CN102213723A - Doxycycline detection kit and preparation method thereof - Google Patents
Doxycycline detection kit and preparation method thereof Download PDFInfo
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- CN102213723A CN102213723A CN2010101384833A CN201010138483A CN102213723A CN 102213723 A CN102213723 A CN 102213723A CN 2010101384833 A CN2010101384833 A CN 2010101384833A CN 201010138483 A CN201010138483 A CN 201010138483A CN 102213723 A CN102213723 A CN 102213723A
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Abstract
The invention belongs to the technical field of determination of biological immunity methods, and particularly relates to a doxycycline detection kit and a preparation method thereof. The kit comprises a sample pad (1), a colloidal gold pad (2), a nitrocellulose membrane (3), a sample sucking pad (4) and a PVC support plate (5). The colloidal gold pad is colloidal gold labeled anti-doxycycline monoclonal antibody glass fiber (or non-woven fabric), the nitrocellulose membrane is sequentially coated with a doxycycline coupling antigen as a detection line (T line), and a goat anti-mouse IgG antibody as a quality control line (C line). The doxycycline detection kit is prepared by adopting a colloidal gold immunochromatography technology, is simple in preparation method, can be used for detecting doxycycline possibly existing in a sample, and has the characteristics of convenience in use, simplicity in operation, rapidness in reaction, economy, practicability and the like.
Description
Technical field
The present invention relates to the determination techniques field of biology immunization method, particularly relates to a kind of with colloidal gold immunity chromatography fast detecting fortimicin detection kit and preparation method thereof.
Background technology
Fortimicin has another name called doxycycline, claims Doxycycline again, is tetracycline medication, and its proterties is faint yellow or yellow crystalline powder, and is smelly, bitter.Easily molten in water or in the methyl alcohol, slightly soluble in ethanol or acetone, insoluble in chloroform.Be mainly used in the responsive gram positive bacteria and the infection of the upper respiratory tract due to the gram negative bacilli, flat body inflammation, infection of biliary tract, lymphnoditis, honeycomb group inflammation, senile chronic bronchitis etc., also be used for the treatment of typhus, Qiang's parasitosis, Eaton agent pneumonia etc.Still can be used for treating cholera, also can be used for preventing pernicious malaria and leptospiral infection.Therefore in Production of Livestock and Poultry, be widely used as medicine and feed addictive, residual but Irrational Use of Drugs can cause fortimicin can cause fortimicin to be accumulated in edible animal tissue, be detrimental to health then.Therefore, strengthen that the monitoring of fortimicin medicament residue in the animal-derived foods such as meat, egg, milk is had crucial meaning.
At present, the detection of fortimicin medicament residue method commonly used is mainly comprised high performance liquid chromatography (HPLC), liquid chromatograph mass spectrography method (LC-MS), microbial method, enzyme-linked immunosorbent assay (ELISA).High performance liquid chromatography and liquid chromatograph mass spectrography method are highly sensitive, reliable results, but assay method is loaded down with trivial details, time-consuming, and cost is higher, and operating personnel need professional training, is difficult to popularize; Microbial method is simple to operate, but poor specificity, and sense cycle is long and resultant error is bigger.Enzyme-linked immunosorbent assay is a competitive immunization detection method of utilizing the enzyme labeling thing, have that highly sensitive, detection limit is big, low cost and other advantages, but this method still needs special instrument (microplate reader), and analysis time, long (general 1-4 hour) still had certain limitation.Therefore, study a kind of simply more, quick, the testing product that is suitable for rig-site utilization has great importance.
Colloidal gold immunity chromatography (Immunochromatography Assay) is a kind of new immuno analytical method that grows up the eighties in 20th century, is the combination of immune affine technology, printing technology, immunolabelling technique and chromatographic technique.This method has obtained using comparatively widely at present, and it is to utilize colloidal gold-labeled method, as tracer, utilizes the immunologic detection method of the specific reaction between the antigen-antibody with collaurum.The method is simple to operate, reaction is quick, economical and practical, be suitable for on-the-spot the detection.
