CN108152512A - Heparin-binding protein detection kit and preparation method thereof - Google Patents
Heparin-binding protein detection kit and preparation method thereof Download PDFInfo
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- CN108152512A CN108152512A CN201711412733.6A CN201711412733A CN108152512A CN 108152512 A CN108152512 A CN 108152512A CN 201711412733 A CN201711412733 A CN 201711412733A CN 108152512 A CN108152512 A CN 108152512A
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- hbp
- heparin
- binding protein
- detection kit
- latex particulate
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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
- 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
Abstract
The invention discloses heparin-binding proteins（HBP‑Heparin Binding Protein）Detection kit and preparation method thereof, which is latex enhancing immune than turbid detection reagent.The latex enhancing immune is than reaction solution R2 and HBP calibration object and quality-control product that the main component of turbid kit is the dilution R1 containing buffer solution, surfactant, salt and preservative and the present latex particulate marked containing HBP antibody, buffer solution, salt, stabilizer, suspending agent and preservative.The invention also discloses use the kit using heparin-binding protein in transmission or scattering turbidimetry principle detection blood sample（HBP）Concentration method.For the present invention using double reagent, easy to operate, high sensitivity, the range of linearity is wide, various transmissions or scattering analysis instrument is can be widely applied to, including common Biochemical Analyzer, specific protein analyzer etc..
Description
Technical field
The invention belongs to medical immunology diagnostic reagent fields, the more particularly to detection of heparin-binding protein.
Background technology
Heparin-binding protein (Heparin Binding Protein- abbreviation HBP) is a kind of of neutrophil leucocyte secretion
Corpuscular protein has bactericidal effect, while also assists in inflammatory reaction and the adjusting of coagulation process.1984, Shafer et al. was for the first time
It was found that and isolate this albumen, CAP37 (Cationic are named as according to the function of its electropositive feature and sterilization
Antimicrobial Protein, molecular weight 37k).Later again successively someone from the aniline blue particles in polymorphonuclear leukocyte
In isolate the reddish black heparin-binding protein (HBP- for killing plain (Azurocidin) and having very strong binding ability with heparin
Heparin Binding Protein).Further protein structure and gene sequencing research shows that, these three albumen are real
It is same albumen on border, unifies to be represented with heparin-binding protein in the present invention.Heparin-binding protein is a kind of multi-functional
Albumen, can change vasopermeability by combining vascular endothelial, induce plasma leakage and inflammatory reaction.Heparin knot
Hop protein also is able to chemotactic and activated mononuclear leucocyte and macrophage, copes with bacterium infection, participates in adjusting immune response.Recently
Some research work show that the concentration of the heparin-binding protein in blood plasma has very strong phase with pyemia and septic shock
Guan Xing.Pyemia is systemic infection inflammation reaction, the symptoms such as can cause body temperature change, increased heart rate, be short of breath, causing
Whole body Coagulation Dysfunction and immunization inflammatory reaction are unbalance, induce organ failure, septic shock can occur very when serious
To death.Medical field is not also fully aware of to pyemic cause of disease mechanism, early predicts that pyemic generation can be effectively
Organ failure and death caused by preventing pyemia.Linder et al. (Adam Linder et al, Critical Care
Medicine,2015,43:2378-2386) research shows that as participate in inflammatory reaction heparin-binding protein, can be effective
The generation of pyemia and septic shock is predicted on ground, is a good clinical diagnosis index.
Heparin-binding protein (HBP) is enzyme-linked immunosorbent assay by the detection method that national Bureau of Drugs Supervision ratifies
(ELISA), representative products are the enzyme linked immunological kits of Axis-Shield companies of Britain (state's tool is noted into 20162400300).
Enzyme-linked immunosorbent assay is that cladding captures antibody on 96 orifice plates, when the sample containing antigen is cultivated in hole, is coated on hole
Interior antibody reacts the antigen captured in sample by antibody antigen, and the antibody and antigen binding, formation then marked with enzyme resists
The sandwich structure of body-Ag-Ab.The enzyme marked can quantitatively react with corresponding substrate, generate color
Variation.By measuring the absorbance change under specific wavelength, the antigen concentration in detection sample can be quantified.ELISA
Method can accurately detect the heparin-binding protein concentration in sample, but ELISA experiments are time-consuming long in practical operation, need
It is incubated, cleans, the multinomial manual operations such as liquid relief, process is more complicated, and testing result can just be obtained by generally requiring 3 to 5 hours,
The demand quickly detected of outpatient service or emergency treatment can not be met well.The detection method of other reports also has
The method of immunofluorescence chromatography disclosed in colloidal gold method disclosed in CN204882574U and CN204882575U.Colloidal gold method and exempt from
Although epidemic disease fluorescent chromatographic method can be detected quickly, qualitative or sxemiquantitative detection can only be carried out, accuracy is low, Wu Fati
For accurate clinical judgment foundation.Simultaneously be limited to method in itself, the precision of detection is poor so that test repeatability with it is consistent
Property cannot be protected.It discloses in Chinese patent CN107167589A and is detected with latex enhancing immune turbidimetry recently
The method of HBP, but it is only capable of using on Biochemical Analyzer.Latex enhancing immune turbidimetry can be measured with fast quantification in blood
HBP, be that one of existing detection method is supplemented very well.But using survey in method disclosed in CN107167589A
The detection method of amount transmission absorbance, can only use on Biochemical Analyzer, be not suitable for the requirement that outpatient service quickly detects.Simultaneously should
All there are one in the diversity of antibody label latex process, antibody selection, sample conditions, sample comparison correlation etc. for patent
Determine defect.There is also problem in the selection of the concrete composition ingredient of detection reagent, such as may be generated using poly ethyl alcohol
Nonspecific reaction, that is, false positive.Problems to obtain detection reagent in sensitivity, specificity, linear model by the invention
Enclose etc. is all undesirable, and the accuracy of reagent also can not accomplish good correlation with existing listing reagent.
