CN111024958B - Reagent for detecting monoclonal antibody drug and monoclonal antibody drug antibody and application thereof - Google Patents
Reagent for detecting monoclonal antibody drug and monoclonal antibody drug antibody and application thereof Download PDFInfo
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Abstract
The invention relates to blood concentration detection, in particular to a reagent for detecting monoclonal antibody medicines and monoclonal antibody resisting medicine antibodies and application thereof. The reagent comprises dissociation liquid, wherein the dissociation liquid is glycine buffer solution or acetic acid-sodium acetate buffer solution with the pH value of 2-3 and the concentration of 8-10 mM, and the dissociation liquid contains Tween-20 with the volume fraction of 0.05% -0.1%, 10-15 mu g/ml mouse IgG and/or 10-15 mu g/ml human IgG. The reagent can effectively reduce nonspecific reaction, and solves the problems of strong background signal and low signal-to-noise ratio of the existing acid dissociation reagent.
Description
Technical Field
The invention relates to blood concentration detection, in particular to a reagent for detecting monoclonal antibody medicines and monoclonal antibody resisting medicine antibodies and application thereof.
Background
Protein drugs belong to macromolecular drugs, and the immunogenicity of the drugs is determined by the foreign body properties of the Drug components and structures, so that the human body can generate ADA (anti-Drug antibody) against the drugs. ADA production is related to factors such as drug dosage, treatment frequency, pharmacokinetic individual variation, and the like, in addition to the immunogenicity of the drug. Various factors affect the pharmacokinetics of macromolecular drugs, such as physical constitution, pathological state, medication history, etc., of patients, but ADA production is one of the most important causes. ADA directly affects the therapeutic effect of drugs mainly by blocking the binding of the drugs to the target of action. ADA can alter the pharmacokinetic profile of a drug by increasing drug clearance and decreasing blood levels, resulting in a loss of clinical efficacy. Therefore, reasonable monitoring of drug blood concentration and anti-drug antibody has important guiding significance for improving the treatment effect of patients and reducing the medical cost.
At the end of the last ninety decades, therapeutic anti-TNF- α antibodies were developed (e.g., Infliximab.) chimeric monoclonal antibodies containing 30% murine and 70% human (conserved regions of antibodies consisting of human Fc) capable of activating effector cell function and having good specificity and affinity.
Adalimumab (zameprazole) is a fully humanized antibody, plays an important role in rheumatoid treatment due to high activity and low immunogenicity, and is a main clinical treatment medicament at present. However, in adalimus drug and ADA tests, a significant portion of the drug binds to ADA and it is necessary to perform the assay after dissociating the drug antibody from the drug. The existing dissociation modes are acidic dissociation modes, namely, the drug-bound ADA and the drug are dissociated in the sample under an acidic condition, and then the ADA and the drug concentration are measured after neutralization. Since the pH and concentration (salt ionic strength) of the dissociation reagent and the pH and concentration (salt ionic strength) of the neutralization reagent influence the ADA determination, the signal-to-noise ratio of the reaction system is significantly reduced by the peracid, overbased or high-salt reaction system. In addition, because the hydrophilic-hydrophobic balance of protein is broken in the blood sample under the acidic condition, the nonspecific reaction of the sample after acid dissociation in the measurement of ADA and drug concentration is enhanced, the background signal is increased, and the signal-to-noise ratio of the kit is deteriorated.
Disclosure of Invention
Aiming at the defects in the field, the invention provides the reagent and the detection method for detecting the monoclonal antibody medicines and the anti-monoclonal antibody medicines, which can effectively reduce non-specific reaction and solve the problems of strong background signal and low signal-to-noise ratio of the existing acid dissociation reagent.
In one aspect, the present invention provides a reagent or a kit for detecting blood concentration of a monoclonal antibody drug and an anti-monoclonal antibody drug antibody, comprising a dissociation solution, characterized in that: the dissociation liquid is glycine buffer solution or acetic acid-sodium acetate buffer solution with the pH value of 2-3 and the concentration of 8-10 mM, and contains Tween-20 with the volume fraction of 0.05% -0.1%, mouse IgG with the volume fraction of 10-15 mu g/ml and/or human IgG with the volume fraction of 10-15 mu g/ml.
In a preferred embodiment of the present invention, the reagent or kit further comprises a neutralizing solution and/or an antibody hydrophilization treatment solution; the neutralizing solution contains 10-15% of horse serum by volume fraction; the antibody hydrophilization treatment solution contains 10-15 mg/ml of active ester PEG hydroxyl and/or active ester PEG carboxyl.
In a preferred embodiment of the present invention, the neutralizing solution and/or the antibody hydrophilization treating solution is prepared using a phosphate buffer solution of 20mM, pH 7.4.
In some embodiments of the invention, the dissociation liquid is pH2.5, 10mM glycine buffer solution, which contains Tween-20 with volume fraction of 0.05%, 10 μ g/ml mouse IgG and 10 μ g/ml human IgG; and/or the neutralization solution contains 10% by volume of horse serum; and/or the antibody hydrophilization treatment solution contains 10mg/ml of active ester PEG400 hydroxyl, active ester PEG2k hydroxyl, active ester PEG20k hydroxyl, active ester PEG400 carboxyl, active ester PEG2k carboxyl and/or active ester PEG20k carboxyl.
In some embodiments of the invention, the formulation of the glycine buffer is: 0.75g of glycine is contained in each liter of ultrapure water, and the pH is adjusted to 2.5 by NaOH; the formula of the acetic acid-sodium acetate buffer solution is as follows: 0.57mL of acetic acid is contained in each liter of ultrapure water, and the pH is adjusted to 2.5 by NaOH; the formula of the phosphate buffer solution is as follows: the ultrapure water contained 0.6g of sodium dihydrogen phosphate and 5.8g of disodium hydrogen phosphate per liter, and the pH was adjusted to 7.4.
The monoclonal antibody medicaments provided by the invention comprise, but are not limited to adalimumab, infliximab and Yixepu; the anti-monoclonal antibody drug antibody provided by the invention comprises but is not limited to an anti-adalimumab drug antibody, an anti-infliximab drug antibody and an anti-epsiprep drug antibody.
The application of any reagent or kit in the detection of the blood concentration of the monoclonal antibody medicines and the anti-monoclonal antibody medicines also belongs to the protection scope of the invention.
