CN105203768B - Method and kit for fast detection of human chlamydia pneumoniae antigen based on magnetic resolution and quantum dot labelling - Google Patents
Method and kit for fast detection of human chlamydia pneumoniae antigen based on magnetic resolution and quantum dot labelling Download PDFInfo
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Abstract
The invention provides a method for detection of human chlamydia pneumoniae antigen based on magnetic resolution and quantum dot labelling. The method comprises the following steps: (1) preparing anti-human chlamydia pneumoniae immune nano magnetic beads; (2) preparing quantum dot labelled anti-human chlamydia pneumoniae nanoprobes; (3) dissolving a sample to be detected with a PBST buffer solution, adding the anti-human chlamydia pneumoniae immune nano magnetic beads into the dissolved solution, carrying out magnetic separation after sufficient mixing and reaction, washing with the PBST buffer solution, adding the quantum dot labelled anti-human chlamydia pneumoniae nanoprobes into the obtained precipitate, carrying out magnetic separation after a reaction, washing with the PBST buffer solution, and detecting the fluorescence value with a fluorescence micro-plate reader. The method is accurate, fast, and high in sensitivity, and has very high practical value in the aspects of clinical diagnosis, etiological differentiation, epidemiological surveys and the like of human chlamydia pneumoniae.
Description
Technical field
The present invention relates to technical field of medical detection, specially a kind of based on Magnetic Isolation with quantum dot-labeled detection people
The method for quick of mycoplasma pneumoniae antigen and detection kit, and the method for preparation and use of this detection kit.
Background technology
Mycoplasma pneumoniae (mycoplasma pneumoniae, mp) is the pathogen of mankind's mycoplasma pneumonia, and main warp flies
Foam infects, and in 2-3 week incubation period, sickness rate is with teenager highest.The incidence rate that mp infects in infantile pneumonia cause of disease is up to 10-
30%, it is increasingly becoming children in recent years and infect one of main pathogens of respiratory tract disease.This disease easily causes pharyngitis, tonsil
The respiratory tract infection such as inflammation, in some instances it may even be possible to the multiple organ injury such as secondary meningitiss, hepatitis, myocarditiss simultaneously, may also lead to when serious
Infant is dead.
Because mp infection is similar with the respiratory tract infection symptom that other pathogen cause, do not do etiological examination, being difficult to will
The respiratory tract infection that mp is caused with other pathogen distinguishes.Mp is acellular wall, conventional Beta-lactam medicine is invalid to it,
Therefore the treatment of its infection of causing is entirely different with the therapeutic scheme that other antibacterials and virus infect, hence set up simplicity, quickly,
Feasible, energy early diagnosiss mycoplasma pneumoniae infection method is very necessary.
The detection method of mp mainly has 3 classes at present: one is isolated culture, and it is to confirm infection " goldstandard ", but by
Extremely slow in the growth cycle of mp, cultivation cycle is long, leads to this method clinically can not carry out quick diagnosis；Two is serology
Method, that is, adopt euzymelinked immunosorbent assay (ELISA), colloidal gold immunization, micro-Immunofluorescence assay and indirect hemagglutination test etc., detects examinee
Mp antibody horizontal in serum, can point out the mp presence of infection indirectly.However, serological test can only provide a kind of retrospective examining
Disconnected, and sometimes for paired sera.In addition, the opportunity that antibody occurs is difficult to grasp, deposit between children and adolescents and adult again
There is non-spy with other microorganisms and body tissue in the glycolipid antigen in the difference of mp specific antibody, and, mp cell membrane
Different in nature cross reaction, therefore the detection quality of existing serological method is subject to a definite limitation；Three is using Protocols in Molecular Biology inspection
Survey the presence of mp dna, most common of which is polymerase chain reaction (pcr), and the method is quick, sensitive, special, is current
The important means of research people's mycoplasma pneumoniae infection, but because pcr is higher to experimental facilitiess and operation requirement, and easy appearance is false
The positive, can't be used as conventional methods for clinical diagnosis in China.Therefore, set up people's mycoplasma pneumoniae specific antigen diagnosis side
Method is very necessary.At present it has been disclosed that the method for detection people's mycoplasma pneumoniae antigen of report be mainly double antibodies sandwich elisa method,
Quantum dot mark immunity-chromatography method, indirect immunofluorescence etc., although these method sensitivity are higher, but due to people's pneumonia
Mycoplasma is as extremely low in the content in throat swab in clinical samples, leads to its Clinical detection positive rate or relatively low, does not all enter at present
Row clinical practice is applied.
Content of the invention
For these technical problems present in background technology, the invention provides a kind of be based on Magnetic Isolation and quantum dot
The energy of labelling is easy, quick, the detection method of highly sensitive detection people's mycoplasma pneumoniae antigen and test kit, and this reagent
The method of preparation and use of box.
The present invention is achieved through the following technical solutions:
A kind of based on Magnetic Isolation and quantum dot-labeled detection people's mycoplasma pneumoniae antigen method it is characterised in that:
Described the method comprises the following steps:
1) preparation of rabbit anti-human mycoplasma pneumoniae p1 protein polyclone antibody；
2) preparation of Mus anti-human mycoplasma pneumoniae p1 protein polyclone antibody；
3) by step 1) the rabbit anti-human mycoplasma pneumoniae p1 protein polyclone antibody for preparing passed through altogether with nanometer magnetic bead
Valency is coupled, and prepares anti-human mycoplasma pneumoniae immune nanometer magnetic bead；
4) by step 2) the Mus anti-human mycoplasma pneumoniae p1 protein polyclone antibody for preparing passed through with nano-quantum point
Covalent coupling, prepares quantum dot-labeled anti-human mycoplasma pneumoniae nano-probe；
5) take human respiratory secretions sample (including but not limited to throat swab), after pbst buffer solution, add step
The anti-human mycoplasma pneumoniae immune nanometer magnetic bead that rapid 3) prepare, is sufficiently mixed, and carries out Magneto separate after reaction 25-45min, with
After pbst buffer solution 2 times, in the precipitate obtaining to Magneto separate, add step 4) prepare quantum dot-labeled anti-
People's mycoplasma pneumoniae nano-probe, carries out Magneto separate, after pbst buffer solution 2 times, using fluorescence after reaction 25-45min
Microplate reader reads fluorescent value；In described pbst buffer, each component content is as follows: 8g/l nacl, 0.2g/l kcl, 0.24g/l
kh2po4, 1.44g/l na2hpo4, 0.5ml/l tween-20, the ph=7.4 of described pbst buffer；
6) according to step 1)-step 5) method detect that four parts determine for the negative people of people's mycoplasma pneumoniae through clinic respectively
The respiratory secretions sample of group, reads fluorescent value；The respiratory secretions sample of the negative crowd of described people's mycoplasma pneumoniae
Abbreviation people's mycoplasma pneumoniae negative control sample；The meansigma methodss of the fluorescent value of described four parts of people's mycoplasma pneumoniae negative control sample
It is cut-off value with 3 times of standard deviation sums；If step 5) in human respiratory secretions sample detection fluorescent value be more than cut-
Off value, then be judged as that in human respiratory secretions sample, people's mycoplasma pneumoniae antigen is the positive；If step 5) in human respiratory divide
The detection fluorescent value of secretion sample is less than cut-off value, then be judged as people's pneumonia mycoplasma in human respiratory secretions sample
Originally it was negative.
A kind of based on Magnetic Isolation and quantum dot-labeled detection people's mycoplasma pneumoniae antigen test kit, its feature exists
In: described test kit is by anti-human mycoplasma pneumoniae immune nanometer magnetic bead, the quantum with enrichment people's mycoplasma pneumoniae antigen function
Anti-human mycoplasma pneumoniae nano-probe, quality-control product and the pbst buffer of selecting labelling are formed；Described quality-control product includes the positive
Quality-control product and negative quality-control product；Described positive quality control product is attached on swab by the people's mycoplasma pneumoniae drying inactivateing and forms；
Described feminine gender quality-control product is the throat swab determining through clinic for the negative crowd of people's mycoplasma pneumoniae.
