Method for detecting neutralizing antibodies in anti-HBV preS1 monoclonal antibody candidate drugs

Abstract

This application provides a method for detecting neutralizing antibodies against anti-HBV preS1 monoclonal antibody drug candidates. The method comprises sequentially treating a biological sample with an affinity medium, heparin sodium-agarose, and a magnetic bead conjugate containing a specific anti-HBV envelope protein antibody to remove the target. The method effectively mitigates interference from the target, enabling reliable, specific, and sensitive detection of neutralizing antibodies against anti-HBV preS1 monoclonal antibody drug candidates.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and specifically relates to a novel method for detecting neutralizing antibodies of anti-HBV preS1 monoclonal antibody candidate drugs. Background Art

[0002] Chronic hepatitis B, also known as chronic hepatitis B (CHB), is a disease caused by the hepatitis B virus (HBV) and lasts for more than 6 months.

[0003] Hepatitis B virus (HBV) belongs to the Hepadnaviridae family. Its genome is a partially double-stranded circular DNA that encodes HBsAg, HBcAg, HBeAg, viral polymerase, and HBx protein. HBV has three main antigens: surface antigen (HBsAg), e antigen (HBeAg), and core antigen (HBcAg). Hepatitis D virus (HDV), also a member of the Hepadnaviridae family, is a replication-defective RNA virus. It is a satellite HBV virus that replicates using the HBV surface protein as its envelope protein. Therefore, HDV infection is most common in patients infected with HBV. The sole antigenic component of HDV is the HDAg.

[0004] The preS1 protein (preS1) is a key component of the hepatitis B virus capsid protein. It contains hepatocyte membrane receptors and plays a crucial role in HBV / HDV infection of hepatocytes and the body's immune response. It specifically binds to the sodium-taurocholate cotransporter (NTCP) on the hepatocyte membrane, thereby mediating HBV entry into cells. Anti-preS1 monoclonal antibodies can effectively neutralize HBV and block the binding of PreS1 to NTCP. Therefore, the development of anti-preS1 monoclonal antibodies could effectively bind to the preS1 protein (preS1) and block further HBV / HDV infection of hepatocytes.

[0005] However, patients with chronic hepatitis B and chronic hepatitis D virus infection contain a large number of free targets (HBV / HDV viral particles and viral antigens) in their serum. For example, the serum of patients with chronic hepatitis B is positive for hepatitis B surface antigen (HBsAg). The presence of these viral particles and viral antigens, such as HBsAg, can lead to poor specificity in methods for detecting neutralizing antibodies against anti-HBV preS1 monoclonal antibody candidate drugs, interfering with the detection of neutralizing antibodies. Therefore, there is an urgent need to develop suitable methods to remove these targets and thereby improve the specificity of methods for detecting neutralizing antibodies against anti-HBV preS1 monoclonal antibody candidate drugs. Summary of the Invention

[0006] In response to the above problems, after repeated exploration, the inventors of the present application developed a new method for detecting neutralizing antibodies of anti-HBV preS1 monoclonal antibody candidate drugs in biological samples.

[0007] In one aspect, the present application provides a method for detecting neutralizing antibodies of anti-HBV preS1 monoclonal antibody candidate drugs in a biological sample.

[0008] In some embodiments, the method provided herein includes: performing a first treatment on a biological sample using a heparin-agarose affinity medium to remove the target in the biological sample; performing a second treatment on the first-treated biological sample using a magnetic bead conjugate of a specific anti-HBV envelope protein antibody to further remove the target in the biological sample; and detecting neutralizing antibodies of anti-HBV preS1 monoclonal antibody candidate drugs in the second-treated biological sample using a competitive ligand binding assay based on affinity capture-acid dissociation.

[0009] In some embodiments, the first treatment of the biological sample using a heparin-agarose affinity medium includes: diluting the biological sample with a phosphate buffer having a pH of 7.2 to 7.4 at a volume ratio of 1:1; mixing the diluted biological sample with the affinity medium heparin sodium-agarose at a volume ratio of 2:1, and incubating at room temperature with shaking at 200 to 500 rpm for at least 16 hours; and centrifuging at 800 to 1200 rpm for 2 to 5 minutes to collect the filtered biological sample.

[0010] In some embodiments, the second treatment of the biological sample that has been treated for the first time using a magnetic bead conjugate of a specific anti-HBV envelope protein antibody includes: adding the biological sample that has been treated for the first time and the magnetic bead conjugate of the anti-HBV envelope protein antibody to an incubation plate at a volume ratio of 7:3, incubating at room temperature with shaking at 600 to 1000 rpm for 1 to 2 hours, placing the incubation plate in a magnetic separator and letting it stand for at least 2 minutes, and collecting the biological sample after magnetic separation.

