CN105319360A - Chlamydia pneumoniae quantum dot immunochromatographic assay detection card and preparing method and application thereof - Google Patents
Chlamydia pneumoniae quantum dot immunochromatographic assay detection card and preparing method and application thereof Download PDFInfo
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Abstract
The invention provides a chlamydia pneumoniae quantum dot immunochromatographic assay detection card and a preparing method and application thereof. The detection card comprises a bottom board, a sample pad, a water absorbing pad, a combining pad and a detection layer. Chlamydia pneumoniae resisting nanometer probes labeled with quantum dots envelop combining pad. The detection layer is formed by a solid-phase nitrocellulose membrane with a detection line and a quality control line. Anti-mouse chlamydia pneumoniae 98KD membrane protein polyclonal antibodies envelop the detection line. Anti-rabbit IgG is encapsulated in the quality control line. The detection layer is bonded to the bottom board. The combining pad and the water absorbing pad are arranged on the upper portions of the two ends of the detection layer respectively, partially overlapped with the detection layer and then bonded to the detection layer and the bottom board. The sample pad is arranged on the combining pad, partially overlapped with the combining pad and then bonded to the combining pad and the bottom board. The chlamydia pneumoniae quantum dot immunochromatographic assay detection card has the advantages of being easy and convenient to operate, fast in detection, capable of achieving quantification, high in sensitivity and the like.
Description
Technical field
The present invention relates to technical field of medical detection, be specially a kind of people's Chlamydia pneumoniae quantum dot immune chromatography test card and its preparation method and application.
Background technology
Chlamydia pneumoniae (Chlamydiapneumoniae, Cpn) as a kind of important respiratory diseases pathogenic microorganism of chlamydiaceae, since eighties of last century the mid-80 is by people's definite designations such as Grayston, studied persons extensively research in the time subsequently.Now only know that people is the host of this pathogen, mode of infection may for propagate by respiratory secretions between men.Within less than 5 years old, children are seldom contaminted, and more than 8 years old children and youth are easily infected, especially crowd massing place, as easily popular in family, school, military camp, through seroepidemiology investigation, confirm to have at least 40% to be subject to this choamydiae infection in adult, major part is Subclinical.Chlamydia pneumoniae is the entozoic microorganism of special sexual cell, is lodged in the place such as human respiratory and throat, produces the upper respiratory tract and respiratory tract infection, often cause the breathing problem such as pneumonia and bronchitis in children and adult.Current research shows, it is also relevant with diseases such as atherosclerotic syndrome, senile dementia, myocarditis, asthma.The infection symptoms caused due to itself and other respiratory pathogen is clinically similar, is therefore often difficult to reach a conclusion according to clinical manifestation, x-radiological survey X etc., makes a definite diagnosis and often depend on laboratory diagnosis.Diagnostic method should be just can obtain clear and definite diagnosis at the their early stage of disease fast and effectively, is convenient to implement to treat targetedly, stops the development delay of the state of an illness.
Although Chlamydia pneumoniae is propagated in the world, the kind that can be used for the standardization commercially available reagent of laboratory diagnosis is few.At present, the diagnostic method of infection involving chlamydia pneumoniae has serological detection method, nucleic acid detection method and pathogen direct Detection Method.What in detection serum, CPn IgG, IgA, IgM antibody were commonly used has 5 kinds of methods: microimmunofluorescence antibody test (MIF), complement fixation test (CFT) (CF), recombinase immunoassays (rEIA), SC is tested (SeroCF-EIA) in conjunction with an enzyme immunoassay (EIA), OY Enzymoimmune reagent kit (LoY-EIA).These 5 kinds of methods require that Chlamydia pneumoniae heterogeneity is as antigen, and detecting of chlamydia pneumoniae (cp) can only illustrate that this individuality infected Chlamydia pneumoniae, but can not Chlamydia pneumoniae viable bacteria whether be still had to exist in antimer, and serological specificity antibody test often needs to judge according to the dynamic result of IgM antibody, needs the longer time.Meanwhile, these technology all exist that sensitivity is low, operation steps is complicated, need professional to operate, poor repeatability, detection time is long, detection specificity is poor, the more high defect of cost, are thus difficult to meet clinical actual needs.
Detection of nucleic acids comprises nucleic acid hybridization and PCR (PCR), and nucleic acid hybridization detects the high specificity of Chlamydia pneumoniae, but susceptibility is not high, is mainly used in the detection of PCR result, judgement, is not yet directly used in the detection of clinical samples; PCR has higher susceptibility, and a pair Auele Specific Primer of application Chlamydia pneumoniae outer membrane protein gene fragment carries out PCR mensuration to clinical throat swab, and its result and Serologic detection have good consistance.But PCR experiment has particular/special requirement to laboratory, sample disposal, amplification and testing requirement are strict, and easily occur false positive, can't as conventional methods for clinical diagnosis in China.
Antigen detection method has chick embryo yolk sac to be separated cultivation and cell culture method (conventional HL and Hep-2 two kinds of cells), then the Chlamydia in fluorescence labeling or enzymic-labelled antibody detection sample is adopted, but there is operation steps complexity in this method, the open defects such as the cell chulture time is long, and be not suitable for clinical practice.In recent years the inspection directly applying to clinical samples is also attempted, its susceptibility and collection of specimens are about detecting the existence of Chlamydia pneumoniae antigen with EIA after: sputum and throat swab smear, its susceptibility, specificity and repeatability are undesirable, and are only limitted to ARI; Enzyme linked immunosorbent assay (ELISA) also directly can detect pathogen, main detection Chlamydia pneumoniae outer membrane protein-2, antibody used is anti-chlamydiaceae specific antibody, can not Direct Recognition Chlamydia pneumoniae, compared with " goldstandard " (MIF), although very sensitive, specificity is not high.
Therefore, set up tool high sensitivity at present, people's Chlamydia pneumoniae rapid antigen detection method of high specific just seems very necessary to meet clinical detection demand.
Summary of the invention
The present invention is directed to the technical bottleneck that several people's Chlamydia pneumoniae existing in background technology run in detection mode, propose a kind of have easy and simple to handle, detect fast, can quantitatively and people's Chlamydia pneumoniae quantum dot immune chromatography test card of the advantage such as high sensitivity and its preparation method and application.
The object of the invention is to be realized by following technological means:
A kind of people's Chlamydia pneumoniae quantum dot immune chromatography test card, is characterized in that: described people's Chlamydia pneumoniae quantum dot immune chromatography test card comprises base plate, sample pad, pad, detection layers and adsorptive pads; Described pad is coated with quantum dot-labeled anti-human Chlamydia pneumoniae nano-probe; Described detection layers is made up of the solid phase nitrocellulose filter with a detection line and a nature controlling line; Described detection line is coated with mouse-anti people Chlamydia pneumoniae 98KD memebrane protein polyclonal antibody; Described nature controlling line is coated with anti-rabbit IgG; Described detection layers is pasted onto on base plate; Described pad and adsorptive pads be separately positioned on detection layers both ends top and after partly overlapping with detection layers respectively together with detection layers and base plate sticking; Described sample pad to be arranged on above pad and after partially overlapping with pad respectively together with pad and base plate sticking.
As preferably, quantum dot of the present invention is water-soluble CdSe/ZnS quantum dot that carboxylated amphipathic polymer is modified.
As preferably, anti-rabbit IgG of the present invention includes but not limited to goat anti-rabbit igg.
As preferably, the long 2cm of detection layers of the present invention, described detection layers is pasted onto the backplate surface interlude that length is 6.6-7.7cm; Described detection layers be pasted onto the overlapping 0.2-0.4cm of adsorptive pads that on detection layers and base plate and length is 2.5-3cm; Described detection layers be pasted onto the overlapping 0.2-0.4cm of pad that on detection layers and base plate and length is 0.5-0.8cm; Described pad and the length be pasted onto on pad and base plate are the overlapping 0.2-0.4cm of sample pad of 2.5cm; The spacing of described detection line and nature controlling line is 0.5-0.8cm; The width of described base plate is 0.3-0.5cm.
As preferably, adsorptive pads of the present invention is absorbent filter; Described base plate is PVC board.
