CN114295823A - Acidolysis solution and method for separating rHSA-resistant antibody in monkey blood, and detection method for rHSA-resistant antibody in monkey blood - Google Patents
Acidolysis solution and method for separating rHSA-resistant antibody in monkey blood, and detection method for rHSA-resistant antibody in monkey blood Download PDFInfo
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Abstract
The invention provides an acidolysis solution and a method for separating rHSA-resistant antibody in monkey blood, and a detection method for the rHSA-resistant antibody in the monkey blood, wherein the acidolysis solution for separating the rHSA-resistant antibody in the monkey blood comprises a carboxylated protein, a non-ionic surfactant and an acidic pH buffer, and the buffer range of the pH buffer is pH 4-6. The acidolysis solution provided by the invention provides a weak acid environment by using a pH buffering agent with a buffering range of pH 4-6, the dissociation effect of the nonionic surfactant is mild, the acidolysis solution is used for opening the connection between rHSA and simian blood albumin, and the binding capacity of the carboxylated protein to the anti-rHSA with cations is stronger than that of the simian blood albumin, so that the opening of a compound of the rHSA antibody and the simian blood albumin is promoted. Under the conditions of carboxylated protein, non-ionic surfactant and acidic pH buffer, the antibody can realize better dissociation effect, can keep better anti-rHSA antibody activity, and has higher sensitivity and drug resistance. The detection method of the anti-rHSA antibody in the monkey blood by combining the nano magnetic bead chemiluminescence bridging method disclosed by the invention can realize high-sensitivity detection of the anti-rHSA antibody in the monkey blood and standard reaching of milligram-level drug resistance.
Description
Technical Field
The invention belongs to the field of detection of biological medicine immunogenicity, and particularly relates to an acidolysis solution and method for separating rHSA-resistant antibody in monkey blood and a detection method for the rHSA-resistant antibody in the monkey blood.
Background
Since the first recombinant protein drug is approved to be on the market in 1982, more than 260 therapeutic recombinant protein drugs (including more than 70 therapeutic monoclonal antibody drugs) are widely applied clinically at home and abroad at present and are used for treating more than 230 indications (Nat Biotechnol,2014.32(10): p.992-1000; Health Aff (Millwood),2015.34(2): p.210-9). With the research on therapeutic recombinant protein drugs becoming more and more intensive, the research on the Antibody generated during the administration of the relevant drugs, i.e., the Anti-Drug Antibody (ADA), is becoming more and more important. The generation of anti-drug antibodies can affect the safety and effectiveness of therapeutic recombinant protein drugs, so the detection of ADA is an index which needs to be closely concerned in the clinical medication process.
In the preclinical evaluation stage of recombinant human serum albumin drugs, generally monkeys are used for preclinical safety evaluation, and the scheme of immunogenicity analysis for recombinant human serum albumin (rHSA) is that the recombinant human serum albumin drugs are injected into the monkeys, after the drugs are taken for several months, monkey blood is extracted, whether anti-rHSA antibodies (ADA) are generated in the administered monkey blood is detected, and a rabbit antibody or mouse anti-rHSA is diluted in normal monkey blood to serve as a positive control during detection. However, there is no perfect scheme for detecting the antibody against rHSA in monkey blood, and there is no method for detecting milligram-level drug resistance.
Disclosure of Invention
The applicant found that, because the amino acid composition and protein structure of monkey blood albumin are highly similar to those of human serum albumin, the anti-rHSA positive control added to monkey blood can be combined with the monkey blood albumin, and can not be combined with rHSA any more, because the content of monkey blood albumin in monkey blood is as high as 600mg/ml, so that the conventional acidification method can not separate the anti-rHSA in the positive control from the monkey blood albumin, and can not detect the anti-rHSA in the positive control, and can not quantify the anti-rHSA level in the experimental sample.
In order to solve the problems, the invention provides an acidolysis solution for separating rHSA in monkey blood, which comprises a carboxylated protein, a non-ionic surfactant and an acidic pH buffer, wherein the pH buffer has a buffering range of pH 4-6.
Compared with the prior art, the acidolysis solution provided by the invention provides a weak acid environment by using a pH buffering agent with a buffering range of pH 4-6, the dissociation effect of the nonionic surfactant is mild, the acidolysis solution is used for opening the connection between rHSA and simian blood albumin, and the binding capacity of the carboxylated protein to the anti-rHSA with cations is stronger than that of the simian blood albumin, so that the opening of a compound of the anti-rHSA antibody and the simian blood albumin is promoted. Under the conditions of carboxylated protein, non-ionic surfactant and acidic pH buffer, the antibody can realize better dissociation effect, can keep better anti-rHSA antibody activity, and has higher sensitivity and drug resistance.
Further, the carboxylated protein is carboxylated BSA, the non-ionic surfactant is NP40, and the acidic pH buffer is MES. The carboxylated BSA has a structure similar to that of monkey blood albumin, is rich in negative ions, can better play a role in competitively adsorbing the anti-rHSA with the monkey blood albumin, and the combination of NP40 and MES with the carboxylated BSA can generate a synergistic effect to obtain a better dissociation effect.
Further, the fabric comprises 0.2-5% of carboxylated BSA, 0.01-2% of NP40 and 1.065-10.65% of MES in parts by weight. Further, the fabric comprises 0.5-2% of carboxylated BSA, 0.1-1% of NP40 and 1.065-4.26% of MES in parts by weight. Further, the carboxylated BSA content was 1%, the NP40 content was 0.5%, and the MES content was 2.13%, by weight.
In a second aspect, the present invention provides a method for separating an anti-human albumin antibody from monkey blood, comprising dissociating the monkey blood using the above-mentioned acid hydrolysis solution to obtain a first dissociation solution, and then reacting the first dissociation solution while lowering the pH of the first acid hydrolysis solution to obtain a second dissociation solution.
Compared with the prior art, the method for separating the anti-human albumin antibody in the monkey blood uses the dissociation solution containing the carboxylated protein, the non-ionic surfactant and the acidic pH buffer, the dissociation solution with the buffer range of pH 4-6 is used for carrying out preliminary and mild dissociation on the monkey blood to obtain a first dissociation solution, the pH of the first dissociation solution is reduced for carrying out complete dissociation to obtain a second dissociation solution, and through two-step acidolysis, the anti-rHSA-monkey blood albumin complex is fully dissociated, the activity of the anti-rHSA can be well maintained, and the method has higher sensitivity and drug resistance.
The third aspect of the present invention provides a method for detecting an antibody against rHSA in monkey blood, comprising the steps of,
preparation of positive control samples: using monkey blood containing exogenous anti-rHSA antibody as a positive control group;
a dissociation step: adding any one of the acidolysis solutions into the positive control group to perform primary dissociation reaction;
magnetic bead adsorption and separation: performing magnetic bead adsorption on the positive control group treated by the dissociation step by using rHSA magnetic beads, and performing magnetic bead cleaning and magnetic bead separation to obtain an extracting solution from which the monkey blood albumin is removed;
and (6) performing detection on the machine.