[0011] In some embodiments, detecting neutralizing antibodies of anti-HBV preS1 monoclonal antibody candidate drugs in the second-treated biological sample using a competitive ligand binding assay includes: acid-treating the second-treated biological sample using an acid hydrolysis solution at a volume ratio of 2:1; affinity capturing the acid-treated biological sample using an ELISA plate; acid-dissociating the affinity-captured biological sample using an acid hydrolysis solution, and sequentially adding a neutralization buffer, the acid-dissociated biological sample, and a detection reagent to a new incubation plate, incubating at room temperature with shaking at 400 to 800 rpm for 2 to 4 hours to obtain a mixed solution, wherein the volume ratio of the added neutralization buffer, biological sample, and detection reagent is 3:9:1; and detecting neutralizing antibodies of the anti-HBV preS1 monoclonal antibody candidate drugs in the mixed solution using a competitive ligand binding assay.

[0012] In some embodiments, the acid hydrolysis solution is 300 mM acetic acid or glycine with a pH of 2.5 to 3.5; the neutralization buffer is 1 M Trizma neutralization buffer or Tris buffer with a pH of 9.0 to 10.0; and the detection reagent is an anti-HBV preS1 monoclonal antibody candidate drug labeled with ruthenium.

[0013] In some embodiments, the method of detecting neutralizing antibodies of anti-HBVpreS1 monoclonal antibody candidate drugs in biological samples using a competitive ligand binding assay includes: blocking the MSD microplate, washing the plate, coating the plate with biotin-labeled HBVpreS1, and washing the plate again; adding the mixed solution to the pretreated MSD microplate, incubating at room temperature with shaking at 400 to 800 rpm for 2 to 4 hours; and washing and detecting the incubated MSD microplate.

[0014] In some embodiments, the biological sample is human serum or plasma.

[0015] It can be seen that the present application has established a method for accurately, reliably and highly sensitively detecting neutralizing antibodies of anti-HBV preS1 monoclonal antibody candidate drugs.

[0016] The method of the present application can effectively resolve the interference of free targets (HBV / HDV virus particles and viral antigens) in biological samples, thereby accurately obtaining data on neutralizing antibodies of anti-HBV preS1 monoclonal antibody candidate drugs, to support the evaluation of drug efficacy and safety in clinical studies of anti-HBV preS1 monoclonal antibody candidate drugs.

[0017] Specifically, the method of the present application utilizes an affinity medium, sodium heparin-agarose gel, and a specific anti-HBV envelope protein antibody magnetic bead conjugate to sequentially immunodeplete biological samples, effectively removing target interference. On the one hand, the affinity medium, sodium heparin-agarose gel, binds to free targets through electrostatic adsorption, i.e., positive-negative charge interactions, initially reducing target interference. On the other hand, the specific anti-HBV envelope protein antibody magnetic bead conjugate further specifically binds to free targets in the biological sample, further removing the target from the sample and further reducing target interference. After the free targets have been fully and specifically adsorbed, the sample undergoes affinity capture-acid dissociation (ACE) pretreatment. Subsequently, a competitive ligand binding assay (CLBA) is used on an MSD platform to detect neutralizing antibodies against candidate anti-HBV preS1 monoclonal antibodies in human serum. This allows for more sensitive and specific detection of neutralizing antibodies against candidate anti-HBV preS1 monoclonal antibodies, significantly improving the accuracy of the test results. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1The figure shows the sensitivity of the method for secondary target removal established in this application, which is about 200 ng / mL; wherein "Run" represents one analysis plate.

[0019] Figure 2 The figure shows a comparison of the inhibition rates of chronic hepatitis B patient serum (CHBS) with different treatments, where Group 1 indicates that the target was not removed; Group 2 indicates that only heparin-agarose was used for one target removal treatment; Group 3 indicates that heparin-agarose and specific anti-HBV neutralizing antibody magnetic bead conjugates were used for two target removal treatments in sequence. DETAILED DESCRIPTION

[0020] When detecting neutralizing antibodies, they bind to the labeled drug (detection reagent). If free target is present in the biological sample, it will bind to the detection reagent, resulting in a false positive. False positive results can be avoided by sufficiently specific affinity immunodepletion of free target.

[0021] Regarding the detection of neutralizing antibodies for anti-HBV preS1 monoclonal antibody candidate drugs, since the target in the subject's body significantly interferes with the neutralizing antibody method, the inventors of the present application further attempted to use the affinity medium sodium heparin-agarose to remove target interference based on the premise of establishing and optimizing the affinity capture-acid dissociation (ACE) pre-treatment.

