Bevacizumel Fab antibody, detection reagent and preparation method and application of bevacizumel Fab antibody

Abstract

The invention provides a bevacizumel Fab antibody, a detection reagent as well as a preparation method and application of the detection reagent, and belongs to the technical field of drug detection. The bevacizures Fab antibody disclosed by the invention comprises a coated antibody Beva-7E1 and an enzyme labeled antibody Beva-6C11, wherein the coated antibody Beva-7E1 and the enzyme labeled antibody The coated antibody Beva-7E1 is generated by a hybridoma cell strain Beva-7E1 (the preservation number is CGMCC (China General Microbiological Culture Collection Center) No.46305), and the enzyme labeled antibody Beva-6C11 is generated by a hybridoma cell strain Beva-6C11 (the preservation number is CGMCC No.46306). According to the bevacizures Fab antibody and the detection reagent thereof, the bevacizures free blood concentration can be simply and quickly detected with high specificity, high sensitivity and high automation degree.

Description

Technical Field

[0001] The present invention relates to the technical field of drug detection, and in particular to a bevacizumab Fab antibody, a detection reagent, and a preparation method and application thereof. Background Art

[0002] Bevacizumab, also known as Avastin, is a recombinant humanized monoclonal antibody used to treat various types of cancer. Bevacizumab works by targeting vascular endothelial growth factor (VEGF), preventing it from binding to receptors on the cell surface, thereby inhibiting the formation and growth of tumor blood vessels (i.e., angiogenesis). This makes bevacizumab an anti-tumor drug, and its main uses include but are not limited to: colorectal cancer, non-small cell lung cancer, breast cancer, renal cell carcinoma, ovarian cancer, and glioblastoma. In order to ensure the effectiveness and safety of bevacizumab, monitoring its blood drug concentration becomes crucial. Among them, bevacizumab drugs that have bound to VEGF will be cleared faster, so the results of free bevacizumab are more closely related to the efficacy of the drug.

[0003] However, bevacizumab blood concentration monitoring is rarely carried out in clinical practice, which is related to the cumbersome bevacizumab blood concentration detection method and people's lack of awareness of blood concentration monitoring. Bevacizumab detection and analysis mainly include ELISA and LC-MS. Among them, the LC-MS method has a long analysis time and complex pre-treatment operations, which is difficult to meet the requirements of clinical testing for high efficiency, and can usually only measure the total bevacizumab drug concentration; the ELSIA method has a low degree of automation and a long test time, which cannot meet the clinical high-throughput, rapid and accurate detection needs.

[0004] Based on immunological principles and fully automatic chemiluminescence, it is an ideal solution for detecting the concentration of bevacizumab drugs, but it is necessary to develop paired monoclonal antibodies that specifically recognize bevacizumab drugs. CN 108623684 A discloses a non-neutralizing monoclonal antibody that recognizes bevacizumab and its potential application for total bevacizumab determination; CN 108623688 A discloses a monoclonal antibody that neutralizes bevacizumab and its potential use for ADA detection. CN 111366655 A discloses a method of using VEGF immunoaffinity bevacizumab and then performing mass spectrometry detection, but there are many steps and the total detection time exceeds 16 hours. CN 113899894 A discloses a bevacizumab detection scheme based on mass spectrometry, which requires multiple processes such as enzymatic hydrolysis, and the total detection time is greater than 3.5 hours. These mass spectrometry methods are not suitable for clinical high-throughput applications. CN 115725588A discloses a bevacizumab drug detection method based on an electrochemical sensor and a DNA aptamer, but does not disclose the binding sites adapted on the bevacizumab. The method is suitable for rapid detection of a small amount of samples, but is not suitable for high-throughput detection requirements of clinical central laboratories.

[0005] Therefore, there is currently a lack of a highly specific, highly sensitive, simple, rapid, and highly automated method for detecting the free blood concentration of bevacizumab. Summary of the invention

[0006] In view of this, the object of the present invention is to provide a bevacizumab Fab antibody and a detection reagent, which can detect the free blood concentration of bevacizumab with high specificity, high sensitivity, simplicity, rapidity and high automation.

