CN102313809A - Method for detecting brilliant blue and enzyme linked immunosorbent assay (ELISA) kit - Google Patents
Method for detecting brilliant blue and enzyme linked immunosorbent assay (ELISA) kit Download PDFInfo
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Abstract
The invention discloses a method for detecting brilliant blue and an enzyme linked immunosorbent assay (ELISA) kit. The ELISA kit for detecting the brilliant blue, provided by the invention, comprises a brilliant blue hapten and a brilliant blue specific antibody; and the specific antibody is a polyclonal antibody or monoclonal antibody of the brilliant blue. In the kit, by adopting the high-specificity brilliant blue monoclonal antibody, a detection result is ensured to be reliable; experiment results show that the kit has the characteristics of high specificity, high sensitivity, high precision, high accuracy and the like; and main reagents in the kit are all in the form of working solution, so that convenience for use and low cost are achieved. The method for detecting the brilliant blue by using the kit in the invention is simple to operate, has low requirement for pretreatment of a sample, and can be used for detecting a mass of samples at the same time. Therefore, the method for detecting the brilliant blue by using the ELISA kit disclosed by the invention can be used for field monitoring and is suitable for qualitatively and quantitatively screening a large amount of samples.
Description
Technical field
The present invention relates to a kind of method and enzyme linked immunological kit that detects light blue.
Background technology
The light blue synthetic dyestuff is one of six kinds of edible synthesized coloring matters of present China approval use, is widely used in fields such as food, medicine, cosmetics, medical apparatus, tobacco, feed, edible packing, daily use chemicals article, toy.Though at present to the harmfulness of the synthetic dyestuff of the non-azo class of this type of light blue final conclusion not, the nutritive value that they have no also has no help to the healthy of people, so will try not to eat.Also there is clear and definite edible regulation in China to this type of light blue synthetic food colors; Also there is strict restriction in China in food, adding synthetic dyestuff: milk-contained drink; Vegetable protein beverage; Soda, the edible animal easings, limiting the quantity of of Collagent casing for sausages etc. is 0.025g/kg; Limiting the quantity of of cocoa products, chocolate and chocolate, chilled beverage, jelly, seasoning condensed milk (comprising that condensed milk, seasoning breast and other have used the modulation condensed milk of non-dairy milk starting material) etc. is 0.05g/kg, and limiting the quantity of of fruit flavoured syrup, jam, semi-solid compound seasoner etc. is 0.5g/kg.At present, the detection method taked of light blue national standard is tlc analysis, spectrophotometer method and high-efficient liquid phase analysis method.The detection requirement though these methods can be up to state standards, it is relatively more expensive to detect cost, and can not detect fast in enormous quantities, can not satisfy the requirement that detects on the market on the spot.In recent years; China's researcher has been done a large amount of work in the light blue context of detection; But all mainly concentrate on the physics and chemistry context of detection, the report about the ELISA detection method of light blue is not found in literature search as yet, Given this; The present invention studies and has set up the competitive ELISA detection method of light blue, makes and developed light blue enzyme linked immunological kit and detection method thereof.
Summary of the invention
An object of the present invention is to provide a kind of enzyme linked immunological kit that detects light blue.The enzyme linked immunological kit of detection light blue provided by the present invention comprises the specific antibody of light blue haptens and light blue; Said specific antibody is the polyclonal antibody or the monoclonal antibody of light blue; Wherein, the specific antibody of said haptens and light blue can exist with following any form:
1) light blue haptens and carrier protein carry out coupling, obtain the conjugate (below be called the hapten-carrier protein conjugate) of light blue haptens and carrier protein, and as coating antigen, said specific antibody carries out after the enzyme labeling as the enzyme labeling thing with it;
2) said specific antibody is a coating antigen, and said haptens carries out after the enzyme labeling as the enzyme labeling thing.
Said kit can also both comprise the specific antibody of haptens and light blue, comprised antiantibody again; Said antiantibody is sheep anti mouse antiantibody or goat-anti rabbit antiantibody; Wherein, said haptens and antiantibody exist with following arbitrary form:
1) said haptens and carrier protein are carried out coupling, obtain the hapten-carrier protein conjugate, as coating antigen, said antiantibody carries out after the enzyme labeling as the enzyme labeling thing with it;
2) said antiantibody is a coating antigen, and said haptens carries out after the enzyme labeling as the enzyme labeling thing.
