CN104569381A - Method and enzyme linked immunosorbent assay kit for detecting aflatoxin M1 - Google Patents
Method and enzyme linked immunosorbent assay kit for detecting aflatoxin M1 Download PDFInfo
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Abstract
The invention discloses a method and an enzyme linked immunosorbent assay kit for detecting the content of aflatoxin M1 in a sample. The operation steps can be decreased by adopting direct competitive ELISA detection mode through adopting high-specificity and high-affinity antibodies, and the detection sensitivity and accuracy can be enhanced; compared with the antibody coating, the coating of an ELISA plate is carried out by adopting coating antigen, the better coating effect and long storage time can be more favorably achieved, and further the detection precision and stability of the kit can be enhanced; in addition, according to the kit, marking is carried out by adopting ELISA plate labelled antibody technology through adopting an improved periodate oxidization method, enzyme is directly labelled on an aflatoxin M1 specificity antibody, the two most important reactants namely aflatoxin M1 specificity antibody and enzyme are combined into a whole, so that the labelling efficiency is improved, the usage amounts of the enzyme and the antibody are saved, the good activities of the labelled enzyme and the antibody are guaranteed, the antibody is unnecessarily arranged in the kit, and the cost of the kit is greatly lowered.
Description
Technical field
The present invention relates to a kind of method for detecting Aflatoxins M1 and enzyme linked immunological kit.
Background technology
Aflatoxin is the poisonous metabolic product of a class fungi (as aspergillus flavus and aspergillus parasiticus), and they have very strong carcinogenicity, is mainly present in cereal, nut, cottonseed and some and human blood, in the product that animal feed is relevant.Aflatoxins M1 is the hydroxylation metabolism product of aflatoxin B1, is also a kind of strong carcinogen.Cow's milk and goods thereof are one of food being vulnerable to Aflatoxins M1 pollution.The detection method of Aflatoxin M1 has high performance liquid chromatography (HPLC), thin layer chromatography (TLC) etc.Use Aflatoxins M1 ELISA kit then can analyze Aflatoxins M1 in sample fast and accurately to remain.At present, the method that Aflatoxins M1 national standard is taked is thin layer chromatography, spectrophotometer method, fluorescent spectrometry and high performance liquid chromatography.In recent years, China's researcher has done a large amount of work in Aflatoxins M1 context of detection, but all mainly concentrate on Physico-chemical tests aspect, literature search not yet finds the report of the EL ISA detection method about Aflatoxins M1, Given this, the present invention studies and establishes the direct competitive EL ISA detection method of Aflatoxins M1.
Summary of the invention
The object of the invention is for solving current Aflatoxins M1 testing cost costly, and can not detect fast in enormous quantities, the technical matters of testing requirement on the spot can not be met.
In order to solve the problems of the technologies described above, the invention provides a kind of method detecting Aflatoxins M1 content in sample, comprising the following steps:
1) with Aflatoxins M1 specific antibody coated elisa plate;
2) add the Aflatoxins M1 haptens of standard items or testing sample and enzyme labeling, hatch, washing;
3) colour developing of substrate nitrite ion is added;
4) reaction terminating liquid cessation reaction is added;
5) by comparing the color of Aflatoxins M1 standard items and testing sample, the Aflatoxins M1 content in testing sample is inferred; Or, measure the absorbance in each hole, set up Aflatoxins M1 relative concentration in the typical curve of absorbance, and to be extrapolated the Aflatoxins M1 content in testing sample by the absorbance of testing sample according to this typical curve.
The present invention also provides the another kind of method detecting Aflatoxins M1 content in sample, comprises the following steps: 1) with Aflatoxins M1 haptens or Aflatoxins M1 hapten-carrier protein conjugate coated elisa plate;
2) Aflatoxins M1 standard items or testing sample is added;
3) add Aflatoxins M1 specific antibody, hatch, washing;
4) add the antibody of enzyme labeling, hatch, washing;
5) colour developing of substrate nitrite ion is added;
6) reaction terminating liquid cessation reaction is added;
7) by comparing the color of Aflatoxins M1 standard items and testing sample, the Aflatoxins M1 content in testing sample is inferred; Or, measure the absorbance in each hole, set up Aflatoxins M1 relative concentration in the typical curve of absorbance, and to be extrapolated the Aflatoxins M1 content in testing sample by the absorbance of testing sample according to this typical curve.
