An optical sensor for key antibodies of malignant tumors and its preparation method

Abstract

The present invention relates to the field of antibody detection, and specifically discloses a method for preparing an optical sensor for a key antibody of a malignant tumor, including: S1. a washing step, vortexing or rotating the first optical sensor, transferring it to a centrifuge tube, adding the same volume of the first buffer and resuspending it, removing the supernatant using a magnetic frame, then removing the magnetic frame, and resuspending the first optical sensor with the same volume of the first buffer; S2. a step of coupling the target antigen with the first optical sensor, transferring the washed and resuspended first optical sensor to a new centrifuge tube, removing the supernatant; S3. resuspending it in the first buffer containing the target antigen; S4. adding ammonium sulfate buffer, rotating and incubating at 37°C for 12-18 hours; S5. after removing the supernatant, adding a phosphate buffer containing BSA and suspending and incubating it for 1 hour. The technical solution of the present invention is simple to operate, can be used for multiple project coding, can exclude false positives and nonspecific adsorption, and is convenient for equipment to perform single molecule detection.

Description

Technical Field

[0001] The present invention relates to the field of antibody detection, and in particular to an optical sensor for key antibodies of malignant tumors and a preparation method thereof. Background Art

[0002] Early diagnosis and treatment of malignant tumors are crucial to improving patients' survival rate and quality of life. Detection of tumor markers is one of the key means to achieve early diagnosis. Existing detection technologies, such as horseradish peroxidase-antibody optical sensors and time-resolved fluorescent microspheres-antibody optical sensors, have met clinical needs to a certain extent, but still have some limitations.

[0003] The preparation process of the horseradish peroxidase-antibody optical sensor includes multiple steps such as dissolution of horseradish peroxidase, oxidation treatment with sodium periodate, dialysis, coupling of antibodies, reduction treatment with sodium borohydride, etc.

[0004] The preparation of time-resolved fluorescent microsphere-antibody optical sensor involves the steps of cleaning, activation, coupling, blocking, re-cleaning and re-dissolution and storage of microspheres.

[0005] Although existing technologies have played a role in tumor marker detection, they still have the following major shortcomings:

[0006] The sensor signal is single, cannot effectively identify false positive results (needs to distinguish non-specific binding), and cannot be used with high-resolution instruments for high-sensitivity analysis.

[0007] In view of the limitations of the prior art, the present invention aims to develop an optical sensor for key antibodies of malignant tumors and a preparation method thereof, which is used to detect key antibodies of malignant tumors. The sensor aims to overcome the shortcomings of the prior art and provide a solution that is simple to operate, can perform multiple item coding, can exclude false positives and nonspecific adsorption, and facilitates single molecule detection of the device. Summary of the invention

[0008] The present invention provides an optical sensor for key antibodies of malignant tumors and a preparation method thereof, which is simple to operate, can perform multiple item coding, can exclude false positives and nonspecific adsorption, and facilitate single molecule detection by the device.

[0009] In order to solve the above technical problems, this application provides the following technical solutions:

[0010] A method for preparing an optical sensor for a key antibody of a malignant tumor, comprising:

[0011] S1. a washing step, vortexing or spinning the first optical sensor, transferring to a centrifuge tube, adding an equal volume of the first buffer and resuspending, removing the supernatant using a magnetic stand, then removing the magnetic stand, and resuspending the first optical sensor with an equal volume of the first buffer;

[0012] S2. The step of coupling the target antigen to the first optical sensor, transferring the washed and resuspended first optical sensor to a new centrifuge tube, and removing the supernatant;

[0013] S3. Resuspend in the first buffer containing the target antigen;

[0014] S5. After removing the supernatant, add phosphate buffer containing BSA and incubate for 1 hour;

[0015] S6. Repeat the supernatant removal and resuspension steps, and finally resuspend with phosphate buffer containing BSA;

[0016] S7. The target secondary antibody is coupled to the second optical sensor, and S1-S6 are repeated for the second optical sensor;

[0017] Wherein, the first optical sensor and the second optical sensor are multiple fluorescent p-toluenesulfonyl magnetic beads with a diameter of 1-20 μm.

[0018] The basic scheme principle and beneficial effects are as follows: After the configuration is completed, the final product includes a first optical sensor labeled with a target antigen, a second optical sensor labeled with a secondary antibody, and a suitable buffer system.

[0019] The first optical sensor and the second optical sensor can have multiple fluorescence codes after simple labeling mainly by adjusting the pH, and can exclude false positives and nonspecific adsorption.

