CN117003882B - Hybridoma cell strain combination and antibody combination for detecting golimumab and application of hybridoma cell strain combination and antibody combination - Google Patents
Hybridoma cell strain combination and antibody combination for detecting golimumab and application of hybridoma cell strain combination and antibody combination Download PDFInfo
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- CN117003882B CN117003882B CN202311278940.2A CN202311278940A CN117003882B CN 117003882 B CN117003882 B CN 117003882B CN 202311278940 A CN202311278940 A CN 202311278940A CN 117003882 B CN117003882 B CN 117003882B
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention belongs to the technical field of biology, and particularly relates to a hybridoma cell strain combination and antibody combination for detecting golimumab and application thereof. The invention prepares golimumab (Fab) 2 fragment immunogen, immunizes mice, screens anti-golimumab (Fab) 2 fragment idiotype antibody, and finally discovers that a pairing mode which takes antibody Gol-E-4 as a capture antibody and Gol-G-11 as a detection antibody has higher signal to noise ratio, namely better sensitivity, and the pairing method is adopted to establish the detection method of the free blood concentration of the golimumab, and has no cross reaction with a plurality of other TNF-alpha targeted drugs, and has high specificity and high accuracy.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a hybridoma cell strain combination and antibody combination for detecting golimumab and application thereof.
Background
Golimumab (golimumab) is a humanized monoclonal antibody used as tumor necrosis factor alpha (TNF-alpha) immunosuppressive drug. European drug administration (EMA) guidelines used golian monoclonal antibodies for the treatment of rheumatoid arthritis, psoriasis arthritis (psoriatic arthritis) and ankylosing spondylitis. Golian monoclonal antibodies were approved by the U.S. Food and Drug Administration (FDA) and european medicines administration in 2013 for the treatment of ulcerative colitis. The clinical efficacy of anti-TNF-alpha treatment is typically associated with therapeutic antibody trough levels. Monitoring the levels of golimumab drug provides a reference basis for achieving optimal therapeutic regimens.
However, the domestic clinical practice has few monitoring schemes aiming at the blood concentration of the golimumab, which are related to the complicated blood concentration detection method of the golimumab, the lack of consciousness of blood concentration monitoring of people and the like. The detection analysis of golimumab mainly comprises ELISA and LC-MS, but the LC-MS has longer analysis time and complex operation, is difficult to meet the requirement of clinical examination on high efficiency, and has low automation degree and long test time, and can not meet the requirements of clinical high-throughput, rapid and accurate detection.
CN111024958A discloses a reagent for detecting monoclonal antibody and anti-monoclonal antibody and application thereof, the method adopts antibody aiming at antibody drug targeting TNF-alpha as capture peptide and biotinylated TNF-alpha protein as detection object, which results in interference of detection of golimumab antibody by other drugs aiming at TNF-alpha (such as infliximab, adalimus, etanercept, etc.), and has lower specificity and sensitivity.
Disclosure of Invention
The invention aims to make up the defects of the prior art and improve the sensitivity and the specificity of the detection of the golian antibody.
In order to achieve the above object, the present invention provides a hybridoma cell line combination for detecting golimumab, the hybridoma cell line combination including a hybridoma cell line Gol-E-4 and a hybridoma cell line Gol-G-11;
the hybridoma cell strain Gol-E-4 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.45639;
the hybridoma cell strain Gol-G-11 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.45638.
The invention also provides an antibody combination for detecting golimumab, wherein the antibody combination comprises a capture antibody and a detection antibody;
the capture antibody comprises an antibody generated by a hybridoma cell strain Gol-E-4; the hybridoma cell strain Gol-E-4 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.45639;
the detection antibody comprises an antibody generated by a hybridoma cell strain Gol-G-11; the hybridoma cell strain Gol-G-11 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.45638.
The invention also provides application of the hybridoma cell line combination or the antibody combination in preparation of a product for detecting golimumab.
