CN107121416A - A kind of fluorescence polarization assay method detected for immune-active peptides immunocompetence - Google Patents

A kind of fluorescence polarization assay method detected for immune-active peptides immunocompetence Download PDF

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Publication number
CN107121416A
CN107121416A CN201710252890.9A CN201710252890A CN107121416A CN 107121416 A CN107121416 A CN 107121416A CN 201710252890 A CN201710252890 A CN 201710252890A CN 107121416 A CN107121416 A CN 107121416A
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immune
solution
concentration
active peptides
influenza virus
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曹慧
艾青
张宇军
徐斐
袁敏
于劲松
叶泰
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University of Shanghai for Science and Technology
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6445—Measuring fluorescence polarisation
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4703—Regulators; Modulating activity
    • G01N2333/4706—Regulators; Modulating activity stimulating, promoting or activating activity

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  • General Physics & Mathematics (AREA)
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  • Urology & Nephrology (AREA)
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  • Biochemistry (AREA)
  • Analytical Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
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  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
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Abstract

The invention provides a kind of fluorescence polarization method detected for immune-active peptides immunocompetence, including the step of determination influenza virus fluorescent marker working concentration；One determines HLA DRB4The step of compound protein working concentration；One set up immune-active peptides Percentage bound curve the step of；One the step of prepare immune-active peptides sample；The step of one detection immune-active peptides immunocompetence, immune-active peptides are diluted to series concentration with distilled water, take the solution that cumulative volume is 200 μ L, the HLA DRB4 compound proteins solution and 100 μ L immunocompetence peptide solution of influenza virus fluorescent marker, 50 μ L containing 50 μ L in the solution, with ELIASA in absorbing wavelength it is that 492nm, launch wavelength are to detect its fluorescence polarization luminous intensity under 520nm after the solution is incubated, then Computation immunity active peptide IC50 values.The present invention is a kind of easy, quick immunocompetence detection method.