Said light blue polyclonal antibody or light blue monoclonal antibody all obtain as immunogene with said light blue hapten-carrier protein conjugate; Said carrier protein can be thyroprotein, mouse haemocyanin, human albumin, bovine serum albumin, rabbit anteserum albumen, hemocyanin or ovalbumin etc.
Said monoclonal antibody obtains through hybridoma cell technology.Said light blue polyclonal antibody can be mouse source, Ma Yuan, Yang Yuan, rabbit source or cavy source antibody.
Detect for ease, said kit also can comprise ELISA Plate, when said light blue specific antibody or antiantibody carry out mark with enzyme, is coated with said light blue haptens or light blue hapten-carrier protein conjugate on the said ELISA Plate; When said haptens carries out mark with enzyme, be coated with said light blue specific antibody or said antiantibody on the said ELISA Plate.
For more convenient on-site supervision and great amount of samples examination, said kit also can comprise light blue standard solution, colour developing liquid, stop buffer, concentrated cleaning solution, concentrate and redissolve at least a in the liquid;
Wherein, said concentrated cleaning solution is that the pH value is 6-9, the final concentration that contains in said concentrated cleaning solution is that 0.02-0.05% (quality percentage composition) sodium azide, the final concentration in said concentrated cleaning solution are the phosphate buffer of 1.0-2.0% (quality percentage composition) Tween-20,0.1-0.2mol/L; Said concentrated redissolution liquid is that to contain final concentration in said concentrated redissolution liquid be that DMSO, the pH of 5-10% (quality percentage composition) is the phosphate buffer of 6-8,0.1-0.2mol/L.
Used marker enzyme is horseradish peroxidase or alkaline phosphatase in the said enzyme labeling; When marker enzyme was horseradish peroxidase, said substrate colour developing liquid was hydrogen peroxide or urea peroxide, tetramethyl benzidine sulfate mixed solution, and said stop buffer is 1-2mol/L sulfuric acid solution or hydrochloric acid solution.When being labeled as alkaline phosphatase, colour developing liquid is the nitro phosphate buffer, and stop buffer is the 1-2mol/L sodium hydroxide solution.
Said concentrated cleaning solution can be 7.4 for the pH value specifically, contain final concentration 0.03% antiseptic, the final concentration in said concentrated cleaning solution in said concentrated cleaning solution is 1.50% Tween-20,0.2mol/L phosphate buffer; Said concentrated redissolution liquid can be that 8% DMSO, pH are 7.4, the 0.2mol/L phosphate buffer for containing final concentration in said concentrated redissolution liquid specifically; Said substrate colour developing liquid is hydrogen peroxide or urea peroxide, tetramethyl benzidine sulfate mixed solution, and said stop buffer is 2mol/L sulfuric acid solution or hydrochloric acid solution.
Light blue is a small-molecule substance, has only immunoreactivity, does not have immunogenicity, can not bring out body and produce immune response, must with the macromolecular carrier albumen coupling after just have immunogenicity.Adopt carbodiimide method to carry out coupling light blue haptens and bovine serum albumin and obtain immunogene, the ratio of light blue haptens and carrier protein is crossed low or too high all unfavorable to immunity, and haptens is 23-28 with the mol ratio that combines of bovine serum albumin: 1 is more suitable.In preparation during coating antigen, the mole proportioning of light blue haptens and said carrier protein be 26: 1 proper.When making was coated with the ELISA Plate of coating antigen, the said damping fluid that encapsulates can be 9.0-9.6,0.05mol/L carbonate buffer solution for the pH value, and confining liquid is pH6-8, contains 5% lowlenthal serum, the caseic 0.02mol/L phosphate buffer of 10g/L.
Another object of the present invention provides a kind of method that detects light blue.
The method of the light blue content in a kind of test sample provided by the present invention may further comprise the steps:
1) with light blue specific antibody coated elisa plate;
2) the light blue haptens of adding standard items or testing sample and enzyme labeling is hatched, washing;
3) add the colour developing of substrate colour developing liquid;
4) add the reaction terminating liquid cessation reaction;
5) through comparing the color of light blue standard items and testing sample, infer the light blue content that in the testing sample; Perhaps, measure the absorbance in each hole, set up the typical curve of light blue concentration, and extrapolate the light blue content in the testing sample by the absorbance of testing sample according to this typical curve with respect to absorbance.
