CN117187189B - Hybridoma cell strain combination and antibody combination for detecting Wu Sinu monoclonal antibody and application thereof - Google Patents
Hybridoma cell strain combination and antibody combination for detecting Wu Sinu monoclonal antibody and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a hybridoma cell strain combination and antibody combination for detecting Wu Sinu monoclonal antibodies and application thereof. The invention prepares Wu Sinu (Fab) 2 fragment immunogen, immunizes mice, screens anti-Wu Sinu (Fab) 2 fragment idiotype antibody, and finally discovers that the pairing mode of taking antibody Ust-B-1 as a coated antibody and using Ust-G-10 as a detection antibody has higher signal to noise ratio, namely better sensitivity, and the pairing method is adopted to establish Wu Sinu monoclonal antibody free blood concentration detection method, and has no cross reaction with various other commonly used antibody medicines, 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 Wu Sinu monoclonal antibodies and application thereof.
Background
Wu Sinu monoclonal antibody (Ustekinumab), called Wunumab for short, is a humanized monoclonal antibody, and aims at interleukin 12 and interleukin 23 to regulate the immune system and the occurrence of immune-mediated inflammatory diseases. Ustekinumab was shown to be safe and effective in two phase III clinical trials for moderately severe psoriasis, for a maximum of 76 weeks. Ustekinumab is also useful for the treatment of moderate to severe plaque psoriasis, active psoriasis and Crohn's disease, and generally requires higher injection doses.
Clinical efficacy is typically related to the concentration level of therapeutic antibody, and monitoring the concentration of therapeutic antibody may provide a reference for achieving an optimal therapeutic regimen. However, the domestic clinical practice has few monitoring schemes aiming at the blood concentration of Wu Sinu monoclonal antibody, which are related to the complicated blood concentration detection method of Wu Sinu monoclonal antibody, lack of consciousness of blood concentration monitoring and the like. The detection analysis of Wu Sinu monoclonal antibody mainly comprises an ELISA method and an LC-MS method, but the LC-MS method has longer analysis time and complex operation, is difficult to meet the requirement of clinical examination on high efficiency, has low automation degree and long test time, and cannot meet the requirements of clinical high-throughput, rapid and accurate detection.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a method for simply and rapidly detecting a Wu Sinu monoclonal antibody hybridoma cell strain combination and an antibody combination so as to improve the sensitivity, the specificity and the precision of Wu Sinu monoclonal antibody detection.
In order to achieve the above object, the present invention provides a hybridoma cell line combination for detecting Wu Sinu monoclonal antibodies, the hybridoma cell line combination comprising a hybridoma cell line Ust-B-1 and a hybridoma cell line Ust-G-10;
the hybridoma cell strain Ust-B-1 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.45641;
the hybridoma cell strain Ust-G-10 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.45640.
The invention also provides an antibody combination for detecting the ulinunimab, which comprises a coated antibody and a detection antibody;
the coated antibody comprises an antibody generated by a hybridoma cell strain Ust-B-1; the hybridoma cell strain Ust-B-1 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.45641;
the detection antibody comprises an antibody generated by a hybridoma cell strain Ust-G-10; the hybridoma cell strain Ust-G-10 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.45640.
The invention also provides application of the hybridoma cell line combination or the antibody combination in preparation of a product for detecting Wu Sinu monoclonal antibody.
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 Wu Sinu monoclonal antibody.
The invention also provides a kit for detecting the ulinunimab, which comprises a magnetic particle working solution, an antibody working solution and an indicator solution;
the magnetic particle working solution comprises coated antibody coupled magnetic particles and a magnetic bead buffer solution; the coated antibody comprises an antibody generated by a hybridoma cell strain Ust-B-1; the hybridoma cell strain Ust-B-1 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.45641;
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 Ust-G-10; the hybridoma cell strain Ust-G-10 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.45640.
Preferably, the concentration of the coated 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 in the coated antibody-coupled magnetic particles to the coated antibody 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 a TBST buffer;
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 water;
the indication liquid comprises luminous liquid;
the tracer includes alkaline phosphatase.