Based on a preparation method for above-mentioned people's Chlamydia pneumoniae quantum dot immune chromatography test card, it is characterized in that: described preparation method comprises the following steps:
1) preparation of pad:
1.1) to recombinate the preparation of M98-His fusion, purifying:
1.1.1) bioinformatic analysis is carried out to people Chlamydia pneumoniae 98KD surface protein, obtain the peptide section that epitope in its ectodomain enriches the most;
1.1.2) find step 1.1.1) in institute obtain the gene coded sequence of peptide section correspondence, and at 5 ' end of sequence and 3 ' end introducing restriction enzyme site and chemosynthesis complete genome sequence, be designated as m98 with tense marker; Its gene order is as shown in sequence table;
1.1.3) by step 1.1.2) in the m98 that obtains be cloned into expression vector pET-28a (+) by molecular biology method after proceed to expression in escherichia coli restructuring M98-His fusion; Described restructuring M98-His fusion is present in thalline with inclusion body expression way;
1.1.4) with ni-sepharose purification step 1.1.3) recombinant protein that obtains, after SDS-PAGE detects its purity, measure protein concentration with Bradford method, adjustment protein concentration is for subsequent use after 0.2mg/mL;
1.2) preparation of rabbit and mouse-anti people Chlamydia pneumoniae 98KD memebrane protein polyclonal antibody IgG:
1.2.1) with step 1.1.4) in the restructuring M98-His fusion that obtains be comlete antigen, immunize New Zealand White Rabbit and cavy respectively; Prepare rabbit anti-human Chlamydia pneumoniae 98KD memebrane protein antiserum and mouse-anti people Chlamydia pneumoniae 98KD memebrane protein antiserum respectively; Described rabbit anti-human Chlamydia pneumoniae 98KD memebrane protein antiserum and the sero-fast indirect ELISA titer of mouse-anti people Chlamydia pneumoniae 98KD memebrane protein are all greater than 1 × 10
5;
1.2.2) the polyclonal antibody IgG in ProteinG affinity column difference purified rabbit anti-human's Chlamydia pneumoniae 98KD memebrane protein antiserum and mouse-anti people Chlamydia pneumoniae 98KD memebrane protein antiserum is adopted;
1.2.3) with triumphant base Braford protein content detection kit determination step 1.2.2) concentration of two kinds of polyclonal antibody IgG that obtains, for subsequent use after its protein concentration is all adjusted to 3mg/mL;
1.3) preparation and purification of quantum dot-labeled anti-human Chlamydia pneumoniae nano-probe:
1.3.1) in microcentrifugal tube, 2nmol quantum dot, 300nmolN-N-Hydroxysuccinimide and 300nmol carbodiimide is added successively, with phosphate buffer constant volume for 2ml, in rotary mixer, with 15rpm/min, after 37 DEG C of reaction 30min, excessive N-hydroxy-succinamide and carbodiimide are removed in dialysis; In described phosphate buffer, each component concentration is respectively: 2.9g/L sodium hydrogen phosphate, 0.295g/L sodium dihydrogen phosphate and 2g/L sodium chloride, the pH=7.3 of described phosphate buffer;
1.3.2) in the quantum dot of activation, add the step 1.2 of 4-12nmol) prepared by rabbit anti-human Chlamydia pneumoniae 98KD memebrane protein polyclonal antibody IgG, lucifuge reaction 2h, adding Amino End Group polyethylene glycol to final concentration is 1.5%, close unreacted activated carboxyl site, continue lucifuge reaction 1h; With 0.2 μm of PES frit removing antibody aggregation thing, then filtrate transferred in 50000MW ultra-filtration centrifuge tube, with 8000g centrifugal force centrifugal 15min at 4 DEG C, there is not the antibody of coupling reaction and the accessory substance in reacting in removing; Collect super filter tube filter membrane upper strata quantum dot-antibody coupling matter solution, be dissolved in 2ml phosphate cleansing solution, again this solution is transferred in 50000MW ultra-filtration centrifuge tube, with 8000g centrifugal force centrifugal 15min at 4 DEG C, collect super filter tube filter membrane upper strata quantum dot-antibody coupling matter solution, be dissolved in 1ml phosphate conserving liquid, so far obtained quantum dot-labeled anti-human Chlamydia pneumoniae nano-probe;
In described phosphate cleansing solution, each component concentration is respectively: 2.9g/L sodium hydrogen phosphate, 0.295g/L sodium dihydrogen phosphate, 2g/L sodium chloride, 5ml/L Tween-20 and 1g/L Sodium azide; The pH=7.3 of described phosphate cleansing solution; In described phosphate conserving liquid, each component concentration is respectively: 2.9g/L sodium hydrogen phosphate, 0.295g/L sodium dihydrogen phosphate, 2g/L sodium chloride, 10g/LBSA and 1g/L Sodium azide; The pH=7.3 of described phosphate conserving liquid;
1.4) load of quantum dot-labeled antibody:
Dacron film is immersed step 1.3.2) 1h in the quantum dot-labeled anti-human Chlamydia pneumoniae nano-probe solution that obtains, take out, 25 DEG C of rear 4 DEG C of sealings of drying save backup, so far obtained pad;
2) preparation of sample pad:
Get glass fibre element film one, glass fibre element film is soaked at least more than 3h in sample pad treating fluid, then is placed in Biohazard Safety Equipment after 37 DEG C of aeration-dryings, 25 DEG C of hermetically dryings are preserved; So far obtained sample pad;
In described sample pad treating fluid, each component concentration is respectively: 2.9g/L sodium hydrogen phosphate, 0.295g/L sodium dihydrogen phosphate, 2g/L sodium chloride, 20g/L bovine serum albumin(BSA) (BSA), 10ml/L Tween-20,20g/L sucrose and 5g/L polyvinylpyrrolidone, the pH=7.3 of described sample pad treating fluid;
3) preparation of detection layers:
3.1) by step 1.2) to be all adjusted to final concentration be for subsequent use after 0.5-2.5mg/mL for the mouse-anti people Chlamydia pneumoniae 98KD memebrane protein polyclonal antibody prepared and anti-rabbit IgG phosphate buffer; In described phosphate buffer, each component concentration is respectively: 2.9g/L sodium hydrogen phosphate, 0.295g/L sodium dihydrogen phosphate and 2g/L sodium chloride, the pH=7.3 of described phosphate buffer;
3.2) the mouse-anti people Chlamydia pneumoniae 98KD memebrane protein polyclonal antibody diluted is loaded BIODOT to draw in film instrument shower nozzle, the amount arranging 0.8-2.5 μ l/cm is sprayed on nitrocellulose filter, forms detection line; The anti-rabbit IgG diluted is loaded BIODOT draw in film instrument shower nozzle, the amount that 0.8-2.5 μ l/cm is set according to and the interval of detection line 0.5-0.8cm be sprayed on nitrocellulose filter as nature controlling line;
3.3) will be sprayed with the nitrocellulose filter of detection line and nature controlling line at 37 DEG C of dry 2h, 4 DEG C of hermetically dryings are preserved; So far obtained detection layers;
4) preparation of base plate
It is for subsequent use after the base plate of PVC material is pressed actual requirement cutting;
5) preparation of adsorptive pads
For subsequent use after absorbent filter being pressed actual requirement cutting;
6) assembling of people's Chlamydia pneumoniae quantum dot immune chromatography test 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 pastes the central region of base plate, and floating face;
6.3) by step 5) preparation-obtained adsorptive pads is assembled on base plate, and the left side of adsorptive pads and detection layers are overlapped, its right hand edge is alignd with the right hand edge of base plate to glue and floating simultaneously;
6.4) by step 1) preparation-obtained pad is overlapped in the left hand edge place of nitrocellulose filter by partly overlapping mode, sticks on base plate by pad simultaneously;
6.5) by step 2) prepared by obtain sample pad is then overlapped in pad left hand edge place by partly overlapping mode, another side aligns with the left hand edge of base plate, to stick on base plate and floating;
6.6) the people's Chlamydia pneumoniae quantum dot immune chromatography test card assembled is carried out cutting, 4 DEG C of hermetically dryings keep in Dark Place;
Described step 6.1) to step 6.6) be all operate in Biohazard Safety Equipment.
As preferably, step 1.3.2 of the present invention) in add the step 1.2 of 6nmol) prepared by rabbit anti-human Chlamydia pneumoniae 98KD memebrane protein polyclonal antibody IgG;
As preferably, step 3.1 of the present invention) in by step 1.2) the mouse-anti people Chlamydia pneumoniae 98KD memebrane protein polyclonal antibody prepared and anti-rabbit IgG phosphate buffer be adjusted to final concentration and be respectively 1.5-2.0mg/mL and 0.5-1.5mg/mL;
As preferably, step 3.2 of the present invention) in, the mouse-anti people Chlamydia pneumoniae 98KD memebrane protein polyclonal antibody diluted is loaded BIODOT and draws in film instrument shower nozzle, the amount arranging 1.0-2.0 μ l/cm is sprayed on nitrocellulose filter, forms detection line; The anti-rabbit IgG diluted is loaded BIODOT draw in film instrument shower nozzle, the amount that 1.0-2.0 μ l/cm is set according to and the interval of detection line 0.5-0.8cm be sprayed on nitrocellulose filter as nature controlling line.
A kind of application detecting people's Chlamydia pneumoniae based on above-mentioned people's Chlamydia pneumoniae quantum dot immune chromatography test card as nondiagnostic.
