CN116626298A

CN116626298A - Reagent for detecting omalizumab drug-resistant antibody

Info

Publication number: CN116626298A
Application number: CN202310889774.3A
Authority: CN
Inventors: 闫倩; 刘洋洋; 高雪; 刘运龙; 欧伦
Original assignee: Junke Zhengyuan Tianjin Biomedical Technology Co ltd; United Power Pharma Tech Co ltd
Current assignee: Junke Zhengyuan Tianjin Biomedical Technology Co ltd; United Power Pharma Tech Co ltd
Priority date: 2023-07-19
Filing date: 2023-07-19
Publication date: 2023-08-22

Abstract

The application provides a reagent for detecting an omalizumab drug-resistant antibody in a sample, a preparation method and related application thereof. The reagent contains a capture reagent capable of binding to the omalizumab drug-resistant antibody, a detection reagent for detection, and an IgE binding molecule. The reagent can well solve the influence of endogenous IgE interference on the immunogenicity detection of the injection omalizumab.

Description

Reagent for detecting omalizumab drug-resistant antibody

Technical Field

The application relates to the field of biological detection, in particular to a reagent for detecting an amazuki drug-resistant antibody in a sample, especially human blood sample, capable of removing endogenous IgE interference.

Background

One major challenge faced in the detection of drug-resistant antibodies (ADA) against omalizumab-type drugs for injection is the interference of target molecules (IgE). Although the serum IgE content is the lowest in 5 immunoglobulins, the serum IgE content of healthy people is about 250-800ng/mL, but the serum IgE of patients with allergic constitution and allergic diseases such as asthma, allergic rhinitis, urticaria and the like can be obviously increased, and the serum IgE of asthmatic patients can be as high as 2400ng/mL. In one aspect, in a clinical study, serum free IgE levels were dose-dependently reduced within 1 hour after administration of the first dose of omalizumab (Xolair) for injection and remained unchanged between doses, with a mean serum free IgE reduction of greater than 96% at the recommended dose; on the other hand, due to the formation of Xolair-IgE complexes, serum total IgE levels (i.e. bound and unbound) increased after the first dose, the rate of bound IgE elimination was slower compared to free IgE, and the average serum total IgE levels were 5-fold higher than before treatment, measured using standard assays, 16 weeks after the first dose. In the ADA detection process, an ADA-drug complex is required to be dissociated in an acidolysis mode and the like to form free ADA for detection, but the process can release the IgE molecules combined with the drug at the same time, so that the free IgE level in a sample is obviously increased, and the free IgE can be directly bridged with a capture reagent and a detection reagent to generate false positive signals.

The prior art method for solving the problems is mainly to adopt an anti-IgE antibody to remove interference. However, this technique has major technical drawbacks and disadvantages. Firstly, since the removed reagent is an antibody against IgE and the oxomalizumab for drug injection is also an antibody against IgE, a positive control (an antibody against the oxomalizumab for injection) needs to be set in the methodological study, and the positive control may cross-react with the antibody for removing IgE, thereby reducing the signal of the positive control and leading to the possibility of false negative result generation; second, the reactivity of the monoclonal antibody reagent generally involved in IgE removal is only close to the affinity of the oxuzumab for injection to IgE, and the concentration level of the oxuzumab for injection after administration is high, so that a large amount of antibody removal reagent needs to be added, and the effect is not ideal.

Disclosure of Invention

In response to the above problems, the inventors of the present application have repeatedly made studies to develop a reagent for detecting the resistance of omalizumab to endogenous IgE interference in human blood samples (e.g., blood, plasma, serum, etc.).

Specifically, the application provides the following technical scheme:

in a first aspect, the present application provides a reagent for detecting an omalizumab-resistant antibody in a sample, comprising a capture reagent capable of binding to said omalizumab-resistant antibody, a detection reagent for detection, and an IgE binding molecule.

