CN104198710B - Based on the anti-human Chlamydia pneumoniae IgM of magnetic resolution and color quantum point mark, the IgG antibody method of altogether inspection and kit fast - Google Patents

Based on the anti-human Chlamydia pneumoniae IgM of magnetic resolution and color quantum point mark, the IgG antibody method of altogether inspection and kit fast Download PDF

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CN104198710B
CN104198710B CN201410405739.0A CN201410405739A CN104198710B CN 104198710 B CN104198710 B CN 104198710B CN 201410405739 A CN201410405739 A CN 201410405739A CN 104198710 B CN104198710 B CN 104198710B
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chlamydia pneumoniae
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杨波
胡征
董俊
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Hubei Hualong Biological Pharmaceutical Co., Ltd.
Hubei University of Technology
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Hubei University of Technology
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    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens

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Abstract

The invention discloses a kind of anti-human Chlamydia pneumoniae IgM based on magnetic resolution and color quantum point mark, the IgG antibody method of altogether inspection and kit fast, this kit is made up of the anti-human IgM having the anti-human Chlamydia pneumoniae IgM of enrichment, the anti-human chlamydia pneumoniae (cp) of IgG antibody function catches nanometer magnetic bead, color quantum point marks respectively, IgG antibody nano-probe, quality-control product and PBST damping fluid; Quality-control product comprises positive quality control product and negative quality-control product; The anti-human Chlamydia pneumoniae IgM of positive quality control product behaviour infection involving chlamydia pneumoniae person, IgG antibody are respectively positive serum; Negative quality-control product is that anti-human Chlamydia pneumoniae IgM, IgG are negative human serum.The present invention has easy, quick, highly sensitive advantage, can realize the synchronous detection to anti-human Chlamydia pneumoniae IgM, IgG antibody.

Description

Based on the anti-human Chlamydia pneumoniae IgM of magnetic resolution and color quantum point mark, the IgG antibody method of altogether inspection and kit fast
Technical field
The present invention relates to technical field of medical detection, be specially a kind of anti-human Chlamydia pneumoniae IgM based on magnetic resolution and color quantum point mark, the IgG antibody detection method of altogether inspection and detection kit fast, and the method for preparation and use of this detection kit.
Background technology
Chlamydia pneumoniae (Chlamydiapneumoniae, Cpn) as a kind of important respiratory diseases pathogenic microorganism of chlamydiaceae, since eighties of last century the mid-80 is by people's definite designations such as Grayston, studied persons extensively research in the time subsequently.Now only know that people is the host of this pathogen, mode of infection may for propagate by respiratory secretions between men.Within less than 5 years old, children are seldom contaminted, and more than 8 years old children and youth are easily infected, especially crowd massing place, as easily popular in family, school, military camp, through seroepidemiology investigation, confirm to have at least 40% to be subject to this choamydiae infection in adult, major part is Subclinical.Chlamydia pneumoniae is the entozoic microorganism of special sexual cell, is lodged in the place such as human respiratory and throat, produces the upper respiratory tract and respiratory tract infection, often cause the breathing problem such as pneumonia and bronchitis in children and adult.Current research shows, it is also relevant with diseases such as atherosclerotic syndrome, senile dementia, myocarditis, asthma.The infection symptoms caused due to itself and other respiratory pathogen is clinically similar, is therefore often difficult to reach a conclusion according to clinical manifestation, x-radiological survey X etc., makes a definite diagnosis and often depend on laboratory diagnosis.Diagnostic method should be just can obtain clear and definite diagnosis at the their early stage of disease fast and effectively, is convenient to implement to treat targetedly, stops the development delay of the state of an illness.
In the laboratory diagnosis of people's infection involving chlamydia pneumoniae, in examinee's serum, anti-human chlamydia pneumoniae (cp) index is then one of important evidence of diagnostic result.
Immunoglobulin M (ImmunoglobulinM, IgM) and immunoglobulin G (ImmunoglobulinG, IgG) are most important two kinds of antibody in human body.After people's Chlamydia pneumoniae enters body, the immunoglobulin (Ig) occurred the earliest is IgM antibody, occurs IgG afterwards, and by the existence of detection bodies internal specific IgM, IgG antibody, diagnosable human body is to the immune response state of people's Chlamydia pneumoniae.If detect specific IgM antibodies, show the generation having recent infection, but the IgM antibody half life period is shorter, the detection feminine gender of IgM can not prove body uninfection, and also need to detect the half life period longer, the IgG antibody that content is the highest, to clarify a diagnosis.
At present, in serum, the detection method of specific IgM and IgG antibody mainly contains the experiment of condensation collection, gelatin particle aggegation experiment, enzyme-linked immunosorbent assay, Immunogold-labeling method and gold-marking immunity chromatography.Specificity and the susceptibility of the experiment of condensation collection are minimum, and therefore its diagnostic value is little; Though Immunogold-labeling method and gold-marking immunity chromatography simple and efficient to handle, susceptibility is lower slightly, and the patient that antagonist level is lower has the possibility of failing to pinpoint a disease in diagnosis; Gelatin aggegation experiment reagent prepares with operating process comparatively loaded down with trivial details, and needs professional operator.Enzyme linked immunosorbent assay has the advantages such as special, responsive, for checking various antibody or antigen, but there is following problem in the method: (1) every a collection of test from application of sample, temperature bath, to wash the steps such as version more, complex operation, time longer (altogether needing 2-4 hour); (2) need to add color composition A liquid and B liquid respectively in detecting, and also will complete reading at the appointed time, adds somewhat to the possibility of labour intensity and misoperation; (3) this method cannot accomplish to detect while IgM and IgG two kinds of antibody.Although after detecting IgM and IgG antibody separately, more comprehensively the diagnosis of analysis testing result on disease there is no impact, and operating process is loaded down with trivial details, detects convenient and swift not, and testing cost comparatively altogether inspection also can increase.
Therefore, set up to possess and highly sensitively can carry out to the specific IgM of anti-human Chlamydia pneumoniae, IgG antibody the detection method that Fast synchronization examines altogether and seem very necessary.
Summary of the invention
For these technical matterss existed in background technology, the invention provides that a kind of energy based on magnetic resolution and color quantum point mark is easy, quick, highly sensitive synchronously examines anti-human Chlamydia pneumoniae IgM, the detection method of IgG antibody and kit altogether, and the method for preparation and use of this kit.
The present invention is achieved through the following technical solutions:
Based on anti-human Chlamydia pneumoniae IgM, the IgG antibody common method examined fast that magnetic resolution and color quantum point mark, it is characterized in that: said method comprising the steps of:
1) preparation of recombined human Chlamydia pneumoniae 98KD Membrane protein antigen;
2) by step 1) the recombined human Chlamydia pneumoniae 98KD Membrane protein antigen for preparing and nanometer magnetic bead are by covalent coupling, and obtained anti-human chlamydia pneumoniae (cp) catches nanometer magnetic bead;
3) by mouse-anti people IgM monoclonal antibody and emission wavelength be the nano-quantum point of 520nm by covalent coupling, prepare quantum dot-labeled anti-human IgM nano-probe; By mouse-anti human IgG monoclonal antibody and emission wavelength be the nano-quantum point of 600nm by covalent coupling, prepare quantum dot-labeled anti-human igg nano-probe; By anti-human IgM, IgG nano-probe that quantum dot-labeled anti-human IgM nano-probe and quantum dot-labeled anti-human igg nano-probe mixed in equal amounts i.e. obtained color quantum point mark respectively;
4) human serum sample is got, after suitably diluting with PBSA damping fluid, step 2 is added respectively in serum dilution) the anti-human chlamydia pneumoniae (cp) for preparing catches nanometer magnetic bead and step 3) anti-human IgM, IgG nano-probe of marking respectively of the color quantum point for preparing, abundant mixing, Magneto separate is carried out after reaction 5-45min, after PBST buffer solution 2 times, use fluorescence microplate reader, under emission wavelength (Em) is respectively 520nm and 600nm, reads fluorescent value respectively; In described PBSA damping fluid, each component concentration is respectively 8g/LNaCL, 0.2g/LKCl, 0.24g/LKH 2pO 4, 1.44g/LNa 2hPO 4, 5g/LBSA (bovine serum albumin(BSA)), the pH=7.4 of described PBSA damping fluid; In described PBST damping fluid, each component concentration is respectively 8g/LNaCL, 0.2g/LKCl, 0.24g/LKH 2pO 4, 1.44g/LNa 2hPO 4, 5g/LBSA (bovine serum albumin(BSA)), 0.3g/LNaN 3, 0.5ml/LTween-20, the pH=7.4 of described PBST damping fluid;
5) according to step 4) method detect that at least 30 parts are all defined as anti-human Chlamydia pneumoniae IgM through clinical various method, IgG antibody is negative human serum sample, read respectively emission wavelength (Em) be respectively 520nm and 600nm under fluorescent value; Described anti-human Chlamydia pneumoniae IgM, IgG antibody are negative human serum sample and are called for short people's chlamydia pneumoniae (cp) negative control sample; Described people's chlamydia pneumoniae (cp) negative control sample is designated as CUT-OFF1 value at the mean value of emission wavelength (Em) fluorescent value that is 520nm and 3 times of standard deviation sums; Described people's chlamydia pneumoniae (cp) negative control sample is designated as CUT-OFF2 value at the mean value of emission wavelength (Em) fluorescent value that is 600nm and 3 times of standard deviation sums; If step 4) in human serum sample be greater than CUT-OFF1 value at the detection fluorescent value that emission wavelength (Em) is 520nm, to be then judged as in human serum sample that anti-human chlamydia pneumoniae IgM antibody is for the positive; If step 4) in human serum sample be less than CUT-OFF1 value at the detection fluorescent value that emission wavelength (Em) is 520nm, to be then judged as in human serum sample that anti-human chlamydia pneumoniae IgM antibody is for feminine gender; If step 4) in human serum sample be greater than CUT-OFF2 value at the detection fluorescent value that emission wavelength (Em) is 600nm, to be then judged as in human serum sample that anti-human CPn IgG antibody is for the positive; If step 4) in human serum sample be less than CUT-OFF2 value at the detection fluorescent value that emission wavelength (Em) is 600nm, to be then judged as in human serum sample that anti-human CPn IgG antibody is for feminine gender.