Compared with the prior art, the detection method of the anti-rHSA antibody in the monkey blood disclosed by the invention has the advantages that the protective acidolysis solution is used for carrying out primary dissociation reaction on the positive control group, so that the exogenous anti-rHSA antibody combined with the monkey blood albumin in the positive control group is separated from the monkey blood albumin, the activity of the exogenous anti-rHSA antibody cannot be influenced, then the rHSA magnetic beads (preferably the rHSA magnetic beads with OH groups) are used for enriching and separating the anti-rHSA, and the monkey blood albumin in a sample to be detected is removed, so that the problem that the anti-rHSA antibody in the positive control group cannot be detected is solved, and therefore, the anti-rHSA antibody in the positive control group can be accurately quantified, the experiment group containing the endogenous anti-rHSA can be quantitatively detected, and the detection method has higher sensitivity and drug resistance.
Further, the pH of the positive control group was decreased after the primary dissociation reaction to perform a secondary dissociation reaction. The monkey blood is subjected to primary and mild primary dissociation through the dissociation liquid, and the anti-rHSA-monkey blood albumin complex is subjected to complete dissociation through secondary dissociation in a lower pH environment, and the combination of the two dissociation steps can well maintain the activity of the anti-rHSA while ensuring the full dissociation of the anti-rHSA-monkey blood albumin complex.
Further, the magnetic beads of rHSA comprise OH groups and NH2 groups for labeling rHSA. OH groups are negatively charged in the protective acidolysis solution, and anti-rHSA is positively charged, so that the OH groups have adsorption effect on the anti-rHSA, and can enhance the capacity of competitively extracting the anti-rHSA from monkey blood; the rHSA molecule contains more COOH which can be covalently bonded with NH2 in the presence of EDC, so NH2 is selected as a bonding group for labeling rHSA, and the labeling efficiency is much higher than that of other groups. The magnetic beads containing both OH groups and NH2 groups can generate synergistic effect, and better play the role of enrichment and extraction of the anti-rHSA.
Further, the detection on the computer is carried out by using a nano magnetic bead chemiluminescence bridging method. The nano magnetic bead chemiluminescence bridging method has high specificity and sensitivity, and can further improve the detection effect of the anti-rHSA anti-drug antibody. The nano magnetic bead chemiluminescence bridging method for detecting the anti-rHSA antibody has higher specificity and sensitivity, and has synergistic effect with other steps.
Detailed Description
In order that the invention may be more fully understood, a more particular description of the invention will now be rendered by reference to specific embodiments thereof that are illustrated in the appended drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items. Unless otherwise indicated, each set of reagents used herein is commercially available. The following samples protected the acidified solution, i.e., the acidolysis solution.
Example 1
In the first step, various reagents were prepared.
1. And preparing a sample protection acidizing fluid.
The formula is as follows: 1% COOH-BSA, 0.5% NP40 and 2.13% MES, using ultrapure water as solvent.
2. Preparing the separated magnetic beads.
The rHSA-labeled magnetic beads were prepared by one-step EDC method, using EDC at a concentration of 1% and comprising both OH and NH2 groups.
3. Preparing rHSA-ABEI.
The rHSA-ABEI conjugate was prepared by one-step EDC method, using EDC at 2 mg/ml.
Preparation of rHSA-biotin.
NHS-biotin was used to prepare rHSA-biotin, the molecular ratio of biotin to rHSA was 10: 1.
5. preparation of acidified liquid 1.
Analytically pure acetic acid was diluted to 0.3M with purified water.
6. And (4) preparing acidizing fluid 2.
0.1MpH2.4 buffer was prepared with glycine-HCl.
7. And preparing a bridging mixed solution.
Is prepared by mixing rHSA-biotin and rHSA-ABEI with the same molecular number.
8. And (4) preparing a neutralization solution.
A solution of 1MpH11 was prepared using analytically pure tris and hydrochloric acid.
In the second step, the detection method of the anti-rHSA anti-drug antibody comprises the following steps:
1. preparing a sample: adding rabbit anti-rHSA into monkey blood with a final concentration of 250ng/ml to serve as a positive control group sample; adding rHSA into a 250ng/ml rabbit anti-rHSA sample, wherein the final concentration is 1mg/ml, and using the sample as a drug resistance test group; and monkey blood without rabbit anti-rHSA added was used as a blank substrate.
2. Mixing 15 μ L of sample and 15 μ L of sample protective acidizing fluid, vortexing, and standing at room temperature for 30 minutes, which is a primary dissociation reaction.
3. Adding 200 mul of acidification liquid 1, vortexing uniformly, and standing at room temperature for 20 minutes, which is a secondary dissociation reaction.
4. mu.L of rHSA was added to separate the magnetic beads, followed by blowing, adding 50. mu.L of the neutralizing solution, and vortexing.
5. Then transferring the mixture into a No. 1 deep hole plate, putting down a magnetic rod sleeve by a blending instrument, rotating at the speed of 60 minutes for 40 minutes (separating the magnetic rod sleeve from the magnetic rod), putting down a magnetic rod for absorbing 10 minutes, and rotating for 3 minutes.
6. The beads were transferred to a deep well plate No. 2, 900. mu.L of the wash solution was added, the bar cover was put down for 90 minutes for 6 minutes (the bar cover and the bar were separated), and then the bar was put down for 10 minutes for 5 minutes.
7. Transferring the magnetic beads to a deep-well plate No. 3, adding 310 mu L of acidizing fluid 2, putting down a magnetic rod sleeve for 90 minutes and 8 minutes (separating the magnetic rod sleeve from the magnetic rod), then putting down a magnetic rod for 10 minutes and 14 minutes, and removing the magnetic beads.
8. After 200. mu.L of the acidified sample was allowed to stand at room temperature for 20 minutes, 55. mu.L of the mixed solution and 50. mu.L of the neutralized solution were added and allowed to stand at room temperature for 10 minutes.
9. The above full-automatic molecular luminescence bioanalyzer (rightflare60) tests, and the parameters of the apparatus are set as follows: sample 100. mu.L, SA magnetic beads 20. mu.L "one-step" incubation time 5 minutes.
Example 2
In the first step, various reagents were prepared.
1. And preparing a sample protection acidizing fluid.
The formula is as follows: 1% carboxylated BSA, 0.5% Triton X-100 and 0.1M pH7.2PBS, using ultrapure water as solvent.
2. Preparing the separated magnetic beads.
The rHSA-labeled magnetic beads were prepared by one-step EDC method, using EDC at a concentration of 1% and comprising both OH and NH2 groups.
3. Preparing rHSA-ABEI.
The rHSA-ABEI conjugate was prepared by one-step EDC method, using EDC at 2 mg/ml.
