CN113687065A

CN113687065A - Thionine labeled antibody immunochromatographic test strip for visually and rapidly detecting transgenic protein

Info

Publication number: CN113687065A
Application number: CN202111001726.3A
Authority: CN
Inventors: 王金斌; 曾海娟; 杨倩雯
Original assignee: Shanghai Ruifeng Agricultural Technology Co ltd; Shanghai Academy of Agricultural Sciences
Current assignee: Shanghai Ruifeng Agricultural Technology Co ltd; Shanghai Academy of Agricultural Sciences
Priority date: 2021-08-30
Filing date: 2021-08-30
Publication date: 2021-11-23
Anticipated expiration: 2041-08-30
Also published as: CN113687065B

Abstract

A thionine labeled antibody immunochromatographic test strip for visually and rapidly detecting transgenic protein comprises a bottom plate, a sample pad, a combination pad, a nitrocellulose membrane and absorbent paper which are fixed on the bottom plate and sequentially connected, wherein a thionine labeled antibody is fixed in the combination pad, and the thionine labeled antibody is a Thi-mAb1 compound formed by coupling thionine with a monoclonal antibody mAb1 through glutaraldehyde. The immunochromatographic test strip takes Thi as a signal marker, does not need a nano material, does not need a complex synthesis process, utilizes a conventional cross-linking agent to be quickly coupled with an antibody through a covalent bond, breaks through a complicated preparation process of colloidal gold, greatly shortens the time for marking the antibody, simplifies the preparation process, can quickly detect transgenic protein, and has high sensitivity and good specificity, stability and reproducibility.

Description

Thionine labeled antibody immunochromatographic test strip for visually and rapidly detecting transgenic protein

Technical Field

The invention belongs to the technical field of rapid immunoassay, and particularly relates to a thionine labeled antibody immunochromatographic test strip for visually and rapidly detecting transgenic protein.

Background

At present, more and more detection technologies are applied to the detection of transgenic crops. PCR is the main method for detecting the specific DNA of the transgenic crops, but the operation is complicated, the time consumption is long, expensive instruments are needed, and the PCR method is not suitable for field detection. LAMP and RPA overcome the defects of PCR, can detect quickly, sensitively and highly specifically, but adopt agar gel electrophoresis, and have the disadvantages of long time consumption, easy pollution and certain limitation.

Although the above detection based on the nucleic acid level has an ultra-high sensitivity, there still exists a certain disadvantage in the field detection of actual samples. The detection technology based on the level of the exogenous protein can visually detect the sample, and has advantages in the field detection of the actual sample. ELISA is a conventional detection method based on foreign proteins, is simple to operate and has strong specificity, but takes long time. An Immunochromatographic test strip (ICS) has the advantages of low cost, simple operation, short detection time, simple sample treatment and the like, and is widely applied to rapid detection in various fields. At present, an ICS method based on protein detection is very limited, only a few protein test strips can be selected, and further research is continuously needed.

With the continuous development of immunology technology, more and more materials are used as signal markers for the construction of ICS, such as nanoparticles, quantum dots, fluorescent microspheres, etc. The continuous updating of the markers improves the detection sensitivity of ICS, but most of the materials need complex synthesis and modification processes.

The nano materials such as colloidal gold and the like are widely applied to the construction of test strips as color development signals, but complex antibody marking and material modification processes are required, the sensitivity is low generally, and meanwhile, the traditional colloidal gold test strips are easily influenced by sample matrixes such as blood and blood color samples in actual detection and have certain limitations; fluorescent materials such as cadmium telluride quantum dots and up-conversion nanomaterials are also numerous, and have high cost, and the materials need to be visualized by means of small light-emitting sources.

Therefore, further improvement on the labeled signal substance of the test strip is needed, and the establishment of an ICS which has high sensitivity, simple operation and short-time detection has important significance for the development of a rapid immunoassay method.

Disclosure of Invention

The thionine labeled antibody immunochromatographic test strip for visually and rapidly detecting the transgenic protein provided by the invention takes thionine Thi as a signal marker, synthesis is not required, direct labeling is carried out, the labeling process is only 10min, the experimental process is simplified, nano materials are not required, the test strip is ultra-simple, the transgenic protein CP4-EPSPS can be rapidly detected, the limitation of the traditional colloidal gold test strip in actual sample detection is broken, and a new direction is provided for the construction of the immunochromatographic test strip.

