Method for detecting anti-HBV preS1 monoclonal antibody candidate drug neutralizing antibody

Abstract

The invention provides a method for detecting an anti-HBV preS1 monoclonal antibody candidate drug neutralizing antibody. The method provided by the invention comprises the step of sequentially treating a biological sample by utilizing a magnetic bead conjugate of an affinity medium heparin sodium-agarose and a specific anti-HBV envelope protein antibody so as to remove a target object. According to the method provided by the invention, the interference of a target object can be well solved, so that the anti-HBV preS1 monoclonal antibody candidate drug neutralizing antibody can be reliably, specifically and sensitively detected.

Description

Technical Field

[0001] The present application belongs to the field of biological detection, and specifically relates to a new 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 partially double-stranded circular DNA, encoding 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 belongs to the Hepadnaviridae family. It is a replication-deficient RNA virus. It is a HBV satellite virus that replicates with the help of HBV surface protein as its envelope protein. Therefore, hepatitis D virus infection is mostly seen in people infected with hepatitis B virus. The only antigenic component of HDV is hepatitis D virus antigen (HDAg).

[0004] PreS1 protein (preS1) is an important component of the hepatitis B virus capsid protein. It contains liver cell membrane receptors and plays an important role in HBV / HDV infection of liver cells and the body's immune response. It can specifically bind to the sodium ion-sodium taurocholate cotransporter (NTCP) on the liver cell 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 can effectively bind to preS1 protein (preS1) and block HBV / HDV from further infecting liver cells.

[0005] However, there are a large number of free targets (HBV / HDV virus particles and viral antigens) in the serum of patients with chronic hepatitis B and chronic hepatitis D virus infection. For example, the hepatitis B surface antigen (HBsAg) in the serum of patients with chronic hepatitis B is positive. Due to the presence of these virus particles and viral antigens such as HBsAg, the specificity of the method for detecting neutralizing antibodies of anti-HBV preS1 monoclonal antibody candidate drugs is poor, which interferes with the detection of neutralizing antibodies. Therefore, it is urgent to develop a suitable method to remove the target, so as to improve the specificity of the method for detecting neutralizing antibodies of anti-HBV preS1 monoclonal antibody candidate drugs. Summary of the invention

[0006] In view of the above problems, after repeated exploration, the inventors of the present application have 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 in the present application 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 biological sample that has been subjected to the first treatment using a magnetic bead conjugate of a specific anti-HBV envelope protein antibody to further remove the target in the biological sample; and detecting the neutralizing antibodies of the anti-HBV preS1 monoclonal antibody candidate drug in the biological sample that has been subjected to the second treatment 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, 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 in 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: treating the second treated biological sample with acid 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, the biological sample, and the detection reagent is 3:9:1; and detecting neutralizing antibodies of 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 anti-HBVpreS1 monoclonal antibody candidate drug neutralizing antibodies in biological samples using a competitive ligand binding assay includes: pre-treating the MSD microplate by blocking, washing, coating with biotin-labeled HBVpreS1, and washing again; adding the mixed solution to the pre-treated 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 solve 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-HBVpreS1 monoclonal antibody candidate drugs.

[0017] Specifically, the method of the present application uses affinity medium heparin sodium-agarose gel and specific anti-HBV envelope protein antibody magnetic bead conjugates to immunodeplete biological samples in sequence, which can more efficiently remove the interference of the target. On the one hand, the affinity medium heparin sodium-agarose gel can bind to the free target through electrostatic adsorption, that is, the interaction of positive and negative charges, and initially reduce the interference of the target; on the other hand, the specific anti-HBV envelope protein antibody magnetic bead conjugate can further specifically bind to the free target in the biological sample, thereby further removing the target in the sample and reducing the interference of the target again. The sample after the free target is fully and specifically adsorbed by affinity is then pre-treated with affinity capture-acid dissociation (ACE), and then the competitive ligand binding assay (CLBA) is used on the MSD platform to detect the neutralizing antibodies of the anti-HBV preS1 monoclonal antibody candidate drug in human serum, which can more sensitively and specifically detect the neutralizing antibodies of the anti-HBV preS1 monoclonal antibody candidate drug, and significantly improve 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 the present application, which is about 200 ng / mL; wherein "Run" represents one analysis plate.

[0019] Figure 2 The comparison of inhibition rates of serum (CHBS) of chronic hepatitis B patients with different treatments is shown, wherein 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, the neutralizing antibodies will bind to the labeled drug (detection reagent). If there is free target in the biological sample, it will bind to the detection reagent, causing a false positive. False positive results can be avoided by fully and specifically depleting the free target with affinity immunodepletion.

