CN102621318A - Immune chromatography test paper for detecting mycobacterium tuberculosis secretory proteins and manufacture method thereof - Google Patents
Immune chromatography test paper for detecting mycobacterium tuberculosis secretory proteins and manufacture method thereof Download PDFInfo
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Abstract
The invention discloses immune chromatography test paper for fast detecting mycobacterium tuberculosis, which comprises an upper sample pad (1), a colloidal gold pad (2) containing a gold-marked anti-CFP-10 monoclonal antibody TBCA6, a nitrocellulose membrane (5) wrapping a quality control line (3) and a detection line (6), a sample sucking pad (4) and a support plate (7). The detection line (6) is an anti-CFP-10 monoclonal antibody TBCD6 wrapped on the nitrocellulose membrane, and the quality control line (3) is a goat anti mouse IgG antibody wrapped on the nitrocellulose membrane. The colloidal gold pad (2) and the sample sucking pad (4) are respectively arranged on the upper surface of the nitrocellulose membrane (5), and the upper sample pad (1) is arranged on the upper surface of the colloidal gold pad (2). A manufacture method of the immune chromatography test paper which is suitable for fast detecting mycobacterium tuberculosis in culture at basic scene is further provided.
Description
Technical field
The invention belongs to the immunochromatography technique field of Clinical microorganism fast detecting, particularly relate to a kind of method of utilizing the relevant immunochromatography colour developing of anti-CFP-10 protein antibodies to come Much's bacillus in the early stage evaluation culture.
Background technology
According to World Health Organization's statistics, the population in the whole world existing nearly 1/3 has infected Much's bacillus, and the Much's bacillus carrying rate is up to 80% in some adult of developing country, and wherein about 5%-10% carrier can develop into active tuberculosis.China tuberculosis number occupies second in the whole world, and the twice of dying from people lungy every year and be all kinds of infectious disease death toll summations is many.Recent two decades is because AIDS popular, and the possibility that the Much's bacillus carrier who has infected human immunodeficiency virus (HIV) develops into active tuberculosis than the high 30-50 of infected by HIV person not doubly.Tuberculosis becomes the global hygienic issues that threatens human health again, influences the great society and the economic problems of China's development especially.
Laboratory diagnosis lungy mainly relies on the bacteriology smear and cultivates inspection.Because method is limit, and has had a strong impact on timely diagnosis lungy.Therefore, for many years Chinese scholars seeking fast always, responsive, special, easy laboratory diagnostic method.
The Much's bacillus culture of isolated is the goldstandard of present diagnosis of tuberculosis, but uses common Russell medium to need the 4-8 time-of-week, and improveing acid Luo-Qiong Shi cultivation on average also needs 25 days, can't fully satisfy the needs of clinical quick diagnosis.Fast culture to Much's bacillus; The fluid nutrient medium and the auto culturing system (Ma Jun that have occurred various improvement at present; Wang Zili. the progress of Much's bacillus fast culture and clinical meaning thereof [J]. JISUI ZAZHI AUTHOR AND, 2007,17 (5) .388-390).The liquid colour-changeable nutrient culture media by the development of German Heipha/Biotest company, is called the MBRedox system at first; Auto culturing system comprises with Bactec MGIT 960 being the quick manual cultivation of second generation mycobacterium fast culture, susceptibility detection system and BBL MGIT mycobacterium, evaluation, susceptibility detection system of representative etc.No matter be fluid nutrient medium or auto culturing system, all also there is the high problem of pollution rate, be judged as positive sample for instrument, still must carry out smear, acid-fast stain, microscopically and can judge the positive after finding acid-fast bacilli, bothersome effort.Auto culturing system more because instrument and reagent cost an arm and a leg, is difficult to popularized.Therefore the quick detection reagent of researching and developing Much's bacillus of new generation is very necessary.