Preparation of rHSA-biotin
NHS-biotin was used to prepare rHSA-biotin, the molecular ratio of biotin to rHSA was 10: 1.
5. preparation of acidified liquid 1.
Analytically pure acetic acid was diluted to 0.3M with purified water.
6. And (4) preparing acidizing fluid 2.
0.1MpH2.4 buffer was prepared with glycine-HCl.
7. And preparing a bridging mixed solution.
Is prepared by mixing rHSA-biotin and rHSA-ABEI with the same molecular number.
8. And (4) preparing a neutralization solution.
A solution of 1MpH11 was prepared using analytically pure tris and hydrochloric acid.
In the second step, the detection method of the anti-rHSA anti-drug antibody comprises the following steps:
1. preparing a sample: adding rabbit anti-rHSA into monkey blood with a final concentration of 250ng/ml to serve as a positive control group sample; adding rHSA into a 250ng/ml rabbit anti-rHSA sample, wherein the final concentration is 1mg/ml, and using the sample as a drug resistance test group; and monkey blood without rabbit anti-rHSA added was used as a blank substrate.
2. Mixing 15 μ L of sample and 15 μ L of sample protective acidizing fluid, vortexing, and standing at room temperature for 30 minutes, which is a primary dissociation reaction.
3. Adding 200 mul of acidification liquid 1, vortexing uniformly, and standing at room temperature for 20 minutes, which is a secondary dissociation reaction.
4. mu.L of rHSA was added to separate the magnetic beads, followed by blowing, adding 50. mu.L of the neutralizing solution, and vortexing.
5. Then transferring the mixture into a No. 1 deep hole plate, putting down a magnetic rod sleeve by a blending instrument, rotating at the speed of 60 minutes for 40 minutes (separating the magnetic rod sleeve from the magnetic rod), putting down a magnetic rod for absorbing 10 minutes, and rotating for 3 minutes.
6. The beads were transferred to a deep well plate No. 2, 900. mu.L of the wash solution was added, the bar cover was put down for 90 minutes for 6 minutes (the bar cover and the bar were separated), and then the bar was put down for 10 minutes for 5 minutes.
7. Transferring the magnetic beads to a deep-well plate No. 3, adding 310 mu L of acidizing fluid 2, putting down a magnetic rod sleeve for 90 minutes and 8 minutes (separating the magnetic rod sleeve from the magnetic rod), then putting down a magnetic rod for 10 minutes and 14 minutes, and removing the magnetic beads.
8. After 200. mu.L of the acidified sample was allowed to stand at room temperature for 20 minutes, 55. mu.L of the mixed solution and 50. mu.L of the neutralized solution were added and allowed to stand at room temperature for 10 minutes.
9. The above full-automatic molecular luminescence bioanalyzer (rightflare60) tests, and the parameters of the apparatus are set as follows: sample 100. mu.L, SA magnetic beads 20. mu.L "one-step" incubation time 5 minutes.
Example 3
In the first step, various reagents were prepared.
1. And preparing a sample protection acidizing fluid.
The formula is as follows: 0.4% COOH-BSA, 0.04% NP40 and 8% MES, using ultrapure water as solvent.
2. Preparing the separated magnetic beads.
The rHSA-labeled magnetic beads were prepared by one-step EDC method, using EDC at a concentration of 1% and comprising both OH and NH2 groups.
3. Preparing rHSA-ABEI.
The rHSA-ABEI conjugate was prepared by one-step EDC method, using EDC at 2 mg/ml.
Preparation of rHSA-biotin
NHS-biotin was used to prepare rHSA-biotin, the molecular ratio of biotin to rHSA was 10: 1.
5. preparation of acidified liquid 1.
Analytically pure acetic acid was diluted to 0.3M with purified water.
6. And (4) preparing acidizing fluid 2.
0.1MpH2.4 buffer was prepared with glycine-HCl.
7. And preparing a bridging mixed solution.
Is prepared by mixing rHSA-biotin and rHSA-ABEI with the same molecular number.
8. And (4) preparing a neutralization solution.
A solution of 1MpH11 was prepared using analytically pure tris and hydrochloric acid.
In the second step, the detection method of the anti-rHSA anti-drug antibody comprises the following steps:
1. preparing a sample: adding rabbit anti-rHSA into monkey blood with a final concentration of 250ng/ml to serve as a positive control group sample; adding rHSA to a rabbit anti-rHSA sample of 250ng/ml to a final concentration of 1mg/ml to serve as a drug resistance test group; and monkey blood without rabbit anti-rHSA added was used as a blank substrate.
2. Mixing 15 μ L of sample and 15 μ L of sample protective acidizing fluid, vortexing, and standing at room temperature for 30 minutes, which is a primary dissociation reaction.
3. Adding 200 mul of acidification liquid 1, vortexing uniformly, and standing at room temperature for 20 minutes, which is a secondary dissociation reaction.
4. mu.L of rHSA was added to separate the magnetic beads, followed by blowing, adding 50. mu.L of the neutralizing solution, and vortexing.
5. Then transferring the mixture into a No. 1 deep hole plate, putting down a magnetic rod sleeve by a blending instrument, rotating at the speed of 60 minutes for 40 minutes (separating the magnetic rod sleeve from the magnetic rod), putting down a magnetic rod for absorbing 10 minutes, and rotating for 3 minutes.
6. The beads were transferred to a deep well plate No. 2, 900. mu.L of the wash solution was added, the bar cover was put down for 90 minutes for 6 minutes (the bar cover and the bar were separated), and then the bar was put down for 10 minutes for 5 minutes.
7. Transferring the magnetic beads to a deep-well plate No. 3, adding 310 mu L of acidizing fluid 2, putting down a magnetic rod sleeve for 90 minutes and 8 minutes (separating the magnetic rod sleeve from the magnetic rod), then putting down a magnetic rod for 10 minutes and 14 minutes, and removing the magnetic beads.
8. After 200. mu.L of the acidified sample was allowed to stand at room temperature for 20 minutes, 55. mu.L of the mixed solution and 50. mu.L of the neutralized solution were added and allowed to stand at room temperature for 10 minutes.
9. The above full-automatic molecular luminescence bioanalyzer (rightflare60) tests, and the parameters of the apparatus are set as follows: sample 100. mu.L, SA magnetic beads 20. mu.L "one-step" incubation time 5 minutes.
Example 4
In the first step, various reagents were prepared.
1. And preparing a sample protection acidizing fluid.
The formula is as follows: 0.5% COOH-BSA, 0.1% NP40 and 1.2% MES, using ultrapure water as solvent.
2. Preparing the separated magnetic beads.
The rHSA-labeled magnetic beads were prepared by one-step EDC method, using EDC at a concentration of 1% and comprising both OH and NH2 groups.
3. Preparing rHSA-ABEI.
The rHSA-ABEI conjugate was prepared by one-step EDC method, using EDC at 2 mg/ml.
Preparation of rHSA-biotin
NHS-biotin was used to prepare rHSA-biotin, the molecular ratio of biotin to rHSA was 10: 1.
