CN105198990B - Antibody and the preparation method and application thereof for the detection of Bt Cry1 toxoid wide spectrums - Google Patents

Abstract

The present invention discloses a kind of antibody and the preparation method and application thereof for the detection of Bt Cry1 toxoid wide spectrums, which is after being coupled by antigen polypeptide SEQ ID NO.2 and carrier protein KLH, and after Balb/c mouse are immunized, the preserving number of acquisition is CCTCC NO:The secretion of C2015145 hybridomas generates;The OD of six kinds of Bt Cry1 toxoids of the antibody pair₄₅₀Value and negative hole（PBS solution）Ratio be much larger than 2.1, potency is higher, has broad spectrum activity detection result to Bt Cry1 toxoids, makes up the blank of Bt Cry1 toxoid wide spectrum detection fields.

Description

Antibody and the preparation method and application thereof for the detection of Bt Cry1 toxoid wide spectrums

Technical field

The present invention relates to biochemistry, especially a kind of antibody and its preparation for the detection of Bt Cry1 toxoid wide spectrums Method and application.

Background technology

It reports to succeed in developing for the first time from Hilder in 1987 etc. and turns bacillus thuringiensis（Bacillus thuringiensis, Bt）Since toxin gene zoophobous, 400 Bt toxin genes have been had more than at present and has been cloned and surveys Sequence can serve as commercial formulation or turn existing more than ten classes of Bt Cry family gene crops（Including Cry1Aa, Cry1Ab, Cry1Ac, Cry1B, Cry1C, Cry1D, Cry1E, Cry1F, Cry2Aa, Cry2Ab, Cry3A, Cry3B, Cry34, Cry35 etc.）, turn Bt Toxin gene insect resistace crop continuously emerges and obtains the popularization of large area, produces highly significant economic, society and ecology Benefit, but with the large-scale promotion of transgenic Bt plants, to ecological safety risk and to the mankind and other lactations The security risk of animal gradually attracts attention.Currently, be repeatedly reported in China's rice be found that Bt Cry toxin genes and Toxin production, and serious harmful effect is produced to the outlet of China's rice, with greatly developing for transgenic technology, quilt The Bt Cry toxin gene types of application are more and more, the skill of the government safety supervision and food processing enterprises raw material management and control that bring Art restriction is more and more apparent, when especially genetic test cannot obtain promising result in for some netically modified foods, Checking to toxin expression product safe screen, it is particularly urgent to survey.

At present for the detection method of toxin protein in transgenic product mainly using the antibody test for single toxin Technology needs to prepare corresponding antibody due to being directed to different toxin, and antibody preparation process is tediously long, of high cost, cannot meet The requirement of wide spectrum screening detection.Therefore, the similar three-dimensional conformation feature for how utilizing Bt Cry toxin to have, exploitation are based on general character The Bt Cry toxin wide spectrums screening immunoassay technology of structure is the work for having very much application value.

About 90% monoclonal antibody is directed to according to the literature, and there is the antigen of general character structure all there is certain intersection Reaction, and for list, polyclonal antibody or the single-chain antibody of a certain kind Bt Mycotoxin identifications for the higher toxin of other homologys Also there is certain cross reaction, although this is based on having larger difference on different Cry toxin amino acid sequences, it is three-dimensional Structure closely similar principle, currently, for the detection of Bt Cry1 toxoids with wide spectrum antibody there is not yet relevant report.

Invention content

In view of the above-mentioned problems, the present invention by mouse immune, is obtained for Bt Cry1 toxoids detection wide spectrum antibody, The invention is realized in this way：

A kind of antibody for the detection of Bt Cry1 toxoid wide spectrums, which is characterized in that the antibody is to be by preserving number CCTCC NO:What the hybridoma secretion of C2015145 generated.

One plant of preserving number is CCTCC NO:The hybridoma of C2015145, the antibody of secretion is for Bt Cry1 class poison Plain wide spectrum detection.

