CN114044828A - Mouse anti-monkey IgG species specific antibody, application thereof and universal detection method of anti-drug antibody based on mouse anti-monkey IgG species specific antibody - Google Patents
Mouse anti-monkey IgG species specific antibody, application thereof and universal detection method of anti-drug antibody based on mouse anti-monkey IgG species specific antibody Download PDFInfo
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Abstract
The invention relates to the technical field of biology, in particular to a mouse anti-monkey IgG species specific antibody, application thereof and a general detection method of an anti-drug antibody based on the same. The preparation method of the mouse anti-monkey IgG species specific antibody provided by the invention comprises the following steps: immunizing a cynomolgus monkey by using a monoclonal antibody to obtain an anti-drug antibody derived from the monkey; immunizing a mouse by using an anti-drug antibody derived from a monkey to obtain a mouse anti-monkey antibody; further screening of the murine anti-monkey antibody resulted in a murine anti-monkey IgG species specific antibody. The mouse anti-monkey IgG species specific antibody provided by the invention can be used as the basis for detecting a general anti-drug antibody, and the platform technology constructed by the reagent can greatly simplify the development of a detection method of a monkey anti-human anti-drug antibody.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a mouse anti-monkey IgG species specific antibody and a universal monkey anti-human anti-drug antibody detection method constructed based on the antibody reagent.
Background
When applied for clinical use, monoclonal antibody drugs (hereinafter referred to as single-antibody drugs) need to provide data of relevant immunogenicity (such as drug-resistant antibodies generated after monoclonal antibody drugs are used) and are used for analyzing results of preclinical drug substitution, drug effect research, safety and the like. Immunogenicity generally refers to the ability of a therapeutic protein product (e.g., a single drug) to elicit an immune response or immune-related response to itself.
The drugs that treat the protein-like products are recognized as foreign substances (antigens) to generate immune responses. The unexpected immune response may damage the safety of the drug and reduce the expected curative effect, mainly because the generated Anti-drug antibodies (ADA) have potential side effects, which may affect the biological activity and pharmacokinetic parameters of the drug to further reduce the efficacy of the drug, and more seriously have a safety hazard in terms of medication.
In preclinical studies, the analysis of anti-drug antibodies can describe the time point of occurrence of anti-drug antibodies that produce immune responses after drug administration by using certain detection data, and provide corresponding analysis factors for the pharmaco-drug, toxicology, pharmacodynamic and toxicology results.
However, the development of the detection method for obtaining the detection data of the anti-drug antibody in the prior art is time-consuming and labor-consuming, and in practical application, the corresponding method is difficult to develop due to the overlong time for preparing the positive control antibody and the lack of a platform-type detection technology, so that the development time of the drug is delayed.
Disclosure of Invention
In order to solve the above problems, the present invention provides a general-purpose detection method for a murine anti-monkey IgG species-specific antibody and a monkey anti-human anti-drug antibody based thereon. The mouse anti-monkey IgG species specific antibody and the detection technology constructed by the same provided by the invention make up for the defect that a set of detection method needs to be specially developed for the detection of a small amount of samples in the prior art, simplify the detection steps, shorten the detection time, greatly reduce the development time and the development cost, and provide powerful guarantee for more quickly acquiring data declaration clinical application. At present, the industry has no general analysis method for the anti-drug antibody, and the invention also fills the blank of the field.
The invention provides a specific antibody of a mouse anti-monkey IgG species, and the preparation method of the specific antibody of the mouse anti-monkey IgG species comprises the following steps:
immunizing a cynomolgus monkey with a monoclonal antibody to obtain an anti-drug antibody (ADA) derived from the monkey;
immunizing a mouse by using an anti-drug antibody derived from a monkey to obtain a mouse anti-monkey antibody;
screening the mouse anti-monkey antibody to obtain the specific antibody of the mouse anti-monkey IgG species.
The invention also provides application of the mouse anti-monkey IgG species specific antibody in detection of anti-drug antibodies.
The invention also provides application of the mouse anti-monkey IgG species specific antibody in preparation of a kit for detecting an anti-drug antibody.
The invention provides a general detection kit for a monkey anti-human anti-drug antibody, which comprises the following components: the mouse anti-monkey IgG species specific antibody, ADA positive control, antibody drug, magnetic beads and buffer solution;
the ADA positive control includes a cynomolgus monkey-derived anti-drug antibody.