Based on the anti-human Chlamydia pneumoniae IgM of magnetic resolution and color quantum point mark, an IgG antibody altogether check reagent box fast, it is characterized in that: the described anti-human Chlamydia pneumoniae IgM based on magnetic resolution and color quantum point mark, IgG antibody fast altogether check reagent box be made up of the anti-human IgM that there is the anti-human Chlamydia pneumoniae IgM of enrichment, the anti-human chlamydia pneumoniae (cp) of IgG antibody function catches nanometer magnetic bead, color quantum point marks respectively, IgG antibody nano-probe, quality-control product and PBST damping fluid; Described quality-control product comprises positive quality control product and negative quality-control product; Described positive quality control product is that positive serum forms by the serum of the anti-human chlamydia pneumoniae IgM antibody positive of two parts of people's infection involving chlamydia pneumoniae persons and two parts of anti-human CPn IgG antibody; Described negative quality-control product is four parts of anti-human Chlamydia pneumoniae IgM, IgG antibody is negative human serum.
Be total to a method for check reagent box for the preparation of the anti-human Chlamydia pneumoniae IgM marked based on magnetic resolution and color quantum point, IgG antibody fast, it is characterized in that: said method comprising the steps of:
1) anti-human chlamydia pneumoniae (cp) catches the preparation of nanometer magnetic bead:
1.1) to recombinate the preparation of M98-His fusion, purifying:
1.1.1) bioinformatic analysis is carried out to people Chlamydia pneumoniae 98KD outer membrane protein, obtain the peptide section that in people Chlamydia pneumoniae 98KD memebrane protein ectodomain, epitope enriches the most, find the gene order of its correspondence;
1.1.2) in step 1.1.1) in the 5 ' end of gene order that obtains and 3 ' end introduce restriction enzyme site chemosynthesis complete genome sequence respectively, be designated as M98 with tense marker; Its sequence is see sequence table;
1.1.3) by step 1.1.2) in the M98 that obtains be cloned into expression vector pET-28a (+) by molecular biology method after proceed to expression in escherichia coli restructuring M98-His fusion; Described restructuring M98-His fusion is present in genetic engineering thalline with inclusion body expression-form;
1.1.4) with ni-sepharose purification step 1.1.3) the restructuring M98-His fusion that obtains, after SDS-PAGE detects its purity, again renaturation is carried out to restructuring M98-His fusion, carrying out after determination of protein concentration through bradford kit, is 0.2mg/mL by PBS damping fluid adjustment concentration; In described PBS damping fluid, each component content is as follows: 8g/LNaCL, 0.2g/LKCl, 0.24g/LKH 2pO 4, 1.44g/LNa 2hPO 4, the pH=7.4 of described PBS damping fluid;
1.2) the bag quilt of nanometer magnetic bead:
1.2.1) get 5mg magnetic bead, with 1mlMES buffer solution three times, be placed in after nano magnetic separation vessel carries out Magneto separate and remove supernatant; Described magnetic bead is with superparamagnetism Fe 3o 4for the carboxyl magnetic bead that kernel, particle diameter are 1150nm; 2-(N-morpholino) ethyl sulfonic acid of described MES damping fluid to be mass concentration be 2g/L; The pH=6.0 of described MES damping fluid; The magnetic intensity of described nano magnetic separation vessel is 0.4T;
1.2.2) add successively use step 1.2.1) in the concentration of MES buffer be the EDC solution of 8-12mg/ml and use step 1.2.1) in the concentration of MES buffer be each 0.5ml of sulfo-NHS solution of 6-10mg/ml, in rotary mixer, 1hr is activated with 10-40rpm, be placed in after nano magnetic separation vessel carries out Magneto separate and remove supernatant, with 1ml step 1.2.1) in MES damping fluid resuspended, obtain activate after magnetic bead;
1.2.3) 5 centrifuge tubes are got, 200 μ L step 1.2.2 are added in each centrifuge tube) magnetic bead after the activation that obtains, be placed in after nano magnetic separation vessel carries out Magneto separate and remove supernatant, adding by the concentration of PBS damping fluid dilution in each centrifuge tube is 50-200 μ g/ml by step 1.1.4) prepared by each 1ml of restructuring M98-His fusion solution, in rotary mixer, 2-6h is reacted with 15rpm under room temperature, be placed in after removing supernatant after nano magnetic separation vessel carries out Magneto separate, respectively add the above-mentioned PBS damping fluid of 1ml containing 1mg/ml monoethanolamine, under room temperature with 15rpm react in rotary mixer 2h with on closed magnetic bead not with the carboxyl of antibody response, in described PBS damping fluid, each component content is as follows: 8g/LNaCL, 0.2g/LKCl, 0.24g/LKH 2pO 4, 1.44g/LNa 2hPO 4, the pH=7.4 of described PBS damping fluid,
1.2.4) after capping completes, these 5 centrifuge tubes are placed in after nano magnetic separation vessel carries out Magneto separate and remove supernatant, respectively wash three times with 1ml lavation buffer solution; In described lavation buffer solution, each component content is as follows: 8g/LNaCL, 0.2g/LKCl, 0.24g/LKH 2pO 4, 1.44g/LNa 2hPO 4, 0.5ml/LTween-20, the pH=7.4 of described lavation buffer solution;
1.2.5) in each centrifuge tube, add 1ml respectively preserve the resuspended magnetic bead of damping fluid, be placed in 4 DEG C and save backup; So far obtained anti-human chlamydia pneumoniae (cp) catches nanometer magnetic bead; In described preservation damping fluid, each component content is as follows: 8g/LNaCL, 0.2g/LKCl, 0.24g/LKH 2pO 4, 1.44g/LNa 2hPO 4, 0.3g/LNaN 3, 5g/L bovine serum albumin(BSA) (BSA), the pH=7.4 of described preservation damping fluid;
2) preparation of anti-human IgM, IgG nano-probe that marks respectively of color quantum point:
Its concrete preparation method comprises:
2.1) in microcentrifugal tube, add 2nmol carboxyl water-soluble quantum dot, 300nmolN-N-Hydroxysuccinimide sulfo-NHS and 300nmol carbodiimide EDC successively, with phosphate buffer constant volume for 5ml, mixed solution, after 37 DEG C of reaction 30min, excessive sulfo-NHS and EDC as activator is removed in dialysis, obtains the quantum dot after activating; The emission wavelength of described carboxyl water-soluble quantum dot is 520nm; In described phosphate buffer, each component content is as follows: 2.9g/L sodium 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) in the quantum dot of activation that obtains, add the mouse-anti people IgM monoclonal antibody of 2-12nmol, lucifuge reaction 2h, adds single-ended amination polyglycol PEG2000-NH 2be 1% to final concentration, close unreacted activated carboxyl site, continue lucifuge reaction 1h;
2.3) by 0.2 μm of PES frit removing step 2.2) in antibody aggregation thing, then filtrate is transferred in 50000MW ultra-filtration centrifuge tube, with 8000g centrifugal force centrifugal 15min at 4 DEG C, there is not the antibody of coupling reaction and the accessory substance in reacting in removing;
2.4) step 2.3 is collected) middle ultra-filtration centrifuge tube filter membrane upper strata quantum dot-antibody coupling matter solution, be dissolved in 2ml phosphate cleansing solution, again this solution is transferred in a new 50000MW ultra-filtration centrifuge tube, with 8000g centrifugal force centrifugal 15min at 4 DEG C, collect ultra-filtration centrifuge tube filter membrane upper strata quantum dot-antibody coupling matter solution, be dissolved in 1ml phosphate conserving liquid, be placed in 4 DEG C and save backup, so far obtained quantum dot-labeled anti-human IgM nano-probe; In described phosphate cleansing solution, each component content is as follows: 2.9g/L sodium hydrogen phosphate, 0.295g/L sodium dihydrogen phosphate, 4g/L sodium chloride, 5ml/L Tween-20,0.3g/L Sodium azide, the pH=7.4 of described phosphate cleansing solution; In described phosphate conserving liquid, each component content is as follows: 2.9g/L sodium hydrogen phosphate, 0.295g/L sodium dihydrogen phosphate, 2g/L sodium chloride, 10g/L bovine serum albumin(BSA), 0.3g/L Sodium azide; The pH=7.4 of described phosphate conserving liquid;
2.5) by and step 2.1)-2.4) identical method utilizes the carboxyl water-soluble quantum dot that mouse-anti human IgG monoclonal antibody and emission wavelength are 600nm to obtain quantum dot-labeled anti-human igg nano-probe; By the nano-probe solution 1:1 mixing by volume of these two amounts point mark, namely obtain anti-human IgM, IgG nano-probe that color quantum point marks respectively;
3) preparation of PBST damping fluid:
Its concrete compound method comprises:
Get 8gNaCL, 0.2gKCl, 0.24KH 2pO 4, 1.44gNa 2hPO 4, 5gBSA, 0.3gNaN 3, 0.5mlTween-20 is dissolved in 900ml distilled water, adjusts pH to 7.4, then be settled to 1000ml with 5MNaOH;
4) preparation of quality-control product:
4.1) positive quality control product:
4.1.1) anti-human chlamydia pneumoniae IgM antibody positive quality control product: determine that anti-human chlamydia pneumoniae IgM antibody is that positive serum is formed by 0.5ml people's infection involving chlamydia pneumoniae person;
4.1.2) anti-human CPn IgG antibody positive quality-control product: determine that anti-human CPn IgG antibody is that positive serum is formed by 0.5ml people's infection involving chlamydia pneumoniae person;
4.2) negative quality-control product: be negative human serum by the anti-human Chlamydia pneumoniae IgM of 0.5ml, IgG antibody and form.
