CN106970226A - The kit of the ECD concentration levels of HER 2 in a kind of measure human serum - Google Patents
The kit of the ECD concentration levels of HER 2 in a kind of measure human serum Download PDFInfo
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- CN106970226A CN106970226A CN201710174151.2A CN201710174151A CN106970226A CN 106970226 A CN106970226 A CN 106970226A CN 201710174151 A CN201710174151 A CN 201710174151A CN 106970226 A CN106970226 A CN 106970226A
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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
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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
- G01N33/536—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
- G01N33/538—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody by sorbent column, particles or resin strip, i.e. sorbent materials
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Abstract
The present invention relates to determine the extracellular region protein of human serum epidermal growth factor receptor 2 the present invention relates to one kind（The hereinafter referred to as ECD of HER 2）The kit that concentration level is determined, technical problem to be solved is to provide a kind of kit determined suitable for the ECD concentration levels of human serum HER 2 that automatic clinical chemistry analyzer and special protein instrument are used, and the kit is made up of three parts that are mutually related：Reagent R1, reagent R2, calibration object Cal, directly test clinical serum sample, obtained result is the ECD concentration levels of HER 2 in human serum after above agent combination is calibrated on specific instrument.Present invention result compared with chemoluminescence method has higher uniformity, and cost is lower, it is more suitable for the utilization and popularization of clinical detection, the kit for determining the ECD concentration levels of HER 2 in human serum that technical scheme can provide a kind of affinity of antibody and specificity is good, method sensitivity is high, stability is good.
Description
Technical field
The present invention relates to HER-2 ECD concentration levels in technical field of biological, more particularly to a kind of measure human serum
Kit.
Background technology
HER-2 ECD are HER-2 protein extracellulars domains by protease cracking, from cell surface drop to blood in formed
Soluble glycoprotein.Serum HER-2 ECD detections have detects that correlation is good and it is dynamic in real time to be easy to tumor tissues HE reagents R2
The advantage of state detection, is a kind of important supplement of histology.Detection serum HER-2 ECD method is main in the market
It is chemoluminescence method, Siemens's ADVIA HER-2/neu serology chemiluminescence detection systems, which are that the current whole world is unique, obtains beautiful
Food and medicine Surveillance Authority of state（FDA）The Serologic detection product of certification, is more than for the initial serum levels of monitoring management
15ng/ml metastatic breast cancer patient.This method is closed due to reagent instrument system, and import reagent instrument cost is high, behaviour
Make cumbersome, limit its clinical extensive utilization and promote.
The present invention is a kind of brand-new human serum HER-2 ECD detection kits, different for HER-2 ECD using 4 plants
Latex enhancing immune turbidimetry detection kit prepared by the monoclonal antibody of epitope, sensitivity is high, and specificity is good, and with
Siemens's testing result very high uniformity.The open detecting system of present invention system, can be in automatic clinical chemistry analyzer and spy
Determine to carry out on protein analyzer, greatly improve ease-to-operate, reduce testing cost.
The content of the invention
A kind of affinity of antibody and specific good, method are provided the invention aims to overcome the deficiencies in the prior art
The kit for determining HER-2 ECD concentration levels in human serum that sensitivity is high, stability is good.
The detection method, is to be coupled by the anti-human HER-2 ECD monoclonal antibodies of mouse and latex particle, using latex intensified
Turbidimetry determines the content of HER-2 ECD in human serum, and sample, operation letter need not be pre-processed with the reagent of this method
Single, the degree of accuracy is high, reproducible, and can be used on automatic clinical chemistry analyzer or special proteins instrument.
To reach above-mentioned purpose, present invention employs following technical scheme.
The kit of HER-2 ECD concentration levels in a kind of measure human serum, includes reagent R1, reagent R2 and calibration object
Cal, the reagent R1 components include purified water, biological buffer, coagulant, the solidifying agent of suppression and preservative；The reagent R2 components
Including purified water, biological buffer, the sensitizing latex particle of coating mouse anti-human HER-2 ECD collaboration monoclonal antibody, stabilizer,
Suspending agent, surfactant and preservative；The calibration object Cal components include purified water, biological buffer, stabilizer, anti-corrosion
Agent and the restructuring HER-2 ECD albumen for the finite concentration gradient for needing to add according to assignment.
