CN105277693B - Human parainfluenza virus quantum dot immunochromatography typing detection card, preparation method and applications - Google Patents
Human parainfluenza virus quantum dot immunochromatography typing detection card, preparation method and applications Download PDFInfo
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Abstract
The invention provides a human parainfluenza virus quantum dot immunochromatography typing detection card, a preparation method and applications. The detection card comprises a base plate, a sample pad, a water absorption pad, a combination pad and a detection layer. The combination pad is coated with a mixture of rabit-anti I-type, II-type and III-type human parainfluenza virus HN protein polyclonal antibodies labeled with quantum dots respectively. The detection layer is composed of a solid phase nitrocellulose membrane with three detection lines and a quality control line. The three detection lines are coated with rabit-anti I-type, II-type and III-type human parainfluenza virus polyclonal antibodies respectively. The quality control line is coated with anti-rabit IgG. The detection layer is pasted on the base plate. The combination pad and the water absorption pad are arranged above two ends of the detection layer respectively, overlap with part of the detection layer and are pasted with the detection layer and the base plate respectively. The sample pad is arranged on the combination pad, overlaps with part of the combination pad and is pasted with the combination pad and the base plate. The provided detection card has advantages of simple operation, rapid detection, quantification, high sensitivity and the like.
Description
Technical field
The present invention relates to technical field of medical detection, specially a kind of human parainfluenza viruseses' quantum dot immune chromatography typing inspection
Survey card and its preparation method and application.
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.Hpiv infection is in global distribution, is the cause of disease of common Community Acquired Respiratory Tract Infection, and hpiv infects in children child
The incidence rate of acute respiratory infection is up to 30-40%, and it is to lead to children's lower respiratory tract severe infections, be only second to respiratory tract
The pathogen of syncytial viruses.This pathogen is found in the end of the fifties in last century first, and original name Sendai virus, initially from Japanese celestial platform
Separate in the infant lung liquid that one, city dies of pneumonia.4 types can be divided into according to hereditism and serology human parainfluenza viruseses, its
Middle 1-3 type is most commonly seen in clinic, and the main structure of every kind of hypotype is similar with biological property, but epidemiology and being caused
The Clinical symptoms of disease are variant.The relevant disease that hemadsorption virus type 2 and 2 types are led to mainly have croup,
Upper respiratory tract and lower respiratory infection；Hemadsorption virus type 1 mainly results in bronchiolitises and pneumonia；Human parainfluenza viruseses 4
Type is clinically rare, typically only results in mild infection.
Clinical patient due to different respiratory pathogen (as human parainfluenza viruseses, hemophilus influenza, influenza virus,
Respiratory syncytial virus, adenoviruss etc.) infection cause disease symptomses quite similar, which results in popular diagnosis more tired
Difficulty, makes a definite diagnosis and tends to rely on laboratory diagnosiss.Quick and effective diagnostic method should be just permissible in the their early stage of disease
Clearly diagnosed, 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 a following several method:
1st, Routine Test Lab detection
1.1) 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 the method wastes time and energy, generally
Need just to obtain final result within one week, just have certain limitation in clinicing aspect to the treatment of patient.
1.2) Serologic detection
Adopt euzymelinked immunosorbent assay (ELISA), colloidal gold immunization, micro-Immunofluorescence assay and indirect hemagglutination test etc., detection is tested
Hpiv antibody horizontal in person's serum, can point out the hpiv presence of infection indirectly.However, serological test can only provide a kind of review
The diagnosis of property, needs to detect the Acute Stage and convalescent paired sera, if anti-human parainfluenza viruses in convalescent period simultaneously
Higher 4 times or more than 4 times than acute stage of antibody titer just has diagnostic significance.In addition, the opportunity that antibody occurs is difficult to grasp, child,
There is the difference of hpiv specific antibody between teenager and adult again, and it was reported that the recall rate of infant igm only about
50%, 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 have quickly,
Sensitive and special advantage, is the important means of research hpiv infection at present, but because pcr will to experimental facilitiess and operation
Ask higher, and false positive easily occurs, can't be used as conventional methods for clinical diagnosis in China.Therefore, set up " one-step method "
Hpiv specific antigen fast typing method of diagnosis is very necessary.
Content of the invention
The present invention is directed to the technical bottleneck that in background technology, existing several human parainfluenza viruseses run in detection mode,
Propose and a kind of have the advantages that easy and simple to handle, detection is quick, can human parainfluenza viruseses' quantum dot of quantitation and high sensitivity exempt from
Epidemic disease chromatography typing detection card and its preparation method and application.
The purpose of the present invention is realized by following technological means:
A kind of human parainfluenza viruseses' quantum dot immune chromatography typing detection card it is characterised in that: described human parainfluenza viruseses
Quantum dot immune chromatography typing detection card includes base plate, sample pad, pad, detection layers and adsorptive pads；Described pad bag
There is the mixing of the rabbit anti-i type, ii type and iii type human parainfluenza viruseses' hn protein polyclone antibody of quantum dot difference labelling
Thing；Described detection layers are by the solid phase cellulose nitrate with the first detection line, the second detection line, the 3rd detection line and nature controlling line
Plain film is constituted；Described first detection line, the second detection line and the 3rd detection line are coated with Mus anti-i type, ii type and iii respectively
Type human parainfluenza viruseses' polyclonal antibody；Described nature controlling line is coated with anti-rabbit igg；Described detection layers are pasted onto on base plate；Described
Pad and adsorptive pads be separately positioned on the top at detection layers both ends and partly overlap with detection layers after respectively with detection layers
And base plate sticking is together；Described sample pad be arranged on above pad and partially overlap with pad after respectively with pad
And base plate sticking is together.
Preferably, the water solublity cdse/zns amount that quantum dot of the present invention is carboxylated amphipathic polymer to be modified
Sub- point.
Preferably, anti-rabbit igg of the present invention includes but is not limited to goat-anti rabbit igg.
Preferably, the long 4cm of described detection layers of the present invention, it is 8.6- that described detection layers are pasted onto length
The backplate surface interlude of 9.5cm；Described detection layers and the suction that being pasted onto on detection layers and base plate and length are 2.5-3cm
Water cushion overlap 0.2-0.4cm；Described detection layers and the combination that being pasted onto on detection layers and base plate and length are 0.5-0.8cm
The overlapping 0.2-0.4cm of pad；Described pad and the length being pasted onto on pad and base plate are that the sample pad of 2.5cm is overlapping
0.2—0.4cm；Spacing between adjacent two lines in described first detection line, the second detection line, the 3rd detection line and nature controlling line
It is all 0.5-0.8cm；The width of described base plate is 0.3-0.5cm.
Preferably, adsorptive pads of the present invention are absorbent filters；Described base plate is pvc plate.
