CN105277714B - Rapid detection method and kit for human parainfluenza virus based on magnetic separation and quantum dot labeling - Google Patents
Rapid detection method and kit for human parainfluenza virus based on magnetic separation and quantum dot labeling Download PDFInfo
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- CN105277714B CN105277714B CN201410406305.2A CN201410406305A CN105277714B CN 105277714 B CN105277714 B CN 105277714B CN 201410406305 A CN201410406305 A CN 201410406305A CN 105277714 B CN105277714 B CN 105277714B
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Abstract
The invention discloses a rapid detection method and a kit for human parainfluenza virus based on magnetic separation and quantum dot labeling. The kit is composed of anti-human-parainfluenza-virus immunonanomagnetic beads with a I, II and III human parainfluenza virus enrichment function, anti-human-parainfluenza-virus nanoprobes labeled with quantum dots, quality control products and a PBST buffer solution. The quality control products comprise positive quality control products and negative quality control products. The positive quality control products are prepared after inactivated human parainfluenza virus is dried and combined on swabs. The negative quality control products are throat swabs of people who are determined to be negative in human parainfluenza virus. The detection method and the kit which can detect human parainfluenza virus antigens simply, rapidly and with high sensitivity are provided, and preparation and usage methods for the kit are provided.
Description
Technical field
The present invention relates to technical field of medical detection, specially a kind of based on Magnetic Isolation with quantum dot-labeled detection people
The method for quick of parainfluenza virus antigens and detection kit, and the method for preparation and use of this detection kit.
Background technology
Human parainfluenza viruseses (human parainfluenza virus, HPIV) are the weights of children with acute respiratory tract infection
Want pathogen, mainly through droplet infection, also can be propagated by the mucosal contact of eye, mouth or nose, sickness rate is with infant
High.Human parainfluenza viruseses' infection is in global distribution, is the cause of disease of common Community Acquired Respiratory Tract Infection, human parainfluenza disease
Poison infection is up to 30-40% in the incidence rate of children's children with acute respiratory tract infection, and it is to lead to children's lower respiratory tract seriously to be felt
Pathogen contaminating, being only second to respiratory syncytial virus.This pathogen is found in the end of the fifties in last century, original name celestial platform disease first
Poison, initially separates from the one infant lung liquid dying of pneumonia in Japanese celestial platform city.4 can be divided into according to hereditism and serology
Type, and 1-3 type is most commonly seen in clinic, the main structure of every kind of hypotype is similar with biological property, but epidemiology and institute
Diseases induced Clinical symptoms are variant.Parainfluenza viruses 1, the relevant disease that 2 types are led to mainly have croup, on exhale
Inhale road and lower respiratory infection；3 types mainly result in bronchiolitises and pneumonia；4 types are clinically rare, typically only result in light
Micro- infection.
Clinical patient (as parainfluenza viruses, hemophilus influenza, influenza virus, is exhaled due to different respiratory pathogens
Inhale road syncytial viruses, adenoviruss etc.) infection causes disease symptomses quite similar, which results in the popular of human parainfluenza viruseses
Diagnosis is relatively difficult, makes a definite diagnosis and tends to rely on laboratory diagnosiss.Fast and effectively diagnostic method should be at the beginning of the morbidity of disease
Phase can be obtained by clearly diagnosing, the development delay targetedly treated, stop the state of an illness convenient to carry out.
Although human parainfluenza viruseses propagate in the world, the infection of infant is especially universal, can be used for laboratory
The species of the standardization commercially available reagent of diagnosis is few.At present, the detection of human parainfluenza viruseses mainly has following several method：
First, Routine Test Lab detection
1st, virus purification
The goldstandard of laboratory diagnosiss human parainfluenza viruseses is to separate human parainfluenza viruseses' strain.Made using nasopharyngeal secretionses
For the specimen of virus purification, the method isolated viral of LCC-MK2 cell culture can be used.But this method has serious defect, because
Waste time and energy for it, it usually needs just can obtain within one week final result, so in clinicing aspect, the treatment of patient just be had necessarily
Limitation.
2nd, Serologic detection
Adopt euzymelinked immunosorbent assay (ELISA), colloidal gold immunization, micro-Immunofluorescence assay and indirect hemagglutination test etc., detection is tested
Human parainfluenza viruseses' antibody horizontal in person's serum, can prompter's parainfluenza virus infection indirectly presence.However, serological test
A kind of retrospective diagnosis can only be provided, it needs to detect the Acute Stage and convalescent paired sera simultaneously, if recovered
Higher 4 times or more than 4 times than acute stage of interim anti-human parainfluenza viruses antibody titer just has diagnostic significance.In addition, what antibody occurred
Opportunity is difficult to grasp, and there is the difference of human parainfluenza viruseses' specific antibody between children and adolescents and adult again, and, according to
The recall rate only about 50% of report infant IgM, therefore the detection quality of existing serological method is subject to a definite limitation.
2nd, quick diagnosis
Directly check that human parainfluenza viruseses' proteantigen and viral nucleic acid can reach the purpose of quick diagnosis, mainly have at present
Immunofluorescence, immunoenzyme method and PCR method etc..Immunofluorescence and immunoenzyme method all can not carry out a step detection, all there is behaviour
Make step complicated, need professional to operate, detection time length (more than 2h), the shortcomings of relatively costly.PCR method is quick, clever
Quick, special, it is the important means of research human parainfluenza viruseses infection at present, but because PCR requires to experimental facilitiess and operation
Higher, and false positive easily occurs, can't be used as conventional methods for clinical diagnosis in China.Therefore, set up and possess ultra-high sensitive
Human parainfluenza viruseses' specific antigen quick diagnosis method of degree is very necessary.
Content of the invention
For these technical problems present in background technology, the invention provides a kind of be based on Magnetic Isolation and quantum dot
The energy of labelling is easy, quick, the detection method of highly sensitive detection human parainfluenza viruseses' antigen and test kit, and this reagent
The method of preparation and use of box.
The present invention is achieved through the following technical solutions：
A kind of based on Magnetic Isolation and quantum dot-labeled detection human parainfluenza viruseses' antigen method it is characterised in that：
Described the method comprises the following steps：
1) preparation of rabbit anti-I, II and type III human parainfluenza viruseses' HN protein polyclone antibody；
2) preparation of Mus anti-I, II and type III human parainfluenza viruseses' HN protein polyclone antibody；
3) by step 1) rabbit anti-I, II of preparing and type III human parainfluenza viruseses' HN protein polyclone antibody respectively with nanometer
Magnetic bead passes through covalent coupling, prepares anti-I, II and type III human parainfluenza viruseses' immune nanometer magnetic bead respectively, by above-mentioned three kinds of immunity
Nanometer magnetic bead mixed in equal amounts is obtained anti-human parainfluenza viruses immune nanometer magnetic bead；
4) by step 2) Mus anti-I, II of preparing and type III human parainfluenza viruseses' HN protein polyclone antibody respectively with nanometer
Quantum dot passes through covalent coupling, prepares quantum dot-labeled anti-I, II and type III human parainfluenza viruseses' nano-probe respectively, will be upper
State three kinds of nano-probe mixed in equal amounts and quantum dot-labeled anti-human parainfluenza viruses nano-probe is obtained；
5) take human respiratory secretions sample (including but not limited to throat swab), after PBST buffer solution, add step
The anti-human parainfluenza viruses immune nanometer magnetic bead that rapid 3) prepare, is sufficiently mixed, and carries out Magneto separate after reaction 15-45min, with
After PBST buffer solution 2 times, in the precipitate obtaining to Magneto separate, add step 4) prepare quantum dot-labeled anti-
Human parainfluenza viruseses' nano-probe, carries out Magneto separate, after PBST buffer solution 2 times, using fluorescence after reaction 25-45min
Microplate reader reads fluorescent value；In described PBST buffer, each constituent content is respectively 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 PBST buffer；
6) according to step 1)-step 5) method detect that four parts are defined as the negative people of human parainfluenza viruseses through clinic respectively
The respiratory secretions sample of group, reads fluorescent value；The respiratory secretions sample of the negative crowd of described human parainfluenza viruseses
Abbreviation human parainfluenza viruseses' negative control sample；The meansigma methodss of the fluorescent value of described four parts of human parainfluenza viruseses' negative control sample
It is CUT-OFF value with 3 times of standard deviation sums；If step 5) in human respiratory secretions sample detection fluorescent value be more than CUT-
OFF value, then be judged as that in human respiratory secretions sample, human parainfluenza viruseses' antigen is the positive；If step 5) in human respiratory divide
The detection fluorescent value of secretion sample is less than CUT-OFF value, then be judged as that in human respiratory secretions sample, human parainfluenza viruseses resist
Originally it was negative.