A kind of test kit for preparation based on Magnetic Isolation and quantum dot-labeled detection people's mycoplasma pneumoniae antigen
Method it is characterised in that: described preparation method comprises the following steps:
1) preparation of anti-human mycoplasma pneumoniae immune nanometer magnetic bead:
1.1) preparation of rabbit and Mus anti-human mycoplasma pneumoniae p1 protein polyclone antibody igg
1.1.1) the preparation of restructuring p1-his fusion protein, purification:
1.1.1.1) bioinformatic analysis are carried out to people mycoplasma pneumoniae memebrane protein p1, obtain anti-in its ectodomain
Former epi-position peptide fragment the abundantest；
1.1.1.2) find step 1.1.1.1) in the corresponding gene coded sequence of obtained peptide fragment, according in escherichia coli
The Preference of codon, carries out codon optimization to obtained gene coded sequence；
1.1.1.3) in step 1.1.1.2) in 5 ' ends of obtained gene order and 3 ' ends introduce restriction enzyme sites and change
Learn synthesis complete genome sequence, labelling is designated as p1 simultaneously；Its gene order is as shown in sequence table；
1.1.1.4) by step 1.1.1.3) in obtained p1 be cloned into expression vector pet- by molecular biology method
28a (+) after proceed to expression in escherichia coli restructuring p1-his fusion protein；Described restructuring p1-his fusion protein is with solubility table
The mode of reaching is present in thalline；
1.1.1.5) use ni-sepharose purification step 1.1.1.4) obtained by recombiant protein, after sds-page detects its purity,
Protein concentration is measured with bradford method, adjustment protein concentration is standby after 0.2mg/ml；
1.1.2) the preparation of rabbit and Mus anti-human mycoplasma pneumoniae p1 protein polyclone antibody igg:
1.1.2.1) with step 1.1.1.5) in obtained restructuring p1-his fusion protein as complete antigen, immunity respectively
New zealand white rabbit and Cavia porcelluss；Prepare rabbit anti-human mycoplasma pneumoniae p1 protein antiserum and Mus anti-human mycoplasma pneumoniae p1 egg respectively
White antiserum；The anti-human mycoplasma pneumoniae p1 protein antiserum of described rabbit and Mus anti-human mycoplasma pneumoniae p1 protein antiserum indirect
Elisa potency is all higher than 1 × 105；
1.1.2.2) adopt protein g affinity column respectively purified rabbit anti-human's mycoplasma pneumoniae p1 protein antiserum and
Polyclonal antibody igg in Mus anti-human mycoplasma pneumoniae p1 protein antiserum；
1.1.2.3) with triumphant base braford protein content detection kit determination step 1.1.2.2) obtained by more than two kinds
The concentration of clonal antibody igg, its protein concentration is all adjusted to standby after 1mg/ml；
1.2) being coated of immune nanometer magnetic bead:
1.2.1) take 5mg magnetic bead, with 1ml mes buffer solution three times, be placed in nanometer magnetic separator and carry out Magneto separate
After remove supernatant；Described magnetic bead is with superparamagnetism fe3o4It is the carboxyl magnetic bead of 180nm for kernel, particle diameter；Described mes buffer
It is 2- (n- morpholino) ethyl sulfonic acid that mass concentration is 2g/l；The ph=6.0 of described mes buffer；Described nanometer magnetic separator
Magnetic intensity be 0.4t；
1.2.2) sequentially add and use step 1.2.1) in mes buffer concentration be 8-12mg/ml edc solution
And use step 1.2.1) in mes buffer concentration be 6-10mg/ml each 0.5ml of sulfo-nhs solution, with
10-40rpm activates 1hr in rotary mixer, is placed in and removes supernatant after carrying out Magneto separate in nanometer magnetic separator, is walked with 1ml
Rapid 1.2.1) in mes buffer resuspended, magnetic bead after being activated；
1.2.3) take 5 centrifuge tubes, each centrifuge tube add 200 μ l steps 1.2.2) obtained by activation after magnetic
Pearl, is placed in and removes supernatant after carrying out Magneto separate in nanometer magnetic separator, adds use the dense of pbs buffer dilution in each centrifuge tube
Spend for 50-200 μ g/ml by step 1.1) prepared by rabbit anti-human mycoplasma pneumoniae p1 protein polyclone antibody igg solution each
1ml, reacts 2-6h with 15rpm under room temperature in rotary mixer, is placed in and removes after carrying out Magneto separate in nanometer magnetic separator
After clear, each above-mentioned pbs buffer adding 1ml to contain 1mg/ml ethanolamine, reacts 2h with 15rpm under room temperature in rotary mixer
To close the not carboxyl with antibody response on magnetic bead；In described pbs buffer, each component content is as follows: 8g/l nacl, 0.2g/l
Kcl, 0.24g/l kh2po4, 1.44g/l na2hpo4, the ph=7.4 of described pbs buffer；
1.2.4) after the completion of capping, this 5 centrifuge tubes are placed in nanometer magnetic separator and carry out removing after Magneto separate
Supernatant, respectively washs three times with 1ml lavation buffer solution；In described lavation buffer solution, each component content is as follows: 8g/l nacl, 0.2g/
L kcl, 0.24g/l kh2po4, 1.44g/l na2hpo4, 0.5ml/l tween-20, the ph=7.4 of described lavation buffer solution；
1.2.5 in each centrifuge tube) it is separately added into 1ml preserves the resuspended magnetic bead of buffer, be placed in 4 DEG C and save backup；Institute
State each component content in preservation buffer as follows: 8g/l nacl, 0.2g/l kcl, 0.24g/l kh2po4, 1.44g/l
na2hpo4, 0.3g/l nan3, 5g/l bovine serum albumin (bsa), the described ph=7.4 preserving buffer；
2) preparation of quantum dot-labeled anti-human mycoplasma pneumoniae nano-probe:
Its concrete preparation method includes:
2.1) 2nmol carboxyl water-soluble quantum dot, 300nmol n- hydroxy amber are sequentially added in microcentrifugal tube
Amber acid imide sulfo-nhs and 300nmol carbodiimide edc, with phosphate buffer constant volume as 2ml, mixed solution, 37 DEG C
After reaction 30min, dialysis removes excessive sulfo-nhs and edc as activator, the quantum dot after being activated；Described
In phosphate buffer, each component content is as follows: 2.9g/l disodium hydrogen phosphate, 0.295g/l sodium dihydrogen phosphate, 4g/l sodium chloride；
The ph=7.4 of described phosphate buffer；
2.2) in step 2.1) obtained by the quantum dot of activation in, add 4-12nmol step 1.1) in prepared
Mus anti-human mycoplasma pneumoniae p1 protein polyclone antibody igg, lucifuge reacts 2h, adds single-ended amino polyethylene glycol peg2000-
nh2To final concentration of 1%, close unreacted activated carboxyl site, continue lucifuge reaction 1h；
2.3) be filtered to remove step 2.2 with 0.2 μm of pes filter) in antibody aggregation thing, then filtrate be transferred to
In 50000mw ultra-filtration centrifuge tube, 15min is centrifuged at 4 DEG C with 8000g centrifugal force, remove the antibody that coupling reaction does not occur and
By-product in reaction；
2.4) collection step 2.3) in ultra-filtration centrifuge tube filter membrane upper strata quantum dot-antibody coupling matter solution, be dissolved in 2ml phosphoric acid
In salt cleaning mixture, then this solution is transferred in a new 50000mw ultra-filtration centrifuge tube, with 8000g centrifugal force at 4 DEG C from
Heart 15min, collects ultra-filtration centrifuge tube filter membrane upper strata quantum dot-antibody coupling matter solution, is dissolved in 1ml phosphate and preserves in liquid, puts
Save backup in 4 DEG C；In described phosphate cleaning mixture, each component content is as follows: 2.9g/l disodium hydrogen phosphate, 0.295g/l phosphoric acid
