CN105585635B - The immune chromatography reagent kit of anti-human mycoplasma pneumoniae p1 protein antibody and the application antibody - Google Patents
The immune chromatography reagent kit of anti-human mycoplasma pneumoniae p1 protein antibody and the application antibody Download PDFInfo
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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
- C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
- C07K16/1253—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Mycoplasmatales, e.g. Pleuropneumonia-like organisms [PPLO]
-
- 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
- G01N33/56933—Mycoplasma
-
- 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
- G01N2333/30—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Mycoplasmatales, e.g. Pleuropneumonia-like organisms [PPLO]
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- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Analytical Chemistry (AREA)
- Food Science & Technology (AREA)
- Pathology (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- General Physics & Mathematics (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Organic Chemistry (AREA)
- Pulmonology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
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Abstract
The present invention relates to anti-human mycoplasma pneumoniae p1 protein antibody and the immune chromatography reagent kits of application antibody test people's mycoplasma pneumoniae, anti-human mycoplasma pneumoniae p1 protein antibody is the antibody for identifying two linear epitopes composed by people's mycoplasma pneumoniae p1 protein 504-517 and 535-548 amino acids respectively, and people's mycoplasma pneumoniae p1 protein is ACO25351.1 in GenBank sequence number；The amino acid sequence that people's mycoplasma pneumoniae p1 protein is 504-517 and 535-548 is respectively KPKKVIQSDKLDDD and FGTDHSTQPQPQSL.Two kinds of rabbit-antis people's mycoplasma pneumoniae p1 protein antibody provided by the present invention has 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 mycoplasma pneumoniae p1 protein antibody and application antibody inspection
Survey the immune chromatography reagent kit of people's mycoplasma pneumoniae.
Background technique
Mycoplasma pneumoniae (Mycoplasma pneumoniae, Mp) is the pathogen of mankind's Eaton agent pneumonia, main through flying
Foam infects, and incubation period 2-3 weeks, disease incidence was with teenager's highest.Mp infection is up to 10- in the incidence of infantile pneumonia cause of disease
30%, it is increasingly becoming one of the main pathogens that children infect respiratory disease in recent years.The disease easily causes pharyngitis, tonsillotome
The respiratory tract infection such as inflammation, in some instances it may even be possible to while the multiple organ injuries such as secondary meningitis, hepatitis, myocarditis, it may also lead to when serious
Infant is dead.
Due to Mp infect it is similar with symptoms of respiratory tract infection caused by other pathogen, do not do etiological examination, be difficult by
Mp is distinguished with respiratory tract infection caused by other pathogen.Mp is cell-free wall, common Beta-lactam medicine is invalid to its,
Therefore the treatment infected caused by it is entirely different with the therapeutic scheme of other bacteriums and virus infection, therefore establish it is easy, quick,
Method that is feasible, can early diagnosing mycoplasma pneumoniae infection is very necessary.
The detection method of Mp mainly has 3 classes at present: first is that isolated culture, is confirmation infection " goldstandard ", but by
Extremely slow in the growth cycle of Mp, cultivation cycle is long, and the method is caused not can be carried out quick diagnosis clinically；Second is that serology
Method detects examinee that is, using enzyme-linked immunization, colloidal gold immunization, micro-Immunofluorescence assay and indirect hemagglutination test etc.
Mp antibody level in serum, the presence that Mp can be prompted to infect indirectly.However, serological test can only provide, one kind is retrospective to be examined
It is disconnected, and sometimes for paired sera.In addition, the opportunity that antibody occurs is not easy to grasp, deposited again between children and adolescents and adult
In the difference of Mp specific antibody, also, there are non-spies for the glycolipid antigen on Mp cell membrane and other microorganisms and body tissue
Anisotropic cross reaction, therefore the detection quality of existing serological method is subject to certain restrictions；Third is that being examined using Protocols in Molecular Biology
The presence of MpDNA is surveyed, the most commonly used is polymerase chain reaction (PCR), and this method is quick, sensitive, special, are to grind at present
Study carefully the important means of Mp infection, but since PCR is more demanding to experimental facilities and operation, and false positive easily occur, in China
Common methods for clinical diagnosis can't be used as.Therefore, it is very necessary to establish Mp specific antigen diagnostic method.Currently, public
The method for opening the detection Mp antigen of report is mainly double crush syndrome method, indirect immunofluorescence, quantum dot-labeled immune layer
Analysis method etc., but these methods cannot carry out detection by bed, need specific occasion using specific instrument (such as microplate reader,
Luminoscope etc.) it detects, the not only not convenient and fast but also time is longer, clinical application is more inconvenient.
