CN105859884A - Anti-human mycoplasma pneumoniae P30 protein antibody and immunochromatographic kit applying same - Google Patents
Anti-human mycoplasma pneumoniae P30 protein antibody and immunochromatographic kit applying same Download PDFInfo
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- CN105859884A CN105859884A CN201610317213.6A CN201610317213A CN105859884A CN 105859884 A CN105859884 A CN 105859884A CN 201610317213 A CN201610317213 A CN 201610317213A CN 105859884 A CN105859884 A CN 105859884A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
- C07K16/1253—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Mycoplasmatales, e.g. Pleuropneumonia-like organisms [PPLO]
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/10—Detection of antigens from microorganism in sample from host
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Abstract
The invention relates to an anti-human mycoplasma pneumoniae P30 protein antibody and an immunochromatographic kit for detecting human mycoplasma pneumoniae by applying the antibody. The anti-human mycoplasma pneumoniae P30 protein antibody is used for identifying a linear epitope formed by 56th-69th amino acids of human mycoplasma pneumoniae P30 protein; the sequence number of the human mycoplasma pneumoniae P30 protein in a GenBank is ABR09215.1; the sequence of each of the 56th-69th amino acids of the human mycoplasma pneumoniae P30 protein is AEEDTVQIQGKPIT. The rabbit anti-human mycoplasma pneumoniae P30 protein antibody provided by the invention has the characteristics of being good in specificity, high in purity, high in potency and low in preparation cost.
Description
Technical field
The invention belongs to field of biomedicine technology, relate to anti-human mycoplasma pneumoniae P30 protein antibodies and application should
The immune chromatography reagent kit of antibody test people's mycoplasma pneumoniae.
Background technology
Mycoplasma pneumoniae (M.Pneumonia) is the pathogen of mankind's mycoplasma pneumonia.The pathology of mycoplasma pneumonia
Change based on interstitial pneumonia, sometimes concurrent bronchopneumonia, referred to as primary atypical pneumonia.Mainly
Through droplet infection, incubation period 2～3 weeks, sickness rate is the highest with teenager.Clinical symptoms is headache, pharyngalgia, sends out
The general respiratory symptoms such as heat, cough, the most all can occur, but many in time autumn and winter.Mp infects
The incidence rate of infantile pneumonia cause of disease is up to 10-30%, is increasingly becoming children's in recent years and infects the main of respiratory tract disease
One of pathogen.This disease easily causes the respiratory tract infection such as pharyngitis, tonsillitis, in some instances it may even be possible to secondary brain simultaneously
The multiple organ injuries such as film inflammation, hepatitis, myocarditis, may also lead to infant dead time serious.
Owing to Mp infects similar with the respiratory tract infection symptom that other pathogen causes, do not do etiological examination, very
Difficult respiratory tract infection Mp and other pathogen caused distinguishes.Mp is acellular wall, conventional beta-lactam
Class medicine is invalid to it, therefore the treatment of its infection caused is complete with the therapeutic scheme that other antibacterial and virus infect
Difference, hence set up simplicity, quick, feasible, can the method for early diagnosis mycoplasma pneumoniae infection the most necessary.
The detection method of Mp mainly has 3 classes at present: one is isolated culture, and it is to confirm to infect " goldstandard ",
But owing to the growth cycle of Mp is extremely slow, cultivation cycle is long, causes this method can not quickly examine clinically
Disconnected；Two is serological method, i.e. use euzymelinked immunosorbent assay (ELISA), colloidal gold immunization, micro-Immunofluorescence assay and
Connect hemagglutination test etc., Mp antibody horizontal in detection examinee's serum, can indirectly point out the Mp existence infected.But,
Serological test can only provide a kind of retrospective diagnosis, and sometimes for paired sera.It addition, antibody goes out
It is difficult to existing opportunity grasp, between children and adolescents and adult, there is again the difference of Mp specific antibody, and,
There is non-specific cross-reaction with other microorganisms and body tissue in the glycolipid antigen on Mp cell membrane, therefore existing
The detection quality of serological method is by a definite limitation；Three is to utilize depositing of Protocols in Molecular Biology detection Mp DNA
, most common of which is polymerase chain reaction (PCR), and the method is quick, sensitive, special, is current
The important means that research Mp infects, but owing to experimental facilities and operation are required higher by PCR, and vacation easily occurs
The positive, can't be as conventional methods for clinical diagnosis in China.Therefore, Mp specific antigen diagnosis side is set up
Method is the most necessary.At present, it has been disclosed that the method for the detection Mp antigen of report is mainly double crush syndrome method,
Indirect immunofluorescence, quantum dot mark immunity-chromatography method etc., but these methods all can not carry out the other detection of bed,
Need to specific occasion to utilize specific instrument (such as microplate reader, luminoscope etc.) to detect, the most square
Just quick and the time is longer, clinical practice is the most inconvenient.
Therefore, the fast diagnosis method setting up people's mycoplasma pneumoniae specific antigen is the most necessary.Due to human influenza
Well-conserved on mycoplasma pneumoniae P30 protein sequence so that it is become an important examination target.Cause
This anti-human mycoplasma pneumoniae P30 protein antibodies obtaining high specific is exactly a highly important job.At present,
About anti-human mycoplasma pneumoniae P30 protein antibodies report at most for corresponding monoclonal antibody and Anti-TNF-α
Body.The advantage of monoclonal antibody maximum is exactly that specificity is high, but preparation method is loaded down with trivial details, and production cost is high, limit
Make its application.Polyclonal antibody is mainly by animal preparations such as the P30 protein immunization rabbit of gene engineering expression
Become.Its preparation method is simple, low cost, but the P30 albumen of gene engineering expression is inclusion body structure, no
Tool native protein structure, with its antibody prepared, it has the defects such as specificity is low, titer is low.Therefore, low one-tenth
This prepare high specific, the anti-human mycoplasma pneumoniae P30 protein antibodies of high-titer just seems particularly significant.
Summary of the invention
For these technical problems present in background technology, it is an object of the invention to provide identification people's pneumonia and prop up
The antibody of the linear epitope that substance P30 albumen 56-69 amino acids is formed and apply the immunity of this antibody
Chromatography test kit.
Anti-human mycoplasma pneumoniae P30 protein antibodies, it is characterised in that: described anti-human mycoplasma pneumoniae P30 albumen resists
Body is the antibody of the linear epitope that identification people's mycoplasma pneumoniae P30 albumen 56-69 amino acids is formed, institute
Stating people's mycoplasma pneumoniae P30 albumen is ABR09215.1 at GenBank serial number；Described people mycoplasma pneumoniae P30
14 aminoacid sequences of albumen 56-69 position are AEEDTVQIQGKPIT；By people's mycoplasma pneumoniae P30 albumen
14 amino acid whose sequence designations of 56-69 position are P30Line；Described anti-human mycoplasma pneumoniae P30 protein antibodies
It is AbP30Line.
A kind of immunochromatography reagent formed based on foregoing anti-human mycoplasma pneumoniae P30 protein antibodies
Box, it is characterised in that: described test kit is immune chromatography reagent kit based on quantum dot-labeled technology or based on glue
The immune chromatography reagent kit of body gold labelling technique.