Further, hybridoma of the present invention is obtained by：

1）Using MBS method synthetic immunogens, i.e., by the KLH-NH2 and amino acid sequence by desalting processing of equimolar number It is mixed for the polypeptide of SEQ ID NO.2,4 DEG C of reaction 2h are subsequently placed in PBS solution the 72h that dialyses, and adaptive immune is former；

2）By the immunogene of a concentration of 1mg/ml and Fu Shi Freund's complete adjuvants by volume 1:1 ratio mixes, only with 100ug/ Dose immunization mouse；Booster immunization is primary every two weeks later, replaces Freund complete with incomplete Freund's adjuvant when booster immunization Full adjuvant mix with immunogene, the 4th time it is immune after take a blood sample within one week survey potency, potency reaches 10000 times of serum dilution, OD₄₅₀When value is more than 1.0, cell fusion is carried out with murine myeloma cell SP2/0；Then indirect elisa method is used to screen cell Culture supernatant selects positive colony hybridoma wells to be subcloned, limiting dilution assay continuous cloning 2-3 times is used in combination, i.e., Obtain the hybridoma.

Application of the antibody of the present invention in the detection of Bt Cry1 toxoid wide spectrums.

Further, application of the present invention, specially：

A）It is CCTCC NO by preserving number:The hybridoma of C2015145 is inoculated in Mice Body, to induce method in vivo Ascites is prepared, then caprylic acid-ammonium is utilized to purify ascites, the antibody of a concentration of 25mg/ml is obtained, then with PBS solution Antibody is diluted 1000-16000 times；

B）Antibody is coated in solid phase carrier, 4 °C overnight；Next day PBST board-washing, is added 3% MPBS, 200 holes μ L/, 37 ° C is incubated 2h；PBST board-washings, are added product to be detected, and 100 holes μ L/ are incubated 1h；Enzyme labelled antibody is added and is incubated 1h；PBST board-washings, add Substrate TMB- carbamide peroxides, 100 holes μ L/, 37 °C are incubated after 15min per hole a concentration of 2mol/L H of 50 μ L of addition₂SO₄, terminate Reaction；Simultaneously using PBS solution as negative control；

C）Measure reaction solution OD₄₅₀Value, if the OD of product to be detected and negative control₄₅₀Ratio is more than 2.1, then kind to be detected Contain Bt Cry1 toxoids.

The present invention is according to the amino acid sequence of Cry1Aa, Cry1Ab, Cry1Ac, Cry1B, Cry1C and Cry1F, from level-one Sequence similarity analysis, secondary antigen and hydrophilicity analysis and three-dimensional structural analysis, the antigen polypeptide SEQ ID of design After being coupled it with carrier protein KLH, Balb/c mouse are immunized, having successfully been obtained can secrete for Bt Cry1 classes in NO.2 The hybridoma cell strain of Mycotoxin identification broad-spectrum monoclonal antibody, the cell strain preserving number are CCTCC NO:C2015145；Experiment After showing that the antibody of a concentration of 25mg/ml dilutes 16000 times, to the OD of six kinds of Bt Cry1 toxoids₄₅₀Value and negative hole （PBS solution）Ratio be much larger than 2.1, it is seen that its potency is higher, and to Bt Cry1 toxoids have broad spectrum activity detection effect Fruit makes up the blank of Bt Cry1 toxoid wide spectrum detection fields.

Description of the drawings

Fig. 1 is Amino acid sequences alignment's result schematic diagram of selected peptide fragment；

Fig. 2 is the antigenicity and hydrophilicity analysis result schematic diagram of selected peptide fragment；

Fig. 3 is the result schematic diagram that the 3 d structure model of six kinds of toxin is overlapped；

Fig. 4 is the three dimensional structure diagram of selected peptide fragment；

Fig. 5 is monoclonal antibody ELISA testing result schematic diagrames.

Specific implementation mode

The preparation method of solution involved in embodiment：

（1）Saturated ammonium sulfate solution：

500g ammonium sulfate is added in 500mL distilled water, is heated to being completely dissolved；Ambient temperature overnight is precipitated crystallization and is allowed to stay In bottle, required amount is taken before use, with NaOH tune pH to 7.8；

（2）Acetate buffer solution（0.06mol/L pH4.8）：

NaAc 0.29g, HAc 0.141mL are dissolved in deionized water, are settled to 100mL, adjust pH value to 4.8.