5. preparation of acidified liquid 1.
Analytically pure acetic acid was diluted to 0.3M with purified water.
6. And (4) preparing acidizing fluid 2.
0.1MpH2.4 buffer was prepared with glycine-HCl.
7. And preparing a bridging mixed solution.
Is prepared by mixing rHSA-biotin and rHSA-ABEI with the same molecular number.
8. And (4) preparing a neutralization solution.
A solution of 1MpH11 was prepared using analytically pure tris and hydrochloric acid.
In the second step, the detection method of the anti-rHSA anti-drug antibody comprises the following steps:
1. preparing a sample: adding rabbit anti-rHSA into monkey blood with a final concentration of 250ng/ml to serve as a positive control group sample; adding rHSA to a rabbit anti-rHSA sample of 250ng/ml to a final concentration of 1mg/ml to serve as a drug resistance test group; and monkey blood without rabbit anti-rHSA added was used as a blank substrate.
2. Mixing 15 μ L of sample and 15 μ L of sample protective acidizing fluid, vortexing, and standing at room temperature for 30 minutes, which is a primary dissociation reaction.
3. Adding 200 mul of acidification liquid 1, vortexing uniformly, and standing at room temperature for 20 minutes, which is a secondary dissociation reaction.
4. mu.L of rHSA was added to separate the magnetic beads, followed by blowing, adding 50. mu.L of the neutralizing solution, and vortexing.
5. Then transferring the mixture into a No. 1 deep hole plate, putting down a magnetic rod sleeve by a blending instrument, rotating at the speed of 60 minutes for 40 minutes (separating the magnetic rod sleeve from the magnetic rod), putting down a magnetic rod for absorbing 10 minutes, and rotating for 3 minutes.
6. The beads were transferred to a deep well plate No. 2, 900. mu.L of the wash solution was added, the bar cover was put down for 90 minutes for 6 minutes (the bar cover and the bar were separated), and then the bar was put down for 10 minutes for 5 minutes.
7. Transferring the magnetic beads to a deep-well plate No. 3, adding 310 mu L of acidizing fluid 2, putting down a magnetic rod sleeve for 90 minutes and 8 minutes (separating the magnetic rod sleeve from the magnetic rod), then putting down a magnetic rod for 10 minutes and 14 minutes, and removing the magnetic beads.
8. After 200. mu.L of the acidified sample was allowed to stand at room temperature for 20 minutes, 55. mu.L of the mixed solution and 50. mu.L of the neutralized solution were added and allowed to stand at room temperature for 10 minutes.
9. The above full-automatic molecular luminescence bioanalyzer (rightflare60) tests, and the parameters of the apparatus are set as follows: sample 100. mu.L, SA magnetic beads 20. mu.L "one-step" incubation time 5 minutes.
Example 5
In the first step, various reagents were prepared.
1. And preparing a sample protection acidizing fluid.
The formula is as follows: 1% COOH-BSA, 0.5% NP40 and 2.13% MES, using ultrapure water as solvent.
2. Preparing the separated magnetic beads.
The rHSA-labeled magnetic beads were prepared by one-step EDC method, using EDC at a concentration of 1% and comprising both OH and NH2 groups.
3. Preparing rHSA-ABEI.
The rHSA-ABEI conjugate was prepared by one-step EDC method, using EDC at 2 mg/ml.
Preparation of rHSA-biotin
NHS-biotin was used to prepare rHSA-biotin, the molecular ratio of biotin to rHSA was 10: 1.
5. and (4) preparing acidizing fluid.
0.1MpH2.4 buffer was prepared with glycine-HCl.
6. And preparing a bridging mixed solution.
Is prepared by mixing rHSA-biotin and rHSA-ABEI with the same molecular number.
7. And (4) preparing a neutralization solution.
A solution of 1MpH11 was prepared using analytically pure tris and hydrochloric acid.
In the second step, the detection method of the anti-rHSA anti-drug antibody comprises the following steps:
1. preparing a sample: adding rabbit anti-rHSA into monkey blood with a final concentration of 250ng/ml to serve as a positive control group sample; adding rHSA to a rabbit anti-rHSA sample of 250ng/ml to a final concentration of 1mg/ml to serve as a drug resistance test group; and monkey blood without rabbit anti-rHSA added was used as a blank substrate.
2. Mixing 15 mu L of sample and 15 mu L of sample protection acidizing fluid, uniformly vortexing, standing at room temperature for 30 minutes to react, wherein the reaction is a dissociation reaction, and detecting the pH value of a mixed solution of the dissociation reaction.
3. Adding 200 mu L double distilled water, uniformly vortexing, and standing at room temperature for reaction for 20 minutes.
4. mu.L of rHSA was added to separate the magnetic beads, followed by blowing, adding 15. mu.L of the neutralizing solution, and vortexing.
5. Then transferring the mixture into a No. 1 deep hole plate, putting down a magnetic rod sleeve by a blending instrument, rotating at the speed of 60 minutes for 40 minutes (separating the magnetic rod sleeve from the magnetic rod), putting down a magnetic rod for absorbing 10 minutes, and rotating for 3 minutes.
6. The beads were transferred to a deep well plate No. 2, 900. mu.L of the wash solution was added, the bar cover was put down for 90 minutes for 6 minutes (the bar cover and the bar were separated), and then the bar was put down for 10 minutes for 5 minutes.
7. Transferring the magnetic beads to a deep-well plate No. 3, adding 310 mu L of acidizing fluid 2, putting down a magnetic rod sleeve for 90 minutes and 8 minutes (separating the magnetic rod sleeve from the magnetic rod), then putting down a magnetic rod for 10 minutes and 14 minutes, and removing the magnetic beads.
8. After 200. mu.L of the acidified sample was allowed to stand at room temperature for 20 minutes, 55. mu.L of the mixed solution and 50. mu.L of the neutralized solution were added and allowed to stand at room temperature for 10 minutes.
9. The above full-automatic molecular luminescence bioanalyzer (rightflare60) tests, and the parameters of the apparatus are set as follows: sample 100. mu.L, SA magnetic beads 20. mu.L "one-step" incubation time 5 minutes.
Example 6
In the first step, various reagents were prepared.
1. And preparing a sample protection acidizing fluid.
The formula is as follows: 1% COOH-BSA, 0.5% NP40 and 2.13% MES, using ultrapure water as solvent.
2. Preparing the separated magnetic beads.
The rHSA-labeled magnetic beads were prepared by one-step EDC method, using EDC at a concentration of 1% and comprising both OH and COOH groups, bound by COOH of the magnetic beads and NH2 of rHSA.
3. Preparing rHSA-ABEI.
The rHSA-ABEI conjugate was prepared by one-step EDC method, using EDC at 2 mg/ml.
Preparation of rHSA-biotin.
NHS-biotin was used to prepare rHSA-biotin, the molecular ratio of biotin to rHSA was 10: 1.