A kind of based on Magnetic Isolation and quantum dot-labeled detection human parainfluenza viruseses' antigen test kit, its feature exists
In：Described test kit is by the anti-human parainfluenza viruses immune nano magnetic with enrichment I, II, type III human parainfluenza viruseses' function
Pearl, quantum dot-labeled anti-human parainfluenza viruses 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 attached on swab by the human parainfluenza viruseses' drying inactivateing
Form；Described feminine gender quality-control product is the throat swab being defined as the negative crowd of human parainfluenza viruseses through clinic.
A kind of test kit for preparation based on Magnetic Isolation and quantum dot-labeled detection human parainfluenza viruseses' antigen
Method it is characterised in that：Described preparation method comprises the following steps：
1) preparation of anti-human parainfluenza viruses immune nanometer magnetic bead：
1.1) preparation of rabbit and Mus anti-I, II and type III human parainfluenza viruseses HN protein polyclone antibody IgG
1.1.1) the preparation of restructuring HN1-His, HN2-His and HN3-His fusion protein, purification：
1.1.1.1) bioinformatic analysis are carried out to I, II and type III human parainfluenza viruseses' HN albumen, respectively obtain I,
Epitope peptide fragment the abundantest in II and type III human parainfluenza viruseses' HN ectodomain；
1.1.1.2) find step 1.1.1.1) in the corresponding gene coded sequence of obtained peptide fragment, according in escherichia coli
The Preference of codon, to step 1.1.1.1) in obtained gene coded sequence carry out codon optimization；
1.1.1.3) in step 1.1.1.2) in 5 ' ends of obtained gene order and 3 ' ends introduce restriction enzyme site respectively
And distinguish chemosynthesis complete genome sequence, labelling is designated as hn1, hn2 and hn3 simultaneously；Its gene order is referring to sequence table；
1.1.1.4) by step 1.1.1.3) in obtained hn1, hn2 and hn3 press molecular biology method respectively gram
Grand enter expression vector pET-28a (+) after proceed to expression in escherichia coli restructuring HN1-His, HN2-His and HN3-His merge egg
In vain；Described restructuring HN1-His, HN2-His and HN3-His fusion protein is all present in genetic engineering thalline with inclusion bodies
In；
1.1.1.5) use ni-sepharose purification step 1.1.1.4) obtained by three kinds of inclusion body proteins, SDS-PAGE detects that it is pure
After degree, more respectively renaturation is carried out to inclusion body protein, protein concentration is measured with Bradford method, three kinds of protein concentrations of adjustment are equal
For standby after 0.2mg/mL；
1.1.2) the preparation of rabbit and Mus anti-I, II and type III human parainfluenza viruseses HN protein polyclone antibody IgG：
1.1.2.1) with step 1.1.1.5) in obtained restructuring HN1-His, HN2-His and HN3-His through renaturation
Fusion protein is complete antigen, respectively immune new zealand white rabbit and Cavia porcelluss；Prepare rabbit anti-restructuring HN1-His, HN2-His respectively
And HN3-His fusion protein antiserum and Mus anti-restructuring H1-His, HN2-His and HN3-His fusion protein antiserum；Described rabbit
Anti- restructuring HN1-His, HN2-His and HN3-His fusion protein antiserum and Mus anti-restructuring HN1-His, HN2-His and HN3-
The sero-fast indirect ELISA titer of His fusion protein is all higher than 1 × 105；
1.1.2.2) adopt Protein G affinity column purified rabbit anti-restructuring HN1-His, HN2-His and HN3- respectively
Anti-TNF-α in His fusion protein antiserum and Mus anti-restructuring HN1-His, HN2-His and HN3-His fusion protein antiserum
Body IgG；
1.1.2.3) with triumphant base Braford protein content detection kit determination step 1.1.2.2) obtained by more than six kinds
The concentration of clonal antibody IgG, its protein concentration is all adjusted to standby after 1mg/mL, and this six kinds of polyclonal antibody IgG distinguish
For rabbit anti-I, II and type III human parainfluenza viruseses HN protein polyclone antibody IgG and Mus anti-I, II and type III human parainfluenza viruseses
HN protein polyclone antibody IgG；
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 nanometer magnetic separator and carry out Magneto separate
After remove supernatant；Described magnetic bead is with superparamagnetism Fe3O4It is the carboxyl magnetic bead of 350nm for kernel, particle diameter；Described MES buffer
It is 2- (N- morpholino) ethyl sulfonic acid that mass concentration is 2g/L；The pH=6.0 of described MES buffer；Described nanometer magnetic separator
Magnetic intensity be 0.4T；
1.2.2) sequentially add and use step 1.2.1) in MES buffer concentration be 8-12mg/ml EDC solution
And use step 1.2.1) in MES buffer concentration be 6-10mg/ml each 0.5ml of sulfo-NHS solution, with
10-40rpm activates 1hr in rotary mixer, is placed in and removes supernatant after carrying out Magneto separate in nanometer magnetic separator, is walked 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 and removes supernatant after carrying out Magneto separate in nanometer magnetic separator, in each centrifuge tube addition PBS dilute dense
Spend for 50-200 μ g/ml by step 1.1) prepared by rabbit anti-I type human parainfluenza viruseses' HN protein polyclone antibody IgG solution
Each 1ml, reacts 2-6h with 15rpm under room temperature in rotary mixer, is placed in and removes after carrying out Magneto separate in nanometer magnetic separator
After supernatant, each above-mentioned PBS adding 1ml to contain 1mg/ml ethanolamine, is reacted in rotary mixer with 15rpm under room temperature
2h is to close on magnetic bead the not carboxyl with 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 the completion of capping, this 5 centrifuge tubes are placed in nanometer magnetic separator and carry out removing after Magneto separate
Supernatant, respectively 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 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, extremely
This prepared anti-I type human parainfluenza viruseses' immune nanometer magnetic bead；In described preservation 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, 5g/L bovine serum albumin (BSA), institute
State the pH=7.4 preserving buffer；
1.2.6) as with step 1.2.1) -1.2.5) identical method is utilized respectively by step 1.1) prepared by the anti-II of rabbit
Type human parainfluenza viruseses HN protein polyclone antibody IgG and rabbit anti-type III human parainfluenza viruseses HN protein polyclone antibody IgG divides
Zhi get not anti-II, type III human parainfluenza viruseses' immune nanometer magnetic bead；By above-mentioned three kinds of suspension containing magnetic beads by volume 1:1:1 mixing,
Anti-human parainfluenza viruses immune nanometer magnetic bead is obtained；
2) preparation of quantum dot-labeled anti-human parainfluenza viruses nano-probe：
Its concrete preparation method includes：
2.1) 2nmol carboxyl water-soluble quantum dot, 300nmol N- hydroxy amber are sequentially added in microcentrifugal tube
Amber acid imide sulfo-NHS and 300nmol carbodiimide EDC, with phosphate buffer constant volume as 2ml, mixed solution, 37 DEG C
After reaction 30min, dialysis removes excessive sulfo-NHS and EDC as activator, the quantum dot after being activated；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 the quantum dot of activation in, add 4-12nmol step 1.1) in prepared
Mus anti-I type human parainfluenza viruseses HN protein polyclone antibody IgG, lucifuge reacts 2h, adds single-ended amino 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 of PES filter) in antibody aggregation thing, then filtrate be transferred to
In 50000MW ultra-filtration centrifuge tube, 15min is centrifuged at 4 DEG C with 8000g centrifugal force, remove the antibody that coupling reaction does not occur and
By-product in reaction；
2.4) collection 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, quantum dot-labeled anti-I type human parainfluenza viruseses nano-probe is so far obtained；Described phosphate cleaning mixture
In each component content as follows：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, the pH=7.4 of described phosphate cleaning mixture；It is as follows that described phosphate 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；
2.5) as with step 2.1) -2.4) identical method is utilized respectively by step 1.1) prepared by Mus anti-II type people's pair
Influenza virus HN protein polyclone antibody IgG and Mus anti-type III human parainfluenza viruseses HN protein polyclone antibody IgG is obtained respectively
Quantum dot-labeled anti-II, type III human parainfluenza viruseses' nano-probe；By above-mentioned three kinds quantum dot-labeled nano-probe solution
By volume 1:1:1 mixing, that is, be obtained anti-human parainfluenza viruses nano-probe；
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 attached on swab by the human parainfluenza viruseses' drying inactivateing and forms；
4.2) negative quality-control product：Negative quality-control product is wiped through the pharynx that clinic is defined as the negative crowd of human parainfluenza viruseses
Son；
Preferably, the present invention is in step 1.2.2) in, sequentially add and use step 1.2.1) in MES buffer
Concentration is the EDC solution of 10mg/ml and uses step 1.2.1) in MES buffer concentration be 8mg/ml sulfo-
The each 0.5ml of NHS solution, activates 1hr with 15rpm in rotary mixer, is placed in nanometer magnetic separator and moves after carrying out Magneto separate
Except supernatant, with 1ml step 1.2.1) in MES buffer resuspended, magnetic bead after being activated；
Described step 1.2.3) in, in each centrifuge tube, the concentration of addition PBS dilution is 80 μ g/ml by step
The each 1ml of rabbit anti-I type human parainfluenza viruseses' HN protein polyclone antibody IgG solution prepared by rapid 1.1), under room temperature with 15rpm in
React 3h in rotary mixer, be placed in after removing supernatant after carrying out Magneto separate in nanometer magnetic separator, each addition 1ml contains 1mg/ml
The above-mentioned PBS of ethanolamine；
Described step 2.2) in, in step 2.1) obtained by the quantum dot of activation in, add 6nmol step 1.1) in
Prepared Mus anti-I type human parainfluenza viruseses HN protein polyclone antibody IgG, lucifuge reacts 2h.