[0007] A bevacizumab Fab antibody is produced by hybridoma cell lines Beva-7E1 and Beva-6C11; the deposit number of the hybridoma cell line Beva-7E1 is CGMCC No.46305, and the deposit number of the hybridoma cell line Beva-6C11 is CGMCC No.46306.

[0008] Preferably, the antibodies include coating antibody Beva-7E1 and enzyme-labeled antibody Beva-6C11; the coating antibody Beva-7E1 is produced by hybridoma cell line Beva-7E1, and the enzyme-labeled antibody Beva-6C11 is produced by hybridoma cell line Beva-6C11.

[0009] The present invention also provides a bevacizumab free drug detection reagent, comprising the bevacizumab Fab antibody.

[0010] The present invention also provides a method for preparing the detection reagent, comprising the following steps: Obtaining the magnetic particle working solution: diluting the magnetic particles in a borate buffer to obtain a magnetic particle solution, adding the coated antibody Beva-7E1 to the magnetic particle solution, shaking the reaction, and obtaining a magnetic particle-antibody solution; magnetically removing the supernatant from the magnetic particle-antibody solution, adding TBST buffer, reacting for 10-40 hours, magnetically removing the supernatant from the obtained solution again, and adding TBST buffer to obtain a magnetic particle working solution; Obtaining the enzyme-labeled working solution: diluting alkaline phosphatase in PBS buffer to obtain a marker solution; adding the enzyme-labeled antibody Beva-6C11 to the marker solution, and then adding a coupling agent for coupling reaction to obtain a solution containing a conjugate; dialyzing the solution containing the conjugate with PBS buffer, and diluting the obtained dialyzate to obtain the enzyme-labeled working solution.

[0011] Preferably, the mass concentration of the magnetic particles in the magnetic particle working solution is 0.2-0.6 mg / mL.

[0012] Preferably, the mass ratio of the alkaline phosphatase, the enzyme-labeled antibody Beva-6C11 and the coupling agent is 1:0.2-1:0.2-1; the concentration of the alkaline phosphatase in the enzyme-labeled working solution is 0.1-2 μg / mL. More preferably, the coupling agent is glutaraldehyde.

[0013] Preferably, the method further comprises obtaining an indicator solution.

[0014] The present invention also provides the use of the detection reagent in detecting bevacizumab or its biosimilars.

[0015] Preferably, bevacizumab or its biosimilar is detected by magnetic particle luminescence method; the reaction procedure of the magnetic particle luminescence method is: 5-100 μL of the sample to be detected, add 20-100 μL of magnetic particle working solution, react for 1-30 minutes, then wash, then add 20-100 μL of enzyme label working solution, incubate at 37°C for 1-30 minutes, wash, and add indicator solution for color development.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The bevacizumab Fab antibody of the present invention includes the coating antibody Beva-7E1 and the enzyme-labeled antibody Beva-6C11, which can accurately identify bevacizumab or its biosimilars, and has no cross-reaction with a variety of endogenous proteins (such as human IgG protein and human albumin). It has high specificity and can effectively avoid false positive results, providing a reliable basis for accurate clinical judgment of bevacizumab drug concentration.

[0017] The present invention can accurately measure bevacizumab concentrations as low as 0.5 μg / mL, has a strong ability to detect trace drugs, and is helpful for early monitoring of drug concentration changes and timely adjustment of treatment plans.

[0018] The preparation method of the detection reagent of the present invention is relatively simple, and the reaction procedure is simple when the magnetic microparticle luminescence method is used for detection. The entire detection process is short in time, and can be completed in a relatively short time from sample addition to result acquisition, thereby meeting the clinical demand for rapid detection and improving detection efficiency.

[0019] The detection reagent of the present invention has good precision within different concentration ranges, and the detection results are stable and reliable with strong repeatability, and can provide accurate and consistent detection data for clinical use; it can not only be used to detect the original bevacizumab drug, but also can accurately measure a variety of bevacizumab biosimilars (such as Arelto, Puxinting, Probexi, etc.), which makes the detection reagent have broad application prospects in the clinical monitoring of bevacizumab drugs.

