CN202614766U - Immunological detection kit for aflatoxin B1 - Google Patents
Immunological detection kit for aflatoxin B1 Download PDFInfo
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- CN202614766U CN202614766U CN 201220252484 CN201220252484U CN202614766U CN 202614766 U CN202614766 U CN 202614766U CN 201220252484 CN201220252484 CN 201220252484 CN 201220252484 U CN201220252484 U CN 201220252484U CN 202614766 U CN202614766 U CN 202614766U
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Abstract
The utility model relates to an immunological detection kit for aflatoxin B1. According to the key point of the technical scheme, the immunological detection kit for the aflatoxin B1 comprises a box body, and is characterized in that an elisa plate, a cover plate film, 14 reagent bottles and a die with downwards sunken bottle positions are arranged in the box body; the elisa plate consists of an outer frame supporting rack and detachable elisa reaction micro hole strips arranged on the outer frame supporting rack; according to the elisa plate, a 96-hole reagent plate is used as a solid phase carrier; an aflatoxin B1 coupling antigen wraps the micro hole strips, and the micro hole strips are fixed on the outer frame supporting rack of the elisa plate; each detachable elisa reaction micro hole strip is provided with 8 reaction holes; 14 downwards sunken bottle positions are formed on the die; and the 14 reagent bottles are sequentially put into the corresponding downwards sunken bottle positions. The immunological detection kit can quantitatively and qualitatively detect residues of the aflatoxin B1 in a food quickly, easily, sensitively and accurately.
Description
Technical field
The utility model relates to the kit of toxin residue detection in animal food safety and the animal husbandry production, particularly a kind of immunology detection kit that is used for detecting animal food and Feed Manufacturing AFB1.
Background technology
Aflatoxin is the toxic metabolite product of one type of fungi (like aspergillus flavus and aspergillus parasiticus), and they have very strong carcinogenicity, mainly is present in the relevant product of cereal, nut, cottonseed and some and human blood, animal feed.Kind surplus aflatoxin (AFT) has found 20 at present, wherein the toxicity of AFB1 (AFB1), carcinogenicity, pollution frequency all occupy the first.Aflatoxin M 1 is the hydroxylation metabolism product of AFB1, also is a kind of strong carcinogen.Take in the feed of AFB1 pollution when mammal after, can aflatoxin M 1 be secreted in the middle of milk through hydroxylation in vivo, just contain aflatoxin M 1 in the milk of milk cow output behind the feed of taking in the AFB1 pollution.Because aflatoxin M 1 quite stable, pasteurization also can't be eliminated it, not only will detect the animal feed raw material so detect aflatoxin M 1, and will detect final products.For this reason countries in the world stipulated all that the maximum of aflatoxin M 1 in cow's milk and the goods thereof allows content and with it as compulsory standard, be 0.5ng/ml like the highest permission content of aflatoxin M 1 in Chinese Government's regulation cow's milk and the goods (sterilized milk, fresh raw milk, full-fat cow milk, milk wet goods) thereof.
Aflatoxin residue detection commonly used in the prior art mainly contains TLC (TLC), high performance liquid chromatography (HPLC) and immune analysis method etc.Though adopting the physico-chemical analysis method is a kind of comparatively ideal medicament residue analytical approach; But do not have ultra-violet light-emitting group and fluorophore in the AFB1 molecule, in chemical analysis, need change it into ultra-violet light-emitting group or fluorophore derivant and just can analyze, this has brought very big difficulty to testing; And has a sample pre-treatments complex steps; Time and effort consuming, instrument costs an arm and a leg, and needs shortcomings such as the professional operates.The immune analysis method has high specificity, sample pre-treatments is simple, cost is low, can carry out advantages such as batch detection.It is the immunological analysis method of using always that ELISA detects, but domestic to be used for the research that medicament residue detects less, and the ELISA kit that is used to detect AFB1 is a blank especially.
