CN116874596B - Monoclonal antibody of anti S100 beta protein, preparation method and application thereof - Google Patents

Monoclonal antibody of anti S100 beta protein, preparation method and application thereof Download PDF

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CN116874596B
CN116874596B CN202311140628.7A CN202311140628A CN116874596B CN 116874596 B CN116874596 B CN 116874596B CN 202311140628 A CN202311140628 A CN 202311140628A CN 116874596 B CN116874596 B CN 116874596B
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芮兵
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Nanjing Baikang Biotechnology Co ltd
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Abstract

The invention discloses a monoclonal antibody for resisting S100 beta protein, a preparation method and application thereof, comprising S100 beta-Clone 1 and S100 beta-Clone 2, wherein the amino acid sequence of a heavy chain variable region of S100 beta-Clone 1 is shown as SEQ ID NO.1, and the amino acid sequence of a light chain variable region of S100 beta-Clone 1 is shown as SEQ ID NO. 2; the amino acid sequence of the heavy chain variable region of the S100 beta-Clone 2 is shown as SEQ ID NO.3, and the amino acid sequence of the light chain variable region of the S100 beta-Clone 2 is shown as SEQ ID NO. 4. The preparation method comprises the following steps: and (3) carrying out equal volume mixing emulsification on the S100 beta and Freund' S adjuvant after affinity purification, collecting blood serum of a mouse, screening the mouse with better immunity, carrying out hybridoma fusion experiments, taking the S100 beta and the S100 alpha as ELISA plate coating antigens, taking supernatant to carry out ELISA, screening out high-quality positive cell strains, and carrying out two rounds of cell subcloning. Meanwhile, the early diagnosis and detection kit for the brain injury of the antibody is also provided, so that the specificity and sensitivity of the diagnosis reagent are improved.

Description

Monoclonal antibody of anti S100 beta protein, preparation method and application thereof
Technical Field
The invention belongs to the field of antibody preparation and sequence determination, and particularly relates to a monoclonal antibody of an anti-S100deg.P protein.
The invention also relates to a preparation method and application of the monoclonal antibody against the S100deg.P protein.
Background
The S100 protein was originally discovered by Moore equal to 1965 in bovine brain tissue first, and was named because of its 100% solubilization in neutral saturated ammonium sulfate. The S100 protein has a relatively small molecular weight (9-14 kDa) and exists in a physiologically distinct homodimeric form.
The S100 family consists of 25 members and belongs to the calcium ion binding protein. When calcium ions are combined with S100, the calcium ions are exposed on the surface of the hydrophobic group, thereby participating in information transmission. The S100 family comprises various combinations of alpha and beta subunits, most commonly found in the S100A (alpha-alpha) or S100B (alpha-beta and beta-beta) subtypes.
S100B is the most active member of this family, 96% of which is present in the brain and is therefore considered a brain tissue-specific protein, as well as the most studied member of this family.
The S100B protein is mainly expressed in astrocytes and schwann cells of the nervous system, and acts inside and outside the cells, the effect of which depends on the concentration. Under physiological level, due to the integrity of brain cells and blood brain barrier, the level of S100B in blood and cerebrospinal fluid is extremely low and relatively stable, and the S100B protein promotes neurite extension and protects neuron survival; when the structure or function of the blood brain barrier is damaged, S100B enters cerebrospinal fluid from inside cells, and then enters blood through the damaged blood brain barrier, so that the S100B content in the blood is increased, the expression of proinflammatory cytokines is stimulated, apoptosis is caused, and the neurotoxic effect is exerted.
Clinical significance: because the S100B protein has small molecular weight and is easy to pass through the blood brain barrier, the damage degree of the central nervous system can be sensitively reflected in serum, plasma or cerebrospinal fluid, so that the detection of the S100B protein content has important significance for early diagnosis and disease assessment of brain injury of patients.
As a biochemical marker of brain injury, the S100B protein has a certain time change rule after brain injury, and is closely related to the degree and prognosis of brain injury. Can be clinically used for early diagnosis, disease assessment and treatment guidance of neonatal ischemia and hypoxia encephalopathy (HIE), acute cerebral apoplexy (cerebral infarction and cerebral hemorrhage), traumatic craniocerebral injury, central nervous system infection and other diseases.
