CN101609094A - Be used for residual fast detecting colloid gold test paper of betamethasone and preparation method thereof - Google Patents
Be used for residual fast detecting colloid gold test paper of betamethasone and preparation method thereof Download PDFInfo
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- CN101609094A CN101609094A CNA2009101580944A CN200910158094A CN101609094A CN 101609094 A CN101609094 A CN 101609094A CN A2009101580944 A CNA2009101580944 A CN A2009101580944A CN 200910158094 A CN200910158094 A CN 200910158094A CN 101609094 A CN101609094 A CN 101609094A
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- colloid gold
- collaurum
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Abstract
The invention discloses a kind of residual fast detecting colloid gold test paper of betamethasone and preparation method thereof that is used for, this test paper comprises box body, test strips, sponge bracket and support, wherein is adsorbed with the betamethasone polyclonal antibody of envelope antigen and colloid gold label on the test strips.The present invention has overcome betamethasone physico-chemical analysis method complicated operation, has detected the cost height, speed is slow and shortcoming such as secondary environmental pollution, it is residual accurately to detect in water, meat products and the dairy produce betamethasone delicately, the pre-treatment process of sample is simple, consuming time few, can a large amount of sample of while fast detecting.
Description
Technical field
The present invention relates to a kind of residual fast detecting colloid gold test paper of betamethasone and preparation method thereof that is used for.
Background technology
Veterinary drug betamethasone (Betamethasone) is a kind of glucocorticoid medicine, has good antiinflammatory action, can resist caused inflammation such as a variety of causes such as physics, chemistry, physiology, immunity, but glucocorticoid is except therapeutic action, can also improve food conversion ratio, thereby the increase the weight of animals, and and beta-agonist and other steroid hormones synergy is arranged.And glucocorticoid as unreasonable uses such as growth accelerators, or do not notice that off-drug period etc. may finally cause the residual of glucocorticoid in the animal derived food when using as veterinary drug, constituted potential hazard to human health.
The residual conventional method of analysis of glucocorticoid betamethasone mainly is instrumental methods such as liquid phase chromatography, this traditional analytical approach not only needs expensive gas chromatograph, analysis speed is slow, cost is high, is not suitable for a large amount of samples is carried out rapid screening and applies in basic unit.And sample pre-treatments program more complicated, need to use a large amount of organic solvents, caused secondary pollution.Along with sample to be checked, particularly require increasing sharply of field quick detection sample size, traditional residue of veterinary drug analysis means is difficult to adapt to requirement.
Summary of the invention
At the problem that prior art exists, the object of the present invention is to provide that a kind of to have a high specific, high sensitivity, pin-point accuracy, pinpoint accuracy, method of operating simple, and can be used for the residual colloid gold test paper of betamethasone of batch samples fast detecting.Another object of the present invention provides the preparation method of above-mentioned colloid gold test paper.
For achieving the above object, a kind of preparation method who is used for the residual fast detecting colloid gold test paper of betamethasone of the present invention is specially:
1) prepares artificial immunogen after utilizing active ester method with betamethasone and bovine serum albumin(BSA) (BSA) coupling, go out the betamethasone polyclonal antibody with this immunogen preparing;
2) with mixed anhydride method with betamethasone and ovalbumin (OVA) coupling, its compound as envelope antigen, and is fixed in cellulose acetate film (NC) as detection line (T line) with this envelope antigen;
3) the betamethasone polyclonal antibody is fixed in step 2) on the cellulose acetate film (NC) after handling, as control line line (C line);
4) prepare collaurum with the sodium citrate improved method, use its markers step 1 simultaneously) the middle betamethasone polyclonal antibody for preparing, and be fixed on the collaurum pad;
5) the collaurum pad is fitted on the cellulose acetate film of handling well in the step 3) (NC), and glues absorbent material and all-glass paper, promptly make colloid gold test paper.
Further, described betamethasone polyclonal antibody is made by the new zealand white rabbit of immunogen immune described in the step 1).
Further, prepare the method for betamethasone polyclonal antibody in the described step 1), be specially: with betamethasone-BSA conjugate as immunogene, by setting requirement White Rabbit is carried out repeatedly inoculation, after finishing, the blood sampling separation of serum is slightly carried rabbit anti-serum with the saturated ammonium sulfate method earlier, be further purified with albumin A-agarose affinity chromatography method again, obtain purer betamethasone polyclonal antibody.
