CN118894930A - A rapid screening method for nanoantibodies based on biological activity evaluation - Google Patents

Abstract

The invention relates to the technical field of biology, in particular to a rapid screening method of a nano antibody based on biological activity evaluation, which adopts high-efficiency multi-step purification technologies such as affinity chromatography, ion exchange chromatography, gel filtration chromatography and the like to ensure the high purity of the nano antibody; the purity and activity detection means are adopted to comprehensively and accurately control the quality of the product, and provide high purity and high activity for the nano antibody; in the aspects of high affinity and high specificity, a high-throughput screening system is introduced, so that a large number of candidate nano antibody samples can be processed simultaneously, and the screening efficiency is greatly improved; according to the established standardized affinity and specificity evaluation flow, the screened antibodies have high affinity and high specificity; by utilizing phage display technology, a display library containing a large number of candidate antibodies is constructed, and the antibodies with high affinity and high specificity with the target antigen are obtained through efficient screening.

Description

A rapid screening method for nanoantibodies based on biological activity evaluation

Technical Field

The present invention relates to the field of biological technology, and in particular to a method for rapid screening of nano antibodies based on biological activity evaluation.

Background Art

In the development process of nanoantibodies, purity and activity are important factors affecting their clinical application and commercialization.

The current preparation methods of nano-antibodies are complex in process and cumbersome in operation, which makes it difficult to ensure the purity and activity of the final product, seriously restricting its wide application in the field of biomedicine. The purification process of the more common nano-antibodies currently involves multiple steps, such as precipitation, centrifugation, chromatography, etc., and impurities may be introduced in each step; the traditional method relies on simple purity detection methods, which makes it difficult to comprehensively and accurately evaluate the purity of nano-antibodies. At the same time, during the purification process, the activity of nano-antibodies is easily affected by physical and chemical factors, such as pH and temperature, resulting in reduced activity. Nano-antibodies have high requirements for storage conditions, and the slightest carelessness may lead to loss of activity;

In addition, high affinity and high specificity are the key characteristics of nano-antibodies that play an effective role in clinical diagnosis and treatment. However, current screening methods make it difficult to efficiently screen out nano-antibodies that possess both of these characteristics. They mostly rely on low-throughput, manual screening, which is inefficient and difficult to process a large number of samples, resulting in a low screening success rate for high-affinity antibodies. Therefore, a rapid screening method for nano-antibodies based on biological activity evaluation is urgently needed to solve such problems.

Summary of the invention

To this end, the present invention provides a method for rapid screening of nano antibodies based on biological activity evaluation, which is used to overcome the problems in the prior art that it is difficult to comprehensively and accurately evaluate the purity of nano antibodies and to efficiently screen out nano antibodies that simultaneously possess these two characteristics, and that the prior art mostly relies on low-throughput, manual screening, which is inefficient and difficult to process a large number of samples, resulting in a low screening success rate for high-affinity antibodies.

To achieve the above objectives, the present invention provides a method for rapid screening of nanobodies based on biological activity evaluation, comprising:

Step S1, antigen immunization, using camelid animals for antigen immunization to produce heavy chain antibodies against the target antigen, the immunization cycle is 4-6 weeks, and the interval between each immunization is 1 week;

The heavy chain antibody refers to a special type of antibody produced in camelids, which is composed only of heavy chains and lacks the light chain part of traditional antibodies; the variable region (VHH) of the heavy chain antibody is the basis of nano antibodies, with a small molecular weight and high stability;

Step S2, antibody discovery and preparation, collecting blood from the immunized animal, isolating B cells, and amplifying the variable region (VHH) gene of the heavy chain antibody by RT-PCR technology;

Step S3, antibody screening, screening nanoantibodies with high affinity to the target antigen through phage display technology;

Step S4, biological activity evaluation, performing a systematic biological activity evaluation on the screened nanobodies;

Step S5, preparation of nanobodies, selecting the nanobodies with the best biological activity evaluation results for large-scale production;

Step S6, the dosage form and yield of the nanobody, the prepared nanobody is prepared into an injection or lyophilized powder dosage form, each dose containing 1-10 mg of the nanobody.

Furthermore, in step S2, the amplified VHH gene is cloned into a phage display vector to construct a phage display library; the RT-PCR amplification refers to:

Reverse transcription polymerase chain reaction (RT-PCR) is a molecular biology technique that can reverse transcribe mRNA in B cells into cDNA, and then amplify the cDNA using PCR technology; the specific steps include:

Extract total RNA from B cells using TRIzol reagent or other RNA extraction kits;

Reverse transcriptase and specific primers are used to reverse transcribe RNA into cDNA;

Design specific primers for the variable region (VHH) of heavy chain antibodies and amplify cDNA by polymerase chain reaction (PCR);

Purify the amplified product for subsequent cloning and library construction;

Furthermore, the specific steps of antibody screening in step S3 include:

Coating antigen: dilute the target antigen X-VLP to a concentration of 5 μg/ml, use Coating buffer (15 mmol/L NaCO ₃ , 35 mmol/L NaHCO ₃ , pH 9.6) for coating, add 50 μl to each well, and incubate at 4°C overnight in the dark;

