CN105585634B - The immune chromatography reagent kit of anti-human streptococcus pneumonia fam2 family PspA protein antibodies and the application antibody - Google Patents
The immune chromatography reagent kit of anti-human streptococcus pneumonia fam2 family PspA protein antibodies and the application antibody Download PDFInfo
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- CN105585634B CN105585634B CN201610129975.3A CN201610129975A CN105585634B CN 105585634 B CN105585634 B CN 105585634B CN 201610129975 A CN201610129975 A CN 201610129975A CN 105585634 B CN105585634 B CN 105585634B
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Classifications
-
- 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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- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Pathology (AREA)
- Microbiology (AREA)
- Analytical Chemistry (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- General Physics & Mathematics (AREA)
- Food Science & Technology (AREA)
- Organic Chemistry (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Peptides Or Proteins (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The present invention relates to anti-human streptococcus pneumonia fam2 family PspA protein antibodies and the immune chromatography reagent kits of application antibody test people streptococcus pneumonia, anti-human streptococcus pneumonia fam2 family PspA protein antibodies are the antibody for identifying two linear epitopes composed by 34-47, people streptococcus pneumonia fam2 family PspA albumen and 274-287 amino acids respectively, and people streptococcus pneumonia fam2 family's PspA albumen is WP_054380072.1 in GenBank sequence number；The amino acid sequence of 34-47 and 274-287, people streptococcus pneumonia fam2 family PspA albumen is respectively SPQVVEKSSLEKKY and KLLDSLDPEGKTQD.Two kinds of rabbit-antis people streptococcus pneumonia fam2 provided by the present invention family PspA protein antibodies have the characteristics that specificity is good, with high purity, potency is high, preparation cost is cheap.
Description
Technical field
The invention belongs to field of biomedicine technology, are related to anti-human streptococcus pneumonia fam2 family PspA protein antibodies and answer
With the immune chromatography reagent kit of antibody test people streptococcus pneumonia.
Background technique
People streptococcus pneumonia (Streptococcus pneumoniae, Sp) is the important pathogen of childrens respiratory tract infection
Body.Mainly enter respiratory tract through droplet transmission, to parasitize the pharynx nasalis of human body, or the position of intrusion human body not easy-clear
Cause a series of disease, such as lobar pneumonia, meningitis, bronchitis, tympanitis etc..It is that all age groups in the whole world are high-incidence
The main pathogenic fungi of sick rate and case fatality rate.Wherein, in developing country, infant, the elderly and immune deficiency crowd especially
Seriously.Streptococcus pneumonia in 1881 for the first time by Pasteur (Louis Pasteur) and G.M.Sternberg respectively France and
It is isolated from patient's sputum in the U.S..It is Gram-positive, and thallus is like spearhead shape, pair in pairs or at short catenation
Coccus, it is the pod membrane of polysaccharide that toxic strain thallus has chemical component outside.Its pod membrane have antigenicity, be streptococcus pneumonia parting according to
According to.Pneumococcus is divided into 91 serotypes according to the difference of capsular polysaccharide antigen.Its somatic antigen is mainly C polysaccharide,
It is present in pneumococcal cell wall, there is species specificity, is common to various bacterial strain.C polysaccharide can be reacted by C- in serum
Albumen precipitation.In the presence of calcium ion, C polysaccharide can be with referred to as C reactive protein (C reactive in normal human serum
Protein, CRP) beta Globulin combine, precipitate.At present for people's streptococcus pneumoniae antigen detection also primarily directed to
This antigen, and the non-streptococcus pneumonia of the antigen is exclusive, such as mitigates streptococcus and also contains the antigen.And the purification process of C polysaccharide
Difficulty causes production cost higher.In S. pneumonia surface, there are also a kind of important antigens relevant to virulence, are pneumonia streptococcus
Bacterium surface protein A (PspA), is present in all S. pneumoniae serotypes, is the specific antigen of streptococcus pneumonia.
PspA point is 3 families, wherein what the people streptococcus pneumonia with fam1 or fam2 family PspA albumen had accounted for being clinically separated
99% or more of people's streptococcus pneumonia type.
Clinical patient is since different respiratory pathogens (haemophilus influenzae, influenza virus, exhale by such as mycoplasma pneumoniae
Inhale road syncytial virus, adenovirus etc.) infection causes disease symptoms quite similar, which results in prevalence diagnosis is relatively difficult,
It makes a definite diagnosis and tends to rely on laboratory diagnosis.Quickly and effectively diagnostic method should be disease early stage can be obtained by it is bright
True diagnosis, it is convenient to carry out targetedly to treat, prevent the development of the state of an illness from delaying.
Although streptococcus pneumonia is propagated in the world, the infection of infant is especially universal, can be used for laboratory and examines
The type of disconnected standardization commercially available reagent is few.Currently, the detection of streptococcus pneumonia is mainly the following method:
One, Routine Test Lab detects
1, bacterium separates
The goldstandard of laboratory diagnosis streptococcus pneumonia is separation people's S. pneumoniae strain.Using nasopharyngeal secretions conduct
The sample of pathogen isolation can use the method isolated pathogen of blood culture.But the method has serious defect, because of pneumonia chain
Coccus is severe bacteria, and nutritional requirement is high, long the time required to culture, and positive rate is low, it is often more important that, if patient used before sampling
Antibacterials will cause the false positive of cultivation results.Just there is certain limitation to the treatment of patient in clinicing aspect in this way.
2, Serologic detection
I.e. using enzyme-linked immunization, radioimmunoassay, micro-Immunofluorescence assay etc., it is anti-to detect streptococcus pneumonia in examinee's serum
Body is horizontal, can prompt the presence of streptococcus pneumoniae infection indirectly.However, serological test can only provide, one kind is retrospective to be examined
Disconnected, it needs while detecting the paired sera of the Acute Stage and convalescence, if anti-human pneumococci antibody in convalescence
High 4 times or 4 times of potency ratio acute stage or more just have diagnostic significance.In addition, the opportunity that antibody occurs is not easy to grasp, and because thallus
Serotype type is excessive, and the anti-capsular polysaccharide antibody type for causing it to induce is excessive, causes very big be stranded to the detection of antibody
Difficulty, therefore the detection quality of existing serological method is subject to certain restrictions.
Two, quick diagnosis
Direct scrutineer's Streptococcus pneumoniae protein antigen and thallus nucleic acid can reach the purpose of quick diagnosis, mainly have at present
Immunofluorescence technique, immunoenzyme and PCR method etc..Immunofluorescence technique and immunoenzyme not can be carried out step detection, there is behaviour
Make step complexity, professional is needed to operate, the disadvantages of detection time is long (2h or more), higher cost.PCR method is quick, clever
It is quick, special, be the important means of current research streptococcus pneumoniae infection, but due to PCR to experimental facilities and operation require compared with
Height, and easily there is false positive, common methods for clinical diagnosis can't be used as in China.Currently, detection people streptococcus pneumonia is anti-
Former method is mainly that colloidal gold method detects its C polysaccharide antigen, but the method susceptibility is lower, wants to measuring samples quality of materials
Ask higher, at the same there is also with other streptococcus as mitigate streptococcus there are cross reactions the defects of.PspA albumen is then one
The examination target of great specificity.Currently, reporting at most to be about anti-human streptococcus pneumonia PspA protein antibodies corresponding
Polyclonal antibody.Polyclonal antibody is mainly prepared by animals such as the PspA protein immunization rabbit of gene engineering expression.It is made
Preparation Method is simple, at low cost, but it has the defects such as specificity is low, potency is low, purity is low.Therefore, inexpensive preparation is high
Specific, high-titer anti-human streptococcus pneumonia PspA protein antibodies just seem particularly significant.