Further, the method for Aflatoxins M1 content in detection sample provided by the present invention, can also comprise sample pre-treatments step:
When described sample is milk, described sample-pretreating method is: get subnatant after sample is centrifugal to be measured;
When described sample is milk powder, described sample-pretreating method is: be 1: 4-6 to mix sample and deionized water with mass volume ratio, gets subnatant to be measured after centrifugal.
The present invention also provides a kind of enzyme linked immunological kit detecting Aflatoxins M1, comprises the specific antibody of Aflatoxins M1 haptens and Aflatoxins M1; Described specific antibody is polyclonal antibody or the monoclonal antibody of Aflatoxins M1.
Further, described Aflatoxins M1 haptens and carrier protein couplet, the Aflatoxins M1 hapten-carrier protein conjugate that obtains is as coating antigen, and described specific antibody is enzyme labeling; Described specific antibody is as coating antigen, and described haptens is enzyme labeling.
Further, described Aflatoxins M1 haptens and carrier protein couplet, the Aflatoxins M1 hapten-carrier protein conjugate obtained is as coating antigen; Described haptens is enzyme labeling.
Further, at least one in ELISA Plate, Aflatoxins M1 standard solution, substrate solution, substrate buffer solution, reaction terminating liquid, concentrated cleaning solution, sample diluting liquid is also comprised.
Further, described concentrated cleaning solution is pH value is 7.2-7.6, be the Tween-20 of 4.0-6.0% (quality), the phosphate buffer of 0.1-0.2mol/L containing final concentration, preferably, described concentrated cleaning solution is pH value is 7.4, be the 0.1mol/L phosphate buffer of the Tween-20 of 5% (quality) containing final concentration; Described reaction terminating liquid is the sulfuric acid solution of 1-2mol/L or the hydrochloric acid solution of 2mol/L.
Further, marker enzyme used in described enzyme labeling is horseradish peroxidase or alkaline phosphatase;
When marker enzyme is horseradish peroxidase, described substrate solution is for containing 3,3,5, phosphoric acid-the citric acid solution of the pH=5.0 of 5-tetramethyl benzidine or o-phenylenediamine, described substrate buffer solution is the phosphoric acid-citric acid solution of the pH=5.0 containing hydrogen peroxide or urea peroxide;
When marker enzyme is alkaline phosphatase, described substrate solution is to nitro phosphate buffer, and described substrate buffer solution is the phosphoric acid-citric acid solution of pH=5.0 containing hydrogen peroxide or oxidation urea.
Further, any one of the specific antibody of described Aflatoxins M1 haptens or Aflatoxins M1 hapten-carrier protein conjugate, described Aflatoxins M1 has been coated in ELISA Plate.
The present invention adopts direct competive ELISA detecting pattern, adopts the antibody of high specific, high affinity, decreases operation steps, improve the sensitivity of detection, accuracy, adopt envelope antigen to carry out the bag quilt of ELISA Plate, relative to antibody bag quilt, be more conducive to reaching and wrap by effect and longer holding time preferably, thus improve precision and the stability of kit detection, this kit utilizes ELISA Plate labelled antibody technology in addition, and adopt the periodates oxidizing process after improvement to mark, enzyme is directly marked on Aflatoxins M1 specific antibody, Aflatoxins M1 specific antibody and enzyme two kinds of most important reactants are united two into one, improve labeling effciency, save the consumption of enzyme and antibody, after ensureing mark, enzyme and antibody have good activity, not only enormously simplify operation steps and reaction time, decrease the error because complicated operation causes, and without the need to configuring antiantibody again in kit, also save the consumption of Aflatoxins M1 specific antibody and enzyme simultaneously, thus greatly reduce the cost of kit.Based on above advantage, the present invention is highly suitable for the residual trace analysis of Aflatoxins M1 and batch detection, has important practical significance.
Accompanying drawing explanation
Fig. 1 is the ELISA examination criteria curve of Aflatoxins M1.
Embodiment
In conjunction with the accompanying drawings, the present invention is further detailed explanation.These accompanying drawings are the schematic diagram of simplification, only basic structure of the present invention are described in a schematic way, and therefore it only shows the formation relevant with the present invention, and it should not be construed as limitation of the present invention.The experimental technique used in following embodiment if no special instructions, is conventional method.