Further, the sodium citrate improved method prepares collaurum in the described step 4), be specially: adopt the sodium citrate reducing process to prepare collaurum, prepare the gold chloride of 0.01% concentration with the new system deionized water, place in the conical flask, be heated to and boil, accurately draw the trisodium citrate of 1% concentration, add in the conical flask rapidly, rock evenly after, continue to boil, after becoming claret, its color continues the heating setpoint time, stop heating, return to original volume with deionized water after naturally cooling to room temperature, promptly obtain collaurum.
Further, the colloid gold label of betamethasone polyclonal antibody in the described step 4), be specially: the collaurum that is adjusted to optimal pH 8.5, the anti-betamethasone antibody of rabbit that adds 0.2mg/ml concentration, the lucifuge gentle agitation is reacted 1h on magnetic stirring apparatus, adds the OVA of 10% concentration, behind the sealing 30min, 4 ℃ * 1500rpm * 15min is centrifugal, removes the collaurum that dissociates; The centrifugal precipitation of staying of 4 ℃ * 12000rpm * 20min is removed supernatant, and is resuspended with the resuspended liquid of collaurum, room temperature reaction 10min; The centrifugal again precipitation of staying of 4 ℃ * 12000rpm * 20min is removed supernatant, and is resuspended with the resuspended liquid of collaurum; Centrifugal in 4 ℃ * 1000rpm * 10min again, go precipitation, supernatant is the betamethasone polyclonal antibody behind the described mark.
A kind of residual fast detecting colloid gold test paper of betamethasone that is used for comprises test strips, sponge bracket and support, wherein is adsorbed with the betamethasone polyclonal antibody of envelope antigen and colloid gold label on the test strips.
Further, described test strips comprises special-purpose plastic plate, and the upper surface of plastic plate is pasted thieving paper, cellulose acetate membrane, collaurum absorption glass fibre membrane, sample pad glass fibre membrane from top to bottom successively.
Further, bag is arranged by the coupled complex and the goat anti-rabbit antibody of betamethasone and ovalbumin (OVA) on the described cellulose acetate film (NC); Be adsorbed with the anti-betamethasone polyclonal antibody of rabbit of colloid gold label on the collaurum absorption glass fibre membrane.
Further, include the concentrating sample extract in the described support, this concentrating sample extract is by Na
2HPO
42H
2O, NaH
2PO
42H
2O, Tween-20, methyl alcohol, distilled water are formulated.
The present invention has overcome betamethasone physico-chemical analysis method complicated operation, has detected the cost height, speed is slow and shortcoming such as secondary environmental pollution, it is residual accurately to detect in water, meat products and the dairy produce betamethasone delicately, the pre-treatment process of sample is simple, consuming time few, can a large amount of sample of while fast detecting.
Description of drawings
Fig. 1 is the test strips front view;
Fig. 2 is the test strips vertical view;
Fig. 3 is the uv-vis spectra of colloidal gold solution;
Fig. 4 is the collaurum transmission electron microscope picture;
Fig. 5 is collaurum-betamethasone antibody conjugates transmission electron microscope picture;
Fig. 6 is collaurum-betamethasone antibody conjugates uv-vis spectra;
Fig. 7 is a test strips interpretation synoptic diagram;
Fig. 8 is the mensuration (250～0ng/mL) of sensitivity;
Fig. 9 is the mensuration of cross reaction;
Figure 10 is the test strips stability test;
Figure 11 is a sample detection.
Embodiment
(1) use of test strips: it is standby that sample extracting solution in the box is diluted to 5ml.Get working sample 1.0g, add 2ml extract milling and extracting, drop on the sample pad after getting supernatant 50 μ l and sample-loading buffer mixing, room temperature is carried out interpretation after placing 10min.
(2) as negative, betamethasone content＜8ng/mL in the sample then, otherwise＞8ng/mL
1, betamethasone and carrier protein couplet:
Adopt mixed anhydride method, at D ring side chain, on the C21 position of molecule-OH, succinylation, activation succinic acid terminal carboxyl group, with in BSA or the OVA molecule-NH2 links to each other, synthesizes betamethasone immunogene and coating antigen.Preparing former concentration is 20% ethanolic solution of 50 μ g/mL, by UV scanning maximum absorption wavelength as can be known.Preparing the aqueous solution of 200 μ g/mL bovine serum albumins, coupled product is diluted to about 200 μ g/mL with deionized water, survey light absorption value at the 240nm place, is blank with the deionized water, measures light absorption value.
With Bet, Bet-BSA, the mass concentration of BSA is converted to volumetric molar concentration according to its relative molecular mass, calculates separately molar extinction coefficient according to formula (1) then:
Molar extinction coefficient
Wherein, A is an absorbance, and C is a volumetric molar concentration.