Preferably, biological buffer is 2- (N- morpholines) ethyl sulfonic acid in the reagent R1 components, and coagulant is polyethylene
Pyrrolidones K30, the solidifying agent of suppression is sodium chloride, and preservative is Sodium azide.
Biological buffer of the present invention can make reaction system maintain certain pH value, various to make reaction system PH
The material of value stabilization could act as the biological buffer of the present invention.In order that the properties of kit are optimal, in the present invention
Biological buffer is preferably 2- (N- morpholines) ethyl sulfonic acid.
Coagulant of the present invention is to refer to promote latex particle to occur agglutinating reaction to improve reagent sensitivity
A class material, a variety of high molecular polymers may be used as coagulant, such as Macrogol 6000, polyethylene glycol 2000, poly- second
Enol etc., in order that the properties of kit are optimal, the coagulant in the present invention is preferably PVP K30.
The solidifying agent of suppression of the present invention is to refer to suppress latex particle generation agglutinating reaction so as to reduce non-specific anti-
The class material answered, such as monovalent salt, divalent salts, glycerine, poloxamer, in order that the properties of kit are optimal, this hair
It is preferably sodium chloride that agent is coagulated in suppression in bright.
Preservative of the present invention is to refer to suppress microorganism in liquid preparation to grow and breed to prevent reagent dirty
Dye causes a class material of performance change, such as benzoic acid, sodium benzoate, sorbic acid, potassium sorbate, Sodium azide, Proclin 300
Deng in order that the properties of kit are optimal, the preservative in the present invention is preferably Sodium azide.
Further, 2- (N- morpholines) ethyl sulfonic acids 5 ~ 10g, PEG20000 are 1-10g, chlorination in 1L reagents R1 components
Sodium is 1-100g, and Sodium azide is 1g, and remaining is purified water, and reagent R1 sodium hydroxides or watery hydrochloric acid regulation PH are 5.0 ~ 7.0.
Anti-human HER-2 ECD antibody of the present invention is to refer to occur specifically with HER-2 ECD albumen in human serum
The material of the antigen-antibody immunological response of property, including mouse or rabbit monoclonal antibodies, sheep or rabbit polyclonal antibody.In order that reagent
The properties of box are optimal, and the anti-human HER-2 ECD in the present invention are preferably the anti-human grand antibody of HER-2 ECD monoclonal antibodies of mouse.
Latex enhancing immune turbidimetry of the present invention refers to connect specific antibody and microballoon, with serum sample
In target antigen occur immunological response, be further crosslinked by antigen between the microballoon of coated antibody and produce turbidity,
The concentration direct proportionality of turbidity size and target antigen, and this turbidity can be by automatic clinical chemistry analyzer and specific protein analysis
Instrument is detected.Because the present invention uses monoclonal antibody, one plant of monoclonal antibody is just for an epitope, it is impossible to handed over by antigen formation
Connection.In order that the properties of kit are optimal, the preferably 4 plants monoclonal antibodies for different epitopes connect micro- respectively in the present invention
Mixing and the antigen-reactive in serum, reach the effect of many plants of monoclonal antibody collaborations, it is ensured that can form turbidity to be detected by instrument after ball
Arrive.
Latex microsphere of the present invention refers to the polystyrene microsphere of carboxyl modified, and its particle size is to final reagent
The performance indications such as sensitivity, the range of linearity and stability have a great impact, in order that the properties of kit are optimal, this
The carboxyl microballoon of 250 ~ 300nm particle sizes is preferably in invention.
Further, the anti-human HER-2 ECD of coating mouse of the present invention cooperate with the sensitizing latex particle of monoclonal antibody
It is to be prepared by the method for chemical coupling, technique is more controllable, reagent stability more preferably, is comprised the following steps that：
Step one, the cleaning of latex particle：The carboxyl microballoon for taking particle diameter to be 250 ~ 300nm is scattered in 50mmol/L, PH's=6.0
In 2- (N- morpholines) ethanesulfonic acid buffer, the concentration of latex particle is 0.5% ~ 1.0% in buffer solution, and centrifugation is abandoned supernatant and will washed
The latex particle crossed is scattered in the above-mentioned 2- of equal volume (N- morpholines) ethanesulfonic acid buffer.