The invention also provides application of the hybridoma cell line combination or the antibody combination in preparation of a product for monitoring the blood concentration level of golimumab.
The invention also provides a kit for detecting the golimumab, which comprises a magnetic particle working solution, an antibody working solution and an indicator solution;
the magnetic particle working solution comprises capture antibody coupled magnetic particles and a magnetic bead buffer solution; the capture antibody comprises an antibody generated by a hybridoma cell strain Gol-E-4; the hybridoma cell strain Gol-E-4 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.45639;
the antibody working solution comprises a tracer-labeled detection antibody and an antibody diluent; the detection antibody comprises an antibody generated by a hybridoma cell strain Gol-G-11; the hybridoma cell strain Gol-G-11 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.45638.
Preferably, the concentration of the capture antibody coupled magnetic particles in the magnetic particle working solution is 0.2-0.6 mg/mL;
the concentration of the tracer-labeled detection antibody in the antibody working solution is 0.1-2 mug/mL.
Preferably, the mass ratio of the magnetic beads to the capture antibodies in the capture antibody coupled magnetic particles is (50-500): 1.
preferably, the tracer-labeled detection antibody comprises a tracer, a detection antibody and a coupling agent;
the mass ratio of the tracer to the detection antibody to the coupling agent is 1: (0.2-1): (0.2-1).
Preferably, the magnetic bead buffer comprises TBST;
preferably, the antibody diluent comprises 50mM 2-morpholinoethanesulfonic acid, 5mg/mL bovine serum albumin, 0.9mg/mL sodium chloride and 1mM MgCl 2 The method comprises the steps of carrying out a first treatment on the surface of the The pH of the antibody diluent is 6.7, and the solvent is PBS;
the indication liquid comprises luminous liquid;
the tracer includes alkaline phosphatase.
The invention also provides a method for detecting the free blood concentration of the golimumab for non-diagnosis and treatment purposes, which comprises the following steps:
mixing a sample to be detected and standard substances with different concentrations with a magnetic particle working solution respectively, incubating at 37 ℃ for 5min, and removing the supernatant to obtain coated magnetic beads; mixing the coated magnetic beads with antibody working solution, incubating for 5min at 37 ℃, and removing the supernatant to obtain detection magnetic beads; mixing the detection magnetic beads with luminous liquid, and counting luminous values;
establishing a standard curve by utilizing the luminescence values measured by the standard substances with different concentrations, and bringing the detection result of the sample to be detected into the standard curve, namely the free blood concentration of the golimumab in the sample to be detected;
the magnetic particle working solution is the magnetic particle working solution in the kit according to the technical scheme;
the antibody working solution is the antibody working solution in the kit according to the technical scheme.
The beneficial effects are that:
the invention provides a hybridoma cell strain combination for detecting golimumab, which comprises a hybridoma cell strain Gol-E-4 and a hybridoma cell strain Gol-G-11; the hybridoma cell strain Gol-E-4 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.45639; the hybridoma cell strain Gol-G-11 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.45638. The antibody produced by the hybridoma cell line combination can be used for specifically detecting the golimumab, the blood concentration level of the golimumab is monitored, and the results of the embodiment show that the antibody produced by the hybridoma cell line combination has higher signal to noise ratio (26) and high sensitivity when being used for detection, and has no cross reaction with various other TNF-alpha targeted drugs in the process of detecting the golimumab, and has high specificity and high accuracy.
Biological preservation information
The hybridoma cell strain Gol-E-4, classified and named as hybridoma cells, was deposited in China general microbiological culture Collection center, address: the national institute of microbiology, national institute of sciences, 1, 3, north chen, western region, beijing, city, zip code: 100101, the preservation number is CGMCC No.45639;
the hybridoma cell strain Gol-G-11, classified and named as hybridoma cell, is preserved in China general microbiological culture Collection center, address: the national institute of microbiology, national institute of sciences, 1, 3, north chen, western region, beijing, city, zip code: 100101 and the preservation number is CGMCC No.45638.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below.
