CN105585635A - Antibodies for resisting human mycoplasma pneumoniae P1 protein and immunochromatographic kit applying antibodies - Google Patents
Antibodies for resisting human mycoplasma pneumoniae P1 protein and immunochromatographic kit applying antibodies Download PDFInfo
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Abstract
The invention relates to antibodies for resisting human mycoplasma pneumoniae P1 protein and an immunochromatographic kit applying the antibodies to detection on human mycoplasma pneumoniae. The antibodies for resisting the human mycoplasma pneumoniae P1 protein are used for recognizing two linear antigenic epitopes composed of 504<th>-517<th> amino acids and 535<th>-548<th> amino acids of the human mycoplasma pneumoniae P1 protein respectively, and the sequence number of the human mycoplasma pneumoniae P1 protein in GenBank is ACO25351.1; the sequences of the 504<th>-517<th> amino acids and the 535<th>-548<th> amino acids of the human mycoplasma pneumoniae P1 protein are KPKKVIQSDKLDDD and FGTDHSTQPQPQSL respectively. The two rabbit antibodies for resisting the human mycoplasma pneumoniae P1 protein have the advantages of being good in specificity, high in purity and titer and low in preparation cost.
Description
Technical field
The invention belongs to field of biomedicine technology, relate to anti-human mycoplasma pneumoniae p1 protein antibody and apply that this is anti-The immune chromatography reagent kit of people's mycoplasma pneumoniae is surveyed in health check-up.
Background technology
Mycoplasma pneumoniae (Mycoplasmapneumoniae, Mp) is the pathogen of mankind's Eaton agent pneumonia,Mainly, through droplet infection, in 2-3 week in incubation period, the incidence of disease is the highest with teenager. Mp infects in infantile pneumonia cause of diseaseIncidence up to 10-30%, become gradually in recent years children's and infect one of main pathogens of breathing problem.This disease very easily causes the respiratory tract infection such as pharyngitis, tonsillitis, even simultaneously secondary meningitis, hepatitis,The multiple organ injuries such as myocarditis, also can cause infant death when serious.
Because the respiratory tract infection symptom that Mp infects and other pathogen causes is similar, do not do etiological examination, veryThe difficult respiratory tract infection that Mp and other pathogen are caused distinguishes. Mp is acellular wall, conventional beta-lactamClass medicine is invalid to it, therefore the therapeutic scheme of the treatment of its infection causing and other bacterium and virus infections is completeDifference, therefore sets up method easy, quick, feasible, energy early diagnosis mycoplasma pneumoniae infection very necessary.
The detection method of Mp mainly contains 3 classes at present: the one, and isolated culture, it is " goldstandard " that confirms infection,But because the growth cycle of Mp is very slow, cultivation cycle is long, causes this method can not examine fast clinicallyDisconnected; The 2nd, serological method, adopt ELISA, colloidal gold immunization, microimmunofluorescence method andConnect hemagglutination test etc., detect Mp antibody horizontal in examinee's serum, the existence that can indirectly point out Mp to infect. But,Serological test can only provide a kind of retrospective diagnosis, and sometimes needs paired sera. In addition, antibody goes outBe difficult for existing opportunity grasping, between children and adolescents and adult, have again the difference of Mp specific antibody, and,There is non-specific cross-reaction in the glycolipid antigen on Mp cell membrane and other microorganisms and body tissue, therefore existingThe detection quality of serological method is subject to certain limitation; The 3rd, utilize Protocols in Molecular Biology to detect depositing of MpDNA, wherein that the most frequently used is PCR (PCR), the method is quick, sensitive, special, is currentThe important means that research Mp infects, but because PCR is higher to experimental facilities and operation requirements, and be prone to vacationThe positive, can't serve as conventional methods for clinical diagnosis in China. Therefore, set up Mp specific antigen diagnosis sideMethod is very necessary. At present, the method for the detection Mp antigen of open report is mainly double antibodies sandwich ELISA method,IIF, quantum dot mark immunity-chromatography method etc., but these methods all can not be carried out the other detection of bed,Need to arrive the specific instrument of specific occasion utilization (as ELIASA, luminoscope etc.) and detect, not only square notJust quick and the time is longer, clinical practice is inconvenience comparatively.
Therefore, set up the fast diagnosis method of people's mycoplasma pneumoniae specific antigen very necessary. Due to human influenzaHigh conservative in mycoplasma pneumoniae p1 protein sequence, has become an important detection target. ThereforeThe anti-human mycoplasma pneumoniae p1 protein antibody that obtains high specific is exactly a very important job. At present, closeWhat report at most in anti-human mycoplasma pneumoniae p1 protein antibody is corresponding monoclonal antibody and polyclonal antibody.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, has limitedIts application. Polyclonal antibody is mainly prepared from by animals such as the P1 protein immunization rabbit of gene engineering expression. ItsPreparation method is simple, and cost is low, but it has, specificity is low, low, the defect such as purity is low of tiring. Therefore,The anti-human mycoplasma pneumoniae p1 protein antibody of preparing cheaply high specific, high-titer just seems very important.
Summary of the invention
For these technical problems that exist in background technology, the object of the present invention is to provide identification people pneumonia to prop upTwo kinds of two linear epitopes that substance P1 albumen 504-517 position and 535-548 amino acids formAntibody and apply the immune chromatography reagent kit of this antibody.
Anti-human mycoplasma pneumoniae p1 protein antibody, is characterized in that: described anti-human mycoplasma pneumoniae p1 protein antibodyTo identify respectively two that people's mycoplasma pneumoniae p1 protein 504-517 position and 535-548 amino acids formTwo kinds of antibody of linear epitope, described people's mycoplasma pneumoniae p1 protein at GenBank sequence number isACO25351.1; 14 amino acid of described people's mycoplasma pneumoniae p1 protein 504-517 position and 535-548 position14 amino acid whose amino acid sequences are respectively KPKKVIQSDKLDDD and FGTDHSTQPQPQSL; People's pneumonia is propped up formerThe 14 amino acid whose sequences called afters respectively of 14 amino acid of body P1 albumen 504-517 position and 535-548 positionP1A and P1B; Described anti-human mycoplasma pneumoniae p1 protein antibody is AbP1A and AbP1B.
The immune chromatography reagent kit forming based on foregoing anti-human mycoplasma pneumoniae p1 protein antibody,It is characterized in that: described kit is immune chromatography reagent kit based on quantum dot-labeled technology or based on collaurumThe immune chromatography reagent kit of labelling technique.
