CN116047047B - Kit and method for quantitative detection of insect-resistant protein Cry1F enzyme-linked immunity - Google Patents
Kit and method for quantitative detection of insect-resistant protein Cry1F enzyme-linked immunity Download PDFInfo
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Abstract
The ELISA kit consists of a micropore reaction plate coated with a capture antibody, a sample treatment solution, a biotin-marked detection antibody, a standard substance, horseradish peroxidase-marked avidin, a biotin-marked antibody diluent, a horseradish peroxidase-marked avidin diluent, a concentrated washing solution, a substrate solution, a stopping solution and a plate patch, so that the ELISA kit has the characteristics of simple operation, capability of simultaneously and rapidly detecting a large number of samples, high sensitivity and high specificity.
Description
Technical Field
The invention relates to the field of bioengineering, in particular to an enzyme-linked immunosorbent assay kit for quantitatively detecting insect-resistant protein Cry1F in transgenic corn and a detection method thereof.
Background
Bacillus thuringiensis (Bacillus thuringiensis, bt) is a widely occurring gram-positive bacterium, whose secreted anti-insect crystal protein is the current major biopesticide; secreted insect-resistant proteins are classified into two classes according to amino acid sequence similarity: cry and Cry delta-endotoxins. Wherein the Cry proteins are toxic to larvae of a variety of insect pests (e.g., lepidoptera, diptera, coleoptera, nematodes, protozoa, etc.). Cry toxins have been transformed into a variety of crops to render them insect resistant. The Cry1F protein is one of the Cry toxins. At present, crops transformed with Cry genes mainly comprise corn, potato, corn, cotton and the like.
Advances and developments in transgenic technology have driven the development of biology. Although the transgenic food can meet the requirements of people on yield, insect resistance and the like, the transgenic food brings potential threats to the life of people, for example, certain genes can cause toxicity to the food after being introduced into a host, the transgenic food generates allergen, the people generate drug resistance, the nutritional value of the food is changed and the like. In order to comprehensively evaluate the safety of the transgenic food while researching, developing and commercializing the transgenic food, consumers can rapidly distinguish the transgenic food from natural food, and a proper method is established for identifying and detecting the transgenic ingredients in the transgenic food, so that the safety management of agricultural transgenic organisms can be promoted, the safety of people, animals and microorganisms can be ensured, the ecological environment can be protected, and the further research of agricultural transgenic biotechnology can be promoted. In order to carry out rapid quantitative analysis on Cry1F proteins in transgenic crops or derivatives thereof, the research and development of the Cry1F ELISA kit has great significance.
Disclosure of Invention
The invention aims to provide an enzyme-linked immunosorbent assay kit for quantitatively detecting insect-resistant protein Cry1F in transgenic corn and a detection method thereof, which have the characteristics of simple operation, capability of simultaneously and rapidly detecting a large number of samples, high sensitivity and high specificity.
In order to achieve the aim, the invention provides an enzyme-linked immunosorbent assay kit for quantitatively detecting insect-resistant protein Cry1F in transgenic corn, which consists of a microporous reaction plate coated with a capture antibody, a sample treatment solution, a biotin-labeled detection antibody, a standard substance, a horseradish peroxidase-labeled avidin, a biotin-labeled antibody diluent, a horseradish peroxidase-labeled avidin diluent, a concentrated washing solution, a substrate solution, a stop solution and a plate patch.