In a second aspect, the present application provides a method of preparing the reagent of the first aspect, comprising:

diluting the IgE binding molecules with a buffer, such as PBS, e.g., PBS containing 1% BSA fatty acid-free, to a screening dilution having a final concentration of at least about 1 μg/mL;

diluting the capture reagent with the screening diluent to a capture reagent working solution having a final concentration of about 250 ng/mL;

diluting the detection reagent with the screening diluent to a detection reagent working solution with a final concentration of about 250 ng/mL; and

and mixing the capture reagent working solution and the detection reagent working solution according to a ratio of 1:1 to form the reagent.

In a third aspect, the present application provides the use of a reagent according to the first aspect or a reagent prepared according to the method of the second aspect for detecting an omalizumab-resistant antibody in a sample.

A Master Mix working solution for detecting omalizumab-resistant antibodies in human blood samples was developed. The Master Mix working solution can well solve the influence of endogenous IgE interference on the immunogenicity detection of the omalizumab for injection, and has the advantages of one or more of the following:

the IgE receptor is added into the Master Mix working solution, so that the IgE interference removal efficiency is higher. The serum IgE of the asthmatic patient can reach 2400ng/mL, so that the IgE specificity needs to be at least 2400ng/mL, the IgE side does not interfere with the detection of ADA, when the IgE receptor is not used for interference, the detection of the ADA is interfered when the concentration of the endogenous IgE is 1000 ng/m, the detection of the serum sample of the asthmatic patient cannot be met, and when the IgE receptor is used for interference, the detection of the ADA is not influenced under the condition that the signal of the positive control is not reduced, and the detection requirement of the serum sample of the asthmatic patient is completely met when the concentration of the endogenous IgE reaches 8000 ng/mL.

Detailed Description

The omalizumab for injection is a recombinant humanized monoclonal antibody, is an anti-IgE targeted biological agent, and is a targeted therapeutic drug for treating moderate to severe asthma, which is approved for the first time worldwide. 77 allergic asthma studies have been completed. The Chinese biological preparation is the first biological preparation for treating patients suffering from moderate to severe asthma, has definite curative effect and safety, is marketed, and brings more personalized medicine treatment options for patients suffering from moderate to severe allergic asthma in China.

Discontinuation of this treatment typically results in restoration of free IgE levels to higher levels and recurrence of the associated symptoms. Total IgE levels increased during treatment and total IgE remained high for one year of discontinuation of treatment. Therefore, the dose of the product cannot be redetermined based on the re-measured IgE levels during the treatment period of the product. When the treatment is discontinued for less than one year, the dosage administered should be determined based on the serum IgE levels measured at the first dose determination. Only when the treatment has been discontinued for one year or more, the dosage can be determined based on the re-measured total serum IgE levels.

As used herein, "IgE binding molecule" includes any natural or synthetic substance capable of binding free IgE, such as various anti-IgE antibodies and IgE receptors, such as natural or synthetic receptors. There are two types of natural IgE receptors (fcer): one class is the high affinity receptor, namely fcs R I (dissociation constant reaches 1X 10-10M), and the stability of the receptor after being combined with Fc is far higher than that of other Fc receptors; the other class is the low affinity receptor, called fceri, which binds IgE with 100-1 to 1-000 times lower affinity than fceri 1. Fcs R I was found to have two more isoforms expressed on the surface of IL-4 stimulated B cells, monocytes and eosinophils, respectively.

Specifically, the application provides the following technical scheme:

In specific embodiments, the IgE binding molecule is an IgE receptor.

In particular embodiments, the sample is a human body fluid, such as blood, serum, or plasma.

In a specific embodiment, the capture reagent is biotin-labeled omalizumab, e.g., biotin-labeled Xolair.

In a specific embodiment, the detection reagent is ruthenium element labeled omalizumab, e.g., ruthenium element labeled Xolair.

In particular embodiments, the weight percentages of the capture reagent, the detection reagent, and the IgE binding molecule are about 1:1:4.

in a preferred embodiment, the concentration of IgE binding molecules in the reagent is not less than 1 μg/mL.

diluting said capture reagent with said screening diluent to a capture reagent working solution having a final concentration of about 250 ng/mL;

In specific embodiments, the IgE binding molecule is an IgE receptor.