On the other hand, the invention also provides a method for detecting the blood concentration of the monoclonal antibody medicament, which comprises the following steps:
(1) adding dissociation liquid into serum sample, and mixing; the dissociation liquid is glycine buffer solution or acetic acid-sodium acetate buffer solution with the pH value of 2-3 and the concentration of 8-10 mM, wherein the dissociation liquid contains Tween-20 with the volume fraction of 0.05% -0.1%, mouse IgG with the volume fraction of 10-15 mu g/ml and/or human IgG with the volume fraction of 10-15 mu g/ml;
(2) reacting at 37 deg.C for 5-10 min;
(3) adding the neutralization solution, and uniformly mixing; the neutralization solution contains an acridinium ester labeled anti-monoclonal antibody drug antibody;
(4) adding biotinylated TNF- α, and mixing;
(5) adding streptavidin magnetic beads and mixing uniformly;
(6) reacting at 37 deg.C for 10-15 min;
(7) washing the magnetic beads with a washing solution;
(8) adding exciting liquid, measuring the luminous value, and bringing the luminous value into a standard curve to obtain the concentration of the monoclonal antibody drug in the serum sample.
In still another aspect, the present invention provides a method for detecting the blood concentration of an anti-monoclonal antibody drug antibody, comprising the following steps:
(1) adding dissociation liquid into serum sample, and mixing; the dissociation liquid is glycine buffer solution or acetic acid-sodium acetate buffer solution with the pH value of 2-3 and the concentration of 8-10 mM, wherein the dissociation liquid contains Tween-20 with the volume fraction of 0.05% -0.1%, mouse IgG with the volume fraction of 10-15 mu g/ml and/or human IgG with the volume fraction of 10-15 mu g/ml;
(2) reacting at 37 deg.C for 5-10 min;
(3) adding the neutralization solution, and uniformly mixing; the neutralizing solution contains a monoclonal antibody drug marked by acridinium ester;
(4) adding the biotinylated monoclonal antibody medicine and mixing evenly;
(5) adding streptavidin magnetic beads and mixing uniformly;
(6) reacting at 37 deg.C for 10-15 min;
(7) washing the magnetic beads with a washing solution;
(8) adding exciting liquid, measuring the luminous value, and bringing the luminous value into a standard curve to obtain the concentration of the monoclonal antibody drug-resistant antibody in the serum sample.
In a preferred embodiment of the method of the invention, the neutralizing solution further contains 10-15% by volume of horse serum; and/or in the step (3), the acridinium ester labeled antibody is subjected to hydrophilization treatment; the hydrophilization treatment refers to mixing the acridinium ester labeled antibody with an antibody hydrophilization treatment solution and then placing the mixture at room temperature for reaction for 2 to 3 hours; the antibody hydrophilization treatment solution contains 10-15 mg/ml of active ester PEG hydroxyl and/or active ester PEG carboxyl.
In a preferred embodiment of the method of the present invention, the neutralizing solution and/or the antibody hydrophilization treating solution is prepared using a phosphate buffer solution of 20mM at pH 7.4.
In some embodiments of the methods of the invention, the dissociation solution is a glycine buffer solution with a pH of 2.5 and a concentration of 10mM, and contains Tween-20, 10. mu.g/ml mouse IgG and 10. mu.g/ml human IgG in a volume fraction of 0.05%; and/or the neutralization solution contains 10% by volume of horse serum; and/or the hydrophilization treatment solution contains 10mg/ml of active ester PEG400 hydroxyl, active ester PEG2k hydroxyl, active ester PEG20k hydroxyl, active ester PEG400 carboxyl, active ester PEG2k carboxyl and/or active ester PEG20k carboxyl.
In some embodiments of the method of the present invention, the method for making the standard curve comprises: preparing a monoclonal antibody medicament with gradient concentration or a standard anti-monoclonal antibody medicament antibody, measuring the luminous value of each standard according to the method for detecting the blood concentration of the monoclonal antibody medicament or the method for detecting the blood concentration of the anti-monoclonal antibody medicament, and fitting a double-logarithm straight line between the concentration value of the standard and the luminous value thereof to obtain a standard curve.
In the methods of the invention, the monoclonal antibody drugs include, but are not limited to, adalimumab, infliximab, and Yixepu; the anti-monoclonal antibody drug antibody includes but is not limited to an anti-adalimumab drug antibody, an anti-infliximab drug antibody, and an anti-epsiprep drug antibody.
The invention carries out optimized selection from the aspects of the type, concentration, pH value, protein protective agent, surfactant and the like of the dissociation agent, and successfully screens the dissociation agent which can be used for medicine and ADA detection. Further, in the process of neutralizing the dissociating agent, the hydrophilic-hydrophobic balance of the reaction components in the neutralizing agent is affected by the dissociating agent to cause nonspecific binding, and for this reason, the inventors have performed hydrophilization treatment on the acridinium ester labeled antibody in the neutralizing agent to effectively reduce nonspecific reactions. The dissociation liquid, the neutralization liquid and the hydrophilization treatment liquid selected by the invention are adopted to carry out blood concentration detection on the monoclonal antibody drug or the monoclonal antibody drug antibody, the specificity is up to 98-100%, and the accuracy is up to 97.3%.
Detailed Description
The present invention is further illustrated below by reference to examples, which are to be understood as being illustrative and explanatory only and not limiting the scope of the invention in any way.
Reagents and solutions
Adalimumab (Adalimumab) was purchased from the conderle pharmacia, manufacturer AbbVie Ltd, registration number S20160019. anti-Adalimumab antibody (anti-Adalilimumab) was purchased from Bio-Rad, cat # HCA 207. Infliximab (Infliximab) was purchased from the great Condele pharmacy, company Cilag AG, registration number S20120012. anti-Infliximab drug antibody (anti-Infliximab) was purchased from Bio-Rad under the cat number HCA 233. Yisaipu (recombinant human type ii tumor necrosis factor receptor antibody fusion protein for injection, YSP) was purchased from congle pharmacia, manufacturer: san Sheng Guo Jian Yao (Shanghai) GmbH, national Standard S20050059. Anti-pessulant antibodies were purchased from Bio-Rad, cat # HCA 279.
Phosphate buffer (20 mM PB, ph 7.4): weighing sodium dihydrogen phosphate (NaH)2PO4) (national medicine, 20040799) 0.6g, disodium hydrogen phosphate (Na)2HPO4) (national medicine, 100203008) 5.8 g. 600ml of ultrapure water is added, after complete dissolution, the pH value is adjusted to 7.4, and the volume is adjusted to 1000 ml.
Cleaning solution: 20mM PB, pH 7.4: weighing sodium dihydrogen phosphate (NaH)2PO4) (national medicine, 20040799) 0.6g, disodium hydrogen phosphate (Na)2HPO4) 5.8g (Chinese medicine, 100203008), 8g of sodium chloride (NaCl) (Chinese medicine, 10019308) and 500 mu L of Tween-20 (sigma, 44112). 600ml of ultrapure water is added, after complete dissolution, the pH value is adjusted to 7.4, and the volume is adjusted to 1000 ml.