The invention also provides a method for detecting the concentration of Wu Sinu monoclonal antibody free blood 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 luminescence value of the sample to be detected into the standard curve, namely the concentration of Wu Sinu monoclonal antibody free blood 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 Wu Sinu monoclonal antibodies, which comprises a hybridoma cell strain Ust-B-1 and a hybridoma cell strain Ust-G-10; the hybridoma cell strain Ust-B-1 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.45641; the hybridoma cell strain Ust-G-10 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.45640. The antibody produced by the hybridoma cell line combination can be used for specifically detecting Wu Sinu monoclonal antibodies and monitoring the blood concentration level of Wu Sinu monoclonal antibodies, and the results of the examples 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 common antibody medicines in the process of detecting Wu Sinu monoclonal antibodies, and has high specificity and high accuracy.
Biological preservation information
Hybridoma cell line Ust-B-1, classified and named as hybridoma cell, was deposited in China general microbiological culture Collection center, address: in China, beijing, chaoyang area, north Chen Xiyu No. 1, 3, institute of microbiology, academy of sciences, post code: 100101, the preservation number is CGMCC No.45641;
hybridoma cell line Ust-G-10, classified and named as hybridoma cell, was deposited in China general microbiological culture Collection center, address: in China, beijing, chaoyang area, north Chen Xiyu No. 1, 3, institute of microbiology, academy of sciences, post code: 100101 and the preservation number is CGMCC No.45640.
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 is a SDS-PAGE electrophoresis of Wu Sinu (Fab) 2 fragments;
FIG. 2 is a standard curve of the magnetic particle luminescence method for detecting the free blood concentration of Wu Sinu antibody.
Detailed Description
The invention provides a hybridoma cell strain combination for detecting Wu Sinu monoclonal antibodies, which comprises a hybridoma cell strain Ust-B-1 and a hybridoma cell strain Ust-G-10;
the hybridoma cell strain Ust-B-1 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.45641;
the hybridoma cell strain Ust-G-10 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.45640.
The invention also provides an antibody combination for detecting the ulinunimab, which comprises a coated antibody and a detection antibody;
the coated antibody comprises an antibody generated by a hybridoma cell strain Ust-B-1; the hybridoma cell strain Ust-B-1 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.45641;
the detection antibody comprises an antibody generated by a hybridoma cell strain Ust-G-10; the hybridoma cell strain Ust-G-10 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.45640.
The antibody generated by the hybridoma cell strain Ust-B-1 is used as a coating antibody, the antibody generated by the hybridoma cell strain Ust-G-10 is used as a detection antibody for specifically detecting the Wu Sinu monoclonal antibody, the signal to noise ratio is higher, namely the sensitivity is better, and in the process of detecting the Wu Sinu monoclonal antibody, the antibody has no cross reaction with various other clinically commonly used antibody medicines and the specificity is high.
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 Wu Sinu monoclonal antibody and/or a product for monitoring the blood concentration level of Wu Sinu monoclonal antibody also 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 ulinunimab, which comprises a magnetic particle working solution, an antibody working solution and an indicator solution;
the magnetic particle working solution comprises coated antibody coupled magnetic particles and a magnetic bead buffer solution; the coated antibody comprises an antibody generated by a hybridoma cell strain Ust-B-1; the hybridoma cell strain Ust-B-1 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.45641;
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 Ust-G-10; the hybridoma cell strain Ust-G-10 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.45640.
In the invention, the concentration of the coated 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 in the coated antibody coupled magnetic particles to the coated antibody is preferably (50-500): 1, more preferably (50 to 100): 1, 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 a TBST buffer.
In the present invention, the preparation method of the coated antibody-coupled magnetic particles preferably comprises: mixing 50mM borate buffer solution with pH of 8.0 with magnetic beads, mixing with the coated 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 coated 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 water, and more preferably ultrapure water. The indicator liquid according to the present invention preferably includes a luminescent liquid, and more preferably an AMPPD luminescent liquid.
The invention also provides a method for detecting the concentration of Wu Sinu monoclonal antibody free blood 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 luminescence value of the sample to be detected into the standard curve, namely the concentration of Wu Sinu monoclonal antibody free blood 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 invention preferably mixes the detection sample and the magnetic particle working solution, incubates for 5min at 37 ℃, and then removes the supernatant to obtain the 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 the luminous liquid, and counting the luminous value.