FIG. 1 shows SDS-PAGE of golian (Fab) 2 fragments;
FIG. 2 is a calibration curve of the detection of the free blood concentration of golian antibodies by the magnetic particle luminescence method.
Detailed Description
The invention provides a hybridoma cell strain combination for detecting golimumab, which comprises a hybridoma cell strain Gol-E-4 and a hybridoma cell strain Gol-G-11;
the hybridoma cell strain Gol-E-4 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.45639;
the hybridoma cell strain Gol-G-11 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.45638.
The invention also provides an antibody combination for detecting golimumab, wherein the antibody combination comprises a capture antibody and a detection antibody;
the capture antibody comprises an antibody generated by a hybridoma cell strain Gol-E-4; the hybridoma cell strain Gol-E-4 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.45639;
the detection antibody comprises an antibody generated by a hybridoma cell strain Gol-G-11; the hybridoma cell strain Gol-G-11 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.45638.
The invention uses the antibody produced by the hybridoma cell strain Gol-E-4 as a capture antibody, uses the antibody produced by the hybridoma cell strain Gol-G-11 as a detection antibody to specifically detect the golimumab, has higher signal to noise ratio, namely better sensitivity, and has no cross reaction with various other TNF-alpha targeted drugs in the process of detecting the golimumab and high specificity.
According to the advantages of the hybridoma cell line combination and the antibody combination, the application of the hybridoma cell line combination or the antibody combination in preparing a product for detecting the golimumab and/or a product for monitoring the blood concentration level of the golimumab belongs to the protection scope of the invention. In the present invention, the product preferably comprises a kit, further preferably a magnetic particle chemiluminescent detection kit.
The invention also provides a kit for detecting the golimumab, which comprises a magnetic particle working solution, an antibody working solution and an indicator solution;
the magnetic particle working solution comprises capture antibody coupled magnetic particles and a magnetic bead buffer solution; the capture antibody comprises an antibody generated by a hybridoma cell strain Gol-E-4; the hybridoma cell strain Gol-E-4 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.45639;
the antibody working solution comprises a tracer-labeled detection antibody and an antibody diluent; the detection antibody comprises an antibody generated by a hybridoma cell strain Gol-G-11; the hybridoma cell strain Gol-G-11 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.45638.
In the invention, the concentration of the capture antibody coupled magnetic particles in the magnetic particle working solution is preferably 0.2-0.6 mg/mL, and more preferably 0.4mg/mL; the mass ratio of the magnetic beads to the capture antibodies in the capture antibody coupled magnetic particles is preferably (50-500): 1, more preferably (50 to 100:1), still more preferably 50:1. the magnetic beads of the present invention preferably comprise Dynal beads M280 Tosyl magnetic beads, purchased from Simer's Biochemical pharmaceutical Co., ltd. The magnetic particle buffer of the present invention preferably comprises TBST.
In the present invention, the preparation method of the capture antibody-coupled magnetic particles preferably includes: mixing 50mM borate buffer solution with pH of 8.0 with magnetic beads, mixing with the capture antibody after obtaining the magnetic bead buffer solution, carrying out shake reaction for 8 hours at 37 ℃, and removing the supernatant by magnetic attraction to obtain the capture antibody coupled magnetic particles.
In the present invention, the concentration of the tracer-labeled detection antibody in the antibody working solution is preferably 0.1 to 2. Mu.g/mL, more preferably 0.5 to 1.5. Mu.g/mL, and most preferably 1. Mu.g/mL.
In the present invention, the tracer-labeled detection antibody preferably includes a tracer, a detection antibody, and a coupling agent; the mass ratio of the tracer to the detection antibody to the coupling agent is preferably 1: (0.2-1): (0.2 to 1), more preferably 1:0.5:0.5. the tracer of the invention preferably comprises alkaline phosphatase; the coupling agent preferably comprises glutaraldehyde. The tracer, the detection antibody and the coupling agent are preferably mixed and uniformly mixed for 2 hours at room temperature to obtain the tracer-labeled detection antibody.