Examples

The following examples are illustrative of the application and are not intended to limit the scope of the application. Modifications and substitutions to methods, procedures, or conditions of the present application without departing from the spirit and nature of the application are intended to be within the scope of the present application.

Unless otherwise indicated, all reagents used in the examples were conventional commercial reagents and all technical means used in the examples were conventional means well known to those skilled in the art.

Example 1 method for detecting resistance to omalizumab

The analytical method is described as follows:

1) First, neutralizing agent (1M Tris (pH 9.5)) and all samples after acidolysis were added to the solution of previously captured omalizumab for injection (manufacturer: novartis Pharma Stenin AG/Switzerland trade name: xolair) 96-well single removable elisa plate (manufacturer: CORNING; cargo number: 42592 Incubation overnight at room temperature with shaking at 350RPM in the dark, ensures that ADA in the sample forms ADA-drug complexes with the omalizumab for injection in the solid support. After washing the plate, adding acidolysis solution (300 mM HAc) to dissociate ADA-drug complex;

2) The neutralizing agent (1M Tris (pH 9.5)) and Master Mix working solution (formulation method as in step 8 below) were added to round hole polypropylene plate (manufacturer: greiner Bio-one; cargo number: 650201 Then adding the sample to be tested after acidolysis or the sample for verifying the suitability of the sample or the system into the plate. The sample to be tested is a human serum sample collected in a clinical test; validation samples refer to ADA positive control samples of different concentrations formulated using 100% pooled human serum, e.g., sensitivity samples, precision samples, drug resistance samples, selectivity samples, specificity samples, etc.; the system applicability sample refers to a negative control sample (100% mixed human serum), a low-concentration positive control sample and a high-concentration positive control sample (25 ng/mL and 10000ng/mL of positive control sample are prepared in 100% mixed human serum), the system applicability sample is used for monitoring whether an analysis batch passes or not, the sample is subjected to shaking incubation at 600RPM in a dark place at room temperature after a sealing plate is closed, then the sample mixture is transferred to a closed MSD micro-pore plate (manufacturer: meso Scale Discovery; product number: L15 SA-1), and the sample mixture is subjected to shaking incubation at 600RPM in a dark place at room temperature after the sealing plate is closed;

3) Finally, the prepared MSD Read Buffer T (2 x) working fluid (MSD Read Buffer T (4 x) was purchased from Meso Scale Discovery; cargo number: r92TC-1, MSD Read Buffer T (4X) was diluted 1:1 to MSD Read Buffer T (2X) working fluid using ultrapure water, and the instrument signal was read at MESO QUICKPLEX SQ120, the magnitude of the instrument signal value being proportional to the concentration of the drug-resistant antibody. ADA positive control samples of different concentrations (e.g., 200ng/mL,100ng/mL,50ng/mL,25ng/mL,12.5ng/mL,6.25ng/mL,3.13ng/mL, and 1.56 ng/mL) were prepared with 100% pooled human serum (pooled from domestic healthy human subjects recruited by HuSe05Nov 2020), such as the rabbit anti-Margaritimab polyclonal antibody (available from Shanghai Mitaijun Biotechnology Co., ltd., cat. No. ZJ-06-039) in this example. In combination with experience and related reports of other clinical studies, target protein IgE would be eliminated at a slower rate after administration due to the formation of omalizumab-IgE complex, resulting in an increase in total IgE in the acid-treated sample, so that high concentrations of IgE receptors were added to Master Mix during sample pretreatment, which effectively removed the interference of endogenous IgE and did not affect the performance of ADA PC (Positive Control), i.e., positive quality Control sample.

The specific operation flow steps are as follows:

1. capturing

Xolair is diluted into ELISA plate capturing reagent working solution with the final concentration of about 10 mug/mL by using 1 XCBS, the prepared ELISA plate capturing reagent working solution is added into an ELISA plate at 100 mug/hole, and the ELISA plate capturing reagent working solution is kept stand at 2-8 ℃ for overnight after sealing.