The invention provides a general detection method of monkey anti-human anti-drug antibodies in preclinical research, which comprises an avidin-biotin method and an antibody method.
Preferably, the avidin-biotin method comprises the steps of:
(1) mixing the magnetic beads marked with the avidin with the single antibody drug marked with the biotin to obtain magnetic beads combined with the single antibody drug;
(2) placing the magnetic beads combined with the single drug resistance in a plate hole of an enzyme label plate to obtain the enzyme label plate containing the magnetic beads;
(3) adding a sample into the ELISA plate containing the magnetic beads to obtain the ELISA plate containing the sample;
(4) using buffer solution to blow and suck the ELISA plate containing the sample evenly;
(5) after the blowing, sucking and uniformly mixing are finished, performing a binding reaction to obtain a streptavidin magnetic bead compound combined with a specific antibody of the mouse anti-monkey IgG species;
the binding reaction comprises: adding the mouse anti-monkey IgG species specific antibody of claim 1 with a chromogenic label to an ELISA plate containing a streptavidin magnetic bead and sample complex, and performing incubation treatment;
(6) adding a chromogenic substrate into the magnetic bead compound combined with the mouse anti-monkey IgG species specific antibody for chromogenic reaction to obtain a chromogenic reaction magnetic bead compound;
(7) placing a magnetic plate at the bottom of the chromogenic reaction magnetic bead compound, attracting the magnetic beads to the edges of the micropores, and then adding a stop solution;
(8) and (5) detecting by using a microplate reader, and comparing with a blank control group to obtain a detection result.
Preferably, the magnetic beads labeled with avidin include streptavidin magnetic beads.
Preferably, the antibody method comprises the steps of:
(1) mixing the magnetic beads marked with the mouse anti-human IgG and the monoclonal antibody to obtain magnetic beads combined with the monoclonal antibody;
(2) placing the magnetic beads combined with the monoclonal antibody in a plate hole of an enzyme label plate to obtain the enzyme label plate containing the magnetic beads;
(3) adding a sample into the ELISA plate containing the magnetic beads to obtain the ELISA plate containing the sample;
(4) using buffer solution to blow and suck the ELISA plate containing the sample evenly;
(5) after the blowing, sucking and uniformly mixing are finished, carrying out a binding reaction to obtain a magnetic bead compound combined with a mouse anti-monkey IgG species specific antibody;
the binding reaction comprises: adding the mouse anti-monkey IgG species specific antibody of claim 1 with a chromogenic label to an ELISA plate containing a complex of magnetic beads and a sample, and performing incubation treatment;
(6) adding a chromogenic substrate into the magnetic bead compound combined with the mouse anti-monkey IgG species specific antibody for chromogenic reaction to obtain a chromogenic reaction magnetic bead compound;
(7) placing a magnetic plate at the bottom of the chromogenic reaction magnetic bead compound, attracting the magnetic beads to the edges of the micropores, and then adding a stop solution;
(8) and (5) detecting by using a microplate reader, and comparing with a blank control group to obtain a detection result.
The invention provides a specific antibody of a mouse anti-monkey IgG species, and the preparation method of the specific antibody of the mouse anti-monkey IgG species comprises the following steps: immunizing a cynomolgus monkey by using a monoclonal antibody to obtain an anti-drug antibody derived from the monkey; immunizing a mouse by using an anti-drug antibody derived from a monkey to obtain a mouse anti-monkey antibody; further screening the mouse anti-monkey antibody to obtain a mouse anti-monkey IgG species specific antibody. The invention achieves the detection of ADA of different single antibody molecules by using a universal reagent (a mouse anti-monkey IgG species specific antibody is used as a detection reagent, and a monkey-derived anti-drug antibody is used as an ADA positive control), thereby realizing the universality of the method. Meanwhile, the detection method provided by the invention simplifies the ADA detection step by adopting the magnetic bead technology, simplifies the processes of acidification, coating and detection originally needed into the processes of cleaning and then can detect the antibody, reduces the operation time and the operation difficulty, improves the success rate of the detection of the anti-drug antibody, solves the difficulties in the field and fills the blank in the technical aspect.