Summary of the invention
For these technical problems present in background technique, the purpose of the present invention is to provide identification people streptococcus pneumonias
Two kinds of antibody of two linear epitopes composed by 34-47, fam2 family PspA albumen and 274-287 amino acids
And the immune chromatography reagent kit using the antibody.
Anti-human streptococcus pneumonia fam2 family PspA protein antibodies, it is characterised in that: the anti-human streptococcus pneumonia fam2
Family's PspA protein antibodies are to identify 34-47 and 274-287 ammonia of people streptococcus pneumonia fam2 family PspA albumen respectively
Two kinds of antibody of two linear epitopes composed by base acid, people streptococcus pneumonia fam2 family PspA albumen exist
GenBank sequence number is WP_054380072.1；Described 34-47, people streptococcus pneumonia fam2 family PspA albumen 14 ammonia
The amino acid sequence of base acid and 274-287 14 amino acid is respectively SPQVVEKSSLEKKY and KLLDSLDPEGKTQD；
By the sequence of 34-47, family PspA albumen 14 amino acid of people streptococcus pneumonia fam2 and 274-287 14 amino acid
Column are respectively designated as F2P1 and F2F2P2；Anti-human streptococcus pneumonia fam2 family PspA protein antibodies be AbF2P1 and
AbF2P2。
One kind being formed by immunochromatography based on foregoing anti-human streptococcus pneumonia fam2 family PspA protein antibodies
Kit, it is characterised in that: the kit is immune chromatography reagent kit based on quantum dot-labeled technology or based on colloidal gold
The immune chromatography reagent kit of labelling technique.
Preferably, when kit provided by the present invention is the immune chromatography reagent kit based on quantum dot-labeled technology,
The preparation method of the kit is:
1) quantum dot-labeled antibody A bF2P1 solution:
0.4nmol carboxyl water-soluble quantum dot and 800nmol carbodiimide EDC are sequentially added into microcentrifugal tube, with
MES buffer constant volume is 1ml, and mixed solution after 37 DEG C of reaction 5min, adds the antibody A bF2P1 of 0.4mg being prepared,
It is protected from light 2h, single-ended amino-polyethyleneglycols PEG2000-NH2 to final concentration of 1% (m/v) is added, closes unreacted activation
Carboxyl site continues to be protected from light 1h；Sample after reaction is centrifuged (molecular cut off 100k) with super filter tube, 6500g centrifugation
5min, until volume 200ul, sample after ultrafiltration is transferred in common EP pipe, centrifugation obtains upper clear supernate and lower part except reuniting
It precipitates, is centrifuged 3min under the conditions of 10000g；Upper clear supernate is added on splitter Superdex-200 and is purified, certainly to upper clear supernate
It so flows into cylinder, is then rinsed with PBS, with the position of ultraviolet light cylinder observation sample, start to flow from lower part to sample
Start to collect when out, stops collecting after collecting 1ml；By sample after purification with super filter tube (molecular cut off 100k) with 6500g
Centrifugation in common EP pipe is transferred to after centrifugal concentrating to 200ul, and, except reuniting, the condition being centrifuged to common EP pipe is 10000g,
3min；Liquid is saved with phosphate after acquisition supernatant and dilutes 200 times, 4 DEG C save backup；So far quantum dot-labeled antibody is made
AbF2P1 solution；
Each component content is respectively in the MES buffer: 10.66g/L MES and 0.74g/L EDTA, the MES
The pH 7.4 of buffer；
The phosphate save liquid preparation method be weigh 0.29g disodium hydrogen phosphate, 0.0295g sodium dihydrogen phosphate,
0.2g sodium chloride, 1g bovine serum albumin(BSA) BSA and 0.1gNaN3, it is dissolved in the deionized water of 90ml, with 1mol/L NaOH
100ml is settled to deionized water after tune pH to 7.3；
2) bonding pad is prepared
Polyester fiber film is immersed into 1h in the obtained quantum dot-labeled antibody A bF2P1 solution of step 1), is taken out, 25 DEG C
Be cut into after drying rear specification be 4cm × 0.6cm/ item after, 4 DEG C be sealed it is spare, so far be made bonding pad；
3) sample pad is prepared
It takes glass fibre element film one to open, glass fibre element film is impregnated at least 3h in sample pad treatment fluid, then be placed in life
In object safety cabinet after 37 DEG C of aeration-dryings, specification is cut into after 4cm × 2.5cm/ item, to obtain sample pad, 25 DEG C of sealings are protected
It deposits；
The preparation method of the sample pad treatment fluid be weigh 0.29g disodium 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, it is molten
Solution is settled to 100ml with deionized water in the deionized water of 90ml, with after 1mol/L NaOH tune pH to 7.3；
4) detection layers are prepared
Nitrocellulose filter is cut into 4cm × 4cm size；By the antibody A bF2P2 being prepared and goat anti-rabbit igg phosphorus
It is respectively 2.0mg/mL and 1.0mg/mL that phthalate buffer, which is adjusted to final concentration,；The antibody A bF2P2 diluted is packed into BIODOT
It draws in film instrument spray head, the amount of 1.0 μ l/cm of setting is sprayed on nitrocellulose filter, forms detection line；The anti-rabbit IgG that will have been diluted
BIODOT is fitted into draw in film instrument spray heads, the amount of 1.0 μ l/cm of setting is sprayed on nitrocellulose filter as nature controlling line, nature controlling line with
Detection line spacing is 0.7cm；37 DEG C of the nitrocellulose filter dry 2h that will have been sprayed are cut into the specification of 4cm × 4cm, 4 DEG C of sealings
Kept dry；So far detection layers are made；
The preparation method of the phosphate buffer is: weigh 0.29g disodium hydrogen phosphate, 0.0295g sodium dihydrogen phosphate with
And 0.2g sodium chloride, it is dissolved in the deionized water of 90ml, is settled to after 1mol/L NaOH tune pH to 7.3 with deionized water
100ml；
5) assembling detection card
5.1) bottom plate is cut into 4cm × 7.3cm size, it is spare；
5.2) absorbent filter is cut into 4cm × 3cm size, it is spare as water absorption pad；
5.3) adhered protection film on bottom plate that step 5.1) is prepared is taken off, is by detection layers described in step 4)
Nitrocellulose filter with nature controlling line and detection line pastes on bottom plate, and smoothes out film surface；
5.4) the ready water absorption pad of step 5.2) is assembled on bottom plate, makes the left side of water absorption pad and the right end of detection layers
There is the overlapping of 0.2cm, the right hand edge of water absorption pad is then aligned with the right hand edge of bottom plate and glues and smooth out；It is again that step 2) is described
Bonding pad is overlapped at the left edge of detection layers by 0.3cm, and 0.3cm is sticked on bottom plate；
5.5) sample pad described in step 3) is then overlapped at the left edge of bonding pad by one side 0.3cm, it is another
Side is aligned with the left edge of bottom plate, is sticked on bottom plate and is smoothed out；Assembled detection plate is cut into 4.0mm's wide under cutting machine
Detection card, 4 DEG C of hermetically dryings are kept in dark place.