As preferably, kit provided by the present invention is the immune chromatography reagent kit based on quantum dot-labeled technologyTime, the preparation method of described kit is:
1) quantum dot-labeled antibody A bP1A solution:
In microcentrifugal tube, add successively 0.4nmol carboxyl water-soluble quantum dot and 800nmol carbodiimideEDC, taking MES buffer solution constant volume as 1ml, mixed solution, after 37 DEG C of reaction 5min, then adds 0.34mgThe antibody A bP1A preparing, lucifuge reaction 2h, adds single-ended amino polyethylene glycol PEG2000-NH2 extremelyFinal concentration is 1% (m/v), seals unreacted activated carboxyl site, continues lucifuge reaction 1h; ReactedSample super filter tube centrifugal (molecular cut off 100k), the centrifugal 5min of 6500g, to volume 200ul, by after ultrafiltrationSample is transferred in common EP pipe, centrifugal except reuniting, and obtains upper clear supernate and bottom precipitation, 10000g conditionLower centrifugal 3min; Upper clear supernate is added to the upper purifying of splitter Superdex-200, treats that upper clear supernate flows into naturallyIn cylinder, then rinse with PBS, observe the position of sample with UV-irradiation cylinder, treat sample start underPortion starts while outflow to collect, and after collection 1ml, stops collecting; Super filter tube for sample after purifying (is held back to moleculeAmount 100k) to 200ul, be transferred to the interior centrifugal reunion that removes of common EP pipe with 6500g centrifugal concentrating, to common EP pipeCarrying out centrifugal condition is 10000g, 3min; After obtaining supernatant, preserve 200 times of liquid dilutions, 4 DEG C of guarantors with phosphateDeposit for subsequent use; So far make quantum dot-labeled antibody A bP1A solution;
In described MES buffer solution, each constituent content is respectively: 10.66g/LMES and 0.74g/LEDTA,The pH7.4 of described MES buffer solution;
The preparation method that described phosphate is preserved liquid is to take 0.29g sodium hydrogen phosphate, 0.0295g biphosphateSodium, 0.2g sodium chloride, 1g bovine serum albumin(BSA) BSA and 0.1gNaN3, be dissolved in the deionization of 90mlIn water, adjust with 1mol/LNaOH that pH to 7.3 is rear is settled to 100ml by deionized water;
2) prepare pad
Polyester fiber film is immersed to step 1) 1h in the quantum dot-labeled antibody A bP1A solution that obtains, take out,25 DEG C be cut into rear specification after dry and be 4cm × 0.6cm/ bar after, 4 DEG C of sealings save backup, and so far make pad;
3) prepare sample pad
Get one of glass fibre element film, glass fibre element film is soaked at least 3h in sample pad treatment fluid, thenBe placed in Biohazard Safety Equipment after 37 DEG C of aeration-dryings, after being cut into specification and being 4cm × 2.5cm/ bar, make sampleProduct pad, 25 DEG C of sealings are preserved;
The preparation method of described sample pad treatment fluid is to take 0.29g sodium hydrogen phosphate, 0.0295g biphosphateSodium, 0.2g sodium chloride, 2g bovine serum albumin(BSA) BSA, 1ml Tween-20,2g sucrose and 0.5g are poly-Vinylpyrrolidone PVP-10, is dissolved in the deionized water of 90ml, with 1mol/LNaOH tune pH to 7.3Be settled to 100ml by deionized water afterwards;
4) prepare detection layers
Nitrocellulose filter is cut into 4cm × 4cm size; By the antibody A bP1B preparing and goat anti-rabbit iggBe adjusted to final concentration with phosphate buffer and be respectively 2.0mg/mL and 1.0mg/mL; By the antibody having dilutedAbP1B packs BIODOT into and draws in film instrument shower nozzle, and the amount that 1.0 μ l/cm are set is sprayed on nitrocellulose filter, shapeBecome detection line; Pack the anti-rabbit igg having diluted into BIODOT and draw in film instrument shower nozzle, the amount of 1.0 μ l/cm is setBe sprayed on nitrocellulose filter as nature controlling line, nature controlling line and detection line spacing are 0.7cm; By the nitric acid having sprayed37 DEG C of dry 2h of cellulose membrane, are cut into the specification of 4cm × 4cm, and 4 DEG C of hermetically dryings are preserved; So far make inspectionSurvey layer;
The preparation method of described phosphate buffer is: take 0.29g sodium hydrogen phosphate, 0.0295g di(2-ethylhexyl)phosphateHydrogen sodium and 0.2g sodium chloride, be dissolved in the deionized water of 90ml, with 1mol/LNaOH tune pH to 7.3Be settled to 100ml by deionized water afterwards;
5) assembling test card
5.1) base plate is cut into 4cm × 7.3cm size, for subsequent use;
5.2) absorbent filter is cut into 4cm × 3cm size, as adsorptive pads, for subsequent use;
5.3) by step 5.1) viscosity diaphragm on the base plate for preparing takes off, by step 4) describedDetection layers pastes on base plate with the nitrocellulose filter of nature controlling line and detection 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 padsRight end has the overlapping of 0.2cm, and the right hand edge of adsorptive pads aligns with the right hand edge of base plate and glues and floating; AgainBy step 2) described pad is overlapped in the left hand edge place of detection layers by 0.3cm, and 0.3cm sticks on base plate;
5.5) by step 3) described sample pad is overlapped in the left hand edge place of pad by one side 0.3cm,Another side aligns with the left hand edge of base plate, sticks on base plate also floating; By the check-out console assembling under cutting cutterBe cut into the wide test card of 4.0mm, 4 DEG C of hermetically dryings keep in Dark Place.
