CN114371295B - ELISA kit for quantitatively detecting insect-resistant protein Cry 3Bb and detection method thereof - Google Patents
ELISA kit for quantitatively detecting insect-resistant protein Cry 3Bb and detection method thereof Download PDFInfo
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
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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, a horseradish peroxidase-marked avidin, a biotin-marked antibody diluent, a horseradish peroxidase-marked avidin diluent, a concentrated washing solution, a substrate solution, a termination 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 Cry 3Bb 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 currently the predominant 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 Cry 3Bb 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 Cry 3Bb proteins in transgenic crops or derivatives thereof, the research and development of the Cry 3Bb enzyme-linked immunosorbent assay kit has great significance.
Disclosure of Invention
The invention aims to provide an enzyme-linked immunosorbent assay kit for quantitatively detecting insect-resistant protein Cry 3Bb 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 an insect-resistant protein Cry3Bb 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 Cry 3bb 2h11 1h1 murine mab (PBS, 50% glycerol) solution;
Standard substance: 50ng of His-Cry 3Bb recombinant protein (freeze-dried powder), wherein the His-Cry 3Bb recombinant protein is obtained by amplifying and sequencing Cry 3Bb coding genes, connecting the recombinant expression vector pET28a-Cry 3Bb with pET28a plasmid after identification, transforming competent cells of escherichia coli BL21 (DE 3) with a recombinant expression vector pET28a-Cry 3Bb, resuscitating and culturing a preserved recombinant protein Cry 3Bb expression strain, inducing the expressed 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 2O2 are mixed and prepared on site;
Stop solution: 1M H 2SO4.
Characteristics of the kit:
1) The sensitivity is: 0.972ng/mL
2) Precision: intra-batch difference CV% <8%, inter-batch difference CV% <10%
3) Specificity: the kit specifically detects Cry 3Bb 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 strain 1d 71 g8 with a collection number of CGMCC No.23865, and the 1d 71 g8 is used as a capture antibody detection range: 1.5625ng/mL-50ng/mL.
In another embodiment of the enzyme-linked immunosorbent assay kit, the detection antibody is secreted by a hybridoma cell strain 2H11 1H1 with a preservation number of CGMCC No. 23866.
The hybridoma cell strains 1D7 1G8 and 2H11 1H1 are obtained by immunizing a BALB/c mouse with an insect-resistant protein Cry 3Bb in transgenic corn, fusing the immunized mouse 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 Cry 3Bb 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 values of the blank control (S0) holes from the standard substance and the sample value, drawing a curve, if a compound hole is arranged, taking the average value of the compound holes for calculation, taking the concentration of the standard substance as an ordinate and the OD value as an abscissa, drawing a standard curve on logarithmic coordinate paper, 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 respectively, the mixture is gently shaken and mixed, the plate is attached, and the mixture is 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, and repeating 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, and adding 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, and the mixture is gently shaken and mixed, attached to a plate, 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, and repeating a new plate patch for incubation for 1 hour at 37 ℃; 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, and carrying out new plate paste on the mixture to incubate for 1 hour at 37 ℃; 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 Cry 3Bb in transgenic corn at present, domestically and internationally.
Preservation information
The hybridoma cell strain 2H11 1H1 and the hybridoma cell strain 1D7 1G8 provided by the invention are sequentially preserved in the China general microbiological culture Collection center (China general microbiological culture Collection center) at 11 month 08 of 2021, and the preservation addresses are as follows: the korean district North Star, beijing city, part No.1, no. 3. Post code: 100101, and the preservation numbers are CGMCC No.23866 and CGMCC No.23865 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 application quantitatively detects the insect-resistant protein Cry 3Bb in transgenic maize
An ELISA kit for quantitatively detecting an insect-resistant protein Cry 3Bb 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 Cry 3bb 2h11 1h1 murine mab (PBS, 50% glycerol) solution;
Standard substance: 50ng of His-Cry 3Bb recombinant protein (freeze-dried powder), wherein the His-Cry 3Bb recombinant protein is obtained by amplifying and sequencing Cry 3Bb coding genes, connecting the recombinant expression vector pET28a-Cry 3Bb with pET28a plasmid after identification, transforming competent cells of escherichia coli BL21 (DE 3) with a recombinant expression vector pET28a-Cry 3Bb, resuscitating and culturing a preserved recombinant protein Cry 3Bb expression strain, inducing the expressed 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 2O2 are mixed and prepared on site;
Stop solution: 1M H 2SO4.