A kind of preparation method chromatographing typing detection card based on above-mentioned human parainfluenza viruseses' quantum dot immune, its feature exists
In: described preparation method comprises the following steps:
1) preparation of pad:
1.1) preparation of restructuring hn1-his, hn2-his and hn3-his fusion protein, purification and renaturation:
1.1.1) bioinformatic analysis are carried out to i, ii and iii type human parainfluenza viruseses' hn albumen, respectively obtain i,
Epitope peptide fragment the abundantest in the ectodomain of ii and iii type human parainfluenza viruseses' hn albumen；
1.1.2) find step 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) in obtained gene coded sequence carry out codon optimization；
1.1.3) in step 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；Its gene order is as shown in sequence table；
1.1.4) by step 1.1.3) in obtained hn1, hn2 and hn3 be cloned into respectively by molecular biology method
Expression vector pet-28a (+) express recombination fusion protein hn1-his, hn2-his and hn3-his afterwards；Described recombination fusion protein
Hn1-his, hn2-his and hn3-his are all present in genetic engineering bacterium thalline with inclusion bodies；
1.1.5) use nickel post respectively purification step 1.1.4) obtained by inclusion body protein, more respectively inclusion body protein is entered
Row renaturation obtains desired recombinant protein；
1.2) preparation of restructuring hn1-his, hn2-his and hn3-his fusion protein polyclonal antibody igg:
1.2.1) with step 1.1.5) in restructuring hn1-his, hn2-his and hn3-his of obtained renaturation merge
Albumen is complete antigen, immune new zealand white rabbit and Cavia porcelluss, prepares rabbit anti-restructuring hn1-his, hn2-his and hn3- respectively
His fusion protein antiserum and Mus anti-restructuring hn1-his, hn2-his and hn3-his fusion protein antiserum；Described rabbit is anti-heavy
Group hn1-his, hn2-his and hn3-his fusion protein antiserum and Mus anti-restructuring hn1-his, hn2-his and hn3-his melt
The sero-fast indirect elisa potency of hop protein is all higher than 1 × 105；
1.2.2) adopt with 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；
1.2.3) with triumphant base braford protein content detection kit determination step 1.2.2) obtained by antibody concentration,
This concentration is adjusted standby to 2mg/ml simultaneously；
1.3) rabbit anti-restructuring hn1-his, hn2-his and hn3-his fusion protein Anti-TNF-α of quantum dot difference labelling
The preparation of body igg and purification:
1.3.1) sequentially add in microcentrifugal tube 2nmol quantum dot, 300nmol n- N-Hydroxysuccinimide and
300nmol carbodiimide, with phosphate buffer constant volume as 2ml, in rotary mixer, with 15rpm/min, 37 DEG C of reactions
After 30min, dialysis removes excessive n- N-Hydroxysuccinimide and carbodiimide；Described phosphate buffer is by 2.9g/l
Disodium hydrogen phosphate, 0.295g/l sodium dihydrogen phosphate and the configuration of 2g/l sodium chloride form, the ph=of described phosphate buffer
7.3；
1.3.2) activation quantum dot in, add 4-8nmol step 1.2) prepared by rabbit anti-restructuring hn1-his melt
Hop protein polyclonal antibody igg, lucifuge reacts 2h, adds Amino End Group polyethylene glycol to final concentration of 1.5%, closes unreacted
Activated carboxyl site, continue lucifuge reaction 1h；It is filtered to remove antibody aggregation thing with 0.2 μm of pes filter, then filtrate is turned
Move on in 50000mw ultra-filtration centrifuge tube, 15min is centrifuged at 4 DEG C with 8000g centrifugal force, remove and the anti-of coupling reaction does not occur
By-product in body and reaction；Collect ultra-filtration centrifuge tube filter membrane upper strata quantum dot-antibody coupling matter solution, be dissolved in 2ml phosphate
In cleaning mixture, then this solution is transferred in 50000mw ultra-filtration centrifuge tube, 15min is centrifuged at 4 DEG C with 8000g centrifugal force, receive
Collection ultra-filtration centrifuge tube filter membrane upper strata quantum dot-antibody coupling matter solution, is dissolved in 1ml phosphate and preserves in liquid；
Described phosphate cleaning mixture be by 2.9g/l disodium hydrogen phosphate, 0.295g/l sodium dihydrogen phosphate, 2g/l sodium chloride,
5ml/l tween 20 and the configuration of 1g/l sodium azide form；The ph=7.3 of described phosphate cleaning mixture；Described phosphate preserves liquid
It is to be joined by 2.9g/l disodium hydrogen phosphate, 0.295g/l sodium dihydrogen phosphate, 2g/l sodium chloride, 10g/l bsa and 1g/l sodium azide
Put and form；Described phosphate preserves the ph=7.3 of liquid；
1.3.3) according to step 1.3.1) and step 1.3.2) identical method, be utilized respectively step 1.2) prepared
Rabbit anti-restructuring hn2-his and hn3-his fusion protein polyclonal antibody igg quantum dot-labeled rabbit anti-restructuring is obtained respectively
Hn2-his and hn3-his fusion protein polyclonal antibody igg；
1.3.4) by quantum dot-labeled rabbit anti-restructuring hn1-his, hn2-his and hn3-his fusion protein Anti-TNF-α
Standby after body igg 1:1:1 mixing by volume；
1.4) load of quantum dot-labeled antibody:
Polyester fiber film is immersed step 1.3.4) obtained by quantum dot respectively the rabbit anti-restructuring hn1-his of labelling,
1h in hn2-his and hn3-his fusion protein polyclonal antibody igg mixed liquor, takes out, the rear 4 DEG C of sealing preserves of 25 DEG C of dryings
Standby, pad is so far obtained；
2) preparation of sample pad:
Take glass fibre element film one, glass fibre element film is soaked at least more than 3h in sample pad treatment fluid, then puts
After 37 DEG C of aeration-dryings in the Biohazard Safety Equipment, 25 DEG C of hermetically dryings preserve；So far sample pad is obtained；
Described sample pad treatment fluid be by 2.9g/l disodium hydrogen phosphate, 0.295g/l sodium dihydrogen phosphate, 2g/l sodium chloride,
20g/l bovine serum albumin (bsa), 10ml/l tween 20,20g/l sucrose and the configuration of 5g/l Polyvinylpyrrolidone form,
The ph=7.3 of described sample pad treatment fluid；
3) preparation of detection layers:
3.1) by step 1) the middle Mus prepared anti-restructuring hn1-his, hn2-his and hn3-his fusion protein Anti-TNF-α
Body igg and anti-rabbit igg phosphate buffer adjust to final concentration of 0.5-2.5mg/ml, described phosphate buffer be by
2.9g/l disodium hydrogen phosphate, 0.295g/l sodium dihydrogen phosphate and the configuration of 2g/l sodium chloride form, described phosphate buffer
Ph=7.3；
3.2) anti-for the Mus having diluted restructuring hn1-his, hn2-his and hn3-his fusion protein polyclonal antibody igg is divided
Zhuan Ru not draw in film instrument shower nozzle biodot, the amount of setting 0.8-2.5 μ l/cm is sprayed on nitrocellulose filter successively according to certain intervals
On, form the first detection line, the second detection line and the 3rd detection line；Anti-rabbit igg having diluted loading biodot is drawn film instrument
In shower nozzle, the amount of setting 0.8-2.5 μ l/cm is sprayed on nitrocellulose filter as nature controlling line；
3.3) by being sprayed with the first detection line, the nitrocellulose filter of the second detection line, the 3rd detection line and nature controlling line exists
37 DEG C are dried 2h, and 4 DEG C of hermetically dryings preserve；So far detection layers are obtained；
4) preparation of base plate
The base plate of pvc material is pressed standby after actual requirement cutting；
5) preparation of adsorptive pads
Absorbent filter is pressed standby after actual requirement cutting；
6) assembling of human parainfluenza viruseses' quantum dot immune chromatography typing detection card:
6.1) by step 4) adhered protection film on preparation-obtained base plate takes off；
6.2) by step 3) preparation-obtained detection layers paste the central region of base plate, and floating face；
6.3) by step 5) preparation-obtained adsorptive pads are assembled on base plate, make the left side of adsorptive pads and detection layers have portion
Divide overlap, its right hand edge is alignd with the right hand edge of base plate simultaneously and glue and floating；
6.4) by step 1) preparation-obtained pad is overlapped in a left side for nitrocellulose filter by partly overlapping mode
Edge, sticks at pad on base plate simultaneously；
6.5) by step 2) prepared by obtain sample pad and be then overlapped at the left hand edge of pad by partly overlapping mode,
Another side is alignd with the left hand edge of base plate, sticks on base plate and floating；
6.6) human parainfluenza viruseses assembling quantum dot immune chromatography typing detection card is carried out cutting, 4 DEG C of sealings are dry
Dry keep in Dark Place；
Described step 6.1) to step 6.6) it is all to be operated in Biohazard Safety Equipment.
Preferably, step 1.3.2 of the present invention) in add the step 1.2 of 6nmol) prepared by rabbit anti-heavy
Group hn1-his fusion protein polyclonal antibody igg；
Preferably, step 3.1 of the present invention) in by step 1) in preparation Mus anti-restructuring hn1-his, hn2-
It is 0.5-1.0mg/ml that his and hn3-his fusion protein polyclonal antibody igg phosphate buffer adjusts to final concentration；Institute
State step 3.1) in adjust anti-rabbit igg phosphate buffer to final concentration be 0.5-1.5mg/ml；
Preferably, step 3.2 of the present invention) in, by anti-for the Mus having diluted restructuring hn1-his, hn2-his and
Hn3-his fusion protein polyclonal antibody igg is respectively charged into biodot and draws in film instrument shower nozzle, and the amount of setting 1.0-2.0 μ l/cm is pressed
It is sprayed on successively on nitrocellulose filter according to certain intervals, form the first detection line, the second detection line and the 3rd detection line；Will be dilute
Anti-rabbit igg released loads biodot and draws in film instrument shower nozzle, and the amount of setting 1.0-2.0 μ l/cm is sprayed on to be made on nitrocellulose filter
For nature controlling line.
A kind of detection based on above-mentioned human parainfluenza viruseses' quantum dot immune chromatography typing is blocked as nondiagnostic detection people
The application of parainfluenza viruses.