The method of the light blue content in the another kind of test sample provided by the present invention may further comprise the steps:
1) with light blue haptens or light blue hapten-carrier protein conjugate coated elisa plate;
2) add light blue standard items or testing sample;
3) add the light blue specific antibody, hatch, washing;
4) antiantibody of adding enzyme labeling is hatched, washing;
5) add the colour developing of substrate colour developing liquid;
6) add the reaction terminating liquid cessation reaction;
7) through comparing the color of light blue standard items and testing sample, infer the light blue content that in the testing sample; Perhaps, measure the absorbance in each hole, set up the typical curve of light blue concentration, and extrapolate the light blue content in the testing sample by the absorbance of testing sample according to this typical curve with respect to absorbance.
The method of the light blue content in test sample provided by the present invention can also comprise the sample pre-treatments step:
When said sample was liquid food, said sample-pretreating method was: liquid food is removed gas, with sample and sample dilution with volume ratio 1: 3-8 mixes, and gets to mix the back sample and be used for analyzing;
When said sample was cocoa, jellies, said sample-pretreating method was: with thing water-bath to be checked dissolving, with the sample dilution be 1 with mass volume ratio: 10-15, add the normal hexane degreasing of certain volume again, centrifugal, get supernatant and detect.
When said sample was jam, fruits seasoning syrup, said sample-pretreating method was: is 1 with determinand and sample dilution with volume ratio: the 20-25 mixing, add a certain amount of normal hexane degreasing again, and get supernatant after centrifugal and detect.
The enzyme linked immunological kit that the present invention detects light blue adopts direct competitive and the indirect competitive ELISA method is qualitative or the detection by quantitative sample in the content of light blue.Adopt the light blue monoclonal antibody of high specific in this kit, guaranteed the reliability of testing result.Experimental result shows that this kit has specificity height, highly sensitive, characteristics such as degree of accuracy is high, accuracy height; The main agents of this kit all adopts the working fluid form, and is easy to use, with low cost.Detect the method for light blue with kit of the present invention, easy and simple to handle, simplified the step of traditional detection method, shortened the time of detecting, the pre-treatment of sample is required low, fast detecting gross sample simultaneously.Therefore, the method for utilizing enzyme linked immunological kit of the present invention to detect can be carried out the qualitative and quantitative examination of on-site supervision and suitable a large amount of samples, will in the detection of light blue, play a significant role.
Description of drawings
Fig. 1 is a light blue ELISA examination criteria curve.
Embodiment
Employed experimental technique is conventional method like no specified otherwise among the following embodiment.
Embodiment 1, be that coating antigen, antigen are the preparation and the ELISA detection method of the kit of enzyme labeling thing with the light blue specific antibody
One, be that coating antigen, antigen are the ELISA detection principle of enzyme labeling thing with the light blue specific antibody:
When the coating antigen on the ELISA Plate capillary strip is the light blue specific antibody; After in enzyme plate micropore, adding standard solution or sample solution, and the enzyme labeling thing, the light blue in the sample combines light blue specific antibody on the microwell plate with the competition of enzyme labeling thing; Wash plate; Colour developing, the content of light blue is negative correlation in sample light absorption value and sample or the standard items, relatively can draw the content of light blue in the sample with typical curve.Simultaneously also can be according to the depth of color on the ELISA Plate, through with the more rough judgement sample of series concentration light blue standard solution color in the content of light blue.
Two, be that coating antigen, antigen are that the kit of enzyme labeling thing generally can comprise with the light blue specific antibody:
1, be coated with the ELISA Plate of light blue specific antibody, the concentration of coating antigen can be 0.15-0.25 μ g/ml.
2, enzyme-labelled antigen working fluid: enzyme-labelled antigen is the light blue antigen with horseradish peroxidase-labeled; The dilution of enzyme-labelled antigen is that to contain final concentration in dilution be that lowlenthal serum, the pH of 6-8% (quality percentage composition) is 6-8,0.2-0.3mol/L phosphate buffer, and the enzyme-labelled antigen working dilution is 1: 1000.
3, the light blue standard solution is 6 bottles, and concentration is respectively 0 μ g/mL, 1 μ g/mL, 3 μ g/mL, 9 μ g/mL, 27 μ g/mL, 81 μ g/mL.The preparation standard items solution be contain the preparation standard items solution in final concentration be that pH is the phosphate buffer of 7.0-7.5,0.1-0.2mol/L.
4, substrate colour developing liquid: substrate colour developing liquid is hydrogen peroxide or urea peroxide, tetramethyl benzidine sulfate mixed solution, 7ml/ bottle, 1 bottle.
