CN113687065B - Thionine-labeled antibody immunochromatography test strip for visual rapid detection of transgenic protein - Google Patents
Thionine-labeled antibody immunochromatography test strip for visual rapid detection of transgenic protein Download PDFInfo
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G01N33/531—Production of immunochemical test materials
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Abstract
A thionine-labeled antibody immunochromatographic test strip for visual rapid detection of transgenic proteins comprises a bottom plate, a sample pad, a binding pad, a nitrocellulose membrane and absorbent paper which are sequentially connected on the bottom plate, wherein a thionine-labeled antibody is fixed in the binding pad, and is a Thi-mAb1 compound formed by coupling thionine with a monoclonal antibody mAb1 through glutaraldehyde. The immunochromatographic test strip provided by the invention uses Thi as a signal marker, does not need a nano material or a complex synthesis process, and is quickly coupled with an antibody by a covalent bond by using a conventional cross-linking agent, so that the complex preparation process of colloidal gold is broken through, the time for labeling the antibody is greatly shortened, the preparation process is simplified, the transgenic protein can be quickly detected, and the sensitivity is high, and the specificity, stability and reproducibility are good.
Description
Technical Field
The invention belongs to the technical field of rapid immunodetection, and particularly relates to a thionine-labeled antibody immunochromatography test strip for visual rapid detection of transgenic proteins.
Background
Currently, more and more detection techniques are applied to the detection of transgenic crops. PCR is the main method for detecting the specific DNA of transgenic crops, but the operation is complex, the time consumption is long, expensive instruments are needed, and the PCR is not suitable for field detection. LAMP and RPA overcome the defects of PCR, can rapidly, sensitively and highly specifically detect, but adopt agarose gel electrophoresis, and have long time consumption, easy pollution and certain limitations.
The detection based on the nucleic acid level has ultrahigh sensitivity, but has certain disadvantages in the field detection of actual samples. Based on the detection technology of the exogenous protein level, the sample can be intuitively detected, and the method has advantages in the field detection of the actual sample. ELISA is a conventional detection method based on foreign proteins, and has the advantages of simple operation, strong specificity, and long time consumption. The immunochromatography test strip (Immunochromatographic strip, ICS) has the advantages of low cost, simplicity in operation, short detection time, simplicity in sample treatment and the like, and is widely applied to rapid detection in various fields. At present, ICS methods based on protein detection are very limited, and only a few test strips of protein can be selected, so that further research is still needed.
With the continuous development of immunological technology, more and more materials are used as signal markers in the construction of ICS, such as nanoparticles, quantum dots, fluorescent microspheres, and the like. The continuous update of the labels improves the detection sensitivity of ICS, but most of these materials require complex synthesis and modification processes.
The nanometer materials such as colloidal gold are widely applied to the construction of test strips as chromogenic signals, but complex antibody labeling and material modification processes are needed, and the general sensitivity is low, meanwhile, in the actual detection of the traditional colloidal gold test strips, the traditional colloidal gold test strips are easily influenced by sample matrixes such as blood and blood color samples, and have certain limitation; fluorescent materials, such as cadmium telluride quantum dots and up-conversion nanomaterials, are also expensive, and require the use of a small light source for visualization.
Therefore, the label signal of the test strip needs to be further improved, and the ICS with high sensitivity, simple operation and short detection has important significance for the development of a rapid immunoassay.
Disclosure of Invention
The invention aims to provide a thionine-labeled antibody immunochromatographic test strip for visual rapid detection of transgenic proteins, which takes thionine Thi as a signal marker, does not need synthesis, directly carries out labeling, simplifies the experimental process by only 10min, does not need nano materials, is ultra-simple, rapidly detects the transgenic protein CP4-EPSPS, breaks the limitation of the traditional colloidal gold test strip in actual sample detection, and provides a new direction for the construction of the immunochromatographic test strip.
