Trace hydroxyputrescine lysine protein detection method

Abstract

The invention relates to a trace hydroxyputrescine lysine protein detection method, which comprises: 1) preparing a sample to be detected, and slicing cells, body fluid or tissue; (2) sealing the sample by using Duolink sealing liquid; (3) diluting an anti-rabbit PLUS probe and an anti-mouse MINUS probe Merck and DUO92101 / DUO92102 in proportion by using a Duolinkantibody diluent, incubating with a product obtained in the previous step, and respectively combining a rabbit polyclonal antibody and a mouse monoclonal antibody through the PLUS / MINUS probe to provide a basis for a ligation reaction; (4) diluting ligase by using a Duolink ligation buffer solution according to the ratio, and incubating with the product in the step (3) at the temperature; 5) diluting an amplification enzyme with a Duolink amplification buffer solution in proportion, and incubating with a product obtained in the previous step; 6, a mounting medium containing DAPI is adopted for mounting, and imaging analysis is carried out through a fluorescence or confocal microscopy. The problem that due to the fact that the affinity of the antibody is insufficient and the protein expression quantity is low, the omission ratio of the trace hydroxyputrescine lysine modified protein is high can be well solved.

Description

Technical Field

[0001] This invention belongs to the field of trace protein index detection technology, and relates to a method for detecting trace amounts of hydroxycorrosive lysine-modified protein. Background Technology

[0002] Currently, there are various techniques for detecting trace amounts of hydroxyputrescine-lysine-modified proteins, mainly including liquid chromatography-mass spectrometry (LC-MS), Western blotting (radioactive elemental analysis), amino acid analyzers, high-sensitivity sensor devices, and high-throughput enrichment combined with computer-aided prediction. Taking Western blotting as an example, after obtaining patient tissue or body fluid samples, proteins are extracted, electrophoresed, and then chemiluminescently analyzed using commercially available anti-hydroxyputrescine-lysine antibodies, with primary and secondary antibodies conjugated. The problems and drawbacks of this existing technology are its poor sensitivity and high false negative rate for detecting trace amounts of hydroxyputrescine-lysine-modified proteins. We believe the main reason is that this detection method is highly dependent on the affinity of existing antibodies, which have low affinity. Simultaneously, the expression level of hydroxyputrescine-lysine-modified proteins is extremely low, resulting in a low capture rate for trace amounts of these proteins, insufficient detection signal, and a relatively high false negative rate, ultimately leading to a high false negative rate in early cancer detection.

[0003] To address this issue, a method for detecting trace amounts of hydroxycorrosive lysine-modified protein was designed. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a method for detecting trace amounts of hydroxyproline-lysine-modified proteins. This invention can significantly improve the detection sensitivity of trace amounts of hydroxyproline-lysine-modified proteins, thereby solving the problem of high false negative rates of trace amounts of hydroxyproline-lysine-modified proteins due to insufficient antibody affinity and low protein expression levels.

[0005] This invention is achieved through the following technical solution: a method for detecting trace amounts of hydroxyputrescine-lysine-modified protein, the method comprising the following steps: 1) Prepare the sample to be tested, such as cell, body fluid, or tissue sections; 2) After blocking the sample with Duolink® blocking solution, mix and incubate overnight with mouse monoclonal antibody against eIF5A2 and rabbit polyclonal antibody against hydroxyputrescine lysine to allow for full binding. The mouse monoclonal antibody specifically binds to the eIF5A2 protein, while the rabbit polyclonal antibody recognizes the hydroxyputrescine lysine modified epitope for further binding with the probe. 3) Anti-rabbit PLUS probe and anti-mouse MINUS probe Merck, DUO92101 / DUO92102, were diluted with Duolink® antibody diluent according to the ratio and incubated with the product from the previous step. The PLUS / MINUS probes then bound to the rabbit polyclonal antibody and the mouse monoclonal antibody, respectively. When the two antibodies bind to the same target protein, the oligonucleotide ends of the PLUS and MINUS probes are spatially adjacent (<40nm), providing a basis for the ligation reaction. 4) Dilute the ligase with Duolink® ligation buffer according to the specified ratio and incubate it with the product from step 3). This allows the ligase to catalyze the complementary pairing of the oligonucleotide ends of the PLUS and MINUS probes to form a closed circular DNA template. The probe distance only meets the ligation condition when both antibodies bind to the same protein molecule, thus eliminating false positives. 5) Dilute the amplification enzyme with Duolink® amplification buffer according to the ratio, and incubate it with the product from the previous step. The amplification enzyme is used to perform rolling circle amplification of RCA using circular DNA as a template, amplifying the signal by >1000 times. 6) Mount the slides with DAPI-containing mounting medium and analyze them using fluorescence or confocal microscopy to determine the results through fluorescence signals.