1% BSA solution: 20mM PB, pH 7.4: weighing sodium dihydrogen phosphate (NaH)2PO4) (national medicine, 20040799) 0.6g, disodium hydrogen phosphate (Na)2HPO4) (national medicine, 100203008) 5.8g, bovine serum albumin (amresco, S12003C 01) 10 g. 600ml of ultrapure water is added, after complete dissolution, the pH value is adjusted to 7.4, and the volume is adjusted to 1000 ml.
Glycine buffer (Gly): at pH2.5, a certain amount of glycine (Chinese medicine, 62011516) (5 mM:0.38g, 10mM:0.75g, 20mM:1.5g, 50mM:3.75g, 100mM:7.5 g) was weighed out and dissolved in 900ml of pure water, after mixing well, the pH was adjusted to 2.5 with NaOH, and the volume was adjusted to 1000 ml.
Acetic acid-sodium acetate (HAC-NaAC): pH2.5, a certain amount of acetic acid (Chinese medicine, 10000208) (5 mM:0.286mL, 10mM:0.57mL, 20mM:1.14mL, 50mM:2.86mL, 100mM:5.71 mL) was measured, and dissolved in 900mL of pure water, and after mixing, the pH was adjusted to 2.5 with NaOH, and the volume was adjusted to 1000 mL.
Citric acid-sodium hydroxide (HCit-NaOH): pH2.5, a certain amount of citric acid monohydrate (Chinese medicine, 10007108) (5 mM:1.0g, 10mM:2.1g, 20mM:4.2g, 50mM:10.5g, 100mM:21 g) was weighed, dissolved in 900ml of pure water, mixed well, adjusted to pH2.5 with NaOH, and made to volume 1000 ml.
Excitation liquid A: 8mL of concentrated hydrochloric acid (mass fraction of 37%) (national medicine, 10011018), 5mL of hydrogen peroxide (mass fraction of 30%) (national medicine, 10011208), and the volume is adjusted to 1000mL by pure water.
Excitation liquid B: NaOH 8g (Chinese medicine, 10019762) and Tween-20 (sigma, 44112) 10mL, and the volume is adjusted to 1000mL with pure water.
The health examination serum samples used in the following examples originated from the Sterculia tumor Hospital. Unless otherwise specified, the reagents used in the following examples are conventional in the art, are commercially available or are formulated according to methods conventional in the art, and are of laboratory pure grade. Unless otherwise specified, the methods used in the following examples are conventional in the art, and reference may be made to the relevant laboratory manuals or manufacturer's instructions.
Example 1 reagent for detection of monoclonal antibody drug and anti-monoclonal antibody drug antibody
(I) preparation of related reagents
1. Biotinylated tumor necrosis factor α (TNF- α -Biotin) was prepared by adding 0.1mg Biotin-LC-NHS (Cat #21338, Thermo) (10 mg/ml, 20mM PB, pH 7.4) to 1ml TNF- α solution (Cat: 10602-HNAE, sinobiological) (1 mg/ml, 20mM PB, pH 7.4), RT (room temperature), 1h, and dialyzing to remove unreacted Biotin.
2. Biotinylated Adalimumab (Adalilimumab-Biotin) was prepared by the same method as biotinylated TNF- α (TNF- α -Biotin).
3. Preparation of acridinium ester labeled antibody: mu.g of ME-DMAE-NHS (HS-11015006, heliosense) (10 mg/ml, DMSO) was added to 1ml of an antibody solution (1 mg/ml, 20mM PB, pH 7.4), and the mixture was dialyzed at RT (room temperature) for 1 hour to remove the unreacted acridinium ester. The method is adopted to prepare the acridinium ester labeled Adalimumab antibody (anti-Adalimumab-MDN) and the acridinium ester labeled Adalimumab antibody (Adalimumab-MDN).
(II) dissociation buffer is preferably
1. Dissociation buffer screening
The type, pH and concentration of the dissociation agent are closely related to the dissociation effect of the drug-conjugated ADA, the neutralization effect of the neutralization reagent and the reactivity of the detection reagent, so that the test compares various dissociation agents, buffers with different pH values and different concentrations, and the prepared dissociation buffer 1-15# and its dissociation effect are shown in Table 1. The result shows that the pH value of the dissociation buffer solution 2# and 7# is changed slightly after the serum sample is added, so that a better dissociation effect can be ensured, and the pH value is close to neutral after the neutralization reagent is added, so that the determination of the medicine and the ADA is facilitated.
TABLE 1 dissociation Effect of different dissociation buffers
| Buffer name | Buffer sequence Number (C) | Concentration of | pH | pH after addition of serum sample (1/10, blood) Clear sample/dissociation buffer) | pH (10/11, Medium) after addition of neutralizing reagent (20 mM PB) And reagent/dissociation buffer + serum sample) |
| Glycine buffer Gly | 1# | 5mM | 2.5 | 4.3 | 7.1 |
| Glycine buffer Gly | 2# | 10mM | 2.5 | 3.1 | 6.8 |
| Glycine buffer Gly | 3# | 20mM | 2.5 | 2.9 | 6.3 |
| Glycine buffer Gly | 4# | 50mM | 2.5 | 2.8 | 4.2 |
| Glycine buffer Gly | 5# | 100mM | 2.5 | 2.7 | 3.5 |
| Acetic acid-sodium acetate HAC-NaAC | 6# | 5mM | 2.5 | 4.3 | 6.9 |
| Acetic acid-sodium acetateHAC-NaAC | 7# | 10mM | 2.5 | 3.2 | 6.6 |
| Acetic acid-sodium acetate HAC-NaAC | 8# | 20mM | 2.5 | 3.1 | 6.1 |
| Acetic acid-sodium acetate HAC-NaAC | 9# | 50mM | 2.5 | 2.8 | 4.0 |
| Acetic acid-sodium acetate HAC-NaAC | 10# | 100mM | 2.5 | 2.7 | 3.3 |
| Citric acid-sodium hydroxide HCit- NaOH | 11# | 5mM | 2.5 | 4.3 | 6.3 |
| Citric acid-sodium hydroxide HCit- NaOH | 12# | 10mM | 2.5 | 3.1 | 5.8 |
| Citric acid-sodium hydroxide HCit- NaOH | 13# | 20mM | 2.5 | 2.9 | 5.2 |
| Citric acid-sodium hydroxide HCit- NaOH | 14# | 50mM | 2.5 | 2.8 | 4.1 |
| Citric acid-sodium hydroxide HCit- NaOH | 15# | 100mM | 2.5 | 2.7 | 3.3 |
Note: the serum sample used in the test was a health examination serum sample from Sterculia tumor hospital. The volume ratio of serum sample to dissociation buffer was 1: 10. The neutralising agent used in this assay was 20mM PB, ph7.4, neutralising agent: dissociation buffer: serum samples were 10:10:1 (by volume).
