CN105585634A - Antibodies for resisting human streptococcus pneumoniae fam2 family PspA protein and immunochromatographic kit applying antibodies - Google Patents

Antibodies for resisting human streptococcus pneumoniae fam2 family PspA protein and immunochromatographic kit applying antibodies Download PDF

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CN105585634A
CN105585634A CN201610129975.3A CN201610129975A CN105585634A CN 105585634 A CN105585634 A CN 105585634A CN 201610129975 A CN201610129975 A CN 201610129975A CN 105585634 A CN105585634 A CN 105585634A
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fam2
antibody
base plate
pspa
cut
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CN105585634B (en
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胡征
董俊
杨波
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HUBEI HUALONG BIOLOGICAL PHARMACEUTICAL CO Ltd
Hubei University of Technology
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HUBEI HUALONG BIOLOGICAL PHARMACEUTICAL CO Ltd
Hubei University of Technology
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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
    • C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
    • C07K16/1275—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Streptococcus (G)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/56944—Streptococcus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/315—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Streptococcus (G), e.g. Enterococci
    • G01N2333/3156—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Streptococcus (G), e.g. Enterococci from Streptococcus pneumoniae (Pneumococcus)

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Abstract

The invention relates to antibodies for resisting human streptococcus pneumoniae fam2 family PspA protein and an immunochromatographic kit applying the antibodies to detection on human streptococcus pneumoniae. The antibodies for resisting the human streptococcus pneumoniae fam2 family PspA protein are used for recognizing two linear antigenic epitopes composed of 34<th>-47<th> amino acids and 274<th>-287<th> amino acids of the human streptococcus pneumoniae fam2 family PspA protein respectively, and the sequence number of the human streptococcus pneumoniae fam2 family PspA protein in GenBank is WP_054380072.1; the sequences of the 34<th>-47<th> amino acids and the 274<th>-287<th> amino acids of the human streptococcus pneumoniae fam2 family PspA protein are SPQVVEKSSLEKKY and KLLDSLDPEGKTQD respectively. The two rabbit antibodies for resisting the human streptococcus pneumoniae fam2 family PspA protein have the advantages of being good in specificity, high in purity and titer and low in preparation cost.

Description

The anti-human streptococcus pneumonia fam2 PspA of family protein antibodies and apply the immune chromatography reagent kit of this antibody
Technical field
The invention belongs to field of biomedicine technology, relate to the anti-human streptococcus pneumonia fam2 PspA of family protein antibodiesAnd apply the immune chromatography reagent kit of this antibody test people streptococcus pneumonia.
Background technology
People streptococcus pneumonia (Streptococcuspneumoniae, Sp) is the important disease of childrens respiratory tract infectionSubstance. Mainly enter respiratory tract through droplet transmission, thereby parasitize the pharynx nasalis of human body, or invade human body notThe position of easy-clear causes a series of disease, as lobar pneumonia, and meningitis, bronchitis, tympanitis etc.It is the main pathogenic fungi of global all age group high incidences and case fatality rate. Wherein, in developing country, babyParticularly serious in child, the elderly and immune deficiency crowd. Streptococcus pneumonia is in 1881 first by Pasteur(LouisPasteur) and G.M.Sternberg isolate from patient's sputum in France and the U.S. respectively.It is Gram-positive, and thalline is like spearhead shape, becomes two or becomes the diplococcus of short catenation, toxic strain bacteriumIt is external that to have chemical composition be the pod membrane of polysaccharide. Its pod membrane has antigenicity, is the foundation of streptococcus pneumonia somatotype.According to the antigenic difference of capsular polysaccharide, pneumococcus is divided into 91 serotypes. It is many that its somatic antigen is mainly CSugar, it is present in pneumococcal cell wall, has species specificity, for various bacterial strain common. C polysaccharideCan be precipitated by C reactive protein in serum. In the time that calcium ion exists, C polysaccharide can with normal human serum in be called C-The beta Globulin combination of reactive protein (Creactiveprotein, CRP), precipitates. At present for peopleThe detection of streptococcus pneumoniae antigen is mainly also for this antigen, and the non-streptococcus pneumonia of this antigen is exclusive, as slowAlso contain this antigen with streptococcus. And the purification process difficulty of C polysaccharide, causes production cost higher. At lungScorching streptococcus surface also has a kind of important antigen relevant to virulence, be pneumococcal surface protein A (PspA),It is present in all streptococcus pneumonia serotype, is the specific antigen of streptococcus pneumonia. PspA is divided into 3Family, the people streptococcus pneumonia wherein with fam1 or the PspA of fam2 family albumen has accounted for people's lung of clinical separationThe more than 99% of scorching streptococcus kind.