[0021] Regarding the detection of neutralizing antibodies of anti-HBV preS1 monoclonal antibody candidate drugs, since the target in the subject's body has a great interference 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 on the premise of establishing and optimizing the pre-treatment of affinity capture-acid dissociation (ACE).

[0022] First, we tried to use heparin sodium-agarose affinity medium alone to remove target interference, and found that there was still a lot of interference after one treatment. Although the target interference was removed by increasing the number of treatments, the sensitivity of the method also decreased. Therefore, on the basis of using heparin sodium-agarose affinity medium to remove target interference, we further added magnetic bead conjugates of specific anti-HBV envelope protein antibodies to remove endogenous targets. The results showed that heparin sodium-agarose treatment once and magnetic bead conjugates of specific anti-HBV envelope protein antibodies once could completely remove 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 incubated 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 incubated 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-immunodeplete free targets (HBV / HDV virus particles and viral antigens), greatly removes the interference of endogenous free targets, and significantly improves 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 method for anti-HBV preS1 monoclonal antibody candidate drugs provided in the present application is as follows: the biological sample (including quality control / verification sample / test sample) is treated and adsorbed once with a heparin-agarose affinity medium (Heparin Bestarose HP) pre-filled in a microporous filter plate or filter tube to remove free target substances, and after recovering the filtered solution, a magnetic bead conjugate of a specific anti-HBV envelope protein antibody is added to further affinity-immunoexhaust free target substances; the depleted sample is pre-treated with affinity capture-acid dissociation (ACE), and then mixed with a neutralizing agent and a detection agent in an incubation plate for oscillation reaction; thereafter, it is added to an MSD microplate pre-coated with a target antigen peptide (HBV preS1), incubated with oscillation at room temperature, and after washing the plate, 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 monoclonal antibody candidate drug" refers to a monoclonal antibody candidate drug that is developed to bind to HBV

[0029] Antibodies that specifically bind to preS1.

[0030] As used herein, "anti-HBV preS1 monoclonal antibody candidate drug 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 make the neutralizing antibodies free; incubating the acid-treated biological sample with a drug-coated plate can have a similar purification effect. In some embodiments, the acid treatment of the biological sample can reduce the matrix effect. In some embodiments, the acid treatment of the biological sample can improve drug tolerance. In some embodiments, the acid treatment of the biological sample can reduce the amount of sample used.

[0032] As used herein, "affinity capture" refers to capture based on the principle of specific binding of antibodies and antigens, such as using specific antigens to capture antibodies. In some embodiments of the present application, the anti-HBV preS1 monoclonal antibody candidate drug neutralizing antibody in the sample after acid treatment binds to the anti-HBV preS1 monoclonal antibody (equivalent to the antigen) coated on the microplate under neutral pH conditions, thereby achieving affinity capture of the neutralizing antibody.

[0033] As used herein, "acid dissociation" refers to dissociating the drug-anti-drug neutralizing antibody complex in the 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 candidate drug and the neutralizing antibody coated on the microplate, so that the neutralizing antibody originally bound to the drug is dissociated into a free state. In some embodiments of the present application, the anti-HBV preS1 monoclonal antibody candidate drug neutralizing antibody obtained by "affinity capture" is dissociated from the anti-HBV preS1 monoclonal antibody candidate drug-anti-HBV preS1 monoclonal antibody candidate drug neutralizing antibody complex, and the dissociated neutralizing antibody can reflect the neutralizing antibody level through the signal of the detection reagent after incubation with the detection reagent.

[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, and the like.

[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 and centrifugation of the liquid biological sample using a heparin-agarose affinity medium; adding a magnetic bead conjugate of a specific anti-HBV envelope protein antibody to the filtered liquid biological sample for further affinity immunodepletion of free targets; performing affinity capture-acid dissociation (ACE) pretreatment 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 volume of the affinity medium sodium heparin-agarose added is 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 of 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 centrifugal 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 value 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 pH 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, the detecting of anti-HBV preS1 monoclonal antibody candidate drug neutralizing antibodies in the biological sample comprises using a competitive ligand binding assay using an MSD platform to determine the anti-HBV preS1 monoclonal antibody candidate drug neutralizing antibodies in the liquid biological sample.

[0056] As an exemplary embodiment, the method for detecting neutralizing antibodies of anti-HBV preS1 monoclonal antibody candidate drugs comprises the following steps: 1) Sample pretreatment The sample was pre-treated 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).