Based on a nondiagnostic detection method for above-mentioned people's Chlamydia pneumoniae quantum dot immune chromatography test card, it is characterized in that: described detection method comprises the following steps:
1), after measuring samples fully being dissolved by the sample treatment liquid of 500 μ l, take out 120 μ L and drip in the sample pad of test card, after 15 minutes, under uviol lamp, observe testing result; In described sample treatment liquid, each component concentration is respectively: sodium hydrogen phosphate 2.9g/L, sodium dihydrogen phosphate 0.295g/L, NonidetP-4010ml/L, SDS1ml/L and sodium chloride 2g/L, the pH=7.3 of described sample treatment liquid;
2) if containing people's Chlamydia pneumoniae antigen in measuring samples, quantum dot-labeled anti-human Chlamydia pneumoniae nano-probe then in pad is combined, under ultraviolet excites, a macroscopic fluoroscopic examination line can be formed at detection line place after being combined by the mouse-anti people Chlamydia pneumoniae 98KD memebrane protein polyclonal antibody of chromatography effect first on nitrocellulose filter, continue to excite lower formation macroscopic Article 2 fluorescence nature controlling line at ultraviolet after chromatography is combined with anti-rabbit IgG in conjunction with complete quantum dot-labeled antibody;
If unmanned Chlamydia pneumoniae antigen in measuring samples, then only there is a fluorescence nature controlling line; If fluorescence nature controlling line does not occur, then this test card lost efficacy.
As preferably, measuring samples of the present invention includes but not limited to throat swab.
Compared with prior art, tool of the present invention has the following advantages:
1, the method for detection people Chlamydia pneumoniae antigen of the present invention is by immunochromatography and quantum dot-labeled technological synthesis, the high-titer utilizing the present invention to prepare, high specific polyclonal antibody, by fluorescence excitation, sample is detected, there is highly sensitive feature (it is 1ng/ml to the detection bottom line of people's Chlamydia pneumoniae), with its to the testing result of clinical sample with at present to no difference of science of statistics compared with detection " the goldstandard "-cultivation of this pathogen.
2, the antibody that the present invention is used is all the polyclonal antibodies identifying the outer conserved region of people's Chlamydia pneumoniae specificity 98KD surface antigen born of the same parents, its specificity is high, belong to strain specific antibodies, cross reaction is not played with chlamydia trachomatis, chlamydia psittaci, its the most widely used comparatively current monoclonal antibody preparation cost is cheap simultaneously, therefore, testing cost of the present invention is lower.
3, detection method is simple, detect fast, be easy to judge, result judges to complete in 20 minutes, both qualitative detection can be carried out with Ultraviolet Detector, also quantitatively can detect in conjunction with technology such as CCD scan, testing cost is cheap, overcomes that prior art (as ELISA) testing cost is high, complicated operation is loaded down with trivial details, length consuming time and required professional just operable deficiency.
What 4, detect due to test card is people's Chlamydia pneumoniae antigen and non-antibody (appearance of antibody needs to infect several days to a few Zhou Yihou), therefore the method has very high practical value in the early clinical diagnosis and control, pathogen neuraminidase, epidemiology survey etc. of people's Chlamydia pneumoniae.
5, the present invention is first using the exclusive 98KD surface membrane protein of people's Chlamydia pneumoniae as target antigen, and its detection specificity is high, and any Chlamydia does not play cross reaction with other.And the examination target of traditional E LISA method is Cpn outer membrane protein-2 (OMP-2), antibody used is anti-chlamydiaceae specific antibody, can not Direct Recognition Cpn, and it exists the shortcoming playing cross reaction with chlamydia psittaci, chlamydia trachomatis etc.
6, the clinical sample that detection method is used be respiratory secretions as sputum etc., and non-blood, can exempt the psychological burden of misery that infant patient takes a blood sample and the head of a family, therefore comparatively be easy to promote.
Accompanying drawing explanation
Fig. 1 is the longitudinal profile structural representation of test card provided by the present invention;
Fig. 2 is the structural representation after completing assembling of test card provided by the present invention;
Wherein:
1-sample pad; 2-pad; 3-detection layers; 4-detection line; 5-nature controlling line; 6-adsorptive pads; 7-base plate.
Embodiment
Principle of work of the present invention is: the present invention is under the prerequisite of immunochormatography (double-antibody sandwich), based on polyclonal antibody, adopt quantum dot-labeled probe technique, detected the quantum dot immune chromatography test card of people's Chlamydia pneumoniae antigen by the development of quantum dot labelling technique.First be the preparation of rabbit anti-human Chlamydia pneumoniae 98KD memebrane protein polyclonal antibody and mouse-anti people Chlamydia pneumoniae 98KD memebrane protein polyclonal antibody, purifying and quantum dot-labeled, secondly be spray film, then each for test card constituent is assembled, finally prepare people's Chlamydia pneumoniae quantum dot immune chromatography test card.Test card provided by the present invention has sensitivity, fast and the feature such as specificity is good, and can quantitatively detect, and can carry out the high flux examination of sample, have good market application foreground.
As shown in Figure 1, a kind of people's Chlamydia pneumoniae quantum dot immune chromatography test card provided by the present invention, comprises sample pad 1, pad 2, detection layers 3, adsorptive pads 6 and base plate 7 and forms.Pad 2 is coated with quantum dot-labeled anti-human Chlamydia pneumoniae nano-probe; Detection layers 3 is that the solid phase nitrocellulose filter being sprayed with detection line 4 and nature controlling line 5 is called for short NC film; Detection line 4 is coated with mouse-anti people Chlamydia pneumoniae 98KD memebrane protein polyclonal antibody; Nature controlling line 5 is coated with anti-rabbit IgG; Quantum dot is water-soluble CdSe/ZnS quantum dot that carboxylated amphipathic polymer is modified; Adsorptive pads 6 material is absorbent filter; Base plate 7 material is PVC.
Its concrete structure is: the long 2cm of detection layers, and detection layers is pasted onto the backplate surface interlude that length is 6.6-7.7cm; Detection layers be pasted onto the overlapping 0.2-0.4cm of adsorptive pads that on detection layers and base plate and length is 2.5-3cm; Detection layers be pasted onto the overlapping 0.2-0.4cm of pad that on detection layers and base plate and length is 0.5-0.8cm; Pad and the length be pasted onto on pad and base plate are the overlapping 0.2-0.4cm of sample pad of 2.5cm; Detection line and nature controlling line spacing are 0.5-0.8cm; The width of base plate is 0.3-0.5cm.
Wherein, above-mentioned parameter preferred version is: the long 2cm of detection layers 3, is pasted onto base plate 7 long 7.3cm surface interlude, this detection layers right-hand member and the overlapping 0.2cm of the long 3cm of adsorptive pads 6 being pasted onto the right end of base plate 7, its other end and the overlapping 0.3cm of the long 0.6cm of pad 2; Pad 2 and the overlapping 0.3cm of sample pad (1) long 2.5cm being pasted onto base plate 7 left end; Detection line 4 in detection layers 3 and nature controlling line 5 spacing 0.7cm.The width of whole piece test card is 0.4cm.
Prepare the method for above-mentioned people's Chlamydia pneumoniae quantum dot immune chromatography test card, its key step comprises:
One, the preparation of pad
(1) to recombinate the preparation of M98-His fusion, purifying:
Bioinformatic analysis is carried out to people Chlamydia pneumoniae 98KD memebrane protein, obtains the peptide section that in people Chlamydia pneumoniae 98KD memebrane protein ectodomain, epitope enriches the most, find the gene order of its correspondence; At 5 ' end and 3 ' the end introducing restriction enzyme site also difference chemosynthesis complete genome sequence respectively of this two gene sequence, be designated as m98 with tense marker.Its gene order is see sequence table.This gene order is cloned into according to a conventional method expression vector pET-28a (+) and expresses restructuring M98-His fusion afterwards.This fusion is present in genetic engineering thalline with inclusion body expression-form.With restructuring M98-His, fusion in ni-sepharose purification genetic engineering bacterium thalline, after SDS-PAGE detects its purity, then measure protein concentration with Bradford method, adjustment protein concentration is for subsequent use after 0.2mg/mL;
(2) preparation of rabbit and mouse-anti people Chlamydia pneumoniae 98KD memebrane protein polyclonal antibody IgG:
With the restructuring M98-His fusion of purifying for comlete antigen, immunize New Zealand White Rabbit and cavy according to a conventional method, prepares rabbit anti-human Chlamydia pneumoniae 98KD memebrane protein antiserum and mouse-anti people Chlamydia pneumoniae 98KD memebrane protein antiserum respectively.These two kinds of sero-fast indirect ELISA titers are all greater than 1 × 10
5, with the polyclonal antibody IgG in ProteinG affinity column respectively purifying two kinds of antiserums, for subsequent use after being also all adjusted to 3mg/mL by triumphant base Braford protein content detection kit mensuration antibody concentration.Wherein rabbit anti-human Chlamydia pneumoniae 98KD memebrane protein polyclonal antibody IgG is used as quantum dot-labeled test; Mouse-anti people Chlamydia pneumoniae 98KD memebrane protein polyclonal antibody IgG is used as the bag quilt of detection line.
