CN105223352B - Method and kit for rapidly detecting human haemophilus influenzae based on magnetic separation and quantum dot labelling - Google Patents
Method and kit for rapidly detecting human haemophilus influenzae based on magnetic separation and quantum dot labelling Download PDFInfo
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Abstract
The invention provides a method for detecting a human haemophilus influenzae antigen based on magnetic separation and quantum dot labelling. The method includes the steps: (1) preparing anti-human haemophilus influenzae immune nano magnetic beads; (2) preparing quantum dot labelled anti-human haemophilus influenzae nano probe; and (3) after dissolving a to-be-tested sample in a PBST buffer solution, adding the anti-human haemophilus influenzae immune nano magnetic beads to the dissolved solution, fully mixing, carrying out a reaction, then carrying out magnetic separation, washing with a PBST buffer solution, adding the quantum dot labelled anti-human haemophilus influenzae nano probe to the obtained precipitate, carrying out a reaction, then carrying out magnetic separation, washing with a PBST buffer solution, and then detecting a fluorescent value by using a fluorescence microplate reader. The accurate, rapid and high-sensitivity method for detecting human haemophilus influenzae is established, and has quite high practical value in clinical diagnosis, etiology identification and epidemiological investigation of human haemophilus influenzae.
Description
Technical field
The present invention relates to technical field of medical detection, a kind of based on Magnetic Isolation with quantum dot-labeled detection people
The method for quick of hemophilus influenza (Haemophilus influenzae, Hi) antigen and detection kit, and should
The method of preparation and use of detection kit.
Background technology
Hemophilus influenza (Haemophilus influenzae, Hi) is a kind of important respiratory tract disease infecting the mankind
Pathogenic microorganism, this bacterium is found in an influenza pestilence of 1892 by Poland bacteriologist doctor Fei Fo, with
After time in studied persons widely studied.The most only know that people is the host of this pathogen.Old man that immunity is poor and child
For Susceptible population, the infant of particularly less than 5 years old.Hi can cause pneumonia, conjunctivitis, otitis media, meningitis and bacteremia etc.,
In the whole world, annual at least 3,000,000 several cases occur, and infant can be caused to disable even dead.Hi be divided into encapsulated a, b, c,
D, e, f totally 6 serotypes and acapsular indecomposable form hemophilus influenza (nontypeable Haemophilus
Influenzae, NTHi), once the most popular with pod membrane b type Hi.The Hi Serological testing the most also pin that China carries out at present
The pod membrane b type stronger to aggressivity.And due to the successful research and development of this type bacterial strain capsular polysaccharide vaccine and application, it is popular is had
Effect controls.Recent research more show, infect Hi patient in modal bacterial strain be NTHi, its separation rate reached 50% with
On.
Similar with the infection symptoms that other respiratory pathogens cause due to Hi clinically, the most often it is difficult to according to clinical table
Existing, x-radiological survey X etc. is reached a conclusion, and makes a definite diagnosis and tends to rely on laboratory diagnosis.The feature of infant disease is that onset is anxious, turns
Returning fast, the most sensitive, quick, practical Hi detection has very important significance to carrying out effective clinical intervention early.
Although hemophilus influenza is propagated in the world, but can be used for the standardization commercially available reagent of laboratory diagnosis
Kind is few.At present, the diagnostic method that Hi infects has serological detection method, nucleic acid detection method and pathogen direct Detection Method.Inspection
The method that in survey serum, Hi IgG, IgA, IgM antibody are commonly used has: microimmunofluorescence antibody test (MIF), and complement combines examination
Testing (CF), recombinase immunoassay (rEIA), SC combines enzyme immunoassay (EIA) test (SeroCF EIA) etc..But
The detection of Hi antibody can only illustrate that this individuality infected Hi, but can not be the most still with the presence of Hi viable bacteria in antimer, and serum
Learning detection of specific antibody often needs the dynamic result according to IgM antibody to judge, needs the longer time.Prior
It is that the SD object that predominantly detects is anti-capsular antibody, and NTHi is not owing to having pod membrane, then can cause missing inspection.Meanwhile,
These technology all exist that sensitivity is low, operating procedure is complicated, need professional to operate, poor repeatability, detection time length, detection
Poor specificity, the defect such as relatively costly, thus be difficult to meet clinical being actually needed.
Detection of nucleic acids includes nucleic acid hybridization and polymerase chain reaction (PCR), and nucleic acid hybridization detects the high specificity of Hi, but
Sensitivity is the highest, is mainly used in the detection of PCR result, judgement, is not yet directly used in the detection of clinical samples；PCR has higher
Sensitivity, but PCR experiment has particular/special requirement to laboratory, and sample disposal, amplification and testing requirement are strict, and false sun easily occurs
Property, can't be as conventional methods for clinical diagnosis in China.
Pathogen detection method is mainly Isolation and culture of agent method, by specimen inoculation in the chocolate that with the addition of V and the X factor
On blood agar culture-medium, after 24 hours cultivate, the suspicious bacterium colony of picking is after Morphological Identification, with Hi identification card, Vilek 1
Antibacterial automatic analysis system identifies kind, and system carries out biological typing automatically.But it is complicated that this method exists operating procedure, cell is cultivated
The open defects such as time length, are not appropriate for clinical practice.The more important thing is, capsular swelling experiment therein etc. is only limitted to there being pod
The bacterial strain of film is identified, without Buccal mucosa flap (NTHi) then all missing inspections.
Therefore, set up at present tool high sensitivity, high specific human influenza influenzae antigens fast detection method to meet
Clinical detection demand just seems the most necessary.
Summary of the invention
For these technical problems present in background technology, the invention provides a kind of based on Magnetic Isolation and quantum dot
The detection method of the energy of labelling detection easy, quick, highly sensitive human influenza influenzae antigens and test kit, and this examination
The method of preparation and use of agent box.
The present invention is achieved through the following technical solutions:
A kind of based on Magnetic Isolation with the method for quantum dot-labeled detection human influenza influenzae antigens, its feature exists
In: said method comprising the steps of:
1) preparation of rabbit anti-human hemophilus influenza memebrane protein P6 polyclonal antibody；
2) preparation of mouse-anti human influenza haemophilus memebrane protein P6 polyclonal antibody；
3) by step 1) the rabbit anti-human hemophilus influenza memebrane protein P6 polyclonal antibody for preparing leads to nanometer magnetic bead
Cross covalent coupling, prepare anti-human hemophilus influenza immune nanometer magnetic bead；
4) by step 2) the mouse-anti human influenza haemophilus memebrane protein P6 polyclonal antibody for preparing and nano-quantum point
By covalent coupling, prepare quantum dot-labeled anti-human hemophilus influenza nano-probe；
5) take human respiratory secretions sample (including but not limited to throat swab), after PBST buffer solution, add step
Rapid 3) the anti-human hemophilus influenza immune nanometer magnetic bead prepared, is sufficiently mixed, and carries out Magneto separate after reaction 10-45min,
After PBST buffer solution 2 times, in the precipitate that Magneto separate obtains add step 4) prepare quantum dot-labeled
Anti-human hemophilus influenza nano-probe, carries out Magneto separate after reaction 10-45min, after above-mentioned PBST buffer solution 2 times,
Fluorescence microplate reader is used to read fluorescent value；In described PBST buffer, each component content is as follows: 8g/L NaCl, 0.2g/L KCl,
0.24g/L KH2PO4, 1.44g/L Na2HPO4, 0.3g/L NaN3, 0.5ml/L Tween-20；The pH of described PBST buffer
=7.4；
6) according to step 1)-step 5) method detect four parts to be respectively defined as human influenza haemophilus through clinic negative
The respiratory secretions sample of crowd, reads fluorescent value；The respiratory secretions of the crowd that described human influenza haemophilus is negative
Sample is called for short human influenza haemophilus negative control sample；The fluorescent value of described four parts of human influenza haemophilus negative control sample
Meansigma methods and 3 times of standard deviation sums be CUT-OFF value；If step 5) in the detection fluorescent value of human respiratory secretions sample
More than CUT-OFF value, then it is judged as in human respiratory secretions sample that human influenza influenzae antigens is the positive；If step 5) in
The detection fluorescent value of human respiratory secretions sample is less than CUT-OFF value, then be judged as artificial abortion in human respiratory secretions sample
Haemophilus influenza antigen is negative.