The example results show that the mouse anti-monkey IgG species specific antibody provided by the invention has the characteristics of combining with monkey IgG but not being interfered by human antibodies (such as single-antibody), can remove interfering substance signals through acidification, simplifies the detection steps, does not influence the detection result, and has extremely high accuracy and feasibility.
Advantages of the invention
(1) The detection of the general monkey anti-human anti-drug antibody is realized by adopting a special antibody reagent and a magnetic bead technology;
(2) according to the invention, the magnetic beads of the avidin and the monoclonal antibody drug marked with the biotin in a specific ratio are adopted, so that the grabbing result can be effectively improved, and the detection quality is improved;
(3) the universal positive control ADA is provided by the kit, so that the time cost of materials such as investigation, purchase and the like of experimenters is greatly reduced, excessive reagent operation is not required, and the universal positive control ADA is a universal ADA detection method;
(4) and interference substances can be effectively removed by enriching ADA with magnetic beads, so that stability guarantee is provided for the next detection. Greatly reduces the operation time, and simplifies the original steps of acidification separation detection into only cleaning detection.
(5) The magnetic bead enrichment method is adopted, so that the later operation can be automatically realized.
The detection of anti-drug antibodies is a necessary detection project in the development of macromolecular drugs, and because the quantity of preclinical projects is large and the quantity of detection samples related to each project is small, the development of methods aiming at the respective projects inevitably causes a large amount of personnel materials and time cost waste. The detection method provided by the invention skillfully utilizes the physicochemical characteristics of the magnetic beads, the special antibody and the universal positive control ADA, and develops a universal monkey anti-human ADA analysis detection method, so that the problems are solved, the ADA separation, enrichment and later detection are integrated by the method, the detection is not required after separation, the operation flow of the test is greatly simplified, and the method is an ADA analysis platform technology with great innovation.
Drawings
FIG. 1 is a schematic diagram of the avidin-biotin method;
FIG. 2 is a schematic diagram of the operation of the antibody method;
FIG. 3 shows the anti-interference specific detection results of the specific antibody of the murine anti-monkey IgG species;
FIG. 4 shows the results of the detection of the specificity and feasibility of the murine anti-monkey IgG species-specific antibodies.
Detailed Description
For further illustration of the present invention, the following detailed description will be made of the specific antibody of mouse anti-monkey IgG species and its application and general detection method of monkey anti-human anti-drug antibody based on the specific antibody, but they should not be construed as limiting the scope of the present invention.
Example 1
Preparation of murine anti-monkey IgG species-specific antibodies
Injecting single-antibody (including IgG1 subtype antibody medicine, IgG2 subtype antibody medicine and IgG4 subtype antibody medicine in a mass ratio of 1: 1: 1, namely 2mg of each subtype antibody) into a cynomolgus monkey body to cause immune reaction to generate anti-drug antibody, and purifying monkey blood to obtain corresponding monkey-derived anti-drug antibody; injecting a monkey-derived anti-drug antibody (about 2mg) into the abdominal cavity of a mouse to allow the mouse to produce a mouse anti-monkey-specific antibody; hybridoma cell lines which only express the mouse anti-monkey IgG species specific antibody (hereinafter abbreviated as MBD-Mam01) are obtained by screening and removing hybridoma cell lines of the mouse monoclonal antibody which reacts with the humanized IgG in the mouse anti-monkey specific antibody. Murine anti-monkey IgG species specific antibodies were produced from this hybridoma cell line.
Example 2
Detection method flow
1. Avidin-biotin method
(1) Streptavidin magnetic beads and Biotin-labeled single-Drug (Biotin-Drug) were mixed at a mass ratio of 50:1, incubated at room temperature for 1h with a constant temperature shaker, and washed three times with 0.05% tween 20 Phosphate Buffer (PBST).
(2) After the magnetic beads are combined with the single-drug-resistant, PBST is used for cleaning, the magnetic beads combined with the single-drug-resistant are added into a plate hole of an enzyme label plate according to 180 mu L/hole, and the magnetic plate is used for adsorbing and removing liquid.
(3) Samples (monkey blood containing ADA samples, diluted with 1% Casein), 100. mu.L/well, were added to wells of the microplate, incubated for 1h at room temperature in a constant temperature shaker, and washed three times with PBST.