In order to achieve the above purpose, the technical scheme provided by the invention is as follows:
the utility model provides a visual quick detection transgenic protein's thionine mark antibody immunochromatography test strip, its includes, bottom plate, fix sample pad, binding pad, nitrocellulose membrane and the absorbent paper that connects gradually on the bottom plate, wherein, fixed with the mark antibody by thionine mark in the binding pad, mark antibody is the capture antibody of transgenic protein, is the Thi-mAb1 complex that carries out the coupling formation through glutaraldehyde and monoclonal antibody mAb1 amino with covalent bonding mode;
the nitrocellulose membrane is provided with a detection line and a quality control line, the near-binding pad end of the nitrocellulose membrane is coated with a monoclonal antibody mAb2, and the monoclonal antibody mAb2 is used as the detection line and is a detection antibody of the transgenic protein; the nitrocellulose membrane near the end of the water absorption paper is coated with goat anti-mouse IgG antibody as a quality control line.
Preferably, the monoclonal antibodies mAb1 and mAb2 are paired antibodies to the transgenic protein CP 4-EPSPS.
Preferably, the mass of the labeled antibody on the conjugate pad is 0.3-0.4 μg.
And the sample pad, the bonding pad, the nitrocellulose membrane and the absorbent paper are overlapped by 1-2mm.
Further, the test strip also comprises a shell sleeved outside the bottom plate; the shell comprises a shell body, wherein a sample adding hole and an observation window are formed in the shell body of the shell, the sample adding hole is formed in the shell body corresponding to the upper part of the sample pad, and the observation window is formed in the shell body corresponding to the upper part of the nitrocellulose membrane.
The invention firstly selects small-molecule thionine Thi as a signal marker, establishes a rapid detection of CP4-EPSPS protein in transgenic crops, and the Thi is rapidly coupled with monoclonal antibody mAb1 through glutaraldehyde crosslinking without complex modification, so that the preparation process is greatly shortened, meanwhile, the limitation that the result of the traditional colloidal gold test strip in detection of an actual sample (such as a blood color sample) is influenced by the color of the sample is broken due to deep purple Thi, and a new direction is provided for the construction of an immunochromatographic test strip.
According to the invention, a novel thionine chromogenic immunochromatographic test strip is constructed based on a classical double-antibody sandwich mode, a monoclonal antibody mAb1 and a monoclonal antibody mAb2 are paired antibodies of CP4-EPSPS protein, a sample is dripped above a sample pad, if the sample pad contains the target protein CP4-EPSPS protein, the CP4-EPSPS protein is combined with a labeled antibody in a binding pad to form a coupling substance to continue to migrate along with the migration of the sample, the coupling substance is captured by an antibody immobilized on a T line, the excessive coupling substance is combined with a C line antibody successively, clear two macroscopic purple strips are displayed in the test strip, and the experimental result is judged to be positive. If the sample does not contain the CP4-EPSPS protein, the labeled antibody cannot be captured by the T line antibody to accumulate, is directly combined with the C line antibody, presents a clear band, and is judged as negative. In summary, the detection result can be read quickly by naked eyes, and meanwhile, the detection result is quantitatively analyzed by Image J software.
A preparation method of a thionine-labeled antibody immunochromatographic test strip for visually and rapidly detecting transgenic proteins comprises the following steps:
1) Preparing a PVC base plate, a sample pad, a bonding pad, a nitrocellulose membrane and absorbent paper, pretreating the sample pad and the bonding pad, and drying for later use;
2) The goat anti-mouse IgG antibody and the monoclonal antibody mAb2 are respectively immobilized on a nitrocellulose membrane and used as a quality control line C line and a detection line T line;
3) Adding thionine into a centrifuge tube, and coupling the activated amino group of glutaraldehyde with a monoclonal antibody mAb1 to form a Thi-mAb1 complex, wherein the final concentration is 0.05-0.1mg/mL;
4) The Thi-mAb1 complex is used as a labeled antibody to be dripped on a binding pad, and then baked for 40min-60min at 37 ℃, and then a nitrocellulose membrane, the binding pad, a sample pad and absorbent paper are assembled on a bottom plate in sequence to be overlapped for 1-2mm, and then cut.
Preferably, in step 3), the concentration of thionine is 0.5-1mg/mL, the final concentration of Thi-mAb1 complex is 0.05mg/mL, and the volume is 6-8. Mu.L; in the step 4), the test paper is dripped on a bonding pad and then baked for 40min at 37 ℃, and the assembled test paper is cut into strips with the width of 2.5-3 mm.