Summary of the invention
The object of the present invention is to provide a kind of high specificity, highly sensitive, simple to operate, reaction fast, accurately, be suitable for on-the-spot use be specifically designed to kit that detects fortimicin and preparation method thereof, detect fortimicin medicament residue in the animal-derived foods such as meat, egg, milk, be used for the auxiliary monitoring of food quality.
A kind of fortimicin detection kit comprises sample pad, collaurum pad, nitrocellulose filter, suction sample pad and PVC back up pad, and tight adhesion has sample pad, collaurum pad, nitrocellulose filter and suction sample pad successively on the PVC back up pad.Described collaurum pad is the anti-fortimicin antibody glass fibre (or nonwoven fabrics) of colloid gold label; Bag is by two lines on the described nitrocellulose filter, be respectively bag by the detection line (T line) of fortimicin-carrier protein (bovine serum albumin) conjugate and bag quilt the nature controlling line of sheep anti-mouse igg (C line).
The invention provides a kind of preparation method of fortimicin detection kit, may further comprise the steps:
(1) preparation fortimicin coupled antigen
With fortimicin and bovine serum albumin coupling, synthetic fortimicin-bovine serum albumin conjugate is as the fortimicin coupled antigen.
(2) the anti-fortimicin monoclonal antibody of preparation
Adopting fortimicin-bovine serum albumin conjugate is the immunogen immune BALB/C mice, by hybridoma technology, obtains secreting the hybridoma cell strain of anti-fortimicin monoclonal antibody; Prepare antibody in a large number to induce the ascites method in the body, use Protein G post to carry out purifying, obtain anti-fortimicin monoclonal antibody.
(3) preparation collaurum
Get 0.01% aqueous solution of chloraurate aqueous solution 100ml, heated and boiled.Add 1% citric acid, three sodium water solution 1ml as required rapidly, continue to boil about 5min, occur orange red.The size of the colloid gold particle of making like this is 20-40nm.
(4) preparation collaurum pad
Getting grain size is the colloidal gold solution 5ml of 20-40nm, uses 0.1mol/L K
2CO
3The pH value of colloidal gold solution is transferred to 8.0, and room temperature was placed 10 minutes; Dropwise adding protein concentration is the anti-fortimicin antibody 0.15ml of 2.0mg/ml, mixes, and room temperature was placed 30 minutes; Add 0.075ml 10% bovine serum albumin (BSA) solution, mix, room temperature was placed 10 minutes; 11000 left the heart 30 minutes, carefully drew supernatant, discarded, and the borate buffer solution 5mL redissolution with the 0.002mol/L pH 8.0 that contains 5% sucrose repeats 2 times; Be dissolved to 3mL with borate buffer solution at last, obtain anti-fortimicin antibody-colloid gold label thing; To resist fortimicin antibody-colloid gold label thing to spread 56cm by 1mL
2Ratio evenly be layered on the nonwoven fabrics, put drying room again, 38 ℃ of temperature, humidity was made the collaurum pad less than dry 2-4 under 30% the condition hour.
(5) bag is by fortimicin coupled antigen, sheep anti-mouse igg
Set and draw film instrument coating parameters 1 μ L/cm, get 0.2mL fortimicin coupled antigen, goat anti-rabbit igg with microsyringe respectively, receive A, the B pipe joint of drawing the film instrument in order.The PVC plate that posts nitrocellulose filter is placed on the to-and-fro movement platform of drawing the film instrument, open and draw the film instrument, on nitrocellulose filter, apply fortimicin coupled antigen (T line), goat anti-rabbit igg (C line).After the line with in the nitrocellulose filter baking oven, 38 ℃ of temperature, dry 24 hours, standby.
(6) assembling kit
Adhere to sample pad, collaurum pad, nitrocellulose filter and suction sample pad in order successively on the PVC back up pad, obtain the described test strips that is used to detect fortimicin, test strips can be packed in the plastic clip, is assembled into test card.Wherein said sample pad is a glass fibre, and inhaling the sample pad is thieving paper.
