CN103163297A - Multifunctional fluorescent immunochromatographic rapid quantitative detection card - Google Patents
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Abstract
The invention relates to a multifunctional fluorescent immunochromatographic rapid quantitative detection card. The detection card comprises: a) a sample treatment fluid A containing fluorescein-labeled to-be-detected antigens and a fluorescein-labeled contrast, b) a treatment fluid B containing biotin-labeled monoclonal antibodies aiming at the to-be-detected antigens, and c) a detection card comprising a detection line area and a quality control line area, wherein the detection line area is fixed with avidin and the quality control line area is fixed with antibodies aiming at the contrast. The invention further relates a preparation method of the multifunctional fluorescent immunochromatographic rapid quantitative detection card, an application of the card in food safety testing, and a method of detecting the to-be-detected antigens in samples by using the detection card. The invention is characterized in that (1) the detection card is general and can detect different food additives; (2) the product is long in shelf life and good in stability, and (3) the card improves sensitivity and stability of the detection, is not susceptible to interference and can achieve purposes of quantitative detection.
Description
Technical field
The present invention relates to the immunoassays fields.Particularly, the present invention relates to Multifunction fluorescent immunochromatography Quantitative detection card for detection of determined antigen in sample.The invention still further relates to the preparation method of this quantitative test card and the application in food safety detection thereof.
Background technology
Present immunochromatography rapid detection card is mainly with collaurum, color latex particulate or the fluorescein thing that serves as a mark.
Have sensitivity based on the fast detecting product of colloidal gold-labeled method exploitation low, can only qualitative or sxemiquantitative, the problem such as differences between batches are larger.
Although color latex particulate difference between batch improves to some extent, sensitivity is still lower, also can only qualitative or sxemiquantitative.
Immunochromatography sensitivity based on fluorescein-labelled technology is greatly improved, and also can quantitatively detect.
In recent years, the food security accident occurs frequently, and food security more and more is subject to government and broad masses' attention.The event such as melamine, clenbuterol hydrochloride has been exposed to food-safety problem in front of the people.
For little Molecular Detection such as melamine, clenobuterol hydrochloride, Ractopamines, detection method mostly is stratographic analysis and immunoassay.Stratographic analysis comprises high performance liquid chromatography, gas chromatography mass spectrometry chromatogram etc.And immunoassay mainly contains euzymelinked immunosorbent assay (ELISA) (ELISA), collaurum quick diagnosis etc.
One or two projects of the detection that quick detection test paper on the market can only be fixed can only be for detection of melamine as the test card that detects melamine, and the test card that detects clenobuterol hydrochloride can only detect clenobuterol hydrochloride.When needs detect a plurality of project, need to prepare multiple test card.
Therefore this area in the urgent need to the novel test card highly sensitive, that detection applications is wide, especially needs to realize the test card of one card for multiple uses.
Summary of the invention
The present invention has developed a kind of immunofluorescence Quantitative detection card, as long as change corresponding sample preparation liquid for different test items, can detect different materials, and obtain the accurate quantification result, has really realized the purpose of one card for multiple uses.
Of the present inventionly provide a kind of novel multi-functional rapid quantitative detection reagent based on immunochromatography technique, be characterised in that sample preparation liquid is fluorescein-labeled antigen and biotin labeled antibody, is fixed with Avidin (Streptavidin) and sheep anti-mouse antibody on the immunochromatography film.
Fluorescence immune chromatography Quantitative detection card of the present invention, this test card is to overlap successively sample pad, nitrocellulose filter and thieving paper on end liner, detection line (the T line is fixed with Avidin) and nature controlling line (the C line is fixed with goat anti-rabbit antibody) are arranged on nitrocellulose filter.
For different test items, be equipped with the different sample preparation liquid of crossing, sample preparation liquid comprises A, two components of B, contains fluorescein-labeled determined antigen molecule and fluorescein-labeled rabbit in A liquid anti-, contains biotin labeled mouse monoclonal antibody for determined antigen in B liquid.
As detect and contain fluorescein-labeled clenobuterol hydrochloride antigen in the sample preparation liquid A of clenobuterol hydrochloride and fluorescein-labeled rabbit is anti-, contain biotin labeled clenobuterol hydrochloride monoclonal mouse-anti in B liquid.
