CN104181302A - Method and kit for performing quick co-detection on anti-Sp (Streptococcus pneumoniae) IgM (Immunoglobulin M) and IgG (Immunoglobulin G) antibodies based on magnetic separation and multi-color quantum dot labeling - Google Patents

Method and kit for performing quick co-detection on anti-Sp (Streptococcus pneumoniae) IgM (Immunoglobulin M) and IgG (Immunoglobulin G) antibodies based on magnetic separation and multi-color quantum dot labeling Download PDF

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CN104181302A
CN104181302A CN201410404733.1A CN201410404733A CN104181302A CN 104181302 A CN104181302 A CN 104181302A CN 201410404733 A CN201410404733 A CN 201410404733A CN 104181302 A CN104181302 A CN 104181302A
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human
igm
streptococcus pneumonia
antibody
damping fluid
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CN104181302B (en
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胡征
杨波
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Hubei Hualong Biological Pharmaceutical Co., Ltd.
Hubei University of Technology
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Hubei University of Technology
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • 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
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
    • 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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/56944—Streptococcus
    • 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
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/315—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Streptococcus (G), e.g. Enterococci
    • G01N2333/3156—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Streptococcus (G), e.g. Enterococci from Streptococcus pneumoniae (Pneumococcus)

Abstract

The invention discloses a method and a kit for performing quick co-detection on anti-Sp (Streptococcus pneumoniae) IgM (Immunoglobulin M) and IgG (Immunoglobulin G) antibodies based on magnetic separation and multi-color quantum dot labeling. The kit consists of anti-Sp antibody capturing nano magnetic beads with an anti-Sp IgM and IgG antibody gathering function, anti-human IgM and IgG antibody nano probes labeled by multi-color quantum dots, quality control substances and a PBST buffering solution, wherein the quality control substance comprise a positive quality control substance and a negative quality control substance; the positive quality control substance is serum, in which anti-Sp IgM and IgG antibodies of Sp infected people are respectively positive; the negative quality control substance is serum, in which anti-Sp IgM and IgG are respectively negative. The kit and the method have the advantages of simplicity, quickness and high sensitivity, and can be used for carrying out synchronous detection on the anti-Sp IgM and IgG antibodies.

Description

Method and kit that anti-human streptococcus pneumonia IgM based on magnetic resolution and color quantum point mark, IgG antibody are examined fast altogether
Technical field
The present invention relates to technical field of medical detection, be specially a kind of anti-human streptococcus pneumonia IgM, IgG antibody based on magnetic resolution and color quantum point mark and be total to fast detection method and the detection kit of inspection, and the method for preparation and use of this detection kit.
Background technology
People streptococcus pneumonia (Streptococcus pneumoniae, Sp) is the important pathogen body of childrens respiratory tract infection.Mainly through droplet transmission, enter respiratory tract, thereby parasitize the pharynx nasalis of human body, or invade human body and do not cause a series of disease in the position of easy-clear, as lobar pneumonia, meningitis, bronchitis, tympanitis etc.It is the main pathogenic fungi of global all age group high incidences and case fatality rate.Wherein, in developing country, particularly serious in infant, the elderly and immunodeficiency crowd.Streptococcus pneumonia was isolated from patient's sputum in France and the U.S. respectively by Pasteur (Louis Pasteur) and G.M.Sternberg first in 1881.It is Gram-positive, and thalline is like spearhead shape, becomes two or becomes the diplococcus of short catenation, and it is the pod membrane of polysaccharide that toxic strain thalline has chemical composition outward.Its pod membrane has antigenicity, is the foundation of streptococcus pneumonia somatotype.According to the antigenic difference of capsular polysaccharide, pneumococcus is divided into 91 serotypes.Streptococcus pneumonia somatic antigen is mainly C polysaccharide, and it is present in pneumococcal cell wall, has species specificity, for various bacterial strain common.C polysaccharide can be precipitated by C reactive protein in serum.When calcium ion exists, C polysaccharide can with normal human serum in be called the beta Globulin combination of C reactive protein (C reactive protein, CRP), precipitate.Detection for people's streptococcus pneumoniae antigen is mainly also for this antigen at present, and the non-streptococcus pneumonia of this antigen is exclusive, as relaxed streptococcus, also contains this antigen.On streptococcus pneumonia surface, also have a kind of important antigen relevant to virulence, be pneumococcal surface protein A (PspA), it is present in all serotype of streptococcus pneumonia, is the specific antigen of streptococcus pneumonia.But the variation of PspA molecular structure height, has antigen diversity, and the domain upstream of its Pro-rich is called as CDR territory, has diversity.According to the difference in CDR territory, PspA is divided into 3 family's 6 subclass: Clade1, Clade2, Clade3, Clade4, Clade5, Clade6.Clade1 wherein, Clade2 belongs to Fam1 family; Clade3, Clade4, Clade5 belongs to Fam2 family, and Clade6 belongs to Fam3 family.The people streptococcus pneumonia with Fam1 or the PspA of Fam2 family albumen has accounted for the more than 99% of people streptococcus pneumonia kind of clinical separation.China yet there are no the clinical separated report of the streptococcus pneumonia with the PspA of fam3 family albumen.Research show between PspA albumen, to have antibody and antigen cross-reaction widely between same family subclass, between Er Yi family subclass without this cross reaction.
Clinical patient causes that because different respiratory pathogen (as parainfluenza virus, haemophilus influenzae, influenza virus, Respiratory Syncytial Virus(RSV), adenovirus etc.) infects disease symptoms can be quite similar, this has caused popular diagnosis more difficult, makes a definite diagnosis and often depends on laboratory diagnosis.Diagnostic method should be that their early stage in disease just can obtain clear and definite diagnosis fast and effectively, is convenient to implement treatment targetedly, stops the development delay of the state of an illness.
In the laboratory diagnosis of people's streptococcus pneumoniae infection, in examinee's serum, anti-pneumonia streptococcus bacteria antibody index is one of important evidence of diagnostic result.
Immunoglobulin M (Immunoglobulin M, IgM) and immunoglobulin G (Immunoglobulin G, IgG) are most important two kinds of antibody in human body.When people streptococcus pneumonia enters after body, the immunoglobulin (Ig) occurring is the earliest IgM antibody, occurs afterwards IgG, by the existence of detection bodies internal specific IgM, IgG antibody, and the immune response state of diagnosable human body to people streptococcus pneumonia.If detect specific IgM antibodies, show to have the generation of recent infection, but IgM is shorter antibody half life, the detection feminine gender of IgM can not prove body uninfection, also needs to detect the half life period longer, the IgG antibody that content is the highest, to clarify a diagnosis.
At present, in serum, the detection method of specific IgM and IgG antibody mainly contains the experiment of condensation collection, gelatin particle aggegation experiment, enzyme-linked immunosorbent assay, ' Jinbiao ' immunization spot method and gold-marking immunity chromatography.The specificity of condensation collection experiment and susceptibility are minimum, so its diagnostic value is little; Though ' Jinbiao ' immunization spot method and gold-marking immunity chromatography are simple and efficient to handle, susceptibility is lower slightly, and the patient that antagonist level is lower has the possibility of failing to pinpoint a disease in diagnosis; Gelatin aggegation experiment reagent is prepared with operating process comparatively loaded down with trivial details, and needs professional operating personnel.Enzyme linked immunosorbent assay has the advantages such as special, responsive, for checking various antibody or antigen, but there is following problem in the method: (1) every a collection of test from application of sample, that the steps such as version are bathed, washed to temperature is more, complex operation, the time is grown (altogether needing 2-4 hour); (2) in detection, need to add respectively color composition A liquid and B liquid, and also will complete at the appointed time reading, increased to a certain extent the possibility of labour intensity and misoperation; (3) when this method cannot be accomplished IgM and two kinds of antibody of IgG, detect.Although detect separately after IgM and IgG antibody, more comprehensively analyze testing result the diagnosis of disease be there is no to impact, operating process is loaded down with trivial details, detect convenient and swiftly not, testing cost altogether inspection also can increase.
Therefore, set up and to possess high sensitivity, can carry out the detection method that Fast synchronization examines altogether to the specific IgM of anti-human streptococcus pneumonia, IgG antibody and seem very necessary.
Summary of the invention
For these technical matterss that exist in background technology, the invention provides easy, quick, highly sensitive detection method and the kit of synchronously examining altogether anti-human streptococcus pneumonia IgM, IgG antibody of a kind of energy based on magnetic resolution and color quantum point mark, and the method for preparation and use of this kit.
The present invention is achieved through the following technical solutions:
The method that anti-human streptococcus pneumonia IgM based on magnetic resolution and color quantum point mark, IgG antibody are examined fast altogether, is characterized in that: said method comprising the steps of:
1) preparation of recombinant human streptococcus pneumonia PspA albumen;
2) by step 1) the recombinant human streptococcus pneumonia PspA albumen for preparing and nanometer magnetic bead be by covalent coupling, makes anti-human streptococcus pneumonia antibody capture nanometer magnetic bead;
3) nano-quantum point that is 520nm by mouse-anti people IgM monoclonal antibody and emission wavelength, by covalent coupling, is prepared quantum dot-labeled anti-human IgM nano-probe; The nano-quantum point that is 600nm by mouse-anti human IgG monoclonal antibody and emission wavelength, by covalent coupling, is prepared quantum dot-labeled anti-human IgG nano-probe; Quantum dot-labeled anti-human IgM nano-probe and quantum dot-labeled anti-human IgG nano-probe mixed in equal amounts are made to color quantum point anti-human IgM, the IgG nano-probe of mark respectively;
4) get human serum sample, after suitably diluting with PBSA damping fluid, in serum dilution, add step 2 respectively) the anti-human streptococcus pneumonia antibody capture nanometer magnetic bead and the step 3 that prepare) color quantum point for preparing anti-human IgM, the IgG nano-probe of mark respectively, fully mix, after reaction 10-45min, carry out magnetic separation, after PBST damping fluid washing 2 times, use fluorescence microplate reader, at emission wavelength (Em), be respectively and under 520nm and 600nm, read respectively fluorescent value; In described PBSA damping fluid, each component concentration is respectively: 8g/LNaCL, 0.2g/L KCl, 0.24g/L KH 2pO 4, 1.44g/L Na 2hPO 4, 5g/L BSA (bovine serum albumin(BSA)); The pH=7.4 of described PBSA damping fluid; In described PBST damping fluid, each component concentration is respectively: 8g/LNaCL, 0.2g/L KCl, 0.24g/L KH 2pO 4, 1.44g/L Na 2hPO 4, 5g/L BSA (bovine serum albumin(BSA)), 0.3g/L NaN 3, 0.5ml/L Tween-20; The pH=7.4 of described PBST damping fluid;
5) according to step 4) method detect at least 30 parts and be all defined as all negative human serum samples of anti-human streptococcus pneumonia IgM, IgG antibody through clinical the whole bag of tricks, read respectively emission wavelength (Em) and be respectively the fluorescent value under 520nm and 600nm; Described anti-human streptococcus pneumonia IgM, IgG antibody all negative human serum sample are called for short people streptococcus pneumonia negative antibody control sample; Mean value and 3 times of standard deviation sums of the fluorescent value that described people streptococcus pneumonia negative antibody control sample is 520nm at emission wavelength (Em) are designated as CUT-OFF1 value; Mean value and 3 times of standard deviation sums of the fluorescent value that described people streptococcus pneumonia negative antibody control sample is 600nm at emission wavelength (Em) are designated as CUT-OFF2 value; If step 4) in, human serum sample, at emission wavelength (Em) for the detection fluorescent value of 520nm is greater than CUT-OFF1 value, is judged as in human serum sample anti-human streptococcus pneumonia IgM antibody positive; If step 4) in, human serum sample, at emission wavelength (Em) for the detection fluorescent value of 520nm is less than CUT-OFF1 value, is judged as in human serum sample anti-human streptococcus pneumonia IgM antibody negative; If step 4) in, human serum sample, at emission wavelength (Em) for the detection fluorescent value of 600nm is greater than CUT-OFF2 value, is judged as in human serum sample anti-human streptococcus pneumonia IgG antibody positive; If step 4) in, human serum sample, at emission wavelength (Em) for the detection fluorescent value of 600nm is less than CUT-OFF2 value, is judged as in human serum sample anti-human streptococcus pneumonia IgG antibody negative.