A kind of nondiagnostic detection method chromatographing typing detection card based on above-mentioned human parainfluenza viruseses' quantum dot immune,
It is characterized in that: described detection method comprises the following steps:
1), after measuring samples fully being dissolved with the sample treatment liquid of 500 μ l, take out 120 μ l and drip in the sample pad of detection card
On, observe testing result under uviol lamp within 15 minutes；In described sample treatment liquid, each constituent content is respectively disodium hydrogen phosphate
2.9g/l, sodium dihydrogen phosphate 0.295g/l, triton x-100 10ml/l and sodium chloride 2g/l, described sample treatment liquid
Ph=7.3；
2) if the quantum dot-labeled rabbit containing in measuring samples in i type human parainfluenza viruseses' antigen, with pad resists
Restructuring hn1-his fusion protein polyclonal antibody igg combines, by the anti-restructuring of Mus first and on nitrocellulose filter for the chromatography effect
Hn1-his fusion protein polyclonal antibody can form macroscopic one after combining under ultraviolet excites at the first detection line
Bar fluoroscopic examination line, the quantum dot-labeled antibody being not associated with continues to excite lower shape in ultraviolet after chromatography is combined with anti-rabbit igg
Become macroscopic Article 2 fluorescence nature controlling line；
If there being ii type human parainfluenza viruseses' antigen in measuring samples, with the quantum dot-labeled anti-restructuring of rabbit in pad
Hn2-his fusion protein polyclonal antibody igg combines, by Mus anti-restructuring hn2- first and on nitrocellulose filter for the chromatography effect
His fusion protein polyclonal antibody combine after can be formed at the second detection line under ultraviolet excites macroscopic one glimmering
Light detection line, the quantum dot-labeled antibody being not associated with continues to excite lower formation meat in ultraviolet after chromatography is combined with anti-rabbit igg
The visible Article 2 fluorescence nature controlling line of eye；
If there being iii type human parainfluenza viruseses' antigen in measuring samples, anti-heavy with the quantum dot-labeled rabbit in pad
Group hn3-his fusion protein polyclonal antibody igg combines, by the anti-restructuring of Mus first and on nitrocellulose filter for the chromatography effect
Hn3-his fusion protein polyclonal antibody can form macroscopic one after combining under ultraviolet excites at the 3rd detection line
Bar fluoroscopic examination line, the fluorescent-labeled antibody being not associated with continues chromatography and combines to form macroscopic Article 2 with anti-rabbit igg
Fluorescence nature controlling line；
If no i type human parainfluenza viruseses antigen, ii type human parainfluenza viruseses' antigen and iii type people's sidestream in measuring samples
, then only a fluorescence nature controlling line in Influenza Virus antigen；If fluorescence nature controlling line does not occur, this detection card lost efficacy.
Preferably, measuring samples of the present invention are including but not limited to throat swabs.
Compared with prior art, the present invention has the advantage that
1st, the method for detection human parainfluenza viruseses' antigen of the present invention is by immunochromatography and quantum dot-labeled technological synthesiss
Get up, using the high-titer of present invention preparation, high specific polyclonal antibody, by exciting fluorescence that sample is detected,
Have the characteristics that sensitivity is high, it is respectively 5ng/ml to the detection bottom line of i, ii, iii type human parainfluenza viruseses, 5ng/ml and
3ng/ml.
2nd, the antibody used by the present invention is all the polyclonal antibody of the identification extracellular conserved region of human parainfluenza viruseses' hn antigen, its
Specificity is high, and for its more most widely used monoclonal antibody, preparation cost is cheap simultaneously, and therefore, the present invention detects into
This is relatively low.
3rd, detection method is simple, and detection is quickly it is easy to judge, result judgement completed in 20 minutes, both permissible
Carry out qualitative detection with Ultraviolet Detector, also the technology such as can scan in conjunction with ccd and carry out detection by quantitative, testing cost is cheap, overcomes
Prior art (as elisa) testing cost is high, complex operation is loaded down with trivial details, time-consuming and required professional just operable not
Foot.
4th, due to being human parainfluenza viruseses' antigen of detection card detection non-antibody (appearance of antibody needs to infect several days extremely
Several Zhou Yihou), and this detection card can three types viruses examine altogether, therefore the method is in the early clinical diagnosis of human parainfluenza viruseses and anti-
Control, pathogen neuraminidase, the aspect such as 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.
Brief description
Fig. 1 is the longitudinal profile structure schematic of detection card provided by the present invention；
Fig. 2 is the structural representation after completing assembling of detection card provided by the present invention；
Wherein:
1- sample pad；2- pad；3- detection layers；4- detection line；5- detection line；6- detection line；7- nature controlling line；8- absorbs water
Pad；9- base plate.
Specific embodiment
The operation principle of the present invention is: the present invention is on the premise of immunochormatography (double-antibody sandwich), with many
Based on clonal antibody, using quantum dot-labeled probe technique, typing is developed by quantum dot labelling technique and detects human parainfluenza
The quantum dot immune chromatography detection card of virus antigen.It is that rabbit anti-i, ii, iii type human parainfluenza viruseses' hn protein polyclone resists first
The preparation of body and Mus anti-i, ii, iii type human parainfluenza viruseses' hn protein polyclone antibody, purification and quantum dot-labeled, secondly
For spraying film, then detection is blocked each constituent and assembled, finally prepare the detection card that typing detects human parainfluenza viruseses.This
Invent the features such as provided detection card has sensitivity, quick and specificity is good, and can quantitative typing detect, sample can be carried out
High flux examination, there is preferable market application foreground.
As shown in Figure 1, a kind of human parainfluenza viruseses' quantum dot immune chromatography typing detection card provided by the present invention, bag
Include sample pad 1, pad 2, detection layers 3, adsorptive pads 8 and base plate 9 to form.Quantum dot difference labelling is coated with pad 2
The mixture of rabbit anti-i, ii, iii type human parainfluenza viruseses' hn protein polyclone antibody；Detection layers 3 are to be sprayed with detection line 4, detection
The solid phase nitrocellulose filter abbreviation nc film of line 5, detection line 6 and nature controlling line 7；Detection line 4, detection line 5 and 6 points of detection line
It is not coated with Mus anti-i type, ii type and iii type human parainfluenza viruseses' polyclonal antibody；Nature controlling line 7 is coated with anti-rabbit igg；Quantum
The water solublity cdse/zns quantum dot that point is modified for carboxylated amphipathic polymer；Adsorptive pads 8 material is absorbent filter；Base plate 9 material
Matter is pvc.
Its concrete structure is: the long 4cm of detection layers, and detection layers are pasted onto the backplate surface interlude that length is 8.6-9.5cm；
Detection layers and the overlapping 0.2-0.4cm of adsorptive pads that being pasted onto on detection layers and base plate and length are 2.5-3cm；Detection layers with
It is pasted onto the pad overlap 0.2-0.4cm that on detection layers and base plate and length is 0.5-0.8cm；Pad be pasted onto
Length on pad and base plate is sample pad overlap 0.2 0.4cm of 2.5cm；Detection line 4, detection line 5, detection line 6 with
And nature controlling line 7 spacing is all 0.5-0.8cm；The width of base plate is 0.3-0.5cm.
Wherein, above-mentioned parameter preferred version is: the long 4cm of detection layers 3, is pasted onto base plate 9 long 9.3cm surface interlude, this
Detection layers right-hand member and the overlapping 0.2cm of the long 3cm of adsorptive pads 8 being pasted onto the right end of base plate 9, its other end and the long 0.6cm of pad 2
Overlapping 0.3cm；Pad 2 then with the overlapping 0.3cm of the long 2.5cm of sample pad (1) being pasted onto base plate 9 left end；Inspection in detection layers 3
Survey line 4,5,6 and nature controlling line 7 spacing 0.7cm.The width of whole piece detection card is 0.4cm.
Prepare the method that above-mentioned typing detects the quantum dot immune chromatography detection card of human parainfluenza viruseses' antigen, it mainly walks
Rapid inclusion:
First, the preparation of pad
(1) preparation of restructuring hn1-his, hn2-his, hn3-his fusion protein, purification and renaturation
Bioinformatic analysis are carried out to i, ii, iii type human parainfluenza viruseses' hn albumen, obtains its ectodomain respectively
Middle epitope peptide fragment the abundantest, finds its corresponding gene coded sequence, further according in escherichia coli codon inclined
Good property, is carried out to it after codon optimization, the difference full base of chemosynthesis after its sequence 5 ' and 3 ' end introduces restriction enzyme site respectively
Because of sequence, it is designated as hn1, hn2, hn3.Its gene order is respectively referring to sequence table.This three sections of gene orders are divided according to a conventional method
Be not cloned into expression vector pet-28a (+) afterwards express recombination fusion protein hn1-his, hn2-his and hn3-his.This three kinds of weights
Histone is all existed with inclusion bodies in escherichia coli.Distinguish purification inclusion body protein with nickel post, more respectively to inclusion body
Albumen carries out renaturation and obtains desired recombinant protein.
(2) preparation of restructuring hn1-his, hn2-his, hn3-his fusion protein polyclonal antibody igg
With the recombiant protein of renaturation as complete antigen, immune new zealand white rabbit and Cavia porcelluss, prepare respectively according to a conventional method
Rabbit anti-restructuring hn1-his, hn2-his and hn3-his fusion protein antiserum and Mus anti-restructuring hn1-his, hn2-his and hn3-
His fusion protein antiserum.This six kinds sero-fast indirect elisa potency are all higher than 1 × 105, with protein g affinity chromatograph
Polyclonal antibody igg in post difference six kinds of antiserums of purification, measures antibody with triumphant base braford protein content detection kit
Concentration is simultaneously standby after being all adjusted to 3mg/ml.Wherein rabbit anti-restructuring hn1-his, hn2-his and hn3-his fusion protein polyclone
Antibody igg is used as quantum dot-labeled test；Mus anti-restructuring hn1-his, hn2-his and hn3-his fusion protein polyclonal antibody
Igg is used as being coated of detection line.
