CN114524875B - Nano silicon sphere coupled with IL-1 beta monoclonal antibody and application thereof - Google Patents
Nano silicon sphere coupled with IL-1 beta monoclonal antibody and application thereof Download PDFInfo
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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Abstract
The invention provides a nano silicon sphere coupled with an IL-1 beta monoclonal antibody and application thereof, belonging to the technical field of nano material preparation and biological diagnosis; in the invention, a nano silicon sphere with a uniform sphere shape and a very regular straight-through pore canal structure is synthesized; the nanometer silicon sphere can be coupled with an anti-IL-1 beta monoclonal antibody to obtain an antibody-nanometer silicon sphere, the antibody-nanometer silicon sphere can be used for detecting IL-1 beta protein antigen, the Lowest Detection Limit (LDL) is 15.31pg/mL, the lower limit of quantification (LLOQ) is 57.24pg/mL, the half maximum effect concentration (EC 50) is 95.67ng/mL, and the detection range (Dynamic range) is 10-1,000,000pg/mL.
Description
Technical Field
The invention belongs to the technical field of nano material preparation and biological diagnosis, and particularly relates to a nano silicon sphere coupled with an IL-1 beta monoclonal antibody and application thereof.
Background
The homogeneous phase light excitation chemiluminescence immunoassay technology (amplified luminescent proximity homogeneous assay linked immunosorbent assay, alphalisa) is an immunology research technology taking the interaction between biomolecules as a principle, taking fluorescence resonance energy transfer luminescence as a basis, taking silica gel microspheres as a carrier and taking time resolution fluorescence as a detection mode. The technology originates from the singlet oxygen molecular energy transfer luminescence principle discovered by research in 1994, and is based on the established photoexcitation chemiluminescence technology, the photosensitizer generates singlet oxygen through irradiation of excitation light, and the luminophor receives singlet oxygen energy transfer to generate fluorescence. In 1999, this technology was improved by Perkin Elmer company, and the photosensitive agent and the luminescent agent are coated on the surface of silica gel microspheres coated with an affinity coating, and the microspheres are used as carriers of immunoadsorption for immunological analysis, so that the AlphaLISA technology is produced. The homogeneous phase light excitation chemiluminescence immunoassay technology has the advantages of no washing, easy automation and mechanization, has the technical characteristics of high specificity, high sensitivity and high flux, becomes a hot spot of immune research analysis in recent years, is a development trend of the present and future analysis detection technology, and can be widely applied to biomedical research and disease diagnosis.
The alpha LISA adopts surface chemistry to treat silica gel microspheres, and the surfaces of donor microspheres and receptor microspheres are coated with an affinity coating, and common affinity factors include avidin, streptavidin, protein A, protein G and the like, so that the alpha LISA can be combined with biomolecules to be detected. Intermolecular interactions will pull the donor and acceptor microspheres closer to within the singlet oxygen diffusion range (200 nm), thereby exciting the cascade amplified chemiluminescent reaction. The luminescence principle of the chemiluminescence reaction is light excitation chemiluminescence, the surface of a donor microsphere can be coated with a photosensitizer of xylylene blue, the surface of an acceptor microsphere is coated with a luminescent agent of dimethylthiophene derivative and chelated with rare earth atom europium, when the laser is irradiated by 680nm, the photosensitizer on the surface of the donor microsphere decomposes oxygen in the environment to form monomer oxygen molecules (oxygen free radicals), the monomer oxygen molecules are diffused into the acceptor microsphere, energy is finally transmitted to the rare earth atom europium, and the excitation light with the excitation wavelength of 615nm and the half-life of 0.3s can be detected by a fluorescence scanner.
However, the AlphaLISA technology has some technical difficulties, such as the production of nano-microspheres with uniform morphology and stable properties, which can be used for antibody coupling, and how to better couple the nano-microspheres with antibodies, so that the better implementation of the technology also needs a nano-microsphere with uniform morphology and stable properties, which is the key point for solving the problem.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a nano silicon sphere coupled with an IL-1 beta monoclonal antibody and application thereof. In the invention, a nano silicon ball which is in a uniform sphere shape and has a very regular straight-through pore structure is prepared; the nano silicon sphere is modified with a modifier, and can be coupled with an IL-1 beta monoclonal antibody to obtain an antibody-nano silicon sphere, and the antibody-nano silicon sphere can be used for detecting IL-1 beta protein antigen.