Wash the plate, take out the coated ELISA plate, and wash it 4 times with 300 μl/well of Washing buffer (PBS, 0.05% Tween-20, pH 7.4);

Block, add 100 μl/well of Blocking buffer (2% BSA in Washing buffer, pH 7.4), incubate at 37°C for 1.5 hours in the dark, then wash 4 times with Washing buffer, shake off and pat dry;

For sample incubation, add 50 μl/well of the diluted phage display library or nanobody sample, incubate at 37°C for 1 hour in the dark, then wash 4 times with Washing buffer, shake off and pat dry;

For secondary antibody incubation, add 50 μl/well of the diluted secondary antibody (Peroxidase Affini Pure Goat Anti-Human IgG (H+L), 1:20,000 dilution), incubate at 37°C for 1 hour in the dark, then wash 4 times with Washing Buffer, shake off and pat dry;

For color development, add 75 μl/well of color development solution (TMB solution), incubate at 37°C for 15 minutes, remove, add 75 μl/well of stop solution (2MH ₂ SO ₄ ), and place in a microplate reader to set the OD450 reading;

Furthermore, the ELISA plate refers to a multi-well plate used for enzyme-linked immunosorbent assay (ELISA), each well of which can react independently. Common ELISA plates have two specifications: 96 wells and 384 wells. The ELISA plate is made of polystyrene material and has good protein adsorption performance and chemical stability.

Furthermore, the specific steps of biological activity evaluation include:

The binding ability, specificity and affinity of nanobodies were evaluated through high-throughput screening systems;

Use standardized biological activity evaluation methods, such as cell proliferation inhibition assay and cell apoptosis assay, to detect the biological effects of nanobodies;

Record and analyze various biological parameters to screen out nanobodies with the best activity;

Furthermore, in the preparation process of step S5, the culture conditions are controlled (such as temperature 37° C., culture time 48 hours), and an optimized purification process is used to ensure the high purity and high activity of the nanobody; the preparation steps of the nanobody include,

Cloning and construction: clone the selected high-quality nanoantibody genes into expression vectors, and select Escherichia coli and yeast as expression hosts;

Transformation and screening: transform the constructed expression vector into host cells, and screen out high-expressing clones through antibiotic selection and expression detection;

Small-scale expression test: select the high-expressing clones screened out, conduct small-scale expression test, and determine the optimal expression conditions, including inducer concentration, temperature, and time;

Large-scale culture: according to the optimized expression conditions, large-scale culture is carried out in a fermenter, the culture temperature is controlled at 37°C, and the culture time is 48 hours;

Cell collection: After the culture is completed, the cells are collected by centrifugation and the culture medium is removed;

Cell disruption: using an ultrasonic disruptor or a high-pressure homogenizer to disrupt the collected cells to release the nanobodies;

Coarse filtration and clarification, where cell debris is removed by coarse filtration (e.g., filter cloth or paper), and the supernatant is then further clarified using centrifugation or ultrafiltration systems;

Furthermore, in the preparation process of step S5, the preparation step of the Nanobody also includes:

Affinity chromatography, using an affinity column (such as a ProteinA/G column) that specifically binds to the nanobody to perform preliminary purification of the nanobody;

Ion exchange chromatography, using the charge characteristics of nanobodies, further purifies through ion exchange chromatography columns to remove impurity proteins;

Gel filtration chromatography, through a gel filtration chromatography column (such as Superdex75 or Superdex200), separation according to molecular weight to obtain high-purity nanobodies;

Concentration and dialysis: concentrating the purified nanobody solution to an appropriate volume and dialyzing it into a storage buffer (such as PBS);

Sterile filtration: sterile filter the nanobody solution through a 0.22 μm sterile filter membrane;

Quality testing: Conduct quality testing on the final product, including purity testing (such as SDSPAGE, HPLC) and activity testing (such as ELISA, functional experiments) to ensure the high purity and high activity of the nanoantibody;

Packaging and storage: Packaging the qualified nano-antibody solution;

Furthermore, during the preparation of the injection,

Prepare a solution, dissolve the purified nanobody in a buffer and adjust the pH to 7.4;

Filter sterilize by filtering the solution through a 0.22 μm sterile filter membrane;

Packaging: Packaging the sterilized nanobody solution into sterile injection bottles, each bottle containing 1-10 mg of nanobody;

Sealing: using a sealing device to seal the injection bottle;

Testing: quality testing of the prepared injections, including sterility testing, purity testing, and activity testing;

Packaging: Pack qualified injection bottles and mark the batch number, dosage and production date;

Furthermore, the freeze-dried powder preparation process is as follows:

Prepare a solution, dissolve the purified nanobody in a buffer and adjust the pH to 7.4;

Filter sterilize by filtering the solution through a 0.22 μm sterile filter membrane;

Packaging: Packaging the sterilized nanobody solution into sterile lyophilized bottles, each bottle containing 1-10 mg of nanobody;

Freeze drying: Place the packaged freeze-dried bottles in a freeze dryer and perform freeze drying according to the preset program.