The formula of the sample treatment fluid is as follows:
biotin-labeled detection antibody: 100 μg/mL biotin-labeled Cry1f 7 1d8 murine mab (PBS, 50% glycerol) solution;
standard substance: 2.5ng of His-Cry 1F recombinant protein (freeze-dried powder), wherein the His-Cry 1F recombinant protein is obtained by amplifying and sequencing a Cry1F coding gene, connecting the recombinant protein with a pET28a plasmid after identification is correct, transforming a recombinant expression vector pET28a-Cry 1F into competent cells of escherichia coli BL21 (DE 3), resuscitating and culturing a preserved recombinant protein Cry1F expression strain, inducing the expression protein with IPTG at 16 ℃ overnight, and purifying the recombinant protein;
horseradish peroxidase-labeled avidin: 1:40 horseradish peroxidase (Horseradish peroxidase) labeled avidin (avidin) stock solution (Jin Kairui company), working solution diluted 1:100 fold;
biotin-labeled antibody dilution/horseradish peroxidase-labeled avidin dilution formulation:
concentrating the washing liquid: 10 XPBS with 1.0% Tween-20;
substrate solution: 0.5mL of 2mg/mL TMB absolute ethanol solution, 10mL of substrate buffer, 32. Mu.L of 30% H 2 O 2 Mixing, and preparing in situ;
stop solution: 1M H 2 SO 4 。
Characteristics of the kit:
1) The sensitivity is: 0.112ng/mL
2) Precision: intra-batch difference CV% <8%, inter-batch difference CV% <10%
3) Specificity: the kit specifically detects Cry1F and has no cross reaction with other related proteins.
In another embodiment of the enzyme-linked immunosorbent assay kit, the capture antibody is secreted by a hybridoma cell line 1d7 1c6 with a collection number of CGMCC No.45008, and the detection range of the capture antibody is 1d7 1c 6: 0.3125ng/mL-2.5ng/mL.
In another embodiment of the enzyme-linked immunosorbent assay kit, the detection antibody is secreted by a hybridoma cell strain 1F7 1D8 with a collection number of CGMCC No. 45009.
The hybridoma cell strains 1D7 1C6 and 1F7 1D8 are obtained by immunizing BALB/c mice with insect-resistant proteins Cry1F in transgenic corn, fusing immunized mice spleen cells with commercial mouse hybridoma cells SP2/0, and screening by using a HAT culture medium.
The invention also provides a detection method of the enzyme-linked immunosorbent assay kit for quantitatively detecting the insect-resistant protein Cry1F in transgenic corn, which comprises the following steps:
1) Placing the sample treatment solution, biotin-labeled detection antibody, standard substance, horseradish peroxidase-labeled avidin, biotin-labeled antibody diluent, horseradish peroxidase-labeled avidin diluent, concentrated washing solution, substrate solution and stop solution at 18-25deg.C, and balancing for at least 30 min;
2) Setting a standard substance hole and a sample hole to be detected respectively, adding a standard substance or a sample to be detected into each hole for incubation, then discarding liquid, adding a biotin-marked detection antibody working solution for incubation, discarding liquid in the holes, adding a horseradish peroxidase-marked avidin working solution into each hole after spin-drying, incubating, discarding liquid in the holes, adding a substrate solution into each hole after spin-drying, adding a stop solution into each hole after light-shielding and color development, and sequentially measuring the optical density OD value of each hole at a wavelength of 450nm by using an enzyme-labeled instrument within 5 minutes after the reaction is terminated;
3) And (3) data processing: and (3) subtracting the value of the S0 hole from the standard substance and the sample value, drawing a curve, if a compound hole is arranged, taking the average value of the compound hole to calculate, drawing a standard curve on logarithmic coordinate paper by taking the concentration of the standard substance as an ordinate and the OD value as an abscissa, and finding out the corresponding concentration from the standard curve according to the OD value of the sample.
In another embodiment, the working solution of the biotin-labeled detection antibody is obtained by diluting the biotin-labeled detection antibody with a biotin-labeled antibody diluent at a ratio of 1:100, for example, 10. Mu.L of biotin-labeled antibody plus 990. Mu.L of biotin-labeled antibody diluent, and mixing gently, and then adding the mixture within 10 minutes immediately before use.
Concentrating the washing liquid: the washing liquid is preserved at low temperature and has salt precipitation, and the washing liquid can be heated in water bath to assist dissolution when diluted; washing liquid working solution: the concentrated wash was diluted 1:25 times with deionized water. For example, 240mL of deionized water is measured by a measuring cylinder, poured into a concentrated beaker or other clean container, 10mL of concentrated washing liquid is measured again, and the mixture is evenly added, stirred and mixed well and then is prepared just before use.
