）生物科技有限公司 | Laminin nano-magnetic particle chemiluminescent immunity quantitative detection kit and preparation method thereof |
| CN105973878A (en) * | 2016-05-17 | 2016-09-28 | 北京豪迈生物工程有限公司 | Laminin (LN) testing kit and testing method thereof |
| CN106018830A (en) * | 2016-06-30 | 2016-10-12 | 深圳市亚辉龙生物科技股份有限公司 | Laminin chemiluminescence immunoassay kit and preparation method thereof |
| CN108490166A (en) * | 2018-02-28 | 2018-09-04 | 广州市丰华生物工程有限公司 | A kind of improved experimental buffer solution and its application |
| CN108603882A (en) * | 2015-09-28 | 2018-09-28 | 雅培日本有限公司 | Laminin 2 is used for the purposes of diagnosing hepatocellular carcinoma and cancer of pancreas |
-
2018
- 2018-12-13 CN CN201811521732.XA patent/CN109444429A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103364568A (en) * | 2013-07-18 | 2013-10-23 | 博奥赛斯（天津）生物科技有限公司 | Laminin nano-magnetic particle chemiluminescent immunity quantitative detection kit and preparation method thereof |
| CN108603882A (en) * | 2015-09-28 | 2018-09-28 | 雅培日本有限公司 | Laminin 2 is used for the purposes of diagnosing hepatocellular carcinoma and cancer of pancreas |
| CN105973878A (en) * | 2016-05-17 | 2016-09-28 | 北京豪迈生物工程有限公司 | Laminin (LN) testing kit and testing method thereof |
| CN106018830A (en) * | 2016-06-30 | 2016-10-12 | 深圳市亚辉龙生物科技股份有限公司 | Laminin chemiluminescence immunoassay kit and preparation method thereof |
| CN108490166A (en) * | 2018-02-28 | 2018-09-04 | 广州市丰华生物工程有限公司 | A kind of improved experimental buffer solution and its application |
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