[0022] Initially, attempts were made to remove target interference using heparin sodium-agarose affinity media alone. However, significant interference was still observed after a single treatment. Increasing the number of treatments removed target interference, but the sensitivity of the method also decreased. Therefore, in addition to using heparin sodium-agarose affinity media to remove target interference, a magnetic bead conjugate containing a specific anti-HBV envelope protein antibody was added to remove endogenous targets. The results showed that a single heparin sodium-agarose treatment followed by a single magnetic bead conjugate containing a specific anti-HBV envelope protein antibody completely removed endogenous target interference while maintaining good sensitivity and drug resistance.

[0023] Secondly, through a large number of experimental optimizations, the optimal conditions for treatment with heparin sodium-agarose affinity medium were determined to be: the volume ratio of heparin sodium-agarose to biological sample was 1:2, and incubation was performed at room temperature at 200 to 500 rpm, preferably 350 rpm, with shaking for at least 16 hours; the optimal conditions for treatment with magnetic bead conjugates of anti-HBV envelope protein antibodies were determined to be: the volume ratio of biological sample to anti-HBV envelope protein antibodies coupled to magnetic beads was 7:3, and incubation was performed at room temperature at 600 to 1000 rpm, preferably 800 rpm, with shaking for 1 to 3 hours, preferably 1.5 hrs±10 min.

[0024] The method provided in the present application utilizes a heparin-agarose affinity medium and a magnetic bead conjugate of an anti-HBV envelope protein antibody to sequentially immunodeplete a biological sample (e.g., human serum), sufficiently and specifically affinity-immunodepleting free targets (HBV / HDV viral particles and viral antigens), greatly eliminating the interference of endogenous free targets, and significantly improving the specificity of the method for detecting neutralizing antibodies of anti-HBV preS1 monoclonal antibody candidate drugs.

[0025] The principle of the neutralizing antibody detection technology for anti-HBV preS1 monoclonal antibody candidate drugs provided in the present application is as follows: a biological sample (including quality control / verification sample / test sample) is treated and adsorbed once using a heparin-agarose affinity medium (Heparin Bestarose HP) pre-filled in a microporous filter plate or filter tube to remove free target substances, and a magnetic bead conjugate of a specific anti-HBV envelope protein antibody is added after the filtered solution is recovered to further affinity-immunodeplete the free target substances; the depleted sample is pre-treated with affinity capture-acid dissociation (ACE), mixed with a neutralizing reagent and a detection reagent in an incubation plate for oscillation reaction; then, it is added to an MSD microplate pre-coated with a target antigen peptide (HBV preS1), incubated with oscillation at room temperature, washed, and then MSD Read Buffer T (2×) is added and incubated in a MESO QUICKPLEX The instrument signal is read on SQ120; if the sample contains neutralizing active antibodies, the instrument response value (electrochemiluminescence unit, ECLU value) read on the electrochemiluminescence detection instrument is low, and its ratio to the instrument response value (ECLU) of the negative control sample (100% mixed healthy human serum) is also low, and the signal inhibition rate is increased.

[0026] Unless otherwise indicated, this application employs conventional molecular biology, biochemistry, and immunology techniques within the skill of the art.

[0027] Unless otherwise specified, the terms used in this application have the meanings commonly understood by those skilled in the art, which will be exemplified below.

[0028] As used herein, "anti-HBV preS1 mAb candidate drug" refers to a mAb candidate drug that can bind to HBV

[0029] Antibodies that specifically bind to preS1.

[0030] As used herein, "anti-HBV preS1 monoclonal antibody drug candidate neutralizing antibody" refers to an antibody that can specifically bind to the Fab fragment of the anti-HBV preS1 monoclonal antibody.

[0031] As used herein, "acid treatment" refers to the acid treatment of a biological sample prior to affinity capture. Since neutralizing antibodies may bind to pre-existing drugs in the body, acid treatment can free them. Incubating the acid-treated biological sample with a drug-coated plate can achieve a similar purification effect. In some embodiments, acid treatment of the biological sample can reduce matrix effects. In some embodiments, acid treatment of the biological sample can improve drug tolerance. In some embodiments, acid treatment of the biological sample can reduce sample volume.

[0032] As used herein, "affinity capture" refers to capture based on the principle of specific antibody-antigen binding, for example, using a specific antigen to capture an antibody. In some embodiments of the present application, neutralizing antibodies against HBV preS1 monoclonal antibody drug candidates in an acid-treated sample bind to anti-HBV preS1 monoclonal antibodies (equivalent to antigens) coated on a microplate under neutral pH conditions, thereby achieving affinity capture of the neutralizing antibodies.