[0020] During detection, the target antibody being detected specifically binds to the target antigen on the first optical sensor, and at the same time, the target secondary antibody-second optical sensor structure binds to the other end of the target antibody to form a sandwich structure, so that the entire structure has the characteristic spectra of both the first optical sensor and the second optical sensor.

[0021] In this scheme, the coupling method is simple, does not require complex operations and expensive drugs, and can be coupled only by adjusting the pH. Two optical sensors (the first optical sensor and the second optical sensor) form 4-10 multi-dimensional fluorescence encoding, which can be used for multiple project encoding. By combining the characteristic spectrum through multiple encoding on two antibodies (i.e., the target antibody), false positives and nonspecific adsorption can be excluded. The optical sensor composed of multiple fluorescent microspheres can use the equipment for single molecule detection to improve the detection sensitivity.

[0022] In addition, by selecting highly specific antibodies and preparing microspheres with p-toluenesulfonyl modification on the surface, labeling can be completed by adjusting the pH, which effectively solves the problem that the labeling process requires delicate operation and large batch differences. By preparing microspheres containing multiple fluorescent codes, multiple fluorescence can be identified after labeling antibodies to form a unique spectrum. The spectrum can be analyzed by instruments to distinguish whether there are false positives and non-specific binding. Since a single optical sensor is larger, a single fluorescence is stronger than chemiluminescence and Elisa. A single sensor can be resolved by appropriate equipment, effectively improving the detection sensitivity. A single sensor signal can be read without signal amplification, thus meeting the requirements of single molecule detection.

[0023] In summary, this solution does not require complicated operations and expensive drugs. It only needs to adjust the pH for coupling, which can achieve the effect of simple operation. By forming 4-10 multi-dimensional fluorescent codes with two microspheres, multiple project coding can be performed. The characteristic spectrum is combined by the multiple codes on the two antibodies. Inconsistent characteristic spectra mean false positives or nonspecific adsorption, which can eliminate false positives and nonspecific adsorption. The optical sensor composed of multiple fluorescent microspheres can use the equipment for single molecule detection to improve the detection sensitivity.

[0024] Furthermore, the pH value of the first buffer solution is 7.4 to 9.

[0025] Further, the S3 comprises resuspending in 100 μg, adding the first buffer to 150 μL, and mixing evenly.

[0026] Furthermore, 100 μL of ammonium sulfate buffer solution is added to S4, and the pH value of the ammonium sulfate buffer solution is 7.4-9.

[0027] Furthermore, 1 mL of 0.01-0.2% BSA phosphate buffer solution is added to S5, and the pH value of the phosphate buffer solution is 7-8.

[0028] Furthermore, the phosphate buffer containing BSA in the S6 is a 0.01-0.2% BSA phosphate buffer solution, and the pH value is 7-8.

[0029] Furthermore, the first optical sensor and the second optical sensor are p-toluenesulfonyl magnetic beads with a diameter of 10 μm and three kinds of fluorescence.

[0030] Further, after incubation, the supernatant was removed and 1 mL of 0.01-0.2% BSA and 0.01-0.1% phycoerythrin in phosphate buffer solution was added.

[0031] Furthermore, the target antigen is GBU4-5 antigen or MEGA-A1 antigen or PGP9.5 antigen or SOX2 antigen or P53 antigen or CAGE antigen or CAGE7 antigen.

[0032] An optical sensor for a key antibody of a malignant tumor comprises a first optical sensor labeled with a target antigen, a second optical sensor labeled with a secondary antibody, and a buffer system with a pH of 7.4-9 and free of free amino groups.

[0033] Furthermore, the buffer system is a carbonate buffer with a pH of 9. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Schematic diagram of the structure of an optical sensor for key antibodies in malignant tumors. DETAILED DESCRIPTION

[0035] The following is further described in detail through specific implementation methods:

[0036] The symbols in the drawings of the specification include: a first optical sensor 1 , a second optical sensor 2 .

[0037] Example 1

[0038] A method for preparing an optical sensor for a key antibody of a malignant tumor, comprising:

[0039] The first optical sensor 1 is swirled or rotated.

[0040] 2. Transfer to a centrifuge tube, add the same volume of buffer (pH 7.4-9) and resuspend.

[0041] 3. Place on a magnetic rack and remove the supernatant.

[0042] 4. Remove the magnetic stand and resuspend with an equal volume of buffer.

[0043] The GBU4-5 antigen is then coupled to the first optical sensor 1:

[0044] 1. Transfer the washed and resuspended beads to a new centrifuge tube and discard the supernatant.