In step 3), the coupling time between the monoclonal antibody mAb1 and Thi was 10min.
The thionin Thi is used as a marking signal in an experiment, the marking condition of the thionin Thi and a specific monoclonal antibody mAb1 directly influence the signal intensity of a detection line and the sensitivity of a test strip, when the concentration of the thionin is too low, the detection signal is weaker, and when the concentration of the thionin is too high, a background interference phenomenon can be generated, and meanwhile, excessive thionin is accumulated below an NC film to prevent sample migration from forming errors and prevent signal response of the detection line, so that the optimal marking concentration of the thionin is selected in combination with the signal intensity of an observation detection area, the signal generating capacity and the transverse migration behavior are combined, and the strip is clear and uniform and has no background interference phenomenon.
Compared with the prior art, the invention has the following beneficial effects:
in the immunochromatography test strip, thi is used as a small molecular marker, can be quickly coupled with target protein, is used as a substrate to mark and capture monoclonal antibodies, can quickly determine whether target detection objects exist in a sample, can be applied to specific detection of respective proteins by changing the coupled antibodies, becomes a potential marking signal to be applied to a quick detection method, and opens up a new way for signal marking of the immunochromatography test strip.
The invention realizes the real-time detection of the sample by a simple colorimetric method, and the detection only needs 5-10min. Compared with the nano material labeling method used by the traditional test strip, the Thi does not need a complex synthesis process, and the conventional cross-linking agent is used for quickly coupling with the antibody through a covalent bond, so that the complex preparation process of colloidal gold is broken through, the time for labeling the antibody is greatly shortened, and the preparation process is simplified.
The immunochromatographic test strip can successfully detect the CP4-EPSPS proteins in different crops, has high detection sensitivity, is not interfered by other proteins, and can accurately detect soybean, beet and cotton containing the CP4-EPSPS proteins, wherein the vLOD respectively reaches 0.05%,0.1% and 0.1%, and the result is consistent with the expected result after quantitative verification of detection results by Image J software.
The immunochromatographic test strip has good stability and reproducibility, and the vLOD of soybeans, beets and cotton is kept unchanged after the test strip is stored for 3 months at 4 ℃.
Drawings
FIGS. 1-3 show the color development and signal scanning results of test strips of labeled antibodies with different thionine Thi concentrations in example 1 of the present invention.
FIGS. 4-6 show the color development and signal scanning results of test strips of different Thi-mAb volumes in example 1 of the present invention.
FIGS. 7-9 show the color development and signal scanning results of test strips with different labeling times according to example 1 of the present invention.
Fig. 10 is a schematic diagram of the test strip structure in embodiment 1 of the present invention.
Fig. 11 is a schematic diagram of the test strip in embodiment 1 of the present invention.
FIGS. 12-17 show the sensitivity test results of soybean, beet and cotton of example 1 of the present invention, wherein FIGS. 13, 15 and 17 show the peak areas of T lines in FIGS. 12, 14 and 16, respectively, and the error bars show the standard deviation SD of 3 parallel tests.
FIG. 18 shows the results of specific assays in example 1, 1-9:0.01M PBS,10% blank soybean, 10% soybean RRS (CP 4-EPSPS), 1% soybean A2704-12 (PAT), 5% soybean MON87701 (BT-Cry 1 Ac), 5% blank corn, 5% corn MIR162 (BT-VIP 3 Aa), 5% corn MON89034 (BT-Cry 1A105/Cry2 Ab), 5% corn MIR604 (BT-Cry 3A).
FIGS. 19-21 are test strip stability results for soybean, beet and cotton of example 2 of the present invention.
FIG. 22 shows the results of the test of 6 actual samples of common soybeans in example 2 of the present invention, wherein (S) 10% soybean RRS and (N1) blank soybean were used as controls.
FIG. 23 shows the results of the test of 6 actual samples of common corn in example 2 of the present invention, wherein (M) 5% corn NK603 and (N2) blank corn were used as controls.
FIGS. 24-25 show the PCR detection results for CP4-EPSPS in actual samples in example 2 of the present invention, wherein "M": DL2000 DNA marker, "+": positive control, "-": negative control, 1-6: samples of actual soybean and corn were drawn.
