CN105277713B - Rapid detection method and kit for streptococcus pneumoniae based on magnetic separation and quantum dot labeling - Google Patents

Abstract

The invention discloses a rapid detection method and a kit for streptococcus pneumoniae based on magnetic separation and quantum dot labeling. The kit is composed of anti-streptococcus-pneumoniae immunonanomagnetic beads with a streptococcus pneumoniae enrichment function, anti-streptococcus-pneumoniae nanoprobes labeled with quantum dots, quality control products and a PBST buffer solution. The quality control products comprise positive quality control products and negative quality control products. The positive quality control products are prepared after inactivated streptococcus pneumoniae is dried and combined on swabs. The negative quality control products are throat swabs of people who are determined to be negative in streptococcus pneumoniae. The detection method and the kit which can detect streptococcus pneumoniae antigens simply, rapidly and with high sensitivity are provided, and preparation and usage methods for the kit are provided.

Description

Based on Magnetic Isolation and quantum dot-labeled people streptococcus pneumoniae method for quick and Test kit

Technical field

It is the present invention relates to technical field of medical detection, specially a kind of based on Magnetic Isolation and quantum dot-labeled detection people The method for quick of streptococcus pneumoniae antigen and detection kit, and the method for preparation and use of the detection kit.

Background technology

People streptococcus pneumoniae (Streptococcus pneumoniae, Sp) is the important pathogen of childrens respiratory tract infection Body.Main Jing droplet transmission and enter respiratory tract, so as to parasitize the nasopharynx part of human body, or the position for invading human body not easy-clear Cause a series of disease, such as lobar pneumonia, meningitiss, bronchitis, otitis media etc..It is that global all age group are occurred frequently The main pathogenic fungi of sick rate and case fatality rate.Wherein, in developing country, in infant, old people and immunodeficiency crowd particularly Seriously.Streptococcus pneumoniae in 1881 first by Pasteur (Louis Pasteur) and G.M.Sternberg respectively in France and Isolate from patient's sputum in the U.S..Which is Gram-positive, thalline like spearhead shape, into double or into the double of short catenation Coccus, has the pod membrane that chemical composition is polysaccharide outside toxic strain thalline.Its pod membrane has antigenicity, be streptococcus pneumoniae typing according to According to.Streptococcus pneumoniae is divided into by 91 serotypes according to the difference of capsular polysaccharide antigen.Streptococcus pneumoniae somatic antigen is mainly C Polysaccharide, which is present in pneumococcal cell wall, with species specificity, is that various bacterial strain is common.C polysaccharide can be by serum C reactive protein is precipitated.In the presence of calcium ion, C polysaccharide can be with referred to as C reactive protein (C reactive in normal human serum Protein, CRP) beta Globulin combine, occur precipitation.Detection currently for people's streptococcus pneumoniae antigen is also primarily directed to This antigen, and the non-streptococcus pneumoniae of the antigen is exclusive, such as relaxes streptococcus and also contains the antigen.Also have in S. pneumonia surface A kind of important antigen related to virulence, is pneumococcal surface protein A (PspA), and which is present in all blood of streptococcus pneumoniae In clear type, it is the specific antigen of streptococcus pneumoniae.But PspA molecular structures highly make a variation, with antigen diversity, which is rich Domain upstream containing proline is referred to as CDR domains, with multiformity.PspA point is 3 families 6 by the difference according to CDR domains Subclass：Clade1, Clade2, Clade3, Clade4, Clade5, Clade6.Wherein Clade1, Clade2 belong to Fam1 families； Clade3, Clade4, Clade5 belong to Fam2 families, and Clade6 belongs to Fam3 families.With Fam1 or Fam2 families PspA eggs White people streptococcus pneumoniae has accounted for more than the 99% of the people's streptococcus pneumoniae species being clinically separated.China yet there are no to be had The streptococcus pneumoniae of fam3 families PspA albumen is clinically separated report.Research shows to deposit between PspA albumen between same family's subclass In extensive antibody and antigen cross-reaction, and then without this cross reaction between different family's subclass.

Clinical patient (such as parainfluenza viruses, hemophilus influenza, influenza virus, is exhaled due to different respiratory pathogen Inhale road syncytial viruses, adenoviruss etc.) infection causes the disease symptomses can be with quite similar, and it is relatively difficult to which results in popular diagnosis, Make a definite diagnosis and tend to rely on laboratory diagnosiss.Fast and effectively diagnostic method should be disease their early stage can be obtained by it is bright True diagnosis, it is convenient to carry out targetedly to treat, prevent the development delay of the state of an illness.

Although streptococcus pneumoniae is propagated in the world, the infection of infant is especially universal, can be used for laboratory and examines The species of disconnected standardization commercially available reagent is few.At present, the detection of streptococcus pneumoniae mainly has following several method：

First, Routine Test Lab detection

1st, bacteria distribution

The goldstandard of laboratory diagnosiss streptococcus pneumoniae is to separate people's S. pneumoniae strain.Using nasopharyngeal secretionses conduct The specimen of pathogen isolation, can be with the method isolated pathogen of hemoculture.But the method has serious defect, because pneumonia chain Coccus is severe bacteria, and nutritional requirement height, length the time required to culture, positive rate are low, it is often more important that, if the front patient of sampling used Antibacterials, can cause the false positive of cultivation results.Treatment so in clinicing aspect to patient just has certain limitation.

2nd, Serologic detection

I.e. using euzymelinked immunosorbent assay (ELISA), the method for exempting from, micro-Immunofluorescence assay etc. is put, streptococcus pneumoniae resists in detection examinee's serum Body level, can point out the presence of streptococcus pneumoniae infection indirectly.However, serological test can only provide a kind of retrospective examining Disconnected, it needs to detect the Acute Stage and convalescent paired sera simultaneously, if anti-human pneumococci antibody in convalescent period , just there is diagnostic significance high 4 times or more than 4 times of potency ratio acute stage.In addition, the opportunity that antibody occurs is difficult to grasp, and because thalline Serotype species is excessive, causes the anti-capsular polysaccharide antibody type which induces excessive, and the detection of antagonist causes very big being stranded Difficulty, therefore the detection quality of existing serological method is subject to a definite limitation.

2nd, quick diagnosis

Directly scrutineer's Streptococcus pneumoniae protein antigen and thalline nucleic acid can reach the purpose of quick diagnosis, mainly have at present Colloidal gold immunity chromatography, immunofluorescence, immunoenzyme method and PCR methods etc..There is operation in immunofluorescence and immunoenzyme method Step is complicated, needs professional to operate, and detection time length (more than 2h), the shortcomings of relatively costly.PCR method is quick, sensitive, Specifically, be at present study streptococcus pneumoniae infection important means, but due to PCR to experimental facilitiess and operation require it is higher, And easily there is false positive, can't be used as conventional methods for clinical diagnosis in China.The detection target of colloidal gold immunity chromatography Antigen is C polysaccharide antigens, but colloidal gold method sensitivity is relatively low, higher to specimen material prescription, while also existing and which His streptococcus such as relax streptococcus and there is the defects such as cross reaction.Therefore, set up and possess highly sensitive people streptococcus pneumoniae spy Specific Antigen quick diagnosis method is very necessary.

The 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 streptococcus pneumoniae antigen and test kit, and the reagent The method of preparation and use of box.

The present invention is achieved through the following technical solutions：

It is a kind of based on Magnetic Isolation and quantum dot-labeled detection people's streptococcus pneumoniae antigen method, it is characterised in that： Described the method is comprised the following steps：

1) rabbit-anti people streptococcus pneumoniae Fam1 PspA, the preparation of Fam2 PspA protein polyclone antibodies；

2) the anti-human streptococcus pneumoniae Fam1 PspA of Mus, the preparation of Fam2 PspA protein polyclone antibodies；

3) by step 1) the rabbit-anti people streptococcus pneumoniae Fam1 PspA for preparing, Fam2 PspA protein polyclone antibodies difference With nanometer magnetic bead by covalent coupling, anti-human streptococcus pneumoniae Fam1 PspA protein immunizations nanometer magnetic beads and anti-human are prepared respectively Streptococcus pneumoniae Fam2 PspA protein immunization nanometer magnetic beads, by anti-human streptococcus pneumoniae Fam1 PspA protein immunization nanometer magnetic beads And anti-human streptococcus pneumoniae Fam2 PspA protein immunization nanometer magnetic bead mixed in equal amounts is obtained anti-human streptococcus pneumoniae immune nano Magnetic bead；

4) by step 2) the anti-human streptococcus pneumoniae Fam1 PspA of Mus for preparing, Fam2 PspA protein polyclone antibodies difference With nano-quantum point by covalent coupling, quantum dot-labeled people streptococcus pneumoniae Fam1 PspA protein nanos are prepared respectively and is visited Pin and quantum dot-labeled people's streptococcus pneumoniae Fam2 PspA protein nano probes, by quantum dot-labeled people's pneumonia streptococcus Bacterium Fam1 PspA protein nanos probes and quantum dot-labeled people's streptococcus pneumoniae Fam2 PspA protein nano probe equivalent Mixing is obtained quantum dot-labeled anti-human streptococcus pneumoniae nano-probe；