Much's bacillus culturing filtrate protein 10 (CFP-10) is evaluations such as Berthet a kind of low-molecular-weight of coming out, secreted protein (the BehrM A of high degree of specificity; Wilson M A, GillW P, et al.Comparative genomics of BCG vaccines by whole-genome DNA microarray [J] .Science; 1999; 284 (5419): 1520-1523.), have another name called MTBb11, MTSA-10, be positioned at the upper reaches of the early stage secreted antigen of Much's bacillus 6kDa target protein (ESAT-6) gene; Transcribe by same operon and promoter adjusting; Belong to the ESAT-6 family protein, both homologys of about 40% have identical immunological characteristic.CFP-10 is present in the early stage culturing filtrate of MTB, only is distributed in the various tubercle bacillus, and BCG and other non-pathogenic mycobacterium disappearance.Therefore in the process that diagnosing tubercle bacillus infects, can distinguish m tuberculosis infection or non-pathogenic infection due to Mycobacterium tuberculosis.
Summary of the invention
The immune chromatography test paper that the technical matters that the present invention will solve provides a kind of quick, sensitivity easy to use and specificity is high, be suitable for Much's bacillus in basic unit's field quick detection culture.
In order to address the above problem; The present invention provides a kind of immune chromatography test paper (immune chromatography test paper of double antibody sandwich method fast detecting Much's bacillus) of fast detecting Much's bacillus, comprise appearance pad, contain the collaurum pad of the anti-CFP-10 monoclonal antibody TBCA6 of golden mark, the nitrocellulose filter that encapsulates nature controlling line and detection line is inhaled the appearance pad and and back up pad; Detection line is the anti-CFP-10 monoclonal antibody TBCD6 that is coated on the nitrocellulose filter, and nature controlling line is the sheep anti-mouse igg antibody that is coated on the nitrocellulose filter; Upper surface at nitrocellulose filter is provided with the collaurum pad respectively and inhales the appearance pad, and last appearance pad is positioned at the upper surface of collaurum pad, and the lower surface of nitrocellulose filter closely links to each other with back up pad.
The present invention also provides the preparation method of above-mentioned immune chromatography test paper simultaneously, may further comprise the steps:
The preparation of A, anti-CFP-10 monoclonal antibody TBCD6 and TBCA6:
Be used to encapsulate the anti-CFP-10 monoclonal antibody TBCD6 of detection line, the aa 23-36GDLKTQIDQVESTA antigenic peptide sequence of ability specific recognition CFP-10, by China's typical culture collection center preservation, deposit number is CCTCCNO:C201082;
The anti-CFP-10 monoclonal antibody TBCA6 that is used for the golden mark of collaurum pad, the aa53-66AAVVRFQEAANKQK antigenic peptide sequence of ability specific recognition CFP-10, by China's typical culture collection center preservation, deposit number is CCTCC NO:C201083;
The preparation method is following:
1) inducing of odd contradictive hydroperitoneum:, give every 0.5ml of BALB/C mice lumbar injection norphytane, then every inoculation 5 * 10 in inoculation hybridoma the last week
6The positive hybridoma cell of TBCD6 or TBCA6, collection ascites is centrifugal after 10 days, measures antibody titer.