5, stop buffer: 1-2mol/L hydrochloric acid or sulfuric acid.
6, concentrated cleaning solution: the pH value is 6-9, contain final concentration in cleansing solution is that antiseptic and the final concentration in cleansing solution of 0.02-0.05% (quality percentage composition) is the Tween-20 of 1.0-2.0% (quality percentage composition), the phosphate buffer of 0.1-0.2mol/L; The 40ml/ bottle, 1 bottle.
7, concentrate to redissolve liquid: containing redissolving final concentration in the liquid is that DMSO, the pH of 5-10% (quality percentage composition) is the phosphate buffer of 6-8,0.1-0.2mol/L; The 200ml/ bottle, 1 bottle.
Three, be that coating antigen, antigen are the concrete composition and the preparation thereof of the kit of enzyme labeling thing with the light blue specific antibody:
(1) forms
1, be coated with the ELISA Plate of light blue specific antibody, the concentration of coating antigen can be 0.20 μ g/ml.
2, enzyme-labelled antigen working fluid: enzyme-labelled antigen is with the light blue of horseradish peroxidase-labeled and the conjugate of carrier protein; The dilution of enzyme-labelled antigen is that to contain final concentration in dilution be that lowlenthal serum, the pH of 6% (quality percentage composition) is 7.4, the 0.2mol/L phosphate buffer, and enzyme mark antiantibody working dilution is 1: 1000.
3, the light blue standard solution is 6 bottles; Concentration is respectively 0 μ g/mL, 1 μ g/mL, 3 μ g/mL, 9 μ g/mL, 27 μ g/mL, 81 μ g/mL, and the solution of preparation standard items is that pH is 7.2, the phosphate buffer of 0.1mol/L for final concentration in the preparation standard solution.
4, substrate colour developing liquid: substrate colour developing liquid is hydrogen peroxide or urea peroxide, tetramethyl benzidine sulfate mixed solution, 7ml/ bottle, 1 bottle.
5, stop buffer: 2mol/L hydrochloric acid.
6, concentrated cleaning solution: the pH value is 7.4, and the antiseptic, the final concentration in cleansing solution that contain final concentration in cleansing solution and be 0.03% (quality percentage composition) are 1.5% (quality percentage composition) Tween-20,0.2mol/L phosphate buffer; The 40ml/ bottle, 1 bottle.
7, concentrate to redissolve liquid: containing redissolving final concentration in the liquid is that DMSO, the pH of 8% (quality percentage composition) is 7.4, the 0.2mol/L phosphate buffer; The 200ml/ bottle, 1 bottle.
(2) preparation
1, light blue MONOCLONAL ANTIBODIES SPECIFIC FOR
1) immunogenic preparation
Light blue is a small-molecule substance, has only immunoreactivity, does not have immunogenicity, can not bring out body and produce immune response, must with the macromolecular carrier albumen coupling after just have immunogenicity.
The above-mentioned haptens that obtains and the following method of ovalbumin are carried out coupling obtain immunogene, the body step is following:
A.10mg the light blue of purifying and 1.4mg Anhydrous potassium carbonate are dissolved in the 0.5ml anhydrous propanone, add 3mg 6-bromine ethyl hexylate, and mixing is spent the night, and nitrogen dries up;
B. above-mentioned sample is used the 3ml acetic acid ethyl dissolution, uses 1mol/L NaOH solution successively, and 4mol/L solution and distilled water repeatedly wash, and get supernatant liquid, and room temperature nitrogen dries up;
C. above-mentioned residue is again with 3ml 1mol/L NaOH dissolving, and 50 ℃ were stirred 30 minutes, and 3ml ethyl acetate extraction twice is used again with the acidifying of 5ml concentrated hydrochloric acid in the back, gets organic layer nitrogen and dries up;
D. above-mentioned hexabromo ethyl hexylate method is synthetic haptens is dissolved in 1ml DMF, adds 50 μ molDCC and 50 μ mol NHS, reacts under 4 ℃ of conditions and spends the night.
E. above-mentioned reactant liquor is centrifugal, get supernatant and slowly splash in the 4ml 0.25mg/ml ovalbumin phosphate buffer, 4 ℃ are reacted 4h down, and the PBS with 0.1mol/L pH7.2 dialysed 3 days then, changed dislysate every day 3 times, after the packing freeze-drying ,-20 ℃ of preservations.