(3) preparation and purification of quantum dot-labeled anti-human Chlamydia pneumoniae nano-probe
Its operation steps is as follows: in microcentrifugal tube, add 2nmol quantum dot, 300nmolN-N-Hydroxysuccinimide (sulfo-NHS) and 300nmol carbodiimide (EDC) successively, with phosphate buffer (2.9g/L sodium hydrogen phosphate, 0.295g/L sodium dihydrogen phosphate, 2g/L sodium chloride, pH7.3) constant volume is 2ml, in rotary mixer, with 15rpm/min, after 37 DEG C of reaction 30min, excessive activator (sulfo-NHS and EDC) is removed in dialysis.In the quantum dot of activation, add the rabbit anti-human Chlamydia pneumoniae 98KD memebrane protein polyclonal antibody IgG prepared by step (2) of 4-12nmol (being preferably 6nmol), lucifuge reaction 2h, adds Amino End Group polyethylene glycol (PEG2000-NH
2) to final concentration be 1.5%, close unreacted activated carboxyl site, continue lucifuge reaction 1h.With 0.2 μm of PES frit removing antibody aggregation thing, then filtrate transferred in 50000MW ultra-filtration centrifuge tube, with 8000g centrifugal force centrifugal 15min at 4 DEG C, there is not the antibody of coupling reaction and the accessory substance in reacting in removing.Collect super filter tube filter membrane upper strata quantum dot-antibody coupling matter solution, be dissolved in 2ml phosphate cleansing solution (2.9g/L sodium hydrogen phosphate, 0.295g/L sodium dihydrogen phosphate, 2g/L sodium chloride, 5ml/L Tween-20, 1g/L Sodium azide, pH7.3) in, again this solution is transferred in 50000MW ultra-filtration centrifuge tube, with 8000g centrifugal force centrifugal 15min at 4 DEG C, collect super filter tube filter membrane upper strata quantum dot-antibody coupling matter solution, be dissolved in 1ml phosphate conserving liquid (2.9g/L sodium hydrogen phosphate, 0.295g/L sodium dihydrogen phosphate, 2g/L sodium chloride, 10g/LBSA, 1g/L Sodium azide, pH7.3) in, obtained quantum dot-labeled anti-human Chlamydia pneumoniae nano-probe.
(4) load of quantum dot-labeled antibody
1h in the quantum dot-labeled anti-human Chlamydia pneumoniae nano-probe solution obtain dacron film immersion step (3), take out, after being cut into the specification of 4cm*0.6cm after 25 DEG C of dryings, 4 DEG C of sealings save backup, so far obtained pad.
Two, the preparation of sample pad
Get glass fibre element film one, by it at sample pad treating fluid (2.9g/L sodium hydrogen phosphate, 0.295g/L sodium dihydrogen phosphate, 2g/L sodium chloride, 20g/L bovine serum albumin(BSA) (BSA), 10ml/L Tween-20,20g/L sucrose, 5g/L polyvinylpyrrolidone (PVP-10), pH7.3) at least more than 3h is soaked in, be placed in Biohazard Safety Equipment after 37 DEG C of aeration-dryings, be cut into the specification of 4cm*2.5cm, 25 DEG C of hermetically dryings are preserved again.So far obtained sample pad.Confirm that glass fibre element film is after this kind of method process, considerably improves the release rate of quantum dot-labeled antibody through test.
Three, the preparation of detection layers
The preparation of detection layers is by forming detection line and control line by by the special Membrane jetter of mouse-anti people Chlamydia pneumoniae 98KD memebrane protein polyclonal antibody IgG and anti-rabbit IgG prepared in step one on nitrocellulose filter respectively; Its concrete preparation method comprises the steps:
By mouse-anti people Chlamydia pneumoniae 98KD memebrane protein polyclonal antibody IgG and anti-rabbit IgG phosphate buffer (the 2.9g/L sodium hydrogen phosphate of preparation in step one, 0.295g/L sodium dihydrogen phosphate, 2g/L sodium chloride, pH7.3) being all adjusted to final concentration is respectively 0.5-2.5mg/mL, wherein mouse-anti people Chlamydia pneumoniae 98KD memebrane protein polyclonal antibody IgG preferably dilutes final concentration is that preferably to dilute final concentration be 0.5-1.5mg/mL for 1.5-2.0mg/mL, anti-rabbit IgG.The mouse-anti people Chlamydia pneumoniae 98KD memebrane protein polyclonal antibody IgG diluted is loaded BIODOT draw in film instrument shower nozzle, arrange 0.8-2.5 μ l/cm, the amount being preferably 1.0-2.0 μ l/cm is sprayed on nitrocellulose filter, forms detection line; The anti-rabbit IgG diluted is loaded BIODOT draw in film instrument shower nozzle, arrange 0.8-2.5 μ l/cm, the amount being preferably 1.0-2.0 μ l/cm is sprayed on as nature controlling line on nitrocellulose filter, and itself and detection line spacing are 0.7cm.By the nitrocellulose filter 37 DEG C of dry 2h sprayed, be cut into the specification of 4cm*4cm, 4 DEG C of hermetically dryings are preserved.So far obtained detection layers.
Four, the processing of base plate
For subsequent use after the base plate of PVC material being cut into the specification of 4cm*7.3cm.
Five, the preparation of adsorptive pads
Absorbent filter is cut into the specification of 4cm*3cm, namely makes adsorptive pads, for subsequent use.
Six, the assembling of test card
Assembly working operates in Biohazard Safety Equipment; first the adhered protection film on the base plate described in step 4 is taken off; detection layers (namely with the nitrocellulose filter of 1 nature controlling line and 1 detection line) above described in step 3 is pasted the central region of base plate, and careful floating face.Secondly, be assembled on base plate by the adsorptive pads above described in step 5, its left side and detection layers are had, and 0.2cm's is overlapping, is alignd by its right hand edge to glue and carefully floating with the right hand edge of base plate simultaneously.The pad above described in step one is overlapped in the left hand edge place of nitrocellulose filter by 0.3cm, 0.3cm sticks on base plate again.Finally the sample pad above described in step 2 is then overlapped in the left hand edge place of pad by one side 0.3cm, another side aligns with the left hand edge of base plate, sticks on base plate also carefully floating.The check-out console assembled is cut under cutting cutter the wide test card of 4.0mm, 4 DEG C of hermetically dryings keep in Dark Place.So far obtained people's Chlamydia pneumoniae quantum dot immune chromatography test card.
The using method of above-mentioned people's Chlamydia pneumoniae quantum dot immune chromatography test card, step is as follows:
By sample treatment liquid (the 2.9g/L sodium hydrogen phosphate of measuring samples (as throat swab etc.) with 500 μ l, 0.295g/L sodium dihydrogen phosphate, NonidetP-4010ml/L, SDS1ml/L, 2g/L sodium chloride, pH7.3), after fully dissolving, take out 120 μ L and drip in the sample pad of test card, after 15 minutes, under uviol lamp, observe testing result.If containing people's Chlamydia pneumoniae antigen in measuring samples, quantum dot-labeled anti-human Chlamydia pneumoniae nano-probe then in pad is combined, under ultraviolet excites, a macroscopic fluoroscopic examination line can be formed at detection line place after being combined by the mouse-anti people Chlamydia pneumoniae 98KD memebrane protein polyclonal antibody of chromatography effect first on nitrocellulose filter, continue to excite lower formation macroscopic Article 2 fluorescence nature controlling line at ultraviolet after chromatography is combined with anti-rabbit IgG in conjunction with complete quantum dot-labeled antibody; If without related antigen in measuring samples, then only there is a fluorescence nature controlling line.If fluorescence nature controlling line does not occur, then this test card lost efficacy.
Water-soluble CdSe/ZnS quantum dot that carboxylated amphipathic polymer required for the present invention is modified can arrive the professional companies such as such as Wuhan Jia Yuan technology of quantum dots development corporation, Ltd. and buy; Required PVC material base plate, absorbent filter, nitrocellulose filter, dacron film, glass fibre element film etc. can arrive the professional company such as Millipore and Shanghai Jinbiao Bio-Tech Co., Ltd. and buy, and other required conventional instruments, equipment, biochemical drug all have commercially available.
The present invention is further described in detail by following examples.