[0020] Description of biological material deposit Hybridoma cell line Beva-7E1: Classified and named as hybridoma cell line Mus musculus The depository is the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC), the address of the depository is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the deposit number is CGMCC No.46305, and the deposit date is December 17, 2024.

[0021] Hybridoma cell line Beva-6C11: Classified and named as hybridoma cell line Mus musculus The depository is the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC), the address of the depository is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the deposit number is CGMCC No.46306, and the deposit date is December 17, 2024. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the SDS-PAGE electrophoresis of bevacizumab (Fab) 2 fragment; Figure 2 This is the calibration curve for the magnetic particle luminescence method for detecting the free blood concentration of bevacizumab. DETAILED DESCRIPTION

[0023] The present invention provides a bevacizumab Fab antibody produced by a hybridoma cell line Beva-7E1 and a hybridoma cell line Beva-6C11; the hybridoma cell line Beva-7E1 has a deposit number of CGMCC No.46305 and was deposited in the General Microbiology Center of the China National Microbiological Culture Collection on December 17, 2024; the hybridoma cell line Beva-6C11 has a deposit number of CGMCC No.46306 and was deposited in the General Microbiology Center of the China National Microbiological Culture Collection on December 17, 2024.

[0024] In the present invention, the preferred antibodies include coating antibody Beva-7E1 and enzyme-labeled antibody Beva-6C11; coating antibody Beva-7E1 is produced by hybridoma cell line Beva-7E1, and enzyme-labeled antibody Beva-6C11 is produced by hybridoma cell line Beva-6C11.

[0025] The present invention also provides a bevacizumab free drug detection reagent, comprising the bevacizumab Fab antibody; preferably comprising a magnetic particle working solution containing the coated antibody Beva-7E1, and an enzyme-labeled working solution containing the enzyme-labeled antibody Beva-6C11; further preferably also comprising an indicator solution, and more preferably the indicator solution is a commercially available AMPPD and APS-5 substrate.

[0026] The present invention also provides a method for preparing the above detection reagent, comprising the following steps: 1. Obtaining magnetic particle working fluid: 1-1: Magnetic particles are diluted in borate buffer to obtain a magnetic particle solution, coated antibody Beva-7E1 is added to the magnetic particle solution, and the mixture is shaken to react to obtain a magnetic particle-antibody solution; 1-2: Remove the supernatant of the magnetic particle antibody solution by magnetic attraction, add TBST buffer, react for 10-40 hours, preferably for 16 hours, remove the supernatant of the obtained solution by magnetic attraction, add TBST buffer to obtain a magnetic particle working solution; preferably, the mass concentration of the magnetic particles in the magnetic particle working solution is 0.2-0.6 mg / mL, and more preferably 0.4 mg / mL.

[0027] As an implementable method, the steps are as follows: 1-1: 50 mg Dynal beads M280 Tosyl magnetic beads were diluted in 2 mL 50 mM BB pH 8.0 buffer, 1 mg coated antibody Beva-7E1 was added, mixed, and shaken at 37 °C for 8 h; 1-2: Remove the supernatant by magnetic suction, add TBST (50mM Tris, 0.9wt% NaCl, 0.1wt% TW20, pH 7.4) and react at 37℃ for 12h; remove the supernatant by magnetic suction, add TBST, dilute to 0.4mg / mL, name it magnetic particle working solution, and store it at 2~8℃ for later use.

[0028] 2. Obtaining the enzyme labeling working solution: 2-1: Dilute alkaline phosphatase in PBS buffer to obtain a marker solution; 2-2: Add enzyme-labeled antibody Beva-6C11 to the marker solution and mix well, then add a coupling agent to carry out coupling reaction to obtain a solution containing a conjugate; preferably, the mass ratio of alkaline phosphatase, enzyme-labeled antibody Beva-6C11 and coupling agent is 1:0.2-1:0.2-1, more preferably 1:1:0.5; more preferably, the coupling agent is glutaraldehyde; 2-3: The solution containing the conjugate is dialyzed with PBS buffer, and the dialyzate is diluted to obtain an enzyme-labeled working solution; preferably, a mixed buffer solution of 50 mM MES + 0.9% NaCl + 5 mg / mL BSA + 1 mM MgCl 2 , pH 6.7 for dilution; preferably, the concentration of the marker in the enzyme label working solution is 0.1-2 μg / mL, and more preferably 1 μg / mL.