In order to achieve the purpose, the technical scheme provided by the invention is as follows:

a thionine labeled antibody immunochromatographic test strip for visually and rapidly detecting transgenic protein comprises a bottom plate, a sample pad, a combination pad, a nitrocellulose membrane and absorbent paper, wherein the sample pad, the combination pad, the nitrocellulose membrane and the absorbent paper are fixed on the bottom plate and are sequentially connected, a thionine labeled antibody is fixed in the combination pad, the labeled antibody is a capture antibody of the transgenic protein, and is a Thi-mAb1 compound formed by coupling thionine with a monoclonal antibody mAb1 amino group in a covalent binding mode through glutaraldehyde;

the nitrocellulose membrane is provided with a detection line and a quality control line, the end, close to the binding pad, of the nitrocellulose membrane is coated with a monoclonal antibody mAb2 to serve as the detection line, and the monoclonal antibody mAb2 is a detection antibody of the transgenic protein; the nitrocellulose membrane near the absorbent paper end is coated with goat anti-mouse IgG antibody as a quality control line.

Preferably, the monoclonal antibody mAb1 and mAb2 are the partner antibodies of the transgenic protein CP 4-EPSPS.

Preferably, the mass of the labeled antibody on the conjugate pad is 0.3-0.4. mu.g.

And the sample pad, the combination pad, the nitrocellulose membrane and the absorbent paper are overlapped by 1-2 mm.

Further, the test strip also comprises a shell sleeved outside the bottom plate; the shell of the shell is provided with a sample adding hole and an observation window, the sample adding hole is arranged at the position corresponding to the shell above the sample pad, and the observation window is arranged at the position corresponding to the shell above the nitrocellulose membrane.

The invention selects a proper signal marker to be very important for the construction of ICS, the invention firstly selects micromolecule thionine Thi as the signal marker to establish the ICS for the rapid detection of CP4-EPSPS protein in transgenic crops, the Thi does not need a complex modification process, and is rapidly coupled with monoclonal antibody mAb1 through glutaraldehyde crosslinking, only 10min is needed, the preparation process is greatly shortened, and meanwhile, because the Thi is dark purple, the limitation that the result of the traditional colloidal gold test strip is influenced by the color of a sample in the detection of an actual sample (such as a hemochromaticsample) is broken through, and a new direction is provided for the construction of the immunochromatography test strip.

According to the invention, a novel thionine color development immunochromatographic test strip is constructed based on a classical double-antibody sandwich mode, the monoclonal antibody mAb1 and the monoclonal antibody mAb2 are paired antibodies of CP4-EPSPS protein, a sample is dripped above a sample pad, if target protein CP4-EPSPS protein is contained, the CP4-EPSPS protein is combined with a labeled antibody in a binding pad to form a coupler to continuously migrate along with the migration of the sample, the coupler is captured by an antibody immobilized on a T line, excessive coupler is combined with a C line antibody successively, two clear purple bands visible to naked eyes are presented in the test strip, and the experimental result is judged to be positive. If the sample does not contain CP4-EPSPS protein, the labeled antibody can not be captured by the T line antibody and can be accumulated, and the labeled antibody is directly combined with the C line antibody, a clear band is shown, and the sample is judged to be negative. In conclusion, the detection result can be rapidly read by naked eyes, and meanwhile, the detection result is quantitatively analyzed by using Image J software.

A preparation method of a thionine labeled antibody immunochromatographic test strip for visually and rapidly detecting transgenic protein comprises the following steps:

1) preparing a PVC base plate, a sample pad, a combination pad, a nitrocellulose membrane and absorbent paper, and pretreating and drying the sample pad and the combination pad for later use;

2) respectively fixing and loading a goat anti-mouse IgG antibody and a monoclonal antibody mAb2 on a nitrocellulose membrane to be 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 to form a Thi-mAb1 complex with the final concentration of 0.05-0.1 mg/mL;

4) dripping the Thi-mAb1 complex as a labeled antibody on a binding pad, baking at 37 ℃ for 40-60 min, sequentially assembling the nitrocellulose membrane, the binding pad, the sample pad and absorbent paper on a bottom plate, overlapping for 1-2mm, and cutting.

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 μ L; in the step 4), the test paper is dripped on a bonding pad and then dried 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 of monoclonal antibody mAb1 to Thi was 10 min.

The thionine Thi is used as a marking signal in an experiment, the signal intensity of a detection line and the sensitivity of a test strip are directly influenced with the marking condition of a specific monoclonal antibody mAb1, when the concentration of Thi is too low, the detection signal is weaker, when the concentration is too high, a background interference phenomenon can be generated, meanwhile, excessive Thi is accumulated below an NC membrane, sample migration is prevented from forming errors, and the signal response of the detection line is prevented.

