CN103852581A - 3,4-benzopyrene enzyme-linked immune detection kit - Google Patents
3,4-benzopyrene enzyme-linked immune detection kit Download PDFInfo
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- CN103852581A CN103852581A CN201410088941.5A CN201410088941A CN103852581A CN 103852581 A CN103852581 A CN 103852581A CN 201410088941 A CN201410088941 A CN 201410088941A CN 103852581 A CN103852581 A CN 103852581A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
Abstract
The invention relates to an enzyme-linked immune detection kit which is used for detecting content of 3,4-benzopyrene in food. The kit comprises a 96-hole elisa plate, a 3,4-benzopyrene 9-concentration standard substance solution, a high-concentration 3,4-benzopyrene monoclonal antibody solution, a high-concentration HRP (Horse Radish Peroxidase)-3,4-benzopyrene solution, a substrate developing solution, a stop solution, a sample diluent, a concentrated washing solution, a cover template, a self-sealing bag, an instruction book and a quality inspection report. Due to the adoption of an indirect competition ELISA (Enzyme Linked Immunosorbent Assay), a micropore strip of the elisa plate is pre-coated with a goat-anti-mouse second antibody; after combining with the goat-anti-mouse second antibody with a 3,4-benzopyrene monoclonal antibody at a working concentration, the 3,4-benzopyrene and a 3,4-benzopyrene antigen in a sample commonly complete limited loca on the monoclonal antibody; the substrate is used for developing after reaction is balanced; and carrying out antilog process on a value, corresponding to a standard curve, of a sample absorbency and then multiplying the value by corresponding dilution ratios to obtain the content of the 3,4-benzopyrene in the sample.
Description
One, technical field:
The present invention relates to a kind of kit, especially relate to a kind of 3,4-benzopyrene enzyme-linked immunologic detecting kit.
Two, technical background:
Benzopyrene exists extensively in environment, and is one of three universally acknowledged large carcinogens, industrial without producing and use value, general only as the accessory substance forming in production run with toxic emission., early than 1933, the people such as British scientist J.W.Cook separate and obtain benzopyrene sterling from pitch, synthetic its chemical constitution that proved, and carry out zoopery, inducing mouse has produced cutaneum carcinoma, and thus, it is first chemical environment carcinogenic substance that benzopyrene is confirmed to be.Afterwards, researchist adopts the mode administrations such as oral, intravenous injection, suction, tracheal instillation to animal, prove benzopyrene also can be lung cancer caused, the kinds cancer such as cancer of the stomach, carcinoma of urinary bladder and digestive system cancer.In recent years, the food-safety problem that benzopyrene causes is more and more serious, according to country's " GB2716-2005 edible vegetable oil hygienic standard ", the maximum of benzopyrene from edible oil content is 10 μ g/kg, and State General Administration for Quality Supervision shows about the Examined of the great tea oil of Hunan Province's gold before this, benzopyrene content has reached 60 μ g/kg, 6 times of set upper limit, therefore, fully realize 3, the character of 4-benzopyrene, works out quickly and easily detection method, sets up effective monitoring system and control criterion is very necessary.
At present, the residual detection method of 3,4-benzopyrene mainly contains fluorescence spectrophotometry, high performance liquid chromatography (HPLC), coupling technique etc.But these method instrument costlinesses, sample pre-treatments complexity, detection time is long, and testing cost is high, needs those skilled in the art to operate, and has greatly limited its widespread use.Therefore develop special, responsive, quick, easyly 3,4-benzopyrene detection method tool is of great significance.
Three, summary of the invention:
Object of the present invention: a kind of quick, cost is low, highly sensitive, specificity is good testing tool easy to detect is provided, this kind of testing tool do not need professional to operate, can quick and precisely detect 3 of sample, 4-benzopyrene content, has made up the deficiency of chromatogram detection technique.
Technical scheme of the present invention: 3,4-benzopyrene enzyme linked immunological quick detection kit, comprise box body, 17 reagent bottles, 1 ELISA Plate, wherein, plastic foam plate is arranged in box body, plastic foam plate is provided with multiple reagent bottle grooves, and the height of multiple reagent bottle low grooves is 1/3-3/4 of corresponding reagent bottle height, and each group reagent bottle is placed in the groove under multiple reagent bottles.
Above-mentioned a kind of 3,4-benzopyrene enzyme linked immunological quick detection kit, comprises box body, 17 reagent bottles, and an ELISA Plate, a cover mold plate, a valve bag, puts the low groove of reagent bottle for one, a instructions, a quality inspection report.