Detect and to contain fluorescein-labeled Ractopamine antigen in the sample preparation liquid A of Ractopamine and fluorescein-labeled rabbit is anti-, contain biotin labeled Ractopamine monoclonal mouse-anti in B liquid.
Detect and to contain fluorescein-labeled melamine antigen in the sample preparation liquid A of melamine and fluorescein-labeled rabbit is anti-, contain biotin labeled melamine monoclonal mouse-anti in B liquid.
The fluorescein that mark is used can be commercially available various fluorescent markers, as Alexa Fluor fluorescent marker series of FITC, rhodamine, Invitrogen company etc.
The present invention can be used for detecting various little molecular antigens or haptens, detects principle with the Immune competition method.
Detecting step:
Sample to be tested is added in sample preparation liquid A, and then add sample treatment solution B in sample treatment solution, antigen in sample to be tested is combined with the biotin labeled monoclonal antibody that fluorescein-labeled antigen is vied each other in B liquid, in sample, determined antigen concentration is higher, and the fluorescein-labeled antigen amount of being combined with biotin labeled antibody is fewer.
Mixed liquor is dripped in test card, and successively by C, T line, be combined with the Avidin of T line by biotin labeled monoclonal antibody by the chromatography effect for antigen antibody complex, and the fluorescein-labelled rabbit in mixed liquor is anti-to be caught by the goat anti-rabbit antibody of C line.
Antigen concentration in sample to be tested is higher, the amount of fluorescein-labeled antigen and biotin labeled antibody amount formation antigen antibody complex is just fewer, the amount of the fluorescence immunoassay compound of being caught by Avidin on the T line is also just fewer, and in sample to be tested, the concentration of antigen becomes negative correlation with T line signal.
The fluorescein-labelled rabbit antinoise signal value of catching on the C line is relatively fixing.
By fluorescence detector, test card is scanned, become negative correlation according to antigen concentration with fluorescence signal, obtain the accurate concentration of antigen in sample to be tested.
Another object of the present invention is to provide a kind of preparation method of novel multi-functional Quantitative detection card based on immunochromatography technique.
The preparation method of multi-functional Quantitative detection card of the present invention comprises the following steps:
(1) fluorescein-labelled antigen
(2) fluorescein-labelled rabbit is anti-
(3) biotin labeling is for the monoclonal antibody of specific antigen
(4) sample preparation liquid A preparation
(5) sample preparation liquid B preparation
(6) spray film
Avidin is diluted to 0.5～2mg/ml, quantitatively is sprayed on nitrocellulose filter detection zone T with spray film instrument.
Goat anti-rabbit antibody is diluted to 0.5～2mg/ml, quantitatively is sprayed on nitrocellulose filter Quality Control district C with spray film instrument.
C, T line spray film liquid measure are 0.5～2 μ l/cm.
(7) kilocalorie is pasted
Paste successively nitrocellulose filter, sample pad, thieving paper etc. on the base plate of gum.
(8) drying
With above-mentioned sprayed reagent be stuck in greatly in constant temperature oven or drying room 37 degree oven dry 6～24 hours.
(9) slitting, be installed
Dried kilocalorie is cut into the test card of certain width with cutting cutter or hobboing cutter, the test card of well cutting is contained in plastic casing, and with aluminium foil bag or packaging plastic bags, adds drying agent in packaging bag.
Therefore, one aspect of the present invention provides a kind of Multifunction fluorescent immunochromatography Quantitative detection card, and this test card comprises:
A) sample preparation liquid A, it contains fluorescein-labeled determined antigen and fluorescein-labeled contrast;
B) treating fluid B, it contains biotin labeled monoclonal antibody for determined antigen;
C) test card, it comprise detection line zone and nature controlling line regional, described detection line zone is fixed with Avidin, described nature controlling line zone is fixed with the antibody for described contrast.
In the present invention, fluorescein-labeled determined antigen concentration is preferably 1～100ng/ml, and the final concentration of contrast is preferably 1～20ng/ml, and the monoclonal antibody final concentration is preferably 5～50 μ g/ml.
In some embodiments of the present invention, contrast can be that rabbit is anti-, can be goat anti-rabbit antibody for the antibody of this contrast.