Compared with the prior art, the invention has the following beneficial effects:

in the immunochromatographic test strip, Thi is used as a small molecular marker and can be quickly coupled with target protein, Thi is used as a substrate to mark and capture a monoclonal antibody, whether the target detection object exists in a sample can be quickly determined, and the coupled antibody is changed, so that the immunochromatographic test strip can be applied to the specific detection of respective proteins, becomes a potential marker signal and is applied to a quick detection method, and opens up a new way for the signal marking of the immunochromatographic test strip.

The invention realizes the real-time detection of the sample by a simple colorimetric method, and the detection only needs 5-10 min. Compared with the nanometer material marking method used by the traditional test strip, the Thi does not need a complex synthesis process, utilizes the conventional cross-linking agent to be quickly coupled with the antibody through a covalent bond, breaks through the complicated preparation process of the colloidal gold, greatly shortens the time for marking the antibody, and simplifies the preparation process.

The immunochromatographic test strip can successfully detect CP4-EPSPS protein in different crops, has high detection sensitivity, is not interfered by other proteins, for example, soybean, beet and cotton containing CP4-EPSPS protein can be accurately detected, the vLOD respectively reaches 0.05%, 0.1% and 0.1%, and the result is consistent with the expectation after the detection result is quantitatively verified by using Image J software.

The immunochromatographic test strip has good stability and reproducibility, and the vLOD of soybeans, beet and cotton is kept unchanged after the test strip is stored for 3 months at 4 ℃.

Drawings

Fig. 1 to 3 show the test strip color development and signal scanning results of the antibodies labeled with different thionine Thi concentrations in example 1 of the present invention.

FIGS. 4 to 6 show the results of color development and signal scanning of test strips with different volumes of Thi-mAb in example 1.

Fig. 7 to 9 show the test strip color development and signal scanning results at different marking times in example 1 of the present invention.

Fig. 10 is a schematic structural diagram of a test strip 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 to 17 are the results of the sensitivity measurements of soybean, beet and cotton in example 1 of the present invention, wherein FIGS. 13, 15 and 17 are the peak areas of the T-lines in FIGS. 12, 14 and 16, respectively, and the error bars are the standard deviation SD of 3 replicates.

FIG. 18 shows the results of the specificity test in example 1 of the present invention, in which 1 to 9: 0.01M PBS, 10% blank soybean, 10% soybean RRS (CP4-EPSPS), 1% soybean A2704-12(PAT), 5% soybean MON87701(BT-Cry1Ac), 5% blank corn, 5% corn MIR162(BT-VIP3Aa), 5% corn MON89034(BT-Cry1A105/Cry2Ab), 5% corn MIR604(BT-Cry 3A).

FIGS. 19-21 show the stability results of the test strips for soybean, beet and cotton in example 2 of the present invention.

FIG. 22 shows the results of testing 6 general soybean samples in example 2 of the present invention, wherein (S) 10% soybean RRS and (N1) blank soybean are used as controls.

FIG. 23 shows the results of testing 6 general corn samples in example 2 of the present invention, wherein (M) 5% corn NK603 and (N2) blank corn are used as controls.

FIGS. 24 to 25 are the results of PCR detection of CP4-EPSPS in the actual sample in example 2 of the present invention, in which "M": DL2000 DNA marker, "+": positive control, "-": negative control, 1-6: actual soybean and corn samples were drawn.

Detailed Description

The present invention will be further described with reference to the following embodiments.

Thionine (Thionine, Thi), glutaraldehyde, is commercially available from the Shanghai Macklin; the paired antibodies against CP4-EPSPS protein were purchased from Shanghai Holly Biopsies; NC membranes, sample pads, conjugate pads, absorbent paper and PVC backing plates were all purchased from Millipore (Bedford, MA, USA); bovine Serum Albumin (BSA) purchased from Sigma (st. louis, MO, USA); CP4-EPSPS gene primer was 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 rural parts of agriculture, see table 1.

TABLE 1

The seed treatment method comprises the following steps:

seed powder standards for transgenic crops were prepared using 0.01M PBS buffer (pH 7.4) at a 1: 3, adding the seed powder into PBS, violently shaking for 3-5 min, centrifuging at 6000rpm for 5min, collecting supernatant in a layered manner, diluting the supernatant into transgenic protein solutions with different concentrations for detection, and taking the supernatant of corresponding non-transgenic crops as experiment blank control.