As preferably, the present invention is in step 1.2.2) in, add successively and use step 1.2.1) in the concentration of MES buffer be the EDC solution of 10mg/ml and use step 1.2.1) in the concentration of MES buffer be each 0.5ml of sulfo-NHS solution of 8mg/ml, in rotary mixer, 1hr is activated with 15rpm, be placed in after nano magnetic separation vessel carries out Magneto separate and remove supernatant, with 1ml step 1.2.1) in MES damping fluid resuspended, obtain activate after magnetic bead;
Described step 1.2.3) in, adding by the concentration of PBS damping fluid dilution in each centrifuge tube is 150 μ g/ml by step 1.1.4) prepared by each 1ml of restructuring M98-His fusion solution, in rotary mixer, 3h is reacted with 15rpm under room temperature, be placed in after removing supernatant after nano magnetic separation vessel carries out Magneto separate, respectively add the above-mentioned PBS damping fluid of 1ml containing 1mg/ml monoethanolamine;
Described step 2.2) in, in step 2.1) in the quantum dot of activation that obtains, add the mouse-anti people IgM monoclonal antibody of 6nmol, lucifuge reaction 2h.
As preferably, quantum dot of the present invention is water-soluble CdSe/ZnS quantum dot that carboxylated amphipathic polymer is modified.
As preferably, magnetic bead of the present invention is with superparamagnetism Fe 3o 4for the carboxyl magnetic bead that kernel, shell material are polystyrene, surface functional group is carboxyl, particle diameter is 1150nm.
Based on a using method for the anti-human Chlamydia pneumoniae IgM of magnetic resolution and color quantum point mark, IgG antibody altogether check reagent box fast, it is characterized in that: described using method comprises the following steps:
1) serum sample 5 μ l to be checked is got with proceeding in the common centrifuge tube of 1.5ml by dilution after 995 μ lPBSA damping fluid dilutions; In described PBSA damping fluid, each component content is as follows: 8g/LNaCL, 0.2g/LKCl, 0.24g/LKH 2pO 4, 1.44g/LNa 2hPO 4, 5g/LBSA; The pH=7.4 of described PBSA damping fluid;
2) to step 1) in centrifuge tube in add successively based on magnetic resolution and color quantum point mark anti-human Chlamydia pneumoniae IgM, the anti-human chlamydia pneumoniae (cp) of IgG antibody fast altogether in check reagent box catches nanometer magnetic bead 10-100 μ l and the anti-human Chlamydia pneumoniae IgM based on magnetic resolution and color quantum point mark, IgG antibody is total to the anti-human IgM that the color quantum point in check reagent box marks respectively fast, IgG nano-probe 50 μ l, take off after reacting 5-25min with 10rpm on rotary mixer under room temperature, centrifuge tube is inserted nano magnetic separation vessel Magneto separate 3min, with pipettor sucking-off supernatant,
3) add and wash twice based on the anti-human Chlamydia pneumoniae IgM of magnetic resolution and color quantum point mark, the IgG antibody PBST damping fluid 1ml fast altogether in check reagent box, sucking-off cleansing solution after employing nano magnetic separation vessel Magneto separate, finally use the resuspended magnetic bead of 0.2mlPBS damping fluid, obtained nanometer magnetic bead-people's chlamydia pneumoniae (cp)-quantum dot " sandwich " compound; In described PBS damping fluid, each component content is as follows: 8g/LNaCL, 0.2g/LKCl, 0.24g/LKH 2pO 4, 1.44g/LNa 2hPO 4; The pH=7.4 of described PBS damping fluid;
4) by step 3) obtain nanometer magnetic bead-people's chlamydia pneumoniae (cp)-quantum dot compound is loaded in ELISA Plate, use fluorescence microplate reader to be all 365nm in excitation wavelength (Ex), emission wavelength (Em) carries out difference reading to its fluorescent value under being respectively the parameter of 520nm and 600nm respectively;
5) detect by above-mentioned same method that at least 30 parts are all defined as anti-human Chlamydia pneumoniae IgM through clinical various method, IgG antibody is negative human serum sample, read respectively emission wavelength (Em) be respectively 520nm and 600nm under fluorescent value; Describedly all be defined as anti-human Chlamydia pneumoniae IgM through clinical various method, IgG antibody is negative human serum sample and is designated as CUT-OFF1 value at the mean value of emission wavelength (Em) fluorescent value that is 520nm and 3 times of standard deviation sums; Describedly all be defined as anti-human Chlamydia pneumoniae IgM through clinical various method, IgG antibody is negative human serum sample and is designated as CUT-OFF2 value at the mean value of emission wavelength (Em) fluorescent value that is 600nm and 3 times of standard deviation sums; The four parts of negative quality-control product samples simultaneously provided in detection kit and four parts of positive quality control product samples, read respectively emission wavelength (Em) be respectively 520nm and 600nm under fluorescent value; Four parts of negative quality-control product samples are designated as NCX1 at the mean value of the fluorescent value that emission wavelength (Em) is 520nm; Four parts of negative quality-control product samples are designated as NCX2 value at the mean value of the fluorescent value that emission wavelength (Em) is 600nm; Two parts of anti-human chlamydia pneumoniae IgM antibody positive quality control product samples are PCX1 at the fluorescence mean value that emission wavelength (Em) is 520nm; Two parts of anti-human CPn IgG antibody positive quality-control product samples in emission wavelength (Em) are and fluorescent value under 600nm is designated as PCX2; If step 4) in serum sample sample to be checked be greater than CUT-OFF1 value and PCX1/NCX1 >=2.1 at the detection fluorescent value that emission wavelength (Em) is 520nm, to be then judged as in serum sample to be checked that anti-human chlamydia pneumoniae IgM antibody is for positive; If step 4) in serum sample to be checked be less than CUT-OFF1 value and PCX1/NCX1 >=2.1 at the detection fluorescent value that emission wavelength (Em) is 520nm, then the anti-human chlamydia pneumoniae IgM antibody in serum sample to be checked that judges to behave is feminine gender; If step 4) in serum sample to be checked be greater than CUT-OFF2 value and PCX2/NCX2 >=2.1 at the detection fluorescent value that emission wavelength (Em) is 600nm, to be then judged as in serum sample to be checked that anti-human CPn IgG antibody is for positive; If step 4) in serum sample to be checked be less than CUT-OFF2 value and PCX2/NCX2 >=2.1 at the detection fluorescent value that emission wavelength (Em) is 600nm, to be then judged as in serum sample to be checked that anti-human CPn IgG antibody is for negative; If PCX1/NCX1 < 2.1 or PCX2/NCX2 < 2.1, all show that kit lost efficacy.