Detailed Description
The invention is further described below in conjunction with the detailed description.
Thionine (Thi), glutaraldehyde is commercially available from Macklin, shanghai; paired antibodies against CP4-EPSPS proteins were purchased from shanghai Rong Hui biosystems; NC membrane, sample pad, conjugate pad, absorbent paper and PVC base plate were all purchased from Millipore (Bedford, mass., USA); bovine serum albumin (Bovine serum albumin, BSA) was purchased from Sigma (St.Louis, MO, USA); the CP4-EPSPS gene primer is synthesized by Sangon (Shanghai); CM4000 cutter (Bio-Dot, CA, USA); all chemical reagents were of analytical reagent grade.
Transgenic crop seed powder standards with 100% and 5% protein content for the experiments were purchased from AOCS (Urbana, IL, USA) and transgenic crop seed powder standards with 1% protein content were purchased from agricultural rural sectors, see table 1.
TABLE 1
The seed treatment method comprises the following steps:
seed powder standard for transgenic crops with 0.01M PBS buffer (pH 7.4) at 1:3, after adding PBS into seed powder, shaking vigorously for 3-5 min, centrifuging at 6000rpm for 5min, layering, collecting supernatant, diluting into transgenic protein solutions with different concentrations, detecting, and taking supernatant of corresponding non-transgenic crops as experimental blank control.
Grinding actual crop seeds into powder by a food grinder or a mortar according to the following ratio of 1:5, adding double distilled water in proportion, shaking and mixing uniformly, centrifuging at 8000rpm for 5min, and taking the supernatant for detection.
Example 1 preparation method of thionine-labeled antibody immunochromatographic test strip for visual rapid detection of transgenic protein
Thi optimum marker concentration
The thionin Thi is used as a labeling signal in an experiment, and the labeling condition of the thionin Thi and a specific monoclonal antibody mAb1 directly influence the signal intensity of a detection line and the sensitivity of a test strip, so that the concentration, the labeling time and the volume of a finally required labeled antibody of Thi are optimized.
Under the condition of room temperature, thi with the concentration of 0.1mg/mL, 0.25mg/mL, 0.5mg/mL, 1mg/mL, 1.5mg/mL and 2mg/mL is respectively selected for marking the antibody, the marking time is 10min,5 mu L glutaraldehyde (5%) is incubated, and the Thi with clear and uniform bands and no background interference phenomenon is selected as the optimal marking concentration by observing the signal intensity of a detection area.
As shown in FIG. 1, with increasing Thi concentration, the detection signal was continuously enhanced, and when the concentration was 0.5mg/mL, two bands were clearly and brightly seen. By scanning the peak intensity of the strip, the variation trend of the detection signal is verified (fig. 2), when the concentration is increased, the background interference phenomenon is generated, and meanwhile, excessive Thi is accumulated below the NC film, so that the sample migration is prevented from forming errors. As can be seen from FIG. 3, the peak area increases with the Thi concentration, and after the concentration exceeds 1mg/mL, the peak area decreases and tends to stabilize, which indicates that accumulation of Thi with too high concentration on the test strip obstructs the signal response of the detection line, and therefore, the concentration of 0.5-1.0mg/mL is selected as the optimal concentration of Thi by combining the signal generating capability and the lateral migration behavior, and the labeled antibody concentration after labeling is 0.05-0.1mg/mL.
The volume of Thi-mAb1 is another important factor affecting the sensitivity of the test strip, the Thi labeled antibody with the concentration of 0.5mg/mL is selected, 3 mu L, 4 mu L, 5 mu L, 6 mu L, 7 mu L and 8 mu L of labeled antibodies are respectively selected for experiments, and the lowest antibody amount with clear strip, no accumulation and no background interference phenomenon is selected as the optimal antibody addition amount.
As shown in FIG. 4, the signal of the detection line is continuously enhanced with the increasing volume of Thi-mAb1, the peak intensity and peak area obtained by scanning have the same trend (FIGS. 5-6), when the volume of Thi-mAb1 is 6 mu L, clear and bright results can be obtained, and when the concentration is increased, the background interference and antibody accumulation phenomena start to appear with the enhancement of the signal.
