CN109030817A - HMGB1 detection kit and preparation method thereof - Google Patents
HMGB1 detection kit and preparation method thereof Download PDFInfo
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- CN109030817A CN109030817A CN201810721943.1A CN201810721943A CN109030817A CN 109030817 A CN109030817 A CN 109030817A CN 201810721943 A CN201810721943 A CN 201810721943A CN 109030817 A CN109030817 A CN 109030817A
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Classifications
-
- 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
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
-
- 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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- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The present invention relates to a kind of HMGB1 detection kits and preparation method thereof, wherein HMGB1 detection kit includes: reagent 1 and reagent 2；The reagent 1 includes the latex microsphere of HMGB1 antibody label, suspending agent, bovine serum albumin(BSA), the first buffer；The reagent 2 includes sodium chloride, dispersing agent, surfactant, the second buffer.The HMGB1 detection kit is a kind of kit based on latex enhancing immune turbidimetry detection HMGB1, by carrying out antigen, antibody response in homogeneous reaction system when use, after antigen is in conjunction with antibody, measure the absorbance value of reaction solution directly to react the concentration of tested antigen, it is fast, at low cost, time saving and energy saving to detect speed, and stability and reproducible, it can more accurately reflect the content of measured matter.
Description
Technical field
The present invention relates to technical field of medical detection, more particularly to a kind of HMGB1 detection kit and preparation method thereof.
Background technique
High mobility group box-1 (high mobility group protein, HMGB1) is to be present in eukaryocyte core
Interior nonhistone chromosomal binding protein, because its in polyacrylamine gel electrophoresis (PAGE) migration velocity it is fast due to gain the name.
HMGB1 is prevalent in mammalian tissue cell, is the inflammatory factor being of great significance.HMGB1 is as late phase inflammation
The factor is expressed in the long period relative to the Earlier period of inflammation factor and is stablized, can preferably there is a situation where right for inflammation in reactant
The determination of clinical treatment has more importantly meaning.
A large number of studies show that the inflammation as caused by bacterium infection (such as endotoxemia and pyemia, it is especially acute
Inflammation) in, the Earlier period of inflammation factor (such as TNF) stimulates relevant cell (such as macrophage), starts to secrete HMGB1, certain
(such as 18h) reaches peak value after time.For acute inflammation after treatment, the Earlier period of inflammation factor is returned to normal level, if treatment
Ineffective, the late phase inflammation factor (HMGB1) will generate.The late phase inflammation factor is monitored at any time, judges therapeutic effect, and guidance is used
Medicine takes measures early, and it is particularly important to the injury of body to reduce inflammation.
HMGB1 detection is used widely, but existing method is uncomfortable since the operating time is long or sensitivity problem at present
In the development of Clinical laboratory test.Currently, on the market HMGB1 detection mainly using ELISA method (enzyme linked immunosorbent assay (ELISA),
Enzyme linked immunosorbent assay), it is cumbersome, time-consuming.Moreover, domestic HMGB1ELISA detection examination
Agent box quality is irregular, and import reagent price general charged is expensive.Therefore, it is at low cost to need one kind, detection speed is fast, is suitble to face
The technology of bed pattern detection project occurs.
Summary of the invention
Based on this, it is necessary to provide a kind of HMGB1 detection kit that detection speed is fast, at low cost.
A kind of HMGB1 detection kit, comprising: reagent 1 and reagent 2；
The reagent 1 includes latex microsphere, suspending agent, bovine serum albumin(BSA) and the first buffer of HMGB1 antibody label；
The reagent 2 includes sodium chloride, dispersing agent, surfactant and the second buffer.
When being detected using above-mentioned HMGB1 detection kit, takes reagent 1 and reagent 2 to mix, be then added to test sample
Product by carrying out antigen, antibody response in homogeneous reaction system, and measure the absorbance value of reaction solution to react tested
The concentration of HMGB1.It is by the surface-crosslinked HMGB1 antibody of latex microsphere, when antigen and the latex that is crosslinked with HMGB1 antibody are micro-
After chou closes, it can flock together rapidly in a short time, thus change the absorbance of reaction solution, it is anti-by directly measuring
The absorbance value of solution is answered to react the concentration of tested antigen, compared to ELISA method, ELISA method is eliminated and is incubated for and washes repeatedly
The troublesome operations step such as plate, a few minutes can obtain as a result, time saving and energy saving；Also, the simplification of operating procedure also correspondingly avoids
The interference of the extraneous factors such as cumbersome manual operation factor and reagent, environment, stability and repeatability are all preferable, can be relatively true
The content of ground reflection measured matter.