In another embodiment, the working solution of horseradish peroxidase-labeled avidin is obtained by diluting horseradish peroxidase-labeled avidin with horseradish peroxidase-labeled avidin diluent by 1:100 times, for example, 10. Mu.L of horseradish peroxidase-labeled avidin is added with 990. Mu.L of horseradish peroxidase-labeled avidin diluent, and the mixture is gently mixed and prepared within 10 minutes immediately before use.
In another embodiment, in the step 2), 80-120 mu L of standard substance or sample to be detected is added into each hole, the mixture is gently shaken and mixed, covered with a plate and then incubated for 1.5-2.5 hours at 37 ℃; discarding the liquid, spin-drying without washing; adding 80-120 mu L of biotin-labeled antibody working solution into each hole, coating a new plate patch, and incubating at 37 ℃ for 0.5-1.5 hours; discarding the liquid in the holes, spin-drying, washing the plate for 2-4 times, soaking for 1-3 minutes each time, and spin-drying at 150-250 mu L/hole; adding 80-120 mu L of horseradish peroxidase labeled avidin working solution into each hole, coating a new plate patch, and incubating at 37 ℃ for 0.5-1.5 hours; discarding the liquid in the holes, spin-drying, washing the plate for 4-6 times, soaking for 1-3 minutes each time, and spin-drying at 150-250 mu L/hole; adding 80-100 mu L of substrate solution into each hole in sequence, and developing for 15-30 minutes at 37 ℃ in a dark place; the reaction was terminated by adding 50. Mu.L of the termination solution to each well in this order.
In another embodiment, in the step 2), 100 μl of standard or sample to be tested is added to each well, the mixture is gently shaken and mixed, covered with a patch, and incubated at 37deg.C for 2 hours; discarding the liquid, spin-drying without washing; adding 100 mu L of biotin-labeled antibody working solution into each hole, coating a new plate patch, and incubating at 37 ℃ for 1 hour; discarding the liquid in the holes, spin-drying, washing the plate for 3 times, soaking for 2 minutes each time, and spin-drying at 200 mu L/hole; adding 100 mu L of horseradish peroxidase-labeled avidin working solution into each hole, coating a new plate patch, and incubating at 37 ℃ for 1 hour; discarding the liquid in the holes, spin-drying, washing the plate for 5 times, soaking for 2 minutes each time, and spin-drying at 200 mu L/hole; 90. Mu.L of primer solution was added to each well in sequence.
In another embodiment, the detection method is an ELISA double antibody sandwich method.
Compared with the prior art, the invention has the following beneficial effects: the kit is a detection kit based on an ELISA double-antibody sandwich method, has low cost, is provided with main reagents in the form of working solutions, is simple and convenient to operate, and can simultaneously and rapidly detect a large number of samples; the method has the characteristics of high sensitivity and high specificity; also a kit capable of quantitatively and specifically detecting the insect-resistant protein Cry1F in transgenic corn at present, domestically and internationally.
Preservation information
The Cry1Fa monoclonal antibody hybridoma cell strain 1F7 1D8 and the Cry1Fa monoclonal antibody hybridoma cell strain 1D7 1C6 provided by the invention are sequentially preserved in the China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) on 12 months of 2021, and the preservation addresses are as follows: no. 1 and No. 3 of the north cinquefoil of the morning sun area of beijing city. Post code: 100101, and the preservation numbers are CGMCC No.45009 and CGMCC No.45008 in sequence.
Drawings
FIG. 1 is a standard curve of an ELISA method according to the invention
Detailed Description
The following detailed description of embodiments of the invention is, therefore, to be taken in conjunction with the accompanying drawings, and it is to be understood that the scope of the invention is not limited to the specific embodiments.
The experimental methods used in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1 an enzyme-linked immunosorbent assay kit according to the present application quantitatively detects the insect-resistant protein Cry1F in transgenic maize
An ELISA kit for quantitatively detecting an insect-resistant protein Cry1F in transgenic corn comprises a micropore reaction plate coated with a capture antibody, a sample treatment solution, a biotin-marked detection antibody, a standard substance, a horseradish peroxidase-marked avidin, a biotin-marked antibody diluent, a horseradish peroxidase-marked avidin diluent, a concentrated washing solution, a substrate solution, a stop solution and a plate patch.