[0006] Preferably, the mouse monoclonal antibody in step 2) has the catalog number 67907-1-lg; the rabbit polyclonal antibody containing hydroxycorrosive lysine has the catalog number ABS1064, and the incubation environment is 4°C.

[0007] Preferably, the ratio in step 3) is 1:5.

[0008] Preferably, the ratio of ligase to uolink® ligation buffer in step 3) is 1:40.

[0009] Preferably, the incubation environment in step 3) is 37°C and the incubation time is 60 minutes.

[0010] Preferably, the incubation environment in step 4) is 37°C and the incubation time is 30 minutes.

[0011] Preferably, the incubation environment in step 5) is 37°C and the incubation time is 100 minutes.

[0012] Preferably, the ratio of amplification enzyme to Duolink® amplification buffer in step 5) is 1:80.

[0013] The beneficial effects of this invention are as follows: The method for detecting trace amounts of hydroxyproline-lysine-modified proteins designed in this invention can significantly improve the detection sensitivity of trace amounts of hydroxyproline-lysine-modified proteins, thereby solving the problem of high false negative rates of trace amounts of hydroxyproline-lysine-modified proteins due to insufficient antibody affinity and low protein expression levels. Attached Figure Description

[0014] Figure 1 The image shows the Hypusine and eIF5A2 signals diluted 10,000 times using the present invention. Figure 2 This is an image analysis diagram obtained using fluorescence or confocal microscopy. Detailed Implementation

[0015] To enable those skilled in the art to more clearly understand the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0016] In the description of this invention, it should be understood that the orientation or positional relationship indicated by terms such as "upper", "lower", "left", "right", "inner", "outer", "lateral", and "vertical" is based on the orientation or positional relationship shown in the accompanying drawings and is only for the convenience of describing this invention, and is not intended to indicate or imply that the device or component referred to must have a specific orientation, and therefore should not be construed as a limitation of this invention.

[0017] The present invention will now be described in detail with reference to the accompanying drawings: A method for detecting trace amounts of hydroxyputrescine-lysine-modified protein, the method comprising the following steps: 1) Prepare the sample to be tested, such as cell, body fluid, or tissue sections; 2) After blocking the sample with Duolink® blocking solution, mix the mouse monoclonal antibody against eIF5A2 and the rabbit polyclonal antibody against hydroxyputrescine and incubate overnight at 4°C to allow them to fully bind. The mouse monoclonal antibody specifically binds to the eIF5A2 protein, and the rabbit polyclonal antibody recognizes the hydroxyputrescine modified epitope for further binding with the probe. 3) Anti-rabbit PLUS probe and anti-mouse MINUS probe Merck, DUO92101 / DUO92102, were diluted with Duolink® antibody diluent at a ratio of 1:5 and incubated with the product from the previous step at 37°C for 60 minutes. The PLUS / MINUS probes then bound to the rabbit polyclonal antibody and the mouse monoclonal antibody, respectively. When the two antibodies bind to the same target protein, the oligonucleotide ends of the PLUS and MINUS probes are spatially adjacent (<40 nm), providing a basis for the ligation reaction. 4) Dilute the ligase with Duolink® ligation buffer at a ratio of 1:40, and incubate it with the product from step 3) at 37°C for 30 minutes. This allows the ligase to catalyze the complementary pairing of the oligonucleotide ends of the PLUS and MINUS probes to form a closed circular DNA template. The probe distance only meets the ligation condition when both antibodies bind to the same protein molecule, thus eliminating false positives. 5) Dilute the amplification enzyme with Duolink® amplification buffer at a ratio of 1:80, and incubate it with the product from the previous step at 37°C for 100 minutes. The amplification enzyme will then use circular DNA as a template to perform rolling circle amplification of RCA, amplifying the signal by >1000 times. 6) Mount the slides with DAPI-containing mounting medium and analyze them using fluorescence or confocal microscopy to determine the results through fluorescence signals.

[0018] Example 1 This invention can significantly improve the detection sensitivity of trace amounts of hydroxyproline-lysine modified proteins. The following experimental data illustrates this: Lung cancer cell lines were collected and proteins were prepared. These proteins were then diluted with standard protein solutions at 1, 10, 100, 1000, 10000, and 100000 times, respectively. Equal volumes of these proteins were subjected to electrophoresis and transferred to nitrocellulose membranes. The expression levels of eIF5A2 protein and hydroxyproline-lysine were tested using Western blotting and the detection technique of this invention, respectively. The detection sensitivity of the kit was preliminarily verified by comparing the conventional Western blotting method with the trace lysine-modified protein detection kit method. The results showed that after a 100-fold dilution, Western blotting essentially failed to detect hydroxyproline and eIF5A2 signals, while the technique of this invention could still detect them even after a 10000-fold dilution. The detection results are shown below. Figure 1 As shown.