Sealing: After the freeze-drying process is completed, the freeze-dried bottle is immediately vacuum-sealed;

Testing: Conduct quality testing on the prepared freeze-dried powder, including sterility testing, purity testing and activity testing;

Packaging: Pack qualified freeze-dried powder bottles and mark the batch number, dosage and production date;

The output is adjusted according to the needs;

Furthermore, the specific steps of freeze-drying treatment include:

Prefreeze the nanobody solution at -40°C for 2-4 hours;

For primary drying, the pre-frozen samples were heated to -20 °C under vacuum conditions for 20 h to remove most of the water;

Secondary drying: Continue to heat to -20°C under vacuum conditions and maintain for 10 hours to further remove residual moisture.

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

The present invention adopts multi-step purification technologies such as efficient affinity chromatography, ion exchange chromatography and gel filtration chromatography to ensure the high purity of nano antibodies, introduces automated equipment to reduce human operation errors, and ensures the consistency of each batch of products; adopts purity and activity detection methods to conduct comprehensive and accurate quality control of the products, and provides nano antibodies with high purity and high activity;

The present invention, in terms of high affinity and high specificity, introduces a high-throughput screening system, which can process a large number of candidate nanoantibody samples at the same time, greatly improving the screening efficiency; according to the established standardized affinity and specificity evaluation process, the screened antibodies have high affinity and high specificity; using phage display technology, a display library containing a large number of candidate antibodies is constructed, and antibodies with high affinity and high specificity to the target antigen are obtained through efficient screening.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a schematic flow chart of the rapid screening method of nanoantibodies based on biological activity evaluation of the present invention.

DETAILED DESCRIPTION

In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

It should be noted that the terms "first", "second", etc. in the specification and claims of the present invention and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the data used in this way can be interchanged where appropriate, so that the embodiments of the present invention described herein can be implemented in an order other than those illustrated or described herein. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions, for example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units that are clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

The present invention is further described in detail below in conjunction with the accompanying drawings:

Example 1

This embodiment provides a method for preparing a nanobody injection, comprising:

1. Prepare the solution by dissolving the purified nanobody in PBS buffer, adjusting the pH to 7.4 and the concentration to 10 mg/mL;

2. Filter sterilize and filter the solution through a 0.22 μm sterile filter membrane;

3. Packaging: Pack the sterilized nanobody solution into sterile injection bottles, each bottle containing 10 mg of nanobody;

4. Sealing: Use a sealing device to seal the injection bottle;

5. Testing: Conduct sterility testing, purity testing and activity testing on the prepared injection;

6. Packaging: Package the qualified injection bottles and mark the batch number, dosage and production date.

Example 2

This embodiment provides a method for preparing a lyophilized nanobody powder, comprising:

1. Prepare the solution by dissolving the purified nanobody in PBS buffer, adjusting the pH to 7.4 and the concentration to 5 mg/mL;

2. Filter sterilize and filter the solution through a 0.22 μm sterile filter membrane;

3. Packaging: Pack the sterilized nanobody solution into sterile lyophilized bottles, each bottle containing 5 mg of nanobody;

4. Freeze-drying: Place the packaged freeze-dried bottles in the freeze dryer and perform freeze-drying according to the preset program.

4.1. Prefreeze, prefreeze the Nanobody solution at -40°C for 3 hours;

4.2. Primary drying: the pre-frozen sample is heated to -20°C under vacuum conditions and kept for 20 hours to remove most of the water;

4.3. Secondary drying: heating to -20°C under vacuum conditions and maintaining for 10 hours to further remove residual moisture;

5. Sealing: After the freeze-drying process is completed, the freeze-dried bottle is immediately vacuum-sealed;

6. Testing: Conduct sterility testing, purity testing and activity testing on the prepared freeze-dried powder;

7. Packaging: Pack the qualified freeze-dried powder bottles and mark the batch number, dosage and production date.

Example 3

This embodiment provides a method for preparing nanobody injections of different concentrations, comprising:

1. Prepare the solution by dissolving the purified nanobody in PBS buffer, adjusting the pH to 7.4 and the concentration to 1 mg/mL;

2. Filter sterilize and filter the solution through a 0.22 μm sterile filter membrane;

3. Packaging: Pack the sterilized nanobody solution into sterile injection bottles, each bottle containing 1 mg of nanobody;

4. Sealing: Use a sealing device to seal the injection bottle;

5. Testing: Conduct sterility testing, purity testing and activity testing on the prepared injection;

6. Packaging: Package the qualified injection bottles and mark the batch number, dosage and production date.

Example 4

This embodiment provides a method for preparing a high-concentration nanobody lyophilized powder, comprising:

1. Prepare the solution by dissolving the purified nanobody in PBS buffer, adjusting the pH to 7.4 and the concentration to 10 mg/mL;

2. Filter sterilize and filter the solution through a 0.22 μm sterile filter membrane;

3. Packaging: Pack the sterilized nanobody solution into sterile lyophilized bottles, each bottle containing 10 mg of nanobody;

4. Freeze-drying: Place the packaged freeze-dried bottles in the freeze dryer and perform freeze-drying according to the preset program.