The labeling time is further optimized, thi and mAb1 are respectively incubated for 1min, 2min, 5min, 10min, 20min, 30min and 40min, and detection is carried out, and the minimum time for generating clear and bright bands is selected as the optimal incubation time.
Referring to fig. 7, mab1 was coupled rapidly with Thi, and after 10min, a stable and clear band was formed that was visible to the naked eye, and the peak intensities obtained were substantially uniform (fig. 8). Different test strip scans have certain errors, so that peak areas obtained at different coupling times show certain differences (figure 9), and in general, the stabilizing effect can be achieved by coupling mAb1 with Thi for 10min.
2. Preparation of thionine labeled antibody immunochromatography test strip
The two monoclonal antibodies mAb1 and mAb2 used in this example are paired antibodies of CP4-EPSPS, which are obtained by adopting the conventional hybridoma technology, positive clones screened by ELISA method after cell fusion are subjected to expansion culture, and ascites fluid of mice is prepared, and the monoclonal antibodies mAb1 and mAb2 are obtained after the ascites fluid antibodies are purified by protein-G column affinity chromatography, wherein the subtype of the monoclonal antibodies mAb1 is IgG1, and the subtype of the monoclonal antibodies mAb2 is IgG2a.
The immunochromatography test strip mainly comprises a PVC bottom plate, a sample pad 1, a bonding pad 2, an NC film 3 and absorbent paper 6, wherein the sample pad 1 and the bonding pad 2 are pretreated and dried for standby.
Sheep anti-mouse IgG antibody and CP4mAb2 were immobilized on NC membrane 3 (2.5X10 cm) as a quality control line (C line) 4 and a detection line (T line) 5, respectively.
Thionine was added to the centrifuge tube and incubated with monoclonal antibody for 10min at room temperature by 5. Mu.L glutaraldehyde (5%) to form Thi-mAb1 complex, with a final labeled antibody concentration of 0.05mg/mL.
And (3) dripping the marked compound on a bonding pad 2, drying for 1h at 37 ℃, sequentially assembling an NC film 3, the bonding pad 2, a sample pad 1 and a water absorbing paper 6 on a PVC bottom plate, overlapping by 1-2mm, finally cutting into test strips of 3mm by a cutting machine, and sealing and preserving under a dry condition.
As shown in fig. 11, in the detection, a sample is dropped above the sample pad, if the target protein CP4-EPSPS protein is contained, as the sample migrates, the CP4-EPSPS protein and the thionine-labeled antibody in the binding pad 2 combine to form a conjugate, and migrate continuously, the conjugate is captured by the CP4mAb2 antibody immobilized on the T line 4, the excessive conjugate is combined with the C line 5 goat anti-mouse IgG antibody successively, two clear macroscopic purple bands are displayed in the test strip, and the test result is judged to be positive. If the sample does not contain the CP4-EPSPS protein, the thionine labeled antibody cannot be captured and accumulated by the T line 4 antibody, is directly combined with the C line 5 antibody, presents a clear band, and is judged as negative.
3. Performance detection of thionine labeled antibody immunochromatography test strip
3.1 sensitivity
Transgenic crop seed powder standard products of soybean RRS, beet H7-1 and cotton MON88913 with different protein concentrations are sequentially detected, corresponding non-transgenic blank crop supernatant (C) and 0.01MPBS buffer (P) are used as blank control, and each batch of samples is subjected to 3 times of repetition, wherein the protein concentrations of the soybean RRS are respectively as follows: 10%, 5%, 1%,0.5%, 0.1%, 0.05%, 0.01% and 0.005%; the protein concentration of beet H7-1 is respectively: 10%, 1%,0.5%, 0.1%, 0.05% and 0.01%; the protein concentration of cotton MON88913 was: 10%, 1%,0.5%, 0.1%, 0.05%, 0.01% and 0.005%.
After a period of reaction, reading the T line signal intensity by Image J software, performing all tests at least three times, constructing a histogram by taking the abscissa as the sample concentration and the ordinate as the peak area of the T line gray value, and quantitatively analyzing the lowest detection concentration of different crops.