[0057] 2) First target removal treatment of samples

[0058] 2.1) Pretreatment of heparin sodium-agarose affinity medium

[0059] Balance of Heparin-agarose affinity medium (Heparin beads, Heparin bestarose HP): Take an appropriate amount (depending on the actual amount required for the experiment) of Heparin beads, add about 5 times the volume of water, centrifuge at 1800 to 2100 rpm for 2 to 5 minutes, discard the water, repeat 2 times, and finally resuspend with 1 x PBS (pH 7.2~7.4) to the original volume of the affinity medium.

[0060] 2.2) First target removal treatment of samples

[0061] Treat the sample with the pretreated affinity medium: add the pretreated Heparin beads in step 2.1) to the DV Free 96-well filter plate, centrifuge at 800 to 1200 rpm for 3 to 6 minutes, remove the liquid by centrifugation, add the diluted sample obtained in step 1), and incubate at room temperature with shaking at 350 rpm overnight for at least 16 hours; the volume ratio of the added Heparin beads to the sample is 1:2.

[0062] After the 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.

[0063] 3) Second target removal treatment of samples

[0064] 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, and then add the sample obtained in step 2.2) after the first treatment, the volume ratio of the added anti-HBV envelope protein antibody magnetic bead conjugate to the sample treated for the first time is 3:7. After sealing the plate with a sealing film, incubate at room temperature with shaking at 600 to 900 rpm, and stand in a magnetic separator for at least 2 minutes before proceeding to the next experiment.

[0065] 4) Affinity capture-acid dissociation treatment

[0066] 4.1) Acid treatment of samples: Take the sample obtained in step 3) after the second treatment, add acid hydrolysis solution (300mM acetic acid, pH 3.0), and acid hydrolyze at room temperature at 200 to 500 rpm for 30 to 40 minutes for acid treatment, and the volume ratio of the added sample to the acid hydrolysis solution is 1:2.

[0067] 4.2) Pretreatment of ELISA plates: Coating: Coat the ELISA plate capture reagent working solution of CBS (anti-HBV preS1 monoclonal antibody working solution) at 80 to 120 μL / well on the ELISA plate and incubate the plate at 2 to 8° C. overnight.

[0068] Washing: Wash the ELISA plate three times with 1 x PBST at no less than 300 μL / well.

[0069] Blocking: Add 300 μL / well of I-Block Blocking Buffer to the ELISA plate and block at room temperature with shaking at 500 to 800 rpm for at least 2 hr.

[0070] Washing: Wash the ELISA plate three times with 1 x PBST at no less than 300 μL / well.

[0071] 4.3) ELISA plate affinity capture of samples: Add neutralization buffer (1 M Trizma, H 9.5) to the ELISA plate in advance; add the pretreated sample obtained in 4.1) into the wells according to the sample plate diagram, the volume ratio of the added neutralization buffer to the pretreated sample volume is 4:150, seal the plate with a sealing film, and incubate at room temperature with shaking at 200 to 500 rpm for at least 16 hours.

[0072] 4.4) Washing the plate: Take out the ELISA plate on which the neutralizing antibody (Nab) has been captured, and wash the plate three times with 1xPBST at a volume of no less than 300 μL / well.

[0073] 4.5) Acid dissociation of samples: 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.

[0074] 4.6) Preparation of sample mixture: Prepare the sample mixture (Master Mix working solution): add neutralization buffer (1 M Trizma, pH 9.5) to the round-well polypropylene plate, then add the dissociated sample obtained in step 4.5) to neutralize, then add the prepared detection reagent working solution (anti-HBV preS1 monoclonal antibody labeled with ruthenium) to the neutralization solution, mix well, and incubate at room temperature at 500 to 800 rpm for 2 to 4 hours. The volume ratio of the added neutralization buffer, sample and detection reagent working solution is 3:9:1.

[0075] 5) Sample testing

[0076] 5.1) Pretreatment of MSD microplate

[0077] Blocking: Add 120 to 160 μL / well of MSD microplate blocking solution (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.

[0078] Wash the plate: Wash the plate three times with at least 300 μL / well of washing buffer (1xPBST).

[0079] MSD microplate capture: Coat the prepared MSD microplate capture reagent working solution (HBV preS1 working solution) in the MSD microplate at 40 to 60ul / well and incubate at 35 ~ 39℃ for about 1 hour.

[0080] Wash the plate: Wash the plate three times with at least 300 μL / well of washing buffer (1 x PBST).

[0081] 5.2) Sample testing

[0082] Transfer plate and add samples: According to the plate map, add 40 to 60ul / 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.