2) light blue MONOCLONAL ANTIBODIES SPECIFIC FOR
Animal immune: with the hapten-carrier protein conjugate is that immunogene is carried out the interval immunity to the Balb/c mouse, and indirect ELISA detects and obtain containing in the blood mouse spleen of light blue specific antibody.
Fusion of Cells and clone: get the Balb/c mouse boosting cell and the myeloma cell SP2/0 that produce specific antibody and merge, adopt the indirect competitive enzyme-linked immunosorbent method to measure cell conditioned medium liquid, screen positive hole.Utilize limiting dilution assay or microclone method that cloning is carried out in positive hole, obtain and set up the hybridoma cell strain that produces monoclonal antibody.
Cell cryopreservation and recovery: get the hybridoma that is in exponential phase and process cell suspension, be sub-packed in frozen pipe, in the medium-term and long-term preservation of liquid nitrogen with cryopreserving liquid.Take out frozen pipe during recovery, put into 37 ℃ of water-bath middling speeds immediately and melt, behind the centrifugal removal cryopreserving liquid, move in the culture flask and cultivate.
MONOCLONAL ANTIBODIES SPECIFIC FOR and purifying: adopt in the body and induce method, Balb/c mouse (8 age in week) abdominal cavity is injected the sterilization paraffinum liquidum, 7～14 days pneumoretroperitoneum injection hybridomas were gathered ascites after 7～10 days.Carry out the ascites purifying through sad-saturated ammonium sulfate method or affinity chromatography. purity is identified through the SDS-PAGE electrophoresis, bottle packing ,-20 ℃ of preservations.
2, light blue Polyclonal Antibody Preparation
Adopting new zealand white rabbit as immune animal, is immunogene with haptens-thyroprotein conjugate, and immunizing dose is 1.5m g/kg; Freund's complete adjuvant with immunogene and equivalent when head exempts from is mixed and made into emulsifying agent; The subcutaneous multi-point injection of femoribus internus, getting the same dose immunogene 3-4 week adds equivalent incomplete Freund mixing and emulsifying at interval, and booster immunization is once; Immunity is 5 times altogether, does not add adjuvant for the last time.Serum antibody titer is measured in last immunity blood sampling after 10 days, and heart is taken a blood sample, and obtains the polyclonal antibody of purifying with ammonium sulfate precipitation.
3, be coated with the preparation of the ELISA Plate of light blue specific antibody
With encapsulating damping fluid antibody dilution is become 0.15-0.25 μ g/ml, every hole adds 100 μ l, 37 ℃ of incubations 2 hours; 4 ℃ are spent the night again, and the coating buffer that inclines is with cleansing solution washing 5 times; Each 30 seconds, clap and do, in every hole, go into 200-300 μ l confining liquid then; 37 ℃ incubation 1-2 hour, liquid clap to be done in the hole of inclining, deposit with the password protection of aluminium film vacuum dry back.
Wherein, the used damping fluid that encapsulates is that the pH value is 9.5, the 0.05mol/L carbonate buffer solution; Used confining liquid is pH7.4, contains 5% lowlenthal serum, the caseic 0.02mol/L phosphate buffer of 10g/L.
4, horseradish peroxidase-labeled antigen:
Adopt the sodium periodate method to carry out coupling light blue haptens and horseradish peroxidase (HRP).Concrete grammar is:
A) take by weighing the 3mg light blue, be dissolved in the 400ul water, add 100 μ l 1M HCL in this solution, pH＜1.5
B) take by weighing 3mg NaNO
2, be dissolved in the 200 μ l water
C) with b) slowly add a), reaction is 1 hour under water bath condition, solution by colourless slowly become light yellow
D) claim 25mg HRP, be dissolved among the 5ml PBS,
E) with c) gained solution is added drop-wise to d in batches) in, keep between the pH7-8
F) PBS dialysed overnight
G) add preservation liquid, packing ,-20 ℃ are subsequent use.
Use horseradish peroxidase-labeled monoclonal antibody or polyclonal antibody.
Four, with the light blue specific antibody be the application of the enzyme linked immunological kit of coating antigen
1, the present invention provides the testing sample pre-treating method to be:
1) the beverage based food (extension rate: 5):
Like carbonated drink, beverage etc., get the 2ml heating and remove alcohol or carbon dioxide, add 8ml sample dilution, mixing is got 50 μ l and is detected
2) cocoa, jellies food (extension rate: 10)
Get the 1g sample, place 60 ℃ of water-baths to dissolve, add 9ml sample dilution, mixing adds the 5ml normal hexane again, and fully mixing is 10 minutes; 5000rpm centrifugal 10 minutes, gets middle layer 50 μ l and detects.