Invention content
In order to solve the deficiency of above-mentioned existing various methods, the object of the present invention is to provide it is easy to operate, can quickly examine
Survey heparin-binding protein detection kit of heparin-binding protein (HBP) concentration in blood sample and preparation method thereof, the inspection
Test agent box is latex enhancing immune than turbid detection reagent.The kit can both be surveyed on large-scale automatic biochemistry analyzer
Examination, can also be tested in the small-sized POCT equipment such as special protein instrument.
A kind of technical solution provided by the invention is a kind of heparin-binding protein (HBP) latex based on double reagent form
Enhance immunoturbidimetry detection kit.
According to an aspect of the present invention, a kind of heparin-binding protein detection kit, heparin-binding protein detection are provided
Kit is liquid double reagent, and heparin-binding protein detection kit includes dilution R1 and reaction solution R2 and calibration object and matter
Control product, the reaction solution R2 include the present latex particulate of HBP antibody label.
Kit provided by the invention is for latex enhancing immune turbidimetry (Particles Enhanced Turbidity
Immuno-Assay-PETIA it) uses, this method is the HBP in the present latex particulate of tens to hundreds of nanometers of diameter using absorption
Heparin-binding protein (HBP) in antibody capture test sample, formed particle between crosslinking, change solution nephelometric turbidity unit or
Person transmits absorbance.By measuring the nephelometric turbidity unit of solution or transmiting the variation of absorbance, quantitatively calculated according to calibration curve
Go out the concentration of heparin-binding protein in test sample.Kit provided by the invention both can be in general large-scale automatic biochemical point
It is used in analyzer, can also be used in the small-sized POCT such as specific protein analyzer (detection immediately) equipment, there is very strong fit
With property, China can be met well from large-scale Grade A hospital to the use demand of community's basic hospital.It is of the invention and enzyme-linked exempt from
Epidemic disease kit is compared, and due to using latex enhancing immune turbidimetry, kit of the invention has easy to operate, inspection in use
Survey the advantages that quick, test result is reproducible, the measurement range of linearity is wide.
According to an aspect of the present invention, a kind of heparin-binding protein detection kit is provided, dilution R1 includes buffering
Solution, surfactant, salt and preservative, reaction solution R2 include buffer solution, salt, stabilizer, suspending agent and preservative, calibration
Product and quality-control product include heparin-binding protein, buffer solution, salt, stabilizer and preservative.
According to an aspect of the present invention, a kind of heparin-binding protein detection kit, the latex of HBP antibody label are provided
Particle resists for the present latex particulate of HBP monoclonal antibodies label or the present latex particulate of the how anti-labels of HBP or HBP monoclonal antibodies with the HBP combinations more resisted
The present latex particulate of body label, wherein HBP monoclonal antibodies are a pair of or several HBP monoclonal antibodies to pairing.
According to an aspect of the present invention, a kind of heparin-binding protein detection kit, the latex of HBP antibody label are provided
Mixture of the particle for the present latex particulate of HBP monoclonal antibodies label and the present latex particulate of the how anti-labels of HBP, the latex of HBP antibody label
Particle is 2 for the quality ratio range of the present latex particulate of monoclonal antibody label and the present latex particulate of how anti-label：8 to 8：2.Monoclonal antibody can be promoted
Reaction solution R2 improves the sensitivity and specificity of reagent to the recognition capability of antigen well, how anti-that reactant can be substantially improved
The range of linearity of system.
According to an aspect of the present invention, a kind of heparin-binding protein detection kit, the latex of HBP antibody label are provided
A diameter of 100nm~the 400nm of present latex particulate used in particle, present latex particulate are chemical crosslinking type present latex particulate or physical absorption
Type present latex particulate.
According to an aspect of the present invention, a kind of heparin-binding protein detection kit is provided, chemical crosslinking type latex is micro-
Grain, surface modification group are-COOH ,-NH2,-SH ,-OH, one kind in-CHO.
According to an aspect of the present invention, a kind of heparin-binding protein detection kit is provided, in kit of the invention
The present latex particulate used is polystyrene latex microparticles of the surface with carboxyl, a diameter of 200~350nm, and surface modification
Group is-COOH.
According to an aspect of the present invention, a kind of heparin-binding protein detection kit, the latex of HBP antibody label are provided
A concentration of the 0.01%~1% of particle.
According to an aspect of the present invention, a kind of heparin-binding protein detection kit is provided, stabilizer is pure for ox blood
One kind in albumen, casein, gelatin；Salt is sodium chloride, potassium chloride, sodium sulphate, one kind in potassium sulfate, two kinds or two kinds with
On combination；Surfactant is NP40, triton x-100, polysorbas20, polysorbate40, Tween 80, lauryl sodium sulfate
(SDS), one kind in span 40, sorbester p18, two or more combination；Suspending agent for sucrose, glucose, maltose,
One kind, two or more combination in trehalose, mannitol, ethylene glycol, glycerine, lactose；Preservative is Azide
One kind in sodium, thimerosal, Proclin-300.
According to an aspect of the present invention, a kind of heparin-binding protein detection kit is provided, salt is sodium chloride.
According to an aspect of the present invention, a kind of heparin-binding protein detection kit is provided, Surfactant carries out
Preferably, preferred surfactant be triton x-100, one kind in NP40, polysorbas20, a concentration of 0.1%~1%.
Under the combination and concentration of the optimization, it is non-specific that there is surfactant reduction antigen to be generated with the present latex particulate that HBP antibody marks
Property reaction effect.
According to an aspect of the present invention, a kind of heparin-binding protein detection kit is provided, stabilizer is pure for ox blood
Albumen, bovine serum albumin(BSA) concentration 0.2%-2%；Suspending agent be concentration trehalose, trehalose concentration 0.2%-5%；Preservative
For Proclin-300, concentration of preservatives 0.05%-2%；A concentration of the 0.1%~2% of surfactant.