A kind of based on Magnetic Isolation with the test kit of quantum dot-labeled detection human influenza influenzae antigens, its feature exists
In: described test kit by have enrichment human influenza influenzae antigens function anti-human hemophilus influenza immune nanometer magnetic bead,
Quantum dot-labeled anti-human hemophilus influenza nano-probe, quality-control product and PBST buffer are formed；Described quality-control product bag
Include positive quality control product and negative quality-control product；Described positive quality control product is dried by the human influenza haemophilus inactivated and is attached to swab
On form；Described negative quality-control product is the throat swab being defined as the negative crowd of human influenza haemophilus through clinic.
A kind of for preparing based on Magnetic Isolation and the test kit of quantum dot-labeled detection human influenza influenzae antigens
Method, it is characterised in that: described preparation method comprises the following steps:
1) preparation of anti-human hemophilus influenza immune nanometer magnetic bead:
1.1) rabbit and the preparation of mouse-anti human influenza haemophilus memebrane protein P6 polyclonal antibody IgG
1.1.1) the restructuring preparation of P6-His fusion protein, purification:
1.1.1.1) human influenza haemophilus memebrane protein P6 is carried out bioinformatic analysis, obtain the outer conserved structure of its born of the same parents
The peptide fragment that in territory, epitope enriches the most；
1.1.1.2) find step 1.1.1.1) in obtained by gene coded sequence corresponding to peptide fragment, in said gene sequence
5 ' ends and 3 ' ends introduce restriction enzyme site chemosynthesis complete genome sequences, labelling is designated as P6 simultaneously；Its gene order such as sequence
Shown in table；
1.1.1.3) by step 1.1.1.2) in obtained by P6 be cloned into expression vector pET-by molecular biology method
28a (+) after proceed to expression in escherichia coli restructuring P6-His fusion protein；Described restructuring P6-His fusion protein is with solubility table
The mode of reaching is present in thalline；
1.1.1.4) by ni-sepharose purification step 1.1.1.3) obtained by recombiant protein, after SDS-PAGE detects its purity,
Measuring protein concentration with Bradford method, it is standby after 0.2mg/mL for adjusting protein concentration；
1.1.2) rabbit and the preparation of mouse-anti human influenza haemophilus memebrane protein P6 polyclonal antibody IgG:
1.1.2.1) with step 1.1.1.4) in obtained by restructuring P6-His fusion protein as complete antigen, respectively immunity
New zealand white rabbit and Cavia porcellus；Prepare rabbit anti-human hemophilus influenza memebrane protein P6 antiserum and the bloodthirsty bar of mouse-anti human influenza respectively
Mycoderma albumen P6 antiserum；Described rabbit anti-human hemophilus influenza memebrane protein P6 antiserum and mouse-anti human influenza haemophilus film egg
The white sero-fast indirect ELISA titer of P6 is all higher than 1 × 105；
1.1.2.2) Protein G affinity column purified rabbit anti-human's hemophilus influenza anti-blood of memebrane protein P6 respectively is used
Polyclonal antibody IgG clearly and in mouse-anti human influenza haemophilus memebrane protein P6 antiserum；
1.1.2.3) with triumphant base Bradford protein content detection kit determination step 1.1.2.2) obtained by two kinds
The concentration of polyclonal antibody IgG, standby after its protein concentration is all adjusted to 1mg/mL；
1.2) being coated of immune nanometer magnetic bead:
1.2.1) take 5mg magnetic bead, with 1ml MES buffer solution three times, be placed in nano magnetic separator and carry out Magneto separate
After remove supernatant；Described magnetic bead is with superparamagnetism Fe3O4For the carboxyl magnetic bead that kernel, particle diameter are 350nm；Described MES buffer
Be mass concentration be 2-(N-morpholino) ethyl sulfonic acid of 2g/L；The pH=6.0 of described MES buffer；Described nano magnetic separator
Magnetic intensity be 0.4T；
1.2.2) be sequentially added into use step 1.2.1) in the concentration of MES buffer be the EDC solution of 8-12mg/ml
And use step 1.2.1) in the concentration of MES buffer be each 0.5ml of sulfo-NHS solution of 6-12mg/ml, with
10-40rpm activates 1hr in rotary mixer, is placed in after carrying out Magneto separate in nano magnetic separator and removes supernatant, walks with 1ml
Rapid 1.2.1) in MES buffer resuspended, magnetic bead after being activated；
1.2.3) take 5 centrifuge tubes, each centrifuge tube add 200 μ L steps 1.2.2) obtained by activation after magnetic
Pearl, is placed in after carrying out Magneto separate in nano magnetic separator and removes supernatant, and in each centrifuge tube, the dilution of addition PBS is dense
Degree for 50-200 μ g/ml by step 1.1) prepared by rabbit anti-human hemophilus influenza memebrane protein P6 polyclonal antibody IgG molten
The each 1ml of liquid, reacts 2-6h with 15rpm under room temperature in rotary mixer, is placed in nano magnetic separator after carrying out Magneto separate and moves
After supernatant, each add the 1ml above-mentioned PBS containing 1mg/ml ethanolamine, under room temperature with 15rpm in rotary mixer anti-
Answer 2h with close on magnetic bead not with the carboxyl of antibody response；In described PBS, each component content is as follows: 8g/L NaCl,
0.2g/L KCl, 0.24g/L KH2PO4, 1.44g/L Na2HPO4, the pH=7.4 of described PBS；
1.2.4), after capping completes, these 5 centrifuge tubes are placed in after nano magnetic separator carries out Magneto separate and remove
Supernatant, each with 1ml lavation buffer solution washing three times；In described lavation buffer solution, each component content is as follows: 8g/L NaCl, 0.2g/
L KCl, 0.24g/L KH2PO4, 1.44g/L Na2HPO4, 0.5ml/L Tween-20, the pH=7.4 of described lavation buffer solution；
1.2.5) in each centrifuge tube, it is separately added into 1ml preserves the resuspended magnetic bead of buffer, be placed in 4 DEG C and save backup；Institute
State each component content in preservation buffer as follows: 8g/L NaCl, 0.2g/L KCl, 0.24g/L KH2PO4, 1.44g/L
Na2HPO4, 0.3g/L NaN3, 5g/L bovine serum albumin (BSA), the pH=7.4 of described preservation buffer；
2) preparation of quantum dot-labeled anti-human hemophilus influenza nano-probe:
Its concrete preparation method includes:
2.1) in microcentrifugal tube, it is sequentially added into 2nmol carboxyl water-soluble quantum dot, 300nmol N-hydroxy amber
Amber acid imide sulfo-NHS and 300nmol carbodiimide EDC, with phosphate buffer constant volume as 2ml, mixed solution, 37 DEG C
After reaction 30min, sulfo-NHS and EDC as activator of excess, the quantum dot after being activated are removed in dialysis；Described
In phosphate buffer, each component content is as follows: 2.9g/L disodium 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) obtained by activation quantum dot in, add the step 1.1 of 4-12nmol) in prepared
Mouse-anti human influenza haemophilus memebrane protein P6 polyclonal antibody IgG, lucifuge reaction 2h, add single-ended amination Polyethylene Glycol
PEG2000-NH2To final concentration of 1%, close unreacted activated carboxyl site, continue lucifuge reaction 1h；
2.3) be filtered to remove step 2.2 with 0.2 μm PES filter) in antibody aggregation thing, then filtrate be transferred to
In 50000MW ultra-filtration centrifuge tube, with 8000g centrifugal force centrifugal 15min at 4 DEG C, remove do not occur coupling reaction antibody and
By-product in reaction；
2.4) collect step 2.3) in ultra-filtration centrifuge tube filter membrane upper strata quantum dot-antibody coupling matter solution, be dissolved in 2ml phosphoric acid
In salt cleaning mixture, then this solution is transferred in a new 50000MW ultra-filtration centrifuge tube, with 8000g centrifugal force at 4 DEG C from
Heart 15min, collects ultra-filtration centrifuge tube filter membrane upper strata quantum dot-antibody coupling matter solution, is dissolved in 1ml phosphate and preserves in liquid, puts
Save backup in 4 DEG C；In described phosphate cleaning mixture, each component content is as follows: 2.9g/L disodium hydrogen phosphate, 0.295g/L phosphoric acid
Sodium dihydrogen, 4g/L sodium chloride, 5ml/L tween 20,0.3g/L sodium azide, the pH=7.4 of described phosphate cleaning mixture；Described phosphorus
It is as follows that hydrochlorate preserves each component content in liquid: 2.9g/L disodium hydrogen phosphate, 0.295g/L sodium dihydrogen phosphate, 2g/L sodium chloride,
10g/L bovine serum albumin, 0.3g/L sodium azide；Described phosphate preserves the pH=7.4 of liquid；
3) preparation of PBST buffer:
Its concrete compound method includes:
Take 8g NaCl, 0.2g KCl, 0.24KH2PO4, 1.44g Na2HPO4, 0.3g NaN3, 0.5ml Tween-20 is molten
Solution, in 800ml distilled water, adjusts pH to 7.4 with 5M NaOH, then is settled to 1000ml；
4) preparation of quality-control product:
4.1 positive quality control product: positive quality control product is dried by the human influenza haemophilus inactivated and is attached on swab form；
4.2 negative quality-control products: the pharynx that negative quality-control product is i.e. defined as the negative crowd of human influenza haemophilus through clinic is wiped
Son.