2) TBCD6 or TBCA6 Purification of Monoclonal Antibodies: utilize TBCD6 or TBCA6 monoclonal antibody in the Protein G affinity purification difference purifying ascites;
B, making encapsulate the nitrocellulose filter (being about to TBCD6 monoclonal anti liquid solution and sheep anti-mouse igg solution is sprayed onto on the nitrocellulose filter respectively) of detection line and nature controlling line; After drying its (that is, encapsulating the nitrocellulose filter of detection line and nature controlling line) sticked on the back up pad;
The cellulose nitrate film production that encapsulates detection line and nature controlling line is following:
1) uses 0.01mol/L PH7.4 phosphate PBS damping fluid will resist CFP-10 monoclonal antibody TBCD6 to be mixed with the solution of concentration, rule with the parameter of 0.8～1.2ul/cm at the lower surface of nitrocellulose filter, encapsulate into detection line with spray film appearance as 0.3mg/ml;
2) with 0.01mol/L PH7.4 phosphate PBS damping fluid sheep anti-mouse igg antibody is mixed with the solution of 2.4mg/ml, in the parameter line of the upper surface of nitrocellulose filter, encapsulates into nature controlling line with 0.8～1.2ul/cm with spray film appearance;
3) nitrocellulose filter after will ruling is in the temperature of 36.5～37.5 ℃ (best 37 ℃) and under less than the condition of 30% humidity (relative humidity for example is 20～29%) dry 8～10 hours, must encapsulate the nitrocellulose filter of detection line and nature controlling line;
C, preparation contain the collaurum pad of the anti-CFP-10 monoclonal antibody TBCA6 of golden mark:
TBCA6 monoclonal antibody colloidal gold probe solution is added to glass fibre membrane or polyester film, after the drying, gets the collaurum pad; The collaurum pad is sticked on the left side of the upper surface of the nitrocellulose filter that step B obtains;
The preparation method of TBCA6 monoclonal antibody colloidal gold probe solution is following:
1), with mass concentration 0.01%HAuCL
4WS heated and boiled, every 50ml HAuCL
4Mass concentration 1% trisodium citrate aqueous solution of solution rapid disposable adding 1.7～1.8ml (the best is 1.75ml) under magnetic force heating (in 100-110 ℃) is stirred; Continue heating (in 100-110 ℃) and stir 8～12 minutes (the best is 10 minutes), get colloidal gold solution;
2), regulate colloidal gold solution to pH5.5, with the ratio coupling of antibody TBCA6, be the immune colloid gold probe solution of TBCA6 behind the mixing by 0.7～0.75 (the best 0.72) the every 100ml colloidal gold solution of mg;
3), with supernatant discarded behind 4 ℃ of centrifugal 30min of 15000r/min of immune colloid gold probe solution of above-mentioned TBCA6, with the resuspended sediment of resuspended liquid, TBCA6 monoclonal antibody colloidal gold probe solution; Said resuspended liquid is step 2) 0.9～1.2 times of colloidal gold solution volume;
The preparation method of resuspended liquid is following: in concentration is 0.01mol/L, adds the BSA of 9～11g, the sucrose of 33～37g and the PEG20000 of 0.28～0.32g in one liter of the PB damping fluid of PH7.4;
D, paste the appearance pad at the upper surface of the collaurum pad of step C gained; Overlap joint is inhaled appearance pad (4) on the right side of the upper surface of nitrocellulose filter, the immune chromatography test paper of fast detecting Much's bacillus (that is, obtains detecting the immune chromatography test paper of CFP-10.)。
Improvement as the preparation method of the immune chromatography test paper of fast detecting Much's bacillus of the present invention: among the step C: TBCA6 monoclonal antibody colloidal gold probe solution is pressed 1ml solution shop 23～27cm
2Ratio be layered on equably on glass fibre membrane or the dacron film.
In the preparation method of immune chromatography test paper of the present invention, confirmed that the final material width of said single test paper is controlled at 2.5-5mm; The material of said collaurum pad is hydrophilic glass tunica fibrosa or hydrophilic relatively polyester fiber film (thickness is about 0.25-0.8mm); The said appearance pad of going up is hydrophilic glass tunica fibrosa (thickness is about 0.25-0.8mm); The said material of inhaling the appearance pad is absorbent filter (thickness is about 0.45-1.9mm); The optimal decision time of testing result is in the 5-30min.
The invention has the beneficial effects as follows: utilize anti-CFP-10 monoclonal antibody TBCD6 and TBCA6 to prepare colloidal gold immune chromatography test; But thereby the mycobacterium tuberculosis secretory property antigen target protein CFP-10 in the fast detecting fluid nutrient medium identifies whether be people's pathogenic Much's bacillus; Even the layman also can operate to specifications; Observations in 5-30 minute, the minimum CFP-10 albumen 1.2ng/ml that detects.This CFP-10 immunity test strip has the advantage of simplicity, susceptibility, specificity and rapidity, and low price is suitable for, and basic unit is on-the-spot to be used.