（3）Phosphate buffer（PBS, pH7.4）：

NaCl 8.0g, KCl 0.2g, Na₂HPO4·12H₂O 2.9g, KH₂PO₄0.2g adds distilled water to be settled to 1L；

（4）Cleaning solution（PBST）：PBS containing 0.05%Tween；

（5）Confining liquid（MPBS）：PBS containing 3% skimmed milk power；

（6）Citrate buffer（CPBS, substrate buffer solution, pH5.5）：

C₆H₇O₈（Citric acid）21g, Na₂HPO₄·12H₂O 71.6g, add distilled water to be settled to 1L；

（7）Substrate：TMB the and 25uL 0.65%H of 10mg/mL₂O₂It is dissolved in 9.875mLCPBS, it is now with the current；

（8）Tetramethyl benzidine（TMB）Mother liquor：

It weighs 10mg tetramethyl benzidines and is dissolved in 1mL dimethyl sulfoxide (DMSO)s, 4 DEG C of preservations；

（9）Terminate liquid（2M H₂SO₄）：

Take the concentrated sulfuric acid of 11.8mL 98%（18M）Be dissolved in 80mL distilled water, it is cooling after constant volume to 100mL；

（10）Carbonate buffer solution（CBS）：

Na₂CO₃ 1.59g；NaHCO₃ 2.93g, constant volume to 1L；

Involved reagent in embodiment：

Not formula Freund's complete adjuvant and Fu Shi Freund's incomplete adjuvants are purchased from Sigma companies；

Cry1Aa and Cry1B antibody is purchased from the Shanghai bio tech ltd You Long；

Involved amino acid sequence in embodiment：

SEQ ID NO.1:SFREWEADPTNPALREEMRI；

SEQ ID NO.2：CSFREWEADPTNPALREEMRI.

Embodiment 1 prepares monoclonal antibody

（1）The ammonia of Cry1Aa, Cry1Ab, Cry1Ac, Cry1B, Cry1C and Cry1F are retrieved in GenBank databases Base acid sequence；

（2）Using DNAMAN softwares by the amino acid sequence of Cry1Aa, Cry1Ab, Cry1Ac, Cry1B, Cry1C and Cry1F Row are compared, and the highest several polypeptide sequences of similitude are found out, and select Amino acid sequences alignment's result such as Fig. 1 institutes of peptide fragment Show；

（3）It is found out using DNAStar softwares antigenic in Cry1Aa, Cry1Ab, Cry1Ac, Cry1B, Cry1C and Cry1F With the stronger part of hydrophily, the results are shown in Figure 2 with hydrophilicity analysis for the antigenicity of peptide fragment；

（4）Finally utilize SWISS-MODEL Website constructions Cry1Aa, Cry1Ab, Cry1Ac, Cry1B, Cry1C and Cry1F The 3 d structure model of six kinds of toxin is analyzed 3 d structure model with Swiss-PdbViewer 4.1.0 softwares, is found out The region of six kinds of toxin three-dimensional structures overlapping, the 3 d structure models of six kinds of toxin is overlapped, and the results are shown in Figure 3；

（5）By primary sequence similarity analysis result, secondary antigen and hydrophilicity analysis result and three-dimensional structure point It analyses result and carries out Integrated comparative, select sequence similarity higher, antigenic similar compared with strong, space conformation with hydrophily more than one Peptide fragment is the most possible antigen polypeptide for generating broad spectrum activity immune effect, and as immune haptens, the peptide fragment three-dimensional structure is such as Shown in Fig. 4, in Fig. 4, A is selected peptide fragment, and amino acid sequence is as shown in SEQ ID NO.1；Transfer to Nanjing golden the sequence The bio tech ltd Si Rui synthesize, in order to carrier protein KLH, peptide chain one end add Cys residues, synthetic peptide Duan Xulie is as shown in SEQ ID NO.2；

（6）Prepared by immunogene, with hemocyanin（KLH）For carrier protein, using MBS method synthetic immunogens, specific steps It is as follows：Weigh 5mg KLH-NH₂It is dissolved in the PBS of 1ml 0.1mM, is then added the Sulfo-MB of final concentration of 1mM, at 4 DEG C React 2h；Desalination, which is carried out, after G25 Sephadex gel permeation chromatography columns removes extra crosslinking agent；

Then by the KLH-NH after desalting processing₂With the polypeptide SEQ ID NO.2 mixing of equimolar number, 2h is reacted in 4 DEG C Afterwards, then it is placed in PBS solution the 72h that dialyses, is changed the liquid once every 9-12h during dialysis, is i.e. adaptive immune at a temperature of 4 DEG C Then original dispenses, frozen in -20 DEG C spare；