Step 2, the activation of latex particle：Carbodiimides and N- hydroxy thiosuccinimides is taken to use 2- (N- respectively
Coffee quinoline) add in latex solution after ethanesulfonic acid buffer dissolving, it is well mixed to be placed inOn constant-temperature table reaction 10 ~
30min, the latex particle activated.
Step 3, the antibody sensitized of latex particle：The anti-human HER-2 of mouse is added in the latex solution completed respectively to activation
ECD monoclonal antibodies, concussion is well mixed, is placed inReacted 1 ~ 3 hour on constant-temperature table.
Step 4, the closing and washing of sensitizing latex particle：1% bovine serum albumin plain boiled water is added into above-mentioned reaction system
Solution, is well mixed, is placed inReacted 1 ~ 3 hour on constant-temperature table, centrifuge and be resuspended with storing liquid, be diluted to 0.1%
~ 0.5% final concentration.
Preferably, biological buffer 2- (N- morpholines) ethyl sulfonic acid in reagent R2 components, stabilizer is bovine serum albumin
In vain, suspending agent is trehalose, and surfactant is Tween-20, and preservative is Sodium azide Sodium azide.
Stabilizer of the present invention refers to the class unrelated protein to the specific stabilization of sensitizing latex particle, such as ox blood
Pure albumen, casein etc..In order that the properties of kit are optimal, the stabilizer in the present invention is preferably bovine serum albumin
In vain.
Suspending agent of the present invention refers to that storing liquid density can be improved, has the scattered work of suspending agent to sensitizing latex particle
One class material, such as PEG400, glycerine, trehalose sucrose.In order that the properties of kit are optimal, in the present invention
Stabilizer is preferably trehalose.
Surfactant of the present invention refers to the class nonionic surface active agent that can improve colloid solubility, such as
TWEEN Series, Qula lead to series, Nonidet P40 etc..In order that the properties of kit are optimal, in the present invention
Stabilizer is preferably Tween-20.
Further, 2- (N- morpholines) 5 ~ 10g of ethyl sulfonic acid in 1L reagents R2 components, bovine serum albumin(BSA) is 1-10g,
Trehalose is 10-100g, and Sodium azide is 1g, and Tween-20 is 1 ~ 10ml, 1 ~ 5g antibody sensitized latex particle, and remaining is purifying
Water.Reagent R2 PH are 5.0 ~ 7.0.
Preferably, biological buffer is 20mmol/L phosphate buffers PH=7.5, stabilizer in calibration object Cal components
For 1% bovine serum albumin(BSA), preservative is 0.1% Sodium azide, the restructuring HER-2 ECD albumen of finite concentration gradient, and remaining is pure
Change water.
HER-2 ECD albumen of the present invention refers to specific immune response can occur with corresponding antibody and can be used to
An albuminoid of calibration object is configured, it is originated can be divided into restructuring HER-2 full length proteins, restructuring HER-2 ECD, naturally extract
HER-2 albumen etc., in order that the properties of kit are optimal and reduce kit cost, in the present invention is preferably restructuring
HER-2 ECD。
The principle that the present invention is detected is：Specific antibody and microballoon are connected by chemical bond, in specific solution
With the target antigen generation immunological response in serum sample in system, it is crosslinked simultaneously by antigen antibody complex between microballoon
The concentration direct proportionality of turbidity, turbidity size and target antigen is produced, and this turbidity can be by automatic clinical chemistry analyzer and spy
Determine protein analyzer to detect, calculated by the calibration curve calibrated in advance, we can obtain single sera sample
Middle HER-2 ECD concentration levels.