The formula of the sample treatment fluid is as follows:
biotin-labeled detection antibody: 100 μg/mL biotin-labeled Cry1f 7 1d8 murine mab (PBS, 50% glycerol) solution;
standard substance: 2.5ng of His-Cry 1F recombinant protein (freeze-dried powder), wherein the His-Cry 1F recombinant protein is obtained by amplifying and sequencing a Cry1F coding gene, connecting the recombinant protein with a pET28a plasmid after identification is correct, transforming a recombinant expression vector pET28a-Cry 1F into competent cells of escherichia coli BL21 (DE 3), resuscitating and culturing a preserved recombinant protein Cry1F expression strain, inducing the expression protein with IPTG at 16 ℃ overnight, and purifying the recombinant protein;
horseradish peroxidase-labeled avidin: 1:40 horseradish peroxidase (Horseradish peroxidase) labeled avidin (avidin) stock solution (Jin Kairui company), working solution diluted 1:100 fold;
biotin-labeled antibody dilution/horseradish peroxidase-labeled avidin dilution formulation:
concentrating the washing liquid: 10 XPBS with 1.0% Tween-20;
substrate solution: 0.5mL of 2mg/mL TMB absolute ethanol solution, 10mL of substrate buffer, 32. Mu.L of 30% H 2 O 2 Mixing, and preparing in situ;
stop solution: 1M H 2 SO 4 。
Characteristics of the kit:
1) The sensitivity is: 0.112ng/mL
2) Precision: intra-batch difference CV% <8%, inter-batch difference CV% <10%
3) Specificity: the kit specifically detects Cry1F and has no cross reaction with other related proteins.
In another embodiment of the enzyme-linked immunosorbent assay kit, the capture antibody is secreted by a hybridoma cell line 1d7 1c6 with a collection number of CGMCC No.45008, and the detection range of the capture antibody is 1d7 1c 6: 0.3125ng/mL-2.5ng/mL.
In another embodiment of the enzyme-linked immunosorbent assay kit, the detection antibody is secreted by a hybridoma cell strain 1F7 1D8 with a collection number of CGMCC No. 45009.
The hybridoma cell strains 1D7 1C6 and 1F7 1D8 are obtained by immunizing BALB/c mice with insect-resistant proteins Cry1F in transgenic corn, fusing immunized mice spleen cells with commercial mouse hybridoma cells SP2/0, and screening by using a HAT culture medium.
The invention also provides a detection method of the enzyme-linked immunosorbent assay kit for quantitatively detecting the insect-resistant protein Cry1F in transgenic corn, which comprises the following steps:
1) Placing the sample treatment solution, biotin-labeled detection antibody, standard substance, horseradish peroxidase-labeled avidin, biotin-labeled antibody diluent, horseradish peroxidase-labeled avidin diluent, concentrated washing solution, substrate solution and stop solution at 18-25deg.C, and balancing for at least 30 min;
2) Setting a standard substance hole and a sample hole to be detected respectively, adding a standard substance or a sample to be detected into each hole for incubation, then discarding liquid, adding a biotin-marked detection antibody working solution for incubation, discarding liquid in the holes, adding a horseradish peroxidase-marked avidin working solution into each hole after spin-drying, incubating, discarding liquid in the holes, adding a substrate solution into each hole after spin-drying, adding a stop solution into each hole after light-shielding and color development, and sequentially measuring the optical density OD value of each hole at a wavelength of 450nm by using an enzyme-labeled instrument within 5 minutes after the reaction is terminated;
3) And (3) data processing: and (3) subtracting the value of the S0 hole from the standard substance and the sample value, drawing a curve, if a compound hole is arranged, taking the average value of the compound hole to calculate, drawing a standard curve on logarithmic coordinate paper by taking the concentration of the standard substance as an ordinate and the OD value as an abscissa, and finding out the corresponding concentration from the standard curve according to the OD value of the sample.
In another embodiment, the working solution of the biotin-labeled detection antibody is obtained by diluting the biotin-labeled detection antibody with a biotin-labeled antibody diluent at a ratio of 1:100, for example, 10. Mu.L of biotin-labeled antibody plus 990. Mu.L of biotin-labeled antibody diluent, and mixing gently, and then adding the mixture within 10 minutes immediately before use.