As preferably, kit provided by the present invention is the immune chromatography reagent kit based on colloidal gold-labeled methodTime, the preparation method of described kit is:
1) colloidal gold labeled monoclonal antibody AbP1A
1.1) prepare 30nm colloidal gold solution:
Get a 250ml triangular flask that silication is good, add 99ml ultra-pure water, by 1ml1% (m/v) HAuCl4MoltenLiquid adds in 250ml triangular flask and with ultra-pure water and mixes, and oil bath is heated and is stirred to boiling; To 250ml triangular flaskIn add fast 2ml1% (m/v) trisodium citrate aqueous solution, solution continue boiling 10min, treat 250ml triangleSolution in bottle stops heating while changing redness into by blueness, the solution in 250ml triangular flask is naturally cooled toRoom temperature then adds ultra-pure water polishing to 100ml in 250ml triangular flask;
1.2) colloidal gold labeled monoclonal antibody AbP1A:
1.2.1) get a 50ml triangular flask that silication is good, add 10ml step 1.1) prepared collaurumSolution adds 240ul0.2mol/LK in collaurum liquid2CO3Regulate pH to 8.5;
1.2.2) under magnetic stirrer stirs, antibody A bP1A is added in colloidal gold solution, dense eventually to antibodyDegree, for 10ug/ml, dropwise adds while adding antibody, adds rear continuation and stirs 45min～60min;
1.2.3) reacted add 2.5ml5% (m/v) bovine serum albumin(BSA) BSA to final concentration be 1% (m/v),Stir 15～30 minutes, 4 DEG C save backup;
1.2.4) antibody A bP1A that will be good mark packs 50ml centrifuge tube into after taking out, 2500g, 4 DEG C centrifugal 5 minutes,Obtain lower sediment and supernatant liquor, discard lower sediment, supernatant liquor is transferred to another 50ml centrifuge tube,12000g, 4 DEG C centrifugal 30 minutes, obtain lower sediment and supernatant liquor, discard supernatant liquor, by heavy lower floorForm sediment by 10ml collaurum buffer solution resuspended precipitation, and then 12000g, 4 DEG C centrifugal 30 minutes, again obtain downLayer precipitation and supernatant liquor, finally use 3ml collaurum buffer solution resuspended lower sediment, and 4 DEG C save backup;
In described collaurum buffer solution, each constituent content respectively: 10mMTris, 1%m/vBSA, 1%v/vTween-20,5%m/v sucrose and 3 ‰ m/v polyvinylpyrrolidones, the pH of described collaurum buffer solutionBe 10.5;
2) prepare pad
Polyester fiber film is immersed to step 1) 1h in the colloidal gold labeled monoclonal antibody AbP1A solution that obtains, take out,25 DEG C be cut into rear specification after dry and be 4cm × 0.6cm/ bar after, 4 DEG C of sealings save backup, and so far make pad;
3) prepare sample pad
Get one of glass fibre element film, glass fibre element film is soaked at least 2h in sample pad treatment fluid, thenBe placed in Biohazard Safety Equipment after 37 DEG C of aeration-dryings, after being cut into specification and being 4cm × 1.5cm/ bar, make sampleProduct pad, 25 DEG C of sealings are preserved;
The preparation method of described sample pad treatment fluid is to take 0.242gTris, 1g bovine serum albumin(BSA) BSA, 1mlTween-20,5g sucrose and 0.3g polyvinylpyrrolidone PVP-10, be dissolved in the deionized water of 90ml,Adjust with 1mol/LNaOH that pH to 11 is rear is settled to 100ml by deionized water;
4) prepare detection layers
Nitrocellulose filter is cut into 4cm × 2cm size; By the antibody A bP1B preparing and goat anti-rabbit iggBe adjusted to final concentration with phosphate buffer and be respectively 2.0mg/mL and 1.0mg/mL; By the antibody having dilutedAbP1B packs BIODOT into and draws in film instrument shower nozzle, and the amount that 1.0 μ l/cm are set is sprayed on nitrocellulose filter, shapeBecome detection line; Pack the anti-rabbit igg having diluted into BIODOT and draw in film instrument shower nozzle, the amount of 1.0 μ l/cm is setBe sprayed on nitrocellulose filter as nature controlling line, nature controlling line and detection line spacing are 0.5cm; By the nitric acid having sprayed37 DEG C of dry 18h of cellulose membrane, are cut into the specification of 4cm × 2cm, and 4 DEG C of hermetically dryings are preserved; So far make inspectionSurvey layer;
The preparation method of described phosphate buffer is: take 0.29g sodium hydrogen phosphate, 0.0295g di(2-ethylhexyl)phosphateHydrogen sodium and 0.2g sodium chloride, be dissolved in the deionized water of 90ml, with 1mol/LNaOH tune pH to 7.3Be settled to 100ml by deionized water afterwards;
5) assembling test card
5.1) base plate is cut into 4cm × 6cm size, for subsequent use;
5.2) absorbent filter is cut into 4cm × 2.5cm size, as adsorptive pads, for subsequent use;
5.3) by step 5.1) viscosity diaphragm on the base plate for preparing takes off, by step 4) describedDetection layers pastes on base plate with the nitrocellulose filter of nature controlling line and detection 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 padsRight end has the overlapping of 0.2cm, and the right hand edge of adsorptive pads aligns with the right hand edge of base plate and glues and floating; AgainBy step 2) described pad is overlapped in the left hand edge place of detection layers by 0.2cm, and 0.4cm sticks on base plate;
5.5) by step 3) described sample pad is overlapped in the left hand edge place of pad by one side 0.2cm,Another side aligns with the left hand edge of base plate, sticks on base plate also floating; By the check-out console assembling under cutting cutterBe cut into the wide test card of 4.0mm, 4 DEG C of hermetically dryings keep in Dark Place.
Advantage of the present invention is:
The invention provides anti-human mycoplasma pneumoniae p1 protein antibody, this anti-human mycoplasma pneumoniae p1 protein antibody is known14 amino acid of others' mycoplasma pneumoniae p1 protein 504-517 position and 14 amino acid institute groups of 535-548 positionTwo linear epitopes that become, people's mycoplasma pneumoniae p1 protein is ACO25351.1 at GenBank sequence number;14 amino acid of people's mycoplasma pneumoniae p1 protein 504-517 position and 14 amino acid whose amino of 535-548 positionAcid sequence is respectively KPKKVIQSDKLDDD and FGTDHSTQPQPQSL; By people's mycoplasma pneumoniae p1 protein14 amino acid whose sequences called after P1A and the P1B respectively of 14 amino acid of 504-517 position and 535-548 position;Anti-human mycoplasma pneumoniae p1 protein antibody is AbP1A and AbP1B. Based on people's mycoplasma pneumoniae single linear antigenThe prepared anti-human mycoplasma pneumoniae p1 protein antibody of above-mentioned two kinds of rabbits of epi-position has that specificity is good, purity is high, effectThe feature that valency is high, preparation cost is cheap, can be used for producing the Gao Ling of the various principles based on antigen-antibody reactionSensitivity detects the detection kit of people's mycoplasma pneumoniae. Meanwhile, based on this anti-human mycoplasma pneumoniae p1 protein antibodyTwo kinds of different immune chromatography reagent kits are also prepared. Two kinds of different immune chromatography reagent kits can be fast,The accurately people's mycoplasma pneumoniae in detection of biological sample, it includes two kinds of antibody of the present invention; Exempt from for two kindsEpidemic disease chromatography kit all can be used for the auxiliary diagnosis of people's mycoplasma pneumoniae infection, has higher sensitivity and specialProperty, taken into account the advantages such as simple, quick, stable and low cost of manufacture simultaneously, be applicable to the inspection of clinical samplesLook into, and owing to can carrying out large batch of quick inspection, be also suitable for epidemiology survey. Therefore, thisThe bright described anti-human mycoplasma pneumoniae p1 protein antibody of two kinds of rabbits, two kinds of immune chromatography reagent kits all have widelyApplication prospect and practical value.
Detailed description of the invention
In order to contribute to those of ordinary skill in the art further to understand the present invention, preferred embodiment cited below particularlyDescribe the present invention in detail.
The source of various materials and the preparation of related reagent that the present invention uses or adopts
1, sample pad treatment fluid: take 0.242gTris, 1g bovine serum albumin(BSA) (BSA), 1ml Tween-20,5g sucrose, 0.3g polyvinylpyrrolidone (PVP-10), is dissolved in the deionized water of 90ml, uses 1mol/LNaOH adjusts that pH to 11 is rear is settled to 100ml by deionized water.