5) human respiratory secretions sample (including but not limited to throat swab) is taken, after PBST buffer solutions, step is added The rapid anti-human streptococcus pneumoniae immune nanometer magnetic bead for 3) preparing, is sufficiently mixed, and carries out Magneto separate after reaction 15-45min, with After PBST buffer solutions 2 times, in the precipitate obtained to Magneto separate, add step 4) prepare it is quantum dot-labeled anti- People's streptococcus pneumoniae nano-probe, reaction 25-45min after carry out Magneto separate, with PBST buffer solutions 2 times after, using fluorescence Microplate reader reads fluorescent value；In the PBST buffer, each component content is respectively 8g/L NaCl, 0.2g/L KCl, 0.24g/L KH₂PO₄, 1.44g/L Na₂HPO₄, 0.5ml/L Tween-20, the pH=7.4 of the PBST buffer；

6) according to step 1)-step 5) method detect that four parts of Jing are clinical respectively and determine for the negative people of people streptococcus pneumoniae The respiratory secretions sample of group, reads fluorescent value；The respiratory secretions sample of the negative crowd of the people streptococcus pneumoniae Abbreviation people's streptococcus pneumoniae negative control sample；The meansigma methodss of the fluorescent value of four parts of people streptococcus pneumoniae negative control sample CUT-OFF values are with 3 times of standard deviation sums；If step 5) in human respiratory secretions sample detection fluorescent value be more than CUT- OFF values, then in being judged as human respiratory secretions sample, people's streptococcus pneumoniae antigen is the positive；If step 5) in human respiratory point The detection fluorescent value of secretion sample is less than CUT-OFF values, then in being judged as human respiratory secretions sample, people streptococcus pneumoniae resists Originally it was negative.

It is a kind of based on Magnetic Isolation and quantum dot-labeled detection people's streptococcus pneumoniae antigen test kit, its feature exists In：It is described based on Magnetic Isolation and quantum dot-labeled detection people's streptococcus pneumoniae antigen test kit by with enrichment people's pneumonia The anti-human streptococcus pneumoniae immune nanometer magnetic bead of streptococcus function, quantum dot-labeled anti-human streptococcus pneumoniae nano-probe, matter Control product and PBST buffer are constituted；The quality-control product includes positive quality control product and negative quality-control product；The positive quality control Product are dried to be attached on swab by the people streptococcus pneumoniae for inactivating and are formed；The negative quality-control product is that Jing is clinical to be determined for people's pneumonia The throat swab of the negative crowd of streptococcus.

It is a kind of for preparing the test kit based on Magnetic Isolation and quantum dot-labeled detection people's streptococcus pneumoniae antigen Method, it is characterised in that：The preparation method is comprised the following steps：

1) preparation of anti-human streptococcus pneumoniae immune nanometer magnetic bead：

1.1) rabbit and the anti-human streptococcus pneumoniae Fam1 PspA of Mus, the preparation of Fam2 PspA protein polyclone antibody IgG

1.1.1) preparation of restructuring PspA1-His, PspA2-His fusion protein, purification：

1.1.1.1 bioinformatic analysis are carried out to people streptococcus pneumoniae Fam1 PspA, Fam2 PspA albumen), respectively Epitope peptide fragment the abundantest in people streptococcus pneumoniae Fam1 PspA, Fam2 PspA ectodomains is obtained, is looked for To its corresponding gene order；

1.1.1.2) in step 1.1.1.1) in obtained by gene order 5 ' end and 3 ' end introduce restriction enzyme site respectively And difference chemosynthesis complete genome sequence, while labelling is designated as PspA1, PspA2；Its sequence is referring to sequence table；

1.1.1.3) by step 1.1.1.2) in obtained by PspA1, PspA2 be cloned into by molecular biology method respectively Expression in escherichia coli restructuring PspA1-His, PspA2-His fusion protein is proceeded to after expression vector pET-28a (+)；It is described heavy Group PspA1-His, PspA2-His fusion protein is present in genetic engineering thalline with solubility expression form；

1.1.1.4) use ni-sepharose purification step 1.1.1.3) obtained by restructuring PspA1-His, PspA2-His fusion egg In vain, after SDS-PAGE detects its purity, then protein concentration is determined with Bradford methods, two kinds of protein concentrations of adjustment are It is standby after 0.2mg/mL；

1.1.2) rabbit and the anti-human streptococcus pneumoniae Fam1 PspA of Mus, the preparation of Fam2 PspA protein polyclone antibody IgG：

1.1.2.1) with step 1.1.1.4) in obtained by restructuring PspA1-His, PspA2-His fusion protein be complete Antigen, the immune new zealand white rabbit of difference and Cavia porcelluss；Prepare rabbit-anti restructuring PspA1-His, PspA2-His fusion protein respectively to resist Serum and anti-restructuring PspA1-His, PspA2-His fusion protein antiserum of Mus；Described rabbit-anti restructuring PspA1-His, PspA2- His fusion protein antiserum and the sero-fast indirect ELISA titer of anti-restructuring PspA1-His, PspA2-His fusion protein of Mus are equal More than 1 × 10⁵；

1.1.2.2) using Protein G affinity columns, the restructuring of purification rabbit-anti PspA1-His, PspA2-His melt respectively Polyclonal antibody IgG in hop protein antiserum and anti-restructuring PspA1-His, PspA2-His fusion protein antiserum of Mus；

1.1.2.3) with triumphant base Braford protein contents detection kit determination step 1.1.2.2) obtained by more than four kinds Its protein concentration is adjusted to standby after 1mg/mL by the concentration of clonal antibody IgG, and this four kinds of polyclonal antibody IgG distinguish For rabbit-anti people streptococcus pneumoniae Fam1 PspA, Fam2 PspA protein polyclone antibodies IgG and the anti-human streptococcus pneumoniae Fam1 of Mus PspA, Fam2 PspA protein polyclone antibody IgG；

1.2) coating of immune nanometer magnetic bead：

1.2.1 5mg magnetic beads) are taken, with 1ml MES buffer solutions three times, being placed in nanometer magnetic separator carries out Magneto separate After remove supernatant；The magnetic bead is with superparamagnetism Fe₃O₄For kernel, the carboxyl magnetic bead that particle diameter is 180nm；The MES buffer Be mass concentration be 2g/L 2- (N- morpholinoes) ethyl sulfonic acid；The pH=6.0 of the MES buffer；The nanometer magnetic separator Magnetic intensity be 0.4T；

1.2.2) sequentially add and use step 1.2.1) in MES buffers concentration be 8-12mg/ml EDC solution And use step 1.2.1) in MES buffers concentration be 6-10mg/ml each 0.5ml of sulfo-NHS solution, with 10-40rpm activates 1hr in rotary mixer, is placed in after carrying out Magneto separate in nanometer magnetic separator and removes supernatant, is walked with 1ml Rapid 1.2.1) in MES buffer it is resuspended, the magnetic bead after being activated；

1.2.3 5 centrifuge tubes) are taken, 200 μ L steps 1.2.2 are added in each centrifuge tube) obtained by activation after magnetic Pearl, is placed in after carrying out Magneto separate in nanometer magnetic separator and removes supernatant, in each centrifuge tube addition PBS dilute it is dense Spend for 50-200 μ g/ml by step 1.1) prepared by rabbit-anti people streptococcus pneumoniae Fam1 PspA protein polyclone antibody IgG The each 1ml of solution, reacts 2-6h with 15rpm in rotary mixer under room temperature, is placed in after carrying out Magneto separate in nanometer magnetic separator It is after removing supernatant, each to add above-mentioned PBSs of the 1ml containing 1mg/ml ethanolamine, under room temperature with 15rpm in rotary mixer React 2h to close the carboxyl on magnetic bead not with antibody response；In the PBS, each component content is as follows：8g/L NaCl, 0.2g/L KCl, 0.24g/L KH₂PO₄, 1.44g/L Na₂HPO₄, the pH=7.4 of the PBS；

1.2.4) after the completion of capping, 5 centrifuge tubes are placed in after Magneto separate being carried out in nanometer magnetic separator and are removed Supernatant, respectively washs three times with 1ml lavation buffer solutions；In the 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 the lavation buffer solution；

1.2.5) 1ml is separately added into in each centrifuge tube and preserves the resuspended magnetic bead of buffer, be placed in 4 DEG C and save backup；Extremely This is obtained anti-human streptococcus pneumoniae Fam1 PspA protein immunization nanometer magnetic beads；In the preservation buffer, each component content is as follows： 8g/L NaCl, 0.2g/L KCl, 0.24g/L KH₂PO₄, 1.44g/L Na₂HPO₄, 0.3g/L NaN₃, 5g/L bovine serum albumins In vain (BSA), the pH=7.4 for preserving buffer；