Concentrating the washing liquid: the washing liquid is preserved at low temperature and has salt precipitation, and the washing liquid can be heated in water bath to assist dissolution when diluted; washing liquid working solution: the concentrated wash was diluted 1:25 times with deionized water. For example, 240mL of deionized water is measured by a measuring cylinder, poured into a concentrated beaker or other clean container, 10mL of concentrated washing liquid is measured again, and the mixture is evenly added, stirred and mixed well and then is prepared just before use.
In another embodiment, the working solution of horseradish peroxidase-labeled avidin is obtained by diluting horseradish peroxidase-labeled avidin with horseradish peroxidase-labeled avidin diluent by 1:100 times, for example, 10. Mu.L of horseradish peroxidase-labeled avidin is added with 990. Mu.L of horseradish peroxidase-labeled avidin diluent, and the mixture is gently mixed and prepared within 10 minutes immediately before use.
In the step 2), 100 mu L of standard substance or sample to be detected is added into each hole respectively, the mixture is gently shaken and mixed evenly, covered with a plate patch, and incubated for 2 hours at 37 ℃; discarding the liquid, spin-drying without washing; adding 100 mu L of biotin-labeled antibody working solution into each hole, coating a new plate patch, and incubating at 37 ℃ for 1 hour; discarding the liquid in the holes, spin-drying, washing the plate for 3 times, soaking for 2 minutes each time, and spin-drying at 200 mu L/hole; adding 100 mu L of horseradish peroxidase-labeled avidin working solution into each hole, coating a new plate patch, and incubating at 37 ℃ for 1 hour; discarding the liquid in the holes, spin-drying, washing the plate for 5 times, soaking for 2 minutes each time, and spin-drying at 200 mu L/hole; 90. Mu.L of primer solution was added to each well in sequence.
Diluting standard substance from 2.5ng into serial concentration standard substance, adding standard substance into each hole according to the detection method of the application for incubation, then discarding liquid, adding biotin-marked detection antibody working solution for incubation, discarding liquid in the hole, drying, adding horseradish peroxidase-marked avidin working solution into each hole for incubation, discarding liquid in the hole, drying, adding substrate solution into each hole after drying, adding stop solution into each hole after light-proof color development, sequentially measuring optical density OD value of each hole at 450nm wavelength by using enzyme-labeled instrument within 5 minutes after reaction termination to make standard curve, see figure 1 for details, R of standard curve 2 >0.99, linear detection range is 0.3125ng/mL-2.5ng/mL, and a sample concentration calculation formula is deduced: x= (y-0.0103)/0.407.
Grinding transgenic corn leaves by liquid nitrogen, fully mixing the corn leaves with a sample extracting solution, standing on ice for 30min, taking a supernatant, properly diluting, adding a standard substance into each hole for incubation according to the detection method disclosed by the patent, discarding liquid, adding a biotin-marked detection antibody working solution for incubation, discarding liquid in the hole, spin-drying, adding a horseradish peroxidase-marked avidin working solution into each hole for incubation, discarding liquid in the hole, spin-drying, adding a substrate solution into each hole, adding a stop solution into each hole after light-shielding development, sequentially measuring the optical density OD value of each hole at a wavelength of 450nm by using an enzyme-labeled instrument within 5 min after the reaction is terminated, and calculating the content x=0.95+/-0.13 mu g/mL of Cry1F in the transgenic corn leaves.
Key points of operation
1. In order to ensure the accuracy of the detection result, it is suggested that both the standard and the sample are provided with double-hole measurement. Standard curves are needed for each detection.
2. If the content of the substance to be detected in the sample is too high, the sample is diluted by the sample diluent to ensure that the sample accords with the detection range of the kit, and finally, the sample is multiplied by the corresponding dilution multiple during calculation.
3. Sample adding: during sample addition, a disposable clean suction head is required to be used, so that cross contamination is avoided. The sample should be added as slowly as possible to avoid foaming, and the sample is added at the bottom of the ELISA plate hole without adding the sample along the hole wall. The time for one sample application is preferably controlled within 10 minutes, for example, the number of samples is large, and the gun is recommended for sample application.