Due to the utilization of above-mentioned technical proposal, the advantageous effects that the technical program has：
（1）4 plants used are prepared than turbid reagent for the mouse resource monoclonal antibody of HER-2 ECD albumen different epitopes, fully hair
The cooperative effect between many plants of monoclonal antibodies is waved, specificity and sensitivity has both been can guarantee that, has prevented the detection leakage phenomenon of single monoclonal antibody, can also protect
The range of linearity is demonstrate,proved, more anti-false positive issue is overcome, makes result more reliable；
（2）The antigen of use is Escherichia coli restructuring HER-2 ECD albumen so that calibration object raw material stability is good, and difference between batch is small,
And cost is well below the full-length proteins and native protein for being typically easier to obtain；
（3）The method that antibody sensitized particle uses chemical coupling, rather than physical absorption method, between antibody and microballoon lead to
Cross Covalent bonding together so that reagent has preferably corkage stability and heat endurance,It can store more than 12 months, side
Just promote and use for a long time；
（4）The present invention is a kind of brand-new human serum HER-2 ECD detection kits, compared with chemoluminescence method method, is had
Good uniformity, but the open detecting system of present invention system, can be in automatic clinical chemistry analyzer and specific protein analyzer
Upper to carry out, use is more easy, goes out result faster, only needs 3 ~ 10 minutes, performance is more stable, and the present invention is not high to instrument requirements,
So that holistic cost is greatly reduced.
With reference to specific embodiment, the present invention is described in further detail.
Brief description of the drawings:
Fig. 1 is human serum HER-2 ECD detection kit calibration curve schematic diagrames；
Fig. 2 for the present invention and Siemens chemiluminescence detection system clinical sample test result correlation result schematic diagram；
Fig. 3 is the result schematic diagram that kit of the present invention is opened and 37 DEG C of heat damages are tested；
Embodiment
With reference to specific embodiment, the present invention is described in further detail.
First, in human serum Epidermal growth factor-recepor-2 extracellular region protein detection kit reagent R1 components preparation
2- (N- morpholines) ethyl sulfonic acid 5.0g, PEG20000 10.0g, sodium chloride 100.0g is accurately weighed with electronic balance, is folded
Nitrogen sodium 1.0g is placed in 1L beakers, plus purified water 800ml, and stirring makes it fully dissolve, and is adjusted with 1M sodium hydroxides or hydrochloric acid
PH is 6.0 ± 0.2, is settled to 1L with purified water, with 0.45um miillpore filter suction filtrations, takes subsequent filtrate to be placed in 2-8 DEG C and store for future use.
2nd, in human serum Epidermal growth factor-recepor-2 extracellular region protein detection kit reagent R2 components preparation
Step 1, coating buffer 50mmol/L PH=6.0 are prepared：2- (N- morpholines) ethyl sulfonic acid is accurately weighed with electronic balance
10.7g, is placed in 1L beakers, plus purified water 800ml, and stirring makes it fully dissolve, and is with 1M sodium hydroxides or hydrochloric acid regulation PH
6.0 ± 0.2,1L is settled to purified water, with 0.45um miillpore filter suction filtrations, takes subsequent filtrate to be placed in 2-8 DEG C and stores for future use.
Step 2, latex storing liquid is prepared：2- (N- morpholines) ethyl sulfonic acid 5.0g, cow's serum is accurately weighed with electronic balance
Albumin 1.0g, trehalose 10.0g, Sodium azide are that 1g, pipettor accurate measuring Tween-20 1.0ml are placed in 1L beakers, plus
Purified water 800ml, stirring makes it fully dissolve, and is 6.5 ± 0.2 with 1M sodium hydroxides or hydrochloric acid regulation PH, uses purified water constant volume
To 1L, with 0.45um miillpore filter suction filtrations, subsequent filtrate is taken to be placed inStore for future use.
Step 3, the cleaning of latex particle：The carboxyl microballoon 4ml that particle diameter is 280nm, concentration is 10% w/v is taken to be scattered in
In 400ml coating buffers, 4 DEG C of centrifugation 30min of 13000r/min abandon supernatant, the latex particle in precipitation are resuspended in into equal volume
Coating buffer in.
Step 4, the activation of latex particle：Take carbodiimides（EDC）26.5mg and N- hydroxy thiosuccinimides
15.6mg is well mixed to be placed in respectively with being all rapidly added after the above-mentioned coating buffer dissolvings of 2.65ml and 1.56ml in latex solution30min is reacted on constant-temperature table, the latex particle activated.
Step 5, the antibody sensitized of latex particle：Above-mentioned activation latex is divided into 4 parts, 4 plants are added thereto respectively
The anti-human each 10mg of HER-2 ECD monoclonal antibodies of mouse, concussion is well mixed, is placed inReacted 1 hour on constant-temperature table.