Embodiment 1, with the preparation of the Aflatoxins M1 haptens kit that is coating antigen and ELISA detection method
One, Cleaning Principle:
First by antigen coated for Aflatoxins M1 in solid phase carrier, such as, in ELISA Plate, then standard items or testing sample is added, add enzyme labeling Aflatoxins M1 antibody again, Aflatoxins M1 in envelope antigen and standard items/testing sample competes enzyme labelled antibody, during standard items/testing sample Aflatoxins M1 content height, the enzyme labelled antibody be then combined with solid phase antigen is just few, otherwise the enzyme labelled antibody be combined on solid phase antigen is just many, add substrate solution after reaction to carry out developing the color being measured, when enzyme labelled antibody amount one timing, the testing sample added is more containing Aflatoxins M1, fewer with solid phase antigen desmoenzyme labeling antibody, color reaction weakens, percentage absorbance is low, otherwise, then color reaction strengthens, percentage absorbance increases, thus with percentage absorbance for ordinate, with Aflatoxins M1 standard concentration for horizontal ordinate, map at semilog coordinate axle and obtain typical curve, again according to the typical curve of Aflatoxins M1 and the percentage absorbance of measuring samples, the concentration of Aflatoxins M1 in testing sample can be extrapolated.
Two, kit generally can comprise:
1, the haptenic ELISA Plate of Aflatoxins M1 is coated with, ELISA Plate adopts 96 or 40 hole ELISA Plate, coating buffer used is the carbonate buffer solution of pH7.4,0.05mol/L, carbonate buffer solution is containing 1-2g sodium carbonate, 2-4g sodium bicarbonate and distilled water 1L, confining liquid is pH7.4, containing 5% lowlenthal serum, the caseic 0.02mol/L phosphate buffer of 10g/L.
2, enzyme labeling Aflatoxins M1 antibody working fluid: adopting periodates method enzyme and Aflatoxins M1 antibody to be carried out, coupling obtains.Marker enzyme used can be the alkaline phosphatase extracted in horseradish peroxidase or bacterium, the present invention is preferably horseradish peroxidase, and adopt the periodates oxidizing process after improvement to mark, improve labeling effciency, save the consumption of enzyme and antibody, after ensureing mark, enzyme and antibody have good activity.
3, Aflatoxins M1 specific antibody working fluid: can be Aflatoxins M1 polyclonal antibody or Aflatoxins M1 monoclonal antibody working fluid; With dilution, Aflatoxins M1 monoclonal antibody is diluted 3000 times, obtains specific antibody working fluid, to be pH value be described dilution 7.4, the phosphate buffer of 0.2mol/L.
4, Aflatoxins M1 standard solution 6 bottles, 1ml/ bottle, concentration is respectively 0 μ g/mL, 0.1 μ g/mL, 0.3 μ g/mL, 0.9 μ g/mL, 2.7 μ g/mL, 8.1 μ g/mL.Be final concentration to be pH be the solution of preparation standard items 7.4, the phosphate buffer of 0.1mol/L.
5, substrate nitrite ion: substrate nitrite ion is hydrogen peroxide or urea peroxide, tetramethyl benzidine sulfate mixed solution, 7ml/ bottle, 1 bottle.
The sulfuric acid solution of 6, stop buffer: 2mol/L or sodium hydroxide solution, 7ml/ bottle, 1 bottle.
7, concentrated cleaning solution: pH value is 7.4, containing final concentration in cleansing solution be 0.03% (mass percentage) sodium azide, final concentration in cleansing solution is 0.05% (mass percentage) Tween-20,0.02mol/L phosphate buffer; 50ml/ bottle, 1 bottle.
8, sample diluting liquid: pH is 7.4,0.01mol/L phosphate buffer, 70ml/ bottle, 1 bottle.
Embodiment 2, with Aflatoxins M1 specific antibody be coating antigen, the preparation of the antigen kit that is enzyme marker and ELISA detection method
One, with Aflatoxins M1 specific antibody be coating antigen, the antigen ELISA Cleaning Principle that is enzyme marker:
When the coating antigen on ELISA Plate capillary strip is Aflatoxins M1 specific antibody, add standard solution or sample solution in enzyme plate micropore after, and enzyme marker, Aflatoxins M1 specific antibody on Aflatoxins M1 in sample and enzyme marker competition binding microwell plate, wash plate, colour developing, in sample light absorption value and sample or standard items, the content of Aflatoxins M1 is negative correlation, compares the content that can draw Aflatoxins M1 in sample with typical curve.Simultaneously also can according to the depth of color in ELISA Plate, by the content with Aflatoxins M1 in the more rough judgement sample of series concentration Aflatoxins M1 standard solution color.
