CN117825686A - Sample pretreatment method for detecting denominator drug-resistant antibody and application thereof - Google Patents
Sample pretreatment method for detecting denominator drug-resistant antibody and application thereof Download PDFInfo
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- Investigating Or Analysing Biological Materials (AREA)
Abstract
The application provides a sample pretreatment method for detecting a denominator drug-resistant antibody and application thereof. According to the sample pretreatment method, for example, the sample of the denominator drug-resistant antibody to be detected is pretreated by the phosphate buffer solution containing 1wt percent of fatty acid-free bovine serum albumin, so that the interference of free RANKL in the sample can be effectively reduced, and the problem that the free RANKL affects the detection of the immunogenicity of the denominator drug is simply and effectively solved.
Description
Technical Field
The application relates to the field of biological detection, in particular to a pretreatment method for detecting a sample, and particularly relates to a sample pretreatment method for detecting a denomab drug-resistant antibody and application thereof.
Background
With the development of biotechnology, monoclonal antibodies (mabs) have become important therapeutic drugs in modern medicine. mabs have the potential to treat a variety of diseases and conditions, but repeated use of mabs may result in a high degree of immunogenicity. Drug immunogenicity is manifested by the stimulation of the production of drug-resistant antibodies (ADA). ADA can alter the pharmacokinetics and efficacy of a drug, thereby reducing the efficacy of the drug, possibly even counteracting the therapeutic effects of the drug or causing serious adverse events to the patient. Thus, accurate ADA detection is necessary.
Denosumab (also known as AMC-162), a humanized IgG2 monoclonal antibody with high affinity for NF- κb receptor activator ligand (receptor activator of NF- κb ligand, RANKL), acts as a bone resorption inhibitor with a specific mechanism of action, and is capable of inhibiting osteoclast activation and development, reducing bone resorption and increasing bone density.
One major challenge faced in the process of denomab ADA detection is interference of the free target RANKL. This is because in the ADA detection process, the ADA-drug complex needs to be dissociated by acidolysis or the like to form free ADA for detection; however, the dissociation also releases the drug-bound RANKL, which causes a significant increase in the level of free RANKL in the sample, and RANKL can exist in the form of a trimer, so that the RANKL trimer can directly bridge with the capture reagent and the detection reagent in the reaction system to generate false positive signals.
The prior art method for solving the problems mainly adopts an anti-RANKL antibody to remove interference. However, this technique has major technical drawbacks and disadvantages. Firstly, because the reagent adopted is an antibody against RANKL, and the denomab is also an antibody against RANKL, cross reaction can be generated, so that the signal of the local denomab drug resistance antibody is reduced, and the possibility of false negative result generation is caused; secondly, the reactivity of anti-RANKL antibody reagents generally involved in reducing RANKL is also only close to the affinity of denomab for RANKL, whereas the concentration level of denomab after administration is high, resulting in the need to add a large amount of anti-RANKL antibody reagent to reduce RANKL, high cost and less desirable effect.
Thus, there remains a need to develop methods for effectively reducing free RANKL interference in a sample to be tested using denominator-resistant antibodies.
Disclosure of Invention
In view of the above problems, the inventors of the present application have repeatedly made a search for a sample pretreatment method for detecting a denominator resistance antibody in a body fluid sample of an animal (e.g., a body fluid sample of a human such as blood, plasma, serum, etc.), thereby developing a method for detecting a denominator resistance antibody that can reduce free RANKL interference in a sexual sample. In addition, the application also provides the related products prepared thereby and application thereof.
Specifically, the application provides the following technical scheme:
in a first aspect, the present application provides a sample pretreatment method for detecting denominator drug-resistant antibodies, comprising the step of pretreating a sample with a buffer comprising 0.5wt.% to 3 wt wt.% fatty acid-free bovine serum albumin.
In a second aspect, the present application provides a method for detecting a denominator drug-resistant antibody comprising the step of pre-treating a sample using the sample pre-treatment method provided in the first aspect.
In a third aspect, the present application provides the use of a buffer comprising 0.5wt.% to 3 wt wt.% fatty acid-free bovine serum albumin for the preparation of a reagent or kit for detecting a denominator resistant antibody.