Preferably, the water-soluble CdSe that quantum dot of the present invention is carboxylated amphipathic polymer to be modified/ZnS amount
Sub- point.
Preferably, magnetic bead of the present invention is with superparamagnetism Fe3O4It is polystyrene, table for kernel, shell material
Face functional group is carboxyl, particle diameter is the carboxyl magnetic bead of 350nm.
A kind of based on Magnetic Isolation and quantum dot-labeled detection human parainfluenza viruseses' antigen test kit using method,
It is characterized in that：Described using method comprises the following steps：
1) sample to be checked is proceeded to lysate in the common centrifuge tube of 1.5ml with after 0.5ml PBST buffer solution；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 quantum dot-labeled detection human parainfluenza viruseses
Anti-human parainfluenza viruses immune nanometer magnetic bead 60-200 μ l in the test kit of antigen, with 10rpm in rotary mixer under room temperature
Take off after upper reaction 15-45min, centrifuge tube is inserted nanometer magnetic separator Magneto separate 3min, suctions out supernatant with pipettor；
3) the PBST buffer 1ml adding in test kit washs twice, is washed using suctioning out after nanometer magnetic separator Magneto separate
Wash liquid, finally use the resuspended magnetic bead of 1ml PBS, prepared immune nanometer magnetic bead-virus antigen complex；Described PBS buffering
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) 100 μ l steps 3 are taken) immune nanometer magnetic bead-virus antigen complex of obtaining, in another centrifuge tube, adds
Quantum dot-labeled resisting in the test kit based on Magnetic Isolation and quantum dot-labeled detection human parainfluenza viruseses' antigen for the 100 μ l
Human parainfluenza viruseses' nano-probe, reacts 25-45min with 15rpm under room temperature on rotary mixer, by anti-on quantum dot
Body is combined with the immunity of the virus antigen on immune nanometer magnetic bead, and quantum dot is tagged to virus antigen surface, forms magnetic bead-disease
Malicious antigen-quantum dot " sandwich " complex；
5), after the completion of reacting, using nanometer magnetic separator Magneto separate 3min, remove unnecessary quantum dot-labeled anti-human pair
Influenza virus nano-probe, cleans 2 times with the PBST buffer liquid in test kit, and complex is again dispersed in 100 μ l PBS and delays
Rush in liquid, using fluorescence microplate reader, 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) the negative quality-control product sample of four parts providing in above-mentioned same method detection kit and a positive quality control are provided
Product sample, reads fluorescent value respectively；The meansigma methodss of fluorescence reading of four parts of negative quality-control product samples are 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 judged as people's pair in sample to be checked more than CUT-OFF value
Influenza antigen is the positive, otherwise is then judged as that in sample to be checked, human parainfluenza viruseses' antigen is feminine gender；If positive quality control product
The fluorescent value of sample is less than CUT-OFF value, then show that test kit lost efficacy.
Preferably, step 2 proposed by the invention) in, to step 1) in centrifuge tube in add and be based on Magnetic Isolation
With anti-human parainfluenza viruses immune nanometer magnetic bead 140 μ in the test kit of quantum dot-labeled detection human parainfluenza viruseses' antigen
L, is reacted on rotary mixer with 10rpm under room temperature and takes off after 20min, centrifuge tube insertion magnetic frame is separated 3min, with moving
Liquid device suctions out supernatant；
Described step 4) in, take 100 μ l steps 3) immune nanometer magnetic bead-virus antigen complex of obtaining more in another from
In heart pipe, add in the test kit based on Magnetic Isolation and quantum dot-labeled detection human parainfluenza viruseses' antigen for the 100 μ l
Quantum dot-labeled anti-human parainfluenza viruses nano-probe, reacts 30min with 15rpm on rotary mixer under room temperature, passes through
Antibody on quantum dot is combined with the immunity of the human parainfluenza viruseses' antigen on immune nanometer magnetic bead, and quantum dot is tagged to people's pair
Influenza antigen surface, forms magnetic bead-virus antigen-quantum dot " sandwich " complex.
Preferably, sample to be checked provided by the present invention includes but is 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 are bought or are customized；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, detached speed is fast, efficiency high the advantages of,
In combination with characteristics such as quantum dot light chemical stability height, fluorescence intensity height, so that detection system possesses, multiple signal is collaborative to be put
Big effect, thus having the detection sensitivity of superelevation, (it is respectively to the detection bottom line of I, II, type III human parainfluenza viruseses
0.3ng/ml, 0.4ng/ml, 0.3ng/ml).
2nd, the antibody used by the present invention is to identify I, II, the extracellular guarantor of type III human parainfluenza viruseses' specificity HN antigen respectively
The polyclonal antibody of defending zone, its specificity is high, and for its more most widely used monoclonal antibody, preparation cost is low simultaneously
Honest and clean, therefore, testing cost of the present invention is relatively low.
3rd, detection method is simple, and detection is quick, and it is easy to judge, testing cost is cheap, overcomes prior art inspection
Survey positive rate is low, high cost, complex operation are loaded down with trivial details, time-consuming, cannot be carried out the deficiency of clinical practice.
4th, due to being human parainfluenza viruseses' antigen of detection kit detection non-antibody (appearance of antibody needs to infect several
Zhou Yihou), so early diagnosiss and preventing and treating can be carried out, clinical diagnosises coincidence rate is high.The method is in the clinic of human parainfluenza viruseses
The aspects such as diagnosis, nosetiology discriminating, Epidemiological study have very high practical value.
5th, 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.
Specific embodiment
The present invention is according to the double antibodies sandwich principle in immunology, using immune nanometer magnetic bead to sample can enriching,
Detached speed is fast, efficiency high the advantages of, the characteristic such as incorporating quantum point photochemical stability height, fluorescence intensity height, the one of foundation
Set possess multiple signal work in coordination with amplification, have hypersensitivity and high degree of specificity quick detection human parainfluenza viruseses new side
Method, has wide market application foreground.