As preferably, step 2 proposed by the invention) in, to step 1) in centrifuge tube in add successively based on magnetic resolution and color quantum point mark anti-human Chlamydia pneumoniae IgM, the anti-human chlamydia pneumoniae (cp) of IgG antibody fast altogether in check reagent box catches nanometer magnetic bead 20 μ l and the anti-human Chlamydia pneumoniae IgM based on magnetic resolution and color quantum point mark, IgG antibody is total to the anti-human IgM that the color quantum point in check reagent box marks respectively fast, IgG nano-probe 50 μ l, take off after reacting 15min with 10rpm on rotary mixer under room temperature, centrifuge tube is inserted nano magnetic separation vessel Magneto separate 3min, with pipettor sucking-off supernatant.
Carboxyl water-soluble nano quantum dot required for the present invention, 1150nm carboxyl magnetic bead, mouse-anti people IgM monoclonal antibody, mouse-anti human IgG monoclonal antibody etc. can arrive research unit, company's purchase of relevant speciality or customize; Required instrument, equipment, medicine all have commercially available.
The present invention compared to existing technology tool has the following advantages:
1, present invention utilizes nanometer magnetic bead to sample can enriching, separation fast, the efficiency advantages of higher of speed, the characteristics such as incorporating quantum point photochemical stability is high simultaneously, fluorescence intensity is high, detection system is possessed effect that multiple signal works in coordination with amplification, thus there is detection sensitivity and the accuracy of superelevation.
2, the capture antigen that the present invention is used includes the recombinant antigen that people's Chlamydia pneumoniae specificity 98KD Membrane protein antigen extracellular region is rich in epitope, and high for its antibody horizontal produced in patients serum, therefore capture effect is good.
3, surface antigen specific to the capture antigen behaviour Chlamydia pneumoniae that the present invention is used, its specificity is high, and without antibody and antigen cross-reaction between the antibody of other common respiratory pathogens (as chlamydia psittaci, chlamydia trachomatis, first, B-mode human influenza virus, human influenza haemophilus, moraxelle catarrhalis, people's mycoplasma pneumoniae, human influenza haemophilus etc.).
4, the present invention's antigen used is genetic engineering recombinant antigen, and protein expression amount is high, and protein renaturation method is simple, therefore comparatively cheap and easy to get.
5, detection method is simple, detects fast (within 30min), and be easy to judge, testing cost is cheap, overcome that prior art Positive rate is low, cost is high, complicated operation is loaded down with trivial details, length consuming time, clinical practice inconvenience deficiency.
6, the present invention can realize the synchronous detection of anti-human Chlamydia pneumoniae IgM, IgG antibody, solving subitem, to detect the operating process that brings loaded down with trivial details, detect convenient and swift not, the problems such as testing cost is higher, and also do not seen the product of anti-human Chlamydia pneumoniae IgM, IgG antibody synchronously detection altogether or the appearance of related science report at present on the market.
Embodiment
The present invention is according to the antibody antigen reaction principle in immunology, utilize nanometer magnetic bead to sample can enriching, separation fast, the efficiency advantages of higher of speed, the characteristics such as incorporating quantum point photochemical stability is high, fluorescence intensity is high, that sets up a set ofly possesses multiple signal and works in coordination with amplification, has the new method that the Fast synchronization of hypersensitivity and high degree of specificity detects anti-human Chlamydia pneumoniae IgM, IgG antibody, has wide market application foreground.
The present invention is further described in detail by following examples.
The preparation of various reagent and the explanation of material requested
1.PBSA damping fluid: take 1.44g sodium hydrogen phosphate, 0.24g potassium dihydrogen phosphate, 8g sodium chloride, 0.2g potassium chloride, 5g bovine serum albumin(BSA) is dissolved in the deionized water of 900ml, with deionized water is settled to 1000ml after adjusting pH to 7.4 with 1mol/LNaOH.
2.PBS damping fluid: take 1.44g sodium hydrogen phosphate, 0.24g potassium dihydrogen phosphate, 8g sodium chloride, 0.2g potassium chloride is dissolved in the deionized water of 900ml, with deionized water is settled to 1000ml after adjusting pH to 7.4 with 1mol/LNaOH.
3. restructuring M98-His fusion: be the present invention's self-control, with the dilution of PBS damping fluid, shake up, make the weight percent concentration of recombinant protein in solution be 0.2mg/ml.
4. mouse-anti people IgM monoclonal antibody: be Abcam Products, article No. ab99741, with the dilution of PBS damping fluid, shakes up, makes monoclonal antibody weight percent concentration in solution be 1mg/ml.
5. mouse-anti human IgG monoclonal antibody: be Abcam Products, article No. ab99757, with the dilution of PBS damping fluid, shakes up, makes monoclonal antibody weight percent concentration in solution be 1mg/ml.
6. quantum dot: two amounts point used in the present invention is water-soluble CdSe/ZnS quantum dot that carboxylated amphipathic polymer is modified, its emission wavelength is respectively 520nm and 600nm, buy from Wuhan Jia Yuan technology of quantum dots development corporation, Ltd., name of product is carboxyl water-soluble quantum dot-520 and carboxyl water-soluble quantum dot-600.
7. magnetic bead: in the present invention, magnetic bead used is with superparamagnetism Fe 3o 4for kernel, shell material be polystyrene, surface functional group is carboxyl, particle diameter is be respectively 50nm, 180nm, 350nm, 1150nm, the carboxyl magnetic bead of 3 μm, can buy from Shanxi North America Gene Co., Ltd, Aorun Weina New Material Science and Technology Co., Ltd., Shanghai.
Embodiment 1 is recombinated the expression of M98-His fusion, purifying and renaturation
1. the clone of related gene
Bioinformatic analysis is carried out to people Chlamydia pneumoniae 98KD memebrane protein (accessionnumber in its NCBI Protein Data Bank is CAA04672), obtain the peptide section that in the outer conserved domain of its born of the same parents, epitope enriches the most, find the DNA encoding sequence of its correspondence, introduce restriction enzyme site NdeI in sequence 5 ' simultaneously, after 3 ' end introduces termination signal TAA and restriction enzyme site XhoI, (complete sequence synthesis transfers to Jin Sirui bio tech ltd to complete to chemosynthesis complete genome sequence, during delivery, the genetic fragment of Prof. Du Yucang is connected on carrier pUC57), be designated as M98.Its gene complete sequence is as shown in sequence table.Specifically, the protein sequence of M98 gene code is the 130-305aa of natural human Chlamydia pneumoniae 98KD memebrane protein (accessionnumber:CAA04672).Object fragment is reclaimed according to a conventional method after carrier pUC57 NdeI and XhoI of the DNA fragmentation containing this section of Prof. Du Yucang is carried out double digestion, for subsequent use.Adopt NdeI and XhoI to carry out double digestion to carrier pET-28a (+) simultaneously, and according to a conventional method the m98 gene obtained after double digestion is connected in pET-28a (+) carrier, and transformation of E. coli TOP10, build pET-M98 expression vector.Cut through enzyme and confirm that expression vector establishment is errorless with sequencing.This vector expression restructuring M98-His fusion.
2. the expression and purification of restructuring M98-His fusion
Extract plasmid after being cultivated by positive colony bacterium correct for qualification, technology proceeds in competence E.coliBL21 (DE3) routinely, is coated by bacterium liquid on the LB flat board containing 50 μ g/mL kanamycins, screen expression strain according to a conventional method after having transformed.The single bacterium colony with exogenous protein expression ability that picking pET-M98 transforms also is inoculated in 100mLLB nutrient culture media, in 37 DEG C of overnight incubation.After taking out bacterium liquid, be inoculated in 100mL by 1:100 and contain in the LB nutrient culture media of 50 μ g/mL kanamycins, be cultured to OD in 37 DEG C 600when=0.6, adding 1mol/LIPTG to final concentration is 1mmol/L, shakes bacterium and cultivates, induced fusion protein expression in 37 DEG C.After induction 4h, under 8000r/min, centrifugal 10min collects thalline.By this thalline 20mL phosphate buffer (8g/LNaCL, 0.2g/LKCl, 0.24g/LKH 2pO 4, 1.44g/LNa 2hPO 4pH=7.4) wash 3 times and use 10mL sample-loading buffer (20mMNa 3pO 4, 0.5MNaCl; 30mM imidazoles, pH7.4) resuspended after carry out ultrasonication, operating conditions is: 50HZ, 200W, ultrasonic 3S, and interval 5S, works 100 times.Ultrasonic complete after, the centrifugal 15min of 12000g carries out electrophoresis detection after collecting precipitation and supernatant respectively.Find that restructuring M98-His fusion is present in thalline in inclusion body mode.