2, phosphate is preserved liquid: take 0.29g sodium hydrogen phosphate, 0.0295g sodium dihydrogen phosphate, 0.2gSodium chloride, 1g bovine serum albumin(BSA) BSA and 0.1gNaN3, be dissolved in the deionized water of 90ml, with 1Mol/LNaOH adjusts that pH to 7.3 is rear is settled to 100ml by deionized water;
3, phosphate buffer (PBS): take 0.29g sodium hydrogen phosphate, 0.0295g sodium dihydrogen phosphate, 0.2G sodium chloride, is dissolved in the deionized water of 90ml, with using deionized water after 1mol/LNaOH tune pH to 7.3Be settled to 100ml.
4, sample treatment liquid: take 0.0121g trishydroxymethylaminomethane, 0.17g sodium chloride, 0.025gLysozyme is dissolved in 90ml deionized water, adjusts that pH to 8.0 is rear is settled to 100ml by deionized water with hydrochloric acid.
5, antibody A bP1A: be the present invention's self-control, with PBS dilution, shake up, making AC in solution is 3mg/ml。
6, antibody A bP1B: be the present invention's self-control, with PBS dilution, shake up, making AC in solution is 3mg/ml。
7, goat anti-rabbit igg: be Wuhan Boster Biological Technology Co., Ltd.'s product, with PBS dilution, 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 modifiedQuantum dot, its emission wavelength is 565nm, buys from Wuhan Jia Yuan technology of quantum dots development corporation, Ltd., productName is called carboxyl water-soluble quantum dot-565.
9, glass fibre element film: thickness is 0.4mm, and water absorption is 42mg/cm2, glass fiber diameter is0.6-3 μ m, has good hydrophily, buys that (model is in Shanghai Jinbiao Bio-Tech Co., Ltd.BT40)。
10, polyester fiber film: thickness is 0.48mm, and absorption speed is 18s/4cm, has fabulous hydrophilicProperty, for the preparation of pad, buy in Shanghai Jinbiao Bio-Tech Co., Ltd. (model is DL42).
11, nitrocellulose filter: model is MilliporeCorpSHF135, has liner plate, buys in MilliporeCompany.
12, absorbent filter: thickness is 0.95mm, absorption speed is 60s/4cm, water absorption is 700mg/cm2，There is good water imbibition, as the material of making adsorptive pads. Buy in Shanghai Jinbiao Bio-Tech Co., Ltd.(model is CH37K).
13, base plate: be high whiteness PVC material, surface-coated individual layer high polymer pressure sensitive adhesive SM31-40, buysIn Shanghai Jinbiao Bio-Tech Co., Ltd..
14, people's mycoplasma pneumoniae: purchased from American type culture collection (ATCC), be numbered ATCC15531.
15, the present invention's microbiological specimens used is all purchased from American type culture collection (ATCC).
Below in conjunction with embodiment, technical scheme provided by the present invention is elaborated:
The preparation of 1 two kinds of anti-human mycoplasma pneumoniae p1 protein antibody of rabbit of embodiment
The preparation method of two kinds of anti-human mycoplasma pneumoniae p1 protein antibody of rabbit is as follows:
1) after structure biology analysis and Related Experimental Study, selected people's mycoplasma pneumoniae p1 protein (GenBankSequence number ACO25351.1) small peptide of 14 amino acid of 504-517 position and 14 amino acid compositions of 535-548 positionRespectively as two linear epitopes preparing the anti-human mycoplasma pneumoniae p1 protein antibody of rabbit, these two sections of amino acidSequence is respectively KPKKVIQSDKLDDD and FGTDHSTQPQPQSL, by this two sequence respectively called after P1A andP1B；
2) by step 1) the N end of described amino acid sequence P1A, the N end of P1B add respectively a cysteineAfter, with polypeptide automatic synthesizer synthetic polypeptide purifying respectively, two polypeptide after purifying respectively with carrier proteinKLH coupling, forms P1A-KLH compound protein and P1B-KLH compound protein;
3) respectively by step 2) two kinds of compound protein emulsifications of synthesized, many in rabbit subcutaneous abdomen respectively after emulsificationPoint injection, successively injects every minor tick 7-10 days three times;
4) inject for the third time after 10-12 days, collect respectively, separate and obtain two kinds and contain the anti-human mycoplasma pneumoniae of rabbitThe serum of P1 protein antibodies, ELISA detects respectively tiring of the anti-human mycoplasma pneumoniae p1 protein antibody of rabbit in serum,Tiring all more than 1:60000 of two kinds of described anti-human mycoplasma pneumoniae p1 protein antibody of rabbit;
5) by step 2) two polypeptide of synthetic and purifying respectively with the Sepharose4B coupling of cyanogen bromide-activated,Form two groups of polypeptide affinity columns;
6) by step 4) two kinds of serum that contain the anti-human mycoplasma pneumoniae p1 protein antibody of rabbit obtaining are respectively correspondingJoin step 5) in two kinds of affinity columns preparing, and after 4 DEG C of overnight incubation, wash-out antibody,To two kinds of anti-human mycoplasma pneumoniae p1 protein antibody of rabbit; Identify that through SDS-PAGE its purity is all more than 97%, willThe antibody of these two kinds of purifying is called after AbP1A and AbP1B respectively.
Step 2 in the present embodiment)-6) be all existing mature technology, Duo Jia biotechnology company can provideThe technological service of sequencing. The concrete enforcement of related experiment link in above-mentioned steps in the present embodiment is to entrust southJing Jinsirui bio tech ltd completes.
" antibody " of the present invention should be interpreted as containing any spy with required specific binding structural domainOpposite sex binding factor. Thereby this term has been contained the antibody fragment of homology, derivative, humanization with it and has been resistedFunction coordinate and the homologue of body and antibody. The example of antibody be immunoglobulin (Ig) hypotype (as IgG, IgE,IgM, IgD and IgA) and hypotype subclass; Also can be the fragment that comprises antigen binding structural domain as Fab, scFv,Fv, dAb, Fd and double-chain antibody.