As preferably, test kit provided by the present invention is immune chromatography reagent kit based on quantum dot-labeled technology
Time, the preparation method of described test kit is:
1) quantum dot-labeled antibody A bP30Line:
0.4nmol carboxyl water-soluble quantum dot and 800nmol carbodiimide it is sequentially added in microcentrifugal tube
EDC, with MES buffer constant volume as 1ml, mixed solution, after 37 DEG C of reaction 5min, add 0.34mg
Antibody A bP30Line prepared, lucifuge reaction 2h, add single-ended amino-polyethyleneglycols PEG2000-NH2
To final concentration of 1% (m/v), close unreacted activated carboxyl site, continue lucifuge reaction 1h；After reaction
Sample with super filter tube centrifugal (molecular cut off 100k), 6500g is centrifuged 5min, to volume 200ul, by ultrafiltration
Rear sample is transferred in common EP pipe, centrifugal except reuniting, and obtains upper clear supernate and bottom precipitation, 10000g
Under the conditions of centrifugal 3min；Upper clear supernate is added to purification on detached dowel Superdex-200, treats that upper clear supernate is natural
Flow in cylinder, then rinse with PBS, irradiate cylinder with ultraviolet light and observe the position of sample, treat that sample starts
Start when flowing out from bottom to collect, stop collecting after collecting 1ml；Sample super filter tube after purification (is retained
Molecular weight 100k) with centrifugal except reuniting, to commonly in being transferred to common EP pipe after 6500g centrifugal concentrating to 200ul
The condition that EP pipe is centrifuged is 10000g, 3min；Preserve liquid with phosphate after obtaining supernatant and dilute 200 times, 4 DEG C
Save backup；So far the solution containing quantum dot traget antibody AbP30Line is prepared；
In described MES buffer, each constituent content is respectively: 10.66g/L MES and 0.74g/L EDTA,
The pH 7.4 of described MES buffer；
It is to weigh 0.29g disodium hydrogen phosphate, 0.0295g biphosphate that described phosphate preserves the preparation method of liquid
Sodium, 0.2g sodium chloride, 1g bovine serum albumin BSA and 0.1gNaN3, it is dissolved in the deionization of 90ml
In water, after adjusting pH to 7.3 with 1mol/L NaOH, it is settled to 100ml with deionized water；
2) pad is prepared
By polyester fiber film immerse step 1) obtained by the solution containing quantum dot traget antibody AbP30Line in
1h, takes out, and 25 DEG C are cut into after rear specification is 4cm × 0.6cm/ bar after drying, and 4 DEG C of sealings save backup, extremely
This prepared pad；
3) sample pad is prepared
Take glass fibre element film one, glass fibre element film is soaked in sample pad treatment fluid at least 3h, then
It is placed in Biohazard Safety Equipment after 37 DEG C of aeration-dryings, is cut into after specification is 4cm × 2.5cm/ bar, i.e. prepares sample
Product pad, 25 DEG C seal preservation；
The preparation method of described sample pad treatment fluid is to weigh 0.29g disodium hydrogen phosphate, 0.0295g biphosphate
Sodium, 0.2g sodium chloride, 2g bovine serum albumin BSA, 1ml tween 20,2g sucrose and 0.5g are poly-
Vinylpyrrolidone PVP-10, is dissolved in the deionized water of 90ml, adjusts pH to 7.3 with 1mol/L NaOH
It is settled to 100ml afterwards with deionized water；
4) detection layers is prepared
With invention entitled " the people's mycoplasma pneumoniae gold label silver stain immunochromatography inspection of Application No. 201410405275.3
Test agent box and its preparation method and application " description in [0134th] section to disclosed in [0158] section
Content prepares rabbit anti-restructuring P1-His fusion protein polyclonal antibody IgG.
Nitrocellulose filter is cut into 4cm × 4cm size；The anti-restructuring of rabbit that will prepare as stated above
P1-His fusion protein polyclonal antibody IgG and goat anti-rabbit igg phosphate buffer adjust to final concentration respectively
For 2.0mg/mL and 1.0mg/mL；The rabbit anti-restructuring P1-His fusion protein polyclonal antibody IgG that will have diluted
Loading BIODOT to draw in film instrument shower nozzle, the amount arranging 1.0 μ l/cm is sprayed on nitrocellulose filter, forms detection
Line；Being drawn in film instrument shower nozzle by anti-rabbit IgG diluted loading BIODOT, the amount arranging 1.0 μ l/cm is sprayed on nitre
As nature controlling line on acid cellulose film, nature controlling line is 0.7cm with detection distance between centers of tracks；The celluloid that will have sprayed
37 DEG C of dry 2h of film, are cut into the specification of 4cm × 4cm, and 4 DEG C of hermetically dryings preserve；So far detection layers is prepared；
The preparation method of described phosphate buffer is: weigh 0.29g disodium hydrogen phosphate, 0.0295g di(2-ethylhexyl)phosphate
Hydrogen sodium and 0.2g sodium chloride, be dissolved in the deionized water of 90ml, adjusts pH to 7.3 with 1mol/L NaOH
It is settled to 100ml afterwards with deionized water；
5) detection card is assembled
5.1) base plate is cut into 4cm × 7.3cm size, standby；
5.2) absorbent filter is cut into 4cm × 3cm size, as adsorptive pads, standby；
5.3) by step 5.1) adhered protection film on the base plate for preparing takes off, by step 4) described in
Detection layers i.e. pastes on base plate with nature controlling line and the nitrocellulose filter detecting line, and floating face；
5.4) by step 5.2) ready adsorptive pads is assembled on base plate, makes the left side and the detection layers of adsorptive pads
Right end has the overlap of 0.2cm, and the right hand edge of adsorptive pads then aligns with the right hand edge of base plate and glues and floating；Again
By step 2) described in pad be overlapped at the left hand edge of detection layers by 0.3cm, 0.3cm sticks on base plate；
5.5) by step 3) described in sample pad be then overlapped at the left hand edge of pad by one side 0.3cm, separately
While aliging with the left hand edge of base plate, stick on base plate and floating；The detection plate assembled is cut out under cutting cutter
Becoming detection card wide for 4.0mm, 4 DEG C of hermetically dryings keep in Dark Place.