The core raw material of the S100deg.P chemiluminescent kit with excellent development performance is a monoclonal antibody of mouse anti-S100, and the antibody is required to recognize beta subunits of two subtypes of S100B (alpha-beta) and S100B (beta-beta) while maintaining high affinity, thereby improving the specificity and sensitivity of diagnostic reagent and having important significance for early diagnosis and disease assessment of brain injury of patients.
Disclosure of Invention
In order to solve the problems of sensitivity and specificity, the invention provides a monoclonal antibody of an anti-S100deg.P protein. The antibody has excellent performance and high accuracy.
Meanwhile, the S100.beta.chemiluminescence kit is provided, so that the content of S100.beta.protein in blood of a patient can be accurately detected, and a basis is provided for diagnosing brain injury of the patient.
The invention provides the following technical scheme:
monoclonal antibodies against the S100deg.P protein, including S100deg.P-Clone 1 and S100deg.P-Clone 2,
heavy chain variable region amino acid sequence of s100deg.P-Clone 1:
EVQLQQSGAEPVKPGASVKLSCTASGFNIKDTYIHWVKQRPEQGLEWIGRIDPADGDTKYDPKFQGKATITADTSSNTAYLQLSRLTSEDTAVYYCARPSLYDGYYPFAYWGQGTLVTVSA
light chain variable region amino acid sequence of s100deg.P-Clone 1:
DVVMTQTPLSLSVSLGDQASISCRSSQSLIYSNRHTYLHWYLQKPGQSPKLLIFQVSNRFSGVPDRFSGSGSGTDFTLKINRVEAEDLGVYFCSQSTHLPFTFGSGTKLEIK
heavy chain variable region amino acid sequence of s100deg.P-Clone 2:
EVQLQQSGAELVKPGASVKLSCTASGFNIKDYYMHWMKQRTEQGLDWIGRIDPEDGETKYAPEFQGKATITADTSSNTAYLQLSTLTSEDTAVYYCARYYSSYVPFVYWGQGTLVTASA
light chain variable region amino acid sequence of s100deg.P-Clone 2:
ETTVTQSPASLSMAIGEKVTIRCITSTDIDDDMNWYQQKAGEPPKLLISEGNTLRPGVPSRFSSSGYGTDFVFTIENMFSEDVADYYCLRSDKLPYTFGGGTKLEIK
a method for preparing monoclonal antibody of anti S100 beta protein,
mixing and emulsifying the S100 beta and Freund' S adjuvant in equal volume after affinity purification, performing booster immunization after 3 weeks, performing immunization measurement at 50 mug/mouse, collecting mouse blood, separating serum each time after 2 weeks, measuring antibody titer by enzyme-linked immunosorbent assay, screening the immunized mouse for hybridoma fusion experiment, screening and culturing by using a HAT screening culture medium, taking S100 beta and S100 alpha as ELISA plates to coat antigen, taking supernatant for enzyme-linked immunosorbent assay, screening out high-quality positive cell strains, performing two rounds of cell subcloning, cloning monoclonal cell strains, culturing and fermenting cell strains by using no serum, and collecting culture supernatant.
Affinity purification of s100deg.beta: the bacterial cells are fully resuspended by phosphate buffer solution, after high-pressure homogenization by a high-pressure homogenizer, supernatant is centrifugally taken, affinity purification is carried out by a pre-packed column, washing and elution are carried out by gradient imidazole concentration, SDS-PAGE running gel is carried out to detect whether protein purity and Native PAGE detect whether protein spontaneously forms dimer, namely S100deg.beta.or not, then the protein is dialyzed into the phosphate buffer solution, and after sterile filtration by a 0.22 mu m filter, BCA is used for measuring the protein concentration.