4.1. Prefreeze, prefreeze the Nanobody solution at -40°C for 2 hours;

4.2. Primary drying: the pre-frozen sample is heated to -20°C under vacuum conditions and kept for 20 hours to remove most of the water;

4.3. Secondary drying: heating to -20°C under vacuum conditions and maintaining for 10 hours to further remove residual moisture;

5. Sealing: After the freeze-drying process is completed, the freeze-dried bottle is immediately vacuum-sealed;

6. Testing: Conduct sterility testing, purity testing and activity testing on the prepared freeze-dried powder;

7. Packaging: Pack the qualified freeze-dried powder bottles and mark the batch number, dosage and production date.

Example 5

This embodiment provides a method for small-batch production of nanobodies, comprising:

1. Prepare the solution by dissolving the purified nanobody in PBS buffer, adjusting the pH to 7.4 and the concentration to 2 mg/mL;

2. Filter sterilize and filter the solution through a 0.22 μm sterile filter membrane;

3. Packaging: Pack the sterilized nanobody solution into sterile injection bottles, each bottle containing 2 mg of nanobody;

4. Sealing: Use a sealing device to seal the injection bottle;

5. Testing: Conduct sterility testing, purity testing and activity testing on the prepared injection;

6. Packaging: Package the qualified injection bottles and mark the batch number, dosage and production date.

Comparative Example 1

This comparative example 1 provides a nanobody screened by phage display technology;

1. Prepare the solution by dissolving the nanoantibodies screened by phage display technology in PBS buffer, adjusting the pH to 7.4 and the concentration to 10 mg/mL;

2. Filter sterilize: filter the solution through a 0.22 μm sterile filter membrane;

3. Packaging: Pack the sterilized nanobody solution into sterile injection bottles, each bottle containing 10 mg of nanobody;

4. Sealing: Use a sealing device to seal the injection bottle;

5. Detection,

Sterility test, qualified

Purity test, 95%

Active detection, 85%

6. Packaging: Pack qualified injection bottles and mark the batch number, dosage and production date;

7. Comprehensive comparative analysis,

Table 1: Comprehensive comparative analysis data 1

Purity comparison,

The purity of Example 1 and Example 3 is the highest (95%), ensuring the safety and effectiveness of the product;

The purity of Example 2 and Example 4 is slightly lower (92%), but still within an acceptable range and suitable for different application scenarios;

The purity of Example 5 is the lowest (90%), which is slightly inferior to that of other examples;

Activity comparison,

Examples 1 and 3 have the highest activity (85%) and are suitable for acute treatment and situations where rapid effects are required;

The activity of Examples 2 and 4 was slightly lower (83%), but the lyophilized form provided the advantage of better stability and ease of transportation;

Example 5 has the lowest activity (80%) and is inferior to the other examples in terms of activity;

Application scenarios,

Examples 1 and 3 are suitable for acute and chronic treatments requiring high-purity, high-activity injections;

Examples 2 and 4 are suitable for treatment plans that require long-term storage and convenient transportation, and high-concentration lyophilized powder is particularly suitable for clinical applications that require high doses;

Example 5, although the purity and activity are slightly lower, the high concentration and wide applicability still have certain advantages and are suitable for a variety of treatment needs;

By comparison, it can be seen that Example 1 and Example 3 have obvious advantages in purity and activity, and are suitable for acute treatment needs and treatments requiring high purity and high activity;

Although Examples 2 and 4 are slightly inferior in purity and activity, the freeze-dried powder form provides the advantages of stability and ease of transportation;

Although the data of Example 5 is slightly inferior, its high concentration and wide application prospects still have certain clinical value;

Comparative Example 2

This comparative example 2 provides a nanobody screened by yeast display technology;

1. Prepare the solution by dissolving the nanoantibodies screened by yeast display technology in PBS buffer, adjusting the pH to 7.4 and the concentration to 5 mg/mL;

2. Filter sterilize and filter the solution through a 0.22 μm sterile filter membrane;

3. Packaging: Pack the sterilized nanobody solution into sterile lyophilized bottles, each bottle containing 5 mg of nanobody;

4. Freeze-drying: Place the packaged freeze-dried bottles in the freeze dryer and perform freeze-drying according to the preset program.

4.1. Prefreeze, prefreeze the Nanobody solution at -40°C for 3 hours;

4.2. Primary drying: the pre-frozen sample is heated to -20°C under vacuum conditions and kept for 20 hours to remove most of the water;

4.3 Secondary drying: heating to -20°C under vacuum conditions and maintaining for 10 hours to further remove residual moisture;

5. Sealing: After the freeze-drying process is completed, the freeze-dried bottle is immediately vacuum-sealed;

6. Detection:

Sterility test, qualified

Purity test, 92%

Active detection, 83%

7. Packaging: Pack qualified freeze-dried powder bottles and mark the batch number, dosage and production date;

Table 2: Comprehensive comparative analysis data 2

Comprehensive comparison

Purity comparison,

The purity of Example 1 and Example 3 is the highest (95%), ensuring the safety and effectiveness of the product;

The purity of Example 2 and Example 4 is slightly lower (92%), but still within an acceptable range and suitable for different application scenarios;

The purity of Example 5 is the lowest (90%), which is slightly inferior to that of other examples;

Activity comparison,

Examples 1 and 3 have the highest activity (85%) and are suitable for acute treatment and situations where rapid effects are required;

The activity of Examples 2 and 4 was slightly lower (83%), but the lyophilized form provided the advantage of better stability and ease of transportation;

Example 5 has the lowest activity (80%) and is inferior to the other examples in terms of activity;