The present invention utilizes colloidal gold immunochromatographimethod technology and antigen and antibody specific reaction, and the principle that the fortimicin that may contain in utilization fortimicin coupled antigen and sample competition joining gold is marked anti-fortimicin monoclonal antibody detects fortimicin.During test sample, sample because of capillarity to inhaling sample pad one end chromatography.If contain fortimicin in the sample, they will and detection line (T line) go up the limited antibody combining site of fortimicin coupled antigen competition of bag quilt, when the fortimicin in the sample reaches finite concentration, also saturated fully with the anti-fortimicin antibody generation immune response of colloid gold label, this moment, colloidal gold composite did not have the fortimicin coupled antigen combination of bag quilt on vacant site and the detection line, this moment, the T line did not develop the color this positive result.If do not contain fortimicin in the sample, mark the colloid gold particle of anti-fortimicin antibody will be to the T line position in company with the sample chromatography, immune association reaction takes place with the fortimicin coupled antigen of bag quilt on the T line, colloid gold particle is piled up at the T line position and is made the T line present a macroscopic red stripes, this negative result.No matter whether contain fortimicin in the sample, the colloid gold label thing all can combine with the sheep anti-mouse igg on being coated on nature controlling line (C line) and develop the color, C line colour developing is to judge whether enough samples are arranged, and whether the chromatography process normal standard, simultaneously also as the inner quality standard of reagent.
The detection method of kit of the present invention is: with the test sample balance to room temperature; Take out the fortimicin pick-up unit, horizontal positioned; In sample pad, add 2-3 and drip sample, observe and write down the colour developing situation of C, T line in the time of 10 minutes, judge testing result.
Kit of the present invention adopts colloidal gold immunochromatographimethod technical measurement fortimicin, during detection, sample is added on the sample pad on the test strips (card), can observe directly immunoreactive result, finishes sample detection.The present invention can be used for detecting the fortimicin that may exist in the sample, have easy to use, simple to operate, be swift in response, characteristics such as economical and practical.
Description of drawings
Fig. 1 fortimicin detection kit structural representation;
The reference numeral explanation:
1: sample pad;
2: the collaurum pad that contains underlined anti-fortimicin monoclonal antibody;
3: (T: bag is by the detection line of fortimicin coupled antigen for nitrocellulose filter; C: bag is by the nature controlling line of sheep anti-mouse igg);
4: inhale the sample pad;
The 5:PVC back up pad;
The testing result synoptic diagram of Fig. 2 kit of the present invention.
Be followed successively by line positive test symbol of C from left to right, two line positive test symbol of T, C; Two line negative result of T, C; Invalid.
Embodiment:
Embodiment 1: the preparation of fortimicin detection kit
1. prepare the fortimicin coupled antigen
With fortimicin and bovine serum albumin coupling, synthetic fortimicin-bovine serum albumin conjugate is as the fortimicin coupled antigen.
2. prepare anti-fortimicin monoclonal antibody
Adopting the fortimicin coupled antigen is the immunogen immune BALB/C mice, by hybridoma technology, obtains secreting the hybridoma cell strain of anti-fortimicin monoclonal antibody; Prepare antibody in a large number to induce the ascites method in the body, use Protein G post to carry out purifying, obtain anti-fortimicin monoclonal antibody.
3. preparation collaurum
Get 0.01% aqueous solution of chloraurate aqueous solution 100ml, heated and boiled.Add 1% citric acid, three sodium water solution 1ml as required rapidly, continue to boil about 5min, occur orange red.The size of the colloid gold particle of making like this is 20-40nm.