In a fourth aspect, the present application provides a reagent or kit for detecting denominator resistance antibody comprising a buffer comprising 0.5wt.% to 3 wt wt.% fatty acid free bovine serum albumin.
In addition, the application also provides the application of the reagent or the kit provided in the fourth aspect in detecting the denominator drug resistance antibody.
In combination with experience and related reports of other clinical studies, acidolysis of a sample releases drug-bound RANKL molecules during detection of denominator drug-resistant antibodies, thereby generating false positive signals. However, the method for avoiding acidolysis is adopted, and the sample of the denomab drug resistance antibody to be detected is pretreated by a phosphate buffer solution containing 1wt percent of fatty acid-free bovine serum albumin in a certain volume ratio, so that the existence of free RANKL in the sample can be effectively reduced, the interference of the free RANKL is effectively reduced, and the problem of detecting the immunogenicity of the denomab drug affected by the free RANKL is well solved.
In addition, the sample pretreatment method provided by the application is simple and convenient to operate, and the reagent is easy to obtain. The pretreated sample (such as human serum sample) can be qualitatively detected for the drug-resistant antibodies of the dinotefuran by using a Bridging-electrochemical luminescence technology (Briding-ECLIA), and the performance of the subsequent analysis method is not affected. That is, the method for detecting the denominator drug-resistant antibody provided by the application can effectively reduce the interference of free RANKL molecules, and sensitively and accurately detect the denominator drug-resistant antibody in the sample. Based on the method provided by the application, related detection products, such as a reagent or a kit for detecting the denominator drug resistance antibody, can be formed, so that the denominator drug resistance antibody can be conveniently and rapidly detected qualitatively.
Detailed Description
As used herein, "sample" means a body fluid sample from an animal, including blood, serum, plasma, culture supernatant, cell/tissue lysate, cerebrospinal fluid, urine, sweat, lavage fluid, and like biological samples, particularly a body fluid sample from a human, such as human blood, serum, plasma, and the like.
As used herein, "buffer" means any solution that maintains the pH of the solution relatively stable upon addition of small amounts of acid or base and is suitable for use with biological samples, including any buffers conventionally used in the art, such as phosphate, tris, HEPES, and the like.
As used herein, "fatty acid-free bovine serum albumin (BAS)" is a natural protein extracted from bovine serum, which has been subjected to a degreasing purification treatment to make its fatty acid content very low (0.02%) and almost negligible.
Specifically, the application provides the following technical scheme:
in a first aspect, the present application provides a sample pretreatment method for detecting denominator drug-resistant antibodies comprising the step of pretreating a sample with a buffer comprising 0.5 wt% to 3 wt%, preferably 1 wt% fatty acid free bovine serum albumin.
In a specific embodiment, the buffer is a phosphate buffer, tris buffer or HEPES buffer, preferably a phosphate buffer.
In a specific embodiment, the volume ratio of the sample to the buffer is from 1:10 to 1:50, preferably 1:20.
In a specific embodiment, the conditions of the sample pretreatment method provided herein are: the sample is incubated at a temperature of 20℃to 30℃i.e.room temperature for 20min to 40min.
In particular embodiments, the conditions of the sample pretreatment methods provided herein further comprise: the sample is oscillated at a speed of 500 rpm to 700 rpm, preferably 600 rpm.
In a specific embodiment, the sample in the sample pretreatment methods provided herein is a body fluid sample of an animal, such as blood, serum, or plasma.
In a specific embodiment, the sample in the sample pretreatment methods provided herein is human serum.
In a second aspect, the present application provides a method for detecting a denominator drug-resistant antibody comprising the step of pre-treating a sample using the sample pre-treatment method provided in the first aspect.
In particular embodiments, the methods provided herein for detecting a denominator resistance antibody further comprise the step of qualitatively detecting the denominator resistance antibody in the sample using a bridged electrochemiluminescence immunoassay.
In particular embodiments, the sample in the methods provided herein for detecting denominator resistant antibodies is a body fluid sample of an animal, such as blood, serum, or plasma.
In a specific embodiment, the sample in the methods provided herein for detecting denominator resistant antibodies in a sample is human serum.
In a third aspect, the present application provides the use of a buffer comprising 0.5. 0.5wt to 3. 3 wt% fatty acid free bovine serum albumin in the preparation of a reagent or kit for detecting a denominator resistance antibody.