3) jam, fruits seasoning syrup (extension rate: 20)
Get the 1g sample, add 19ml sample dilution, mixing adds the 10ml normal hexane again, and fully mixing is 10 minutes; 5000rpm centrifugal 10 minutes, gets supernatant 50ul and detects.
2, detect
In the ELISA Plate micropore that is coated with the light blue specific antibody, add light blue standard solution or sample solution 50 μ l, add enzyme labeling light blue antigen working fluid 50 μ l again, with cover plate mould shrouding; Reaction is 30 minutes in 25 ℃ of constant temperature; Pour out liquid in the hole, every hole adds 300 μ l cleansing solutions, pours out liquid in the hole after 30 seconds; Repetitive operation 5 times is clapped dried with thieving paper; Add chromogenic substrate liquid 100 μ l, the mixing that vibrates gently, reaction is 30 minutes in 25 ℃ of constant temperature, and every hole adds 50 μ l 2mol/L stop buffer sulfuric acid, and mixing at the 450nm place, is measured every hole absorbance (OD value) with the ELIASA wavelength set.
3, testing result analysis
The absorbance mean value (B) of standard solution of using each concentration that is obtained is divided by the absorbance (B of first standard items liquid (0 standard)
0), multiply by 100% again, obtain the percentage absorbance.
B is the mean light absorbency value of standard solution or sample solution in the formula, B
0It is the mean light absorbency value of 0 μ g/L standard solution.
With light blue standard items concentration (μ g/mL) value is the X axle, and the percentage absorbance is the Y axle, the drawing standard curve map.With the percentage absorbance of same way calculation sample solution, the concentration of corresponding each sample then can be read the content of light blue the sample from typical curve.The analysis of testing result also can be adopted regression equation method among the present invention, calculates sample solution concentration.The be more convenient for express-analysis of a large amount of samples of the analysis of testing result computer professional software also capable of using among the present invention, this method, whole testing process only needed just can accomplish in 60 minutes.
Embodiment 2, be that coating antigen, ELIAS secondary antibody are the preparation and the ELISA detection method of the kit of enzyme labeling thing with the hapten-carrier protein conjugate
One, be that coating antigen, ELIAS secondary antibody are the detection principle of the ELISA kit of enzyme labeling thing with light blue haptens and carrier protein couplet thing:
When the coating antigen on the ELISA Plate capillary strip is light blue hapten-carrier protein conjugate, in the ELISA Plate micropore, behind adding standard solution or the sample solution, add the light blue specific antibody; Light blue coupled antigen competition light blue specific antibody on light blue in the sample and the ELISA Plate; Wash plate, add enzyme mark antiantibody again and carry out amplification, with the colour developing of colour developing liquid; The content of sample absorbance and light blue is negative correlation, relatively can draw the content of light blue in the sample with typical curve.Simultaneously also can be according to the depth of color on the ELISA Plate, through with the more rough judgement sample of series concentration light blue standard solution color in the concentration range of light blue.
Two, be that coating antigen, ELIAS secondary antibody are that the kit of enzyme labeling thing generally can comprise with light blue hapten-carrier protein conjugate:
1, be coated with the ELISA Plate of light blue hapten-carrier protein conjugate, the concentration of coating antigen can be 0.15 μ g/ml.
2, enzyme mark antiantibody working fluid: ELIAS secondary antibody is sheep anti mouse antiantibody or goat-anti rabbit antiantibody with horseradish peroxidase-labeled; The dilution of ELIAS secondary antibody is that the final concentration that contains in dilution is lowlenthal serum, pH7.4, the 0.2mol/L phosphate buffer of 8% (quality percentage composition), and enzyme mark antiantibody working fluid dilutability is 1: 500.
3, light blue specific antibody working fluid: can be light blue polyclonal antibody or light blue monoclonal antibody working fluid; With 3000 times of light blue monoclonal antibody dilutions, obtain the specific antibody working fluid with dilution, said dilution is that the pH value is 7.4, the phosphate buffer of 0.2mol/L.
4, the light blue standard solution is 6 bottles, and concentration is respectively 0 μ g/mL, 1 μ g/mL, 3 μ g/mL, 9 μ g/mL, 27 μ g/mL, 81 μ g/mL.The solution of preparation standard items is that pH is 7.4, the phosphate buffer of 0.1mol/L for final concentration in the preparation standard solution.