4. prepare the collaurum pad
Getting grain size is the colloidal gold solution 5ml of 20-40nm, uses 0.1mol/L K
2CO
3The pH value of colloidal gold solution is transferred to 8.0, and room temperature was placed 10 minutes; Dropwise adding protein concentration is the anti-fortimicin antibody 0.15ml of 2.0mg/ml, mixes, and room temperature was placed 30 minutes; Add 0.075ml 10% bovine serum albumin (BSA) solution, mix, room temperature was placed 10 minutes; 11000 left the heart 30 minutes, carefully drew supernatant, discarded, and the borate buffer solution 5mL redissolution with the 0.002mol/L pH 8.0 that contains 5% sucrose repeats 2 times; Be dissolved to 3mL with borate buffer solution at last, obtain anti-fortimicin antibody-colloid gold label thing; To resist fortimicin antibody-colloid gold label thing to spread 56cm by 1mL
2Ratio evenly be layered on the nonwoven fabrics, put drying room again, 38 ℃ of temperature, humidity was made the collaurum pad less than dry 2-4 under 30% the condition hour.
5. the processing of sample pad
Glass fibre (or nonwoven fabrics) is soaked in 30min among the PBS of 50ml 0.01mol/L pH 7.5, wherein contains 5ml 10% BSA among the PBS, 2.5ml 10% Tweeen-2,37 ℃ of oven dry in drying baker, Vacuum Package is put 4 ℃ of preservations, and is standby
6. bag is by fortimicin coupled antigen, sheep anti-mouse igg
Set and draw film instrument coating parameters 1 μ L/cm, get 0.2mL fortimicin coupled antigen, goat anti-rabbit igg with microsyringe respectively, receive A, the B pipe joint of drawing the film instrument in order.The PVC plate that posts nitrocellulose filter is placed on the to-and-fro movement platform of drawing the film instrument, open and draw the film instrument, on nitrocellulose filter, apply fortimicin coupled antigen (T line), goat anti-rabbit igg (C line).After the line with in the nitrocellulose filter baking oven, 38 ℃ of temperature, dry 24 hours, standby.
7. assembling kit
Adhere to sample pad, collaurum pad, nitrocellulose filter and suction sample pad in order successively on the PVC back up pad, obtain the described test strips that is used to detect fortimicin, test strips can be packed in the plastic clip, is assembled into test card.Wherein said sample pad is a glass fibre, and inhaling the sample pad is thieving paper.
Embodiment 2: the applicating evaluating of fortimicin detection kit
1. negative reference material coincidence rate
With phosphate buffer solution (PBS:0.01mol/L, pH 7.5) compound concentration is that the chloromycetin standard solution of 100ng/mL carries out 10 parallel testings, observes testing result in the time of 10 minutes, and T, C line all present red stripes, and the result is negative.
With phosphate buffer solution (PBS:0.01mol/L, pH 7.5) compound concentration is that the streptomysin standard solution of 100ng/mL carries out 10 parallel testings, observes testing result in the time of 10 minutes, and T, C line all present red stripes, and the result is negative.
2. positive reference material coincidence rate
With phosphate buffer solution (PBS:0.01mol/L, pH 7.5) compound concentration be 15,30, the fortimicin standard items of 45ng/mL, each concentration is carried out 10 parallel testings respectively, observes testing result in the time of 10 minutes, and the T line does not develop the color, the C line all presents redness, and the result is positive.
3. limit of identification
With phosphate buffer solution (PBS:0.01mol/L, pH 7.5) compound concentration be 0,3,5,10,15, the fortimicin standard items of 20ng/mL, each concentration is carried out 10 parallel testings respectively, observe testing result in the time of 10 minutes, the testing result of the fortimicin standard items of 0ng/mL, 3ng/mL is that T, C line all develop the color and colour developing degree homogeneous negative result; The testing result of the fortimicin standard items of 5ng/mL, 10ng/mL is that T, C line all develop the color and the T line color obviously is shallower than the C line color, is judged to be positive test symbol; The testing result of the fortimicin standard items of 15ng/mL, 20ng/mL is that the T line does not develop the color, the colour developing of C line, positive result.Therefore the limit of identification of kit of the present invention is not higher than 5ng/mL.