In the present invention, there is provided an IL-1β monoclonal antibody comprising Ab2 or Ab3.
Furthermore, the amino acid sequence of the heavy chain variable region of Ab2 is shown as SEQ ID NO. 1, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 2;
the amino acid sequence of the heavy chain variable region of Ab3 is shown as SEQ ID NO. 3, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 4.
Further, the preparation method of the IL-1 beta monoclonal antibody comprises the following steps:
immunizing BALB/c mice with IL-1 beta protein fragments, fusing spleen lymphocytes of the mice which are immunized successfully with myeloma cells, screening positive clones to obtain hybridoma cell strains which secrete anti-IL-1 beta monoclonal antibodies specifically, inoculating the cells to abdominal cavities of the BALB/c mice sensitized in advance with liquid paraffin, producing anti-IL-1 beta monoclonal antibodies in a large quantity, and preparing the high-purity anti-IL-1 beta monoclonal antibodies by a protein purification mode; the protein immunization is three times of protein immunization; the myeloma cells are SP2/0 myeloma cells.
The invention also provides a nano silicon sphere coupled with the IL-1 beta monoclonal antibody, and the nano silicon sphere coupled with the IL-1 beta monoclonal antibody is obtained by coupling the IL-1 beta monoclonal antibody and the nano silicon sphere.
Further, the IL-1 beta monoclonal antibodies are Ab2 and Ab3.
The invention also provides application of the nano silicon spheres coupled with the IL-1 beta monoclonal antibody in detection of IL-1 beta protein antigen.
The invention also provides application of the nano silicon spheres coupled with the IL-1 beta monoclonal antibody in preparation of an IL-1 beta protein antigen detection reagent.
The invention also provides application of the nano silicon spheres coupled with the IL-1 beta monoclonal antibody in preparation of anti-IL-1 beta biological diagnostic reagents.
The invention also provides a nano silicon for coupling IL-1 beta monoclonal antibodyThe nano silicon spheres are modified by a modifier to form SiO (silicon oxide) 2 The COOH microspheres are obtained and are uniform spheres with very regular through pore structures;
the modified modifier comprises A, C or B, C:
(A) N, N-dimethyl-4- (3-phenyl-5, 6-dihydro-1, 4-dioxin-2-yl) aniline, eu (TTA) 3 Trioctylphosphine oxide;
(B) N, N-dimethyl-4- (2-phenyl-5, 6-dihydro-1, 4-oxathiolin-3-yl) aniline, eu (TTA) 3 1, 10-phenanthroline;
(C) Chlorophyll a and (Z) -4- (4- (dibutyl-l 4-aza-subunit) -2-hydroxycyclohexyl-2, 5-diene-1-subunit) -2- (4- (dibutyl amino) -2-hydroxyphenyl) -3-oxocyclobutyl-1-ene-1-carboxylate.
Further, the SiO 2 The preparation method of the-COOH microspheres comprises the following steps:
drying the SiO 2 Mixing with anhydrous toluene, heating, adding 3-aminopropyl triethoxysilane (APTES), refluxing under nitrogen protection, centrifuging, washing, and drying to obtain NH 2 -SiO 2 A microsphere;
NH is added to 2 -SiO 2 Dispersing the microspheres in N, N-dimethylformamide DMF solvent, stirring at 80 ℃ for 1-4h, then adding DMF solution containing succinic anhydride under vigorous stirring, stirring and reacting for 8-24h, washing after the reaction is finished to obtain nano silicon spheres which are marked as SiO 2 -COOH microspheres.