Therefore, the fast diagnosis method for establishing people's mycoplasma pneumoniae specific antigen is very necessary.Due to human influenza pneumonia
It is well-conserved on mycoplasma P1 protein sequence, become an important examination target.Therefore high specific is obtained
Anti-human mycoplasma pneumoniae p1 protein antibody be exactly a highly important job.Currently, about anti-human mycoplasma pneumoniae P1 egg
Bai Kangti reports at most to be corresponding monoclonal antibody and polyclonal antibody.The biggest advantage is to special for monoclonal antibody
Property it is high, but preparation method is cumbersome, high production cost, limits its application.Polyclonal antibody is mainly by gene engineering expression
The animals such as P1 protein immunization rabbit are prepared.Preparation method is simple, at low cost, but it is low with specificity, potency is low,
The defects such as purity is low.Therefore, inexpensive prepare high specific, the anti-human mycoplasma pneumoniae p1 protein antibody of high-titer just seems
It is 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's mycoplasma pneumoniaes
Two kinds of antibody of two linear epitopes composed by 504-517, P1 albumen and 535-548 amino acids and application should
The immune chromatography reagent kit of antibody.
Anti-human mycoplasma pneumoniae p1 protein antibody, it is characterised in that: the anti-human mycoplasma pneumoniae p1 protein antibody is point
It Shi Bie not two linear epitopes composed by people's mycoplasma pneumoniae p1 protein 504-517 and 535-548 amino acids
Two kinds of antibody, people's mycoplasma pneumoniae p1 protein is ACO25351.1 in GenBank sequence number；People's mycoplasma pneumoniae
The amino acid sequence of 504-517, P1 albumen 14 amino acid and 535-548 14 amino acid is respectively
KPKKVIQSDKLDDD and FGTDHSTQPQPQSL；By people's mycoplasma pneumoniae p1 protein 504-517 14 amino acid and
The sequence of 535-548 14 amino acid is respectively designated as P1A and P1B；The anti-human mycoplasma pneumoniae p1 protein antibody is
AbP1A and AbP1B.
One kind being formed by immune chromatography reagent kit based on foregoing anti-human mycoplasma pneumoniae p1 protein antibody, special
Sign is: the kit is immune chromatography reagent kit based on quantum dot-labeled technology or is exempted from based on colloidal gold-labeled method
Epidemic disease chromatographs kit.
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 bP1A 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 bP1A of 0.34mg 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
AbP1A 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 bP1A 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) 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 bP1B being prepared and goat anti-rabbit igg phosphoric acid
It is respectively 2.0mg/mL and 1.0mg/mL that salt buffer, which is adjusted to final concentration,；The antibody A bP1B diluted is packed into 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；The anti-rabbit IgG 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 as nature controlling line, nature controlling line and inspection
Survey 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, 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 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；Again by knot described in 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
It is aligned, sticks on bottom plate and smoothes out with the left edge of bottom plate；Assembled detection plate is cut into the inspection of 4.0mm wide under cutting machine
Card is surveyed, 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 AbP1A
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 AbP1A:
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 bP1A 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 bP1A marked, 2500g, 4 DEG C are centrifuged 5 minutes, obtain
Lower sediment and supernatant liquor, discard lower sediment, and supernatant liquor is transferred to another 50ml centrifuge tube, 12000g, 4 DEG C of centrifugations
30 minutes, 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/vBSA, 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 AbP1A 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) 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 bP1B being prepared and goat anti-rabbit igg phosphoric acid
It is respectively 2.0mg/mL and 1.0mg/mL that salt buffer, which is adjusted to final concentration,；The antibody A bP1B diluted is packed into 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；The anti-rabbit IgG 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 as nature controlling line, nature controlling line and inspection
Survey 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, 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 × 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；Again by knot described in 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
It is aligned, sticks on bottom plate and smoothes out with the left edge of bottom plate；Assembled detection plate is cut into the inspection of 4.0mm wide under cutting machine
Card is surveyed, 4 DEG C of hermetically dryings are kept in dark place.