Application scenarios,

Examples 1 and 3 are suitable for acute and chronic treatments requiring high-purity, high-activity injections;

Examples 2 and 4 are suitable for treatment plans that require long-term storage and convenient transportation, and high-concentration lyophilized powder is particularly suitable for clinical applications that require high doses;

Example 5, although the purity and activity are slightly lower, the high concentration and wide applicability still have certain advantages and are suitable for a variety of treatment needs;

By comparison, it can be seen that Example 1 and Example 3 have obvious advantages in purity and activity, and are suitable for acute treatment needs and treatments requiring high purity and high activity; although Example 2 and Example 4 are slightly inferior in purity and activity, the lyophilized powder form provides the advantages of stability and easy transportation; although Example 5 is slightly inferior in data, its high concentration and wide application prospects still have certain clinical value.

Experimental Example 1: Evaluation of biological activity of antigen X antibody

Experimental methods

Table 3: Preparation method of experimental solution 1

Sample dilution

Use Dilutionbuffer to dilute the anti-antigen X antibody into multiple concentration gradients;

Procedure

1. Coating antibody,

Coat the antibody in the Stripwell ^TM Microplate according to the number of samples;

Use Coating buffer to dilute the antigen XFullLengthProteinVLP to 5 μg/mL and add 50 μL/well;

Incubate at 4°C overnight in the dark;

2. Wash the plate,

Take out the coated ELISA plate and wash it 4 times with 300 μL/well of Washing buffer;

3. Closed,

Add 100 μL/well of Blocking buffer and incubate at 37°C for 1.5 hours in the dark;

Wash 4 times with 300 μL/well Washing buffer, shake off and pat dry;

4. Add standards and samples,

Add 50 μL/well of the diluted standard and sample and incubate at 37°C for 1 hour in the dark;

Wash 4 times with 300 μL/well Washing buffer, shake off and pat dry;

5. Add secondary antibody,

Add 50 μL/well of the diluted secondary antibody and incubate at 37°C for 1 hour in the dark;

Wash 4 times with 300 μL/well Washing buffer, shake off and pat dry;

6. Color rendering,

Add 75 μL/well of color development solution, incubate at 37°C for 15 minutes, then remove;

Add 75 μL/well of stop solution;

7. Read the OD value,

Place the ELISA plate into the ELISA reader and set the OD450 reading

Experimental Results

Through the above steps, the activity evaluation results of antigen X antibody at different concentration gradients can be obtained. The specific data include:

Table 4: Activity evaluation results 1

Sample concentration (μg/mL) OD450 reading 0.1 0.112 0.5 0.324 1.0 0.576 2.0 0.789 5.0 1.234 10.0 1.678

in conclusion

Through this experiment, the biological activity of antigen X antibodies at different concentrations can be effectively evaluated. The data show that antibodies exhibit stronger activity at high concentrations. This method has high sensitivity and high specificity, providing a reliable basis for the rapid screening of nanoantibodies.

Experimental Example 2: Antibody Functional Experiment

1. Anti-antigen X antibody blocks chemokine-induced migration assay

This experiment evaluates the blocking effect of multiple different human antigen X antibodies on the migration of cells expressing human antigen X through a chemokine-induced migration experiment; in the evaluation of the Anti-antigen X antibody blocking chemokine-induced migration, the antibodies of the present invention all have the ability to block the migration of cells expressing human antigen X;

Experimental methods

Preparation of experimental solution

Table 5: Preparation method of experimental solution 2

Procedure

1. Count cells, mix the cell suspension, take 10 μL of the cell suspension and spot it on the spotting platform of the cell counter, repeat three times and calculate the average value

2. For cell treatment, add 4×10 ⁵ cells to each well, centrifuge at 500g, 4°C for 5 minutes, and discard the supernatant;

3. Antibody preparation and incubation: dilute the antibody to the corresponding concentration with migration buffer, add 100 μL/well to the cells, mix well, and incubate in a 37°C incubator for 30 minutes;

4. Ligands preparation and migration: Ligands were diluted to the corresponding concentration with migration buffer and added to the lower chamber of the transwell at 300 μL/well. After the cells were incubated for 30 minutes, they were transferred to the upper chamber of the transwell. The cells migrated in a 37°C incubator for 4 hours.

5. Collect the migrated cells. After the migration is completed, transfer the cells in the lower chamber of the transwell to the flow tube. Add a certain amount of reference cells to each tube, centrifuge and discard the supernatant.

Add 100 μL MACS buffer to resuspend, prepare for flow cytometry, and analyze the number of cells that migrated to the lower chamber;

2. Anti-antigen X antibody-mediated cell killing (ADCC) activity assessment

Experimental methods

Preparation of experimental solutions

Table 6: Preparation method of experimental solution 3

Procedure

1. Effector cell and target cell treatment: resuspend effector cells (such as NK cells) and target cells (cells expressing human antigen X) in corresponding buffers and adjust the cell concentration;

2. Antibody preparation and incubation: dilute the antibody to the corresponding concentration with target buffer, add 100 μL/well to the target cells, mix well, and incubate in a 37°C incubator for 30 minutes;

3. Add effector cells: add effector cells to each well at a certain effector cell/target cell ratio (e.g., 10:1) and mix well;