Step 6, the closing and washing of sensitizing latex particle：10% w/v cow's serums are added into above-mentioned reaction system respectively
Albumin aqueous solution 10ml, is well mixed, is placed on 37 DEG C of constant-temperature tables anti-1 hour,Centrifuge 30min simultaneously
It is resuspended respectively with 100ml storing liquids.
Step 7, ultrasonication and mixing：Above-mentioned emulsion reagent is all mixed into common 400ml, ultrasonic cell disintegration machine is visited
Head is placed in one, ultrasonic 30min（30% power, ultrasonic 5s, interval 5s）.Probe is taken out, reagent is placed inStorage is standby
With.
3rd, the preparation of human serum Epidermal growth factor-recepor-2 extracellular region protein detection kit alignment product Cal components
Step 1, calibration object dilution is configured：0.5014g NaH2PO4,6.025gNa2 HPO4 are accurately weighed with electronic balance
12 H2 O are placed in 1L beakers, plus purified water 800ml, and stirring makes it fully dissolve, and PH is adjusted with 1M sodium hydroxides or hydrochloric acid
For 7.5 ± 0.2；Weigh 10g bovine serum albumin(BSA)s, 1g Sodium azides are added thereto stirring it is fully dissolved, it is fixed with purified water
Hold to 1L, with 0.45um miillpore filter suction filtrations, take subsequent filtrate to be placed in 2-8 DEG C and store for future use.
Step 2：The restructuring HER-2 ECD solution of measured amounts, is added in above-mentioned calibration object dilution, makes its concentration
For 400ng/ml, gradient dilution is carried out to it with calibration object dilution, the calibration object of 5 various concentrations gradients is prepared altogether：
0.0ng/ml, 50ng/ml, 100ng/ml, 200ng/ml, 400ng/mml.This calibration object is placed in into 2-8 DEG C to store for future use.
4th, human serum Epidermal growth factor-recepor-2 extracellular region protein detection kit is determined
The tester that this measure is used is the automatic clinical chemistry analyzer of Hitachi 7170, set factors：Dominant wavelength/complementary wave/examination
Agent R1/ reagents R2/S=700nm/0/150ul/50ul/15ul；Calibrating mode：Spline；Read point：Reagent R1 and sample（Calibration
Product）It is incubated after addition 5 minutes, then adds reagent R2, immediately read point A1, is reacted 5 minutes read point A2 again, calculate the change of absorbance
Change, △ ABS=A2-A1.
Reagent R1/ reagent R2/ calibration object Cal threes are combined, is calibrated, finally obtained by said procedure
Human serum HER-2 ECD detection kit calibration curves, refer to accompanying drawing 1.
5th, human serum Epidermal growth factor-recepor-2 extracellular region protein detection kit Main Analysis Performance Evaluation（This is assessed
The tester used is the automatic clinical chemistry analyzer of Hitachi 7170）
5.1 reagent blank absorbances
Using distilled water as blank solution, absorbance △ ABS ＜ 1.2, as reagent blank extinction are tested according to the method for testing of regulation
Degree.
5.2 sensitivity for analysis（Also known as minimum detection limit）Determine
With kit of the present invention to dummy（Physiological saline containing 5% bovine serum albumin(BSA)）Replication 20 times,
Lowest detection limit adds 20 standard deviations for the average concentration of blank, obtains lowest detection limit for 2ng/ml.
5.3 the range of linearity
（1）Take the serum sample dilution close to range of linearity lower limit close to the serum sample of the range of linearity upper limit, be mixed into five
Diluted concentration（xi）.Detect that each concentration determination 3 times obtains the average of each Concentration Testing result respectively with necessary instrument
（yi）, with five concentration（xi）For independent variable, the average of each Concentration Testing result（yi）Linear regression side is obtained for dependent variable
Journey, correlation coefficient r is calculated by formula m.
Collected because the serum sample of range of linearity bound is more difficult, available reference serum or recombinant protein in exfactory inspection
The calibration object for being configured to 5 various concentrations levels is tested.Detect that each concentration determination 3 times is obtained respectively with necessary instrument
The average of each Concentration Testing result（yi）, with five concentration（xi）For independent variable, the average of each Concentration Testing result（yi）
Equation of linear regression is obtained for dependent variable, by formula（1）Calculate correlation coefficient r.