The present invention uses the colloidal gold immunochromatographimethod technology and comes across phase early 1980s, because of it has susceptibility height, high specificity, advantage such as simple to operate, quick, in medical test, is used widely.Its ultimate principle with the immune colloid gold probe in detecting of another pairing, macroscopic precipitation line occurs then for being used on film with the antibody sandwich of thing CFP-10 pairing to be checked catching after positive 2-5 minute.This technology and additive method compare, and advantage is that sample preparation is simple, does not need specialized equipment and staff training, and the layman can operate to specifications, and rapid observations, are fit to basic unit and use.
Description of drawings
Do further explain below in conjunction with the accompanying drawing specific embodiments of the invention.
Fig. 1 is the colloid gold particle (* 100000) under the transmission electron microscope;
Fig. 2 is the explosion synoptic diagram of the main TV structure of immune chromatography test paper of the present invention;
Fig. 3 is the schematic top plan view of Fig. 2.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the present invention.
Gold chloride (HAuCL
44H
2O), analyze pure, Sigma company;
Trisodium citrate (Na
3C
6H
5O
72H
2O), analyze pure, Shanghai chemical reagent main office of Chinese Medicine group;
Sheep anti-mouse igg antibody, Shanghai gold mark company;
Polyglycol (PEG) 20000, analyze pure, Sigma company;
Sucrose (C12H19Cl3O8), analyze pure, Sigma company;
Bovine serum albumin(BSA) (BSA), worker bio-engineering corporation is given birth in Shanghai;
Cellulose nitrate (NC) film, Millipore company;
It is pure that other common agents are analysis.
Step:
1, preparation and purifying anti-CFP-10 mouse monoclonal antibody TBCD6 and TBCA6
1), the inducing of odd contradictive hydroperitoneum:, give every 0.5ml of BALB/C mice lumbar injection norphytane, then every inoculation 5 * 10 in inoculation hybridoma the last week
6Can produce the positive hybridoma cell of TBCD6 or TBCA6, collection ascites is centrifugal after 10 days, measures antibody titer.
The above-mentioned positive hybridoma cell that can produce mouse monoclonal antibody TBCD6 or TBCA6 derives from Chinese typical culture collection center; Deposit number is respectively CCTCC NO:C201082; CCTCC NO:C201083.
2) Purification of Monoclonal Antibodies: utilize TBCD6 or TBCA6 monoclonal antibody in the Protein G affinity purification purifying ascites.
2, make the nitrocellulose filter 5 that encapsulates detection line 6 and nature controlling line 3; That is, TBCD6 monoclonal anti liquid solution and sheep anti-mouse igg solution are sprayed onto respectively on the nitrocellulose filter 5, after drying its (encapsulating the nitrocellulose filter 5 of detection line 6 and nature controlling line 3) are sticked on the back up pad 7;
The method for making of nitrocellulose filter 5 that encapsulates detection line 6 and nature controlling line 3 is following:
1), use 0.01mol/L PH7.4 phosphate PBS damping fluid will resist CFP-10 monoclonal antibody TBCD6 to be mixed with the solution of concentration as 0.3mg/ml; Rule with the parameter of 0.8～1.2ul/cm (the best is 1ul/cm) at the lower surface of nitrocellulose filter 5 with spray film appearance, encapsulate into detection line 6;
2), sheep anti-mouse igg antibody is mixed with the solution of 2.4mg/ml, rule with the parameter of 0.8～1.2ul/cm (the best is 1ul/cm) at the upper surface of nitrocellulose filter 5, encapsulate into nature controlling line 3 with spray film appearance with 0.01mol/LPH7.4 phosphate PBS damping fluid;
3) nitrocellulose filter 5 after will ruling is in 37 ℃ temperature and under less than the condition of the relative humidity of 30% (for example being 20%～29%) dry 8～10 hours, must encapsulate the nitrocellulose filter 5 of detection line 6 and nature controlling line 3.