Sodium dihydrogen, 4g/l sodium chloride, 5ml/l tween 20,0.3g/l sodium azide, the ph=7.4 of described phosphate cleaning mixture；Described phosphorus
It is as follows that hydrochlorate preserves each component content in liquid: 2.9g/l disodium hydrogen phosphate, 0.295g/l sodium dihydrogen phosphate, 2g/l sodium chloride,
10g/l bovine serum albumin, 0.3g/l sodium azide；Described phosphate preserves the ph=7.4 of liquid；
3) preparation of pbst buffer:
Its concrete compound method includes:
Take 8g nacl, 0.2g kcl, 0.24kh2po4, 1.44g na2hpo4, 0.3g nan3, 0.5ml tween-20 is molten
Solution, in 800ml distilled water, adjusts ph to 7.4 with 5m naoh, then is settled to 1000ml；
4) preparation of quality-control product:
4.1 positive quality control product: positive quality control product is attached on swab by the people's mycoplasma pneumoniae drying inactivateing and forms；
4.2 negative quality-control products: negative quality-control product determines the throat swab for the negative crowd of people's mycoplasma pneumoniae through clinic；
Preferably, the present invention is in step 1.2.2) in, sequentially add and use step 1.2.1) in mes buffer
Concentration is the edc solution of 10mg/ml and uses step 1.2.1) in mes buffer concentration be 8mg/ml sulfo-
The each 0.5ml of nhs solution, activates 1hr with 15rpm in rotary mixer, is placed in nanometer magnetic separator and moves after carrying out Magneto separate
Except supernatant, with 1ml step 1.2.1) in mes buffer resuspended, magnetic bead after being activated；
Described step 1.2.3) in, add in each centrifuge tube with pbs buffer dilution concentration be 100 μ g/ml by
Step 1.1) prepared by each 1ml of rabbit anti-human mycoplasma pneumoniae p1 protein polyclone antibody igg solution, under room temperature with 15rpm in
React 3h in rotary mixer, be placed in after removing supernatant after carrying out Magneto separate in nanometer magnetic separator, each addition 1ml contains 1mg/ml
The above-mentioned pbs buffer of ethanolamine；
Described step 2.2) in, in step 2.1) obtained by the quantum dot of activation in, add 6nmol step 1.1) in
Prepared Mus anti-human mycoplasma pneumoniae p1 protein polyclone antibody igg, lucifuge reacts 2h.
Preferably, the water solublity cdse/zns amount that quantum dot of the present invention is carboxylated amphipathic polymer to be modified
Sub- point.
Preferably, magnetic bead of the present invention is with superparamagnetism fe3o4It is polystyrene, table for kernel, shell material
Face functional group is carboxyl, particle diameter is the carboxyl magnetic bead of 180nm.
A kind of based on Magnetic Isolation and quantum dot-labeled detection people's mycoplasma pneumoniae antigen test kit using method,
It is characterized in that: described using method comprises the following steps:
1) sample to be checked is proceeded to lysate in the common centrifuge tube of 1.5ml with after 0.5ml pbst buffer solution；Institute
State each component content in pbst buffer as follows: 8g/l nacl, 0.2g/l kcl, 0.24g/l kh2po4, 1.44g/l
na2hpo4, 0.3g/l nan3, 0.5ml/l tween-20；The ph=7.4 of described pbst buffer；
2) to step 1) in centrifuge tube in add based on Magnetic Isolation and quantum dot-labeled detection people's mycoplasma pneumoniae
Anti-human mycoplasma pneumoniae immune nanometer magnetic bead 50-150 μ l in the test kit of antigen, with 10rpm in rotary mixer under room temperature
Take off after upper reaction 25-45min, centrifuge tube is inserted nanometer magnetic separator Magneto separate 3min, suctions out supernatant with pipettor；
3) the pbst buffer 1ml adding in test kit washs twice, is washed using suctioning out after nanometer magnetic separator Magneto separate
Wash liquid, finally use the resuspended magnetic bead of 1ml pbs buffer, prepared immune nanometer magnetic bead-mycoplasma antigen complex；Described pbs delays
Rush each component content in liquid as follows: 8g/l nacl, 0.2g/l kcl, 0.24g/l kh2po4, 1.44g/l na2hpo4；Described
The ph=7.4 of pbs buffer；
4) take 100 μ l steps 3) immune nanometer magnetic bead-mycoplasma antigen complex of obtaining in another centrifuge tube, then plus
Enter quantum dot-labeled in the test kit based on Magnetic Isolation and quantum dot-labeled detection people's mycoplasma pneumoniae antigen for the 100 μ l
Anti-human mycoplasma pneumoniae nano-probe, reacts 25-45min with 15rpm under room temperature, by quantum dot on rotary mixer
Antibody is combined with the immunity of the mycoplasma antigen on immune nanometer magnetic bead, and quantum dot is tagged to mycoplasma surface, formed magnetic bead-
Mycoplasma antigen-quantum dot " sandwich " complex；
5), after the completion of reacting, using nanometer magnetic separator Magneto separate 3min, remove unnecessary quantum dot-labeled anti-human lung
Scorching mycoplasma nano-probe, cleans 2 times with the pbst buffer liquid in test kit, and complex is again dispersed in 100 μ l pbs and delays
Rush in liquid, using fluorescence microplate reader, its fluorescent value is detected；In described pbs buffer, each component content is as follows: 8g/l
Nacl, 0.2g/l kcl, 0.24g/l kh2po4, 1.44g/l na2hpo4；The ph=7.4 of described pbs buffer；
6) the negative quality-control product sample of four parts providing in above-mentioned same method detection kit and a positive quality control are provided
Product sample, reads fluorescent value respectively；The meansigma methodss of fluorescence reading of four parts of negative quality-control product samples are with 3 times of standard deviation sums
Cut-off value；If step 5) if in the detection fluorescent value of sample to be checked be judged as people's lung in sample to be checked more than cut-off value
Scorching mycoplasma antigen is the positive, otherwise is then judged as that in sample to be checked, people's mycoplasma pneumoniae antigen is feminine gender；If positive quality control product
The fluorescent value of sample is less than cut-off value, then show that test kit lost efficacy.
Preferably, step 2 proposed by the invention) in, to step 1) in centrifuge tube in add and be based on Magnetic Isolation
With anti-human mycoplasma pneumoniae immune nanometer magnetic bead 100 μ in the test kit of quantum dot-labeled detection people's mycoplasma pneumoniae antigen
L, is reacted on rotary mixer with 10rpm under room temperature and takes off after 30min, centrifuge tube insertion magnetic frame is separated 3min, with moving
Liquid device suctions out supernatant；
Described step 4) in, take 100 μ l steps 3) immune nanometer magnetic bead-mycoplasma antigen complex of obtaining in another from
In heart pipe, add in the test kit based on Magnetic Isolation and quantum dot-labeled detection people's mycoplasma pneumoniae antigen for the 100 μ l
Quantum dot-labeled anti-human mycoplasma pneumoniae nano-probe, reacts 30min with 15rpm on rotary mixer under room temperature, passes through
Antibody on quantum dot is combined with the immunity of the mycoplasma antigen on immune nanometer magnetic bead, and quantum dot is tagged to mycoplasma table
Face, forms magnetic bead-mycoplasma antigen-quantum dot " sandwich " complex.
Preferably, sample to be checked provided by the present invention includes but is not limited to throat swab.
Carboxyl water-soluble nano quantum dot needed for the present invention, 180nm carboxyl magnetic bead, can arrive the research list of relevant speciality
Position, company are bought or are customized；Required instrument, equipment, medicine are commercially available.