The purification step of restructuring M98-His fusion is as follows:
By above-mentioned deposited components lavation buffer solution (20mMNa 3pO 4, 0.5MNaCl; 3M urea, 30mM imidazoles, pH7.4) wash twice after, the centrifugal 15min collecting precipitation of 12000g.By precipitation Bindingbuffer (20mMNa 3pO 4, 0.5MNaCl; 8M urea, 30mM imidazoles, pH7.4) dissolve under room temperature after, the centrifugal 15min of 12000g, the supernatant filter membrane of 0.45 μm filters.Recombinant protein in lysate, with HisTrapaffinitycolumns (GEhealthcare Products), uses the same method to specifications and carries out purifying.Concrete grammar is as follows:
1) be filled distilled water with 5mL syringe, turn on the stopper of post, with the joint provided, post is connected with syringe, wash post with 1mL/min flow velocity.
2) by 10mLBindingbuffer balance, 1mL/min flow velocity.
3) by fusion loading, 1mL/min flow velocity.
4) with 10mLBindingbuffer, post is washed with 1mL/min flow velocity.
5) with 10mLElutionbuffer (20mMNa 3pO 4, 0.5MNaCl; 8M urea, 500mM imidazoles, pH7.4), with 1mL/min flow velocity wash-out, be in charge of collection, often pipe 1ml, 12%SDS-PAGE detect, and merge the sample containing destination protein in elution fraction.Carry out after determination of protein concentration through bradford kit, adjustment concentration is 0.5mg/mL.
3. the renaturation of restructuring M98-His fusion
By the above-mentioned restructuring M98-His fusion through HisTrapaffinitycolumns purifying, first by the PBS damping fluid dialysed overnight containing 4mol/L urea, and then each respectively with containing 2,1,0.6, the PBS buffer gradient of 0.2mol/L urea to dialyse each 4h, finally with PBS damping fluid dialysis 4h.Dislysate is respectively used the centrifugal 15min of 12000rpm, supernatant is the restructuring M98-His fusion of renaturation.Carry out after determination of protein concentration through bradford kit, adjust its concentration with PBS damping fluid and be 0.2mg/mL, for nanometer magnetic bead bag by.
The anti-human chlamydia pneumoniae (cp) of embodiment 2 catches the preparation of nanometer magnetic bead
1. the optimization of restructuring M98-His fusion coupled bead reaction conditions:
Using coupling, the magnetic bead of restructuring M98-His fusion is as solid phase carrier, and quantum dot-labeled mouse-anti people IgM monoclonal antibody, as detection antibody, detects anti-human chlamydia pneumoniae IgM antibody positive serum, observes the coupling situation of magnetic bead and recombinant protein.Respectively to the particle diameter of magnetic bead, and the coupling condition such as EDC/NHS activator concentration, coupled antibody concentration, coupling time, sealer kind has carried out a series of optimum choice.
The selection of 1.1 magnetic bead particle diameters
Selection particle diameter is 50nm, 180nm, 350nm, 1150nm, the carboxyl nanometer magnetic bead of 3 μm, after the PBS damping fluid all added containing 4mg/mlEDC and 4mg/mlNHS carries out priming reaction, respectively with restructuring M98-His fusion coupling reaction.Respectively the carboxyl nanometer magnetic bead prepared is detected anti-human chlamydia pneumoniae IgM antibody positive serum, observations under fluorescent microscope, select fluorescence intensity large, background fluorescence interference is few, and the very fast person of velocity of separation is the suitableeest magnetic bead particle diameter under magnetic fields.Result shows the magnetic bead requirement the most according to the invention of particle diameter 1150nm, determines that the suitableeest carboxyl magnetic bead particle diameter is 1150nm.
The selection of 1.2EDC/NHS activator concentration
Carry out concentration gradient combination after EDC and NHS concentration in reaction system is set to l ~ 10mg/ml separately, activate the carboxyl nanometer magnetic bead of particle diameter 1150nm respectively.The carboxyl nanometer magnetic bead prepared is detected anti-human chlamydia pneumoniae IgM antibody positive serum, selects the most powerhouse of fluorescence to be the suitableeest activation concentration of EDC and NHS solution.Result shows that coupling effect is best when EDC concentration be 5mg/ml, NHS concentration is 4mg/ml.
The selection of 1.3 coupled antigen concentration
The restructuring M98-His fusion of 10 μ g, 50 μ g, 100 μ g, 120 μ g, 150 μ g, 200 μ g, 250 μ g, 300 μ g is carried out coupling with 1mg by the magnetic bead that the particle diameter that above-mentioned best practice activates is 1150nm respectively.The carboxyl nanometer magnetic bead prepared is detected anti-human chlamydia pneumoniae IgM antibody positive serum respectively, found that, when the injected volume of recombinant protein is less than 150 μ g/mg, fluorescence intensity increases along with the concentration increase of antibody, and when the injected volume of recombinant protein is greater than 150 μ g/mg, fluorescence intensity is substantially constant even slightly to be reduced, and therefore the present embodiment selects the coupling amount of restructuring M98-His recombinant protein to be 150 μ g/mg.
The selection of 1.4 coupling times
After determining the particle diameter of magnetic bead, EDC/NHS activator concentration and antibody coupling amount, the coupling reaction time of restructuring M98-His fusion and magnetic bead is set to 0.5h, 1h, 2h, 3h, 4h, 5h respectively.The carboxyl nanometer magnetic bead prepared is detected anti-human chlamydia pneumoniae IgM antibody positive serum respectively, found that, when coupled during >3h, fluorescence intensity tends towards stability, and after this extends coupling time again, and fluorescence no longer strengthens.Therefore, the suitableeest coupling reaction time of M98-His fusion and magnetic bead determined to recombinate is 3h.Coupling time is far fewer than the 24h of traditional E LISA method.
The selection of 1.5 sealers
Coupling reaction is carried out after selecting the particle diameter of magnetic bead, EDC/NHS activator concentration, antibody coupling amount and coupling time according to the above-mentioned optimal conditions determined.After coupling terminates, select BSA, monoethanolamine, Tris and D-Glucosamine Hydrochloride, as nanometer magnetic bead sealer, obtain finished product carboxyl nanometer magnetic bead.The nanometer magnetic bead prepared is detected anti-human chlamydia pneumoniae IgM antibody positive serum respectively, found that, adopt monoethanolamine the highest as the detection fluorescent value of the nanometer magnetic bead of sealer.Infer because the molecule of monoethanolamine is less, can consume preferably due to the sterically hindered surface carboxyl groups be not combined with antibody, make closed more complete, and effectively reduce space steric effect to the structure influence connecting antibody.
Simultaneously, using coupling, the carboxyl nanometer magnetic bead of restructuring M98-His fusion is as solid phase carrier, corresponding quantum dot-labeled mouse-anti human IgG monoclonal antibody is as detection antibody, set up the detection system of anti-human CPn IgG antibody, the reaction conditions of the recombinant protein coupled bead in corresponding detection system is optimized.Found that, the best coupling condition in this detection system is all completely the same with the result given by above-mentioned steps 1.1-1.5.
2. coupling process:
Get 5mg magnetic bead (with superparamagnetism Fe 3o 4carboxyl magnetic bead for kernel, particle diameter are 1150nm) in the common centrifuge tube of 1.5ml, with 1mlMES damping fluid (2g/LMES, pH6.0) wash three times, be placed in after nano magnetic separation vessel carries out Magneto separate (0.4T) and remove supernatant, to add by the concentration of above-mentioned MES buffer be successively the EDC solution of 10mg/ml and be each 0.5ml of sulfo-NHS solution of 8mg/ml by the concentration of above-mentioned MES buffer, in rotary mixer, 1hr is activated with 15rpm, remove supernatant after Magneto separate, the MES damping fluid above-mentioned with 1ml is resuspended; Get 5 centrifuge tubes, in each centrifuge tube, add the magnetic bead of the above-mentioned activation of 200 μ L, sucking-off supernatant after Magneto separate, add in each pipe with PBS damping fluid (8g/LNaCL, 0.2g/LKCl, 0.24g/LKH 2pO 4, 1.44g/LNa 2hPO 4the restructuring M98-His fusion solution each 1ml of the concentration of pH7.4) diluting prepared by the embodiment 1 of 150 μ g/ml, in rotary mixer, 3h is reacted with 15rpm under room temperature, after Magneto separate removes supernatant, respectively add 1ml containing the above-mentioned PBS damping fluid of 1mg/ml monoethanolamine, under room temperature with 15rpm react in rotary mixer 2h with on closed magnetic bead not with the carboxyl of antibody response.Each pipe supernatant is removed after Magneto separate, each with 1ml lavation buffer solution (8g/LNaCL, 0.2g/LKCl, 0.24g/LKH 2pO 4, 1.44g/LNa 2hPO 4, 0.5ml/LTween-20, pH7.4) and wash three times, finally each 1ml preserves damping fluid (8g/LNaCL, 0.2g/LKCl, 0.24g/LKH 2pO 4, 1.44g/LNa 2hPO 4, 0.3g/LNaN 3, 5g/LBSA, pH7.4) and resuspended magnetic bead, be placed in 4 DEG C and save backup, so far obtained anti-human chlamydia pneumoniae (cp) catches nanometer magnetic bead.