3, anti-CFP-10 immune colloid gold probe of preparation and corresponding TBCA6 antibody gold label pad:
1) with 0.01% (mass concentration) HAuCL
4WS heated and boiled, every 50ml HAuCL
4The WS stirs 1% (mass concentration) trisodium citrate aqueous solution of rapid disposable adding 1.75ml down in magnetic force heating (in 100-110 ℃), continues heating (in 100 ℃) and stirs 10 minutes, gets colloidal gold solution.
2) electron microscopic observation of colloid gold particle
Colloid gold particle in the down visible colloidal gold solution of transmission electron microscope is rounded or oval, and big or small uniformity is counted 100 gold grains, and particle diameter is about 19.5nm.The result shows, and is qualified according to the colloid gold particle of above technology path preparation.
3) confirm minimum coupling AC: regulate colloidal gold solution and be used to dilute antibody TBCA6 to pH5.5 (weak acid, the weak caustic solution of routine capable of using are regulated).Get 5ul respectively, 10ul, 15ul; 20ul; The isopyknic antibody TBCA6 of 25ul adds in each self-corresponding 1ml colloidal gold solution, in room temperature held 5min, leaves standstill behind adding 10% (mass concentration) NaCL WS 0.1ml mixing behind the mixing; Contained minimum antibody amount when the colloidal gold solution color is constant behind the 10-20min is for stablizing the required minimum coupling antibody amount of 1ml colloidal gold solution;
What 4) in the present embodiment, minimum antibody amount was corresponding is the ratio of the every 100ml colloidal gold solution of 0.6mg.
5) the antibody TBCA6 of 120% times above-mentioned minimum coupling antibody amount is joined in the 50ml colloidal gold solution in proportion (be about to the ratio coupling of antibody TBCA6 in the every 100ml colloidal gold solution of 0.72mg; This colloidal gold solution adjusted is to pH5.5); Room temperature left standstill 1 hour behind the mixing, got the immune colloid gold probe solution of TBCA6;
With supernatant discarded gently behind 4 ℃ of centrifugal 30min of 15000r/min of immune colloid gold probe solution of TBCA6, with the 1 volume resuspended sediment of resuspended liquid of (being 50ml) doubly, TBCA6 monoclonal antibody colloidal gold probe solution;
The preparation method of resuspended liquid is following: in concentration is 0.01mol/L, adds BSA, the sucrose of 35g and the PEG20000 of 0.3g of 10g in 1 liter of the PB damping fluid of PH7.4;
6) with above-mentioned 5) solution (TBCA6 monoclonal antibody colloidal gold probe solution) press 1ml solution shop 25cm
2Ratio be layered on the hydrophilic glass tunica fibrosa equably or relatively on the hydrophilic polyesters tunica fibrosa (thickness is about 0.25-0.8mm); Put drying room again; In 20-25 ℃ less than the condition of 30% humidity (for example be 20% relative humidity) under dry 2-4 hour, it is subsequent use to process collaurum pad 2.This collaurum pad 2 is the collaurum pad that contains the anti-CFP-10 monoclonal antibody TBCA6 of golden mark.
4, detect the assembling of CFP-10 immune chromatography test paper (immune chromatography test paper of fast detecting Much's bacillus):
Detecting the immune chromatography test paper of anti-CFP-10 is made up of last kind of pad 1, the nitrocellulose filter 5 (hereinafter to be referred as the nitrocellulose filter 5 of coated antibody) that has encapsulated detection line 6 and nature controlling line 3, collaurum pad 2, suction appearance pad 4 and back up pad 7 these five parts.