The invention has the advantages that
The present invention provides anti-human mycoplasma pneumoniae p1 protein antibody, which identifies people
Composed by mycoplasma pneumoniae p1 protein 504-517 14 amino acid and 535-548 14 amino acid two it is linear
Epitope, people's mycoplasma pneumoniae p1 protein are ACO25351.1 in GenBank sequence number；People's mycoplasma pneumoniae p1 protein 504-
The amino acid sequence of 517 14 amino acid and 535-548 14 amino acid be respectively KPKKVIQSDKLDDD and
FGTDHSTQPQPQSL；By people's mycoplasma pneumoniae p1 protein 504-517 14 amino acid and 535-548 14 amino
The sequence of acid is respectively designated as P1A and P1B；Anti-human mycoplasma pneumoniae p1 protein antibody is AbP1A and AbP1B.Based on people's lung
Above two rabbit-anti people mycoplasma pneumoniae p1 protein antibody prepared by scorching mycoplasma single linear epitope has specificity
Feature good, with high purity, potency is high, preparation cost is cheap can be used for producing the height of the various principles based on antigen-antibody reaction
The detection kit of sensitivity technique people's mycoplasma pneumoniae.Meanwhile it also being prepared based on the anti-human mycoplasma pneumoniae p1 protein antibody
Two different immune chromatography reagent kits are obtained.Two different immune chromatography reagent kits can quickly, accurately detect biological sample
People's mycoplasma pneumoniae in product includes two kinds of antibody of the present invention；Two kinds of immune chromatography reagent kits are used equally for people
The auxiliary diagnosis of mycoplasma pneumoniae infection has higher sensitivity and specificity, combined it is simple, quickly, stablize with
And the advantages such as manufacturing cost is low are also fitted suitable for the inspection of clinical samples, and due to that can carry out large batch of quick inspection
Together in epidemiological survey.Therefore, two kinds of rabbit-antis people's mycoplasma pneumoniae p1 protein antibody of the present invention, two kinds of immunochromatographies
Kit all has broad 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/LNaOH 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 bP1A: for present invention self-control, is diluted, is shaken up with PBS, make antibody concentration 3mg/ml in solution.
6, it antibody A bP1B: 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, it people's mycoplasma pneumoniae: is purchased from American type culture collection (ATCC), number ATCC15531.
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's mycoplasma pneumoniae p1 protein antibody of embodiment
Two kinds of rabbit-anti people's mycoplasma pneumoniae p1 protein antibody the preparation method is as follows:
1) after structure biology analysis and Related Experimental Study, people's mycoplasma pneumoniae p1 protein (GenBank sequence is selected
Number ACO25351.1) 504-517 14 amino acid and 535-548 14 amino acid composition small peptide respectively as preparation
Two linear epitopes of rabbit-anti people's mycoplasma pneumoniae p1 protein antibody, two sections of amino acid sequences are respectively
This two sequence is respectively designated as P1A and P1B by KPKKVIQSDKLDDD and FGTDHSTQPQPQSL；
2) it after the N-terminal of the N-terminal of amino acid sequence P1A, P1B described in step 1) being added a cysteine respectively, uses
Polypeptide automatic synthesizer is respectively synthesized polypeptide and purifies, and two polypeptides after purification with carrier protein KLH, are formed respectively
P1A-KLH compound protein and P1B-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 mycoplasma pneumoniae p1 protein
The serum of antibody, ELISA detect the potency of rabbit-anti people mycoplasma pneumoniae p1 protein antibody in serum, two kinds of rabbit-antis respectively
The potency of people's mycoplasma pneumoniae p1 protein antibody 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's mycoplasma pneumoniae p1 protein antibody obtained step 4) are corresponding to be added
Enter in the two kinds of affinity columns prepared to step 5), and after 4 DEG C are incubated overnight, antibody elution obtains two kinds of rabbit-anti people's lungs
Scorching mycoplasma P1 protein antibodies；Identify that 97% or more, both antibody purified are ordered respectively for its purity through SDS-PAGE
Entitled AbP1A and AbP1B.