As preferably, the present invention is in step 1.2.2) in, be sequentially added into and use step 1.2.1) in MES buffer
Concentration is the EDC solution of 10mg/ml and uses step 1.2.1) in the concentration of MES buffer be 10mg/ml
The each 0.5ml of sulfo-NHS solution, activates 1hr with 15rpm in rotary mixer, is placed in nano magnetic separator and carries out magnetic and divide
Remove supernatant after from, by 1ml step 1.2.1) in MES buffer resuspended, magnetic bead after being activated；
Described step 1.2.3) in, in each centrifuge tube addition PBS dilution concentration be 100 μ g/ml by
Step 1.1) prepared by the rabbit anti-human hemophilus influenza memebrane protein P6 polyclonal antibody each 1ml of IgG solution, under room temperature with
15rpm reacts 3h in rotary mixer, is placed in after removing supernatant after carrying out Magneto separate in nano magnetic separator, respectively adds 1ml
Above-mentioned PBS containing 1mg/ml ethanolamine；
Described step 2.2) in, in step 2.1) obtained by activation quantum dot in, add 6nmol step 1.1) in
Prepared mouse-anti human influenza haemophilus memebrane protein P6 polyclonal antibody IgG, lucifuge reaction 2h.
As preferably, quantum dot of the present invention is water-soluble CdSe/ZnS amount that carboxylated amphipathic polymer is modified
Sub-point.
As preferably, magnetic bead of the present invention is with superparamagnetism Fe3O4It is polystyrene, table for kernel, shell material
Face functional group be carboxyl, particle diameter be the carboxyl magnetic bead of 350nm.
A kind of based on Magnetic Isolation with the user of test kit of quantum dot-labeled detection human influenza influenzae antigens
Method, it is characterised in that: described using method comprises the following steps:
1) after sample 0.5ml PBST buffer solution to be checked, lysate is proceeded in the common centrifuge tube of 1.5ml；Institute
State each component content in PBST buffer as follows: 8g/L NaCl, 0.2g/L KCl, 0.24g/L KH2PO4, 1.44g/L
Na2HPO4, 0.3g/L NaN3, 0.5ml/L Tween-20；The pH=7.4 of described PBST buffer；
2) to step 1) in centrifuge tube in add based on Magnetic Isolation and the quantum dot-labeled bloodthirsty bar of detection human influenza
Anti-human hemophilus influenza immune nanometer magnetic bead 50-150 μ l in the test kit of bacterium antigen, mixed in rotating with 10rpm under room temperature
Close and take off after reacting 10-45min on instrument, centrifuge tube is inserted nano magnetic separator Magneto separate 3min, with pipettor sucking-off supernatant；
3) the PBST buffer 1ml added in test kit washs twice, and after using nano magnetic separator Magneto separate, sucking-off is washed
Wash liquid, finally with the resuspended magnetic bead of 1ml PBS, prepare immune nanometer magnetic bead-somatic antigen complex；Described PBS buffers
In liquid, each component content is as follows: 8g/L NaCl, 0.2g/L KCl, 0.24g/L KH2PO4, 1.44g/L Na2HPO4；Described PBS
The pH=7.4 of buffer；
4) take 100 μ l steps 3) immune nanometer magnetic bead-somatic antigen complex of obtaining in another centrifuge tube, add
Quantum dot-labeled in 100 μ l test kit based on Magnetic Isolation and quantum dot-labeled detection human influenza influenzae antigens
Anti-human hemophilus influenza nano-probe, reacts 10-45min on rotary mixer with 15rpm under room temperature, passes through quantum dot
On antibody and immune nanometer magnetic bead on the immunity combination of somatic antigen, quantum dot is tagged to somatic antigen surface, is formed
Magnetic bead-somatic antigen-quantum dot " sandwich " complex；
5), after having reacted, use nano magnetic separator Magneto separate 3min, remove unnecessary quantum dot-labeled anti-human stream
Haemophilus influenza nano-probe, cleans 2 times with the PBST buffer liquid in test kit, and complex is dispersed in 100 μ l PBS again
In buffer, use fluorescence microplate reader that its fluorescent value is detected；In described PBS, each component content is as follows: 8g/L
NaCl, 0.2g/L KCl, 0.24g/L KH2PO4, 1.44g/L Na2HPO4；The pH=7.4 of described PBS；
6) by the four parts of negative quality-control product samples provided in above-mentioned same method detection kit and a positive quality control
Product sample, reads fluorescent value respectively；The meansigma methods of the fluorescence reading of four parts of negative quality-control product samples is with 3 times of standard deviation sums
CUT-OFF value；If step 5) if in the detection fluorescent value of sample to be checked be i.e. judged as artificial abortion in sample to be checked more than CUT-OFF value
Haemophilus influenza antigen is positive, otherwise is then judged as in sample to be checked that human influenza influenzae antigens is feminine gender；If positive matter
The fluorescent value of control product sample is less than CUT-OFF value, then show that test kit lost efficacy.
As preferably, step 2 proposed by the invention) in, to step 1) in centrifuge tube in add based on Magnetic Isolation
With the anti-human hemophilus influenza immune nanometer magnetic bead in the test kit of quantum dot-labeled detection human influenza influenzae antigens
100 μ l, take off after reacting 20min with 10rpm under room temperature on rotary mixer, centrifuge tube inserts magnetic frame and separates 3min,
With pipettor sucking-off supernatant；
Described step 4) in, taking 100 μ l steps 3) immune nanometer magnetic bead-somatic antigen complex of obtaining is centrifuged in another
Guan Zhong, adds in 100 μ l test kit based on Magnetic Isolation and quantum dot-labeled detection human influenza influenzae antigens
Quantum dot-labeled anti-human hemophilus influenza nano-probe, reacts 20min with 15rpm under room temperature on rotary mixer, logical
Crossing the immunity combination of the antibody on quantum dot and the somatic antigen on immune nanometer magnetic bead, quantum dot is tagged to somatic antigen table
Face, forms magnetic bead-somatic antigen-quantum dot " sandwich " complex.
As preferably, sample to be checked provided by the present invention includes but not limited to throat swab.
Carboxyl water-soluble nano quantum dot needed for the present invention, 350nm carboxyl magnetic bead, can arrive the research list of relevant speciality
Position, company buy or customization；Required instrument, equipment, medicine are commercially available.