Moreover, by the way that HMGB1 antibody label on latex particle, is being guaranteed condition of the antibody in conjunction with antigentic specificity
Under, also improve the sensibility of reagent detection.
The reagent 1 includes the component of following final concentration: 60 μ of μ g/mL~120 g/mL in one of the embodiments,
HMGB1 antibody, 0.1v/v%~0.15v/v% latex microsphere, 30g/L~60g/L suspending agent, 5g/L~20g/L ox blood are pure
The first buffer of albumen and 40mM/mL~60mM/mL, the pH value of the reagent 1 are 7~8.
The latex microsphere is the polystyrene latex microspheres of surface active, the polyphenyl in one of the embodiments,
The surface active group of ethylene latex microballoon is carboxyl, and the particle diameter of the latex microsphere is 20nm~50nm.
Using the nano pipe/polyhenylethylene microballoon of carboxyl modified, is combined, improved by way of covalent bond with HMGB1 antibody
While with antibody coupling efficiency, reduction antibody usage amount, suitable 3D structure also is provided for HMGB1 antibody and antigen carries out
In conjunction with, and then the sensibility and specificity of detection can be promoted.And the polystyrene microsphere of 20nm~50nm has Gao Bibiao
Area can accelerate the time and the adsorption equilibrium stability that reach adsorption equilibrium when detection, reaction speed and result can be improved
Stability.
In one of the embodiments, the suspending agent in trehalose, glucose, lactose and sucrose at least one
Kind.
More preferably, the suspending agent is trehalose.Trehalose is added in reagent 1, can not only promote HMGB1 antibody mark
The latex microsphere of note suspends, and also easily prevents protein denaturation and removes OH free radical caused by α and β ray, improves kit
Stability.
First buffer is selected from PB (phosphate) buffer, MES (2- morpholine second sulphur in one of the embodiments,
Acid) buffer, Tris-HCl (orthoformic acid hydrochloric acid) at least one of buffer and glycine buffer.
Further, the pH value of first buffer is 7~8.In this way, can by the pH value of first buffer compared with
It is easy to get to the pH value of the reagent 1.
More preferably, first buffer is PB buffer.
The reagent 2 includes the component of following final concentration: 5g/L~9g/L sodium chloride, 5g/ in one of the embodiments,
The second buffer of L~10g/L dispersing agent, 0.2g/L~0.8g/L surfactant and 40mM/mL~60mM/mL, the reagent 2
PH value be 7~8.
In one of the embodiments, the surfactant in the reagent 2 be selected from Tween-20, Tween-80,
At least one of TritonX-100 (Triton X-100), span 40 and sorbester p18.
Second buffer is selected from PB buffer, MES buffer, Tris-HCl buffering in one of the embodiments,
At least one of liquid and glycine buffer.
Further, the pH value of second buffer is 7~8.
The dispersing agent is selected from polyethylene glycols or stearates dispersing agent in one of the embodiments,.
More preferably, the dispersing agent is Macrogol 6000.
The dosage volume ratio of the reagent 1 and the reagent 2 is 1:(1~3 in one of the embodiments).
More preferably, the dosage volume ratio of the reagent 1 and the reagent 2 is 1:3.It should be understood that using above-mentioned HMGB1
When detection kit is detected, the dosage volume ratio of the reagent 1 and the reagent 2 is 1:3.
Another object of the present invention is to provide a kind of preparation method of above-mentioned HMGB1 detection kit, including the reagent 1
Preparation step and the reagent 2 preparation step；
Wherein, the reagent 1 preparation step the following steps are included:
The latex that the suspending agent, the bovine serum albumin(BSA), first buffer and the HMGB1 antibody are marked
Microballoon mixing, and ultrasound suspending, obtain ultrasound suspending liquid；
The ultrasonic suspension is subjected to ultraviolet sterilization, sterile packaged, obtains the reagent 1；
The preparation step of the reagent 2: each component of the reagent 2 is uniformly mixed, and obtains the reagent 2.