In the present invention, the antibody diluent preferably comprises 50mM MES, 5mg/mL bovine serum albumin, 0.9mg/mL sodium chloride and 1mM MgCl 2 The method comprises the steps of carrying out a first treatment on the surface of the The pH of the antibody dilution is preferably 6.7 and the solvent is preferably PBS. The indicator liquid according to the present invention preferably comprises a luminescent liquid.
The invention also provides a method for detecting the free blood concentration of the golimumab for non-diagnosis and treatment purposes, which comprises the following steps:
mixing a sample to be detected and standard substances with different concentrations with a magnetic particle working solution respectively, incubating at 37 ℃ for 5min, and removing the supernatant to obtain coated magnetic beads; mixing the coated magnetic beads with antibody working solution, incubating for 5min at 37 ℃, and removing the supernatant to obtain detection magnetic beads; mixing the detection magnetic beads with luminous liquid, and counting luminous values;
establishing a standard curve by utilizing the luminescence values measured by the standard substances with different concentrations, and bringing the detection result of the sample to be detected into the standard curve, namely the free blood concentration of the golimumab in the sample to be detected;
the magnetic particle working solution is the magnetic particle working solution in the kit according to the technical scheme;
the antibody working solution is the antibody working solution in the kit according to the technical scheme.
According to the invention, the sample to be detected is incubated by using the magnetic particle working solution, the adalimumab in the sample to be detected is coated by the coating Gol-E-4 in the magnetic particle working solution, and then the adalimumab free blood concentration is detected by pairing the capture antibody Gol-G-11 in the antibody working solution with the coating Gol-E-4 and detecting a luminescence value.
The kit and the detection method provided by the invention can specifically detect the golimumab, and the results of the examples show that the golimumab is detected without cross reaction with various other TNF-alpha targeted drugs, and the kit and the detection method have high specificity and high precision.
For further explanation of the present invention, a hybridoma cell line combination and an antibody combination for detecting golimumab and applications thereof, which are provided in the present invention, are described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Preparation of golian (Fab) 2 fragment immunogen, consisting of the following steps:
(1) Diluting 20mg of golian antibody to 5mg/mL with 20mM phosphate buffer (PB, pH 7.4), adding 2mg of Idez protease (purchased from Beijing AoTide Biotechnology Co., ltd.) to a total volume of 4mL, and incubating at 37℃for 30min to obtain an incubated product;
(2) Adsorbing unreacted full-length antibody and cut Fc segment in the incubation product obtained in the step (1) by using a ProteinA column to obtain a first product; the IdeZ enzyme in the first product was removed by Ni affinity chromatography, the flow-through was collected, the concentration was determined by OD280, SDS-PAGE identification and cryopreservation at-20 ℃. SDS-PAGE detection results are shown in FIG. 1.
As can be seen from fig. 1, a golian (Fab) 2 fragment can be obtained after cleavage of the full length golian antibody. The molecular weights of the light chain Fab and the heavy chain Fab were similar on SDS-PAGE, thus showing coincident bands of electrophoresis.
Example 2
Preparation of anti-golimu (Fab) 2 fragment idiotype antibody
The preparation of the anti-golian idiotype monoclonal antibody adopts a hybridoma cell fusion technology, and comprises the following specific steps:
diluting the golian (Fab) 2 fragment immunogen prepared in the example 1 to 1mg/mL with PBS buffer solution respectively, adding an equal volume of Freund's complete adjuvant, emulsifying completely, and performing primary immunization on mice according to a dose of 0.1 mg/mouse; after 4 weeks of interval, taking 1mg of the golian (Fab) 2 fragment immunogen prepared in the example 1, mixing and emulsifying the same volume of Freund's incomplete adjuvant, and performing secondary immunization on a mouse after one immunization according to the dosage of 0.1 mg/mouse;
after 3 days, taking the second immune spleen cells to fuse with Sp2/0 cells, obtaining a plurality of hybridoma cells and numbering; ELISA 96-well plates are coated with golian drugs (2 mug/mL), the culture supernatant of hybridoma cells is subjected to titer determination, and clones positive in titer detection are subjected to competitive screening:
competitive screening ELISA 96-well plates were coated with golian drug (2. Mu.g/mL), 50. Mu.L per well, 10. Mu.g/mL TNF-. Alpha.protein was added, and the culture supernatants of hybridoma cells were assayed for OD450 values by conventional ELISA methods. Cell titers were selected to measure strong positives, while clones with weak positives or negatives were competitively screened, and finally 9 hybridoma cells producing competitive anti-golian idiotype antibodies were screened, with specific information shown in table 1.