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 bP1A
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 bP1A prepared by the embodiment 1 of 0.34mg 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 bP1A.
2. the preparation of bonding pad
Polyester fiber film is immersed into 1h in the obtained quantum dot-labeled antibody A bP1A 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 bP1B 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 bP1B 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, forms detection line；It is anti-by what is diluted
Rabbit igg is fitted into BIODOT and draws in film instrument spray head, and the amount of 1.0 μ l/cm of setting, which is sprayed on nitrocellulose filter, is used as 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.
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 edge of detection layers by 0.3cm again
Place, 0.3cm are sticked on bottom plate 7.Sample pad described in step 3 is finally then overlapped in by one side 0.3cm to the left side of bonding pad
At 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 under cutting machine
It is cut into the detection card of 4.0mm wide, 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.If pneumonia mycoplasma containing someone is former in throat swab, in conjunction with the quantum dot-labeled antibody A bP1A in bonding pad,
It will form at detection line under ultraviolet light excitation after acting on elder generation in conjunction with the antibody A bP1B on nitrocellulose filter by chromatography
A macroscopic fluorescence detection line, the quantum dot-labeled antibody being not associated with continue chromatography in conjunction with goat anti-rabbit igg after
Macroscopic Article 2 fluorescence nature controlling line is formed under ultraviolet light excitation；If only occurring in throat swab to be checked without related antigen
One fluorescence nature controlling line.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), stream of people's haemophilus influenza
(ATCC 53781), people's chlamydia pneumoniae (AR-39 plants, ATCC number 53592), 3 type of adenovirus hominis (GB plants, ATCC number VR-
3), 7 type of adenovirus hominis (Gomen plants, ATCC number VR-7), influenza virus A hominis (H1N1, ATCC number VR-1743), people
Influenza B virus (ATCC number VR-790), human respiratory syncytial virus (ATCC number VR26), people streptococcus pneumonia (ATCC
Number 700670) etc. detect instead of people's mycoplasma pneumoniae, it is dilute that kit detects the phosphate buffer containing these microorganisms
It is negative for releasing liquid all.
2) clinical trial example
Using people's mycoplasma pneumoniae detection " goldstandard "-cultivation as reference, 120 division of respiratory disease respiratory tract infection persons are taken
Kit described in oropharyngeal swab specimen step 6 is detected, and cultivation positive rate is 13% (13/100), this kit is
12% (12/100), the coincidence rate of 2 kinds of methods are 97% (97/100).Concrete outcome is as shown in table 1.
The testing result of 1 clinical samples of table
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 AbP1A
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 AbP1A
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 bP1A 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 bP1A marked, 2500g, 4 DEG C are centrifuged 5 minutes, obtain lower layer
Precipitating and supernatant liquor discard lower sediment, and supernatant liquor is transferred to another 50ml centrifuge tube, and 12000g, 4 DEG C are centrifuged 30 points
Clock obtains lower sediment and supernatant liquor, discards supernatant liquor, and lower sediment is resuspended with 10ml colloidal gold buffer and is precipitated,
Then 12000g again, 4 DEG C are centrifuged 30 minutes, obtain lower sediment and supernatant liquor again, lower sediment is finally used to 3ml colloid
Golden 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 colloidal gold buffering
The pH of liquid is 10.5.
2. the preparation of bonding pad
Polyester fiber film is immersed into 1h in the obtained colloidal gold labeled monoclonal antibody AbP1A 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 × 2.5cm/ item.