The present invention has the advantage that compared to existing technology
1 present invention utilizes immune nanometer magnetic bead to sample can enriching, detached speed is fast, efficiency high the advantages of,
In combination with characteristics such as quantum dot light chemical stability height, fluorescence intensity height, so that detection system possesses, multiple signal is collaborative to be put
Big effect, thus having the detection sensitivity of superelevation, (it is 0.2ng/ to the detection bottom line of people's mycoplasma pneumoniae p1 memebrane protein
Ml, less than the detection method of current any report, such as 2 μ g/ml of elisa method), with its detection knot to clinical sample
Fruit no difference of science of statistics compared with detection " the goldstandard "-culture method to this pathogen at present.
2nd, the antibody used by the present invention is the Anti-TNF-α of the identification extracellular conserved region of people's mycoplasma pneumoniae specificity p1 antigen
Body, its specificity is high, and for its more most widely used monoclonal antibody, preparation cost is cheap simultaneously, therefore, the present invention
Testing cost is relatively low.
3rd, detection method is simple, and detection is quick, and it is easy to judge, testing cost is cheap, overcomes prior art inspection
Survey positive rate is low, high cost, complex operation are loaded down with trivial details, time-consuming, cannot be carried out the deficiency of clinical practice.
4th, it is that (appearance of antibody needs to infect several for people's mycoplasma pneumoniae antigen rather than antibody due to detection kit detection
Zhou Yihou), so early diagnosiss and preventing and treating can be carried out, clinical diagnosises coincidence rate is high.The method is in the clinic of people's mycoplasma pneumoniae
The aspects such as diagnosis, nosetiology discriminating, Epidemiological study have very high practical value.
5th, the clinical sample used by detection method is respiratory secretions such as sputum etc., and non-blood, can exempt
The misery that infant patient takes a blood sample and the psychological burden of the head of a family, therefore be relatively easy to promote.
Specific embodiment
The present invention is according to the double antibodies sandwich principle in immunology, using immune nanometer magnetic bead to sample can enriching,
Detached speed is fast, efficiency high the advantages of, the characteristic such as incorporating quantum point photochemical stability height, fluorescence intensity height, the one of foundation
Set possess multiple signal work in coordination with amplification, have hypersensitivity and high degree of specificity quick detection people's mycoplasma pneumoniae new side
Method, has wide market application foreground.
The present invention is further described in detail by following examples.
The preparation of various reagents and the explanation of material requested
1.pbs buffer: weigh 1.44g disodium hydrogen phosphate, 0.24g potassium dihydrogen phosphate, 8g sodium chloride, 0.2g potassium chloride,
It is dissolved in the deionized water of 900ml, adjust deionized water after ph to 7.4 to be settled to 1000ml with 1mol/l naoh.
2. rabbit anti-human mycoplasma pneumoniae p1 protein polyclone antibody igg: for present invention self-control, with the dilution of pbs buffer, shake
Even, make polyclonal antibody weight percent concentration in solution be 1mg/ml.
3. Mus anti-human mycoplasma pneumoniae p1 protein polyclone antibody igg: for present invention self-control, with the dilution of pbs buffer, shake
Even, make polyclonal antibody weight percent concentration in solution be 1mg/ml.
4. quantum dot: in the present invention, quantum dot used is the water solublity cdse/zns quantum that carboxylated amphipathic polymer is modified
Point, its launch wavelength is 565nm, buys from Wuhan Jia Yuan technology of quantum dots development corporation, Ltd., and name of product is that carboxyl is water-soluble
Property quantum dot -565.
5. magnetic bead: in the present invention, magnetic bead used is with superparamagnetism fe3o4It is polystyrene, surface official for kernel, shell material
Can roll into a ball for carboxyl, particle diameter be respectively 50nm, 180nm, 350nm, 1150nm, 3 μm of carboxyl magnetic bead, can be from Shaanxi North America gene
Limited company, Aorun Weina New Material Science and Technology Co., Ltd., Shanghai buy.
6. people's mycoplasma pneumoniae: purchased from American type culture collection (atcc), numbering is atcc15531.
7. the microbiological specimens used in the present invention are purchased from American type culture collection (atcc).
Embodiment 1 rabbit and the preparation of Mus anti-human mycoplasma pneumoniae p1 protein polyclone antibody igg
(1) preparation of restructuring p1-his fusion protein, purification
1. the clone of related gene
To people mycoplasma pneumoniae memebrane protein p1, (the accession number in its ncbi Protein Data Bank is
Aak92040) carry out bioinformatic analysis, obtain epitope peptide fragment the abundantest in its extracellular conserved domain, find
Its corresponding dna coded sequence, further according to e. coli codon Preference, carries out codon optimization to it, simultaneously its 5 '
Introduce restriction enzyme site ndei, 3 ' ends introduce chemosynthesis complete genome sequence (complete sequence after termination signal taa and restriction enzyme site xhoi
Synthesis transfers to Jin Sirui bio tech ltd to complete, and during delivery, the genetic fragment of synthetic is connected on carrier puc57),
It is designated as p1.Its gene complete sequence is as shown in sequence table.Specifically, the protein sequence of p1 gene code props up for natural human pneumonia
The 1310-1523aa of substance memebrane protein p1 (accession number:aak92040).By the dna containing this section of synthetic
The carrier puc57 of fragment ndei and xhoi carries out reclaiming purpose fragment according to a conventional method after double digestion, standby.Adopt simultaneously
Ndei and xhoi to carrier pet-28a (+) carry out double digestion, and according to a conventional method the p1 gene obtaining after double digestion is connected into
Pet-28a (+) in carrier, and convert escherichia coli top10, build pet-p1 expression vector.Confirm through enzyme action and sequencing
Expression vector establishment is errorless.This vector expression restructuring p1-his fusion protein.
2. the expression and purification of restructuring p1-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.What picking pet-p1 converted has the single bacterium colony of exogenous protein expression ability and inoculates in 100ml lb culture medium,
In 37 DEG C of overnight incubation.After taking out bacterium solution, it is inoculated in the lb culture medium that 100ml contains 50 μ g/ml kanamycin by 1:100,
Cultivate to od in 37 DEG C600When=0.6, add 1mol/l iptg to final concentration of 1mmol/l, shake bacterium culture, induction in 37 DEG C
Expressing fusion protein.Induction 4h is centrifuged 10min collects thalline under 8000r/min.By this thalline 20ml phosphate-buffered
Liquid (8g/l nacl, 0.2g/l kcl, 0.24g/l kh2po4, 1.44g/l na2hpo4Ph7.4) washing 3 times and with 10ml
Sample buffer (20mm na3po4,0.5m nacl；30mm imidazoles, ph7.4) resuspended after carry out ultrasonication, operating condition is:
50hz, 200w, ultrasonic 3s, interval 5s, work 100 times.After the completion of ultrasonic, 12000g centrifugation 15min respectively collect precipitation and on
Carry out electrophoresis detection after clear.Find that restructuring p1-his fusion protein is present in thalline in solubility expression mode.
The purification step of restructuring p1-his fusion protein is as follows:
His trap affinity is used with 0.45 μm of filter membrane after the ultrasonication supernatant of above-mentioned acquisition is filtered
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 sample-loading buffer is used to balance, 1ml/min flow velocity.
3) by fusion protein loading, 1ml/min flow velocity.
4) use 10ml sample-loading buffer, post is washed with 1ml/min flow velocity.
5) use 10ml elution buffer (20mm na3po4, 0.5m nacl, 300mm imidazoles, ph7.4), with 1ml/min stream
Fast eluting, is in charge of collection, often pipe 1ml, and 12%sds-page detects, merges the sample containing destination protein in elution fraction.Warp
After bradford test kit carries out determination of protein concentration, adjustment concentration is 0.2mg/ml.