The visual limit (visual Limit of detection, vLOD) was defined as the concentration value at which the T-line signal was weaker than the blank (C) and 0.01M PBS (P).
And detecting the prepared test strip by using different crop standard powder supernatant dilutions containing the CP4-EPSPS protein. Soybean RRS ranging from 0.005% -10%, beet H7-1 ranging from 0.01% -10% and cotton MON88913 ranging from 0.005% -10% were dropped onto the sample pads, respectively, with the corresponding blank crop supernatant dilutions (C) and 0.01M PBS (P) as control groups, and each batch of samples was repeated 3 times.
The results showed that the T line signal gradually decreased until it disappeared as the concentration of CP4-EPSPS protein was decreased, and it was found from the results of the test that vloD of soybean RRS (FIG. 12) was 0.05%, vloD of beet H7-1 (FIG. 14) was 0.1%, and vloD of cotton MON88913 (FIG. 16) was 0.1%. The method has thus been demonstrated to be useful for detecting a variety of transgenic crops, including soybean, beet and cotton. Quantitative analysis of the results was performed using Image J software, and the T line peak area was again confirmed, and the method detected soybean containing CP4-EPSPS protein (FIG. 13), beet (FIG. 15), and cotton (FIG. 17) at the lowest concentrations of 0.05%,0.1%, and 0.1%, respectively.
3.2 specificity assay
In order to evaluate whether the established method can specifically select CP4-EPSPS proteins without interference from other proteins, crop seed powder standards containing different transgenic proteins were selected for analysis, all seed powders were tested by extracting supernatants with PBS buffer, no. 1-9 in order: 0.01M PBS buffer, 10% blank soybean, 10% soybean RRS (CP 4-EPSPS), 1% soybean A2704-12 (PAT), 5% soybean MON87701 (BT-Cry 1 Ac); 5% blank corn, 5% corn MIR162 (BT-VIP 3 Aa), 5% corn MON89034 (BT-Cry 1A105/Cry2 Ab), and 5% corn MIR604 (BT-Cry 3A).
As shown in FIG. 18, the method can successfully detect the soybean standard containing the CP4-EPSPS protein, is not interfered by other proteins, and the substrates of different samples have a certain influence on the visual detection effect, but do not influence the final detection sensitivity. The sensitivity of the antibody to different proteins is different to some extent, so that the detection signal intensity of the different proteins is different to some extent. In conclusion, the test strip provided by the invention has good specificity and applicability, and can meet the requirement of rapid screening of the CP4-EPSPS protein in a sample.
3.3 stability and reproducibility of test strips
The test strip prepared in the embodiment is sealed, dried and preserved for 3 months at 4 ℃ for stability analysis.
In order to further verify that the developed test strip has good stability and reproducibility, the test strips stored at 4 ℃ for 3 months are used for detecting the soybean RRS, beet H7-1 and cotton MON88913 standard substances with different concentrations.
The test strip detected vLOD of soybean, beet and cotton at 0.05%,0.1% and 0.1%, respectively (fig. 19-21). After the signal intensity and the peak area of the T line are scanned, the same result is obtained, and the developed test strip is proved to have good reproducibility and can be successfully applied to the detection of CP4-EPSPS proteins of different crops. Because of certain errors in the manufacture of different batches, the indentation of the T line causes a small amount of thionine to accumulate, and the experimental result is not influenced, so that the experimental result can be ignored.
Example 2
In order to further verify the practicability and accuracy of the test strip in an actual sample, 6 soybean and 6 corn seed samples are randomly selected from a local market, the samples are treated and protein extracted by the same method, the supernatant with the protein concentration of 10% is used for detection, the corresponding standard 10% soybean RRS (S), the 5% corn NK603 (M) and the corresponding blank crop supernatant (N) are used as controls, the current standard PCR method is used for verifying the experimental result, the transgenic soybean RRS and the corn NK603 are selected as positive controls, and the blank soybean and the corn are negative controls.
The DNA of randomly purchased soybean and corn samples was extracted using a kit for use. The primer sequence of the EPSPS gene is selected from: f1:5'-GACTTGCGTGTTCGTTCTTC-3' and R1:5'-AACACCGTTGAGCTTGAGAC-3'.