In some embodiments of the present invention, determined antigen includes but not limited to clenobuterol hydrochloride, Ractopamine, melamine.In addition, determined antigen also comprises little molecular antigen and haptens.
In the present invention, fluorescein can use any suitable fluorescein, such as FITC, rhodamine, Alexa Fluor fluorescent marker series etc.
The present invention also provides the application of test card of the present invention in food safety detection on the other hand.
Another aspect of the invention provides the method for using test card of the present invention to detect determined antigen in sample, and it comprises the steps:
I) sample to be tested is added in sample preparation liquid A, and then add sample treatment solution B in sample treatment solution, obtain mixed liquor, wherein, antigen in sample to be tested is vied each other to be combined with monoclonal antibody described in treating fluid B with fluorescein-labeled determined antigen described in treating fluid A and is formed antigen antibody complex, in sample, determined antigen concentration is higher, and the amount of being combined with monoclonal antibody described in treating fluid B of fluorescein-labeled determined antigen described in treating fluid A is fewer;
Ii) with i) in the mixed liquor that obtains drip in test card, described antigen antibody complex is regional by nature controlling line zone, detection line successively by the chromatography effect, biotin labeled monoclonal antibody is combined with the Avidin in detection line zone, and the fluorescein-labelled contrast in mixed liquor is by the antibody capture of this contrast of nature controlling line; Antigen concentration in sample to be tested is higher, the amount of fluorescein-labeled antigen and biotin labeled antibody amount formation antigen antibody complex is just fewer, the amount of the fluorescence immunoassay compound of being caught by Avidin on detection line is also just fewer, and in sample to be tested, the concentration of antigen becomes negative correlation with the detection line signal; The signal value of the fluorescein-labeled contrast of catching on nature controlling line is relatively fixing;
Iii) by fluorescence detector, test card is scanned, become negative correlation according to antigen concentration with fluorescence signal, obtain the accurate concentration of antigen in sample to be tested.
Another aspect of the invention provides the preparation method of test card, and it comprises the following steps:
I) prepare fluorescein-labeled determined antigen, fluorescein-labeled contrast, biotin labeled monoclonal antibody for determined antigen;
Ii) preparation treating fluid A, treating fluid B;
Iii) spray film, it comprises Avidin is diluted to 0.5～2mg/ml, quantitatively is sprayed on the carrier film detection line with spray film instrument regional;
Iv) will for the antibody dilution to 0.5 that contrasts～2mg/ml, quantitatively be sprayed on the nature controlling line zone of carrier film with spray film instrument; The spray film liquid measure of nature controlling line, detection line is 0.5～2 μ l/cm;
V) kilocalorie is pasted, and glues successively note carrier film, sample pad, thieving paper on the base plate of gum;
Vi) drying has been sprayed being stuck in greatly of reagent and was dried in constant temperature oven or drying room 6～24 hours above-mentioned;
Vii) slitting, be installed, dried kilocalorie is cut into test card with cutting cutter or hobboing cutter, the test card of well cutting is contained in plastic casing, and with aluminium foil bag or packaging plastic bags, adds drying agent in packaging bag.
In the present invention, can use any suitable carrier film, as nitrocellulose filter.
Description of drawings
Fig. 1: film array mode and zone are divided
1: base plate
2: sample pad
3: adsorptive pads
4: nitrocellulose filter
5: detection line (T line) zone
6: nature controlling line (C line) zone.
Fig. 2: clenobuterol hydrochloride examination criteria curve.
Fig. 3: Ractopamine examination criteria curve.
Fig. 4: melamine examination criteria curve.
Embodiment
Below the present invention will be further elaborated by specific embodiment, but embodiment and non-limiting protection scope of the present invention.
Embodiment 1: fluorescein-labelled albumen
(1) fluorescein is (take fluorescein Alexa Fluor 647 as example, available from Invitrogen) mark clenobuterol hydrochloride (Ractopamine, melamine) antigen (is connected with the macromolecular carriers such as BGG, BSA, clenobuterol hydrochloride-BSA synthetic antigen is biological available from the grand base in Hangzhou, Ractopamine-BSA synthetic antigen is biological available from the grand base in Hangzhou, and melamine-BSA synthetic antigen is biological available from Beijing Bo Aosen) or goat anti-rabbit antibody (biological available from the grand base in Hangzhou)
100 μ l (1mg/ml, PBS) add 10 μ l 1mol/L sodium bicarbonate buffer liquid (pH 8.3) in clenobuterol hydrochloride (Ractopamine, melamine) antigen or goat anti-rabbit antibody, add again 1 μ l Alexa Fluor 647 (10mg/ml), then react under 4 ℃ and spend the night.