Specifically, the suspending agent, the bovine serum albumin(BSA) and second buffer are uniformly mixed, are suspended
Liquid；Then the latex microsphere ultrasound suspending of HMGB1 antibody label is obtained into ultrasound suspending liquid in suspension.
Above-mentioned preparation method, ultrasound suspending is while the latex microsphere that dispersion, suspension HMGB1 antibody mark, to liquid
Good sterilization functions are played in inside, and ultraviolet sterilization can play good sterilization effect to liquid surface and air, by super
Sound and ultraviolet sterilization, which combine, can play good sterilization effect, so as to avoid in kit be added as sodium azide,
The preservatives such as phenol, P-hydroxybenzoic acid, ethyl-para-hydroxybenzoate avoid such preservative from endangering human body and environment bring
Evil.Moreover, such preservative Nature comparison is active, although antisepsis can be played, to the long-time stability of latex system
It will cause and seriously affect.
Ultrasound suspending acts on the latex system stepless action simultaneously to reagent 1 by ultrasonic wave homogeneous, keeps suspended particulate fast
Speed reaches micron order or hereinafter, avoids because using other dispersing methods to need the time is longer, generates biggish fuel factor to make HMGB1
The structure of antibody is affected.
The preparation step of the reagent 1 further includes that the latex of the HMGB1 antibody label is micro- in one of the embodiments,
The preparation of ball, includes the following steps:
It takes latex microsphere to be added in buffer, surfactant and activator is then added, activates the latex microsphere,
Mixed liquor A is made；The activator is carboxyl activator or amino group activating reagents；
HMGB1 antibody is added into the mixed liquor A, carries out coupling label reaction, the glue of the label of antibody containing HMGB1 is made
The solution B of newborn microballoon；
It is added confining liquid into the solution B, after the completion of capping, is centrifuged, gained precipitating is the HMGB1
The latex microsphere of antibody label.
The latex microsphere is that the polystyrene latex that particle diameter is 20nm~50nm is micro- in one of the embodiments,
Ball.
One embodiment wherein, the volumetric concentration of latex microsphere is 0.1%~0.15% in the mixed liquor A.
The surfactant in the preparation method of the reagent 1 is selected from TritonX- in one of the embodiments,
100, at least one of Tween-20 and methyl sodiosul foaliphatate.
More preferably, the surfactant is methyl sodiosul foaliphatate.Selected fatty acid methyl esters when activating latex group
Sulfonate surfactants can greatly reduce the interference to latex labeling process, improve labeling effciency.
The activator is N-HS (n-hydroxysuccinimide) solution and/or EDC (1- in one of the embodiments,
Ethyl -3 (3- dimethyl aminopropyl)-carbodiimides) solution.It is activated by N-HS, EDC, so that latex microsphere surface is formed
Carboxy activating group.
Further, when activating the latex microsphere, the surfactant, N-HS and EDC are successively dripped, in 37 DEG C
React 0.5h~1.5h.
Buffer used in the preparation of the latex microsphere of the HMGB1 antibody label is in one of the embodiments,
MES buffer.
The present invention also provides a kind of sides using the content of HMGB1 in above-mentioned HMGB1 detection kit detection sample to be tested
Method.
Specifically, it is by volume 1:(1~3) by after the reagent 1 and the mixing of the reagent 2, it is added described to test sample
Product measure absorbance after mixing reaction, obtain the content of HMGB1 in the sample to be tested.
Mentioned reagent box of the present invention has the advantage that
1) at low cost, it is easy to operate, the content of HMGB1, high sensitivity can be quickly detected using mentioned reagent box.
2) by the optimal setting matched to reagent, it is effectively reduced the usage amount of HMGB1 antibody, makes HMGB1 antibody
Usage amount only in 100 μ g/mL or so, can save reagent cost.
3) volume final concentration of 0.1%~0.15% for controlling latex microsphere in reagent 1, can reduce the background that reagent uses
Level improves testing result precision.