[0033] As used herein, "acid dissociation" refers to the dissociation of a drug-anti-drug neutralizing antibody complex in a sample into the drug and the anti-drug neutralizing antibody by acidifying the sample. In some embodiments of the present application, the purpose of acid dissociation is to disconnect the anti-HBV preS1 monoclonal antibody drug candidate from the neutralizing antibody coated on the microplate, thereby dissociating the neutralizing antibody previously bound to the drug into a free state. In some embodiments of the present application, the anti-HBV preS1 monoclonal antibody drug candidate neutralizing antibody obtained by "affinity capture" is dissociated from the anti-HBV preS1 monoclonal antibody drug candidate-anti-HBV preS1 monoclonal antibody drug candidate neutralizing antibody complex. The dissociated neutralizing antibody is then incubated with a detection reagent, and the detection reagent signal can be used to reflect the neutralizing antibody level.

[0034] As used herein, "room temperature" refers to a suitable ambient temperature, such as 10 to 40°C, 20 to 30°C, or 20 to 25°C, etc.

[0035] Therefore, the present application provides a method for detecting neutralizing antibodies of anti-HBV preS1 monoclonal antibody candidate drugs in a liquid biological sample, the method comprising: affinity adsorption of the liquid biological sample using a heparin-agarose affinity medium and centrifugation; adding a magnetic bead conjugate of a specific anti-HBV envelope protein antibody to the filtered liquid biological sample to further affinity immunodeplete free targets; performing affinity capture-acid dissociation (ACE) pre-treatment on the depleted sample, and then adding a neutralizing reagent; and detecting neutralizing antibodies of anti-HBV preS1 monoclonal antibody candidate drugs in the liquid biological sample.

[0036] In some embodiments, the centrifugation condition is: 800 to 1200 rpm for 2 to 5 minutes.

[0037] In some embodiments, the centrifugation condition is: 800, 850, 900, 950, 1000, 1050, 1100, 1150, or 1200 rpm, or a range between any of the above values.

[0038] In some embodiments, the centrifugation condition is: centrifugation for 2, 3, 4 or 5 minutes, or a range between any of the above values.

[0039] In some embodiments, the centrifugation condition is: centrifugation at 1000 rpm for 4 minutes.

[0040] In some embodiments, the affinity medium sodium heparin-agarose is added in a volume of 50 to 150 μL / well.

[0041] In some embodiments, the volume of the affinity medium heparin sodium-agarose added is 50, 60, 70, 80, 90, 100, 110, 120, 130, 140 or 150 μL / well, or a range between any of the above values.

[0042] In some embodiments, the affinity medium sodium heparin-agarose is added in a volume of 100 μL / well.

[0043] In some embodiments, the incubation time of the biological sample and the magnetic bead conjugate after centrifugation and filtration is 1-2 h.

[0044] In some embodiments, the incubation time between the biological sample and the magnetic bead conjugate after centrifugation and filtration is 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0 h, or a range between any of the above values.

[0045] In some embodiments, the incubation time of the biological sample and the magnetic bead conjugate after centrifugation and filtration is 1.5 h.

[0046] In some embodiments, the pH value of the acetic acid solution during affinity capture of the sample is 2.5-3.5.

[0047] In some embodiments, the pH value of the acetic acid solution during affinity capture of the sample is 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4 or 3.5, or a range between any of the above values.

[0048] In some embodiments, the pH value of the acetic acid solution during affinity capture of the sample is 3.0.

[0049] In some embodiments, the pH value of the acetic acid solution during acid dissociation of the sample is 2.5-3.5.

[0050] In some embodiments, the pH of the acetic acid solution during acid dissociation of the sample is 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4 or 3.5, or a range between any of the above values.

[0051] In some embodiments, the pH value of the acetic acid solution during acid dissociation of the sample is 3.5.

[0052] In some embodiments, the neutralizing agent is 1 M Trizma neutralizing buffer at a pH of 9.5.

[0053] In some embodiments, the biological sample is serum or plasma.

[0054] In some embodiments, the biological sample is human serum.

[0055] In some embodiments, detecting neutralizing antibodies against the anti-HBV preS1 monoclonal antibody drug candidate in the biological sample comprises performing a competitive ligand binding assay using an MSD platform to determine neutralizing antibodies against the anti-HBV preS1 monoclonal antibody drug candidate in the liquid biological sample.

[0056] As an exemplary embodiment, a method for detecting neutralizing antibodies in anti-HBV preS1 monoclonal antibody candidate drugs comprises the following steps:

[0057] 1) Sample pretreatment

[0058] The sample was pretreated by diluting it with 1 x PBS (pH 7.2 ~ 7.4) at a volume ratio of 1:1 (e.g., 110 μL sample + 110 μL PBS).

