CN1818656A - Verifying test paper of clenbuterol hydrochloride gel by gold method and production thereof - Google Patents
Verifying test paper of clenbuterol hydrochloride gel by gold method and production thereof Download PDFInfo
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- CN1818656A CN1818656A CN 200610010727 CN200610010727A CN1818656A CN 1818656 A CN1818656 A CN 1818656A CN 200610010727 CN200610010727 CN 200610010727 CN 200610010727 A CN200610010727 A CN 200610010727A CN 1818656 A CN1818656 A CN 1818656A
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- clenbuterol
- monoclonal antibody
- nitrocellulose filter
- adsorptive pads
- gold
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Abstract
A method for preparing test paper of clenbuterol hydrochloride colloidal gold detection technique includes anchoring chenbuterol single resistance being labeled with colloidal gold and chenbuterol coupled antigen as well as resisting mouse IgG antibody on nitrocellulose film and on base plate, using chenbuterol antigen as detection belt and using resisting mouse IgG antibody as reference belt. The prepared test paper can detect out residual amount of chenbuterol in serum sample or in urine sample quickly and accurately.
Description
Affiliated technical field
The invention belongs to the mensuration test paper and the preparation method of biology immunization method.
Background technology
Clenobuterol hydrochloride (Clenbuterol Hydrochloride), claim clenbuterol hydrochloride again, it is the beta-stimulants similar drug that feed and breeding production ban use of, this medicine is residual savings in livestock products, cause human produce acute poisoning and other influences by food chain, completely forbidden growth accelerator as edible animal.According to Ministry of Agriculture's " veterinary drug and other compound inventory of food animal forbidding " regulation, 10
-9Must not detect on the ppb order of magnitude.What use was more in detecting at present is euzymelinked immunosorbent assay (ELISA) (ELISA) kit, it has the Ag-Ab atopy, can qualitative sample result, false negative and the least possible false positive promptly do not appear, this method detects as the screening of a large amount of samples usually, need instruments such as microplate reader, hydro-extractor and analytical balance, analysis and determination step are all more loaded down with trivial details.For qualitative or quantitative test detection several different methods and technology are arranged, as use the high performance liquid chromatography HPLC or the vapor-phase chromatography GC of two or more detection principles, or band hyphen (hyphenated) detection technique, be chromatogram and mass spectrometric hyphenated technique-GC-MS or LC-MSn, obtain testing compound structure essence mass spectrogram thus.But this method needs large-scale instrument, needs special place and professional, the analysis cost height, and pre-treatment is more time-consuming.
For easy clinical medicine antibody, antigen or haptens check, before this, the applicant has obtained " a kind of infertile detection test paper and detection method thereof " (00112780.2 Chinese invention patent communique), " the total ash area value of a kind of Human Whole Serum or serum detects and carcinoma of prostate detects test paper " multinomial patents for invention such as (01129174.5 Chinese invention patent communiques).
Summary of the invention
The purpose of this invention is to provide a kind of can half-quantitative detection Clenbuterol, the preparation method of colloidal gold method test paper and test paper fast and accurately.
The present invention realizes above-mentioned purpose by following approach:
Clenobuterol hydrochloride detects test paper, comprises the nitrocellulose filter 6 that is fixed on the base plate 7, it is characterized in that gold mark anti-clenbuterol monoclonal antibody 2, Clenbuterol coupled antigen 3, anti-mouse IgG antibody 4, and the compartment of terrain order forms composite bed with nitrocellulose filter 6 surfaces.
Described base plate 7 middle parts are nitrocellulose filter 6 fixedly, and nitrocellulose filter 6 one ends are connected with last adsorptive pads 5; And the other end or connect with following adsorptive pads 1, perhaps overlapping by gold mark anti-clenbuterol monoclonal antibody 2 parts that are fixed in nitrocellulose filter 6 one ends links to each other with following adsorptive pads 1 again.
Described adsorptive pads 1 down, gold mark anti-clenbuterol monoclonal antibody 2 reach upward, and there is self-adhesive paper on adsorptive pads 5 surfaces.
Above-mentioned film bar width is divided into three types of 2.5mm, 3.0mm, 4.0mm.