The source of various materials that the present invention uses or adopts and the preparation of related reagent
1, sample pad treating fluid: take 0.29g sodium hydrogen phosphate, 0.0295g sodium dihydrogen phosphate, 0.2g sodium chloride, 2g bovine serum albumin(BSA) (BSA), 1ml Tween-20,2g sucrose, 0.5g polyvinylpyrrolidone (PVP-10), be dissolved in the deionized water of 90ml, after adjusting pH to 7.3 with 1mol/LNaOH, be settled to 100ml with deionized water.
2, phosphate cleansing solution: take 0.29g sodium hydrogen phosphate, 0.0295g sodium dihydrogen phosphate, 0.2g sodium chloride, 0.5ml Tween-20,0.1g sodium azide, is dissolved in the deionized water of 90ml, with deionized water is settled to 100ml after adjusting pH to 7.3 with 1mol/LNaOH.
3, phosphate conserving liquid: take 0.29g sodium hydrogen phosphate, 0.0295g sodium dihydrogen phosphate, 0.2g sodium chloride, 1g bovine serum albumin(BSA) (BSA), 0.1gNaN
3, be dissolved in the deionized water of 90ml, after adjusting pH to 7.3 with 1mol/LNaOH, be settled to 100ml with deionized water.
4, phosphate buffer (PBS): take 0.29g sodium hydrogen phosphate, 0.0295g sodium dihydrogen phosphate, 0.2g sodium chloride, be dissolved in the deionized water of 90ml, with deionized water is settled to 100ml after adjusting pH to 7.3 with 1mol/LNaOH.
5, rabbit anti-human Chlamydia pneumoniae 98KD memebrane protein polyclonal antibody IgG: be the present invention's self-control, with PBS dilution, shake up, make Anti-TNF-α bulk concentration in solution be 3mg/ml.
6, mouse-anti people Chlamydia pneumoniae 98KD memebrane protein polyclonal antibody IgG: be the present invention's self-control, with PBS dilution, shake up, make Anti-TNF-α bulk concentration in solution be 3mg/ml.
7, goat anti-rabbit igg: be doctor's moral Products, with PBS dilution, shakes up, makes Anti-TNF-α bulk concentration in solution be 1mg/ml.
8, quantum dot: in the present invention, quantum dot used is water-soluble CdSe/ZnS quantum dot that carboxylated amphipathic polymer is modified, its emission wavelength is 565nm, buy from Wuhan Jia Yuan technology of quantum dots development corporation, Ltd., name of product is carboxyl water-soluble quantum dot-565.
9, glass fibre element film: thickness is 0.4mm, and water absorbing capacity is 42mg/cm
2, glass fiber diameter is 0.6-3 μm, has good water wettability, buys in Shanghai Jinbiao Bio-Tech Co., Ltd. (model is BT40).
10, dacron film: thickness is 0.48mm, and absorption speed is 18s/4cm, has fabulous water wettability, for the preparation of pad, buys in Shanghai Jinbiao Bio-Tech Co., Ltd. (model is DL42).
11, nitrocellulose filter: model is MilliporeCorpSHF135, has liner plate, buys in Millipore company.
12, absorbent filter: thickness is 0.95mm, absorption speed is 60s/4cm, and water absorbing capacity is 700mg/cm
2, there is good water absorptivity, as the material making adsorptive pads.Buy in Shanghai Jinbiao Bio-Tech Co., Ltd. (model is CH37K).
13, base plate: be high whiteness PVC material, surface coating individual layer high polymer pressure sensitive adhesive SM31, buys in Shanghai Jinbiao Bio-Tech Co., Ltd..
14, people's Chlamydia pneumoniae bacterial strain: purchased from American Type Tissue Collection (ATCC), is numbered ATCC53592.
15, the equal purchased from American Type Tissue Collection (ATCC) of the microbiological specimens used by the present invention.
Below in conjunction with embodiment, technical scheme provided by the present invention is described in detail:
Embodiment 1 (preparation embodiment)
The preparation of pad
(1) to recombinate the preparation of M98-His fusion, purifying
1. the clone of related gene
Bioinformatic analysis is carried out to people Chlamydia pneumoniae 98KD surface protein (accessionnumber in its NCBI Protein Data Bank is CAA04672), obtain the peptide section that in the outer conserved domain of its born of the same parents, epitope enriches the most, find the DNA encoding sequence of its correspondence, simultaneously at its 5 ' introducing restriction enzyme site NdeI, after 3 ' end introduces termination signal TAA and restriction enzyme site XhoI, (complete sequence synthesis transfers to Jin Sirui bio tech ltd to complete to chemosynthesis complete genome sequence, during delivery, the genetic fragment of Prof. Du Yucang is connected on carrier pUC57), be designated as m98.Its gene complete sequence is as shown in sequence table.Specifically, the protein sequence of m98 gene code is the 130-305aa of natural human Chlamydia pneumoniae 98KD surface protein (accessionnumber:CAA04672).Object fragment is reclaimed according to a conventional method after carrier pUC57 NdeI and XhoI of the DNA fragmentation containing this section of Prof. Du Yucang is carried out double digestion, for subsequent use.Adopt NdeI and XhoI to carry out double digestion to carrier pET-28a (+) simultaneously, and according to a conventional method the m98 gene obtained after double digestion is connected in pET-28a (+) carrier, and transformation of E. coli TOP10, build pET-M98 expression vector.Cut through enzyme and confirm that expression vector establishment is errorless with sequencing.This vector expression restructuring M98-His fusion.
2. the expression and purification of restructuring M98-His fusion
Extract plasmid after being cultivated by positive colony bacterium correct for qualification, technology proceeds in competence E.coliBL21 (DE3) routinely, is coated by bacterium liquid on the LB flat board containing 50 μ g/mL kanamycins, screen expression strain according to a conventional method after having transformed.The single bacterium colony with exogenous protein expression ability that picking pET-M98 transforms also is inoculated in 100mLLB nutrient culture media, in 37 DEG C of overnight incubation.After taking out bacterium liquid, be inoculated in 100mL by 1:100 and contain in the LB nutrient culture media of 50 μ g/mL kanamycins, be cultured to OD in 37 DEG C
600when=0.6, adding 1mol/LIPTG to final concentration is 1mmol/L, shakes bacterium and cultivates, induced fusion protein expression in 37 DEG C.Thalline is collected respectively at 10min centrifugal under 8000r/min after induction 4h.Carry out ultrasonication after by thalline 20mLPBS buffer solution 3 times and again resuspended, operating conditions is: 50HZ, 200W, ultrasonic 3S, and interval 5S, works 100 times.Ultrasonic complete after, the centrifugal 15min collecting precipitation of 12000g, is inclusion body.By this inclusion body lavation buffer solution (20mMNa
3pO
4, 0.5MNaCl; 3M urea, 30mM imidazoles, pH7.4) wash twice after, the centrifugal 15min collecting precipitation of 12000g.By precipitation Bindingbuffer (20mMNa
3pO
4, 0.5MNaCl; 8M urea, 30mM imidazoles, pH7.4) dissolve under room temperature after, the centrifugal 15min of 12000g, the supernatant filter membrane of 0.45 μm filters.Recombinant protein in this lysate is with HisTrapaffinitycolumns (GEhealthcare Products), and method to specifications carries out purifying.Concrete grammar is as follows:
1) be filled distilled water with 5mL syringe, turn on the stopper of post, with the joint provided, post is connected with syringe, wash post with 1mL/min flow velocity.
2) by 10mLBindingbuffer balance, 1mL/min flow velocity.
3) by fusion loading, 1mL/min flow velocity.
4) with 10mLBindingbuffer, post is washed with 1mL/min flow velocity.
5) with 10mLElutionbuffer (20mMNa
3pO
4, 0.5MNaCl; 8M urea, 500mM imidazoles, pH7.4), with 1mL/min flow velocity wash-out, be in charge of collection, often pipe 1ml, 12%SDS-PAGE detect, and merge the sample containing destination protein in elution fraction.Carry out after determination of protein concentration through bradford kit, adjustment concentration is 0.2mg/mL.