As preferably, test kit provided by the present invention is immune chromatography reagent kit based on colloidal gold-labeled method
Time, the preparation method of described test kit is:
1) colloidal gold labeled monoclonal antibody AbP30Line:
1.1) 30nm colloidal gold solution is prepared:
Take a good 250ml triangular flask of silication, add 99ml ultra-pure water, by 1ml 1% (m/v) HAuCl4Molten
Liquid adds in 250ml triangular flask and mixes with ultra-pure water, and oil bath is heated and stirs to boiling；To 250ml triangular flask
In rapidly join 2ml 1% (m/v) trisodium citrate aqueous solution, solution continues boiling 10min, treats 250ml triangle
Solution in Ping stops heating when being changed into redness by blueness, is naturally cooled to by the solution in 250ml triangular flask
Room temperature, then adds ultra-pure water polishing to 100ml in 250ml triangular flask；
1.2) colloidal gold labeled monoclonal antibody AbP30Line:
1.2.1) take a good 50ml triangular flask of silication, add 10ml step 1.1) prepared by gold colloidal
Solution, adds 240ul 0.2mol/L K in gold colloidal liquid2CO3Regulation pH to 8.5；
1.2.2) under magnetic stirrer, antibody A bP30Line is added in colloidal gold solution, to antibody
Final concentration of 10ug/ml, is added dropwise over when adding antibody, continues stirring 45min～60min after adding；
1.2.3) reacted and added 5% (m/v) bovine serum albumin BSA to final concentration of 1% (m/v), stirred
Mixing 15～30 minutes, 4 DEG C save backup；
1.2.4) loading 50ml centrifuge tube, 2500g after antibody A bP30Line good for labelling being taken out, 4 DEG C are centrifuged 5
Minute, obtaining lower sediment and the supernatant, discard lower sediment, the supernatant is transferred to another 50ml
Centrifuge tube, 12000g, 4 DEG C are centrifuged 30 minutes, obtain lower sediment and the supernatant, discard the supernatant,
By the lower sediment 10ml resuspended precipitation of gold colloidal buffer, 12000g the most again, 4 DEG C are centrifuged 30 minutes, then
Secondary obtain lower sediment and the supernatant, lower sediment is finally resuspended with 3ml gold colloidal buffer, 4 DEG C of preservations
Standby, obtain the solution containing colloidal gold labeled monoclonal antibody AbP30Line；
In described gold colloidal buffer, each constituent content is respectively: 10mM Tris, 1%m/vBSA, 1%v/v
Tween-20,5%m/v sucrose and 3 ‰ m/v polyvinylpyrrolidone PVP-10, described gold colloidal buffer
PH be 10.5；
2) pad is prepared
By polyester fiber film immerse step 1) obtained by the solution containing colloidal gold labeled monoclonal antibody AbP30Line in
1h, takes out, and 25 DEG C are cut into after rear specification is 4cm × 0.6cm/ bar after drying, and 4 DEG C of sealings save backup, extremely
This prepared pad；
3) sample pad is prepared
Take glass fibre element film one, glass fibre element film is soaked in sample pad treatment fluid at least 2h, then
It is placed in Biohazard Safety Equipment after 37 DEG C of aeration-dryings, is cut into after specification is 4cm × 1.5cm/ bar, i.e. prepares sample
Product pad, 25 DEG C seal preservation；
The preparation method of described sample pad treatment fluid is to weigh 0.242g Tris, 1g bovine serum albumin BSA, 1ml
Tween 20,5g sucrose and 0.3g polyvinylpyrrolidone PVP-10, be dissolved in the deionized water of 90ml,
It is settled to 100ml with deionized water after adjusting pH to 11 with 1mol/L NaOH；
4) detection layers is prepared
With invention entitled " the people's mycoplasma pneumoniae gold label silver stain immunochromatography inspection of Application No. 201410405275.3
Test agent box and its preparation method and application " description in [0134th] section to disclosed in [0158] section
Content prepares rabbit anti-restructuring P1-His fusion protein polyclonal antibody IgG.
Nitrocellulose filter is cut into 4cm × 2cm size；The anti-restructuring of rabbit that will prepare as stated above
P1-His fusion protein polyclonal antibody IgG and goat anti-rabbit igg phosphate buffer adjust to final concentration respectively
For 2.0mg/mL and 1.0mg/mL；The rabbit anti-restructuring P1-His fusion protein polyclonal antibody IgG that will have diluted
Loading BIODOT to draw in film instrument shower nozzle, the amount arranging 1.0 μ l/cm is sprayed on nitrocellulose filter, forms detection
Line；Being drawn in film instrument shower nozzle by anti-rabbit IgG diluted loading BIODOT, the amount arranging 1.0 μ l/cm is sprayed on nitre
As nature controlling line on acid cellulose film, nature controlling line is 0.5cm with detection distance between centers of tracks；The celluloid that will have sprayed
37 DEG C of dry 18h of film, are cut into the specification of 4cm × 2cm, and 4 DEG C of hermetically dryings preserve；So far detection layers is prepared；
The preparation method of described phosphate buffer is: weigh 0.29g disodium hydrogen phosphate, 0.0295g di(2-ethylhexyl)phosphate
Hydrogen sodium and 0.2g sodium chloride, be dissolved in the deionized water of 90ml, adjusts pH to 7.3 with 1mol/L NaOH
It is settled to 100ml afterwards with deionized water；
5) detection card is assembled
5.1) base plate is cut into 4cm × 6cm size, standby；
5.2) absorbent filter is cut into 4cm × 2.5cm size, as adsorptive pads, standby；
5.3) by step 5.1) adhered protection film on the base plate for preparing takes off, by step 4) described in
Detection layers i.e. pastes on base plate with nature controlling line and the nitrocellulose filter detecting line, and floating face；
5.4) by step 5.2) ready adsorptive pads is assembled on base plate, makes the left side and the detection layers of adsorptive pads
Right end has the overlap of 0.2cm, and the right hand edge of adsorptive pads then aligns with the right hand edge of base plate and glues and floating；Again
By step 2) described in pad be overlapped at the left hand edge of detection layers by 0.2cm, 0.4cm sticks on base plate；
5.5) by step 3) described in sample pad be then overlapped at the left hand edge of pad by one side 0.2cm, separately
While aliging with the left hand edge of base plate, stick on base plate and floating；The detection plate assembled is cut out under cutting cutter
Becoming detection card wide for 4.0mm, 4 DEG C of hermetically dryings keep in Dark Place.
The invention have the advantage that
The invention provides anti-human mycoplasma pneumoniae P30 protein antibodies, this anti-human mycoplasma pneumoniae P30 protein antibodies
Identify the linear epitope that 14 aminoacid of people mycoplasma pneumoniae P30 albumen 56-69 position is formed, people's pneumonia
Mycoplasma P30 albumen is ABR09215.1 at GenBank serial number；People mycoplasma pneumoniae P30 albumen 56-69 position
14 amino acid whose aminoacid sequences are AEEDTVQIQGKPIT；By people mycoplasma pneumoniae P30 albumen 56-69 position
14 amino acid whose sequence designations are P30Line；Anti-human mycoplasma pneumoniae P30 protein antibodies is AbP30Line.
Resist based on the above-mentioned rabbit anti-human mycoplasma pneumoniae P30 albumen prepared by people's mycoplasma pneumoniae single linear epitope
Body has the advantages that specificity is good, purity is high, titer is high, preparation cost is cheap, can be used for producing various based on
The detection kit of high-sensitivity detection people's mycoplasma pneumoniae of the principle of antigen-antibody reaction.Meanwhile, based on
This anti-human mycoplasma pneumoniae P30 protein antibodies has also prepared two kinds of different immune chromatography reagent kits.Two kinds
Different immune chromatography reagent kits quickly, accurately can detect the people's mycoplasma pneumoniae in biological sample, and it all includes
Antibody of the present invention；The auxiliary that two kinds of immune chromatography reagent kits are used equally to people's mycoplasma pneumoniae infection is examined
Disconnected, there is higher susceptiveness and specificity, taken into account simple, quick, stable and low cost of manufacture simultaneously
Etc. advantage, it is adaptable to the inspection of clinical samples, and owing to large batch of quick inspection can be carried out, it also is adapted for
In Epidemiological study.Therefore, rabbit of the present invention anti-human mycoplasma pneumoniae P30 protein antibodies, exempt from for two kinds
Epidemic disease chromatography test kit is respectively provided with the prospect of being widely applied and practical value.