4. repeated
With phosphate buffer solution (PBS:0.01mol/L, pH 7.5) compound concentration is the fortimicin standard items of 15ng/mL, carries out 10 parallel testings, observes testing result in the time of 10 minutes, the result is the T line and does not develop the color, the colour developing of C line, positive result, colour developing degree homogeneous.
5. stable
Place after 10 days for 37 ℃, every index all meets above requirement.
Embodiment 3: the detection method of fortimicin detection kit
1. The pretreatment
Serum: extract serum, centrifugal or leave standstill after get transparent supernatant and use; If the excessive haemolysis of serum, serum is experimentized with behind one times of the distilled water diluting again, otherwise the analoids redness is too dark, influences test result.
Urine: directly test with urine, if urine is visible muddy shape, need centrifugal earlier, filter or treat that its post precipitation gets supernatant and detect.
Milk: 4 ℃, the centrifugal 15min of 5000r/min, detect behind the fat of removal upper strata.
Tissue sample: after getting the 5-10g tissue sample and smashing to pieces, add 20-30ml (0.01mol/L, pH 7.5) PBS, 80 ℃ hatch 30min after, water-bath 10min in 4 ℃, the centrifugal 15min of 5000r/min, removes upper strata fat, gets supernatant and detects.
Feed: get the feed to be measured that 0.5-2g grinds, add 8-20ml (0.01mol/L, pH 7.5) PBS, 80 ℃ hatch 30min after, water-bath 10min in 4 ℃, the centrifugal 15min of 5000r/min, gets supernatant and detects.
2. operation
Take out the fortimicin pick-up unit, horizontal positioned; On sample pad, splash into 3 samples, observe and write down the colour developing situation of C, T line after 10 minutes, judge testing result.
3. the result judges
Positive:
The T line does not develop the color, and the C line presents red stripes, is judged to be positive findings, illustrates that the content of fortimicin is higher than 15ng/mL in the sample.
T line, C line all develop the color, and the T line color is shallower than the C line color, are judged to be positive findings, and the content that fortimicin in the sample is described is between 5-15ng/mL.
Negative:
T line, C line all present red stripes, and the T line color equals or be deeper than the C line color, and the content of fortimicin is lower than 5ng/mL in the interpret sample.
Invalid:
The C line does not develop the color, and the rotten damage of maloperation or kit is described.
Claims (7)
1. fortimicin detection kit and preparation method thereof, it is characterized in that forming by sample pad, collaurum pad, nitrocellulose filter, suction sample pad and PVC back up pad, sample pad, collaurum pad, nitrocellulose filter, suction sample pad stick on the PVC back up pad successively, the collaurum pad is the anti-fortimicin monoclonal antibody glass fibre or the nonwoven fabrics of colloid gold label, on the nitrocellulose filter successively the bag by the fortimicin coupled antigen as detection line (T line), sheep anti-mouse igg antibody is as nature controlling line (C line).
2. described fortimicin detection kit of claim 1 and preparation method thereof is characterized in that described sample pad is glass fibre or nonwoven fabrics, and inhaling the sample pad is absorbent filter.
3. described fortimicin detection kit of claim 1 and preparation method thereof is characterized in that described fortimicin coupled antigen is the conjugate of fortimicin and bovine serum albumin.Anti-fortimicin monoclonal antibody is obtained by the immunity of fortimicin coupled antigen.
4. described fortimicin detection kit of claim 1 and preparation method thereof is characterized in that described collaurum is by gold chloride (HAuCl
4) under the effect of reductive agent citric acid trisodium, make size and be the colloid gold particle of 20-40nm.