Affinity purification of s100deg.alpha.beta: the bacterial cells are fully resuspended by phosphate buffer solution, the bacterial cells are subjected to high-pressure homogenization by a high-pressure homogenizer, the supernatant is obtained by centrifugation, the pre-packed column is subjected to affinity purification, the phosphate buffer solution is used for washing impurities, then the 2.5 mM desulfurization biotin is used for eluting, SDS-PAGE gel is used for detecting the purity of the protein, the protein is dialyzed into the phosphate buffer solution, a 0.22 mu m filter is used for sterile filtration, BCA is used for measuring the protein concentration, the purified S100deg.C subunit and the beta subunit are mixed in equal quantity, the mixture is placed in a shaking table at 2-5 ℃ for incubation overnight, the mixture of the S100deg.C, S100deg.C and S100deg.C is obtained, the mixture of the three is subjected to affinity purification by Ni Smart-6FF beads, the mixture of the S100deg.C and the S100 alpha is obtained by washing impurities and elution by gradient concentration of imidazole, the mixture of the S100deg.C and the S100 alpha is subjected to affinity purification by the pre-packed column, and the phosphoric acid is used for phosphoric acidBuffer solutionThe solution was washed and eluted with 2.5. 2.5 mM desulphated biotin, and SDS-PAGE running was performed to determine protein purity and Native PAGE was performed to determine whether the protein formed dimer S100deg.alpha.beta.
Expression production of s100deg.beta.subunit: transforming the pET30a-S100 beta expression plasmid into escherichia coli competent cells, picking single colony for induction expression, culturing in LB culture medium containing kanamycin, adding IPTG for induction expression when the OD is 600nm to 0.6-0.8, and collecting thalli.
Expression production of S100 alpha subunit: transforming the pET30a-S100 alpha expression plasmid into escherichia coli competent cells, picking single colony for induction expression, culturing in LB culture medium containing kanamycin, adding IPTG for induction expression when the OD is 600nm to 0.6-0.8, and collecting thalli.
Application of monoclonal antibody of anti S100 beta protein in preparing early diagnosis reagent for brain injury of patient.
Monoclonal antibodies were used in the s100deg.C chemiluminescent kit. The kit uses S100deg.P-Clone 1 and S100deg.P-Clone 2 as coating antibody and detection antibody, respectively.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides an anti-S100deg.P protein which is used for preparing monoclonal antibody for diagnosis, the monoclonal antibody for resisting S100deg.P protein has the characteristics of specificity and high affinity, and simultaneously provides a chemiluminescent detection kit based on the antibody, thereby improving the specificity and sensitivity of diagnostic reagent and having important significance for early diagnosis and disease assessment of brain injury of clinical patients.
Drawings
FIG. 1 is a graph showing the correlation between the detection kit of the present invention and the similar products.
Description of the embodiments
The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
EXAMPLE 1 construction of s100deg.alpha and S100deg.beta subunit expression vectors
Searching the amino acid sequence of human S100deg.beta.subunit on NCBI functional network, adding histidine tag to beta-subunit protein amino acid, optimizing codon in colibacillus expression system by utilizing codon optimizing software, utilizing NdeI & XhoI restriction enzyme cutting site after gene synthesis, cloning gene into pET30a expression vector to construct pET30 a-S100deg.beta expression plasmid, and sequencing to verify gene sequence.
Searching the amino acid sequence of human S100deg.alpha.subunit on NCBI functional network, adding Strep-tag II tag to alpha subunit protein amino acid, optimizing codon in colibacillus expression system by utilizing codon optimizing software, utilizing NdeI & XhoI restriction enzyme cutting site after gene synthesis, cloning gene into pET30a expression vector to construct pET30 a-S100deg.alpha expression plasmid, and sequencing to verify gene sequence.
Example 2 expression production of s100deg.beta.subunit
Transforming competent cells of Escherichia coli BL21 (DE 3) with pET30 a-S100deg.C expression plasmid, picking single colony for induction expression, culturing in LB culture medium containing 50 μg/ml kanamycin, culturing at 37deg.C and 220 rpm until OD600nm to 0.6-0.8, adding 0.2mM IPTG for induction expression, inducing at 37deg.C for 6 hr, 8000 g, centrifuging for 10 min, and collecting thallus.
Example 3 expression production of the s100.alpha.subunit
Transforming competent cells of Escherichia coli BL21 (DE 3) with pET30a-S100 alpha expression plasmid, picking single colony for induction expression, culturing in LB culture medium containing 50 mug/ml kanamycin, culturing to OD600nm to 0.6-0.8 at 37 ℃ and 220 rpm, adding 0.2mM IPTG for induction expression, inducing at 37 ℃ for 6 hours, 8000 g, centrifuging for 10 min, and collecting thalli.