The corresponding system was added to the PCR reaction tube for amplification, and each sample was repeated 3 times. The reaction procedure is: denaturation at 95℃for 5min; amplification was performed 35 cycles (denaturation at 94℃for 1min, annealing at 56℃for 30s, and extension at 72℃for 7 min). And taking out after the reaction is finished, and performing agarose gel electrophoresis detection.
As a result, as shown in FIGS. 22 to 23, all purchased seed samples were judged negative, and all sample detection results were consistent with the PCR amplification results (FIGS. 24 to 25).
Therefore, after simple sample treatment, the CP4-EPSPS protein can be successfully detected by using the test strip provided by the invention, and the test strip has good stability and repeatability.
Claims (9)
1. A thionine-labeled antibody immunochromatographic test strip for visual rapid detection of transgenic proteins comprises a bottom plate, a sample pad, a binding pad, a nitrocellulose membrane and absorbent paper which are fixed on the bottom plate and are connected in sequence,
the binding pad is fixed with a labeled antibody marked by thionine, the labeled antibody is a capturing antibody of transgenic protein, and is a Thi-mAb1 complex formed by coupling thionine and monoclonal antibody mAb1 amino in a covalent binding mode through glutaraldehyde;
the nitrocellulose membrane is provided with a detection line and a quality control line, the near-binding pad end of the nitrocellulose membrane is coated with a monoclonal antibody mAb2, and the monoclonal antibody mAb2 is used as the detection line and is a detection antibody of the transgenic protein; the nitrocellulose membrane near the end of the water absorption paper is coated with goat anti-mouse IgG antibody as a quality control line.
2. The thionine-labeled antibody immunochromatographic test strip according to claim 1, wherein the monoclonal antibody mAb1 and the monoclonal antibody mAb2 are paired antibodies of the transgenic protein CP 4-EPSPS.
3. The thionine-labeled antibody immunochromatographic test strip according to claim 1 or 2, in which the mass of the labeled antibody on the binding pad is 0.3 to 0.4 μg.
4. The thionine-labeled antibody immunochromatographic test strip of claim 1, wherein the sample pad, the conjugate pad, the nitrocellulose membrane, and the absorbent paper are overlapped by 1-2mm between adjacent.
5. The thionine-labeled antibody immunochromatographic test strip of claim 1, further comprising a housing sleeved outside the bottom plate; the shell comprises a shell body, wherein a sample adding hole and an observation window are formed in the shell body of the shell, the sample adding hole is formed in the shell body corresponding to the upper part of the sample pad, and the observation window is formed in the shell body corresponding to the upper part of the nitrocellulose membrane.
6. A preparation method of a thionine-labeled antibody immunochromatographic test strip for visually and rapidly detecting transgenic proteins comprises the following steps:
1) Preparing a PVC base plate, a sample pad, a bonding pad, a nitrocellulose membrane and absorbent paper, pretreating the sample pad and the bonding pad, and drying for later use;
2) The goat anti-mouse IgG antibody and the monoclonal antibody mAb2 are respectively immobilized on a nitrocellulose membrane and used as a quality control line C line and a detection line T line;
3) Adding thionine into a centrifuge tube, and coupling the thionine with a monoclonal antibody mAb1 through glutaraldehyde activated amino group to form a Thi-mAb1 complex, wherein the final concentration of the Thi-mAb1 complex is 0.05-0.1mg/mL;
4) And (3) dripping the Thi-mAb1 complex serving as a labeled antibody on a binding pad, baking at 37 ℃ for 40-60 min, sequentially assembling a nitrocellulose membrane, the binding pad, a sample pad and absorbent paper on a bottom plate, overlapping for 1-2mm, and cutting into test strips with the width of 2.5-3 mm.
7. The method according to claim 6, wherein the concentration of thionine in step 3) is 0.5-1mg/mL.
8. The method according to claim 6, wherein in step 3), the monoclonal antibody mAb1 is coupled to Thi for a period of 10min.
9. The method of claim 6, wherein the monoclonal antibodies mAb1 and mAb2 are a mating antibody to CP 4-EPSPS.
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