5. preparation of acidified liquid 1.
Analytically pure acetic acid was diluted to 0.3M with purified water.
6. And (4) preparing acidizing fluid 2.
0.1MpH2.4 buffer was prepared with glycine-HCl.
7. And preparing a bridging mixed solution.
Is prepared by mixing rHSA-biotin and rHSA-ABEI with the same molecular number.
8. And (4) preparing a neutralization solution.
A solution of 1MpH11 was prepared using analytically pure tris and hydrochloric acid.
In the second step, the detection method of the anti-rHSA anti-drug antibody comprises the following steps:
1. preparing a sample: adding rabbit anti-rHSA into monkey blood with a final concentration of 250ng/ml to serve as a positive control group sample; adding rHSA into a 250ng/ml rabbit anti-rHSA sample, wherein the final concentration is 1mg/ml, and using the sample as a drug resistance test group; and monkey blood without rabbit anti-rHSA added was used as a blank substrate.
2. Mixing 15 μ L of sample and 15 μ L of sample protective acidizing fluid, vortexing, and standing at room temperature for 30 minutes, which is a primary dissociation reaction.
3. Adding 200 mul of acidification liquid 1, vortexing uniformly, and standing at room temperature for 20 minutes, which is a secondary dissociation reaction.
4. mu.L of rHSA was added to separate the magnetic beads, followed by blowing, adding 50. mu.L of the neutralizing solution, and vortexing.
5. Then transferring the mixture into a No. 1 deep hole plate, putting down a magnetic rod sleeve by a blending instrument, rotating at the speed of 60 minutes for 40 minutes (separating the magnetic rod sleeve from the magnetic rod), putting down a magnetic rod for absorbing 10 minutes, and rotating for 3 minutes.
6. The beads were transferred to a deep well plate No. 2, 900. mu.L of the wash solution was added, the bar cover was put down for 90 minutes for 6 minutes (the bar cover and the bar were separated), and then the bar was put down for 10 minutes for 5 minutes.
7. Transferring the magnetic beads to a deep-well plate No. 3, adding 310 mu L of acidizing fluid 2, putting down a magnetic rod sleeve for 90 minutes and 8 minutes (separating the magnetic rod sleeve from the magnetic rod), then putting down a magnetic rod for 10 minutes and 14 minutes, and removing the magnetic beads.
8. After 200. mu.L of the acidified sample was allowed to stand at room temperature for 20 minutes, 55. mu.L of the mixed solution and 50. mu.L of the neutralized solution were added and allowed to stand at room temperature for 10 minutes.
9. The above full-automatic molecular luminescence bioanalyzer (rightflare60) tests, and the parameters of the apparatus are set as follows: sample 100. mu.L, SA magnetic beads 20. mu.L "one-step" incubation time 5 minutes.
Example 7
In the first step, various reagents were prepared.
1. And preparing a sample protection acidizing fluid.
The formula is as follows: 1% COOH-BSA, 0.5% NP40 and 2.13% MES, using ultrapure water as solvent.
2. Preparing the separated magnetic beads.
The rHSA-labeled magnetic beads were prepared by one-step EDC method, using EDC at a concentration of 1% and a magnetic bead concentration of 1%, the magnetic beads carrying only NH2 groups.
3. Preparing rHSA-ABEI.
The rHSA-ABEI conjugate was prepared by one-step EDC method, using EDC at 2 mg/ml.
Preparation of rHSA-biotin.
NHS-biotin was used to prepare rHSA-biotin, the molecular ratio of biotin to rHSA was 10: 1.
5. preparation of acidified liquid 1.
Analytically pure acetic acid was diluted to 0.3M with purified water.
6. And (4) preparing acidizing fluid 2.
0.1MpH2.4 buffer was prepared with glycine-HCl.
7. And preparing a bridging mixed solution.
Is prepared by mixing rHSA-biotin and rHSA-ABEI with the same molecular number.
8. And (4) preparing a neutralization solution.
A solution of 1MpH11 was prepared using analytically pure tris and hydrochloric acid.
In the second step, the detection method of the anti-rHSA anti-drug antibody comprises the following steps:
1. preparing a sample: adding rabbit anti-rHSA into monkey blood with a final concentration of 250ng/ml to serve as a positive control group sample; adding rHSA into a 250ng/ml rabbit anti-rHSA sample, wherein the final concentration is 1mg/ml, and using the sample as a drug resistance test group; and monkey blood without rabbit anti-rHSA added was used as a blank substrate.
2. Mixing 15 μ L of sample and 15 μ L of sample protective acidizing fluid, vortexing, and standing at room temperature for 30 minutes, which is a primary dissociation reaction.
3. Adding 200 mul of acidification liquid 1, vortexing uniformly, and standing at room temperature for 20 minutes, which is a secondary dissociation reaction.
4. mu.L of rHSA was added to separate the magnetic beads, followed by blowing, adding 50. mu.L of the neutralizing solution, and vortexing.
5. Then transferring the mixture into a No. 1 deep hole plate, putting down a magnetic rod sleeve by a blending instrument, rotating at the speed of 60 minutes for 40 minutes (separating the magnetic rod sleeve from the magnetic rod), putting down a magnetic rod for absorbing 10 minutes, and rotating for 3 minutes.
6. The beads were transferred to a deep well plate No. 2, 900. mu.L of the wash solution was added, the bar cover was put down for 90 minutes for 6 minutes (the bar cover and the bar were separated), and then the bar was put down for 10 minutes for 5 minutes.
7. Transferring the magnetic beads to a deep-well plate No. 3, adding 310 mu L of acidizing fluid 2, putting down a magnetic rod sleeve for 90 minutes and 8 minutes (separating the magnetic rod sleeve from the magnetic rod), then putting down a magnetic rod for 10 minutes and 14 minutes, and removing the magnetic beads.
8. After 200. mu.L of the acidified sample was allowed to stand at room temperature for 20 minutes, 55. mu.L of the mixed solution and 50. mu.L of the neutralized solution were added and allowed to stand at room temperature for 10 minutes.
9. The above full-automatic molecular luminescence bioanalyzer (rightflare60) tests, and the parameters of the apparatus are set as follows: sample 100. mu.L, SA magnetic beads 20. mu.L "one-step" incubation time 5 minutes.
Comparative example 1
In the first step, various reagents were prepared.
1. And preparing a sample protection acidizing fluid.
The formula is as follows: 1% BSA, 0.5% NP40, and 2.13% MES, using ultrapure water as the solvent.
2. Preparing the separated magnetic beads.
The rHSA-labeled magnetic beads were prepared by one-step EDC method, using EDC at a concentration of 1% and comprising both OH and NH2 groups.
3. Preparing rHSA-ABEI.
The rHSA-ABEI conjugate was prepared by one-step EDC method, using EDC at 2 mg/ml.