TABLE 1 anti-golimumab (Fab) 2 fragment idiotype antibody titre assay
Note that: OD450 > 1 is strong positive, OD450 is less than or equal to 0.3 is weak positive or negative.
Example 3
Screening of paired antibodies;
the antibody obtained in example 2 was used as a capture antibody or a detection antibody, respectively, wherein the detection antibody was conjugated with HRP (horseradish peroxidase) to obtain a golian idiotype antibody-HRP conjugate, and the paired antibody was screened by checkerboard method:
coating ELISA 96-well plates with detection antibodies (2 mug/mL), adding 5 mug/mL of golian drugs, 50 mug/mL of golian drugs in each well, washing 3 times with PBST after reacting for 30min, adding 2 mug/mL of anti-golian idiotype antibody-HRP conjugate, 50 mug/mL of anti-golian drugs in each well, washing 3 times with PBST after reacting for 30min, adding TMB color developing solution, reading OD450 value with an enzyme-labeled instrument, and marking as S5; meanwhile, a treatment mode without adding the golian drugs is set as a control, an OD450 value is read by an enzyme-labeling instrument, the result is marked as S0, and the signal-to-noise ratio (S5/S0) is calculated. The screening has only 2 pairing modes, and specific information is shown in table 2.
TABLE 2 screening results for paired antibodies
As can be seen from Table 2, the pairing mode using the antibody Gol-E-4 as the capture antibody and the antibody Gol-G-11 as the detection antibody has higher signal to noise ratio, i.e. better sensitivity, and the subsequent establishment of the method for detecting the free blood concentration of the golimumab by using the pairing will preserve the hybridoma cell Gol-E-4 producing the antibody Gol-E-4 and the hybridoma cell Gol-G-11 producing the antibody Gol-G-11, respectively.
Example 4
Establishment of method for detecting free blood concentration of golian antibody (particle luminescence method)
1. Preparation of magnetic particle working liquid
50mg Dynal beads M280 Tosyl magnetic beads are diluted in 2mL of 50mM borate buffer solution with pH of 8.0, 1mg of anti-golimu Gol-E-4 antibody generated by the hybridoma cell Gol-E-4 in example 2 is added, and after being uniformly mixed, the mixture is subjected to shaking reaction at 37 ℃ for 8 hours; removing the supernatant by magnetic attraction, adding TBST (50 mM Tris, 0.9wt% NaCl, 0.1wt% TW20 and the balance of ultrapure water, pH 7.4), and reacting at 37 ℃ for 12 hours; removing the supernatant by magnetic attraction, adding TBST, diluting to 0.2-0.6 mg/mL, and obtaining a magnetic particle working solution, and preserving at 2-8 ℃ for later use.
2. Preparation of antibody working solution
1mg alkaline phosphatase was dissolved in 1mL of PBS, 0.5mg of the anti-golimu Gol-G-11 antibody produced by the hybridoma cell Gol-G-11 of example 2 was added, mixed well, 10. Mu.L of 50wt.% glutaraldehyde solution was added, mixed well at room temperature for 2 hours, dialyzed into PBS, and diluted with antibody (50 mM MES, 0.9 wt.% NaCl, 5mg/mL BSA, 1mM MgCl) 2 And the balance of ultrapure water, pH 6.7), diluted to 1. Mu.g/mL to obtain an antibody working solution.
