CN102313810A - Chemiluminescent immunoassay method for rapidly detecting zearalenone toxin - Google Patents
Chemiluminescent immunoassay method for rapidly detecting zearalenone toxin Download PDFInfo
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Abstract
A chemiluminescence immunoassay method for detecting zearalenone toxin in the technical field of biological detection engineering comprises the following steps of: linking ZEN semiantigen with OVA to obtain ZEN-OVA; extracting a sample to be measured to obtain an extract; coating ZEN-OVA on a 96-well white plate at the optimum coating concentration, followed by sealing, respectively adding a known solution of the ZEN pure product and anti-zearalenone monoclonal antibody and a mixed solution of the extract of the sample to be measured and anti-zearalenone monoclonal antibody at different concentrations, followed by incubation; adding HRP-labeled secondary antibody after incubation, and adding a luminescence substrate for luminescent determination to obtain a result; making a standard curve; and acquiring the ZEN concentration in the extract of the sample to be measured through the standard curve. The method provided by the invention can be used to rapidly detect the amount of ZEN in the sample to be measured; in the meanwhile, the method has strong pertinency of detection object and results in high accuracy.
Description
Technical field
The present invention relates to a kind of method of biological detection field of engineering technology, specifically is a kind of immunochemiluminescence method of fast detecting zearalenone toxin.
Background technology
(Zearalenone ZEN) is the sickle-like bacteria secondary metabolite that breeding is produced under certain humidity and temperature conditions to the zearalenone toxin.The zearalenone toxin can be present in cereal such as corn, wheat, barley, Chinese sorghum, rye, also can be present in edible containing in the animal tissue of ZEN feed, comprises milk, egg etc.Zearalenone toxin and spontaneous breast cancer, fallopian tubal and uterus oedema, hyperplasia, diseases such as spermatid distortion, apoptosis are relevant.The zearalenone toxin have distribute extensively, the residence time is long, hard to manage and other toxin have the phenomenon of enhancing toxicity together.After the accession to WTO; The quantum of international trade of agricultural product and relevant food increases day by day; Also increasingly high to the requirement of the biological safety of importing and exporting product thereupon; For the smooth listing that guarantees this series products and eater's health, departments such as entry and exit quarantine, customs, manufacturing enterprise, superintendent office press for a kind of fast and convenient zearalenone toxins checking method.
Chemiluminescence immune assay (Chemiluminescence Immunoassay; CLIA); Be to have highly sensitive chemical luminescent detecting technology to combine, be used for the check and analysis technology of various antigens, haptens, antibody, hormone, enzyme, fatty acid, vitamin and medicine etc. with the immune response of high specific.It is a up-to-date immunoassay of exempting to analyze, growing up after fluoroimmunoassay and the time resolved fluoro-immunoassay continue radioimmunology analysis, enzyme.
(Chemiluminescence just obtains confirming CL) that promptly moment is emitted energy with the form that discharges photon in chemical reaction process as far back as the eighties in 19th century in chemiluminescence.After develop and radiating immuning analysis technology (RIA), because of its high sensitivity and high specific receive widespread usage.CLIA is on the basis of RIA basic theories, is a kind of non-radioactive labelled immune analytic approach that the tracer signal is set up with the mark luminous agent.Development in recent years is swift and violent; Be to develop at present and apply the fastest immune analysis method; Also be present state-of-the-art label immunoassay technology, sensitivity and degree of accuracy are exempted from method, the high several magnitude of fluorescence method than enzyme, significantly are superior to radiommunoassay and ELISA.
Chemiluminescence enzyme immunoassay (Chemiluminescent Enzyme Immunoassay; CLEIA) belong to EIA enzyme immunoassay; Just the substrate of enzyme reaction is a luminous agent, and operation steps and EIA enzyme immunoassay are identical: carry out immune response with enzyme labeling bioactivator (like the antigen or the antibody of enzyme labeling), the enzyme on the immune response compound remakes and is used for luminous substrate; Luminous under the signal reagent effect, carry out luminescence assays with the luminous signal analyzer.In enzymatic chemical luminous immunoassay, can supply the enzyme of mark to have a lot, marker enzyme commonly used at present is horseradish peroxidase (HRP) and alkaline phosphatase (ALP).