5, substrate colour developing liquid: substrate colour developing liquid is hydrogen peroxide or urea peroxide, tetramethyl benzidine sulfate mixed solution, 7ml/ bottle, 1 bottle.
6, stop buffer: 2mol/L hydrochloric acid or sulfuric acid.
7, concentrated cleaning solution: the pH value is 7.4, and the final concentration that contains in cleansing solution is that 0.03% (quality percentage composition) sodium azide, the final concentration in cleansing solution are 0.05% (quality percentage composition) Tween-20,0.02mol/L phosphate buffer; The 40ml/ bottle, 1 bottle.
8, concentrate to redissolve liquid: containing redissolving final concentration in the liquid is that DMSO, the pH of 8% (quality percentage composition) is 7.4, the 0.2mol/L phosphate buffer; The 200ml/ bottle, 1 bottle.
Embodiment 3, kit precision, accuracy, keeping quality test
1, the precision of kit experiment
(1) standard solution replica test
From 3 batches of ELISA Plates according to the preparation of the method the embodiment 1, each extracts 10 micropores out, measures the absorbance (OD value) of 20 μ g/mL standard solutions, repeats 10 times, calculates coefficient of variation CV, and the result sees table 1.
Table 1 standard solution replica test
| cv％ | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| 01 batch | 8.5 | 6.2 | 6.4 | 6.5 | 7.0 | 8.1 | 8.2 | 8.3 | 7.9 | 8.4 |
| 02 batch | 7.7 | 7.3 | 7.9 | 8.2 | 8.0 | 9.0 | 8.3 | 7.8 | 7.6 | 7.3 |
| 03 batch | 7.6 | 7.2 | 7.3 | 8.7 | 6.5 | 7.2 | 6.3 | 8.1 | 8.2 | 8.5 |
The result shows variation within batch coefficient scope that the kit standard items detect between 6.2～9.0%, and interassay coefficient of variation is 9.1%, meet the coefficient of variation criticize in less than 10%, between batch less than 20% regulation
(2) sample replica test
Add standard items to negative fruit juice, solid soup class, pulp based food, adding final concentration is 20 μ g/ml.Get each five of the kits of three different batches respectively, each concentration repeats 3 times, calculates the coefficient of variation respectively, and the result sees table 2-4.
The repeatable test of table 2 fruit juice sample
The repeatable test of table 3 solid soup class
The repeatable test of table 4 jam
2, the accuracy test of kit
Sample is added recovery test, and adding final concentration is 10 μ g/ml, 30 μ g/ml.It is parallel that each concentration is done 3 holes, and calculate recovery rate is seen table 5 respectively.
Table 5
3, kit storage life test
(1) kit is positioned over 2～8 ℃; Get 0,2,4,6,8,9,10,11 and 12 months kit respectively, to absorbance, 50% inhibition concentration of light blue standard model (1 μ g/ml), add the recovery, each parameter of variation within batch coefficient is measured.
(2) with kit the condition held of 37 ℃ of preservations 12 days, every day to absorbance, 5096 inhibition concentrations of light blue standard model (1 μ g/ml), add the recovery, each parameter of variation within batch coefficient is measured.
(3) kit was preserved 12 days at-20 ℃ of refrigerators, measure absorbance, 50% inhibition concentration, the interpolation recovery, each parameter of variation within batch coefficient of light blue standard model (1 μ g/ml) every day.
Can find out that from the result preserve test through three kinds of conditions, the absorbance of light blue standard model (1 μ g/ml) descends less than 5%, and OD is not less than 1.5; 50% inhibiting rate is between 20～40 μ g/ml; Add the recovery between 70～105%; Variation within batch coefficient coefficient is less than 10%; Each item index all conforms to quality requirements, and therefore, kit can be preserved 12 months at 2～8 ℃.
Claims (10)
1. enzyme linked immunological kit that detects light blue comprises the specific antibody of light blue haptens and light blue; Said specific antibody is the polyclonal antibody or the monoclonal antibody of light blue.
2. kit according to claim 1 is characterized in that:
1) said light blue haptens and carrier protein couplet, the hapten-carrier protein conjugate that obtains be as coating antigen, and said specific antibody is an enzyme labeling;
2) said specific antibody is as coating antigen, and said haptens is an enzyme labeling.