4) using the microsphere particles raw material of Nanoscale Surface modification, it is anti-to improve HMGB1 by particle diameter 20nm-50nm
Body marks the detection sensitivity of latex particle Contrast agent box, and detection sensitivity is made to reach 1.0ng/mL.
5) ultrasound suspending and ultraviolet sterilization combine, it is ensured that kit reaches germ-free condition, avoids making for preservative
With and its harm of the bring to human body and environment.
Detailed description of the invention
Fig. 1 is that the latex of the embodiment of the present invention 3 reacts linear relationship chart；
Fig. 2 is the latex reaction normal curve graph of the embodiment of the present invention 10.
Specific embodiment
To facilitate the understanding of the present invention, below will to invention is more fully described, and give it is of the invention compared with
Good embodiment.But the invention can be realized in many different forms, however it is not limited to embodiment described herein.Phase
Instead, purpose of providing these embodiments is makes the disclosure of the present invention more thorough and comprehensive.
Unless otherwise defined, all technical and scientific terms used herein and belong to technical field of the invention
The normally understood meaning of technical staff is identical.Term as used herein in the specification of the present invention is intended merely to description tool
The purpose of the embodiment of body, it is not intended that in the limitation present invention.Term as used herein "and/or" includes one or more phases
Any and all combinations of the listed item of pass.
Embodiment 1 prepares HMGB1 antigen
Construction recombination plasmid screens positive recombinant plasmid, cultivates the thin of high efficient expression His-SBP-HMGB1 fusion protein
Bacterium.Under conditions of 6000 × g, 4 DEG C, it is centrifuged bacteria-containing culture solution 30min, collect bacterium and is saved backup in -20 DEG C；It takes suitable
Amount thallus is resuspended in brokenly bacterium buffer (Tris-HCL containing 50mmol/L, 250mmol/L NaCl and 10mmol/L imidazoles, pH value
In 8.0), ice-bath ultrasonic, it is spare to collect supernatant by 10000 × g, 4 DEG C of centrifugation 20min；Parent is carried out with peristaltic pump loading nickel column
And chromatography, first with broken bacterium buffer solution for cleaning non-specific binding albumen, then with elution buffer (Tris-HCl containing 50mmol/L,
250mmol/L NaCl and 300mmol/L imidazoles, pH value 8.0) elution, and collect destination protein --- HMGB1, as
HMGB1 antigen is spare.The protein concentration collected with ultraviolet specrophotometer test, identifies that its is pure with 12% agarose gel electrophoresis
Degree.
Embodiment 2 prepares HMGB1 polyclonal antibody
By the HMGB1 antigen concentration of normal saline dilution to 0.5mg/ml~2mg/ml, with Freund's adjuvant in 1:1 ratio
It mixes.By mixture to multi-point injection and hind leg muscle injection is carried out under rabbit skin, injection dosage is 0.5mg~1mg for the first time.Often
1~2 week acquisition serum carries out serum titer measurement by agar gel diffusion test, and such as not up to expected potency carries out reinforcing exempting from
Epidemic disease, adding injection dosage is 1/2 injected for the first time, is generally needed booster immunization 2~4 times.After serum titer reaches demand, to rabbit
Serum collection is carried out, the serum of collection is saved backup in -20 DEG C.
100mgHMGB1 is taken to be added to the active agarose medium of 3g cyanogen bromide (CNBr-activated Sepharose
In 4B), it is prepared into volume about 10mlHMGB1 affinity column under alkaline condition, it is spare.It is anti-using affine column purification HMGB1
Body serum obtains HMGB1 polyclonal antibody.
Embodiment 3 marks latex particle
A, prepare latex solution
The latex solution of the final concentration of 0.11v/v% of 1mL is prepared with latex mother liquor (5v/v%, 20nm~50nm partial size)
(+978 μ L50mM MES buffer of 22 μ L latex mother liquor), pH value 6.0.
B, latex group is activated
Three kinds of solution are sequentially added into 1mL latex solution: 4.5 μ L surfactants are (with 50mM MES, pH value 6.0
Solution or pure water be configured to 5% mother liquor)；4.5 μ L N-HS solution (concentration 10mg/mL)；(concentration is 4.5 μ L EDC solutions
10mg/ml).After solution is added, latex solution is statically placed in 37 DEG C of reaction 1h.