Anti-human streptococcus pneumonia IgM based on magnetic resolution and color quantum point mark, IgG antibody are total to a check reagent box fast, it is characterized in that: described anti-human streptococcus pneumonia IgM, IgG antibody based on magnetic resolution and color quantum point mark is total to check reagent box fast, and by anti-human streptococcus pneumonia antibody capture nanometer magnetic bead, the color quantum point with the anti-human streptococcus pneumonia IgM of enrichment, IgG antibody function, anti-human IgM, IgG antibody nano-probe, quality-control product and the PBST damping fluid of mark are formed respectively; Described quality-control product comprises positive quality control product and negative quality-control product; Described positive quality control product is comprised of serum and two parts of positive serum of anti-human streptococcus pneumonia IgG antibody of two parts of people's streptococcus pneumoniae infection persons' anti-human streptococcus pneumonia IgM antibody positive; Described negative quality-control product is all negative human serums of four parts of anti-human streptococcus pneumonia IgM, IgG antibody.
For the preparation of the anti-human streptococcus pneumonia IgM based on magnetic resolution and color quantum point mark, IgG antibody, be total to fast a method for check reagent box, it is characterized in that: described preparation method comprises the following steps:
1) preparation of anti-human streptococcus pneumonia antibody capture nanometer magnetic bead:
1.1) preparation, the purifying of recombinant human streptococcus pneumonia PspA albumen:
1.1.1) people streptococcus pneumonia Fam1 PspA, Fam2 PspA albumen are carried out to bioinformatic analysis, obtain respectively the abundantest peptide section of epitope in people streptococcus pneumonia Fam1 PspA, Fam2 PspA albumen ectodomain, find its corresponding gene order;
1.1.2) at step 1.1.1) in 5 ' end of resulting gene order and 3 ' end introduce respectively restriction enzyme site chemosynthesis complete genome sequence respectively, with tense marker, be designated as PspA1, PspA2; Its sequence is referring to sequence table;
1.1.3) by step 1.1.2) in resulting PspA1, PspA2 by molecular biology method, be cloned into respectively and proceed to expression in escherichia coli restructuring PspA1-His, PspA2-His fusion after expression vector pET-28a (+); Described restructuring PspA1-His, PspA2-His fusion are all present in genetic engineering thalline with solubility expression form;
1.1.4) use ni-sepharose purification step 1.1.3) resulting restructuring PspA1-His, PspA2-His fusion, SDS-PAGE detects after its purity, by PBS damping fluid difference dialysed overnight; Collect dislysate, after bradford kit carries out determination of protein concentration, with PBS damping fluid, adjust respectively concentration and be 2mg/mL; In described PBS damping fluid, each component content is as follows: 8g/L NaCL, 0.2g/L KCl, 0.24g/L KH 2pO 4, 1.44g/LNa 2hPO 4, the pH=7.4 of described PBS damping fluid;
1.1.5) by step 1.1.4) two kinds of recombinant protein solution obtaining by volume 1:1 mix and make recombinant human streptococcus pneumonia PspA albumen;
1.2) nanometer magnetic bead is coated:
1.2.1) get 5mg magnetic bead, with 1ml MES damping fluid washing three times, be placed in nano magnetic separation vessel and carry out removing supernatant after magnetic separation; Described magnetic bead is with superparamagnetism Fe 3o 4for kernel, the particle diameter carboxyl magnetic bead that is 1150nm; Described MES damping fluid is that mass concentration is 2-(N-morpholino) ethyl sulfonic acid of 2g/L; The pH=6.0 of described MES damping fluid; The magnetic intensity of described nano magnetic separation vessel is 0.4T;
1.2.2) add successively and use step 1.2.1) in the concentration of MES damping fluid preparation be the EDC solution of 8-12mg/ml and use step 1.2.1) in the concentration prepared of MES damping fluid be each 0.5ml of sulfo-NHS solution of 6-10mg/ml, with 10-40rpm/min, in rotation mixed instrument, activate 1hr, being placed in nano magnetic separation vessel carries out removing supernatant after magnetic separation, with 1ml step 1.2.1) in MES damping fluid resuspended, the magnetic bead after being activated;
1.2.3) get 5 centrifuge tubes, in each centrifuge tube, add 200 μ L step 1.2.2) magnetic bead after resulting activation, being placed in nano magnetic separation vessel carries out removing supernatant after magnetic separation, to add in each centrifuge tube concentration with the dilution of PBS damping fluid be 100-400 μ g/ml by step 1.1.5) prepared each 1ml of recombinant human streptococcus pneumonia PspA protein solution, under room temperature, with 15rpm/min, in rotation mixed instrument, react 2-6h, being placed in nano magnetic separation vessel carries out removing after supernatant after magnetic separation, respectively add 1ml to contain the above-mentioned PBS damping fluid of 1mg/ml monoethanolamine, under room temperature with 15rpm/min in rotation react in mixed instrument 2h with sealing magnetic bead on not with the carboxyl of antibody response, in described PBS damping fluid, each component content is as follows: 8g/L NaCL, 0.2g/L KCl, 0.24g/L KH 2pO 4, 1.44g/L Na 2hPO 4, the pH=7.4 of described PBS damping fluid,
1.2.4) after capping completes, these 5 centrifuge tubes are placed in to nano magnetic separation vessel and carry out removing supernatant after magnetic separation, each washs three times with 1ml lavation buffer solution; In described lavation buffer solution, each component content is as follows: 8g/L NaCL, 0.2g/L KCl, 0.24g/L KH 2pO 4, 1.44g/L Na 2hPO 4, 0.5ml/L Tween-20, the pH=7.4 of described lavation buffer solution;
1.2.5) in each centrifuge tube, add respectively 1ml to preserve the resuspended magnetic bead of damping fluid, be placed in 4 ℃ and save backup; So far make anti-human streptococcus pneumonia antibody capture nanometer magnetic bead; In described preservation damping fluid, each component content is as follows: 8g/L NaCL, 0.2g/L KCl, 0.24g/L KH 2pO 4, 1.44g/L Na 2hPO 4, 0.3g/LNaN 3, 5g/L bovine serum albumin(BSA) (BSA), the pH=7.4 of described preservation damping fluid;
2) the color quantum point difference anti-human IgM of mark is, the preparation of IgG nano-probe:
Its concrete preparation method comprises:
2.1) in microcentrifugal tube, add successively 4nmol carboxyl water-soluble quantum dot, 600nmol N-hydroxy-succinamide sulfo-NHS and 600nmol carbodiimide EDC, take phosphate buffer constant volume as 5ml, mixed solution, after 37 ℃ of reaction 30min, the excessive sulfo-NHS as activator and EDC, the quantum dot after being activated are removed in dialysis; The emission wavelength of described carboxyl water-soluble quantum dot is 520nm; In described phosphate buffer, each component content is as follows: 2.9g/L sodium hydrogen phosphate, 0.295g/L sodium dihydrogen phosphate, 4g/L sodium chloride; The pH=7.4 of described phosphate buffer;
2.2) in step 2.1) in the quantum dot of resulting activation, add the mouse-anti people IgM monoclonal antibody of 8-16nmol, lucifuge reaction 2h, adds single-ended amination polyglycol PEG2000-NH 2to final concentration be 1%, seal unreacted activated carboxyl site, continue lucifuge reaction 1h;
2.3) with 0.2 μ m PES filter, remove by filter step 2.2) in antibody aggregation thing, then filtrate is transferred in 50000MW ultra-filtration centrifuge tube, with 8000g centrifugal force centrifugal 15min at 4 ℃, remove the antibody of coupling reaction and the accessory substance in reaction do not occur;
2.4) collect step 2.3) middle ultra-filtration centrifuge tube filter membrane upper strata quantum dot-antibody coupling matter solution, be dissolved in 2ml phosphate cleansing solution, again this solution is transferred in a new 50000MW ultra-filtration centrifuge tube, with 8000g centrifugal force centrifugal 15min at 4 ℃, collect ultra-filtration centrifuge tube filter membrane upper strata quantum dot-antibody coupling matter solution, be dissolved in 1ml phosphate and preserve in liquid, be placed in 4 ℃ and save backup, so far make quantum dot-labeled anti-human IgM nano-probe; In described phosphate cleansing solution, each component content is as follows: 2.9g/L sodium hydrogen phosphate, 0.295g/L sodium dihydrogen phosphate, 4g/L sodium chloride, 5ml/L Tween-20,0.3g/L Sodium azide, the pH=7.4 of described phosphate cleansing solution; In described phosphate preservation liquid, each component content is as follows: 2.9g/L sodium hydrogen phosphate, 0.295g/L sodium dihydrogen phosphate, 2g/L sodium chloride, 10g/L bovine serum albumin(BSA), 0.3g/L Sodium azide; Described phosphate is preserved the pH=7.4 of liquid;
2.5) by and step 2.1)-2.4) to utilize mouse-anti human IgG monoclonal antibody and emission wavelength be that the carboxyl water-soluble quantum dot of 600 nm makes quantum dot-labeled anti-human IgG nano-probe for identical method; By the 1:1 mixing by volume of above-mentioned two kinds of quantum dot-labeled nano-probe solution, make color quantum point anti-human IgM, the IgG nano-probe of mark respectively;
3) preparation of PBST damping fluid:
Its concrete compound method comprises:
Get 8g NaCL, 0.2g KCl, 0.24 KH 2pO 4, 1.44g Na 2hPO 4, 5g BSA, 0.3g NaN 3, 0.5ml Tween-20 is dissolved in 900ml distilled water, adjusts pH to 7.4, then be settled to 1000ml with 5M NaOH;
4) preparation of quality-control product:
4.1) positive quality control product:
4.1.1) anti-human streptococcus pneumonia IgM antibody positive quality-control product: the definite positive serum of anti-human streptococcus pneumonia IgM antibody by 0.5ml people's streptococcus pneumoniae infection person forms;
4.1.2) anti-human streptococcus pneumonia IgG antibody positive quality-control product: the definite positive serum of anti-human streptococcus pneumonia IgG antibody by 0.5ml people's streptococcus pneumoniae infection person forms;
4.2) negative quality-control product: by the anti-human streptococcus pneumonia IgM of 0.5ml, IgG antibody all negative human serum form.