Clinical patient is because different respiratory pathogens is (as mycoplasma pneumoniae, haemophilus influenzae, influenza diseasePoison, Respiratory Syncytial Virus(RSV), adenovirus etc.) infect cause that disease symptoms can be quite similar, this has caused streamRow diagnosis is more difficult, makes a definite diagnosis and often depends on laboratory diagnosis. Diagnostic method should be in disease fast and effectivelySick early stage just can obtain clear and definite diagnosis, is convenient to implement treatment targetedly, stops the development of the state of an illnessDelay.
Although streptococcus pneumonia is propagated in the world, infant's infection is especially general, can be used for experimentThe kind of the standardization commercially available reagent of chamber diagnosis is few. At present, the detection of streptococcus pneumonia mainly contains following severalMethod:
One, Routine Test Lab detects
1, bacterium separates
The goldstandard of laboratory diagnosis streptococcus pneumonia is to separate people streptococcus pneumonia strain. Adopt nasopharyngeal secretionsAs the sample of pathogen isolation, can use the method bacterial isolate body of blood culture. But this method has serious lackingFall into, because streptococcus pneumonia is severe bacteria, nutritional requirement is high, cultivates required time long, and positive rate is low, heavierWant, if patient used antibacterials before sampling, can cause the false positive of cultivation results. Like this clinicalJust there is certain limitation aspect to patient's treatment.
2, serology detects
Adopt ELISA, put the method for exempting from, microimmunofluorescence method etc., detect pneumonia chain in examinee's serumCoccus antibody horizontal, can point out the existence of streptococcus pneumoniae infection indirectly. But serological test can only provideA retrospective diagnosis, it need to detect the Acute Stage and convalescent paired sera simultaneously, if recoveredHigher 4 times or 4 times than acute stage of interim anti-human streptococcus pneumonia antibody titers just have diagnostic significance above. In addition, anti-Be difficult for the opportunity that body occurs grasping, and because thalline serotype kind is too much, the anti-capsular polysaccharide that causes it to induceAntibody type is too much, and the detection of antagonist has caused very large difficulty, therefore the detection quality of existing serological methodBe subject to certain limitation.
Two, quick diagnosis
Directly scrutineer streptococcus pneumonia proteantigen and thalline nucleic acid can reach the object of quick diagnosis, main at presentThere are immunofluorescence technique, immunoenzyme and PCR method etc. Immunofluorescence technique and immunoenzyme all can not carry out a stepDetect, all have operating procedure complexity, need professional to operate, detection time long (more than 2h), costThe shortcoming such as higher. PCR method is quick, sensitive, special, is the important hand of studying at present streptococcus pneumoniae infectionSection, but because PCR is higher to experimental facilities and operation requirements, and be prone to false positive, can't in ChinaAs conventional methods for clinical diagnosis. At present, the method for detection people streptococcus pneumoniae antigen is mainly colloidal gold methodDetect its C polysaccharide antigen, but this method susceptibility is lower, treats sample product material quality requirement higher, simultaneouslyAlso exist with other streptococcus and have the defects such as cross reaction as relaxed streptococcus. PspA albumen is one to be hadSpecific detection target. What at present, report at most about anti-human streptococcus pneumonia PspA protein antibodies is phaseThe polyclonal antibody of answering. Polyclonal antibody is mainly by animal systems such as the PspA protein immunization rabbit of gene engineering expressionStandby forming. Its preparation method is simple, and cost is low, but its have specificity low, tire low, purity is low etc. lacksFall into. Therefore, prepare cheaply the anti-human streptococcus pneumonia PspA protein antibodies of high specific, high-titer just aobviousObtain 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 chainTwo linear antigen tables that the coccus fam2 PspA of family albumen 34-47 position and 274-287 amino acids formTwo kinds of antibody of position and apply the immune chromatography reagent kit of this antibody.