Two, with Aflatoxins M1 specific antibody be coating antigen, antigen is enzyme marker kit generally can comprise:
1, be coated with the ELISA Plate of Aflatoxins M1 specific antibody, the concentration of coating buffer can be 0.15-0.25 μ g/ml.
2, enzyme-labelled antigen working fluid: enzyme-labelled antigen is the Aflatoxins M1 antigen by horseradish peroxidase-labeled; The dilution of enzyme-labelled antigen be containing the final concentration in dilution be the lowlenthal serum of 6-8% (mass percentage), pH is 6-8,0.2-0.3mol/L phosphate buffer, enzyme-labelled antigen working dilution is 1: 1000.
3, Aflatoxins M1 standard solution 6 bottles, concentration is respectively 0 μ g/mL, 0.1 μ g/mL, 0.3 μ g/mL, 0.9 μ g/mL, 2.7 μ g/mL, 8.1 μ g/mL.The phosphate buffer of the solution of preparation standard items be final concentration to be pH be 7.0-7.5,0.1-0.2mol/L.
4, substrate nitrite ion: substrate nitrite ion 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: pH value is 7.2-7.6, the final concentration contained in cleansing solution is the Tween-20 of 1.0-2.0% (mass percentage), the phosphate buffer of 0.1-0.2mol/L; 40ml/ bottle, 1 bottle.
7, sample diluting liquid: pH is 7.4,0.01mol/L phosphate buffer, 70ml/ bottle, 1 bottle.
Three, with Aflatoxins M1 specific antibody be coating antigen, the concrete composition of the antigen kit that is enzyme marker and preparation thereof:
(1) form
1, be coated with the ELISA Plate of Aflatoxins M1 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 Aflatoxins M1 of horseradish peroxidase-labeled and the conjugate of carrier protein; The dilution of enzyme-labelled antigen be containing the final concentration in dilution be the lowlenthal serum of 6% (mass percentage), pH is 7.4,0.2mol/L phosphate buffer, enzymic-labelled antibody working dilution is 1: 1000.
3, Aflatoxins M1 standard solution 6 bottles, concentration is respectively 0 μ g/mL, 1 μ Wagtail _ %3_jg/mL, 3 μ g/mL, 9 μ g/mL, 27 μ g/mL, 81 μ g/mL, the solution of preparation standard items be in preparation standard solution final concentration to be pH be 7.2, the phosphate buffer of 0.1mol/L.
4, substrate nitrite ion: substrate nitrite ion is hydrogen peroxide or urea peroxide, tetramethyl benzidine sulfate mixed solution, 7ml/ bottle, 1 bottle.
5, stop buffer: 2mol/L sulfuric acid.
6, concentrated cleaning solution: pH value is 7.4, the final concentration in cleansing solution is 1.5% (mass percentage) Tween-20,0.2mol/L phosphate buffer; 40ml/ bottle, 1 bottle.
7, sample diluting liquid: pH is 7.4,0.01mol/L phosphate buffer, 70ml/ bottle, 1 bottle.
(2) prepare
1, the preparation of Aflatoxins M1 antigen:
A.200mg the amino Aflatoxins M1 of purifying adds the ratio of 0.3mol/L HCL 12mL, by the amino Aflatoxins M1 stirring and dissolving of purifying;
B. under the condition of ice bath stirring, 10g/L NaN02 is slowly added, make amino Aflatoxins M1 diazotising, whether excessively monitor NaN02 simultaneously, detection method: get starch/liquor kalii iodide and drip on white dried glass sheet, drip 1 above-mentioned diazo solution, after mixing, in 30s, present the black-and-blue diazotising namely completing amino Aflatoxins M1;
C. above-mentioned diazotizing amino Aflatoxins M1 continues reaction 20min, takes the BSA of 800mg, is dissolved in carbonate buffer solution, make 20g/L protein solution, slowly adds the amino Aflatoxins M1 solution of diazotising under ice bath stirring condition; Edged 0.01mol/L NaOH in limit regulates pH to 7.5 ~ 8.0, and acquisition Aflatoxins M1 and bovine serum albumin(BSA) obtain conjugate, are placed in 4 DEG C of refrigerator overnight;
D. under 4 DEG C of conditions, first with rare HCL (0.01mol/L) the solution dialysis that pH value is lower, then with the PBS dialysis 3d of 0.1mol/L pH7.2, dislysate is changed every day 3 times, after packing freeze-drying ,-20 DEG C of preservations.