Above-mentioned reactant liquor is removed free Alexa dyestuff with Sephadex G25 chromatographic column (available from Phamarcia), obtained fluorescein-labeled clenobuterol hydrochloride (Ractopamine, melamine) antigen.
Embodiment 2: biotinylated protein
(1) biotin labeling clenobuterol hydrochloride (Ractopamine, melamine) monoclonal antibody, (the clenobuterol hydrochloride monoclonal antibody is biological available from the grand base in Hangzhou, and the Ractopamine monoclonal antibody is biological available from the grand base in Hangzhou; The melamine monoclonal antibody is biological available from Beijing Bo Aosen)
With 0.1M NaHCO3, antibody to be marked is mixed with 1mg/ml solution, adopts DMSO configuration Biotin-X-X-NHS solution to 16.172mg/ml, get in 5.4ul Biotin-X-X-NHS to 1mg antibody-solutions to be marked, mix and place under 4 ℃ and spend the night.
Above-mentioned reactant liquor is removed free Biotin-X-X-NHS with Sephadex G25 chromatographic column, obtained biotin labeled antibody to be marked.
Embodiment 3: sample preparation liquid A preparation
(1) the sample preparation liquid A of clenobuterol hydrochloride
Prepare fluorescein-labelled clenobuterol hydrochloride antigen and the anti-mixed liquor of fluorescein-labelled rabbit, fluorescein-labeled clenobuterol hydrochloride final concentration 1～100ng/ml wherein, the anti-final concentration of fluorescein-labelled rabbit is 1～20ng/ml.
This mixed liquor is clenobuterol hydrochloride sample preparation liquid A
(2) the sample preparation liquid A of Ractopamine
Prepare fluorescein-labelled Ractopamine antigen and the anti-mixed liquor of fluorescein-labelled rabbit, fluorescein-labeled Ractopamine final concentration 1～100ng/ml wherein, the anti-final concentration of fluorescein-labelled rabbit is 1～20ng/ml.
This mixed liquor is Ractopamine sample preparation liquid A
(3) the sample preparation liquid A of melamine
Prepare fluorescein-labelled melamine antigen and the anti-mixed liquor of fluorescein-labelled rabbit, fluorescein-labeled Ractopamine final concentration 1～100ng/ml wherein, the anti-final concentration of fluorescein-labelled rabbit is 1～20ng/ml.
This mixed liquor is melamine sample preparation liquid A
Embodiment 4: sample preparation liquid B preparation
(1) the sample preparation liquid B of clenobuterol hydrochloride
Prepare biotin labeled clenobuterol hydrochloride monoclonal antibody, final concentration 5～50 μ g/ml are as clenobuterol hydrochloride sample preparation liquid B.
(2) the sample preparation liquid B of Ractopamine
Prepare biotin labeled Ractopamine monoclonal antibody, final concentration 5～50 μ g/ml are as Ractopamine sample preparation liquid B.
(3) the sample preparation liquid B of melamine
Prepare biotin labeled melamine monoclonal antibody, final concentration 5～50 μ g/ml are as melamine sample preparation liquid B.
Embodiment 5: the preparation of multifunction immunity chromatography quantitative test card
(1) T line solution preparation
With 10mM PB (containing 3% methyl alcohol) solution, Avidin (Streptavidin is available from sigma) is diluted to 1mg/ml.
(2) C line solution preparation
With 10mM PB (containing 3% methyl alcohol) solution, goat anti-rabbit antibody is diluted to 1mg/ml.
(3) spray film
C, T line solution are sprayed on the CT zone (in accompanying drawing, difference corresponding 6,5) of nitrocellulose filter (available from Millipore company, model HFl35)
(4) the sticking note of kilocalorie
Successively at the upper sticking note nitrocellulose filter of base plate (in accompanying drawing 1) (in accompanying drawing 4), sample pad (in accompanying drawing 2), adsorptive pads (in accompanying drawing 3).