Compared with the prior art, the invention has the beneficial effects that:
the invention uses IL-1 beta protein fragment as immunogen to immunize BALB/c mouse, uses cell fusion technique to fuse the successfully immunized mouse spleen lymphocyte with mouse myeloma cell to screen hybridoma cell strain which can stably secrete anti-IL-1 beta monoclonal antibody, and largely produces IL-1 beta monoclonal antibody, and obtains high purity anti-IL-1 beta monoclonal antibody after purification, and the obtained anti-IL-1 beta monoclonal antibody has high potency and good specificity, can be directly applied to molecular immunology research, or can be made into various in vitro diagnostic kits for detecting the immunodetection tool of IL-1 beta antigen. Can be used for preparing the research and development of anti-IL-1 beta biological diagnostic reagent.
The invention prepares stable and uniform nano silicon spheres, and the provided anti-IL-1 beta monoclonal antibody can specifically bind IL-1 beta protein, thereby reducing possible cross reaction and having high specificity. Detection tools comprising the anti-IL-1β monoclonal antibodies of the invention can be used for detection of the protein. The minimum detection limit (LDL) of the invention is 15.31pg/mL, the lower limit of quantification (LLOQ) is 57.24pg/mL, the half maximum effect concentration (EC 50) is 95.67ng/mL, and the detection range (Dynamic range) is 10-1,000,000pg/mL, which is improved and improved compared with other technologies.
According to the invention, the nano silicon spheres are coupled with the IL-1 beta monoclonal antibody, and then the nano silicon spheres are used for detecting the IL-1 beta antigen by the alpha LISA, and the detection method has the advantages of high sensitivity, uniformity and wide detection range, is simple and convenient to operate, has low sample demand, and reduces the total detection time; meanwhile, the method effectively avoids the fluorescent interference of the sample, has low requirement on the quality of the sample to be detected, is easy to optimize a detection system, and truly realizes the uniformity of detection.
Drawings
FIG. 1 is an SDS-PAGE electrophoresis of fractions of purified IL-1β monoclonal antibodies.
FIG. 2 shows the results of the immunotiter assays for different dilutions of IL-1β monoclonal antibodies Ab2 and Ab3.
FIG. 3 shows the specificity and cross-reaction identification results of IL-1β monoclonal antibodies Ab2 and Ab3.
FIG. 4 is a graph showing the detection result of IL-1β.
Detailed Description
The invention discloses preparation and application of a nano silicon sphere coupled with a specific IL-1 beta antibody, and a person skilled in the art can refer to the content of the nano silicon sphere and properly improve the technological parameters. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the methods and applications described herein, and in the practice and application of the techniques of this invention, without departing from the spirit or scope of the invention. The methods, apparatus, materials, and so forth in the following examples, unless otherwise indicated, are all conventional in the art and are commercially available.
Example 1: preparation of nano silicon spheres
1g of SiO 2 Vacuum drying at 110deg.C overnight, adding 50mL of anhydrous toluene to thoroughly mix the solution, heating to 115deg.C at constant speed, adding 1.6mL of 3-aminopropyl triethoxysilane (APTES), refluxing under nitrogen protection for 24h, centrifuging, washing with anhydrous ethanol for 3-4 times, and drying in oven at 80deg.C to obtain NH 2 -SiO 2 And (3) microspheres.
NH is added to 2 -SiO 2 Microspheres (0.5 g) were dispersed in N, N-Dimethylformamide (DMF) solvent and stirred at 80℃for 2h. Then 1g of succinic anhydride (dissolved in DMF) is mixed with the solution under the condition of intense stirring, after stirring for 12 hours, the mixture is purified by ethanol and deionized water, and the mixture is frozen and dried to obtain nano silicon spheres which are marked as SiO 2 -COOH microspheres.
The SiO prepared in this example 2 Representing the carboxyl modified nano silicon sphere SiO of the-COOH microsphere by using a scanning electron microscope 2 The COOH microspheres and the synthesized mesoporous silica have uniform spherical structures and very regular straight-through pore structures.
The SiO is also modified with a modifier in this example 2 The preparation method of the-COOH microsphere is characterized in that the nano silicon microsphere is prepared by the following modification steps:
SiO is made of 2 Adding the-COOH microspheres into A or B to obtain a mixed system, wherein A is: containing 3.3mM of the compound N, N-dimethyl-4- (3-phenyl-5, 6-dihydro-1, 4-dioxin-2-yl) aniline, 15.5mM Eu (TTA) 3 And 20mM trioctylphosphine oxide (TOPO);
b is: containing 3.3mM N, N-dimethyl-4- (2-phenyl-5, 6-dihydro-1, 4-oxothiooct-3-yl) aniline, 15.5mM Eu (TTA) 3 And 15.5mM 1, 10-phenanthroline (Phen).