The present invention has the advantage that compared to existing technology
1, present invention utilizes immune nanometer magnetic bead to sample can enriching, separation speed is fast, efficiency advantages of higher,
The characteristic such as high in combination with quantum dot light chemical stability, fluorescence intensity is high so that detection system possesses that multiple signal is collaborative to be put
Big effect, thus (it is 1 × 10 to the detection bottom line of human influenza haemophilus to have the detection sensitivity of superelevation3CFU/ml,
Less than the most any detection method reported, with it to the testing result of clinical sample and the current inspection to this pathogen
That surveys " goldstandard "-culture method compares no difference of science of statistics.
2, the antibody used by the present invention identifies that the outer conserved region of human influenza haemophilus specificity P6 surface antigen born of the same parents is many
Clonal antibody, its specificity is high, and for its more most widely used monoclonal antibody, preparation cost is cheap simultaneously, therefore,
Testing cost of the present invention is relatively low.
3, detection method is simple, and detection is quickly, it is easy to judging, testing cost is cheap, overcomes prior art inspection
Positive rate is low, cost is high in survey, operate complicated length loaded down with trivial details, time-consuming, cannot be carried out the deficiency of clinical practice.
4, due to being human influenza influenzae antigens of detection kit detection non-antibody (appearance of antibody needs to infect
Several Zhou Yihou), so early diagnosis and preventing and treating can be carried out, clinical diagnosis coincidence rate is high.The method is at human influenza haemophilus
The aspects such as clinical diagnosis, nosetiology discriminating, Epidemiological study have the highest practical value.
5, due to the present invention first using the exclusive outer membrane protein P6 of human influenza haemophilus as antigen target, and this resists
Former it is not only present in all types of hemophilus influenza cell surface, includes pod membrane and without Buccal mucosa flap, and the high (bacterium of conservative
Between strain, protein conservative is 100%), belong to hemophilus influenza specific antigen, therefore the present invention can high degree of specificity
The infection of detection all types human influenza haemophilus.And have no any on market at present and can quickly examine as the present invention
The instrument surveying all types human influenza haemophilus exists.
6, the clinical sample used by detection method is respiratory secretions such as sputum etc., and non-blood, can exempt
The misery that infant patient takes a blood sample and the psychological burden of the head of a family, therefore be relatively easy to promote.
Detailed description of the invention
The present invention is according to the double antibodies sandwich principle in immunology, utilize immune nanometer magnetic bead to sample can enriching,
The characteristics such as the speed separated is fast, efficiency advantages of higher, and incorporating quantum point photochemical stability is high, fluorescence intensity is high, the one of foundation
Set possess multiple signal work in coordination with amplifications, there is hypersensitivity and high degree of specificity quickly detect human influenza haemophilus
New method, has wide market application foreground.
The present invention is further described in detail by following example.
The preparation of various reagent and the explanation of material requested
1.PBS buffer: weigh 1.44g disodium hydrogen phosphate, 0.24g potassium dihydrogen phosphate, 8g sodium chloride, 0.2g potassium chloride,
It is dissolved in the deionized water of 900ml, after adjusting pH to 7.4 with 1mol/L NaOH, is settled to 1000ml with deionized water.
2. rabbit anti-human hemophilus influenza memebrane protein P6 polyclonal antibody IgG: make by oneself for the present invention is dilute with PBS
Releasing, shake up, making polyclonal antibody weight percent concentration in solution is 1mg/ml.
3. mouse-anti human influenza haemophilus memebrane protein P6 polyclonal antibody IgG: make by oneself for the present invention is dilute with PBS
Releasing, shake up, making polyclonal antibody weight percent concentration in solution is 1mg/ml.
4. quantum dot: in the present invention, quantum dot used is water-soluble CdSe/ZnS quantum that carboxylated amphipathic polymer is modified
Point, a length of 565nm of its transmitted wave, to buy from Wuhan Jia Yuan technology of quantum dots development corporation, Ltd., name of product is that carboxyl is water-soluble
Property quantum dot-565.
5. magnetic bead: in the present invention, magnetic bead used is with superparamagnetism Fe3O4It is polystyrene, surface official for kernel, shell material
Can roll into a ball for carboxyl, particle diameter be respectively 50nm, the carboxyl magnetic bead of 180nm, 350nm, 1150nm, 3 μm, can be from North America, Shaanxi gene
Limited company, Aorun Weina New Material Science and Technology Co., Ltd., Shanghai buy.
6. human influenza haemophilus: purchased from American type culture collection (ATCC), numbered ATCC53781.
7. the microbiological specimens used in the present invention is purchased from American type culture collection (ATCC).
Embodiment 1 rabbit and the preparation of mouse-anti human influenza haemophilus memebrane protein P6 polyclonal antibody IgG
(1) the restructuring preparation of P6-His fusion protein, purification
1. the clone of related gene
To human influenza haemophilus memebrane protein P6, (the accession number in its NCBI Protein Data Bank is
AAA24994) carry out bioinformatic analysis, obtain the peptide fragment that in the outer conserved domain of its born of the same parents, epitope enriches the most, find
The DNA encoding sequence of its correspondence, introduces restriction enzyme site NdeI, 3 ' end introducing termination signal TAA and enzyme action positions in sequence 5 ' simultaneously
After some XhoI, (complete sequence synthesis transfers to Jin Sirui bio tech ltd to complete to chemosynthesis complete genome sequence, people during delivery
The genetic fragment of work synthesis is connected on carrier pUC57), it is designated as P6.Its gene complete sequence is as shown in sequence table.Specifically, P6
The protein sequence of gene code is natural human hemophilus influenza memebrane protein P6 (accession number:AAA24994)
48-153aa.Carrier pUC57 NdeI and XhoI of the DNA fragmentation containing this section of synthetic is carried out after double digestion routinely
Method reclaims purpose fragment, standby.Use simultaneously NdeI and XhoI to carrier pET-28a (+) carry out double digestion, and side routinely
Method the P6 gene obtained after double digestion is connected into pET-28a (+) in carrier, and convert escherichia coli TOP10, build pET-P6
Expression vector.Confirm that expression vector establishment is errorless through enzyme action and sequencing.This vector expression restructuring P6-His fusion protein.
2. the expression and purification of restructuring P6-His fusion protein
To identify that correct positive colony bacterium extracts plasmid after cultivating, technology proceeds to competence E.coli BL21 routinely
(DE3), in plysS, after having converted, bacterium solution is coated on the LB flat board containing 50 μ g/mL kanamycin, screen according to a conventional method
Expression strain.Picking pET-P6 convert the single bacterium colony with exogenous protein expression ability and inoculate into 100mL LB culture medium
In, in 30 DEG C of overnight incubation.After taking out bacterium solution, it is inoculated in 100mL by 1:100 and contains the LB culture medium of 50 μ g/mL kanamycin
In, cultivate to OD in 30 DEG C600When=0.6, add 1mol/L IPTG to final concentration of 1mmol/L, shake bacterium in 30 DEG C and cultivate, lure
Lead expressing fusion protein.After induction 4h, under 8000r/min, centrifugal 10min collects thalline.This thalline 20mL phosphate is delayed
Rush liquid (8g/L NaCl, 0.2g/L KCl, 0.24g/L KH2PO4, 1.44g/L Na2HPO4PH=7.4) wash 3 times and use
10mL sample-loading buffer (20mM Na3PO4, 0.5M NaCl；30mM imidazoles, pH7.4) resuspended after carry out ultrasonication, operate bar
Part is: 50HZ, 200W, ultrasonic 3S, intermittently 5S, works 100 times.Ultrasonic complete after, 12000g is centrifuged 15min and collects precipitation respectively
With carry out electrophoresis detection after supernatant.Find that restructuring P6-His fusion protein is present in thalline in solubility expression mode.
With His Trap affinity after the ultrasonication supernatant of above-mentioned acquisition is filtered with the filter membrane of 0.45 μm
Columns (GE healthcare Products), method to specifications carries out the purification of restructuring P6-His fusion protein.
Concrete grammar is as follows:
1) it is filled distilled water with 5mL syringe, turns on the stopper of post, with the joint provided, post and syringe are connected,
Post is washed with 1mL/min flow velocity.
2) balance with 10mL sample-loading buffer, 1mL/min flow velocity.
3) by fusion protein loading, 1mL/min flow velocity.
4) use 10mL sample-loading buffer, wash post with 1mL/min flow velocity.