2. Washing plate

The captured ELISA plates were removed, plate washed 3 times with plate wash (1 xPBST) at not less than 300. Mu.L/Kong Xi, and blotted dry on clean paper.

3. Closure

Blocking solution I was added to the captured ELISA plates at 300. Mu.L/well and blocked by shaking at 350RPM at room temperature in the absence of light for at least 1.0 hr.

4. Acid treatment

All samples were acid treated with acidolysis solution (i.e., 300mM HAc)/confirmed acidolysis solution (i.e., confirmed acidolysis solution diluted with 300mM HAc to a final concentration of about 400. Mu.g/mL) at 1:50 times and shaken at 600RPM at room temperature for 10-20 min in the absence of light.

5. Washing plate

After the ELISA plate was blocked, plates were 3 times with plate wash (1 xPBST) at not less than 300. Mu.L/Kong Xi and were dried on clean paper.

6. Sample incubation

Neutralizing agents (1M Tris (pH 9.5)) were added to the washed ELISA plates in this order according to the plate pattern, 30. Mu.L/well; acid treated sample, 100 μl/well; after sealing the plates, incubations were performed at least 2.0. 2.0 hr at room temperature with shaking at 350RPM in the dark and allowed to extend overnight.

7. Sample dissociation

The ELISA plate with captured ADA was removed, washed with plate washing solution (1 xPBST) at 300. Mu.L/Kong Xi plate for 3 times, then acidolysis solution (300 mM HAc) was added, 80. Mu.L/well, and after sealing, the plate was dissociated by shaking at 600RPM at room temperature in the absence of light for 10-20 min.

Preparation of Master Mix working solution

Diluting IgE receptors (name: recombinant human IgE receptor-Fc fusion protein; manufacturer: shanghai Mitaijun Aureobiological technology Co., ltd.; product number: ZJ-01-061) with a diluent, namely PBS containing 1% BSA and no fatty acid, to a screening diluent with a final concentration of about 0 [ mu ] g/mL, 0.1 [ mu ] g/mL, 0.2 [ mu ] g/mL, 0.5 [ mu ] g/mL,1 [ mu ] g/mL,2 [ mu ] g/mL and 5 [ mu ] g/mL; the capture reagent concentrated solution (Stock Bio-Xolair) (obtained by using biotin labeling Xolair by the department of forward biological analysis of the military) was diluted with each concentration of screening dilution to obtain a capture reagent working solution with a final concentration of about 250 ng/mL; and diluting the detection reagent concentrated solution (Stock Ru-Xolair) (obtained by using ruthenium element labeled Xolair by the Junko forward biological analysis department) with screening diluent of each concentration to obtain detection reagent working solution with the final concentration of about 250 ng/mL; finally, mixing the capture reagent working solution and the detection reagent working solution into a Master Mix working solution according to the volume ratio of 1:1, wherein the finally prepared Master Mix working solution contains 7 IgE receptors with different concentrations, namely 0 [ mu ] g/mL, 0.1 [ mu ] g/mL, 0.2 [ mu ] g/mL, 0.5 [ mu ] g/mL,1 [ mu ] g/mL,2 [ mu ] g/mL and 5 [ mu ] g/mL of IgE receptors.

9. Incubation of rotating plate

1) Adding a neutralizing agent (1M Tris (pH 9.5)) into a round hole polypropylene plate, wherein the neutralizing agent is 20 mu L/hole;

2) Adding the Master Mix working solution prepared in the step 8 into a round hole polypropylene plate, wherein the volume of the Master Mix working solution is 90 mu L/hole;

3) Adding the dissociated sample in the step 7 into a round hole polypropylene plate in a single hole according to the sample sequence in the plate diagram, wherein the sample is 50 mu L/hole;

4) After sealing the plate, the plate is incubated at room temperature for 2.0-2.5 hr at 600RPM in the dark.

MSD microplate closure

Blocking solution II, 150 mL/well, was added to the MSD microplate and the plate was closed by shaking at 350RPM at room temperature for at least 0.5hr.

11. Washing plate

The plate was washed 3 times with plate washing solution (1 XPBST) at not less than 300. Mu.L/Kong Xi and dried on clean paper.