Detailed description of the invention
It is further appreciated by the present invention, preferred embodiment cited below particularly to contribute to those of ordinary skill in the art
Describe the present invention in detail.
" m/v " in concentration unit in present specification, when m unit is g, v unit is mL, belongs to ability
The general knowledge in territory.
The source of the various materials that the present invention uses or uses and the preparation of related reagent
1, sample pad treatment fluid: weigh 0.242g Tris, 1g bovine serum albumin (BSA), 1ml tween 20,
5g sucrose, 0.3g polyvinylpyrrolidone (PVP-10), it is dissolved in the deionized water of 90ml, uses 1mol/L
NaOH is settled to 100ml with deionized water after adjusting pH to 11.
2, phosphate preserves liquid: weigh 0.29g disodium hydrogen phosphate, 0.0295g sodium dihydrogen phosphate, 0.2g
Sodium chloride, 1g bovine serum albumin BSA and 0.1gNaN3, it is dissolved in the deionized water of 90ml, with 1
Mol/L NaOH is settled to 100ml with deionized water after adjusting pH to 7.3；
3, phosphate buffer (PBS): weigh 0.29g disodium hydrogen phosphate, 0.0295g sodium dihydrogen phosphate, 0.2
G sodium chloride, is dissolved in the deionized water of 90ml, uses deionized water after adjusting pH to 7.3 with 1mol/L NaOH
It is settled to 100ml.
4, sample treatment liquid: weigh 0.0121g trishydroxymethylaminomethane, 0.17g sodium chloride, 0.025g
Lysozyme is dissolved in 90ml deionized water, is settled to 100ml with deionized water after adjusting pH to 8.0 with hydrochloric acid.
5, antibody A bP30Line: make by oneself for the present invention, dilutes with PBS, shakes up, make antibody concentration in solution
For 3mg/ml.
6, rabbit anti-restructuring P1-His fusion protein polyclonal antibody IgG: make by oneself for the present invention, dilutes with PBS,
Shaking up, making antibody concentration in solution is 3mg/ml.
7, goat anti-rabbit igg: for Wuhan Boster Biological Technology Co., Ltd.'s product, dilute with PBS, shake up,
Making Anti-TNF-α bulk concentration in solution is 1mg/ml.
8, quantum dot: in the present invention, quantum dot used is water-soluble CdSe/ZnS that carboxylated amphipathic polymer is modified
Quantum dot, a length of 565nm of its transmitted wave, buys from Wuhan Jia Yuan technology of quantum dots development corporation, Ltd., product
Entitled carboxyl water-soluble quantum dot-565.
9, glass fibre element film: thickness is 0.4mm, water absorption is 42mg/cm2, glass fiber diameter is
0.6-3 μm, has good hydrophilic, buys in Shanghai Jinbiao Bio-Tech Co., Ltd. that (model is
BT40)。
10, polyester fiber film: thickness is 0.48mm, absorption speed is 18s/4cm, has fabulous hydrophilic
Property, for the preparation of pad, buy in Shanghai Jinbiao Bio-Tech Co., Ltd. (model is DL42).
11, nitrocellulose filter: model is Millipore Corp SHF135, has liner plate, buys in Millipore
Company.
12, absorbent filter: thickness is 0.95mm, absorption speed is 60s/4cm, and water absorption is 700mg/cm2,
There is good water absorption, as the material making adsorptive pads.Buy in Shanghai Jinbiao Bio-Tech Co., Ltd.
(model is CH37K).
13, base plate: for high whiteness PVC material, surface coating monolayer high polymer pressure sensitive adhesive SM31-40, buy
In Shanghai Jinbiao Bio-Tech Co., Ltd..
14, people's mycoplasma pneumoniae: purchased from American type culture collection (ATCC), numbered ATCC15531.
15, the microbiological specimens used in the present invention is purchased from American type culture collection (ATCC).
Below in conjunction with embodiment, technical scheme provided by the present invention is described in detail:
The preparation of embodiment 1 rabbit anti-human mycoplasma pneumoniae P30 protein antibodies
The preparation method of rabbit anti-human mycoplasma pneumoniae P30 protein antibodies is as follows:
1) after structure biology analysis and Related Experimental Study, selected people's mycoplasma pneumoniae P30 albumen
14 aminoacid of (GenBank serial number ABR09215.1) 56-69 position are as the preparation anti-human mycoplasma pneumoniae of rabbit
The linear epitope of P30 protein antibodies, this section of aminoacid sequence is AEEDTVQIQGKPIT, by this sequence designations is
P30Line；
2) by step 1) described in the C end of aminoacid sequence P30Line add after a cysteine, use polypeptide
Automatic synthesizer synthesis polypeptide purification, polypeptide after purification and carrier protein KLH, formed
P30Line-KLH compound protein；
3) by step 2) synthesized by compound protein emulsifying, at rabbit subcutaneous abdomen multi-point injection after emulsifying, successively
Inject three times, every minor tick 7-10 days；
4) after third time is injected 10-12 days, collect, isolated contains rabbit anti-human mycoplasma pneumoniae P30 albumen
The serum of antibody, the titer of rabbit anti-human mycoplasma pneumoniae P30 protein antibodies, described rabbit in ELISA detection serum
The titer of anti-human mycoplasma pneumoniae P30 protein antibodies is all at more than 1:60000；
5) by step 2) synthesize the polypeptide of also purification and the Sepharose 4B coupling of cyanogen bromide-activated, formation is many
Peptide affinity column；
6) by step 4) serum containing rabbit anti-human mycoplasma pneumoniae P30 protein antibodies that obtains joins step 5)
In the affinity column of preparation, and after 4 DEG C of overnight incubation, antibody elution, obtain the anti-human mycoplasma pneumoniae of rabbit
P30 protein antibodies；Through SDS-PAGE identify its purity all more than 97%, by named for the antibody of this purification
AbP30Line。
Step 2 in the present embodiment)-6) it is all existing mature technology, Duo Jia biotechnology company can provide
The technical service of sequencing.Being embodied as of related experiment link in above-mentioned steps in the present embodiment, is to entrust south
Jing Jinsirui bio tech ltd completes.
" antibody " of the present invention should be construed to contain any spy with required specific binding structural domain
The anisogamy factor.Thus, this term covers the antibody fragment of homology, derivant, humanization therewith and resists
The function coordinate of body and antibody and congener.The example of antibody be immunoglobulin hypotype (as IgG, IgE,
IgM, IgD and IgA) and isotype sub-classes；Can also be the fragment comprising antigen-binding domains such as Fab, scFv,
Fv, dAb, Fd and double-chain antibody.