Incubate in 37°C incubator for 4 hours;

4. Collection and detection: After the incubation, collect the supernatant and detect the killing of target cells by LDH release method or flow cytometry;

Experimental Results

Chemokine-induced migration assay

The number of cells that migrated to the lower chamber was analyzed by flow cytometry, and the results are shown in the table below:

Table 7: Migration cell number detection results 1

ADCC activity assessment

The killing of target cells was detected by LDH release method or flow cytometry, and the results are shown in the table below:

Table 8: Target cell survival rate test results 2

Sample No. Antibody concentration (μg/mL) Target cell survival rate (%) Sample 1 0.1 85 Sample 2 0.5 70 Sample 3 1.0 55 Sample 4 2.0 40 Sample 5 5.0 25 Sample 6 10.0 10

in conclusion

1. Chemokine-induced migration assay. The experimental results showed that Anti-antigen X antibody can significantly block the migration of cells expressing human antigen X; with the increase of antibody concentration, the number of cell migration decreased significantly, which verified the effectiveness of the antibody in blocking cell migration;

2. ADCC activity evaluation. The experimental results show that Anti-antigen X antibody can mediate the effective killing of target cells expressing human antigen X by effector cells; the higher the antibody concentration, the lower the survival rate of target cells, which verifies the effectiveness of the antibody in ADCC activity.

Experimental Example 3: Antibody Functional Experiment

1. Anti-antigen X antibody blocks chemokine-induced migration assay

This experiment evaluates the blocking effect of multiple different human antigen X antibodies on the migration of cells expressing human antigen X through a chemokine-induced migration experiment; in the evaluation of the Anti-antigen X antibody blocking chemokine-induced migration, the antibodies of the present invention all have the ability to block the migration of cells expressing human antigen X;

Experimental methods

Preparation of experimental solutions

Table 9: Preparation method of experimental solution 4

Procedure

1. Count cells, mix the cell suspension, take 10 μL of the cell suspension and spot it on the spotting platform of the cell counter, repeat three times and calculate the average value;

2. For cell treatment, add 4×10 ⁵ cells to each well, centrifuge at 500g, 4°C for 5 minutes, and discard the supernatant;

3. Antibody preparation and incubation: dilute the antibody to the corresponding concentration with migration buffer, add 100 μL/well to the cells, mix well, and incubate in a 37°C incubator for 30 minutes;

4. Ligands preparation and migration: ligands were diluted to the corresponding concentration with migration buffer and added to the lower chamber of the transwell at 300 μL/well;

After incubation for 30 minutes, the cells were transferred to the upper chamber of the transwell and allowed to migrate in a 37°C incubator for 4 hours;

5. Collect the migrated cells. After the migration is completed, transfer the cells in the lower chamber of the transwell to the flow tube. Add a certain amount of reference cells to each tube, centrifuge and discard the supernatant; add 100 μL MACS buffer to resuspend, prepare for flow cytometry, and analyze the number of cells that migrated to the lower chamber;

2. Anti-antigen X antibody-mediated cell killing (ADCC) activity assessment

Preparation of experimental solutions

Table 10: Preparation method of experimental solution 5

Procedure

1. Effector cell and target cell processing,

Resuspend effector cells (such as NK cells) and target cells (cells expressing human antigen X) in corresponding buffers and adjust the cell concentration;

2. Antibody preparation and incubation: dilute the antibody to the corresponding concentration with target buffer, add 100 μL/well to the target cells, mix well, and incubate in a 37°C incubator for 30 minutes;

3. Add effector cells to each well at a certain effector cell/target cell ratio (e.g., 10:1), mix well, and incubate in a 37°C incubator for 4 hours;

4. Collection and detection: After the incubation, collect the supernatant and detect the killing of target cells by LDH release method or flow cytometry;

Experimental Results

Chemokine-induced migration assay

The number of cells that migrated to the lower chamber was analyzed by flow cytometry, and the results are shown in the table.

Table 11: Migration cell number detection results 2:

Sample No. Antibody concentration (μg/mL) Number of migrated cells Sample 1 0.1 1500 Sample 2 0.5 1200 Sample 3 1.0 900 Sample 4 2.0 600 Sample 5 5.0 300 Sample 6 10.0 100

ADCC activity assessment

The killing of target cells was detected by LDH release method or flow cytometry. The results are shown in the table.

Table 12: Migration cell number detection results 2:

Sample No. Antibody concentration (μg/mL) Target cell survival rate (%) Sample 1 0.1 85 Sample 2 0.5 70 Sample 3 1.0 55 Sample 4 2.0 40 Sample 5 5.0 25 Sample 6 10.0 10

in conclusion

1. Chemokine-induced migration assay. The experimental results showed that Anti-antigen X antibody can significantly block the migration of cells expressing human antigen X; with the increase of antibody concentration, the number of cell migration decreased significantly, which verified the effectiveness of the antibody in blocking cell migration;

2. ADCC activity evaluation. The experimental results show that Anti-antigen X antibody can mediate the effective killing of target cells expressing human antigen X by effector cells; the higher the antibody concentration, the lower the survival rate of target cells, which verifies the effectiveness of the antibody in ADCC activity.