2, Aflatoxins M1 mouse monoclonal antibody preparation:
Animal immune program: adopt Balb/c mouse as immune animal, with Aflatoxins M1 haptens and bovine serum albumin(BSA) conjugate for immunogene, immunizing dose is 60 μ g/, during first immunisation, the Freund's complete adjuvant of immunogene and equivalent is mixed and made into emulsifying agent, lumbar injection, interval is got same dose immunogene for 3 weeks and is added equivalent incomplete Freund's adjuvant wet conjunction emulsification, and booster immunization is once, four immune pneumoretroperitoneum booster immunizations once, extracting spleen cell after 3 days.
Fusion of Cells and cloning: get immune Balb/c mouse boosting cell, in 4:1 ratio and SP2/0 myeloma cell fusion, adopt indirect competitive enzyme-linked immunosorbent method to measure cell conditioned medium liquid, screen positive hole.Microclone method is utilized to carry out cloning to positive hole, until obtain the hybridoma cell strain of stably excreting monoclonal antibody.
Cell cryopreservation and recovery: get the cell suspension that the hybridoma cryopreserving liquid being in exponential phase makes 5 × 106/mL, be sub-packed in cryopreservation tube, preserve for a long time in-70 DEG C of ultra low temperature freezers.Take out cryopreservation tube during recovery, put into 37 DEG C of water-bath middling speeds immediately and melt, after centrifugal segregation cryopreserving liquid, move into and cultivate culture in glassware.
The preparation and purification of monoclonal antibody: adopt in body and induce method, by the mouse peritoneal injection hybridoma 5 × 106 in 8 weeks age of Balb/c/, gather ascites after 7-10 days.Carry out ascites with immunochromatographic method to purify, bottle packing ,-20 DEG C of preservations.
3, the extraction of horseradish peroxidase or alkaline phosphatase
1), the extraction of horseradish peroxidase
A. water extraction is got: take the 20kg fresh horseradish of wash clean or horseradish skin, be cut into small pieces, rub in comminutor.Grinding residue-pulp adds 10kg water stirring and leaching 8h at low temperatures, with 3000r/min component velocity centrifugal 10 minutes, collects supernatant.
B. ammonium sulfate fractionation is separated: often liter of filtrate adds 226g ammonium sulfate powder, and limit edged stirs, and puts overnight at room temperature.Next day, Aspirate supernatant, then added 258g ammonium sulfate powder by often going up clear liquid, with adding with stirring, after ammonium sulfate dissolving completely, putting cold house and spending the night.Next day sucks supernatant, sediment fraction in refrigerated centrifuge with the centrifugal 20min of 13000r/min, abandoning supernatant, collecting precipitation.Precipitation be dissolved in 200 ~ 300mL distilled water, be sub-packed in bag filter, dialyse l-2d in circulating water, until the water appeared add barium chloride solution without precipitation generate till.And then the 8h that dialyses in distilled water.Merge dislysate, with the centrifugal 15min of 4000r/min in refrigerated centrifuge, collect supernatant.
C. acetone classification is separated: poured into by supernatant in the bath of beaker juxtaposition cryosel, under constantly stirring, add along wall of cup the acetone that equal-volume is chilled to-15 DEG C in advance with dropper, with the centrifugal 15min of 4000r/min in refrigerated centrifuge, collect supernatant, add-15 DEG C of acetone of former supernatant volume 0.8 times again, centrifugal (condition is the same) collecting precipitation.Precipitation be dissolved in a small amount of distilled water, dialysis (method is the same) removing acetone, obtains thick HRP.