According to an aspect of the present invention, a kind of heparin-binding protein detection kit is provided, buffer solution delays for glycine
Fliud flushing, 4- hydroxyethyl piperazineethanesulfonic acids (HEPES) buffer solution, 3- (N- morpholines) -2- hydroxy-propanesulfonic acids (MOPSO) buffer solution, three
Hydroxyl amino methane hydrochloride (Tris-HCl) buffer solution, phosphoric acid physiological saline (PBS) buffer solution and 2-morpholine ethane sulfonic acid (MES)
Any of which of buffer solution, the range of buffer concentration are 10~200mmol/L.
According to an aspect of the present invention, heparin-binding protein standard items and Quality Control are included in kit provided by the invention
Product, the standard items of heparin-binding protein of the invention are a series of low value concentration to the HBP antigenic solutions of high level concentration, concentration
Range tests required calibration curve from 0 to 240ng/mL, for establishing.
According to another aspect of the present invention, a kind of preparation method of heparin-binding protein detection kit, the party are provided
Method uses any one above-mentioned formula composition heparin-binding protein detection kit, HBP antibody in this test agent box preparation method
The method of the present latex particulate labelled antibody of label is any one in intermediate ester process, one-step method, two-step method or physisorphtion.
According to another aspect of the present invention, a kind of preparation method of heparin-binding protein detection kit is provided, it is intermediate
Ester process is the carboxyl using Carbodiimides and succinimide ester activation of catalyst present latex particulate surface, forms surface and carries
The present latex particulate of succinimide ester intermediate adds HBP antibody, amino and the present latex particulate surface of HBP antibody surfaces
HBP antibody labeling process is completed in succinimide ester condensation.
According to another aspect of the present invention, a kind of preparation method of heparin-binding protein detection kit, a step are provided
Method is that HBP antibody is first mixed with present latex particulate, and HBP antibody is adsorbed on present latex particulate surface, then adds in Carbodiimides
Catalyst, the carboxyl or amino of HBP antibody surfaces are condensed with the carboxyl on polystyrene latex microparticles surface or amino, complete HBP
Antibody labeling process.
According to another aspect of the present invention, a kind of preparation method of heparin-binding protein detection kit, two steps are provided
Method is the carboxyl using Carbodiimides activation of catalyst present latex particulate surface, then adds in HBP antibody, the amino of the two
With carboxyl direct polycondensation, HBP antibody labeling process is completed.
According to another aspect of the present invention, a kind of preparation method of heparin-binding protein detection kit, physics are provided
Absorption method is to be used without the ps particle of any surface active groups, then adds in HBP antibody, and the absorption of HBP antibody exists
HBP antibody labeling process is completed on ps particle surface.
It is according to the present invention to also have on the one hand, it provides a kind of latex enhancing immune using double reagent and is detected than turbid reagent
The method of heparin-binding protein, any of the above-described kind of heparin-binding protein detection kit, in Biochemical Analyzer or specific protein
By the way that transmittance is turbid or the method for scattering turbidimetry measures the HBP contents in sample to be tested on analyzer.
It is according to the present invention to also have on the one hand, it provides a kind of latex enhancing immune using double reagent and is detected than turbid reagent
The method of heparin-binding protein, used detection sample is the fresh serum or plasma sample extracted within 6 hours.
When detecting sample, the HBP antibody in reagent provided by the invention in present latex particulate is sent out with the HBP antigens in sample
Raw antibody antigen reaction, so that latex gradually agglomerates, changes the turbidity of test fluid.The variation of turbidity can pass through transmission
Instrument or scatterometer measure.Usually, using the method that transmittance is turbid in the biochemical instruments of large automatic, advantage is certainly
Dynamicization degree is high, and the range of linearity is wide, reproducible.The side of scattering turbidimetry is used in the POCT equipment such as specific protein analyzer
Method has many advantages, such as that detection speed is fast, high sensitivity.The absorbance or scattered light intensity of transmission and the HBP concentration in sample
It is linear in the range of a certain concentration.By reference to HBP standard items establish calibration curve, can quantitatively calculate by
The concentration of HBP in sample.
Heparin-binding protein detection kit provided by the invention is adapted to various transilluminators or scatterometer etc. and sets
It is standby.
Beneficial effects of the present invention further include：
1. the composition of detection reagent is simple, operating procedure is few.Kit provided by the invention only has double reagent, anti-without diluting
Original does not need to carry out antigen diluent, multistep cleaning plus substrate in test as existing import ELISA reagents in the market
With the operation of terminate liquid and control each step reaction time, test program is simplified, is brought great convenience for operating personnel,
The accuracy of test has also preferably been ensured simultaneously.
2. detection is quick, the method for the present invention can be completed to detect for 1-3 minutes or so on small-sized POCT, automatically give birth to
Changing can also complete to detect for 10-12 minutes on analyzer.And existing ELISA method needs 3 to 5 hours to complete detection.
3. reagent broad quantum, the range of linearity is wide, can not have to dilution directly sample of the measurement at concentrations up to 240ng/mL,
Antigen concentration, which is up to 600ng/mL, simultaneously hook-shaped (Hook) effect will not occurs.
4. reagent applicability is good, can be used in transmission or scatterometer.Latex enhancing immune provided by the present invention
Turbidimetry detection kit can both use in fully-automatic large-scale biochemical instruments, can also be used in small-sized POCT equipment.It can
To meet the detection needs of large hospital clinical laboratory, small hospital's outpatient service and emergency treatment section office simultaneously.