1.2.6) as with step 1.2.1) -1.2.5) identical method is using by step 1.1) prepared by rabbit-anti people's pneumonia Streptococcus Fam2 PspA protein polyclone antibody IgG are obtained anti-human streptococcus pneumoniae Fam2 PspA protein immunization nanometer magnetic beads. By above two immune nanometer magnetic bead suspension by volume 1:1 mixing, that is, be obtained anti-human streptococcus pneumoniae immune nanometer magnetic bead；

2) preparation of quantum dot-labeled anti-human streptococcus pneumoniae nano-probe：

Its concrete preparation method includes：

2.1) 4nmol carboxyl water-soluble quantum dots, 600nmol N- hydroxy ambers is sequentially added in microcentrifugal tube Amber acid imide sulfo-NHS and 600nmol carbodiimide EDC, with phosphate buffer constant volume as 5ml, mixed solution, 37 DEG C After reaction 30min, dialysis removes excessive sulfo-NHS and EDC as activator, the quantum dot after being activated；It is described In phosphate buffer, each component content is as follows：2.9g/L disodium hydrogen phosphates, 0.295g/L sodium dihydrogen phosphate, 4g/L Sodium Chloride； The pH=7.4 of the phosphate buffer；

2.2) in step 2.1) obtained by activation quantum dot in, the step of add 8-16nmol 1.1) in it is prepared The anti-human streptococcus pneumoniae Fam1 PspA protein polyclone antibody IgG of Mus, lucifuge reaction 2h, add single-ended amino polyethylene glycol PEG2000-NH₂To final concentration of 1%, unreacted activated carboxyl site is closed, 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 tubes, 15min is centrifuged at 4 DEG C with 8000g centrifugal force, removing do not occur the antibody of coupling reaction and By-product in reaction；

2.4) collection step 2.3) in ultra-filtration centrifuge tube filter membrane upper strata quantum dot-antibody coupling matter solution, be dissolved in 2ml phosphoric acid In salt cleaning mixture, then this solution is transferred in a new 50000MW ultra-filtration centrifuge tube, with 8000g centrifugal force at 4 DEG C from Heart 15min, collects ultra-filtration centrifuge tube filter membrane upper strata quantum dot-antibody coupling matter solution, is dissolved in 1ml phosphate and preserves in liquid, puts Save backup in 4 DEG C, quantum dot-labeled anti-human streptococcus pneumoniae Fam1 PspA protein nano probes are so far obtained；The phosphorus In hydrochlorate cleaning mixture, each component content is as follows：2.9g/L disodium hydrogen phosphates, 0.295g/L sodium dihydrogen phosphate, 4g/L Sodium Chloride, 5ml/L tween 20s, 0.3g/L sodium azides, the pH=7.4 of the phosphate cleaning mixture；The phosphate preserves each composition in liquid Content is as follows：2.9g/L disodium hydrogen phosphates, 0.295g/L sodium dihydrogen phosphate, 2g/L Sodium Chloride, 10g/L bovine serum albumin, 0.3g/L sodium azides；The phosphate preserves the pH=7.4 of liquid；

2.5) as with step 2.1) -2.4) identical method is using by step 1.1) prepared by the anti-human streptococcus pneumoniae of Mus Fam2 PspA protein polyclone antibody IgG are obtained quantum dot-labeled anti-human streptococcus pneumoniae Fam2 PspA protein nanos and visit Pin.By the nano-probe solution of above two quantum point labelling by volume 1:1 mixing, that is, be obtained anti-human streptococcus pneumoniae nanometer Probe；

3) preparation of PBST buffer：

Its concrete compound method includes：

Take 8g NaCl, 0.2g KCl, 0.24KH₂PO₄, 1.44g Na₂HPO₄, 0.3g NaN₃, 0.5ml Tween-20 are molten Solution in 800ml distilled water adjusts pH to 7.4 with 5M NaOH, then is settled to 1000ml；

4) preparation of quality-control product：

4.1) positive quality control product：Positive quality control product is dried to be attached on swab by the people streptococcus pneumoniae for inactivating and is formed；

4.2) negative quality-control product：Negative quality-control product is that Jing is clinical determines that the pharynx for the negative crowd of people streptococcus pneumoniae is wiped Son.

Preferably, the present invention is in step 1.2.2) in, sequentially add and use step 1.2.1) in MES buffers Concentration is the EDC solution of 10mg/ml and uses step 1.2.1) in MES buffers concentration be 8mg/ml sulfo- The each 0.5ml of NHS solution, activates 1hr in rotary mixer with 15rpm, is placed in nanometer magnetic separator after carrying out Magneto separate and moves Except supernatant, with 1ml steps 1.2.1) in MES buffer it is resuspended, the magnetic bead after being activated；

Step 1.2.3) in, in each centrifuge tube addition PBS dilution concentration be 100 μ g/ml by Step 1.1) prepared by each 1ml of rabbit-anti people's streptococcus pneumoniae Fam1 PspA protein polyclone antibody IgG solution, under room temperature with 15rpm reacts 3h in rotary mixer, is placed in after removing supernatant after carrying out Magneto separate in nanometer magnetic separator, respectively adds 1ml Above-mentioned PBS containing 1mg/ml ethanolamine；

The step 2.2) in, in step 2.1) obtained by activation quantum dot in, add 12nmol the step of 1.1) In the prepared anti-human streptococcus pneumoniae Fam1 PspA protein polyclone antibody IgG of Mus, lucifuge reaction 2h.

Preferably, quantum dot of the present invention is the water-soluble CdSe/ZnS amounts of carboxylated amphipathic polymer modification Sub- point.

Preferably, magnetic bead of the present invention is with superparamagnetism Fe₃O₄It is polystyrene, table for kernel, shell material The carboxyl magnetic bead that face functional group is carboxyl, particle diameter is 180nm.

It is a kind of based on Magnetic Isolation and quantum dot-labeled detection people's streptococcus pneumoniae antigen test kit using method, It is characterized in that：The using method is comprised the following steps：

1) by sample to be checked with after 0.5ml PBST buffer solutions proceeding to lysate in the common centrifuge tubes of 1.5ml；Institute In stating PBST buffer, each component content is as follows：8g/L NaCl, 0.2g/L KCl, 0.24g/L KH₂PO₄, 1.44g/L Na₂HPO₄, 0.3g/L NaN₃, 0.5ml/L Tween-20；The pH=7.4 of the PBST buffer；

2) to step 1) in centrifuge tube in add based on Magnetic Isolation and quantum dot-labeled detection people streptococcus pneumoniae Anti-human streptococcus pneumoniae immune nanometer magnetic bead 60-200 μ l in the test kit of antigen, with 10rpm in rotary mixer under room temperature Remove after upper reaction 15-45min, centrifuge tube is inserted into nanometer magnetic separator Magneto separate 3min, supernatant is suctioned out with pipettor；

3) the PBST buffer 1ml added in test kit is washed twice, is washed using suctioning out after nanometer magnetic separator Magneto separate Liquid is washed, finally with the resuspended magnetic bead of 1ml PBSs, immune nanometer magnetic bead-streptococcal antigen complex is obtained；The 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；It is described The pH=7.4 of PBS；

4) 100 μ l steps 3 are taken) immune nanometer magnetic bead-streptococcal antigen complex for obtaining in another centrifuge tube, then plus Enter quantum dot-labeled in test kits of the 100 μ l based on Magnetic Isolation and quantum dot-labeled detection people's streptococcus pneumoniae antigen Anti-human streptococcus pneumoniae nano-probe, reacts 25-45min with 15rpm under room temperature, on rotary mixer by quantum dot Antibody is combined with the immunity of the streptococcal antigen on immune nanometer magnetic bead, and quantum dot is tagged to streptococcal antigen surface, is formed Magnetic bead-streptococcal 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 streptococcus nano-probe, is cleaned 2 times with the PBST buffer liquid in test kit, and complex is dispersed in again 100 μ l PBS and delays Rush in liquid, its fluorescent value is detected using fluorescence microplate reader；In the PBS, each component content is as follows：8g/L NaCl, 0.2g/L KCl, 0.24g/L KH₂PO₄, 1.44g/L Na₂HPO₄；The pH=7.4 of the PBS；

6) by four parts provided in above-mentioned same method detection kit negative quality-control product samples and a positive quality control Product sample, reads fluorescent value respectively；The meansigma methodss of the fluorescence reading of four parts of negative quality-control product samples are with 3 times of standard deviation sums CUT-OFF values；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 values Scorching streptococcal antigen is the positive, otherwise is then judged as in sample to be checked that people's streptococcus pneumoniae antigen is feminine gender；If positive quality control product The fluorescent value of sample is less than CUT-OFF values, then show that test kit fails.