C, antibody coupling marks
100 μ g HMGB1 antibody (the 5mg/mL antibody mother liquors of 20 μ L) are added, are placed in 37 DEG C of reaction 2h, and be slowly stirred.
D, latex group is closed
100 μ L confining liquids (1M Tris-HCl, pH value 8.0) are added, are statically placed in 37 DEG C of reaction 30min.
E, latex precipitating is resuspended
After capping, 6000 × g be centrifuged 5min~10min (it should be noted that as far as possible with minimum centrifugal force and
Shortest time centrifugation latex, not muddy for centrifugation terminal to be centrifuged supernatant)；Centrifugation (contains 50g/L with 1mL suspension
Trehalose, 10g/L BSA, 50mM PB, pH value 7.4) and ultrasound suspending (with small probe ice-bath ultrasonic, stop 5s, ultrasonic 10s, it is heavy
4min~5min is handled again).
F, preservative free detection kit is prepared
Ultraviolet irradiation ultrasound suspending liquid kills the bacterium around liquid surface and air, sterile packaged.
G, test agent performance
Different time sections take 250 μ L latex re-suspension liquids as detection reagent 1, be added 750 μ L detection liquid (NaCl containing 9g/L,
10g/L PEG 6000,0.5g/L Tween-20,50mM PB, pH value 7.4) it is used as detection reagent 2, in 546nm Detection wavelength
Under, taking 10 μ l concentration is the HMGB1 sample of 500ng/ml, with the suction in the visible ultraviolet spectrophotometer measurement 5min reaction time
Light varience.
As a result as shown in Figure 1, it will be seen from figure 1 that background of the emulsion reagent in reaction is 0.5 or so, when HMGB1 is dense
When degree is 500ng/ml, absorbance difference is 1 after reacting 5min, can reach the desired effect of detection.
4 HMGB1 detection kit of embodiment
Reagent 1: composed of the following components: polystyrene latex microspheres, trehalose, the ox blood of HMGB1 antibody label are pure
Albumen (BSA), phosphate；Each substance is final concentration of: 0.11v/v% polystyrene latex microspheres (particle diameter be 20nm~
50nm), 100 μ g/mL HMGB1 antibody, 50g/L trehalose, 10g/L BSA, 50mM PB, pH value 7.4.
The specific process for preparation of reagent 1 is as follows:
(1) with 5v/v% polystyrene latex mother liquor (partial size of polystyrene is 20nm~50nm) preparation 1mL final concentration
For the latex solution (+980 μ l 50mM MES solution of 20 μ l latex mother liquor, pH value 6.0) of 0.11v/v%.
(2) three kinds of solution: 4.5 μ L surfactant (5% fatty acid methyl ester sulfonic acid are sequentially added into 1mL latex solution
Sodium), 4.5 μ L N-HS solution (10mg/mL), 4.5 μ L EDC solutions (10mg/mL).After solution is added, latex solution is quiet
It is placed in 37 DEG C of reaction 1h.
(3) 100 μ g HMGB1 antibody (the 5mg/mL antibody mother liquors of 20 μ L) are added, are placed in 37 DEG C of reaction 2h, and slowly stir
It mixes.
(4) 100 μ L confining liquids (1M Tris-HCl, pH value 8.0) are added, are statically placed in 37 DEG C of reaction 30min.
(5) after capping, 6000 × g is centrifuged 5min~10min；Centrifugation (contains 50g/L with 1mL suspension
Trehalose, 10g/L BSA, 50mM PB, pH value 7.4) after ultrasound suspending, ultraviolet sterilization is to get reagent 1.
Contain in reagent 2: sodium chloride, Macrogol 6000, Tween-20, phosphate, each substance are final concentration of: 9g/L
NaCl, 10g/L PEG 6000,0.5g/L Tween-20,50mM PB, pH value 7.4.
The selection of 5 latex particle of embodiment optimizes with latex concentration
Compare two kinds of polystyrene microsphere label effects of diameter 20nm~50nm and 50nm~100nm, and it is dense to configure volume
It spends and is marked for 0.05%, 0.1%, 0.2%, 0.3% 4 kind of various concentration, observe the variation of latex state in labeling process
With final label result.