When measuring, envelope antigen ZEN-OVA is coated on the 96 hole blanks, the sealing back adds anti-ZEN monoclonal antibody and testing sample; Antibody meeting and encapsulate the specific combination of ZEN onboard, and if contain ZEN in the testing sample, so can with the ZEN competition antibody that encapsulates onboard; The content that is ZEN in the testing sample is high more; It is few more that antibody combines with ZEN on the plate, otherwise, then many more.Adding two of sheep anti mouse HRP mark or ALP mark after hatching again resists; Two anti-can combinations with the antibody that adds before (one is anti-); After adding luminous substrate; Enzyme on the immune response compound remakes and is used for luminous substrate, and is luminous under the signal reagent effect, carries out luminescence assays with the luminous signal analyzer.The content of ZEN is high more in the testing sample at this moment, and it is low more to measure the luminous value that obtains; Otherwise, then high more (as shown in Figure 1).
Detect about the zearalenone toxin, set up several different methods both at home and abroad.The method that detects ZEN at present is mainly high performance liquid chromatography (HPLC), GC-MS (GC-MS), liquid spectrum and mass spectrometry method (LC-MS).Yet these methods need be carried out strict pre-service to test sample, also need expensive instruments such as high performance liquid chromatograph, require to have the operating personnel of specialty simultaneously, are unfavorable for on-the-spot conventional sense use.The ELISA kit of existing detection zearalenone toxin; Too late chemiluminescence ELISA method of the present invention on detection sensitivity; Therefore immunochemiluminescence will have higher detection sensitivity, and the safety that ensures cereals is had more important role.
Summary of the invention
The objective of the invention is to overcome the deficiency of prior art, a kind of immunochemiluminescence method of fast detecting zearalenone toxin is provided.Method of the present invention can fast detecting goes out the amount of the ZEN that contains in the testing sample; Simultaneously, method detected object of the present invention is with strong points, and accuracy rate is high, and the more commercially available ELISA detection kit of sensitivity is high; Method of the present invention adopts anti-zearalenone monoclonal antibody, and with the mycotoxin no cross reaction in other cereal, more commercially available is that basic ELISA kit has stronger specificity with the polyclonal antibody, has reduced false positive rate.
The immunochemiluminescence method of the fast detecting zearalenone toxin that the present invention relates to comprises the steps:
Step 1 with ZEN haptens and OVA coupling, obtains conjugate ZEN-OVA;
Step 2 extracts testing sample, obtains extract;
Step 3; ZEN-OVA is encapsulated concentration with the best to be coated on the 96 hole blanks; Add the pure article of ZEN of known variable concentrations and the mixed solution of anti-zearalenone monoclonal antibody after the sealing respectively, the mixed solution of testing sample extract and anti-zearalenone monoclonal antibody is hatched;
Step 4 is hatched two of back adding HRP mark and is resisted, and adds luminous substrate afterwards again, carries out luminescence assays, obtains the result;
Step 5 is according to the luminous value of the mixed solution of known pure article of variable concentrations ZEN and anti-zearalenone monoclonal antibody, production standard curve;
Step 6, the luminous value according to the mixed solution of testing sample extract and anti-zearalenone monoclonal antibody obtains the ZEN concentration in the testing sample extract through typical curve.
Preferably, in the step 1, the method for said coupling is an active ester method.
Preferably, in the step 2, said extraction is: get the 0.3g testing sample, add the 3ml volume fraction and be methyl alcohol-PBS solution of 70%, concuss 15min leaves standstill 30min, and supernatant is through the fast qualitative filter paper filtering, dilute 5 times after, must extract.
Preferably, in the step 3, said hatching is 37 ℃ and hatched 4 hours.
The present invention has following beneficial effect:
When containing the ZEN toxin in the test sample article; Because ZEN and anti-ZEN monoclonal antibody specificity combine, monoclonal antibody is closed with two resistive connections of HRP enzyme or ALP enzyme labeling again then, and enzyme remakes and is used for luminous substrate; Luminous under the signal reagent effect, carry out luminescence assays with the luminous signal analyzer; Because amount and the luminous detection result of the ZEN that contains in the testing sample have linear relationship, therefore can calculate the amount of the ZEN that contains in the testing sample through typical curve;
Method detected object of the present invention is with strong points, and accuracy rate is high, and the more commercially available ELISA detection kit of sensitivity is high;
Method of the present invention adopts anti-zearalenone monoclonal antibody, and with the mycotoxin no cross reaction in other cereal, more commercially available is that basic ELISA kit has stronger specificity with the polyclonal antibody, has reduced false positive rate.