[0045] 2. Resuspend in 100 μg, add buffer (pH 7.4-9) to 150 μL, and mix well.

[0046] 3. Add 100 μL of ammonium sulfate buffer (pH 7.4-9) and mix.

[0047] 4. Incubate at 37°C with rotation for 12-18 hours.

[0048] 5. Remove the supernatant, add 1 mL of 0.01-0.2% BSA phosphate buffer solution (pH 7-8), and incubate for 1 hour.

[0049] 6. Repeat the supernatant removal and resuspension steps, and finally resuspend in 0.01-0.2% BSA phosphate buffer solution (pH 7-8).

[0050] 7. The second optical sensor 2 is labeled with the secondary antibody using the same method as above.

[0051] During specific use, the GBU4-5 antibody specifically binds to the GBU4-5 antigen on the first optical sensor 1, and at the same time, the secondary antibody-second optical sensor 2 structure binds to the other end of the GBU4-5 antibody to form a sandwich structure, so that the entire optical sensor has the characteristic spectra of the first optical sensor 1 and the second optical sensor 2 at the same time.

[0052] It mainly consists of a first optical sensor 1 labeled with anti-GBU4-5 antigen, a second optical sensor 2 labeled with a secondary antibody, and a suitable buffer system.

[0053] Optical sensors can have multiple fluorescence codes after simple labeling by adjusting pH, and can eliminate false positives and nonspecific adsorption.

[0054] The first optical sensor 1 is a p-toluenesulfonyl magnetic bead with a diameter of 10 μm and three kinds of fluorescence.

[0055] The second optical sensor 2 is a p-toluenesulfonyl magnetic bead with a diameter of 0.3 μm and three kinds of fluorescence.

[0056] An optical sensor for key antibodies of malignant tumors comprises a first optical sensor marked by a target antigen, a second optical sensor marked by a secondary antibody and a buffer system, wherein the buffer system is a carbonate buffer with a pH of 9.

[0057] In this embodiment, by selecting a high-specificity antibody and preparing a microsphere coupling with a p-toluenesulfonyl group on the surface, the labeling can be completed by adjusting the pH, which effectively solves the problem that the labeling process requires delicate operation and large batch differences. Through the prepared microspheres containing multiple fluorescent codes, multiple fluorescences can be identified after the labeled antibodies to form a unique spectrum. The spectrum can be analyzed by an instrument to distinguish whether there are false positives and non-specific binding. Because the single optical sensor is larger, the single fluorescence is stronger than chemiluminescence and Elisa. The single sensor can be resolved by suitable equipment, effectively improving the detection sensitivity. The single sensor signal can be read out without signal amplification, meeting the requirements of single molecule detection.

[0058] Example 2

[0059] This embodiment provides a method for preparing an optical sensor, which is particularly suitable for detecting key antibodies related to malignant tumors, such as MEGA-A1 antibody.

[0060] 1. Cleaning of the optical sensor: The first optical sensor 1 needs to be cleaned to remove impurities before use. First, place the first optical sensor 1 in a centrifuge tube and clean it by vortexing or spinning. Then, add a buffer (pH 7.4-9) of the same volume as the first optical sensor 1 and resuspend the first optical sensor 1. Next, place the centrifuge tube on a magnetic stand to remove the supernatant, remove the magnetic stand, and resuspend the optical sensor again with the same volume of buffer.

[0061] 2. Coupling of MEGA-A1 antigen to the first optical sensor 1: The washed and resuspended first optical sensor 1 was transferred to a new centrifuge tube and the supernatant was removed. The first optical sensor 1 was resuspended in a buffer (pH 7.4-9) containing 100 μg of MEGA-A1 antigen to a total volume of 150 μL and mixed evenly. Then, 100 μL of ammonium sulfate buffer (pH 7.4-9) was added and incubated with rotation at 37°C for 12 to 18 hours to promote coupling. After incubation, the supernatant was removed and 1 mL of phosphate buffer solution (pH 7-8) containing 0.01-0.2% BSA and 0.01-0.1% phycoerythrin (the function is to indirectly determine whether the protein is ineffective through the fluorescence provided by phycoerythrin) was added, and the suspension was incubated for 1 hour to block the unreacted sites. The supernatant removal and suspension steps were repeated, and finally the first optical sensor 1 was resuspended in 0.01-0.2% BSA phosphate buffer solution (pH 7-8).

[0062] 3. Secondary Antibody Labeling of Second Optical Sensor 2: The second optical sensor 2 is labeled with a secondary antibody using the same method as the first optical sensor 1 labeled with MEGA-A1 antigen.