The present invention provides a method for rapid screening of nano antibodies based on biological activity evaluation; the method can quickly and accurately screen out nano antibodies with high affinity and high specificity through a high-throughput screening system combined with a standardized biological activity evaluation process;

High-throughput system refers to the use of automated equipment and technology to achieve parallel processing and screening of a large number of candidate nanoantibody samples, thereby significantly improving screening efficiency and shortening screening time;

Automated equipment refers to, but is not limited to, liquid handling workstations, high-throughput ELISA testers, automated cell culture systems, automated data analysis software, etc. These devices can realize the full process automation from sample preparation, processing, testing to analysis;

Standardized biological activity evaluation refers to the use of validated and standardized experimental methods and processes to systematically and reliably evaluate the biological activity of nanobodies; these methods include cell proliferation inhibition experiments, cell apoptosis experiments, ELISA assays, etc., to ensure the accuracy and repeatability of the results;

The evaluation process refers to a series of standardized steps from the initial screening of nanobodies to the final biological activity evaluation; specifically, it includes antigen immunization, antibody discovery and preparation, antibody screening, biological activity evaluation and nanobody preparation;

Screening refers to screening a large number of candidate nanobodies through high-throughput screening systems and standardized biological activity evaluation methods to determine their binding ability, specificity and affinity with the target antigen, and ultimately screen out the nanobodies with the best biological activity;

Candidate nanoantibodies refer to nanoantibodies with potential biological activity that are isolated from immunized animals and obtained through phage display technology or other antibody discovery technologies; these candidate nanoantibodies need to be further screened and verified to determine whether they have application value;

Phage display technology refers to a technology that uses phage display vectors to insert foreign antibody genes into the phage genome, allowing the phage to display foreign antibody proteins. Through this technology, phage display libraries can be constructed, and antibodies with high affinity to the target antigen can be screened in the library;

Exogenous antibody proteins refer to antibody fragments obtained from other organisms (such as camelids) through genetic engineering technology, including but not limited to heavy chain variable regions (VHH) or single-chain antibodies (scFv), etc. These exogenous antibody proteins have specific antigen binding capabilities and can be correctly folded and displayed in phage display systems;

High affinity refers to the extremely high binding force between the nanobody and the target antigen, which is quantified by measuring the dissociation constant (Kd); nanobodies with high affinity have better biological effects and application potential;

High specificity means that nanoantibodies can specifically bind to target antigens without obvious cross-reactions with other non-target molecules, thereby ensuring their accuracy and reliability in applications.

So far, the technical solutions of the present invention have been described in conjunction with the preferred embodiments shown in the accompanying drawings. However, it is easy for those skilled in the art to understand that the protection scope of the present invention is obviously not limited to these specific embodiments. Without departing from the principle of the present invention, those skilled in the art can make equivalent changes or substitutions to the relevant technical features, and the technical solutions after these changes or substitutions will fall within the protection scope of the present invention.

Claims (10)