The preparation of embodiment 2 immune chromatography reagent kit based on quantum dot-labeled technology and application
The most quantum dot-labeled antibody A bP30Line
0.4nmol carboxyl water-soluble quantum dot and 800nmol carbodiimide it is sequentially added in microcentrifugal tube
(EDC), with MES buffer (10.66g/L MES, 0.74g/L EDTA pH 7.4) constant volume for 1ml,
Ceaselessly mixed solution, after 37 DEG C of reaction 5min, adds the antibody prepared by embodiment 1 of 0.34mg
AbP30Line, lucifuge reaction 2h, add single-ended amino-polyethyleneglycols (PEG2000-NH2) to final concentration of
1% (m/v), closes unreacted activated carboxyl site, continues lucifuge reaction 1h.Reacted sample is with super
Chimney filter is centrifuged (molecular cut off 100k), and 6500g is centrifuged 5min, to volume 200ul, is turned by sample after ultrafiltration
Move in common EP pipe, centrifugal except reuniting (10000g, 3min).Upper clear supernate is added to detached dowel
(Superdex-200) upper purification, treats that it flows in cylinder naturally, then rinses (liquid flows down naturally) with PBS,
Irradiate cylinder with ultraviolet light and observe the position of sample at any time, start to collect when sample starts and flows out from bottom, receive
Stop collecting after collection 1ml.Sample super filter tube (molecular cut off 100k) after purification is centrifuged dense with 6500g
In being transferred to common EP pipe after being reduced to 200ul, centrifugal (10000g, 3min) is except reuniting.With phosphoric acid after acquisition supernatant
Salt preserves liquid and dilutes 200 times, and 4 DEG C save backup.So far prepare containing quantum dot traget antibody AbP30Line's
Solution.
2. the preparation of pad
By in the solution containing quantum dot traget antibody AbP30Line obtained by polyester fiber film immersion step 11
H, takes out, and 25 DEG C are cut into after rear specification is 4cm × 0.6cm/ bar after drying, and 4 DEG C of sealings save backup, so far
Prepare pad.
3. the preparation of sample pad
Take glass fibre element film one, it is soaked in sample pad treatment fluid at least 3h, then is placed in biological peace
In full cabinet after 37 DEG C of aeration-dryings, it is cut into after specification is 4cm × 2.5cm/ bar, i.e. prepares sample pad, 25 DEG C
Seal and preserve.
4. the preparation of detection layers
With invention entitled " the people's mycoplasma pneumoniae gold label silver stain immunochromatography inspection of Application No. 201410405275.3
Test agent box and its preparation method and application " description in [0134th] section to disclosed in [0158] section
Content prepares rabbit anti-restructuring P1-His fusion protein polyclonal antibody IgG.
Nitrocellulose filter is cut into 4cm × 4cm size；The anti-restructuring of rabbit that will prepare as stated above
P1-His fusion protein polyclonal antibody IgG and goat anti-rabbit igg phosphate buffer adjust to final concentration respectively
For 2.0mg/mL and 1.0mg/mL；The rabbit anti-restructuring P1-His fusion protein polyclonal antibody IgG that will have diluted
Loading BIODOT to draw in film instrument shower nozzle, the amount arranging 1.0 μ l/cm is sprayed on nitrocellulose filter, forms detection
Line；Being drawn in film instrument shower nozzle by anti-rabbit IgG diluted loading BIODOT, the amount arranging 1.0 μ l/cm is sprayed on nitre
As nature controlling line on acid cellulose film, nature controlling line is 0.7cm with detection distance between centers of tracks；The celluloid that will have sprayed
37 DEG C of dry 2h of film, are cut into the specification of 4cm × 4cm, and 4 DEG C of hermetically dryings preserve；So far detection layers is prepared；
5. the assembling of detection card
Base plate is cut into 4cm × 7.3cm size, standby.
Absorbent filter is cut into 4cm × 3cm size, as adsorptive pads, standby.
Assembly working operates in Biohazard Safety Equipment, first the adhered protection film on base plate is taken off, by step 4
Described detection layers i.e. pastes on base plate with nature controlling line and the nitrocellulose filter detecting line, and the most floating
Face.Secondly, the adsorptive pads cut out in advance is assembled on base plate so that it is the left side and the right end of detection layers have 0.2
The overlap of cm, its right hand edge then aligns with the right hand edge of base plate and glues and the most floating.Again by described in step 2
Pad is overlapped at the left hand edge of detection layers by 0.3cm, and 0.3cm sticks on base plate 7.Finally by step 3
Described sample pad is then overlapped at the left hand edge of pad by one side 0.3cm, another side and the left hand edge of base plate
Alignment, sticks on base plate and the most floating.The detection plate assembled is cut into the wide inspection of 4.0mm under cutting cutter
Surveying card, 4 DEG C of hermetically dryings keep in Dark Place.
6. the composition of immune chromatography reagent kit based on quantum dot-labeled technology
Immune chromatography reagent kit based on quantum dot-labeled technology is by the detection card described in step 5 and sample treatment liquid
Formed.
7. the using method of immune chromatography reagent kit based on quantum dot-labeled technology
Obtain the throat swab of person to be checked according to a conventional method, be inserted into soft the moulding equipped with 500 μ l sample treatment liquid
In material pipe, squeezable plastic tube wall, make the sample on swab after fully dissolving, take out 120 μ L and drip in detection card
In sample pad, after 15 minutes under uv analyzer (model is WD-9403A, Liuyi Instruments Plant, Beijing produce,
Burst of ultraviolel wavelength 365nm) observe testing result.If containing people's mycoplasma pneumoniae antigen in throat swab, then with
Quantum dot-labeled antibody A bP30Line in pad combines, by chromatography effect elder generation and nitrocellulose filter
On rabbit anti-restructuring P1-His fusion protein polyclonal antibody IgG combine after under ultraviolet excites detection line at
Can form a macroscopic fluoroscopic examination line, the quantum dot-labeled antibody being not associated with continues chromatography and goat-anti
Rabbit igg excites lower formation macroscopic Article 2 fluorescence nature controlling line at ultraviolet after combining；If throat swab to be checked
Middle without related antigen, a fluorescence nature controlling line the most only occurs.If fluorescence nature controlling line does not occurs, then this detection card
Lost efficacy.
8. the application effect citing of immune chromatography reagent kit based on quantum dot-labeled technology
The using method reference of the immune chromatography reagent kit based on quantum dot-labeled technology of indication in the present embodiment
Operating procedure described in step 7.
1) specific test
By respiratory tract common causative such as legionella pneumophilia (ATCC 33152), human III type parainfluenza virus virus
(ATCC VR-93), human influenza haemophilus (ATCC 53781), people's Chlamydia pneumoniae (AR-39 strain, ATCC
Numbering 53592), adenovirus hominis 3 type (GB strain, ATCC numbering VR-3), adenovirus hominis 7 type (Gomen strain, ATCC
Numbering VR-7), influenza virus A hominis's (H1N1, ATCC numbering VR-1743), people Influenza B virus (ATCC
Numbering VR-790), human respiratory syncytial virus's (ATCC numbering VR26), people streptococcus pneumoniae (ATCC number
700670) people's mycoplasma pneumoniae etc. is replaced to detect, the test kit detection phosphate-buffered containing these microorganisms
Liquid diluent is all negative.
2) clinical trial example
Using people's mycoplasma pneumoniae detection " goldstandard "-culture method as reference, take 100 example Pneumology Department respiratory tract senses
The test kit described in oropharyngeal swab specimen step 6 of dye person detects, and culture method positive rate is 13%
(13/100), this test kit is 13% (13/100), and the coincidence rate of 2 kinds of methods is 98% (98/100).Specifically
Result is as shown in table 1.