[0063] 4. Formation of characteristic spectrum of optical sensor: When in use, the MEGA-A1 antibody specifically binds to the MEGA-A1 antigen on the first optical sensor 1, and at the same time, the secondary antibody-second optical sensor 2 binds to the other end of the MEGA-A1 antibody to form a sandwich structure (such as Figure 1 This structure enables the optical sensor to have the characteristic spectra of the first optical sensor 1 and the second optical sensor 2 at the same time.

[0064] 5. Composition of the optical sensor: The optical sensor is mainly composed of a first optical sensor 1 labeled with anti-MEGA-A1 antigen, a second optical sensor 2 labeled with a secondary antibody, and a suitable buffer system. The buffer system is a carbonate buffer with a pH of 9.

[0065] 6. Characteristics of optical sensors: The first optical sensor 1 is a 10 μm diameter toluenesulfonyl magnetic bead with three fluorescent colors. The second optical sensor 2 is a 0.3 μm diameter toluenesulfonyl magnetic bead also with three fluorescent colors. After simple labeling by adjusting pH, the optical sensor can have multiple fluorescent codes, effectively eliminating false positives and nonspecific adsorption.

[0066] 7. Application of optical sensors: By preparing microspheres containing multiple fluorescent codes, multiple fluorescence can be identified after labeling antibodies to form a unique spectrum. By analyzing the spectrum with instruments, it is possible to distinguish whether there are false positives and non-specific binding. Since a single optical sensor is large and a single fluorescence is stronger than chemiluminescence and ELISA, a single sensor can be distinguished by appropriate equipment, effectively improving the detection sensitivity and meeting the requirements of single molecule detection.

[0067] In the above process, phycoerythrin may be used to detect and improve the blocking effect during the preparation stage. Because phycoerythrin has fluorescent properties, it can be used as a tracer to help researchers observe and evaluate whether the blocking agent (such as BSA) is evenly covered on the surface of the optical sensor, thereby ensuring that unreacted sites are effectively blocked and reducing nonspecific binding in subsequent reactions.

[0068] During the storage stage, phycoerythrin may help maintain the stability of the optical sensor. Due to its fluorescent properties, phycoerythrin can be used as a protective agent to monitor the integrity of the sensor surface through its fluorescence changes (both are proteins, and if they are in conditions that are prone to inactivation, phycoerythrin will also be inactivated at the same time), and it can also prevent non-specific binding between the first optical sensor and the second optical sensor during the storage process.

[0069] The fluorescent properties of phycoerythrin make it an effective indicator. During the preparation and use of the optical sensor, the fluorescence intensity of phycoerythrin can be used as an indicator of sensor activity and effectiveness. If the fluorescence intensity is within the expected range, this indicates that the sensor remains in good functional condition and is suitable for subsequent detection. Due to the strong fluorescent properties of phycoerythrin, it can provide a detectable signal even at very low concentrations, which helps to improve the sensitivity of the optical sensor to detect MEGA-A1 antibodies.

[0070] If phycoerythrin is used in combination with other fluorescent markers, multiplexed detection can be performed. Different fluorescent codes can detect multiple targets at the same time, increasing the throughput and efficiency of detection. The addition of phycoerythrin may help improve the stability of optical sensors under different environmental conditions, including temperature, pH changes, etc., thereby extending the shelf life and service life of the sensor. By using phycoerythrin labeling and fluorescence detection, specific binding and nonspecific binding can be more clearly distinguished, thereby reducing background noise and improving the accuracy of detection.

[0071] The above are only embodiments of the present invention. The invention is not limited to the fields involved in this implementation case. The common sense such as the known specific structures and characteristics in the scheme is not described in detail here. The ordinary technicians in the relevant field know all the common technical knowledge in the technical field to which the invention belongs before the application date or the priority date, can obtain all the existing technologies in the field, and have the ability to apply the conventional experimental means before that date. The ordinary technicians in the relevant field can improve and implement this scheme in combination with their own abilities under the enlightenment given by this application. Some typical known structures or known methods should not become obstacles for ordinary technicians in the relevant field to implement this application. It should be pointed out that for those skilled in the art, without departing from the structure of the present invention, several deformations and improvements can be made, which should also be regarded as the protection scope of the present invention, which will not affect the effect of the implementation of the present invention and the practicality of the patent. The protection scope required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to explain the content of the claims.