Clenobuterol hydrochloride detects the preparation method of test paper:
Comprise the nitrocellulose filter 6 that applies on base plate 7, it is characterized in that:
(1) Clenbuterol and the high molecular weight protein clone that is combined into Clenbuterol coupled antigen 3 immunized mice cells obtains the anti-clenbuterol monoclonal antibody,
(2) colloid gold particle mark anti-clenbuterol monoclonal antibody forms gold mark anti-clenbuterol monoclonal antibody 2,
(3) the different animal of anti-clenbuterol monoclonal antibody immunity kind obtains anti-mouse IgG antibody 4,
(4) with colloid gold label anti-clenbuterol monoclonal antibody 2, Clenbuterol coupled antigen 3, anti-mouse IgG antibody 4 compartment of terrains order attached on the nitrocellulose filter 6,
Wherein, serve as to detect band with Clenbuterol coupled antigen 3, serve as with reference to band with anti-mouse IgG antibody 4.
The anti-clenbuterol monoclonal antibody is by Clenbuterol coupled antigen immunized mice cell, and with the preparation of mouse ascites fluid form, antibody titer is not less than 15000, or potent antibodies content is greater than 0.5mg/ml.
Described base plate 7 middle parts are nitrocellulose filter 6 fixedly, and following adsorptive pads 1 and last adsorptive pads 5 are affixed on base plate 7 two ends respectively and connect with nitrocellulose filter 6; Following adsorptive pads 1 or overlap with gold mark anti-clenbuterol monoclonal antibody 2 parts that are fixed in nitrocellulose filter 6 one ends.
Described adsorptive pads 1 down, gold are marked anti-clenbuterol monoclonal antibody 2 and are gone up adsorptive pads 5 surfaces and post self-adhesive paper.
It is the capillary siphoning effect of utilizing adsorptive pads to form that the present invention detects principle, make detected antigen at first with the combining of the anti-clenbuterol monoclonal antibody generation competition form of colloid gold label, its consequence is, when the anti-clenbuterol monoclonal antibody of colloid gold label is excessive, unnecessary monoclonal antibody floats to and detects band, combines with the Clenbuterol coupled antigen and colour developing; And the anti-clenbuterol monoclonal antibody of the colloid gold label that combines with antigen, its V district detected antigen of binding site occupies, can only cross over the detection band and float to, detect band together and carry out colorimetric and obtain testing result with reference to being with C position point and anti-mouse IgG antibody non-specific binding.
The present invention uses one step of chromatography type competition law principle, come residual of kelengtelu amount in half-quantitative detection serum or the urine sample with colorimetric up and down in the test paper, with the precision of 1ppb (1ng/ml), in 5-10min, detect sample rapidly and accurately and whether contain clenobuterol hydrochloride, to determine whether clenobuterol hydrochloride exceeds standard, can satisfy food security the residual of kelengtelu amount is detected demand, be applicable to feed, meat producing plant and testing agency of government.
The present invention compared with prior art, its effect is self-evident, promptly have easy to use, economical quick, make characteristics easy, with low cost.
Description of drawings
Fig. 1 is a vertical view of the present invention.
Fig. 2 is that adsorptive pads 1 is marked the side view that anti-clenbuterol monoclonal antibody 2 parts overlap with gold under the present invention.
Fig. 3 is the side view that adsorptive pads 1 is connected with nitrocellulose filter 6 under the present invention.
Embodiment
Be described further below in conjunction with accompanying drawing.
1, clenobuterol hydrochloride detects test paper
Comprise that adsorptive pads 1, gold are marked anti-clenbuterol monoclonal antibody 2, Clenbuterol coupled antigen 3, anti-mouse IgG antibody 4, gone up adsorptive pads 5, nitrocellulose filter 6, base plate 7 down.
Wherein,, be with as reference as detecting band with Clenbuterol coupled antigen 3 with anti-mouse IgG antibody 4.
Film bar width is not less than 2.5mm, and flat appearance, the material adhesion-tight, and the liquid speed of dividing a word with a hyphen at the end of a line is not less than 5mm/min.
2, clenobuterol hydrochloride detects the preparation of test paper
Clenbuterol and bovine serum albumin molecule are combined into Clenbuterol coupled antigen 3, and the clone who uses antigen 3 immunized mice cells is with the preparation of mouse ascites fluid form, and antibody titer is 15000, thereby obtains the anti-clenbuterol monoclonal antibody; Form gold mark anti-clenbuterol monoclonal antibody 2 with colloid gold particle mark anti-clenbuterol monoclonal antibody; Anti-clenbuterol monoclonal antibody immune sheep obtains sheep anti-mouse igg antibody 4.
With gold mark anti-clenbuterol monoclonal antibody 2, Clenbuterol coupled antigen 3, sheep anti-mouse igg antibody 4 at interval 3-5mm spread in proper order base plate 7 middle parts nitrocellulose filter 6 on, form composite beds with nitrocellulose filter 6 surfaces.