Preferably, when kit provided by the present invention is the immune chromatography reagent kit based on colloidal gold-labeled method,
The preparation method of the kit is:
1) colloidal gold labeled monoclonal antibody AbF2P1
1.1) 30nm colloidal gold solution is prepared:
99ml ultrapure water is added, by 1ml 1% (m/v) HAuCl in the 250ml triangular flask for taking a silication good4Solution is added
It is mixed in 250ml triangular flask and with ultrapure water, oil bath heating is simultaneously stirred to boiling；2ml is rapidly joined into 250ml triangular flask
1% (m/v) trisodium citrate aqueous solution, solution continue the 10min that boils, are changed into the solution in 250ml triangular flask by blue
Stop heating when red, then the solution cooled to room temperature in 250ml triangular flask is added super into 250ml triangular flask
Pure water polishing is to 100ml；
1.2) colloidal gold labeled monoclonal antibody AbF2P1:
1.2.1 colloidal gold solution prepared by 10ml step 1.1) is added in the 50ml triangular flask for) taking a silication good, to
240ul 0.2mol/L K is added in colloidal gold liquid2CO3Adjust pH to 8.5；
1.2.2) under magnetic stirrer, antibody A bF2P1 is added in colloidal gold solution, until antibody is final concentration of
10ug/ml is added dropwise when antibody is added, and continues to stir 45min~60min after adding；
1.2.3) reaction completes that (m/v) the bovine serum albumin(BSA) BSA of 2.5ml 5% to final concentration of 1% (m/v) is added, and stirs
It mixes 15~30 minutes, 4 DEG C save backup；
1.2.4 50ml centrifuge tube is packed into after) taking out the antibody A bF2P1 marked, 2500g, 4 DEG C are centrifuged 5 minutes, obtain
To lower sediment and supernatant liquor, discard lower sediment, supernatant liquor is transferred to another 50ml centrifuge tube, 12000g, 4 DEG C from
The heart 30 minutes, lower sediment and supernatant liquor are obtained, supernatant liquor is discarded, lower sediment 10ml colloidal gold buffer is resuspended
Precipitating, then 12000g again, 4 DEG C are centrifuged 30 minutes, obtain lower sediment and supernatant liquor again, lower sediment is finally used
3ml colloidal gold buffer is resuspended, and 4 DEG C save backup；
Each component content is respectively in the colloidal gold buffer: 10mM Tris, 1% (m/v) BSA, 1% (v/v)
Tween-20,5% (m/v) sucrose and 3 ‰ (m/v) polyvinylpyrrolidones, the pH of the colloidal gold buffer are 10.5；
2) bonding pad is prepared
Polyester fiber film is immersed into 1h in the obtained colloidal gold labeled monoclonal antibody AbF2P1 solution of step 1), is taken out, 25 DEG C
Be cut into after drying rear specification be 4cm × 0.6cm/ item after, 4 DEG C be sealed it is spare, so far be made bonding pad；
3) sample pad is prepared
It takes glass fibre element film one to open, glass fibre element film is impregnated at least 2h in sample pad treatment fluid, then be placed in life
In object safety cabinet after 37 DEG C of aeration-dryings, specification is cut into after 4cm × 1.5cm/ item, to obtain sample pad, 25 DEG C of sealings are protected
It deposits；
The preparation method of the sample pad treatment fluid be weigh 0.242g Tris, 1g bovine serum albumin(BSA) BSA, 1ml are spat
Temperature -20,5g sucrose and 0.3g polyvinylpyrrolidone PVP-10, are dissolved in the deionized water of 90ml, with 1mol/L NaOH
100ml is settled to deionized water after tune pH to 11；
4) detection layers are prepared
Nitrocellulose filter is cut into 4cm × 2cm size；By the antibody A bF2P2 being prepared and goat anti-rabbit igg phosphorus
It is respectively 2.0mg/mL and 1.0mg/mL that phthalate buffer, which is adjusted to final concentration,；The antibody A bF2P2 diluted is packed into BIODOT
It draws in film instrument spray head, the amount of 1.0 μ l/cm of setting is sprayed on nitrocellulose filter, forms detection line；The anti-rabbit IgG that will have been diluted
BIODOT is fitted into draw in film instrument spray heads, the amount of 1.0 μ l/cm of setting is sprayed on nitrocellulose filter as nature controlling line, nature controlling line with
Detection line spacing is 0.7cm；37 DEG C of the nitrocellulose filter dry 18h that will have been sprayed, are cut into the specification of 4cm × 2cm, 4 DEG C close
Seal kept dry；So far detection layers are made；
The preparation method of the phosphate buffer is: weigh 0.29g disodium hydrogen phosphate, 0.0295g sodium dihydrogen phosphate with
And 0.2g sodium chloride, it is dissolved in the deionized water of 90ml, is settled to after 1mol/L NaOH tune pH to 7.3 with deionized water
100ml；
5) assembling detection card
5.1) bottom plate is cut into 4cm × 6cm size, it is spare；
5.2) absorbent filter is cut into 4cm × 2.5cm size, it is spare as water absorption pad；
5.3) adhered protection film on bottom plate that step 5.1) is prepared is taken off, is by detection layers described in step 4)
Nitrocellulose filter with nature controlling line and detection line pastes on bottom plate, and smoothes out film surface；
5.4) the ready water absorption pad of step 5.2) is assembled on bottom plate, makes the left side of water absorption pad and the right end of detection layers
There is the overlapping of 0.2cm, the right hand edge of water absorption pad is then aligned with the right hand edge of bottom plate and glues and smooth out；It is again that step 2) is described
Bonding pad is overlapped at the left edge of detection layers by 0.2cm, and 0.4cm is sticked on bottom plate；
5.5) sample pad described in step 3) is then overlapped at the left edge of bonding pad by one side 0.2cm, it is another
Side is aligned with the left edge of bottom plate, is sticked on bottom plate and is smoothed out；Assembled detection plate is cut into 4.0mm's wide under cutting machine
Detection card, 4 DEG C of hermetically dryings are kept in dark place.
The invention has the advantages that
The present invention provides anti-human streptococcus pneumonia fam2 family PspA protein antibodies, the anti-human streptococcus pneumonia fam2 men
14 amino acid and 274-287 of race's PspA protein antibodies 34-47, people streptococcus pneumonia fam2 family PspA albumen of identification
Two linear epitopes composed by 14 amino acid of position, people streptococcus pneumonia fam2 family PspA albumen is in GenBank sequence
Row number is AAB59852.1；People streptococcus pneumonia fam2 34-47, family PspA albumen 14 amino acid and 274-287
The amino acid sequences of 14 amino acid be respectively SKYTIQRSTGDSID and FGIAQSSTRGGSRV；By people streptococcus pneumonia fam2
The sequence of 34-47, family PspA albumen 14 amino acid and 274-287 14 amino acid be respectively designated as F2P1 and
F2P2；Anti-human streptococcus pneumonia fam2 family PspA protein antibodies are AbF2P1 and AbF2P2.Based on people streptococcus pneumonia
Above two rabbit-anti people streptococcus pneumonia fam2 family PspA prepared by fam2 family PspA albumen single linear epitope
Protein antibodies have the characteristics that specificity is good, with high purity, potency is high, preparation cost is cheap, can be used for producing various based on antigen-
The detection kit of the highly sensitive detection people streptococcus pneumonia of the principle of antibody response.Meanwhile being based on the anti-human pneumonia streptococcus
Two different immune chromatography reagent kits have also been prepared in bacterium fam2 family PspA protein antibodies.Two different immunochromatographies
Kit can quickly, accurately detect the people streptococcus pneumonia with fam2 family PspA albumen in biological sample, include
Two kinds of antibody of the present invention；Two kinds of immune chromatography reagent kits are used equally for the auxiliary diagnosis of people's streptococcus pneumoniae infection, tool
There are higher sensitivity and specificity, the advantages such as simple, quick, stable and manufacturing cost is low has been combined, suitable for facing
The inspection of bed sample, and since large batch of quick inspection can be carried out, it is also suitable for epidemiological survey.Therefore, this hair
Bright two kinds of rabbit-anti people streptococcus pneumonia fam2 family PspA protein antibodies, two kinds of immune chromatography reagent kits all have extensively
Application prospect and practical value.