The present invention is further described in detail by following examples.
The preparation of various reagents 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, adjust deionized water after pH to 7.4 to be settled to 1000ml with 1mol/L NaOH.
2. rabbit anti-I type human parainfluenza viruseses HN protein polyclone antibody IgG：Make by oneself for the present invention, dilute with PBS
Release, shake up, make polyclonal antibody weight percent concentration in solution be 1mg/ml.
3. rabbit anti-II type human parainfluenza viruseses HN protein polyclone antibody IgG：Make by oneself for the present invention, dilute with PBS
Release, shake up, make polyclonal antibody weight percent concentration in solution be 1mg/ml.
4. rabbit anti-type III human parainfluenza viruseses HN protein polyclone antibody IgG：Make by oneself for the present invention, dilute with PBS
Release, shake up, make polyclonal antibody weight percent concentration in solution be 1mg/ml.
5. Mus anti-I type human parainfluenza viruseses HN protein polyclone antibody IgG：Make by oneself for the present invention, dilute with PBS
Release, shake up, make polyclonal antibody weight percent concentration in solution be 1mg/ml.
6. Mus anti-II type human parainfluenza viruseses HN protein polyclone antibody IgG：Make by oneself for the present invention, dilute with PBS
Release, shake up, make polyclonal antibody weight percent concentration in solution be 1mg/ml.
7. Mus anti-type III human parainfluenza viruseses HN protein polyclone antibody IgG：Make by oneself for the present invention, dilute with PBS
Release, shake up, make polyclonal antibody weight percent concentration in solution be 1mg/ml.
8. quantum dot：In the present invention, quantum dot used is water-soluble CdSe/ZnS quantum that carboxylated amphipathic polymer is modified
Point, its launch wavelength is 585nm, buys from Wuhan Jia Yuan technology of quantum dots development corporation, Ltd., and name of product is that carboxyl is water-soluble
Property quantum dot -585.
9. 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, 180nm, 350nm, 1150nm, 3 μm of carboxyl magnetic bead, can be from Shaanxi North America gene
Limited company, Aorun Weina New Material Science and Technology Co., Ltd., Shanghai buy.
10th, I type human parainfluenza viruseses：For ATCC type strain C35, purchased from American type culture collection (ATCC),
Numbering is ATCC VR-94.
11st, II type human parainfluenza viruseses：For ATCC type strain Greer, purchased from American type culture collection
(ATCC), numbering is ATCC VR-92.
12nd, type III human parainfluenza viruseses：For ATCC type strain C243, purchased from American type culture collection
(ATCC), numbering is ATCC VR-93.
Microbiological specimens used in 13. present invention are purchased from American type culture collection (ATCC).
Embodiment 1 rabbit and the preparation of Mus anti-I, II and type III human parainfluenza viruseses HN protein polyclone antibody IgG
(1) preparation of restructuring HN1-His, HN2-His, HN3-His fusion protein, purification and renaturation
1. the clone of related gene
To I, II, type III human parainfluenza viruseses' HN albumen (accession number in its NCBI Protein Data Bank
It is respectively AAC23946.1, BAA00739.1 and ACF28540.1) carry out bioinformatic analysis, obtain its extracellular structure respectively
Epitope peptide fragment the abundantest in domain, finds its corresponding DNA encoding sequence, further according to e. coli codon preference
Property, it is carried out with codon optimization, introduces termination signal TAA and enzyme action position at its 5 ' introducing restriction enzyme site NdeI, 3 ' ends simultaneously
After point XhoI, (complete sequence synthesis transfers to Jin Sirui bio tech ltd to complete to difference chemosynthesis complete genome sequence, delivery
When synthetic genetic fragment be respectively connected on carrier pUC57), be designated as hn1, hn2, hn3.Its gene complete sequence such as sequence
Shown in table.Wherein, the protein sequence of hn1 gene code is natural Type I human parainfluenza viruseses HN albumen (accession
number:AAC23946.1 214-508aa).The protein sequence of hn2 gene code is native type II human parainfluenza viruseses HN
Albumen (accession number:BAA00739.1 312-552aa).The protein sequence of hn3 gene code is natural III
Type human parainfluenza viruseses HN albumen (accession number:ACF28540.1 243-512aa).Contained this three sections respectively
The carrier pUC57 of the DNA fragmentation of synthetic carries out being separately recovered according to a conventional method mesh respectively after double digestion with NdeI and XhoI
Fragment, standby.Adopt simultaneously NdeI and XhoI to carrier pET-28a (+) carry out double digestion, and according to a conventional method respectively will be through
The hn1 obtaining after double digestion, hn2 and hn3 gene be connected into pET-28a (+) in carrier, and convert escherichia coli TOP10, build
PET-HN1, pET-HN2, pET-HN3 expression vector.Confirm that expression vector establishment is errorless through enzyme action and sequencing.This carrier divides
Biao Da not recombinate HN1-His, HN2-His, HN3-His fusion protein.
2. the expression and purification of restructuring HN1-His, HN2-His, HN3-His fusion protein
Plasmid will be extracted, routinely technology proceeds to competence E.coli BL21 after correct positive colony bacterium culture will be identified
(DE3) in, after the completion of conversion, bacterium solution is coated on the LB flat board containing 50 μ g/mL kanamycin, according to a conventional method screening expression
Bacterial strain.Picking pET-HN1, pET-HN2, pET-HN3 conversion has the single bacterium colony of exogenous protein expression ability and divides respectively
Do not inoculate in 100mL LB culture medium, in 37 DEG C of overnight incubation.After taking out bacterium solution respectively, by 1：100 are inoculated in 100mL respectively
In LB culture medium containing 50 μ g/mL kanamycin, cultivate to OD in 37 DEG C600When=0.6, add 1mol/L IPTG dense to end
Spend for 1mmol/L, shake bacterium culture, induced fusion protein expression in 37 DEG C.It is centrifuged under 8000r/min after induction 4h
10min collects thalline.This three parts of thalline are washed 3 times with 20mL PBS respectively and carry out after resuspended again ultrasonic broken
Broken, operating condition is：50HZ, 200W, ultrasonic 3S, interval 5S, work 100 times.After the completion of ultrasonic, 12000g centrifugation 15min divides
Shou Ji not precipitate, as inclusion body.Respectively by this three parts of inclusion bodys lavation buffer solution (20mM Na3PO4, 0.5M NaCl；3M
Carbamide, 30mM imidazoles, pH7.4) wash twice after, 12000g centrifugation 15min collects precipitation.Three parts of precipitations are used respectively
Binding buffer(20mM Na3PO4,0.5M NaCl；8M carbamide, 30mM imidazoles, pH7.4) dissolve at room temperature after,
12000g is centrifuged 15min, and supernatant is filtered with 0.45 μm of filter membrane.This three parts of lysates are all with His Trap affinity
Columns (GE healthcare Products), carries out purification with same method to specifications.Concrete grammar is as follows：
1) it is filled distilled water with 5mL syringe, turns on the stopper of post, with the joint providing, post and syringe are connected,
Post is washed with 1mL/min flow velocity.
2) 10mL Binding buffer is used to balance, 1mL/min flow velocity.
3) by fusion protein loading, 1mL/min flow velocity.
4) use 10mL Binding buffer, post is washed with 1mL/min flow velocity.
5) use 10mL Elution buffer (20mM Na3PO4, 0.5M NaCl；8M carbamide, 500mM imidazoles, pH7.4),
With 1mL/min flow velocity eluting, it is in charge of collection, often pipe 1ml, 12%SDS-PAGE detects, merge and in elution fraction, contain purposeful egg
White sample.