The testing result of table 1 clinical samples
The preparation of embodiment 3 immune chromatography reagent kit based on colloidal gold-labeled method and application
1. colloidal gold labeled monoclonal antibody AbP30Line
A.30nm the preparation of gold colloidal
Take the 250ml triangular flask that a silication is good, add 99ml ultra-pure water, by molten for 1ml 1% (m/v) HAuCl4
Liquid is added thereto mixing, and oil bath is heated and stirs to boiling.Rapidly join 2ml 1% (m/v) Fructus Citri Limoniae wherein
Acid three sodium water solutions, solution continues boiling 10min (during this, solution is changed into redness by blueness).Stop
Heating, allows solution naturally cool to room temperature, is then added thereto to ultra-pure water polishing to 100ml.
B. colloidal gold labeled monoclonal antibody AbP30Line
1) take the 50ml triangular flask that a silication is good, add the colloidal gold liquid prepared by 10ml step a, Xiang Jin
Liquid adds 240ul 0.2mol/L K2CO3Regulation pH to 8.5；
2) under magnetic stirrer, antibody A bP30Line is added in colloidal gold solution, the denseest to antibody
Degree is 10ug/ml, should be added dropwise over when adding antibody, continues stirring 45min～60min after adding；
3) addition 2.5ml 5% (m/v) bovine serum albumin (BSA) extremely final concentration of 1% (m/v) has been reacted,
Stirring 15～30 minutes, 4 DEG C save backup.
4) loading 50ml centrifuge tube, 2500g after antibody A bP30Line good for labelling being taken out, 4 DEG C are centrifuged 5 minutes,
Obtaining lower sediment and the supernatant, discard lower sediment, the supernatant is transferred to another 50ml centrifuge tube,
12000g, 4 DEG C are centrifuged 30 minutes, obtain lower sediment and the supernatant, discard the supernatant, lower floor sunk
The shallow lake 10ml resuspended precipitation of gold colloidal buffer, 12000g the most again, 4 DEG C are centrifuged 30 minutes, under again obtaining
Layer precipitation and the supernatant, lower sediment is finally resuspended with 3ml gold colloidal buffer, and 4 DEG C save backup,
To the solution containing colloidal gold labeled monoclonal antibody AbP30Line；
In above-mentioned gold colloidal buffer, each constituent content is respectively: 10mM Tris, 1% (m/v) BSA, 1% (v/v)
Tween-20,5% (m/v) sucrose and 3 ‰ (m/v) polyvinylpyrrolidone PVP-10, described gold colloidal delays
The pH rushing liquid is 10.5.
2. the preparation of pad
By in the solution containing colloidal gold labeled monoclonal antibody AbP30Line obtained by polyester fiber film immersion step 11
H, takes out, and 25 DEG C are cut into after rear specification is 4cm × 0.6cm/ bar after drying, and 4 DEG C of sealings save backup, so far
Prepare pad.
3. the preparation of sample pad
Take glass fibre element film one, it is soaked in sample pad treatment fluid at least 2h, then is placed in biological peace
In full cabinet after 37 DEG C of aeration-dryings, it is cut into after specification is 4cm × 2.5cm/ bar, i.e. prepares sample pad, 25 DEG C
Seal and preserve.
4. the preparation of detection layers
With invention entitled " the people's mycoplasma pneumoniae gold label silver stain immunochromatography inspection of Application No. 201410405275.3
Test agent box and its preparation method and application " description in [0134th] section to disclosed in [0158] section
Content prepares rabbit anti-restructuring P1-His fusion protein polyclonal antibody IgG.
Nitrocellulose filter is cut into 4cm × 4cm size；The anti-restructuring of rabbit that will prepare as stated above
P1-His fusion protein polyclonal antibody IgG and goat anti-rabbit igg phosphate buffer adjust to final concentration respectively
For 2.0mg/mL and 1.0mg/mL；The rabbit anti-restructuring P1-His fusion protein polyclonal antibody IgG that will have diluted
Loading BIODOT to draw in film instrument shower nozzle, the amount arranging 1.0 μ l/cm is sprayed on nitrocellulose filter, forms detection
Line；Being drawn in film instrument shower nozzle by anti-rabbit IgG diluted loading BIODOT, the amount arranging 1.0 μ l/cm is sprayed on nitre
As nature controlling line on acid cellulose film, nature controlling line is 0.7cm with detection distance between centers of tracks；The celluloid that will have sprayed
37 DEG C of dry 2h of film, are cut into the specification of 4cm × 4cm, and 4 DEG C of hermetically dryings preserve；So far detection layers is prepared.
5. the assembling of detection card
Base plate is cut into 4cm × 6cm size, standby.
Absorbent filter is cut into 4cm × 2.5cm size, as adsorptive pads, standby.
Assembly working operates in Biohazard Safety Equipment, first the adhered protection film on base plate is taken off, by step 4
Described detection layers i.e. pastes on base plate with nature controlling line and the nitrocellulose filter detecting line, and the most floating
Face.Secondly, the adsorptive pads cut out in advance is assembled on base plate so that it is the left side and the right end of detection layers have 0.2
The overlap of cm, its right hand edge then aligns with the right hand edge of base plate and glues and the most floating.Again by described in step 2
Pad is overlapped at the left hand edge of detection layers 3 by 0.2cm, and 0.4cm sticks on base plate.Finally by step 3 institute
The sample pad stated then is overlapped at the left hand edge of pad by one side 0.2cm, another side and the left hand edge pair of base plate
Together, stick on base plate and the most floating.The detection plate assembled is cut into the wide detection of 4.0mm under cutting cutter
Card, 4 DEG C of hermetically dryings keep in Dark Place.
6. the composition of immune chromatography reagent kit based on colloidal gold-labeled method
Immune chromatography reagent kit based on colloidal gold-labeled method is by the detection card described in step 5 and sample treatment liquid
Formed.
7. the using method of immune chromatography reagent kit based on colloidal gold-labeled method
Obtain the throat swab of person to be checked according to a conventional method, be inserted into soft the moulding equipped with 500 μ l sample treatment liquid
In material pipe, squeezable plastic tube wall, make the sample on swab fully dissolve and after 50 DEG C of water-bath 20min, take out afterwards
120 μ L drip in the sample pad of detection card, perusal testing result after 15 minutes.If throat swab contains
People's mycoplasma pneumoniae antigen, then antibody A bP30Line of the colloid gold label in pad is combined, by layer
Analysis effect after first the rabbit anti-restructuring P1-His fusion protein polyclonal antibody IgG on nitrocellulose filter is combined
Can form a macroscopic red detection line at detection line, the colloidal gold labeled monoclonal antibody being not associated with continues
Chromatography forms macroscopic Article 2 redness nature controlling line after being combined with goat anti-rabbit igg；If nothing in throat swab to be checked
, the most only there is a red nature controlling line in related antigen.If red nature controlling line does not occurs, then this detection card lost efficacy.
8. the application effect citing of immune chromatography reagent kit based on colloidal gold-labeled method
The using method reference of the immune chromatography reagent kit based on colloidal gold-labeled method of indication in the present embodiment
Operating procedure described in step 7.