(3) rabbit anti-restructuring hn1-his, hn2-his, hn3-his fusion protein polyclonal antibody of quantum dot difference labelling
The preparation of igg and purification
Its operating procedure is as follows: sequentially adds 2nmol quantum dot, 300nmol n- hydroxysuccinimidyl acyl 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, 2g/l sodium chloride, ph 7.3) constant volume is 2ml, in rotary mixer, with 15rpm/min, 37 DEG C of reactions
After 30min, dialysis removes excessive activator (sulfo-nhs and edc).In the quantum dot of activation, add 4-8nmol (excellent
Elect 6nmol as) the anti-restructuring hn1-his fusion protein polyclonal antibody igg of the rabbit prepared by step (2), lucifuge react 2h, plus
Enter Amino End Group polyethylene glycol (peg2000-nh2) to final concentration of 1.5%, close unreacted activated carboxyl site, continue
Lucifuge reacts 1h.It is filtered to remove antibody aggregation thing with 0.2 μm of pes filter, then filtrate be transferred to 50000mw ultrafiltration centrifugation
Guan Zhong, is centrifuged 15min with 8000g centrifugal force at 4 DEG C, removes the by-product in the antibody that coupling reaction does not occur and reaction.
Collect ultra-filtration centrifuge tube filter membrane upper strata quantum dot-antibody coupling matter solution, be dissolved in 2ml phosphate cleaning mixture (2.9g/l phosphoric acid hydrogen
Disodium, 0.295g/l sodium dihydrogen phosphate, 2g/l sodium chloride, 5ml/l tween 20,1g/l sodium azide, ph 7.3) in, then this is molten
Liquid is transferred in 50000mw ultra-filtration centrifuge tube, is centrifuged 15min with 8000g centrifugal force at 4 DEG C, collects ultra-filtration centrifuge tube filter membrane
Upper strata quantum dot-antibody coupling matter solution, is dissolved in 1ml phosphate and preserves liquid (2.9g/l disodium hydrogen phosphate, 0.295g/l di(2-ethylhexyl)phosphate
Hydrogen sodium, 2g/l sodium chloride, 10g/l bsa, 1g/l sodium azide, ph 7.3) in.
According to above-mentioned same procedure, rabbit anti-restructuring hn2-his and hn3-his prepared by step (2) is used to merge egg respectively
White polyclonal antibody igg is obtained quantum dot-labeled rabbit anti-restructuring hn2-his and hn3-his fusion protein polyclonal antibody igg.
Will be standby after the 1:1:1 mixing by volume of three kinds of quantum dot-labeled antibody.
(4) load of quantum dot-labeled antibody
Rabbit anti-restructuring hn1-his, hn2- of quantum dot difference labelling obtained by polyester fiber film is immersed step (3)
1h in his, hn3-his fusion protein polyclonal antibody igg mixed liquor, takes out, is cut into the specification of 4cm*0.6cm after 25 DEG C of dryings
4 DEG C of sealing preserves are standby afterwards, and pad is so far obtained.
2nd, the preparation of sample pad
Take glass fibre element film one, by it in sample pad treatment fluid (2.9g/l disodium hydrogen phosphate, 0.295g/l di(2-ethylhexyl)phosphate
Hydrogen sodium, 2g/l sodium chloride, 20g/l bovine serum albumin (bsa), 10ml/l tween 20,20g/l sucrose, 5g/l polyvinyl pyrrole
Alkanone (pvp-10), ph 7.3) in soak at least more than 3h, then after being placed in 37 DEG C of aeration-dryings in Biohazard Safety Equipment, be cut into
The specification of 4cm*2.5cm, 25 DEG C of hermetically dryings preserve.So far sample pad is obtained.It is experimentally verified that glass fibre element film through this kind
After method is processed, considerably improve the release rate of quantum dot-labeled antibody.
3rd, the preparation of detection layers
The preparation of detection layers, be by respectively by by Mus prepared in step one anti-restructuring hn1-his, hn2-his and
Hn3-his fusion protein polyclonal antibody igg and the special Membrane jetter of anti-rabbit igg form detection line on nitrocellulose filter
And control line；Its concrete preparation method comprises the steps:
Respectively by anti-for the Mus of preparation in above-mentioned steps one many grams of hn1-his, hn2-his and hn3-his fusion protein of restructuring
Grand antibody igg and anti-rabbit igg phosphate buffer (2.9g/l disodium hydrogen phosphate, 0.295g/l sodium dihydrogen phosphate, 2g/l chlorination
Sodium, ph 7.3) adjust to final concentration of 0.5-2.5mg/ml, wherein Mus anti-restructuring hn1-his, hn2-his and hn3-his merges
Protein polyclone antibody igg preferably dilutes final concentration of 0.5-1.0mg/ml, and anti-rabbit igg preferably dilutes final concentration of 0.5-
1.5mg/ml.By anti-for the Mus having diluted restructuring hn1-his, hn2-his and hn3-his fusion protein polyclonal antibody igg respectively
Load biodot to draw in film instrument shower nozzle, 0.8-2.5 μ l/cm is set, and preferably the amount of 1.0-2.0 μ l/cm is according to the interval of 0.7cm
It is sprayed on successively on nitrocellulose filter, form three detections line；Anti-rabbit igg having diluted loading biodot is drawn film instrument shower nozzle
In, arrange 0.8-2.5 μ l/cm, the amount of preferably 1.0-2.0 μ l/cm is sprayed on as nature controlling line on nitrocellulose filter, its with
A near detection distance between centers of tracks is also 0.7cm.By the nitrocellulose filter having sprayed, 37 DEG C are dried 2h, are cut into the rule of 4cm*4cm
Lattice, 4 DEG C of hermetically dryings preserve.So far detection layers are obtained.
4th, the processing of base plate
The base plate of pvc material is cut into standby after the specification of 4cm*9.3cm.
5th, the preparation of adsorptive pads
Absorbent filter is cut into the specification of 4cm*3cm, that is, makes adsorptive pads, standby.
6th, the assembling of detection card
Assembly working operates in Biohazard Safety Equipment, takes the adhered protection film on the base plate described in step 4 off first,
Detection layers (carrying the nitrocellulose filter of 1 nature controlling line and 3 detection lines) described in above step three are pasted base plate
Central region, and carefully floating face.Secondly, the adsorptive pads described in above step five are assembled on base plate so as to the left side
Have with detection layers that 0.2cm's is overlapping, its right hand edge is alignd with the right hand edge of base plate simultaneously and glue and carefully floating.Again will above
Pad described in step one is overlapped at the left hand edge of nitrocellulose filter by 0.3cm, and 0.3cm sticks on base plate.Finally will
Sample pad described in above step two is then overlapped at the left hand edge of pad by one side 0.3cm, the left side of another side and base plate
Edge aligns, and sticks on base plate and carefully floating.The detection plate assembling is cut into the wide detection card of 4.0mm under cutting cutter, 4 DEG C
Hermetically drying keeps in Dark Place.So far it is obtained and can detect that the quantum dot immune chromatography of human parainfluenza viruseses' antigen detects card by typing.
Above-mentioned typing detects the using method of the quantum dot immune chromatography detection card of human parainfluenza viruseses' antigen, and step is such as
Under:
By measuring samples (as throat swab etc.) sample treatment liquid (2.9g/l disodium hydrogen phosphate, the 0.295g/l phosphorus of 500 μ l
Acid dihydride sodium, 10ml/l triton x-100,2g/l sodium chloride, ph 7.3) fully dissolve after, take out 120 μ l and drip in detection card
Sample pad on, 15 minutes under uviol lamp observe testing result.If containing i type human parainfluenza viruseses' antigen in sample,
Be combined with the quantum dot-labeled rabbit anti-restructuring hn1-his fusion protein polyclonal antibody igg in pad, acted on by chromatography
Lower formation is excited in ultraviolet after being first combined with the anti-restructuring hn1-his fusion protein polyclonal antibody of the Mus on nitrocellulose filter
Macroscopic fluoroscopic examination line 1, the quantum dot-labeled antibody being not associated with continues after chromatography is combined with anti-rabbit igg in ultraviolet
Excite the macroscopic Article 2 fluorescence nature controlling line of lower formation；If having ii type human parainfluenza viruseses' antigen in sample, with combination
After quantum dot-labeled accordingly antibodies in pad, by Mus anti-restructuring hn2- first and on nitrocellulose filter for the chromatography effect
His fusion protein polyclonal antibody excites the macroscopic fluoroscopic examination line 2 of lower formation in ultraviolet after combining, and has been not associated with
Quantum dot-labeled antibody continues to excite the macroscopic Article 2 fluorescent substance of lower formation in ultraviolet after chromatography is combined with anti-rabbit igg
Control line；If having iii type human parainfluenza viruseses' antigen in sample, also quantum dot-labeled antibodies corresponding with pad,
In ultraviolet after chromatography effect is first combined with the anti-restructuring hn3-his fusion protein polyclonal antibody of the Mus on nitrocellulose filter
Line excites the macroscopic fluoroscopic examination line 3 of lower formation, and the fluorescent-labeled antibody being not associated with is continued chromatography and is combined with anti-rabbit igg
Form macroscopic Article 2 fluorescence nature controlling line；If no related antigen in sample, a fluorescence nature controlling line only occurs.If
Fluorescence nature controlling line does not occur, then this detection card lost efficacy.