As preferably, the present invention is at step 1.2.2) in, add successively and use step 1.2.1) in the concentration of MES damping fluid preparation be the EDC solution of 10mg/ml and use step 1.2.1) in the concentration prepared of MES damping fluid be each 0.5ml of sulfo-NHS solution of 8mg/ml, with 15rpm/min, in rotation mixed instrument, activate 1hr, being placed in nano magnetic separation vessel carries out removing supernatant after magnetic separation, with 1ml step 1.2.1) in MES damping fluid resuspended, the magnetic bead after being activated;
Described step 1.2.3) in, to add in each centrifuge tube concentration with the dilution of PBS damping fluid be 200 μ g/ml by step 1.1.5) prepared each 1ml of recombinant human streptococcus pneumonia PspA protein solution, under room temperature, with 15rpm/min, in rotation mixed instrument, react 3h, be placed in nano magnetic separation vessel and carry out removing after supernatant after magnetic separation, respectively add 1ml to contain the above-mentioned PBS damping fluid of 1mg/ml monoethanolamine;
Described step 2.2) in, in step 2.1) in the quantum dot of resulting activation, add the mouse-anti people IgM monoclonal antibody of 12nmol, lucifuge reaction 2h.
As preferably, quantum dot of the present invention is water-soluble CdSe/ZnS quantum dot that carboxylated amphipathic polymer is modified.
As preferably, magnetic bead of the present invention is with superparamagnetism Fe 3o 4for kernel, shell material are that polystyrene, surface functional group are the carboxyl magnetic bead that carboxyl, particle diameter are 1150nm.
Anti-human streptococcus pneumonia IgM based on magnetic resolution and color quantum point mark, IgG antibody are total to a using method for check reagent box fast, it is characterized in that: described using method comprises the following steps:
1) get serum sample 5 μ l to be checked with after 995 μ l PBSA damping fluid dilutions, dilution being proceeded in the common centrifuge tube of 1.5ml; In described PBSA damping fluid, each component content is as follows: 8g/L NaCL, 0.2g/L KCl, 0.24g/L KH 2pO 4, 1.44g/L Na 2hPO 4, 5g/L BSA; The pH=7.4 of described PBSA damping fluid;
2) to step 1) in centrifuge tube in add successively the anti-human streptococcus pneumonia IgM based on magnetic resolution and color quantum point mark, IgG antibody is total to anti-human streptococcus pneumonia antibody capture nanometer magnetic bead 10-100 μ l and the anti-human streptococcus pneumonia IgM based on magnetic resolution and color quantum point mark in check reagent box fast, IgG antibody is total to the anti-human IgM of the color quantum point difference mark in check reagent box fast, IgG nano-probe 50 μ l, after reacting 5-25min with 10rpm/min on rotation mixed instrument under room temperature, take off, centrifuge tube is inserted to the separated 3min of nano magnetic separation vessel magnetic, with pipettor sucking-off supernatant,
3) adding the anti-human streptococcus pneumonia IgM based on magnetic resolution and color quantum point mark, the PBST damping fluid 1ml that IgG antibody is total in check reagent box fast washs twice, sucking-off cleansing solution after the separation of employing nano magnetic separation vessel magnetic, finally use the resuspended magnetic bead of 0.2ml PBS damping fluid, make nanometer magnetic bead-streptococcus antibody-quantum dot " sandwich " compound; In described PBS damping fluid, each component content is as follows: 8g/L NaCL, 0.2g/L KCl, 0.24g/L KH 2pO 4, 1.44g/L Na 2hPO 4; The pH=7.4 of described PBS damping fluid;
4) by step 3) nanometer magnetic bead-streptococcus antibody-quantum dot compound of obtaining is loaded in ELISA Plate, use fluorescence microplate reader to be all 365nm in excitation wavelength (Ex), emission wavelength (Em) is respectively under the parameter of 520nm and 600nm distinguishes reading to its fluorescent value respectively;
5) by above-mentioned same method, detect at least 30 parts and be all defined as all negative human serum samples of anti-human streptococcus pneumonia IgM, IgG antibody through clinical the whole bag of tricks, read respectively emission wavelength (Em) and be respectively the fluorescent value under 520nm and 600nm; Described mean value and the 3 times of standard deviation sums that are all defined as anti-human streptococcus pneumonia IgM, the IgG antibody fluorescent value that all negative human serum sample is 520nm at emission wavelength (Em) through clinical the whole bag of tricks are designated as CUT-OFF1 value; Described mean value and the 3 times of standard deviation sums that are all defined as anti-human streptococcus pneumonia IgM, the IgG antibody fluorescent value that all negative human serum sample is 600nm at emission wavelength (Em) through clinical the whole bag of tricks are designated as CUT-OFF2 value; The four parts of negative quality-control product samples and the four parts of positive quality control product samples that in detection kit, provide read respectively emission wavelength (Em) and are respectively the fluorescent value under 520nm and 600nm simultaneously; The mean value of the fluorescent value that four parts of negative quality-control product samples are 520nm at emission wavelength (Em) is designated as NCX1; The mean value of the fluorescent value that four parts of negative quality-control product samples are 600nm at emission wavelength (Em) is designated as NCX2 value; Two parts of anti-human streptococcus pneumonia IgM antibody positive quality-control product samples emission wavelength (Em) for the fluorescence mean value of 520nm be PCX1; Two parts of anti-human streptococcus pneumonia IgG antibody positive quality-control product samples emission wavelength (Em) for and 600nm under fluorescent value be designated as PCX2; If step 4) in, serum sample to be checked, at emission wavelength (Em) for the detection fluorescent value of 520nm is greater than CUT-OFF1 value and PCX1/NCX1 >=2.1, is judged as in serum sample to be checked anti-human streptococcus pneumonia IgM antibody positive; If step 4) in, sample to be checked, at emission wavelength (Em) for the detection fluorescent value of 520nm is less than CUT-OFF1 value and PCX1/NCX1 >=2.1, is judged as in serum sample to be checked anti-human streptococcus pneumonia IgM antibody negative; If step 4) in, serum sample to be checked, at emission wavelength (Em) for the detection fluorescent value of 600nm is greater than CUT-OFF2 value and PCX2/NCX2 >=2.1, is judged as in serum sample to be checked anti-human streptococcus pneumonia IgG antibody positive; If step 4) in, serum sample to be checked, at emission wavelength (Em) for the detection fluorescent value of 600nm is less than CUT-OFF2 value and PCX2/NCX2 >=2.1, is judged as in serum sample to be checked anti-human streptococcus pneumonia IgG antibody negative; If PCX1/NCX1 < 2.1 or PCX2/NCX2 < 2.1, all show that kit lost efficacy.
As preferably, step 2 proposed by the invention) in, to step 1) in centrifuge tube in add successively the anti-human streptococcus pneumonia IgM based on magnetic resolution and color quantum point mark, IgG antibody is total to anti-human streptococcus pneumonia antibody capture nanometer magnetic bead 20 μ l and the anti-human streptococcus pneumonia IgM based on magnetic resolution and color quantum point mark in check reagent box fast, IgG antibody is total to the anti-human IgM of the color quantum point difference mark in check reagent box fast, IgG nano-probe 50 μ l, after reacting 15min with 10rpm/min on rotation mixed instrument under room temperature, take off, centrifuge tube is inserted to the separated 3min of nano magnetic separation vessel magnetic, with pipettor sucking-off supernatant.
Carboxyl water-soluble nano quantum dot required for the present invention, 1150nm carboxyl magnetic bead, research unit, company that mouse-anti people IgM monoclonal antibody, mouse-anti human IgG monoclonal antibody etc. can arrive relevant speciality buy or customization; Required instrument, equipment, medicine all have commercially available.
The present invention compared to existing technology tool has the following advantages:
1, the present invention utilized nanometer magnetic bead to sample can enriching, fast, the efficiency advantages of higher of separated speed, the characteristics such as the while is high in conjunction with quantum dot photochemical stability, fluorescence intensity is high, make detection system possess the collaborative effect of amplifying of multiple signal, thereby there is detection sensitivity and the accuracy of superelevation.
2, the present invention's capture antigen used is to include the hybrid antigen that people streptococcus pneumonia specificity Fam1 PspA and Fam2PspA proteantigen extracellular region are rich in epitope, and the antibody horizontal for its generation in patients serum is high, so capture effect is good.
3, the present invention's capture antigen specificity used is high, and between the antibody of other common respiratory pathogens (as first, B-mode human influenza virus, human influenza haemophilus, moraxelle catarrhalis, people's mycoplasma pneumoniae, people's Chlamydia pneumoniae etc.) without antibody and antigen cross-reaction.
4, the present invention's antigen used is genetic engineering recombinant antigen, and protein expression amount is high, and is solubility expression, without renaturation, therefore comparatively cheap and easy to get.
5, detection method of the present invention is simple, detects (within 30min) fast, is easy to judge, testing cost is cheap, has overcome that prior art Positive rate is low, cost is high, complicated operation is loaded down with trivial details, the deficiency of length consuming time, clinical practice inconvenience.