Summary of the invention
The purpose of the utility model has provided a kind of immunology detection kit of AFB1 of small volume and less weight; Use this kit easy and simple to handle, detect fast, accurate, highly sensitive, cost is low; Good stability can carry out the detection of AFB1 in a plurality of samples of one-time continuous.
The technical scheme of the utility model is: a kind of immunology detection kit of AFB1; Comprise box body; It is characterized in that: include ELISA Plate, cover plate film, 14 bottles of reagent in the box body and have the mould of recessed bottle position; Described ELISA Plate is made up of outer frame support and the dismantled and assembled enzyme mark reaction capillary strip of placing on it; ELISA Plate adopts 96 hole agent plate as solid phase carrier, is fixed on the outer frame support of ELISA Plate after encapsulating the AFB1 coupled antigen on the capillary strip in advance, and each dismantled and assembled enzyme mark reaction capillary strip has 8 reacting holes; Described cover plate film size is consistent with ELISA Plate; Recessed bottle position on the described mould is respectively titer bottle position, high concentration titer bottle position, ELIAS secondary antibody working fluid bottle position, aflatoxin b1 antibody bottle position, colour developing liquid A liquid bottle position, colour developing liquid B liquid bottle position, stop buffer bottle position, concentrated cleaning solution bottle position, concentrating sample dilution bottle position.Described box body is a carton box; Described ELISA Plate is processed by polystyrene, is put in the vacuum aluminide-coating bag; Described cover plate film is the transparent plastic dura mater, is put in the aluminide-coating bag; Described 14 bottles of reagent are respectively 6 bottles of titers, 1 bottle of high concentration titer, 1 bottle of ELIAS secondary antibody working fluid, 1 bottle of aflatoxin b1 antibody, 1 bottle of colour developing liquid A liquid, 1 bottle of colour developing liquid B liquid, 1 bottle of stop buffer, 1 bottle of concentrated cleaning solution, 1 bottle of concentrating sample dilution; Titer is all used the milky plastic bottle splendid attire of white valve protection cap; The ELIAS secondary antibody working fluid is with the white plastic bottle splendid attire of red valve protection cap; Aflatoxin b1 antibody is with the white plastic bottle splendid attire of yellow valve protection cap; Developer A liquid is with the black plastic bottle splendid attire of black valve protection cap; Developer B liquid is with the white plastic bottle splendid attire of green valve protection cap, and stop buffer is with the white plastic bottle splendid attire of green valve protection cap, and concentrated cleaning solution and sample diluting liquid are with the translucent plastic bottle splendid attire of white valve protection cap; Described mould is processed by plastic foam, has 14 recessed bottle positions, and 14 reagent bottles are put into corresponding recessed bottle position successively.Described 6 bottles of titers are the 1ml/ bottle, and concentration is respectively 0ng/ml, 0.02ng/ml, 0.06ng/ml, 0.1ng/ml, 0.3ng/ml, 0.8ng/ml; 1 bottle of high concentration standard solution, the 1ml/ bottle; 1 bottle of ELIAS secondary antibody working fluid, the 6ml/ bottle; 1 bottle of aflatoxin b1 antibody working fluid, the 6ml/ bottle; 1 bottle of colour developing liquid A liquid, the 7ml/ bottle; 1 bottle of colour developing liquid B liquid, the 7ml/ bottle; 1 bottle of stop buffer, the 7ml/ bottle; 1 bottle of concentrated cleaning solution (10 *), 40 ml/ bottles; 1 bottle of concentrating sample dilution (2 *), 40 ml/ bottles.