Preferably, step 2 proposed by the invention) in, to step 1) in centrifuge tube in add based on Magnetic Isolation With anti-human 160 μ of streptococcus pneumoniae immune nanometer magnetic bead in the test kit of quantum dot-labeled detection people's streptococcus pneumoniae antigen L, removes after reacting 30min with 10rpm under room temperature on rotary mixer, centrifuge tube insertion magnetic frame is separated 3min, with shifting Liquid device suctions out supernatant；

The step 4) in, taking 100 μ l steps 3) immune nanometer magnetic bead-streptococcal antigen complex for more obtaining is in another In centrifuge tube, add in test kits of the 100 μ l based on Magnetic Isolation and quantum dot-labeled detection people's streptococcus pneumoniae antigen Quantum dot-labeled anti-human streptococcus pneumoniae nano-probe, 30min is reacted with 15rpm on rotary mixer under room temperature, lead to The antibody crossed on quantum dot is combined with the immunity of the people's streptococcus pneumoniae antigen on immune nanometer magnetic bead, and quantum dot is tagged to people Streptococcus pneumoniae antigen surface, forms magnetic bead-streptococcal 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, 180nm carboxyl magnetic beads needed for the present invention, can arrive the research list of relevant speciality Position, company's purchase are customized；Required instrument, equipment, medicine are commercially available.

The present invention is had the advantage that compared to existing technology：

1st, present invention utilizes immune nanometer magnetic bead to sample can enriching, detached speed 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 collaboration and puts Big effect, so as to the detection sensitivity with superelevation (as its prevalence persister Sp6B clinical to people streptococcus pneumoniae, Sp19F, The detection bottom line of Sp23F is respectively 2 × 10³CFU/ml, 1 × 10³CFU/ml and 5 × 10³CFU/ml)。

2nd, the antibody used by the present invention is the polyclone for recognizing the extracellular conserved region of people's streptococcus pneumoniae specificity PspA antigens Antibody, its specificity are high, and common respiratory pathogen no cross reaction, while its more most widely used monoclonal For antibody, preparation cost is cheap, therefore, testing cost of the present invention is relatively low.

3rd, detection method is simple, and detection is quick, it is easy to judge, testing cost is cheap, overcomes prior art inspection Survey low positive rate, high cost, complex operation is loaded down with trivial details, time-consuming, cannot carry out the deficiency of clinical practice.

4th, due to being people's streptococcus pneumoniae antigen of detection kit detection non-antibody (appearance of antibody needs infection several Zhou Yihou), so early diagnosiss and preventing and treating can be carried out, clinical diagnosises coincidence rate is high.Clinic of the method in people streptococcus 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 psychological burden of pain and the head of a family that infant patient takes a blood sample, therefore be relatively easy to promote.

Specific embodiment

The present invention be 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 possesses multiple signal collaboration amplification, the new side of the quick detection people streptococcus pneumoniae with hypersensitivity and high degree of specificity Method, with 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 phosphates, 0.24g potassium dihydrogen phosphates, 8g Sodium Chloride, 0.2g potassium chloride, It is dissolved in the deionized water of 900ml, adjusts deionized water after pH to 7.4 to be settled to 1000ml with 1mol/L NaOH.

2. rabbit-anti people streptococcus pneumoniae Fam1 PspA protein polyclone antibody IgG：For present invention self-control, PBS is used Dilution, shakes up, and in making solution, polyclonal antibody weight percent concentration is 1mg/ml.

3. rabbit-anti people streptococcus pneumoniae Fam2 PspA protein polyclone antibody IgG：For present invention self-control, PBS is used Dilution, shakes up, and in making solution, polyclonal antibody weight percent concentration is 1mg/ml.

4. anti-human streptococcus pneumoniae Fam1 PspA protein polyclone antibody IgG of Mus：For present invention self-control, PBS is used Dilution, shakes up, and in making solution, polyclonal antibody weight percent concentration is 1mg/ml.

5. anti-human streptococcus pneumoniae Fam2 PspA protein polyclone antibody IgG of Mus：For present invention self-control, PBS is used Dilution, shakes up, and in making solution, polyclonal antibody weight percent concentration is 1mg/ml.

6. quantum dot：In the present invention, quantum dot used is the water-soluble CdSe/ZnS quantum of carboxylated amphipathic polymer modification Point, its launch wavelength are 585nm, are bought from Wuhan Jia Yuan technology of quantum dots development corporation, Ltd., and name of product is that carboxyl is water-soluble Property quantum dot -585.

7. magnetic bead：In the present invention, magnetic bead used is with superparamagnetism Fe₃O₄It is polystyrene, surface official for kernel, shell material It is respectively 50nm, 180nm, 350nm, 1150nm, 3 μm of carboxyl magnetic bead that can roll into a ball as carboxyl, particle diameter, can be from Shaanxi North America gene Limited company, Aorun Weina New Material Science and Technology Co., Ltd., Shanghai's purchase.

8th, people's Streptococcus pneumoniae subtypes bacterial strain Sp6B：Purchased from American type culture collection (ATCC), numbering is ATCC 700670。

9th, people's Streptococcus pneumoniae subtypes bacterial strain Sp19F：Purchased from American type culture collection (ATCC), numbering is ATCC 49619。

10th, people's Streptococcus pneumoniae subtypes bacterial strain Sp23F：Purchased from American type culture collection (ATCC), numbering is ATCC 700669。

Microbiological specimens used in 11. present invention are purchased from American type culture collection (ATCC).

1 rabbit of embodiment and the anti-human streptococcus pneumoniae Fam1 PspA of Mus, the system of Fam2 PspA protein polyclone antibody IgG It is standby

(1) preparation of restructuring PspA1-His, PspA2-His fusion protein, purification

1. the clone of related gene

To people streptococcus pneumoniae Fam1 PspA, the Fam2 PspA albumen (accession in its NCBI Protein Data Bank Number is respectively AAF27703, AAF27712) bioinformatic analysis are carried out, antigen table in its ectodomain is obtained respectively Position peptide fragment the abundantest, finds its corresponding DNA encoding sequence, while introducing at its 5 ' introducing restriction enzyme site NdeI, 3 ' ends (Jin Sirui biologies section is transferred in complete sequence synthesis to distinguish chemosynthesis complete genome sequence after termination signal TAA and restriction enzyme site XhoI Skill company limited completes, and during delivery, the genetic fragment of synthetic is respectively connected on carrier pUC57), PspA1 is designated as, PspA2.Its gene complete sequence is as shown in sequence table.Wherein, the protein sequence behaviour streptococcus pneumoniae of PspA1 gene codes Fam1 PspA albumen (accession number:AAF27703 29-406aa).The protein sequence of PspA2 gene codes For people streptococcus pneumoniae Fam2 PspA albumen (accession number:AAF27712 26-427aa).Should by containing respectively The carrier pUC57 of the DNA fragmentation of two sections of synthetic is carried out being returned respectively after double digestion according to a conventional method respectively with NdeI and XhoI Purpose fragment is received, it is standby.Double digestion is carried out to carrier pET-28a (+) using NdeI and XhoI simultaneously, and is distinguished according to a conventional method By the PspA1 obtained Jing after double digestion, PspA2 is connected in pET-28a (+) carrier, and converts escherichia coli TOP10, is built PET-PspA1, pET-PspA2 expression vector.Jing enzyme action and sequencing confirm that expression vector establishment is errorless.The carrier distinguishes table Up to restructuring PspA1-His, PspA2-His fusion protein.

2. recombinate PspA1-His, PspA2-His fusion protein expression and purification

Plasmid is extracted after correct positive colony bacterium culture being identified, routinely technology proceeds to competence E.coli BL21 (DE3) in, bacterium solution is coated on the LB flat boards containing 50 μ g/mL kanamycin after the completion of conversion, according to a conventional method screening expression Bacterial strain.Respectively picking pET-PspA1, pET-PspA2 conversion the single bacterium colony with exogenous protein expression ability and connect respectively Plant in 100mL LB culture medium, in 37 DEG C of overnight incubations.After taking out bacterium solution respectively, by 1：100 are inoculated in 100mL respectively contains In the LB culture medium of 50 μ g/mL kanamycin, cultivate to OD in 37 DEG C₆₀₀When=0.6,1mol/L IPTG are added to final concentration of 1mmol/L, shakes bacterium culture, induced fusion protein expression in 37 DEG C.10min is centrifuged under 8000r/min after induction 4h to receive Collection thalline.This two parts of thalline are used 20mL phosphate buffers (8g/L NaCl, 0.2g/L KCl, 0.24g/L KH respectively₂PO₄, 1.44g/L Na₂HPO₄PH=7.4) wash 3 times and use 10mL sample-loading buffers (20mM Na respectively₃PO₄,0.5M NaCl； 30mM imidazoles, pH7.4) it is resuspended after carry out ultrasonication, operating condition is：50HZ, 200W, ultrasonic 3S, interval 5S, work 100 It is secondary.After the completion of ultrasound, 12000g centrifugation 15min carry out electrophoresis detection after collecting precipitation and supernatant respectively.It was found that restructuring PspA1- His, PspA2-His fusion protein is present in thalline in solubility expression mode.Collect this two parts of cytoclastic supernatants Liquid, respectively with His Trap affinity columns (GE healthcare Products), to specifications with same Method carries out purification.Concrete grammar is as follows：

1) distilled water is filled with 5mL syringes, turns on the stopper of post, post and syringe are connected with the joint for providing, Post is washed with 1mL/min flow velocitys.

2) balanced with 10mL sample-loading buffers, 1mL/min flow velocitys.