The invention preferably mixes the standard substances with different concentrations with the magnetic particle working solution respectively, incubates for 5min at 37 ℃, and then removes the supernatant to obtain the 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 the luminous liquid, and counting the luminous value.
The invention establishes a standard curve by utilizing the luminescence values measured by the standard substances with different concentrations and the corresponding concentrations, and brings the luminescence value of the sample to be detected into the standard curve, namely the concentration of Wu Sinu monoclonal antibody free blood in the sample to be detected.
According to the invention, the sample to be detected is incubated by using the magnetic particle working solution, the coated antibody Ust-B-1 in the magnetic particle working solution can coat the Wu Sinu monoclonal antibody in the sample to be detected, and then the detection antibody Ust-G-10 in the antibody working solution is paired with the coated antibody Ust-B-1, so that the detection of the concentration of Wu Sinu monoclonal antibody free blood is realized by detecting a luminescence value.
The kit and the detection method provided by the invention can be used for specifically detecting Wu Sinu monoclonal antibody, and the results of the examples show that the Wu Sinu monoclonal antibody has no cross reaction with various other clinically common antibody medicines at the same time, and has high specificity and high precision.
For further explanation of the present invention, a hybridoma cell line combination and an antibody combination for detecting Wu Sinu monoclonal antibody and applications thereof, which are provided by the present invention, will be described in detail with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of the present invention.
Example 1
An antigen binding fragment of Wu Sinu mab ((Fab) 2 fragment, i.e. light chain fab+heavy chain Fab fragment) was used as immunogen, wherein the preparation of the (Fab) 2 fragment immunogen consisted of the following steps:
(1) Diluting 20mg Wu Sinu antibody to 5mg/mL with 20mM phosphate buffer (PB, pH 7.4), adding 2mg IdeZ protease (purchased from Beijing AoTide Biotechnology Co., ltd.) to total volume of 4mL, and incubating at 37deg.C 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; removing IdeZ enzyme in the first product by Ni affinity chromatography, collecting flow-through liquid, measuring the concentration by OD280, and freezing and preserving at-20 ℃;
SDS-PAGE was performed using Wu Sinu antibody (labeled antibody), ideZ protease (labeled IdeZ) and fluid flow-through (labeled cleavage product) as samples, and the results are shown in FIG. 1.
As can be seen from FIG. 1, the cleavage product obtained after cleavage of the full-length Wu Sinu antibody contains a fragment of Wunu (Fab) 2. Light chain Fab and heavy chain Fab can be seen on SDS-PAGE.
Example 2
Preparation of antibodies against the idiotype of the Wu Sinu (Fab) 2 fragment
The preparation of the anti-Wu Sinu idiotype monoclonal antibody adopts a hybridoma cell fusion technology, and comprises the following specific steps:
collecting Wu Sinu (Fab) 2 fragments in the digestion product of the example 1, diluting Wu Sinu (Fab) 2 fragments to 1mg/mL by using PBS buffer, adding an equal volume of Freund's complete adjuvant, emulsifying completely, and performing primary immunization on mice according to the dosage of 0.1mg Wu Sinu (Fab) 2 fragments/only; after 4 weeks of interval, taking 1mg/mL of Wu Sinu (Fab) 2 fragment diluent, mixing and emulsifying the diluent with Freund's incomplete adjuvant in an equal volume, and performing secondary immunization on a mice after one immunization according to the dosage of 0.1mg Wu Sinu (Fab) 2 fragment/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 Wu Sinu monoclonal antibody (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 a Wunu antibody drug (2. Mu.g/mL), 50. Mu.L per well, 5. Mu.g/mL IL-23 protein and 5. Mu.g/mL IL-12 protein were 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 competitively screening for weakly positive or negative clones, and finally 9 hybridoma cells producing antibodies against the competitive antigen Wu Sinu idiotype were selected, with specific information shown in table 1.