5. described fortimicin detection kit of claim 1 and preparation method thereof, it is characterized in that the preparation method of described collaurum pad is: getting grain size is the colloidal gold solution 5ml of 20-40nm, uses 0.1mol/L K
2CO
3The pH value of colloidal gold solution is transferred to 8.0, and room temperature was placed 10 minutes; Dropwise adding protein concentration is the anti-fortimicin antibody 0.15ml of 2.0mg/ml, mixes, and room temperature was placed 30 minutes; Add 0.075ml 10% bovine serum albumin (BSA) solution, mix, room temperature was placed 10 minutes; 11000 left the heart 30 minutes, carefully drew supernatant, discarded, and the borate buffer solution 5mL redissolution with the 0.002mol/L pH 8.0 that contains 5% sucrose repeats 2 times; Be dissolved to 3mL with borate buffer solution at last, obtain anti-fortimicin antibody-colloid gold label thing; To resist fortimicin antibody-colloid gold label thing to spread 56cm by 1mL
2Ratio evenly be layered on the nonwoven fabrics, put drying room again, 38 ℃ of temperature, humidity was made the collaurum pad less than dry 2-4 under 30% the condition hour.
6. described fortimicin detection kit of claim 1 and preparation method thereof, the method for coating that it is characterized in that two lines on the described nitrocellulose filter is: set and draw film instrument coating parameters 1 μ L/cm, get 0.2mL fortimicin coupled antigen, goat anti-rabbit igg with microsyringe respectively, receive A, the B pipe joint of drawing the film instrument in order.The PVC plate that posts nitrocellulose filter is placed on the to-and-fro movement platform of drawing the film instrument, open and draw the film instrument, on nitrocellulose filter, apply fortimicin coupled antigen (T line), goat anti-rabbit igg (C line).After the line with in the nitrocellulose filter baking oven, 38 ℃ of temperature, dry 24 hours, standby.
7. described fortimicin detection kit of claim 1 and preparation method thereof, the assemble method that it is characterized in that described fortimicin detection kit is: sample pad, collaurum pad, nitrocellulose filter, suction sample pad are attached on the PVC back up pad successively by an end, can form the reagent strip that detects fortimicin, reagent strip also can be packed into and be formed card type packing in the plastic clip.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010101384833A CN102213723A (en) | 2010-04-02 | 2010-04-02 | Doxycycline detection kit and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010101384833A CN102213723A (en) | 2010-04-02 | 2010-04-02 | Doxycycline detection kit and preparation method thereof |
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| Publication Number | Publication Date |
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| CN102213723A true CN102213723A (en) | 2011-10-12 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN2010101384833A Pending CN102213723A (en) | 2010-04-02 | 2010-04-02 | Doxycycline detection kit and preparation method thereof |
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| CN103630692A (en) * | 2013-06-02 | 2014-03-12 | 刘辉宇 | Colloidal gold immunochromatography kit for rapidly detecting urine C peptide and detecting method thereof |
| CN106053798A (en) * | 2016-07-26 | 2016-10-26 | 鲁东大学 | Doxycycline field test paper and methods of preparing and using same |
| CN106290927A (en) * | 2016-07-26 | 2017-01-04 | 鲁东大学 | Doxycycline quick detection kit and preparation, using method |
| CN106771157A (en) * | 2016-11-30 | 2017-05-31 | 百奥森（江苏）食品安全科技有限公司 | A kind of fortimicin detection method and detection card |
| CN107525921A (en) * | 2017-08-11 | 2017-12-29 | 太原瑞盛生物科技有限公司 | A kind of chemiluminescence detection kit of fortimicin and preparation method thereof |
| CN108709994A (en) * | 2018-05-29 | 2018-10-26 | 吉林大学 | A kind of NeuGc ALPHA2-3Gal quick detection test paper and detection method |
| CN110824162A (en) * | 2019-10-16 | 2020-02-21 | 佛山科学技术学院 | Tetracycline antibiotic six-joint detection card |
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| CN106290927A (en) * | 2016-07-26 | 2017-01-04 | 鲁东大学 | Doxycycline quick detection kit and preparation, using method |
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| CN108709994A (en) * | 2018-05-29 | 2018-10-26 | 吉林大学 | A kind of NeuGc ALPHA2-3Gal quick detection test paper and detection method |
| CN110824162A (en) * | 2019-10-16 | 2020-02-21 | 佛山科学技术学院 | Tetracycline antibiotic six-joint detection card |
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Application publication date: 20111012 |