Grinding actual crop seeds into powder by a food grinder or a mortar according to the weight ratio of 1: and 5, adding double distilled water according to a proportion of 5, shaking and uniformly mixing, centrifuging at 8000rpm for 5min, and taking the supernatant for detection.

Embodiment 1 preparation method of thionine labeled antibody immunochromatographic test strip for visually and rapidly detecting transgenic protein

Optimal marker concentration for Thi

The thionine Thi is used as a labeling signal in an experiment, the labeling condition of the thionine Thi and a specific monoclonal antibody mAb1 directly influences the signal intensity of a dye detection line and the sensitivity of a test strip, and the concentration of Thi, the labeling time and the volume of a finally required labeling antibody 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 to mark the antibody, the marking time is 10min, 5 mu L of 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, the detection signal increases with the increase of the concentration of Thi, and two clear and bright bands appear at a concentration of 0.5 mg/mL. The trend of the detection signal was verified by scanning the peak intensity of the band (fig. 2), and as the concentration increased, a background interference phenomenon was generated, while excessive Thi accumulated below the NC film, hindering sample migration from forming errors. As can be seen from FIG. 3, the peak area increases with the concentration of Thi, and when the concentration exceeds 1mg/mL, the peak area decreases and becomes stable, which indicates that the accumulation of the Thi with too high concentration on the test strip hinders the signal response of the detection line, therefore, the concentration of 0.5-1.0mg/mL is selected as the optimum concentration of Thi, and the labeled antibody concentration after labeling is 0.05-0.1mg/mL, in combination with the signal generation capacity and the lateral migration behavior.

The volume of the Thi-mAb1 is another important factor influencing the sensitivity of the test strip, the Thi labeled antibody with the concentration of 0.5mg/mL is selected, the labeled antibodies with the concentrations of 3 muL, 4 muL, 5 muL, 6 muL, 7 muL and 8 muL are respectively selected for experiments, and the lowest antibody amount with clear bands, no accumulation and no background interference phenomenon is selected as the optimal antibody adding amount.

As shown in FIG. 4, with the increasing volume of Thi-mAb1, the signal of the detection line is strengthened, the peak intensity and peak area obtained by scanning the detection line have the same trend (FIGS. 5-6), when the volume of Thi-mAb1 is 6 μ L, a clear and bright result can be obtained, and when the concentration is increased, the background interference and antibody accumulation phenomenon begin to appear with the strengthening of the signal.

And (3) further optimizing the labeling time, incubating the Thi and the mAb1 for 1min, 2min, 5min, 10min, 20min, 30min and 40min respectively, and detecting, wherein the optimal incubation time is selected as the minimum time for generating clear and bright bands.

Referring to fig. 7, mAb1 was conjugated to Thi rapidly, and after 10min, a visually stable clear band was formed with essentially uniform peak intensity (fig. 8). Different test strip scans have certain errors, so peak areas obtained at different coupling times show certain differences (fig. 9), and in general, the stable effect can be achieved by coupling the mAb1 with Thi for 10 min.

2. Immunochromatographic test strip for preparing thionine labeled antibody

The two monoclonal antibodies mAb1 and mAb2 used in this example are the conjugated antibodies of CP4-EPSPS, both obtained by conventional hybridoma technology, after cell fusion, the positive clones screened by ELISA method were expanded and cultured, and mouse ascites was prepared, the ascites antibody was purified by protein-G column affinity chromatography to obtain mAb1 and mAb2, the subtype of mAb1 was IgG1, and the subtype of mAb2 was IgG2 a.

The immunochromatographic test strip is shown in fig. 10 and fig. 11 in structure and principle, and mainly comprises a PVC base plate, a sample pad 1, a combination pad 2, an NC membrane 3 and absorbent paper 6, wherein the sample pad 1 and the combination pad 2 are pretreated and dried for later use.

Goat anti-mouse IgG antibody and CP4mAb2 were immobilized on NC membrane 3 (2.5X 30cm) as quality control line (C line) 4 and detection line (T line) 5, respectively.

Thionine was added to the centrifuge tube and incubated with 5. mu.L glutaraldehyde (5%) and monoclonal antibody for 10min at room temperature to form a Thi-mAb1 complex with a final concentration of 0.05mg/mL of labeled antibody.

And dripping the marked compound on a bonding pad 2, drying at 37 ℃ for 1h, assembling the NC membrane 3, the bonding pad 2, the sample pad 1 and the absorbent paper 6 on a PVC (polyvinyl chloride) base plate in sequence, overlapping by 1-2mm, cutting into test strips with the thickness of 3mm by using a cutting machine, and sealing and preserving under a dry condition.