The renaturation of 3.HN1-His, HN2-His, HN3-His fusion protein
Above-mentioned restructuring HN1-His, HN2-His, HN3-His through His Trap affinity columns purification is melted
Hop protein, first each respectively with the phosphate buffer dialysed overnight containing 4mol/L carbamide, then more each respectively with containing 2,1,0.6,
The phosphate buffer gradient of 0.2mol/L carbamide is dialysed each 4h, is finally dialysed 4h with phosphate buffer.Dialysis solution is respectively used
12000rpm is centrifuged 15min, and each supernatant is restructuring HN1-His, HN2-His, HN3-His fusion protein of renaturation.Warp
After bradford test kit carries out determination of protein concentration, adjust its concentration and be 0.2mg/mL.
(2) preparation of rabbit and Mus anti-I, II and type III human parainfluenza viruseses HN protein polyclone antibody IgG
1. the preparation of rabbit anti-I, II and type III human parainfluenza viruseses HN protein polyclone antibody IgG
Mixed with 1mL Freund's complete adjuvant according to 200 μ g (1mL) with the restructuring HN1-His fusion protein of step (one) purification
Immune Male New Zealand White Rabbit (being provided by Disease Prevention Control Center, Hubei Prov) after even emulsifying, in dorsal sc multiple spot note
Penetrate, after the 7d of interval again immunity once, with the restructuring HN1-His fusion protein of above-mentioned purification according to 200 μ g (1mL) after 14d
Mix with 1mL incomplete Freund's adjuvant and carry out booster immunization after emulsifying, strengthen again in same way as described above again after booster immunization 7d
Immunity is once.Haemanalysises antibody titer is taken after 7d.If dissatisfied, may be repeated one and arrive booster immunization twice, to antibody titer
Satisfaction (measures antibody titer with ELISA method and is more than 1 × 105).If satisfied, Culling heart blood, separate serum, with Protein G parent
With chromatographic column (GE healthcare Products), in strict accordance with operating instruction purified polyclonal antibodies IgG, use triumphant base
Braford protein content detection kit measure antibody concentration and with phosphate buffer (8g/L NaCl, 0.2g/L KCl,
0.24g/L KH2PO4, 1.44g/L Na2HPO4PH=7.4) it is adjusted to 1mg/mL, -20 DEG C of preservations are standby, the anti-I of rabbit is so far obtained
Type human parainfluenza viruseses HN protein polyclone antibody IgG.Restructuring HN2-His, HN3-His fusion protein with step (one) purification
Rabbit anti-II, type III human parainfluenza viruseses HN protein polyclone antibody IgG are obtained as antigen according to above-mentioned same method respectively.
Westen blot test shows, this three kinds of polyclonal antibody IgG all specific recognition I, II of energy correspondence, type III people's sidestream
Influenza Virus total length HN albumen.
2. the preparation of Mus anti-I, II and type III human parainfluenza viruseses HN protein polyclone antibody IgG
With the restructuring HN1-His fusion protein of step (one) purification as complete antigen immune guinea pig (by Hubei Province's disease
Prevention and control center provides), injections of antigens 200 μ g/ under omoplate.Fundamental immunity is isopyknic antigen and Freund's complete adjuvant
Carry out emulsifying, carried out a booster immunization every 2 weeks, booster immunization equal-volume antigen and equal-volume incomplete Freund's adjuvant enter
Row emulsifying, immunity 4 times altogether.Haemanalysises antibody titer is taken after final immunization 10d.If dissatisfied, may be repeated one and arrive twice
Booster immunization, (measures antibody titer with ELISA method and is more than 1 × 10 to antibody titer is satisfied5).If satisfied, put to death Cavia porcelluss 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 with triumphant base Braford 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, -20 DEG C of preservations are standby
With Mus anti-I type human parainfluenza viruseses HN protein polyclone antibody IgG is so far obtained.Restructuring HN2- with step (one) purification
His, HN3-His fusion protein is obtained the anti-II of Mus, type III human parainfluenza viruseses HN according to above-mentioned same method respectively as antigen
Protein polyclone antibody IgG.Westen blot test shows, the specificity of this three kinds of polyclonal antibody IgG all energy correspondences is known
Other I, II, type III human parainfluenza viruseses' total length HN albumen.
The preparation of the anti-human parainfluenza viruses immune nanometer magnetic bead of embodiment 2
1. the optimization of anti-human parainfluenza viruses HN protein polyclone antibody coupled bead reaction condition：
To be coupled the magnetic bead of anti-I type human parainfluenza viruseses HN protein polyclone antibody IgG as solid phase carrier, quantum dot
The anti-human parainfluenza viruses HN protein polyclone antibody of labelling, as detection antibody, detects I type people by double-antibody method principle
Parainfluenza virus antigens, observe the coupling situation of magnetic bead and multi-resistance.The activator of particle diameter to magnetic bead, and EDC/NHS respectively is dense
Degree, the coupling condition such as coupled antibody concentration, coupling time, sealer species have carried out a series of optimized choice.
The selection of 1.1 magnetic bead particle diameters
Selection particle diameter be 50nm, 180nm, 350nm, 1150nm, 3 μm of carboxyl magnetic bead, all add EDC containing 4mg/ml and
After the PBS of 4mg/ml NHS carries out priming reaction, respectively with rabbit anti-I type human parainfluenza viruseses' HN protein polyclone antibody
Carry out coupling reaction.Respectively the immune nanometer magnetic bead preparing is detected the I type human parainfluenza viruseses of 10ng/mL, fluorescence microscopy
Microscopic observation result, selects fluorescence intensity big, and background fluorescence interference is few, and the very fast person of separating rate is under the action of a magnetic field
Suitable magnetic bead particle diameter.Result shows that the magnetic bead of particle diameter 350nm best suits the requirement of the present invention, determines that the most suitable magnetic microsphere particle diameter is
350nm.
The selection of 1.2EDC/NHS activator concentration
EDC and NHS concentration in reaction system is each set to after l～10mg/ml carry out Concentraton gradient combination, activates respectively
The carboxyl magnetic bead of particle diameter 350nm.The immune nanometer magnetic bead preparing is detected respectively the I type human parainfluenza viruseses of 10ng/mL, choosing
Select the most suitable activation concentration that fluorescence powerhouse is EDC and NHS solution.Result shows
During 4mg/ml, coupling effect is best.
The selection of 1.3 coupled antibody concentration
Will be many for rabbit anti-I type human parainfluenza viruseses' HN albumen of 20 μ g, 40 μ g, 60 μ g, 80 μ g, 100 μ g, 120 μ g, 140 μ g
Clonal antibody IgG is coupled by the magnetic bead that the particle diameter that above-mentioned best practice activates is 350nm with 1mg respectively.By prepare
Immune nanometer magnetic bead detects the I type human parainfluenza viruseses of 10ng/mL respectively, it was found that when the injected volume of antibody is less than 80 μ g/
During mg, fluorescence intensity increases with the concentration increase of antibody, and when the mass concentration of antibody is more than 80 μ g/mg, fluorescence is strong
Degree is basically unchanged or even slightly reduces, and therefore the present embodiment selects rabbit anti-I type human parainfluenza viruseses' HN protein polyclone antibody
Coupling amount is 80 μ g/mg.
The selection of 1.4 coupling times
After determining particle diameter, EDC/NHS activator concentration and the antibody coupling amount of magnetic bead, by rabbit anti-I type human parainfluenza viruseses
HN protein polyclone antibody IgG is set to 0.5h, 1h, 2h, 3h, 4h, 5h with the coupling reaction time of magnetic bead.By prepare
Immune nanometer magnetic bead detects the I type human parainfluenza viruseses of 10ng/mL respectively, it was found that between when coupled>During 3h, fluorescence is strong
Degree tends towards stability, and hereafter extends coupling time again, and fluorescence no longer strengthens.Accordingly, it is determined that rabbit anti-I type human parainfluenza viruseses' HN albumen
Polyclonal antibody IgG is 3h with the most suitable coupling reaction time of magnetic bead.Coupling time is far fewer than the 24h of traditional ELISA method.
The selection of 1.5 sealers
Optimal conditionss according to above-mentioned determination select particle diameter, EDC/NHS activator concentration, antibody coupling amount and the idol of magnetic bead
Carry out coupling reaction after the connection time.After coupling terminates, select BSA, ethanolamine, Tris and D-Glucosamine Hydrochloride are as exempting from
Epidemic disease nanometer magnetic bead sealer, finished product immune nanometer magnetic bead.The immune nanometer magnetic bead preparing is detected 10ng/mL's respectively
I type human parainfluenza viruseses, it was found that adopt ethanolamine as the detection fluorescent value highest of the immune nanometer magnetic bead of sealer.