1) specific test
With respiratory tract common causative such as legionella pneumophilia (ATCC 33152), I type human parainfluenza viruses (ATCC
VR-94), II type human parainfluenza viruses (ATCC VR-92), type III human parainfluenza viruses virus (ATCC VR-93),
Human influenza haemophilus (ATCC 53781), people's Chlamydia pneumoniae (AR-39 strain, ATCC numbering 53592), people
Adenovirus type III (GB strain, ATCC numbering VR-3), adenovirus hominis 7 type (Gomen strain, ATCC numbering VR-7),
(ATCC numbers for influenza virus A hominis's (H1N1, ATCC numbering VR-1743), people's Influenza B virus
VR-790), human respiratory syncytial virus's (ATCC numbering VR26), people streptococcus pneumoniae (ATCC numbering 700670)
Detect Deng replacement people's mycoplasma pneumoniae, the test kit detection phosphate buffer diluent containing these microorganisms
It is all negative.
2) clinical trial example
Using people's mycoplasma pneumoniae detection " goldstandard "-culture method as reference, take 100 example Pneumology Department respiratory tract senses
The test kit described in oropharyngeal swab specimen step 6 of dye person detects, and culture method positive rate is 13%
(13/100), this test kit is 12% (12/100), and the coincidence rate of 2 kinds of methods is 97% (97/100).Specifically
Result is as shown in table 1.
The testing result of table 1 clinical samples
It is pointed out that and the foregoing is only presently preferred embodiments of the present invention, not in order to limit this
Invention, all any amendment, equivalents etc. made within present invention spirit and principle should be included in this
Within bright protection domain.
Claims (4)
1. an anti-human mycoplasma pneumoniae P30 protein antibodies, it is characterized in that: described anti-human mycoplasma pneumoniae P30 protein antibodies is the antibody of the linear epitope that identification people's mycoplasma pneumoniae P30 albumen 56-69 amino acids is formed, and described people's mycoplasma pneumoniae P30 albumen is ABR09215.1 at GenBank serial number；14 aminoacid sequences of described people mycoplasma pneumoniae P30 albumen 56-69 position are AEEDTVQIQGKPIT；It is P30Line by 14 amino acid whose sequence designations of people mycoplasma pneumoniae P30 albumen 56-69 position；Described anti-human mycoplasma pneumoniae P30 protein antibodies is AbP30Line.
2. the immune chromatography reagent kit formed based on anti-human mycoplasma pneumoniae P30 protein antibodies as claimed in claim 1, it is characterised in that: described test kit is immune chromatography reagent kit based on quantum dot-labeled technology or immune chromatography reagent kit based on colloidal gold-labeled method.
The immune chromatography reagent kit that anti-human mycoplasma pneumoniae P30 protein antibodies the most according to claim 2 is formed, it is characterised in that: when described test kit is immune chromatography reagent kit based on quantum dot-labeled technology, the preparation method of described test kit is:
1) quantum dot-labeled antibody A bP30Line:
0.4 it is sequentially added in microcentrifugal tube
Nmol carboxyl water-soluble quantum dot and 800
Nmol carbodiimide EDC, it is 1 ml with MES buffer constant volume, mixed solution, after 37 DEG C of reaction 5 min, adding antibody A bP30Line prepared of 0.34 mg, lucifuge reacts 2 h, adds single-ended amino-polyethyleneglycols PEG2000-NH2 to final concentration of 1%m/v, close unreacted activated carboxyl site, continue lucifuge and react 1 h；Reacted sample super filter tube is centrifuged, and 6500g is centrifuged 5min, to volume 200ul, is transferred to by sample after ultrafiltration in common EP pipe, centrifugal except reuniting, and obtains upper clear supernate and bottom precipitation, centrifugal 3min under the conditions of 10000g；Upper clear supernate is added to purification on detached dowel Superdex-200, treats that upper clear supernate flows in cylinder naturally, then rinse with PBS, irradiate cylinder with ultraviolet light and observe the position of sample, start to collect when sample starts and flows out from bottom, stop collecting after collecting 1 ml；By sample super filter tube after purification with centrifugal except reuniting in being transferred to common EP pipe after 6500g centrifugal concentrating to 200ul, the condition being centrifuged common EP pipe is 10000g, 3min；Preserving liquid with phosphate after obtaining supernatant and dilute 200 times, 4 DEG C save backup；So far the solution containing quantum dot traget antibody AbP30Line is prepared；
In described MES buffer, each constituent content is respectively: 10.66 g/L MES and 0.74 g/L EDTA, the pH 7.4 of described MES buffer；
It is to weigh 0.29 g disodium hydrogen phosphate, 0.0295 g sodium dihydrogen phosphate, 0.2 g sodium chloride, 1 g bovine serum albumin BSA and 0.1 gNaN that described phosphate preserves the preparation method of liquid3, it is dissolved in the deionized water of 90 ml, after adjusting pH to 7.3 with 1 mol/L NaOH, is settled to 100 with deionized water
ml；
2) pad is prepared
Polyester fiber film being immersed 1 h in the solution containing quantum dot traget antibody AbP30Line obtained by step 1), takes out, 25 DEG C are cut into after rear specification is 4cm × 0.6cm/ bar after drying, and 4 DEG C of sealings save backup, and so far prepare pad；
3) sample pad is prepared
Taking glass fibre element film one, glass fibre element film soaks in sample pad treatment fluid at least 3 h, then be placed in Biohazard Safety Equipment after 37 DEG C of aeration-dryings, be cut into after specification is 4cm × 2.5cm/ bar, i.e. prepare sample pad, 25 DEG C seal and preserve；
The preparation method of described sample pad treatment fluid is to weigh 0.29 g disodium hydrogen phosphate, 0.0295 g sodium dihydrogen phosphate, 0.2 g sodium chloride, 2 g bovine serum albumin BSA, 1 ml tween 20,2 g sucrose and 0.5 g polyvinylpyrrolidone PVP-10, it is dissolved in the deionized water of 90 ml, after adjusting pH to 7.3 with 1 mol/L NaOH, is settled to 100 with deionized water
ml；
4) detection layers is prepared
4.1) rabbit anti-restructuring P1-His fusion protein polyclonal antibody IgG is prepared；
4.2) nitrocellulose filter is cut into 4cm × 4cm size；By step 4.1) the rabbit anti-restructuring P1-His fusion protein polyclonal antibody IgG for preparing and goat anti-rabbit igg phosphate buffer adjust to final concentration and be respectively 2.0 mg/mL and 1.0 mg/mL；Being drawn in film instrument shower nozzle by anti-for the rabbit diluted restructuring P1-His fusion protein polyclonal antibody IgG loading BIODOT, the amount arranging 1.0 μ l/cm is sprayed on nitrocellulose filter, forms detection line；Being drawn in film instrument shower nozzle by anti-rabbit IgG diluted loading BIODOT, the amount arranging 1.0 μ l/cm is sprayed on nitrocellulose filter as nature controlling line, and nature controlling line is 0.7 cm with detection distance between centers of tracks；The nitrocellulose filter 37 DEG C sprayed being dried 2 h, is cut into the specification of 4cm × 4cm, 4 DEG C of hermetically dryings preserve；So far detection layers is prepared；
The preparation method of described phosphate buffer is: weighs 0.29 g disodium hydrogen phosphate, 0.0295 g sodium dihydrogen phosphate and 0.2 g sodium chloride, is dissolved in the deionized water of 90 ml, is settled to 100 with deionized water after adjusting pH to 7.3 with 1 mol/L NaOH
ml；
5) detection card is assembled
5.1) base plate is cut into 4cm × 7.3cm size, standby；
5.2) absorbent filter is cut into 4cm × 3cm size, as adsorptive pads, standby；
5.3) by step 5.1) adhered protection film on the base plate for preparing takes off, and by the detection layers described in step 4), i.e. nitrocellulose filter with nature controlling line and detection line pastes on base plate, and floating face；
5.4) by step 5.2) ready adsorptive pads is assembled on base plate, and making the left side end right with detection layers of adsorptive pads have, 0.2 cm's is overlapping, and the right hand edge of adsorptive pads then aligns with the right hand edge of base plate and glues and floating；Again by step 2) described in pad be overlapped at the left hand edge of detection layers by 0.3 cm, 0.3 cm sticks on base plate；
5.5) by the sample pad described in step 3) then by one side 0.3
Cm is overlapped at the left hand edge of pad, and another side aligns with the left hand edge of base plate, sticks on base plate and floating；The detection plate assembled is cut under cutting cutter detection card wide for 4.0 mm, and 4 DEG C of hermetically dryings keep in Dark Place.