(5) oven dry
With above-mentioned sprayed reagent be stuck in greatly in constant temperature oven or drying room 37 degree oven dry 24 hours.
(6) slitting and being installed
The kilocalorie of oven dry is cut into the paper slip of 3～5mm width, is assembled in plastic housing, aluminium foil bag or packaging plastic bags, in join drying agent, form multifunction immunity fluorescent quantitation check-out console.Coordinate with corresponding sample preparation liquid A, B and can detect corresponding antigen.
Embodiment 6: Specification Curve of Increasing
(1) clenobuterol hydrochloride examination criteria Drawing of Curve
With phosphate buffer (PBS:0.01M, p H 7.4) compound concentration is 0,0.2,1,10,50, the clenobuterol hydrochloride standard items of 100ng/ml carry out Parallel testing, each concentration is carried out 10 experiments, reaction time is 10min, the Instrument measuring testing result with concentration and signal drawing standard curve, is stored in data card, as the clenobuterol hydrochloride calibration curve of this batch reagent, typical curve is seen Fig. 2.
Table 1 clenobuterol hydrochloride standard items testing result
(2) Ractopamine examination criteria Drawing of Curve
With phosphate buffer (PBS:0.01M, p H 7.4) compound concentration is 0,0.5,1,10,50, the Ractopamine standard items of 100ng/ml carry out Parallel testing, each concentration is carried out 10 experiments, reaction time is 10min, the Instrument measuring testing result with concentration and signal drawing standard curve, is stored in data card, as the Ractopamine calibration curve of this batch reagent, typical curve is seen Fig. 3.
Table 2 Ractopamine standard items testing result
(3) melamine examination criteria Drawing of Curve
With phosphate buffer (PBS:0.01M, p H 7.4) compound concentration is 0,0.5,1,10,50, the melamine standard items of 100ng/ml carry out Parallel testing, each concentration is carried out 10 experiments, reaction time is 10min, the Instrument measuring testing result with concentration and signal drawing standard curve, is stored in data card, as the Ractopamine calibration curve of this batch reagent, typical curve is seen Fig. 4.
Table 3 melamine standard items testing result
Embodiment 7: pattern detection
(1) clenobuterol hydrochloride pattern detection
Tissue samples: first adopt sample pretreatment solution (containing the surfactants such as SDS) to carry out cracking the fritter tissue samples, then the tissue samples after cracking is added in clenobuterol hydrochloride sample preparation liquid A, add again clenobuterol hydrochloride sample treatment solution B, fully mix.
Mixed liquor is dripped on check-out console, with fluorescence chart scanner reading, calculate clenobuterol hydrochloride concentration in sample according to the calibration curve that stores in data card after 10min.
Urine: a certain amount of urine is added in clenobuterol hydrochloride sample preparation liquid A, then add clenobuterol hydrochloride sample treatment solution B, fully mix.
Mixed liquor is dripped on check-out console, with fluorescence chart scanner reading, calculate clenobuterol hydrochloride concentration in sample according to the calibration curve that stores in data card after 10min.
Feed: a certain amount of feed is added fully concussion mixing in the sample pre-treatments liquid that contains the surfactants such as tween, then a certain amount of pretreatment liquid is added in clenobuterol hydrochloride sample preparation liquid A, add again clenobuterol hydrochloride sample treatment solution B, fully mix.
Mixed liquor is dripped on check-out console, with fluorescence chart scanner reading, calculate clenobuterol hydrochloride concentration in sample according to the calibration curve that stores in data card after 10min.
(2) Ractopamine pattern detection
Tissue samples: first adopt sample pretreatment solution (containing the surfactants such as SDS) to carry out cracking the fritter tissue samples, then the tissue samples after cracking is added in Ractopamine sample preparation liquid A, add again Ractopamine sample treatment solution B, fully mix.
Mixed liquor is dripped on check-out console, with fluorescence chart scanner reading, calculate Ractopamine concentration in sample according to the calibration curve that stores in data card after 10min.
Urine: a certain amount of urine is added in Ractopamine sample preparation liquid A, then add Ractopamine sample treatment solution B, fully mix.
Mixed liquor is dripped on check-out console, with fluorescence chart scanner reading, calculate Ractopamine concentration in sample according to the calibration curve that stores in data card after 10min.