3. Drawing of calibration curves
The golimumab is diluted by human serum matrix which is not treated by the golimumab, and the golimumab calibrator with different concentrations is prepared (the concentrations of the golimumab are 0, 0.5, 2, 5, 20 and 50 mug/mL in sequence)
Diluting the calibrator 10 times by using PBS, mixing 20 mu L of diluted calibrator with 50 mu L of the magnetic particle working solution obtained in the step 1 for 5min, cleaning, removing the supernatant, adding 50 mu L of the antibody working solution obtained in the step 2, incubating at 37 ℃ for 5min, cleaning, adding the AMPPD luminescent solution for color development, counting the luminescent value, and drawing a standard curve, wherein the results are shown in Table 3 and figure 2.
TABLE 3 data on calibration curves of free blood concentration of Fagolimumab by particle luminescence
According to table 3 and fig. 2, the calibration curve obtained by the magnetic particle luminescence method is: four parameter equations: y= (A-D)/[ 1+ (X/C) B]+D; wherein: a= 8951.481445; b= 0.9546268; c= 65950.0625; d= 9142602752; correlation coefficient R 2 ：0.999。
4. And (3) precision detection: diluting golimumab with human serum matrix which is not treated by the golimumab, preparing a high-concentration golimumab solution and a low-concentration golimumab solution, measuring 10 holes at each concentration point in one experiment, and calculating an intra-batch variation Coefficient (CV) according to SD/Mean, wherein the concentration of the golimumab in the high-concentration golimumab solution is 10.0 mug/mL; the concentration of the golimumab in the low-concentration golimumab solution is 2.0 mug/mL;
mixing 20 mu L of the sample to be detected and 50 mu L of the magnetic particle working solution obtained in the step 1 for 5min, then cleaning, removing the supernatant, adding 50 mu L of the antibody working solution obtained in the step 2, incubating at 37 ℃ for 5min, cleaning, adding the AMPPD luminescent solution for color development, counting the luminescent value, substituting the luminescent value into the calibration curve of the step 3, and calculating the concentration of golimumab, wherein the result is shown in Table 4.
TABLE 4 precision measurement results
As can be seen from Table 4, the magnetic particle luminescence method prepared by using the Gol-E-4/Gol-G-11 paired antibody has high precision in measuring the concentration of the golimumab, and can be used for measuring the content of the golimumab.
5. Cross-reaction detection
5 cross-reactive species (human IgG1 protein, etanercept protein, infliximab, wu Sinu mab and adalimumab) were selected and diluted to 50 μg/mL respectively using human serum matrix not treated with golimumab; diluting the golimumab by using a human serum matrix which is not treated by the golimumab, and preparing a high-concentration golimumab solution and a low-concentration golimumab solution, wherein the concentration of the golimumab in the high-concentration golimumab solution is 30 mug/mL; the concentration of the golimumab in the low-concentration golimumab solution is 8 mug/mL; calculating the cross reaction rate;
mixing 20 mu L of the sample to be detected and 50 mu L of the magnetic particle working solution obtained in the step 1 for 5min, then cleaning, removing the supernatant, adding 50 mu L of the antibody working solution obtained in the step 2, incubating at 37 ℃ for 5min, cleaning, adding the AMPPD luminescent solution for color development, counting the luminescent value, substituting the luminescent value into the calibration curve of the step 3, calculating the concentration of golimumab, and calculating the cross reaction rate according to the following formula, wherein the result is shown in Table 5;
cross reaction Rate= (measured concentration-theoretical concentration)/Cross reactant concentration X100%
TABLE 5 Cross reaction results
As can be seen from Table 5, the magnetic particle luminescence method established by Gol-E-4/Gol-G-11 paired antibody is used for detecting the concentration of the golimumab, and has no cross reaction with various other TNF-alpha targeted drugs and high specificity.