Experiments have shown that the effect that 0.1% latex concentration and diameter are 20nm~50nm is preferable, latex state in labeling process
Stablize.
6 HMGB1 antibody labelled amount of embodiment and reaction condition optimization
According to 40 μm of ol/g~100 μm ol/g of microsphere surface group content, calculate amount of antibody about need 150 μ g/mL~
300 μ g/mL are (according to antibody molecule amount 150kd, latex concentration 0.1%, 100 μm of ol/g of group content, group activation mark rate
1%~2% calculates).It is carried out respectively according to the HMGB1 antibody concentration of 100 μ g/mL, 150 μ g/mL, 200 μ g/mL, 300 μ g/mL
Mark test.
The results show that latex system becomes very unstable when antibody concentration is greater than 150 μ g/mL.Optimize reaction condition,
37 DEG C of constant temperature are reacted and are constantly slowly shaken up, and antibody concentration is that the 100 μ g/mL effects of μ g/mL~150 are preferable.To avoid reagent unrestrained
Take, preferably 100 μ g/mL antibody usage amounts.
The centrifugation of 7 latex of embodiment optimizes with floating condition
The present embodiment is to implement step (5) latex centrifugation in 4 reagent, 1 process for preparation to optimize with floating condition.It is preferred that 20nm
~50nm ps particle, volumetric concentration 0.1%, 100 μ g/mL of amount of antibody are marked, are centrifuged after closing.Respectively
Label latex particle is collected by centrifugation with 3000 × g, 6000 × g, 9000 × g, 12000 × g.
Label latex cannot be centrifuged completely by the low centrifugal force of the results show (such as 3000 × g) in 5min~10min
Precipitating, high centrifugal force keep latex precipitating hardened, and subsequent suspension effect is bad, selects 6000 × g centrifugal effect preferable.In suspension
The preferred 50g/L trehalose of ingredient, 10g/L BSA and 50mM PB effect are preferable.
8 reagent of embodiment, 2 ratio optimization
The present embodiment mainly investigates influence of the PEG6000 concentration to reaction effect.Respectively prepare 5g/L, 10g/L, 15g/L,
The reagent 2 of the PEG6000 of 20g/L, 1 proportion optimizing of preferred reagent, and the HMGB1 antigenic solution of debita spissitudo is prepared, compare
Reaction effect in 5min.
The results show that UV absorption variation is small when PEG6000 concentration is lower；When PEG6000 concentration is higher, cause to react
System is unstable.PEG6000 concentration is that 10g/L is more preferably to select.
Additional proportion determines in the detection for 9 reagent 1 of embodiment and reagent 2
Influence of the reagent dosage to reaction system stability is investigated in the present embodiment.In 1mL reaction system, investigate
500 μ L reagent 1+500 μ L reagents 2 (1:1), 333 μ L reagent 1+667 μ L reagents 2 (1:2), 250 μ L reagent 1+750 μ L reagents 2
(1:3) in the case of these three, reaction system is stablized and reacts background problem.
Test result is as shown in table 1 below, shows to cause reaction system background to rise when 1 additional amount of reagent increases, right
Testing result causes a deviation.Preferably 250 μ L reagent 1+750 μ L reagents 2 (1:3) in three kinds of schemes.
Influence of the 1 reagent additional proportion of table to absorbance
| Serial number | Reagent 1: reagent 2 (volume ratio) | Initial absorbance | Absorbance after 5min | Absorbance difference |
| 1 | 1:3 | 0.523 | 0.534 | 0.011 |
| 2 | 1:2 | 0.702 | 0.852 | 0.15 |
| 3 | 1:1 | 1.197 | 1.547 | 0.35 |
The foundation of 10 standard curve of embodiment and the determination of the range of linearity
Determination condition: full automatic biochemical apparatus, 250 μ L reagent, 1,750 μ L reagent 2, Detection wavelength 546nm.
Measuring method (Two point end assay): the reagent 1 and 2,250 μ L reagent 1 of reagent of Example 4 are added to 750 μ L examination
In agent 2,10 μ L samples are then added, start read point measurement absorbance A 1 after reacting 5s, read point measures extinction to 5min again later
A2 is spent, absorbance difference Δ A=A2-A1 is calculated.