[0083] Wash the plate: Wash the plate three times with at least 300 μL / well of washing buffer (1xPBST).

[0084] Detection: Add 140 to 160 μL / well of the prepared MSD Read Buffer T (2x) working solution into the MSD microplate. Place the MSD microplate in the MESO QUICKPLEX SQ120 (set the equipment parameters according to the manufacturer) for detection within 15 min. Example

[0085] 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 non-critical parameters that can be changed or modified to produce substantially the same result.

[0086] 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.

[0087] Example 1

[0088] This example provides a method for detecting neutralizing antibodies of anti-HBV preS1 monoclonal antibody candidate drugs using secondary target removal treatment.

[0089] The specific steps are as follows: 1. Sample pretreatment: All samples were diluted with 1xPBS (pH 7.2-7.4) at a volume ratio of 1:1 (e.g. 110 μL sample + 110 μL PBS) for pretreatment.

[0090] 2. Heparin-agarose (Heparin bestarose HP) balance: Take an appropriate amount (depending on the actual amount required for the experiment) of heparin-agarose, then add about 5 times the volume of water, then centrifuge, discard the water, add about 5 times the volume of 1× PBS, wash twice (add about 5 times the volume of 1× PBS, then centrifuge, and discard the water), and finally resuspend to the original volume with 1× PBS (pH 7.2~7.4).

[0091] 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 min, remove the liquid by filtration, add 200 μL / well of diluted serum sample (single well loading), shake at 350 rpm overnight at room temperature for at least 16 hours.

[0092] 4. Coating: Coat the ELISA plate with the prepared ELISA plate capture reagent working solution, 100ul / well, and incubate at 2~8℃ overnight for at least 16 hours.

[0093] The ELISA plate capture reagent working solution was prepared by the following method: 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 about 20 µg / mL of the ELISA plate capture reagent working solution.

[0094] 5. Wash the plate: Wash the ELISA plate three times with washing buffer (1 x PBST) at a volume of no less than 300 μL / well.

[0095] 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 hr.

[0096] 7. Sample collection: After incubation, centrifuge the DV Free 96-well filter plate at 1000 rpm for 4 minutes and collect the samples after centrifugation.

[0097] 8. Samples treated with magnetic bead conjugates of specific anti-HBV preS1 antibodies: 1) The magnetic bead conjugate concentrate of the specific anti-HBV preS1 antibody was vortexed for about 20 seconds to resuspend the magnetic beads; 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 1 x PBS (pH 7.2 ~ 7.4) (about 4 times the volume) and gently shake and wash, let it stand in the magnetic separator 2 / 15 for at least 2 minutes, and then remove the washing solution; 3) Add about 5 times 1 x PBS (pH 7.2 ~ 7.4) and fully shake and resuspend to obtain the magnetic bead conjugate working solution of specific anti-HBVpreS1 antibody for standby use; 4) The resuspended specific anti-HBV preS1 antibody magnetic bead conjugate working solution was fully shaken again and added to a non-adsorbed 96-well polypropylene plate according to the plate map, 45 μL / well; 5) Add the sample treated with heparin-agarose, 105 μL / well; seal the plate with a sealing film, resuspend the magnetic beads and shake at about 800 rpm at room temperature for 1.5 hrs ± 10 min for immunodepletion; let stand in the magnetic separator 96 I for at least 2 min before proceeding to the next step of the experiment.

[0098] 9. Sample acid treatment: Take the sample treated with magnetic bead conjugate and treat it with acid solution I (300mM HAc, pH 3.0) at a volume ratio of 1:2, and shake at 350 rpm at room temperature for 30 to 40 min.

[0099] 10. Wash the plate: Wash the ELISA plate three times with washing buffer (1 x PBST) at a volume of no less than 300 μL / well.

[0100] 11. ELISA plate loading capture: Add Trizma neutralization buffer (1 M pH 9.5) to the ELISA plate in advance, 4 μL / well; add all pre-treated samples into the wells according to the sample loading plate diagram, load each well, 150 μL / well; seal the plate with a sealing film, and incubate at room temperature with shaking at 350 rpm overnight (at least 16 hr).

[0101] 12. Wash the plate: Take out the ELISA plate with captured neutralizing antibody (Nab) and wash the plate three times with washing buffer (1xPBST) at a volume of no less than 300 μL / well.

[0102] 13. Sample acid dissociation: Add acid dissociation solution II (300mM HAc, pH 3.5) to the ELISA plate, 110 μL / well, and shake at room temperature for 30 to 40 minutes.