Fig. 2 is pasted on base plate 7 by gold mark anti-clenbuterol monoclonal antibody 2 one ends with following adsorptive pads 1, and marks anti-clenbuterol monoclonal antibody 2 parts overlapping with the gold on nitrocellulose filter 6 surfaces; Last adsorptive pads 5 is pasted on base plate 7 and is with 4 one ends by reference, and overlaps with nitrocellulose filter 6 one ends.
Fig. 3 is pasted on base plate 7 by gold mark anti-clenbuterol monoclonal antibody 2 one ends with following adsorptive pads 1, and overlaps with nitrocellulose filter 6 parts; Last adsorptive pads 5 is pasted same Fig. 2.
Following adsorptive pads 1, gold mark anti-clenbuterol monoclonal antibody 2 and on adsorptive pads 5 surfaces self-adhesive paper is arranged.
3, test paper requirement
(1) negative reference material coincidence rate
With phosphate buffer (PBS:0.01mol/L, Ph7.4) compound concentration is that the adrenaline national standard product solution of 100ng/ml carries out 10 parallel detections, with normal or correct the visual observation result, the reaction time is observations when 5min, should be negative.
With phosphate buffer (PBS:0.01mol/L, Ph7.4) compound concentration is that the Terbutaline national standard product solution of 100ng/ml carries out 10 parallel detections, with normal or correct the visual observation result, the reaction time is observations when 5min, should be negative.
With phosphate buffer (PBS:0.01mol/L, Ph7.4) compound concentration is that the salbutamol national standard product solution of 100ng/ml carries out 10 parallel detections, with normal or correct the visual observation result, the reaction time is observations when 5min, should be negative.
(2) positive reference material coincidence rate
With phosphate buffer (PBS:0.01mol/L, Ph7.4) compound concentration is 10,50, the Clenbuterol national standard product of 100ng/ml carry out parallel detection, each concentration is carried out 10 parallel laboratory tests, the reaction time is observations when 5min, feminine gender must not occur.
(3) limit of identification
With phosphate buffer (PBS:0.01mol/L; Ph7.4) respectively compound concentration be 0,0.5,1,2,5, the Clenbuterol national standard product solution of 10ng/ml; each concentration is carried out 10 parallel laboratory tests; reaction time is when 5min; with normal or rectification visual observation result, limit of identification is not higher than 1ng/ml.
(4) repeatability
With phosphate buffer (PBS:0.01mol/L, Ph7.4) compound concentration is the Clenbuterol national standard product solution of 10ng/ml respectively, carries out 10 parallel laboratory tests; reaction time is when 5min; with normal or correct the visual observation result, unanimity as a result, colour developing degree homogeneous.
(5) stability test 37 placed after 10 days, and every index should meet above requirement.
4, test paper using method
(1) specimen preparation
Serum: extract animal blood to be checked, centrifugal or leave standstill after get transparent supernatant and use; If serum has excessive haemolysis, 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, as muddiness, the dilution in 1: 1 of first centrifuging and taking supernatant or distilled water.
Feed: get the feed adding 10ml dilution to be measured that 1g one grinds, therebetween every firmly concussion of 2min; During 10min, leave standstill 1min after shaking all and get supernatant 100 μ l, analyze with test paper after doing 10 times of dilutions with dilution.
(2) operation
Test paper arrow end is immersed in the sample to be tested, does not surpass adsorptive pads 1 down.Immerse the 30sec taking-up and set level, 10min reads the result.
(3) testing result
Negative:
Two bands all develop the color, and detect and be with color to be deeper than with reference to band color, not hydrochloric Clenbuterol in the interpret sample.
Two bands all develop the color, and detect the band color with identical or approaching with reference to the band color, and the clenobuterol hydrochloride content of interpret sample is lower than 1ppb (1ng/ml).
Positive:
Two bands all develop the color, and are shallower than with reference to the band color but detect the band color, illustrate that clenobuterol hydrochloride content surpasses 1ppb (1ng/ml) in the test sample.
Detect band and do not develop the color, with reference to the band colour developing, the clenobuterol hydrochloride content in the interpret sample is higher than 5ppb (5ng/ml).
Above result is as shown in the table:
| The detection band＞ | Detect band ≈ | The detection band＜ | Detection is with 0 | |
| With reference to the band colour developing | Do not contain | 1＜ | ＞1 | ＞5 |
Last table is the unit of detection limit with ppb or ng/ml all.Detect band＞, ≈,＜, the 0th, and relatively with reference to the band color.