…………………………m
（2）With diluted concentration in 5.6.1 methods（xi）Equation of linear regression is substituted into, yi estimate and yi and estimate is calculated
Absolute deviation or relative deviation.
It is computed, the range of linearity of the invention is 1-400 ng/mL, in this range of linearity：
A, linearly dependent coefficient r are not less than 0.990；
B, in the range of 1-30 ng/mL, linear absolute deviation be no more than ± 5ng/mL；In the range of 30-400 ng/mL, linearly
Relative deviation is no more than ± 10%
5.4 correlation
The fresh specimens of 50 parts of Siemens's ADVIA HER-2/neu serology chemiluminescence detection system definite values are collected, are tried with this
Agent box is tested, obtained result, the present invention and Siemens's chemiluminescence detection system clinical sample test result correlation, please be detailed
It is shown in Table 1 and accompanying drawing 2.
Table 1：The present invention and Siemens's chemiluminescence detection system clinical sample test result correlation
As can be seen from the above results, the present invention has fine with Siemens's chemiluminescence detection system clinical sample test result
Correlation, overall correlation coefficient r ＞ 0.99（n=50）.
5.5 precision
With replication at least 10 times under similarity condition to reference serum of the kit of same lot number（n≥10）, based on formula n
Calculate the coefficient of variation（CV）.
………………………………n
In formula：
CV-the coefficient of variation
S-standard deviation
The average value of-measured value
Precision of measurement result such as table 2 below of the present invention：
Table 2：Precision of measurement of the present invention
Result above shows coefficient of variation ＜ 5% of the present invention, goes out value very stable.
5.6 stability
Kit of the present invention corkage is placed in biochemical instruments and simulates carrier aircraft experiment, it is closed to be placed inIn baking oven, respectively the 0th
My god, 1 day, 7 days, 14 days, 30 days test reference serum 1 and 2, investigate out whether value is stablized, kit corkage and 37 DEG C of heat damages
Experiment, obtained result refers to accompanying drawing 3.
Data above shows that kit of the present invention is very stable, not only meets reagent use demand, and be easy to long term storage.
It the above is only the concrete application example of the present invention, protection scope of the present invention be not limited in any way.It is all to use
Technical scheme formed by equivalent transformation or equivalent replacement, all falls within rights protection scope of the present invention.
Claims (7)
1. a kind of kit for determining HER-2 ECD concentration levels in human serum, it is characterised in that：Include reagent R1, reagent R2
And tri- kinds of reagents of calibration object Cal, the reagent R1 components include purified water, biological buffer, coagulant, the solidifying agent of suppression and anti-corrosion
Agent；The reagent R2 components include the cause that purified water, biological buffer, the anti-human HER-2 ECD of coating mouse cooperate with monoclonal antibody
Quick latex particle, stabilizer, suspending agent, surfactant and preservative；The calibration object Cal components include purified water, biology
Buffer, stabilizer, preservative and restructuring HER-2 ECD albumen.
2. a kind of kit for determining HER-2 ECD concentration levels in human serum according to claim 1, its feature exists
In：Biological buffer is 2- (N- morpholines) ethyl sulfonic acid in the reagent R1 components, and coagulant is PVP K30,
The solidifying agent of suppression is sodium chloride, and preservative is Sodium azide.