The immune chromatography reagent kit that anti-human mycoplasma pneumoniae P30 protein antibodies the most according to claim 2 is formed, it is characterised in that: when described test kit is immune chromatography reagent kit based on colloidal gold-labeled method, the preparation method of described test kit is:
1) colloidal gold labeled monoclonal antibody AbP30Line:
1.1) 30 nm colloidal gold solutions are prepared:
Take a good 250ml triangular flask of silication, add 99ml ultra-pure water, by 1 ml 1% (m/v) HAuCl4Solution adds in 250ml triangular flask and mixes with ultra-pure water, and oil bath is heated and stirs to boiling；2ml 1%m/v trisodium citrate aqueous solution is rapidly joined in 250ml triangular flask, solution continues boiling 10min, solution in 250ml triangular flask stops heating when being changed into redness by blueness, solution in 250ml triangular flask is naturally cooled to room temperature, in 250ml triangular flask, then adds ultra-pure water polishing to 100ml；
1.2) colloidal gold labeled monoclonal antibody AbP30Line:
1.2.1) take a good 50ml triangular flask of silication, add 10 ml steps 1.1) prepared by colloidal gold solution, in gold colloidal liquid add 240ul 0.2 mol/L K2CO3Regulation pH to 8.5；
1.2.2) under magnetic stirrer, antibody A bP30Line is added in colloidal gold solution, to final concentration of 10 ug/ml of antibody, be added dropwise over when adding antibody, after adding, continue stirring 45 min～60 min；
1.2.3) having reacted addition 5%m/v bovine serum albumin BSA to final concentration of 1%m/v, stirred 15～30 minutes, 4 DEG C save backup；
1.2.4) 50ml centrifuge tube is loaded after antibody A bP30Line good for labelling being taken out, 2500g, 4 DEG C are centrifuged 5 minutes, obtain lower sediment and the supernatant, discard lower sediment, the supernatant is transferred to another 50ml centrifuge tube, 12000g, 4 DEG C are centrifuged 30 minutes, obtain lower sediment and the supernatant, discard the supernatant, by lower sediment by the 10 ml resuspended precipitations of gold colloidal buffer, 12000g the most again, 4 DEG C are centrifuged 30 minutes, again obtain lower sediment and the supernatant, lower sediment is finally resuspended with 3ml gold colloidal buffer, 4 DEG C save backup, obtain the solution containing colloidal gold labeled monoclonal antibody AbP30Line；
In described gold colloidal buffer, each constituent content is respectively: 10mM Tris, 1%m/vBSA, 1% v/v Tween-20,5% m/v sucrose and 3 ‰ m/v polyvinylpyrrolidone PVP-10, the pH of described gold colloidal buffer is 10.5；
2) pad is prepared
Polyester fiber film being immersed 1 h in the solution containing colloidal gold labeled monoclonal antibody AbP30Line obtained by step 1), takes out, 25 DEG C are cut into after rear specification is 4cm × 0.6cm/ bar after drying, and 4 DEG C of sealings save backup, and so far prepare pad；
3) sample pad is prepared
Taking glass fibre element film one, glass fibre element film soaks in sample pad treatment fluid at least 2 h, then be placed in Biohazard Safety Equipment after 37 DEG C of aeration-dryings, be cut into after specification is 4cm × 1.5cm/ bar, i.e. prepare sample pad, 25 DEG C seal and preserve；
The preparation method of described sample pad treatment fluid is to weigh 0.242g Tris, 1g bovine serum albumin BSA, 1 ml tween 20,5g sucrose and 0.3g polyvinylpyrrolidone PVP-10, it is dissolved in the deionized water of 90 ml, after adjusting pH to 11 with 1 mol/L NaOH, is settled to 100 with deionized water
ml；
4) detection layers is prepared
4.1) rabbit anti-restructuring P1-His fusion protein polyclonal antibody IgG is prepared；
4.2) nitrocellulose filter is cut into 4cm × 2cm size；By step 4.1) the rabbit anti-restructuring P1-His fusion protein polyclonal antibody IgG for preparing and goat anti-rabbit igg phosphate buffer adjust to final concentration and be respectively 2.0 mg/mL and 1.0 mg/mL；Being drawn in film instrument shower nozzle by anti-for the rabbit diluted restructuring P1-His fusion protein polyclonal antibody IgG loading BIODOT, the amount arranging 1.0 μ l/cm is sprayed on nitrocellulose filter, forms detection line；Being drawn in film instrument shower nozzle by anti-rabbit IgG diluted loading BIODOT, the amount arranging 1.0 μ l/cm is sprayed on nitrocellulose filter as nature controlling line, and nature controlling line is 0.5cm with detection distance between centers of tracks；The 37 DEG C of dry 18h of nitrocellulose filter that will have sprayed, are cut into the specification of 4cm × 2cm, and 4 DEG C of hermetically dryings preserve；So far detection layers is prepared；
The preparation method of described phosphate buffer is: weighs 0.29 g disodium hydrogen phosphate, 0.0295 g sodium dihydrogen phosphate and 0.2 g sodium chloride, is dissolved in the deionized water of 90 ml, is settled to 100 with deionized water after adjusting pH to 7.3 with 1 mol/L NaOH
ml；
5) detection card is assembled
5.1) base plate is cut into 4cm × 6cm size, standby；
5.2) absorbent filter is cut into 4cm × 2.5cm size, as adsorptive pads, standby；
5.3) by step 5.1) adhered protection film on the base plate for preparing takes off, and by the detection layers described in step 4), i.e. nitrocellulose filter with nature controlling line and detection line pastes on base plate, and floating face；
5.4) by step 5.2) ready adsorptive pads is assembled on base plate, and making the left side end right with detection layers of adsorptive pads have, 0.2 cm's is overlapping, and the right hand edge of adsorptive pads then aligns with the right hand edge of base plate and glues and floating；Again by step 2) described in pad be overlapped at the left hand edge of detection layers by 0.2cm, 0.4 cm sticks on base plate；
5.5) by the sample pad described in step 3) then by one side 0.2
Cm is overlapped at the left hand edge of pad, and another side aligns with the left hand edge of base plate, sticks on base plate and floating；The detection plate assembled is cut under cutting cutter detection card wide for 4.0 mm, and 4 DEG C of hermetically dryings keep in Dark Place.
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| 王成红等: "肺炎支原体黏附蛋白P30的高效表达及初步应用", 《生物技术通讯》 * |
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