TABLE 1 detection of idiotype antibody titers against Wu Sinu (Fab) 2 fragments
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;
using the antibody obtained in example 2 as a sample, as a coating antibody or a detection antibody, respectively, screening the paired antibodies by using a chessboard method, wherein the total of 9*8 =72 paired methods; wherein the detection antibody is conjugated with HRP (horseradish peroxidase) to give a Wu Sinu idiotype antibody-HRP conjugate:
coating ELISA 96-well plates by using coating antibodies (2 mug/mL), adding 5 mug/mL of Wu Sinu monoclonal antibodies into each well by 50 mug/mL, washing 3 times by using PBST after reacting for 30min, adding 2 mug/mL of anti-Wu Sinu idiotype antibody-HRP conjugate into each well by 50 mug/mL, washing 3 times by using PBST after reacting for 30min, adding TMB color developing solution, reading OD450 value by using an enzyme-labeled instrument, and marking as S5; meanwhile, a treatment mode without adding Wu Sinu medicine is set as a control, an OD450 value is read by an enzyme-labeling instrument, the result is marked as S0, and a signal-to-noise ratio (S/N) is calculated. The screening has only 2 pairing modes to realize effective pairing, 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 Ust-B-1 as the coating antibody and the Ust-G-10 as the detection antibody has higher signal to noise ratio, i.e. better sensitivity, and the hybridoma cell Ust-B-1 producing the antibody Ust-B-1 and the hybridoma cell Ust-G-10 producing the antibody Ust-G-10 are respectively preserved by subsequently adopting the pairing to establish the method for detecting the concentration of the free blood of the ulinumab.
Example 4
Establishment of method for detecting free blood concentration of Wu Jiu antibody (microparticle 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-Wu Sinu Ust-B-1 antibody generated by hybridoma cell Ust-B-1 in example 3 is added, and after uniform mixing, shaking reaction is carried out 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 0.4mg/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 (ALP) was dissolved in 1mL of PBS, 0.5mg of anti-Wu Sinu antibody Ust-G-10 produced by hybridoma cell Ust-G-10 of example 3 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, 0.9wt.% NaCl, 5mg/mL BSA, 1mM MgCl) 2 And the balance of ultrapure water, pH 6.7) to 1. Mu.g/mL to obtain an antibody working solution.
3. Drawing of a Standard Curve
Diluting Wu Sinu monoclonal antibody by using a human serum matrix which is not treated by the ulinunimab, and preparing Wu Sinu calibrator with different concentrations (the concentration of Wu Sinu monoclonal antibody is 0, 0.5, 2, 5, 20 and 50 mug/mL in sequence);
and (2) diluting the calibrator 10 times by using PBS, mixing 10 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, removing the supernatant, 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 standard curve data for free blood concentration of monoclonal antibody Wu Sinu by microparticle luminescence
According to Table 3 and FIG. 2, the standard curve obtained by the magnetic particle luminescence methodThe method comprises the following steps: four parameter equations: y= (A-D)/[ 1+ (X/C) B]+D; wherein: a= 12980.69043; b= 0.97545528; c= 104691.0234; d= 21257211904; correlation coefficient R 2 ：0.999。
4. And (3) precision detection: diluting Wu Sinu monoclonal antibody by using a human serum matrix which is not treated by the ulinunimab, preparing Wu Sinu monoclonal antibody solution with high concentration and low concentration, detecting the luminescence values of Wu Sinu monoclonal antibody solution with high concentration and low concentration in a mode of step 3, measuring 10 holes (numbered 1-10 in sequence) at each concentration point in one experiment, and calculating the intra-batch variation Coefficient (CV) according to SD/Mean, wherein the concentration of Wu Sinu monoclonal antibody in the high concentration Wu Sinu monoclonal antibody solution is 10.0 mug/mL; the concentration of Wu Sinu mab in the low concentration Wu Sinu mab solution is 2.0 μg/mL;
mixing 10 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, removing the supernatant, adding the AMPPD luminescent solution for color development, counting the luminescent value, substituting the luminescent value into a standard curve of the step 3, and calculating the concentration of Wu Sinu monoclonal antibodies, 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 using the use-B-1/use-G-10 conjugate antibody provides high precision in measuring the concentration of Wu Sinu monoclonal antibody, and can be used for measuring the content of Wu Sinu monoclonal antibody.
