CN1818655A - Verifying test paper of chloromycetin gel by gold method and production thereof - Google Patents
Verifying test paper of chloromycetin gel by gold method and production thereof Download PDFInfo
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- CN1818655A CN1818655A CN 200610010726 CN200610010726A CN1818655A CN 1818655 A CN1818655 A CN 1818655A CN 200610010726 CN200610010726 CN 200610010726 CN 200610010726 A CN200610010726 A CN 200610010726A CN 1818655 A CN1818655 A CN 1818655A
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- chloromycetin
- monoclonal antibody
- chloramphenicol resistance
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Abstract
A method for preparing test paper of chloromycetin colloidal gold detection technique includes anchoring chloromycetin single resistance being labeled with colloidal gold and chloromycetin coupled antigen as well as resisting mouse IgG antibody on nitrocellulose film and on base plate, using chloromycetin coupled antigen as detectionbelt and using resisting mouse IgG antibody as reference belt .The prepared test paper can detect out residual amount of chloromycetin 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 detection test substance.
Background technology
Chloromycetin is that a class has the antimicrobial agents that suppresses the synthetic broad spectrum antibiotic activity of bacterioprotein, owing to its good antibiotic and pharmacological kinetics characteristic, is widely used in the medicine for treatment of feed addictive and bacteriosis.Chloromycetin has bigger toxicity to marrow and neonate, easily causes the human body blood poisoning, causes irreversible alpastic anemia.The people is long-term to take in that residual chloromycetin can produce chronic or savings toxicity in animal derived food such as animal muscle, internal organ, milk etc., therefore, it directly or indirectly threatens consumer's health, be put into Ministry of Agriculture's " food animal forbidding veterinary drug and other compound inventory ", be defined in chloramphenicol residue in all food animals 10
-9Must not detect on the ppb order of magnitude.At present, the method of measuring chloromycetin mainly contains methods such as high performance liquid chromatography (HPLC) and enzyme linked immunological, the HPLC method requires to have special instrument and equipment and professional and technical personnel, and sample screening amount less, need the pre-treatment of sample, therefore it is long that it detects cost height, detection time, these effects limit the HPLC method to the carrying out of chloromycetin test item, can not satisfy the needs that present food security detects chloramphenicol residue fully.And the main dependence on import of ELISA reagent, it is higher to detect cost.
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
It is easy to the purpose of this invention is to provide a kind of using method, time saving and energy saving saving money, the colloidal gold method test paper and the method for preparing test paper of energy half-quantitative detection chloromycetin.
The present invention realizes above-mentioned purpose by following approach:
Chloromycetin detects test paper, comprises the nitrocellulose filter 6 that is fixed on the base plate 7, it is characterized in that gold mark chloramphenicol resistance monoclonal antibody 2, chloromycetin 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 chloramphenicol resistance 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 chloramphenicol resistance 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.
Chloromycetin detects the preparation method of test paper:
Comprise the nitrocellulose filter 6 that applies on base plate 7, it is characterized in that:
(1) chloromycetin and the animal high molecular weight protein clone that is combined into chloromycetin coupled antigen 3 immunized mice cells obtains the chloramphenicol resistance monoclonal antibody,
(2) colloid gold particle mark chloramphenicol resistance monoclonal antibody forms gold mark chloramphenicol resistance monoclonal antibody 2,
(3) the different animal of chloramphenicol resistance monoclonal antibody immunity kind obtains anti-mouse IgG antibody 4,
(4) with colloid gold label chloramphenicol resistance monoclonal antibody 2, chloromycetin 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 chloromycetin coupled antigen 3, serve as with reference to band with anti-mouse IgG antibody 4.
The chloramphenicol resistance monoclonal antibody is by chloromycetin 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 chloramphenicol resistance monoclonal antibody 2 parts that are fixed in nitrocellulose filter 6 one ends.
Described adsorptive pads 1 down, gold are marked chloramphenicol resistance 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 chloramphenicol resistance monoclonal antibody generation competition form of colloid gold label, its consequence is, when the chloramphenicol resistance monoclonal antibody of colloid gold label is excessive, unnecessary monoclonal antibody floats to and detects band, combine and colour developing with the chloromycetin coupled antigen, form the colour band that naked eyes can conveniently be observed; And the chloramphenicol resistance 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 chloramphenicol residue in half-quantitative detection serum or the urine sample with colorimetric up and down in the test paper, sensitivity can reach 1ppb (1ng/ml), in 5-20min, fast, detect to sxemiquantitative sample and whether contain chloromycetin, to determine whether chloromycetin exceeds standard, food security be can satisfy to the primary demand that chloramphenicol residue detects, feed, meat producing plant and testing agency of government are applicable to.
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 chloramphenicol resistance 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, chloromycetin detects test paper
Comprise that adsorptive pads 1, gold are marked chloramphenicol resistance monoclonal antibody 2, chloromycetin 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 chloromycetin 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, chloromycetin detects the preparation of test paper
Chloromycetin and bovine serum albumin molecule are combined into chloromycetin 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 chloramphenicol resistance monoclonal antibody; Form gold mark chloramphenicol resistance monoclonal antibody 2 with colloid gold particle mark chloramphenicol resistance monoclonal antibody; Chloramphenicol resistance monoclonal antibody immune sheep obtains sheep anti-mouse igg antibody 4.