Preparation and the application of the immune chromatography reagent kit of embodiment 2 based on quantum dot-labeled technology
1. quantum dot-labeled antibody A bP1A
In microcentrifugal tube, add successively 0.4nmol carboxyl water-soluble quantum dot and 800nmol carbodiimide(EDC), taking MES buffer solution (10.66g/LMES, 0.74g/LEDTApH7.4) constant volume as 1ml,Ceaselessly mixed solution, 37 DEG C are reacted after 5min, then add the prepared antibody of embodiment 1 of 0.34mgAbP1A, lucifuge reaction 2h, adding single-ended amino polyethylene glycol (PEG2000-NH2) to final concentration is 1% (m/v),Seal unreacted activated carboxyl site, continue lucifuge reaction 1h. Reacted sample (cuts with super filter tube is centrifugalStay molecular weight 100k), the centrifugal 5min of 6500g, to volume 200ul, is transferred to common EP by sample after ultrafiltrationIn pipe, centrifugal except reunite (10000g, 3min). Upper clear supernate is added on splitter (Superdex-200)Purifying, treats that it flows in cylinder naturally, then rinses (liquid flows down naturally) with PBS, uses UV-irradiation postBody is observed the position of sample at any time, starts to start to collect while outflow from bottom until sample, after collection 1ml, stops receivingCollection. Super filter tube for sample after purifying (molecular cut off 100k) is turned to 200ul with 6500g centrifugal concentratingMove to centrifugal (10000g, 3min) in common EP pipe and remove reunion. After obtaining supernatant, preserve liquid dilution 200 with phosphateDoubly, 4 DEG C save backup. So far make quantum dot-labeled antibody A bP1A.
2. the preparation of pad
1h in the quantum dot-labeled antibody A bP1A solution that polyester fiber film immersion step 1 is obtained, takes out,25 DEG C be cut into rear specification after dry and be 4cm × 0.6cm/ bar after, 4 DEG C of sealings save backup, and so far make pad.
3. the preparation of sample pad
Get one of glass fibre element film, it is soaked in sample pad treatment fluid at least 3h, then be placed in biological peaceIn full cabinet after 37 DEG C of aeration-dryings, after being cut into specification and being 4cm × 2.5cm/ bar, make sample pad, 25 DEG CSealing is preserved.
4. the preparation of detection layers
Nitrocellulose filter is cut into 4cm × 4cm size. By antibody A bP1B prepared in embodiment 1 and goat-antiRabbit igg is adjusted to final concentration with phosphate buffer and is respectively 2.0mg/mL and 1.0mg/mL. By what dilutedAntibody A bP1B packs BIODOT into and draws in film instrument shower nozzle, and the amount that 1.0 μ l/cm are set is sprayed on nitrocellulose filter,Form detection line; Pack the anti-rabbit igg having diluted into BIODOT and draw in film instrument shower nozzle, arrange 1.0 μ l/cm'sAmount is sprayed on nitrocellulose filter as nature controlling line, and itself and detection line spacing are 0.7cm. By the nitric acid fibre having sprayedTie up 37 DEG C of dry 2h of plain film, be cut into the specification of 4cm × 4cm, 4 DEG C of hermetically dryings are preserved. So far make detectionLayer.
5. the assembling of test card
Base plate is cut into 4cm × 7.3cm size, for subsequent use.
Absorbent filter is cut into 4cm × 3cm size, as adsorptive pads, for subsequent use.
Assembly working operates in Biohazard Safety Equipment, first the viscosity diaphragm on base plate is taken off, by step 4Described detection layers pastes on base plate with the nitrocellulose filter of nature controlling line and detection line, and carefully floatingFace. Secondly, the adsorptive pads cutting out is in advance assembled on base plate, makes the right end of its left side and detection layers have 0.2Cm's is overlapping, and its right hand edge aligns with the right hand edge of base plate and glues and carefully floating. Again by described in step 2Pad is overlapped in the left hand edge place of detection layers by 0.3cm, 0.3cm sticks on base plate 7. Finally by step 3Described sample pad is overlapped in the left hand edge place of pad, the left side of another side and base plate by one side 0.3cmEdge alignment, sticks on base plate also carefully floating. The check-out console assembling is cut into 4.0mm under cutting cutter wideTest card, 4 DEG C of hermetically dryings keep in Dark Place.
6. the composition of the immune chromatography reagent kit based on quantum dot-labeled technology
Immune chromatography reagent kit based on quantum dot-labeled technology is by the test card described in step 5 and sample treatment liquidInstitute forms.
7. the using method of the immune chromatography reagent kit based on quantum dot-labeled technology
Obtain according to a conventional method person's to be checked throat swab, be inserted into soft the moulding that 500 μ l sample treatment liquid are housedIn material pipe, extruding plastic tube wall, makes sample on swab fully dissolve that rear taking-up 120 μ L drip in test cardIn sample pad, after 15 minutes under uv analyzer (model is WD-9403A, Liuyi Instruments Plant, Beijing produce,Burst of ultraviolel wavelength 365nm) observation testing result. If contain people's mycoplasma pneumoniae antigen in throat swab, withQuantum dot-labeled antibody A bP1A combination in pad, by chromatography effect first and on nitrocellulose filterAfter antibody A bP1B combination, under ultraviolet ray excited, can form a macroscopic fluoroscopic examination at detection line placeLine, after not continuing chromatography and be not combined with goat anti-rabbit igg in conjunction with complete quantum dot-labeled antibody ultraviolet ray excited lower shapeBecome macroscopic Article 2 fluorescence nature controlling line; If in throat swab to be checked without related antigen, only occur one glimmeringLight nature controlling line. If fluorescence nature controlling line does not occur, this test card inefficacy.
8. the effect of the immune chromatography reagent kit based on quantum dot-labeled technology for example
The using method reference of the immune chromatography reagent kit based on quantum dot-labeled technology of indication in the present embodimentOperating procedure described in step 7.
1) specific test
As bloodthirsty in human III type parainfluenza virus virus (ATCCVR-93), human influenza with respiratory tract common disease substanceBacillus (ATCC53781), people's CPN (AR-39 strain, ATCC numbering 53592), adenovirus hominis 3 types(GB strain, ATCC numbers VR-3), adenovirus hominis 7 types (Gomen strain, ATCC numbers VR-7), human influenza AVirus (H1N1, ATCC numbers VR-1743), people's influenza B virus (ATCC numbers VR-790), people breatheRoad syncytial virus (ATCC numbers VR26), people streptococcus pneumonia (ATCC numbering 700670) etc. replace people's pneumoniaMycoplasma detects, and kit detects containing the phosphate buffer dilution of these microorganisms all negative.
2) clinical trial example
Detect " goldstandard "-cultivation as reference using people's mycoplasma pneumoniae, get 120 routine division of respiratory disease respiratory tract sensesThe person's of dying oropharyngeal swab specimen detects with the kit described in step 6, and cultivation positive rate is 13%(13/100), this kit is 12% (12/100), and the coincidence rate of 2 kinds of methods is 97% (97/100). SpecificallyResult is as shown in table 1.
The testing result of table 1 clinical samples
Preparation and the application of the immune chromatography reagent kit of embodiment 3 based on colloidal gold-labeled method
1. colloidal gold labeled monoclonal antibody AbP1A
A.30nm the preparation of collaurum
Get the 250ml triangular flask that a silication is good, add 99ml ultra-pure water, by molten 1ml1% (m/v) HAuCl4Liquid adds wherein and mixes, and oil bath is heated and is stirred to boiling. Add fast wherein 2ml1% (m/v) lemonAcid three sodium water solutions, solution continues boiling 10min (in this process, solution changes redness into by blueness). StopHeating, allows solution naturally cool to room temperature, then adds wherein ultra-pure water polishing to 100ml.