In the detection process, as shown in fig. 11, a sample is dripped on the sample pad, if the sample contains the target protein CP4-EPSPS protein, as the sample migrates, the CP4-EPSPS protein is combined with the thionine labeled antibody in the binding pad 2 to form a coupling substance which continuously migrates, the coupling substance is captured by the CP4mAb2 antibody immobilized on the T-line 4, the excessive coupling substance is combined with the C-line 5 goat anti-mouse IgG antibody successively, two clear purple bands which can be seen with naked eyes are presented in the test strip, and the test result is judged to be positive. If the sample does not contain CP4-EPSPS protein, the thionine labeled antibody can not be captured by the T line 4 antibody and can be accumulated, and the thionine labeled antibody is directly combined with the C line 5 antibody, presents a clear band and is judged to be negative.

3. Performance detection of thionine labeled antibody immunochromatographic test strip

3.1 sensitivity

Sequentially detecting transgenic crop seed powder standard products of soybean RRS, beet H7-1 and cotton MON88913 with different protein concentrations, taking corresponding non-transgenic blank crop supernatant (C) and 0.01MPBS buffer solution (P) as blank controls, and repeating for 3 times for each batch of samples, 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 concentrations of beet H7-1 were: 10%, 1%, 0.5%, 0.1%, 0.05% and 0.01%; the protein concentrations of cotton MON88913 were: 10%, 1%, 0.5%, 0.1%, 0.05%, 0.01% and 0.005%.

And after reacting for a period of time, reading the signal intensity of the T line by using 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 gray value of the T line, and quantitatively analyzing the lowest detection concentration of different crops.

The visual Limit of detection (vLOD) is defined as the concentration value at which the T-line signal is weaker than that of the blank control (C) and 0.01M PBS (P).

The prepared test strip is used for detecting supernatant diluent of different crop standard product powder containing CP4-EPSPS protein. Soybean RRS at a concentration range of 0.005% -10%, beet H7-1 at a concentration range of 0.01% -10% and cotton MON88913 at a concentration range of 0.005% -10% were dropped on the sample pad, and the corresponding blank crop supernatant dilutions (C) and 0.01M PBS (P) were used as control groups, and each batch of samples was repeated 3 times.

The results showed that with the increasing decrease of CP4-EPSPS protein concentration, the signal of T line gradually decreased until disappeared, and from the detection results, it can be seen that the vLOD of soybean RRS (FIG. 12) reached 0.05%, the vLOD of beet H7-1 (FIG. 14) reached 0.1%, and the vLOD of cotton MON88913 (FIG. 16) reached 0.1%. This demonstrates that the method can be used to detect a variety of transgenic crops, including soybean, sugar beet and cotton. The results were quantified using Image J software and the peak areas of the T-lines confirmed again that the method detected the lowest concentrations of CP4-EPSPS protein in soybean (FIG. 13), beet (FIG. 15) and cotton (FIG. 17) as 0.05%, 0.1% and 0.1%, respectively.

3.2 specificity test

In order to evaluate whether the established method can specifically select the CP4-EPSPS protein without being interfered by other proteins, crop seed powder standard products containing different transgenic proteins are selected for analysis, all seed powder is extracted from supernatant by PBS buffer solution for experiment, and No. 1-No. 9 are as follows: 0.01M PBS buffer, 10% blank soybean, 10% soybean RRS (CP4-EPSPS), 1% soybean A2704-12(PAT), 5% soybean MON87701(BT-Cry1 Ac); 5% maize blanc, 5% maize MIR162(BT-VIP3Aa), 5% maize MON89034(BT-Cry1A105/Cry2Ab) and 5% maize MIR604(BT-Cry 3A).

As shown in FIG. 18, the method can successfully detect the soybean standard containing CP4-EPSPS protein, and is not interfered by other proteins, and the matrixes of different samples have certain influence on the visual detection effect, but do not influence the final detection sensitivity. The sensitivity of the antibody to different proteins has certain difference, so that the detection signal intensity of different proteins has certain difference. In conclusion, the test strip disclosed by the invention has good specificity and applicability, and can meet the requirement of rapid screening of CP4-EPSPS protein in a sample.

3.3 stability and reproducibility of the test strip

The test strips prepared in this example were stored in a sealed and dried condition at 4 ℃ for 3 months for stability analysis.