Claims (6)
1, the clenobuterol hydrochloride colloidal gold method detects test paper, comprise the nitrocellulose filter (6) that is fixed in base plate (7), it is characterized in that gold mark anti-clenbuterol monoclonal antibody (2), clenobuterol hydrochloride coupled antigen (3), anti-mouse IgG antibody (4), the compartment of terrain order forms composite bed with nitrocellulose filter (6).
2, clenobuterol hydrochloride colloidal gold method according to claim 1 detects test paper, it is characterized in that fixedly nitrocellulose filter (6) of base plate (7) middle part, and nitrocellulose filter (6) one ends are connected with last adsorptive pads (5); And the other end or connect with following adsorptive pads (1) perhaps with by the gold mark anti-clenbuterol monoclonal antibody (2) that is fixed in nitrocellulose filter (6) one ends links to each other with following adsorptive pads (1).
3, detect the preparation method of test paper according to claim 1,2 described clenobuterol hydrochloride colloidal gold methods, adsorptive pads (1), gold are marked anti-clenbuterol monoclonal antibody (2) and gone up adsorptive pads (5) surface under it is characterized in that self-adhesive paper.
4, the clenobuterol hydrochloride colloidal gold method detects the preparation method of test paper, comprises the nitrocellulose filter (6) that is connected with base plate (7), it is characterized in that
(1) Clenbuterol and animal high molecular weight protein are combined into Clenbuterol coupled antigen (3), obtain the anti-clenbuterol monoclonal antibody with the clone of antigen (3) immunized mice cell,
(2) respectively the anti-clenbuterol monoclonal antibody is formed gold mark anti-clenbuterol monoclonal antibody (2) and obtains anti-mouse IgG antibody (4) with the different animal of anti-clenbuterol monoclonal antibody immunity kind with the colloid gold particle mark,
(3) gold is marked anti-clenbuterol monoclonal antibody (2), Clenbuterol coupled antigen (3), anti-mouse IgG antibody (4) compartment of terrain and is attached in proper order on the nitrocellulose filter (6),
Wherein,, be with as reference as detecting band with Clenbuterol coupled antigen (3) with anti-mouse IgG antibody (4).
5, clenobuterol hydrochloride colloidal gold method according to claim 4 detects the preparation method of test paper, it is characterized in that fixedly nitrocellulose filter (6) of base plate (7) middle part, last adsorptive pads (5) and following adsorptive pads (1) are affixed on base plate (7) respectively and upward connect with nitrocellulose filter (6); Following adsorptive pads (1) or overlap with gold mark anti-clenbuterol monoclonal antibody (2) part that is fixed in nitrocellulose filter (6) one ends.
6, detect the preparation method of test paper according to claim 4,5 described clenobuterol hydrochloride colloidal gold methods, it is characterized in that adsorptive pads (1), gold are marked anti-clenbuterol monoclonal antibody (2) and gone up adsorptive pads (5) surface and post self-adhesive paper down.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200610010727 CN1818656A (en) | 2006-03-06 | 2006-03-06 | Verifying test paper of clenbuterol hydrochloride gel by gold method and production thereof |
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| CN 200610010727 CN1818656A (en) | 2006-03-06 | 2006-03-06 | Verifying test paper of clenbuterol hydrochloride gel by gold method and production thereof |
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| CN1818656A true CN1818656A (en) | 2006-08-16 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102590518A (en) * | 2012-02-08 | 2012-07-18 | 上海交通大学 | Immune colloidal gold test paper and method for quantitatively detecting configurable logic block (CLB) by matching with photoelectric sensor thereof |
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2006
- 2006-03-06 CN CN 200610010727 patent/CN1818656A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102590518A (en) * | 2012-02-08 | 2012-07-18 | 上海交通大学 | Immune colloidal gold test paper and method for quantitatively detecting configurable logic block (CLB) by matching with photoelectric sensor thereof |
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Effective date of registration: 20070216 Address after: 650091 Yunnan province Kunming City Lake Road No. 2 Applicant after: Yunnan University Co-applicant after: Yunnan Agricultural University Co-applicant after: Yunda Biological Technology Co., Ltd., Kunming Address before: 650091 Yunnan province Kunming City Lake Road No. 2 Applicant before: Yunnan University Co-applicant before: Yunda Biological Technology Co., Ltd., Kunming |
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