6, the present invention can realize the synchronous detection of anti-human streptococcus pneumonia IgM, IgG antibody, having solved subitem, to detect the operating process bringing loaded down with trivial details, detect convenient and swift not, the problems such as testing cost is higher, and also do not seen on the market the appearance that anti-human streptococcus pneumonia IgM, the synchronous product detecting of IgG antibody or related science are reported at present.
Embodiment
The present invention is according to the antibody antigen reaction principle in immunology, utilize nanometer magnetic bead to sample can enriching, fast, the efficiency advantages of higher of separated speed, the characteristic such as high in conjunction with quantum dot photochemical stability, fluorescence intensity is high, that sets up a set ofly possesses the new method that the collaborative Fast synchronization that amplifies, has hypersensitivity and high degree of specificity of multiple signal detects anti-human streptococcus pneumonia IgM, IgG antibody, has wide market application foreground.
The present invention is further described in detail by following examples.
The preparation of various reagent and the explanation of material requested
1.PBSA damping fluid: take 1.44g sodium hydrogen phosphate, 0.24g potassium dihydrogen phosphate, 8g sodium chloride, 0.2g potassium chloride, 5g bovine serum albumin(BSA) is dissolved in the deionized water of 900ml, adjusts that pH to 7.4 is rear is settled to 1000ml with deionized water with 1mol/L NaOH.
2.PBS damping fluid: take 1.44g sodium hydrogen phosphate, 0.24g potassium dihydrogen phosphate, 8g sodium chloride, 0.2g potassium chloride is dissolved in the deionized water of 900ml, adjusts that pH to 7.4 is rear is settled to 1000ml with deionized water with 1mol/L NaOH.
3. recombinant human streptococcus pneumonia PspA albumen: be the present invention's self-control, the restructuring PspA1-His fusion that the homemade concentration of the present invention is to 0.2mg/ml and restructuring PspA2-His fusion by volume 1:1 shake up after mixing, and make recombinant human streptococcus pneumonia PspA albumen.
4. mouse-anti people IgM monoclonal antibody: be Abcam company product, article No. ab99741, with the dilution of PBS damping fluid, shakes up, and making monoclonal antibody weight percent concentration in solution is 1mg/ml.
5. mouse-anti human IgG monoclonal antibody: be Abcam company product, article No. ab99757, with the dilution of PBS damping fluid, shakes up, and making monoclonal antibody weight percent concentration in solution is 1mg/ml.
6. quantum dot: in the present invention, two kinds of quantum dots used are water-soluble CdSe/ZnS quantum dot that carboxylated amphipathic polymer is modified, its emission wavelength is respectively 520nm and 600nm, from Wuhan Jia Yuan technology of quantum dots development corporation, Ltd., buy, name of product is carboxyl water-soluble quantum dot-520 and carboxyl water-soluble quantum dot-600.
7. magnetic bead: in the present invention, magnetic bead used is with superparamagnetism Fe 3o 4for kernel, shell material are that polystyrene, surface functional group are that carboxyl, particle diameter are the carboxyl magnetic bead that is respectively 50nm, 180nm, 350nm, 1150nm, 3 μ m, Ke Cong Shanxi North America Gene Co., Ltd, Aorun Weina New Material Science and Technology Co., Ltd., Shanghai buy.
The preparation and purification of embodiment 1 recombinant human streptococcus pneumonia PspA albumen
1. the clone of related gene
To people streptococcus pneumonia Fam1 PspA, (the accession number in its NCBI Protein Data Bank is respectively AAF27703 to Fam2 PspA albumen, AAF27712) carry out bioinformatic analysis, obtain respectively the abundantest peptide section of epitope in its ectodomain, find its corresponding DNA encoding sequence, simultaneously at its 5 ' introducing restriction enzyme site NdeI, after 3 ' end is introduced termination signal TAA and restriction enzyme site XhoI, (complete sequence is synthetic transfers to Jin Sirui bio tech ltd to complete to difference chemosynthesis complete genome sequence, during delivery, artificial synthetic genetic fragment is connected on carrier pUC57 respectively), be designated as PspA1, PspA2.Its gene complete sequence is as shown in sequence table.Wherein, the 29-406aa of the protein sequence behaviour streptococcus pneumonia Fam1 PspA albumen (accession number:AAF27703) of PspA1 gene code.The 26-427aa of the protein sequence behaviour streptococcus pneumonia Fam2 PspA albumen (accession number:AAF27712) of PspA2 gene code.The carrier pUC57 that contains respectively these two sections of artificial synthetic DNA fragmentations is carried out reclaiming respectively according to a conventional method object fragment after double digestion with NdeI and XhoI respectively, standby.Adopt NdeI and XhoI to carry out double digestion to carrier pET-28a (+) simultaneously, and according to a conventional method respectively by the PspA1 obtaining after double digestion, PspA2 is connected in pET-28a (+) carrier, and transforms Escherichia coli TOP10, builds pET-PspA1, pET-PspA2 expression vector.Through enzyme, cut with sequencing and confirm that expression vector establishment is errorless.This carrier is expressed respectively restructuring PspA1-His, PspA2-His fusion.
2. the expression and purification of restructuring PspA1-His, PspA2-His fusion
By identifying after correct positive colony bacterium is cultivated, extract plasmid, technology proceeds in competence E.coliBL21 (DE3) routinely, after having transformed, bacterium liquid is coated containing on the LB flat board of 50 μ g/mL kanamycins, screens according to a conventional method expression strain.The single bacterium colony with exogenous protein expression ability that picking pET-PspA1, pET-PspA2 transform respectively is also inoculated respectively in 100mL LB nutrient culture media, in 37 ℃ of overnight incubation.Take out respectively after bacterium liquid, by 1:100, be inoculated in respectively in the LB nutrient culture media that 100mL contains 50 μ g/mL kanamycins, in 37 ℃, be cultured to OD 600=0.6 o'clock, add 1mol/L IPTG to final concentration be 1mmol/L, in 37 ℃, shake bacterium and cultivate, induction expressing fusion protein.After induction 4h, respectively at centrifugal 10min under 8000r/min, collect thalline.These two parts of thalline are used respectively to 20mL phosphate buffer (8g/L NaCL, 0.2g/L KCl, 0.24g/LKH 2pO 4, 1.44g/L Na 2hPO 4pH=7.4) washing is also used respectively 10mL sample-loading buffer (20mM Na 3 times 3pO 4, 0.5M NaCl; 30mM imidazoles, carries out ultrasonication after pH7.4) resuspended, and operating conditions is: 50HZ, and 200W, ultrasonic 3S, intermittently 5S, works 100 times.After ultrasonic completing, the centrifugal 15min of 12000g carries out electrophoresis detection after collecting precipitation and supernatant respectively.Find that restructuring PspA1-His, PspA2-His fusion are all present in thalline in solubility expression mode.Collect this two portions of cytoclastic supernatants, use respectively His Trap affinity columns (GE healthcare company product), use the same method to specifications and carry out purifying.Concrete grammar is as follows:
1) with 5mL syringe, be filled distilled water, turn on the stopper of post, with the joint providing, post is connected with syringe, with 1mL/min flow velocity, wash post.
2) with 10mL sample-loading buffer balance, 1mL/min flow velocity.
3) by fusion loading, 1mL/min flow velocity.
4) use 10mL sample-loading buffer, with 1mL/min flow velocity, wash post.
5) with 10mL elution buffer (20mM Na 3pO 4, 0.5M NaCl, 300mM imidazoles, pH7.4), with 1mL/min flow velocity wash-out, is in charge of collection, every pipe 1ml, 12%SDS-PAGE detects, and merges the sample that contains destination protein in elution fraction, by PBS damping fluid dialysed overnight.Collect dislysate after bradford kit carries out determination of protein concentration, with PBS damping fluid, adjusting concentration is 0.2mg/mL.
3. the preparation of recombinant human streptococcus pneumonia PspA fusion
The PBS damping fluid that contains respectively restructuring PspA1-His, the PspA2-His fusion 1:1 mixing by volume of above-mentioned steps 2 preparations is made to recombinant human streptococcus pneumonia PspA albumen, coated for nanometer magnetic bead.
The preparation of the anti-human streptococcus pneumonia antibody capture of embodiment 2 nanometer magnetic bead
1. the optimization of recombinant human streptococcus pneumonia PspA albumen coupling magnetic bead reaction conditions:
Using coupling the magnetic bead of recombinant human streptococcus pneumonia PspA albumen as solid phase carrier, quantum dot-labeled mouse-anti people IgM monoclonal antibody, as detecting antibody, detects anti-human streptococcus pneumonia IgM Positive Sera, observes the coupling situation of magnetic bead and recombinant protein.The coupling conditions such as the activator concentration of the particle diameter to magnetic bead, and EDC/NHS respectively, coupling antibody concentration, coupling time, sealer kind have carried out a series of optimization and have selected.
The selection of 1.1 magnetic bead particle diameters
Selection particle diameter is the carboxyl nanometer magnetic bead of 50nm, 180nm, 350nm, 1150nm, 3 μ m, all adds the PBS damping fluid containing 4mg/ml EDC and 4mg/ml NHS to carry out after priming reaction, reacts respectively with recombinant human streptococcus pneumonia PspA albumen coupling.Respectively the carboxyl nanometer magnetic bead preparing is detected to anti-human streptococcus pneumonia IgM Positive Sera, observations under fluorescent microscope, selects fluorescence intensity large, and background fluorescence disturbs few, and the very fast person of velocity of separation is the suitableeest magnetic bead particle diameter under magnetic fields.Result shows the magnetic bead requirement the most according to the invention of particle diameter 1150nm, determines that the suitableeest carboxyl magnetic bead particle diameter is 1150nm.
The selection of 1.2 EDC/NHS activator concentrations
After EDC in reaction system and NHS concentration are made as to 1～10mg/ml separately, carry out concentration gradient combination, activate respectively the carboxyl nanometer magnetic bead of particle diameter 1150nm.The carboxyl nanometer magnetic bead preparing is detected to anti-human streptococcus pneumonia IgM Positive Sera, and selecting the powerhouse of fluorescence is the suitableeest activation concentration of EDC and NHS solution.Result show when EDC concentration be 5mg/ml, NHS concentration while being 4mg/ml coupling effect best.