2. Validation of the Effect of the dissociation buffer
The concentrations of adalimumab and anti-adalimumab drug-resistant antibody in samples P1-P12 were measured by the following methods 1 and 2, respectively, using the preferred dissociation buffers #2 and # 7 in step 1 as sample dissociation solutions and 1% BSA as dissociation solution control, and the effect of the dissociation buffers was examined. The samples P1-P12 were prepared from fetal bovine serum, and the concentrations of adalimumab and anti-adalimumab drug antibodies in each sample are shown in Table 2.
TABLE 2 concentrations of adalimumab and anti-adalimumab drug antibodies in P1-P20 samples
| Anti-Adalilimumab Antibody concentration (Bio- Rad，HCA204） | Anti-Adalilimumab Antibody concentration (Bio- Rad，HCA204） | Anti-Adalilimumab Antibody concentration (Bio- Rad，HCA204） | |
| Adalimumab Concentration of | 0ng/ml | 10ng/ml | 1000ng/ml |
| 0ng/ml | P1 | P2 | P3 |
| 10ng/ml | P4 | P5 | P6 |
| 100ng/ml | P7 | P8 | P9 |
| 1000ng/ml | P10 | P11 | P12 |
Note: the sample is prepared by adding the medicine and the anti-medicine antibody into fetal calf serum (ilex purpurea Hassk, Commodity No. 11011-: (Adalilimumab/Anti-Adalilimumab Antibody): fetal bovine serum 1: 500 (volume ratio).
The method comprises the following steps: method for measuring adalimumab drug blood concentration
(1) Sample dilution: taking 10 mu L of serum sample/calibrator (the calibrator is prepared by adopting fetal bovine serum (four seasons green, product number 11011-;
(2) and (3) incubation: reacting for 6min at 37 ℃;
(3) sample adding: add 100. mu.L of neutralizing solution (containing 0.05. mu.g/ml acridinium ester labeled anti-adalimumab antibody (Biorad, HCA 207) (containing 1% BSA, 20mM PB, pH 7.4) and mix well;
(4) adding sample, adding 100 μ L of 1.0 μ g/ml Biotin labeled TNF- α (TNF- α -Biotin) (1% BSA for dilution), and mixing;
(5) sample adding: adding 20 μ L of 1.0 μ g/ml streptavidin magnetic beads (MP-SA) (GE, cat # 30-1029-61) (1% BSA for dilution), and mixing;
(6) and (3) incubation: reacting for 10min at 37 ℃;
(7) washing: washing with cleaning solution for 3 times;
(8) and (3) detection: adding 200 μ L of exciting solution A and 200 μ L of exciting solution B, respectively, and measuring luminescence value (Nanjing Diegos ACL 180);
(9) and (3) calculating: and performing double-logarithmic straight line fitting (LogX-LogY) on the concentration value of the calibrator and the luminous value of the calibrator to obtain a standard curve, and then substituting the luminous value of the serum sample into the standard curve to obtain the concentration of the adalimumab in the serum sample.
The method 2 comprises the following steps: method for determining anti-adalimumab drug antibody
(1) Sample dilution: taking 10 mu L of serum sample/calibrator (the calibrator is prepared by fetal bovine serum (four seasons green, product number 11011-;
(2) and (3) incubation: reacting for 6min at 37 ℃;
(3) sample adding: adding 100 μ L of neutralizing solution (containing 0.05 μ g/ml acridinium ester-labeled adalimumab) (containing 1% BSA, 20mM PB, pH 7.4), and mixing;
(4) sample adding: adding 100 μ L of 1.0 μ g/ml Biotin-labeled Adalimumab (1% BSA for dilution), and mixing;
(5) sample adding: adding 20 μ L of 1.0 μ g/ml streptavidin magnetic beads (MP-SA) (GE, cat # 30-1029-61) (1% BSA for dilution), and mixing;
(6) and (3) incubation: reacting for 10min at 37 ℃;
(7) washing: washing with cleaning solution for 3 times;
(8) and (3) detection: mu.L of the exciting liquid A and 200. mu.L of the exciting liquid B were added, respectively, to measure the luminescence value (Nanjing Diegos ACL 180).
(9) And (3) calculating: and performing double-logarithmic straight line fitting (LogX-LogY) on the concentration value of the calibrator and the luminous value of the calibrator to obtain a standard curve, and bringing the luminous value of the serum sample into the standard curve to obtain the concentration of the anti-adalimumab drug antibody in the serum sample.
Finally, the deviation between the measurement result and the theoretical result is calculated, the theoretical result is the concentration values of adalimumab and anti-adalimumab drug antibodies in the sample P1-P12 listed in Table 2, the calculation formula is that the deviation = (measurement result/theoretical result-1) × 100%, the results are shown in Table 3, the 2# and 7# buffer systems have good dissociation effects, and the 2# dissociation buffer is preferred in consideration of the volatility of acetic acid in the acetic acid buffer.
TABLE 3 deviation of the measured results from the theoretical results
| Adalimumab | Adalimumab | Adalimumab | Anti-Adalimumab | Anti-Adalimumab | Anti-Adalimumab | |
| 1%BSA | 2# | 7# | 1%BSA | 2# | 7# | |
| P1 | 3.5% | - | - | 4.5% | - | - |
| P2 | 5.0% | - | - | -4.2% | -2.8% | -4.2% |
| P3 | 2.6% | - | - | 2.5% | -5.2% | -3.5% |
| P4 | 3.4% | 4.5% | 4.1% | -4.6% | - | - |
| P5 | -90.3% | -3.1% | -5.4% | -80.7% | -3.2% | -3.5% |
| P6 | -98.2% | -8.1% | -8.2% | -6.8% | -5.7% | -3.1% |
| P7 | 2.5% | 3.8% | 3.6% | 3.4% | - | - |
| P8 | -9.6% | -4.7% | -5.2% | -85% | -5.6% | -5.2% |
| P9 | -96.9% | -6.8% | -7.3% | -25% | -6.7% | -6.2% |
| P10 | 2.6% | 2.3% | 2.5% | 5.6% | - | - |
| P11 | -1.2% | -7.8% | -7.1% | -93.2% | -8.1% | -8.0% |
| P12 | -86.3% | -9.2% | -9.3% | -87.5% | -5.8% | -6.3% |
(III) surfactant type is preferred
Under the condition of acid dissociation, the acid hydrolysis balance of the protein in the serum is broken, and the protein is separated out and agglomerated, so that the non-specific binding of the reaction is enhanced. The surfactant can improve the solubility of the protein under acidic conditions to reduce non-specific reactions. Candidate surfactants and their use concentrations are shown in table 4. The surfactants were dissolved in 2# dissociation buffer solutions, respectively, to obtain 6 kinds of dissociation solutions. The surfactant was screened by measuring the positive rate of Adalimumab and the positive rate of Anti-Adalimumab (the positive rates are percentages determined as positive samples) in 100 serum samples of the physical examination by the method 1 and the method 2, respectively, using 6 types of dissociation solutions with the 2# dissociation buffer as the dissociation solution control. The results show that a volume fraction of 0.05% Tween-20 can improve nonspecific reactions (Table 4).