(2) preparation of rabbit and Mus anti-human mycoplasma pneumoniae p1 protein polyclone antibody igg
1. the preparation of rabbit anti-human mycoplasma pneumoniae p1 protein polyclone antibody igg
Mixed with 1ml Freund's complete adjuvant according to 200 μ g (1ml) with the restructuring p1-his fusion protein of step (one) purification
Immune Male New Zealand White Rabbit (being provided by Disease Prevention Control Center, Hubei Prov) after emulsifying, in dorsal sc multi-point injection,
After the 7d of interval again immunity once, with the restructuring p1-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
Elisa method measures antibody titer and is more than 1 × 105).If satisfied, Culling heart blood, separate serum, with protein g affinity chromatograph
Post (ge healthcare Products), in strict accordance with operating instruction purified polyclonal antibodies igg, with triumphant base braford egg
White reagent box for detecting content measures antibody concentration and with phosphate buffer (8g/l nacl, 0.2g/l kcl, 0.24g/l
kh2po4, 1.44g/l na2hpo4Ph=7.4) it is adjusted to 1mg/ml, -20 DEG C of preservations are standby, the anti-human pneumonia of rabbit is so far obtained and props up
Substance p1 protein polyclone antibody igg.Westen blot test shows, this polyclonal antibody igg can specific recognition people's pneumonia
Mycoplasma total length p1 albumen.
2. the preparation of Mus anti-human mycoplasma pneumoniae p1 protein polyclone antibody igg
Restructuring p1-his fusion protein with step (one) purification is (pre- by Hubei Province's disease as complete antigen immune guinea pig
Anti- control centre provides), injections of antigens 200 μ g/ under omoplate.Fundamental immunity is entered with Freund's complete adjuvant for isopyknic antigen
Row emulsifying, carried out a booster immunization every 2 weeks, and booster immunization is carried out with equal-volume incomplete Freund's adjuvant with equal-volume antigen
Emulsifying, immunity 4 times altogether.Haemanalysises antibody titer is taken after final immunization 10d.If dissatisfied, may be repeated one arrive twice plus
Immunity by force, (measures antibody titer with elisa method and is more than 1 × 10 to antibody titer is satisfied5).If satisfied, put to death Cavia porcelluss and take blood
Clearly, with protein g affinity column (ge healthcare Products), in strict accordance with operating instruction purified polyclonal
Antibody igg, measures antibody concentration and with phosphate buffer (8g/l with triumphant base braford protein content detection kit
Nacl, 0.2g/l kcl, 0.24g/l kh2po4, 1.44g/l na2hpo4Ph=7.4) it is adjusted to 1mg/ml, standby, so far make
Obtain Mus anti-human mycoplasma pneumoniae p1 protein polyclone antibody igg.Westen blot test shows, this polyclonal antibody igg can be special
Opposite sex identification people's mycoplasma pneumoniae total length p1 albumen.
The preparation of the anti-human mycoplasma pneumoniae immune nanometer magnetic bead of embodiment 2
1. the optimization of anti-human mycoplasma pneumoniae polyclonal antibody coupled bead reaction condition:
To be coupled the magnetic bead of anti-human mycoplasma pneumoniae polyclonal antibody as solid phase carrier, quantum dot-labeled anti-human lung
Scorching mycoplasma p1 protein polyclone antibody, as detection antibody, detects people's mycoplasma pneumoniae antigen by double-antibody method principle,
Observe the coupling situation of magnetic bead and multi-resistance.The activator concentration of particle diameter to magnetic bead, and edc/nhs respectively, coupled antibody concentration,
The coupling conditions such as coupling time, sealer species have carried out a series of optimized choice.
The selection of 1.1 magnetic bead particle diameters
Selection particle diameter be 50nm, 180nm, 350nm, 1150nm, 3 μm of carboxyl magnetic bead, all add edc containing 4mg/ml and
After the pbs buffer of 4mg/ml nhs carries out priming reaction, respectively with the anti-human mycoplasma pneumoniae p1 egg of the rabbit described by embodiment 1
White polyclonal antibody igg carries out coupling reaction.Respectively the immune nanometer magnetic bead preparing is detected 104People's pneumonia of cfu/ml is propped up
Substance (atcc numbering 15531), observed result under fluorescence microscope, select fluorescence intensity big, background fluorescence interference is few, Yi Ji
Under the action of a magnetic field, the very fast person of separating rate is the most suitable magnetic bead particle diameter.Result shows that the magnetic bead of particle diameter 180nm best suits the present invention's
Require, the most suitable magnetic microsphere particle diameter of determination is 180nm.
The selection of 1.2edc/nhs activator concentration
Edc and nhs concentration in reaction system is each set to after l～10mg/ml carry out Concentraton gradient combination, activates respectively
The carboxyl magnetic bead of particle diameter 180nm.The immune nanometer magnetic bead preparing is detected 104(atcc compiles people's mycoplasma pneumoniae of cfu/ml
Numbers 15531), select the most suitable activation concentration that fluorescence powerhouse is edc and nhs solution.Result shows that when edc concentration be 5mg/
Ml, nhs concentration is that during 4mg/ml, coupling effect is best.
The selection of 1.3 coupled antibody concentration
By 20 μ g, 40 μ g, 60 μ g, 80 μ g, 100 μ g, 120 μ g, 140 μ g rabbit anti-human mycoplasma pneumoniae p1 protein polyclone
Antibody igg is coupled by the magnetic bead that the particle diameter that above-mentioned best practice activates is 180nm with 1mg respectively.By the immunity preparing
Nanometer magnetic bead detection 104People's mycoplasma pneumoniae (atcc numbering 15531) of cfu/ml, it was found that when the injected volume of antibody is little
When 100 μ g/mg, fluorescence intensity increases with the concentration increase of antibody, and the mass concentration working as antibody is more than 100 μ g/mg
When, fluorescence intensity is basically unchanged or even slightly reduces, and therefore the present embodiment selects rabbit anti-human mycoplasma pneumoniae p1 protein polyclone
The coupling amount of antibody igg is 100 μ g/mg.
The selection of 1.4 coupling times
After determining particle diameter, edc/nhs activator concentration and the antibody coupling amount of magnetic bead, by the coupling reaction of antibody and magnetic bead
Time is set to 0.5h, 1h, 2h, 3h, 4h, 5h, and the immune nanometer magnetic bead preparing is detected 104People's pneumonia of cfu/ml is propped up
Substance (atcc numbering 15531).It was found that between when coupled > 3h when, fluorescence intensity tends towards stability, and hereafter extends coupling again
Time, fluorescence no longer strengthens.Accordingly, it is determined that the most suitable idol of rabbit anti-human mycoplasma pneumoniae p1 protein polyclone antibody igg and magnetic bead
The connection response time is 3h.Coupling time is far fewer than the 24h of traditional elisa method.
The selection of 1.5 sealers
Optimal conditionss according to above-mentioned determination select particle diameter, edc/nhs activator concentration, antibody coupling amount and the idol of magnetic bead
Carry out coupling reaction after the connection time.After coupling terminates, select bsa, ethanolamine, tris and d- glucosamine hydrochloride is as exempting from
Epidemic disease nanometer magnetic bead sealer, finished product immune nanometer magnetic bead.The immune nanometer magnetic bead preparing is detected 104The people of cfu/ml
Mycoplasma pneumoniae (atcc numbering 15531).It was found that it is glimmering as the detection of the immune nanometer magnetic bead of sealer using ethanolamine
Light value highest.It is presumed that because the molecule of ethanolamine is less, can preferably consume due to sterically hindered not with antibodies
Surface carboxyl groups, make closing more complete, and effectively reduce space steric effect to the structure influence connecting antibody.