Description of the drawings
Fig. 1 is reaction normal curve of the embodiment 1,2 and 3 in biochemical instruments；
Fig. 2 is reaction normal curve of the embodiment 4 in POCT equipment；
Fig. 3 is reaction normal curve of the embodiment 5 in POCT equipment；
Fig. 4 is the range of linearity of embodiment 1；
Fig. 5 is the range of linearity of embodiment 4；
Fig. 6 is the hook effect curve of embodiment 9；
Fig. 7 is the correlation analysis of embodiment 10.
Specific embodiment
The present invention is further illustrated below in conjunction with the accompanying drawings.
Following embodiments is some preferred embodiments in order to further illustrate the present invention, and not all embodiments.This
Field professional made under the premise of no progress creative work based on the other embodiment of the present invention, belong to this
The rights protection scope of invention.
Embodiment 1
1) preparation of monoclonal antibody and the emulsion reagent (333nm, reaction solution R2) of how anti-composite marking
Polystyrene latex microparticles (classification number P0323, producer JSR of the surface with carboxyl that intermediate ester process label grain size is 333nm
Life Sciences Corporatiion).Particle of the grain size for 333nm surface carboxyl groups is diluted in MES buffer solutions
To 1%, stir at room temperature.EDC and Sulfo-NHS solid powders are put into, 10000RPM is centrifuged 20 minutes after stirring 2 hours.Glue
Breast is cleaned with MES buffer solutions and is resuspended afterwards twice, is divided into two parts.Portion adds in HBP monoclonal antibodies, is stirred to react 2 hours, separately
Portion adds in HBP polyclonal antibodies, is stirred to react 2 hours.Then two parts of latex 10000RPM centrifuge 20 minutes, remove on
Clear liquid.The latex of two parts of precipitations is resuspended in storing liquid and ultrasonic disperse, stirs 1 hour respectively, then is mixed in following ratios
The present latex particulate of HBP monoclonal antibodies label and the present latex particulate of how anti-label.It is molten to include present latex particulate, buffering by reaction solution R2 in this example
A concentration of 0.15%, the HBP monoclonal antibodies label of present latex particulate of liquid, salt, stabilizer, suspending agent and preservative, wherein HBP antibody label
The mass ratio of present latex particulate of present latex particulate and how anti-label be 2 respectively：8、3：7、4：6、5：5 and 4：6, buffer solution is
50mM MES pH=6.5, stabilizer are 0.5% bovine serum albumin(BSA)s, and preservative is 0.1%ProClin 300, and suspending agent is
3% trehalose, salt are 2.7% sodium chloride.
2) preparation of dilution R1
The reagent includes buffer solution, surfactant, salt and preservative, and buffer solution is 50mM MOPSO pH=7.0,
0.1%ProClin 300, surfactant are 0.5% triton x-100s, and salt is 0.9% sodium chloride.
3) preparation of standard items
Commercially available HBP antigens are configured to series of standards product with standard dilutions, concentration is respectively 0ng/mL, 5ng/mL,
20ng/mL, 50ng/mL, 100ng/mL, 240ng/mL, the compositions of standard dilutions are 50mM PBS pH7.4,0.1%
Proclin 300,0.1% triton x-100,1.8% sodium chloride, 1% bovine serum albumin(BSA).
4) preparation of quality-control product
The HBP samples (80ng/mL) of high concentration are diluted to 8ng/mL and 40ng/mL with standard dilutions, respectively as low
It is worth quality-control product and high level quality-control product.
5) calibration curve is established in biochemical instruments
By 6 HBP standard items, concentration is put in biochemical instruments (using auspicious full automatic biochemical apparatus BS- advanced in years in this example from 0 to 240ng/mL
400) it in sample disc, is reacted respectively with the HBP dilutions R1 of preparation and reaction solution R2, sample：R1：R2=10 μ l:160μl:
40 μ l, are detected, and record degree of reaction, and reagent parameter setting is completed after being placed correctly with reagent sites, and Biochemical Analyzer is automatic
It completes to draw reagent and sampling step, whole process about takes 10-12 minutes.Detection wavelength is 700nm, using 2 terminals
Method, 300 seconds reaction time calculate 2 degree of reaction differences.Calibration curve can be established according to concentration and degree of reaction difference.Fig. 1
It is the calibration curve that emulsion reagent is established in embodiment 1.
Embodiment 2
1) preparation of the emulsion reagent (333nm, reaction solution R2) of how anti-label
Polystyrene latex microparticles (classification number P0323, producer JSR of the surface with carboxyl that intermediate ester process label grain size is 333nm
Life Sciences Corporatiion).Particle of the grain size for 333nm surface carboxyl groups is diluted in MES buffer solutions
To 1%, stir at room temperature.EDC and Sulfo-NHS solid powders are put into, 10000RPM is centrifuged 20 minutes after stirring 2 hours.So
Latex centrifuges 10 minutes in 10000RPM afterwards, removes supernatant.The latex of precipitation is resuspended in storing liquid and ultrasonic disperse, stirring
It is spare after 1 hour.Reaction solution R2 includes present latex particulate, buffer solution, salt, stabilizer, suspending agent and preservative in this example, wherein
The present latex particulate a concentration of 0.15% of HBP antibody label, buffer solution is 50mM MES pH=6.5, and stabilizer is 0.5% N
Seralbumin, preservative are 0.1%ProClin 300, and suspending agent is 3% trehalose, and salt is 2.7% sodium chloride.
R1 and calibration object, the preparation of quality-control product and detection process are same as Example 1, in auspicious full automatic biochemical apparatus advanced in years
Calibration curve is established on BS-400.Fig. 1 includes the calibration curve that emulsion reagent is established in embodiment 2.