The water solublity cdse/zns quantum dot that carboxylated amphipathic polymer needed for the present invention is modified can arrive such as Wuhan Ka
The professional company such as source technology of quantum dots development corporation, Ltd. buys；Required pvc material base plate, absorbent filter, cellulose nitrate
It is professional that plain film, polyester fiber film, glass fibre element film etc. can arrive millipore and Shanghai Jinbiao Bio-Tech Co., Ltd. etc.
Company buys, and required other conventional instruments, equipment, biochemical drug are commercially available.
The present invention is further described in detail by following examples.
The present invention uses or the source of various materials of employing and the preparation of related reagent
1st, sample pad treatment fluid: weigh 0.29g disodium hydrogen phosphate, 0.0295g sodium dihydrogen phosphate, 0.2g sodium chloride, 2g cattle
Serum albumin (bsa), 1ml tween 20,2g sucrose, 0.5g Polyvinylpyrrolidone (pvp-10), be dissolved in 90ml go from
In sub- water, deionized water after ph to 7.3 is adjusted to be settled to 100ml with 1mol/l naoh.
2nd, phosphate cleaning mixture: weigh 0.29g disodium hydrogen phosphate, 0.0295g sodium dihydrogen phosphate, 0.2g sodium chloride, 0.5ml
Tween 20,0.1g Hydrazoic acid,sodium salt, it is dissolved in the deionized water of 90ml, adjusted with 1mol/l naoh and use deionization after ph to 7.3
Water is settled to 100ml.
3rd, phosphate preserves liquid: weighs 0.29g disodium hydrogen phosphate, 0.0295g sodium dihydrogen phosphate, 0.2g sodium chloride, 1g cattle
Serum albumin (bsa), 0.1g nan3, it is dissolved in the deionized water of 90ml, adjusted with 1mol/l naoh and spend after ph to 7.3
Ionized water is settled to 100ml.
4th, phosphate buffer (pbs): weigh 0.29g disodium hydrogen phosphate, 0.0295g sodium dihydrogen phosphate, 0.2g sodium chloride,
It is dissolved in the deionized water of 90ml, adjust deionized water after ph to 7.3 to be settled to 100ml with 1mol/l naoh.
5th, rabbit anti-restructuring hn1-his fusion protein polyclonal antibody igg: for present invention self-control, with pbs dilution, shake up, make
In solution, Anti-TNF-α bulk concentration is 2mg/ml.
6th, rabbit anti-restructuring hn2-his fusion protein polyclonal antibody igg: for present invention self-control, with pbs dilution, shake up, make
In solution, Anti-TNF-α bulk concentration is 2mg/ml.
7th, rabbit anti-restructuring hn3-his fusion protein polyclonal antibody igg: for present invention self-control, with pbs dilution, shake up, make
In solution, Anti-TNF-α bulk concentration is 2mg/ml.
8th, Mus anti-restructuring hn1-his fusion protein polyclonal antibody igg: for present invention self-control, with pbs dilution, shake up, make
In solution, Anti-TNF-α bulk concentration is 1mg/ml.
9th, Mus anti-restructuring hn2-his fusion protein polyclonal antibody igg: for present invention self-control, with pbs dilution, shake up, make
In solution, Anti-TNF-α bulk concentration is 1mg/ml.
10th, Mus anti-restructuring hn3-his fusion protein polyclonal antibody igg: for present invention self-control, with pbs dilution, shake up,
Anti-TNF-α bulk concentration in solution is made to be 1mg/ml.
11st, goat-anti rabbit igg: for doctor's moral Products, with pbs dilution, shake up, make the Anti-TNF-α bulk concentration in solution be
1mg/ml.
12nd, quantum dot: in the present invention, quantum dot used is the water solublity cdse/zns amount that carboxylated amphipathic polymer is modified
Sub-, its launch wavelength is 565nm, buys from Wuhan Jia Yuan technology of quantum dots development corporation, Ltd., name of product is carboxyl water
Dissolubility quantum dot -565.
13rd, glass fibre element film: thickness is 0.4mm, water absorption is 42mg/cm2, glass fiber diameter be 0.6-3 μm, tool
There is good hydrophilic, buy in Shanghai Jinbiao Bio-Tech Co., Ltd. (model bt40).
14th, polyester fiber film: thickness is 0.48mm, absorption speed is 18s/4cm, has fabulous hydrophilic, for tying
Close the preparation of pad, buy in Shanghai Jinbiao Bio-Tech Co., Ltd. (model dl42).
15th, nitrocellulose filter: model millipore corp shf135, there is liner plate, buy public in millipore
Department.
16th, absorbent filter: thickness is 0.95mm, absorption speed is 60s/4cm, and water absorption is 700mg/cm2, have good
Water absorption, as make adsorptive pads material.Buy in Shanghai Jinbiao Bio-Tech Co., Ltd. (model ch37k).
17th, base plate: for high whiteness pvc material, surface is coated with monolayer high polymer pressure sensitive adhesive sm31, buys in Shanghai gold mark
Bio tech ltd.
18th, i type human parainfluenza viruseses: for atcc type strain c35, purchased from American type culture collection (atcc),
Numbering is atcc vr-94.
19th, ii type human parainfluenza viruseses: for atcc type strain greer, purchased from American type culture collection
(atcc), numbering is atcc vr-92.
20th, iii type human parainfluenza viruseses: for atcc type strain c243, purchased from American type culture collection
(atcc), numbering is atcc vr-93.
21st, the microbiological specimens used in the present invention are purchased from American type culture collection (atcc).
With reference to embodiment, technical scheme provided by the present invention is described in detail:
Embodiment 1 (preparation embodiment)
The preparation of pad
(1) preparation of restructuring hn1-his, hn2-his, hn3-his fusion protein, purification and renaturation
(1) clone of related gene
To i, ii, iii type 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 coded 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 i type human parainfluenza viruseses hn albumen (accession
Number:aac23946.1 214-508aa).The protein sequence of hn2 gene code is natural ii type human parainfluenza viruseses hn
The 312-552aa of albumen (accession number:baa00739.1).The protein sequence of hn3 gene code is natural iii
The 243-512aa of type human parainfluenza viruseses' hn albumen (accession number:acf28540.1).Contained this three sections respectively
The carrier puc57 of the dna fragment 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) expression and purification of 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, it is inoculated in 100ml respectively by 1:100
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 phosphate buffer respectively and carry out after resuspended again ultrasonic broken
Broken, operating condition is: 50hz, 200w, ultrasonic 3s, interval 5s, works 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.
(3) renaturation of 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 restructuring hn1-his, hn2-his, hn3-his fusion protein polyclonal antibody igg
(1) preparation of rabbit anti-restructuring hn1-his, hn2-his, hn3-his fusion protein polyclonal antibody igg
Mix breast with the restructuring hn1-his fusion protein of above-mentioned purification according to 200 μ g (1ml) with 1ml Freund's complete adjuvant
Immune Male New Zealand White Rabbit (being provided by Disease Prevention Control Center, Hubei Prov) after change, in dorsal sc multi-point injection,
After 7d again immunity once, with the restructuring hn1-his fusion protein of above-mentioned purification according to 200 μ g (1ml) and 1ml after 14d
Incomplete Freund's adjuvant carries out booster immunization after mixing emulsifying, booster immunization one more in same way as described above again after booster immunization 7d
Secondary.Haemanalysises antibody titer is taken after 7d.If dissatisfied, may be repeated one and arrive booster immunization twice, (use to antibody titer is satisfied
Indirect Elisa measures antibody titer and is more than 1 × 105).If satisfied, Culling heart blood, separate serum, affine with protein g
Chromatographic column (ge healthcare Products), in strict accordance with operating instruction purified polyclonal antibodies igg, uses triumphant base
Braford protein content detection kit measures antibody concentration and is adjusted to 2mg/ml with phosphate buffer, and -20 DEG C of preservations are standby
With rabbit anti-restructuring hn1-his fusion protein polyclonal antibody igg is so far obtained.
With above-mentioned same method, prepared respectively with restructuring hn2-his, hn3-his fusion protein of above-mentioned purification respectively
Rabbit anti-restructuring hn2-his, hn3-his fusion protein polyclonal antibody igg.This three kinds of antibody are as quantum dot-labeled use.