(2) preparation of rabbit and mouse-anti people Chlamydia pneumoniae 98KD memebrane protein polyclonal antibody IgG
1. the preparation of rabbit anti-human Chlamydia pneumoniae 98KD memebrane protein polyclonal antibody IgG
Immune Male New Zealand White Rabbit (being provided by Disease Prevention Control Center, Hubei Prov) after mixing emulsification according to 200 μ g (1mL) with 1mL Freund's complete adjuvant with the restructuring M98-His fusion of step (one) purifying, in dorsal sc multi-point injection, after the 7d of interval, immunity is once again, carry out booster immunization mix emulsification with the restructuring M98-His fusion of above-mentioned purifying according to 200 μ g (1mL) and 1mL incomplete Freund's adjuvant after 14d after, booster immunization is once again to press above-mentioned same method after booster immunization 7d again.Haemanalysis antibody titer is got after 7d.If dissatisfied, one to twice booster immunization can be repeated, (measure antibody titer by ELISA method to antibody titer is satisfied and be greater than 1 × 10
5).If satisfied, Culling heart blood, separation of serum, with ProteinG affinity column (GEhealthcare Products), in strict accordance with operational manual purified polyclonal antibodies IgG, measure antibody concentration by triumphant base Braford protein content detection kit and use phosphate buffer (8g/LNaCL, 0.2g/LKCl, 0.24g/LKH
2pO
4, 1.44g/LNa
2hPO
4pH7.4) be adjusted to 1mg/mL ,-20 DEG C of preservations are for subsequent use, so far obtained rabbit anti-human Chlamydia pneumoniae 98KD memebrane protein polyclonal antibody IgG.Westenblot test shows, this polyclonal antibody IgG can specific recognition people Chlamydia pneumoniae total length 98KD memebrane protein.
2. the preparation of mouse-anti people Chlamydia pneumoniae 98KD memebrane protein polyclonal antibody IgG
With the restructuring M98-His fusion of step (one) purifying as comlete antigen immune guinea pig (being provided by Disease Prevention Control Center, Hubei Prov), omoplate hemostasis antigen 200 μ g/ only.Fundamental immunity is that isopyknic antigen and Freund's complete adjuvant carry out emulsification, and carried out a booster immunization every 2 weeks, booster immunization equal-volume antigen and equal-volume incomplete Freund's adjuvant carry out emulsification, altogether immunity 4 times.Haemanalysis antibody titer is got after final immunization 10d.If dissatisfied, one to twice booster immunization can be repeated, (measure antibody titer by ELISA method to antibody titer is satisfied and be greater than 1 × 10
5).If satisfied, put to death cavy and get serum, with ProteinG affinity column (GEhealthcare Products), in strict accordance with operational manual purified polyclonal antibodies IgG, measure antibody concentration by triumphant base Braford protein content detection kit and use phosphate buffer (8g/LNaCL, 0.2g/LKCl, 0.24g/LKH
2pO
4, 1.44g/LNa
2hPO
4pH=7.4) 1mg/mL is adjusted to, for subsequent use, so far obtained mouse-anti people Chlamydia pneumoniae 98KD memebrane protein polyclonal antibody IgG.Westenblot test shows, this polyclonal antibody IgG can specific recognition people Chlamydia pneumoniae total length 98KD memebrane protein.
(3) preparation of quantum dot-labeled anti-human Chlamydia pneumoniae nano-probe
1. the optimization of the quantum dot-labeled rabbit of nanometer carboxylic anti-human Chlamydia pneumoniae 98KD memebrane protein polyclonal antibody IgG reaction conditions:
1.1, the determination of the quantum dot-labeled antibody probe optimum mark pH of carboxyl
Phosphate buffer pH in labeled reactant is set to 5,6,7,8,9 respectively, utilizes full spectrometer to carry out fluorescent strength determining to marked product, observe the impact of different pH value on coupling reaction, determining the Optimal pH that quantum dot-labeled many anti-reflective answer is 7.0-8.0.This experimental selection pH7.4.
1.2, the determination of carboxyl quantum dot-labeled antibody probe optimum mark amount
Quantum dot volumetric molar concentration and the ratio of how anti-concentration are set to 1:1 respectively, 1:2,1:3 and 1:4, after carrying out labeled reactant, full spectrometer is utilized to carry out fluorescent strength determining to marked product, the impact of both observations variable concentrations comparison coupling reaction, determines that optimum molar concentration ratio that quantum dot-labeled rabbit anti-human Chlamydia pneumoniae 98KD memebrane protein polyclonal antibody IgG reacts be quantum dot and antibody molar ratio is 1:3.This optimal concentration ratio of this experimental selection determines labelled amount.
1.3, the determination of the best sealer kind of the quantum dot-labeled antibody probe of carboxyl
With monoethanolamine, Tris, PEG2000-NH
2or BSA is as sealer, after the condition determined by step 1.1 and 1.2 carries out labeled reactant, utilize full spectrometer to carry out fluorescent strength determining to marked product, observe the impact of different sealers for labeled reactant, found that, PEG2000-NH
2for best sealer, it can significantly improve colloidal stability and the immunocompetence of labeled complex.
2. labeling process:
2nmol carboxyl water-soluble quantum dot, 300nmolN-N-Hydroxysuccinimide (sulfo-NHS) and 300nmol carbodiimide (EDC) is added successively in microcentrifugal tube, with phosphate buffer (2.9g/L sodium hydrogen phosphate, 0.295g/L sodium dihydrogen phosphate, 4g/L sodium chloride, pH7.4) constant volume is 2ml, ceaselessly mixed solution, after 37 DEG C of reaction 30min, excessive sulfo-NHS and the EDC as activator is removed in dialysis.Activation quantum dot in, the step (two) adding 6nmol prepare rabbit anti-human Chlamydia pneumoniae 98KD memebrane protein polyclonal antibody IgG, lucifuge reaction 2h, add single-ended amination polyglycol (PEG2000-NH
2) to final concentration be 1%, close unreacted activated carboxyl site, continue lucifuge reaction 1h.With 0.2 μm of PES frit removing antibody aggregation thing, then filtrate transferred in 50000MW ultra-filtration centrifuge tube, with 8000g centrifugal force centrifugal 15min at 4 DEG C, there is not the antibody of coupling reaction and the accessory substance in reacting in removing.Collect super filter tube filter membrane upper strata quantum dot-antibody coupling matter solution, be dissolved in 2ml phosphate cleansing solution (2.9g/L sodium hydrogen phosphate, 0.295g/L sodium dihydrogen phosphate, 4g/L sodium chloride, 5ml/L Tween-20, 0.3g/L Sodium azide, pH7.4) in, again this solution is transferred in 50000MW ultra-filtration centrifuge tube, with 8000g centrifugal force centrifugal 15min at 4 DEG C, collect super filter tube filter membrane upper strata quantum dot-antibody coupling matter solution, be dissolved in 1ml phosphate conserving liquid (2.9g/L sodium hydrogen phosphate, 0.295g/L sodium dihydrogen phosphate, 2g/L sodium chloride, 10g/LBSA, 0.3g/L Sodium azide, pH7.4) in, so far obtained quantum dot-labeled anti-human Chlamydia pneumoniae nano-probe, be placed in 4 DEG C to save backup.
(4) load of quantum dot-labeled anti-human Chlamydia pneumoniae nano-probe
1h in the quantum dot-labeled anti-human Chlamydia pneumoniae nano-probe solution obtain dacron film immersion step (three), take out, being cut into rear specification after 25 DEG C of dryings is after 4cm*0.6cm/ bar, and 4 DEG C of sealings save backup, so far obtained pad.
Embodiment 2 (preparation embodiment)
The preparation of sample pad
The sample pad treating fluid of preparation different formulations, the releasing effect of observation of quantum point labelled antibody, by repeatedly optimization of orthogonal test, obtains optimum sample pad prescription for the treatment of liquid (namely of the present invention).Get glass fibre element film one, it is soaked at least 3h in sample pad treating fluid, then is placed in Biohazard Safety Equipment after 37 DEG C of aeration-dryings, being cut into specification is after 4cm*2.5cm/ bar, i.e. obtained sample pad, and 25 DEG C of hermetically dryings are preserved.Confirm the use of this sample pad through test, substantially increase the release rate of quantum dot-labeled antibody on pad, reach good effect.
Embodiment 3 (preparation embodiment)
The preparation of detection layers
Nitrocellulose filter is cut into 4cm*4cm size.Mouse-anti people Chlamydia pneumoniae 98KD memebrane protein polyclonal antibody IgG prepared in embodiment 1 and anti-rabbit IgG phosphate buffer are adjusted to final concentration and are respectively 2.0mg/mL and 1.0mg/mL.The mouse-anti people Chlamydia pneumoniae 98KD memebrane protein polyclonal antibody IgG diluted is loaded BIODOT draw in film instrument shower nozzle, the amount arranging 1.0 μ l/cm is sprayed on nitrocellulose filter, forms detection line; The anti-rabbit IgG diluted is loaded BIODOT draw in film instrument shower nozzle, the amount arranging 1.0 μ l/cm is sprayed on as nature controlling line on nitrocellulose filter, and itself and detection line spacing are 0.7cm.By the nitrocellulose filter 37 DEG C of dry 2h sprayed, be cut into the specification of 4cm*4cm, 4 DEG C of hermetically dryings are preserved.So far obtained detection layers.
Embodiment 4 (preparation embodiment)
The assembling of test card
Assembling below in conjunction with accompanying drawing 1 and accompanying drawing 2 pairs of test card is described further.
Base plate is cut into 4cm*7.3cm size, for subsequent use.