3. kit according to claim 1 is characterized in that: said kit also comprises antiantibody, like sheep anti mouse antiantibody or goat-anti rabbit antiantibody; And:
1) said light blue haptens and carrier protein couplet, the hapten-carrier protein conjugate that obtains be as coating antigen, and said antiantibody is an enzyme labeling;
2) said antiantibody is as coating antigen, and said haptens is an enzyme labeling.
4. according to each described kit among the claim 1-3, it is characterized in that: said kit also comprises ELISA Plate, light blue standard solution, substrate colour developing liquid, stop buffer, concentrated cleaning solution, concentrate and redissolve at least a in the liquid.
5. according to each described kit among the claim 1-3, it is characterized in that: any in said light blue haptens or light blue hapten-carrier protein conjugate, said light blue specific antibody, the said antiantibody is coated on the ELISA Plate.
6. according to each described kit among the claim 1-3, it is characterized in that: used marker enzyme is horseradish peroxidase or alkaline phosphatase in the said enzyme labeling; When marker enzyme was horseradish peroxidase, the substrate that uses colour developing liquid was hydrogen peroxide or urea peroxide, tetramethyl benzidine sulfate mixed solution, and stop buffer is 1-2mol/L sulfuric acid solution or hydrochloric acid solution; When marker enzyme was alkaline phosphatase, the colour developing liquid that uses was the nitro phosphate buffer, and stop buffer is the 1-2mol/L sodium hydroxide solution.
7. kit according to claim 4; It is characterized in that: said concentrated cleaning solution is the pH value for 6-9, to contain antiseptic, the final concentration that final concentration is 0.02-0.05% (quality) be the Tween-20 of 1.0-2.0% (quality), the phosphate buffer of 0.1-0.2mol/L; Preferably, said concentrated cleaning solution is that the pH value is 7.4, to contain at final concentration be the antiseptic of 0.03% (quality), the 0.2mol/L phosphate buffer of Tween-20 that final concentration is 1.5% (quality); Said concentrated redissolution liquid is for containing the DMSO that final concentration is 5-10% (quality), the 0.1-0.2mol/L phosphate buffer that pH is 6-8; Preferably, said concentrated redissolution liquid is 7.4 0.2mol/L phosphate buffer for containing DMSO, pH that final concentration is 8% (quality); Said substrate colour developing liquid is hydrogen peroxide or urea peroxide, tetramethyl benzidine sulfate mixed solution; Said stop buffer is 2mol/L sulfuric acid solution or hydrochloric acid solution.
8. the method for the light blue content in the test sample may further comprise the steps:
1) with light blue specific antibody coated elisa plate;
2) the light blue haptens of adding standard items or testing sample and enzyme labeling is hatched, washing;
3) add the colour developing of substrate colour developing liquid;
4) add the reaction terminating liquid cessation reaction;
5) through comparing the color of light blue standard items and testing sample, infer the light blue content that in the testing sample; Perhaps, measure the absorbance in each hole, set up the typical curve of light blue concentration, and extrapolate the light blue content in the testing sample by the absorbance of testing sample according to this typical curve with respect to absorbance.
9. the method for the light blue content in the test sample may further comprise the steps:
1) with light blue haptens or light blue hapten-carrier protein conjugate coated elisa plate;
2) add light blue standard items or testing sample;
3) add the light blue specific antibody, hatch, washing;
4) antiantibody of adding enzyme labeling is hatched, washing;
5) add the colour developing of substrate colour developing liquid;
6) add the reaction terminating liquid cessation reaction;
7) through comparing the color of light blue standard items and testing sample, infer the light blue content that in the testing sample; Perhaps, measure the absorbance in each hole, set up the typical curve of light blue concentration, and extrapolate the light blue content in the testing sample by the absorbance of testing sample according to this typical curve with respect to absorbance.
10. the method for light blue content according to Claim 8 or in the 9 described test sample also comprises the sample pre-treatments step, wherein:
When said sample was liquid food, said sample-pretreating method was: liquid food is removed gas, with sample and sample dilution with volume ratio 1: 3-8 mixes, and gets to mix the back sample and be used for analyzing;
When said sample was cocoa, jellies, said sample-pretreating method was: with thing water-bath to be checked dissolving, with the sample dilution be 1 with mass volume ratio: 10-15, add the normal hexane degreasing of certain volume again, centrifugal, get supernatant and detect.
When said sample was jam, fruits seasoning syrup, said sample-pretreating method was: is 1 with determinand and sample dilution with volume ratio: the 20-25 mixing, add a certain amount of normal hexane degreasing again, and get supernatant after centrifugal and detect.
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