[0059] 2) First target removal treatment of the sample

[0060] 2.1) Pretreatment of Heparin Sodium-Sepharose Affinity Medium

[0061] Equilibration of Heparin-agarose affinity matrix (Heparin beads, Heparin bestarose HP): Take an appropriate amount (depending on the actual experimental amount) of Heparin beads, add approximately 5 times the volume of water, centrifuge at 1800-2100 rpm for 2-5 minutes, discard the water, repeat twice, and finally resuspend in 1x PBS (pH 7.2-7.4) to the original volume of affinity matrix.

[0062] 2.2) First target removal treatment of samples

[0063] Treat the sample with the pretreated affinity medium: Add the pretreated Heparin beads from step 2.1) to a DV Free 96-well filter plate. Centrifuge at 800-1200 rpm for 3-6 minutes. Remove the liquid by centrifugation and add the diluted sample from step 1). Incubate at room temperature with shaking at 350 rpm overnight for at least 16 hours. The volume ratio of Heparin beads to sample should be 1:2.

[0064] After incubation, the DV Free 96-well filter plate was centrifuged at 800 to 1200 rpm for 2 to 5 minutes, and the filtrate was collected.

[0065] 3) Second target removal treatment of samples

[0066] Treat samples with anti-HBV envelope protein antibodies: Add the anti-HBV envelope protein antibody magnetic bead conjugate to a non-adsorbed 96-well polypropylene plate, then add the first treated sample obtained in step 2.2) in a volume ratio of 3:7. Seal the plate with sealing film and incubate at room temperature with shaking at 600 to 900 rpm. Let it stand in a magnetic separator for at least 2 minutes before proceeding to the next step of the experiment.

[0067] 4) Affinity capture-acid dissociation treatment

[0068] 4.1) Acid treatment of samples:

[0069] The sample obtained in step 3) after the second treatment was added with an acid hydrolysis solution (300 mM acetic acid, pH 3.0), and acid hydrolyzed at room temperature with shaking at 200 to 500 rpm for 30 to 40 minutes for acid treatment. The volume ratio of the added sample to the acid hydrolysis solution was 1:2.

[0070] 4.2) Pretreatment of ELISA plates:

[0071] Coating: Coat the ELISA plate capture reagent working solution (anti-HBV preS1 monoclonal antibody working solution) of CBS at 80 to 120 μL / well on the ELISA plate and incubate at 2 to 8°C overnight.

[0072] Wash the plate: Wash the ELISA plate three times with 1 x PBST at a volume of not less than 300 μL / well.

[0073] Blocking: Add 300 μL / well of I-Block Blocking Buffer to the ELISA plate and shake at 500 to 800 rpm at room temperature for at least 2 hours.

[0074] Wash the plate: Wash the ELISA plate three times with 1 x PBST at a volume of not less than 300 μL / well.

[0075] 4.3) Sample affinity capture on ELISA plate:

[0076] Pre-add neutralization buffer (1 M Trizma, H 9.5) to the ELISA plate; add the pretreated sample obtained in 4.1) to the wells according to the sample loading diagram, with the volume ratio of the added neutralization buffer to the pretreated sample being 4:150. Seal the plate with sealing film and incubate at room temperature with shaking at 200 to 500 rpm for at least 16 hours.

[0077] 4.4) Washing the plate:

[0078] Remove the ELISA plate with captured neutralizing antibody (Nab) and wash the plate three times with 1xPBST at a volume of not less than 300 μL / well.

[0079] 4.5) Acid dissociation of samples:

[0080] To the washed ELISA plate, add 90 to 120 μL / well of acid dissociation solution (300 mM acetic acid, pH 3.5) and shake at 500 to 800 rpm at room temperature for 30 to 40 minutes.

[0081] 4.6) Preparation of sample mixture:

[0082] Prepare the sample master mix: Add neutralization buffer (1 M Trizma, pH 9.5) to a round-well polypropylene plate. Then, add the dissociated sample obtained in step 4.5 to neutralize the sample. Add the prepared detection reagent working solution (ruthenium-labeled anti-HBV preS1 monoclonal antibody) to the neutralized solution. Mix thoroughly and incubate at room temperature with shaking at 500-800 rpm for 2-4 hours. The volume ratio of neutralization buffer, sample, and detection reagent working solution should be 3:9:1.

[0083] 5) Sample testing

[0084] 5.1) Pretreatment of MSD microplates

[0085] Blocking: Add 120 to 160 μL / well of MSD microplate blocking buffer (PBS containing 3% BSA) to the MSD plate and incubate at room temperature with shaking at 500 to 800 rpm in the dark for at least 1 hr.