Specific embodiment
The present invention is further understood in order to facilitate those skilled in the art, it is detailed that preferred embodiment is cited below particularly
Illustrate the present invention.
The source for a variety of materials that the present invention is used or used and the preparation of related reagent
1, sample pad treatment fluid: weighing 0.242g Tris, 1g bovine serum albumin(BSA) (BSA), 1ml Tween-20,5g sucrose,
0.3g polyvinylpyrrolidone (PVP-10), is dissolved in the deionized water of 90ml, with spending after 1mol/L NaOH tune pH to 11
Ionized water is settled to 100ml.
2, phosphate saves liquid: weighing 0.29g disodium hydrogen phosphate, 0.0295g sodium dihydrogen phosphate, 0.2g sodium chloride, 1g ox
Seralbumin BSA and 0.1gNaN3, it is dissolved in the deionized water of 90ml, with being spent after 1mol/L NaOH tune pH to 7.3
Ionized water is settled to 100ml；
3, phosphate buffer (PBS): weighing 0.29g disodium hydrogen phosphate, 0.0295g sodium dihydrogen phosphate, 0.2g sodium chloride,
It is dissolved in the deionized water of 90ml, is settled to 100ml with deionized water with after 1mol/L NaOH tune pH to 7.3.
4,0.0121g trishydroxymethylaminomethane, 0.17g sodium chloride, the dissolution of 0.025g lysozyme sample treatment liquid: are weighed
In 90ml deionized water, 100ml is settled to deionized water with after hydrochloric acid tune pH to 8.0.
5, it antibody A bF2P1: for present invention self-control, is diluted, is shaken up with PBS, make antibody concentration 3mg/ml in solution.
6, it antibody A bF2P2: for present invention self-control, is diluted, is shaken up with PBS, make antibody concentration 3mg/ml in solution.
7, it goat anti-rabbit igg: for Wuhan Boster Biological Technology Co., Ltd.'s product, is diluted, is shaken up with PBS, made in solution
Anti-TNF-α bulk concentration is 1mg/ml.
8, quantum dot: quantum dot used is water-soluble CdSe/ZnS quantum of carboxylated amphipathic polymer modification in the present invention
Point, launch wavelength 565nm, from Wuhan Jia Yuan technology of quantum dots development corporation, Ltd., name of product is that carboxyl is water-soluble for purchase
Property quantum dot -565.
9, glass fibre element film: with a thickness of 0.4mm, water absorption 42mg/cm2, glass fiber diameter is 0.6-3 μm, tool
There is good hydrophily, buys in Shanghai Jinbiao Bio-Tech Co., Ltd. (model BT40).
10, polyester fiber film: with a thickness of 0.48mm, absorption speed 18s/4cm, there is fabulous hydrophily, for tying
The preparation of pad is closed, is bought in Shanghai Jinbiao Bio-Tech Co., Ltd. (model DL42).
11, nitrocellulose filter: model Millipore Corp SHF135 has liner plate, and purchase is in Millipore public affairs
Department.
12, absorbent filter: with a thickness of 0.95mm, absorption speed 60s/4cm, water absorption 700mg/cm2, have good
Water imbibition, as production water absorption pad material.It buys in Shanghai Jinbiao Bio-Tech Co., Ltd. (model CH37K).
13, bottom plate: for high whiteness PVC material, surface is coated with single layer high polymer pressure sensitive adhesive SM31-40, buys in Shanghai
Jin Biao Biotechnology Co., Ltd.
14, people's Streptococcus pneumoniae subtypes bacterial strain Sp23F: being purchased from American type culture collection (ATCC), and number is
ATCC 700669。
15, the microbiological specimens used in the present invention are purchased from American type culture collection (ATCC).
Technical solution provided by the present invention is described in detail below with reference to embodiment:
The preparation of 1 two kinds of rabbit-anti people streptococcus pneumonia fam2 family PspA protein antibodies of embodiment
Two kinds of rabbit-anti people streptococcus pneumonia fam2 family PspA protein antibodies the preparation method is as follows:
1) after structure biology analysis and Related Experimental Study, people streptococcus pneumonia fam2 family PspA albumen is selected
14 amino acid and 274-287 14 amino acid that (GenBank sequence number WP_054380072.1) is 34-47 form short
Two linear epitopes of the peptide respectively as preparation rabbit-anti people streptococcus pneumonia fam2 family PspA protein antibodies, two sections of ammonia
Base acid sequence is respectively SPQVVEKSSLEKKY and KLLDSLDPEGKTQD, this two sequence is respectively designated as F2P1 and F2P2；
2) after the N-terminal of the C-terminal of amino acid sequence F2P1, F2P2 described in step 1) being added a cysteine respectively,
Polypeptide is respectively synthesized with polypeptide automatic synthesizer and is purified, and two polypeptides after purification with carrier protein KLH, are formed respectively
F2P1-KLH compound protein and F2P2-KLH compound protein；
3) two kinds of compound protein emulsification synthesized by step 2) is infused in rabbit subcutaneous abdomen multiple spot respectively after emulsification respectively
It penetrates, successively injects three times, every minor tick 7-10 days；
4) third time injection 10-12 days after, respectively collect, it is isolated two kinds contain rabbit-anti people streptococcus pneumonia fam2 man
The serum of race's PspA protein antibodies, ELISA detect rabbit-anti people streptococcus pneumonia fam2 family PspA protein antibodies in serum respectively
Potency, the potency of two kinds of rabbit-anti people streptococcus pneumonia fam2 family PspA protein antibodies is in 1:60000 or more；
5) two polypeptides that step 2) is synthesized and purified are coupled with the Sepharose 4B of cyanogen bromide-activated respectively, are formed
Two groups of polypeptide affinity columns；
6) the two kinds of serum containing rabbit-anti people streptococcus pneumonia fam2 family PspA protein antibodies point obtained step 4)
In not corresponding two kinds of affinity columns for being added to step 5) preparation, and after 4 DEG C are incubated overnight, antibody elution obtains two
Kind rabbit-anti people streptococcus pneumonia fam2 family PspA protein antibodies；Its purity is identified 97% or more, by this through SDS-PAGE
The antibody of two kinds of purifying is respectively designated as AbF2P1 and AbF2P2.
Step 2) -6 in the present embodiment) it is all existing mature technology, Duo Jia biotechnology company can provide sequencing
Technological service.In the present embodiment in above-mentioned steps related experiment link specific implementation, entrust Nanjing Jin Sirui biology section
Skill Co., Ltd completes.
" antibody " of the present invention should be construed to cover any specificity of the binding structural domain with required specificity
Binding factor.Thus, this term covers the function of antibody fragment homologous therewith, derivative, humanized antibody and antibody
It can coordinate and homologue.The example of antibody is that immunoglobulin hypotype (such as IgG, IgE, IgM, IgD and IgA) and its hypotype are sub-
Class；It is also possible to segment such as Fab, scFv, Fv, dAb, Fd and double-chain antibody comprising antigen-binding domains.