The selection of 1.3 coupled antigen concentration
By the recombinant human streptococcus pneumonia PspA albumen of 10 μ g, 50 μ g, 100 μ g, 150 μ g, 200 μ g, 250 μ g, 300 μ g, being 1150 nm respectively with 1mg by the particle diameter of above-mentioned best practice activation, magnetic bead carries out coupling.The carboxyl nanometer magnetic bead preparing is detected respectively to anti-human streptococcus pneumonia IgM Positive Sera, found that, when the injected volume of recombinant protein is less than 200 μ g/mg, fluorescence intensity is along with the concentration of antibody increases and increases, and when the injected volume of recombinant protein is greater than 200 μ g/mg, fluorescence intensity is substantially constant even slightly to be reduced, so the coupling amount of the present embodiment selection recombinant human streptococcus pneumonia PspA albumen is 200 μ g/mg.
The selection of 1.4 coupling times
Determine after particle diameter, EDC/NHS activator concentration and the antibody coupling amount of magnetic bead, the coupling reaction time of recombinant human streptococcus pneumonia PspA albumen and magnetic bead is made as respectively to 0.5h, 1h, 2h, 3h, 4h, 5h.The carboxyl nanometer magnetic bead preparing is detected respectively to anti-human streptococcus pneumonia IgM Positive Sera, found that, when coupling time >3h, fluorescence intensity tends towards stability, and after this extends coupling time again, and fluorescence no longer strengthens.Therefore the suitableeest coupling reaction time of, determining recombinant human streptococcus pneumonia PspA albumen and magnetic bead is 3h.Coupling time is far fewer than the 24h of traditional E LISA method.
The selection of 1.5 sealers
According to carrying out coupling reaction after particle diameter, EDC/NHS activator concentration, antibody coupling amount and the coupling time of above-mentioned definite optimal conditions selection magnetic bead.After coupling finishes, select BSA, monoethanolamine, Tris and D-Glucosamine Hydrochloride, as nanometer magnetic bead sealer, make finished product carboxyl nanometer magnetic bead.The nanometer magnetic bead preparing is detected respectively to anti-human streptococcus pneumonia IgM Positive Sera, found that, adopt monoethanolamine the highest as the detection fluorescent value of the nanometer magnetic bead of sealer.Supposition, because the molecule of monoethanolamine is less, can consume the surperficial carboxyl of not being combined with antibody due to sterically hindered preferably, makes sealing more complete, and effectively reduces space steric effect to connecting the structure influence of antibody.
Simultaneously, using coupling the carboxyl nanometer magnetic bead of recombinant human streptococcus pneumonia PspA albumen as solid phase carrier, corresponding quantum dot-labeled mouse-anti human IgG monoclonal antibody is as detecting antibody, set up the detection system of anti-human streptococcus pneumonia IgG antibody, the reaction conditions of the recombinant protein coupling magnetic bead in corresponding detection system is optimized.Found that, the best coupling condition in this detection system is all in full accord with the given result of above-mentioned steps 1.1-1.5.
2. coupling process:
Get 5mg magnetic bead (with superparamagnetism Fe 3o 4for kernel, the particle diameter carboxyl magnetic bead that is 1150nm) in the common centrifuge tube of 1.5ml, with 1ml MES damping fluid (2g/L MES, pH6.0) washing is three times, being placed in nano magnetic separation vessel carries out removing supernatant after magnetic separation (0.4T), add successively the EDC solution that the concentration with the preparation of above-mentioned MES damping fluid is 10mg/ml and each 0.5ml of sulfo-NHS solution that is 8mg/ml by the concentration that above-mentioned MES damping fluid is prepared, with 15rpm/min, in rotation mixed instrument, activate 1hr, after magnetic separation, remove supernatant, resuspended with the MES damping fluid that 1ml is above-mentioned; Get 5 centrifuge tubes, add the magnetic bead of the above-mentioned activation of 200 μ L in each centrifuge tube, sucking-off supernatant after magnetic separation adds with PBS damping fluid (8g/L NaCL, 0.2g/LKCl, 0.24g/L KH in each pipe 2pO 4, 1.44g/L Na 2hPO 4pH7.4) concentration of dilution is prepared each 1ml of recombinant human streptococcus pneumonia PspA protein solution of embodiment 1 of 200 μ g/ml, under room temperature, with 15rpm/min, in rotation mixed instrument, react 3h, magnetic separation removes after supernatant, respectively add 1ml to contain the above-mentioned PBS damping fluid of 1mg/ml monoethanolamine, under room temperature, with 15rpm/min, in rotation mixed instrument, react 2h with on sealing magnetic bead not with the carboxyl of antibody response.After magnetic separation, remove and respectively manage supernatant, each uses 1ml lavation buffer solution (8g/L NaCL, 0.2g/L KCl, 0.24g/L KH 2pO 4, 1.44g/L Na 2hPO 4, 0.5ml/L Tween-20, pH7.4) wash three times, last each preserved damping fluid (8g/L NaCL, 0.2g/L KCl, 0.24g/L KH with 1ml 2pO 4, 1.44g/L Na 2hPO 4, 0.3g/L NaN 3, 5g/L BSA, pH7.4) and resuspended magnetic bead, be placed in 4 ℃ and save backup, so far make anti-human streptococcus pneumonia antibody capture nanometer magnetic bead.
The embodiment 3 color quantum points difference anti-human IgM of mark are, the preparation of IgG nano-probe
1. the optimization of the quantum dot-labeled mouse-anti people of nanometer carboxylic IgM monoclonal antibody reactive condition:
1.1, the quantum dot-labeled antibody probe optimum mark of carboxyl pH's determines
Phosphate buffer pH in labeled reactant is made as respectively to 5,6,7,8,9, the full spectrometer of marked product utilization is carried out to fluorescent strength determining, observe the impact of different pH values on coupling reaction, determined that the best pH of quantum dot-labeled monoclonal antibody reaction is 7.0-8.0.This experimental selection pH7.4.
1.2, the quantum dot-labeled antibody probe optimum mark of carboxyl amount determines
Quantum dot volumetric molar concentration and the ratio of monoclonal antibody concentration are set to respectively to 1:1,1:2,1:3 and 1:4, carry out after labeled reactant, the full spectrometer of marked product utilization is carried out to fluorescent strength determining, observe the impact of the two variable concentrations comparison coupling reaction, the optimum molar concentration ratio of determining quantum dot-labeled mouse-anti people IgM monoclonal antibody reactive is that quantum dot and antibody mol ratio are 1:3.This optimal concentration of this experimental selection is recently determined labelled amount.
1.3, the best sealer kind of the quantum dot-labeled antibody probe of carboxyl determines
With monoethanolamine, Tris, PEG2000-NH 2or BSA, as sealer, is undertaken after labeled reactant by step 1.1 and 1.2 definite conditions, and the full spectrometer of marked product utilization is carried out to fluorescent strength determining, observes different sealers for the impact of labeled reactant, found that PEG2000-NH 2for best sealer, it can significantly improve colloidal stability and the immunocompetence of labeled complex.
The optimum results of the quantum dot-labeled mouse-anti people of the nanometer carboxylic IgM monoclonal antibody reactive correlated condition that the condition optimizing result of the quantum dot-labeled mouse-anti human IgG of nanometer carboxylic monoclonal antibody reactive and step 1.1-1.3 describe is all in full accord.
2. labeling process:
In microcentrifugal tube, add successively 4nmol carboxyl water-soluble quantum dot (emission wavelength 520nm), 600nmol N-hydroxy-succinamide (sulfo-NHS) and 600nmol carbodiimide (EDC), with phosphate buffer (2.9g/L sodium hydrogen phosphate, 0.295g/L sodium dihydrogen phosphate, 4g/L sodium chloride, pH 7.4) constant volume is 2ml, ceaselessly mixed solution, after 37 ℃ of reaction 30min, dialyses and removes the excessive sulfo-NHS as activator and EDC.In the quantum dot of activation, add the mouse-anti people IgM monoclonal antibody of 12nmol, lucifuge reaction 2h, adds single-ended amination polyglycol (PEG2000-NH 2) to final concentration be 1%, seal unreacted activated carboxyl site, continue lucifuge reaction 1h.With 0.2 μ m PES filter, remove by filter antibody aggregation thing, then filtrate is transferred in 50000MW ultra-filtration centrifuge tube, with 8000g centrifugal force centrifugal 15min at 4 ℃, remove the antibody of coupling reaction and the accessory substance in reaction do not occur.Collect super filter tube filter membrane upper strata quantum dot-antibody coupling matter solution, be dissolved in 2ml phosphate cleansing solution (2.9g/L sodium hydrogen phosphate, 0.295g/L sodium dihydrogen phosphate, 4g/L sodium chloride, 5ml/L Tween-20, 0.3g/L Sodium azide, pH 7.4) in, again this solution is transferred in 50000MW ultra-filtration centrifuge tube, with 8000g centrifugal force centrifugal 15min at 4 ℃, collect super filter tube filter membrane upper strata quantum dot-antibody coupling matter solution, be dissolved in 1ml phosphate and preserve liquid (2.9g/L sodium hydrogen phosphate, 0.295g/L sodium dihydrogen phosphate, 2g/L sodium chloride, 10g/L BSA, 0.3g/L Sodium azide, pH 7.4) in, so far make quantum dot-labeled anti-human IgM nano-probe, being placed in 4 ℃ saves backup.
By above-mentioned same procedure, utilize the carboxyl water-soluble quantum dot that mouse-anti human IgG monoclonal antibody and emission wavelength are 600nm to make quantum dot-labeled anti-human IgG nano-probe.By the 1:1 mixing by volume of above-mentioned two kinds of quantum dot-labeled nano-probe solution, make color quantum point anti-human IgM, the IgG nano-probe of mark respectively;
Embodiment 4 carboxyl nanometer magnetic beads carry out the optimization of immunocapture condition to people's streptococcus pneumoniae antigen
Using coupling the magnetic bead of recombinant human streptococcus pneumonia PspA albumen as solid phase carrier, the color quantum point respectively anti-human IgM, IgG nano-probe of mark, as detecting antibody, is set up the detection system of people's pneumonia streptococcus bacteria antibody.Consumption to carboxyl nanometer magnetic bead in detection system respectively, the conditions such as capture time have been carried out a series of optimization and have been selected.