[0086] Wash the plate: Wash the plate three times with at least 300 μL / well of 1xPBST.

[0087] MSD microplate capture: Coat the prepared MSD microplate capture reagent working solution (HBV preS1 working solution) at 40 to 60 μl / well on an MSD microplate and incubate at 35 to 39°C for approximately 1 hour.

[0088] Wash the plate: Wash the plate three times with at least 300 μL / well of 1 x PBST.

[0089] 5.2) Sample testing

[0090] Transfer and add samples: According to the plate map, add 40 to 60 μl / well of the sample mixture prepared in step 4.6) to the MSD microplate and incubate at room temperature with shaking at 500 to 800 rpm for about 2 hours.

[0091] Wash the plate: Wash the plate three times with at least 300 μL / well of 1xPBST.

[0092] Detection: Add 140 to 160 μL / well of the prepared MSD Read Buffer T (2x) working solution to the MSD microplate. Within 15 minutes, place the MSD microplate in the MESO QUICKPLEX SQ120 (set the equipment parameters according to the manufacturer) for detection. Example

[0093] The application will be described in more detail by way of specific examples. The following examples are provided for illustrative purposes only and are not intended to limit the application in any way. Those skilled in the art will readily recognize various noncritical parameters that can be changed or modified to produce substantially the same result.

[0094] Unless otherwise specified, the reagents and consumables used in the examples are all commercially available, and the technical means used in the examples are all conventional means well known to those skilled in the art.

[0095] Example 1

[0096] This example provides a method for detecting neutralizing antibodies in anti-HBV preS1 monoclonal antibody candidate drugs using secondary target removal treatment.

[0097] The specific steps are as follows:

[0098] 1. Sample pretreatment: All samples were diluted with 1xPBS (pH 7.2-7.4) at a 1:1 volume ratio (e.g., 110 μL sample + 110 μL PBS).

[0099] 2. Heparin-agarose (Heparin bestarose HP) equilibration: Take an appropriate amount (depending on the actual experimental amount) of heparin-agarose, then add approximately 5 times the volume of water, centrifuge, discard the water, add approximately 5 times the volume of 1× PBS, wash twice (add approximately 5 times the volume of 1× PBS, then centrifuge and discard the water), and finally resuspend to the original volume in 1× PBS (pH 7.2-7.4).

[0100] 3. Heparin-agarose treated samples: Add 100 μL / well of washed Heparin beads to a DV Free 96-well filter plate. Centrifuge at 1000 rpm for 4 minutes. Remove the liquid by filtration. Add 200 μL / well of diluted serum sample (single well). Shake at 350 rpm at room temperature overnight for at least 16 hours.

[0101] 4. Coating: Coat the ELISA plate with the prepared ELISA plate capture reagent working solution at 100 μl / well. Incubate at 2-8°C overnight for at least 16 hours.

[0102] The ELISA plate capture reagent working solution was prepared as follows: the test drug (anti-HBV preS1 monoclonal antibody) was diluted with coating buffer (carbonate buffer 1x CBS, pH 9.4-9.8) to a final concentration of approximately 20 µg / mL of ELISA plate capture reagent working solution.

[0103] 5. Wash the plate: Wash the ELISA plate three times with at least 300 μL / well of 1 x PBST.

[0104] 6. Blocking: Add blocking buffer (0.2% I-Block in PBS) to the ELISA plate at 300 μL / well and shake at 600 rpm at room temperature for at least 2 hours.

[0105] 7. Sample collection: Centrifuge the DV Free 96-well filter plate at 1000 rpm for 4 minutes after incubation and collect the sample.

[0106] 8. Samples treated with magnetic bead conjugates of specific anti-HBV preS1 antibodies:

[0107] 1) Vortex the concentrated solution of the specific anti-HBV preS1 antibody magnetic bead conjugate for approximately 20 seconds to resuspend the magnetic beads;

[0108] 2) Take an appropriate amount (depending on the actual amount required for the experiment, such as 10 mg / mL) of the magnetic bead conjugate concentrate of the specific anti-HBV preS1 antibody and add it to a suitable EP tube. Add 1x PBS (pH 7.2-7.4) (approximately 4 times the volume) and gently shake to wash. Let it stand in a magnetic separator 2 / 15 for at least 2 minutes before removing the wash solution.

[0109] 3) Add approximately 5 times 1x PBS (pH 7.2-7.4) and thoroughly vortex to resuspend to obtain a working solution of the magnetic bead conjugate of the specific anti-HBV preS1 antibody for later use.