The utility model is used the plastic bottle and the vial splendid attire of different sizes, different colours and is fixed in the polyfoam mould and together is encapsulated in the carton box with ELISA Plate, cover plate film, valve bag, instructions and Quality Control report, so that carry and transport.Reagent in each kit enough carries out 96 parts of mensuration (comprising the standard analysis hole), can carry out the detection of a plurality of samples of one-time continuous, also can the ELISA Plate capillary strip be taken apart repeatedly and use.At this moment, remaining capillary strip is put back in the aluminium foil bag with the valve bag sealing accuracy and stability that like this can guarantee reagent box testing result.During detection; Residue AFB1 in the sample will be striven aspergillus flavus resisting toxin B1 antibody unexpectedly with the AFB1 coupled antigen that encapsulates in advance on the capillary strip; After adding ELIAS secondary antibody, with the tmb substrate colour developing, the sample light absorption value becomes negative correlation with the content of its contained residue AFB1; Relatively can draw the content of corresponding residue AFB1 with typical curve, it is easy to operation.
Show that through experiment this kit has very high sensitivity: lowest detection is limited to 0.02ng/ml; Cereals such as recovery peanut, corn are 90 ± 15%, the feed sample is 85 ± 15%, edible oil is 90 ± 15%, fresh milk class and goods thereof are 92 ± 15%, and the Variation Lines number average of kit is less than 10%.Detect the residual method of AFB1 with respect to other, the needed instrument of this kit is less, only needs ELIASA, even matter device, vibrating machine and micro sample adding appliance, and generally all there is outfit in similar laboratory, and operation is simple, and required cost is lower.The beneficial effect of the utility model is: can be used for animal derived food AFB1 residual quantitative, qualitative detection quick, easy, sensitive, exactly.
Description of drawings
Fig. 1 is the front view of ELISA Plate in the utility model;
Fig. 2 is along the longitudinal sectional view of A-A among Fig. 1;
Fig. 3 is the front view of the utility model cover plate film;
Fig. 4 is the front view of reagent bottle in the utility model;
Fig. 5 is the structural representation that has recessed bottle position mould in the utility model;
Fig. 6 is a structural representation of putting into recessed bottle position mould box body in the utility model.
Drawing explanation: 1, bracing frame; 2, capillary strip; 3, AFB1 coupled antigen; 4, cover plate film; 5, white valve protection cap; 6, translucent plastic reagent bottle; 7, yellow valve protection cap or red valve protection cap; 8, black valve protection cap; 9, black plastic reagent bottle; 10, green valve protection cap; 11, white plastic reagent bottle; 12, white valve protection cap; 13,6ml aflatoxin b1 antibody working fluid bottle position; 14,6ml ELIAS secondary antibody working fluid bottle position; 15,7ml substrate developer A liquid bottle position; 16,1ml high concentration AFB1 standard solution bottle position; 17,7ml substrate developer B liquid and 7ml stop buffer bottle position; 18, the titer bottle position of the various concentration of 1ml/ bottle; 19,40ml concentrated cleaning solution bottle position; 20,40ml concentrating sample dilution bottle position; 21, the mould that has recessed bottle position; 22, box body.
Embodiment
Describe embodiment in detail in conjunction with accompanying drawing.Be fixed on the outer frame support (1) of ELISA Plate after enzyme mark reaction capillary strip (2) encapsulates AFB1 coupled antigen (3) in advance, and enzyme mark reaction capillary strip (2) can be with requiring dismounting; Cover plate film (4) is used for ELISA Plate capping enzyme mark reaction capillary strip (2) when putting water-bath or constant temperature oven internal reaction; The translucent plastic reagent bottle (6) of white valve protection cap (5) is used to encapsulate 40ml concentrated cleaning solution or 40ml concentrating sample dilution; The white plastic reagent bottle of yellow valve protection cap (7) is used to encapsulate 6ml aflatoxin b1 antibody working fluid, and the white plastic reagent bottle of red valve protection cap (7) is used to encapsulate 6ml ELIAS secondary antibody working fluid; The black plastic reagent bottle (9) of black valve protection cap (8) is used to encapsulate 7ml substrate developer A liquid, and the white plastic reagent bottle (11) of green valve protection cap (10) is used to encapsulate 7ml substrate developer B liquid or 7ml stop buffer; The translucent plastic reagent bottle of white valve protection cap (12) is used to encapsulate the standard solution of 1ml/ bottle; The built-in novel mould (21) of utility model box body (22) with recessed bottle position; On 14 bottle positions are arranged, placement location is followed successively by: 6ml aflatoxin b1 antibody working fluid bottle position (13), 6ml ELIAS secondary antibody working fluid bottle position (14); 7ml substrate developer A liquid bottle position (15); 1ml high concentration AFB1 standard solution bottle position (16), 7ml substrate developer B liquid and 7ml stop buffer bottle position (17), the titer bottle position (18) of 6 kinds of various concentration of 1ml/ bottle; 40ml concentrated cleaning solution bottle position (19), 40ml concentrating sample dilution bottle position (20).