Test the selection of 1. carboxyl nanometer magnetic bead additions
5 μ l, 10 μ l, 20 μ l, 30 μ l, 50 μ l, 70 μ l are joined respectively to 0.5ml containing in the human serum dilution of anti-human streptococcus pneumonia IgG antibody by the made anti-human streptococcus pneumonia antibody capture nanometer magnetic bead of getting ready of embodiment 2, carry out immunocapture, again by the described color quantum point of embodiment 3 respectively anti-human IgM, the IgG nano-probe of mark detect, record fluorescent value.Found that, along with the increase of carboxyl nanometer magnetic bead addition, fluorescent value increases gradually, and when carboxyl nanometer magnetic bead addition reaches 20 μ l, it is maximum that fluorescent value reaches.Continue to increase again the amount of carboxyl nanometer magnetic bead, fluorescent value reduces on the contrary.Therefore this experimental selection 20 μ l are as the optimal addn of nanometer magnetic bead.
Test the selection of 2. immunocapture times
Determine after the addition of magnetic bead, get four parts of made nanometer magnetic beads of getting ready of embodiment 2, at room temperature with 10r/min, anti-human streptococcus pneumonia IgM Positive Sera is carried out to the immunocapture of 5min, 10min, 15min, 20min, 30min and 40min, by the described quantum dot-labeled probe of embodiment 3, detected again, record fluorescent value.Found that, fluorescent value shows maximal value when immunocapture 15min, and along with the prolongation of time, numerical value declines to some extent.Therefore this experimental selection 15min is as the Best Times of immunocapture.
Simultaneously, using coupling the carboxyl nanometer magnetic bead of recombinant human streptococcus pneumonia PspA albumen as solid phase carrier, corresponding quantum dot-labeled mouse-anti human IgG monoclonal antibody is as detecting antibody, the detection system of setting up anti-human streptococcus pneumonia IgG antibody, is optimized the contact conditions in corresponding detection system.Found that, the best capture condition in above-mentioned detection system is all in full accord with above-mentioned experiment 1 and experiment 2 given results.
The preparation of embodiment 5 PBST damping fluids
Get 8g NaCL, 0.2g KCl, 0.24 KH 2pO 4, 1.44g Na 2hPO 4, 5g BSA, 0.3g NaN 3, 0.5ml Tween-20 is dissolved in 900ml distilled water, adjusts pH to 7.4, then be settled to 1000ml with 5M NaOH.
The preparation of embodiment 6 quality-control products
6.1) positive quality control product:
6.1.1) anti-human streptococcus pneumonia IgM antibody positive quality-control product: the definite positive serum of anti-human streptococcus pneumonia IgM antibody by 0.5ml people's streptococcus pneumoniae infection person forms;
6.1.2) anti-human streptococcus pneumonia IgG antibody positive quality-control product: the definite positive serum of anti-human streptococcus pneumonia IgG antibody by 0.5ml people's streptococcus pneumoniae infection person forms;
6.2) negative quality-control product: by the anti-human streptococcus pneumonia IgM of 0.5ml, IgG antibody all negative human serum form.
The preparation of embodiment 7 kits
A set of common anti-human streptococcus pneumonia IgM, the IgG antibody forming based on magnetic resolution and color quantum point mark of anti-human IgM, IgG nano-probe solution 5ml, the described PBST damping fluid of embodiment 5 200ml, the described quality-control product of embodiment 6 by the described color quantum point difference of the described anti-human streptococcus pneumonia antibody capture nanometer magnetic bead solution 2ml of embodiment 2, embodiment 3 mark is total to check reagent box fast.
Determining of embodiment 8 serum dilution to be checked
With clinical the whole bag of tricks, be all defined as anti-human streptococcus pneumonia IgM antibody and be respectively each 20 parts, negative and positive human serum sample, with PBSA damping fluid, carry out respectively 1:50,1:100,1:200,1:400,1:800,1:1600 doubly dilutes, and selects the high dilution of ratio >3 of positive serum and the detection fluorescence numerical value of negative serum as the suitableeest working concentration of serum to be checked.Found that the best results of 200 times of dilutions.
Equally, with clinical the whole bag of tricks, be all defined as anti-human streptococcus pneumonia IgG antibody and be respectively each 20 parts, negative and positive human serum sample, with PBSA damping fluid, carry out respectively 1:50,1:100,1:200,1:400,1:800,1:1600 doubly dilutes, and selects the high dilution of ratio >3 of positive serum and the detection fluorescence numerical value of negative serum as the suitableeest working concentration of serum to be checked.Result is also found, the best results of 200 times of dilutions.
The using method of embodiment 9 kits
1) get after serum sample 5 μ l to be checked dilute (200 times of dilutions) with 995 μ l PBSA damping fluids dilution is proceeded in the common centrifuge tube of 1.5ml;
2) to step 1) in centrifuge tube in add successively the anti-human streptococcus pneumonia IgM based on magnetic resolution and color quantum point mark, IgG antibody is total to anti-human streptococcus pneumonia antibody capture nanometer magnetic bead 20 μ l and the anti-human streptococcus pneumonia IgM based on magnetic resolution and color quantum point mark in check reagent box fast, IgG antibody is total to the anti-human IgM of the color quantum point difference mark in check reagent box fast, IgG nano-probe 50 μ l, after reacting 15min with 10rpm/min on rotation mixed instrument under room temperature, take off, centrifuge tube is inserted to the separated 3min of nano magnetic separation vessel magnetic, with pipettor sucking-off supernatant,
3) adding the anti-human streptococcus pneumonia IgM based on magnetic resolution and color quantum point mark, the PBST damping fluid 1ml that IgG antibody is total in check reagent box fast washs twice, sucking-off cleansing solution after the separation of employing nano magnetic separation vessel magnetic, finally use the resuspended magnetic bead of 0.2ml PBS damping fluid, make nanometer magnetic bead-streptococcus antibody-quantum dot " sandwich " compound;
4) by step 3) 0.2ml nanometer magnetic bead-streptococcus antibody-quantum dot compound of obtaining is loaded in ELISA Plate, use fluorescence microplate reader to be all 365nm in excitation wavelength (Ex), emission wavelength (Em) is respectively under the parameter of 520nm and 600nm distinguishes reading to its fluorescent value respectively;
5) determining of CUT-OFF value: by and above-mentioned steps 1)-4) same method detects 100 parts and is all defined as all negative human serum samples of anti-human streptococcus pneumonia IgM, IgG antibody through clinical the whole bag of tricks, read respectively emission wavelength (Em) and be respectively the fluorescent value under 520nm and 600nm; Described 100 parts of mean values (58.3) that are all defined as anti-human streptococcus pneumonia IgM, the IgG antibody fluorescent value that all negative human serum sample is 520nm at emission wavelength (Em) through clinical the whole bag of tricks are designated as CUT-OFF1 value (93.28) with 3 times of standard deviations (11.66) sum; Described 100 parts of mean values (42.5) that are all defined as anti-human streptococcus pneumonia IgM, the IgG antibody fluorescent value that all negative human serum sample is 600nm at emission wavelength (Em) through clinical the whole bag of tricks are designated as CUT-OFF2 value (71.9) with 3 times of standard deviations (9.8) sum;
6) by and above-mentioned steps 1)-4) four parts of negative quality-control product samples and four parts of positive quality control product samples that identical method provides in detection kit simultaneously, read respectively emission wavelength (Em) and be respectively the fluorescent value under 520nm and 600nm; The mean value of the fluorescent value that four parts of negative quality-control product samples are 520nm at emission wavelength (Em) is designated as NCX1; The mean value of the fluorescent value that four parts of negative quality-control product samples are 600nm at emission wavelength (Em) is designated as NCX2 value; Two parts of anti-human streptococcus pneumonia IgM antibody positive quality-control product samples emission wavelength (Em) for the fluorescence mean value of 520nm be PCX1; Two parts of anti-human streptococcus pneumonia IgG antibody positive quality-control product samples emission wavelength (Em) for and 600nm under fluorescent value be designated as PCX2; If step 4) in, serum sample to be checked, at emission wavelength (Em) for the detection fluorescent value of 520nm is greater than CUT-OFF1 value (93.28) and PCX1/NCX1 >=2.1, is judged as in serum sample to be checked anti-human streptococcus pneumonia IgM antibody positive; If step 4) in, serum sample to be checked is at emission wavelength (Em) for the detection fluorescent value of 520nm is less than CUT-OFF1 value (93.28) and PCX1/NCX1 >=2.1, and in judgement behaviour serum sample to be checked, anti-human streptococcus pneumonia IgM antibody is negative; If step 4) in, serum sample to be checked, at emission wavelength (Em) for the detection fluorescent value of 600nm is greater than CUT-OFF2 value (71.9) and PCX2/NCX2 >=2.1, is judged as in serum sample to be checked anti-human streptococcus pneumonia IgG antibody positive; If step 4) in, serum sample to be checked, at emission wavelength (Em) for the detection fluorescent value of 600nm is less than CUT-OFF2 value (71.9) and PCX2/NCX2 >=2.1, is judged as in serum sample to be checked anti-human streptococcus pneumonia IgG antibody negative; If PCX1/NCX1 < 2.1 or PCX2/NCX2 < 2.1, all show that kit lost efficacy.
The specific test of embodiment 10 kits
With the serum that respiratory tract common disease substance is positive as human respiratory syncytial virus, people's mycoplasma pneumoniae, people's Chlamydia pneumoniae, adenovirus hominis 3 types, adenovirus hominis 7 types, influenza virus A hominis, people's influenza B virus, human influenza haemophilus, moraxelle catarrhalis the infected's corresponding pathogen antigen, replace people streptococcus pneumonia Positive Sera to detect by the method as described in embodiment 9 with the kit as described in embodiment 7, find that testing result is all negative.
Embodiment 11 clinical trial examples
The anti-human streptococcus pneumonia IgM based on magnetic resolution and color quantum point mark for preparing of application the present invention, IgG antibody fast altogether check reagent box 80 parts, 60 parts clear and definite, anti-human streptococcus pneumonia IgM Positive Sera sample of clinical diagnosis and negative serum sample detected, testing result is as table 1.
The clinical serum test result of the anti-human streptococcus pneumonia IgM of table 1 antibody
The anti-human streptococcus pneumonia IgM based on magnetic resolution and color quantum point mark for preparing of application the present invention, IgG antibody fast altogether check reagent box 50 parts, 30 parts clear and definite, anti-human streptococcus pneumonia IgG Positive Sera sample of clinical diagnosis and negative serum sample detected, testing result is as table 2.
The clinical serum test result of the anti-human streptococcus pneumonia IgG of table 2 antibody
It is pointed out that and the foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any modifications of making within the present invention spirit and principle, be equal within replacement etc. all should be included in protection scope of the present invention.