Again according to formula (2) estimation crosslinking rate:
M
DEX∶M
BAS＝(ε
DEX-BSA-ε
BAS)/ε
DEX (2)
2, betamethasone polyclonal antibody preparation:
With betamethasone-BSA conjugate as immunogene, by the dosage inoculation of body weight 500 μ g/kg 4 times, for the first time with the immunogene that contains the 1.5ml Freund's complete adjuvant, intracutaneous or subcutaneous multi-point injection, after this 4 weeks of every interval are with the antigen booster immunization that contains the 1.5ml incomplete Freund 1 time, antiserum titre is measured in blood sampling before each the reinforcement.Last 1 immunity back 10d slaughters, the blood sampling separation of serum, measure antibody titer, the selection height of tiring, the serum of high specificity is slightly carried rabbit anti-serum with the saturated ammonium sulfate method earlier, is further purified with albumin A-agarose affinity chromatography method again, obtain purer betamethasone polyclonal antibody, it is standby to add equivalent glycerine-20 ℃ refrigeration.
3, Preparation of Colloidal Gold
The sodium citrate improved method prepares collaurum:
Adopt the sodium citrate reducing process to prepare the collaurum of the about 25nm of diameter.With new system deionized water preparation 100ml 0.01% gold chloride, place in the conical flask, electric furnace is heated to and boils.Accurately draw 1% trisodium citrate of 1.5ml, add rapidly in the conical flask, rock evenly after, put into electric furnace immediately and continue to boil about 7min, observe and add behind the liquid color and become blackly earlier, slowly become claret subsequently.Continue to stop heating behind the heating 3-5min after seeing claret.Return to original volume with deionized water after naturally cooling to room temperature, what obtain is the collaurum that diameter is 25nm, 4 ℃ of preservations of sterile sealing.Whether the colloid gold particle size of observing preparation under transmission electron microscope uniformity, has or not ellipse, triangle and polygon, and according to Electronic Speculum tape measure colloid gold particle.Wherein Fig. 3 has shown the uv-vis spectra of colloidal gold solution, and Fig. 4 has shown the collaurum transmission electron microscope picture.
4, the colloid gold label of betamethasone antibody
The collaurum 4ml that is adjusted to optimal pH 8.5 places the little burning of 10ml, adds the anti-betamethasone antibody 50 μ l of rabbit of 0.2mg/ml, lucifuge gentle agitation reaction 1h on the magnetic stirring apparatus, add 10%OVA 400 μ l, behind the sealing 30min, 4 ℃ * 1500rpm * 15min is centrifugal, removes free gold; The centrifugal precipitation of staying of 4 ℃ * 12000rpm * 20min is removed supernatant, and is resuspended with the resuspended liquid of 4ml collaurum, room temperature reaction 10min; The centrifugal again precipitation of staying of 4 ℃ * 12000rpm * 20min is removed supernatant, and is resuspended with the resuspended liquid of 1ml collaurum; Centrifugal in 4 ℃ * 1000rpm * 10min again, go precipitation, supernatant is colloidal gold probe, and 4 ℃ keep in Dark Place.Wherein Fig. 5 has shown collaurum-betamethasone antibody conjugates transmission electron microscope picture, and Fig. 6 has shown collaurum-betamethasone antibody conjugates uv-vis spectra.
5, colloidal gold strip assembling
(1) with an amount of envelope antigen and goat anti-rabbit antibody bag by in the NC film (on the 4mm * 3cm).
(2) an amount of gold mark betamethasone antibody is fixed in glass fibre (on the 4mm * 1cm).
(3) get the PVC plate of making the test strips special use, tear the paper slip of pasting the nitrocellulose filter position earlier, the nitrocellulose filter that cuts respective length sticks on the PVC plate, the glass fibre of getting the good golden labeling antibody of absorption then is affixed on corresponding site, push down nitrocellulose filter, glue absorbent material again and do not have the all-glass paper of ADSORPTION OF GOLD labeling antibody.
Its structure as shown in Figure 1 and Figure 2.
6, test strips detection sensitivity
With betamethasone concentration is the standard solution of 1 μ g/ml, with 10 series concentration of 0.01mol/L PBS dilution, after sample preparation liquid equal-volume mixes, final concentration is 250ng/ml, 125ng/ml, 62.5ng/ml, 31.3ng/ml, 15.6ng/ml, 7.8ng/ml, 3.9ng/ml, 1.9ng/ml, 0.95ng/ml, 0.48ng/ml, be mixed into negative control with PBS and sample preparation liquid equal-volume, behind the point sample, determine the toe-in fruit in the 10min.Detect and judgement; As Fig. 7, shown in Figure 11, betamethasone sample extracting solution to be measured is dripped on sample pad, after room temperature is placed 10min, observe.As T line colour developing then negative (being that betamethasone content is less than controlled quentity controlled variable in the sample), otherwise positive, promptly the T line does not develop the color (betamethasone content is higher than controlled quentity controlled variable in the sample).