Feed: a certain amount of feed is added fully concussion mixing in the sample pre-treatments liquid that contains the surfactants such as tween, then a certain amount of pretreatment liquid is added in Ractopamine sample preparation liquid A, then add Ractopamine sample treatment solution B, fully mix.
Mixed liquor is dripped on check-out console, with fluorescence chart scanner reading, calculate Ractopamine concentration in sample according to the calibration curve that stores in data card after 10min.
(3) melamine pattern detection
Liquid milk sample: directly a certain amount of liquid milk is added in melamine sample preparation liquid A, then add melamine sample treatment solution B, fully mix.
Mixed liquor is dripped on check-out console, with fluorescence chart scanner reading, calculate melamine concentration in sample according to the calibration curve that stores in data card after 10min.
Milk powder sample: get a certain amount of milk powder and add in melamine sample preparation liquid A, then add melamine sample treatment solution B, fully mix.
Mixed liquor is dripped on check-out console, with fluorescence chart scanner reading, calculate melamine concentration in sample according to the calibration curve that stores in data card after 10min.
Other samples: sample to be tested is mixed with certain density solution, gets a certain amount of solution and add in melamine sample preparation liquid A, then add melamine sample treatment solution B, fully mix.
Mixed liquor is dripped on check-out console, with fluorescence chart scanner reading, calculate melamine concentration in sample according to the calibration curve that stores in data card after 10min.
The immunochromatography that above embodiment can also be applicable to other all employing competition law patterns detects, and comprises medicine, food additives, drugs etc.
Embodiment 8: the performance test of Multifunction fluorescent immunochromatography Quantitative detection card
(1) clenobuterol hydrochloride performance test
With test card (lot number: 20090522) carry out performance test, measure the precision of test card as sample with 1ng/ml and 10ng/ml antigen.
Table 4 test card is criticized interpolation
Adopt the Quantitative detection card detection clenobuterol hydrochloride scope of the method preparation can reach 0～100ng/ml, sensitivity is below 0.2ng/ml, and withinrun precision can reach 10% left and right.
On market, similar quick diagnosis product can only carry out qualitative detection, sensitivity generally can only reach 3ng/ml, the good 1ng/ml that also can only reach, our Multifunction fluorescent immunochromatography Quantitative detection card not only can access quantitative result, also has higher sensitivity.
(2) Ractopamine performance test
With test card (lot number: 20090522) carry out performance test, measure the precision of test card as sample with 1ng/ml and 10ng/ml antigen.
Table 5 test card is criticized interpolation
Adopt the Quantitative detection card detection Ractopamine scope of the method preparation can reach 0～100ng/ml, sensitivity is below 0.5ng/mg, and withinrun precision can reach 10% left and right.
On market, similar quick diagnosis product can only carry out qualitative detection, and the sensitivity majority can only reach 5ng/ml, good 1ng/ml, and our Multifunction fluorescent immunochromatography Quantitative detection card not only can access quantitative result, also has higher sensitivity.
(3) melamine performance test
With test card (lot number: 20090522) carry out performance test, measure the precision of test card as sample with 1ng/ml and 10ng/ml antigen.
Table 6 test card is criticized interpolation
Adopt the Quantitative detection card detection Ractopamine scope of the method preparation can reach 0～100ng/ml, sensitivity is below 0.5ng/mg, and withinrun precision can reach 10% left and right.
On market, similar quick diagnosis product can only carry out qualitative detection, and the sensitivity majority can only reach 5ng/ml, and our Multifunction fluorescent immunochromatography Quantitative detection card not only can access quantitative result, also has higher sensitivity.
Claims (10)
1. Multifunction fluorescent immunochromatography Quantitative detection card, this test card comprises:
A) sample preparation liquid A, it contains fluorescein-labeled determined antigen and fluorescein-labeled contrast;
B) treating fluid B, it contains biotin labeled monoclonal antibody for determined antigen;
C) test card, it comprise detection line zone and nature controlling line regional, described detection line zone is fixed with Avidin, described nature controlling line zone is fixed with the antibody for described contrast.