Preparation of rHSA-biotin
NHS-biotin was used to prepare rHSA-biotin, the molecular ratio of biotin to rHSA was 10: 1.
5. preparation of acidified liquid 1.
Analytically pure acetic acid was diluted to 0.3M with purified water.
6. And (4) preparing acidizing fluid 2.
0.1MpH2.4 buffer was prepared with glycine-HCl.
7. And preparing a bridging mixed solution.
Is prepared by mixing rHSA-biotin and rHSA-ABEI with the same molecular number.
8. And (4) preparing a neutralization solution.
A solution of 1MpH11 was prepared using analytically pure tris and hydrochloric acid.
In the second step, the detection method of the anti-rHSA anti-drug antibody comprises the following steps:
1. preparing a sample: adding rabbit anti-rHSA into monkey blood with a final concentration of 250ng/ml to serve as a positive control group sample; adding rHSA into a 250ng/ml rabbit anti-rHSA sample, wherein the final concentration is 1mg/ml, and using the sample as a drug resistance test group; and monkey blood without rabbit anti-rHSA added was used as a blank substrate.
2. Mixing 15 μ L of sample and 15 μ L of sample protective acidizing fluid, vortexing, and standing at room temperature for 30 minutes, which is a primary dissociation reaction.
3. Adding 200 mul of acidification liquid 1, vortexing uniformly, and standing at room temperature for 20 minutes, which is a secondary dissociation reaction.
4. mu.L of rHSA was added to separate the magnetic beads, followed by blowing, adding 50. mu.L of the neutralizing solution, and vortexing.
5. Then transferring the mixture into a No. 1 deep hole plate, putting down a magnetic rod sleeve by a blending instrument, rotating at the speed of 60 minutes for 40 minutes (separating the magnetic rod sleeve from the magnetic rod), putting down a magnetic rod for absorbing 10 minutes, and rotating for 3 minutes.
6. The beads were transferred to a deep well plate No. 2, 900. mu.L of the wash solution was added, the bar cover was put down for 90 minutes for 6 minutes (the bar cover and the bar were separated), and then the bar was put down for 10 minutes for 5 minutes.
7. Transferring the magnetic beads to a deep-well plate No. 3, adding 310 mu L of acidizing fluid 2, putting down a magnetic rod sleeve for 90 minutes and 8 minutes (separating the magnetic rod sleeve from the magnetic rod), then putting down a magnetic rod for 10 minutes and 14 minutes, and removing the magnetic beads.
8. After 200. mu.L of the acidified sample was allowed to stand at room temperature for 20 minutes, 55. mu.L of the mixed solution and 50. mu.L of the neutralized solution were added and allowed to stand at room temperature for 10 minutes.
9. The above full-automatic molecular luminescence bioanalyzer (rightflare60) tests, and the parameters of the apparatus are set as follows: sample 100. mu.L, SA magnetic beads 20. mu.L "one-step" incubation time 5 minutes.
Comparative example 2
In the first step, various reagents were prepared.
1. And preparing a sample protection acidizing fluid.
The formula is as follows: 1% COOH-BSA, 0.5% SDS and 2.13% MES, using ultrapure water as solvent.
2. Preparing the separated magnetic beads.
The rHSA-labeled magnetic beads were prepared by one-step EDC method, using EDC at a concentration of 1% and comprising both OH and NH2 groups.
3. Preparing rHSA-ABEI.
The rHSA-ABEI conjugate was prepared by one-step EDC method, using EDC at 2 mg/ml.
Preparation of rHSA-biotin
NHS-biotin was used to prepare rHSA-biotin, the molecular ratio of biotin to rHSA was 10: 1.
5. preparation of acidified liquid 1.
Analytically pure acetic acid was diluted to 0.3M with purified water.
6. And (4) preparing acidizing fluid 2.
0.1MpH2.4 buffer was prepared with glycine-HCl.
7. And preparing a bridging mixed solution.
Is prepared by mixing rHSA-biotin and rHSA-ABEI with the same molecular number.
8. And (4) preparing a neutralization solution.
A solution of 1MpH11 was prepared using analytically pure tris and hydrochloric acid.
In the second step, the detection method of the anti-rHSA anti-drug antibody comprises the following steps:
1. preparing a sample: adding rabbit anti-rHSA into monkey blood with a final concentration of 250ng/ml to serve as a positive control group sample; adding rHSA to a rabbit anti-rHSA sample of 250ng/ml to a final concentration of 1mg/ml to serve as a drug resistance test group; and monkey blood without rabbit anti-rHSA added was used as a blank substrate.
2. Mixing 15 μ L of sample and 15 μ L of sample protective acidizing fluid, vortexing, and standing at room temperature for 30 minutes, which is a primary dissociation reaction.
3. Adding 200 mul of acidification liquid 1, vortexing uniformly, and standing at room temperature for 20 minutes, which is a secondary dissociation reaction.
4. mu.L of rHSA was added to separate the magnetic beads, followed by blowing, adding 50. mu.L of the neutralizing solution, and vortexing.
5. Then transferring the mixture into a No. 1 deep hole plate, putting down a magnetic rod sleeve by a blending instrument, rotating at the speed of 60 minutes for 40 minutes (separating the magnetic rod sleeve from the magnetic rod), putting down a magnetic rod for absorbing 10 minutes, and rotating for 3 minutes.
6. The beads were transferred to a deep well plate No. 2, 900. mu.L of the wash solution was added, the bar cover was put down for 90 minutes for 6 minutes (the bar cover and the bar were separated), and then the bar was put down for 10 minutes for 5 minutes.
7. Transferring the magnetic beads to a deep-well plate No. 3, adding 310 mu L of acidizing fluid 2, putting down a magnetic rod sleeve for 90 minutes and 8 minutes (separating the magnetic rod sleeve from the magnetic rod), then putting down a magnetic rod for 10 minutes and 14 minutes, and removing the magnetic beads.
8. After 200. mu.L of the acidified sample was allowed to stand at room temperature for 20 minutes, 55. mu.L of the mixed solution and 50. mu.L of the neutralized solution were added and allowed to stand at room temperature for 10 minutes.
9. The above full-automatic molecular luminescence bioanalyzer (rightflare60) tests, and the parameters of the apparatus are set as follows: sample 100. mu.L, SA magnetic beads 20. mu.L "one-step" incubation time 5 minutes.
Comparative example 3
In the first step, various reagents were prepared.
1. And preparing a sample protection acidizing fluid.
The formula is as follows: 1% COOH-BSA, 0.5% NP40, and 0.1M acetate buffer, using ultrapure water as a solvent.
2. Preparing the separated magnetic beads.
rHSA-labeled magnetic beads were prepared in a conventional two-step method using glutaraldehyde at a concentration of 10% and 1% and containing both OH and NH2 groups.
3. Preparing rHSA-ABEI.
The rHSA-ABEI conjugate was prepared by one-step EDC method, using EDC at 2 mg/ml.