The preparation and application of immune chromatography reagent kit of the embodiment 2 based on quantum dot-labeled technology
1. quantum dot-labeled antibody A bF2P1
0.4nmol carboxyl water-soluble quantum dot and 800nmol carbodiimide (EDC) are sequentially added into microcentrifugal tube,
With MES buffer (10.66g/L MES, 0.74g/L EDTA pH 7.4) constant volume for 1ml, ceaselessly mixed solution, 37 DEG C anti-
After answering 5min, antibody A bF2P1 prepared by the embodiment 1 of 0.4mg is added, 2h is protected from light, the poly- second two of single-ended amino is added
Alcohol (PEG2000-NH2) closes unreacted activated carboxyl site, continues to be protected from light 1h to final concentration of 1% (m/v).Instead
Sample after answering is centrifuged (molecular cut off 100k) with super filter tube, and 6500g is centrifuged 5min, until volume 200ul, by sample after ultrafiltration
It is transferred in common EP pipe, centrifugation is except reunion (10000g, 3min).Upper clear supernate is added on splitter (Superdex-200)
Purifying, flows into cylinder naturally to it, then rinses (liquid flows down naturally) with PBS, is observed at any time with ultraviolet light cylinder
The position of sample starts to collect since when lower part is flowed out, stops collecting after collecting 1ml when sample.Sample after purification is used
Super filter tube (molecular cut off 100k) be transferred to after 6500g centrifugal concentrating to 200ul centrifugation in common EP pipe (10000g,
3min) except reunion.Liquid is saved with phosphate after acquisition supernatant and dilutes 200 times, 4 DEG C save backup.So far it is made quantum dot-labeled
Antibody A bF2P1.
2. the preparation of bonding pad
Polyester fiber film is immersed into 1h in the obtained quantum dot-labeled antibody A bF2P1 solution of step 1, is taken out, 25 DEG C dry
Be cut into after dry rear specification be 4cm × 0.6cm/ item after, 4 DEG C be sealed it is spare, so far be made bonding pad.
3. the preparation of sample pad
It takes glass fibre element film one to open, it is impregnated at least 3h in sample pad treatment fluid, then be placed in Biohazard Safety Equipment
After 37 DEG C of aeration-dryings, specification is cut into obtain sample pad, 25 DEG C are sealed after 4cm × 2.5cm/ item.
4. the preparation of detection layers
Nitrocellulose filter is cut into 4cm × 4cm size.By antibody A bF2P2 prepared in embodiment 1 and goat-anti rabbit
It is respectively 2.0mg/mL and 1.0mg/mL that IgG, which is adjusted with phosphate buffer to final concentration,.The antibody A bF2P2 diluted is filled
Enter BIODOT to draw in film instrument spray head, the amount of 1.0 μ l/cm of setting is sprayed on nitrocellulose filter, forms detection line；By what is diluted
Anti-rabbit IgG is fitted into BIODOT and draws in film instrument spray head, and the amount of 1.0 μ l/cm of setting is sprayed on nitrocellulose filter as nature controlling line,
It is 0.7cm with detection line spacing.37 DEG C of the nitrocellulose filter dry 2h that will have been sprayed, are cut into the specification of 4cm × 4cm, 4 DEG C close
Seal kept dry.So far detection layers are made.
5. detecting the assembling of card
Bottom plate is cut into 4cm × 7.3cm size, it is spare.
Absorbent filter is cut into 4cm × 3cm size, it is spare as water absorption pad.
Assembly working first takes the adhered protection film on bottom plate off in operating in Biohazard Safety Equipment, will be described in step 4
Detection layers have nature controlling line and the nitrocellulose filter of detection line pastes on bottom plate, and carefully smooth out film surface.Secondly, by thing
The water absorption pad first cut out is assembled on bottom plate, and having its left side and detection layers right end, 0.2cm's is overlapping, right hand edge then with bottom
The right hand edge alignment of plate is glued and is carefully smoothed out.Bonding pad described in step 2 is overlapped in the left side of detection layers by 0.3cm again
At edge, 0.3cm is sticked on bottom plate 7.Sample pad described in step 3 is then finally overlapped in bonding pad by one side 0.3cm
At left edge, another side is aligned with the left edge of bottom plate, is sticked on bottom plate and is carefully smoothed out.By assembled detection plate in slitting
The detection card of 4.0mm wide is cut under machine, 4 DEG C of hermetically dryings are kept 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 detection card as described in step 5 and sample treatment liquid institute group
At.
7. the application method of the immune chromatography reagent kit based on quantum dot-labeled technology
The throat swab for obtaining person to be checked according to a conventional method is inserted into the flexible plastic pipe equipped with 500 μ l sample treatment liquids
In, squeezable plastic tube wall, make the sample on swab take out after completely dissolution 120 μ L drops in detection card sample pad on, 15 minutes
(model WD-9403A, Liuyi Instruments Plant, Beijing's production, burst of ultraviolel wavelength 365nm) the observation detection under uv analyzer afterwards
As a result.It is and quantum dot-labeled anti-in bonding pad if the people streptococcus pneumonia containing tool fam2PspA antigen in throat swab
Body AbF2P1 combine, by chromatography effect first with the antibody A bF2P2 on nitrocellulose filter in conjunction with after ultraviolet light excite under
It will form a macroscopic fluorescence detection line at detection line, the quantum dot-labeled antibody being not associated with continues chromatography and goat-anti
Rabbit igg forms macroscopic Article 2 fluorescence nature controlling line under ultraviolet light excitation after combining；If without correlation in throat swab to be checked
Then only there is a fluorescence nature controlling line in antigen.If fluorescence nature controlling line does not occur, detection card failure.
8. the application effect of the immune chromatography reagent kit based on quantum dot-labeled technology is illustrated
The application method of the signified immune chromatography reagent kit based on quantum dot-labeled technology is referring to step 7 in the present embodiment
The operating procedure.
1) specific test
With respiratory tract common causative such as human III type parainfluenza virus virus (ATCC VR-93), people's mycoplasma pneumoniae
(GB plants, ATCC is numbered for (ATCC number 15531), people's chlamydia pneumoniae (AR-39 plants, ATCC number 53592), 3 type of adenovirus hominis
VR-3), 7 type of adenovirus hominis (Gomen plants, ATCC number VR-7), influenza virus A hominis (H1N1, ATCC number VR-1743),
People's influenza B virus (ATCC number VR-790), haemophilus influenzae (ATCC number 53781), human respiratory syncytial virus
(ATCC number VR26) etc. is detected instead of people streptococcus pneumonia, and kit detects the phosphate buffer containing these microorganisms
Dilution is all negative.
2) sensitivity tests
Study of Sensitivity is done by measurement culture of streptococcus pneumonia object dilution.There to be fam2PspA antigen people's pneumonia
After streptococcus subtype strains Sp23F (ATCC number 700669) sample is serially diluted with phosphate buffer, with step 6 institute
It states kit to be detected, the results showed that it is 2 × 10 that it, which detects the lowest limit,4CFU/ml.And it is used to from the entitled of manufacturer Binax
The kit of Binax NOW Streptococcus pneumoniae test (colloidal gold method) is detected, and finds its detection
The lowest limit is 5 × 105CFU/ml.The Monitoring lower-cut of kit of the present invention is substantially reduced compared with it.
The preparation and application of immune chromatography reagent kit of the embodiment 3 based on colloidal gold-labeled method
1. colloidal gold labeled monoclonal antibody AbF2P1
A.30nm the preparation of colloidal gold
The 250ml triangular flask that a silication is good is taken, 99ml ultrapure water is added, it is added in 1ml 1% (m/v) HAuCl4 solution
Middle mixing, oil bath heating are simultaneously stirred to boiling.Rapidly join 2ml 1% (m/v) trisodium citrate aqueous solution thereto, solution after
Continuous boiling 10min (solution is changed into red by blue during this).Stop heating, allow solution cooled to room temperature, so
Ultrapure water polishing is added thereto afterwards to 100ml.