[0019] Example 2 1. After blocking lung cancer monoclonal cells with Duolink® blocking solution, mix them with mouse monoclonal antibody against eIF5A2 (1:200 dilution) and rabbit polyclonal antibody against hydroxyputrescine (1:100 dilution) and incubate overnight at 4°C to allow them to fully bind. 2. Anti-rabbit PLUS probe and anti-mouse MINUS probe (Merck, DUO92101 / DUO92102) were diluted with Duolink® antibody diluent at a ratio of 1:5 and incubated with the product from the previous step at 37°C for 1 hour. 3. Dilute the ligase with Duolink® ligation buffer at a ratio of 1:40, and incubate it with the product from the previous step at 37°C for 30 minutes. 4. Dilute the amplification enzyme with Duolink® amplification buffer at a ratio of 1:80, and incubate it with the product from the previous step at 37°C for 100 minutes. 5. Mount the slide with a mounting medium containing DAPI, and analyze the image using fluorescence or confocal microscopy. The results are as follows: Figure 2 As shown.

[0020] The specific embodiments described herein are merely illustrative of the principles and effects of the invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in this invention should still be covered by the claims of this invention.

Claims

1. A method for detecting trace amounts of hydroxyputrescine-lysine-modified protein, characterized in that, The method includes the following steps: 1) Prepare the sample to be tested, such as cell, body fluid, or tissue sections; 2) After blocking the sample with Duolink® blocking solution, mix and incubate overnight with mouse monoclonal antibody against eIF5A2 and rabbit polyclonal antibody against hydroxyputrescine lysine to allow for full binding. The mouse monoclonal antibody specifically binds to the eIF5A2 protein, while the rabbit polyclonal antibody recognizes the hydroxyputrescine lysine modified epitope for further binding with the probe. 3) Anti-rabbit PLUS probe and anti-mouse MINUS probe Merck, DUO92101 / DUO92102, were diluted with Duolink® antibody diluent according to the ratio and incubated with the product from the previous step. The PLUS / MINUS probes then bound to the rabbit polyclonal antibody and the mouse monoclonal antibody, respectively. When the two antibodies bind to the same target protein, the oligonucleotide ends of the PLUS and MINUS probes are spatially adjacent (<40 nm), providing a basis for the ligation reaction. 4) Dilute the ligase with Duolink® ligation buffer according to the specified ratio and incubate it with the product from step 3). This allows the ligase to catalyze the complementary pairing of the oligonucleotide ends of the PLUS and MINUS probes to form a closed circular DNA template. The probe distance only meets the ligation condition when both antibodies bind to the same protein molecule, thus eliminating false positives. 5) Dilute the amplification enzyme with Duolink® amplification buffer according to the ratio, and incubate it with the product from the previous step. The amplification enzyme is used to perform rolling circle amplification of RCA using circular DNA as a template, amplifying the signal by >1000 times. 6) Mount the slides with DAPI-containing mounting medium and analyze them using fluorescence or confocal microscopy to determine the results through fluorescence signals.

2. The method for detecting trace amounts of hydroxyputrescine-lysine-modified protein according to claim 1, characterized in that, The mouse monoclonal antibody in step 2) has the catalog number 67907-1-lg; the rabbit polyclonal antibody containing hydroxycorrosive lysine has the catalog number ABS1064, and the incubation environment is 4°C.

3. The method for detecting trace amounts of hydroxyputrescine-lysine-modified protein according to claim 1, characterized in that, The ratio in step 3) is 1:

5.

4. The method for detecting trace amounts of hydroxyputrescine-lysine-modified protein according to claim 1, characterized in that, In step 3), the ratio of ligase to uolink® ligation buffer is 1:

40.

5. The method for detecting trace amounts of hydroxyputrescine-lysine-modified protein according to claim 1, characterized in that, The incubation environment in step 3) is 37°C and the incubation time is 60 minutes.

6. The method for detecting trace amounts of hydroxyputrescine-lysine-modified protein according to claim 1, characterized in that, The incubation environment in step 4) is 37°C for 30 minutes.

7. The method for detecting trace amounts of hydroxyputrescine-lysine-modified protein according to claim 1, characterized in that, The incubation environment in step 5) is 37°C for 100 minutes.

8. The method for detecting trace amounts of hydroxycorrosive lysine-modified protein according to claim 1, characterized in that, In step 5), the ratio of amplification enzyme to Duolink® amplification buffer is 1:80.

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