AFB1 Determination on content in the animal feed sample:
1) sample pre-treatment: get the 3g sample that grinds fodder, add the 9ml sample extracting solution; Thermal agitation 10min; The centrifugal 5min of 4000r/min, or use the quantitative test filter paper filtering; Supernatant or filtrating are pressed 1:9 dilution proportion (+9 parts of deionized waters of 1 part of filtrating) with deionized water; It is to be measured to get dilution back liquid.Extension rate: 30 times.
2) operation steps of use kit: 1. required reagent is taken out from cold storage environment, place room temperature (20～25 ℃) more than the balance 30min, note to shake up before every kind of liquid reagent uses; 2. taking-up needs the capillary strip of quantity, no capillary strip is put in the former aluminium foil bag and with the drying agent that provides reseal, and is stored in 2～8 ℃; 3. washing working fluid also need rise again before use; 4. numbering: the corresponding micropore of sample and standard items is numbered according to the order of sequence, and it is parallel that each sample and standard items are done 2 holes, and the position that belongs to of record standard hole and sample aperture; 5. add standard items/sample: add standard items/sample 50 μ l in the micropore of correspondence; Add ELIAS secondary antibody working fluid 50 μ l/holes immediately; Add aflatoxin b1 antibody working fluid 50 μ l/holes again, the mixing that vibrates gently is with reacting 30min in the rearmounted 37 ℃ of lucifuge environment of cover plate membrane cover plate; 6. wash plate: carefully open the cover plate film, liquid in the hole is dried, use washing working fluid 300 μ l/ holes, fully wash 5 times, each 10s at interval claps dried (clapping the bubble that is not eliminated after doing can poke with original rifle head) with thieving paper; 7. colour developing: add substrate developer A liquid 50 μ l/ holes, add substrate developer B liquid 50 μ l/ holes again, the mixing that vibrates gently is with the rearmounted room temperature lucifuge of cover plate membrane cover plate environment reaction 15～20min; 8. measure: add stop buffer 50 μ l/ holes, the mixing that vibrates is gently set ELIASA in the 450nm place (suggestion detects with dual wavelength 450/630nm, in 5min, runs through data), measures every hole OD value.(if no ELIASA does not then add stop buffer and can judge with ocular estimate).
3) result judges has two kinds of methods, judges available the 1st kind of method roughly, and quantitatively judges with the 2nd kind of method.Notice that the sample light absorption value becomes negative correlation with its contained AFB1.
1. mean light absorbency value and the standard value with sample relatively can draw its concentration range.The absorbance of supposing sample 1 is 0.659, and the absorbance of sample 2 is 1.525, and the titer absorbance is respectively: 0ng/ml is 2.101; 0.02ng/ml be 1.738; 0.06ng/ml be 1.313; 0.1ng/ml be 0.831; 0.3ng/ml be 0.262.Then the concentration range of sample 1 is 0.1ng/ml～0.3ng/ml; The concentration range of sample 2 is 0.02ng/ml～0.06ng/ml, multiply by the concentration range that its corresponding extension rate can draw AFB1 in the sample again.