[0029] As an implementable method, the steps are as follows: 2-1: 1 mg ALP was dissolved in 1 mL PBS; 2-2: Add 0.5 mg of enzyme-labeled antibody Beva-6C11, mix well, add 10 μl of 50% glutaraldehyde solution, and mix well at room temperature for 2 hours; 2-3: Dialyze into PBS with 50mM MES 0.9% NaCl 5mg / mL BSA 1mM MgCl 2 Dilute to 1 μg / mL at pH 6.7 and name it as enzyme labeling working solution.

[0030] 3. Obtain the indicator solution.

[0031] The present invention also provides the use of the detection reagent in detecting bevacizumab or its biosimilars, preferably the biosimilars include Arituo, Puxinting, Pubeixi, Dayoutong, Hanbetai, Beianting, Boyunuo, Ankeda and Ambis.

[0032] The present invention preferably uses a magnetic particle luminescence method to detect bevacizumab or its biosimilars; further preferably, the reaction procedure of the magnetic particle luminescence method is: 5-100 μL of the sample to be detected, add 20-100 μL of magnetic particle working solution, react for 1-30 minutes, then wash, then add 20-100 μL of enzyme label working solution, incubate at 37°C for 1-30 minutes, wash, and add indicator solution for color development; more preferably, 10 μL of the sample to be detected (automatically diluted 20 times with PBS) is added with 50 μL of magnetic particle liquid working solution for 5 minutes, then wash, then add 50 μL of enzyme label working solution, incubate at 37°C for 5 minutes, wash, and add AMPPD luminescent solution for color development.

[0033] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0034] Example 1 Preparation of Bevacizumab (Fab)2 fragment immunogen 1. Dilute 20 mg of bevacizumab antibody to 5 mg / mL with 20 mM PB7.4 buffer, total volume 4 mL; 2. Add 1 mL of IdeZ protease affinity column (Beijing Deaoping Biotechnology), incubate at 37°C for 30 min, centrifuge at 2000 rpm for 10 min, and take the supernatant; 3. The supernatant reaction products were passed through the Protein A column to adsorb the unreacted full-length antibody and the cleaved Fc segment, and the flow-through was collected. 4. Use OD280 to measure the concentration and store at -20℃.

[0035] 5. Perform SDS-PAGE identification, the results are as follows Figure 1 As shown, the full-length bevacizumab was not completely cleaved, but the (Fab)2 fragment was obtained after adsorption of impurity fragments by Protein A.

[0036] Example 2 Preparation of Bevacizumab (Fab) 2 fragment antibody Using hybridoma cell fusion technology, refer to "Development and characterization of mouse hybridomas", the steps are as follows: The bevacizumab (Fab) 2 fragment antigen prepared in Example 1 was diluted to 1 mg / mL with PBS buffer, an equal volume of Freund's complete adjuvant was added, and the mixture was completely emulsified. The mice were first immunized at a dose of 0.1 mg / mouse. After an interval of 4 weeks, 1 mg of the bevacizumab (Fab) 2 fragment antigen prepared in Example 1 was mixed with an equal volume of Freund's incomplete adjuvant and emulsified, and the mice were secondly immunized at a dose of 0.1 mg / mouse. After 3 days, spleen cells from the second immunization were fused with Sp2 / 0 cells, and ELISA 96-well plates were coated with bevacizumab drug 2 μg / mL, and the supernatant of the fused cells was titered, and clones with positive titer detection were competitively screened again.