4. Incubation: in order to prevent sample evaporation or contamination, the ELISA plate must be covered and attached during incubation, and the ELISA plate should be prevented from being in a dry state during the experiment. During the incubation process, whether the temperature of the incubator is constant at 37 ℃ or not should be observed at any time, and the temperature should be adjusted in time. During the incubation, the incubator is not easily opened too many times to avoid affecting the temperature balance.
5. Washing: the washing process is very important, and insufficient washing is prone to false positives.
(1) The manual plate washing method comprises the following steps: sucking (without touching the hole wall and the hole bottom) or throwing away the liquid in the ELISA plate; laying several layers of absorbent paper on the experiment table, and beating the ELISA plate downwards for several times; the recommended wash buffer was injected into the hole at 200. Mu.L/well and immersed for 2 minutes. This process was repeated several times, as described in the procedure.
(2) Automatic plate washing: if an automatic plate washer is available, the plate washer should be used in the formal experiment process after the plate washer is used by the skilled person.
6. Color development: in order to ensure the accuracy of the experimental result, the stop solution should be added as soon as possible after the substrate reaction time. The color development can be observed at intervals after the substrate solution is added to control the reaction time (e.g., at intervals of 10 minutes). When the front 3-4 holes of the standard product are visible to naked eyes and have obvious gradient blue, and the color development of the rear 3-4 holes is not obvious, stopping solution can be added to stop the reaction, and the blue color immediately turns yellow. The order of addition of the stop solution should be as similar as possible to the order of addition of the substrate solution.
7. The substrate solution should be bluish or colorless and must be discarded if the color becomes severely darkened. The substrate solution is easy to be polluted and is required to be preserved properly in dark place.
Claims (7)
1. The ELISA kit for quantitatively detecting the insect-resistant protein Cry1F in transgenic corn is characterized by comprising a micropore reaction plate coated with a capture antibody, a sample treatment solution, a biotin-labeled detection antibody, a standard substance, horseradish peroxidase-labeled avidin, a biotin-labeled antibody diluent, a horseradish peroxidase-labeled avidin diluent, a concentrated washing solution, a substrate solution, a stop solution and a plate patch;
wherein, the sample treatment fluid is: 1M Tris, pH 7.5 500uL,1M NaCL 1.5 mL,0.5M EDTA 20 uL,50% glycerol 2 mL,10% SDS 1mL, 10mL with double distilled water, 1 sheet protease inhibitor and 50uL 1mM phenylmethylsulfonyl fluoride were added at the time of use;
biotin-labeled detection antibody: 100 μg/mL biotin-labeled Cry1F 7 1D8 murine mab in PBS,50% glycerol solution;
standard substance: 2.5ng of His-Cry 1F recombinant protein;
the avidin marked by horseradish peroxidase is a 1:40 avidin solution marked by horseradish peroxidase;
biotin-labeled antibody dilutions and horseradish peroxidase-labeled avidin dilutions were formulated with 1% BSA 1g,0.05% Tween 20.05 mL,0.1% NaN 3 0.1mL, and finally, 1 XPBS was used to determine the volume to 100mL;
concentrating the washing liquid: 10 XPBS with 1.0% Tween-20;
substrate solution: 0.5mL 2mg/mL TMB absolute ethanol solution, 10mL bottomBuffer solution, 32. Mu.L 30% H 2 O 2 Mixing, and preparing in situ;
stop solution: 1M H 2 SO 4 ，
The capture antibody is secreted by a hybridoma cell strain 1D7 1C6 with a preservation number of CGMCC No.45008, and the detection range of the capture antibody is 0.3125ng/mL-2.5 ng/mL; the detection antibody is secreted by a hybridoma cell strain 1F7 1D8 with a preservation number of CGMCC No. 45009.
2. The ELISA kit for quantitatively detecting herbicide-resistant protein Cry1F in transgenic corn according to claim 1, wherein the recombinant protein of His-Cry 1F is prepared by amplifying and sequencing Cry1F coding genes, connecting the recombinant protein with pET28a plasmid after identification, and recombining the expression vector pET28a-Cry 1FCompetent cells of escherichia coli BL21 (DE 3) are transformed, the preserved recombinant protein Cry1F expression strain is subjected to resuscitative culture, the expression protein is induced by IPTG at 16 ℃ overnight, and the recombinant protein is purified.