B. colloidal gold labeled monoclonal antibody AbP1A
1) get the 50ml triangular flask that a silication is good, add the prepared collaurum gold liquid of 10ml step a, Xiang JinIn liquid, add 240ul0.2mol/LK2CO3Regulate pH to 8.5;
2) under magnetic stirrer stirs, antibody A bP1A is added in colloidal gold solution, to antibody final concentration be10ug/ml, should dropwise add while adding antibody, adds rear continuation and stirs 45min～60min;
3) reacted that to add 2.5ml5% (m/v) bovine serum albumin(BSA) (BSA) to final concentration be 1% (m/v),Stir 15～30 minutes, 4 DEG C save backup.
4) antibody A bP1A that will be good mark packs 50ml centrifuge tube into after taking out, 2500g, 4 DEG C centrifugal 5 minutes,To lower sediment and supernatant liquor, discard lower sediment, supernatant liquor is transferred to another 50ml centrifuge tube,12000g, 4 DEG C centrifugal 30 minutes, obtain lower sediment and supernatant liquor, discard supernatant liquor, by heavy lower floorForm sediment by 10ml collaurum buffer solution resuspended precipitation, and then 12000g, 4 DEG C centrifugal 30 minutes, again obtain downLayer precipitation and supernatant liquor, finally use 3ml collaurum buffer solution resuspended lower sediment, and 4 DEG C save backup;In above-mentioned collaurum buffer solution, each constituent content respectively: 10mMTris, 1% (m/v) BSA, 1% (v/v)Tween-20,5% (m/v) sucrose and 3 ‰ (m/v) polyvinylpyrrolidone, described collaurum buffer solutionPH is 10.5.
2. the preparation of pad
1h in the colloidal gold labeled monoclonal antibody AbP1A solution that polyester fiber film immersion step 1 is obtained, takes out,25 DEG C be cut into rear specification after dry and be 4cm × 0.6cm/ bar after, 4 DEG C of sealings save backup, and so far make pad.
3. the preparation of sample pad
Get one of glass fibre element film, it is soaked in sample pad treatment fluid at least 2h, then be placed in biological peaceIn full cabinet after 37 DEG C of aeration-dryings, after being cut into specification and being 4cm × 2.5cm/ bar, make sample pad, 25 DEG CSealing is preserved.
4. the preparation of detection layers
Nitrocellulose filter is cut into 4cm × 2cm size. By antibody A bP1B prepared in embodiment 1 and goat-antiRabbit igg is adjusted to final concentration with phosphate buffer and is respectively 2.0mg/mL and 1.0mg/mL. By what dilutedAntibody A bP1B packs BIODOT into and draws in film instrument shower nozzle, and the amount that 1.0 μ l/cm are set is sprayed on nitrocellulose filter,Form detection line; Pack the anti-rabbit igg having diluted into BIODOT and draw in film instrument shower nozzle, arrange 1.0 μ l/cm'sAmount is sprayed on nitrocellulose filter as nature controlling line, and itself and detection line spacing are 0.5cm. By the nitric acid fibre having sprayedTie up 37 DEG C of dry 2h of plain film, be cut into the specification of 4cm × 2cm, 4 DEG C of hermetically dryings are preserved. So far make detectionLayer.
5. the assembling of test card
Base plate is cut into 4cm × 6cm size, for subsequent use.
Absorbent filter is cut into 4cm × 2.5cm size, as adsorptive pads, for subsequent use.
Assembly working operates in Biohazard Safety Equipment, first the viscosity diaphragm on base plate is taken off, by step 4Described detection layers pastes on base plate with the nitrocellulose filter of nature controlling line and detection line, and carefully floatingFace. Secondly, the adsorptive pads cutting out is in advance assembled on base plate, makes the right end of its left side and detection layers have 0.2Cm's is overlapping, and its right hand edge aligns with the right hand edge of base plate and glues and carefully floating. Again by described in step 2Pad is overlapped in the left hand edge place of detection layers 3 by 0.2cm, 0.4cm sticks on base plate. Finally by step 3State sample pad be overlapped in the left hand edge place of pad, the left hand edge of another side and base plate by one side 0.2cmAlignment, sticks on base plate also carefully floating. The check-out console assembling is cut into the wide inspection of 4.0mm under cutting cutterSurvey card, 4 DEG C of hermetically dryings keep in Dark Place.
6. the composition of the immune chromatography reagent kit based on colloidal gold-labeled method
Immune chromatography reagent kit based on colloidal gold-labeled method is by the test card described in step 5 and sample treatment liquidInstitute forms.
7. the using method of the immune chromatography reagent kit based on colloidal gold-labeled method
Obtain according to a conventional method person's to be checked throat swab, be inserted into soft the moulding that 500 μ l sample treatment liquid are housedIn material pipe, extruding plastic tube wall, sample on swab is fully dissolved and 50 DEG C of water-bath 20min after, rear taking-up120 μ L drip in the sample pad of test card, after (15) minute, visually observe testing result. If contain in throat swabSomeone mycoplasma pneumoniae antigen, the antibody A bP1A of the colloid gold label in pad is combined, and passes through chromatographyThe effect first antibody A bP1B on nitrocellulose filter can form macroscopic one at detection line place after being combinedThe red detection line of bar, does not continue chromatography in conjunction with complete colloidal gold labeled monoclonal antibody and is combined rear formation meat with goat anti-rabbit iggThe red nature controlling line of the visible Article 2 of eye; If without related antigen, only there is an erythrophane in throat swab to be checkedControl line. If red nature controlling line does not occur, this test card inefficacy.
8. the effect of the immune chromatography reagent kit based on colloidal gold-labeled method for example
The using method reference of the immune chromatography reagent kit based on colloidal gold-labeled method of indication in the present embodimentOperating procedure described in step 7.
1) specific test
With respiratory tract common disease substance as I type human parainfluenza viruses (ATCCVR-94), II type human parainfluenza viruses(ATCCVR-92), III type human parainfluenza viruses virus (ATCCVR-93), human influenza haemophilus (ATCC53781), people's CPN (AR-39 strain, ATCC numbering 53592), adenovirus hominis 3 types (GB strain, ATCCNumbering VR-3), adenovirus hominis 7 types (Gomen strain, ATCC numbers VR-7), influenza virus A hominis (H1N1,ATCC numbers VR-1743), people's influenza B virus (ATCC numbers VR-790), human respiratory syncytial virusThe replacement people mycoplasma pneumoniaes such as (ATCC numbers VR26), people streptococcus pneumonia (ATCC numbering 700670) carry outDetect, kit detects containing the phosphate buffer dilution of these microorganisms all negative.