[0110] 4) The resuspended anti-HBV preS1 antibody magnetic bead conjugate working solution was thoroughly shaken again and added to a non-adsorbed 96-well polypropylene plate according to the plate map, 45 μL / well;

[0111] 5) Add 105 μL / well of the heparin-agarose-treated sample. Seal the plate with sealing film, resuspend the magnetic beads, and shake at approximately 800 rpm at room temperature for 1.5 hours ± 10 minutes for immunodepletion. Allow to stand in a 96 I magnetic separator for at least 2 minutes before proceeding to the next step.

[0112] 9. Sample acid treatment: After magnetic bead conjugate treatment, sample was treated with acid solution I (300mM HAc, pH 3.0) at a volume ratio of 1:2 and shaken at 350 rpm for 30-40 min at room temperature.

[0113] 10. Wash the plate: Wash the ELISA plate three times with at least 300 μL / well of 1 x PBST.

[0114] 11. ELISA plate loading and capture: Pre-add Trizma neutralization buffer (1 M pH 9.5) to the ELISA plate at 4 μL / well. Add all pretreated samples to the wells according to the sample loading plate diagram, loading 150 μL / well per well. Seal the plate with sealing film and incubate at room temperature with shaking at 350 rpm overnight (at least 16 hours).

[0115] 12. Washing: Remove the ELISA plate containing the captured neutralizing antibody (Nab) and wash the plate three times with plate washing buffer (1xPBST) at a volume of at least 300 μL / well.

[0116] 13. Sample acid dissociation: Add 110 μL / well of Acid Dissociation Solution II (300 mM HAc, pH 3.5) to the ELISA plate and shake at room temperature for 30 to 40 minutes.

[0117] 14. Master Mix: Add 30 μL / well of Trizma Neutralization Buffer (1 M, pH 9.5) to a round-well polypropylene plate. Neutralize the sample by adding 90 μL / well of the acid-dissociated sample. Add 10 μL / well of the prepared detection reagent working solution to this neutralized solution, mix thoroughly, and incubate at room temperature with shaking at 600 rpm for 2.5 hr ± 10 min.

[0118] The detection reagent working solution was prepared by the following method: the detection reagent concentrate (ruthenium-labeled anti-HBV preS1 monoclonal antibody) was diluted with PBS containing 1% BSA to a final concentration of approximately 65 ng / mL of the detection reagent working solution.

[0119] 15. Blocking of MSD microplates: Add 150 μL / well of MSD microplate blocking solution (PBS containing 3% BSA) to the MSD plate and incubate at room temperature with shaking at approximately 600 rpm for at least 1 hr in the dark.

[0120] 16. Wash the plate: Wash the plate three times with at least 300 μL / well of 1 x PBST.

[0121] 17. MSD microplate capture: Coat the prepared MSD microplate capture reagent working solution into the MSD microplate, 50 μl / well, and incubate at 35-39°C for 1 hour ± 5 minutes.

[0122] The MSD microplate capture reagent working solution was prepared by the following method: MSD microplate capture reagent concentrate (HBVpreS1) was diluted with DPBS to a final concentration of approximately 50 ng / mL of MSD microplate capture reagent working solution.

[0123] 18. Wash the plate: Wash the plate three times with at least 300 μL / well of 1 x PBST.

[0124] 19. Transfer and add samples: According to the plate map, add the Master Mix prepared in step 14 to the MSD microplate at 50 μL / well in duplicate. Incubate at room temperature with shaking at 600 rpm for 2 hr ± 10 min.

[0125] 20. Wash the plate: Wash the plate three times with at least 300 μL / well of 1 x PBST.

[0126] 21. Detection: Add the prepared MSD Read Buffer T (2x) working solution to the MSD microplate at 150 μL / well. Within 15 minutes, place the MSD microplate in a MESO QUICKPLEX SQ120 plate reader (Model: 1300, purchased from MesoScale Discovery) for detection.

[0127] MSD Read Buffer T (2x) working solution was prepared by mixing MSD Read Buffer T (4×) (purchased from Meso Scale Discovery) with ultrapure water in a 1:1 ratio. After preparation, tighten the cap and use as soon as possible.

[0128] The sensitivity of the above method for detecting neutralizing antibodies of anti-HBV preS1 monoclonal antibody candidate drugs is approximately 200 ng / mL.

[0129] Specifically, the sensitivity curve sets 8 concentration points of neutralizing antibody samples, and the concentrations from large to small are 4000, 2000, 1000, 500, 250, 125, 62.5, and 31.25 ng / mL.

[0130]

[0131] The negative control sample was a pooled serum from healthy individuals that did not contain neutralizing antibodies.