3. The enzyme-linked immunosorbent assay kit for quantitatively detecting the herbicide-resistant protein Cry1F in transgenic corn according to claim 1, wherein the hybridoma cell strains 1D7 1C6 and 1F7 1D8 are obtained by immunizing BALB/c mice with the herbicide-resistant protein Cry1F in transgenic corn, fusing the immunized mice spleen cells with commercial mouse hybridoma cells SP2/0 and screening with HAT medium.
4. A method for the detection of an enzyme-linked immunosorbent assay kit for the quantitative detection of the insect-resistant protein Cry1F in transgenic maize according to any one of claims 1 to 3, characterized by comprising the following steps:
1) Placing the sample treatment solution, biotin-labeled detection antibody, standard substance, horseradish peroxidase-labeled avidin, biotin-labeled antibody diluent, horseradish peroxidase-labeled avidin diluent, concentrated washing solution, substrate solution and stop solution at 18-25deg.C, and balancing for at least 30 min;
2) Setting a standard substance hole and a sample hole to be detected respectively, adding a standard substance or a sample to be detected into each hole for incubation, then discarding liquid, spin-drying, adding a biotin-marked detection antibody working solution for incubation, discarding liquid in the holes, washing and spin-drying, adding a horseradish peroxidase-marked avidin working solution into each hole for incubation, discarding liquid in the holes, washing and spin-drying, adding a substrate solution into each hole, adding a stop solution into each hole after light-shielding development, uniformly mixing, and measuring the optical density OD value of each hole at 450nm wavelength by using an enzyme-labeled instrument within 5 minutes after the reaction is terminated;
3) And (3) data processing: and (3) subtracting the value of the S0 hole from the standard substance and the sample value, drawing a curve, if a compound hole is arranged, taking the average value of the compound hole to calculate, drawing a standard curve by taking the concentration of the standard substance as an ordinate and the OD value as an abscissa, and finding out the corresponding concentration from the standard curve according to the OD value of the sample.
5. The method according to claim 4, wherein the biotin-labeled detection antibody working solution is obtained by diluting a biotin-labeled detection antibody liquid with a biotin-labeled antibody diluent by 1:100 times, and the horseradish peroxidase-labeled avidin working solution is obtained by diluting a horseradish peroxidase-labeled avidin diluent with horseradish peroxidase by 1:100 times.
6. The detection method according to claim 4, wherein in the step 2), 80-120 mu L of standard substance or sample to be detected is added into each hole respectively, the mixture is gently shaken and mixed, covered with a plate patch, and incubated at 37 ℃ for 1.5-2.5 hours; discarding the liquid, spin-drying without washing; adding 80-120 mu L of biotin-labeled antibody working solution into each hole, coating a new plate patch, and incubating at 37 ℃ for 0.5-1.5 hours; discarding the liquid in the holes, spin-drying, washing the plate for 2-4 times, soaking for 1-3 minutes each time, and spin-drying at 150-250 mu L/hole; adding 80-120 mu L of horseradish peroxidase labeled avidin working solution into each hole, coating a new plate patch, and incubating at 37 ℃ for 0.5-1.5 hours; discarding the liquid in the holes, spin-drying, washing the plate for 4-6 times, soaking for 1-3 minutes each time, and spin-drying at 150-250 mu L/hole; adding 90 mu L of substrate solution into each hole in sequence, and developing for 15-30 minutes at 37 ℃ in a dark place; the reaction was terminated by adding 50. Mu.L of the termination solution to each well in this order.
7. A method for the quantitative detection of the enzyme-linked immunosorbent assay kit for the anti-insect protein Cry1F in transgenic maize according to any one of claims 1 to 3, characterized in that said detection method is an ELISA double antibody sandwich method.
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| CN109781993A (en) * | 2018-12-27 | 2019-05-21 | 中国农业科学院生物技术研究所 | A kind of enzyme linked immunological kit of quantitative detection insect resistance protein Cry1C |
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