2. accurately quantitative Analysis is divided two steps: the first step; The calculating of percentage absorptance; The percentage absorptance of standard items or sample equals the mean value (diplopore) of percentage absorbance of standard items or sample divided by the absorbance of first standard (0 standard); Multiply by 100% again, that is: percentage absorbance (%)=B/B
0* 100%
In the formula, the mean light absorbency value of B-standard solution or sample solution; B
0The mean light absorbency value of-0ng/ml standard solution.
In second step, the drafting of typical curve and calculating: with standard items percentage absorptance is ordinate, is horizontal ordinate with the semilog of AFB1 standard items concentration (ng/ml), the drawing standard curve map.In the percentage absorptance substitution typical curve with sample, read the pairing concentration of sample, multiply by its corresponding extension rate and be AFB1 actual content in the sample from typical curve.
4) points for attention:
1. room temperature is lower than 20 ℃ or reagent and sample and does not get back to room temperature (20～25 ℃) and can cause the OD value of all standards on the low side;
2. in washing the plate process if the plate hole dry situation, it is non-linear then can typical curve to occur, the phenomenon that repeatability is bad.After doing, the plate bat should carry out next step operation so wash immediately;
3. need it is shaken up before whenever adding a kind of reagent;
4. reaction terminating liquid is concentration 2 mol/l sulfuric acid, avoids contacting skin;
5. do not use the kit of date of expiration; Do not use any reagent in the kit of the term of validity, the kit that used the term of validity of mixing can cause the reduction of sensitivity; The reagent in the different lot number kits is not used in exchange;
6. preserve kit in 2～8 ℃, can not be freezing, put no ELISA Plate microwell plate into valve bag and reseal.Standard substance and colourless colour former (substrate solution) therefore will be avoided being directly exposed under the light to photaesthesia;
7. the rotten sign of reagent: it is rotten that substrate solution has any color to show, should abandon it.The absorbance of 0 standard (450/630nm) value is less than 0.5 (A
450nm<0.5) time, expression reagent possibly go bad;
8. after adding substrate solution A liquid and substrate solution B liquid, general developing time is that 15～20min gets final product.If lighter color can prolong the reaction time to 30min (or longer).Otherwise, then shorten the reaction time;
9. concentrated cleaning solution belongs to normal phenomenon if any crystallization, please use after the heating for dissolving;
10. this kit optimal reaction temperature is 37 ℃, too high or too low for temperaturely will cause detecting absorbance and sensitivity changes.
Claims (3)
1. the immunology detection kit of an AFB1; Comprise box body; It is characterized in that: include ELISA Plate, cover plate film, 14 bottles of reagent in the box body and have the mould of recessed bottle position; Described ELISA Plate is made up of outer frame support and the dismantled and assembled enzyme mark reaction capillary strip of placing on it; ELISA Plate adopts 96 hole agent plate as solid phase carrier, is fixed on the outer frame support of ELISA Plate after encapsulating the AFB1 coupled antigen on the capillary strip in advance, and each dismantled and assembled enzyme mark reaction capillary strip has 8 reacting holes; Described cover plate film size is consistent with ELISA Plate; Recessed bottle position on the described mould is respectively titer bottle position, high concentration titer bottle position, ELIAS secondary antibody working fluid bottle position, aflatoxin b1 antibody bottle position, colour developing liquid A liquid bottle position, colour developing liquid B liquid bottle position, stop buffer bottle position, concentrated cleaning solution bottle position, concentrating sample dilution bottle position.