Description of drawings
Fig. 1 is an embodiment of the invention principle; Wherein, a is not for adding the synoptic diagram that contains the ZEN sample, and b is for adding the synoptic diagram that contains the ZEN sample;
Fig. 2 is that the embodiment of the invention 96 hole blanks detect synoptic diagram.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.These embodiment only be used to the present invention is described and be not used in the restriction scope of the present invention.The experimental technique of unreceipted actual conditions in the following example; Usually according to normal condition; For example the Sambrook equimolecular is cloned: laboratory manual (New York:Cold Spring Harbor Laboratory Press; 1989) condition described in, or the condition of advising according to manufacturer.
Term " ZEN " is the abbreviation of zearalenone toxin, and " OVA " is the abbreviation of O-ethyloic hydroxyl.
Embodiment
1, the preparation of antigen, the preparation of zearalenone envelope antigen (ZEN-OVA)
Get 0.33ml (3mg/ml) ZEN, be mixed in the 1.2ml pyridine, add 2mg O-ethyloic azanol, stirring at room reaction 24h.After the vacuum drying, add 4ml distilled water, and make its dissolving, adjustment pH to 8.0.Unreacted ZEN removes (3ml benzene, extracting is 3 times altogether) with the benzene extracting, removes the benzene phase, keeps water.Water is transferred pH to 3.0, with ethyl acetate extracting (10ml ethyl acetate, extracting is 4 times altogether), extracts the ester phase out, abandons water.Ester dries up after filtering with anhydrous sodium sulfate.Crystal after drying up is dissolved in the dioxane that the 0.5ml alkali alumina handled.Take by weighing 10mgOVA and be dissolved in the PBS solution of 0.7ml 0.05mol/L (pH7.2), two solution are slowly mixed in 4 ℃.1mgNHS and 2mgDCC are dissolved in the 0.2ml dioxane, and slowly drip this solution.Mixed solution is placed stirring at room reaction 16h.Transfer pH to 6.0, dry up in the fuming cupboard, slowly drip DCC solution (2mgDCC is dissolved in the 0.2ml dioxane and obtains).Stirring at room reaction 48h.At last with PBS solution dialysis 2～3d ,-20 ℃ of preservations.
2, testing sample is extracted, obtain extract
Corn is ground, get 0.3g as testing sample, adding 3ml volume fraction is methyl alcohol-PBS solution of 70%, and concuss 15min leaves standstill 30min.Supernatant is through the fast qualitative filter paper filtering, dilute 5 times after, get 50 μ l and detect.
3, chemiluminescence ELISA detects the testing sample extract
3.1 ZEN-OVA is encapsulated concentration with the best to be coated on the 96 hole blanks; Add the pure article of ZEN of known variable concentrations and the mixed solution of anti-zearalenone monoclonal antibody after the sealing respectively; The mixed solution of testing sample extract and anti-zearalenone monoclonal antibody is hatched;
(1) ZEN-OVA is encapsulated concentration with the best and be coated on the 96 hole blanks, hatched 4 hours for 37 ℃; Fig. 1 is an embodiment of the invention principle; Wherein, a is not for adding the synoptic diagram that contains the ZEN sample, and b is for adding the synoptic diagram that contains the ZEN sample; Fig. 2 is that the embodiment of the invention 96 hole blanks detect synoptic diagram.The ZEN standard solution that contains 100,50,10,5,1,0.1,0 ng/ml in the detection kit; The colour developing back is according to luminous value (3 repetitions of each sample of standard solution; Obtaining mean value) curve settles the standard; Luminous value (each sample repeats for 3 times, obtains mean value) according to testing sample calculates corresponding concentration from typical curve again; For the reliability of test kit, design adds blank, if meet or exceed the half the of the pairing luminous value of 0 ng/mlZEN solution at the luminous value that detects the empty contrast, judges that then kit lost efficacy.
(2) with 0.01 M PBST washing 3 times, add PBST at every turn, shake 3 minutes and pour out liquid in the hole, the adding massfraction is 5% skimmed milk power, hatches 2 hours for 37 ℃;
(3), added the PBST concussion 3 minutes at every turn and poured out liquid in the hole with 0.01 M PBST washing 3 times;
(4) in the typical curve hole, add the pure article 50 μ l of ZEN of 100,50,10,5,1,0.1,0 ng/ml successively, and do 3 repetitions; In treating gaging hole, add the testing sample extract 50 μ l of preparation in the step 2, and do 3 repetitions.More than add the anti-zearalenone monoclonal antibody of 1/40000 dilution of 50 μ l in each hole respectively; Add 100 μ l 0.01M PBS in the blank well, 96 orifice plates are placed 37 ℃ hatched 1 hour;
Resist 3.2 hatch two of back adding HRP mark, add luminous substrate afterwards again, carry out luminescence assays, obtain the result;
(1) with 0.01M PBST washing 3 times, add the PBST concussion 3 minutes at every turn and poured out liquid in the hole, add two of HRP mark and resist, hatched 1 hour for 37 ℃;
(2) with 0.01 M PBST washing 3 times, add the PBST concussion 3 minutes at every turn and poured out liquid in the hole, add 100 μ l luminous substrate; Said luminous substrate is that pH is 8.6 0.1mol/L Tris-HCl, and concrete compound method is used HCl adjustment pH to 8.6 for 0.1mol Tris is dissolved in the 1L water; Add luminol afterwards, p-iodophenol, hydrogen peroxide; Make its final concentration be respectively the 1mmol/L luminol, 2.5mmol/L p-iodophenol, 1mmol/L hydrogen peroxide); Carry out luminescence assays with the luminous signal analyzer, obtain the result.