With gold mark chloramphenicol resistance monoclonal antibody 2, chloromycetin 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 chloramphenicol resistance monoclonal antibody 2 one ends with following adsorptive pads 1, and marks chloramphenicol resistance 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 chloramphenicol resistance 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 chloramphenicol resistance 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 chloramphenicol Base based sols 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 Thiamphenicol 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 chloromycetin national standard product solution of 100ng/ml carries out parallel detection, each concentration is carried out 10 parallel laboratory tests, and 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 chloromycetin 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 chloromycetin 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 is positioned over 37 ℃ ± 0.5 ℃ thermostatic drying chamber with the finished product of Packing Sound and takes out detection 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 the band color, do not contain chloromycetin 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 chloromycetin 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 chloromycetin 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 chloromycetin 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 chloromycetin 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 chloramphenicol resistance monoclonal antibody (2), chloromycetin coupled antigen (3), anti-mouse IgG antibody (4), the compartment of terrain order forms composite bed with nitrocellulose filter (6).
2, chloromycetin 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 chloramphenicol resistance 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 chloromycetin colloidal gold methods, adsorptive pads (1), gold are marked chloramphenicol resistance monoclonal antibody (2) and gone up adsorptive pads (5) surface under it is characterized in that self-adhesive paper.
4, the chloromycetin 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) chloromycetin and animal high molecular weight protein are combined into chloromycetin coupled antigen (3), obtain the chloramphenicol resistance monoclonal antibody with the clone of antigen (3) immunized mice cell,
(2) respectively the chloramphenicol resistance monoclonal antibody is formed gold mark chloramphenicol resistance monoclonal antibody (2) and obtains anti-mouse IgG antibody (4) with the different animal of chloramphenicol resistance monoclonal antibody immunity kind with the colloid gold particle mark,
(3) gold is marked chloramphenicol resistance monoclonal antibody (2), chloromycetin 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 chloromycetin coupled antigen (3) with anti-mouse IgG antibody (4).
5, chloromycetin 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 chloramphenicol resistance 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 chloromycetin colloidal gold methods, it is characterized in that adsorptive pads (1), gold are marked chloramphenicol resistance 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 |
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| CN 200610010726 CN1818655A (en) | 2006-03-06 | 2006-03-06 | Verifying test paper of chloromycetin gel by gold method and production thereof |
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| CN 200610010726 CN1818655A (en) | 2006-03-06 | 2006-03-06 | Verifying test paper of chloromycetin gel by gold method and production thereof |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101839909A (en) * | 2010-05-07 | 2010-09-22 | 杭州南开日新生物技术有限公司 | Method for preparing chloramphenicol detecting reagent plate |
| CN101858907A (en) * | 2010-05-20 | 2010-10-13 | 杭州南开日新生物技术有限公司 | Preparation method of reagent plate for detecting chloramphenicol in cosmetics |
| CN101943701A (en) * | 2010-07-07 | 2011-01-12 | 南昌大学 | Preparation method of colloidal gold test strip for quickly detecting chloramphenicol residues |
| CN101118238B (en) * | 2007-08-29 | 2011-05-04 | 浙江大学 | Composite protecting agent and uses thereof |
| CN102288762A (en) * | 2011-06-29 | 2011-12-21 | 杭州南开日新生物技术有限公司 | Production method of reagent board for detecting chloramphenicol in aquatic product |
| CN102507940A (en) * | 2011-10-25 | 2012-06-20 | 广东省药品检验所 | Method for quickly detecting chloramphenicol in cosmetics |
| CN107110847A (en) * | 2014-05-28 | 2017-08-29 | 快速诊断国际公司 | For detecting or measuring the ethyl glucuronide aldehydic acid glycosides lateral flow calibration tape of ethyl glucuronide aldehydic acid glycosides, immunoassays, apparatus and method |
| CN109752535A (en) * | 2017-11-05 | 2019-05-14 | 镇江华维检测技术有限公司 | The immune colloid of chloramphenicol detects card in a kind of detection tissue |
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2006
- 2006-03-06 CN CN 200610010726 patent/CN1818655A/en active Pending
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101118238B (en) * | 2007-08-29 | 2011-05-04 | 浙江大学 | Composite protecting agent and uses thereof |
| CN101839909A (en) * | 2010-05-07 | 2010-09-22 | 杭州南开日新生物技术有限公司 | Method for preparing chloramphenicol detecting reagent plate |
| CN101858907A (en) * | 2010-05-20 | 2010-10-13 | 杭州南开日新生物技术有限公司 | Preparation method of reagent plate for detecting chloramphenicol in cosmetics |
| CN101943701A (en) * | 2010-07-07 | 2011-01-12 | 南昌大学 | Preparation method of colloidal gold test strip for quickly detecting chloramphenicol residues |
| CN101943701B (en) * | 2010-07-07 | 2014-01-22 | 南昌大学 | Preparation method of colloidal gold test strip for quickly detecting chloramphenicol residues |
| CN102288762A (en) * | 2011-06-29 | 2011-12-21 | 杭州南开日新生物技术有限公司 | Production method of reagent board for detecting chloramphenicol in aquatic product |
| CN102507940A (en) * | 2011-10-25 | 2012-06-20 | 广东省药品检验所 | Method for quickly detecting chloramphenicol in cosmetics |
| CN102507940B (en) * | 2011-10-25 | 2014-04-30 | 广东省药品检验所 | Method for quickly detecting chloramphenicol in cosmetics |
| CN107110847A (en) * | 2014-05-28 | 2017-08-29 | 快速诊断国际公司 | For detecting or measuring the ethyl glucuronide aldehydic acid glycosides lateral flow calibration tape of ethyl glucuronide aldehydic acid glycosides, immunoassays, apparatus and method |
| US10648974B2 (en) | 2014-05-28 | 2020-05-12 | Carehealth America Corporation | Ethyl glucuronide lateral flow test strips, immunoassays, devices and methods for detecting or measuring thereof |
| CN109752535A (en) * | 2017-11-05 | 2019-05-14 | 镇江华维检测技术有限公司 | The immune colloid of chloramphenicol detects card in a kind of detection tissue |
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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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