The nitrocellulose filter 5 of the coated antibody for preparing is sticked on (lower surface of nitrocellulose filter 5 contacts with back up pad 7) on the back up pad 7, the upper surface of nitrocellulose filter 5 left ends of the lower surface of collaurum pad 2 right-hand members and coated antibody be fixedly linked (mode to paste realizes); The lower surface of last appearance pad 1 right-hand member be fixedly linked with the upper surface of collaurum pad 2 left ends (mode with stickup realizes).The lower surface of inhaling appearance pad 4 left ends and the upper surface of nitrocellulose filter 5 right-hand members of coated antibody be fixedly linked (mode with stickup realizes); Press width 0.25cm-0.5cm cutting (as shown in Figure 3) at last, be the immune chromatography test paper that detects anti-CFP-10 albumen, add drying agent after sealing preserve.
1, test paper detects the susceptibility of CFP-10
The recombinant protein c FP-10 that buys with Fitzgerald Industries International company is a standard; Behind the physiological saline limiting dilution; As sample detection liquid, get CFP-10 and detect test paper (being the immune chromatography test paper of embodiment 1 gained), immerse respectively in the different CFP-10 concentration sample detection liquid; Begin observations after 2 minutes, observation in 15 minutes stops.Result's report: it is negative 1 redness (contrast) precipitation line to occur, does not promptly have CFP-10 and detects; It is positive 2 redness (sample and contrast) precipitation line to occur, promptly has CFP-10 to detect.Concrete testing result is as described in Table 1.
CFP-10 test paper provided by the present invention can detect minimum 1.2ng/ml.
Table 1, test strips susceptibility
Annotate: the positive :+; Negative :-
2, CFP-10 detects the discriminating of test paper to pathogenic and non-pathogenic Much's bacillus
With culture supernatant to be checked with physiological saline by the dilution of 1: 10 volume ratio, get 1 of the immune chromatography test paper of the m tuberculosis infection of preparation among the embodiment 1, immerse in the sample of above-mentioned dilution, begin observations behind the 2min, 30min observes termination.1 redness (contrast) precipitation line appears, for Much's bacillus detects feminine gender, i.e. and nontuberculous mycobacteria growth; 2 redness (sample and contrast) precipitation line occurs,, the Much's bacillus growth is arranged promptly for Much's bacillus detects the positive.With detection paper tuberculosis specific antigen CFP-10 level, the result is following with variety classes Much's bacillus culture supernatant:
The positive rate of CFP-10 in table 2, the detection clinical sample culture supernatant
| Sample classification | The example number | The CFP-10 positive detects number |
| Bacillus tuberculosis typus humanus's culture supernatant | 36 | 36 |
| The mycobacterium avium-intracellulare culture supernatant | 10 | 0 |
| The mycobacterium kansasii culture supernatant | 10 | 0 |
3, the CFP-10 in the clinical culture samples of detection
Method of testing is with above-mentioned " 2 ", and the immune chromatography test paper detection media samples result who detects the Much's bacillus growth is as shown in table 3, shows that the testing result of test paper is consistent with the result of final culture identification, has specificity.
Table 3 detects the positive rate of CFP-10 in the clinical sample culture supernatant
| Sample classification | The example number | The CFP-10 positive detects number |
| Bacillus tuberculosis typus humanus's culture supernatant | 36 | 36 |
| Non-bacillus tuberculosis typus humanus's culture supernatant | 20 | 0 |
Comparative Examples 1,
With 0.01% (mass concentration) HAuCL
4The WS places with acid anhydrides and soaks and the dry flask of crossing, and shakes up and is placed on heated and boiled on the magnetic force heating stirrer, seals up bottleneck with the aluminium film and overflows in a large number with preventing hydration steam.Every 50mlHAuCL
4The WS once adds 1% trisodium citrate aqueous solution of 1.75ml rapidly in 100-110 ℃ of magnetic force heated and stirred to boiling back, continue to stop behind heating (100 ℃) certain hour, and the room temperature cooling is settled to 50mL with pure water at last.Be optimized adding for 1% trisodium citrate aqueous solution afterreaction time; The mean grain size of its particle, homogeneity, dispersiveness etc. are carried out phenetic analysis through pattern counts and measure moving particle diameter under Electronic Speculum; Specifically as shown in table 4, to optimize best preparation factor.