Claims (8)

1. the method that the anti-human streptococcus pneumonia IgM based on magnetic resolution and color quantum point mark, IgG antibody are examined fast altogether, is characterized in that: said method comprising the steps of:
1) preparation of recombinant human streptococcus pneumonia PspA albumen;
2) by step 1) the recombinant human streptococcus pneumonia PspA albumen for preparing and nanometer magnetic bead be by covalent coupling, makes anti-human streptococcus pneumonia antibody capture nanometer magnetic bead;
3) nano-quantum point that is 520nm by mouse-anti people IgM monoclonal antibody and emission wavelength, by covalent coupling, is prepared quantum dot-labeled anti-human IgM nano-probe; The nano-quantum point that is 600nm by mouse-anti human IgG monoclonal antibody and emission wavelength, by covalent coupling, is prepared quantum dot-labeled anti-human IgG nano-probe; Quantum dot-labeled anti-human IgM nano-probe and quantum dot-labeled anti-human IgG nano-probe mixed in equal amounts are made to color quantum point anti-human IgM, the IgG nano-probe of mark respectively;
4) get human serum sample, after suitably diluting with PBSA damping fluid, in serum dilution, add step 2 respectively) the anti-human streptococcus pneumonia antibody capture nanometer magnetic bead and the step 3 that prepare) color quantum point for preparing anti-human IgM, the IgG nano-probe of mark respectively, fully mix, after reaction 10-45min, carry out magnetic separation, after PBST damping fluid washing 2 times, use fluorescence microplate reader, at emission wavelength, be respectively and under 520nm and 600nm, read respectively fluorescent value; In described PBSA damping fluid, each component concentration is respectively: 8g/L NaCL, 0.2g/L KCl, 0.24g/L KH 2pO 4, 1.44g/L Na 2hPO 4, 5g/L BSA; The pH=7.4 of described PBSA damping fluid; In described PBST damping fluid, each component concentration is respectively: 8g/L NaCL, 0.2g/L KCl, 0.24g/L KH 2pO 4, 1.44g/L Na 2hPO 4, 5g/L BSA, 0.3g/L NaN 3, 0.5ml/L Tween-20; The pH=7.4 of described PBST damping fluid;
5) according to step 4) method detect at least 30 parts and be all defined as all negative human serums of anti-human streptococcus pneumonia IgM, IgG antibody through clinical the whole bag of tricks, read respectively emission wavelength and be respectively the fluorescent value under 520nm and 600nm; Described anti-human streptococcus pneumonia IgM, IgG antibody all negative human serum are called for short people streptococcus pneumonia negative antibody control sample; Mean value and 3 times of standard deviation sums of the fluorescent value that described people streptococcus pneumonia negative antibody control sample is 520nm at emission wavelength are designated as CUT-OFF1 value; Mean value and 3 times of standard deviation sums of the fluorescent value that described people streptococcus pneumonia negative antibody control sample is 600nm at emission wavelength are designated as CUT-OFF2 value; If step 4) in, human serum sample is 520nm at emission wavelength detection fluorescent value is greater than CUT-OFF1 value, is judged as in human serum sample anti-human streptococcus pneumonia IgM antibody positive; If step 4) in, human serum sample is 520nm at emission wavelength detection fluorescent value is less than CUT-OFF1 value, is judged as in human serum sample anti-human streptococcus pneumonia IgM antibody negative; If step 4) in, human serum sample is 600nm at emission wavelength detection fluorescent value is greater than CUT-OFF2 value, is judged as in human serum sample anti-human streptococcus pneumonia IgG antibody positive; If step 4) in, human serum sample is 600nm at emission wavelength detection fluorescent value is less than CUT-OFF2 value, is judged as in human serum sample anti-human streptococcus pneumonia IgG antibody negative.
2. the anti-human streptococcus pneumonia IgM based on magnetic resolution and color quantum point mark, IgG antibody are total to a check reagent box fast, it is characterized in that: described anti-human streptococcus pneumonia IgM, IgG antibody based on magnetic resolution and color quantum point mark is total to check reagent box fast, and by anti-human streptococcus pneumonia antibody capture nanometer magnetic bead, the color quantum point with the anti-human streptococcus pneumonia IgM of enrichment, IgG antibody function, anti-human IgM, IgG antibody nano-probe, quality-control product and the PBST damping fluid of mark are formed respectively; Described quality-control product comprises positive quality control product and negative quality-control product; Described positive quality control product is comprised of serum and two parts of positive serum of anti-human streptococcus pneumonia IgG antibody of two parts of people's streptococcus pneumoniae infection persons' anti-human streptococcus pneumonia IgM antibody positive; Described negative quality-control product is all negative human serums of four parts of anti-human streptococcus pneumonia IgM, IgG antibody.
3. for the preparation of anti-human streptococcus pneumonia IgM, the IgG antibody based on magnetic resolution and color quantum point mark as claimed in claim 2, be total to fast a method for check reagent box, it is characterized in that: said method comprising the steps of:
1) preparation of anti-human streptococcus pneumonia antibody capture nanometer magnetic bead:
1.1) preparation, the purifying of recombinant human streptococcus pneumonia PspA albumen:
1.1.1) people streptococcus pneumonia Fam1 PspA, Fam2 PspA albumen are carried out to bioinformatic analysis, obtain respectively the abundantest peptide section of epitope in people streptococcus pneumonia Fam1 PspA, Fam2 PspA albumen ectodomain, find its corresponding gene order;
1.1.2) at step 1.1.1) in 5 ' end of resulting gene order and 3 ' end introduce respectively restriction enzyme site chemosynthesis complete genome sequence respectively, with tense marker, be designated as PspA1, PspA2;
1.1.3) by step 1.1.2) in resulting PspA1, PspA2 by molecular biology method, be cloned into respectively and proceed to expression in escherichia coli restructuring PspA1-His, PspA2-His fusion after expression vector pET-28a (+); Described restructuring PspA1-His, PspA2-His fusion are all present in genetic engineering thalline with solubility expression form;
1.1.4) use ni-sepharose purification step 1.1.3) resulting restructuring PspA1-His, PspA2-His fusion, SDS-PAGE detects after its purity, by PBS damping fluid difference dialysed overnight; Collect dislysate, after bradford kit carries out determination of protein concentration, with PBS damping fluid, adjust respectively concentration and be 2mg/mL; In described PBS damping fluid, each component content is as follows: 8g/L NaCL, 0.2g/L KCl, 0.24g/L KH 2pO 4, 1.44g/LNa 2hPO 4, the pH=7.4 of described PBS damping fluid;
1.1.5) by step 1.1.4) two kinds of recombinant protein solution obtaining by volume 1:1 mix and make recombinant human streptococcus pneumonia PspA albumen;
1.2) nanometer magnetic bead is coated:
1.2.1) get 5mg magnetic bead, with 1ml MES damping fluid washing three times, be placed in nano magnetic separation vessel and carry out removing supernatant after magnetic separation; Described magnetic bead is with superparamagnetism Fe 3o 4for kernel, the particle diameter carboxyl magnetic bead that is 1150nm; Described MES damping fluid is that mass concentration is 2-(N-morpholino) ethyl sulfonic acid of 2g/L; The pH=6.0 of described MES damping fluid; The magnetic intensity of described nano magnetic separation vessel is 0.4T;
1.2.2) add successively and use step 1.2.1) in the concentration of MES damping fluid preparation be the EDC solution of 8-12mg/ml and use step 1.2.1) in the concentration prepared of MES damping fluid be each 0.5ml of sulfo-NHS solution of 6-10mg/ml, with 10-40rpm/min, in rotation mixed instrument, activate 1hr, being placed in nano magnetic separation vessel carries out removing supernatant after magnetic separation, with 1ml step 1.2.1) in MES damping fluid resuspended, the magnetic bead after being activated;
1.2.3) get 5 centrifuge tubes, in each centrifuge tube, add 200 μ L step 1.2.2) magnetic bead after resulting activation, being placed in nano magnetic separation vessel carries out removing supernatant after magnetic separation, to add in each centrifuge tube concentration with the dilution of PBS damping fluid be 100-400 μ g/ml by step 1.1.5) prepared each 1ml of recombinant human streptococcus pneumonia PspA protein solution, under room temperature, with 15rpm/min, in rotation mixed instrument, react 2-6h, being placed in nano magnetic separation vessel carries out removing after supernatant after magnetic separation, respectively add 1ml to contain the above-mentioned PBS damping fluid of 1mg/ml monoethanolamine, under room temperature with 15rpm/min in rotation react in mixed instrument 2 h with sealing magnetic bead on not with the carboxyl of antibody response, in described PBS damping fluid, each component content is as follows: 8g/L NaCL, 0.2g/L KCl, 0.24g/L KH 2pO 4, 1.44g/L Na 2hPO 4, the pH=7.4 of described PBS damping fluid,
1.2.4) after capping completes, these 5 centrifuge tubes are placed in to nano magnetic separation vessel and carry out removing supernatant after magnetic separation, each washs three times with 1ml lavation buffer solution; In described lavation buffer solution, each component content is as follows: 8g/L NaCL, 0.2g/L KCl, 0.24g/L KH 2pO 4, 1.44g/L Na 2hPO 4, 0.5ml/L Tween-20, the pH=7.4 of described lavation buffer solution;
1.2.5) in each centrifuge tube, add respectively 1ml to preserve the resuspended magnetic bead of damping fluid, be placed in 4 ℃ and save backup; So far make anti-human streptococcus pneumonia antibody capture nanometer magnetic bead; In described preservation damping fluid, each component content is as follows: 8g/L NaCL, 0.2g/L KCl, 0.24g/L KH 2pO 4, 1.44g/L Na 2hPO 4, 0.3g/LNaN 3, 5g/L BSA, the pH=7.4 of described preservation damping fluid;
2) the color quantum point difference anti-human IgM of mark is, the preparation of IgG nano-probe:
Its concrete preparation method comprises:
2.1) in microcentrifugal tube, add successively 4nmol carboxyl water-soluble quantum dot, 600nmol N-hydroxy-succinamide sulfo-NHS and 600nmol carbodiimide EDC, take phosphate buffer constant volume as 5ml, mixed solution, after 37 ℃ of reaction 30min, the excessive sulfo-NHS as activator and EDC, the quantum dot after being activated are removed in dialysis; The emission wavelength of described carboxyl water-soluble quantum dot is 520nm; In described phosphate buffer, each component content is as follows: 2.9g/L sodium hydrogen phosphate, 0.295g/L sodium dihydrogen phosphate, 4g/L sodium chloride; The pH=7.4 of described phosphate buffer;