The result as shown in Table 1, during the betamethasone titer of test strips detection level more than 8.0ng/mL, the result is positive, does not see sample test strips T line, negative visible obviously T line.Therefore, the detection sensitivity of test strips is defined as 8.0ng/mL.As shown in Figure 8.
The mensuration (250～0ng/mL) of table 1 sensitivity
Annotate: "+" "--" represented positive and negative respectively
7, specificity test (or the test of cross reaction thing)
Betamethasone, dexamethasone, diflucortolone and kanamycins standard solution are diluted to 15.6ng/ml, carry out the cross reaction test.Press the test paper detecting method experiment.The results are shown in Table 2, Fig. 9.
Table 2 test strips cross reaction test
Annotate: "+" "--" represented positive and negative respectively.Respectively 15.6ng/ml betamethasone, dexamethasone, diflucortolone and kanamycins standard solution are tested with test strips.It is all negative that the result shows that betamethasone analog, kanamycins detect, and test strips is positive to the betamethasone testing result, illustrates that this test strips has higher specificity, only is applicable to that betamethasone detects in the sample.
8, test strips stability test
Colloidal gold immuno-chromatography test paper strip is deposited in 37 ℃, and at 3d, 4d, 5d, 6d, 7d, take out with betamethasone standard solution (8ng/ml) and detect, every processing repeats 3 times, and contrast as sample blank with same PBS solution, observe stability test result (the having or not of detection line and nature controlling line, the sharpness of band and the degree that glass fibre discharges golden labelled antibody).
The betamethasone colloidal gold colloidal gold detection test paper strip that is taken at 37 ℃ of placements detects, the testing result of 7d as shown in figure 10, as can be seen from the figure the betamethasone test strips is stable fine, still can specificly detect betamethasone behind 37 ℃ of placement 7d, and susceptibility does not descend.
Advantage of the present invention is accurately to detect delicately betamethasone in water, meat products and the dairy produce Residual, the determination process is simple, and is consuming time few, and energy is a large amount of sample of fast detecting simultaneously, The sample detection cost is far below traditional instrument detection method. The storage life of reagent strip at least 6 months. In the testing process, the specificity of the betamethasone in the sample and gold mark betamethasone antibody (quantitatively) is anti-Should, by remaining gold mark betamethasone antibody pre-coated betamethasone on the test strips cellulose acetate film The specificity of envelope antigen (detection line, T line), and association reaction determines betamethasone content in the sample, Its colour developing is negative, namely is no more than controlled quentity controlled variable; Otherwise then surpass controlled quentity controlled variable. The test strips acetate fiber Pre-coated goat anti-rabbit antibody is as quality control line (C line) on the film, and its colour developing is effective for test strips, Otherwise lost efficacy.
Claims (9)
1, a kind of preparation method who is used for the residual fast detecting colloid gold test paper of betamethasone is specially:
1) prepares artificial immunogen after utilizing active ester method with betamethasone and bovine serum albumin(BSA) (BSA) coupling, go out the betamethasone polyclonal antibody with this immunogen preparing;
2) with mixed anhydride method with betamethasone and ovalbumin (OVA) coupling, its compound as envelope antigen, and is fixed in cellulose acetate film (NC) as detection line (T line) with this envelope antigen;
3) the betamethasone polyclonal antibody is fixed in step 2) on the cellulose acetate film (NC) after handling, as control line line (C line);
4) prepare collaurum with the sodium citrate improved method, use its markers step 1 simultaneously) the middle betamethasone polyclonal antibody for preparing, and be fixed on the collaurum pad;
5) the collaurum pad is fitted on the cellulose acetate film of handling well in the step 3) (NC), and glues absorbent material and all-glass paper, promptly make colloid gold test paper.
2, the preparation method who is used for the residual fast detecting colloid gold test paper of betamethasone as claimed in claim 1 is characterized in that, described betamethasone polyclonal antibody is made by the new zealand white rabbit of immunogen immune described in the step 1).