In a specific embodiment, the buffer is a phosphate buffer, tris buffer or HEPES buffer, preferably a phosphate buffer.
In a fourth aspect, the present application also provides a kit or kit for detecting denominator resistance antibodies comprising a buffer comprising 0.5wt to 3 wt% fatty acid free bovine serum albumin.
In a specific embodiment, the buffer is a phosphate buffer, tris buffer or HEPES buffer, preferably a phosphate buffer.
In addition, in a fifth aspect, the present application further provides the use of the reagent or kit provided in the fourth aspect for detecting a denominator drug resistant antibody.
Examples
The following examples are illustrative of the present application, but are not intended to limit the scope of the present application. Modifications and substitutions to methods, procedures, or conditions of the present application without departing from the spirit and substance of the present 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.
The information on the reagents used in the examples below is shown in table 1 below.
TABLE 1 reagent information
Example 1
In this embodiment, the sample pretreatment method provided in the present application is used to pretreat a human serum sample, and a bridging electrochemiluminescence technology is used on an MSD platform to qualitatively detect the denominator drug-resistant antibody in the sample, and the principle is briefly described as follows:
pretreatment of serum samples with Phosphate Buffer (PBS) containing 1wt.% fatty acid-free Bovine Serum Albumin (BSA); incubating the pretreated serum sample and a Master Mix working solution in a round hole polypropylene plate to form a biotin-drug-resistance antibody-drug-ruthenium element bridged complex, transferring the incubated bridged complex into an MSD streptavidin microplate to capture so as to form the MSD microplate-streptavidin-drug-resistance antibody-drug-ruthenium element bridged complex; after washing the plate, adding MSD plate reading liquid T (2 x) (MSD Read Buffer T (2 x)) to read instrument signals on the MSD plate reading machine, wherein the magnitude of the instrument signal value is in direct proportion to the concentration of the drug-resistant antibody; 100% normal healthy human serum was diluted with a diluent according to the required concentration as a positive control sample.
The specific operation flow steps are as follows:
1. sample pretreatment
The pretreatment of serum samples is carried out by adding sample pretreatment liquid, namely PBS containing 1wt percent of fatty acid-free BSA, into serum samples to be tested in an analysis batch according to the volume ratio of 1:20, and carrying out shaking incubation at 600rpm for 20-40 min at room temperature.
Preparation of Master Mix working solution
The capture reagent concentrate (Stock Bio-Drug) (biotin-labeled denomab) was diluted with a diluent to a capture reagent working solution with a final concentration of about 500 ng/mL, the detection reagent concentrate (Stock Ru-Drug) (ruthenium element-labeled denomab) was diluted with a diluent to a detection reagent working solution with a final concentration of about 500 ng/mL, and then the capture reagent working solution and the detection reagent working solution were mixed at a volume ratio of 1:1 to a Master Mix working solution.
3. Sample incubation
1) In a round hole polypropylene plate (manufacturer: greiner Bio-one; cargo number: 650201 20 mul/hole) of diluent is added;
2) Adding the Master Mix working solution prepared in the step 2 into a round hole polypropylene plate, wherein the volume of the Master Mix working solution is 90 mu L/hole;
3) Adding the pretreated sample in the step 1 into a round hole polypropylene plate in a single hole according to the sample sequence in a plate diagram, wherein the sample is 50 mu L/hole;
4) The plates were then incubated at room temperature with shaking at 600rpm for 2.0hr to 2.5hr in the absence of light.
MSD microplate closure
A blocking solution was added to the MSD microplate (manufacturer: meso Scale Discovery; cat# L15 SA-1), 150. Mu.L/well and incubated at room temperature with shaking at 600rpm in the absence of light for at least 30min.
5. Washing plate
After the completion of the MSD microplate sealing, the plate was washed 3 times with a plate washing liquid, not less than 300. Mu.L/well each time.
6. Rotating plate sample feeding
Samples in a round hole polypropylene plate are transferred into an MSD microplate according to a plate diagram, 50 mu L/well, sealed and incubated at room temperature for 1.0hr to 1.5hr at 600rpm in a dark place.
7. Washing plate
After the MSD microplate incubation is completed, the plate is washed 3 times with no less than 300. Mu.L/well each time.