TABLE 4 surfactant screening
| Name of reagent | Manufacturer/goods number | Concentration of use | Adalilimumab Positive Rate | Anti-Adalilimumab positive rate |
| 2# dissociation buffer | - | - | 23% | 17% |
| Tween-20 | Sigma，44112 | 0.05%（v/v） | 21% | 15% |
| Triton X-100 | Sigma, V900502 | 0.01%（v/v） | 22% | 16% |
| PEG20000 | Sigma, 818897 | 0.1%（w/w） | 23% | 16% |
| Hydroxypropyl methylcellulose-type II | Aladdin, 100G/H108822 | 0.1%（w/v，g/ml） | 23% | 17% |
| Hydroxypropyl methylcellulose-type II | Aladdin, 100G/H108823 | 0.1%（w/v，g/ml） | 24% | 17% |
| Hydroxypropyl methylcellulose-type II | Aladdin, 100G/H108824 | 0.1%（w/v，g/ml） | 24% | 17% |
Note: dissolving surfactant in #2 dissociation buffer solution to obtain dissociation solution.
The protein protecting agent in the dissociation solution is preferably
The protein protective agent can wrap the protein, and the change of acid to the protein structure is reduced to a certain extent, so that the non-specific reaction is reduced. Candidate protein protectants and their use concentrations are shown in table 5. The protein protective agents were dissolved in 2# dissociation buffer (containing 0.05% Tween-20) to obtain 6 dissociation solutions. Using these 6 types of dissociation liquids, the positive rates of Adalilimumab and Anti-Adalilimumab (the positive rates are percentages of samples determined to be positive) in 100 serum samples of the physical examination were measured according to the methods 1 and 2, respectively, and different protein protective agents were screened. The results showed that the dissociation solution contained 10. mu.g/ml mouse IgG and 10. mu.g/ml human IgG to improve the nonspecific reaction (Table 5).
TABLE 5 protein protectant screening in dissociation fluids
| Name of reagent | Manufacturer/goods number | Concentration of use | Adalilimumab Positive Rate | Anti-Adalilimumab positive rate |
| BSA | amresco，S12003C01 | 1.0%（w/v，g/ml） | 21% | 14% |
| Casein protein | Sigma，C6554 | 0.5%（w/v，g/ml） | 23% | 16% |
| Sucrose | Chinese medicine 10021418 | 3%（w/v，g/ml） | 23% | 15% |
| Mouse IgG | Qitai, MG01 | 10μg/ml | 19% | 12% |
| Human IgG | Qitai, HG01 | 10μg/ml | 17% | 13% |
| Mouse IgG + human IgG | MG01+ HG01, Qitai | 10. mu.g/ml of each | 17% | 12% |
Note: the protein protective agent is dissolved by 2# dissociation buffer solution containing 0.05% (v/v) Tween-20 to prepare dissociation solution.
(V) the protein protective agent in the neutralization solution is preferably
Candidate protein protectants and their use concentrations are shown in table 6. Protein protective agents were dissolved in 20mM PB (pH7.4) containing the corresponding antibodies, respectively, to prepare 5 kinds of neutralization solutions containing different protein protective agents. Using 5 kinds of the neutralizing solutions, respectively, using a 2# dissociation buffer solution (containing 0.05% Tween-20, 10. mu.g/ml mouse IgG, 10. mu.g/ml human IgG) as the dissociation solution, the positive rate of Adalilimumab and the positive rate of Anti-Adalilimumab (the positive rates refer to the percentage of samples judged to be positive) in 100 serum samples for health examination were measured, and the protein protective agents in the neutralizing solutions were screened. The results show that 10% by volume of horse serum contained in the neutralization solution is effective in improving nonspecific reactions (Table 6).
TABLE 6 protein protectant screening in neutralization
| Name of reagent | Manufacturer/goods number | Concentration of use | Adalilimumab Positive Rate | Anti-Adalilimumab positive rate |
| Casein protein | Sigma，C6554 | 0.5%（w/v，g/ml） | 23% | 17% |
| Mouse IgG | Qitai, MG01 | 10μg/ml | 18% | 15% |
| Human IgG | Qitai, HG01 | 10μg/ml | 19% | 14% |
| Fetal bovine serum | Chinese holly leaf, 11011- | 10%（v/v） | 11% | 9% |
| Horse serum | Hyclone，SH30074.03 | 10%（v/v） | 8% | 6% |
Note: 1. the dissociation solution is 2# dissociation buffer containing 0.05% (v/v) Tween-20, 10. mu.g/ml mouse IgG and 10. mu.g/ml human IgG. 2. The protein protective agent was dissolved in 20mM PB (pH 7.4) containing the corresponding antibody to prepare a neutralized solution.
(VI) Effect of neutralizing Acridinium ester reactant in solution for hydrophilization treatment
1.1 ml of 1mg/ml acridine ester labeled antibody (20 mM PB, pH 7.4) and 10mg/ml active ester PEG carboxyl (PS 2-HCM-400/2k/20k, Peng large organisms, the addition amount is 0.1mg \0.5mg \5mg respectively) or active ester PEG hydroxyl (PS 2-HO-400/2k/20k, Peng large organisms, the addition amount is 0.1mg \0.5mg \5mg respectively) solution (20 mM PB, pH 7.4) are mixed, and the mixture is reacted for 2 hours at room temperature and stored for later use.
2. Using 2# dissociation buffer (containing 0.05% Tween-20, 10. mu.g/ml murine IgG, 10. mu.g/ml human IgG) as dissociation liquid, 20mM PB (pH7.4) containing 10% (v/v) horse serum as neutralization liquid, diluting the acridine ester labeled antibody (MDN-Anti-Adalimunab-PEG-OH or MDN-Anti-Adalimunab-PEG-COOH, MDN-Adalimunab-PEG-OH or MDN-Adalimunab-PEG-COOH) prepared in step 1 with the neutralization liquid to a final concentration of 0.05. mu.g/ml, and determining the positive rate of Adalimunab and the positive rate of Anti-Adalimunab in 100 serum samples of the health examination samples (the positive rates refer to the percentage of positive samples) according to the above methods 1 and 2, respectively.
As shown in Table 7, the acridine ester labeled antibodies treated with the carboxyl group or the hydroxyl group of the active ester PEG400 had better effects, had smaller molecular weight differences, and preferably had smaller molecular weights such as the hydroxyl group of the active ester PEG400 in view of the use cost.