2.2) in step 2.1) in the quantum dot of resulting activation, add the mouse-anti people IgM monoclonal antibody of 8-16nmol, lucifuge reaction 2h, adds single-ended amination polyglycol PEG2000-NH 2to final concentration be 1%, seal unreacted activated carboxyl site, continue lucifuge reaction 1h;
2.3) with 0.2 μ m PES filter, remove by filter step 2.2) in antibody aggregation thing, then filtrate is transferred in 50000MW ultra-filtration centrifuge tube, with 8000g centrifugal force centrifugal 15min at 4 ℃, remove the antibody of coupling reaction and the accessory substance in reaction do not occur;
2.4) collect step 2.3) middle ultra-filtration centrifuge tube filter membrane upper strata quantum dot-antibody coupling matter solution, be dissolved in 2ml phosphate cleansing solution, again this solution is transferred in 50000 new MW ultra-filtration centrifuge tubes, with 8000g centrifugal force centrifugal 15min at 4 ℃, collect ultra-filtration centrifuge tube filter membrane upper strata quantum dot-antibody coupling matter solution, be dissolved in 1ml phosphate and preserve in liquid, be placed in 4 ℃ and save backup, so far make quantum dot-labeled anti-human IgM nano-probe; In described phosphate cleansing solution, each component content is as follows: 2.9g/L sodium hydrogen phosphate, 0.295g/L sodium dihydrogen phosphate, 4g/L sodium chloride, 5ml/L Tween-20,0.3g/L Sodium azide, the pH=7.4 of described phosphate cleansing solution; In described phosphate preservation liquid, each component content is as follows: 2.9g/L sodium hydrogen phosphate, 0.295g/L sodium dihydrogen phosphate, 2g/L sodium chloride, 10g/L bovine serum albumin(BSA), 0.3g/L Sodium azide; Described phosphate is preserved the pH=7.4 of liquid;
2.5) by and step 2.1)-2.4) identical method utilizes the carboxyl water-soluble quantum dot that mouse-anti human IgG monoclonal antibody and emission wavelength are 600nm to make quantum dot-labeled anti-human IgG nano-probe; By the 1:1 mixing by volume of above-mentioned two kinds of quantum dot-labeled nano-probe solution, make color quantum point anti-human IgM, the IgG nano-probe of mark respectively;
3) preparation of PBST damping fluid:
Its concrete compound method comprises:
Get 8g NaCL, 0.2g KCl, 0.24 KH 2pO 4, 1.44g Na 2hPO 4, 5g BSA, 0.3g NaN 3, 0.5ml Tween-20 is dissolved in 900ml distilled water, adjusts pH to 7.4, then be settled to 1000ml with 5M NaOH;
4) preparation of quality-control product:
4.1) positive quality control product:
4.1.1) anti-human streptococcus pneumonia IgM antibody positive quality-control product: the definite positive serum of anti-human streptococcus pneumonia IgM antibody by 0.5ml people's streptococcus pneumoniae infection person forms;
4.1.2) anti-human streptococcus pneumonia IgG antibody positive quality-control product: the definite positive serum of anti-human streptococcus pneumonia IgG antibody by 0.5ml people's streptococcus pneumoniae infection person forms;
4.2) negative quality-control product: by the anti-human streptococcus pneumonia IgM of 0.5ml, IgG antibody all negative human serum form.
4. method according to claim 3, it is characterized in that: described step 1.2.2), add successively and use step 1.2.1) in the concentration of MES damping fluid preparation be the EDC solution of 10mg/ml and use step 1.2.1) in the concentration prepared of MES damping fluid be each 0.5ml of sulfo-NHS solution of 8mg/ml, with 15rpm/min, in rotation mixed instrument, activate 1hr, being placed in nano magnetic separation vessel carries out removing supernatant after magnetic separation, with 1ml step 1.2.1) in MES damping fluid resuspended, the magnetic bead after being activated;
Described step 1.2.3) in, to add in each centrifuge tube concentration with the dilution of PBS damping fluid be 200 μ g/ml by step 1.1.5) prepared each 1ml of recombinant human streptococcus pneumonia PspA protein solution, under room temperature, with 15rpm/min, in rotation mixed instrument, react 3h, be placed in nano magnetic separation vessel and carry out removing after supernatant after magnetic separation, respectively add 1ml to contain the above-mentioned PBS damping fluid of 1mg/ml monoethanolamine;
Described step 2.2) in, in step 2.1) in the quantum dot of resulting activation, add the mouse-anti people IgM monoclonal antibody of 12nmol, lucifuge reaction 2h.
5. according to the method described in claim 3 or 4, it is characterized in that: described quantum dot is water-soluble CdSe/ZnS quantum dot that carboxylated amphipathic polymer is modified.
6. method according to claim 5, is characterized in that: described magnetic bead is with superparamagnetism Fe 3o 4for kernel, shell material are that polystyrene, surface functional group are the carboxyl magnetic bead that carboxyl, particle diameter are 1150nm.
7. based on anti-human streptococcus pneumonia IgM, the IgG antibody based on magnetic resolution and color quantum point mark as claimed in claim 2, be total to fast a using method for check reagent box, it is characterized in that: described using method comprises the following steps:
1) get serum sample 5 μ l to be checked with after 995 μ l PBSA damping fluid dilutions, dilution being proceeded in the common centrifuge tube of 1.5ml; In described PBSA damping fluid, each component content is as follows: 8g/L NaCL, 0.2g/L KCl, 0.24g/L KH 2pO 4, 1.44g/L Na 2hPO 4, 5g/L BSA; The pH=7.4 of described PBSA damping fluid;
2) to step 1) in centrifuge tube in add successively the anti-human streptococcus pneumonia IgM based on magnetic resolution and color quantum point mark, IgG antibody is total to anti-human streptococcus pneumonia antibody capture nanometer magnetic bead 10-100 μ l and the anti-human streptococcus pneumonia IgM based on magnetic resolution and color quantum point mark in check reagent box fast, IgG antibody is total to the anti-human IgM of the color quantum point difference mark in check reagent box fast, IgG nano-probe 50 μ l, after reacting 5-25min with 10rpm/min on rotation mixed instrument under room temperature, take off, centrifuge tube is inserted to the separated 3min of nano magnetic separation vessel magnetic, with pipettor sucking-off supernatant,
3) adding the anti-human streptococcus pneumonia IgM based on magnetic resolution and color quantum point mark, the PBST damping fluid 1ml that IgG antibody is total in check reagent box fast washs twice, sucking-off cleansing solution after the separation of employing nano magnetic separation vessel magnetic, finally use the resuspended magnetic bead of 0.2ml PBS damping fluid, make nanometer magnetic bead-streptococcus antibody-quantum dot " sandwich " compound; In described PBS damping fluid, each component content is as follows: 8g/L NaCL, 0.2g/L KCl, 0.24g/L KH 2pO 4, 1.44g/L Na 2hPO 4; The pH=7.4 of described PBS damping fluid;
4) by step 3) nanometer magnetic bead-streptococcus antibody-quantum dot compound of obtaining is loaded in ELISA Plate, use fluorescence microplate reader to be all 365nm in excitation wavelength, emission wavelength is respectively under the parameter of 520nm and 600nm distinguishes reading to its fluorescent value respectively;
5) by above-mentioned same method, detect at least 30 parts and be all defined as all negative human serum samples of anti-human streptococcus pneumonia IgM, IgG antibody through clinical the whole bag of tricks, read respectively emission wavelength and be respectively the fluorescent value under 520nm and 600nm; Described mean value and the 3 times of standard deviation sums that are all defined as anti-human streptococcus pneumonia IgM, the IgG antibody fluorescent value that all negative human serum sample is 520nm at emission wavelength through clinical the whole bag of tricks are designated as CUT-OFF1 value; Described mean value and the 3 times of standard deviation sums that are all defined as anti-human streptococcus pneumonia IgM, the IgG antibody fluorescent value that all negative human serum sample is 600nm at emission wavelength through clinical the whole bag of tricks are designated as CUT-OFF2 value; The four parts of negative quality-control product samples and the four parts of positive quality control product samples that in detection kit, provide read respectively emission wavelength and are respectively the fluorescent value under 520nm and 600nm simultaneously; The mean value of the fluorescent value that four parts of negative quality-control product samples are 520nm at emission wavelength is designated as NCX1; The mean value of the fluorescent value that four parts of negative quality-control product samples are 600nm at emission wavelength is designated as NCX2 value; The fluorescence mean value that two parts of anti-human streptococcus pneumonia IgM antibody positive quality-control product samples are 520nm at emission wavelength is PCX1; Two parts of anti-human streptococcus pneumonia IgG antibody positive quality-control product samples emission wavelength for and 600nm under fluorescent value be designated as PCX2;
If step 4) the detection fluorescent value that in, serum sample to be checked is 520nm at emission wavelength is greater than CUT-OFF1 value and PCX1/NCX1 >=2.1, is judged as in serum sample to be checked anti-human streptococcus pneumonia IgM antibody positive;
If step 4) the detection fluorescent value that in, sample to be checked is 520nm at emission wavelength is less than CUT-OFF1 value and PCX1/NCX1 >=2.1, and in judgement behaviour serum sample to be checked, anti-human streptococcus pneumonia IgM antibody is negative;
If step 4) the detection fluorescent value that in, serum sample to be checked is 600nm at emission wavelength is greater than CUT-OFF2 value and PCX2/NCX2 >=2.1, is judged as in serum sample to be checked anti-human streptococcus pneumonia IgG antibody positive;
If step 4) the detection fluorescent value that in, serum sample to be checked is 600nm at emission wavelength is less than CUT-OFF2 value and PCX2/NCX2 >=2.1, is judged as in serum sample to be checked anti-human streptococcus pneumonia IgG antibody negative;
If PCX1/NCX1 < 2.1 or PCX2/NCX2 < 2.1, all show that kit lost efficacy.
8. method according to claim 7, it is characterized in that: described step 2), to step 1) in centrifuge tube in add successively the anti-human streptococcus pneumonia IgM based on magnetic resolution and color quantum point mark, IgG antibody is total to anti-human streptococcus pneumonia antibody capture nanometer magnetic bead 20 μ l and the anti-human streptococcus pneumonia IgM based on magnetic resolution and color quantum point mark in check reagent box fast, IgG antibody is total to the anti-human IgM of the color quantum point difference mark in check reagent box fast, IgG nano-probe 50 μ l, after reacting 15min with 10rpm/min on rotation mixed instrument under room temperature, take off, centrifuge tube is inserted to the separated 3min of nano magnetic separation vessel magnetic, with pipettor sucking-off supernatant.
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