(4) Add glycine buffer pH 3.0 and pipette well, 100 u L/hole, room temperature incubation for 1h, PBST washing three times.
(5) A concentration of murine anti-monkey IgG-HRP (i.e., horseradish peroxidase-conjugated MBD-Mam01, the same below) was added to a final concentration of about 250ng/ml at 100. mu.L/well, incubated for 1h at room temperature with a constant temperature shaker, and washed three times with PBST.
(6) Adding chromogenic substrate 3,3',5,5' -Tetramethylbenzidine (TMB) and blowing and sucking, mixing evenly, incubating at 100 mu L/hole for 10min at room temperature.
(7) And (3) placing the annular magnetic plate at the bottom of the ELISA plate (attracting magnetic beads to the edges of the micropores), adding a stop solution, and standing for 1-3 min.
(8) Detecting by a microplate reader to obtain a result.
The schematic diagram of the avidin-biotin method is shown in FIG. 1.
2. Antibody method
(1) Mixing mouse anti-human IgG magnetic beads and the monoclonal antibody in a mass ratio of 100:1, incubating for 1h at room temperature by a constant-temperature shaking instrument, and washing for three times by PBST.
(2) And (3) washing the magnetic beads combined with the monoclonal antibody by using PBST, adding the magnetic beads combined with the monoclonal antibody into a plate hole of an ELISA plate according to 180 mu L/hole after washing, and adsorbing and removing liquid by using a magnetic plate.
(3) Samples (monkey blood containing ADA samples, diluted with 1% Casein), 100. mu.L/well, were added to wells of the microplate, incubated for 1h at room temperature in a constant temperature shaker, and washed three times with PBST.
(4) Glycine buffer at pH 3.0 was added and mixed by pipetting, 100 μ L/well, incubated for 1h at room temperature and washed three times with PBST.
(5) A final concentration of about 250ng/ml, 100. mu.L/well of murine anti-monkey IgG-HRP was added, incubated for 1h at room temperature in a constant temperature shaker, and washed three times with PBST.
(6) Adding TMB, blowing and sucking, mixing uniformly, incubating at 100 μ L/well for 10min at room temperature.
(7) And (3) placing the annular magnetic plate at the bottom of the ELISA plate (attracting magnetic beads to the edges of the micropores), adding a stop solution, and standing for 1-3 min.
(8) Detecting by a microplate reader to obtain a result.
The schematic diagram of the operation of the antibody method is shown in FIG. 2.
Application example 1
Detection of murine anti-monkey IgG species-specific antibodies
The binding of MBD-Mam01 provided in example 1 to monkey IgG, but not to human antibodies (e.g., single antibody) was assayed and the data shown in figure 3 and table 1.
TABLE 1
As can be seen from fig. 3 and table 1, MBD-Mam01 antibody was incubated with human antibodies (IgG1 subtype antibody drug, IgG2 subtype antibody drug, and IgG4 subtype antibody drug) and monkey-derived ADA in 1% BSA (PBST buffer), but only the detection signal of monkey-derived ADA in fig. 3 showed an S-type curve, and the detection signal of human antibody was straight at the bottom, indicating that MBD-Mam01 provided by the present invention did not react with human antibody non-specifically, and reacted with monkey-derived ADA specifically, indicating that MBD-Mam01 antibody has specific properties of specifically recognizing monkey-derived ADA and not interfering with humanized antibody drug.
Application example 2
Verifying the influence of the acidification of glycine on the test effect
According to the two detection methods provided by the embodiment 2, experimental data of glycine acidification and non-acidification in the detection method are compared, and the influence of glycine acid-washed on the later detection of the ADA to be detected is detected, so that the feasibility of the reagent is confirmed. The results are shown in Table 2.
TABLE 2
As can be seen from table 2, after acidification with glycine, the signal-to-noise ratio (S/N) between each concentration signal value and the blank group is significantly improved compared to the non-acidified group, and detection of two methods, namely a universal antibody and a specific antibody, is performed on the washed supernatant, and no influence is generated on ADA to be detected after acidification with glycine, which indicates that acidification with glycine has an excellent effect of removing interferents without influencing the detection.