The preparation of anti-human IgM, IgG nano-probe that embodiment 3 color quantum point marks respectively
1. the optimization of nanometer carboxylic quantum dot-labeled mouse-anti people IgM monoclonal antibody reaction conditions:
1.1, the determination of the quantum dot-labeled antibody probe optimum mark pH of carboxyl
Phosphate buffer pH in labeled reactant is set to 5,6,7,8,9 respectively, utilizes full spectrometer to carry out fluorescent strength determining to marked product, observe the impact of different pH value on coupling reaction, the Optimal pH determining the reaction of quantum dot-labeled monoclonal antibody is 7.0-8.0.This experimental selection pH7.4.
1.2, the determination of carboxyl quantum dot-labeled antibody probe optimum mark amount
Quantum dot volumetric molar concentration and the ratio of monoclonal antibody concentration are set to 1:1 respectively, 1:2,1:3 and 1:4, after carrying out labeled reactant, full spectrometer is utilized to carry out fluorescent strength determining to marked product, the impact of both observations variable concentrations comparison coupling reaction, determines that optimum molar concentration ratio that quantum dot-labeled mouse-anti people IgM monoclonal antibody is reacted be quantum dot and antibody molar ratio is 1:3.This optimal concentration ratio of this experimental selection determines labelled amount.
1.3, the determination of the best sealer kind of the quantum dot-labeled antibody probe of carboxyl
With monoethanolamine, Tris, PEG2000-NH 2or BSA is as sealer, after the condition determined by step 1.1 and 1.2 carries out labeled reactant, utilize full spectrometer to carry out fluorescent strength determining to marked product, observe the impact of different sealers for labeled reactant, found that, PEG2000-NH 2for best sealer, it can significantly improve colloidal stability and the immunocompetence of labeled complex.
The optimum results that the nanometer carboxylic quantum dot-labeled mouse-anti people IgM monoclonal antibody that the condition optimizing result of nanometer carboxylic quantum dot-labeled mouse-anti human IgG monoclonal antibody reactive and step 1.1-1.3 describe reacts correlated condition is all completely the same.
2. labeling process:
2nmol carboxyl water-soluble quantum dot (emission wavelength 520nm), 300nmolN-N-Hydroxysuccinimide (sulfo-NHS) and 300nmol carbodiimide (EDC) is added successively in microcentrifugal tube, with phosphate buffer (2.9g/L sodium hydrogen phosphate, 0.295g/L sodium dihydrogen phosphate, 4g/L sodium chloride, pH7.4) constant volume is 2ml, ceaselessly mixed solution, after 37 DEG C of reaction 30min, excessive sulfo-NHS and the EDC as activator is removed in dialysis.In the quantum dot of activation, add the mouse-anti people IgM monoclonal antibody of 6nmol, lucifuge reaction 2h, adds single-ended amination polyglycol (PEG2000-NH 2) to final concentration be 1%, close unreacted activated carboxyl site, continue lucifuge reaction 1h.With 0.2 μm of PES frit removing antibody aggregation thing, then filtrate transferred in 50000MW ultra-filtration centrifuge tube, with 8000g centrifugal force centrifugal 15min at 4 DEG C, there is not the antibody of coupling reaction and the accessory substance in reacting in removing.Collect super filter tube filter membrane upper strata quantum dot-antibody coupling matter solution, be dissolved in 2ml phosphate cleansing solution (2.9g/L sodium hydrogen phosphate, 0.295g/L sodium dihydrogen phosphate, 4g/L sodium chloride, 5ml/L Tween-20, 0.3g/L Sodium azide, pH7.4) in, again this solution is transferred in 50000MW ultra-filtration centrifuge tube, with 8000g centrifugal force centrifugal 15min at 4 DEG C, collect super filter tube filter membrane upper strata quantum dot-antibody coupling matter solution, be dissolved in 1ml phosphate conserving liquid (2.9g/L sodium hydrogen phosphate, 0.295g/L sodium dihydrogen phosphate, 2g/L sodium chloride, 10g/LBSA, 0.3g/L Sodium azide, pH7.4) in, so far obtained quantum dot-labeled anti-human IgM nano-probe, be placed in 4 DEG C to save backup.
The carboxyl water-soluble quantum dot that mouse-anti human IgG monoclonal antibody and emission wavelength are 600nm is utilized to obtain quantum dot-labeled anti-human igg nano-probe by above-mentioned same procedure.By the nano-probe solution 1:1 mixing by volume of these two amounts point mark, namely obtain anti-human IgM, IgG nano-probe that color quantum point marks respectively;
Embodiment 4 carboxyl nanometer magnetic bead carries out the optimization of immunocapture condition to people's Chlamydia pneumoniae antigen
Using coupling, the magnetic bead of restructuring M98-His fusion is as solid phase carrier, and anti-human IgM, IgG nano-probe that color quantum point marks respectively, as detection antibody, sets up the detection system of people's chlamydia pneumoniae (cp).Respectively to the consumption of carboxyl nanometer magnetic bead in detection system, the conditions such as capture time have carried out a series of optimum choice.
Test the selection of 1. carboxyl nanometer magnetic bead additions
By 5 μ l, 10 μ l, 15 μ l, 20 μ l, 30 μ l, 50 μ l, 70 μ l by the made anti-human chlamydia pneumoniae (cp) got ready of embodiment 2 catch nanometer magnetic bead join respectively 0.5ml containing anti-human CPn IgG antibody human serum dilution in, carry out immunocapture, anti-human IgM, IgG nano-probe that color quantum point again described by embodiment 3 marks respectively detects, record fluorescent value.Found that, along with the increase of carboxyl nanometer magnetic bead addition, fluorescent value increases gradually, and when carboxyl nanometer magnetic bead addition reaches 20 μ l, fluorescent value reaches maximum.Continue the amount increasing carboxyl nanometer magnetic bead again, fluorescent value reduces on the contrary.Therefore this experimental selection 20 μ l is as the optimal addn of nanometer magnetic bead.
Test the selection of 2. immunocapture times
After determining the addition of magnetic bead, get four parts of made nanometer magnetic beads got ready of embodiment 2, at room temperature with 10r/min, anti-human chlamydia pneumoniae IgM antibody positive serum is carried out to the immunocapture of 5min, 10min, 15min, 20min, 30min and 40min, quantum dot-labeled probe again described by embodiment 3 detects, record fluorescent value.Found that, fluorescent value shows maximal value when immunocapture 15min, and along with the prolongation of time, numerical value is substantially constant.Therefore this experimental selection 15min is as the Best Times of immunocapture.
Simultaneously, using coupling, the carboxyl nanometer magnetic bead of restructuring M98-His fusion is as solid phase carrier, corresponding quantum dot-labeled mouse-anti human IgG monoclonal antibody is as detection antibody, set up the detection system of anti-human CPn IgG antibody, the contact conditions in corresponding detection system is optimized.Found that, the best capture condition in above-mentioned detection system all with above-mentioned experiment 1 and the result of testing given by 2 completely the same.
The preparation of embodiment 5PBST damping fluid
Get 8gNaCL, 0.2gKCl, 0.24KH 2pO 4, 1.44gNa 2hPO 4, 5gBSA, 0.3gNaN 3, 0.5mlTween-20 is dissolved in 900ml distilled water, adjusts pH to 7.4, then be settled to 1000ml with 5MNaOH.
The preparation of embodiment 6 quality-control product
6.1) positive quality control product:
6.1.1) anti-human chlamydia pneumoniae IgM antibody positive quality control product: determine that anti-human chlamydia pneumoniae IgM antibody is that positive serum is formed by 0.5ml people's infection involving chlamydia pneumoniae person;
6.1.2) anti-human CPn IgG antibody positive quality-control product: determine that anti-human CPn IgG antibody is that positive serum is formed by 0.5ml people's infection involving chlamydia pneumoniae person;
6.2) negative quality-control product: be negative human serum by the anti-human Chlamydia pneumoniae IgM of 0.5ml, IgG antibody and form.
The preparation of embodiment 7 kit
Anti-human IgM, IgG nano-probe 5ml that anti-human chlamydia pneumoniae (cp) described by embodiment 2 color quantum point of catching described by nanometer magnetic bead 2ml, embodiment 3 marks respectively, the PBST damping fluid 200ml described by embodiment 5, quality-control product described by embodiment 6 are a set of to be formed jointly based on the anti-human Chlamydia pneumoniae IgM of magnetic resolution and color quantum point mark, IgG antibody check reagent box altogether fast.
The determination of embodiment 8 serum dilution to be checked
All be defined as anti-human chlamydia pneumoniae IgM antibody by clinical various method and be respectively feminine gender and each 20 parts positive of human serum sample, 1:50, l:lOO, 1:200, l:400,1:800 is carried out respectively with PBSA damping fluid, 1:1600 doubly dilutes, and selects the suitableeest working concentration of most high dilution as serum to be checked of the ratio >3 of positive serum and the detection fluorescence values of negative serum.Found that, the best results of 200 times of dilutions.