Preparation of rHSA-biotin
NHS-biotin was used to prepare rHSA-biotin, the molecular ratio of biotin to rHSA was 10: 1.
5. preparation of acidified liquid 1.
Analytically pure acetic acid was diluted to 0.3M with purified water.
6. And (4) preparing acidizing fluid 2.
0.1MpH2.4 buffer was prepared with glycine-HCl.
7. And preparing a bridging mixed solution.
Is prepared by mixing rHSA-biotin and rHSA-ABEI with the same molecular number.
8. And (4) preparing a neutralization solution.
A solution of 1MpH11 was prepared using analytically pure tris and hydrochloric acid.
In the second step, the detection method of the anti-rHSA anti-drug antibody comprises the following steps:
1. preparing a sample: adding rabbit anti-rHSA into monkey blood with a final concentration of 250ng/ml to serve as a positive control group sample; adding rHSA to a rabbit anti-rHSA sample of 250ng/ml to a final concentration of 1mg/ml to serve as a drug resistance test group; and monkey blood without rabbit anti-rHSA added was used as a blank substrate.
2. Mixing 15 μ L of sample and 15 μ L of sample protective acidizing fluid, vortexing, and standing at room temperature for 30 minutes, which is a primary dissociation reaction.
3. Adding 200 mul of acidification liquid 1, vortexing uniformly, and standing at room temperature for 20 minutes, which is a secondary dissociation reaction.
4. mu.L of rHSA was added to separate the magnetic beads, followed by blowing, adding 50. mu.L of the neutralizing solution, and vortexing.
5. Then transferring the mixture into a No. 1 deep hole plate, putting down a magnetic rod sleeve by a blending instrument, rotating at the speed of 60 minutes for 40 minutes (separating the magnetic rod sleeve from the magnetic rod), putting down a magnetic rod for absorbing 10 minutes, and rotating for 3 minutes.
6. The beads were transferred to a deep well plate No. 2, 900. mu.L of the wash solution was added, the bar cover was put down for 90 minutes for 6 minutes (the bar cover and the bar were separated), and then the bar was put down for 10 minutes for 5 minutes.
7. Transferring the magnetic beads to a deep-well plate No. 3, adding 310 mu L of acidizing fluid 2, putting down a magnetic rod sleeve for 90 minutes and 8 minutes (separating the magnetic rod sleeve from the magnetic rod), then putting down a magnetic rod for 10 minutes and 14 minutes, and removing the magnetic beads.
8. After 200. mu.L of the acidified sample was allowed to stand at room temperature for 20 minutes, 55. mu.L of the mixed solution and 50. mu.L of the neutralized solution were added and allowed to stand at room temperature for 10 minutes.
9. The above full-automatic molecular luminescence bioanalyzer (rightflare60) tests, and the parameters of the apparatus are set as follows: sample 100. mu.L, SA magnetic beads 20. mu.L "one-step" incubation time 5 minutes.
Comparative example 4
In the first step, various reagents were prepared.
1. And preparing a sample acidizing fluid.
The formula is as follows: 0.3M acetic acid buffer, using ultrapure water as solvent.
2. ELISA SA coated plates were prepared.
Dissolving 1ug/ml SA protein in 0.05M carbonate buffer solution, adding 100ul per well, and standing at 4-8 deg.C for 16 hr. Blocking with 1% BSA37 ℃ for 1 hour. And (5) spin-drying for later use.
3. rHSA-HRP was prepared.
rHSA-HRP conjugate was prepared by sodium periodate oxidation at a concentration of 0.5 ug/ml.
Preparation of rHSA-biotin
NHS-biotin was used to prepare rHSA-biotin, the molecular ratio of biotin to rHSA was 10: 1.
5. and preparing a bridging mixed solution.
Is prepared by mixing rHSA-biotin and rHSA-HRP with the same molecular number.
6. And (4) preparing a neutralization solution.
A solution of 1MpH11 was prepared using analytically pure tris and hydrochloric acid.
7. Color developing agent is commercially available
8. The terminating agent is commercially available
In the second step, the detection method of the anti-rHSA anti-drug antibody comprises the following steps:
1. preparing a sample: adding rabbit anti-rHSA into monkey blood with a final concentration of 250ng/ml to serve as a positive control group sample; adding rHSA to a rabbit anti-rHSA sample of 250ng/ml to a final concentration of 1mg/ml to serve as a drug resistance test group; and monkey blood without rabbit anti-rHSA added was used as a blank substrate.
2. Mix 30 μ L sample and 270 μ L sample acidified solution, vortex evenly, and stand to react for 30 minutes at room temperature.
3. Add 80. mu.L of neutralization solution to step 2, vortex down, and add 100. mu.L of bridging mixture.
4. And (3) taking 100 mu L of the liquid in the step (3), adding the liquid into an ELISA SA coated plate, and standing at room temperature for reaction for 120 minutes.
5. The plate was washed 5 times, dried by spin, and 100. mu.L of a color-developing agent was added to each well, and reacted at room temperature for 30 minutes.
6. And (5) measuring the absorbance by using a microplate reader.
The experimental data of the above examples and comparative examples are shown in table 1. The following P/N values are the ratios of the mean values of the detection signals of the positive control group and the blank substrate in each experimental group.
Table 1, test results of examples and comparative examples
| Group of | Positive control group (sensitivity P/N) | Drug resistance test group (sensitivity drug resistance P/N) |
| Example 1 | 1.48 | 1.32 |
| Example 2 | 1.25 | 1.09 |
| Example 3 | 1.20 | 1.17 |
| Example 4 | 1.23 | 1.16 |
| Example 5 | 1.20 | 1.15 |
| Example 6 | 1.18 | 1.13 |
| Example 7 | 1.18 | 1.09 |
| Comparative example 1 | 1.08 | 1.05 |
| Comparative example 2 | 1.06 | 0.98 |
| Comparative example 3 | 1.10 | 1.08 |
| Comparative example 4 | 0.97 | 1.01 |
As can be seen from the data in table 1, examples 1 to 7 are significantly better than comparative example 4, because the method for detecting an anti-rHSA antibody in monkey blood according to the present invention separates the monkey blood albumin from the exogenous anti-rHSA antibody bound to the monkey blood albumin in the positive control by performing a primary dissociation reaction on the positive control using a sample protective acidification liquid, and does not affect the activity of the exogenous anti-rHSA antibody. And then, enriching and separating the anti-rHSA by using rHSA magnetic beads with OH groups, and removing monkey blood albumin in a sample to be detected, so that the problem that the anti-rHSA antibody in a positive control group cannot be detected is solved, the anti-rHSA antibody in the positive control group can be accurately quantified, and the quantitative detection can be performed on an experiment group containing endogenous anti-rHSA.