5. Cross-reaction detection
Selecting 5 cross reactants (human IgG1 protein, etanercept protein, adalimumab, golimumab and infliximab), diluting Wu Sinu mab by using human serum matrix which is not treated by the ulinunimab, and preparing Wu Sinu mab solution with high concentration and low concentration, wherein the concentration of Wu Sinu mab in the high-concentration Wu Sinu mab solution is 30 mug/mL; the concentration of Wu Sinu monoclonal antibody in the low-concentration Wu Sinu monoclonal antibody solution is 8 mug/mL; adding any one of the cross reactants into the Wu Sinu monoclonal antibody solution with high concentration and low concentration respectively to enable the final concentration of the cross reactant to be 50 mug/mL, taking the final concentration as a sample to be detected, and calculating the cross reaction rate;
mixing 10 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, removing the supernatant, adding the AMPPD luminescent solution for color development, counting the luminescent value, substituting the luminescent value into a standard curve of the step 3, calculating Wu Sinu monoclonal antibody concentration, calculating the cross reaction rate according to the following formula, and obtaining the result shown in the table 5;
cross-reaction rate = (measured concentration-theoretical concentration)/cross-reactant concentration x 100%;
wherein the measured concentration is the concentration of Wu Sinu monoclonal antibody in the sample to be detected; the theoretical concentration is the concentration of Wu Sinu monoclonal antibody added when preparing a sample to be detected, namely 8 mug/mL or 30 mug/mL; the concentration of the cross-reagent is the concentration of the cross-reagent added in preparing the sample to be tested, i.e., 50. Mu.g/mL.
TABLE 5 Cross reaction results
As can be seen from Table 5, when the concentration of Wu Sinu monoclonal antibody is detected by adopting a magnetic particle luminescence method established by using the Ust-B-1/Ust-G-10 paired antibodies, the monoclonal antibody has no cross reaction with various other clinically common antibody medicines and high specificity.
According to the above, the detection method has higher sensitivity, specificity and precision when detecting the Wu Sinu monoclonal antibody by adopting the Ust-B-1/Ust-G-10 paired antibody, and can meet the detection requirement of Wu Sinu monoclonal antibody.
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 Wu Sinu monoclonal antibodies, wherein the hybridoma cell line combination comprises a hybridoma cell line Ust-B-1 and a hybridoma cell line Ust-G-10;
the hybridoma cell strain Ust-B-1 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.45641;
the hybridoma cell strain Ust-G-10 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.45640.
2. An antibody combination for detecting kusnezoffii mab, wherein said antibody combination comprises a coating antibody and a detecting antibody;
the coated antibody is an antibody generated by a hybridoma cell strain Ust-B-1; the hybridoma cell strain Ust-B-1 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.45641;
the detection antibody is an antibody generated by a hybridoma cell strain Ust-G-10; the hybridoma cell strain Ust-G-10 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.45640.
3. Use of the hybridoma cell line combination of claim 1 or the antibody combination of claim 2 in the preparation of a product for detecting Wu Sinu mab.
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 Wu Sinu mab.
5. The kit for detecting the ulinunimab is characterized by comprising a magnetic particle working solution, an antibody working solution and an indicator solution;
the magnetic particle working solution comprises coated antibody coupled magnetic particles and a magnetic bead buffer solution; the coated antibody is an antibody generated by a hybridoma cell strain Ust-B-1; the hybridoma cell strain Ust-B-1 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.45641;
the antibody working solution comprises a tracer-labeled detection antibody and an antibody diluent; the detection antibody is an antibody generated by a hybridoma cell strain Ust-G-10; the hybridoma cell strain Ust-G-10 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.45640.
6. The kit of claim 5, wherein the concentration of the coated 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.
7. The kit of claim 6, wherein the mass ratio of the magnetic beads to the coated antibodies in the coated 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 buffer;
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 water;
the indication liquid comprises luminous liquid;
the tracer includes alkaline phosphatase.
10. A method for detecting the free blood concentration of Wu Sinu monoclonal antibody 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 luminescence values of the sample to be detected into the standard curve to obtain the concentration of Wu Sinu monoclonal antibody free blood 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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