2) clinical trial example
Detect " goldstandard "-cultivation as reference using people's mycoplasma pneumoniae, get 100 routine division of respiratory disease respiratory tract sensesThe person's of dying oropharyngeal swab specimen detects with the kit described in step 6, and cultivation positive rate is 13%(16/120), this kit is 11% (1/100), and the coincidence rate of 2 kinds of methods is 96% (96/100). SpecificallyResult is as shown in table 1.
The testing result of table 1 clinical samples
It is pointed out that and the foregoing is only preferred embodiment of the present invention, in order to restriction not originallyInvention, all any amendments of making within the present invention spirit and principle, is equal to replacement etc. and all should be included in thisWithin bright protection domain.
Claims (4)
1. an anti-human mycoplasma pneumoniae p1 protein antibody, it is characterized in that: described anti-human mycoplasma pneumoniae p1 protein antibody is two kinds of antibody identifying respectively two linear epitopes that people's mycoplasma pneumoniae p1 protein 504-517 position and 535-548 amino acids form, and described people's mycoplasma pneumoniae p1 protein is ACO25351.1 at GenBank sequence number; 14 amino acid of described people's mycoplasma pneumoniae p1 protein 504-517 position and 14 amino acid whose amino acid sequences of 535-548 position are respectively KPKKVIQSDKLDDD and FGTDHSTQPQPQSL; By 14 amino acid whose sequences called after P1A and the P1B respectively of 14 amino acid of people's mycoplasma pneumoniae p1 protein 504-517 position and 535-548 position; Described anti-human mycoplasma pneumoniae p1 protein antibody is AbP1A and AbP1B.
2. the immune chromatography reagent kit forming based on anti-human mycoplasma pneumoniae p1 protein antibody as claimed in claim 1, is characterized in that: described kit is the immune chromatography reagent kit based on quantum dot-labeled technology or the immune chromatography reagent kit based on colloidal gold-labeled method.
3. the immune chromatography reagent kit that anti-human mycoplasma pneumoniae p1 protein antibody according to claim 2 forms, is characterized in that: when described kit is the immune chromatography reagent kit based on quantum dot-labeled technology, the preparation method of described kit is:
1) quantum dot-labeled antibody A bP1A solution:
In microcentrifugal tube, add successively 0.4nmol carboxyl water-soluble quantum dot and 800nmol carbodiimide EDC, taking MES buffer solution constant volume as 1ml, mixed solution, after 37 DEG C of reaction 5min, add again the antibody A bP1A preparing of 0.34mg, lucifuge reaction 2h, add single-ended amino polyethylene glycol PEG2000-NH2 to final concentration be 1%m/v, seal unreacted activated carboxyl site, continue lucifuge reaction 1h; Reacted sample is centrifugal with super filter tube, and the centrifugal 5min of 6500g, to volume 200ul, is transferred to sample after ultrafiltration in common EP pipe, centrifugal except reuniting, and obtains upper clear supernate and bottom precipitation, centrifugal 3min under 10000g condition; Upper clear supernate is added to above purifying of splitter Superdex-200, treats that upper clear supernate flows in cylinder naturally, then rinse with PBS, observe the position of sample with UV-irradiation cylinder, start to start to collect while outflow from bottom until sample, after collection 1ml, stop collecting; Sample after purifying is transferred to the interior centrifugal reunion that removes of common EP pipe with 6500g centrifugal concentrating with super filter tube to 200ul, and it is 10000g that common EP pipe is carried out to centrifugal condition, 3min; After obtaining supernatant, preserve 200 times of liquid dilutions with phosphate, 4 DEG C save backup; So far make quantum dot-labeled antibody A bP1A solution;
In described MES buffer solution, each constituent content respectively: 10.66g/LMES and 0.74g/LEDTA, the pH7.4 of described MES buffer solution;
The preparation method that described phosphate is preserved liquid is to take 0.29g sodium hydrogen phosphate, 0.0295g sodium dihydrogen phosphate, 0.2g sodium chloride, 1g bovine serum albumin(BSA) BSA and 0.1gNaN3, be dissolved in the deionized water of 90ml, adjust with 1mol/LNaOH that pH to 7.3 is rear is settled to 100ml by deionized water;
2) prepare pad
Polyester fiber film is immersed to 1h in the quantum dot-labeled antibody A bP1A solution that obtains of step 1), takes out, 25 DEG C be cut into rear specification after dry and be 4cm × 0.6cm/ bar after, 4 DEG C of sealings save backup, and so far make pad;
3) prepare sample pad
Get one of glass fibre element film, glass fibre element film is soaked at least 3h in sample pad treatment fluid, then be placed in Biohazard Safety Equipment after 37 DEG C of aeration-dryings, after being cut into specification and being 4cm × 2.5cm/ bar, make sample pad, 25 DEG C of sealings are preserved;
The preparation method of described sample pad treatment fluid is to take 0.29g sodium hydrogen phosphate, 0.0295g sodium dihydrogen phosphate, 0.2g sodium chloride, 2g bovine serum albumin(BSA) BSA, 1ml Tween-20,2g sucrose and 0.5g polyvinylpyrrolidone PVP-10, be dissolved in the deionized water of 90ml, adjust with 1mol/LNaOH that pH to 7.3 is rear is settled to 100ml by deionized water;
4) prepare detection layers
Nitrocellulose filter is cut into 4cm × 4cm size; The antibody A bP1B preparing and goat anti-rabbit igg are adjusted to final concentration with phosphate buffer and are respectively 2.0mg/mL and 1.0mg/mL; Pack the antibody A bP1B having diluted into BIODOT and draw in film instrument shower nozzle, the amount that 1.0 μ l/cm are set is sprayed on nitrocellulose filter, forms detection line; Pack the anti-rabbit igg having diluted into BIODOT and draw in film instrument shower nozzle, the amount that 1.0 μ l/cm are set is sprayed on nitrocellulose filter as nature controlling line, and nature controlling line and detection line spacing are 0.7cm; By 37 DEG C of dry 2h of the nitrocellulose filter having sprayed, be cut into the specification of 4cm × 4cm, 4 DEG C of hermetically dryings are preserved; So far make detection layers;
The preparation method of described phosphate buffer is: take 0.29g sodium hydrogen phosphate, 0.0295g sodium dihydrogen phosphate and 0.2g sodium chloride, be dissolved in the deionized water of 90ml, adjust that pH to 7.3 is rear is settled to 100ml by deionized water with 1mol/LNaOH;
5) assembling test card
5.1) base plate is cut into 4cm × 7.3cm size, for subsequent use;
5.2) absorbent filter is cut into 4cm × 3cm size, as adsorptive pads, for subsequent use;
5.3) by step 5.1) viscosity diaphragm on the base plate for preparing takes off, the detection layers described in step 4) pasted on base plate with the nitrocellulose filter of nature controlling line and detection line, and floating face;
5.4) by step 5.2) ready adsorptive pads is assembled on base plate, makes the left side of adsorptive pads and the right end of detection layers have the overlapping of 0.2cm, and the right hand edge of adsorptive pads aligns with the right hand edge of base plate and glues and floating; Again by step 2) described pad is overlapped in the left hand edge place of detection layers by 0.3cm, and 0.3cm sticks on base plate;
5.5) by described in step 3) sample pad be overlapped in the left hand edge place of pad by one side 0.3cm, another side aligns with the left hand edge of base plate, sticks on base plate and floating; The check-out console assembling is cut into the wide test card of 4.0mm under cutting cutter, and 4 DEG C of hermetically dryings keep in Dark Place.