Make standard curve: using the HMGB1 standard items of the application, concentration is respectively S6:300ng/mL, S5:180ng/
ML, S4:90ng/mL, S3:35ng/mL, S2:10ng/mL, S1:0ng/mL.Difference DELTA A is measured according to above-mentioned steps, draws mark
Directrix curve, as shown in Fig. 2, wherein x-axis represents the concentration of HMGB1, y-axis represents the difference of absorbance.Calculating regression equation is y
=0.0015x+0.0939, coefficient R2It is 0.9996.Kit of the present invention correlation in the 0-300ng/mL range of linearity
Preferably.
The detection of 11 actual sample of embodiment
6 human plasma samples are taken, measure its content roughly with commercially available HMGB1.Each sample is bisected into two parts, and thereto one
HMGB1 standard specimen amount is separately added by table 1 under in part.With the sample and mark-on to 12 parts of non-mark-on samples of the kit of embodiment 4
The sample replication of sample 3 times, measuring method are as follows: take 250 μ L reagents 1 to be added in 750 μ L reagents 2,10 μ L samples are then added
This, after reacting 5s, measures absorbance, the amount of HMGB1 in sample is calculated, to results are averaged.A acquired results of mark-on
Non- mark-on portion acquired results are subtracted, difference calculates sample recovery of standard addition, knot compared with the theoretical value that standard substance is added
Fruit such as the following table 1.Average recovery rate is 99.6% as can be seen from Table 1, illustrates the accuracy that kit detects in actual sample
It is very high.
The rate of recovery after 2 HMGB1 mark-on sample of table
| Number | Sample size (ng) | Standard specimen amount (ng) | Measured amount (ng) | The rate of recovery (%) |
| 1 | 113.6 | 20 | 20.4 | 102.0 |
| 2 | 85 | 20 | 19.8 | 99.1 |
| 3 | 127.4 | 20 | 19.8 | 99.5 |
| 4 | 194.6 | 40 | 39.4 | 98.5 |
| 5 | 198.2 | 40 | 39.6 | 99.0 |
| 6 | 205.2 | 40 | 39.8 | 99.5 |
The measurement of 12 stability of embodiment
The reagent 1 of embodiment 4 and reagent 2 are placed in 4 DEG C of refrigerators to save, continuously observed every other day its state one month, is sent out
Now it is visible by naked eyes variation.It was detected by 10 the method for embodiment every 7 days, reactivity is stablized.Illustrate implementation of the present invention
The stabilization of kit of example is good.
Each technical characteristic of embodiment described above can be combined arbitrarily, for simplicity of description, not to above-mentioned reality
It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, all should be considered as described in this specification.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art
It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection of the invention
Range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Claims (10)
1. a kind of HMGB1 detection kit, which is characterized in that including reagent 1 and reagent 2；The reagent 1 includes HMGB1 antibody
Latex microsphere, suspending agent, bovine serum albumin(BSA) and the first buffer of label；
The reagent 2 includes sodium chloride, dispersing agent, surfactant and the second buffer.
2. HMGB1 detection kit according to claim 1, which is characterized in that the reagent 1 includes following final concentration
Component: the 60 μ g/mL HMGB1 of μ g/mL~120 antibody, 0.1v/v%~0.15v/v% latex microsphere, 30g/L~60g/L suspending
The first buffer of agent, 5g/L~20g/L bovine serum albumin(BSA) and 40mM/mL~60mM/mL, the pH value of the reagent 1 are 7~8.
3. HMGB1 detection kit according to claim 1, which is characterized in that the latex microsphere is surface active
Polystyrene latex microspheres, the surface active group of the polystyrene latex microspheres are carboxyl, the particle of the latex microsphere
Diameter is 20nm~50nm.
4. HMGB1 detection kit according to claim 1, which is characterized in that the suspending agent is selected from trehalose, grape
At least one of sugar, lactose and sucrose.
5. HMGB1 detection kit according to claim 1, which is characterized in that first buffer is buffered selected from PB
At least one of liquid, MES buffer, Tris-HCl buffer and glycine buffer.