D. refine: often liter of crude enzyme liquid adds 1mL 1M solution of zinc sulfate, with the centrifugal 10min of 5000r/min in refrigerated centrifuge, collect supernatant, be sub-packed in bag filter, sulfuric acid of dialysing in flowing water zinc, about needs 1d, and then dialyse 8h in distilled water.Dislysate is merged, carries out vacuum drying and namely obtain refining HRP.Product is ecru threadiness fluffer.
2), alkaline phosphatase
The EColi-317 strain fermentation producing alkaline phosphatase is utilized to cultivate, centrifugal (the 8000r/min of fermentation liquor, 1Omin), the thalline of precipitation uses lysozyme broken wall after 5 × 10-4M EDTA--0.03M pH 8.0Tris--0.5M sucrose height sepage process, upper clear enzyme solution through DEAE cellulose stirring and adsorbing and wash-out, thermal treatment and Sephadex G-100 sieve chromatography purifying.
4, the preparation of enzyme labeling Aflatoxins M1 antibody
The preparation that horseradish peroxidase HRP marks Aflatoxins M1 antibody adopts periodates oxidizing process, and adopt Over-voltage protection to carry out coupling Aflatoxins M1 antibody and horseradish peroxidase (HRP), concrete grammar is:
A. dissolve 5mg HRP in 1mL ultrapure water, the 0.1mol/L sodium periodate 75 μ L. adding new configuration puts room temperature or 4 DEG C of refrigerator reaction 20min or 30min.
B. reacted rear loading bag filter, 0.00lmol/L pH4.0 hac buffer 4 DEG C of dialysed overnight, change dislysate as required.
C. Aflatoxins M1 antibody 0.1mol/L carbonic acid buffer is diluted to 10mg/mL, with 0.1mol/L carbonic acid buffer, the pH value of solution of the HRP activated is adjusted to 9.5 in addition.0.5mL antibody is added in HRP solution, puts room temperature or 4 DEG C of refrigerator reaction 2h.
D. 100 μ L 4mg/mL sodium borohydrides are added, 4 DEG C of refrigerator reaction 2h.
E. to 0.0lmol/L PBS dialysed overnight, it is for subsequent use to add conserving liquid-20 DEG C of preservations.
Four, with the application of the Aflatoxins M1 specific antibody enzyme linked immunological kit that is coating antigen
1, the invention provides testing sample pre-treating method is:
1) milk (extension rate: 1)
Get testing sample, centrifugal under 7000rpm room temperature, 5min;
Get subnatant 50 μ l to be measured.
2) milk powder (extension rate: 5)
Get 1.0g milk powder, add 5ml deionized water, mixing;
Centrifugal under 7000rpm room temperature, 5min;
Get subnatant 50 μ l to be measured.
2, detect
(1) taken out from cold storage environment by kit, be placed in room temperature, namely 20 ~ 24 DEG C, balance 30min, the batten of enough standard items and sample quantity used is fixed on support, and standard items and sample do two parallel laboratory tests, number in order.
(2) add 50 μ L standard items in standard sample wells, sample well adds 50uL testing sample.Then every hole adds 50 μ L enzyme marker, and jog mixes.Cover cover plate film, hatch 15min room temperature or 25 DEG C.
(3) pour out the liquid in hole, be upside down on thieving paper by micropore frame and pat, often wheel is washed plate and is patted 5 times, to ensure the liquid removed completely in hole.Be filled with in hole with 250 μ L cleansing solutions, again outwell the liquid in micropore, then repetitive operation 5 times.
(4) every hole adds 100 μ L nitrite ions (being mixed with substrate solution equal-volume by substrate buffer solution), and pat mixing, cover cover plate film, dark at room temperature hatches 15min.
(5) add 50 μ L reaction terminating liquids in micropore, mixing, at wavelength 450nm or 492nm, is blank with air, measures each hole light absorption value, must read immediately after termination.
Testing result calculates and analyzes:
With the mean value calculation percentage absorbance of obtained standard model light absorption value, with percentage absorbance for ordinate, the semilog of Aflatoxins M1 concentration of standard solution is horizontal ordinate drawing standard curve, obtains straight-line equation.See accompanying drawing l.Y=-14.756X+87.767; R2=0.9974, the percentage absorbance of the calculation sample solution that uses the same method, obtains the Aflatoxins M1 concentration of counter sample according to equation.The calculating formula of described percentage absorbance is:
Percentage absorbance (%)=(B/Bo) × 100
Wherein, B is the average absorbance value of standard solution or sample, and Bo is the mean absorbance values of 0 μ g/L standard solution.