The above mixture was then stirred in an 8:1:1 (volume ratio) ethylene glycol/benzyl alcohol/water for 8-10 minutes. Diluting the particles with an equal amount of ethanol, centrifuging, andwashing with water, and performing ultrasonic treatment to obtain chemiluminescent particles, designated as A/Eu (TTA) 3 TOPO or B/Eu (TTA) 3 /Phen。
The chemiluminescent particles obtained above were added to a mixed solution containing 10mM chlorophyll A (Chl) and 10mM (Z) -4- (4- (dibutyl-l 4-azasubunit) -2-hydroxycyclohexyl-2, 5-diene-1-subunit) -2- (4- (dibutylamino) -2-hydroxyphenyl) -3-oxocyclobutyl-1-ene-1-carboxylate (C) to obtain a mixed system, and the mixed system was stirred in an ethylene glycol/benzyl alcohol/water ratio of 8:1:1 (volume ratio) for 8-10 minutes. Diluting the particles with equal amount of ethanol, centrifuging, washing with water, and performing ultrasonic treatment to obtain the nano silicon spheres.
The prepared nano silicon spheres are characterized by a scanning electron microscope, and after the groups are modified, the morphology and the pore channel structure of the silicon spheres are kept good, because the added reagent is combined with the silicon spheres in the form of Si-O-Si bonds, and the sample is not damaged.
Example 2: IL-1 beta monoclonal antibody preparation
(1) Expression and purification of recombinant IL-1 beta protein fragments:
recombinant plasmid pET-30a-IL-1 beta (purchased from Addgene company), the recombinant plasmid is transformed into escherichia coli BL21 (Shanghai process) by adopting a method of heat shock for 90s at 42 ℃, kanamycin-resistant bacterial plaques are picked up, the bacterial plaques are cultured in 500mL LB culture medium (Shanghai process) until the OD of bacterial liquid is 0.6-0.8, 0.5mM IPTG is added to induce the expression of target protein, after the bacterial cells are collected after the bacterial cells are cultured for 12 hours at the constant temperature of 25 ℃, ultrasonic bacteria breaking is carried out, and the supernatant is collected centrifugally.
The supernatant of the cell lysate was applied to a nickel column (from Thermo Fisher Scientific) to purify the protein, the recombinant protein was eluted with 500mM imidazole buffer, and then the expression of the recombinant protein was detected by SDS-PAGE electrophoresis.
FIG. 1 is an SDS-PAGE electrophoresis of recombinant protein IL-1. Beta. Fraction collection; as shown in figure 1, IL-1 beta protein can obtain higher purity through one-time elution, the eluted protein is collected, and the subsequent animal immunization is carried out after freeze-drying.
(2) Immunization and potency detection of BALB/c mice:
BALB/c mice used in this example were purchased from the university of Nanjing animal model institute. Mouse immunization was performed in a conventional manner (refer to modern antibody technology and applications published by Beijing university Press and antibody preparation and use laboratory Manual by scientific Press). The serum titers of mice after immunization were detected by indirect ELISA, and specific procedures are shown below.
And (2) taking the recombinant IL-1 beta protein prepared in the step (1), diluting the IL-1 beta protein to 1 mu g/mL by using a carbonate buffer solution with the pH of 9.6 and 0.05mol/L, adding a 96-well ELISA plate, coating the ELISA plate at the temperature of 4 ℃ for overnight in each well by 100 mu L, taking out the ELISA plate coated overnight, washing the ELISA plate for 3 times by using a TBS-T buffer solution, drying the ELISA plate, and storing the ELISA plate at the temperature of 4 ℃ for later use.