Description

A kind of fluorescence polarization assay method detected for immune-active peptides immunocompetence
Technical field
The invention belongs to analyze detection field, it is related to a kind of activity test method of functional components, specifically one Planting is used for the fluorescence polarization method that immune-active peptides immunocompetence is detected.
Background technology
Immune-active peptides typically refer to the toleragen epitope peptide or Altered peptide of allogenic gene restructuring or artificial chemistry synthesis. As a kind of small peptide, immune-active peptides have the characteristics of source is wide, activity is high, stability is strong, functional food, health products and There is vast application prospect in the fields such as Medicines.At present, the immune-active peptides overwhelming majority derives from natural products, at this In the screening process of a little immune-active peptides, lymphopoiesis method is conventional immunocompetence detection method.The method operating process Cumbersome, detection time is long, and sensitivity is low, and testing result is originated by lymphocyte, operating personnel's professional standards and operating environment Deng the interference of factors, hence it is imperative that development is a kind of simpler and accurate.
Research in recent years finds that immune-active peptides have different epitopes, when immune-active peptides are offered by antigen When cell (APC) is recognized, these epitopes can be specifically bound with the MHC quasi-molecules on APC surfaces, formation MHC quasi-molecules-exempt from Epidemic disease activity peptide complexes, the compound is specifically bound with T cell surface receptor (TCR) molecule, and then it is anti-to activate itself The immune response of answering property T lymphocytes, makes its activation, propagation.
The content of the invention
For above-mentioned technical problem of the prior art, it is used for the inspection of immune-active peptides immunocompetence the invention provides one kind The fluorescence polarization method of survey, the described this fluorescence polarization method detected for immune-active peptides immunocompetence will solve existing Detect that the immunocompetent method operating process of immune-active peptides is cumbersome in technology, detection time is long, the low technical problem of sensitivity.
The invention provides a kind of fluorescence polarization method detected for immune-active peptides immunocompetence, it is characterised in that bag Include following steps：
1) one the step of determine influenza virus fluorescent marker working concentration, with 100mmol/L, pH5.5 citric acid- Sodium citrate buffer solution takes 200 μ L in 96 orifice plates in 5 times of ratio diluent stream Influenza Virus fluorescent markers, 37 DEG C of incubation 72h, In absorbing wavelength it is 492nm with SpectraMax M5 ELIASAs, launch wavelength is to detect fluorescence polarization luminous intensity under 520nm, with Reach the Cmin corresponding to stable polarized value as the working concentration of influenza virus fluorescent marker；
2) a determination HLA-DRB4The step of compound protein working concentration, with 100mmol/L, pH5.5 citric acid-lemon Lemon acid sodium buffer solution presses 1:2、1:4、1:8、1:80 and 1:800 dilution proportion HLA-DRB4Compound protein solution；Take 100 μ L HLA-DRB4Then compound protein solution add influenza virus fluorescent marker and 100mmol/L, pH5.5 lemon in 96 orifice plates Lemon acid-sodium citrate buffer solution, the working concentration for determining the final concentration of step of influenza virus fluorescent marker (1), makes simultaneously It is 200ul to obtain the volume in 96 orifice plates；Reaction solution is mixed and is incubated 72min after 37 DEG C, with SpectraMax M5 ELIASAs It is that 492nm, launch wavelength are to detect fluorescence polarization luminous intensity under conditions of 520nm in absorbing wavelength, with maximum polarization value 60% Corresponding HLA-DRB4Compound protein concentration is used as its best effort concentration；
3) one set up immune-active peptides Percentage bound curve the step of, using the plain concentration of influenza virus as abscissa, with reference to Rate is the Percentage bound curve that ordinate sets up immune-active peptides, and the calculation formula of Percentage bound is as follows：
In formula：FPSampleFor the fluorescence polarization value of detected sample, FPMark peptideFor influenza virus fluorescent label peptide correspondence Fluorescence polarization value, FPIt is uncontestedFor influenza virus fluorescent marker fluorescence labeling peptide and HLA-DRB4The fluorescence polarization of compound protein Value；
4) one weighs 200mgII collagen types the step of prepare immune-active peptides sample, plus distilled water is configured to 1mg/mL solution, is placed in the enzyme reactor that temperature is 50 DEG C, is that 80U/ml adds neutral protease enzymolysis 3h by enzyme concentration, The enzyme activity of protease is 10000U/g, collects enzymolysis liquid, enzymolysis liquid is concentrated in 40 DEG C, 100rpm Rotary Evaporators；Make Its final concentration of 4mg/ml；
5) the step of detection immune-active peptides immunocompetence, uses double by concentration for 4mg/ml immune-active peptides sample Steam water and be diluted to series concentration by 100 times of extension rates, take the stream containing 50 μ L in the solution that cumulative volume is 200 μ L, the solution The immunocompetence peptide solution of Influenza Virus fluorescent marker, 50 μ L HLA-DRB4 compound proteins solution and 100 μ L, described stream The concentration of Influenza Virus fluorescent marker meets step 1) result, the concentration of described HLA-DRB4 compound protein solution meets step Rapid result 2), the solution is placed in 96 orifice plates, 37 DEG C of incubation 72min after mixing, then is existed with SpectraMax M5 ELIASAs Absorbing wavelength is that 492nm, launch wavelength are to detect its fluorescence polarization luminous intensity under 520nm, then Computation immunity active peptide IC50 Value, wherein, IC50 values are defined as the concentration of immune-active peptides when Percentage bound is 50% in competitive trials.