4, the making and the result that detect the typical curve of ZEN judge
(1) according to the luminous value that obtains after known variable concentrations ZEN and the adding of anti-zearalenone monoclonal antibody; The drawing standard curve; Specific practice is: the logarithm value to add ZEN concentration is a horizontal ordinate; Average to add the resulting different luminous values of variable concentrations ZEN is ordinate divided by the result who does not add the resulting luminous value of ZEN (blank), through Microsoft Excel software, draws scatter diagram and adds Trendline formula and R2;
(2) according to the mean value of the luminous value of testing sample, calculate, obtain corresponding ZEN concentration through the resulting Trendline formula of typical curve.
5, the chemiluminescence detection of mark-on sampleTo known negative sample (corn that does not contain ZEN), after the grinding, add ZEN, preparation mark-on sample, making the ZEN concentration in the sample is 1 μ g/kg, 10 μ g/kg, 50 μ g/kg.Get 0.3g mark-on sample and add 3ml 70% (v/v) methyl alcohol, concuss 15 minutes left standstill 1 hour, and centrifugal 15 minutes of 2500g gets supernatant.Supernatant is through the fast qualitative filter paper filtering, dilute 5 times after, get 50 μ l and detect.
Extract is added in the testing sample hole, add the anti-zearalenone monoclonal antibody of 1/40000 dilution of 50 μ l again, same sample is done 3 repetitions.
At last, the luminous value through the mark-on sample and the typical curve of standard model calculate the zearalenone content of mark-on sample, and its recovery is 80%～120%.
In sum, method of the present invention can fast detecting goes out the amount of the ZEN that contains in the testing sample; Simultaneously, method detected object of the present invention is with strong points, and accuracy rate is high, and the more commercially available ELISA detection kit of sensitivity is high; Method of the present invention adopts anti-zearalenone monoclonal antibody, and with the mycotoxin no cross reaction in other cereal, more commercially available is that basic ELISA kit has stronger specificity with the polyclonal antibody, has reduced false positive rate.
Claims (4)
1. the immunochemiluminescence method of a fast detecting zearalenone toxin is characterized in that, comprises the steps:
Step 1 with ZEN haptens and OVA coupling, obtains conjugate ZEN-OVA;
Step 2 extracts testing sample, obtains extract;
Step 3; ZEN-OVA is encapsulated concentration with the best to be coated on the 96 hole blanks; Add the pure article of ZEN of known variable concentrations and the mixed solution of anti-zearalenone monoclonal antibody after the sealing respectively, the mixed solution of testing sample extract and anti-zearalenone monoclonal antibody is hatched;
Step 4 is hatched two of back adding HRP mark and is resisted, and adds luminous substrate afterwards again, carries out luminescence assays, obtains the result;
Step 5 is according to the luminous value of the mixed solution of known pure article of variable concentrations ZEN and anti-zearalenone monoclonal antibody, production standard curve;
Step 6, the luminous value according to the mixed solution of testing sample extract and anti-zearalenone monoclonal antibody obtains the ZEN concentration in the testing sample extract through typical curve.
2. the immunochemiluminescence method of fast detecting zearalenone toxin according to claim 1 is characterized in that, in the step 1, the method for said coupling is an active ester method.
3. the immunochemiluminescence method of fast detecting zearalenone toxin according to claim 1 is characterized in that, in the step 2; Said extraction is: get the 0.3g testing sample, adding 3ml volume fraction is methyl alcohol-PBS solution of 70%, concuss 15min; Leave standstill 30min; Supernatant is through the fast qualitative filter paper filtering, dilute 5 times after, must extract.
4. the immunochemiluminescence method of fast detecting zearalenone toxin according to claim 1 is characterized in that, in the step 3, said hatching is 37 ℃ and hatched 4 hours.
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