1 week after the second immunization, a proper amount of blood was collected from the tail vein of the mice, serum was separated by centrifugation for 15min at 5000g, serum was diluted with a sample dilution (phosphate buffer containing 0.5% bovine serum albumin) in a gradient of 1:100, 1:1000, 1:10000, 1:100000, 1:1000000, 100. Mu.L of each well was added to the ELISA plate to be tested, 37℃was incubated for 1 hour, and after incubation for 3 times with TBS-T buffer, the ELISA plate was dried by pipetting, 100. Mu.L of 1:5000 diluted HRP-labeled goat anti-mouse secondary antibody (from Jackson Immuno Research Co.) was added to each well, and incubated for 30min at 37 ℃. Taking out the ELISA plate, washing 5 times by TBS-T buffer solution, adding 100 mu L of TMB substrate display solution into each hole, developing at 37 ℃ in dark for 10-15 min, adding 50 mu L of stop solution, and reading the light absorption value at the wavelength of 450nm of the ELISA. Selecting serum titer to reach 1: mice above 105 were immunized for the third time.
(3) Fusion and screening of hybridoma cells:
preparing feeder layer cells, preparing SP2/0 myeloma cells, taking mouse spleen cells for cell fusion 3-4 days after immunization, then plating the fused cells (6 96-well plates), screening hybridoma cells producing monoclonal antibodies by using an indirect ELISA method, and obtaining hybridoma cell strains with best antibody secretion through two rounds of subcloning screening.
(4) Production and purification of anti-IL-1. Beta. Monoclonal antibodies:
the ascites of the mouse monoclonal antibody is prepared according to the conventional method, the ascites antibody is purified by an n-octanoic acid-protein G method, the purity of the antibody is verified by SDS-PAGE electrophoresis, as shown in figure 2, the antibody can obtain higher purity after elution, and the molecular weight of the anti-IL-1 beta monoclonal antibody IgG (H+L) is about 160KD, wherein the IgG heavy chain is about 55KD, and the IgG light chain is about 25KD.
Example 3: IL-1 beta monoclonal antibody titer determination and specificity
(1) Titer determination of monoclonal antibodies:
IL-1. Beta. Protein was diluted to 1. Mu.g/mL with a carbonate buffer at pH 9.6,0.05mol/L, coated with ELISA plate, 100. Mu.L/well, overnight at 4 ℃; the above monoclonal antibodies were diluted 1:10 with sample dilution (phosphate buffer containing 0.5% bovine serum albumin) 3 、1:10 4 、1：10 5 、1：10 6 、1：10 7 Diluting, 100 mu L/well, and incubating at 37 ℃ for 1h; taking out the ELISA plate, washing 3 times by TBS-T, drying the ELISA plate by beating, adding 100 mu L of HRP-marked goat anti-mouse secondary antibody diluted by 1:5000 into each hole, and incubating for 30min at 37 ℃; after washing 5 times by TBS-T, 100 mu L of TMB substrate display liquid is added into each hole, the color development is carried out at 37 ℃ in a dark place for 10-15 min, then 50 mu L of stop solution is added to stop the reaction, and the absorbance value is read at the wavelength of 450nm of an enzyme label instrument.
FIG. 2 is a graph showing the results of the detection of the immunotiter of different dilutions of IL-1β monoclonal antibodies; as shown in FIG. 2, the titers of the purified monoclonal antibodies all reached 1X 10 6 。
(2) Monoclonal antibody specific recognition and cross-reaction identification:
HSP90, HB-EGF, EGFR, TNF-alpha, IL-1 beta and HGF proteins were diluted to 1. Mu.g/mL with carbonate buffer, an ELISA plate was coated, and a blank control was set, which was 1. Mu.g/mL BSA protein, and an indirect ELISA assay was performed using IL-1 beta monoclonal antibody.
FIG. 3 shows the specificity and cross-reaction identification results of IL-1β monoclonal antibodies; as shown in FIG. 3, the IL-1 beta monoclonal antibody has no cross reaction with HSP90, HB-EGF, EGFR, TNF-alpha, IL-1 beta and HGF proteins, which shows that the specificity of the antibody is better.