Embodiment 3
1) preparation of the emulsion reagent (333nm, reaction solution R2) of a pair of of monoclonal antibody
Polystyrene latex microparticles (classification number P0323, producer JSR of the surface with carboxyl that intermediate ester process label grain size is 333nm
Life Sciences Corporatiion).Particle of the grain size for 333nm surface carboxyl groups is diluted in MES buffer solutions
To 1%, stir at room temperature.EDC and Sulfo-NHS solid powders are put into, 10000RPM is centrifuged 20 minutes after stirring 2 hours.Glue
Breast is cleaned with MES buffer solutions and is resuspended afterwards twice, is divided into two parts.It is each to add in HBP monoclonal antibodies, it is stirred to react 2 hours.Then
Two parts of latex centrifuge 20 minutes in 10000RPM, remove supernatant.The latex of two parts of precipitations is resuspended and is surpassed in storing liquid
Sound disperses, and stirs 1 hour respectively.Reaction solution R2 includes present latex particulate, buffer solution, salt, stabilizer, suspending agent and prevents in this example
The present latex particulate a concentration of 0.15% of rotten agent, wherein HBP antibody label, buffer solution is 50mM MES pH=6.5, stabilizer
It is 0.5% bovine serum albumin(BSA), preservative is 0.1%ProClin 300, and suspending agent is 3% trehalose, and salt is 2.7% chlorination
Sodium.
R1 and calibration object, the preparation of quality-control product and detection process are same as Example 1, in auspicious full automatic biochemical apparatus advanced in years
Calibration curve is established on BS-400.Fig. 1 includes the calibration curve that emulsion reagent is established in embodiment 3.
Embodiment 4
1) preparation of how anti-label emulsion reagent (198nm, reaction solution R2)
Two step method marks polystyrene latex microparticles (classification number P0113, producer JSR Life of the surface with carboxyl of 198nm
Sciences Corporatiion).Particle of the grain size for 198nm surface carboxyl groups is diluted to 1% in MES buffer solutions,
It stirs at room temperature.EDC solid powders are put into, 10000RPM is centrifuged 10 minutes after stirring 2 hours.Latex is clear with MES buffer solutions
It washes and is resuspended afterwards twice.HBP polyclonal antibodies are added in, are stirred to react 2 hours.Then latex centrifuges 10 minutes in 10000RPM, goes
Fall supernatant.The latex of precipitation is resuspended in storing liquid and ultrasonic disperse, spare after stirring 1 hour.Reaction solution R2 packets in this example
Present latex particulate, buffer solution, salt, stabilizer, suspending agent and preservative are included, the present latex particulate of wherein HBP antibody label is a concentration of
0.10%, buffer solution is 100mM Tris pH=7.4, and stabilizer is 1% bovine serum albumin(BSA), and preservative is 0.1%
ProClin 300, suspending agent are 1% sucrose, and salt is 3.0% potassium chloride.
2) preparation of dilution R1
The reagent includes buffer solution, surfactant, salt, stabilizer and preservative, and buffer solution is 100mM PBS pH=
7.4, preservative is 0.1%ProClin 300, and surfactant is the combination of 0.1%SDS and 0.1%NP-40, and salt is 1.2%
Sodium chloride.
3) preparation of standard items
Commercially available HBP antigens are configured to series of standards product with standard dilutions, concentration is respectively 0ng/mL, 5ng/mL,
20ng/mL, 50ng/mL, 100ng/mL, 240ng/mL, the constituent of standard dilutions is 50mM PBS pH7.4,
0.1%Proclin 300,0.1% triton x-100,1.8% sodium chloride, 1% bovine serum albumin(BSA).
4) preparation of quality-control product
The HBP samples (80ng/mL) of high concentration are diluted to 8ng/mL and 40ng/mL with standard dilutions, respectively as low
It is worth quality-control product and high level quality-control product.
5) calibration curve is established on small-sized POCT
By 6 HBP standard items, concentration is from 0 to 240ng/mL, after directly being mixed with dilution R1 respectively, is put in the test of POCT
(Laura electronic semiautomatic specific protein analyzer EZ-400 is used in this example) in hole, be eventually adding the latex of HBP antibody label
The reaction solution R2 that particle is formed, is detected, sample：R1：R2=10 μ l:160μl:40 μ l record degree of reaction.Detect wave
A length of 650nm, using two point rate assay, 180 seconds reaction time calculated 2 degree of reaction differences.It is poor according to concentration and degree of reaction
Value can establish calibration curve.Fig. 2 is the calibration curve that emulsion reagent is established in embodiment 4.
Embodiment 5
1) preparation of the emulsion reagent (210nm, reaction solution R2) of a pair of of monoclonal antibody label
Physisorphtion marks polystyrene latex microparticles (the classification number PS02N, producer Bangs of 210nm
Laboratories).The particle that grain size is 210nm in PBS buffer solutions is diluted to 1%, is stirred at room temperature.It adds in a pair of
HBP monoclonal antibodies are stirred to react 2 hours.Then latex centrifuges 10 minutes in 10000RPM, removes supernatant.The glue of precipitation
Breast is resuspended in storing liquid and ultrasonic disperse, spare after stirring 1 hour.It is molten to include present latex particulate, buffering by reaction solution R2 in this example
Liquid, salt, stabilizer, suspending agent and preservative, wherein present latex particulate a concentration of 0.10%, buffer solution are 100mM PBS pH=
7.4, stabilizer is 2% bovine serum albumin(BSA), and preservative is 0.1%ProClin 300, and suspending agent is 4% trehalose, and salt is
1.8% sodium chloride.
2) preparation of dilution R1
The reagent includes buffer solution, surfactant, salt, stabilizer and preservative, and buffer solution is 100mM PBS pH=
7.4, preservative is 0.1%ProClin 300, and surfactant is 0.1% polysorbas20, and salt is 1.8% sodium chloride.
3) preparation of standard items
Commercially available HBP antigens are configured to series of standards product with standard dilutions, concentration is respectively 0ng/mL, 5ng/mL,
20ng/mL, 50ng/mL, 100ng/mL, 240ng/mL, the compositions of standard dilutions are 50mM PBS pH7.4,0.1%
Proclin 300,0.1% triton x-100,1.8% sodium chloride, 1% bovine serum albumin(BSA).