2. coupling process:
Take 5mg magnetic bead (with superparamagnetism fe3o4It is the carboxyl magnetic bead of 180nm for kernel, particle diameter) commonly it is centrifuged in 1.5ml
Guan Zhong, is washed three times with 1ml mes buffer (2g/l mes, ph6.0), is placed in nanometer magnetic separator and carries out Magneto separate
(0.4t) remove supernatant after, sequentially add the edc solution that the concentration with above-mentioned mes buffer is 10mg/ml and with above-mentioned
The concentration of mes buffer is each 0.5ml of sulfo-nhs solution of 8mg/ml, is activated in rotary mixer with 15rpm
1hr, removes supernatant after Magneto separate, resuspended with the above-mentioned mes buffer of 1ml；Take 5 centrifuge tubes, in each centrifuge tube, add 200
The magnetic bead of the above-mentioned activation of μ l, suctions out supernatant after Magneto separate, adds use pbs buffer (8g/l nacl, 0.2g/l in each pipe
Kcl, 0.24g/l kh2po4, 1.44g/l na2hpo4, ph7.4) and the concentration that dilutes is prepared by the embodiment 1 of 100 μ g/ml
The each 1ml of rabbit anti-human mycoplasma pneumoniae p1 protein polyclone antibody igg solution, is reacted in rotary mixer with 15rpm under room temperature
3h, after Magneto separate removes supernatant, each above-mentioned pbs buffer adding 1ml to contain 1mg/ml ethanolamine, with 15rpm in rotation under room temperature
Turn in mixed instrument reaction 2h to close on magnetic bead the not carboxyl with antibody response.Remove each pipe supernatant after Magneto separate, respectively washed with 1ml
Wash buffer (8g/l nacl, 0.2g/l kcl, 0.24g/l kh2po4, 1.44g/l na2hpo4, 0.5ml/l tween-20,
Ph7.4) wash three times, finally respectively preserve buffer (8g/l nacl, 0.2g/l kcl, 0.24g/l kh with 1ml2po4,
1.44g/l na2hpo4, 0.3g/l nan3, 5g/l bsa, ph7.4) and resuspended magnetic bead, it is placed in 4 DEG C and save backup.
The preparation of the quantum dot-labeled anti-human mycoplasma pneumoniae nano-probe of embodiment 3
1. the quantum dot-labeled Mus of nanometer carboxylic anti-human mycoplasma pneumoniae p1 protein polyclone antibody igg reaction condition is excellent
Change:
1.1st, the determination of carboxyl quantum dot-labeled antibody probe optimum mark ph
During labelling is reacted, phosphate buffer ph is set to 5,6,7,8,9, and marked product is entered using full spectrogrph
Row fluorescent strength determining, observe different ph values to coupling reaction impact it is determined that quantum dot-labeled multi-resistance reaction optimal ph
For 7.0-8.0.This experimental selection ph7.4.
1.2nd, the determination of carboxyl quantum dot-labeled antibody probe optimum mark amount
The ratio of quantum dot molar concentration and multi-resistance concentration is respectively set to 1:1,1:2,1:3 and 1:4, 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 Mus anti-human mycoplasma pneumoniae p1 protein polyclone antibody igg reacts is quantum dot
It is 1:3 with antibody molar ratio.This optimal concentration ratio of this experimental selection is determining labelled amount.
1.3rd, the determination of the optimal sealer species of the quantum dot-labeled antibody probe of carboxyl
With ethanolamine, tris, peg2000-nh2Or bsa 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 labeled complex and immunocompetence.
2. labeling process:
2nmol carboxyl water-soluble quantum dot, 300nmol n- hydroxy succinyl is sequentially added in microcentrifugal tube
Imines (sulfo-nhs) and 300nmol carbodiimide (edc), with phosphate buffer (2.9g/l disodium hydrogen phosphate, 0.295g/
L sodium dihydrogen phosphate, 4g/l sodium chloride, ph 7.4) constant volume is 2ml, ceaselessly mixed solution, after 37 DEG C of reaction 30min, dialysis is gone
Except excessive sulfo-nhs and edc as activator.In the quantum dot of activation, add prepared by the embodiment 1 of 6nmol
Mus anti-human mycoplasma pneumoniae p1 protein polyclone antibody igg, lucifuge react 2h, add single-ended amino polyethylene glycol
(peg2000-nh2) to final concentration of 1%, close unreacted activated carboxyl site, continue lucifuge reaction 1h.With 0.2 μm
Pes filter is filtered to remove antibody aggregation thing, then filtrate be transferred to, in 50000mw ultra-filtration centrifuge tube, exist with 8000g centrifugal force
It is centrifuged 15min at 4 DEG C, remove the by-product in the antibody that coupling reaction does not occur and reaction.Collect super filter tube filter membrane upper strata amount
Sub- point-antibody coupling matter solution, be dissolved in 2ml phosphate cleaning mixture (2.9g/l disodium hydrogen phosphate, 0.295g/l sodium dihydrogen phosphate,
4g/l sodium chloride, 5ml/l tween 20,0.3g/l sodium azide, ph 7.4) in, then by this solution transfer to 50000mw ultrafiltration from
In heart pipe, 15min is centrifuged at 4 DEG C with 8000g centrifugal force, collects super filter tube filter membrane upper strata quantum dot-antibody coupling matter solution,
Be dissolved in 1ml phosphate preserve liquid (2.9g/l disodium hydrogen phosphate, 0.295g/l sodium dihydrogen phosphate, 2g/l sodium chloride, 10g/l bsa,
0.3g/l sodium azide, ph 7.4) in, it is placed in 4 DEG C and save backup.
Embodiment 4 immune nanometer magnetic bead carries out the optimization of immunocapture condition to people's mycoplasma pneumoniae antigen
Carried using the immune nanometer magnetic bead being coupled rabbit anti-human mycoplasma pneumoniae p1 protein polyclone antibody igg as solid phase
Body, quantum dot-labeled Mus anti-human mycoplasma pneumoniae p1 protein polyclone antibody is as detection antibody, former by double-antibody method
Reason, sets up the detection system of people's mycoplasma pneumoniae antigen.Consumption to immune nanometer magnetic bead in detection system respectively, capture time
Carry out a series of optimized choice etc. condition.
1. the selection of immune nanometer magnetic bead addition
By 20 μ l, 40 μ l, 60 μ l, 80 μ l, 100 μ l, 120 μ l, 140 μ l by the made immune nano got ready of embodiment 2
Magnetic bead is added separately to 0.5ml and contains 104In the pbs suspension of people's mycoplasma pneumoniae (atcc numbering 15531) of cfu/ml, carry out
Immunocapture, the more quantum dot-labeled probe described by embodiment 3 detected, records fluorescent value.It was found that with exempting from
The increase of epidemic disease nanometer magnetic bead addition, fluorescent value is gradually increased, when immune nanometer magnetic bead addition reaches 100 μ l, fluorescent value
Reach maximum.It is further continued for increasing the amount of immune nanometer magnetic bead, fluorescent value reduces on the contrary.This is likely due to immune nanometer magnetic bead mistake
Many, during Magneto separate, thalline is caused to damage, thus leading to capture rate to decline.Therefore this experimental selection 100 μ l is as immune nano magnetic
The optimal addn of pearl.
2. the selection of immunocapture time
After determining the addition of magnetic bead, take the made immune nanometer magnetic bead got ready of four parts of embodiments 2, at room temperature with 10r/
Min, to 104People's mycoplasma pneumoniae (atcc numbering 15531) of cfu/ml carries out exempting from of 15min, 30min, 45min and 60min
Epidemic disease captures, the more quantum dot-labeled probe described by embodiment 3 is detected, records fluorescent value.It was found that fluorescent value exists
Maximum is shown during immunocapture 30min, prolongation over time, numerical value has declined.Therefore the 30min conduct of this experimental selection
The Best Times of immunocapture.
The preparation of embodiment 5 pbst buffer
Take 8g nacl, 0.2g kcl, 0.24kh2po4, 1.44g na2hpo4, 0.3g nan3, 0.5ml tween-20 is molten
Solution, in 800ml distilled water, adjusts ph to 7.4 with 5m naoh, then is settled to 1000ml.