It is characterized in that: 96 hole ELISA Plate, be coated with sheep anti mouse two above and resist, detachably become 12 enzyme mark capillary strips, can once test multiple samples, also can take gradation test apart, remaining enzyme mark capillary strip can be stored in valve bag, and used next time; Described reagent bottle is divided into eight groups: the first group reagent bottle is the reagent bottle that standard solution is housed, and totally 9 bottles, second group is the reagent bottle that high concentration standard solution is housed, totally 1 bottle; The 3rd group reagent bottle is that enzyme mark 3 is housed, the reagent bottle of 4-benzopyrene concentrate, totally 1 bottle; The 4th group reagent bottle is for the reagent bottle of 3,4-benzopyrene monoclonal antibody concentrate is housed, and totally 1 bottle, the 5th group reagent bottle is the reagent bottle that nitrite ion A liquid and B liquid are housed respectively, and each 1 bottle respectively, totally 2 bottles; The 6th group reagent bottle is the reagent bottle that stop buffer is housed, totally 1 bottle; The 7th group reagent bottle is the reagent bottle that sample diluting liquid is housed, totally 1 bottle; The 8th group reagent bottle is that concentrated cleaning solution reagent bottle is housed, totally 1 bottle.Reagent is further distinguished with bottle cap and the bottle of different colours.
Above-mentioned one 3, 4-benzopyrene enzyme linked immunological quick detection kit, wherein eight group reagent bottles are respectively: standard solution and high concentration standard solution are all used the Brown Glass Brown glass bottles and jars only of black caps, enzyme mark 3, the white PE plastic bottle of red cap for 4-benzopyrene concentrate, 3, the black PE plastic bottle of black caps for 4-benzopyrene monoclonal antibody concentrated solution, the black plastic bottle of white cap for nitrite ion A liquid, the black PE plastic bottle of red cap for nitrite ion B liquid, the white PE plastic bottle of yellow cap for stop buffer, the white PE plastic bottle of hyaline cap for condensing cleansing solution, the white PE plastic bottle of red cap for sample diluting liquid.Be made up of white plastic foam recessed bottle position.
According to claim 1 a kind of 3,4-benzopyrene enzyme-linked immunoassay kit, is characterized in that, described box body is hard box body.
Above-mentioned 3,4-benzopyrene enzyme-linked immunoassay kit, wherein, the size of described multiple reagent bottle low grooves and corresponding reagent bottle size match.
Reagent bottle is placed in plastic foam groove, is encapsulated in rigid packet together with instructions, quality inspection report, cover mold plate, ELISA Plate, valve bag, easy to carry and transport.
When test, adopt indirect competitive ELISA method, on ELISA Plate capillary strip, pre-coated sheep anti mouse two is anti-, with 3 of working concentration, after the combination of 4-benzopyrene monoclonal antibody, in sample, contain 3,4-benzopyrene and enzyme mark 3, limited site in the common competition of 4-benzopyrene antigen 3,4-benzopyrene monoclonal antibody, reaction reaches after balance and develops the color with substrate, the value that sample absorbance is more corresponding with typical curve, after inverse logarithm, be multiplied by again its corresponding extension rate, can draw in sample 3, the content of 4-benzopyrene.
Compared with existing detection technique, the present invention has significant advantage: 1) compare with high performance liquid chromatography-mass-spectrometric technique, this kit is with low cost; 2) compared with routine techniques, this kit does not need special skilled operating personnel, simple to operate, convenient, easy to utilize.3) this kit to sample require lowly, pre-treatment is simple, can be widely used in various food inspection; 4) principle of this kit is enzyme linked immunoassay, and therefore, sensitivity is high compared with additive method, and lowest detectable limit reaches 0.554 μ g/L, and the recovery reaches 89 ± 11%.
Four, accompanying drawing explanation:
Be described further below in conjunction with accompanying drawing and the concrete reagent kit of the present invention of having a try, but do not form any restriction of the present invention.
Fig. 1 is elisa plate crosscut schematic diagram of the present invention;
Fig. 2 is elisa plate rip cutting schematic diagram of the present invention;
Fig. 3 is elisa plate diagrammatic cross-section of the present invention;
Fig. 4 is the cover plate schematic diagram of elisa plate of the present invention;
Fig. 5 is that schematic diagram is looked in the survey of reagent bottle of the present invention;
Fig. 6 is the schematic top plan view of foam formwork of the present invention;
Fig. 7 is the schematic diagram of opening of kit of the present invention.
Five, embodiment
(1) pre-treatment of sample
1. obtain solution: obtain solution 1.0mol/L sodium hydroxide solution, take the NaOH of 4.0g, adding distil water dissolves constant volume to 100mL.