4) preparation of quality-control product
The HBP samples (80ng/mL) of high concentration are diluted to 8ng/mL and 40ng/mL with standard dilutions, respectively as low
It is worth quality-control product and high level quality-control product.
5) calibration curve is established on small-sized POCT
By 6 HBP standard items, concentration is from 0 to 240ng/mL, after directly being mixed with dilution R1 respectively, is put in the test of POCT
(Laura electronic semiautomatic specific protein analyzer EZ-400 is used in this example) in hole, be eventually adding the latex of HBP antibody label
The reaction solution R2 that particle is formed, is detected, sample：R1：R2=10 μ l:160μl:40 μ l record degree of reaction.Detect wave
A length of 650nm, using two point rate assay, 180 seconds reaction time calculated 2 degree of reaction differences.It is poor according to concentration and degree of reaction
Value can establish calibration curve.Fig. 3 is the calibration curve that emulsion reagent is established in embodiment 5.
Embodiment 6
The sensitivity for analysis (blank limit) of reagent
Using embodiment 1, (the present latex particulate particle and the mass ratio of the present latex particulate of how anti-label that HBP monoclonal antibodies mark are 5：5)、2
With the reagent in 3, value standard product is selected to be tested as blank sample, on automatic clinical chemistry analyzer (stepping auspicious BS-400),
37 DEG C of temperature, 700nm wavelength under the conditions of 1cm optical paths, test absorbance value.Retest 20 times, the standard established according to Fig. 1
Curve calculates the average value of 20 test resultsWith standard deviation (SD),Analysis as reagent is sensitive
It spends (blank limit).3 groups of test results are as shown in table 1, and display sensitivity for analysis is respectively 2.745,3.353 and 1.298ng/mL,
Prove the sensitivity highest of the emulsion reagent of monoclonal antibody label, the latex of monoclonal antibody label is added in the latex of how anti-label to be carried
Rise the sensitivity of reagent.The detection clinical reference value of heparin-binding protein (HBP) is in 30ng/mL or so, the spirit of reagent of the present invention
Sensitivity an order of magnitude smaller than reference value, complies fully with the needs used.
The reagent analysis sensitivity test result of 1. embodiment 1,2 and 3 of table
Embodiment 7
The range of linearity of reagent
With the high concentration HBP standard items of zero HBP standard items diluted concentrations 240ng/mL, 240ng/mL, 100ng/ is respectively prepared
ML, 50ng/mL, 20ng/mL, 5ng/mL, 0ng/mL totally 6 different diluted concentration samples.Use (the HBP monoclonal antibody marks of embodiment 1
The present latex particulate of note and the mass ratio of the present latex particulate of how anti-label are 6：4) reagent and in embodiment 4 prepared is respectively to these
Sample is tested, and each diluted concentration is tested 3 times, and the mean value of each diluted concentration testing result is obtained.Measurement result is shown in Table
2.Using diluted concentration as independent variable, equation of linear regression is obtained using testing result mean value as dependent variable, calculates the phase of linear regression
Relationship number (r).Linear correlation curve is shown in Fig. 4 and Fig. 5.
The range of linearity measurement result of 2. embodiment 1 of table and embodiment 4
Embodiment 8
The repeatability of reagent
It is dense with the control substance and HBP of the normal value level of a concentration of 8ng/mL of reagent test HBP prepared in embodiment 2 or so
Spend the control substance of the exceptional value level for 40ng/mL or so, each retest 10 times calculates the average value of measured valueWith standard deviation (SD).The coefficient of variation (CV) is calculated by formula (1).Test result is shown in Table 3.According to measurement result, calculate
Go out coefficient of variation CV=7.66% and 4.32%, meet the technology requirement of reagent C V≤10%.
In formula：
CV --- the coefficient of variation；
SD --- standard deviation；
--- the average value of measured value.
The repetition measurement result of 3. embodiment 2 of table
With the control substance of normal value level of the 5 group reagents test HBP a concentration of 8ng/mL prepared in embodiment 1 or so with
The control substance of the exceptional value level of a concentration of 40ng/mL of HBP or so, each retest 10 times calculate the average value of measured valueWith standard deviation (SD).The coefficient of variation (CV) is calculated by formula (1).Test result is shown in Table 4 and table 5.It is tied according to measuring
Fruit, the coefficient of variation for calculating the control substance of a concentration of 8ng/mL of HBP be respectively CV=10.39%, 12.06%,
8.44%th, 7.98% and 6.44%；The coefficient of variation of the control substance of a concentration of 40ng/mL of HBP be respectively CV=5.12%,
5.42%th, 5.27%, 4.14% and 3.42%, the results showed that monoclonal antibody is 2 with how anti-label latex quality ratio：8 and 3：When 7 not
Meet the technology requirement of reagent C V≤10%, monoclonal antibody is 4 with how anti-label latex quality ratio：6、5：5 and 6：When 4, meet reagent
The technology requirement of CV≤10%.
The repetition measurement result (Quality Control concentration 8ng/mL) of 4. embodiment 1 of table
The repetition measurement result (Quality Control concentration 40ng/mL) of 5. embodiment 1 of table
Embodiment 9
Hook-shaped (Hook) effect
Commercially available HBP antigens are configured to a series of antigenic solution of concentration with standard dilutions, concentration is respectively 240ng/
ML, 3000ng/mL, 400ng/mL, 500ng/mL, 600ng/mL, 700ng/mL, 800ng/mL, 900ng/mL, 1000ng/
mL.Fig. 6 be in embodiment 9 degree of reaction with the variation relation of antigen concentration.Latex enhancing immune provided by the invention is than turbid reagent
Box does not occur Hook effects in 600ng/mL.Fig. 6 is the hook effect curve of embodiment 9.