[0037] Competitive screening used 2 μg / mL bevacizumab to coat ELISA 96-well plates, then added 1 μg / mL VEGF protein and fusion cell supernatant. Cell titer determination was performed to select strong positive clones and competitively screen weak positive or negative clones. Finally, 5 competitive anti-bevacizumab idiotype antibodies were screened, as shown in Table 1.

[0038] Table 1 Competitive anti-bevacizumab idiotypic antibodies screened

[0039] The screening of paired antibodies was performed by the checkerboard method. 2 μg / mL of anti-bevacizumab idiotype antibody was coated on an ELISA 96-well plate. 0 or 20 ng / mL of bevacizumab was added. After reacting for 30 minutes, the plate was washed with PBST three times. 2 μg / mL of anti-bevacizumab idiotype antibody-HRP conjugate was added. After reacting for 30 minutes, the plate was washed with PBST three times. TMB colorimetric solution was added and the results were read using an enzyme reader. One paired result was obtained as shown in Table 2.

[0040] Table 2

[0041] The results showed that Beva-7E1 / Beva-6C11 had a good low signal-to-noise ratio. Therefore, this pairing was used to establish a method for detecting free blood concentration of bevacizumab.

[0042] Example 3 Establishment of a magnetic microparticle chemiluminescence method for detecting free bevacizumab concentration in blood (1) 50 mg Dynal beads M280 Tosyl magnetic beads were diluted in 2 mL 50 mM BB pH 8.0 buffer, and 1 mg anti-bevacizumab Beva-7E1 antibody obtained in Example 2 was added. After mixing, the mixture was shaken at 37°C for 8 h.

[0043] The supernatant was removed by magnetic suction, and TBST (50 mM Tris, 0.9 wt% NaCl, 0.1 wt% TW20, pH 7.4) was added and reacted at 37 °C for 12 h.

[0044] Remove the supernatant by magnetic suction, add TBST, dilute to 0.4 mg / mL, name it magnetic particle working solution, and store it at 2-8°C until use.

[0045] (2) Dissolve 1 mg ALP in 1 mL PBS, add 0.5 mg anti-bevacizumab Beva-6C11 antibody, mix well, add 10 μl 50% glutaraldehyde solution, mix well at room temperature for 2 h; dialyze into PBS, use 50 mM MES 0.9% NaCl 5 mg / mL BSA 1 mM MgCl 2 Dilute to 1 μg / mL at pH 6.7 and name it as enzyme labeling working solution.

[0046] (3) Detection was performed using DanDa's fully automatic DF200i chemiluminescent immunoassay analyzer. Reaction procedure: 10 μL of sample (automatically diluted 20 times with PBS) was added to 50 μL of magnetic microparticle liquid working solution for 5 min and then washed. Then, 50 μL of enzyme label working solution was added and incubated at 37°C for 5 min, washed, and AMPPD luminescent solution was added for color development.

[0047] Example 4 The standard curves were prepared using human serum matrix that had not been treated with bevacizumab and different concentrations of bevacizumab calibrators (0-0.5-2-5-20-50ug / mL) as shown in Table 3 and Figure 2 shown.

[0048] Table 3 Calibration curve data of free blood concentration of bevacizumab by magnetic microparticle luminescence method

[0049] from Figure 2 It can be seen that the calibration curve obtained by the magnetic particle luminescence method is: four-parameter equation: Y=(AD) / [1+(X / C)B]+D; among which: A=12739.811; B=1.18757592; C=64.79017513; D=4528878.2229; correlation coefficient R 2 :0.998.

[0050] Precision was determined by repeating the low and high value quality controls 10 times each and calculating the CV according to SD / Mean. The results are shown in Table 4.

[0051] Table 4 Precision results

[0052] It can be seen from Table 4 that the magnetic particle luminescence bevacizumab blood drug concentration determination reagent prepared using Beva-7E1 / Beva-6C11 paired antibodies has high precision.

[0053] Different concentrations of cross-test substances were added to bevacizumab samples with concentrations of 5 μg / mL and 50 μg / mL for cross-reaction, and the cross-reaction rate was calculated. The results are shown in Table 5.