3) by fusion protein loading, 1mL/min flow velocitys.

4) 10mL sample-loading buffers are used, post is washed with 1mL/min flow velocitys.

5) 10mL elution buffers (20mM Na are used₃PO₄, 0.5M NaCl, 300mM imidazoles, pH7.4), flowed with 1mL/min Fast eluting, is in charge of collection, often pipe 1ml, 12%SDS-PAGE detections, merges the sample containing destination protein in elution fraction.Jing After bradford test kits carry out determination of protein concentration, adjustment concentration is 0.2mg/mL.

(2) rabbit and the anti-human streptococcus pneumoniae Fam1 PspA of Mus, the preparation of Fam2 PspA protein polyclone antibody IgG

1. rabbit-anti people streptococcus pneumoniae Fam1 PspA, the preparation of Fam2 PspA protein polyclone antibody IgG

With the restructuring PspA1-His fusion protein of step (one) purification according to 200 μ g (1mL) and 1mL Freund's complete adjuvants After mixing emulsifying immune Male New Zealand White Rabbit (being provided by Disease Prevention Control Center, Hubei Prov), note in dorsal sc multiple spot Penetrate, after the 7d of interval again immunity once, after 14d with the restructuring P1-His fusion protein of above-mentioned purification according to 200 μ g (1mL) and 1mL incomplete Freund's adjuvants carry out booster immunization after mixing emulsifying, strengthen again in same way as described above again exempting from after booster immunization 7d Epidemic disease is once.Haemanalysises antibody titer is taken after 7d.If dissatisfied, one is may be repeated to booster immunization twice, it is full to antibody titer Meaning (determines antibody titer with ELISA method and is more than 1 × 10⁵).The Culling heart blood if satisfied, separates serum, affine with Protein G Chromatographic column (GE healthcare Products), in strict accordance with operating instruction purified polyclonal antibodies IgG, uses triumphant base Braford protein contents detection kit determine antibody concentration and with phosphate buffer (8g/L NaCl, 0.2g/L KCl, 0.24g/L KH₂PO₄, 1.44g/L Na₂HPO₄PH=7.4 1mg/mL) is adjusted to, -20 DEG C of preservations are standby, rabbit-anti is so far obtained People streptococcus pneumoniae Fam1 PspA protein polyclone antibody IgG.With the restructuring PspA2-His fusion protein of step (one) purification Rabbit-anti people streptococcus pneumoniae Fam2 PspA protein polyclone antibody IgG are obtained as antigen according to above-mentioned same method.Westen Blot is tested and is shown, specific recognition people's streptococcus pneumoniae total length PspA egg of this two kinds of polyclonal antibody IgG energy correspondences In vain.

2. the anti-human streptococcus pneumoniae Fam1 PspA of Mus, the preparation of Fam2 PspA protein polyclone antibody IgG

With the restructuring PspA1-His fusion protein of step (one) purification as complete antigen immune guinea pig (by Hubei diagnosing disease Sick prevention and control center provides), 200 μ g/ of injections of antigens under omoplate.Fundamental immunity is helped with Freund completely for isopyknic antigen Agent carries out emulsifying, carries out a booster immunization, booster immunization equal-volume antigen and equal-volume incomplete Freund's adjuvant every 2 weeks Emulsifying is carried out, altogether immunity 4 times.Haemanalysises antibody titer is taken after final immunization 10d.If dissatisfied, one to two are may be repeated Secondary booster immunization, (determines antibody titer with ELISA method and is more than 1 × 10 to antibody titer satisfaction⁵).Put to death Cavia porcelluss if satisfied to take Serum, with Protein G affinity columns (GE healthcare Products), in strict accordance with many grams of operating instruction purification Grand IgG antibody, determines antibody concentration and with phosphate buffer (8g/L with triumphant base Braford protein contents detection kit NaCl, 0.2g/L KCl, 0.24g/L KH₂PO₄, 1.44g/L Na₂HPO₄PH=7.4 1mg/mL) is adjusted to, -20 DEG C of preservations are standby With Mus anti-human streptococcus pneumoniae Fam1 PspA protein polyclone antibody IgG are so far obtained.With the restructuring of step (one) purification It is many that PspA2-His fusion protein is obtained the anti-human streptococcus pneumoniae Fam2 PspA albumen of Mus as antigen according to above-mentioned same method Clonal antibody IgG.Westen blot are tested and are shown, specific recognition people's lung of this two kinds of polyclonal antibody IgG energy correspondences Scorching streptococcus total length PspA albumen.

The preparation of 2 anti-human streptococcus pneumoniae immune nanometer magnetic bead of embodiment

1. the optimization of anti-human streptococcus pneumoniae Fam1 PspA protein polyclone antibody coupled bead reaction conditions：

To be coupled the magnetic bead of rabbit-anti people streptococcus pneumoniae Fam1 PspA protein polyclone antibody IgG as solid phase carrier, The anti-human streptococcus pneumoniae PspA protein polyclone antibodies of quantum dot-labeled Mus as detection antibody, by double-antibody method principle Detection people's Streptococcus pneumoniae subtypes bacterial strain Sp6B antigens, observe the coupling situation of magnetic bead and multi-resistance.Particle diameter to magnetic bead respectively, with And the coupling condition such as EDC/NHS activator concentrations, coupled antibody concentration, coupling time, sealer species carried out it is 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, add EDC containing 4mg/ml and After the PBS of 4mg/ml NHS carries out priming reaction, respectively with rabbit-anti people's streptococcus pneumoniae Fam1 PspA protein polyclones Antibody carries out coupling reaction.The immune nanometer magnetic bead for preparing is detected into 10 respectively⁴The people Streptococcus pneumoniae subtypes bacterium of CFU/mL Strain Sp6B, fluorescence microscopy Microscopic observation result select fluorescence intensity big, and background fluorescence interference is few, and under the action of a magnetic field point It is most suitable magnetic bead particle diameter from speed person.As a result show that the magnetic bead of particle diameter 180nm best suits the requirement of the present invention, it is determined that most suitable Magnetic microsphere particle diameter is 180nm.

The selection of 1.2 EDC/NHS activator concentrations

EDC and NHS concentration in reaction system is each set to carry out Concentraton gradient combination after l～10mg/ml, is activated respectively The carboxyl magnetic bead of particle diameter 180nm.The immune nanometer magnetic bead for preparing is detected into 10 respectively⁴People's Streptococcus pneumoniae subtypes of CFU/mL Bacterial strain Sp6B, selects fluorescence most powerhouse for the most suitable activation concentration of EDC and NHS solution.As a result show that when EDC concentration be 5mg/ When ml, NHS concentration is 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 people's streptococcus pneumoniae Fam1 PspA eggs White polyclonal antibody IgG is coupled for the magnetic bead of 180nm by the particle diameter that above-mentioned best practice is activated with 1mg respectively.To prepare Good immune nanometer magnetic bead detects 10 respectively⁴The people Streptococcus pneumoniae subtypes bacterial strain Sp6B of CFU/mL, as a result has found, when antibody When injected volume is less than 100 μ g/mg, fluorescence intensity increases as the concentration of antibody increases, and the mass concentration for working as antibody is more than During 100 μ g/mg, fluorescence intensity is basically unchanged and even slightly reduces, therefore the present embodiment selects rabbit-anti people streptococcus pneumoniae Fam1 The coupling amount of PspA protein polyclone antibody IgG is 100 μ g/mg.

The selection of 1.4 coupling times

After determining particle diameter, EDC/NHS activator concentrations and the antibody coupling amount of magnetic bead, by rabbit-anti people streptococcus pneumoniae Fam1 PspA protein polyclone antibodies IgG is set to 0.5h, 1h, 2h, 3h, 4h, 5h with the coupling reaction time of magnetic bead.To prepare Immune nanometer magnetic bead detect 10 respectively⁴The people Streptococcus pneumoniae subtypes bacterial strain Sp6B of CFU/mL, as a result has found, when coupled between >During 3h, fluorescence intensity tends towards stability, and hereafter extends coupling time again, and fluorescence no longer strengthens.Accordingly, it is determined that rabbit-anti people's pneumonia chain Coccus Fam1 PspA protein polyclone antibodies IgG are 3h with the most suitable coupling reaction time of magnetic bead.Coupling time is far fewer than tradition The 24h of ELISA method.

The selection of 1.5 sealers

Optimal conditionss according to above-mentioned determination select particle diameter, EDC/NHS activator concentrations, antibody coupling amount and the idol of magnetic bead Coupling reaction is carried out after the connection time.After coupling terminates, BSA is selected, ethanolamine, Tris and D-Glucosamine Hydrochloride are used as exempting from Epidemic disease nanometer magnetic bead sealer, finished product immune nanometer magnetic bead.The immune nanometer magnetic bead for preparing is detected into 10 respectively⁴CFU/mL People Streptococcus pneumoniae subtypes bacterial strain Sp6B, as a result find, using ethanolamine as the immune nanometer magnetic bead of sealer detection Fluorescent value highest.Speculate due to the molecule of ethanolamine it is less, preferably can consume due to it is sterically hindered not with antibodies Surface carboxyl groups, make closing more complete, and effectively reduce space steric effect to connecting the structure influence of antibody.