（7）The preparation of monoclonal antibody：Utilize step（6）8 week old of immunogen immune of acquisition or so Balb/c females are small Mouse 4, when first immunisation, by the immunogene of a concentration of 1mg/ml and Fu Shi Freund's complete adjuvants by volume 1:After the mixing of 1 ratio, with Mouse is immunized in the mode of intraperitoneal injection, and immunizing dose is 100ug/；

Booster immunization is primary every two weeks later, when booster immunization with incomplete Freund's adjuvant replace Freund's complete adjuvant and Immunogene mixes, the 4th time it is immune after one week blood sampling survey potency, potency reaches ideal value（I.e.:Serum dilute 10000 times and OD₄₅₀Value is more than 1.0）When, according still further to conventional hybridization tumor integration technology（Referring to Wu Jianxiang etc., " Pyricularia oryzae monoclonal antibody Develop and its to shadow noons of note fields " microorganism journal, 2000,40 (6): 638-645）With murine myeloma cell SP2/0 carries out cell fusion, screens cells and supernatant using indirect elisa method, positive colony hybridoma is selected to carry out Subclone, is used in combination limiting dilution assay continuously to clone 2-3 times, obtains the hybridoma cell strain of one plant of stably excreting antibody.

Steps are as follows for indirect elisa method：

1. being coated with：It is with coating buffer CBS that coating antigen Cry1Aa, Cry1Ab, Cry1Ac, Cry1B, Cry1C and Cry1F is dilute It is interpreted into the concentration of 4 μ g/mL, is added separately in 96 hole elisa Plates, per 100 μ L of hole, 4 °C of coatings are overnight；

2. closing：Three times, 3% MPBS is added in PBST board-washings, and 200 holes μ L/ set in 37 °C of incubators and are incubated 2h；

3. plus culture supernatant：PBST board-washings three times, draw the 100 μ L of culture supernatant of cell hole to be detected, are added to 96 holes In ELISA Plate, sets in 37 °C of incubators and be incubated 1h, while setting negative serum and positive serum（1/2000 sample-adding）And acellular Kong Pei Support supernatant control；

4. plus ELIAS secondary antibody：Three times, the sheep anti-mouse igg of HRP labels is added in PBST board-washings（Purchased from KPL companies, work is dense Degree 1/5000, dilution MPBS）, per 100 μ L of hole, set in 37 °C of incubators and be incubated 1h；

5. developing the color：PBST board-washings four times, add substrate（TMB- carbamide peroxides）, per 100 μ L of hole, it is incubated in 37 °C of incubators 15min；

6. terminating：Add 2mol/L H₂SO₄, per 50 μ L of hole；

7. result judgement：OD is measured with microplate reader₄₅₀Value, the hole of P/N >=2.1 is judged to positive hole.

Applicant by the hybridoma cell strain of acquisition from being named as hybridoma cell strain DS2D3, and in September in 2015 16 days It is preserved in China typical culture collection center (China Center for Type CultureCollection, CCTCC), Address：Wuhan City, Hubei Province Wuhan University, postcode 430072, deposit number are CCTCC NO:C2015145.

2 antibody sensitivity technique of embodiment

（1）The hybridoma cell strain obtained to mouse Inoculation embodiment 1 prepares ascites, tool using method is induced in vivo Steps are as follows for body：

Well-grown hybridoma is gently purged, is collected by centrifugation, is resuspended with RPMI-1640 basic culture solutions It counts, cell quantity is controlled 1~2 × 10⁶Within the scope of a/mL, the mouse that intraperitoneal injection has used paraffin oil pretreated in advance, 2 mouse of every plant of cell infusion, every mouse 0.5mL.The health status of close observation mouse and sign of ascites as, about 10d or so, Mouse web portion starts to expand, and when ascites is as more as possible, neck is drawn to put to death mouse, cuts open the belly and draw ascites.

The ascites of collection is centrifuged into 10min in 4 °C of lower 5000rpm, removes fat and haemocyte etc., abdomen after centrifugation Add equivalent glycerine in water, is preserved in -20 °C of refrigerators.