Absorbent filter is cut into 4cm*3cm size, as adsorptive pads, for subsequent use.
Assembly working operates in Biohazard Safety Equipment; first the adhered protection film on base plate 7 is taken off; namely detection layers 3 described in embodiment 3 is pasted the concrete region of accompanying drawing 1 indication on base plate 7 with the nitrocellulose filter of nature controlling line 5 and detection line 4, and careful floating face.Secondly, be assembled on base plate 7 by the adsorptive pads 6 cut out in advance, the right end of its left side and detection layers is had, and 0.2cm's is overlapping, and its right hand edge then aligns with the right hand edge of base plate 7 and to glue and carefully floating.The pad 2 described by embodiment 1 is overlapped in the left hand edge place of detection layers 3 by 0.3cm, 0.3cm sticks on base plate 7 again.Finally by described by embodiment 2 sample pad 1 to be overlapped in the left hand edge place of pad 2 by one side 0.3cm, another side aligns with the left hand edge of base plate 7, to stick on base plate 7 and carefully floating.The check-out console assembled is cut under cutting cutter the wide test card of 4.0mm, 4 DEG C of hermetically dryings keep in Dark Place.
Embodiment 5 (Application Example)
The using method of test card
Obtain the throat swab of person to be checked according to a conventional method, be inserted into sample treatment liquid (the 2.9g/L sodium hydrogen phosphate that 500 μ l are housed, 0.295g/L sodium dihydrogen phosphate, NonidetP-4010ml/L, SDS1ml/L, 2g/L sodium chloride, pH7.3) in nonrigid plastic pipe, extruding plastic tube wall, after sample on swab is fully dissolved, take out 120 μ L and drip in the sample pad of test card, after 15 minutes, under uv analyzer, (model is WD-9403A, Liuyi Instruments Plant, Beijing produces, burst of ultraviolel wavelength 365nm) observe testing result.If containing people's Chlamydia pneumoniae antigen in throat swab, quantum dot-labeled anti-human Chlamydia pneumoniae nano-probe then in pad is combined, under ultraviolet excites, a macroscopic fluoroscopic examination line can be formed at detection line place after being combined by the mouse-anti people Chlamydia pneumoniae 98KD memebrane protein polyclonal antibody of chromatography effect first on nitrocellulose filter, continue to excite lower formation macroscopic Article 2 fluorescence nature controlling line at ultraviolet after chromatography is combined with anti-rabbit IgG in conjunction with complete quantum dot-labeled antibody; If without related antigen in throat swab to be checked, then only there is a fluorescence nature controlling line.If fluorescence nature controlling line does not occur, then this test card lost efficacy.
Embodiment 6 (Application Example)
Effect citing of the present invention
In the present embodiment, the using method of people's Chlamydia pneumoniae quantum dot immune chromatography test card of indication is with reference to the operation steps described in embodiment 5.
1) specific test
With respiratory tract common causative as human respiratory syncytial virus's (Long strain, ATCC numbering VR26), people's mycoplasma pneumoniae (ATCC numbering 15531), (the AR-39 strain of people's Chlamydia pneumoniae, ATCC numbering 53592), (the GB strain of adenovirus hominis 3 type, ATCC numbering VR-3), (the Gomen strain of adenovirus hominis 7 type, ATCC numbering VR-7), influenza virus A hominis (H1N1, ATCC numbering VR-1743), people's influenza B virus (ATCC numbering VR-790), haemophilus influenzae (ATCC numbering 53781), streptococcus pneumonias (ATCC numbering 700670) etc. replace people's Chlamydia pneumoniae to detect, the sample treatment liquid that test card detects containing these microorganisms is all negative.
2) sensitivity tests
Doing Study of Sensitivity by measuring people's Chlamydia pneumoniae culture dilution, determining that the Monitoring lower-cut of test card detection people's Chlamydia pneumoniae bacterial strain (AR-39 strain, ATCC numbering 53592) described in embodiment 4 is 1ng/ml.
3) clinical trial example
Goldstandard-cultivation is detected as reference using people's Chlamydia pneumoniae, the test card described by bronchoalveolar lavage fluid sample embodiment 4 of getting 68 routine division of respiratory disease Patients with Lower Respiratory Tract Infections detects, cultivation positive rate is 27.9% (19/68), this test card is 26.5% (18/68), and the coincidence rate of 2 kinds of methods is 95.6% (65/68).
The testing result of table 1 clinical samples
It is pointed out that and the foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments, equivalent replacement etc. made within the present invention's spirit and principle all should be included within protection scope of the present invention.
Claims (10)
1. people's Chlamydia pneumoniae quantum dot immune chromatography test card, is characterized in that: described people's Chlamydia pneumoniae quantum dot immune chromatography test card comprises base plate, sample pad, pad, detection layers and adsorptive pads; Described pad is coated with quantum dot-labeled anti-human Chlamydia pneumoniae nano-probe; Described detection layers is made up of the solid phase nitrocellulose filter with a detection line and a nature controlling line; Described detection line is coated with mouse-anti people Chlamydia pneumoniae 98KD memebrane protein polyclonal antibody; Described nature controlling line is coated with anti-rabbit IgG; Described detection layers is pasted onto on base plate; Described pad and adsorptive pads be separately positioned on detection layers both ends top and after partly overlapping with detection layers respectively together with detection layers and base plate sticking; Described sample pad to be arranged on above pad and after partially overlapping with pad respectively together with pad and base plate sticking.
2. people's Chlamydia pneumoniae quantum dot immune chromatography test card according to claim 1, is characterized in that: described quantum dot is water-soluble CdSe/ZnS quantum dot that carboxylated amphipathic polymer is modified.
3. people's Chlamydia pneumoniae quantum dot immune chromatography test card according to claim 1 and 2, is characterized in that: described anti-rabbit IgG includes but not limited to goat anti-rabbit igg.
4. people's Chlamydia pneumoniae quantum dot immune chromatography test card according to claim 3, it is characterized in that: the long 2cm of described detection layers, described detection layers is pasted onto the backplate surface interlude that length is 6.6-7.7cm; Described detection layers be pasted onto the overlapping 0.2-0.4cm of adsorptive pads that on detection layers and base plate and length is 2.5-3cm; Described detection layers be pasted onto the overlapping 0.2-0.4cm of pad that on detection layers and base plate and length is 0.5-0.8cm; Described pad and the length be pasted onto on pad and base plate are the overlapping 0.2-0.4cm of sample pad of 2.5cm; The spacing of described detection line and nature controlling line is 0.5-0.8cm; The width of described base plate is 0.3-0.5cm.
5. people's Chlamydia pneumoniae quantum dot immune chromatography test card according to claim 4, is characterized in that: described adsorptive pads is absorbent filter; Described base plate is PVC board.