Supposition is less due to the molecule of ethanolamine, can preferably consume due to the sterically hindered not surface carboxyl groups with antibodies, make
Closing is more complete, and effectively reduces space steric effect to the structure influence connecting antibody.
Anti- II, the optimum results of type III human parainfluenza viruseses' HN protein polyclone antibody IgG coupled bead reaction condition with
Anti-I type human parainfluenza viruseses' HN protein polyclone antibody of above-mentioned steps 1.1-1.5 description and magnetic bead coupling reaction correlated response
The optimum results of condition are all completely the same.
2. coupling process：
Take 5mg magnetic bead (with superparamagnetism Fe3O4It is the carboxyl magnetic bead of 350nm for kernel, particle diameter) commonly it is centrifuged in 1.5ml
Guan Zhong, is washed three times with 1ml MES buffer (2g/L MES, pH6.0), is placed in nanometer magnetic separator and carries out Magneto separate
(0.4T) remove supernatant after, sequentially add the EDC solution that the concentration with above-mentioned MES buffer is 10mg/ml and with above-mentioned
The concentration of MES buffer is each 0.5ml of sulfo-NHS solution of 8mg/ml, is activated in rotary mixer with 15rpm
1hr, removes supernatant after Magneto separate, resuspended with the above-mentioned MES buffer of 1ml；Take 5 centrifuge tubes, in each centrifuge tube, add 200
The magnetic bead of the above-mentioned activation of μ L, suctions out supernatant, addition PBS (8g/L NaCl, 0.2g/L in each pipe after Magneto separate
KCl, 0.24g/L KH2PO4, 1.44g/L Na2HPO4, pH7.4) and the concentration that dilutes is prepared by the embodiment 1 of 80 μ g/ml
Rabbit anti-I type human parainfluenza viruseses' HN protein polyclone antibody IgG solution each 1ml is anti-in rotary mixer with 15rpm under room temperature
Answer 3h, after Magneto separate removes supernatant, each above-mentioned PBS adding 1ml to contain 1mg/ml ethanolamine, under room temperature with 15rpm in
In rotary mixer, reaction 2h is to close on magnetic bead the not carboxyl with 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, finally respectively preserve buffer (8g/L NaCl, 0.2g/L KCl, 0.24g/L KH with 1ml2PO4,
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, be so far obtained anti-
I type human parainfluenza viruseses' immune nanometer magnetic bead.
It is utilized respectively many grams of rabbit anti-II type human parainfluenza viruseses' HN albumen prepared by embodiment 1 as above-mentioned same procedure
Grand IgG antibody and rabbit anti-type III human parainfluenza viruseses HN protein polyclone antibody IgG are obtained anti-II, type III human parainfluenza respectively
Virus immunity nanometer magnetic bead.By three kinds of immune nanometer magnetic beads by volume 1:1:Standby after 1 mixing.
The preparation of the quantum dot-labeled anti-human parainfluenza viruses nano-probe of embodiment 3
1. nanometer carboxylic quantum dot-labeled Mus anti-I type human parainfluenza viruseses HN protein polyclone antibody IgG reaction condition
Optimize：
1.1st, the determination of carboxyl quantum dot-labeled antibody probe optimum mark pH
During labelling is reacted, phosphate buffer pH is set to 5,6,7,8,9, and marked product is entered using full spectrogrph
Row fluorescent strength determining, observes the impact to coupling reaction for the different pH value it is determined that the Optimal pH of quantum dot-labeled multi-resistance reaction
For 7.0-8.0.This experimental selection pH7.4.
1.2nd, the determination of carboxyl quantum dot-labeled antibody probe optimum mark amount
The ratio of quantum dot molar concentration and multi-resistance concentration is respectively set to 1:1,1:2,1:3 and 1:4, it is marked reaction
Afterwards, using full spectrogrph, fluorescent strength determining is carried out to marked product, observes the impact that the two variable concentrations compares coupling reaction,
The optimum molar concentration ratio determining quantum dot-labeled Mus anti-I type human parainfluenza viruseses HN protein polyclone antibody IgG reaction is for measuring
Son point and antibody molar ratio are 1:3.This optimal concentration ratio of this experimental selection is determining labelled amount.
1.3rd, the determination of the optimal sealer species of the quantum dot-labeled antibody probe of carboxyl
With ethanolamine, Tris, PEG2000-NH2Or BSA, as sealer, is entered by the condition of step 1.1 and 1.2 determinations
After line flag reaction, using full spectrogrph, fluorescent strength determining is carried out to marked product, observe different sealers for labelling
The impact of reaction, it was found that PEG2000-NH2For optimal sealer, it is remarkably improved the colloid-stabilised of labeled complex
Property and immunocompetence.
The bar of the anti-II of the quantum dot-labeled Mus of nanometer carboxylic, type III human parainfluenza viruseses HN protein polyclone antibody IgG reaction
Many grams of nanometer carboxylic quantum dot-labeled Mus anti-I type human parainfluenza viruseses' HN albumen that piece optimization result is described with step 1.1-1.3
The optimum results of grand antibody response correlated condition are all completely the same.
2. labeling process：
2nmol carboxyl water-soluble quantum dot, 300nmol N- hydroxy succinyl is sequentially added in microcentrifugal tube
Imines (sulfo-NHS) and 300nmol carbodiimide (EDC), with phosphate buffer (2.9g/L disodium hydrogen phosphate, 0.295g/
L sodium dihydrogen phosphate, 4g/L sodium chloride, pH 7.4) constant volume is 2ml, ceaselessly mixed solution, after 37 DEG C of reaction 30min, dialysis is gone
Except excessive sulfo-NHS and EDC as activator.In the quantum dot of activation, add prepared by the embodiment 1 of 6nmol
Mus anti-I type human parainfluenza viruseses HN protein polyclone antibody IgG, lucifuge react 2h, add single-ended amino polyethylene glycol
(PEG2000-NH2) to final concentration of 1%, close unreacted activated carboxyl site, continue lucifuge reaction 1h.With 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
It is centrifuged 15min at 4 DEG C, remove the by-product in the antibody that coupling reaction does not occur and 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 by this solution transfer to 50000MW ultrafiltration from
In heart pipe, 15min is centrifuged at 4 DEG C with 8000g centrifugal force, collects 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, so far quantum dot-labeled anti-I type human parainfluenza viruseses nano-probe is obtained, is placed in 4 DEG C
Save backup.
It is utilized respectively many grams of Mus anti-II type human parainfluenza viruseses' HN albumen prepared by embodiment 1 as above-mentioned same procedure
Grand IgG antibody and Mus anti-type III human parainfluenza viruseses HN protein polyclone antibody IgG are obtained anti-II, type III human parainfluenza respectively
Virus nano probe.By above-mentioned three kinds anti-human parainfluenza viruses nano-probe solution by volume 1:1:Standby after 1 mixing.
Embodiment 4 immune nanometer magnetic bead carries out the optimization of immunocapture condition to human parainfluenza viruseses' antigen
To be coupled the immune nanometer magnetic bead of rabbit anti-I type human parainfluenza viruseses HN protein polyclone antibody IgG as solid phase
Carrier, quantum dot-labeled Mus anti-I type human parainfluenza viruseses HN protein polyclone antibody IgG as detection antibody, by dual anti-
Sandwich assay principle, sets up the detection system of I type human parainfluenza viruseses' antigen.Use to immune nanometer magnetic bead in detection system respectively
Amount, the condition such as capture time has carried out a series of optimized choice.