The analysis of testing result can also utilize computer professional software to carry out calculating and analyze, and be 0.5 ~ 50 μ g/L to Aflatoxins M1 linear detection range, detect and be limited to 0.5 μ g/L, whole testing process only needs 35min just can complete.
Embodiment 3, kit precision, accuracy, keeping quality test
1, the precision test of kit
In the ELISA Plate prepared according to the method embodiment 4 (6) from 3 batches, respectively extract 10 micropores out, measure the absorbance (OD value) of 9 μ g/mL standard solutions, repeat 10 times, calculating coefficient of variation CV, the results are shown in Table 1.
Table 1 standard solution replica test
| cv％ | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| 01 batch | 8 | 6.5 | 7.3 | 6.6 | 6.9 | 7.1 | 8.2 | 6.8 | 7.4 | 8.1 |
| 02 batch | 8.2 | 6.8 | 6.7 | 6.9 | 7.2 | 7.4 | 8.4 | 6.3 | 6.6 | 8.3 |
| 03 batch | 7.2 | 6.8 | 6.3 | 6.1 | 8 | 7.5 | 6.3 | 7.6 | 8.1 | 7.7 |
Result shows that variation within batch coefficient range that kit standard product detect is between 6.1 ~ 8.4%, and interassay coefficient of variation is 9.6%, meet the coefficient of variation criticize in be less than 10%, be less than the regulation of 20% between batch.
2, sample repeatability and accuracy test
Accuracy refers to the matching degree of measured value and true value, and in ELISA measures, accuracy often represents with the recovery, and precision often represents with the coefficient of variation.In dummy (as milk), Aflatoxins M1 being added into final concentration is 2ug/ml, 10ug/ml, each dense shelf each 10 parallel, measure 3 batches.Calculating mean value, TIANZHU XINGNAO Capsul and batch interior and interassay coefficient of variation.The results are shown in Table 2.
The test of table 2 milk repeatability
3, storage life test
(1) kit is positioned over 2 ~ 8 DEG C, get the kit of 0,2,4,6,8,9,10,11 and 12 months respectively, the absorbance of Aflatoxins M1 standard model (2.5ug/ml), 50% inhibition concentration, TIANZHU XINGNAO Capsul, each parameter of variation within batch coefficient are measured.
(2) placed 12 days under 37 DEG C of conditions of preserving by kit, every day measures the absorbance of Aflatoxins M1 standard model (2.5ug/ml), 50% inhibition concentration, TIANZHU XINGNAO Capsul, each parameter of variation within batch coefficient.
(3) by kit-20 DEG C of Refrigerator stores 12 days, every day measures the absorbance of Aflatoxins M1 standard model (2.5ug/ml), 50% inhibition concentration, TIANZHU XINGNAO Capsul, each parameter of variation within batch coefficient.
Can find out from result. through three kinds of condition food preservation test, the absorbance of Aflatoxins M1 standard model (2.5ug/ml) declines and is less than 5%, and OD is not less than 1.5; 50% inhibiting rate is between 0.5-2.5 μ g/ml; TIANZHU XINGNAO Capsul is between 70 ~ 105%; Variation within batch coefficient coefficient is less than 10%; Indices all conforms to quality requirements, and therefore, kit at 2 ~ 8 DEG C, can be preserved 12 months.
With above-mentioned according to desirable embodiment of the present invention for enlightenment, by above-mentioned description, relevant staff in the scope not departing from this invention technological thought, can carry out various change and amendment completely.The technical scope of this invention is not limited to the content on instructions, must determine its technical scope according to right.
Claims (10)
1. detect a method for Aflatoxins M1 content in sample, it is characterized in that, comprise the following steps:
1) with Aflatoxins M1 specific antibody coated elisa plate;
2) add the Aflatoxins M1 haptens of standard items or testing sample and enzyme labeling, hatch, washing;
3) colour developing of substrate nitrite ion is added;
4) reaction terminating liquid cessation reaction is added;
5) by comparing the color of Aflatoxins M1 standard items and testing sample, the Aflatoxins M1 content in testing sample is inferred; Or, measure the absorbance in each hole, set up Aflatoxins M1 relative concentration in the typical curve of absorbance, and to be extrapolated the Aflatoxins M1 content in testing sample by the absorbance of testing sample according to this typical curve.