3, the preparation method who is used for the residual fast detecting colloid gold test paper of betamethasone as claimed in claim 1, it is characterized in that, prepare the method for betamethasone polyclonal antibody in the described step 1), be specially: with betamethasone-BSA conjugate as immunogene, by setting requirement White Rabbit is carried out repeatedly inoculation, after finishing, the blood sampling separation of serum, earlier slightly carry rabbit anti-serum with the saturated ammonium sulfate method, be further purified with albumin A-agarose affinity chromatography method again, obtain purer betamethasone polyclonal antibody.
4, the preparation method who is used for the residual fast detecting colloid gold test paper of betamethasone as claimed in claim 1, it is characterized in that, the sodium citrate improved method prepares collaurum in the described step 4), be specially: adopt the sodium citrate reducing process to prepare collaurum, prepare the gold chloride of 0.01% concentration with the new system deionized water, place in the conical flask, be heated to and boil, accurately draw the trisodium citrate of 1% concentration, add rapidly in the conical flask, after rocking evenly, continue to boil, after its color becomes claret, continue the heating setpoint time, stop heating, return to original volume with deionized water after naturally cooling to room temperature, promptly obtain collaurum.
5, the preparation method who is used for the residual fast detecting colloid gold test paper of betamethasone as claimed in claim 4, it is characterized in that, the colloid gold label of betamethasone polyclonal antibody in the described step 4), be specially: be adjusted to the collaurum of optimal pH 8.5, add the anti-betamethasone antibody of rabbit of 0.2mg/ml concentration, lucifuge gentle agitation reaction 1h on magnetic stirring apparatus, the OVA that adds 10% concentration, behind the sealing 30min, 4 ℃ * 1500rpm * 15min is centrifugal, removes the collaurum that dissociates; The centrifugal precipitation of staying of 4 ℃ * 12000rpm * 20min is removed supernatant, and is resuspended with the resuspended liquid of collaurum, room temperature reaction 10min; The centrifugal again precipitation of staying of 4 ℃ * 12000rpm * 20min is removed supernatant, and is resuspended with the resuspended liquid of collaurum; Centrifugal in 4 ℃ * 1000rpm * 10min again, go precipitation, supernatant is the betamethasone polyclonal antibody behind the described mark.
6, a kind of residual fast detecting colloid gold test paper of betamethasone that is used for is characterized in that this test paper comprises test strips, sponge bracket and support, wherein is adsorbed with the betamethasone polyclonal antibody of envelope antigen and colloid gold label on the test strips.
7, the residual fast detecting colloid gold test paper of betamethasone that is used for as claimed in claim 6, it is characterized in that, described test strips comprises special-purpose plastic plate, the upper surface of plastic plate is pasted thieving paper, cellulose acetate membrane, collaurum absorption glass fibre membrane, sample pad glass fibre membrane from top to bottom successively.
8, the residual fast detecting colloid gold test paper of betamethasone that is used for as claimed in claim 7 is characterized in that, bag is arranged by the coupled complex and the goat anti-rabbit antibody of betamethasone and ovalbumin (OVA) on the described cellulose acetate film (NC); Be adsorbed with the anti-betamethasone polyclonal antibody of rabbit of colloid gold label on the collaurum absorption glass fibre membrane.
9, the residual fast detecting colloid gold test paper of betamethasone that is used for as claimed in claim 6 is characterized in that include the concentrating sample extract in the described support, this concentrating sample extract is by Na
2HPO
42H
2O, NaH
2PO
42H
2O, Tween-20, methyl alcohol, distilled water are formulated.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103940999A (en) * | 2013-01-19 | 2014-07-23 | 北京勤邦生物技术有限公司 | A dipstick used for testing betamethasone and application thereof |
| CN106443019A (en) * | 2016-08-31 | 2017-02-22 | 广东省药品检验所 | Method for rapidly detecting multiple glucocorticoids in cosmetics |
-
2009
- 2009-07-20 CN CNA2009101580944A patent/CN101609094A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103940999A (en) * | 2013-01-19 | 2014-07-23 | 北京勤邦生物技术有限公司 | A dipstick used for testing betamethasone and application thereof |
| CN103940999B (en) * | 2013-01-19 | 2016-06-01 | 北京勤邦生物技术有限公司 | A kind of test strip detecting Betamethasone Valerate and its preparation method and application |
| CN106443019A (en) * | 2016-08-31 | 2017-02-22 | 广东省药品检验所 | Method for rapidly detecting multiple glucocorticoids in cosmetics |
| CN106443019B (en) * | 2016-08-31 | 2018-09-25 | 广东省药品检验所 | A method of for quickly detecting a variety of glucocorticoids in cosmetics |
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Application publication date: 20091223 |