Example 4: sequencing and identification of heavy and light chain variable regions of IL-1 beta monoclonal antibodies
In order to solve the problems, the invention utilizes molecular biology technology to carry out gene amplification of heavy chain variable region (mVH) and light chain variable region (mVL) on positive monoclonal cell strains by utilizing a Mouse Ig-Primer Set kit (Millipore) and carry out sequence identification.
The amplified sequences are Ab2 and Ab3, wherein the amino acid sequence of the heavy chain variable region of Ab2 is shown as SEQ ID NO. 1, namely:
LDVQVQQSGAELARPGASVKLSCKASGYTFITHWMQWVKQRPGQGLEWIGTIYPGD GDTRYTQKFKGKATLTADKSSSTAYMQLSSLASEDSAVYYCASYDEDLPFAYWGQGTLVTV SA; the amino acid sequence of the light chain variable region sequence of Ab2 is shown as SEQ ID NO. 2, namely:
TQSPSSMYASLGERVTITCKAPQDIRRYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFS GSGSGQDYSLTISSLEYEDMGIYYCLQRDEAPFTFGSGTKLEIK；
ab3 heavy chain variable region amino acid sequence is shown in SEQ ID NO. 3, namely:
QIQAQESGPGLVKPSQSLSLTCAGRPFWILAKGGWNWIRQFPGNKLEWMGALTQILGN GKLFPLKNRISITRDTSKNQFFLKLNSVTPEDTATYFCARSRAGQSFYWGQGTSVIVSSA; ab3 light chain variable region amino acid sequence is shown in SEQ ID NO. 4, namely:
TQTPLSLPVSLGDQASISCGLFQRADILNKAGQAIWFLQKPGQSPKLLIFLKGAQFRGV PDRFSGSESGTDFTLKISRVEAEDLGVYFCYQADLVLEPFGAGTKLELKR。
example 5: preparation of IL-1 beta monoclonal antibody coupled nano silicon ball
In an EP test of 1.5mL, the nano-silica spheres were washed and centrifuged at 15000 Xg for 15 minutes, and the supernatant was discarded for further use; preparation of fresh 400mM NaBH 3 CN aqueous solution for use.
To an EP tube containing 1mg of nanosilicon spheres, 10. Mu.L of 400mM NaBH was added 3 CN aqueous solution, 10mg/ml IL-1. Beta. Monoclonal antibody, 1.25. Mu.L 10% Tween-20 and 100mM HEPES (pH 7.4), and the final total volume was 200. Mu.L. The EP tube was then reacted at 37℃for 24 hours with rotational vibration (6-10 rpm). Next, 10. Mu.L of 65mg/mL fresh carboxymethoxyamine solution was added to the EP tube and reacted under rotary shaking (6-10 rpm) at 37 ℃And 1 hour. After the reaction was completed, the supernatant was removed by centrifugation at 16000 Xg for 15 minutes, the nanosilicon spheres were resuspended, and subjected to a brief sonication (10 short pulses of 1 second) twice, after which the nanosilicon spheres were suspended in a storage buffer (200. Mu.L of PBS+0.05% proclin-300 as preservative) at a concentration of 5mg/mL to give nanosilicon spheres coupled with IL-1. Beta. Monoclonal antibodies, designated as antibody-nanosilicon spheres, and stored in an opaque EP tube at 4℃for use. The IL-1 beta monoclonal antibodies are Ab2 and Ab3 respectively, and the antibody-nano silicon spheres prepared by the method are marked as Bead-Ab2 and Bead-Ab3.
Example 6: antibody pairing reactions
In this example, antibodies Ab2 and Ab3 were first biotinylated for antibody pairing reactions. The antibody biotinylation comprises the following specific steps:
5mg of IL-1. Beta. Monoclonal antibody was dissolved in 0.5mL of PBS, and then a freshly prepared 10mM Biotin reagent solution (2.3 mg of NHS-LC-Biotin (succinimidyl-6- (biotinamide) hexanoate, manufactured) was dissolved in 500. Mu.l of DMSO), and the reaction was incubated on ice for 2 hours to give biotinylated antibodies, noted Biotin-Ab2 and Biotin-Ab3, respectively.