The principle that the present invention can be specifically bound according to MHC quasi-molecules with immune-active peptides, it is proposed that one kind is used to be immunized The fluorescence polarization immunoassay of active peptide immunocompetence detection.Fluorescence polarization immunoassay (fluorescence Polarization immunoassay, FPIA) it is a kind of quantitative immunoassay technology, its general principle is fluorescent material through list After blue polarised light (485nm) irradiation of one plane, absorb luminous energy and leap to excitation state, then return back to ground state, and send single flat The polarized fluorescence in face.
The present invention using influenza virus coupling the synthesizing fluorescently labeled thing of FITC fluoresceins, and using the fluorescent marker as Competitor, make its with immune-active peptides to be measured competitively with HLA-DRB4Compound protein is combined, and passes through the calculating to Percentage bound Establish the fluorescence polarization quantitative detecting method of immunocompetence peptide activity.
The present invention is compared with prior art, and its technological progress is significant.The immune work of immune-active peptides that the present invention is set up Property FPIA method, be that the high frequency zone of immune-active peptides and preparation provide a kind of reliable analysis hand Section.The IC that the method is detected to immune-active peptides immunocompetence50For 98.32ng/mL, linear range is 9.82~9985.43ng/mL (IC20-IC80), with higher sensitivity.The method of the present invention has quick, easy, efficient and reliable advantage, the present invention Method only need to sample-adding, without separation and washing operation.It at home and abroad there is no on using fluorescence polarization immunoassay inspection Survey the immunocompetent method of immune-active peptides.
Brief description of the drawings
Fig. 1 is the Percentage bound curve for the influenza virus element set up with fluorescence polarization method.
Fig. 2 is the immune-active peptides Percentage bound curve set up with fluorescence polarization method.
Embodiment
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention Rather than limitation the scope of the present invention.In addition, it is to be understood that after the content of the invention lectured has been read, people in the art Member can make various changes or modifications to the present invention, and these equivalent form of values equally fall within the application appended claims and limited Scope.In addition, the raw material that the present invention is used is the material that in the market can be bought.
Embodiment 1
The determination of influenza virus fluorescent marker working concentration, is concretely comprised the following steps：
With 100mmol/L, pH5.5 citric acid-sodium citrate buffer solution in 5 times of ratio diluent stream Influenza Virus fluorescence labelings Thing (is purchased from Nanjing Tai Ye bio tech ltd), takes 200 μ L in 96 orifice plates, 37 DEG C of incubation 72h, uses SpectraMax M5 ELIASAs absorbing wavelength be 492nm, launch wavelength be 520nm under detect its fluorescence polarization luminous intensity.When polarization value reaches After stable, the Cmin of corresponding influenza virus fluorescent marker is 5 μM, in this, as influenza virus fluorescent marker Working concentration.
Embodiment 2
The determination of MHC working concentrations, is concretely comprised the following steps：
1 is pressed with 100mmol/L, pH5.5 citric acid-sodium citrate buffer solution:2、1:4、1:8、1:80 and 1:800 ratio Example dilution HLA-DRB4Compound protein solution (is purchased from Wuhan Sanying Bio-Technology Co., Ltd.)；Take 100 μ L HLA-DRB4It is multiple Hop protein solution adds the influenza virus fluorescent marker of equal volume in 96 orifice plates, makes influenza virus fluorescent marker whole Concentration is the working concentration that step (1) is determined；Reaction solution is mixed and is incubated 72min after 37 DEG C, with SpectraMax M5 enzymes Mark instrument is that 492nm, launch wavelength are to detect its fluorescence polarization luminous intensity under conditions of 520nm in absorbing wavelength.Work as fluorescence polarization When luminous intensity reaches the 60% of maximum polarization value, corresponding HLA-DRB4Compound protein concentration is 125nM, and this is HLA-DRB4 The best effort concentration of compound protein.See accompanying drawing 1.
Embodiment 3
Set up the Percentage bound curve of immune-active peptides：Using the plain concentration of influenza virus as abscissa, Percentage bound is built for ordinate The Percentage bound curve of vertical immune-active peptides.The calculation formula of Percentage bound is as follows：
In formula：FPSampleFor the fluorescence polarization value of detected sample, FPMark peptideFor influenza virus fluorescent label peptide correspondence Fluorescence polarization value, FPIt is uncontestedFor influenza virus fluorescent marker fluorescence labeling peptide and HLA-DRB4The fluorescence polarization of compound protein Value.
Embodiment 4
The preparation of immune-active peptides sample：200mgII collagen types are weighed, plus distilled water is configured to the molten of 1mg/mL Liquid, is placed in the enzyme reactor that temperature is 50 DEG C.It is that 80U/ml additions neutral proteinase (is purchased from Chinese medicines group chemistry by enzyme concentration Reagent Co., Ltd, enzyme activity is 10000U/g) enzymolysis 3h, collects enzymolysis liquid.By enzymolysis liquid in 40 DEG C, 100rpm rotary evaporation Concentrated in instrument.
Embodiment 5
The immunocompetent detection of immune-active peptides, is comprised the following steps that：
Concentration is diluted to series concentration with distilled water for 4mg/ml immune-active peptides by 100 times of extension rates.Take total Volume is 200 μ L solution, is containing the influenza virus fluorescent marker, 50 μ L concentration that 50 μ L concentration are 5 μM in the solution 125nM HLA-DRB4The immunocompetence peptide solution of compound protein solution and 100 μ L.The solution is placed in 96 orifice plates, mixed 37 DEG C of incubation 72min after even, then with SpectraMax M5 ELIASAs in absorbing wavelength be under 492nm, launch wavelength are 520nm Detect its fluorescence polarization luminous intensity.Blank control is used as using distilled water.Calculate IC50Value, IC50Value is defined as in competitive trials The concentration of immune-active peptides when middle Percentage bound is 50%.As a result the IC of immune-active peptides is shown50=64.36ng/ml.See accompanying drawing 2。