2. wash solution: amount as required, 20 × thickening and washing solution is diluted according to the volume ratio of 1:19 with distilled water, for the washing of ELISA Plate.
To be added with the food samples treatment step of 3,4-benzopyrene: take food sample 10.0g sample, add 40mL PBS and 5mL1mol/L NaOH solution, stir 30min, filter, filtrate is adjusted pH7.2～7.4 with 1mol/L HCl, solution after treatment still heat be concentrated into 10mL for detection of.
(2) use kit operation steps
1. required kit is comprised to dilution and cleansing solution take out from the environment of refrigeration, be placed on room temperature (20-25 ℃) more than 30 minutes, make it reach room temperature, before use every kind of reagent is shaken up.
2. take out ELISA Plate frame and need the micropore of quantity, unwanted micropore is put into valve bag, refrigeration.
3. will concentrate 3,4-benzopyrene monoclonal antibody and concentrated enzyme mark 3,5 times of dilutions of sample diluting liquid for 4-benzopyrene.
4. numbering: by sample solution and micropore corresponding to standard items working solution numbering, it is parallel that each sample solution and standard items working solution do 2 holes, and the position at record standard hole and sample aperture place.
5. add 3 after dilution, 4-benzopyrene monoclonal antibody, every hole 50 μ L, establish feminine gender and blank, react 15 minutes in 37 ℃.
6. take out microwell plate, throw away solution in hole, with cleansing solution washing 3-4 time, after each washing, pat dry with thieving paper.
7. every hole adds enzyme mark 3,4-benzopyrene working fluid 50 μ L, simultaneously equal-volume adds 3,4-benzopyrene standard items and sample solution, cover cover plate film be placed in 37 ℃ 30 minutes, take out and repeat above-mentioned washing step.
8. by nitrite ion A and by volume 1:1 mixing of B, every hole adds 50 μ L, color development at room temperature 5 minutes.
9. every hole adds stop buffer 50 μ L, in 450nm place, measures every hole OD value (if without enzyme mark liquid instrument, not adding stop buffer ocular estimate can judge) by microplate reader
(3) result is judged
Result is determined with two kinds of methods, judges roughly available the 1st kind of method, quantitatively judges by the 2nd kind of method.
Note the relation of sample light absorption value and 3,4-benzopyrene content.
1. object judgement method: the concentration range of carrying out per sample judgement sample with the depth of the color of standard items.The color of standard items is according to concentration (0 μ g/L, 1 μ g/L, 2 μ g/L, 4 μ g/L, 8 μ g/L, 16 μ g/L, 32 μ g/L, 64 μ g/L, 128 μ g/L) rising color shoal successively to colourless, if the color of sample is in 4 μ g/L, between 8 μ g/L two hole colors, the concentration of sample, just in 4 μ g/L, is multiplied by extension rate between 8 μ g/L more so.
2. quantitative analysis method:
(1) production standard curve:
The calculating of percentage absorptance, the percent of the light absorption value of sample or standard items equals the mean value of standard items or sample divided by the light absorption value in first standard items (hole that standard items concentration is 0), be multiplied by again 100%, it is percentage absorbance (%)=B/B0 × 100%, B is the mean value of the light absorption value of standard items or sample corresponding aperture, and B0 is that standard items concentration is the mean value of the light absorption value in 0 hole.
Take standard items percentage absorptance as ordinate, take the logarithm of standard items concentration (0 μ g/L) as horizontal ordinate, drawing standard curve.The percentage absorptance of sample is brought in typical curve, read the logarithm value of the corresponding sample concentration of sample from typical curve, then inverse logarithm, obtain concentration and be multiplied by again extension rate, obtain in sample 3, the content of 4-benzopyrene.
(4) points for attention while mensuration:
1. before test, reagent used and sample should be got back to normal temperature (20-25 ℃), otherwise cause OD value on the low side.
2. in washing process, can not be interrupted to wash and clap plate and should carry out immediately next step test, otherwise, occur dryly in midair causing typical curve non-linear, poor repeatability, measurement result is inaccurate.
3. reagent mix is wanted evenly, and nitrite ion B will join nitrite ion A, fully mix, otherwise typical curve is non-linear, and repeatability is bad, affects measurement result.
4. the sulfuric acid solution that stop buffer is 2mol/L, avoids contacting skin.