2. Multifunction fluorescent immunochromatography Quantitative detection card according to claim 1, wherein said determined antigen concentration is 1～100ng/ml, and described contrast final concentration is 1～20ng/ml, and described monoclonal antibody final concentration is 5～50 μ g/ml.
3. Multifunction fluorescent immunochromatography Quantitative detection card according to claim 1 and 2, wherein said contrast are that rabbit is anti-, are goat anti-rabbit antibodies for the antibody of described contrast.
4. the described Multifunction fluorescent immunochromatography of any one Quantitative detection card according to claim 1-3, wherein said determined antigen comprises clenobuterol hydrochloride, Ractopamine, melamine.
5. the described Multifunction fluorescent immunochromatography of any one Quantitative detection card according to claim 1-4, wherein said fluorescein comprises FITC, rhodamine, Alexa Fluor fluorescent marker series.
6. the described Multifunction fluorescent immunochromatography of any one Quantitative detection card according to claim 1-5, wherein said determined antigen comprises little molecular antigen and haptens.
7. the described Multifunction fluorescent immunochromatography of any one Quantitative detection is stuck in application in food safety detection according to claim 1-6.
8. method that detects determined antigen in sample by the described Multifunction fluorescent immunochromatography of any one Quantitative detection card in claim 1-6, it comprises the steps:
I) sample to be tested is added in sample preparation liquid A, and then add sample treatment solution B in sample treatment solution, obtain mixed liquor, wherein, antigen in sample to be tested is vied each other to be combined with monoclonal antibody described in treating fluid B with fluorescein-labeled determined antigen described in treating fluid A and is formed antigen antibody complex, in sample, determined antigen concentration is higher, and the amount of being combined with monoclonal antibody described in treating fluid B of fluorescein-labeled determined antigen described in treating fluid A is fewer;
Ii) with i) in the mixed liquor that obtains drip in test card, described antigen antibody complex is regional by nature controlling line zone, detection line successively by the chromatography effect, biotin labeled monoclonal antibody is combined with the Avidin in detection line zone, and the fluorescein-labelled contrast in mixed liquor is by the antibody capture of this contrast of nature controlling line; Antigen concentration in sample to be tested is higher, the amount of fluorescein-labeled antigen and biotin labeled antibody amount formation antigen antibody complex is just fewer, the amount of the fluorescence immunoassay compound of being caught by Avidin on detection line is also just fewer, and in sample to be tested, the concentration of antigen becomes negative correlation with the detection line signal; The signal value of the fluorescein-labeled contrast of catching on nature controlling line is relatively fixing;
Iii) by fluorescence detector, test card is scanned, become negative correlation according to antigen concentration with fluorescence signal, obtain the accurate concentration of antigen in sample to be tested.
9. the preparation method of the described Multifunction fluorescent immunochromatography of any one Quantitative detection card in a claim 1-6, it comprises the following steps:
I) prepare fluorescein-labeled determined antigen, fluorescein-labeled contrast, biotin labeled monoclonal antibody for determined antigen;
Ii) preparation treating fluid A, treating fluid B;
Iii) spray film, it comprises Avidin is diluted to 0.5～2mg/ml, quantitatively is sprayed on the carrier film detection line with spray film instrument regional;
Iv) will for the antibody dilution to 0.5 that contrasts～2mg/ml, quantitatively be sprayed on the nature controlling line zone of carrier film with spray film instrument; The spray film liquid measure of nature controlling line, detection line is 0.5～2 μ l/cm;
V) kilocalorie is pasted, and glues successively note carrier film, sample pad, thieving paper on the base plate of gum;
Vi) drying has been sprayed being stuck in greatly of reagent and was dried in constant temperature oven or drying room 6～24 hours above-mentioned;
Vii) slitting, be installed, dried kilocalorie is cut into test card with cutting cutter or hobboing cutter, the test card of well cutting is contained in plastic casing, and with aluminium foil bag or packaging plastic bags, adds drying agent in packaging bag.
10. preparation method according to claim 9, wherein said carrier film is nitrocellulose filter.
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| CN104714017A (en) * | 2015-02-06 | 2015-06-17 | 北京豪迈生物工程有限公司 | Method for quantitatively detecting procalcitonin |
| CN105988008A (en) * | 2015-02-11 | 2016-10-05 | 四川迈克生物科技股份有限公司 | Measurement device, kit and measurement method |
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