Test the selection of 1. immune nanometer magnetic bead additions
By 20 μ l, 40 μ l, 60 μ l, 80 μ l, 100 μ l, 120 μ l, 140 μ l, 160 μ l, 200 μ l prepared by embodiment 2
Good immune nanometer magnetic bead is added separately in the PBS suspension of I type human parainfluenza viruseses that 0.5ml contains 10ng/mL, is exempted from
Epidemic disease captures, the more quantum dot-labeled probe described by embodiment 3 is detected, records fluorescent value.It was found that with immunity
The increase of nanometer magnetic bead addition, fluorescent value is gradually increased, and when immune nanometer magnetic bead addition reaches 140 μ l, fluorescent value reaches
To maximum.It is further continued for increasing the amount of immune nanometer magnetic bead, fluorescent value reduces on the contrary.Therefore this experimental selection 140 μ l receives as immunity
The optimal addn of rice magnetic bead.
Test the selection of 2. immunocapture times
After determining the addition of magnetic bead, take the made immune nanometer magnetic bead got ready of four parts of embodiments 2, at room temperature with 10r/
The I type human parainfluenza viruseses of 10ng/mL are carried out the immunocapture of 15min, 20min, 30min, 45min and 60min by min, then
Quantum dot-labeled probe described by embodiment 3 is detected, records fluorescent value.It was found that fluorescent value is in immunocapture
Maximum is shown during 20min, prolongation over time, numerical value has declined.Therefore this experimental selection 20min is as immunocapture
Best Times.
Meanwhile, respectively to be coupled the anti-II of rabbit, the immunity of type III human parainfluenza viruseses HN protein polyclone antibody IgG receives
Rice magnetic bead as solid phase carrier, the anti-II of corresponding quantum dot-labeled Mus, type III human parainfluenza viruseses' HN protein polyclone antibody
IgG, as detection antibody, by double-antibody method principle, sets up II and the detection system of type III human parainfluenza viruseses' antigen, right
Contact conditionss in corresponding detection system are optimized.It was found that the best capture condition in above two detection system
All completely the same with the result given by above-mentioned experiment 1 and experiment 2.
The preparation of embodiment 5PBST 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：Swab will be attached to type III human parainfluenza viruseses (the 0.5 μ g) drying of 1% formalin-inactivated
On, as positive quality control product.
2. negative quality-control product：Negative quality-control product is defined as the throat swab sample of the negative crowd of human parainfluenza viruseses through clinic
Product.
The preparation of embodiment 7 test kit
Quantum dot-labeled described by anti-human parainfluenza viruses immune nanometer magnetic bead described by embodiment 2, embodiment 3
Anti-human parainfluenza viruses nano-probe, the PBST buffer described by embodiment 5, the quality-control product described by embodiment 6 common
The test kit based on Magnetic Isolation and quantum dot-labeled detection human parainfluenza viruseses' antigen for the composition.
The using method of embodiment 8 test kit
Routinely clinical means obtain people's throat swab, with 0.5ml test kit PBST buffer (8g/L NaCl,
0.2g/L KCl, 0.24g/L KH2PO4, 1.44g/L Na2HPO4, 0.5ml/L Tween-20, pH7.4) dissolve on throat swab
Clinical sample after, lysate is proceeded in the common centrifuge tube of 1.5ml, in this centrifuge tube add test kit in anti-human sidestream
Influenza Virus immune nanometer magnetic bead 140 μ l, is reacted on rotary mixer with 10rpm under room temperature and takes off after 20min, centrifuge tube is inserted
Enter magnetic frame and separate 3min, suction out supernatant with pipettor.The PBST buffer 1ml adding in test kit washs twice, Magneto separate
Suction out cleaning mixture afterwards, finally use the resuspended magnetic bead of 1mL PBS.Take the above-mentioned immune nanometer magnetic bead-virus antigen of 100 μ l multiple
Compound, in another centrifuge tube, adds the quantum dot-labeled anti-human parainfluenza viruses nano-probe in 100 μ l test kits, room
Under temperature, 30min is reacted on rotary mixer with 15rpm, by the people's sidestream on the antibody on quantum dot and immune nanometer magnetic bead
The immunity of Influenza Virus antigen combines, and quantum dot is tagged to virus surface, forms magnetic bead-virus antigen-quantum dot " sandwich "
Complex.After the completion of reaction, Magneto separate 3min, remove unnecessary quantum dot-labeled probe, and with PBST buffer solution for cleaning 2 times,
Complex is dispersed in 100 μ l PBS again, using fluorescence microplate reader (Ex=405nm, Em=585nm) to its fluorescence
Value is detected.
By the negative quality-control product of four parts providing in above-mentioned same method detection kit and a positive quality control product sample,
Read fluorescent value respectively；The meansigma methodss of fluorescence reading of four parts of negative quality-control product samples are CUT-OFF with 3 times of standard deviation sums
Value；If if the detection fluorescent value of above-mentioned clinic people's throat swab sample is judged as in this part of clinical throat swab more than CUT-OFF value
Human parainfluenza viruseses' antigen is the positive, otherwise is then judged as that in this part of clinical people's throat swab sample, human parainfluenza viruseses' antigen is the moon
Property；If the fluorescent value of positive quality control product sample is less than CUT-OFF value, show that test kit lost efficacy.
The detection sensitivity of embodiment 9 test kit and specific test
I type human parainfluenza viruseses are adjusted to concentration with phosphate buffer and are respectively 100,10,1,0.5,0.4,0.3,
After 0.2,0.1ng/mL concentration, detected with this test kit, result shows that its detection lowest limit is 0.3ng/ml.By same
Method detects to II, type III human parainfluenza viruseses, determines that its Monitoring lower-cut is respectively 0.4ng/mL and 0.3ng/mL.
Propped up former with respiratory tract common causative such as human respiratory syncytial virus (Long strain, ATCC numbering VR26), people's pneumonia
(GB strain, ATCC compiles for body (ATCC numbering 15531), people's Chlamydia pneumoniae (AR-39 strain, ATCC numbering 53592), adenovirus hominiss 3 type
Number VR-3), adenovirus hominiss 7 type (Gomen strain, ATCC numbering VR-7), influenza virus A hominis's (H1N1, ATCC numbering VR-
1743), people's Influenza B viruss (ATCC numbering VR-790), streptococcus pneumoniae (ATCC numbering 49619), hemophilus influenza
(ATCC numbering 53781), moraxelle catarrhalises (ATCC numbering 25238), etc. replace human parainfluenza viruseses detected, test kit examine
It is all negative for surveying the PBST buffer containing these microorganisms.