In order to further verify that the developed test strip has good stability and reproducibility, the test strip stored at 4 ℃ for 3 months is used for detecting soybean RRS, beet H7-1 and cotton MON88913 standards with different concentrations.

The test strip detected vLOD of soybean, beet and cotton as 0.05%, 0.1% and 0.1%, respectively (fig. 19-21). After scanning the signal intensity and the peak area of the T line, 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 the manufacture of different batches has certain errors, the indentation of the T line causes a small amount of thionine to be accumulated, the experimental result is not influenced, and the accumulation can be ignored.

Example 2

In order to further verify the practicability and accuracy of the test strip in actual samples, 6 soybean and 6 corn seed samples are randomly selected from a local market, the samples are treated and extracted with protein by the same method, supernatant with the protein concentration of 10% is used for detection, corresponding standard products of 10% soybean RRS (S), 5% corn NK603(M) and corresponding blank crop supernatant (N) are used as controls, the experimental results are verified by the current standard PCR method, transgenic soybean RRS and corn NK603 are used as positive controls, and blank soybean and corn are used as negative controls.

DNA of randomly purchased soybean and corn samples was extracted using a kit for future use. The primer sequence of the EPSPS gene is selected from: f1: 5'-GACTTGCGTGTTCGTTCTTC-3' and R1: 5'-AACACCGTTGAGCTTGAGAC-3' are provided.

The corresponding system was added to the PCR reaction tube for amplification, and each sample was repeated 3 times. The reaction procedure is as follows: denaturation at 95 deg.C for 5 min; 35 cycles of amplification (denaturation at 94 ℃ for 1min, annealing at 56 ℃ for 30s, and extension at 72 ℃ for 7min) were performed. And taking out after the reaction is finished, and carrying out agarose gel electrophoresis detection.

As shown in FIGS. 22 to 23, all the seed samples purchased were judged as negative, and the results of all the samples were identical to those of the PCR amplification (FIGS. 24 to 25).

Therefore, after simple sample treatment, the test strip can successfully detect the CP4-EPSPS protein, and has good stability and repeatability.

Claims

1. A thionine labeled antibody immunochromatographic test strip for visually and rapidly detecting transgenic protein comprises a bottom plate, a sample pad, a combination pad, a nitrocellulose membrane and absorbent paper which are fixed on the bottom plate and are sequentially connected, wherein,

a labelled antibody labeled by thionine is fixed in the binding pad, the labelled antibody is a capture antibody of transgenic protein, and is a Thi-mAb1 complex formed by coupling thionine and a monoclonal antibody mAb1 amino group in a covalent binding mode through glutaraldehyde;

2. The thionine labeled antibody immunochromatographic test strip according to claim 1, characterized in that the monoclonal antibody mAb1 and mAb2 are the conjugated antibodies of transgenic protein CP 4-EPSPS.

3. The thionine-labeled antibody immunochromatographic test strip according to claim 1 or 2, wherein the mass of the labeled antibody on the conjugate pad is 0.3 to 0.4 μ g.

4. The thionine-labeled antibody immunochromatographic test strip according to claim 1, wherein the sample pad, the conjugate pad, the nitrocellulose membrane and the absorbent paper are overlapped by 1-2 mm.

5. The thionine labeled antibody immunochromatographic test strip according to claim 1, further comprising a housing fitted over the bottom plate; the shell of the shell is provided with a sample adding hole and an observation window, the sample adding hole is arranged at the position corresponding to the shell above the sample pad, and the observation window is arranged at the position corresponding to the shell above the nitrocellulose membrane.

6. A preparation method of a thionine labeled antibody immunochromatographic test strip for visually and rapidly detecting transgenic protein comprises the following steps:

3) adding thionine into a centrifuge tube, and coupling the thionine with a monoclonal antibody mAb1 through activating amino groups by glutaraldehyde to form a Thi-mAb1 complex, wherein the final concentration of the Thi-mAb1 complex is 0.05-0.1 mg/mL;

4) dripping the Thi-mAb1 compound as a labeled antibody on a binding pad, drying at 37 ℃ for 40-60 min, sequentially assembling the nitrocellulose membrane, the binding pad, the sample pad and the 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 thionine is present in the amount of 0.5 to 1mg/mL in the step 3).

8. The method according to claim 6, wherein the coupling time of monoclonal antibody mAb1 to Thi in step 3) is 10 min.

9. The method of claim 6, wherein the monoclonal antibody mAb1 and mAb2 are the partner antibodies of CP 4-EPSPS.