Claims (1)

1. a kind of fluorescence polarization method detected for immune-active peptides immunocompetence, it is characterised in that comprise the following steps：
1) the step of determination influenza virus fluorescent marker working concentration, with 100mmol/L, pH5.5 citric acid-lemon Sour sodium buffer solution takes 200 μ L in 96 orifice plates in 5 times of ratio diluent stream Influenza Virus fluorescent markers, 37 DEG C of incubation 72h, uses SpectraMax M5 ELIASAs absorbing wavelength be 492nm, launch wavelength be 520nm under detect fluorescence polarization luminous intensity, with up to To working concentration of the Cmin corresponding to stable polarized value as influenza virus fluorescent marker；
2) a determination HLA-DRB4The step of compound protein working concentration, with 100mmol/L, pH5.5 citric acid-citric acid Sodium buffer solution presses 1:2、1:4、1:8、1:80 and 1:800 dilution proportion HLA-DRB4Compound protein solution；Take 100 μ L HLA- DRB4Compound protein solution added in 96 orifice plates, then influenza virus fluorescent marker and 100mmol/L, pH5.5 citric acid- Sodium citrate buffer solution, the working concentration for determining the final concentration of step of influenza virus fluorescent marker (1), while so that 96 holes Volume in plate is 200ul；Reaction solution is mixed and is incubated 72min after 37 DEG C, is being absorbed with SpectraMax M5 ELIASAs Wavelength is that 492nm, launch wavelength are to detect fluorescence polarization luminous intensity under conditions of 520nm, corresponding with maximum polarization value 60% HLA-DRB4Compound protein concentration is used as its best effort concentration；
3) one set up immune-active peptides Percentage bound curve the step of, using the plain concentration of influenza virus as abscissa, Percentage bound is Ordinate sets up the Percentage bound curve of immune-active peptides, and the calculation formula of Percentage bound is as follows：
In formula：FPSampleFor the fluorescence polarization value of detected sample, FPMark peptideIt is corresponding glimmering for influenza virus fluorescent label peptide Light polarization value, FPIt is uncontestedFor influenza virus fluorescent marker fluorescence labeling peptide and HLA-DRB4The fluorescence polarization value of compound protein；
4) one weighs 200mgII collagen types the step of prepare immune-active peptides sample, plus distilled water is configured to 1mg/mL Solution, be placed in during temperature is 50 DEG C of enzyme reactor, be that 80U/ml adds neutral protease enzymolysis 3h, protease by enzyme concentration Enzyme activity be 10000U/g, collect enzymolysis liquid, enzymolysis liquid concentrate in 40 DEG C, 100rpm Rotary Evaporators, make its end it is dense Spend for 4mg/ml；
5) the step of detection immune-active peptides immunocompetence, by the immune-active peptides sample distilled water that concentration is 4mg/ml Series concentration is diluted to by 100 times of extension rates, the influenza disease containing 50 μ L in the solution that cumulative volume is 200 μ L, the solution is taken The immunocompetence peptide solution of malicious fluorescent marker, 50 μ L HLA-DRB4 compound proteins solution and 100 μ L, described influenza disease The concentration of malicious fluorescent marker meets step 1) result, the concentration of described HLA-DRB4 compound protein solution meets step 2) Result, the solution is placed in 96 orifice plates, after mixing 37 DEG C incubation 72min, then with SpectraMax M5 ELIASAs absorption Wavelength is that 492nm, launch wavelength are to detect its fluorescence polarization luminous intensity under 520nm, then Computation immunity active peptide IC50 values, its In, IC50 values are defined as the concentration of immune-active peptides when Percentage bound is 50% in competitive trials.
CN201710252890.9A 2017-04-18 2017-04-18 A kind of fluorescence polarization assay method detected for immune-active peptides immunocompetence Pending CN107121416A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111855827A (en) * 2019-04-24 2020-10-30 岛津企业管理（中国）有限公司 Method for determining polysaccharide protein binding site binding rate in polysaccharide protein binding vaccine

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CN1237242A (en) * 1997-09-05 1999-12-01 松下电器产业株式会社 Fluorescence polarization method
CN105136755A (en) * 2015-08-11 2015-12-09 中国农业大学 Fluorescence polarization immunoassay method for detection of erythromycin

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CN1237242A (en) * 1997-09-05 1999-12-01 松下电器产业株式会社 Fluorescence polarization method
CN105136755A (en) * 2015-08-11 2015-12-09 中国农业大学 Fluorescence polarization immunoassay method for detection of erythromycin

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Title
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LIUSONG YIN 等: "A novel method to measure HLA-DM-susceptibility of peptides bound to MHC class II molecules based on peptide binding competition assay and differential IC50 determination", 《JOURNAL OF IMMUNOLOGICAL METHODS》 *
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111855827A (en) * 2019-04-24 2020-10-30 岛津企业管理（中国）有限公司 Method for determining polysaccharide protein binding site binding rate in polysaccharide protein binding vaccine
CN111855827B (en) * 2019-04-24 2022-09-16 岛津企业管理（中国）有限公司 Method for determining polysaccharide protein binding site binding rate in polysaccharide protein binding vaccine

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