2. detect a method for Aflatoxins M1 content in sample, it is characterized in that, comprise the following steps:
1) with Aflatoxins M1 haptens or Aflatoxins M1 hapten-carrier protein conjugate coated elisa plate;
2) Aflatoxins M1 standard items or testing sample is added;
3) add Aflatoxins M1 specific antibody, hatch, washing;
4) add the antibody of enzyme labeling, hatch, washing;
5) colour developing of substrate nitrite ion is added;
6) reaction terminating liquid cessation reaction is added;
7) by comparing the color of Aflatoxins M1 standard items and testing sample, the Aflatoxins M1 content in testing sample is inferred; Or, measure the absorbance in each hole, set up Aflatoxins M1 relative concentration in the typical curve of absorbance, and to be extrapolated the Aflatoxins M1 content in testing sample by the absorbance of testing sample according to this typical curve.
3. the method for Aflatoxins M1 content in detection sample according to claim 1 and 2, is characterized in that, also comprise sample pre-treatments step, wherein:
When described sample is milk, described sample-pretreating method is: get subnatant after sample is centrifugal to be measured;
When described sample is milk powder, described sample-pretreating method is: be 1: 4-6 to mix sample and deionized water with mass volume ratio, gets subnatant to be measured after centrifugal.
4. detect an enzyme linked immunological kit for Aflatoxins M1, it is characterized in that, comprise the specific antibody of Aflatoxins M1 haptens and Aflatoxins M1; Described specific antibody is polyclonal antibody or the monoclonal antibody of Aflatoxins M1.
5. the enzyme linked immunological kit of detection Aflatoxins M1 according to claim 4, is characterized in that:
1) described Aflatoxins M1 haptens and carrier protein couplet, the Aflatoxins M1 hapten-carrier protein conjugate that obtains is as coating antigen, and described specific antibody is enzyme labeling;
2) described specific antibody is as coating antigen, and described haptens is enzyme labeling.
6. the enzyme linked immunological kit of detection Aflatoxins M1 according to claim 4, is characterized in that:
1) described Aflatoxins M1 haptens and carrier protein couplet, the Aflatoxins M1 hapten-carrier protein conjugate obtained is as coating antigen;
2) described haptens is enzyme labeling.
7. the enzyme linked immunological kit of the detection Aflatoxins M1 according to any one of claim 4-6, is characterized in that: also comprise at least one in ELISA Plate, Aflatoxins M1 standard solution, substrate solution, substrate buffer solution, reaction terminating liquid, concentrated cleaning solution, sample diluting liquid.
8. the enzyme linked immunological kit of detection Aflatoxins M1 according to claim 7, it is characterized in that: described concentrated cleaning solution is pH value is 7.2-7.6, be the Tween-20 of 4.0-6.0% (quality), the phosphate buffer of 0.1-0.2mol/L containing final concentration, preferably, described concentrated cleaning solution is pH value is 7.4, be the 0.1mol/L phosphate buffer of the Tween-20 of 5% (quality) containing final concentration; Described reaction terminating liquid is the sulfuric acid solution of 1-2mol/L or the hydrochloric acid solution of 2mol/L.
9. the enzyme linked immunological kit of detection Aflatoxins M1 according to claim 7, is characterized in that:: marker enzyme used in described enzyme labeling is horseradish peroxidase or alkaline phosphatase;
When marker enzyme is horseradish peroxidase, described substrate solution is for containing 3,3,5, phosphoric acid-the citric acid solution of the pH=5.0 of 5-tetramethyl benzidine or o-phenylenediamine, described substrate buffer solution is the phosphoric acid-citric acid solution of the pH=5.0 containing hydrogen peroxide or urea peroxide;
When marker enzyme is alkaline phosphatase, described substrate solution is to nitro phosphate buffer, and described substrate buffer solution is the phosphoric acid-citric acid solution of pH=5.0 containing hydrogen peroxide or oxidation urea.
10. the enzyme linked immunological kit of the detection Aflatoxins M1 according to any one of claim 4-6, is characterized in that: any one of the specific antibody of described Aflatoxins M1 haptens or Aflatoxins M1 hapten-carrier protein conjugate, described Aflatoxins M1 has been coated in ELISA Plate.
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