2. the immunology detection kit of AFB1 according to claim 1, it is characterized in that: described box body is a carton box; Described ELISA Plate is processed by polystyrene, is put in the vacuum aluminide-coating bag; Described cover plate film is the transparent plastic dura mater, is put in the aluminide-coating bag; Described 14 bottles of reagent are respectively 6 bottles of titers, 1 bottle of high concentration titer, 1 bottle of ELIAS secondary antibody working fluid, 1 bottle of aflatoxin b1 antibody, 1 bottle of colour developing liquid A liquid, 1 bottle of colour developing liquid B liquid, 1 bottle of stop buffer, 1 bottle of concentrated cleaning solution, 1 bottle of concentrating sample dilution; Titer is all used the milky plastic bottle splendid attire of white valve protection cap; The ELIAS secondary antibody working fluid is with the white plastic bottle splendid attire of red valve protection cap; Aflatoxin b1 antibody is with the white plastic bottle splendid attire of yellow valve protection cap; Developer A liquid is with the black plastic bottle splendid attire of black valve protection cap; Developer B liquid is with the white plastic bottle splendid attire of green valve protection cap, and stop buffer is with the white plastic bottle splendid attire of green valve protection cap, and concentrated cleaning solution and sample diluting liquid are with the translucent plastic bottle splendid attire of white valve protection cap; Described mould is processed by plastic foam, has 14 recessed bottle positions, and 14 reagent bottles are put into corresponding recessed bottle position successively.
3. the immunology detection kit of AFB1 according to claim 2 is characterized in that described 6 bottles of titers, is the 1ml/ bottle, and concentration is respectively 0ng/ml, 0.02ng/ml, 0.06ng/ml, 0.1ng/ml, 0.3ng/ml, 0.8ng/ml; 1 bottle of high concentration standard solution, the 1ml/ bottle; 1 bottle of ELIAS secondary antibody working fluid, the 6ml/ bottle; 1 bottle of aflatoxin b1 antibody working fluid, the 6ml/ bottle; 1 bottle of colour developing liquid A liquid, the 7ml/ bottle; 1 bottle of colour developing liquid B liquid, the 7ml/ bottle; 1 bottle of stop buffer, the 7ml/ bottle; 1 bottle of concentrated cleaning solution (10 *), 40 ml/ bottles; 1 bottle of concentrating sample dilution (2 *), 40 ml/ bottles.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201220252484 CN202614766U (en) | 2012-05-31 | 2012-05-31 | Immunological detection kit for aflatoxin B1 |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201220252484 CN202614766U (en) | 2012-05-31 | 2012-05-31 | Immunological detection kit for aflatoxin B1 |
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| CN202614766U true CN202614766U (en) | 2012-12-19 |
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| CN 201220252484 Expired - Fee Related CN202614766U (en) | 2012-05-31 | 2012-05-31 | Immunological detection kit for aflatoxin B1 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104569380A (en) * | 2015-01-23 | 2015-04-29 | 天津伯克生物科技有限公司 | Method for detecting aflatoxin B1 and enzyme-linked immunosorbent assay kit |
| CN105067804A (en) * | 2015-07-21 | 2015-11-18 | 内蒙古蒙牛乳业（集团）股份有限公司 | Method for detecting aflatoxin B1 in cereal food |
| CN106525797A (en) * | 2016-11-09 | 2017-03-22 | 百奥森（江苏）食品安全科技有限公司 | Kit for detecting aflatoxin B1 in food |
-
2012
- 2012-05-31 CN CN 201220252484 patent/CN202614766U/en not_active Expired - Fee Related
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104569380A (en) * | 2015-01-23 | 2015-04-29 | 天津伯克生物科技有限公司 | Method for detecting aflatoxin B1 and enzyme-linked immunosorbent assay kit |
| CN105067804A (en) * | 2015-07-21 | 2015-11-18 | 内蒙古蒙牛乳业（集团）股份有限公司 | Method for detecting aflatoxin B1 in cereal food |
| CN106525797A (en) * | 2016-11-09 | 2017-03-22 | 百奥森（江苏）食品安全科技有限公司 | Kit for detecting aflatoxin B1 in food |
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| Date | Code | Title | Description |
|---|---|---|---|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20121219 Termination date: 20130531 |