The optimum results of anti-human streptococcus pneumoniae Fam2 PspA protein polyclone antibodies IgG coupled bead reaction conditions with The anti-human streptococcus pneumoniae Fam1 PspA protein polyclone antibodies of above-mentioned steps 1.1-1.5 description are related to magnetic bead coupling reaction The optimum results of reaction condition are completely the same.

2. coupling process：

5mg magnetic beads are taken (with superparamagnetism Fe₃O₄For kernel, the carboxyl magnetic bead that particle diameter is 180nm) commonly it is centrifuged in 1.5ml Guan Zhong, is washed three times with 1ml MES buffer (2g/L MES, pH6.0), and being placed in nanometer magnetic separator carries out Magneto separate (0.4T) supernatant is removed after, is sequentially added with the EDC solution that the concentration of above-mentioned MES buffers is 10mg/ml and with above-mentioned The concentration of MES buffers 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；5 centrifuge tubes are taken, in each centrifuge tube, 200 is added The magnetic bead of the above-mentioned activation of μ L, suctions out supernatant after Magneto separate, addition PBS (8g/L NaCl, 0.2g/L in each pipe KCl, 0.24g/L KH₂PO₄, 1.44g/L Na₂HPO₄, pH7.4) and the concentration that dilutes is prepared by the embodiment 1 of 100 μ g/ml The each 1ml of rabbit-anti people's streptococcus pneumoniae Fam1 PspA protein polyclone antibody IgG solution, with 15rpm in rotary mixer under room temperature Middle reaction 3h, it is after Magneto separate removes supernatant, each to add above-mentioned PBSs of the 1ml containing 1mg/ml ethanolamine, under room temperature with 15rpm reacts 2h in rotary mixer to close the carboxyl on magnetic bead not with antibody response.Each pipe supernatant is removed after Magneto separate, 1ml lavation buffer solutions (8g/L NaCl, 0.2g/L KCl, 0.24g/L KH are used respectively₂PO₄, 1.44g/L Na₂HPO₄, 0.5ml/L Tween-20, pH7.4) wash three times, last each 1ml preserves buffer (8g/L NaCl, 0.2g/L KCl, 0.24g/L KH₂PO₄, 1.44g/L Na₂HPO₄, 0.3g/L NaN₃, 5g/L BSA, pH7.4) and resuspended magnetic bead, it is placed in 4 DEG C and saves backup, so far Anti-human streptococcus pneumoniae Fam1 PspA protein immunization nanometer magnetic beads are obtained.

As above-mentioned steps with identical method using the rabbit-anti people streptococcus pneumoniae Fam2 PspA by prepared by embodiment 1 Protein polyclone antibody IgG is obtained anti-human streptococcus pneumoniae Fam2 PspA protein immunization nanometer magnetic beads.Above two immunity is received Rice suspension containing magnetic beads by volume 1:1 mixing, that is, be obtained anti-human streptococcus pneumoniae immune nanometer magnetic bead.

The preparation of the quantum dot-labeled anti-human streptococcus pneumoniae nano-probe of embodiment 3

1. the anti-human streptococcus pneumoniae Fam1 PspA protein polyclone antibodies IgG of the quantum dot-labeled Mus of nanometer carboxylic reacts bar The optimization of part：

1.1st, the determination of quantum dot-labeled antibody probe optimum mark pH of carboxyl

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, different impacts of the pH value to coupling reaction of observation, it is determined that the Optimal pH of quantum dot-labeled multi-resistance reaction For 7.0-8.0.This experimental selection pH7.4.

1.2nd, the determination of the quantum dot-labeled antibody probe optimum mark amount of carboxyl

Ratio of the quantum dot molar concentration with multi-resistance concentration is respectively set to into 1:1,1:2,1:3 and 1:4, it is marked reaction Afterwards, fluorescent strength determining is carried out using full spectrogrph to marked product, both observations variable concentrations compare the impact of coupling reaction, Determine the optimum molar concentration ratio of the anti-human streptococcus pneumoniae Fam1 PspA protein polyclone antibodies IgG reactions of quantum dot-labeled Mus It is 1 for quantum dot and antibody molar ratio:3.This experimental selection optimal concentration ratio 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 entered by step 1.1 and 1.2 conditions for determining as sealer After line flag reaction, fluorescent strength determining is carried out using full spectrogrph to marked product, different sealers are observed for labelling The impact of reaction, as a result finds, PEG2000-NH₂For optimal sealer, which is remarkably improved the colloid-stabilised of labeled complex Property and immunocompetence.

The bar of the anti-human streptococcus pneumoniae Fam2 PspA protein polyclone antibodies IgG reactions of the quantum dot-labeled Mus of nanometer carboxylic The anti-human streptococcus pneumoniae Fam1 PspA albumen of the quantum dot-labeled Mus of nanometer carboxylic that piece optimization result is described with step 1.1-1.3 The optimum results of polyclonal antibody IgG reaction correlated conditions are completely the same.

2. labeling process：

4nmol carboxyl water-soluble quantum dots, 600nmol N- hydroxy succinyls is sequentially added in microcentrifugal tube Imines (sulfo-NHS) and 600nmol carbodiimides (EDC), with phosphate buffer (2.9g/L disodium hydrogen phosphates, 0.295g/ L sodium dihydrogen phosphate, 4g/L Sodium Chloride, 7.4) constant volume is 2ml to pH, ceaselessly mixed solution, and 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 12nmol The anti-human streptococcus pneumoniae Fam1 PspA protein polyclone antibody IgG of Mus, lucifuge reaction 2h, add the poly- second of single-ended amination two Alcohol (PEG2000-NH₂) to final concentration of 1%, unreacted activated carboxyl site is closed, continue lucifuge reaction 1h.With 0.2 μm PES filters are filtered to remove antibody aggregation thing, then filtrate be transferred to, in 50000MW ultra-filtration centrifuge tubes, exist with 8000g centrifugal force 15min is centrifuged at 4 DEG C, and removing does not occur the by-product in the antibody of coupling reaction 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 phosphates, 0.295g/L sodium dihydrogen phosphate, 4g/L Sodium Chloride, 5ml/L tween 20s, 0.3g/L sodium azides, pH 7.4) in, then by this solution be transferred 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 phosphates, 0.295g/L sodium dihydrogen phosphate, 2g/L Sodium Chloride, 10g/L BSA, 0.3g/L sodium azides, pH 7.4) in, quantum dot-labeled anti-human streptococcus pneumoniae Fam1 PspA protein nanos are so far obtained and visit Pin, is placed in 4 DEG C and saves backup.

As above-mentioned same procedure using many grams of the anti-human streptococcus pneumoniae Fam2 PspA albumen of Mus by prepared by embodiment 1 Grand IgG antibody is obtained quantum dot-labeled anti-human streptococcus pneumoniae Fam2 PspA protein nano probes.By above two quantum point The nano-probe solution of labelling by volume 1:1 mixing, that is, be obtained anti-human streptococcus pneumoniae nano-probe.

4 immune nanometer magnetic bead of embodiment carries out the optimization of immunocapture condition to people's streptococcus pneumoniae antigen

To be coupled the magnetic bead of people streptococcus pneumoniae Fam1 PspA protein polyclone antibody IgG as solid phase carrier, quantum The anti-human streptococcus pneumoniae PspA protein polyclone antibodies of point labelling, are set up by double-antibody method principle as detection antibody The detection system of people's streptococcus pneumoniae antigen.The conditions such as the consumption to immune nanometer magnetic bead in detection system respectively, capture time A series of optimized choice is carried out.

Test the selection of 1. immune nanometer magnetic bead additions

By 20 μ l, 40 μ l, 60 μ l, 80 μ l, 100 μ l, 120 μ l, 140 μ l, 160 μ l, 200 μ l by prepared by embodiment 2 Good immune nanometer magnetic bead is added separately to 0.5ml containing 10⁴The PBS of the people Streptococcus pneumoniae subtypes bacterial strain Sp6B of CFU/mL suspends In liquid, immunocapture is carried out, then the quantum dot-labeled probe by described by embodiment 3 is detected, record fluorescent value.As a result send out Existing, with the increase of immune nanometer magnetic bead addition, fluorescent value gradually increases, when immune nanometer magnetic bead addition reaches 160 μ l When, fluorescent value reaches maximum.It is further continued for increasing the amount of immune nanometer magnetic bead, fluorescent value is reduced on the contrary.Therefore 160 μ l of this experimental selection As the optimal addn of immune nanometer magnetic bead.

Test the selection of 2. immunocapture times

After determining the addition of magnetic bead, the made immune nanometer magnetic bead got ready of four parts of embodiments 2 is taken, at room temperature with 10r/ Min, to 10⁴The people Streptococcus pneumoniae subtypes bacterial strain Sp6B of CFU/mL carries out the immunity of 15min, 30min, 45min and 60min and catches Obtain, then the quantum dot-labeled probe by described by embodiment 3 is detected, record fluorescent value.As a result find, fluorescent value is in immunity Maximum is shown during capture 30min, prolongation over time, numerical value have declined.Therefore this experimental selection 30min is used as immunity The Best Times of capture.