12. Rotating plate sample feeding

And transferring the samples in the round hole polypropylene plate into an MSD micro-pore plate according to a plate diagram, carrying out 50 mu L/hole, sealing the plate, and carrying out light-proof shaking incubation at 600RPM for 1.0-1.5 hr at room temperature.

13. Washing plate

14. Detection of

The prepared MSD Read Buffer T (2 x) working solution was added to the MSD microplate, 150. Mu.L/well, and the MSD microplate was placed into MESO QUICKPLEX SQ for 15min for detection.

Table 1: reagent information

Example 2 optimization of Master Mix working fluid

Preparing a rabbit anti-omalizumab polyclonal antibody into samples with the concentration of 200-1.56 ng/mL by using 100% mixed healthy human serum, and respectively named as samples STD 01-STD 08; and human IgE antigen (Shanghai Michaelsen Biotechnology Co., ltd., product number: ZJ-01-060) was prepared as samples with a concentration of 8000-600 ng/mL using 100% mixed healthy human serum, and designated as IgE 01-IgE 08, respectively.

Samples STD 01-STD 08 and IgE 01-IgE 08 were tested in MESO QUICKPLEX SQ by the method of example 1, and the test results are shown in Table 2.

TABLE 2

Remarks: ECLU is an instrument response value unit, namely an electrochemiluminescence unit; S/N: signal to noise ratio, i.e., the response value of the target sample divided by the response value of the negative control sample.

The experimental results in Table 2 show that after 7 IgE receptors with different concentrations are added into the Master Mix working solution, the ADA detection sensitivity is consistent and can reach 6.25ng/mL; the detection of serum samples of asthma patients cannot be met by interfering with the detection of ADA when the concentration of IgE receptors is 0 mug/mL, 0.1 mug/mL, 0.2 mug/mL and 0.5 mug/mL and the concentration of endogenous IgE is 1000 ng/mL and 2000 ng/mL, the detection of ADA is still not influenced when the concentration of IgE receptors is 1 mug/mL, 2 mug/mL and 5 mug/mL, the detection requirement of serum samples of asthma patients is completely met, and therefore, the requirement of at least 2400ng/mL of IgE specificity can be met under the condition that the signal of ADA is not reduced by selecting Master Mix working solution containing IgE receptors not lower than 1 mug/mL.

The application has been described in detail with reference to the general description and the specific embodiments thereof, and such modifications and improvements can be made without departing from the spirit of the application, which is within the scope of the application as claimed.

Claims

1. A reagent for detecting an omalizumab-resistant antibody in a sample, comprising a capture reagent capable of binding the omalizumab-resistant antibody, a detection reagent for detection, and an IgE binding molecule.

2. The reagent of claim 1, wherein the IgE binding molecule is an IgE receptor, optionally the sample is a human body fluid, such as blood, serum or plasma.

3. The reagent of claim 1, wherein the capture reagent is biotin-labeled omalizumab, e.g., biotin-labeled Xolair.

4. The reagent of claim 1, wherein the detection reagent is ruthenium element labeled omalizumab, e.g., ruthenium element labeled Xolair.

5. The reagent of any one of claims 1 to 4, wherein the weight percent of the capture reagent, the detection reagent, and the IgE binding molecule is about 1:1:4, optionally, the concentration of IgE binding molecules in the reagent is not less than 1 μg/mL.

6. A method of preparing the reagent of any one of claims 1-5, comprising:

7. The method of claim 6, wherein the IgE binding molecule is an IgE receptor.

8. The method of claim 6, wherein the capture reagent is biotin-labeled omalizumab, e.g., biotin-labeled Xolair.

9. The method of claim 6, wherein the detection reagent is ruthenium element labeled omalizumab, e.g., ruthenium element labeled Xolair.

10. Use of the reagent of any one of claims 1-5 or the reagent prepared according to the method of any one of claims 6-9 for detecting an omalizumab-resistant antibody in a sample, optionally a human body fluid, such as blood, serum or plasma.