Westen blot test shows, this three kinds of polyclonal antibody igg all specific recognition i, ii of energy correspondence, iii type people's sidestream
Influenza Virus total length hn albumen.
(2) preparation of Mus anti-restructuring hn1-his, hn2-his, hn3-his fusion protein polyclonal antibody igg
With the restructuring hn1-his fusion protein of above-mentioned purification as complete antigen immune guinea pig (by Hubei Province's disease prevention
Control centre provides), injections of antigens 200 μ g/ under omoplate.Fundamental immunity is carried out with Freund's complete adjuvant for isopyknic antigen
Emulsifying, carried out a booster immunization every 2 weeks, and booster immunization carries out breast with equal-volume antigen and equal-volume incomplete Freund's adjuvant
Change, altogether immunity 4 times.Haemanalysises antibody titer is taken after final immunization 10d.If dissatisfied, may be repeated one to strengthening twice
Immunity, (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 serum,
With protein g affinity column (ge healthcare Products), in strict accordance with operating instruction purified polyclonal antibodies
Igg, is adjusted to 1mg/ml with triumphant base braford protein content detection kit mensure antibody concentration and with phosphate buffer,
Standby, Mus anti-restructuring hn1-his fusion protein polyclonal antibody igg is so far obtained.
With above-mentioned same method, prepared respectively with restructuring hn2-his, hn3-his fusion protein of above-mentioned purification respectively
Mus anti-restructuring hn2-his, hn3-his fusion protein polyclonal antibody igg.This three kinds of antibody are used as being coated detection line.
Westen blot test shows, this three kinds of polyclonal antibody igg all specific recognition i, ii of energy correspondence, iii type people's sidestream
Influenza Virus total length hn albumen.
(3) rabbit anti-restructuring hn1-his, hn2-his, hn3-his fusion protein polyclonal antibody of quantum dot difference labelling
The preparation of igg and purification
A. the optimization of quantum dot-labeled anti-human parainfluenza viruses polyclonal antibody reaction condition:
1) determination of 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 carried out using full spectrogrph
Fluorescent strength determining, observes the impact to coupling reaction for the different ph values it is determined that the optimal ph of quantum dot-labeled multi-resistance reaction is
7.0-8.0.This experimental selection ph7.3.
2) determination of 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, 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,
Determine that the optimum molar concentration ratio that quantum dot-labeled multi-resistance reacts is quantum dot and antibody molar ratio is 1:3.This experimental selection should
Optimal concentration ratio is determining labelled amount.
3) determination of the optimal sealer species of quantum dot-labeled antibody probe
With ethanolamine, tris, peg2000-nh2Or bsa is as sealer, after being marked reaction, to marked product
Carry out fluorescent strength determining using full spectrogrph, observe the impact that different sealers react for labelling, it was found that
peg2000-nh2For optimal sealer, it is remarkably improved the colloidal stability of complex and immunocompetence.
B. labeling process:
2nmol quantum dot, 300nmol n- N-Hydroxysuccinimide (sulfo-nhs) is sequentially added in microcentrifugal tube
With 300nmol carbodiimide (edc), with phosphate buffer constant volume as 2ml, in rotary mixer, with 15rpm/min, 37
After DEG C reaction 30min, dialysis removes excessive activator (sulfo-nhs and edc).In the quantum dot of activation, add 6nmol
Rabbit anti-restructuring hn1-his fusion protein polyclonal antibody igg, lucifuge react 2h, add Amino End Group polyethylene glycol
(peg2000-nh2) to final concentration of 1.5%, 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 on ultra-filtration centrifuge tube filter membrane
Layer quantum dot-antibody coupling matter solution, is dissolved in 2ml phosphate cleaning mixture, then this solution is transferred to 50000mw ultrafiltration centrifugation
Guan Zhong, is centrifuged 15min with 8000g centrifugal force at 4 DEG C, collects ultra-filtration centrifuge tube filter membrane upper strata quantum dot-antibody coupling matter molten
Liquid, is dissolved in 1ml phosphate and preserves in liquid.
According to above-mentioned same procedure, it is utilized respectively rabbit anti-restructuring hn2-his fusion protein polyclonal antibody igg and rabbit is anti-heavy
Group hn3-his fusion protein polyclonal antibody igg is obtained quantum dot-labeled rabbit anti-restructuring hn2-his and hn3-his respectively and melts
Hop protein polyclonal antibody igg.The volume ratio that three kinds of quantum dot-labeled antibody are pressed 1:1:1 is standby after mixing.
(4) rabbit anti-restructuring hn1-his, hn2-his, hn3-his fusion protein polyclonal antibody of quantum dot difference labelling
The load of igg
1h in quantum dot-labeled multi-resistance igg mixed liquor obtained by polyester fiber film is immersed step (three), takes out, 25
It is cut into after rear specification is 4cm*0.6cm/ bar after DEG C drying, 4 DEG C of sealing preserves are standby, and pad is so far obtained.
Embodiment 2 (preparation embodiment)
The preparation of sample pad
Prepare the sample pad treatment fluid of different formulations, observe the releasing effect of quantum dot traget antibody, by repeatedly orthogonal
Assay optimization, obtains the sample pad prescription for the treatment of liquid (i.e. of the present invention) of optimum.Take glass fibre element film one, by it in sample
Soak at least 3h in product pad treatment fluid, then after being placed in 37 DEG C of aeration-dryings in Biohazard Safety Equipment, being cut into specification is 4cm*
After 2.5cm/ bar, that is, sample pad is obtained, 25 DEG C of hermetically dryings preserve.It is experimentally verified that the use of this sample pad, substantially increase
The release rate of quantum dot-labeled antibody on pad, has reached preferable application effect.
Embodiment 3 (preparation embodiment)
The preparation of detection layers
Nitrocellulose filter is cut into 4cm*4cm size.By Mus anti-restructuring hn1-his, hn2- prepared in embodiment 1
His and hn3-his fusion protein polyclonal antibody igg is respectively charged into biodot and draws in film instrument shower nozzle, the spray speed of setting 1 μ l/cm
It is sprayed on successively on nitrocellulose filter, as three detections line, detect distance between centers of tracks 0.7cm.Equally, goat-anti rabbit igg is loaded
Biodot draws in film instrument shower nozzle, and the spray speed of setting 1 μ l/cm is sprayed on as nature controlling line on nitrocellulose filter, and it is with nearest one
Bar detection distance between centers of tracks is also 0.7cm.By the nitrocellulose filter having sprayed, 37 DEG C are dried 2h, and 4 DEG C of hermetically dryings preserve.
Embodiment 4 (preparation embodiment)
The assembling of detection card
Below in conjunction with the accompanying drawings 1 and accompanying drawing 2 to detection card assembling be described further.
Base plate is cut into 4cm*9.3cm size, standby.
Absorbent filter is cut into 4cm*3cm size, as adsorptive pads, standby.
Assembly working operates in Biohazard Safety Equipment, takes the adhered protection film on base plate 9 off first, by embodiment 3 institute
The detection layers 3 the stated i.e. nitrocellulose filter with nature controlling line 7 and detection line 4,5,6 pastes the tool of accompanying drawing 1 indication on base plate 9
Body region, and carefully floating face.Secondly, the adsorptive pads cutting out in advance 8 are assembled on base plate 9 so as to the left side and detection layers
There is the overlap of 0.2cm right end, and its right hand edge is then alignd with the right hand edge of base plate 9 and glues and carefully floating.Again by embodiment 1 institute
The pad 2 of description is overlapped at the left hand edge of detection layers 3 by 0.3cm, and 0.3cm sticks on base plate 9.Finally by embodiment 2 institute
Description sample pad 1 be then overlapped at the left hand edge of pad 2 by one side 0.3cm, the left hand edge pair of another side and base plate 9
Together, stick on base plate 9 and carefully floating.The detection plate assembling is cut into the wide detection card of 4.0mm under cutting cutter, 4 DEG C close
Envelope drying is kept in Dark Place.