The anti-human streptococcus pneumonia fam2 PspA of family protein antibodies, is characterized in that: described anti-human streptococcus pneumoniaThe PspA of fam2 family protein antibodies be identify respectively the people streptococcus pneumonia fam2 PspA of family albumen 34-47 position andTwo kinds of antibody of two linear epitopes that 274-287 amino acids forms, described people streptococcus pneumoniaThe PspA of fam2 family albumen is WP_054380072.1 at GenBank sequence number; Described people streptococcus pneumonia fam214 amino acid of the PspA of family albumen 34-47 position and 14 amino acid whose amino acid sequences of 274-287 position divideWei SPQVVEKSSLEKKY and KLLDSLDPEGKTQD; By the people streptococcus pneumonia fam2 PspA of family albumen14 amino acid whose sequences of 14 amino acid of 34-47 position and 274-287 position respectively called after F2P1 andF2F2P2; The described anti-human streptococcus pneumonia fam2 PspA of family protein antibodies is AbF2P1 and AbF2P2.
A kind of immunity forming based on the foregoing anti-human streptococcus pneumonia fam2 PspA of family protein antibodiesChromatography kit, is characterized in that: described kit is the immune chromatography reagent kit based on quantum dot-labeled technologyOr immune chromatography reagent kit based on colloidal gold-labeled method.
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 bF2P1 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 0.4mg'sThe antibody A bF2P1 preparing, lucifuge reaction 2h, adds single-ended amino polyethylene glycol PEG2000-NH2 to wholeConcentration is 1% (m/v), seals unreacted activated carboxyl site, continues lucifuge reaction 1h; Reacted sampleProduct super filter tube centrifugal (molecular cut off 100k), the centrifugal 5min of 6500g, to volume 200ul, by sample after ultrafiltrationProduct are transferred in common EP pipe, centrifugal except reuniting, and obtain upper clear supernate and bottom precipitation, under 10000g conditionCentrifugal 3min; Upper clear supernate is added to the upper purifying of splitter Superdex-200, treats that upper clear supernate flows into post naturallyIn body, then rinse with PBS, observe the position of sample with UV-irradiation cylinder, treat that sample starts from bottomWhen outflow, start to collect, after collection 1ml, stop collecting; By the super filter tube (molecular cut off for sample after purifying100k) to 200ul, be transferred to the interior centrifugal reunion that removes of common EP pipe with 6500g centrifugal concentrating, common EP pipe is enteredThe centrifugal condition of row is 10000g, 3min; After obtaining supernatant, preserve 200 times of liquid dilutions, 4 DEG C of preservations with phosphateFor subsequent use; So far make quantum dot-labeled antibody A bF2P1 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 bF2P1 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 bF2P2 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 dilutedAbF2P2 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 at base plateOn;
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 AbF2P1
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) HAuCl4SolutionAdd in 250ml triangular flask and with ultra-pure water and mix, oil bath is heated and is stirred to boiling; In 250ml triangular flaskAdd fast 2ml1% (m/v) trisodium citrate aqueous solution, solution continues boiling 10min, treats 250ml triangular flaskIn solution stop heating while changing redness into by blueness, the solution in 250ml triangular flask is naturally cooled to chamberTemperature then adds ultra-pure water polishing to 100ml in 250ml triangular flask;
1.2) colloidal gold labeled monoclonal antibody AbF2P1:
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 bF2P1 is added in colloidal gold solution, whole to antibodyConcentration is 10ug/ml, while adding antibody, dropwise adds, and 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) will after antibody A bF2P1 taking-up good mark, pack 50ml centrifuge tube into, 2500g, 4 DEG C centrifugal 5 pointsClock, obtains lower sediment and supernatant liquor, discards lower sediment, and it is centrifugal that supernatant liquor is transferred to another 50mlPipe, 12000g, 4 DEG C centrifugal 30 minutes, obtain lower sediment and supernatant liquor, discard supernatant liquor, will underThe resuspended precipitation of 10ml collaurum buffer solution for layer precipitation, and then 12000g, 4 DEG C centrifugal 30 minutes, againTo 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 respectively: 10mMTris, 1% (m/v) BSA, 1% (v/v)Tween-20,5% (m/v) sucrose and 3 ‰ (m/v) polyvinylpyrrolidone, described collaurum buffer solutionPH be 10.5;
2) prepare pad
Polyester fiber film is immersed to step 1) 1h in the colloidal gold labeled monoclonal antibody AbF2P1 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, 1Ml Tween-20,5g sucrose and 0.3g polyvinylpyrrolidone PVP-10, be dissolved in the deionized water of 90mlIn, 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 bF2P2 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 dilutedAbF2P2 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 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 the anti-human streptococcus pneumonia fam2 PspA of family protein antibodies, this anti-human streptococcus pneumonia14 amino of the PspA of the fam2 family protein antibodies identification people streptococcus pneumonia fam2 PspA of family albumen 34-47 positionTwo linear epitopes that 14 amino acid of acid and 274-287 position form, people streptococcus pneumonia fam2 familyThe PspA of family albumen is AAB59852.1 at GenBank sequence number; The people streptococcus pneumonia fam2 PspA of family albumen14 amino acid of 34-47 position and 14 amino acid whose amino acid sequences of 274-287 position are respectivelySKYTIQRSTGDSID and FGIAQSSTRGGSRV; By the people streptococcus pneumonia fam2 PspA of family albumen 34-4714 amino acid whose sequences called after F2P1 and the F2P2 respectively of 14 amino acid of position and 274-287 position; Anti-humanThe streptococcus pneumonia fam2 PspA of family protein antibodies is AbF2P1 and AbF2P2. Based on people streptococcus pneumonia fam2The above-mentioned two kinds of anti-human streptococcus pneumonia fam2 of rabbit families that the PspA of family albumen single linear epitope is preparedPspA protein antibodies has that specificity is good, purity is high, the height of tiring, feature that preparation cost is cheap, can be used for rawProduce the detection kit of the high-sensitivity detection people streptococcus pneumonia of the various principles based on antigen-antibody reaction.Meanwhile, two kinds of different exempting from have also been prepared based on this anti-human streptococcus pneumonia fam2 PspA of family protein antibodiesEpidemic disease chromatography kit. Having in two kinds of different quick, accurate detection of biological samples of immune chromatography reagent kit energyThe people streptococcus pneumonia of the PspA of fam2 family albumen, it includes two kinds of antibody of the present invention; Two kinds of immunityChromatography kit all can be used for the auxiliary diagnosis of people's streptococcus 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 two kinds of described anti-human streptococcus pneumonia fam2 of the rabbit PspA of family protein antibodies, two kinds of immune chromatography reagent kitsAll be with a wide range of applications 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 bF2P1: be the present invention's self-control, with PBS dilution, shake up, making AC in solution is 3mg/ml。
6, antibody A bF2P2: 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 streptococcus pneumonia subtype strains Sp23F: purchased from American type culture collection (ATCC),Be numbered ATCC700669.