The preparation of embodiment 6 quality-control product
1. positive quality control product: will be attached on swab with people's mycoplasma pneumoniae (0.5 μ g) drying of 1% formalin-inactivated, that is,
For positive quality control product.
2. negative quality-control product: negative quality-control product determines the throat swab sample for the negative crowd of people's mycoplasma pneumoniae through clinic
Product.
The preparation of embodiment 7 test kit
Quantum dot-labeled described by anti-human mycoplasma pneumoniae immune nanometer magnetic bead described by embodiment 2, embodiment 3
Anti-human mycoplasma pneumoniae nano-probe, the pbst buffer described by embodiment 5, the quality-control product described by embodiment 6 common
The test kit based on Magnetic Isolation and quantum dot-labeled detection people's mycoplasma pneumoniae antigen for the composition.
The using method of embodiment 8 test kit
Routinely clinical means obtain people's throat swab, with 0.5ml test kit pbst buffer (8g/l nacl,
0.2g/l kcl, 0.24g/l kh2po4, 1.44g/l na2hpo4, 0.5ml/l tween-20, ph7.4) dissolve on throat swab
Clinical sample after, lysate is proceeded in the common centrifuge tube of 1.5ml, in this centrifuge tube add test kit in anti-human pneumonia
Mycoplasma immune nanometer magnetic bead 100 μ l, is reacted on rotary mixer with 10rpm under room temperature and takes off after 30min, centrifuge tube is inserted
Enter magnetic frame and separate 3min, suction out supernatant with pipettor.The pbst buffer 1ml adding in test kit washs twice, Magneto separate
Suction out cleaning mixture afterwards, finally use the resuspended magnetic bead of 1ml pbs buffer.Take the above-mentioned immune nanometer magnetic bead-mycoplasma antigen of 100 μ l
Complex, in another centrifuge tube, adds the quantum dot-labeled anti-human mycoplasma pneumoniae nano-probe in 100 μ l test kits,
Under room temperature, 30min is reacted on rotary mixer with 15rpm, by former on the antibody on quantum dot and immune nanometer magnetic bead
The immunity of body antigen combines, and quantum dot is tagged to mycoplasma surface, forms magnetic bead-mycoplasma antigen-quantum dot " sandwich "
Complex.After the completion of reaction, Magneto separate 3min, remove unnecessary quantum dot-labeled probe, and with pbst buffer solution for cleaning 2 times,
Complex is dispersed in 100 μ l pbs buffer again, using fluorescence microplate reader (ex=405nm, em=565nm) to its fluorescence
Value is detected.
By the negative quality-control product of four parts providing in above-mentioned same method detection kit and a positive quality control product sample,
Read fluorescent value respectively；The meansigma methodss of fluorescence reading of four parts of negative quality-control product samples are cut-off with 3 times of standard deviation sums
Value；If if the detection fluorescent value of above-mentioned clinic people's throat swab sample is judged as in this part of clinical throat swab more than cut-off value
People's mycoplasma pneumoniae antigen is the positive, otherwise is then judged as that in this part of clinical people's throat swab sample, people's mycoplasma pneumoniae antigen is the moon
Property；If the fluorescent value of positive quality control product sample is less than cut-off value, show that test kit lost efficacy.
The detection sensitivity of embodiment 9 test kit and specific test
Recombined human mycoplasma pneumoniae p1 albumen (p1-his) of the artificial expression described in embodiment 1 is entered with pbs buffer
After row is serially diluted, detected with this test kit, result shows that its detection lowest limit is 0.2ng/ml.Meanwhile, 10 are used7cfu/ml
Ureaplasma urealyticum (atcc numbering 27618), mycoplasma hominis (atcc numbering 23114), Chlamydia pneumoniae (ar-39 strain, atcc
Numbering 53592), streptococcus pneumoniae (atcc numbering 49619) etc. detected, the pbs containing these microorganisms is dilute for test kit detection
It is all negative for releasing liquid.
Embodiment 10 clinical trial example
Using detection of mycoplasma " goldstandard " isolated culture as reference, in epidemic season, take 220 clinical throat swab marks
Originally detected, culture method positive rate is 13.1% (29/221), this test kit is 15.4% (34/221), the symbol of 2 kinds of methods
Conjunction rate is 97.7% (216/221).Concrete outcome is as shown in table 1.
The testing result of table 1 clinical samples
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 (4)
1. a kind of side for preparation based on Magnetic Isolation and the test kit of quantum dot-labeled detection people's mycoplasma pneumoniae antigen
Method it is characterised in that: described preparation method comprises the following steps:
1) preparation of anti-human mycoplasma pneumoniae immune nanometer magnetic bead:
1.1) preparation of rabbit and Mus anti-human mycoplasma pneumoniae p1 protein polyclone antibody igg:
1.1.1) the preparation of restructuring p1-his fusion protein, purification:
1.1.1.1) bioinformatic analysis are carried out to people mycoplasma pneumoniae memebrane protein p1, obtain antigen table in its ectodomain
Position peptide fragment the abundantest；
1.1.1.2) find step 1.1.1.1) in the corresponding gene coded sequence of obtained peptide fragment, according to password in escherichia coli
The Preference of son, carries out codon optimization to obtained gene coded sequence；
1.1.1.3) in step 1.1.1.2) in obtained gene order 5 ' ends and 3 ' ends introduce restriction enzyme sites and chemistry closes
Become complete genome sequence, be labeled as simultaneouslyp1；
1.1.1.4) by step 1.1.1.3) in obtainedp1It is cloned into expression vector pet-28a by molecular biology method
Expression in escherichia coli restructuring p1-his fusion protein is proceeded to after (+)；Described restructuring p1-his fusion protein is with solubility expression
Mode is present in thalline；
1.1.1.5) use ni-sepharose purification step 1.1.1.4) obtained by recombiant protein, after sds-page detects its purity, with
Bradford method measures protein concentration, and adjustment protein concentration is standby after 0.2 mg/ml；
1.1.2) rabbit and Mus anti-human mycoplasma pneumoniae p1 protein polyclone antibody igg preparation:
1.1.2.1) with step 1.1.1.5) in obtained restructuring p1-his fusion protein as complete antigen, immunity is newly western respectively
Blue White Rabbit and Cavia porcelluss；Prepare rabbit anti-human mycoplasma pneumoniae p1 protein antiserum respectively and Mus anti-human mycoplasma pneumoniae p1 albumen resists
Serum；The anti-human mycoplasma pneumoniae p1 protein antiserum of described rabbit and Mus anti-human mycoplasma pneumoniae p1 protein antiserum indirect
Elisa potency is all higher than 1 × 105；
1.1.2.2 purified rabbit anti-human's mycoplasma pneumoniae p1 protein antiserum and Mus resist respectively) to adopt protein g affinity column
Polyclonal antibody igg in people's mycoplasma pneumoniae p1 protein antiserum；
1.1.2.3) with triumphant base bradford protein content detection kit determination step 1.1.2.2) obtained by more than two kinds gram
The concentration of grand antibody igg, its protein concentration is all adjusted to standby after 1 mg/ml；
1.2) being coated of immune nanometer magnetic bead:
1.2.1) take 5 mg magnetic beads, with 1 ml mes buffer solution three times, be placed in after carrying out Magneto separate in nanometer magnetic separator
Remove supernatant；Described magnetic bead is with superparamagnetism fe3o4The carboxyl magnetic bead being 180 nm for kernel, particle diameter；Described mes buffer
It is 2- (n- morpholino) ethyl sulfonic acid that mass concentration is 2 g/l；The ph=6.0 of described mes buffer；Described nanometer magnetic separator