1. A method for rapid screening of nanobodies based on biological activity evaluation, comprising: Step S1, antigen immunization, using camelid animals for antigen immunization to produce heavy chain antibodies against the target antigen, the immunization cycle is 4-6 weeks, and the interval between each immunization is 1 week; Step S2, antibody discovery and preparation, collecting blood from the immunized animal, isolating B cells, and amplifying the variable region (VHH) gene of the heavy chain antibody by RT-PCR technology; Step S3, antibody screening, screening nanoantibodies with high affinity to the target antigen through phage display technology; Step S4, biological activity evaluation, performing a systematic biological activity evaluation on the screened nanobodies; Step S5, preparation of nanobodies, selecting the nanobodies with the best biological activity evaluation results for large-scale production; Step S6, the dosage form and yield of the nanobody, the prepared nanobody is prepared into an injection or lyophilized powder dosage form, each dose containing 1-10 mg of the nanobody. 2. A method for rapid screening of nanobodies based on biological activity evaluation according to claim 1, characterized in that in step S2, the amplified VHH gene is cloned into a phage display vector to construct a phage display library; the specific steps of RT-PCR amplification include: Extract total RNA from B cells using TRIzol reagent or other RNA extraction kits; Reverse transcriptase and specific primers are used to reverse transcribe RNA into cDNA; Design specific primers for the variable region (VHH) of heavy chain antibodies and amplify cDNA by polymerase chain reaction (PCR); The amplified product was purified for subsequent cloning and library construction. 3. A method for rapid screening of nanobodies based on biological activity evaluation according to claim 2, characterized in that the specific steps of antibody screening in step S3 include: Coating antigen: dilute the target antigen X-VLP to a concentration of 5 μg/ml, use Coating buffer (15 mmol/L NaCO ₃ , 35 mmol/L NaHCO ₃ , pH 9.6) for coating, add 50 μl to each well, and incubate at 4°C overnight in the dark; Wash the plate, take out the coated ELISA plate, and wash it 4 times with 300 μl/well of Washing buffer (PBS, 0.05% Tween-20, pH 7.4); Block, add 100 μl/well of Blocking buffer (2% BSA in Washing buffer, pH 7.4), incubate at 37°C for 1.5 hours in the dark, then wash 4 times with Washing buffer, shake off and pat dry; For sample incubation, add 50 μl/well of the diluted phage display library or nanobody sample, incubate at 37°C for 1 hour in the dark, then wash 4 times with Washing buffer, shake off and pat dry; For secondary antibody incubation, add 50 μl/well of the diluted secondary antibody (Peroxidase Affini Pure Goat Anti-Human IgG (H+L), 1:20,000 dilution), incubate at 37°C for 1 hour in the dark, then wash 4 times with Washing Buffer, shake off and pat dry; For color development, 75 μl/well of color development solution (TMB solution) was added, incubated at 37°C for 15 minutes, then taken out, 75 μl/well of stop solution (2MH ₂ SO ₄ ) was added, and the plate was placed in an ELISA reader to set the OD450 reading. 4. A method for rapid screening of nanoantibodies based on biological activity evaluation according to claim 3, characterized in that the ELISA plate refers to a multi-well plate used for enzyme-linked immunosorbent assay (ELISA), each well of which can react independently, and common ELISA plates have two specifications of 96 wells and 384 wells; the ELISA plate is made of polystyrene material. 5. A method for rapid screening of nanobodies based on biological activity evaluation according to claim 4, characterized in that the specific steps of biological activity evaluation include: The binding ability, specificity and affinity of nanobodies were evaluated through high-throughput screening systems; Use standardized biological activity evaluation methods to detect the biological effects of nanobodies; Record and analyze various biological parameters to screen out nanoantibodies with optimal activity. 6. A method for rapid screening of nanobodies based on biological activity evaluation according to claim 5, characterized in that in the preparation process of step S5, the step of controlling the culture conditions for preparing nanobodies comprises: Cloning and construction: clone the selected high-quality nanoantibody genes into expression vectors, and select Escherichia coli and yeast as expression hosts; Transformation and screening: transform the constructed expression vector into host cells, and screen out high-expressing clones through antibiotic selection and expression detection; Small-scale expression test: select the high-expressing clones screened out, conduct small-scale expression test, and determine the optimal expression conditions, including inducer concentration, temperature, and time; Large-scale culture: according to the optimized expression conditions, large-scale culture is carried out in a fermenter, the culture temperature is controlled at 37°C, and the culture time is 48 hours; Cell collection: After the culture is completed, the cells are collected by centrifugation and the culture medium is removed; Cell disruption: using an ultrasonic disruptor or a high-pressure homogenizer to disrupt the collected cells to release the nanobodies; Filtration and clarification: Cell debris is removed by filtration and the supernatant is then further clarified using centrifugation or ultrafiltration systems. 7. A method for rapid screening of nanobodies based on biological activity evaluation according to claim 6, characterized in that in the preparation process of step S5, the preparation step of the nanobodies further comprises: Affinity chromatography, using an affinity column that specifically binds to the nanobody to perform preliminary purification of the nanobody; Ion exchange chromatography, using the charge characteristics of nanobodies, further purifies through ion exchange chromatography columns to remove impurity proteins; Gel filtration chromatography: using a gel filtration chromatography column to separate the molecules according to their molecular weight to obtain high-purity nanobodies; Concentration and dialysis: concentrating the purified nanobody solution to an appropriate volume and dialyzing it into a storage buffer; Sterile filtration: sterile filter the nanobody solution through a 0.22 μm sterile filter membrane; Quality testing: quality testing of the final product, including purity testing and activity testing; The nano-antibody solution that has passed the test is packaged and stored. 8. A method for rapid screening of nanobodies based on biological activity evaluation according to claim 7, characterized in that the injection preparation process is: Prepare a solution, dissolve the purified nanobody in a buffer and adjust the pH to 7.4; Filter sterilize by filtering the solution through a 0.22 μm sterile filter membrane; Packaging: Packaging the sterilized nanobody solution into sterile injection bottles, each bottle containing 1-10 mg of nanobody; Sealing: using a sealing device to seal the injection bottle; Testing: quality testing of the prepared injections, including sterility testing, purity testing, and activity testing; Packaging: package qualified injection bottles and mark the batch number, dosage and production date. 9. A method for rapid screening of nanobodies based on biological activity evaluation according to claim 8, characterized in that the freeze-dried powder is prepared by: Prepare a solution, dissolve the purified nanobody in a buffer and adjust the pH to 7.4; Filter sterilize by filtering the solution through a 0.22 μm sterile filter membrane; Packaging: Packaging the sterilized nanobody solution into sterile lyophilized bottles, each bottle containing 1-10 mg of nanobody; Freeze drying: Place the packaged freeze-dried bottles in a freeze dryer and perform freeze drying according to the preset program. Sealing: After the freeze-drying process is completed, the freeze-dried bottle is immediately vacuum-sealed; Testing: Conduct quality testing on the prepared freeze-dried powder, including sterility testing, purity testing and activity testing; Packaging: Pack qualified freeze-dried powder bottles and mark the batch number, dosage and production date; The output is adjusted as required. 10. A method for rapid screening of nanobodies based on biological activity evaluation according to claim 9, characterized in that the specific steps of freeze-drying treatment include: Prefreeze the nanobody solution at -40°C for 2-4 hours; For primary drying, the pre-frozen samples were heated to -20 °C under vacuum conditions for 20 h to remove most of the water; Secondary drying: Continue to heat to -20°C under vacuum conditions and maintain for 10 hours to further remove residual moisture.