（2）By step（1）The ascites of acquisition is purified using caprylic acid-ammonium, and purification step is as follows：

4 °C of 12,000rpm of ascites centrifuge 15min, collect supernatant, abandon precipitation；1 part of ascites and 2~4 parts of acetate salt buffers Liquid is sufficiently mixed, and adjusts pH value to 4.5~4.8；It is slowly added to 33 μ L/mL ascites of caprylic acid under stirring, mixes 30min at room temperature, 4 °C of postposition standing 2h or more；4 °C of 12,000rpm centrifuge 5min, collect supernatant, abandon precipitation；Supernatant is filtered with double-layer filter paper Afterwards, the PBS buffer solution of the 0.1mol/L pH value 7.4 of 1/10 volume is added, with 2mol/L NaOH tune pH value to 7.4,4 °C it is pre- Cold, in ammonium sulfate is added in 30min, under condition of ice bath to final concentration of 0.277g/mL, 4 °C stand 2h；4°C 12,000rpm 30min is centrifuged, supernatant is abandoned；With the 0.01mol/L pH value 7.4 PBS dissolving precipitations of 1/10 former ascites volume；To 0.01mol/L The PBS of pH value 7.4 dialyses one day, and insoluble impurity is removed in centrifugation, and the clear solution obtained is antibody-solutions, a concentration of 25mg/ml is placed in -80 °C and freezes；

（3）The measurement of antibody sensitivity：It is measured using indirect elisa method.

Coating antigen uses six kinds of standard Cry1 toxoids of Cry1Aa, Cry1Ab, Cry1Ac, Cry1B, Cry1C and Cry1F, A concentration of 4ug/ml；

Antibody is step（2）A concentration of 25mg/ml antibody-solutions obtained, antibody is diluted with PBS buffer solution successively 1000 times, 2000 times, 4000 times, 8000 times, 16000 times；

It is as follows：

1. being coated with：By antigens c ry1Aa, Cry1Ab, Cry1Ac, Cry1B, Cry1C and Cry1F toxin proteins are coated with respectively In 96 hole elisa Plates, 100 holes μ L/, 4 °C of coatings are overnight；

2. closing：Three times, 3% MPBS is added in PBST board-washings, and 200 holes μ L/ set in 37 °C of incubators and are incubated 2h；

3. plus antibody：PBST board-washings three times, press the monoclonal antibody that the holes 100uL/ are added after dilution, while with PBS respectively Instead of monoclonal antibody as negative control；

4. plus ELIAS secondary antibody：Three times, the sheep anti-mouse igg of HRP labels is added in PBST board-washings（Purchased from KPL companies, MPBS is used According to 1：5000 dilution proportion）, per 100 μ L of hole, set in 37 °C of incubators and be incubated 1h；

5. developing the color：PBST board-washings four times, add substrate（TMB- carbamide peroxides）, per 100 μ L of hole, it is incubated in 37 °C of incubators 15min；

6. terminating：Add 2mol/L H₂SO₄, per 50 μ L of hole；

7. result judgement：OD is measured with microplate reader₄₅₀Value；

Antibody titer measurement result is as shown in figure 5, as seen from Figure 5, negative control value is respectively less than 0.1, works as antibody When diluting 16000 times, to the OD of Cry1Aa, Cry1Ab, Cry1Ac, Cry1B, Cry1C and Cry1F₄₅₀Value respectively 0.78, 0.535,0.688,0.764,0.744 and 0.71,2.1 are much larger than with the ratio of negative hole, shows the potency of monoclonal antibody It is higher, it can be applied to the broad spectrum activity detection of Bt Cry1 toxoids.

The detection experiment of 3 Bt Cry1 toxoids of embodiment

Experiment is divided into following four groups：

Experimental group：2 step of embodiment（2）The antibody of acquisition, a concentration of 25mg/ml, with 16000 times of dilutions of PBS solution；

Control group 1：Cry1Aa antibody is diluted to and experimental group same concentrations with PBS；

Control group 2：Cry1B antibody is diluted to and experimental group same concentrations with PBS；

Negative control：PBS solution；

Detecting step is as follows：

1. being coated with：Each group antibody is coated in solid phase carrier, 100 holes μ L/ respectively, 4 °C of coatings overnight, make to form solid phase Antibody；

2. closing：Three times, 3% MPBS is added in PBST board-washings, and 200 holes μ L/ set in 37 °C of incubators and are incubated 2h；

3. plus antigen：PBST board-washings three times, are separately added into the antigen of concentration 2ug/ml, and 100 holes μ L/ are incubated 1h, make to resist Original is fully reacted with the antibody on solid phase carrier, forms solid phase antigen antibody complex；