TABLE 7 screening of antibody hydrophilization treatment reagents
| Name of reagent | Manufacturer/goods number | Adalilimumab Positive Rate | Anti-Adalilimumab positive rate |
| Active ester PEG400 carboxyl | PS2-HCM-400 | 0% | 1% |
| Active ester PEG2k carboxyl | PS2-HCM-2k | 0% | 0% |
| Active ester PEG20k carboxyl | PS2-HCM-20k | 1% | 2% |
| Active ester PEG400 hydroxy | PS2-HO-400 | 0% | 0% |
| Active ester PEG2k hydroxy | PS2-HO-2k | 0% | 0% |
| Active ester PEG20k hydroxy | PS2-HO-20k | 1% | 1% |
Note: 1. 2# dissociation buffer containing 0.05% (v/v) Tween-20, 10. mu.g/ml mouse IgG, 10. mu.g/ml human IgG was used as the dissociation solution. 2. 20mM PB (pH7.4) containing 10% (v/v) horse serum was used as a neutralizing solution. 3. The above acridinium ester-labeled antibody was diluted with a neutralizing solution to a final concentration of 0.05. mu.g/ml.
The preferred reagents for monoclonal antibody drug and anti-monoclonal antibody drug antibody detection by the above assay are as follows:
(1) dissociation liquid: 10mM glycine buffer containing 0.05% (v/v) Tween-20, 10. mu.g/ml murine IgG, 10. mu.g/ml human IgG, pH 2.5.
(2) Neutralizing liquid: containing 10% (v/v) horse serum, 20mM PB, pH 7.4.
(3) Antibody hydrophilization treatment solution: 10mg/ml active ester PEG400 hydroxy solution (20 mM PB, pH 7.4).
The concentrations of adalimumab and anti-adalimumab drug-resistant antibody in the P1-P20 samples were measured according to methods 1 and 2 using the preferred dissociation solution, neutralization solution and antibody hydrophilization treatment solution, respectively, and the deviation of the measurement results from the theoretical results was calculated, the theoretical results are the concentration values of adalimumab and anti-adalimumab drug-resistant antibody in the P1-P12 samples listed in table 2, the calculation formula: deviation = (measurement result/theoretical result-1) × 100%, the results are shown in table 8.
TABLE 8 deviation of the measured results from the theoretical results
| Adalimumab | Anti-Adalimumab | |
| P1 | - | - |
| P2 | - | -2.9% |
| P3 | - | -3.2% |
| P4 | 4.3% | - |
| P5 | -3.1% | -3.2% |
| P6 | -7.4% | -3.5% |
| P7 | 3.6% | - |
| P8 | -4.2% | -5.4% |
| P9 | -6.1% | -6.5% |
| P10 | 2.7% | - |
| P11 | -7.1% | -8.3% |
| P12 | -8.7% | -5.4% |
Example 2 application of preferred reagents in the assay of Infliximab (Infliximab) and anti-Infliximab drug antibody (anti-Infliximab)
1. Preparation of reagents
The preparation methods of biotinylated tumor necrosis factor α (TNF- α -Biotin), biotinylated Infliximab (Infliximab-Biotin), acridinium ester-labeled anti-Infliximab antibody (anti-Infliximab-MDN), and acridinium ester-labeled Infliximab (Infliximab-MDN) were the same as those described in example 1 (A).
Hydrophilization treatment methods for acridinium ester-labeled Infliximab (Infliximab-MDN) and acridinium ester-labeled anti-Infliximab antibody (anti-Infliximab-MDN) (i.e., MDN-Infliximab-PEG400-OH and MDN-anti-Infliximab-PEG 400-OH) were as described in (VI) of example 1.
2. Method for measuring blood concentration of infliximab medicament
(1) Sample dilution: taking 10 mu L of sample/calibrator (the calibrator is prepared by adopting fetal bovine serum (ilex purpurea Hassk, Cat. No. 11011-;
(2) and (3) incubation: reacting for 6min at 37 ℃;
(3) sample adding: adding 100 μ L of neutralizing solution (containing 0.05 μ g/ml of hydrophilic acridinium ester labeled anti-Infliximab drug antibody, MDN-anti-Infliximab-PEG 400-OH) (Bio-Rad, HCA 233) (containing 10% horse serum, 20mMPB, pH 7.4), and mixing;
(4) adding sample, adding 100 μ L of 1.0 μ g/ml biotinylated TNF- α (TNF- α -Biotin) (diluted by 1% BSA), and mixing;
(5) sample adding: adding 20 μ L of 1.0 μ g/ml streptavidin magnetic beads (MP-SA) (GE, cat # 30-1029-61) (1% BSA for dilution), and mixing;
(6) and (3) incubation: reacting for 10min at 37 ℃;
(7) washing: washing with cleaning solution for 3 times;
(8) and (3) detection: adding 200 μ L of exciting solution A and 200 μ L of exciting solution B, respectively, and measuring luminescence value (Nanjing Diegos ACL 180);
(9) and (3) calculating: and performing double-logarithmic straight line fitting (LogX-LogY) on the concentration value of the calibrator and the luminous value of the calibrator to obtain a standard curve, and then substituting the luminous value of the serum sample into the standard curve to obtain the concentration of the infliximab in the sample.
3. Method for determining anti-infliximab drug antibody
(1) Sample dilution: taking 10 mu L of sample/calibrator (the calibrator is prepared by adopting fetal bovine serum (ilex purpurea Hassk, cat # 11011-;
(2) and (3) incubation: reacting for 6min at 37 ℃;
(3) sample adding: adding 100 μ L of neutralizing solution (containing 0.05 μ g/ml hydrophilized acridine ester labeled Infliximab, MDN-Infliximab-PEG 400-OH) (containing 10% horse serum, 20mM PB, pH 7.4), and mixing;
(4) sample adding: adding 100 μ L of 1.0 μ g/ml biotinylated Infliximab (Infliximab-Biotin) (1% BSA for dilution), and mixing;
(5) sample adding: adding 20 μ L of 1.0 μ g/ml streptavidin magnetic beads (MP-SA) (GE, cat # 30-1029-61) (1% BSA for dilution), and mixing;
(6) and (3) incubation: reacting for 10min at 37 ℃;
(7) washing: washing with cleaning solution for 3 times;
(8) and (3) detection: adding 200 μ L of exciting solution A and 200 μ L of exciting solution B, respectively, and measuring luminescence value (Nanjing Diegos ACL 180);
(9) and (3) calculating: and performing double-logarithmic straight line fitting (LogX-LogY) on the concentration value of the calibrator and the luminous value of the calibrator to obtain a standard curve, and bringing the luminous value of the serum sample into the standard curve to obtain the concentration of the anti-infliximab drug antibody in the sample.
4. Sample assay
The positive rates of infliximab and anti-infliximab drug antibodies in 100 serum samples of the physical examination (the positive rates refer to the percentage of samples determined to be positive) were determined according to the methods described in examples 2 and 3.