Equally, all be defined as anti-human CPn IgG antibody by clinical various method and be respectively feminine gender and each 20 parts positive of human serum sample, 1:50, l:lOO, 1:200, l:400,1:800 is carried out respectively with PBSA damping fluid, 1:1600 doubly dilutes, and selects the suitableeest working concentration of most high dilution as serum to be checked of the ratio >3 of positive serum and the detection fluorescence values of negative serum.Result also finds, the best results of 200 times of dilutions.
The using method of embodiment 9 kit
1) getting after serum sample 5 μ l 995 μ lPBSA damping fluids to be checked dilute (200 times of dilutions) proceeds in the common centrifuge tube of 1.5ml by dilution;
2) to step 1) in centrifuge tube in add successively based on magnetic resolution and color quantum point mark anti-human Chlamydia pneumoniae IgM, the anti-human chlamydia pneumoniae (cp) of IgG antibody fast altogether in check reagent box catches nanometer magnetic bead 20 μ l and the anti-human Chlamydia pneumoniae IgM based on magnetic resolution and color quantum point mark, IgG antibody is total to the anti-human IgM that the color quantum point in check reagent box marks respectively fast, IgG nano-probe 50 μ l, take off after reacting 15min with 15rpm on rotary mixer under room temperature, centrifuge tube is inserted nano magnetic separation vessel Magneto separate 3min, with pipettor sucking-off supernatant,
3) add and wash twice based on the anti-human Chlamydia pneumoniae IgM of magnetic resolution and color quantum point mark, the IgG antibody PBST damping fluid 1ml fast altogether in check reagent box, sucking-off cleansing solution after employing nano magnetic separation vessel Magneto separate, finally use the resuspended magnetic bead of 0.2mlPBS damping fluid, obtained nanometer magnetic bead-people's chlamydia pneumoniae (cp)-quantum dot " sandwich " compound;
4) by step 3) obtain 0.2ml nanometer magnetic bead-people's chlamydia pneumoniae (cp)-quantum dot compound is loaded in ELISA Plate, use fluorescence microplate reader to be all 365nm in excitation wavelength (Ex), emission wavelength (Em) carries out difference reading to its fluorescent value under being respectively the parameter of 520nm and 600nm respectively;
5) determination of CUT-OFF value: by and above-mentioned steps 1)-4) same method detects that 100 parts are all defined as anti-human Chlamydia pneumoniae IgM through clinical various method, IgG antibody is negative human serum sample, read respectively emission wavelength (Em) be respectively 520nm and 600nm under fluorescent value; The described human serum sample that 100 parts are all defined as anti-human Chlamydia pneumoniae IgM through clinical various method, IgG antibody is feminine gender is designated as CUT-OFF1 value (71.78) at the mean value (42.5) of the fluorescent value that emission wavelength (Em) is 520nm with 3 times of standard deviation (9.76) sums; The described human serum sample that 100 parts are all defined as anti-human Chlamydia pneumoniae IgM through clinical various method, IgG antibody is feminine gender is designated as CUT-OFF2 value (97.92) at the mean value (57.3) of the fluorescent value that emission wavelength (Em) is 600nm with 3 times of standard deviation (13.54) sums;
6) by and above-mentioned steps 1)-4) four parts of negative quality-control product samples simultaneously providing in detection kit of identical method and four parts of positive quality control product samples, read respectively emission wavelength (Em) be respectively 520nm and 600nm under fluorescent value; Four parts of negative quality-control product samples are designated as NCX1 at the mean value of the fluorescent value that emission wavelength (Em) is 520nm; Four parts of negative quality-control product samples are designated as NCX2 value at the mean value of the fluorescent value that emission wavelength (Em) is 600nm; Two parts of anti-human chlamydia pneumoniae IgM antibody positive quality control product samples are PCX1 at the fluorescence mean value that emission wavelength (Em) is 520nm; Two parts of anti-human CPn IgG antibody positive quality-control product samples in emission wavelength (Em) are and fluorescent value under 600nm is designated as PCX2; If step 4) in serum sample to be checked be greater than CUT-OFF1 value (71.78) and PCX1/NCX1 >=2.1 at the detection fluorescent value that emission wavelength (Em) is 520nm, to be then judged as in serum sample to be checked that anti-human chlamydia pneumoniae IgM antibody is for positive; If step 4) in serum sample to be checked be less than CUT-OFF1 value (71.78) and PCX1/NCX1 >=2.1 at the detection fluorescent value that emission wavelength (Em) is 520nm, then the anti-human chlamydia pneumoniae IgM antibody in serum sample to be checked that judges to behave is feminine gender; If step 4) in serum sample to be checked be greater than CUT-OFF2 value (97.92) and PCX2/NCX2 >=2.1 at the detection fluorescent value that emission wavelength (Em) is 600nm, to be then judged as in serum sample to be checked that anti-human CPn IgG antibody is for positive; If step 4) in serum sample to be checked be less than CUT-OFF2 value (97.92) and PCX2/NCX2 >=2.1 at the detection fluorescent value that emission wavelength (Em) is 600nm, to be then judged as in serum sample to be checked that anti-human CPn IgG antibody is for negative; If PCX1/NCX1 < 2.1 or PCX2/NCX2 < 2.1, all show that kit lost efficacy.
The specific test of embodiment 10 kit
Replace the kit as described in people's chlamydia pneumoniae (cp) positive serum embodiment 7 to detect by the method as described in embodiment 9 with the serum that the corresponding pathogen antigen of respiratory tract common causative as human respiratory syncytial virus, people's mycoplasma pneumoniae, chlamydia psittaci, chlamydia trachomatis, adenovirus hominis 3 type, adenovirus hominis 7 type, influenza virus A hominis, people's influenza B virus, human influenza haemophilus, people's streptococcus pneumoniae infection person is positive, find that testing result is feminine gender.
Embodiment 11 clinical trial example
Anti-human Chlamydia pneumoniae IgM based on magnetic resolution and color quantum point mark prepared by application the present invention, IgG antibody fast altogether check reagent box clear and definite anti-human 120 parts, the chlamydia pneumoniae IgM antibody positive serum sample of clinical diagnosis and 70 parts, negative serum sample are detected, testing result is as table 1.
Table 1 anti-human chlamydia pneumoniae IgM antibody clinical serum assay
Anti-human Chlamydia pneumoniae IgM based on magnetic resolution and color quantum point mark prepared by application the present invention, IgG antibody fast altogether check reagent box clear and definite anti-human 70 parts, the CPn IgG Positive Sera sample of clinical diagnosis and 50 parts, negative serum sample are detected, testing result is as table 2.
Table 2 anti-human CPn IgG antibody clinical serology results
It is pointed out that and the foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments, equivalent replacement etc. made within the present invention's spirit and principle all should be included within protection scope of the present invention.