The biotinylated antibodies Biotin-Ab2 and Biotin-Ab3 were subjected to antibody pairing reaction with the antibodies-nanosilicon spheres Bead-Ab2 and Bead-Ab3 from example 5, wherein the reaction steps are as follows: the most suitable antibody pairs were selected by combining Bead-Ab2 and Bead-Ab3 at concentrations of 3, 1, 0.3 and 0.1. Mu.g/mL with Biotin-Ab2 and Biotin-Ab3 at concentrations of 3, 1, 0.3 and 0.1. Mu.g/mL, respectively, and examining the absorbance at the time of the change in IL-1β concentration (0 to 1000. Mu.L).
Through the above experiments, it was found that the combination of Biotin-Ab2 at a concentration of 0.3. Mu.g/mL and Bead-Ab3 at a concentration of 1. Mu.g/mL showed a linear change in absorbance (R) when the IL-1β concentration was varied (0-400 pg/ul) 2 =0.998), and highest among the various combinations. ) The pairing result of 0.3. Mu.g/mL Biotin-Ab2 and 1. Mu.g/mL Bead-Ab3 is shown in Table 1.
Table 1.0.3. Mu.g/mL Biotin-Ab2 and 1. Mu.g/mL Bead-Ab3 pairing results
Example 7: IL-1 beta detection
mu.L of IL-1β diluted in assay buffer (from platinum Elmol) was added to 384-well plates, 20. Mu.L of antibody mixture (Biotin-Ab 2 at a final concentration of 0.3. Mu.g/mL and Bead-Ab3 at a final concentration of 1. Mu.g/mL) was prepared, incubated at 25℃for 1 hour, then 25. Mu.L of streptavidin donor Bead solution (from platinum Elmol) at a final concentration of 40. Mu.g/mL was added, and after 30 minutes of reaction in the dark at 25℃the results were read by a Synergy multifunctional fluorometer (Biotek) and are shown in FIG. 4.
The minimum detection limit (LDL) was calculated to be 15.31pg/mL, the lower limit of quantitation (LLOQ) was calculated to be 57.24pg/mL, and the half maximal Effective Concentration (EC) 50 ) The detection range (Dynamic range) is 10-1,000,000pg/mL, which is 95.67ng/mL, and is improved and improved over other technologies.
The examples are preferred embodiments of the present invention, but the present invention is not limited to the above-described embodiments, and any obvious modifications, substitutions or variations that can be made by one skilled in the art without departing from the spirit of the present invention are within the scope of the present invention.
Sequence listing
<110> Jiangsu Laisen Biotech institute of Endoconcha
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<213> Artificial sequence (Artificial Sequence)
<400> 1
Leu Asp Val Gln Val Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly
1 5 10 15
Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ile Thr
20 25 30
His Trp Met Gln Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp
35 40 45
Ile Gly Thr Ile Tyr Pro Gly Asp Gly Asp Thr Arg Tyr Thr Gln Lys
50 55 60
Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala
65 70 75 80
Tyr Met Gln Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Tyr
85 90 95
Cys Ala Ser Tyr Asp Glu Asp Leu Pro Phe Ala Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ala
115
<210> 2
<211> 103
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 2
Thr Gln Ser Pro Ser Ser Met Tyr Ala Ser Leu Gly Glu Arg Val Thr
1 5 10 15
Ile Thr Cys Lys Ala Pro Gln Asp Ile Arg Arg Tyr Leu Ser Trp Phe
20 25 30
Gln Gln Lys Pro Gly Lys Ser Pro Lys Thr Leu Ile Tyr Arg Ala Asn
35 40 45
Arg Leu Val Asp Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly
50 55 60
Gln Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Tyr Glu Asp Met Gly
65 70 75 80
Ile Tyr Tyr Cys Leu Gln Arg Asp Glu Ala Pro Phe Thr Phe Gly Ser
85 90 95
Gly Thr Lys Leu Glu Ile Lys
100
<210> 3
<211> 118
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 3
Gln Ile Gln Ala Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Ser Leu Ser Leu Thr Cys Ala Gly Arg Pro Phe Trp Ile Leu Ala Lys
20 25 30
Gly Gly Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp
35 40 45
Met Gly Ala Leu Thr Gln Ile Leu Gly Asn Gly Lys Leu Phe Pro Leu
50 55 60
Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe
65 70 75 80
Leu Lys Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Thr Tyr Phe Cys
85 90 95
Ala Arg Ser Arg Ala Gly Gln Ser Phe Tyr Trp Gly Gln Gly Thr Ser
100 105 110
Val Ile Val Ser Ser Ala
115
<210> 4
<211> 109
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 4
Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly Asp Gln Ala Ser
1 5 10 15
Ile Ser Cys Gly Leu Phe Gln Arg Ala Asp Ile Leu Asn Lys Ala Gly
20 25 30
Gln Ala Ile Trp Phe Leu Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu
35 40 45
Ile Phe Leu Lys Gly Ala Gln Phe Arg Gly Val Pro Asp Arg Phe Ser
50 55 60
Gly Ser Glu Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu
65 70 75 80
Ala Glu Asp Leu Gly Val Tyr Phe Cys Tyr Gln Ala Asp Leu Val Leu
85 90 95
Glu Pro Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg
100 105
Claims (7)
1. An IL-1 β monoclonal antibody, wherein the IL-1 β monoclonal antibody comprises Ab2 or Ab3;
the amino acid sequence of the heavy chain variable region of Ab2 is shown as SEQ ID NO. 1, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 2;
the amino acid sequence of the heavy chain variable region of Ab3 is shown as SEQ ID NO. 3, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 4.
2. The IL-1 beta monoclonal antibody-coupled nano-silica spheres is characterized in that the IL-1 beta monoclonal antibody-coupled nano-silica spheres are obtained by coupling the IL-1 beta monoclonal antibody according to claim 1 with nano-silica spheres.
3. Use of the IL-1 β monoclonal antibody-conjugated nanospheres of claim 2 for detection of IL-1 β protein antigens for non-diagnostic and therapeutic purposes.
4. The use of the IL-1 β monoclonal antibody-coupled nanospheres of claim 2 in the preparation of reagents for detecting IL-1 β protein antigens.
5. Use of the IL-1 β monoclonal antibody-conjugated nanospheres of claim 2 in the preparation of anti-IL-1 β biological diagnostic reagents.
6. A nano silicon sphere for coupling IL-1 beta monoclonal antibody is characterized in that the nano silicon sphere is modified by a modifier by SiO 2 The COOH microspheres are obtained and are uniform spheres with very regular through pore structures;
the modifier includes A, C or B, C as follows:
(A) N, N-dimethyl-4- (3-phenyl-5, 6-dihydro-1, 4-dioxin-2-yl) aniline, eu (TTA) 3 Trioctylphosphine oxide;
(B) N, N-dimethyl-4- (2-phenyl-5, 6-dihydro-1, 4-oxathiolin-3-yl) aniline, eu (TTA) 3 1, 10-phenanthroline;
(C) Chlorophyll a and (Z) -4- (4- (dibutyl-l 4-aza-subunit) -2-hydroxycyclohexyl-2, 5-diene-1-subunit) -2- (4- (dibutyl amino) -2-hydroxyphenyl) -3-oxocyclobutyl-1-ene-1-carboxylate;
the IL-1 beta monoclonal antibody comprises Ab2 or Ab3;
the amino acid sequence of the heavy chain variable region of Ab2 is shown as SEQ ID NO. 1, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 2;
the amino acid sequence of the heavy chain variable region of Ab3 is shown as SEQ ID NO. 3, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 4.
7. The nanosilicon sphere for coupling IL-1 β monoclonal antibody of claim 6, wherein the SiO 2 The preparation method of the-COOH microspheres comprises the following steps:
drying the SiO 2 Mixing with anhydrous toluene, heating, adding 3-aminopropyl triethoxysilane APTES, refluxing under nitrogen protection, centrifuging, washing, and drying to obtain NH 2 -SiO 2 A microsphere;
NH is added to 2 -SiO 2 Dispersing the microspheres in N, N-dimethylformamide DMF solvent, stirring for reaction, adding DMF solution containing succinic anhydride under vigorous stirring, stirring for reaction, purifying with ethanol and deionized water to obtain SiO 2 -COOH microspheres.
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