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 the 1 two kinds of anti-human streptococcus pneumonia fam2 of the rabbit PspA of family protein antibodies of embodiment
The preparation method of the two kinds of anti-human streptococcus pneumonia fam2 of the rabbit PspA of family protein antibodies is as follows:
1) after structure biology analysis and Related Experimental Study, the selected people streptococcus pneumonia fam2 PspA of family14 of 14 amino acid of albumen (GenBank sequence number WP_054380072.1) 34-47 position and 274-287 positionThe small peptide of amino acid composition is respectively as two of the anti-human streptococcus pneumonia fam2 of the preparation rabbit PspA of family protein antibodiesIndividual linear epitope, these two sections of amino acid sequences are respectively SPQVVEKSSLEKKY and KLLDSLDPEGKTQD,By this two sequence called after F2P1 and F2P2 respectively;
2) by step 1) the C end of described amino acid sequence F2P1, the N end of F2P2 add respectively one and half Guang ammoniaAfter acid, with polypeptide automatic synthesizer synthetic polypeptide purifying respectively, two polypeptide after purifying respectively with carrier eggWhite KLH coupling, forms F2P1-KLH compound protein and F2P2-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 streptococcus pneumonia of rabbitThe serum of the PspA of fam2 family protein antibodies, ELISA detects respectively the anti-human streptococcus pneumonia of rabbit fam2 family in serumTiring of the PspA of family protein antibodies, the two kinds of described anti-human streptococcus pneumonia fam2 of the rabbit PspA of family protein antibodiesTire all more than 1:60000;
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) obtain two kinds of blood that contain the anti-human streptococcus pneumonia fam2 of the rabbit PspA of family protein antibodiesWhat sorting was corresponding joins step 5) in two kinds of affinity columns preparing, and after 4 DEG C of overnight incubation, washDe-antibody, obtains the two kinds of anti-human streptococcus pneumonia fam2 of the rabbit PspA of family protein antibodies; Identify it through SDS-PAGEPurity is all more than 97%, by the antibody of these two kinds of purifying called after AbF2P1 and AbF2P2 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 bF2P1
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 0.4mgAbF2P1, 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 ultrafiltrationManage centrifugal (molecular cut off 100k), the centrifugal 5min of 6500g, to volume 200ul, shifts sample after ultrafiltrationTo common EP pipe, centrifugal except reunite (10000g, 3min). Upper clear supernate is added to splitter(Superdex-200) upper purifying, treats that it flows in cylinder naturally, then rinses (liquid flows down naturally) with PBS,Observe at any time the position of sample with UV-irradiation cylinder, start to start to collect while outflow from bottom until sample, receiveAfter collection 1ml, stop collecting. By centrifugal dense with 6500g the super filter tube for sample after purifying (molecular cut off 100k)After being reduced to 200ul, be transferred to common EP pipe interior centrifugal (10000g, 3min) except reuniting. Obtain after supernatant with phosphoric acidSalt is preserved 200 times of liquid dilutions, and 4 DEG C save backup. So far make quantum dot-labeled antibody A bF2P1.
2. the preparation of pad
1h in the quantum dot-labeled antibody A bF2P1 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 bF2P2 prepared in embodiment 1 and sheepAnti-rabbit igg is adjusted to final concentration with phosphate buffer and is respectively 2.0mg/mL and 1.0mg/mL. By dilution wellAntibody A bF2P2 pack BIODOT into and draw in film instrument shower nozzle, the amount that 1.0 μ l/cm are set is sprayed on nitrocellulose filterUpper, form detection line; Pack the anti-rabbit igg having diluted into BIODOT and draw in film instrument shower nozzle, 1.0 μ l/cm are setAmount be sprayed on nitrocellulose filter as nature controlling line, itself 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.