3. a kind of kit for determining HER-2 ECD concentration levels in human serum according to claim 2, its feature exists
In：2- (N- morpholines) ethyl sulfonic acids 5 ~ 10g, PEG20000 are 1-10g in the reagent R1 components of the 1L, and sodium chloride is 1-
100g, Sodium azide is 1g, and remaining is purified water, the reagent R1 components using sodium hydroxide or watery hydrochloric acid regulation PH to 5.0 ~
7.0。
4. a kind of kit for determining HER-2 ECD concentration levels in human serum according to claim 1, its feature exists
In：The sensitizing latex particle of the anti-human HER-2 ECD collaborations monoclonal antibody of coating mouse in the reagent R2 components be by with
What lower step was obtained：
Step one, the cleaning of latex particle：The carboxyl microballoon for taking particle diameter to be 250 ~ 300nm is scattered in 50mmol/L, PH's=6.0
In 2- (N- morpholines) ethanesulfonic acid buffer, the concentration of latex particle is 0.5% ~ 1.0% in buffer solution, and centrifugation is abandoned supernatant and will washed
The latex particle crossed is scattered in the above-mentioned 2- of equal volume (N- morpholines) ethanesulfonic acid buffer；
Step 2, the activation of latex particle：Carbodiimides and N- hydroxy thiosuccinimides is taken to use 2- (N- morphines respectively
Quinoline) add in latex solution after ethanesulfonic acid buffer dissolving, it is well mixed to be placed inOn constant-temperature table reaction 10 ~
30min, the latex particle activated；
Step 3, the antibody sensitized of latex particle：It is mono- that the anti-human HER-2 ECD of mouse are added in the latex solution completed respectively to activation
Clonal antibody, concussion is well mixed, is placed inReacted 1 ~ 3 hour on constant-temperature table；
Step 4, the closing and washing of sensitizing latex particle：1% Bovine Serum Albumin in Aqueous Solution is added into above-mentioned reaction system,
It is well mixed, it is placed inReacted 1 ~ 3 hour on constant-temperature table, centrifuge and be resuspended with storing liquid, be diluted to 0.1% ~ 0.5%
Final concentration.
5. a kind of kit for determining HER-2 ECD concentration levels in human serum according to claim 1, its feature exists
In：Biological buffer 2- (N- morpholines) ethyl sulfonic acid in the reagent R2 components, stabilizer is bovine serum albumin(BSA), and suspending agent is
Trehalose, surfactant is Tween-20, and preservative is Sodium azide Sodium azide.
6. a kind of kit for determining HER-2 ECD concentration levels in human serum according to claim 5, its feature exists
In：2- (N- morpholines) 5 ~ 10g of ethyl sulfonic acid in the reagent R2 components of the 1L, bovine serum albumin(BSA) is 1-10g, and trehalose is
10-100g, Sodium azide is 1g, and Tween-20 is 1 ~ 10ml, 1 ~ 5g antibody sensitized latex particle, and remaining is purified water, reagent R2
The PH of component solution is 5.0 ~ 7.0.
7. a kind of kit for determining HER-2 ECD concentration levels in human serum according to claim 1, its feature exists
In：Biological buffer in the calibration object Cal components is 20mmol/L phosphate buffers, and pH value is equal to 7.5, stabilizer
For 1% bovine serum albumin(BSA), preservative is 0.1% Sodium azide.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710174151.2A CN106970226A (en) | 2017-03-22 | 2017-03-22 | The kit of the ECD concentration levels of HER 2 in a kind of measure human serum |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710174151.2A CN106970226A (en) | 2017-03-22 | 2017-03-22 | The kit of the ECD concentration levels of HER 2 in a kind of measure human serum |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106970226A true CN106970226A (en) | 2017-07-21 |
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| CN201710174151.2A Pending CN106970226A (en) | 2017-03-22 | 2017-03-22 | The kit of the ECD concentration levels of HER 2 in a kind of measure human serum |
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Cited By (5)
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| CN107941791A (en) * | 2017-12-21 | 2018-04-20 | 江苏泽成生物技术有限公司 | A kind of cancer base antigen calibration object method of inspection |
| CN111999510A (en) * | 2020-08-20 | 2020-11-27 | 安徽伊普诺康生物技术股份有限公司 | Kit for determining anti-mullerian hormone and preparation and use methods thereof |
| CN112964869A (en) * | 2021-02-01 | 2021-06-15 | 深圳市希莱恒医用电子有限公司 | Kit and detection system for detecting anti-cyclic citrullinated peptide antibody |
| CN114859048A (en) * | 2022-05-27 | 2022-08-05 | 美康生物科技股份有限公司 | A kit of chitinase 3-like protein 1 |
| CN115516312A (en) * | 2020-03-30 | 2022-12-23 | 电化株式会社 | Method for determining target substance by latex agglutination method and reagent thereof |
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| CN112964869A (en) * | 2021-02-01 | 2021-06-15 | 深圳市希莱恒医用电子有限公司 | Kit and detection system for detecting anti-cyclic citrullinated peptide antibody |
| CN114859048A (en) * | 2022-05-27 | 2022-08-05 | 美康生物科技股份有限公司 | A kit of chitinase 3-like protein 1 |
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