Table 4, reaction time are optimized analysis
Above experimental result shows, adds the uniformity coefficient of trisodium citrate afterreaction time appreciable impact colloid gold particle, adds in this example that optimum reacting time is ten minutes behind the trisodium citrate.
Comparative Examples 2,
After the test strips that the resuspended liquid of the golden labeling antibody of different proportionings is made is accomplished, detect the positive criteria article respectively, the situation that the observing colloid gold discharges has or not dead gold, and the colour developing situation of Jinsui River separation case and check point Quality Control point is its result (like table 5) relatively.Under the identical situation of other conditions; Resuspended liquid E dilution back and resuspended liquid be original volume 1 * golden labeling antibody be added drop-wise to the hydrophilic glass tunica fibrosa or the gold mark pad color that obtains on the hydrophilic polyesters tunica fibrosa relatively the darkest; It is the most even to distribute; It is the most clear to develop the color, and absorption and release rate are fast, therefore selects the 1 * dilution of E group.
The result of table 5, the resuspended liquid of golden labeling antibody relatively
Annotate: discharge :+fully, ± small amount of residual ,-residual; Flow :+fully, ± small amount of residual ,-residual; Colour developing :+colour developing is better, and ± colour developing is more shallow, the colour developing of-nothing;
The PB damping fluid of A:0.01mol/L pH7.4; The PB damping fluid of B:0.01mol/L pH7.4 (containing 1%BSA, 5% sucrose); The PB damping fluid of C:0.01mol/L pH7.4 (containing 1%BSA, 3.5% sucrose); The PB damping fluid of D:0.01mol/L pH7.4 (containing 1%BSA, 3.5% sucrose, 0.05%Tween-20,0.3mg/mL PEG20000); The PB damping fluid of E:0.01mol/L pH7.4 (contains 1%BSA; 3.5% sucrose, 0.3mg/mL PEG20000), promptly; The preparation method is: in concentration is 0.01mol/L, adds BSA, the sucrose of 35g and the PEG20000 of 0.3g of 10g in one liter of the PB damping fluid of PH7.4.
At last, it is also to be noted that what more than enumerate only is several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention all should be thought protection scope of the present invention.
Claims (3)
1. the immune chromatography test paper of a fast detecting Much's bacillus is characterized in that: comprise appearance pad (1), contain the anti-CFP-10 monoclonal antibody TBCA6 of golden mark collaurum pad (2), encapsulate nature controlling line (3) and detection line (6) nitrocellulose filter (5), inhale appearance and fill up (4) and a back up pad (7); Said detection line (6) is for being coated on the anti-CFP-10 monoclonal antibody TBCD6 on the nitrocellulose filter, and nature controlling line (3) is for being coated on the sheep anti-mouse igg antibody on the nitrocellulose filter (5); Upper surface at nitrocellulose filter (5) is provided with collaurum pad (2) respectively and inhales appearance pad (4), and last appearance pad (1) is positioned at the upper surface of collaurum pad (2), and the lower surface of nitrocellulose filter (5) and back up pad (7) are fixedly linked.