Embodiment 10
With the correlation analysis of import ELISA kit
In the present embodiment, we have collected 264 clinical serums or plasma sample, using the reagent and POCT in embodiment 2
Equipment is detected, and testing result and the testing result of import reagent box in the market have carried out correlation analysis.Analysis result
As shown in Figure 7.The result of correlation fitting a straight line shows that fit equation is y=0.968x+0.5102, and wherein Y intercept is
0.5102, slope 0.968, coefficient R=0.9817.This is the result shows that kit and import listing in the present invention try
Agent box correlation is high, and accuracy is very consistent.Correlation analysis figure as shown in Figure 7.
Embodiment 11
It is compared with the performance parameter of other kits in the market
The performance parameter of reagent of the present invention and other HBP kits in the market compares situation as listed by table 6.It can from table
Go out, box reagent of the invention is mainly in detection sample type, the amount of reagent needed and type, the range of linearity of reagent, reagent
Detection speed and precision in terms of be substantially better than contrast agent box in the market.
Table 6.HBP kits performance parameter compares
Above-described is only some embodiments of the present invention, it is noted that for those of ordinary skill in the art,
Under the premise of the creation design of the present invention is not departed from, other modification and improvement can also be made, these belong to the present invention's
Protection domain.
Claims (17)
1. heparin-binding protein detection kit, which is characterized in that the heparin-binding protein detection kit includes dilution
R1 and reaction solution R2 and calibration object and quality-control product, the reaction solution R2 include the present latex particulate of HBP antibody label.
2. heparin-binding protein detection kit according to claim 1, which is characterized in that the dilution R1 includes slow
Solution, surfactant, salt and preservative are rushed, the reaction solution R2 includes buffer solution, salt, stabilizer, suspending agent and anti-corrosion
Agent, the calibration object and quality-control product include heparin-binding protein, buffer solution, salt, stabilizer and preservative.
3. heparin-binding protein detection kit according to claim 2, which is characterized in that the HBP antibody label
Present latex particulate be monoclonal antibody label present latex particulate or how anti-label present latex particulate or HBP monoclonal antibodies label present latex particulate with
The mixture of the present latex particulate of the how anti-labels of HBP, the monoclonal antibody are a pair of or several HBP monoclonal antibodies to pairing.
4. heparin-binding protein detection kit according to claim 3, the present latex particulate of the HBP antibody label is single
The mixture of the present latex particulate of anti-label and the present latex particulate of how anti-label, the present latex particulate of the HBP antibody label is monoclonal antibody
The quality of the present latex particulate of label and the present latex particulate of how anti-label ratio range is 2：8 to 8：2.
5. heparin-binding protein detection kit according to claim 3, which is characterized in that the HBP antibody label
A diameter of 100nm ~ the 400nm of present latex particulate used in present latex particulate, the present latex particulate for chemical crosslinking type present latex particulate or
Physisorption type present latex particulate.
6. heparin-binding protein detection kit according to claim 5, which is characterized in that the chemical crosslinking type latex
Its surface modification group of particle is-COOH, one kind in-NH2 ,-SH ,-OH ,-CHO.
7. heparin-binding protein detection kit according to claim 4, which is characterized in that the HBP antibody label
Present latex particulate mass concentration is 0.01% ~ 1%.
8. heparin-binding protein detection kit according to claim 2, which is characterized in that the stabilizer is cow's serum
One kind in albumin, casein, gelatin；The salt for sodium chloride, potassium chloride, sodium sulphate, one kind in potassium sulfate, two kinds or
Two or more combinations；The surfactant is NP40, triton x-100, polysorbas20, polysorbate40, Tween 80, dodecyl
Sodium sulphate（SDS）, span 40, one kind in sorbester p18, two or more combination；The suspending agent is sucrose, grape
One kind, two or more combination in sugar, maltose, trehalose, mannitol, ethylene glycol, glycerine, lactose；It is described
Preservative be sodium azide, thimerosal, one kind in Proclin-300.
9. heparin-binding protein detection kit according to claim 8, which is characterized in that the stabilizer is cow's serum
Albumin, the bovine serum albumin(BSA) concentration 0.2%-2%；The suspending agent be concentration trehalose, the trehalose concentration 0.2%-
5%；The preservative is Proclin-300, and the concentration of preservatives is 0.05%-2%；The surfactant it is a concentration of
0.1%~2%。
10. heparin-binding protein detection kit according to claim 2, which is characterized in that the buffer solution is sweet ammonia
Acid buffer, 4- hydroxyethyl piperazineethanesulfonic acids（HEPES）Buffer solution, 3- (N- morpholines) -2- hydroxy-propanesulfonic acids（MOPSO）Buffering
Liquid, trihydroxy aminomethane hydrochloric acid（Tris-HCl）Buffer solution, phosphoric acid physiological saline（PBS）Buffer solution and 2-morpholine ethane sulfonic acid
（MES）Any of which of buffer solution, the range of buffer concentration are 10-200mmol/L.
11. the preparation method of heparin-binding protein detection kit, which is characterized in that the method uses claim 1 ~ 10
Heparin-binding protein detection kit described in any one formula composition, the present latex particulate labelled antibody of the HBP antibody label
Method be physisorphtion, any one in intermediate ester process, one-step method or two-step method.
12. the preparation method of heparin-binding protein detection kit according to claim 10, which is characterized in that in described
Between ester process be using Carbodiimides and succinimide ester activation of catalyst present latex particulate surface carboxyl, formed surface band
There is the present latex particulate of succinimide ester intermediate, add HBP antibody, amino and the present latex particulate surface of HBP antibody surfaces
Succinimide ester condensation, that is, complete HBP antibody labeling process.