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 step 2 is describedPad be 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, a left side for another side and base plate by one side 0.3cmJustified margin, sticks on base plate also carefully floating. The check-out console assembling is cut into 4.0mm under cutting cutterWide test 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 lung of tool fam2PspA antigen in throat swabScorching streptococcus, the quantum dot-labeled antibody A bF2P1 in pad is combined, by chromatography effect elder generation and nitreAntibody A bF2P2 on acid cellulose film in conjunction with after can to form naked eyes at detection line place under ultraviolet ray excited visibleA fluoroscopic examination line, after not continuing chromatography and be not combined with goat anti-rabbit igg in conjunction with complete quantum dot-labeled antibodyThe macroscopic Article 2 fluorescence of ultraviolet ray excited lower formation nature controlling line; If in throat swab to be checked without related antigen,Only there is a fluorescence 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
Prop up former with respiratory tract common disease substance as human III type parainfluenza virus virus (ATCCVR-93), people's pneumoniaBody (ATCC numbering 15531), people's CPN (AR-39 strain, ATCC numbering 53592), adenovirus hominis 3Type (GB strain, ATCC numbers VR-3), adenovirus hominis 7 types (Gomen strain, ATCC numbers VR-7), people's A type streamInfluenza Virus (H1N1, ATCC numbers VR-1743), people's influenza B virus (ATCC numbers VR-790), influenzaHaemophilus (ATCC numbering 53781), human respiratory syncytial virus's (ATCC numbers VR26) etc. replace people's pneumoniaStreptococcus detects, and kit detects containing the phosphate buffer dilution of these microorganisms all negative.
2) sensitivity tests
Do Study of Sensitivity by measuring culture of streptococcus pneumonia thing dilution. To there is fam2PspA antigenPeople streptococcus pneumonia subtype strains Sp23F (ATCC numbering 700669) sample carries out series with phosphate bufferAfter dilution, detect with kit described in step 6, result shows that it detects lowest limit is 2 × 104CFU/ml。And the BinaxNOWStreptococcuspneumoniaetest (colloid by name being used for from manufacturer BinaxGold method) kit detect, find its detect lowest limit be 5 × 105CFU/ml. The inspection of kit of the present inventionSurveying lower limit obviously reduces compared with it.
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 AbF2P1
A.30nm the preparation of collaurum
Get the 250ml triangular flask that a silication is good, add 99ml ultra-pure water, by 1ml1% (m/v) HAuCl4 solutionAdd wherein and mix, oil bath is heated and is stirred to boiling. Add fast wherein 2ml1% (m/v) citric acidThree sodium water solutions, solution continues boiling 10min (in this process, solution changes redness into by blueness). Stop addingHeat, allows solution naturally cool to room temperature, then adds wherein ultra-pure water polishing to 100ml.
B. colloidal gold labeled monoclonal antibody AbF2P1
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 bF2P1 is added in colloidal gold solution, to antibody final concentrationFor 10ug/ml, while adding antibody, should dropwise add, add rear continuation and stir 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 bF2P1 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 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 be 10.5;
2. the preparation of pad
1h in the colloidal gold labeled monoclonal antibody AbF2P1 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 × 1.5cm/ 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 bF2P2 prepared in embodiment 1 and sheepAnti-rabbit igg is adjusted to final concentration with phosphate buffer and is respectively 2.0mg/mL and 1.0mg/mL. By dilution wellAntibody A bF2P2 pack BIODOT into and draw in film instrument shower nozzle, the amount that 1.0 μ l/cm are set is sprayed on nitrocellulose filterUpper, form detection line; Pack the anti-rabbit igg having diluted into BIODOT and draw in film instrument shower nozzle, 1.0 μ l/cm are setAmount be sprayed on nitrocellulose filter as nature controlling line, itself 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 makeDetection layers.