Claims

1. A method for preparing an optical sensor for detecting key antibodies of malignant tumors, characterized in that: include: A first optical sensor washing step: vortexing or spinning the first optical sensor, transferring to a centrifuge tube, adding an equal volume of the first buffer and resuspending, removing the supernatant using a magnetic stand, then removing the magnetic stand, and resuspending the first optical sensor with an equal volume of the first buffer; The step of coupling the target antigen with the first optical sensor is as follows: the washed and resuspended first optical sensor is transferred to a new centrifuge tube, and the supernatant is removed; the first optical sensor is resuspended in the first buffer containing the target antigen; the ammonium sulfate buffer is added, and the sensor is rotated and incubated at 37°C for 12-18 hours; the supernatant is removed, and 1 mL of a phosphate buffer solution containing 0.01-0.2% BSA and 0.01-0.1% phycoerythrin is added, and the sensor is suspended and incubated for 1 hour; the steps of removing the supernatant and suspending are repeated, and finally the sensor is resuspended with a phosphate buffer containing BSA; The second optical sensor washing step includes vortexing or spinning the second optical sensor, transferring to a centrifuge tube, adding an equal volume of the first buffer and resuspending, removing the supernatant using a magnetic stand, then removing the magnetic stand, and resuspending the second optical sensor with an equal volume of the first buffer; The steps of coupling the secondary antibody to the second optical sensor are as follows: the washed and resuspended second optical sensor is transferred to a new centrifuge tube and the supernatant is removed; the second optical sensor is resuspended in the first buffer containing the secondary antibody; the ammonium sulfate buffer is added and the mixture is rotated and incubated at 37°C for 12-18 hours; the supernatant is removed and 1 mL of a phosphate buffer solution containing 0.01-0.2% BSA and 0.01-0.1% phycoerythrin is added, and the mixture is suspended and incubated for 1 hour; the steps of removing the supernatant and resuspending are repeated, and finally the mixture is resuspended with a phosphate buffer containing BSA; The first optical sensor is a p-toluenesulfonyl magnetic bead with a diameter of 10 μm and three kinds of fluorescence; the second optical sensor is a p-toluenesulfonyl magnetic bead with a diameter of 0.3 μm and three kinds of fluorescence; and Among them, the key antibody for malignant tumor is GBU4-5 antibody or MEGA-A1 antibody; when the key antibody for malignant tumor is GBU4-5 antibody, the target antigen is GBU4-5 antigen, and when the key antibody for malignant tumor is MEGA-A1 antibody, the target antigen is MEGA-A1 antigen.

2. The method for preparing an optical sensor for detecting key antibodies of malignant tumors according to claim 1, characterized in that: The pH value of the first buffer is 7.4 to 9.

3. The method for preparing an optical sensor for detecting key antibodies of malignant tumors according to claim 2, characterized in that: Resuspending the first optical sensor in a first buffer solution containing the target antigen comprises resuspending the first optical sensor in a first buffer solution containing 100 μg of the target antigen, adding the first buffer solution to 150 μL, and mixing evenly; resuspending the second optical sensor in the first buffer solution containing the secondary antibody comprises resuspending the second optical sensor in a first buffer solution containing 100 μg of the secondary antibody, adding the first buffer solution to 150 μL, and mixing evenly.

4. The method for preparing an optical sensor for detecting key antibodies of malignant tumors according to claim 3, characterized in that: The adding of ammonium sulfate buffer is adding 100 μL of ammonium sulfate buffer, and the pH value of the ammonium sulfate buffer is 7.4-9.

5. The method for preparing an optical sensor for detecting key antibodies of malignant tumors according to claim 4, characterized in that: The pH value of the phosphate buffer solution containing 0.01-0.2% BSA and 0.01-0.1% phycoerythrin is 7-8.

6. The method for preparing an optical sensor for detecting key antibodies of malignant tumors according to claim 5, characterized in that: The BSA-containing phosphate buffer is a 0.01-0.2% BSA phosphate buffer solution, and the pH value is 7-8.

7. An optical sensor for detecting key antibodies of malignant tumors, characterized in that: It comprises a first optical sensor labeled with a target antigen, a second optical sensor labeled with a secondary antibody, and a buffer system having a pH of 7.4-9 and containing no free amino groups; wherein the first optical sensor labeled with the target antigen and the second optical sensor labeled with the secondary antibody are prepared by the preparation method according to any one of claims 1 to 6.

8. The optical sensor for detecting key antibodies of malignant tumors according to claim 7, characterized in that: The buffer system without free amino groups is a carbonate buffer with a pH of 9.

Images