[0103] 14. Master Mix Working Solution Mixing: Add 30 μL / well Trizma neutralization buffer (1 M, pH 9.5) to a round-well polypropylene plate, then add 90 μL / well of the acid-dissociated sample to neutralize, then add 10 μL / well of the prepared detection reagent working solution to the neutralization solution, mix well, and incubate at room temperature at 600 rpm for 2.5 hr ± 10 min.

[0104] 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 detection reagent working solution with a final concentration of approximately 65 ng / mL.

[0105] 15. MSD microplate blocking: 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 about 600 rpm for at least 1 hr in the dark.

[0106] 16. Wash the plate: Wash the plate three times with at least 300 μL / well of washing buffer (1 x PBST).

[0107] 17. MSD microplate capture: Coat the prepared MSD microplate capture reagent working solution in the MSD microplate, 50ul / well, and incubate at 35 ~ 39℃ for 1 hr ± 5 min.

[0108] The MSD microplate capture reagent working solution was prepared by the following method: the MSD microplate capture reagent concentrate (HBVpreS1) was diluted with DPBS to a final concentration of approximately 50 ng / mL of the MSD microplate capture reagent working solution.

[0109] 18. Wash the plate: Wash the plate three times with at least 300 μL / well of washing buffer (1 x PBST).

[0110] 19. Transfer plate and add samples: According to the plate map, add the Master Mix prepared in step 14 to the MSD microplate, 50 μL / well, add samples in duplicate, and incubate at room temperature with shaking at 600 rpm for 2 hr ± 10 min.

[0111] 20. Wash the plate: Wash the plate three times with at least 300 μL / well of washing buffer (1 x PBST).

[0112] 21. Detection: Add the prepared MSD Read Buffer T (2x) working solution into the MSD microplate at 150 μL / well. Place the MSD microplate into the plate reader MESO QUICKPLEX SQ120 (model: 1300, purchased from MesoScale Discovery) for detection within 15 min.

[0113] MSD Read Buffer T (2x) working solution was prepared by the following method: MSD Read Buffer T (4×) (purchased from Meso Scale Discovery) was mixed with ultrapure water in a 1:1 ratio. After preparation, the cap was tightened and used as soon as possible.

[0114] The sensitivity of the above method for detecting neutralizing antibodies of anti-HBV preS1 monoclonal antibody candidate drugs is approximately 200 ng / mL.

[0115] Specifically, the sensitivity curve sets 8 concentration points of neutralizing antibody samples, and the concentrations are 4000, 2000, 1000, 500, 250, 125, 62.5, and 31.25 ng / mL from high to low.

[0116]

[0117] The negative control sample was mixed healthy human serum without neutralizing antibodies.

[0118] Figure 1 It is a 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).

[0119] 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 conjugates were used for two target removal treatments in sequence.

[0120] It can be seen that the inhibition rate of serum from patients with chronic hepatitis B detected by the method provided in the present application (affinity medium sodium heparin-agarose gel and double de-interference treatment with 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.

[0121] 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 sodium heparin-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 The anti-HBV preS1 monoclonal antibody candidate drug neutralizing antibodies in the second treated biological sample are detected using a competitive ligand binding assay.

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 a biological sample that has undergone the first treatment.

3. The method according to claim 1 or 2, wherein the specific anti-HBV envelope protein antibody is coupled to magnetic beads, and the second treatment of the biological sample subjected to the first treatment using the specific anti-HBV envelope protein antibody comprises: The biological sample subjected to the first treatment was mixed with the anti-HBV envelope protein antibody coupled to the magnetic beads 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 biological sample subjected to the second treatment.

4. The method according to any one of claims 1 to 3, wherein detecting the anti-HBV preS1 monoclonal antibody candidate drug neutralizing antibody in the second treated biological sample using a competitive ligand binding assay comprises: treating the biological sample subjected to the second treatment with acid using an acid hydrolysis solution at a volume ratio of 2:1; Affinity capture of acid-treated biological samples using ELISA plates; The affinity captured biological sample is again subjected to acid dissociation using the acid hydrolysis solution, and a neutralizing agent and a detection agent 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 the neutralizing buffer, the biological sample and the detection agent is 3:9:1; Competitive ligand binding assay was used to detect neutralizing antibodies of 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 according to claim 4 or 5, wherein the method of detecting the anti-HBV preS1 monoclonal antibody candidate drug neutralizing antibody in the mixed solution by competitive ligand binding assay comprises: The MSD microplate was blocked, washed, and pre-coated with biotin-labeled HBV preS1; The mixture was added to the pre-treated 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 detected.

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

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