B. colloidal gold labeled monoclonal antibody AbF2P1
1) the 50ml triangular flask that a silication is good is taken, colloidal gold liquid prepared by 10ml step a is added, is added into golden liquid
240ul 0.2mol/L K2CO3Adjust pH to 8.5；
2) under magnetic stirrer, antibody A bF2P1 is added in colloidal gold solution, until the final concentration of 10ug/ of antibody
Ml should be added dropwise when antibody is added, and continue to stir 45min~60min after adding；
3) reaction completes to be added 2.5ml 5% (m/v) bovine serum albumin(BSA) (BSA) to final concentration of 1% (m/v), stirring
15~30 minutes, 4 DEG C saved backup.
4) 50ml centrifuge tube is packed into after taking out the antibody A bF2P1 marked, 2500g, 4 DEG C are centrifuged 5 minutes, obtain down
Layer precipitating and supernatant liquor, discard lower sediment, and supernatant liquor is transferred to another 50ml centrifuge tube, 12000g, 4 DEG C of centrifugations 30
Minute, lower sediment and supernatant liquor are obtained, supernatant liquor is discarded, it is heavy that lower sediment 10ml colloidal gold buffer is resuspended
It forms sediment, then 12000g again, 4 DEG C are centrifuged 30 minutes, obtain lower sediment and supernatant liquor again, lower sediment is finally used 3ml
Colloidal gold buffer is resuspended, and 4 DEG C save backup；
Each component content is respectively in above-mentioned colloidal gold buffer: 10mM Tris, 1% (m/v) BSA, 1% (v/v)
Tween-20,5% (m/v) sucrose and 3 ‰ (m/v) polyvinylpyrrolidones, the pH of the colloidal gold buffer are 10.5；
2. the preparation of bonding pad
Polyester fiber film is immersed into 1h in the obtained colloidal gold labeled monoclonal antibody AbF2P1 solution of step 1, is taken out, 25 DEG C dry
Be cut into after dry rear specification be 4cm × 0.6cm/ item after, 4 DEG C be sealed it is spare, so far be made bonding pad.
3. the preparation of sample pad
It takes glass fibre element film one to open, it is impregnated at least 2h in sample pad treatment fluid, then be placed in Biohazard Safety Equipment
After 37 DEG C of aeration-dryings, specification is cut into obtain sample pad, 25 DEG C are sealed after 4cm × 1.5cm/ item.
4. the preparation of detection layers
Nitrocellulose filter is cut into 4cm × 2cm size.By antibody A bF2P2 prepared in embodiment 1 and goat-anti rabbit
It is respectively 2.0mg/mL and 1.0mg/mL that IgG, which is adjusted with phosphate buffer to final concentration,.The antibody A bF2P2 diluted is filled
Enter BIODOT to draw in film instrument spray head, the amount of 1.0 μ l/cm of setting is sprayed on nitrocellulose filter, forms detection line；By what is diluted
Anti-rabbit IgG is fitted into BIODOT and draws in film instrument spray head, and the amount of 1.0 μ l/cm of setting is sprayed on nitrocellulose filter as nature controlling line,
It is 0.5cm with detection line spacing.37 DEG C of the nitrocellulose filter dry 18h that will have been sprayed, are cut into the specification of 4cm × 2cm, and 4 DEG C
Hermetically drying saves.So far detection layers are made.
5. detecting the assembling of card
Bottom plate is cut into 4cm × 6cm size, it is spare.
Absorbent filter is cut into 4cm × 2.5cm size, it is spare as water absorption pad.
Assembly working first takes the adhered protection film on bottom plate off in operating in Biohazard Safety Equipment, will be described in step 4
Detection layers have nature controlling line and the nitrocellulose filter of detection line pastes on bottom plate, and carefully smooth out film surface.Secondly, by thing
The water absorption pad first cut out is assembled on bottom plate, and having its left side and detection layers right end, 0.2cm's is overlapping, right hand edge then with bottom
The right hand edge alignment of plate is glued and is carefully smoothed out.Bonding pad described in step 2 is overlapped in a left side for detection layers 3 by 0.2cm again
Edge, 0.4cm are sticked on bottom plate.Sample pad described in step 3 is then finally overlapped in bonding pad by one side 0.2cm
At left edge, another side is aligned with the left edge of bottom plate, is sticked on bottom plate and is carefully smoothed out.By assembled detection plate in slitting
The detection card of 4.0mm wide is cut under machine, 4 DEG C of hermetically dryings are kept in dark place.
6. the composition of the immune chromatography reagent kit based on colloidal gold-labeled method
Based on the immune chromatography reagent kit of colloidal gold-labeled method detection card as described in step 5 and sample treatment liquid institute group
At.
7. the application method of the immune chromatography reagent kit based on colloidal gold-labeled method
The throat swab for obtaining person to be checked according to a conventional method is inserted into the flexible plastic pipe equipped with 500 μ l sample treatment liquids
In, squeezable plastic tube wall makes the sample on swab after completely dissolution in 50 DEG C of water-bath 20min, takes out 120 μ L drops in detection card
In sample pad, testing result is visually observed after 30 minutes.If the people streptococcus pneumonia containing tool fam2PspA antigen in throat swab,
Then in conjunction with the antibody A bF2P1 of the colloid gold label in bonding pad, pass through antibody of the chromatography effect first and on nitrocellulose filter
AbF2P2 will form macroscopic one red detection line after combining at detection line, and the colloid gold label being not associated with is anti-
Body forms macroscopic Article 2 red nature controlling line after continuing chromatography in conjunction with goat anti-rabbit igg；If without phase in throat swab to be checked
Antigen is closed, then a red nature controlling line only occurs.If red nature controlling line does not occur, detection card failure.
8. the application effect of the immune chromatography reagent kit based on colloidal gold-labeled method is illustrated
The application method of the signified immune chromatography reagent kit based on colloidal gold-labeled method is referring to step 7 in the present embodiment
The operating procedure.
1) specific test
With respiratory tract common causative such as I type human parainfluenza viruses (ATCC VR-94), II type human parainfluenza viruses (ATCC
VR-92), type III human parainfluenza viruses virus (ATCC VR-93), people's mycoplasma pneumoniae (ATCC number 15531), people's pneumonia clothing
Substance (AR-39 plants, ATCC number 53592), 3 type of adenovirus hominis (GB plants, ATCC number VR-3), 7 type (Gomen of adenovirus hominis
Strain, ATCC number VR-7), influenza virus A hominis (H1N1, ATCC number VR-1743), people's influenza B virus (ATCC number
VR-790), haemophilus influenzae (ATCC number 53781), human respiratory syncytial virus (ATCC number VR26) etc. replace people's lung
Scorching streptococcus is detected, and phosphate buffer dilution of the kit detection containing these microorganisms is all negative.
2) sensitivity tests
Study of Sensitivity is done by measurement culture of streptococcus pneumonia object dilution.There to be fam2PspA antigen people's pneumonia
After streptococcus subtype strains Sp23F (ATCC number 700669) sample is serially diluted with phosphate buffer, with step 6 institute
It states kit to be detected, the results showed that it is 6 × 10 that it, which detects the lowest limit,4CFU/ml.And it is used to from the entitled of manufacturer Binax
The kit of Binax NOW Streptococcus pneumoniae test (colloidal gold method) is detected, and finds its detection
The lowest limit is 5 × 105CFU/ml.The Monitoring lower-cut of kit of the present invention is substantially reduced compared with it.