Claims (4)

1. for the preparation of the method based on the anti-human Chlamydia pneumoniae IgM of magnetic resolution and color quantum point mark, IgG antibody altogether check reagent box fast, the described anti-human Chlamydia pneumoniae IgM based on magnetic resolution and color quantum point mark, IgG antibody fast altogether check reagent box be made up of the anti-human IgM that there is the anti-human Chlamydia pneumoniae IgM of enrichment, the anti-human chlamydia pneumoniae (cp) of IgG antibody function catches nanometer magnetic bead, color quantum point marks respectively, IgG antibody nano-probe, quality-control product and PBST damping fluid; Described quality-control product comprises positive quality control product and negative quality-control product; Described positive quality control product is that positive serum forms by the serum of the anti-human chlamydia pneumoniae IgM antibody positive of two parts of people's infection involving chlamydia pneumoniae persons and two parts of anti-human CPn IgG antibody; Described negative quality-control product is four parts of anti-human Chlamydia pneumoniae IgM, IgG antibody is negative human serum; It is characterized in that: said method comprising the steps of:
1) anti-human chlamydia pneumoniae (cp) catches the preparation of nanometer magnetic bead:
1.1) to recombinate the preparation of M98-His fusion, purifying:
1.1.1) bioinformatic analysis is carried out to people Chlamydia pneumoniae 98KD outer membrane protein, obtain the peptide section that in people Chlamydia pneumoniae 98KD memebrane protein ectodomain, epitope enriches the most, find the gene order of its correspondence;
1.1.2) in step 1.1.1) in the 5 ' end of gene order that obtains and 3 ' end introduce restriction enzyme site chemosynthesis complete genome sequence respectively, be designated as M98 with tense marker;
1.1.3) by step 1.1.2) in the M98 that obtains be cloned into expression vector pET-28a (+) by molecular biology method after proceed to expression in escherichia coli restructuring M98-His fusion; Described restructuring M98-His fusion is present in genetic engineering thalline with inclusion body expression-form;
1.1.4) with ni-sepharose purification step 1.1.3) the restructuring M98-His fusion that obtains, after SDS-PAGE detects its purity, again renaturation is carried out to restructuring M98-His fusion, carrying out after determination of protein concentration through bradford kit, is 0.2mg/mL by PBS damping fluid adjustment concentration; In described PBS damping fluid, each component content is as follows: 8g/LNaCL, 0.2g/LKCl, 0.24g/LKH 2pO 4, 1.44g/LNa 2hPO 4, the pH=7.4 of described PBS damping fluid;
1.2) the bag quilt of nanometer magnetic bead:
1.2.1) get 5mg magnetic bead, with 1mlMES buffer solution three times, be placed in after nano magnetic separation vessel carries out Magneto separate and remove supernatant; Described magnetic bead is with superparamagnetism Fe 3o 4for the carboxyl magnetic bead that kernel, particle diameter are 1150nm; 2-(N-morpholino) ethyl sulfonic acid of described MES damping fluid to be mass concentration be 2g/L; The pH=6.0 of described MES damping fluid; The magnetic intensity of described nano magnetic separation vessel is 0.4T;
1.2.2) add successively use step 1.2.1) in the concentration of MES buffer be the EDC solution of 8-12mg/ml and use step 1.2.1) in the concentration of MES buffer be each 0.5ml of sulfo-NHS solution of 6-10mg/ml, in rotary mixer, 1hr is activated with 10-40rpm, be placed in after nano magnetic separation vessel carries out Magneto separate and remove supernatant, with 1ml step 1.2.1) in MES damping fluid resuspended, obtain activate after magnetic bead;
1.2.3) 5 centrifuge tubes are got, 200 μ L step 1.2.2 are added in each centrifuge tube) magnetic bead after the activation that obtains, be placed in after nano magnetic separation vessel carries out Magneto separate and remove supernatant, adding by the concentration of PBS damping fluid dilution in each centrifuge tube is 50-200 μ g/ml by step 1.1.4) prepared by each 1ml of restructuring M98-His fusion solution, in rotary mixer, 2-6h is reacted with 15rpm under room temperature, be placed in after removing supernatant after nano magnetic separation vessel carries out Magneto separate, respectively add the above-mentioned PBS damping fluid of 1ml containing 1mg/ml monoethanolamine, under room temperature with 15rpm react in rotary mixer 2h with on closed magnetic bead not with the carboxyl of antibody response, in described PBS damping fluid, each component content is as follows: 8g/LNaCL, 0.2g/LKCl, 0.24g/LKH 2pO 4, 1.44g/LNa 2hPO 4, the pH=7.4 of described PBS damping fluid,
1.2.4) after capping completes, these 5 centrifuge tubes are placed in after nano magnetic separation vessel carries out Magneto separate and remove supernatant, respectively wash three times with 1ml lavation buffer solution; In described lavation buffer solution, each component content is as follows: 8g/LNaCL, 0.2g/LKCl, 0.24g/LKH 2pO 4, 1.44g/LNa 2hPO 4, 0.5ml/LTween-20, the pH=7.4 of described lavation buffer solution;
1.2.5) in each centrifuge tube, add 1ml respectively preserve the resuspended magnetic bead of damping fluid, be placed in 4 DEG C and save backup; So far obtained anti-human chlamydia pneumoniae (cp) catches nanometer magnetic bead; In described preservation damping fluid, each component content is as follows: 8g/LNaCL, 0.2g/LKCl, 0.24g/LKH 2pO 4, 1.44g/LNa 2hPO 4, 0.3g/LNaN 3, 5g/LBSA, the pH=7.4 of described preservation damping fluid;
2) preparation of anti-human IgM, IgG nano-probe that marks respectively of color quantum point:
Its concrete preparation method comprises:
2.1) in microcentrifugal tube, add 2nmol carboxyl water-soluble quantum dot, 300nmolN-N-Hydroxysuccinimide sulfo-NHS and 300nmol carbodiimide EDC successively, with phosphate buffer constant volume for 5ml, mixed solution, after 37 DEG C of reaction 30min, excessive sulfo-NHS and EDC as activator is removed in dialysis, obtains the quantum dot after activating; The emission wavelength of described carboxyl water-soluble quantum dot is 520nm; In described phosphate buffer, each component content is as follows: 2.9g/L sodium 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) in the quantum dot of activation that obtains, add the mouse-anti people IgM monoclonal antibody of 2-12nmol, lucifuge reaction 2h, adds single-ended amination polyglycol PEG2000-NH 2be 1% to final concentration, close unreacted activated carboxyl site, continue lucifuge reaction 1h;
2.3) by 0.2 μm of PES frit removing step 2.2) in antibody aggregation thing, then filtrate is transferred in 50000MW ultra-filtration centrifuge tube, with 8000g centrifugal force centrifugal 15min at 4 DEG C, there is not the antibody of coupling reaction and the accessory substance in reacting in removing;
2.4) step 2.3 is collected) middle ultra-filtration centrifuge tube filter membrane upper strata quantum dot-antibody coupling matter solution, be dissolved in 2ml phosphate cleansing solution, again this solution is transferred in a new 50000MW ultra-filtration centrifuge tube, with 8000g centrifugal force centrifugal 15min at 4 DEG C, collect ultra-filtration centrifuge tube filter membrane upper strata quantum dot-antibody coupling matter solution, be dissolved in 1ml phosphate conserving liquid, be placed in 4 DEG C and save backup, so far obtained quantum dot-labeled anti-human IgM nano-probe; In described phosphate cleansing solution, each component content is as follows: 2.9g/L sodium hydrogen phosphate, 0.295g/L sodium dihydrogen phosphate, 4g/L sodium chloride, 5ml/L Tween-20,0.3g/L Sodium azide, the pH=7.4 of described phosphate cleansing solution; In described phosphate conserving liquid, each component content is as follows: 2.9g/L sodium hydrogen phosphate, 0.295g/L sodium dihydrogen phosphate, 2g/L sodium chloride, 10g/L bovine serum albumin(BSA), 0.3g/L Sodium azide; The pH=7.4 of described phosphate conserving liquid;
2.5) by and step 2.1)-2.4) identical method utilizes the carboxyl water-soluble quantum dot that mouse-anti human IgG monoclonal antibody and emission wavelength are 600nm to obtain quantum dot-labeled anti-human igg nano-probe; By the nano-probe solution 1:1 mixing by volume of these two amounts point mark, namely obtain anti-human IgM, IgG nano-probe that color quantum point marks respectively;
3) preparation of PBST damping fluid:
Its concrete compound method comprises:
Get 8gNaCL, 0.2gKCl, 0.24KH 2pO 4, 1.44gNa 2hPO 4, 5gBSA, 0.3gNaN 3, 0.5mlTween-20 is dissolved in 900ml distilled water, adjusts pH to 7.4, then be settled to 1000ml with 5MNaOH;
4) preparation of quality-control product:
4.1) positive quality control product:
4.1.1) anti-human chlamydia pneumoniae IgM antibody positive quality control product: determine that anti-human chlamydia pneumoniae IgM antibody is that positive serum is formed by 0.5ml people's infection involving chlamydia pneumoniae person;
4.1.2) anti-human CPn IgG antibody positive quality-control product: determine that anti-human CPn IgG antibody is that positive serum is formed by 0.5ml people's infection involving chlamydia pneumoniae person;
4.2) negative quality-control product: be negative human serum by the anti-human Chlamydia pneumoniae IgM of 0.5ml, IgG antibody and form.
2. method according to claim 1, it is characterized in that: described step 1.2.2) in, add successively and use step 1.2.1) in the concentration of MES buffer be the EDC solution of 10mg/ml and use step 1.2.1) in the concentration of MES buffer be each 0.5ml of sulfo-NHS solution of 8mg/ml, in rotary mixer, 1hr is activated with 15rpm, be placed in after nano magnetic separation vessel carries out Magneto separate and remove supernatant, with 1ml step 1.2.1) in MES damping fluid resuspended, obtain activate after magnetic bead;
Described step 1.2.3) in, adding by the concentration of PBS damping fluid dilution in each centrifuge tube is 150 μ g/ml by step 1.1.4) prepared by each 1ml of restructuring M98-His fusion solution, in rotary mixer, 3h is reacted with 15rpm under room temperature, be placed in after removing supernatant after nano magnetic separation vessel carries out Magneto separate, respectively add the above-mentioned PBS damping fluid of 1ml containing 1mg/ml monoethanolamine;
Described step 2.2) in, in step 2.1) in the quantum dot of activation that obtains, add the mouse-anti people IgM monoclonal antibody of 6nmol, lucifuge reaction 2h.
3. method according to claim 1 and 2, is characterized in that: described quantum dot is water-soluble CdSe/ZnS quantum dot that carboxylated amphipathic polymer is modified.
4. method according to claim 3, is characterized in that: described magnetic bead is with superparamagnetism Fe 3o 4for the carboxyl magnetic bead that kernel, shell material are polystyrene, surface functional group is carboxyl, particle diameter is 1150nm.
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