As can be seen from the data in Table 1, examples 1 to 7 are significantly better than comparative examples 1 to 3 because the acidolysis solution of the present invention provides a weak acid environment using a pH buffer with a buffer range of pH4 to 6, the dissociation effect of the nonionic surfactant is milder for opening the connection between rHSA and simian serum albumin, and the binding ability of the carboxylated protein to the cationic anti-rHSA is stronger than that of simian serum albumin, thereby facilitating the opening of the complex between rHSA antibody and simian serum albumin. Under the conditions of carboxylated protein, non-ionic surfactant and acidic pH buffer, the antibody can realize better dissociation effect, can keep better anti-rHSA antibody activity, and has higher sensitivity and drug resistance. The carboxylated protein, the nonionic surfactant and the acidic pH buffer are absent, otherwise, the synergistic effect of the carboxylated protein, the nonionic surfactant and the acidic pH buffer cannot be realized, and the detection result is influenced.
As can be seen from the data in Table 1, in each example, the drug resistance test group added with rHSA of 1mg/ml still could achieve better detection effect. This shows that the drug resistance of the detection method of the invention reaches 1mg/ml under the condition of 250ng/ml sensitivity.
As can be seen from the data in Table 1, the effect of example 1 is better than that of examples 2 to 4 because the most preferred ratio of the components of example 1 is used. In addition, example 1 is better than example 5 in that example 5 only performed one dissociation reaction, and a part of rHSA-monkey blood albumin complex was not dissociated.
As is clear from the data in table 1, example 1 is superior to example 6 in that NH2 is used as the labeling group of rHSA in example 1, and since rHSA molecules contain a large amount of COOH, which can be covalently bonded to NH2 in the presence of EDC, NH2 is selected as the binding group of labeled rHSA, and the labeling efficiency is much higher than that of other groups. Example 1 is better than example 7 because example 1 uses OH groups, which are negatively charged in the liquid of the detection method described above and positively charged, and thus the OH groups have an adsorption effect on rHSA, which enhances competitive extraction of rHSA from monkey blood. Example 1 is better than examples 6-7 because the use of magnetic beads containing both OH groups and NH2 groups provides a synergistic effect and better adsorption against rHSA.
The above description is only for the purpose of illustrating preferred embodiments of the present invention and is not to be construed as limiting the present invention, and it is apparent to those skilled in the art that various modifications and variations can be made in the present invention.
Claims (10)
1. The acidolysis solution for separating the anti-human albumin antibody in the monkey blood is characterized by comprising a carboxylated protein, a non-ionic surfactant and an acidic pH buffer, wherein the buffering range of the pH buffer is 4-6.
2. The acidolysis solution according to claim 1, wherein the carboxylated protein is carboxylated BSA, the nonionic surfactant is NP40, and the acidic pH buffer is MES.
3. The acidolysis solution according to claim 2, wherein the acidolysis solution comprises 0.2 to 5% by weight of the carboxylated BSA, 0.01 to 2% by weight of the NP40, and 1.065 to 10.65% by weight of the MES.
4. The acidolysis solution according to claim 3, which comprises 0.5 to 2% by weight of the carboxylated BSA, 0.1 to 1% by weight of the NP40, and 1.065 to 4.26% by weight of the MES.
5. The acidolysis solution of claim 4, which comprises 1% of the carboxylated BSA, 0.5% of the NP40, and 2.13% of the MES in terms of weight fraction.
6. A method for separating an anti-human albumin antibody from monkey blood, which comprises dissociating the monkey blood with the acid hydrolysis solution according to any one of claims 1 to 5 to obtain a first dissociation solution, and then reacting the first dissociation solution while lowering the pH of the first acid hydrolysis solution to obtain a second dissociation solution.
7. A method for detecting an antibody against rHSA in monkey blood, comprising the steps of:
preparation of positive control samples: using monkey blood containing exogenous anti-rHSA antibody as a positive control group;
a dissociation step: adding the acidolysis solution of any one of claims 1 to 5 to the positive control group to perform a primary dissociation reaction;
magnetic bead adsorption and separation: performing magnetic bead adsorption on the positive control group treated by the dissociation step by using rHSA magnetic beads, and performing magnetic bead cleaning and magnetic bead separation to obtain an extracting solution from which the monkey blood albumin is removed;
and (6) performing detection on the machine.
8. The method of claim 7, wherein the pH of the positive control is lowered after the primary dissociation reaction to perform a secondary dissociation reaction.
9. The method of claim 7, wherein the magnetic rHSA beads contain an OH group and an NH2 group for labeling rHSA.
10. The method of claim 7, wherein said on-machine detection is performed by using a Nanomagnetic bead chemiluminescence bridging method.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111656758.7A CN114295823B (en) | 2021-12-30 | 2021-12-30 | Acidolysis solution and method for separating anti-rHSA in monkey blood and detection method of anti-rHSA anti-drug antibody in monkey blood |
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| US5185264A (en) * | 1990-11-09 | 1993-02-09 | Abbott Laboratories | Diluent buffer and method for diluting an assay component |
| US20130203616A1 (en) * | 2010-04-26 | 2013-08-08 | Merrimack Pharmaceuticals | Assays for anti-drug antibodies in the presence of abundant endogenous protein counterpart of the drug |
| WO2015121784A1 (en) * | 2014-02-11 | 2015-08-20 | Dr. Reddy's Laboratories Limited | Method for detection of anti-rituximab antibodies |
| CN108318680A (en) * | 2018-02-01 | 2018-07-24 | 北京新艾进生物科技有限公司 | A kind of detection method and detection kit of anti-medicine antibody |
| CN111024958A (en) * | 2020-03-11 | 2020-04-17 | 同昕生物技术（北京）有限公司 | Reagent for detecting monoclonal antibody drug and monoclonal antibody drug antibody and application thereof |
| CN111982615A (en) * | 2019-05-23 | 2020-11-24 | 百奥泰生物制药股份有限公司 | Dissociation liquid for dissociating drug/ADA compound and application thereof |
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| US5185264A (en) * | 1990-11-09 | 1993-02-09 | Abbott Laboratories | Diluent buffer and method for diluting an assay component |
| US20130203616A1 (en) * | 2010-04-26 | 2013-08-08 | Merrimack Pharmaceuticals | Assays for anti-drug antibodies in the presence of abundant endogenous protein counterpart of the drug |
| WO2015121784A1 (en) * | 2014-02-11 | 2015-08-20 | Dr. Reddy's Laboratories Limited | Method for detection of anti-rituximab antibodies |
| CN108318680A (en) * | 2018-02-01 | 2018-07-24 | 北京新艾进生物科技有限公司 | A kind of detection method and detection kit of anti-medicine antibody |
| CN111982615A (en) * | 2019-05-23 | 2020-11-24 | 百奥泰生物制药股份有限公司 | Dissociation liquid for dissociating drug/ADA compound and application thereof |
| CN111024958A (en) * | 2020-03-11 | 2020-04-17 | 同昕生物技术（北京）有限公司 | Reagent for detecting monoclonal antibody drug and monoclonal antibody drug antibody and application thereof |
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