Application example 3
Flow verification: reliability of contrast test
The magnetic beads combined with the sample in the elisa plate containing the sample in step (3) of the avidin-biotin method in example 2 were taken, ADA detection was performed on (Drug a, bispecific antibody Drug, targeting CD3& BCMA) using the detection method provided by the present invention and the molecular specific detection method provided by the prior art, the difference between the two sets of data was compared, and the accuracy and feasibility of the universal method were verified. The results of the measurements are shown in FIG. 4 and Table 3.
TABLE 3
As can be seen from fig. 4 and table 3, the detection results of the universal ADA detection method provided by the present invention and the traditional ADA analysis method are consistent, and the S/N value is higher, so it can be determined that the universal ADA detection method provided by the present invention not only can replace the traditional ADA analysis method, but also has certain advancement.
Application example 4
1. Qualitative and quantitative detection
The avidin-biotin method general-purpose anti-Drug antibody analysis provided in example 2 was performed on the serum samples of the test monkeys injected with the monoclonal antibody Drug, tamela (Drug B), at different sampling time points (since the relationship between the Drug injection amount and injection frequency of the antibody Drug and the experimental results is small, and the method of the present invention is well known to those skilled in the art, the present invention is not described herein in detail). The analysis results are shown in Table 4.
TABLE 4
As can be seen from Table 4, the ADA detection results of the placebo group are negative, and the experimental groups are positive, so that the general detection method provided by the invention can be used for specifically distinguishing the placebo group (the placebo group does not use monoclonal antibody drug injection and cannot generate ADA) from the test group, and the ADA generation trend of the test group conforms to the change rule of the monoclonal antibody drug in the monkey sample, which indicates that the general detection method provided by the invention not only can qualitatively detect ADA, but also can show the quantitative detection effect to a certain extent.
2. Universal detection of different media detection samples
In order to verify the detection effect of the method in other media (such as plasma), the invention performs the same test procedure as the above on the monkey plasma sample corresponding to the monkey serum sample, and verifies the general performance of different media samples. The results are shown in Table 5.
TABLE 5
As can be seen from table 5 above, the placebo group showed ADA negative, i.e. no ADA was detected in the monkey plasma samples; the ADA signal values of the test groups were all high, and positive. That is, in the monkey blood sample, the results of ADA detection in the plasma and serum samples were consistent whether the sample was derived from the plasma or serum sample, demonstrating the feasibility of our developed universal method to detect ADA in the monkey blood sample.
3. Universal testing of different drugs
The same ADA detection was performed on monkey serum samples of bispecific antibody (Drug C, targeting PDL1& TIGIT antibody Drug) and fusion antibody (Drug D, targeting neutralizing antibody for treating new coronavirus), which verifies that the universal detection method provided in example 2 of the present invention is applicable to different projects, and proves the universal performance of the detection method in different antibody drugs. The results of the measurements are shown in tables 6 and 7.
TABLE 6
TABLE 7
As shown in tables 6 and 7, both Drug C (bispecific antibody) and Drug D (fusion antibody) can be detected by the universal ADA detection method provided by the invention, i.e. the placebo group shows negative ADA, the test group shows no positive ADA, and the universal ADA detection method can be applied to ADA detection of different biological antibody drugs and is fully proved to have universality.
In summary, the following steps: the universal ADA detection method developed for the monkey blood specimen completely meets the test requirements, is not only suitable for monkey serum and monkey plasma specimens, but also has no influence on the detection results of different antibody drug projects, has certain reliability of data, has good universality, not only solves the problem of development of the antibody drug ADA detection method before clinical treatment, but also greatly simplifies the experimental operation process, and is an ADA detection method with great innovation.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (8)
1. A preparation method of a mouse anti-monkey IgG species specific antibody comprises the following steps:
immunizing a cynomolgus monkey by using a monoclonal antibody to obtain an anti-drug antibody derived from the monkey;
immunizing a mouse by using an anti-drug antibody derived from a monkey to obtain a mouse anti-monkey antibody;
screening the mouse anti-monkey antibody to obtain the specific antibody of the mouse anti-monkey IgG species.
2. Use of the murine anti-monkey IgG species-specific antibody of claim 1 for the detection of anti-drug antibodies.
3. Use of the murine anti-monkey IgG species-specific antibody of claim 1 in the preparation of a detection kit for anti-drug antibodies.