5) with 10mL elution buffer (20mM Na3PO4, 0.5M NaCl, 300mM imidazoles, pH7.4), flow with 1mL/min
Speed eluting, is in charge of collection, often pipe 1ml, and 12%SDS-PAGE detects, and merges the sample containing destination protein in elution fraction.Warp
After Bradford test kit carries out determination of protein concentration, adjustment concentration is 0.2mg/mL.
(2) rabbit and the preparation of mouse-anti human influenza haemophilus memebrane protein P6 polyclonal antibody IgG
1. the preparation of rabbit anti-human hemophilus influenza memebrane protein P6 polyclonal antibody IgG
Mix with 1mL Freund's complete adjuvant according to 200 μ g (1mL) with the restructuring P6-His fusion protein of step (one) purification
Immunity Male New Zealand White Rabbit (being provided by Disease Prevention Control Center, Hubei Prov) after emulsifying, in dorsal sc multi-point injection,
Interval 7d after again immunity once, with the restructuring P6-His fusion protein of above-mentioned purification according to 200 μ g (1mL) and 1mL after 14d
Booster immunization is carried out, booster immunization one the most again after booster immunization 7d after incomplete Freund's adjuvant mixing emulsifying
Secondary.Haemanalysis antibody titer is taken after 7d.If dissatisfied, may be repeated one to twice booster immunization, (use to antibody titer is satisfied
ELISA method measures antibody titer more than 1 × 105).If satisfied, Culling heart blood, separate serum, with Protein G affinity chromatograph
Post (GE healthcare Products), in strict accordance with operating instruction purified polyclonal antibodies IgG, with triumphant base Bradford
Protein content detection kit measures antibody concentration and with phosphate buffer (8g/L NaCl, 0.2g/L KCl, 0.24g/L
KH2PO4, 1.44g/L Na2HPO4PH=7.4) being adjusted to 1mg/mL ,-20 DEG C of preservations are standby, so far prepare the anti-human influenza of rabbit addicted to
Blood bacillus memebrane protein P6 polyclonal antibody IgG.Westen blot test shows, this polyclonal antibody IgG can specific recognition people
Hemophilus influenza total length memebrane protein P6.
2. the preparation of mouse-anti human influenza haemophilus memebrane protein P6 polyclonal antibody IgG
(pre-by Hubei Province's disease as complete antigen immune guinea pig with the restructuring P6-His fusion protein of step (one) purification
Anti-control centre provides), omoplate hemostasis antigen 200 μ g/ is only.Fundamental immunity is that isopyknic antigen enters with Freund's complete adjuvant
Row emulsifying, carried out a booster immunization every 2 weeks, and booster immunization equal-volume antigen is carried out with equal-volume incomplete Freund's adjuvant
Emulsifying, altogether immunity 4 times.Haemanalysis antibody titer is taken after final immunization 10d.If dissatisfied, may be repeated one to twice and add
Strong immunity, (measures antibody titer more than 1 × 10 by ELISA method to antibody titer is satisfied5).If satisfied, put to death Cavia porcellus and take blood
Clearly, with Protein G affinity column (GE healthcare Products), in strict accordance with operating instruction purified polyclonal
IgG antibody, measures antibody concentration and with phosphate buffer (8g/L by triumphant base Bradford protein content detection kit
NaCl, 0.2g/L KCl, 0.24g/L KH2PO4, 1.44g/L Na2HPO4PH=7.4) it is adjusted to 1mg/mL, standby, so far make
Obtain mouse-anti human influenza haemophilus memebrane protein P6 polyclonal antibody IgG.Westen blot test shows, this polyclonal antibody
IgG can specific recognition human influenza haemophilus total length memebrane protein P6.
The preparation of embodiment 2 anti-human hemophilus influenza immune nanometer magnetic bead
The optimization of the most anti-human hemophilus influenza polyclonal antibody coupled bead reaction condition:
Using coupling, the magnetic bead of anti-human hemophilus influenza memebrane protein P6 polyclonal antibody is as solid phase carrier, quantum dot mark
The anti-human hemophilus influenza memebrane protein P6 polyclonal antibody of note, as detection antibody, detects artificial abortion by double-antibody method principle
Haemophilus influenza antigen, observes the coupling situation of magnetic bead and multi-resistance.The activator of particle diameter to magnetic bead, and EDC/NHS respectively is dense
The coupling condition such as degree, coupled antibody concentration, coupling time, sealer kind has carried out a series of optimized choice.
1.1 the selection of magnetic bead particle diameter
Selection particle diameter is 50nm, the carboxyl magnetic bead of 180nm, 350nm, 1150nm, 3 μm, all add containing 4mg/ml EDC and
After the PBS of 4mg/ml NHS carries out priming reaction, respectively with the rabbit anti-human hemophilus influenza film described by embodiment 1
Albumen P6 polyclonal antibody IgG carries out coupling reaction.The immune nanometer magnetic bead detection 10 that will prepare respectively4The artificial abortion of CFU/mL
Haemophilus influenza (ATCC numbering 53781), observed result under fluorescence microscope, select fluorescence intensity big, background fluorescence interference is few,
And the very fast person of separating rate is the suitableeest magnetic bead particle diameter under the action of a magnetic field.Result shows that the magnetic bead of particle diameter 350nm best suits this
The requirement of invention, determines that the suitableeest magnetic microsphere particle diameter is 350nm.
The selection of 1.2EDC/NHS activator concentration
After EDC and NHS concentration is each set to l～10mg/ml in reaction system, carries out Concentraton gradient combination, activates respectively
The carboxyl magnetic bead of particle diameter 350nm.The immune nanometer magnetic bead detection 10 that will prepare4Human influenza haemophilus (the ATCC of CFU/mL
Numbering 53781), the suitableeest activation concentration selecting fluorescence powerhouse to be EDC and NHS solution.Result shows when EDC concentration is 5mg/
When ml, NHS concentration is 5mg/ml, coupling effect is best.
The selection of 1.3 coupled antibody concentration
By 20 μ g, 40 μ g, 60 μ g, 80 μ g, 100 μ g, 120 μ g, 140 μ g rabbit anti-human hemophilus influenza memebrane protein P6 many
The magnetic bead that particle diameter is 350nm that clonal antibody IgG is activated by above-mentioned best practice with 1mg respectively carries out coupling.By prepare
Immune nanometer magnetic bead detection 104The human influenza haemophilus (ATCC numbering 53781) of CFU/mL, it was found that when the throwing of antibody
Time high-volume less than 100 μ g/mg, fluorescence intensity increases along with the concentration of antibody and increases, and when the mass concentration of antibody is more than 100
During μ g/mg, fluorescence intensity is basically unchanged and the most slightly reduces, and therefore the present embodiment selects rabbit anti-human hemophilus influenza film egg
The coupling amount of white P6 polyclonal antibody IgG is 100 μ g/mg.
The selection of 1.4 coupling times
After determining the particle diameter of magnetic bead, EDC/NHS activator concentration and antibody coupling amount, by the coupling reaction of antibody Yu magnetic bead
Time is set to 0.5h, 1h, 2h, 3h, 4h, 5h, the immune nanometer magnetic bead detection 10 that will prepare4The human influenza of CFU/mL addicted to
Blood bacillus (ATCC numbering 53781).It was found that between when coupled > 3h time, fluorescence intensity tends towards stability, and extends idol the most again
The connection time, fluorescence no longer strengthens.Accordingly, it is determined that rabbit anti-human hemophilus influenza memebrane protein P6 polyclonal antibody IgG and magnetic bead
The suitableeest coupling reaction time is 3h.Coupling time is far fewer than the 24h of traditional E LISA method.