Claims (4)
1. a kind of side for preparation based on Magnetic Isolation and the test kit of quantum dot-labeled detection human parainfluenza viruseses' antigen
Method it is characterised in that：Described preparation method comprises the following steps：
1) preparation of anti-human parainfluenza viruses immune nanometer magnetic bead：
1.1) preparation of rabbit and Mus anti-I, II and type III human parainfluenza viruseses HN protein polyclone antibody IgG：
1.1.1) the preparation of restructuring HN1-His, HN2-His and HN3-His fusion protein, purification：
1.1.1.1) bioinformatic analysis are carried out to I, II and type III human parainfluenza viruseses' HN albumen, respectively obtain I, II and
Epitope peptide fragment the abundantest in type III human parainfluenza viruseses' HN ectodomain；
1.1.1.2) find step 1.1.1.1) in the corresponding gene coded sequence of obtained peptide fragment, according to password in escherichia coli
Son Preference, to step 1.1.1.1) in obtained gene coded sequence carry out codon optimization；
1.1.1.3) in step 1.1.1.2) in 5 ' ends of obtained gene order and 3 ' ends introduce restriction enzyme site respectively and divide
Other chemosynthesis complete genome sequence, simultaneously labelling be designated as hn1, hn2 and hn3；
1.1.1.4) by step 1.1.1.3) in obtained hn1, hn2 and hn3 be cloned into respectively by molecular biology method
Expression vector pET-28a (+) after proceed to expression in escherichia coli restructuring HN1-His, HN2-His and HN3-His fusion protein；Institute
State restructuring HN1-His, HN2-His and HN3-His fusion protein to be all present in genetic engineering thalline with inclusion bodies；
1.1.1.5) use ni-sepharose purification step 1.1.1.4) obtained by three kinds of inclusion body proteins, SDS-PAGE detects its purity
Afterwards, more respectively to inclusion body protein carry out renaturation, protein concentration is measured with Bradford method, three kinds of protein concentrations of adjustment are
Standby after 0.2mg/mL；
1.1.2) the preparation of rabbit and Mus anti-I, II and type III human parainfluenza viruseses HN protein polyclone antibody IgG：
1.1.2.1) with step 1.1.1.5) in obtained restructuring HN1-His, HN2-His and HN3-His through renaturation merge
Albumen is complete antigen, respectively immune new zealand white rabbit and Cavia porcelluss；Prepare respectively rabbit anti-restructuring HN1-His, HN2-His and
HN3-His fusion protein antiserum and Mus anti-restructuring H1-His, HN2-His and HN3-His fusion protein antiserum；Described rabbit resists
Restructuring HN1-His, HN2-His and HN3-His fusion protein antiserum and Mus anti-restructuring HN1-His, HN2-His and HN3-His
The sero-fast indirect ELISA titer of fusion protein is all higher than 1 × 105；
1.1.2.2) using Protein G affinity column, purified rabbit anti-restructuring HN1-His, HN2-His and HN3-His melt respectively
Polyclonal antibody in hop protein antiserum and Mus anti-restructuring HN1-His, HN2-His and HN3-His fusion protein antiserum
IgG；
1.1.2.3) with triumphant base Braford protein content detection kit determination step 1.1.2.2) obtained by six kinds of polyclones
The concentration of IgG antibody, its protein concentration is all adjusted to standby after 1mg/mL, and this six kinds of polyclonal antibody IgG are respectively rabbit
Anti- I, II and type III human parainfluenza viruseses HN protein polyclone antibody IgG and Mus anti-I, II and type III human parainfluenza viruseses' HN egg
White polyclonal antibody IgG；
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 nanometer magnetic separator and move after carrying out Magneto separate
Except supernatant；Described magnetic bead is with superparamagnetism Fe3O4It is the carboxyl magnetic bead of 350nm for kernel, particle diameter；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 nanometer magnetic separator
Property intensity is 0.4T；
1.2.2) sequentially add and use step 1.2.1) in MES buffer concentration be 8-12mg/ml EDC solution and
Use step 1.2.1) in MES buffer concentration be 6-10mg/ml each 0.5ml of sulfo-NHS solution, with 10-
40rpm activates 1hr in rotary mixer, is placed in and removes supernatant after carrying out Magneto separate in nanometer magnetic separator, 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 and remove supernatant after carrying out Magneto separate in nanometer magnetic separator, the concentration that addition is diluted with PBS in each centrifuge tube
For 50-200 μ g/ml by step 1.1) prepared by rabbit anti-I type human parainfluenza viruseses' HN protein polyclone antibody IgG solution each
1ml, reacts 2-6h with 15rpm under room temperature in rotary mixer, is placed in and removes after carrying out Magneto separate in nanometer magnetic separator
After clear, each above-mentioned PBS adding 1ml to contain 1mg/ml ethanolamine, reacts 2h with 15rpm under room temperature in rotary mixer
To close the not carboxyl with antibody response on magnetic bead；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 the completion of capping, this 5 centrifuge tubes are placed in nanometer magnetic separator and carry out removing supernatant after Magneto separate,
Respectively wash 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 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, so far make
Obtain anti-I type human parainfluenza viruseses' immune nanometer magnetic bead；In described preservation 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, 5g/L bovine serum albumin, described preservation
The pH=7.4 of buffer；
1.2.6) as with step 1.2.1) -1.2.5) identical method is utilized respectively by step 1.1) prepared by rabbit anti-II type people
Parainfluenza viruses HN protein polyclone antibody IgG and rabbit anti-type III human parainfluenza viruseses HN protein polyclone antibody IgG makes respectively
II, type III human parainfluenza viruseses' immune nanometer magnetic bead must be resisted；By above-mentioned three kinds of suspension containing magnetic beads by volume 1:1:1 mixing, that is, make
Obtain anti-human parainfluenza viruses immune nanometer magnetic bead；
2) preparation of quantum dot-labeled anti-human parainfluenza viruses nano-probe：
Its concrete preparation method includes：
2.1) 2nmol carboxyl water-soluble quantum dot, 300nmol N- hydroxy succinyl are sequentially added in microcentrifugal tube
Imines sulfo-NHS and 300nmol carbodiimide EDC, with phosphate buffer constant volume as 2ml, mixed solution, 37 DEG C of reactions
After 30min, dialysis removes excessive sulfo-NHS and EDC as activator, the quantum dot after being activated；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 the quantum dot of activation in, the step 1.1 that adds 4-12nmol) in prepared Mus resist
I type human parainfluenza viruseses HN protein polyclone antibody IgG, lucifuge reacts 2h, adds single-ended amino 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 of PES filter) in antibody aggregation thing, then filtrate be transferred to 50000MW
In ultra-filtration centrifuge tube, 15min is centrifuged at 4 DEG C with 8000g centrifugal force, removes in the antibody that coupling reaction does not occur and reaction
By-product；
2.4) collection 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, quantum dot-labeled anti-I type human parainfluenza viruseses nano-probe is so far obtained；Each in described phosphate cleaning mixture
Component content is as follows：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, the pH=7.4 of described phosphate cleaning mixture；It is as follows that described phosphate 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；
2.5) as with step 2.1) -2.4) identical method is utilized respectively by step 1.1) prepared by Mus anti-II type human parainfluenza
Viral HN protein polyclonal antibody IgG and Mus anti-type III human parainfluenza viruseses HN protein polyclone antibody IgG is obtained quantum respectively
The anti-II of point labelling, type III human parainfluenza viruseses' nano-probe；Above-mentioned three kinds quantum dot-labeled nano-probe solution are pressed body
Amass and compare 1:1:1 mixing, that is, be obtained anti-human parainfluenza viruses nano-probe；
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 attached on swab by the human parainfluenza viruseses' drying inactivateing and forms；
4.2) negative quality-control product：Negative quality-control product is defined as the throat swab of the negative crowd of human parainfluenza viruseses through clinic.
2. method according to claim 1 it is characterised in that：Described step 1.2.2) in, sequentially add and use 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 prepared is each 0.5ml of sulfo-NHS solution of 8mg/ml, activates 1hr with 15rpm, be placed in and receive in rotary mixer
Rice magnetic separator in carry out Magneto separate after remove supernatant, with 1ml step 1.2.1) in MES buffer resuspended, after being activated
Magnetic bead；
Described step 1.2.3) in, in each centrifuge tube, the concentration of addition PBS dilution is 80 μ g/ml by step
1.1) each 1ml of rabbit anti-I type human parainfluenza viruseses' HN protein polyclone antibody IgG solution prepared by, with 15rpm in rotation under room temperature
Turn reaction 3h in mixed instrument, be placed in after removing supernatant after carrying out Magneto separate in nanometer magnetic separator, each addition 1ml second containing 1mg/ml
The above-mentioned PBS of hydramine；
Described step 2.2) in, in step 2.1) obtained by the quantum dot of activation in, add 6nmol step 1.1) in made
Standby Mus anti-I type human parainfluenza viruseses HN protein polyclone antibody IgG, lucifuge reacts 2h.
3. method according to claim 2 it is characterised in that：Described quantum dot is the water that carboxylated amphipathic polymer is modified
Dissolubility CdSe/ZnS quantum dot.
4. method according to claim 3 it is characterised in that：Described magnetic bead is with superparamagnetism Fe3O4For kernel, shell material
Expect for polystyrene, surface functional group be carboxyl, particle diameter be 350nm carboxyl magnetic bead.
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| CN101421621A (en) * | 2004-02-17 | 2009-04-29 | 比南股份有限公司 | Methods and kits for detection of multiple pathogens |
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Effective date of registration: 20180425 Address after: 432500 8 Heping Road, Yunmeng Economic Development Zone, Xiaogan, Hubei Patentee after: Hubei numeihua antibody drug Technology Co., Ltd. Address before: 432800 Hubei Hualong bio Pharmaceutical Co., Ltd. 8, Changzheng Road, Chengguan Town, Dawu County, Xiaogan, Hubei Patentee before: Dong Jun |