5. condition of storage: preserving kit should be at 0-4 ℃, not freezing, puts no microwell plate into valve bag and reseals, and standard substance and colourless luminous agent, to photaesthesia, are therefore avoided directly cruelly leaking under light.If nitrite ion has any color to show to go bad, should not re-use.The absorbance of 0 standard should, in 1 left and right, be less than at 0.5 o'clock, represents that reagent may go bad.Do not use the kit of the term of validity, dilution can cause that with doping use experimental result is inaccurate, does not use the reagent in different lot number kits.
Add general colour developing 15min after A, B liquid, if color is more shallow, can extend developing time, but can not exceed 30min.
This kit optimal reaction temperature is 25 ℃, excess Temperature or too lowly will cause detecting absorbance and sensitivity changes.
Claims (7)
1. one kind is detected in food 3, the enzyme linked immunological kit of 4-benzopyrene content, comprise the ELISA Plate in 96 holes in box body and box, a cover plate film, 17 bottles of reagent and the little recessed bottle position of putting reagent, a valve bag, a instructions and a quality inspection report, it is characterized in that: ELISA Plate is to adopt 96 hole agent plate as solid phase carrier, the anti-check-out console of making of pre-coated sheep anti mouse two on kit capillary strip, 17 bottles of reagent are respectively 9 bottles of standard solutions, 1 bottle height concentration standard product solution, 1 bottle 3, 4-benzopyrene monoclonal antibody concentrated solution, 1 bottle of enzyme mark 3, 4-benzopyrene concentrated solution, 1 bottle of nitrite ion A solution, 1 bottle of nitrite ion B solution, 1 bottle of stop buffer, 1 bottle of Sample Dilution solution, 1 bottle of thickening and washing solution, totally 18 of lower recess.
2. kit according to claim 1, is characterized in that: box body is carton box; The polystyrene ELISA Plate in 96 holes, is put in vacuum aluminide-coating bag; Cover plate film is plastics dies; Standard solution and high concentration standard solution are all used the Brown Glass Brown glass bottles and jars only of black caps, enzyme mark 3, the white PE plastic bottle of red cap for 4-benzopyrene concentrated solution, 3, the black PE plastic bottle of black caps for 4-benzopyrene monoclonal antibody concentrated solution, the black plastic bottle of white cap for nitrite ion A liquid, the black PE plastic bottle of red cap for nitrite ion B, the white PE plastic bottle of yellow cap for stop buffer, the white PE plastic bottle of red cap for sample diluting liquid, the white PE plastic bottle of hyaline cap for concentrated cleaning solution; Be made up of white plastic foam recessed bottle position.
3. according to claim 1 a kind of 3,4-benzopyrene enzyme-linked immunoassay kit, it is characterized in that, described elisa plate (2) is 96 removable hole elisa plates, be detachably 12 enzyme mark reaction capillary strips, each enzyme mark reaction capillary strip has 8 reacting holes, is coated with that sheep anti mouse two is anti-makes coated plate on the micropore of elisa plate.
4. cover plate film size is consistent with ELISA Plate size.
5. totally 9 bottles of standard solutions, 1mL/bottle, concentration is respectively 128,64,32,16,8,4,2,1,0 μ g/L, high concentration standard solution (1mg/L), 1 bottle, 1mL; 1 bottle of 3,4-benzopyrene monoclonal antibody concentrate, 2mL; High-concentration enzyme mark 3,1 bottle of 4-benzopyrene, 2mL; 1 bottle of nitrite ion A liquid, 7mL; 1 bottle of nitrite ion B liquid, 7mL; 1 bottle of stop buffer, 7mL; 1 bottle of sample diluting liquid, 30mL; 1 bottle of concentrated cleaning solution, 30mL.
6. according to claim 1 a kind of 3,4-benzopyrene enzyme-linked immunoassay kit, is characterized in that, described box body is hard box body.
7. according to claim 1 a kind of 3,4-benzopyrene enzyme-linked immunoassay kit, is characterized in that, the size of described multiple reagent bottle low grooves and corresponding reagent bottle size match.
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Cited By (4)
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| CN105738611A (en) * | 2015-11-23 | 2016-07-06 | 轻工业环境保护研究所 | Benzo(a)pyrene enzyme-linked immunosorbent assay (ELISA) kit and detection method thereof |
| CN108456661A (en) * | 2017-12-27 | 2018-08-28 | 江南大学 | One plant of anti-BaP monoclonal antibody specific hybridoma cell strain and its application |
| CN108715611A (en) * | 2018-05-31 | 2018-10-30 | 上海交通大学 | A kind of preparation method and its usage of BaP immunogene |
| CN108997103A (en) * | 2018-05-31 | 2018-12-14 | 上海交通大学 | A kind of preparation method and its usage of BaP haptens |
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Application publication date: 20140611 |