According to the above, the invention has higher sensitivity, specificity and precision when the Gol-E-4/Gol-G-11 paired antibody is adopted to detect the golimumab, and can meet the detection requirement of the golimumab.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (10)
1. A hybridoma cell line combination for detecting golimumab, characterized in that the hybridoma cell line combination comprises a hybridoma cell line Gol-E-4 and a hybridoma cell line Gol-G-11;
the hybridoma cell strain Gol-E-4 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.45639;
the hybridoma cell strain Gol-G-11 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.45638.
2. An antibody combination for detecting golimumab, wherein the antibody combination comprises a capture antibody and a detection antibody;
the capture antibody comprises an antibody generated by a hybridoma cell strain Gol-E-4; the hybridoma cell strain Gol-E-4 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.45639;
the detection antibody comprises an antibody generated by a hybridoma cell strain Gol-G-11; the hybridoma cell strain Gol-G-11 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.45638.
3. Use of the hybridoma cell line combination of claim 1 or the antibody combination of claim 2 for the preparation of a product for detecting golimumab.
4. Use of the hybridoma cell line combination of claim 1 or the antibody combination of claim 2 for the preparation of a product for monitoring the blood concentration level of golimumab.
5. A kit for detecting golimumab, which is characterized by comprising a magnetic particle working solution, an antibody working solution and an indicator solution;
the magnetic particle working solution comprises capture antibody coupled magnetic particles and a magnetic bead buffer solution; the capture antibody comprises an antibody generated by a hybridoma cell strain Gol-E-4; the hybridoma cell strain Gol-E-4 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.45639;
the antibody working solution comprises a tracer-labeled detection antibody and an antibody diluent; the detection antibody comprises an antibody generated by a hybridoma cell strain Gol-G-11; the hybridoma cell strain Gol-G-11 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.45638.
6. The kit of claim 5, wherein the concentration of the capture antibody-coupled magnetic particles in the magnetic particle working fluid is 0.2-0.6 mg/mL;
the concentration of the tracer-labeled detection antibody in the antibody working solution is 0.1-2 mug/mL.
7. The kit of claim 6, wherein the mass ratio of the magnetic beads to the capture antibodies in the capture antibody-coupled magnetic particles is (50-500): 1.
8. the kit of claim 6, wherein the tracer-labeled detection antibody comprises a tracer, a detection antibody, and a coupling agent;
the mass ratio of the tracer to the detection antibody to the coupling agent is 1: (0.2-1): (0.2-1).
9. The kit of claim 5, wherein the magnetic bead buffer comprises TBST;
the antibody diluent comprises 50mM 2-morpholinoethanesulfonic acid, 5mg/mL bovine serum albumin, 0.9mg/mL sodium chloride and 1mM MgCl 2 The method comprises the steps of carrying out a first treatment on the surface of the The pH of the antibody diluent is 6.7, and the solvent is PBS;
the indication liquid comprises luminous liquid;
the tracer includes alkaline phosphatase.
10. A method for detecting the free blood concentration of golimumab for non-diagnostic and therapeutic purposes, comprising the steps of:
mixing a sample to be detected and standard substances with different concentrations with a magnetic particle working solution respectively, incubating at 37 ℃ for 5min, and removing the supernatant to obtain coated magnetic beads; mixing the coated magnetic beads with antibody working solution, incubating for 5min at 37 ℃, and removing the supernatant to obtain detection magnetic beads; mixing the detection magnetic beads with luminous liquid, and counting luminous values;
establishing a standard curve by utilizing the luminescence values measured by the standard substances with different concentrations, and bringing the detection result of the sample to be detected into the standard curve, namely the free blood concentration of the golimumab in the sample to be detected;
the magnetic particle working solution is the magnetic particle working solution in the kit according to any one of claims 5-9;
the antibody working solution is the antibody working solution in the kit according to any one of claims 5 to 9.
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