Meanwhile, respectively being coupled the immune nano of rabbit-anti people streptococcus pneumoniae Fam2 PspA protein polyclone antibody IgG Magnetic bead as solid phase carrier, the anti-human streptococcus pneumoniae Fam2 PspA protein polyclone antibody IgG of corresponding quantum dot-labeled Mus As detection antibody, by double-antibody method principle, the detection system of people's streptococcus pneumoniae antigen is set up, to corresponding detection body Contact conditionss in system are optimized.As a result find, the best capture condition in above-mentioned detection system with above-mentioned experiment 1 and real The result tested given by 2 is completely the same.

The preparation of 5 PBST buffer of embodiment

Take 8g NaCl, 0.2g KCl, 0.24KH₂PO₄, 1.44g Na₂HPO₄, 0.3g NaN₃, 0.5ml Tween-20 are molten Solution in 800ml distilled water adjusts pH to 7.4 with 5M NaOH, then is settled to 1000ml.

The preparation of 6 quality-control product of embodiment

1. positive quality control product：People Streptococcus pneumoniae subtypes bacterial strain Sp6B (0.5 μ g) with 1% formalin-inactivated is dried and is combined To on swab, as positive quality control product.

2. negative quality-control product：Negative quality-control product is the clinical throat swab samples determined for the negative crowd of people streptococcus pneumoniae of Jing Product.

The preparation of 7 test kit of embodiment

It is quantum dot-labeled described by anti-human streptococcus pneumoniae immune nanometer magnetic bead, embodiment 3 by described by embodiment 2 Anti-human streptococcus pneumoniae nano-probe, the PBST buffer described by embodiment 5, the quality-control product described by embodiment 6 it is common Constitute the test kit based on Magnetic Isolation and quantum dot-labeled detection people's streptococcus pneumoniae antigen.

The using method of 8 test kit of embodiment

Routinely clinical means obtain people's throat swab, with 0.5ml test kits 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 tubes of 1.5ml, in the centrifuge tube add test kit in anti-human pneumonia 160 μ l of streptococcus immune nanometer magnetic bead, remove after reacting 30min with 10rpm, centrifuge tube is inserted under room temperature on rotary mixers Enter magnetic frame and separate 3min, supernatant is suctioned out with pipettor.PBST buffer 1ml in addition test kit is washed twice, Magneto separate After suction out cleaning mixture, finally with the resuspended magnetic bead of 1mL PBSs.Take the above-mentioned immune nanometer magnetic bead-streptococcal antigen of 100 μ l Complex adds the quantum dot-labeled anti-human streptococcus pneumoniae nano-probe in 100 μ l test kits in another centrifuge tube, 30min is reacted with 15rpm on rotary mixer under room temperature, by the people's lung on the antibody on quantum dot and immune nanometer magnetic bead The immunity of scorching streptococcal antigen is combined, and quantum dot is tagged to S. pneumonia surface, formed magnetic bead-streptococcus pneumoniae antigen- Quantum dot " sandwich " complex.After the completion of reaction, Magneto separate 3min removes unnecessary quantum dot-labeled probe, and uses PBST Buffer solution for cleaning 2 times, complex are dispersed in again in 100 μ l PBSs, using fluorescence microplate reader (Ex=405nm, Em= 585nm) its fluorescent value is detected.

By four parts provided in above-mentioned same method detection kit negative quality-control products and a positive quality control product sample, Fluorescent value is read respectively；The meansigma methodss of the 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 clinical people's throat swab sample is judged as in this part of clinical throat swab more than CUT-OFF values People's streptococcus pneumoniae antigen is the positive, otherwise is then judged as that people's streptococcus pneumoniae antigen is the moon in this part of clinical people's throat swab sample Property；If the fluorescent value of positive quality control product sample is less than CUT-OFF values, show that test kit fails.

The detection sensitivity of 9 test kit of embodiment and specific test

By determining culture of streptococcus pneumonia thing diluent doing Study of Sensitivity, determine described by embodiment 7 based on Test kit detection people's Streptococcus pneumoniae subtypes bacterial strain of Magnetic Isolation and quantum dot-labeled detection people's streptococcus pneumoniae antigen The Monitoring lower-cut of Sp6B is 2 × 10³CFU/ml, and from the entitled Binax NOW Streptococcus of manufacturer Binax The detection of the test kit of pneumoniae test (colloidal gold method) is limited to 5 × 10⁵CFU/ml, under the detection of test kit of the present invention Limit is compared with its obvious reduction.In addition, to the clinical relatively conventional people's pneumonia streptococcus with Fam1 PspA and Fam2 PspA respectively The testing result of bacterium subtype strains Sp19F and Sp23F shows, under detection of the test kit to two subtype strains described in embodiment 7 Limit is respectively 1 × 10³CFU/ml and 5 × 10³CFU/ml。

With respiratory tract common causative such as human respiratory syncytial virus (Long strains, ATCC numberings VR26), people's pneumonia original (AR-39 strains, ATCC numberings are 53592), 3 type of adenovirus hominiss (compile by GB strains, ATCC for body (ATCC numberings 15531), people's Chlamydia pneumoniae Number VR-3), 7 type of adenovirus hominiss (Gomen strains, ATCC numberings VR-7), influenza virus A hominis's (H1N1, ATCC numbering VR- 1743), people's Influenza B viruss (ATCC numberings VR-790), hemophilus influenza (ATCC numberings 53781), moraxelle catarrhalises (ATCC numberings 25238) etc. replaces people streptococcus pneumoniae to be detected, test kit PBST buffer of the detection containing these microorganisms All it is negative.

10 clinical trial example of embodiment

Using streptococcus pneumoniae standard assays-Sputum culturing method as reference, 150 Pneumology Department Patients with Lower Respiratory Tract Infection are taken Bronchoalveolar lavage fluid specimen is detected that with the test kit described by embodiment 7 culture method positive rate is 12% (18/150), this examination Agent box is 13.3% (20/150), and the coincidence rate of 2 kinds of methods is 97.3% (146/150).Two methods testing result difference without Significance.Concrete outcome is as shown in table 1.

The testing result of 1 clinical samples of table

It is pointed out that presently preferred embodiments of the present invention is the foregoing is only, it is not to limit the present invention, all Any modification for being made within spirit of the invention and principle, equivalent etc. should be included in protection scope of the present invention it It is interior.

Claims (4)

1. a kind of side for preparing the test kit based on Magnetic Isolation and quantum dot-labeled detection people's streptococcus pneumoniae antigen Method, it is characterised in that：The preparation method is comprised the following steps：

1）The preparation of anti-human streptococcus pneumoniae immune nanometer magnetic bead：

1.1）Rabbit and the anti-human streptococcus pneumoniae Fam1 PspA of Mus, the preparation of Fam2 PspA protein polyclone antibody IgG

1.1.1）The preparation of restructuring PspA1-His, PspA2-His fusion protein, purification：

1.1.1.1）Bioinformatic analysis are carried out to people streptococcus pneumoniae Fam1 PspA, Fam2 PspA albumen, is obtained respectively In people streptococcus pneumoniae Fam1 PspA, Fam2 PspA ectodomains, epitope peptide fragment the abundantest, finds which Corresponding gene order；

1.1.1.2）In step 1.1.1.1）In obtained by gene order 5 ' end and 3 ' end respectively introduce restriction enzyme site and divide Other chemosynthesis complete genome sequence, while labelling is designated asPspA1、PspA2；

1.1.1.3）By step 1.1.1.2）In obtained byPspA1、PspA2Expression is cloned into respectively by molecular biology method Carrier pET-28a（+）After proceed to expression in escherichia coli restructuring PspA1-His, PspA2-His fusion protein；The restructuring PspA1-His, PspA2-His fusion protein is present in genetic engineering thalline with solubility expression form；

1.1.1.4）With ni-sepharose purification step 1.1.1.3）Resulting restructuring PspA1-His, PspA2-His fusion protein, After SDS-PAGE detects its purity, then protein concentration is determined with Bradford methods, two kinds of protein concentrations of adjustment are 0.2 mg/ It is standby after mL；

1.1.2）Rabbit and the anti-human streptococcus pneumoniae Fam1 PspA of Mus, the preparation of Fam2 PspA protein polyclone antibody IgG：

1.1.2.1）With step 1.1.1.4）In obtained by restructuring PspA1-His, PspA2-His fusion protein for resisting completely Original, the immune new zealand white rabbit of difference and Cavia porcelluss；The anti-blood of rabbit-anti restructuring PspA1-His, PspA2-His fusion protein is prepared respectively Anti- restructuring PspA1-His, PspA2-His fusion protein antiserum of clear and Mus；Described rabbit-anti restructuring PspA1-His, PspA2-His Fusion protein antiserum and the sero-fast indirect ELISA titer of anti-restructuring PspA1-His, PspA2-His fusion protein of Mus are big In 1 × 10⁵；