It should be pointed out that the foregoing is merely illustrative of the preferred embodiments of the present invention, it is not intended to limit the invention, it is all
Any modification, equivalent replacement for being made within spirit of that invention and principle etc. should be included in protection scope of the present invention it
It is interior.
Claims (1)
1. one kind is formed by immune chromatography reagent kit, feature based on anti-human streptococcus pneumonia fam2 family PspA protein antibodies
Be: the kit is the immune chromatography reagent kit or immune based on colloidal gold-labeled method based on quantum dot-labeled technology
Chromatograph kit；
Anti-human streptococcus pneumonia fam2 family PspA protein antibodies include AbF2P1 and AbF2P2, AbF2P1 and AbF2P2
Preparation method is:
1) after structure biology analysis and Related Experimental Study, people streptococcus pneumonia fam2 family PspA albumen 34-47 is selected
The small peptide of 14 amino acid of position and 274-287 14 amino acid composition is respectively as preparation rabbit-anti people streptococcus pneumonia fam2
Two linear epitopes of family's PspA protein antibodies, two sections of amino acid sequences be respectively SPQVVEKSSLEKKY and
This two sequence is respectively designated as F2P1 and F2P2 by KLLDSLDPEGKTQD；
2) after the N-terminal of the C-terminal of amino acid sequence F2P1, F2P2 described in step 1) being added a cysteine respectively, with more
Automatic peptide synthesizer is respectively synthesized polypeptide and purifies, and two polypeptides after purification with carrier protein KLH, are formed respectively
F2P1-KLH compound protein and F2P2-KLH compound protein；
3) respectively by two kinds of compound protein emulsification synthesized by step 2), respectively in rabbit subcutaneous abdomen multi-point injection after emulsification, first
After inject three times, every minor tick 7-10 days；
4) third time injection 10-12 days after, respectively collect, it is isolated two kinds contain rabbit-anti people streptococcus pneumonia fam2 family
The serum of PspA protein antibodies, ELISA detect rabbit-anti people streptococcus pneumonia fam2 family PspA protein antibodies in serum respectively
Potency, the potency of two kinds of rabbit-anti people streptococcus pneumonia fam2 family PspA protein antibodies is in 1:60000 or more；
5) two polypeptides that step 2) is synthesized and purified are coupled with the Sepharose4B of cyanogen bromide-activated respectively, form two groups
Polypeptide affinity column；
6) the two kinds of serum containing rabbit-anti people streptococcus pneumonia fam2 family PspA protein antibodies obtained step 4) are right respectively
That answers is added in two kinds of affinity columns of step 5) preparation, and after 4 DEG C are incubated overnight, antibody elution obtains two kinds of rabbits
Anti-human streptococcus pneumonia fam2 family PspA protein antibodies；Its purity is identified 97% or more, by both through SDS-PAGE
The antibody of purifying is respectively designated as AbF2P1 and AbF2P2；
When the kit is the immune chromatography reagent kit based on quantum dot-labeled technology, the preparation method of the kit is:
1) quantum dot-labeled antibody A bF2P1 solution:
0.4nmol carboxyl water-soluble quantum dot and 800nmol carbodiimide EDC are sequentially added into microcentrifugal tube, it is slow with MES
Fliud flushing constant volume is 1ml, and mixed solution after 37 DEG C of reaction 5min, adds the antibody A bF2P1 of 0.4mg being prepared, is protected from light
2h is reacted, single-ended amino-polyethyleneglycols PEG2000-NH2 to final concentration of 1%m/v is added, closes unreacted activated carboxyl position
Point continues to be protected from light 1h；Sample after reaction is centrifuged with super filter tube, and 6500g is centrifuged 5min, until volume 200ul, after ultrafiltration
Sample is transferred in common EP pipe, and centrifugation obtains upper clear supernate and lower part precipitating, be centrifuged under the conditions of 10000g except reuniting
3min；Upper clear supernate is added on splitter Superdex-200 and is purified, flows into cylinder to upper clear supernate, then uses naturally
PBS is rinsed, and with the position of ultraviolet light cylinder observation sample, is started to collect since when lower part is flowed out when sample, is collected 1ml
Stop collecting afterwards；Sample after purification is transferred to centrifugation in common EP pipe with super filter tube after 6500g centrifugal concentrating to 200ul
Except reunion, the condition being centrifuged to common EP pipe is 10000g, 3min；Liquid dilution 200 is saved with phosphate after acquisition supernatant
Times, 4 DEG C save backup；So far quantum dot-labeled antibody A bF2P1 solution is made；
Each component content is respectively in the MES buffer: 10.66g/L MES and 0.74g/L EDTA, the MES buffering
The pH 7.4 of liquid；
The preparation method that the phosphate saves liquid is to weigh 0.29g disodium hydrogen phosphate, 0.0295g sodium dihydrogen phosphate, 0.2g chlorine
Change sodium, 1g bovine serum albumin(BSA) BSA and 0.1 gNaN3, it is dissolved in the deionized water of 90ml, with 1mol/L NaOH tune pH
100ml is settled to deionized water after to 7.3；
2) bonding pad is prepared
Polyester fiber film is immersed into 1h in the obtained quantum dot-labeled antibody A bF2P1 solution of step 1), is taken out, 25 DEG C of dryings
After be cut into rear specification be 4cm × 0.6cm/ item after, 4 DEG C be sealed it is spare, so far be made bonding pad；
3) sample pad is prepared
It takes glass fibre element film one to open, glass fibre element film is impregnated at least 3h in sample pad treatment fluid, then be placed in biological peace
In full cabinet after 37 DEG C of aeration-dryings, specification is cut into obtain sample pad, 25 DEG C are sealed after 4cm × 2.5cm/ item；
The preparation method of the sample pad treatment fluid is to weigh 0.29g disodium hydrogen phosphate, 0.0295g sodium dihydrogen phosphate, 0.2g chlorine
Change sodium, 2g bovine serum albumin(BSA) BSA, 1ml Tween-20,2g sucrose and 0.5g polyvinylpyrrolidone PVP-10, is dissolved in
In the deionized water of 90ml, 100ml is settled to deionized water with after 1mol/L NaOH tune pH to 7.3；
4) detection layers are prepared
Nitrocellulose filter is cut into 4cm × 4cm size；By the antibody A bF2P2 being prepared and goat anti-rabbit igg phosphate
It is respectively 2.0mg/mL and 1.0mg/mL that buffer, which is adjusted to final concentration,；The antibody A bF2P2 diluted is packed into BIODOT and draws film
In instrument spray head, the amount of 1.0 μ l/cm of setting is sprayed on nitrocellulose filter, forms detection line；The anti-rabbit IgG diluted is packed into
BIODOT is drawn in film instrument spray head, and the amount of 1.0 μ l/cm of setting is sprayed on nitrocellulose filter as nature controlling line, nature controlling line and detection
Line spacing is 0.7cm；37 DEG C of the nitrocellulose filter dry 2h that will have been sprayed, are cut into the specification of 4cm × 4cm, 4 DEG C of hermetically dryings
It saves；So far detection layers are made；
The preparation method of the phosphate buffer is: weigh 0.29g disodium hydrogen phosphate, 0.0295g sodium dihydrogen phosphate and
0.2g sodium chloride, is dissolved in the deionized water of 90ml, is settled to after 1mol/L NaOH tune pH to 7.3 with deionized water
100ml；
5) assembling detection card
5.1) bottom plate is cut into 4cm × 7.3cm size, it is spare；
5.2) absorbent filter is cut into 4cm × 3cm size, it is spare as water absorption pad；