According to the methods described in examples 2 and 3, the concentrations of Infliximab and anti-Infliximab drug antibody in the positive samples (P1 and P2) were measured, respectively, and the deviation between the measurement result and the theoretical result was calculated.the calculation formula: deviation = (measurement result/theoretical result-1) × 100%, wherein the positive sample P1 contained 10ng/ml Infliximab and 1000ng/ml anti-Infliximab drug antibody (anti-Infliximab, Bio-Rad, HCA 213), and the positive sample P2 contained 1000ng/ml Infliximab and 10ng/ml anti-Infliximab drug antibody (anti-Infliximab, Bio-Rad, HCA 213). the positive samples (P1 and P2) were prepared by adding bovine serum (bovine serum albumin) and anti-Infliximab drug to fetal bovine serum (japanese green, product No. 11011: (Infliximab/anti-Infliximab ratio: 500).
The results show that the serum samples of the health examination are negative, and the deviation of the measurement results of the positive samples P1 and P2 from the theoretical results is within 10%.
Example 3 use of preferred reagents in assays for Yissepu and anti-Yissepu antibodies
1. Preparation of reagents
Biotinylated tumor necrosis factor α (TNF- α -Biotin), biotinylated pessary, acridinium ester-labeled anti-pessary antibody (anti-YSP-MDN), and acridinium ester-labeled pessary antibody (YSP-MDN) were prepared as described in example 1 (I).
The hydrophilization treatment methods for acridinium ester-labeled pessary (YSP-MDN) and acridinium ester-labeled anti-pessary antibody (anti-YSP-MDN) (i.e., MDN-YSP-PEG400-OH and MDN-anti-YSP-PEG 400-OH) were the same as those described in (VI) of example 1.
2. Method for measuring blood concentration of Yisaipu medicament
(1) Sample dilution: taking 10 mu L of sample/calibrator (the calibrator is prepared by adopting fetal bovine serum (ilex purpurea Hassk, Cat. No. 11011-;
(2) and (3) incubation: reacting for 6min at 37 ℃;
(3) sample adding: add 100. mu.L of neutralizing solution (containing 0.05. mu.g/ml of hydrophilized acridinium ester labeled anti-pessimian drug antibody, MDN-anti-YSP-PEG 400-OH) (Biorad, HCA 279) (containing 10% horse serum, 20mM PB, pH 7.4) and mix well;
(4) adding sample, adding 100 μ L of 1.0 μ g/ml biotinylated TNF- α (TNF- α -Biotin) (diluted by 1% BSA), and mixing;
(5) sample adding: adding 20 μ L of 1.0 μ g/ml streptavidin magnetic beads (MP-SA) (GE, cat # 30-1029-61) (1% BSA for dilution), and mixing;
(6) and (3) incubation: reacting for 10min at 37 ℃;
(7) washing: washing with cleaning solution for 3 times;
(8) and (3) detection: adding 200 μ L of exciting solution A and 200 μ L of exciting solution B, respectively, and measuring luminescence value (Nanjing Diegos ACL 180);
(9) and (3) calculating: and performing double-logarithmic straight line fitting (LogX-LogY) on the concentration value of the calibrator and the luminous value of the calibrator to obtain a standard curve, and then substituting the luminous value of the serum sample into the standard curve to obtain the concentration of the Yisaipu in the sample.
3. Determination method of anti-Yisaipu drug antibody
(1) Sample dilution: taking 10 mu L of sample/calibrator (the calibrator is prepared by adopting fetal bovine serum (ilex purpurea Hassk, Cat. No. 11011-;
(2) and (3) incubation: reacting for 6min at 37 ℃;
(3) sample adding: adding 100 μ L of neutralizing solution (containing 0.05 μ g/ml hydrophilized acridinium ester labeled pesoprop, MDN-YSP-PEG 400-OH) (containing 10% horse serum, 20mM PB, pH 7.4), and mixing;
(4) sample adding: adding 100 μ L of 1.0 μ g/ml biotinylated Yisaipu (YSP-Biotin) (1% BSA for dilution), and mixing;
(5) sample adding: adding 20 μ L of 1.0 μ g/ml streptavidin magnetic beads (MP-SA) (GE, cat # 30-1029-61) (1% BSA for dilution), and mixing;
(6) and (3) incubation: reacting for 10min at 37 ℃;
(7) washing: washing with cleaning solution for 3 times;
(8) and (3) detection: adding 200 μ L of exciting solution A and 200 μ L of exciting solution B, respectively, and measuring luminescence value (Nanjing Diegos ACL 180);
(9) and (3) calculating: and performing double-logarithmic straight line fitting (LogX-LogY) on the concentration value of the calibrator and the luminous value of the calibrator to obtain a standard curve, and then substituting the luminous value of the serum sample into the standard curve to obtain the concentration of the anti-protest drug antibody in the sample.
4. Sample assay
The positive rate of goserel and the positive rate of anti-goserel drug antibody (the positive rate is the percentage of positive samples) in 100 serum samples of the health examination were measured according to the methods described in examples 2 and 3.
The concentrations of the antibodies to the prebiotic and anti-prebiotic drugs in the positive samples (P1 and P2) were measured, respectively, as described in examples 2 and 3, and the deviation from the theoretical result was calculated, using the equation of deviation = (measurement/theoretical result-1) × 100% where the positive sample P1 contained 10ng/ml prebiotic and 1000ng/ml anti-prebiotic drug antibodies (anti-YSP, Bio-Rad, HCA 277), the positive sample P2 contained 1000ng/ml prebiotic and 10ng/ml anti-prebiotic drug antibodies (anti-YSP, Bio-Rad, HCA 277), and the positive sample was prepared by adding the prebiotic and anti-prebiotic drugs to fetal bovine serum (holly, cat # 11011-.
The results show that the serum samples of the health examination are negative, and the deviation of the measurement results of the positive samples P1 and P2 from the theoretical results is within 10%.
Claims (13)
1. The reagent or the kit for detecting the blood concentration of the monoclonal antibody medicament and the anti-monoclonal antibody medicament antibody comprises dissociation liquid and is characterized in that: the dissociation liquid is glycine buffer solution or acetic acid-sodium acetate buffer solution with the pH value of 2-3 and the concentration of 8-10 mM, wherein the dissociation liquid contains Tween-20 with the volume fraction of 0.05% -0.1%, mouse IgG with the volume fraction of 10-15 mug/ml and human IgG with the volume fraction of 10-15 mug/ml;
also includes neutralizing liquid and antibody hydrophilization treating liquid; the neutralizing solution contains 10-15% of horse serum by volume fraction; the antibody hydrophilization treatment solution contains 10-15 mg/ml of active ester PEG hydroxyl and/or active ester PEG carboxyl.