[0132] Figure 1 The figure shows the fitted curve with the neutralizing antibody concentration as the horizontal axis and the inhibition rate as the vertical axis (curve range: 31.25~4000 ng / mL).

[0133] Furthermore, the above method (after two interference removal treatments) was compared with the results of only one interference removal treatment with heparin sodium-agarose without any interference removal treatment. Figure 2 As shown, group 1 indicates that the target was not removed; group 2 indicates that only heparin-agarose was used for one target removal treatment; group 3 indicates that heparin-agarose and specific anti-HBV neutralizing antibody magnetic bead conjugate were used for two target removal treatments in sequence.

[0134] It can be seen that the inhibition rate of serum from patients with chronic hepatitis B detected by the method provided in this application (double de-interference treatment with affinity medium heparin sodium-agarose gel and specific anti-HBV surface protein antibodies) is greatly reduced, that is, the specificity of the neutralizing antibody detection method is significantly enhanced, and the results are more accurate.

[0135] The exemplary embodiments of the present application are described above. However, without departing from the essence and scope of the present application, those skilled in the art can modify or improve the exemplary embodiments described in the present application, and the resulting variations or equivalent solutions also fall within the scope of the present application.

Claims

1. A method for detecting neutralizing antibodies of anti-HBV preS1 monoclonal antibody candidate drugs in a biological sample, the method comprising: performing a first treatment on the biological sample using a heparin sodium-agarose affinity medium to remove the target substance in the biological sample; performing a second treatment on the biological sample after the first treatment using a specific anti-HBV envelope protein antibody to further remove the target substance in the biological sample; as well as detecting neutralizing antibodies of the anti-HBV preS1 monoclonal antibody candidate drug in the second-treated biological sample using a competitive ligand binding assay; The targets are HBV / HDV virus particles and viral antigens.

2. The method of claim 1, wherein the first treatment of the biological sample using the heparin sodium-agarose affinity medium comprises: Dilute the biological sample with phosphate buffer at a pH of 7.2 to 7.4 at a 1:1 volume ratio; Mix the diluted biological sample with the pretreated sodium heparin-agarose affinity medium at a volume ratio of 2:1 and incubate at room temperature with shaking at 200 to 500 rpm for at least 16 hours; and The mixture is centrifuged at 800 to 1200 rpm for 2 to 5 minutes, filtered, and the filtrate is collected to obtain the biological sample that has undergone the first treatment.

3. The method of claim 2, wherein the specific anti-HBV envelope protein antibody is coupled to magnetic beads, and performing a second treatment on the biological sample after the first treatment using the specific anti-HBV envelope protein antibody comprises: The first-treated biological sample and the anti-HBV envelope protein antibody coupled to magnetic beads were mixed at a volume ratio of 7:3, and incubated at room temperature with shaking at 600 to 1000 rpm for 1 to 3 hours to obtain a second-treated biological sample.

4. The method of claim 3, wherein detecting neutralizing antibodies of the anti-HBV preS1 monoclonal antibody candidate drug in the second treated biological sample using a competitive ligand binding assay comprises: acid-treating the second-treated biological sample using an acid hydrolysis solution at a volume ratio of 2:1; Affinity capture of acid-treated biological samples was performed using ELISA plates; The affinity-captured biological sample is again subjected to acid dissociation using the acid hydrolysis solution. A neutralization reagent and a detection reagent are added to the acid-dissociated biological sample, and the mixture is incubated at room temperature with shaking at 400 to 800 rpm for 2 to 4 hours to obtain a mixed solution, wherein the volume ratio of neutralization buffer, biological sample, and detection reagent is 3:9:1; Competitive ligand binding assay was used to detect neutralizing antibodies against anti-HBV preS1 monoclonal antibody candidate drugs in the mixture.

5. The method according to claim 4, wherein the acid hydrolysis solution is 300 mM acetic acid or glycine with a pH of 2.5 to 3.5; the neutralization reagent is 1 M Trizma neutralization buffer or Tris buffer with a pH of 9.0 to 10.0; and the detection reagent is a ruthenium-labeled anti-HBV preS1 monoclonal antibody candidate drug.

6. The method of claim 4, wherein the detecting the anti-HBVpreS1 monoclonal antibody candidate drug neutralizing antibody in the mixed solution using a competitive ligand binding assay comprises: The MSD microplate was blocked, washed, and pre-coated with biotinylated HBV preS1; The mixture was added to the pretreated MSD microplate and incubated at room temperature with shaking at 400 to 800 rpm for 2 to 4 hours; as well as The incubated MSD microplate was washed and tested.

7. The method of any one of claims 1 to 6, wherein the biological sample is human serum or plasma.

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