5.3) adhered protection film on bottom plate that step 5.1) is prepared is taken off, detection layers described in step 4) is had
Nature controlling line and the nitrocellulose filter of detection line paste on bottom plate, and smooth out film surface；
5.4) the ready water absorption pad of step 5.2) is assembled on bottom plate, there is the left side of water absorption pad with the right end of detection layers
The overlapping of 0.2cm, the right hand edge of water absorption pad are then aligned with the right hand edge of bottom plate and glue and smooth out；Again by the described knot of step 2)
It closes pad to be overlapped at the left edge of detection layers by 0.3cm, 0.3cm is sticked on bottom plate；
5.5) sample pad described in step 3) is then overlapped at the left edge of bonding pad by one side 0.3cm, another side and bottom
The left edge of plate is aligned, and is sticked on bottom plate and is smoothed out；Assembled detection plate is cut into the detection of 4.0mm wide under cutting machine
Card, 4 DEG C of hermetically dryings are kept in dark place；
When the kit is the immune chromatography reagent kit based on colloidal gold-labeled method, the preparation method of the kit is:
1) colloidal gold labeled monoclonal antibody AbF2P1
1.1) 30nm colloidal gold solution is prepared:
99ml ultrapure water is added, by 1ml 1%m/v HAuCl in the 250ml triangular flask for taking a silication good4250ml is added in solution
It is mixed in triangular flask and with ultrapure water, oil bath heating is simultaneously stirred to boiling；2ml 1%m/v is rapidly joined into 250ml triangular flask
Trisodium citrate aqueous solution, solution continue the 10min that boils, stop when the solution in 250ml triangular flask is changed into red by blue
It only heats, by the solution cooled to room temperature in 250ml triangular flask, ultrapure water polishing is then added into 250ml triangular flask
To 100ml；
1.2) colloidal gold labeled monoclonal antibody AbF2P1:
1.2.1 colloidal gold solution prepared by 10ml step 1.1) is added, to colloid in the 50ml triangular flask for) taking a silication good
240ul 0.2mol/L K is added in golden liquid2CO3Adjust pH to 8.5；
1.2.2) under magnetic stirrer, antibody A bF2P1 is added in colloidal gold solution, until the final concentration of 10ug/ of antibody
Ml is added dropwise when antibody is added, and continues to stir 45min~60min after adding；
1.2.3) reaction completes that 2.5ml 5%m/v bovine serum albumin(BSA) BSA to final concentration of 1%m/v, stirring 15~30 is added
Minute, 4 DEG C save backup；
1.2.4 50ml centrifuge tube is packed into after) taking out the antibody A bF2P1 marked, 2500g, 4 DEG C are centrifuged 5 minutes, obtain down
Layer precipitating and supernatant liquor, discard lower sediment, and supernatant liquor is transferred to another 50ml centrifuge tube, 12000g, 4 DEG C of centrifugations 30
Minute, lower sediment and supernatant liquor are obtained, supernatant liquor is discarded, it is heavy that lower sediment 10ml colloidal gold buffer is resuspended
It forms sediment, then 12000g again, 4 DEG C are centrifuged 30 minutes, obtain lower sediment and supernatant liquor again, lower sediment is finally used 3ml
Colloidal gold buffer is resuspended, and 4 DEG C save backup；
Each component content is respectively in the colloidal gold buffer: 10mM Tris, 1%m/v BSA, 1%v/v Tween-20,
5%m/v sucrose and 3 ‰ m/v polyvinylpyrrolidones, the pH of the colloidal gold buffer are 10.5；
2) bonding pad is prepared
Polyester fiber film is immersed into 1h in the obtained colloidal gold labeled monoclonal antibody AbF2P1 solution of step 1), is taken out, 25 DEG C of dryings
After be cut into rear specification be 4cm × 0.6cm/ item after, 4 DEG C be sealed it is spare, so far be made bonding pad；
3) sample pad is prepared
It takes glass fibre element film one to open, glass fibre element film is impregnated at least 2h in sample pad treatment fluid, then be placed in biological peace
In full cabinet after 37 DEG C of aeration-dryings, specification is cut into obtain sample pad, 25 DEG C are sealed after 4cm × 1.5cm/ item；
The preparation method of the sample pad treatment fluid be weigh 0.242g Tris, 1g bovine serum albumin(BSA) BSA, 1ml Tween-20,
5g sucrose and 0.3g polyvinylpyrrolidone PVP-10, are dissolved in the deionized water of 90ml, extremely with 1mol/L NaOH tune pH
100ml is settled to deionized water after 11；
4) detection layers are prepared
Nitrocellulose filter is cut into 4cm × 4cm size；By the antibody A bF2P2 being prepared and goat anti-rabbit igg phosphate
It is respectively 2.0mg/mL and 1.0mg/mL that buffer, which is adjusted to final concentration,；The antibody A bP2 diluted is packed into BIODOT and draws film instrument
In spray head, the amount of 1.0 μ l/cm of setting is sprayed on nitrocellulose filter, forms detection line；The anti-rabbit IgG diluted is packed into
BIODOT is drawn in film instrument spray head, and the amount of 1.0 μ l/cm of setting is sprayed on nitrocellulose filter as nature controlling line, nature controlling line and detection
Line spacing is 0.5cm；37 DEG C of the nitrocellulose filter dry 18h that will have been sprayed, are cut into the specification of 4cm × 2cm, and 4 DEG C of sealings are dry
Dry preservation；So far detection layers are made；
The preparation method of the phosphate buffer is: weigh 0.29g disodium hydrogen phosphate, 0.0295g sodium dihydrogen phosphate and
0.2g sodium chloride, is dissolved in the deionized water of 90ml, is settled to after 1mol/L NaOH tune pH to 7.3 with deionized water
100ml；
5) assembling detection card
5.1) bottom plate is cut into 4cm × 6cm size, it is spare；
5.2) absorbent filter is cut into 4cm × 2.5cm size, it is spare as water absorption pad；
5.3) adhered protection film on bottom plate that step 5.1) is prepared is taken off, detection layers described in step 4) is had
Nature controlling line and the nitrocellulose filter of detection line paste on bottom plate, and smooth out film surface；
5.4) the ready water absorption pad of step 5.2) is assembled on bottom plate, there is the left side of water absorption pad with the right end of detection layers
The overlapping of 0.2cm, the right hand edge of water absorption pad are then aligned with the right hand edge of bottom plate and glue and smooth out；Again by the described knot of step 2)
It closes pad to be overlapped at the left edge of detection layers by 0.2cm, 0.4cm is sticked on bottom plate；
5.5) sample pad described in step 3) is then overlapped at the left edge of bonding pad by one side 0.2cm, another side and bottom
The left edge of plate is aligned, and is sticked on bottom plate and is smoothed out；Assembled detection plate is cut into the detection of 4.0mm wide under cutting machine
Card, 4 DEG C of hermetically dryings are kept in dark place.
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| CN104844712A (en) * | 2015-04-03 | 2015-08-19 | 长春百克生物科技股份公司 | Streptococcus pneumonia protein antigen, and preparation method and application thereof |
| CN105319359A (en) * | 2014-08-18 | 2016-02-10 | 董俊 | Streptococcus pneumoniae quantum dot immunochromatographic assay detection card and preparing method and application thereof |
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| CN104844712A (en) * | 2015-04-03 | 2015-08-19 | 长春百克生物科技股份公司 | Streptococcus pneumonia protein antigen, and preparation method and application thereof |
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| 肺炎链球菌表面粘附素A蛋白抗原表位的预测筛选及确定;古文雅等;《中山大学学报（医学科学版）》;20150131;第36卷(第1期);摘要，第56页 |
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