2. The reagent or kit of claim 1, wherein: the neutralizing solution and/or the antibody hydrophilization treating solution are prepared by using a phosphate buffer solution of 20mM and pH 7.4.
3. The reagent or kit of claim 2, wherein: the dissociation solution is pH2.5, 10mM glycine buffer solution, which contains Tween-20 with volume fraction of 0.05%, 10 mug/ml mouse IgG and 10 mug/ml human IgG; and/or the neutralization solution contains 10% by volume of horse serum; and/or the antibody hydrophilization treatment solution contains 10mg/ml of active ester PEG400 hydroxyl, active ester PEG2k hydroxyl, active ester PEG20k hydroxyl, active ester PEG400 carboxyl, active ester PEG2k carboxyl and/or active ester PEG20k carboxyl.
4. The reagent or kit of claim 3, wherein: the formula of the glycine buffer solution is as follows: 0.75g of glycine is contained in each liter of ultrapure water, and the pH is adjusted to 2.5 by NaOH; the formula of the acetic acid-sodium acetate buffer solution is as follows: 0.57mL of acetic acid is contained in each liter of ultrapure water, and the pH is adjusted to 2.5 by NaOH; the formula of the phosphate buffer solution is as follows: the ultrapure water contained 0.6g of sodium dihydrogen phosphate and 5.8g of disodium hydrogen phosphate per liter, and the pH was adjusted to 7.4.
5. The reagent or kit according to any one of claims 1 to 4, wherein: the monoclonal antibody medicines comprise adalimumab, infliximab and Yixepu; the anti-monoclonal antibody drug antibody comprises an anti-adalimumab drug antibody, an anti-infliximab drug antibody and an anti-epsiprep drug antibody.
6. A method for detecting the plasma concentration of a monoclonal antibody drug for non-diagnostic and therapeutic purposes, comprising the steps of:
(1) adding dissociation liquid into serum sample, and mixing; the dissociation liquid is glycine buffer solution or acetic acid-sodium acetate buffer solution with the pH value of 2-3 and the concentration of 8-10 mM, wherein the dissociation liquid contains Tween-20 with the volume fraction of 0.05% -0.1%, mouse IgG with the volume fraction of 10-15 mug/ml and human IgG with the volume fraction of 10-15 mug/ml;
(2) reacting at 37 deg.C for 5-10 min;
(3) adding the neutralization solution, and uniformly mixing; the neutralization solution contains an acridinium ester labeled anti-monoclonal antibody drug antibody and horse serum with the volume fraction of 10-15%; the acridinium ester marked anti-monoclonal antibody drug antibody is subjected to hydrophilization treatment; the hydrophilization treatment refers to mixing an acridinium ester marked anti-monoclonal antibody drug antibody and an antibody hydrophilization treatment solution and then placing the mixture at room temperature for reaction for 2 to 3 hours; the antibody hydrophilization treatment solution contains 10-15 mg/ml of active ester PEG hydroxyl and/or active ester PEG carboxyl;
(4) adding biotinylated TNF- α, and mixing;
(5) adding streptavidin magnetic beads and mixing uniformly;
(6) reacting at 37 deg.C for 10-15 min;
(7) washing the magnetic beads with a washing solution;
(8) adding exciting liquid, measuring the luminous value, and bringing the luminous value into a standard curve to obtain the concentration of the monoclonal antibody drug in the serum sample.
7. A method for detecting the blood concentration of anti-monoclonal antibody drug antibodies for non-diagnostic and therapeutic purposes, comprising the steps of:
(1) adding dissociation liquid into serum sample, and mixing; the dissociation liquid is glycine buffer solution or acetic acid-sodium acetate buffer solution with the pH value of 2-3 and the concentration of 8-10 mM, wherein the dissociation liquid contains Tween-20 with the volume fraction of 0.05% -0.1%, mouse IgG with the volume fraction of 10-15 mug/ml and human IgG with the volume fraction of 10-15 mug/ml;
(2) reacting at 37 deg.C for 5-10 min;
(3) adding the neutralization solution, and uniformly mixing; the neutralization solution contains an acridinium ester labeled monoclonal antibody drug and horse serum with the volume fraction of 10-15%; the acridinium ester marked monoclonal antibody medicine is subjected to hydrophilization treatment; the hydrophilization treatment refers to mixing the acridinium ester marked monoclonal antibody medicament with an antibody hydrophilization treatment solution and then placing the mixture at room temperature for reaction for 2 to 3 hours; the antibody hydrophilization treatment solution contains 10-15 mg/ml of active ester PEG hydroxyl and/or active ester PEG carboxyl;
(4) adding the biotinylated monoclonal antibody medicine and mixing evenly;
(5) adding streptavidin magnetic beads and mixing uniformly;
(6) reacting at 37 deg.C for 10-15 min;
(7) washing the magnetic beads with a washing solution;
(8) adding exciting liquid, measuring the luminous value, and bringing the luminous value into a standard curve to obtain the concentration of the monoclonal antibody drug-resistant antibody in the serum sample.
8. The method according to claim 6 or 7, characterized in that: the neutralizing solution and/or the antibody hydrophilization treating solution are prepared by using a phosphate buffer solution of 20mM and pH 7.4.
9. The method of claim 8, wherein: the dissociation solution is pH2.5, 10mM glycine buffer solution, which contains Tween-20 with volume fraction of 0.05%, 10 mug/ml mouse IgG and 10 mug/ml human IgG; and/or the neutralization solution contains 10% by volume of horse serum; and/or the hydrophilization treatment solution contains 10mg/ml of active ester PEG400 hydroxyl, active ester PEG2k hydroxyl, active ester PEG20k hydroxyl, active ester PEG400 carboxyl, active ester PEG2k carboxyl and/or active ester PEG20k carboxyl.
10. The method of claim 6, wherein: the manufacturing method of the standard curve comprises the following steps: preparing a monoclonal antibody drug standard substance with gradient concentration, measuring the luminous value of each standard substance according to the method for detecting the blood concentration of the monoclonal antibody drug, and fitting a double-logarithm straight line between the concentration value of the standard substance and the luminous value thereof to obtain a standard curve.
11. The method of claim 7, wherein: the manufacturing method of the standard curve comprises the following steps: preparing a standard substance of the anti-monoclonal antibody drug antibody with gradient concentration, measuring the luminous value of each standard substance according to the method for detecting the blood concentration of the anti-monoclonal antibody drug antibody, and fitting a double-logarithmic straight line between the concentration value of the standard substance and the luminous value thereof to obtain a standard curve.
12. The method of claim 6, wherein: the monoclonal antibody drugs comprise adalimumab, infliximab and Yixepu.
13. The method of claim 7, wherein: the anti-monoclonal antibody drug antibody comprises an anti-adalimumab drug antibody, an anti-infliximab drug antibody and an anti-epsiprep drug antibody.
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