The selection of 1.5 sealers
The particle diameter of magnetic bead, EDC/NHS activator concentration, antibody coupling amount and idol is selected according to the above-mentioned optimal conditions determined
Coupling reaction is carried out after the connection time.After coupling terminates, selecting BSA, ethanolamine, Tris and D-Glucosamine Hydrochloride are as exempting from
Epidemic disease nanometer magnetic bead sealer, prepares finished product immune nanometer magnetic bead.The immune nanometer magnetic bead detection 10 that will prepare4The people of CFU/mL
Hemophilus influenza (ATCC numbering 53781).It was found that use ethanolamine as the detection of the immune nanometer magnetic bead of sealer
Fluorescent value is the highest.It is presumed that owing to the molecule of ethanolamine is less, can preferably consume and not tie with antibody due to sterically hindered
The surface carboxyl groups closed, makes closing the most complete, and effectively reduces the space steric effect structure influence to connecting antibody.
2. coupling process:
Take 5mg magnetic bead (with superparamagnetism Fe3O4Carboxyl magnetic bead for kernel, particle diameter are 350nm) commonly it is centrifuged in 1.5ml
Guan Zhong, washs three times with 1ml MES buffer (2g/L MES, pH6.0), is placed in nano magnetic separator and carries out Magneto separate
(0.4T) remove supernatant after, be sequentially added into the EDC solution that concentration is 10mg/ml with above-mentioned MES buffer and with above-mentioned
The concentration of MES buffer is each 0.5ml of sulfo-NHS solution of 10mg/ml, activates in rotary mixer with 15rpm
1hr, removes supernatant after Magneto separate, resuspended with MES buffer above-mentioned for 1ml；Take 5 centrifuge tubes, each centrifuge tube adds 200
The magnetic bead of the above-mentioned activation of μ L, sucking-off supernatant after Magneto separate, addition PBS (8g/L NaCl, 0.2g/L in each pipe
KCl, 0.24g/L KH2PO4, 1.44g/L Na2HPO4, pH7.4) and prepared by the embodiment 1 that concentration is 100 μ g/ml that dilutes
The rabbit anti-human hemophilus influenza memebrane protein P6 polyclonal antibody each 1ml of IgG solution, under room temperature with 15rpm in rotary mixer
Reaction 3h, after Magneto separate removes supernatant, each 1ml of addition contains the above-mentioned PBS of 1mg/ml ethanolamine, with 15rpm under room temperature
React in rotary mixer 2h with close on magnetic bead not with the carboxyl of antibody response.Remove each pipe supernatant after Magneto separate, respectively use
1ml lavation buffer solution (8g/L NaCl, 0.2g/L KCl, 0.24g/L KH2PO4, 1.44g/L Na2HPO4, 0.5ml/L
Tween-20, pH7.4) wash three times, last each 1ml preserves buffer (8g/L NaCl, 0.2g/L KCl, 0.24g/L
KH2PO4, 1.44g/L Na2HPO4, 0.3g/L NaN3, 5g/L BSA, pH7.4) and resuspended magnetic bead, it is placed in 4 DEG C and saves backup.
The preparation of the anti-human hemophilus influenza nano-probe that embodiment 3 is quantum dot-labeled
1. nanometer carboxylic quantum dot-labeled mouse-anti human influenza haemophilus memebrane protein P6 polyclonal antibody IgG reaction condition
Optimize:
1.1, the determination of carboxyl quantum dot-labeled antibody probe optimum mark pH
In being reacted by labelling, phosphate buffer pH is set to 5, and 6,7,8,9, marked product utilize full spectrogrph enter
Row fluorescent strength determining, observes the different pH value impact on coupling reaction, it is determined that the Optimal pH of quantum dot-labeled multi-resistance reaction
For 7.0-8.0.This experimental selection pH7.4.
1.2, the determination of carboxyl quantum dot-labeled antibody probe optimum mark amount
The ratio of quantum dot molar concentration with multi-resistance concentration is respectively set to 1:1,1:2,1:3 and 1:4, is marked reaction
After, marked product utilizes full spectrogrph carry out fluorescent strength determining, observes the impact of the two variable concentrations comparison coupling reaction,
Determine that the optimum molar concentration ratio that quantum dot-labeled mouse-anti human influenza haemophilus memebrane protein P6 polyclonal antibody IgG reacts is amount
Son point is 1:3 with antibody molar ratio.This optimal concentration ratio of this experimental selection determines labelled amount.
1.3, the determination of the quantum dot-labeled antibody probe of carboxyl optimal sealer kind
With ethanolamine, Tris, PEG2000-NH2Or BSA is as sealer, after being marked reaction, to marked product
Utilize full spectrogrph to carry out fluorescent strength determining, observe the impact that different sealers reacts for labelling, it was found that
PEG2000-NH2For optimal sealer, its colloidal stability being remarkably improved labeled complex and immunocompetence.
2. labeling process:
2nmol carboxyl water-soluble quantum dot, 300nmol N-hydroxy-succinamide it is sequentially added in microcentrifugal tube
(sulfo-NHS) and 300nmol carbodiimide (EDC), with phosphate buffer (2.9g/L disodium hydrogen phosphate, 0.295g/L phosphorus
Acid dihydride sodium, 4g/L sodium chloride, pH 7.4) constant volume is 2ml, ceaselessly mixed solution, after 37 DEG C of reaction 30min, dialysis is removed
Sulfo-NHS and EDC as activator of excess.In the quantum dot of activation, add prepared by the embodiment 1 of 6nmol
Mouse-anti human influenza haemophilus memebrane protein P6 polyclonal antibody IgG, lucifuge reaction 2h, add single-ended amination Polyethylene Glycol
(PEG2000-NH2) to final concentration of 1%, close unreacted activated carboxyl site, continue lucifuge reaction 1h.Use 0.2 μm
PES filter is filtered to remove antibody aggregation thing, then filtrate be transferred to, in 50000MW ultra-filtration centrifuge tube, exist with 8000g centrifugal force
Centrifugal 15min at 4 DEG C, removes the antibody that coupling reaction does not occurs and the by-product in reaction.Collect super filter tube filter membrane upper strata amount
Sub-point-antibody coupling matter solution, be dissolved in 2ml phosphate cleaning mixture (2.9g/L disodium 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, then this solution is transferred to 50000MW ultrafiltration from
In heart pipe, with 8000g centrifugal force centrifugal 15min at 4 DEG C, collect super filter tube filter membrane upper strata quantum dot-antibody coupling matter solution,
Be dissolved in 1ml phosphate preserve liquid (2.9g/L disodium hydrogen phosphate, 0.295g/L sodium dihydrogen phosphate, 2g/L sodium chloride, 10g/L BSA,
0.3g/L sodium azide, pH 7.4) in, it is placed in 4 DEG C and saves backup.
Embodiment 4 immune nanometer magnetic bead carries out the optimization of immunocapture condition to human influenza influenzae antigens
Using coupling, the immune nanometer magnetic bead of rabbit anti-human hemophilus influenza memebrane protein P6 polyclonal antibody IgG is as solid phase
Carrier, quantum dot-labeled mouse-anti human influenza haemophilus memebrane protein P6 polyclonal antibody is as detection antibody, by dual anti-folder
Heart method principle, sets up the detection system of human influenza influenzae antigens.Respectively to the consumption of immune nanometer magnetic bead in detection system,
The conditions such as capture time have carried out a series of optimized choice.
1. the selection of immune nanometer magnetic bead addition
By 20 μ l, 40 μ l, 60 μ l, 80 μ l, 100 μ l, 120 μ l, 140 μ l by the made immune nano got ready of embodiment 2
Magnetic bead is added separately to 0.5ml containing 104PBST buffer (the 8g/ of the human influenza haemophilus (ATCC numbering 53781) of CFU/mL
L NaCl, 0.2g/L KCl, 0.24g/L KH2PO4, 1.44g/L Na2HPO4, 0.3g/L NaN3, 0.5ml/L Tween-20；
PH7.4) in, carry out immunocapture, then detected by the quantum dot-labeled probe described by embodiment 3, record fluorescent value.Knot
Fruit finds, along with the increase of immune nanometer magnetic bead addition, fluorescent value is gradually increased, when immune nanometer magnetic bead addition reaches
During 100 μ l, fluorescent value reaches maximum.Being further continued for increasing the amount of immune nanometer magnetic bead, fluorescent value reduces on the contrary.This be probably by
Too much in immune nanometer magnetic bead, during Magneto separate, thalline is caused damage, thus causes capture rate to decline.Therefore this experimental selection 100 μ
L is as the optimal addn of immune nanometer magnetic bead.