13. the preparation method of heparin-binding protein detection kit according to claim 10, which is characterized in that described one
Footwork is that HBP antibody is first mixed with present latex particulate, and HBP antibody is adsorbed on present latex particulate surface, then adds in carbodiimides
Class catalyst, the carboxyl or amino of HBP antibody surfaces are condensed with the carboxyl on polystyrene latex microparticles surface or amino, are completed
HBP antibody labeling process.
14. the preparation method of heparin-binding protein detection kit according to claim 10, which is characterized in that described two
Footwork is the carboxyl using Carbodiimides activation of catalyst present latex particulate surface, then adds in HBP antibody, the amino of the two
With carboxyl direct polycondensation, HBP antibody labeling process is completed.
15. the preparation method of heparin-binding protein detection kit according to claim 10, which is characterized in that the object
It is to be used without the polystyrene latex microparticles of any surface active groups to manage absorption method, then adds in HBP antibody, HBP antibody
Absorption is in polystyrene latex microparticles surface, completion HBP antibody labeling process.
16. a kind of latex enhancing immune using double reagent is than the method for turbid reagent detection heparin-binding protein, which is characterized in that
Using heparin-binding protein detection kit any in claim 1 ~ 10, in Biochemical Analyzer or specific protein analyzer
On by the way that transmittance is turbid or the method for scattering turbidimetry measures the HBP contents in sample to be tested.
17. detection method according to claim 16, which is characterized in that used detection sample was carried within 6 hours
The fresh serum or plasma sample taken.
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| CN201711412733.6A CN108152512A (en) | 2017-12-25 | 2017-12-25 | Heparin-binding protein detection kit and preparation method thereof |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109613259A (en) * | 2018-12-20 | 2019-04-12 | 北京贝尔生物工程股份有限公司 | A kind of people's heparin-binding protein assay kit of highly sensitive, wide detection range |
| CN110058015A (en) * | 2019-04-08 | 2019-07-26 | 深圳优普生物技术有限公司 | The measuring method and application of reagent, kit, platelet response index |
| CN110716057A (en) * | 2019-11-05 | 2020-01-21 | 安徽大千生物工程有限公司 | Kit for determining HBP (hepatitis B protein) based on latex enhanced immunoturbidimetry and preparation and use methods thereof |
| CN110806487A (en) * | 2019-12-02 | 2020-02-18 | 深圳上泰生物工程有限公司 | Kit for detecting human heparin binding protein and preparation method thereof |
| CN112051401A (en) * | 2020-08-28 | 2020-12-08 | 武汉生之源生物科技股份有限公司 | Pepsinogen's assay kit |
| CN112255416A (en) * | 2020-09-29 | 2021-01-22 | 北京利德曼生化股份有限公司 | Kit for quantitatively detecting HBP (hepatitis B protein) by using magnetic particle chemiluminescence as well as preparation and detection methods thereof |
| CN113189341A (en) * | 2021-02-26 | 2021-07-30 | 苏州优诺康生物技术有限公司 | Human heparin binding protein immunoturbidimetry detection reagent and application thereof |
| CN113945718A (en) * | 2020-03-04 | 2022-01-18 | 上海奥普生物医药股份有限公司 | Heparin binding protein detection kit, preparation method and application |
| WO2022021597A1 (en) * | 2020-07-30 | 2022-02-03 | 中翰盛泰生物技术股份有限公司 | Application of hbp in prognosis and risk warning for covid-19 patient |
| CN114324898A (en) * | 2022-03-11 | 2022-04-12 | 南京岚煜生物科技有限公司 | Binding pad treatment solution for heparin binding protein HBP detection |
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| CN102628864A (en) * | 2011-12-30 | 2012-08-08 | 北京九强生物技术股份有限公司 | Kit for determining heart-type fatty acid binding protein in serum or urine by latex enhanced turbidimetric immunoassay |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109613259A (en) * | 2018-12-20 | 2019-04-12 | 北京贝尔生物工程股份有限公司 | A kind of people's heparin-binding protein assay kit of highly sensitive, wide detection range |
| CN110058015A (en) * | 2019-04-08 | 2019-07-26 | 深圳优普生物技术有限公司 | The measuring method and application of reagent, kit, platelet response index |
| CN110716057A (en) * | 2019-11-05 | 2020-01-21 | 安徽大千生物工程有限公司 | Kit for determining HBP (hepatitis B protein) based on latex enhanced immunoturbidimetry and preparation and use methods thereof |
| CN110806487A (en) * | 2019-12-02 | 2020-02-18 | 深圳上泰生物工程有限公司 | Kit for detecting human heparin binding protein and preparation method thereof |
| CN113945718A (en) * | 2020-03-04 | 2022-01-18 | 上海奥普生物医药股份有限公司 | Heparin binding protein detection kit, preparation method and application |
| WO2022021597A1 (en) * | 2020-07-30 | 2022-02-03 | 中翰盛泰生物技术股份有限公司 | Application of hbp in prognosis and risk warning for covid-19 patient |
| CN112051401A (en) * | 2020-08-28 | 2020-12-08 | 武汉生之源生物科技股份有限公司 | Pepsinogen's assay kit |
| CN112255416A (en) * | 2020-09-29 | 2021-01-22 | 北京利德曼生化股份有限公司 | Kit for quantitatively detecting HBP (hepatitis B protein) by using magnetic particle chemiluminescence as well as preparation and detection methods thereof |
| CN113189341A (en) * | 2021-02-26 | 2021-07-30 | 苏州优诺康生物技术有限公司 | Human heparin binding protein immunoturbidimetry detection reagent and application thereof |
| CN114324898A (en) * | 2022-03-11 | 2022-04-12 | 南京岚煜生物科技有限公司 | Binding pad treatment solution for heparin binding protein HBP detection |
| CN114324898B (en) * | 2022-03-11 | 2022-05-31 | 南京岚煜生物科技有限公司 | Binding pad treatment solution for heparin binding protein HBP detection |
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Application publication date: 20180612 |