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 step 2 is describedPad be overlapped in the left hand edge place of detection layers 3 by 0.2cm, 0.4cm sticks on base plate. Finally by step 3Described sample pad is overlapped in the left hand edge place of pad, a left side for another side and base plate by one side 0.2cmJustified margin, sticks on base plate also carefully floating. The check-out console assembling is cut into 4.0mm under cutting cutterWide test 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 after in 50 DEG C of water-bath 20min, take out 120μ L drips in the sample pad of test card, after 30 minutes, visually observes testing result. If contain tool fam2 in throat swabThe people streptococcus pneumonia of PspA antigen, the antibody A bF2P1 of the colloid gold label in pad is combined, and passes throughChromatography effect first the antibody A bF2P2 on nitrocellulose filter be combined after can form naked eyes at detection line place canA red detection line of seeing, after not being combined with goat anti-rabbit igg in conjunction with complete colloidal gold labeled monoclonal antibody continuation chromatographyForm the red nature controlling line of macroscopic Article 2; If without related antigen, only occur one in throat swab to be checkedRed nature controlling 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), people's mycoplasma pneumoniae (ATCCNumbering 15531), 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), influenza virus A hominis (H1N1,ATCC numbers VR-1743), people's influenza B virus (ATCC numbers VR-790), haemophilus influenzae (ATCCNumbering 53781), the replacement people streptococcus pneumonia such as human respiratory syncytial virus's (ATCC numbers VR26) detects,Kit detects containing the phosphate buffer dilution of these microorganisms all negative.
2) sensitivity tests
Do Study of Sensitivity by measuring culture of streptococcus pneumonia thing dilution. To there is fam2PspA antigenPeople streptococcus pneumonia subtype strains Sp23F (ATCC numbering 700669) sample carries out series with phosphate bufferAfter dilution, detect with kit described in step 6, result shows that it detects lowest limit is 6 × 104CFU/ml。And the BinaxNOWStreptococcuspneumoniaetest (colloid by name being used for from manufacturer BinaxGold method) kit detect, find its detect lowest limit be 5 × 105CFU/ml. The inspection of kit of the present inventionSurveying lower limit obviously reduces compared with it.
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. the anti-human streptococcus pneumonia fam2 PspA of a family protein antibodies, it is characterized in that: the described anti-human streptococcus pneumonia fam2 PspA of family protein antibodies is two kinds of antibody identifying respectively two linear epitopes that the people streptococcus pneumonia fam2 PspA of family albumen 34-47 position and 274-287 amino acids form, and the described people streptococcus pneumonia fam2 PspA of family albumen is WP_054380072.1 at GenBank sequence number; 14 amino acid of the described people streptococcus pneumonia fam2 PspA of family albumen 34-47 position and 14 amino acid whose amino acid sequences of 274-287 position are respectively SPQVVEKSSLEKKY and KLLDSLDPEGKTQD; By 14 amino acid whose sequences called after F2P1 and the F2F2P2 respectively of 14 amino acid of the people streptococcus pneumonia fam2 PspA of family albumen 34-47 position and 274-287 position; The described anti-human streptococcus pneumonia fam2 PspA of family protein antibodies is AbF2P1 and AbF2P2.
2. the immune chromatography reagent kit forming based on the anti-human streptococcus pneumonia fam2 PspA of family protein antibodies 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 the anti-human streptococcus pneumonia fam2 PspA of family protein antibodies according to claim 2 forms, it 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 bF2P1 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 bF2P1 preparing of 0.4mg, 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 bF2P1 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 bF2P1 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 bF2P2 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 bF2P2 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) sample pad described step 3) is overlapped in to 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 the anti-human streptococcus pneumonia fam2 PspA of family protein antibodies according to claim 2 forms, it 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 AbF2P1
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 AbF2P1:
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 bF2P1 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 bF2P1 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 AbF2P1 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 × 4cm size; The antibody A bF2P2 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 bP2 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) sample pad described step 3) is overlapped in to 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.
CN201610129975.3A 2016-03-08 2016-03-08 The immune chromatography reagent kit of anti-human streptococcus pneumonia fam2 family PspA protein antibodies and the application antibody Active CN105585634B (en)

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