2. the selection of immunocapture time
After determining the addition of magnetic bead, take four parts of made immune nanometer magnetic beads got ready of embodiment 2, at room temperature with 10r/
Min, to 104The human influenza haemophilus (ATCC numbering 53781) of CFU/mL carry out 10min, 15min, 20min, 30min,
The immunocapture of 45min and 60min, then detected by the quantum dot-labeled probe described by embodiment 3, record fluorescent value.
It was found that fluorescent value reaches maximum when immunocapture 20min, therefore this experimental selection 20min is as immunocapture
Best Times.
The preparation of embodiment 5 PBST buffer
Take 8g NaCl, 0.2g KCl, 0.24KH2PO4, 1.44g Na2HPO4, 0.3g NaN3, 0.5ml Tween-20 is molten
Solution, in 800ml distilled water, adjusts pH to 7.4 with 5M NaOH, then is settled to 1000ml.
The preparation of embodiment 6 quality-control product
1. positive quality control product: be attached to being dried with the human influenza haemophilus (0.5 μ g) of 1% formalin-inactivated on swab,
It is positive quality control product.
2. negative quality-control product: negative quality-control product is i.e. defined as the throat swab of the negative crowd of human influenza haemophilus through clinic
Sample.
The preparation of embodiment 7 test kit
By the anti-human hemophilus influenza immune nanometer magnetic bead described by embodiment 2, the quantum dot mark described by embodiment 3
PBST buffer described by the anti-human hemophilus influenza nano-probe of note, embodiment 5, the quality-control product described by embodiment 6
Collectively constitute based on Magnetic Isolation and the test kit of quantum dot-labeled detection human influenza influenzae antigens.
The using method of embodiment 8 test kit
Clinical means obtains people's throat swab routinely, with on the PBST buffer solution throat swab in 0.5ml test kit
After clinical sample, lysate is proceeded in the common centrifuge tube of 1.5ml, add in this centrifuge tube anti-human influenza in test kit addicted to
Blood bacillus immune nanometer magnetic bead 100 μ l, takes off after reacting 20min with 10rpm, inserted by centrifuge tube under room temperature on rotary mixer
Enter magnetic frame and separate 3min, with pipettor sucking-off supernatant.Add the PBST buffer 1ml in test kit to wash twice, Magneto separate
Rear sucking-off cleaning mixture, finally with the 1mL resuspended magnetic bead of PBS buffer.Take the above-mentioned immune nanometer magnetic bead-somatic antigen of 100 μ l
Complex is in another centrifuge tube, and the quantum dot-labeled anti-human hemophilus influenza nanometer added in 100 μ l test kits is visited
Pin, reacts 20min with 15rpm under room temperature on rotary mixer, by the antibody on quantum dot and immune nanometer magnetic bead
The immunity combination of somatic antigen, quantum dot is tagged to somatic antigen surface, forms magnetic bead-somatic antigen-quantum dot " Sanming City
Control " complex.After having reacted, Magneto separate 3min, remove unnecessary quantum dot-labeled probe, and by PBST buffer solution for cleaning 2
Time, complex is dispersed in 100 μ l PBS again, uses fluorescence microplate reader (Ex=405nm, Em=565nm) to it
Fluorescent value detects.
By the four parts of negative quality-control products provided in above-mentioned same method detection kit and a positive quality control product sample,
Read fluorescent value respectively；The meansigma methods of the fluorescence reading of four parts of negative quality-control product samples and 3 times of standard deviation sums are CUT-OFF
Value；If if the detection fluorescent value of above-mentioned clinical people's throat swab sample is i.e. judged as in this part of clinical throat swab more than CUT-OFF value
Human influenza influenzae antigens is positive, otherwise is then judged as human influenza influenzae antigens in this part of clinical people's throat swab sample
For feminine gender；If the fluorescent value of positive quality control product sample is less than CUT-OFF value, then show that test kit lost efficacy.
The detection sensitivity of embodiment 9 test kit and specific test
After human influenza haemophilus (ATCC numbering 53781) sample PBST buffer is carried out serial dilution, with enforcement
Test kit described in example 7 detects, and result shows that its detection lowest limit is 1 × 103CFU/ml.Meanwhile, with Ureaplasma urealyticum (ATCC
Numbering 27618), mycoplasma hominis (ATCC numbering 23114), mycoplasma pneumoniae (ATCC numbering 15531), streptococcus pneumoniae
(ATCC numbering 49619) adenovirus hominis 3 type (GB strain, ATCC numbering VR-3), adenovirus hominis 7 type (Gomen strain, ATCC numbering VR-
7), influenza virus A hominis's (H1N1, ATCC numbering VR-1743), people's Influenza B virus (ATCC numbering VR-790), pneumonia clothing
Substance (AR-39 strain, ATCC numbering 53592), moraxelle catarrhalis (ATCC numbering 25238) etc. detect, and test kit detection contains
The PBST buffer of these microorganisms is all negative.
Embodiment 10 clinical trial example
Using hemophilus influenza detection goldstandard-culture method as reference, take 88 example Pneumology Department Patients with Lower Respiratory Tract Infections
Test kit described by oropharyngeal swab specimen embodiment 7 detects, and culture method positive rate is 20.5% (18/88), this reagent
Box is 22.7% (20/88), and the coincidence rate of 2 kinds of methods is 95.5% (84/88).Concrete outcome is as shown in table 1.
The testing result of table 1 clinical samples
It is pointed out that and the foregoing is only presently preferred embodiments of the present invention, not in order to limit the present invention, all
Any amendment of being made within present invention spirit and principle, equivalent etc. should be included in protection scope of the present invention it
In.
Claims (4)
1. one kind is used for preparation test kit based on Magnetic Isolation and quantum dot-labeled detection human influenza influenzae antigens
Method, it is characterised in that: described preparation method comprises the following steps:
1) preparation of anti-human hemophilus influenza immune nanometer magnetic bead:
1.1) rabbit and the preparation of mouse-anti human influenza haemophilus memebrane protein P6 polyclonal antibody IgG
1.1.1) the restructuring preparation of P6-His fusion protein, purification:
1.1.1.1) human influenza haemophilus memebrane protein P6 is carried out bioinformatic analysis, obtain in the outer conserved domain of its born of the same parents
The peptide fragment that epitope enriches the most；
1.1.1.2) find step 1.1.1.1) in obtained by gene coded sequence corresponding to peptide fragment, in the 5 ' of said gene sequence
End and 3 ' ends introduce restriction enzyme site chemosynthesis complete genome sequence, and labelling is designated as P6 simultaneously；
1.1.1.3) by step 1.1.1.2) in obtained by P6 be cloned into expression vector pET-28a by molecular biology method
Expression in escherichia coli restructuring P6-His fusion protein is proceeded to after (+)；Described restructuring P6-His fusion protein is with solubility expression
Mode is present in thalline；
1.1.1.4) by ni-sepharose purification step 1.1.1.3) obtained by recombiant protein, after SDS-PAGE detects its purity, with
Bradford method measures protein concentration, and it is standby after 0.2mg/mL for adjusting protein concentration；
1.1.2) rabbit and the preparation of mouse-anti human influenza haemophilus memebrane protein P6 polyclonal antibody IgG:
1.1.2.1) with step 1.1.1.4) in obtained by restructuring P6-His fusion protein as complete antigen, the respectively new west of immunity
Blue White Rabbit and Cavia porcellus；Prepare rabbit anti-human hemophilus influenza memebrane protein P6 antiserum and mouse-anti human influenza haemophilus film respectively
Albumen P6 antiserum；Described rabbit anti-human hemophilus influenza memebrane protein P6 antiserum and mouse-anti human influenza haemophilus memebrane protein P6
Sero-fast indirect ELISA titer is all higher than 1 × 105；