Characteristics of the kit:
1) The sensitivity is: 0.972ng/mL
2) Precision: intra-batch difference CV% <8%, inter-batch difference CV% <10%
3) Specificity: the kit specifically detects Cry 3Bb 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 strain 1d 71 g8 with a collection number of CGMCC No.23865, and the 1d 71 g8 is used as a capture antibody detection range: 1.5625ng/mL-50ng/mL.
In another embodiment of the enzyme-linked immunosorbent assay kit, the detection antibody is secreted by a hybridoma cell strain 2H11 1H1 with a preservation number of CGMCC No. 23866.
The hybridoma cell strains 1D7 1G8 and 2H11 1H1 are obtained by immunizing a BALB/c mouse with an insect-resistant protein Cry 3Bb in transgenic corn, fusing the immunized mouse 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 Cry 3Bb 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, the plate is attached, and the mixture is 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, and repeating a new plate patch for incubation for 1 hour at 37 ℃; 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, and carrying out new plate paste on the mixture to incubate for 1 hour at 37 ℃; 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 the standard substance from 50ng to a series of concentration standard substances, adding the standard substance into each hole for incubation according to the detection method disclosed by the application, then discarding the liquid, adding a biotin-marked detection antibody working solution for incubation, discarding the liquid in the hole, adding a horseradish peroxidase-marked avidin working solution into each hole after spin-drying, performing incubation, discarding the liquid in the hole, adding a substrate solution into each hole after spin-drying, adding a stop solution into each hole after light-shielding color development, 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 to make a standard curve, and further see the accompanying figure 1, wherein R 2 of the standard curve is more than 0.99, the linear detection range is 1.5625ng/mL-50ng/mL, and the sample concentration calculation formula is deduced: x= (y-0.0433)/0.0466.
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=22.28+/-0.14 mug/g of Cry 3Bb 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: to prevent sample evaporation or contamination, the microplate must be applied during incubation and dried 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 (4)
1. The ELISA kit for quantitatively detecting the insect-resistant protein Cry 3Bb 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 diluent of the biotin-labeled detection antibody, a diluent of the horseradish peroxidase-labeled avidin, a concentrated washing solution, a substrate solution, a stop solution and a plate patch; the capture antibody is secreted by a hybridoma cell strain 1D7 1G8 with a preservation number of CGMCC No.23865, and the detection range of the capture antibody is 1.5625ng/mL-50 ng/mL; the detection antibody is secreted by a hybridoma cell strain 2H11-1H1 with a preservation number of CGMCC No. 23866.
2. A method for quantitatively detecting the insect-resistant protein Cry 3Bb in transgenic corn using the enzyme-linked immunosorbent assay kit of claim 1, comprising the steps of:
1) The sample treatment solution, the biotin-labeled detection antibody, the standard substance, the horseradish peroxidase-labeled avidin, the dilution of the biotin-labeled detection antibody, the dilution of the horseradish peroxidase-labeled avidin, the concentrated washing solution, the substrate solution and the stop solution according to claim 1 are placed at 18-25 ℃ and balanced for at least 30 minutes;
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 working solution of a biotin-marked detection antibody for incubation after spin-drying, discarding liquid in the holes, adding working solution of horseradish peroxidase-marked avidin into each hole after washing and spin-drying, carrying out incubation, discarding liquid in the holes, adding a substrate solution into each hole after washing and spin-drying, adding a stop solution into each hole after light-shielding color 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 values of the blank control holes from the standard substance and the sample value, drawing a curve, if a compound hole is arranged, taking the average value of the compound holes for calculation, 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.
3. The method according to claim 2, wherein the working solution of biotin-labeled detection antibody is obtained by diluting a biotin-labeled detection antibody solution with a biotin-labeled detection antibody diluent by 1:100 times, and the working solution of horseradish peroxidase-labeled avidin is obtained by diluting a horseradish peroxidase-labeled avidin diluent with horseradish peroxidase by 1:100 times.
4. The method according to claim 2, wherein in step 2), 80-120 μl of standard or sample to be tested is added to each well, and the mixture is gently shaken and mixed, and then the plate is attached, and incubated at 37deg.C 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, and repeating 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, and adding 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.
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| CN117147882B (en) * | 2023-10-31 | 2024-01-26 | 中国农业科学院生物技术研究所 | Kit and method for quantitative detection of insect-resistant protein Cry1Ah enzyme-linked immunity |
| CN117169516B (en) * | 2023-11-01 | 2024-02-23 | 中国农业科学院生物技术研究所 | Insect-resistant protein Cry1Ah colloidal gold immunochromatography rapid test strip and detection method thereof |
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