6., based on a preparation method for the people's Chlamydia pneumoniae quantum dot immune chromatography test card as described in claim as arbitrary in claim 1-5, it is characterized in that: described preparation method comprises the following steps:
1) preparation of pad:
1.1) to recombinate the preparation of M98-His fusion, purifying:
1.1.1) bioinformatic analysis is carried out to people Chlamydia pneumoniae 98KD surface protein, obtain the peptide section that epitope in its ectodomain enriches the most;
1.1.2) find step 1.1.1) in institute obtain the gene coded sequence of peptide section correspondence, and at 5 ' end of sequence and 3 ' end introducing restriction enzyme site and chemosynthesis complete genome sequence, be designated as m98 with tense marker;
1.1.3) by step 1.1.2) in the m98 that obtains be cloned into expression vector pET-28a (+) by molecular biology method after proceed to expression in escherichia coli restructuring M98-His fusion; Described restructuring M98-His fusion is present in thalline with inclusion body expression way;
1.1.4) with ni-sepharose purification step 1.1.3) recombinant protein that obtains, after SDS-PAGE detects its purity, measure protein concentration with Bradford method, adjustment protein concentration is for subsequent use after 0.2mg/mL;
1.2) preparation of rabbit and mouse-anti people Chlamydia pneumoniae 98KD memebrane protein polyclonal antibody IgG:
1.2.1) with step 1.1.4) in the restructuring M98-His fusion that obtains be comlete antigen, immunize New Zealand White Rabbit and cavy respectively; Prepare rabbit anti-human Chlamydia pneumoniae 98KD memebrane protein antiserum and mouse-anti people Chlamydia pneumoniae 98KD memebrane protein antiserum respectively; Described rabbit anti-human Chlamydia pneumoniae 98KD memebrane protein antiserum and the sero-fast indirect ELISA titer of mouse-anti people Chlamydia pneumoniae 98KD memebrane protein are all greater than 1 × 10
5;
1.2.2) the polyclonal antibody IgG in ProteinG affinity column difference purified rabbit anti-human's Chlamydia pneumoniae 98KD memebrane protein antiserum and mouse-anti people Chlamydia pneumoniae 98KD memebrane protein antiserum is adopted;
1.2.3) with triumphant base Braford protein content detection kit determination step 1.2.2) concentration of two kinds of polyclonal antibody IgG that obtains, for subsequent use after its protein concentration is all adjusted to 3mg/mL;
1.3) preparation and purification of quantum dot-labeled anti-human Chlamydia pneumoniae nano-probe:
1.3.1) in microcentrifugal tube, 2nmol quantum dot, 300nmolN-N-Hydroxysuccinimide and 300nmol carbodiimide is added successively, with phosphate buffer constant volume for 2ml, in rotary mixer, with 15rpm/min, after 37 DEG C of reaction 30min, excessive N-hydroxy-succinamide and carbodiimide are removed in dialysis; In described phosphate buffer, each component concentration is respectively: 2.9g/L sodium hydrogen phosphate, 0.295g/L sodium dihydrogen phosphate and 2g/L sodium chloride, the pH=7.3 of described phosphate buffer;
1.3.2) in the quantum dot of activation, add the step 1.2 of 4-12nmol) prepared by rabbit anti-human Chlamydia pneumoniae 98KD memebrane protein polyclonal antibody IgG, lucifuge reaction 2h, adding Amino End Group polyethylene glycol to final concentration is 1.5%, close unreacted activated carboxyl site, continue lucifuge reaction 1h; With 0.2 μm of PES frit removing antibody aggregation thing, then filtrate transferred in 50000MW ultra-filtration centrifuge tube, with 8000g centrifugal force centrifugal 15min at 4 DEG C, there is not the antibody of coupling reaction and the accessory substance in reacting in removing; Collect super filter tube filter membrane upper strata quantum dot-antibody coupling matter solution, be dissolved in 2ml phosphate cleansing solution, again this solution is transferred in 50000MW ultra-filtration centrifuge tube, with 8000g centrifugal force centrifugal 15min at 4 DEG C, collect super filter tube filter membrane upper strata quantum dot-antibody coupling matter solution, be dissolved in 1ml phosphate conserving liquid, so far obtained quantum dot-labeled anti-human Chlamydia pneumoniae nano-probe;
In described phosphate cleansing solution, each component concentration is respectively: 2.9g/L sodium hydrogen phosphate, 0.295g/L sodium dihydrogen phosphate, 2g/L sodium chloride, 5ml/L Tween-20 and 1g/L Sodium azide; The pH=7.3 of described phosphate cleansing solution; In described phosphate conserving liquid, each component concentration is respectively: 2.9g/L sodium hydrogen phosphate, 0.295g/L sodium dihydrogen phosphate, 2g/L sodium chloride, 10g/LBSA and 1g/L Sodium azide; The pH=7.3 of described phosphate conserving liquid;
1.4) load of quantum dot-labeled antibody:
Dacron film is immersed step 1.3.2) 1h in the quantum dot-labeled anti-human Chlamydia pneumoniae nano-probe solution that obtains, take out, 25 DEG C of rear 4 DEG C of sealings of drying save backup, so far obtained pad;
2) preparation of sample pad:
Get glass fibre element film one, glass fibre element film is soaked at least more than 3h in sample pad treating fluid, then is placed in Biohazard Safety Equipment after 37 DEG C of aeration-dryings, 25 DEG C of hermetically dryings are preserved; So far obtained sample pad;
In described sample pad treating fluid, each component concentration is respectively: 2.9g/L sodium 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 5g/L polyvinylpyrrolidone, the pH=7.3 of described sample pad treating fluid;
3) preparation of detection layers:
3.1) by step 1.2) to be all adjusted to final concentration be for subsequent use after 0.5-2.5mg/mL for the mouse-anti people Chlamydia pneumoniae 98KD memebrane protein polyclonal antibody prepared and anti-rabbit IgG phosphate buffer; In described phosphate buffer, each component concentration is respectively: 2.9g/L sodium hydrogen phosphate, 0.295g/L sodium dihydrogen phosphate and 2g/L sodium chloride, the pH=7.3 of described phosphate buffer;
3.2) the mouse-anti people Chlamydia pneumoniae 98KD memebrane protein polyclonal antibody diluted is loaded BIODOT to draw in film instrument shower nozzle, the amount arranging 0.8-2.5 μ l/cm is sprayed on nitrocellulose filter, forms detection line; The anti-rabbit IgG diluted is loaded BIODOT draw in film instrument shower nozzle, the amount that 0.8-2.5 μ l/cm is set according to and the interval of detection line 0.5-0.8cm be sprayed on nitrocellulose filter as nature controlling line;
3.3) will be sprayed with the nitrocellulose filter of detection line and nature controlling line at 37 DEG C of dry 2h, 4 DEG C of hermetically dryings are preserved; So far obtained detection layers;
4) preparation of base plate
It is for subsequent use after the base plate of PVC material is pressed actual requirement cutting;
5) preparation of adsorptive pads
For subsequent use after absorbent filter being pressed actual requirement cutting;
6) assembling of people's Chlamydia pneumoniae quantum dot immune chromatography test 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 pastes the central region of base plate, and floating face;
6.3) by step 5) preparation-obtained adsorptive pads is assembled on base plate, and the left side of adsorptive pads and detection layers are overlapped, its right hand edge is alignd with the right hand edge of base plate to glue and floating simultaneously;
6.4) by step 1) preparation-obtained pad is overlapped in the left hand edge place of nitrocellulose filter by partly overlapping mode, sticks on base plate by pad simultaneously;
6.5) by step 2) prepared by obtain sample pad is then overlapped in pad left hand edge place by partly overlapping mode, another side aligns with the left hand edge of base plate, to stick on base plate and floating;
6.6) the people's Chlamydia pneumoniae quantum dot immune chromatography test card assembled is carried out cutting, 4 DEG C of hermetically dryings keep in Dark Place;
Described step 6.1) to step 6.6) be all operate in Biohazard Safety Equipment.
7. method according to claim 6, is characterized in that: described step 1.3.2) in add the step 1.2 of 6nmol) prepared by rabbit anti-human Chlamydia pneumoniae 98KD memebrane protein polyclonal antibody IgG;
Described step 3.1) in by step 1.2) the mouse-anti people Chlamydia pneumoniae 98KD memebrane protein polyclonal antibody prepared and anti-rabbit IgG phosphate buffer be adjusted to final concentration and be respectively 1.5-2.0mg/mL and 0.5-1.5mg/mL;
Described step 3.2) in, the mouse-anti people Chlamydia pneumoniae 98KD memebrane protein polyclonal antibody diluted is loaded BIODOT and draws in film instrument shower nozzle, the amount arranging 1.0-2.0 μ l/cm is sprayed on nitrocellulose filter, forms detection line; The anti-rabbit IgG diluted is loaded BIODOT draw in film instrument shower nozzle, the amount that 1.0-2.0 μ l/cm is set according to and the interval of detection line 0.5-0.8cm be sprayed on nitrocellulose filter as nature controlling line.
8. one kind is detected the application of people's Chlamydia pneumoniae as nondiagnostic based on the people's Chlamydia pneumoniae quantum dot immune chromatography test card as described in claim as arbitrary in claim 1-5.
9., based on a nondiagnostic detection method for the people's Chlamydia pneumoniae quantum dot immune chromatography test card as described in claim as arbitrary in claim 1-5, it is characterized in that: described detection method comprises the following steps:
1), after measuring samples fully being dissolved by the sample treatment liquid of 500 μ l, take out 120 μ L and drip in the sample pad of test card, after 15 minutes, under uviol lamp, observe testing result; In described sample treatment liquid, each component concentration is respectively: sodium hydrogen phosphate 2.9g/L, sodium dihydrogen phosphate 0.295g/L, NonidetP-4010ml/L, SDS1ml/L and sodium chloride 2g/L, the pH=7.3 of described sample treatment liquid;
2) if containing people's Chlamydia pneumoniae antigen in measuring samples, quantum dot-labeled anti-human Chlamydia pneumoniae nano-probe then in pad is combined, under ultraviolet excites, a macroscopic fluoroscopic examination line can be formed at detection line place after being combined by the mouse-anti people Chlamydia pneumoniae 98KD memebrane protein polyclonal antibody of chromatography effect first on nitrocellulose filter, continue to excite lower formation macroscopic Article 2 fluorescence nature controlling line at ultraviolet after chromatography is combined with anti-rabbit IgG in conjunction with complete quantum dot-labeled antibody;
If unmanned Chlamydia pneumoniae antigen in measuring samples, then only there is a fluorescence nature controlling line; If fluorescence nature controlling line does not occur, then this test card lost efficacy.
10. method according to claim 5, is characterized in that: described measuring samples includes but not limited to throat swab.
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Effective date of registration: 20180428 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 |