2. the preparation method of the immune chromatography test paper of fast detecting Much's bacillus as claimed in claim 1 is characterized in that may further comprise the steps:
The preparation of A, anti-CFP-10 monoclonal antibody TBCD6 and TBCA6:
Be used to encapsulate the anti-CFP-10 monoclonal antibody TBCD6 of detection line (6), by China's typical culture collection center preservation, deposit number is CCTCC NO:C201082;
Be used for the anti-CFP-10 monoclonal antibody TBCA6 of the golden mark of collaurum pad (2), by China's typical culture collection center preservation, deposit number is CCTCC NO:C201083;
The preparation method is following:
1) inducing of odd contradictive hydroperitoneum:, give every 0.5ml of BALB/C mice lumbar injection norphytane, then every inoculation 5 * 10 in inoculation hybridoma the last week
6The positive hybridoma cell of TBCD6 or TBCA6, collection ascites is centrifugal after 10 days, measures antibody titer;
2) TBCD6 or TBCA6 Purification of Monoclonal Antibodies: utilize TBCD6 or TBCA6 monoclonal antibody in the Protein G affinity purification difference purifying ascites;
B, making encapsulate the nitrocellulose filter (5) of detection line (6) and nature controlling line (3); The nitrocellulose filter (5) that will encapsulate detection line (6) and nature controlling line (3) then sticks on the back up pad (7);
Nitrocellulose filter (5) method for making that encapsulates detection line (6) and nature controlling line (3) is carried out following steps successively:
1), use 0.01mol/L PH7.4 phosphate PBS damping fluid will resist CFP-10 monoclonal antibody TBCD6 to be mixed with the solution of concentration as 0.3mg/ml; Rule with the parameter of 0.8～1.2ul/cm at the lower surface of nitrocellulose filter (5) with spray film appearance, encapsulate into detection line (6);
2), sheep anti-mouse igg antibody is mixed with the solution of 2.4mg/ml, in the parameter line of the upper surface of nitrocellulose filter (5), encapsulate into nature controlling line (3) with 0.8～1.2ul/cm with spray film appearance with 0.01mol/L PH7.4 phosphate PBS damping fluid;
3) nitrocellulose filter (5) after will ruling is in 36.5～37.5 ℃ temperature and under less than the condition of 30% humidity dry 8～10 hours; Must encapsulate the nitrocellulose filter (5) of detection line (6) and nature controlling line (3);
C, preparation contain the collaurum pad (2) of the anti-CFP-10 monoclonal antibody TBCA6 of golden mark:
TBCA6 monoclonal antibody colloidal gold probe solution is added to glass fibre membrane or polyester film, after the drying, gets collaurum pad (2); Collaurum pad (2) is sticked on the left side of the upper surface of the nitrocellulose filter (5) that step B obtains;
The preparation method of said TBCA6 monoclonal antibody colloidal gold probe solution is following:
1), with the HAuCL of mass concentration 0.01%
4WS heated and boiled, every 50ml HAuCL
4The WS adds the trisodium citrate aqueous solution of the mass concentration 1% of 1.7～1.8ml under 100-110 ℃ magnetic force heated and stirred, continued heated and stirred 8～12 minutes in 100-110 ℃, colloidal gold solution;
2), regulate colloidal gold solution to pH5.5, with the ratio coupling of antibody TBCA6, be the immune colloid gold probe solution of TBCA6 behind the mixing in 0.7～0.75mg antibody TBCA6/100ml colloidal gold solution;
3), with supernatant discarded behind 4 ℃ of centrifugal 30min of 15000r/min of immune colloid gold probe solution of above-mentioned TBCA6, with the resuspended sediment of resuspended liquid, TBCA6 monoclonal antibody colloidal gold probe solution; Said resuspended liquid is step 2) 0.9～1.2 times of colloidal gold solution volume;
The preparation method of said resuspended liquid is following: in concentration is 0.01mol/L, adds the BSA of 9～11g, the sucrose of 33～37g and the PEG20000 of 0.28～0.32g in one liter of the PB damping fluid of PH7.4;
D, paste at the upper surface of the collaurum pad (2) of step C gained and to go up appearance pad (1); Overlap joint is inhaled appearance pad (4) on the right side of the upper surface of nitrocellulose filter (5), the immune chromatography test paper of fast detecting Much's bacillus.
3. the preparation method of the immune chromatography test paper of fast detecting Much's bacillus according to claim 2 is characterized in that among the said step C: TBCA6 monoclonal antibody colloidal gold probe solution is pressed 1ml solution shop 23～27cm
2Ratio be layered on equably on glass fibre membrane or the dacron film.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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