Magnetic intensity be 0.4t；
1.2.2) sequentially add and use step 1.2.1) in mes buffer concentration be 8-12 mg/ml edc solution with
And use step 1.2.1) in mes buffer concentration be 6-10 mg/ml each 0.5 ml of sulfo-nhs solution, with
10-40 rpm activates 1 hr in rotary mixer, is placed in and removes supernatant after carrying out Magneto separate in nanometer magnetic separator, uses 1 ml
Step 1.2.1) in mes buffer resuspended, magnetic bead after being activated；
1.2.3) take 5 centrifuge tubes, each centrifuge tube add 200 μ l steps 1.2.2) obtained by activation after magnetic bead,
It is placed in and removes supernatant after carrying out Magneto separate in nanometer magnetic separator, add the concentration with the dilution of pbs buffer in each centrifuge tube
For 50-200 μ g/ml by step 1.1) prepared by rabbit anti-human mycoplasma pneumoniae p1 protein polyclone antibody igg solution each 1
Ml, reacts 2-6 h with 15 rpm under room temperature in rotary mixer, is placed in and removes after carrying out Magneto separate in nanometer magnetic separator
After clear, each above-mentioned pbs buffer adding 1 ml to contain 1 mg/ml ethanolamine, anti-in rotary mixer with 15 rpm under room temperature
Answer 2 h to close on magnetic bead the not carboxyl with antibody response；In described pbs buffer, each component content is as follows: 8 g/l nacl,
0.2 g/l kcl, 0.24 g/l kh2po4, 1.44 g/l na2hpo4, the ph=7.4 of described pbs buffer；
1.2.4) after the completion of capping, this 5 centrifuge tubes are placed in nanometer magnetic separator and carry out removing supernatant after Magneto separate,
Respectively wash three times with 1 ml lavation buffer solution；In described lavation buffer solution, each component content is as follows: 8 g/l nacl, 0.2 g/l
Kcl, 0.24 g/l kh2po4, 1.44 g/l na2hpo4, 0.5 ml/l tween-20, the ph=7.4 of described lavation buffer solution；
1.2.5 in each centrifuge tube) it is separately added into 1 ml preserves the resuspended magnetic bead of buffer, be placed in 4 DEG C and save backup；Described
Preserve each component content in buffer as follows: 8 g/l nacl, 0.2 g/l kcl, 0.24 g/l kh2po4, 1.44 g/l
na2hpo4, 0.3 g/l nan3, 5 g/l bovine serum albumin, the ph=7.4 of described preservation buffer；
2) preparation of quantum dot-labeled anti-human mycoplasma pneumoniae nano-probe:
Its concrete preparation method includes:
2.1) 2 nmol carboxyl water-soluble quantum dots, 300 nmol n- hydroxy succinums are sequentially added in microcentrifugal tube
Acid imide sulfo-nhs and 300 nmol carbodiimide edc, with phosphate buffer constant volume for 2 ml, mixed solution, 37
After DEG C reaction 30 min, dialysis removes excessive sulfo-nhs and edc as activator, the quantum dot after being activated；Institute
State each component content in phosphate buffer as follows: 2.9 g/l disodium hydrogen phosphates, 0.295 g/l sodium dihydrogen phosphate, 4 g/l chlorine
Change sodium；The ph=7.4 of described phosphate buffer；
2.2) in step 2.1) obtained by the quantum dot of activation in, the step 1.1 that adds 4-12 nmol) in prepared Mus
Anti-human mycoplasma pneumoniae p1 protein polyclone antibody igg, lucifuge reacts 2 h, adds single-ended amino polyethylene glycol peg2000-
nh2To final concentration of 1%, close unreacted activated carboxyl site, continue lucifuge and react 1 h；
2.3) be filtered to remove step 2.2 with 0.2 μm of pes filter) in antibody aggregation thing, then filtrate be transferred to 50000
In mw ultra-filtration centrifuge tube, it is centrifuged 15 min at 4 DEG C with 8000 g centrifugal force, remove antibody and the reaction that coupling reaction does not occur
In by-product；
2.4) collection step 2.3) in ultra-filtration centrifuge tube filter membrane upper strata quantum dot-antibody coupling matter solution, be dissolved in 2 ml phosphate
In cleaning mixture, then this solution is transferred in 50000 new mw ultra-filtration centrifuge tubes, with 8000 g centrifugal force at 4 DEG C
It is centrifuged 15 min, collects ultra-filtration centrifuge tube filter membrane upper strata quantum dot-antibody coupling matter solution, be dissolved in 1 ml phosphate and preserve liquid
In, it is placed in 4 DEG C and save backup；In described phosphate cleaning mixture, each component content is as follows: 2.9 g/l disodium hydrogen phosphates, and 0.295
G/l sodium dihydrogen phosphate, 4 g/l sodium chloride, 5 ml/l tween 20s, 0.3 g/l sodium azide, the ph=of described phosphate cleaning mixture
7.4；It is as follows that described phosphate preserves each component content in liquid: 2.9 g/l disodium hydrogen phosphates, 0.295 g/l sodium dihydrogen phosphate, and 2
G/l sodium chloride, 10 g/l bovine serum albumin, 0.3 g/l sodium azide；Described phosphate preserves the ph=7.4 of liquid；
3) preparation of pbst buffer:
Its concrete compound method includes:
Take 8 g nacl, 0.2 g kcl, 0.24 kh2po4, 1.44 g na2hpo4, 0.3 g nan3, 0.5 ml tween-20
It is dissolved in 800 ml distilled water, adjust ph to 7.4 with 5 m naoh, then be settled to 1000 ml；
4) preparation of quality-control product:
4.1) positive quality control product: positive quality control product is attached on swab by the people's mycoplasma pneumoniae drying inactivateing and forms；
4.2) negative quality-control product: negative quality-control product determines the throat swab for the negative crowd of people's mycoplasma pneumoniae through clinic.
2. according to claim 1 for preparation based on Magnetic Isolation and quantum dot-labeled detection people's pneumonia mycoplasma
The method of former test kit it is characterised in that: described step 1.2.2) in, sequentially add and use step 1.2.1) in mes buffering
The concentration that liquid is prepared is the edc solution of 10 mg/ml and uses step 1.2.1) in the concentration of mes buffer be 8 mg/
Each 0.5 ml of sulfo-nhs solution of ml, activates 1 hr in rotary mixer with 15 rpm, is placed in nanometer magnetic separator
Remove supernatant after row Magneto separate, with 1 ml step 1.2.1) in mes buffer resuspended, magnetic bead after being activated；
Described step 1.2.3) in, add the concentration with the dilution of pbs buffer to be 100 μ g/ml by step in each centrifuge tube
1.1) each 1 ml of the anti-human mycoplasma pneumoniae p1 protein polyclone antibody igg solution of the rabbit prepared by, with 15 rpm in rotation under room temperature
Turn and in mixed instrument, react 3 h, be placed in after removing supernatant after carrying out Magneto separate in nanometer magnetic separator, each addition 1 ml contains 1 mg/
The above-mentioned pbs buffer of ml ethanolamine；
Described step 2.2) in, in step 2.1) obtained by the quantum dot of activation in, add 6 nmol step 1.1) in institute
The Mus anti-human mycoplasma pneumoniae p1 protein polyclone antibody igg of preparation, lucifuge reacts 2 h.
3. according to claim 2 for preparation based on Magnetic Isolation and quantum dot-labeled detection people's pneumonia mycoplasma
The method of former test kit it is characterised in that: described quantum dot is the water solublity cdse/zns that carboxylated amphipathic polymer is modified
Quantum dot.
4. according to claim 3 for preparation based on Magnetic Isolation and quantum dot-labeled detection people's pneumonia mycoplasma
The method of former test kit it is characterised in that: described magnetic bead is with superparamagnetism fe3o4For kernel, shell material be polystyrene,
The carboxyl magnetic bead that surface functional group is carboxyl, particle diameter is 180 nm.
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| CN105842451B (en) * | 2016-05-17 | 2017-10-27 | 郑州大学 | Method based on quantum dot fluorescence immune detection DNMT1 |
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