[0054] Table 5 Cross-reaction results

[0055] Cross-reaction rate = (measured concentration - theoretical concentration) / added cross-reaction concentration × 100%; From the results in Table 5, it can be concluded that the magnetic microparticle luminescence bevacizumab blood drug concentration determination reagent prepared by the present invention has no cross-reaction with a variety of endogenous proteins and possible combined drugs, and has high specificity.

[0056] Example 5 Various bevacizumab biosimilars (Bio similar), Arelto, Puxinting, Pubeixi, Dayutong, Hanbetai, Beianting, Boyunuo, Ankeda, and Abesi, were prepared into 5μg / mL and 50μg / mL using human serum matrix that had not been treated with bevacizumab, and tested using the bevacizumab blood drug concentration determination reagent of the present invention. The results are shown in Table 6.

[0057] Table 6 Test results of bevacizumab biosimilars

[0058] The results showed that the deviations between the measured concentrations and the theoretically configured concentrations of the nine biosimilars were less than ±10%, indicating that the reagent of the present invention can be used to measure a variety of bevacizumab biosimilars.

[0059] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A bevacizumab Fab antibody, characterized in that: From hybridoma cell lines ( Mus musculus ) Beva-7E1 and hybridoma cell lines ( Mus musculus ) Beva-6C11; the deposit number of the hybridoma cell line Beva-7E1 is CGMCC No.46305, and the deposit number of the hybridoma cell line Beva-6C11 is CGMCC No.46306.

2. The bevacizumab Fab antibody according to claim 1, characterized in that The antibodies include coated antibody Beva-7E1 and enzyme-labeled antibody Beva-6C11; the coated antibody Beva-7E1 is produced by hybridoma cell strain Beva-7E1, and the enzyme-labeled antibody Beva-6C11 is produced by hybridoma cell strain Beva-6C11.

3. A bevacizumab free drug detection reagent, characterized in that: Comprising the bevacizumab Fab antibody according to claim 1 or 2.

4. A method for preparing the detection reagent according to claim 3, characterized in that: The following steps are involved: Obtaining the magnetic particle working solution: diluting the magnetic particles in a borate buffer to obtain a magnetic particle solution, adding the coated antibody Beva-7E1 to the magnetic particle solution, shaking the reaction, and obtaining a magnetic particle-antibody solution; magnetically removing the supernatant from the magnetic particle-antibody solution, adding TBST buffer, reacting for 10-40 hours, magnetically removing the supernatant from the obtained solution again, and adding TBST buffer to obtain a magnetic particle working solution; Obtaining the enzyme-labeled working solution: diluting alkaline phosphatase in PBS buffer to obtain a marker solution; adding the enzyme-labeled antibody Beva-6C11 to the marker solution, and then adding a coupling agent for coupling reaction to obtain a solution containing a conjugate; dialyzing the solution containing the conjugate with PBS buffer, and diluting the obtained dialyzate to obtain the enzyme-labeled working solution.

5. The preparation method according to claim 4, characterized in that: The mass concentration of the magnetic particles in the magnetic particle working solution is 0.2-0.6 mg / mL.

6. The preparation method according to claim 4, characterized in that: The mass ratio of the alkaline phosphatase, the enzyme-labeled antibody Beva-6C11 and the coupling agent is 1:0.2-1:0.2-1; the concentration of the alkaline phosphatase in the enzyme-labeled working solution is 0.1-2 μg / mL.

7. The preparation method according to claim 4 or 6, characterized in that: The coupling agent is glutaraldehyde.

8. The preparation method according to claim 4, characterized in that: It also includes the acquisition of indicator solution.

9. Use of the detection reagent according to claim 3 in detecting bevacizumab or its biosimilars.

10. The use according to claim 9, characterized in that: The magnetic particle luminescence method is used to detect bevacizumab or its biosimilars; the reaction procedure of the magnetic particle luminescence method is: 5-100 μL of the sample to be detected, add 20-100 μL of magnetic particle working solution, react for 1-30 minutes, then wash, add 20-100 μL of enzyme label working solution, incubate at 37°C for 1-30 minutes, wash, and add indicator solution for color development.

Images