8. Detection of
The prepared MSD plate reading liquid T (MSD Read Buffer T (2 x)) working liquid is added into an MSD micro-pore plate, 150 mu L/hole is formed, and the MSD micro-pore plate is placed into a hypersensitive multifactorial electrochemiluminescence analyzer (MESO QUICKPLEX SQ) for detection within 15 min.
The results are shown in Table 2 below, in which the conventional detection method is different from the detection method of the present invention in that the conventional detection method pretreats the serum sample using an acidic reagent such as hydrochloric acid (HCl), acetic acid (HAc), glycine (Glycine) or the like as a sample pretreatment liquid, whereas the present invention pretreats the serum sample using 1wt.% of a buffer solution of fatty acid-free bovine serum albumin as a sample pretreatment liquid.
TABLE 2
Critical value: according to the requirements of the drug immunogenicity research technical guidelines, the numerical value for judging the negative and positive of the detection result is obtained through verification test data statistics.
Instrument response value: the values were read by a hypersensitive multifactor electrochemiluminescence analyzer (MESO QUICKPLEX SQ).
Signal-to-noise ratio: ratio of the response value of the sample instrument to the response value of the negative control sample.
As can be seen from Table 2, in the presence of free RANKL, compared with the case of a false positive result that a negative sample is detected as positive by a conventional method, the detection method of the invention can obviously reduce the influence of free RANKL on the detection result of the method and the sample under the condition that the sensitivity and the drug resistance meet the requirements of drug immunogenicity research technical guidelines and clinical detection issued by the drug review center of the national drug administration, and can not cause the case of false positive detection result and abnormally high percent of positive sample.
The present application has been described in detail with reference to general descriptions and specific embodiments thereof, and such modifications and improvements can be made without departing from the spirit of the application, and are intended to be within the scope of the present application.
Claims (10)
1. A sample pretreatment method for detecting a denominator drug-resistant antibody comprising the step of pretreating a sample with a buffer containing 0.5wt.% to 3 wt wt.% fatty acid-free bovine serum albumin, wherein the volume ratio of the sample to the buffer is 1:10 to 1:50.
2. The method of claim 1, wherein the pretreatment conditions are as follows: the samples were incubated with shaking at a temperature of 20℃to 30℃and a rotational speed of 500 rpm to 700 rpm for 20min to 40min.
3. The method of claim 1, wherein the buffer is selected from the group consisting of: phosphate buffer, tris buffer, and HEPES buffer.
4. A method according to any one of claims 1 to 3, wherein the sample is a body fluid sample of an animal, such as blood, serum or plasma.
5. A method for detecting a denominator drug-resistant antibody comprising the step of pre-treating a sample using the sample pre-treatment method of any one of claims 1 to 3.
6. The method of claim 5, further comprising the step of qualitatively detecting the denominator resistance antibody in the sample using a bridged electrochemiluminescent immunoassay.
7. The method of claim 5 or 6, wherein the sample is a body fluid of an animal, such as blood, serum or plasma.
8. Use of a buffer comprising 0.5wt.% to 3 wt wt.% fatty acid free bovine serum albumin for the preparation of a reagent or kit for detecting denominator resistant antibodies.
9. A reagent or kit for detecting a denominator drug-resistant antibody comprising a buffer comprising 0.5wt to 3 wt% fatty acid free bovine serum albumin.
10. Use of the reagent or kit of claim 9 for detecting denominator drug-resistant antibodies.
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| TW202336035A (en) * | 2022-03-03 | 2023-09-16 | 美商伊諾西生命科學公司 | Methods and compositions to treat autoimmune diseases and cancer |
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2024
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| WO2011058993A1 (en) * | 2009-11-16 | 2011-05-19 | 国立大学法人大阪大学 | Method for screening for novel bone resorption inhibitor |
| US20160097777A1 (en) * | 2014-09-12 | 2016-04-07 | Gyros Patent Ab | Immunological method |
| CN112074537A (en) * | 2018-05-01 | 2020-12-11 | 美国安进公司 | Antibodies with modulated glycan profiles |
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| KELLY K. ARTHUR等: "In Vitro Stoichiometry of Complexes between the Soluble RANK Ligand and the Monoclonal Antibody Denosumab", 《BIOCHEMISTRY》, no. 51, 12 January 2021 (2021-01-12), pages 795 - 806 * |
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