6. any HMGB1 detection kit according to claim 1~5, which is characterized in that the reagent 2 includes following
The component of final concentration: 5g/L~9g/L sodium chloride, 5g/L~10g/L dispersing agent, 0.2g/L~0.8g/L surfactant and
The second buffer of 40mM/mL~60mM/mL, the pH value of the reagent 2 are 7~8.
7. HMGB1 detection kit according to claim 6, which is characterized in that the use of the reagent 1 and the reagent 2
Amount volume ratio is 1:(1~3).
8. the preparation method of any HMGB1 detection kit of claim 1~7, which is characterized in that including the reagent
The preparation step of 1 preparation step and the reagent 2；
Wherein, the reagent 1 preparation step the following steps are included:
The latex microsphere that the suspending agent, the bovine serum albumin(BSA), first buffer and the HMGB1 antibody are marked
Mixing, and ultrasound suspending, obtain ultrasound suspending liquid；
The ultrasound suspending liquid is subjected to ultraviolet sterilization, sterile packaged, obtains the reagent 1；
The preparation step of the reagent 2:
The each component of the reagent 2 is uniformly mixed, the reagent 2 is obtained.
9. preparation method according to claim 8, which is characterized in that the preparation step of the reagent 1 further includes described
The preparation of the latex microsphere of HMGB1 antibody label, includes the following steps:
It takes latex microsphere to be added in buffer, surfactant and activator is then added, activates the latex microsphere, is made
Mixed liquor A；The activator is carboxyl activator or amino group activating reagents；
HMGB1 antibody is added into the mixed liquor A, carries out coupling label reaction, the latex that the label of antibody containing HMGB1 is made is micro-
The solution B of ball；
It is added confining liquid into the solution B, after the completion of capping, is centrifuged, gained precipitating is the HMGB1 antibody
The latex microsphere of label.
10. preparation method according to claim 9, which is characterized in that the surface in the preparation method of the reagent 1
Activating agent is selected from least one of TritonX-100, Tween-20 and methyl sodiosul foaliphatate.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012017466A1 (en) * | 2010-08-05 | 2012-02-09 | D.M.G. Italia Srl | Use of hmgb1 as' a biological marker of bowel inflammatory conditions, non-invasive method for its detection in fecal samples and kit thereof |
| CN102375064A (en) * | 2010-08-26 | 2012-03-14 | 杭州华得森生物技术有限公司 | In-vitro diagnostic kit for detecting HMGA2 (High Mobility Group A) content with enzyme-linked immuno sorbent assay |
| JP5055598B2 (en) * | 2001-07-13 | 2012-10-24 | 株式会社シノテスト | Method and reagent for immunological measurement of human HMG-1 using an antibody that specifically binds to human HMG-1 |
| CN205608008U (en) * | 2016-05-12 | 2016-09-28 | 无锡市人民医院 | Albumen B1 of people's high mobility clan optical excitation chemiluminescence detect reagent box |
| CN106093373A (en) * | 2016-05-27 | 2016-11-09 | 安徽伊普诺康生物技术股份有限公司 | A kind of test kit measuring hyaluronic acid |
-
2018
- 2018-07-04 CN CN201810721943.1A patent/CN109030817A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP5055598B2 (en) * | 2001-07-13 | 2012-10-24 | 株式会社シノテスト | Method and reagent for immunological measurement of human HMG-1 using an antibody that specifically binds to human HMG-1 |
| WO2012017466A1 (en) * | 2010-08-05 | 2012-02-09 | D.M.G. Italia Srl | Use of hmgb1 as' a biological marker of bowel inflammatory conditions, non-invasive method for its detection in fecal samples and kit thereof |
| CN102375064A (en) * | 2010-08-26 | 2012-03-14 | 杭州华得森生物技术有限公司 | In-vitro diagnostic kit for detecting HMGA2 (High Mobility Group A) content with enzyme-linked immuno sorbent assay |
| CN205608008U (en) * | 2016-05-12 | 2016-09-28 | 无锡市人民医院 | Albumen B1 of people's high mobility clan optical excitation chemiluminescence detect reagent box |
| CN106093373A (en) * | 2016-05-27 | 2016-11-09 | 安徽伊普诺康生物技术股份有限公司 | A kind of test kit measuring hyaluronic acid |
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Application publication date: 20181218 |