Embodiment 5 (Application Example)
The using method of detection card
Obtain the throat swab of person to be checked according to a conventional method, be inserted into and with the addition of 1%triton x-100 equipped with 500 μ l
In the flexible plastic pipe of phosphate buffer (pbst) of (percent by volume), squeezable plastic tube wall, so that the sample on swab is filled
Point dissolving after, take out 120 μ l drip in detection card sample pad on, 15 minutes under uv analyzer (model wd-9403a,
Liuyi Instruments Plant, Beijing produces, burst of ultraviolel wavelength 365nm) observe testing result.If containing i type human parainfluenza viruseses in sample
Antigen, then be combined with the quantum dot-labeled rabbit anti-restructuring hn1-his fusion protein polyclonal antibody igg in pad, pass through
Chromatography effect is swashed in ultraviolet after being first combined with the anti-restructuring hn1-his fusion protein polyclonal antibody of the Mus on nitrocellulose filter
Give the macroscopic fluoroscopic examination line 1 of formation, after the quantum dot-labeled antibody continuation chromatography being not associated with is combined with anti-rabbit igg
Excite the macroscopic Article 2 fluorescence nature controlling line of lower formation in ultraviolet；If there being ii type human parainfluenza viruseses' antigen in sample,
Then with after the corresponding quantum dot-labeled antibodies in pad, first resisted with the Mus on nitrocellulose filter by chromatography effect
Restructuring hn2-his fusion protein polyclonal antibody excites the macroscopic fluoroscopic examination line 2 of lower formation in ultraviolet, not after combining
Continue to excite lower formation macroscopic second in ultraviolet after chromatography is combined with anti-rabbit igg in conjunction with complete quantum dot-labeled antibody
Bar fluorescence nature controlling line；If there being iii type human parainfluenza viruseses' antigen in sample, also corresponding with pad quantum dot-labeled anti-
Body combines, and is first combined with the anti-restructuring hn3-his fusion protein polyclonal antibody of the Mus on nitrocellulose filter by chromatography effect
Excite the macroscopic fluoroscopic examination line 3 of lower formation in ultraviolet afterwards, the fluorescent-labeled antibody being not associated with continues chromatography and resists
Rabbit igg combines to form macroscopic Article 2 fluorescence nature controlling line；If no related antigen in sample, a fluorescent substance only occurs
Control line.If fluorescence nature controlling line does not occur, this detection card lost efficacy.
Embodiment 6 (Application Example)
The application effect citing of the present invention
In the present embodiment, the typing of indication detects the detection of the quantum dot mark immunity-chromatography detection card of human parainfluenza viruseses
Method is with reference to the operating procedure described in embodiment 5.
1) specific test
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, detection card inspection
It is all negative for surveying the pbst diluent containing these microorganisms.Meanwhile, when detecting i, ii, iii type human parainfluenza viruseses' sample, also not
Find the cross reaction between three kinds of viruses.
2) sensitivity testss
I type human parainfluenza viruseses' antigen phosphate buffer is adjusted to concentration and is respectively 100,10,1,0.1,0.01,
After the concentration of 0.005,0.003,0.001 μ g/ml, take out 120 μ l and drip in dripping in the sample pad of detection card, 15 minutes after purple
Under outer analysis instrument, (model wd-9403a, Liuyi Instruments Plant, Beijing produces, ultraviolet wavelength 365nm) observes testing result.Right
On the premise of fluorescence occurring according to line, if having macroscopic fluorescence in corresponding detection line, it is judged as the positive, otherwise for the moon
Property.Result shows, this detection card detects that the Monitoring lower-cut of i type human parainfluenza viruseses is 0.005 μ g/ml, i.e. 5ng/ml.By same
Method ii, iii type human parainfluenza viruseses are detected, determine its Monitoring lower-cut be respectively 5ng/ml and 3ng/ml.
3) replica test
3.1) batch interior reperformance test of detection card
It is chosen at the same a collection of detection card assembling of 4 DEG C of preservations, detect three variable concentrations type containing i people daily
The sample of parainfluenza viruses.Determine the repetition of detection card by observing the fluorescence intensity change in the upper corresponding detection line of detection card
Property.Experimental result shows, the coefficient of variation repeating in batch is 2.3%.In kind with blocking to containing ii type with a collection of detection
Human parainfluenza viruseses, the sample of iii type human parainfluenza viruseses are detected, find that batch interior coefficient of variation repeating is respectively
2.6% and 2.4%.
3.2) detection card batch between repeatability measure
Be chosen at 3 different batches assembling of 4 DEG C of preservations detection card carefully, be selected in phase one time, measure same containing i
The sample of type human parainfluenza viruseses.Determine detection card by observing the fluorescence intensity change in the upper corresponding detection line of detection card
Repeatability.Experimental result shows, the coefficient of variation repeating between batch is 4.3%.In kind with a collection of detection card to containing
Ii type human parainfluenza viruseses, the sample of iii type human parainfluenza viruseses are detected, find that batch interior coefficient of variation repeating is respectively
3.5% and 3.8%.
3.3) repeatability test
To be entered in different laboratorys in same laboratory and same personnel by different personnel with a batch of detection card
Row operation, determines the repeatability of detection card by detecting the fluorescent effect in card detection line, result shows, this detection card to i,
The testing result of ii, iii type human parainfluenza viruseses is respectively provided with repeatability.
3.4) stability test
Plastic bag and foil sealing will be used respectively with a batch of detection card, plus desiccant will be in 4 DEG C, -20 DEG C, room temperature preservation
Do the test that accelerates the failure with 37 DEG C, when different storage lives is with freshly prepd detection card to containing i type human parainfluenza respectively
The sample (virus of 10ng/ml) of virus is measured, and determines detection by detecting the change of fluorescence intensity in card detection line
The stability of card.
Experimental result shows, is stuck in 4 DEG C of preservations period of 6 months with a batch of detection, and the result measuring weekly shows,
Phosphor strip tape is all high-visible, and intensity has no significant change, illustrates that detection is stuck in 4 DEG C and can preserve more than 6 months, is also continuing
Detection；It is stuck in -20 DEG C of preservations period of 6 months with a batch of detection, per two weeks detection respectively once, detects so far
Also the change that fluorescence intensity has declined in card, and is also continuing detection；It is stuck in 37 DEG C of failure tests with a batch of detection
Period, all detected daily, when the 12nd day, the fluorescence intensity of detection card decreased.To ii, iii type human parainfluenza viruseses
Experimental result identical with the experimental result of above-mentioned ii type human parainfluenza viruseses.Illustrate that detection is stuck in 37 DEG C and can preserve 11 days half,
Be equivalent to 2～8 DEG C and deposit 17 months about, stability meets the index of national defined.
It is pointed out that the foregoing is only presently preferred embodiments of the present invention, not in order to limit the present invention, all
Any modification of being made within present invention spirit and principle, equivalent etc. should be included in protection scope of the present invention it
Interior.
Claims (2)
1. a kind of based on human parainfluenza viruseses' quantum dot immune chromatograph typing detection card preparation method it is characterised in that: described
Preparation method comprises the following steps:
1) preparation of pad:
1.1) preparation of restructuring hn1-his, hn2-his and hn3-his fusion protein, purification and renaturation:
1.1.1) bioinformatic analysis are carried out to i, ii and iii type human parainfluenza viruseses' hn albumen, respectively obtain i, ii with
And epitope peptide fragment the abundantest in the ectodomain of iii type human parainfluenza viruseses' hn albumen；
1.1.2) find step 1.1.1) in the corresponding gene coded sequence of obtained peptide fragment, according to codon in escherichia coli
Preference, to step 1.1.1) in obtained gene coded sequence carry out codon optimization；
1.1.3) in step 1.1.2) in 5 ' ends of obtained gene order and 3 ' ends introduce restriction enzyme site respectively and change respectively
Learn synthesis complete genome sequence, be labeled as hn1, hn2 and hn3 simultaneously；
1.1.4) by step 1.1.3) in obtained hn1, hn2 and hn3 be cloned into expression respectively by molecular biology method
Carrier pet-28a (+) express recombination fusion protein hn1-his, hn2-his and hn3-his afterwards；Described recombination fusion protein hn1-
His, hn2-his and hn3-his are all present in genetic engineering bacterium thalline with inclusion bodies；
1.1.5) use nickel post respectively purification step 1.1.4) obtained by inclusion body protein, more respectively inclusion body protein is carried out multiple
Property obtains desired recombinant protein；
1.2) preparation of restructuring hn1-his, hn2-his and hn3-his fusion protein polyclonal antibody igg:
1.2.1) with step 1.1.5) in obtained renaturation restructuring hn1-his, hn2-his and hn3-his fusion protein
For complete antigen, immune new zealand white rabbit and Cavia porcelluss, prepare rabbit anti-restructuring hn1-his, hn2-his and hn3-his respectively and melt
Hop protein antiserum and Mus anti-restructuring hn1-his, hn2-his and hn3-his fusion protein antiserum；The anti-restructuring hn1- of described rabbit
His, hn2-his and hn3-his fusion protein antiserum and Mus anti-restructuring hn1-his, hn2-his and hn3-his fusion protein
Sero-fast indirect elisa potency is all higher than 1 × 105；
1.2.2) using protein g affinity column, purified rabbit anti-restructuring hn1-his, hn2-his and hn3-his merge respectively
Polyclonal antibody igg in protein antiserum and Mus anti-restructuring hn1-his, hn2-his and hn3-his fusion protein antiserum；
1.2.3) with triumphant base braford protein content detection kit determination step 1.2.2) obtained by antibody concentration, simultaneously
This concentration is adjusted standby to 3mg/ml；
1.3) rabbit anti-restructuring hn1-his, hn2-his and hn3-his fusion protein polyclonal antibody igg of quantum dot difference labelling
Preparation and purification:
1.3.1 in microcentrifugal tube) sequentially add 2nmol quantum dot, 300nmol n- N-Hydroxysuccinimide and 300nmol
Carbodiimide, with phosphate buffer constant volume as 2ml, in rotary mixer, with 15rpm/min, after 37 DEG C of reaction 30min,
Dialysis removes excessive n- N-Hydroxysuccinimide and carbodiimide；In described phosphate buffer, each constituent content is respectively
Disodium hydrogen phosphate 2.9g/l, sodium dihydrogen phosphate 0.295g/l and sodium chloride 2g/l, the ph=7.3 of described phosphate buffer；
1.3.2) activation quantum dot in, add 4-8nmol step 1.2) prepared by rabbit anti-restructuring hn1-his fusion egg
White polyclonal antibody igg, lucifuge reacts 2h, adds Amino End Group polyethylene glycol to final concentration of 1.5%, closes unreacted work
Change carboxyl site, continue lucifuge reaction 1h；It is filtered to remove antibody aggregation thing with 0.2 μm of pes filter, 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；Collect ultra-filtration centrifuge tube filter membrane upper strata quantum dot-antibody coupling matter solution, be dissolved in the washing of 2ml phosphate
In liquid, then this solution is transferred in 50000mw ultra-filtration centrifuge tube, 15min is centrifuged at 4 DEG C with 8000g centrifugal force, collect super
Filter centrifuge tube filter membrane upper strata quantum dot-antibody coupling matter solution, is dissolved in 1ml phosphate and preserves in liquid；
In described phosphate cleaning mixture, each constituent content is respectively disodium hydrogen phosphate 2.9g/l, sodium dihydrogen phosphate 0.295g/l, chlorine
Change sodium 2g/l, tween 20 5ml/l and sodium azide 1g/l, the ph=7.3 of described phosphate cleaning mixture；Described phosphate preserves
In liquid, each constituent content is respectively disodium hydrogen phosphate 2.9g/l, sodium dihydrogen phosphate 0.295g/l, sodium chloride 2g/l, bsa 10g/l
And sodium azide 1g/l, the ph=7.3 of described phosphate preservation liquid；
1.3.3) according to step 1.3.1) and step 1.3.2) identical method, be utilized respectively step 1.2) prepared by rabbit
Anti- restructuring hn2-his and hn3-his fusion protein polyclonal antibody igg is obtained quantum dot-labeled rabbit anti-restructuring hn2- respectively
His and hn3-his fusion protein polyclonal antibody igg；
1.3.4) by quantum dot-labeled rabbit anti-restructuring hn1-his, hn2-his and hn3-his fusion protein polyclonal antibody igg
Standby after 1:1:1 mixing by volume；
1.4) load of quantum dot-labeled antibody:
Polyester fiber film is immersed step 1.3.4) obtained by quantum dot respectively labelling rabbit anti-restructuring hn1-his, hn2-his
And 1h in hn3-his fusion protein polyclonal antibody igg mixed liquor, take out, the rear 4 DEG C of sealing preserves of 25 DEG C of dryings are standby, extremely
This prepared pad；
2) preparation of sample pad:
Take glass fibre element film one, glass fibre element film is soaked at least more than 3h in sample pad treatment fluid, then is placed in life
After 37 DEG C of aeration-dryings in thing safety cabinet, 25 DEG C of hermetically dryings preserve；So far sample pad is obtained；
In described sample pad treatment fluid, each constituent content is respectively disodium hydrogen phosphate 2.9g/l, sodium dihydrogen phosphate 0.295g/l, chlorine
Change sodium 2g/l, bovine serum albumin 20g/l, tween 20 10ml/l, sucrose 20g/l and Polyvinylpyrrolidone 5g/l, institute
State the ph=7.3 of sample pad treatment fluid；
3) preparation of detection layers:
3.1) by step 1.2.2) the middle Mus prepared anti-restructuring hn1-his, hn2-his and hn3-his fusion protein Anti-TNF-α
Body igg and anti-rabbit igg phosphate buffer adjust to final concentration of 0.5-2.5mg/ml, each group in described phosphate buffer
Content is divided to be respectively disodium hydrogen phosphate 2.9g/l, sodium dihydrogen phosphate 0.295g/l and sodium chloride 2g/l, described phosphate-buffered
The ph=7.3 of liquid；
3.2) anti-for the Mus having diluted restructuring hn1-his, hn2-his and hn3-his fusion protein polyclonal antibody igg is filled respectively
Enter biodot to draw in film instrument shower nozzle, the amount of setting 0.8-2.5 μ l/cm is sprayed on nitrocellulose filter successively according to certain intervals,
Form the first detection line, the second detection line and the 3rd detection line；Anti-rabbit igg having diluted loading biodot is drawn film instrument shower nozzle
In, the amount of setting 0.8-2.5 μ l/cm is sprayed on nitrocellulose filter as nature controlling line；
3.3) nitrocellulose filter of the first detection line, the second detection line, the 3rd detection line and nature controlling line will be sprayed with 37 DEG C
2h is dried, 4 DEG C of hermetically dryings preserve；So far detection layers are obtained；
4) preparation of base plate
The base plate of pvc material is pressed standby after actual requirement cutting；
5) preparation of adsorptive pads
Absorbent filter is pressed standby after actual requirement cutting；
6) assembling of human parainfluenza viruseses' quantum dot immune chromatography typing detection card:
6.1) by step 4) adhered protection film on preparation-obtained base plate takes off；
6.2) by step 3) preparation-obtained detection layers paste the central region of base plate, and floating face；
6.3) by step 5) preparation-obtained adsorptive pads are assembled on base plate, make the left side of adsorptive pads and detection layers have part
Its right hand edge is alignd with the right hand edge of base plate simultaneously and glues and floating by overlap；
6.4) by step 1) preparation-obtained pad is overlapped in the left hand edge of nitrocellulose filter by partly overlapping mode
Place, sticks at pad on base plate simultaneously；
6.5) by step 2) prepared by obtain sample pad and be then overlapped at the left hand edge of pad by partly overlapping mode, another
Being alignd with the left hand edge of base plate in side, sticks on base plate and floating；
6.6) human parainfluenza viruseses assembling quantum dot immune chromatography typing detection card is carried out cutting, 4 DEG C of hermetically dryings are kept away
Light preserves；
Described step 6.1) to step 6.6) it is all to be operated in Biohazard Safety Equipment.
2. preparation method according to claim 1 it is characterised in that:
Described step 1.3.2) in add 6nmol step 1.2) prepared by rabbit anti-restructuring hn1-his fusion protein polyclone
Antibody igg；
Described step 3.1) in by step 1) in preparation Mus anti-restructuring hn1-his, hn2-his and hn3-his fusion protein many
It is 0.5-1.0mg/ml that clonal antibody igg phosphate buffer adjusts to final concentration；Described step 3.1) in by anti-rabbit igg use
It is 0.5-1.5mg/ml that phosphate buffer adjusts to final concentration；
Described step 3.2) in, by anti-for the Mus having diluted restructuring hn1-his, hn2-his and hn3-his fusion protein Anti-TNF-α
Body igg is respectively charged into biodot and draws in film instrument shower nozzle, and the amount of setting 1.0-2.0 μ l/cm is sprayed on nitric acid successively according to certain intervals
On cellulose membrane, form the first detection line, the second detection line and the 3rd detection line；Anti-rabbit igg having diluted is loaded
Biodot draws in film instrument shower nozzle, and the amount of setting 1.0-2.0 μ l/cm is sprayed on nitrocellulose filter as nature controlling line.
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| CN105968197B (en) * | 2016-05-13 | 2019-08-16 | 湖北工业大学 | A kind of anti-3 type Ureaplasma urealyticum MB protein antibodies and the immune chromatography reagent kit using the antibody |
| CN106248972A (en) * | 2016-08-24 | 2016-12-21 | 李俊香 | A kind of quantum dot immune chromatograph test strip of quick detection apolipoprotein B48 R and preparation method thereof |
| CN109085349A (en) * | 2018-08-22 | 2018-12-25 | 宁波奥丞生物科技有限公司 | A kind of immune chromatography reagent kit of multinomial joint-detection |
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| CN111879946A (en) * | 2020-07-28 | 2020-11-03 | 江苏扬新生物医药有限公司 | Multi-index rapid detection fluorescence immunochromatography kit |
| CN113804882A (en) * | 2021-08-25 | 2021-12-17 | 广东省实验动物监测所 | Immunofluorescence chromatography test strip for detecting Sendai virus antibody and application thereof |
| CN114264820A (en) * | 2021-12-22 | 2022-04-01 | 广东医科大学附属医院 | Influenza virus A-type and B-type quantum dot joint detection test strip and preparation method and application thereof |
| CN114965761B (en) * | 2022-05-17 | 2024-07-02 | 深圳赛保尔生物药业有限公司 | Detection method of N-hydroxysuccinimide in polyethylene glycol protein medicine |
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