4. enzyme labeling antibody：PBST board-washings three times, remove other unbonded materials, are added enzyme labelled antibody, 100 holes μ L/, It is incubated 1h, makes to form solid phase antibody determined antigen-enzyme labelled antibody sandwich complex, washing, which removes, is not associated with enzyme labelled antibody；

Enzyme labelled antibody is to mix the immune rabbit polyclonal antibody prepared of Cry1 toxoids（Immunization method and 1 step of embodiment （7）Monoclonal antibody is identical, and immunizing dose is every each toxin of rabbit each 100ug, total 600ug/, after being immunized three times, rabbit Son blood sampling detection, after potency reaches ideal value, heart adopts whole blood）, after purification through saturated ammonium sulphate, marked with HRP, enzyme mark Antibody concentration is 20mg/ml, presses 1 with PBS when use：4000 dilutions；

5. developing the color：PBST board-washings four times, add substrate（TMB- carbamide peroxides）, per 100 μ L of hole, it is incubated in 37 °C of incubators 15min；

6. terminating：Add 2mol/L H₂SO₄, per 50 μ L of hole；

7. result judgement：OD is measured with microplate reader₄₅₀Value,

The OD for respectively obtaining experimental group and control group 1,2₄₅₀Value is compared with negative control, and ratio is more than 2.1, note For " √ ", it is otherwise denoted as "×", testing result is as shown in table 1：

Table 1：Bt Cry1 toxoid ELISA testing results

Group

Cry1Aa

Cry1Ab

Cry1Ac

Cry1B

Cry1C

Cry1F

Experimental group

√

√

√

√

√

√

Control group 1

√

×

×

×

×

×

Control group 2

×

×

×

√

×

×

By table 1 as it can be seen that commercial antibody only can detect the Bt Cry1 toxoids of correspondence, and other toxin can not be detected；And The antibody that the present invention obtains is all higher than 2.1 with the ratio of negative hole, can be used for after being reacted with six kinds of Bt Cry1 toxoid antigens Bt Cry1 toxoid wide spectrums detect.

The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered It is considered as protection scope of the present invention.

SEQUENCE LISTING

<110>Jiangsu Province's farm institute

<120>Antibody and the preparation method and application thereof for the detection of Bt Cry1 toxoid wide spectrums

<130> 2

<160> 2

<170> PatentIn version 3.3

<210> 1

<211> 20

<212> PRT

<213>It is artificial synthesized

<400> 1

Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg Glu

1 5 10 15

Glu Met Arg Ile

20

<210> 2

<211> 21

<212> PRT

<213>It is artificial synthesized

<400> 2

Cys Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg

1 5 10 15

Glu Glu Met Arg Ile

20

Claims (4)

1. a kind of antibody for the detection of Bt Cry1 toxoid wide spectrums, which is characterized in that the antibody is to be by preserving number CCTCC NO:What the hybridoma secretion of C2015145 generated.

2. one plant of preserving number is CCTCC NO:The antibody of the hybridoma of C2015145, secretion is used for Bt Cry1 toxoids Wide spectrum detects.

3. application of the antibody as described in claim 1 in the detection of Bt Cry1 toxoid wide spectrums.

4. applying according to claim 3, which is characterized in that the specific steps are：

A) it is CCTCC NO by preserving number:The hybridoma of C2015145 is inoculated in Mice Body, to induce method preparation in vivo Then ascites is utilized caprylic acid-ammonium to purify ascites, obtains the antibody of a concentration of 25mg/ml, then will be resisted with PBS solution Body dilutes 1000-16000 times；

B) antibody after dilution is coated in solid phase carrier, 4 °C overnight；3% MPBS, 200 μ L/ is added in next day PBST board-washing Hole, 37 °C of incubation 2h；PBST board-washings, are added product to be detected, and 100 holes μ L/ are incubated 1h；Enzyme labelled antibody is added and is incubated 1h；PBST is washed Plate adds substrate TMB- carbamide peroxides, and 100 holes μ L/, 37 °C are incubated after 15min per hole 50 a concentration of 2mol/L of μ L of addition H₂SO₄, reaction was completed；Simultaneously using PBS solution as negative control；

C) reaction solution OD is measured₄₅₀Value, if the OD of product to be detected and negative control₄₅₀Ratio is more than 2.1, then kind to be detected contains Bt Cry1 toxoids.