1.1.2.2) use Protein G affinity column respectively purified rabbit anti-human's hemophilus influenza memebrane protein P6 antiserum and
Polyclonal antibody IgG in mouse-anti human influenza haemophilus memebrane protein P6 antiserum；
1.1.2.3) with triumphant base Bradford protein content detection kit determination step 1.1.2.2) obtained by more than two kinds gram
The concentration of grand IgG antibody, standby after its protein concentration is all adjusted to 1mg/mL；
1.2) being coated of immune nanometer magnetic bead:
1.2.1) take 5mg magnetic bead, with 1ml MES buffer solution three times, be placed in nano magnetic separator after carrying out Magneto separate and move
Except supernatant；Described magnetic bead is with superparamagnetism Fe3O4For the carboxyl magnetic bead that kernel, particle diameter are 350nm；Described MES buffer is matter
Amount concentration is 2-(N-morpholino) ethyl sulfonic acid of 2g/L；The pH=6.0 of described MES buffer；The magnetic of described nano magnetic separator
Property intensity is 0.4T；
1.2.2) be sequentially added into use step 1.2.1) in the concentration of MES buffer be 8-12mg/ml EDC solution and
Use step 1.2.1) in the concentration of MES buffer be each 0.5ml of sulfo-NHS solution of 6-12mg/ml, with 10-
40rpm activates 1hr in rotary mixer, is placed in after carrying out Magneto separate in nano magnetic separator and removes supernatant, uses 1ml step
1.2.1) the MES buffer in is resuspended, the magnetic bead after being activated；
1.2.3) take 5 centrifuge tubes, each centrifuge tube add 200 μ L steps 1.2.2) obtained by activation after magnetic bead,
It is placed in after nano magnetic separator carries out Magneto separate and removes supernatant, the concentration of addition PBS dilution in each centrifuge tube
For 50-200 μ g/ml by step 1.1) prepared by rabbit anti-human hemophilus influenza memebrane protein P6 polyclonal antibody IgG solution
Each 1ml, reacts 2-6h with 15rpm under room temperature in rotary mixer, is placed in after carrying out Magneto separate in nano magnetic separator and removes
After supernatant, each 1ml of addition contains the above-mentioned PBS of 1mg/ml ethanolamine, reacts in rotary mixer with 15rpm under room temperature
2h with close on magnetic bead not with the carboxyl of antibody response；In described PBS, each component content is as follows: 8g/L NaCl,
0.2g/L KCl, 0.24g/L KH2PO4, 1.44g/L Na2HPO4, the pH=7.4 of described PBS；
1.2.4), after capping completes, these 5 centrifuge tubes are placed in after nano magnetic separator carries out Magneto separate and remove supernatant,
Each with 1ml lavation buffer solution washing three times；In described lavation buffer solution, each component content is as follows: 8g/L NaCl, 0.2g/L
KCl, 0.24g/L KH2PO4, 1.44g/L Na2HPO4, 0.5ml/L Tween-20, the pH=7.4 of described lavation buffer solution；
1.2.5) in each centrifuge tube, it is separately added into 1ml preserves the resuspended magnetic bead of buffer, be placed in 4 DEG C and save backup；Described guarantor
Deposit each component content in buffer as follows: 8g/L NaCl, 0.2g/L KCl, 0.24g/L KH2PO4, 1.44g/L Na2HPO4,
0.3g/L NaN3, 5g/L bovine serum albumin, the pH=7.4 of described preservation buffer；
2) preparation of quantum dot-labeled anti-human hemophilus influenza nano-probe:
Its concrete preparation method includes:
2.1) in microcentrifugal tube, it is sequentially added into 2nmol carboxyl water-soluble quantum dot, 300nmol N-hydroxy succinyl
Imines sulfo-NHS and 300nmol carbodiimide EDC, with phosphate buffer constant volume as 2ml, mixed solution, 37 DEG C of reactions
After 30min, sulfo-NHS and EDC as activator of excess, the quantum dot after being activated are removed in dialysis；Described phosphoric acid
In salt buffer, each component content is as follows: 2.9g/L disodium hydrogen phosphate, 0.295g/L sodium dihydrogen phosphate, 4g/L sodium chloride；Described
The pH=7.4 of phosphate buffer；
2.2) in step 2.1) obtained by activation quantum dot in, add the step 1.1 of 4-12nmol) in prepared mouse-anti
Human influenza haemophilus memebrane protein P6 polyclonal antibody IgG, lucifuge reaction 2h, add single-ended amination Polyethylene Glycol PEG2000-
NH2To final concentration of 1%, close unreacted activated carboxyl site, continue lucifuge reaction 1h；
2.3) be filtered to remove step 2.2 with 0.2 μm PES filter) in antibody aggregation thing, then filtrate be transferred to 50000MW
In ultra-filtration centrifuge tube, with 8000g centrifugal force centrifugal 15min at 4 DEG C, remove in antibody and reaction that coupling reaction does not occurs
By-product；
2.4) collect step 2.3) in ultra-filtration centrifuge tube filter membrane upper strata quantum dot-antibody coupling matter solution, be dissolved in 2ml phosphoric acid brine wash
Wash in liquid, then this solution is transferred in a new 50000MW ultra-filtration centrifuge tube, be centrifuged at 4 DEG C with 8000g centrifugal force
15min, collects ultra-filtration centrifuge tube filter membrane upper strata quantum dot-antibody coupling matter solution, is dissolved in 1ml phosphate and preserves in liquid, is placed in 4
DEG C save backup；In described phosphate cleaning mixture, each component content is as follows: 2.9g/L disodium hydrogen phosphate, 0.295g/L biphosphate
Sodium, 4g/L sodium chloride, 5ml/L tween 20,0.3g/L sodium azide, the pH=7.4 of described phosphate cleaning mixture；Described phosphate
Preserve each component content in liquid as follows: 2.9g/L disodium hydrogen phosphate, 0.295g/L sodium dihydrogen phosphate, 2g/L sodium chloride, 10g/L cattle
Serum albumin, 0.3g/L sodium azide；Described phosphate preserves the pH=7.4 of liquid；
3) preparation of PBST buffer:
Its concrete compound method includes:
Take 8g NaCl, 0.2g KCl, 0.24KH2PO4, 1.44g Na2HPO4, 0.3g NaN3, 0.5ml Tween-20 is dissolved in
In 800ml distilled water, adjust pH to 7.4 with 5M NaOH, then be settled to 1000ml；
4) preparation of quality-control product:
4.1) positive quality control product: positive quality control product is dried by the human influenza haemophilus inactivated and is attached on swab form；
4.2) negative quality-control product: negative quality-control product is i.e. defined as the throat swab of the negative crowd of human influenza haemophilus through clinic.
Method the most according to claim 1, it is characterised in that: described step 1.2.2) in, it is sequentially added into and uses step
The concentration of the MES buffer in 1.2.1) is the EDC solution of 10mg/ml and uses step 1.2.1) in MES buffer
The concentration of preparation is each 0.5ml of sulfo-NHS solution of 10mg/ml, activates 1hr with 15rpm, be placed in and receive in rotary mixer
Rice magnetic separator in carry out Magneto separate after remove supernatant, by 1ml step 1.2.1) in MES buffer resuspended, after being activated
Magnetic bead；
Described step 1.2.3) in, in each centrifuge tube addition PBS dilution concentration be 100 μ g/ml by step
1.1) the rabbit anti-human hemophilus influenza memebrane protein P6 polyclonal antibody each 1ml of IgG solution prepared by, under room temperature with 15rpm in
Reacting 3h in rotary mixer, be placed in after removing supernatant after carrying out Magneto separate in nano magnetic separator, each 1ml that adds is containing 1mg/ml
The above-mentioned PBS of ethanolamine；
Described step 2.2) in, in step 2.1) obtained by activation quantum dot in, add 6nmol step 1.1) in made
Standby mouse-anti human influenza haemophilus memebrane protein P6 polyclonal antibody IgG, lucifuge reaction 2h.
Method the most according to claim 1 and 2, it is characterised in that: described quantum dot is that carboxylated amphipathic polymer is modified
Water-soluble CdSe/ZnS quantum dot.
Method the most according to claim 3, it is characterised in that: described magnetic bead is with superparamagnetism Fe3O4For kernel, shell material
The carboxyl magnetic bead that material is polystyrene, surface functional group is carboxyl, particle diameter is 350nm.
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