4. the preparation of detection layers
Nitrocellulose filter is cut into 4cm × 2cm size.By antibody A bP1B 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 bP1B 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, forms detection line；It is anti-by what is diluted
Rabbit igg is fitted into BIODOT and draws in film instrument spray head, and the amount of 1.0 μ l/cm of setting, which is sprayed on nitrocellulose filter, is used as nature controlling line, with
Detection line spacing is 0.5cm.37 DEG C of the nitrocellulose filter dry 2h that will have been sprayed are cut into the specification of 4cm × 2cm, 4 DEG C of sealings
Kept dry.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 the left side of detection layers 3 by 0.2cm again
At edge, 0.4cm is sticked on bottom plate.Sample pad described in step 3 is finally then overlapped in by one side 0.2cm to the left side of bonding pad
At 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 under cutting machine
It is cut into the detection card of 4.0mm wide, 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, after so that the sample on swab is sufficiently dissolved simultaneously 50 DEG C of water-bath 20min, rear 120 μ L drops of taking out are blocked in detection
Sample pad on, visually observe testing result after (15) minute.If pneumonia mycoplasma containing someone is former in throat swab, with combination
The antibody A bP1A of colloid gold label in pad is combined, first in conjunction with the antibody A bP1B on nitrocellulose filter by chromatography effect
It will form macroscopic one red detection line at detection line afterwards, the colloidal gold labeled monoclonal antibody being not associated with continues to chromatograph
Macroscopic Article 2 red nature controlling line is formed after in conjunction with goat anti-rabbit igg；If without related antigen in throat swab to be checked, only
There is a red nature controlling line.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), stream of people's haemophilus influenza (ATCC 53781), 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), human respiratory syncytial virus (ATCC number VR26), people streptococcus pneumonia (ATCC number 700670) etc. replace people's lung
Scorching mycoplasma is detected, and phosphate buffer dilution of the kit detection containing these microorganisms is all negative.
2) clinical trial example
Using people's mycoplasma pneumoniae detection " goldstandard "-cultivation as reference, 100 division of respiratory disease respiratory tract infection persons are taken
Kit described in oropharyngeal swab specimen step 6 is detected, and cultivation positive rate is 13% (16/120), this kit is
11% (1/100), the coincidence rate of 2 kinds of methods are 96% (96/100).Concrete outcome is as shown in table 1.
The testing result of 1 clinical samples of table
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 (3)
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| CN110128512B (en) * | 2019-05-06 | 2020-12-25 | 南华大学 | Antigenic determinants and uses thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006083322A2 (en) * | 2004-07-29 | 2006-08-10 | Ligocyte Pharmaceuticals, Inc. | Methods for the treatment and prevention of infection using anti-selectin agents |
| CN103059109A (en) * | 2013-01-16 | 2013-04-24 | 中国人民解放军军事医学科学院基础医学研究所 | Mycoplasma pneumonia antigen, preparation method and immunodetection kit |
| CN104198703A (en) * | 2014-08-18 | 2014-12-10 | 湖北工业大学 | Human mycoplasma pneumoniae gold-marked silver-stained immunochromatographic assay kit and preparation method and application thereof |
| CN105203768A (en) * | 2014-08-18 | 2015-12-30 | 董俊 | Method and kit for fast detection of human chlamydia pneumoniae antigen based on magnetic resolution and quantum dot labelling |
-
2016
- 2016-03-08 CN CN201610130233.2A patent/CN105585635B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006083322A2 (en) * | 2004-07-29 | 2006-08-10 | Ligocyte Pharmaceuticals, Inc. | Methods for the treatment and prevention of infection using anti-selectin agents |
| CN103059109A (en) * | 2013-01-16 | 2013-04-24 | 中国人民解放军军事医学科学院基础医学研究所 | Mycoplasma pneumonia antigen, preparation method and immunodetection kit |
| CN104198703A (en) * | 2014-08-18 | 2014-12-10 | 湖北工业大学 | Human mycoplasma pneumoniae gold-marked silver-stained immunochromatographic assay kit and preparation method and application thereof |
| CN105203768A (en) * | 2014-08-18 | 2015-12-30 | 董俊 | Method and kit for fast detection of human chlamydia pneumoniae antigen based on magnetic resolution and quantum dot labelling |
Non-Patent Citations (2)
| Title |
|---|
| Expression in Spiroplasma citri of an Epitope Carried on the G Fragment of the Cytadhesin P1 Gene from;ARMELLE MARAIS等;《JOURNAL OF BACTERIOLOGY》;19930531;第175卷(第9期);第2783-2787页 |
| 肺炎支原体主要黏附蛋白的研究进展;王丽琼等;《现代生物医学进展》;20140731;第14卷(第19期);第3757-3760页 |
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