1.1.2.2）Using Protein G affinity columns difference purification rabbit-anti restructuring PspA1-His, PspA2-His fusion egg Polyclonal antibody IgG in white antiserum and anti-restructuring PspA1-His, PspA2-His fusion protein antiserum of Mus；

1.1.2.3）With triumphant base Braford protein contents detection kit determination step 1.1.2.2）Four kinds of resulting polyclones Its protein concentration is adjusted to standby after 1 mg/mL by the concentration of IgG antibody, and this four kinds of polyclonal antibody IgG are respectively rabbit The anti-human streptococcus pneumoniae Fam1 of anti-human streptococcus pneumoniae Fam1 PspA, Fam2 PspA protein polyclone antibodies IgG and Mus PspA, Fam2 PspA protein polyclone antibody IgG；

1.2）The coating of immune nanometer magnetic bead：

1.2.1）5 mg magnetic beads are taken, and with 1 ml MES buffer solutions three times, are placed in after Magneto separate being carried out in nanometer magnetic separator Remove supernatant；The magnetic bead is with superparamagnetism Fe₃O₄For the carboxyl magnetic bead that kernel, particle diameter are 180 nm；The MES buffer Be mass concentration be 2 g/L 2- (N- morpholinoes) ethyl sulfonic acid；The pH=6.0 of the MES buffer；The nanometer magnetic separator Magnetic intensity be 0.4T；

1.2.2）Sequentially add and use step 1.2.1）In MES buffers concentration be 8-12 mg/ml EDC solution with And use step 1.2.1）In MES buffers concentration be 6-10 mg/ml each 0.5 ml of sulfo-NHS solution, with 10-40 rpm activate 1 hr in rotary mixer, are placed in after carrying out Magneto separate in nanometer magnetic separator and remove supernatant, use 1 ml Step 1.2.1）In MES buffer it is resuspended, the magnetic bead after being activated；

1.2.3）5 centrifuge tubes are taken, 200 μ L steps 1.2.2 are added in each centrifuge tube）Magnetic bead after resulting activation, Be placed in after Magneto separate being carried out in nanometer magnetic separator remove supernatant, the concentration that addition is diluted with PBS in each centrifuge tube It is 50-200 μ g/ml by step 1.1）Prepared rabbit-anti people streptococcus pneumoniae Fam1 PspA protein polyclone antibody IgG Each 1 ml of solution, reacts 2-6 h with 15 rpm in rotary mixer under room temperature, and being placed in nanometer magnetic separator carries out Magneto separate After remove supernatant after, it is each to add above-mentioned PBSs of 1 ml containing 1 mg/ml ethanolamine, it is mixed in rotation with 15 rpm under room temperature React 2 h to close the carboxyl on magnetic bead not with antibody response in closing instrument；In the PBS, 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 the PBS；

1.2.4）After the completion of capping, 5 centrifuge tubes are placed in after Magneto separate being carried out in nanometer magnetic separator and remove supernatant, Respectively three times are washed with 1 ml lavation buffer solutions；In the 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 the lavation buffer solution；

1.2.5）1 ml is separately added into in each centrifuge tube and preserves the resuspended magnetic bead of buffer, be placed in 4 DEG C and save backup；So far Anti-human streptococcus pneumoniae Fam1 PspA protein immunization nanometer magnetic beads are obtained；In the preservation 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, 0.3 g/L NaN₃, 5 g/L Ox blood serums Albumin, the pH=7.4 of the preservation buffer；

1.2.6）By with step 1.2.1）-1.2.5）Identical method is using by step 1.1）Prepared rabbit-anti people's pneumonia streptococcus Bacterium Fam2 PspA protein polyclone antibody IgG are obtained anti-human streptococcus pneumoniae Fam2 PspA protein immunization nanometer magnetic beads；Will be upper State two kinds of immune nanometer magnetic bead suspensions by volume 1:1 mixing, that is, be obtained anti-human streptococcus pneumoniae immune nanometer magnetic bead；

2）The preparation of quantum dot-labeled anti-human streptococcus pneumoniae nano-probe：

Its concrete preparation method includes：

2.1）4 nmol carboxyl water-soluble quantum dots, 600 nmol N- hydroxy succinums are sequentially added in microcentrifugal tube Acid imide sulfo-NHS and 600 nmol carbodiimide EDC, with phosphate buffer constant volume as 5 ml, mixed solution, 37 After DEG C 30 min of reaction, dialysis removes excessive sulfo-NHS and EDC as activator, the quantum dot after being activated；Institute In stating phosphate buffer, each component content is 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 the phosphate buffer；

2.2）In step 2.1）In the quantum dot of resulting activation, add 8-16 nmol the step of 1.1）In prepared Mus Anti-human streptococcus pneumoniae Fam1 PspA protein polyclone antibody IgG, lucifuge react 2 h, add single-ended amino polyethylene glycol PEG2000-NH₂To final concentration of 1%, unreacted activated carboxyl site is closed, continue lucifuge and react 1 h；

2.3）Step 2.2 is filtered to remove with 0.2 μm of PES filter）In antibody aggregation thing, then filtrate be transferred to 50000 In MW ultra-filtration centrifuge tubes, 15 min are centrifuged at 4 DEG C with 8000 g centrifugal force, removing does not occur antibody and the reaction of coupling reaction In by-product；

2.4）Collection step 2.3）Middle ultra-filtration centrifuge tube filter membrane upper strata quantum dot-antibody coupling matter solution, is 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 15 min are centrifuged, ultra-filtration centrifuge tube filter membrane upper strata quantum dot-antibody coupling matter solution is collected, 1 ml phosphate are dissolved in and are preserved liquid In, it is placed in 4 DEG C and saves backup, quantum dot-labeled anti-human streptococcus pneumoniae Fam1 PspA protein nano probes is so far obtained；Institute In stating phosphate cleaning mixture, each component content is as follows：2.9 g/L disodium hydrogen phosphates, 0.295 g/L sodium dihydrogen phosphate, 4 g/L chlorine Change sodium, 5 ml/L tween 20s, 0.3 g/L sodium azides, the pH=7.4 of the phosphate cleaning mixture；The phosphate preserves liquid In each component content it is as follows：2.9 g/L disodium hydrogen phosphates, 0.295 g/L sodium dihydrogen phosphate, 2 g/L Sodium Chloride, 10 g/L cattle Serum albumin, 0.3 g/L sodium azides；The phosphate preserves the pH=7.4 of liquid；

2.5）By with step 2.1）-2.4）Identical method is using by step 1.1）The anti-human streptococcus pneumoniae Fam2 of prepared Mus PspA protein polyclone antibody IgG are obtained quantum dot-labeled anti-human streptococcus pneumoniae Fam2 PspA protein nano probes；Will be upper State the nano-probe solution by volume 1 of two amounts son point labelling:1 mixing, that is, be obtained anti-human streptococcus pneumoniae nano-probe；

3）The 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 Be dissolved in 800 ml distilled water, pH is adjusted to 7.4 with 5 M NaOH, then be settled to 1000 ml；

4）The preparation of quality-control product：

4.1）Positive quality control product：Positive quality control product is dried to be attached on swab by the people streptococcus pneumoniae for inactivating and is formed；

4.2）Negative quality-control product：Negative quality-control product is the clinical throat swabs determined for the negative crowd of people streptococcus pneumoniae of Jing.

2. method according to claim 1, it is characterised in that：Step 1.2.2）In, sequentially add and use step 1.2.1）In the concentration of MES buffers be the EDC solution of 10 mg/ml and use step 1.2.1）In MES buffer The concentration of preparation is each 0.5 ml of sulfo-NHS solution of 8 mg/ml, activates 1 hr with 15 rpm, put in rotary mixer Supernatant is removed after Magneto separate is carried out in nanometer magnetic separator, with 1 ml steps 1.2.1）In MES buffer it is resuspended, obtain Magnetic bead after activation；

Step 1.2.3）In, in each centrifuge tube, the concentration of addition PBS dilution is 100 μ g/ml by step 1.1）Prepared each 1 ml of rabbit-anti people's streptococcus pneumoniae Fam1 PspA protein polyclone antibody IgG solution, with 15 under room temperature Rpm reacts 3 h in rotary mixer, is placed in after removing supernatant after carrying out Magneto separate in nanometer magnetic separator, each to add 1 ml Above-mentioned PBS containing 1 mg/ml ethanolamine；

The step 2.2）In, in step 2.1）In the quantum dot of resulting activation, add 12 nmol the step of 1.1）Middle institute The anti-human streptococcus pneumoniae Fam1 PspA protein polyclone antibody IgG of Mus of preparation, lucifuge react 2 h.

3. method according to claim 2, it is characterised in that：The quantum dot is the water of carboxylated amphipathic polymer modification Dissolubility CdSe/ZnS quantum dots.

4. method according to claim 3, it is characterised in that：The magnetic bead is with superparamagnetism Fe₃O₄For kernel, shell material Expect that for polystyrene, surface functional group be carboxyl, the carboxyl magnetic bead that particle diameter is 180 nm.