4. the immune chromatography reagent kit that anti-human mycoplasma pneumoniae p1 protein antibody according to claim 2 forms, is characterized in that: when described kit is the immune chromatography reagent kit based on colloidal gold-labeled method, the preparation method of described kit is:
1) colloidal gold labeled monoclonal antibody AbP1A
1.1) prepare 30nm colloidal gold solution:
Get a 250ml triangular flask that silication is good, add 99ml ultra-pure water, by 1ml1%m/vHAuCl4Solution adds in 250ml triangular flask and with ultra-pure water and mixes, and oil bath is heated and is stirred to boiling; In 250ml triangular flask, add fast 2ml1%m/v trisodium citrate aqueous solution, solution continues boiling 10min, solution in 250ml triangular flask stops heating while changing redness into by blueness, solution in 250ml triangular flask is naturally cooled to room temperature, then in 250ml triangular flask, add ultra-pure water polishing to 100ml;
1.2) colloidal gold labeled monoclonal antibody AbP1A:
1.2.1) get a 50ml triangular flask that silication is good, add 10ml step 1.1) prepared colloidal gold solution, in collaurum liquid, add 240ul0.2mol/LK2CO3Regulate pH to 8.5;
1.2.2) under magnetic stirrer stirs, antibody A bP1A is added in colloidal gold solution, to antibody final concentration be 10ug/ml, while adding antibody, dropwise add, add rear continuation and stir 45min～60min;
1.2.3) reacted add 2.5ml5%m/v bovine serum albumin(BSA) BSA to final concentration be 1%m/v, stir 15～30 minutes, 4 DEG C save backup;
1.2.4) will after antibody A bP1A taking-up good mark, pack 50ml centrifuge tube into, 2500g, 4 DEG C centrifugal 5 minutes, obtain lower sediment and supernatant liquor, discard lower sediment, supernatant liquor is transferred to another 50ml centrifuge tube, 12000g, and 4 DEG C are centrifugal 30 minutes, obtain lower sediment and supernatant liquor, discard supernatant liquor, by the resuspended precipitation of 10ml collaurum buffer solution for lower sediment, and then 12000g, 4 DEG C centrifugal 30 minutes, again obtain lower sediment and supernatant liquor, finally use 3ml collaurum buffer solution resuspended lower sediment, 4 DEG C save backup;
In described collaurum buffer solution, each constituent content is respectively: 10mMTris, 1%m/vBSA, 1%v/vTween-20,5%m/v sucrose and 3 ‰ m/v polyvinylpyrrolidones, and the pH of described collaurum buffer solution is 10.5;
2) prepare pad
Polyester fiber film is immersed to 1h in the colloidal gold labeled monoclonal antibody AbP1A solution that obtains of step 1), takes out, 25 DEG C be cut into rear specification after dry and be 4cm × 0.6cm/ bar after, 4 DEG C of sealings save backup, and so far make pad;
3) prepare sample pad
Get one of glass fibre element film, glass fibre element film is soaked at least 2h in sample pad treatment fluid, then be placed in Biohazard Safety Equipment after 37 DEG C of aeration-dryings, after being cut into specification and being 4cm × 1.5cm/ bar, make sample pad, 25 DEG C of sealings are preserved;
The preparation method of described sample pad treatment fluid is to take 0.242gTris, 1g bovine serum albumin(BSA) BSA, 1ml Tween-20,5g sucrose and 0.3g polyvinylpyrrolidone PVP-10, be dissolved in the deionized water of 90ml, adjust with 1mol/LNaOH that pH to 11 is rear is settled to 100ml by deionized water;
4) prepare detection layers
Nitrocellulose filter is cut into 4cm × 2cm size; The antibody A bP1B preparing and goat anti-rabbit igg are adjusted to final concentration with phosphate buffer and are respectively 2.0mg/mL and 1.0mg/mL; Pack the antibody A bP1B having diluted into BIODOT and draw in film instrument shower nozzle, the amount that 1.0 μ l/cm are set is sprayed on nitrocellulose filter, forms detection line; Pack the anti-rabbit igg having diluted into BIODOT and draw in film instrument shower nozzle, the amount that 1.0 μ l/cm are set is sprayed on nitrocellulose filter as nature controlling line, and nature controlling line and detection line spacing are 0.5cm; By 37 DEG C of dry 18h of the nitrocellulose filter having sprayed, be cut into the specification of 4cm × 2cm, 4 DEG C of hermetically dryings are preserved; So far make detection layers;
The preparation method of described phosphate buffer is: take 0.29g sodium hydrogen phosphate, 0.0295g sodium dihydrogen phosphate and 0.2g sodium chloride, be dissolved in the deionized water of 90ml, adjust that pH to 7.3 is rear is settled to 100ml by deionized water with 1mol/LNaOH;
5) assembling test card
5.1) base plate is cut into 4cm × 6cm size, for subsequent use;
5.2) absorbent filter is cut into 4cm × 2.5cm size, as adsorptive pads, for subsequent use;
5.3) by step 5.1) viscosity diaphragm on the base plate for preparing takes off, the detection layers described in step 4) pasted on base plate with the nitrocellulose filter of nature controlling line and detection line, and floating face;
5.4) by step 5.2) ready adsorptive pads is assembled on base plate, makes the left side of adsorptive pads and the right end of detection layers have the overlapping of 0.2cm, and the right hand edge of adsorptive pads aligns with the right hand edge of base plate and glues and floating; Again by step 2) described pad is overlapped in the left hand edge place of detection layers by 0.2cm, and 0.4cm sticks on base plate;
5.5) by described in step 3) sample pad be overlapped in the left hand edge place of pad by one side 0.2cm, another side aligns with the left hand edge of base plate, sticks on base plate and floating; The check-out console assembling is cut into the wide test card of 4.0mm under cutting cutter, and 4 DEG C of hermetically dryings keep in Dark Place.
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| CN110540971A (en) * | 2018-12-20 | 2019-12-06 | 湖北诺美华抗体药物技术有限公司 | Monoclonal antibody of surface protein of mycoplasma hyopneumoniae and application thereof |
| CN110128512A (en) * | 2019-05-06 | 2019-08-16 | 南华大学 | Antigenic determinant and its application |
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