4. A general detection kit for monkey anti-human anti-drug antibodies comprises the following components: the murine anti-monkey IgG species-specific antibody of claim 1, an ADA positive control, an antibody drug, magnetic beads, and a buffer;
the ADA positive control includes a cynomolgus monkey-derived anti-drug antibody.
5. A general detection method for a preclinical monkey anti-human anti-drug antibody comprises an avidin-biotin method and an antibody method.
6. The universal method for detecting a drug-resistant antibody according to claim 5, wherein the avidin-biotin method comprises the steps of:
(1) mixing the magnetic beads marked with the avidin with the single antibody drug marked with the biotin to obtain magnetic beads combined with the single antibody drug;
(2) placing the magnetic beads combined with the single drug resistance in a plate hole of an enzyme label plate to obtain the enzyme label plate containing the magnetic beads;
(3) adding a sample into the ELISA plate containing the magnetic beads to obtain the ELISA plate containing the sample;
(4) using buffer solution to blow and suck the ELISA plate containing the sample evenly;
(5) after the blowing, sucking and uniformly mixing are finished, performing a binding reaction to obtain a streptavidin magnetic bead compound combined with a specific antibody of the mouse anti-monkey IgG species;
the binding reaction comprises: adding the mouse anti-monkey IgG species specific antibody of claim 1 with a chromogenic label to an ELISA plate containing a streptavidin magnetic bead and sample complex, and performing incubation treatment;
(6) adding a chromogenic substrate into the magnetic bead compound combined with the mouse anti-monkey IgG species specific antibody for chromogenic reaction to obtain a chromogenic reaction magnetic bead compound;
(7) placing a magnetic plate at the bottom of the chromogenic reaction magnetic bead compound, attracting the magnetic beads to the edges of the micropores, and then adding a stop solution;
(8) and (5) detecting by using a microplate reader, and comparing with a blank control group to obtain a detection result.
7. The method for universal detection of drug-resistant antibodies according to claim 6, wherein said avidin-labeled magnetic beads comprise streptavidin magnetic beads.
8. The universal method for detecting a drug-resistant antibody according to claim 5, wherein the antibody method comprises the steps of:
(1) mixing the magnetic beads marked with the mouse anti-human IgG and the monoclonal antibody to obtain magnetic beads combined with the monoclonal antibody;
(2) placing the magnetic beads combined with the monoclonal antibody in a plate hole of an enzyme label plate to obtain the enzyme label plate containing the magnetic beads;
(3) adding a sample into the ELISA plate containing the magnetic beads to obtain the ELISA plate containing the sample;
(4) using buffer solution to blow and suck the ELISA plate containing the sample evenly;
(5) after the blowing, sucking and uniformly mixing are finished, carrying out a binding reaction to obtain a magnetic bead compound combined with a mouse anti-monkey IgG species specific antibody;
the binding reaction comprises: adding the mouse anti-monkey IgG species specific antibody of claim 1 with a chromogenic label to an ELISA plate containing a complex of magnetic beads and a sample, and performing incubation treatment;
(6) adding a chromogenic substrate into the magnetic bead compound combined with the mouse anti-monkey IgG species specific antibody for chromogenic reaction to obtain a chromogenic reaction magnetic bead compound;
(7) placing a magnetic plate at the bottom of the chromogenic reaction magnetic bead compound, attracting the magnetic beads to the edges of the micropores, and then adding a stop solution;
(8) and (5) detecting by using a microplate reader, and comparing with a blank control group to obtain a detection result.
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| CN101506659A (en) * | 2006-09-12 | 2009-08-12 | 弗·哈夫曼-拉罗切有限公司 | Anti-drug antibody assay |
| CN108318680A (en) * | 2018-02-01 | 2018-07-24 | 北京新艾进生物科技有限公司 | A kind of detection method and detection kit of anti-medicine antibody |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101506659A (en) * | 2006-09-12 | 2009-08-12 | 弗·哈夫曼-拉罗切有限公司 | Anti-drug antibody assay |
| CN108318680A (en) * | 2018-02-01 | 2018-07-24 | 北京新艾进生物科技有限公司 | A kind of detection method and detection kit of anti-medicine antibody |
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