EXAMPLE 4 affinity purification of S100deg.beta.beta.
The cells were resuspended in 20mM PB,300mM NaCl,10% glycerol, pH8.0 buffer, homogenized under high pressure at 800bar, 12000g, centrifuged for 30min, the supernatant was collected, affinity purified using a pre-packed column Ni Smart-6FF beads, 20mM PB,300mM NaCl,10% glycerol, 50mM imidazole, pH8.0 solution, 20mM PB,300mM NaCl,10% glycerol, 250mM imidazole, pH8.0 solution, eluted, SDS-PAGE running was performed to determine protein purity, native PAGE was performed to determine whether the protein forms dimer S100deg.beta.and the protein was dialyzed to 20mM PB,300mM NaCl,10% glycerol in pH8.0 buffer, sterile filtered through a 0.22 μm filter, and BCA was assayed for protein concentration.
EXAMPLE 5 affinity purification of S100deg.alpha.beta
The cells were resuspended in 20mM PB,300mM NaCl,10% glycerol, pH8.0 buffer, homogenized under high pressure at 800bar, 12000g, centrifuged for 30min, the supernatant was collected, affinity purified by pre-packed column Streptactin Beads FF, washed with 20mM PB,300mM NaCl,pH8.0 solution, eluted with 2.5 mM desulphated biotin, sterile filtered through a 0.22 μm filter, and the protein concentration was measured by BCA. Mixing the purified S100deg.C subunit and the purified beta subunit in equal amount, placing in a shaking table at 2-5deg.C for incubation overnight to obtain a mixture of S100deg.C, S100deg.C beta and S100deg.C beta, subjecting the mixture of the three to affinity purification with Ni Smart-6FF beads, washing and eluting with gradient imidazole concentration to obtain a mixture of S100deg.C beta and S100deg.C beta, subjecting the mixture of the two to affinity purification with a pre-packed column Streptactin Beads FF, washing with PB solution, eluting with 2.5 mM desulphated biotin, detecting protein purity by SDS-PAGE gel and detecting whether protein forms dimer S100deg.C beta by Native PAGE.
EXAMPLE 6 preparation of monoclonal antibodies
Mixing and emulsifying the S100 beta and Freund' S adjuvant in equal volume after affinity purification, performing booster immunization after 3 weeks, performing immunization measurement at 50 mug/mouse, collecting mouse blood, separating serum each time after 2 weeks, measuring antibody titer by enzyme-linked immunosorbent assay, screening the immunized mouse for hybridoma fusion experiment, screening and culturing by HAT screening culture medium, taking S100 beta and S100 alpha as ELISA plates to coat antigen, taking supernatant for enzyme-linked immunosorbent assay, screening out high-quality positive cell strains, performing two rounds of cell subcloning, cloning monoclonal cell strains, culturing and fermenting cell strains by serum-free, collecting culture supernatant, performing antibody purification, and performing diagnostic performance analysis on the purified antibody.
EXAMPLE 7 monoclonal antibody Performance analysis
The kit is prepared based on the principle of a double antibody sandwich method and an acridine ester luminescence method.
1. Preparation of streptavidin magnetic particles
The purchased streptavidin magnetic particles from the company Siemens were diluted to a solid content of 0.02% using a magnetic particle buffer (50 mM PB, 150mM NaCl, 3% Trehalose, 0.1% Proclin 300) as R1 reagent.
2. Acridinium ester labeled antibody
Acridinium ester and S100deg.B antibody are mixed according to the molecular mole ratio of 3:1 in PBS for 2h; after the reaction is finished, a 30kDa ultrafiltration centrifuge tube is used for ultrafiltration centrifugation to remove free acridinium ester; after centrifugation, the markers in the ultrafiltration tube were collected and the concentration of acridinium ester-antibody was detected using BCA method; the acridinium ester-antibody is diluted to 1.5 mug/mL by using an R2 preparation solution, and the diluted solution is the R2 reagent, wherein the R2 preparation solution comprises 20mM Tris, 200mM KCl, 2% BSA, 0.1% Tween20 and 0.1% Proclin300.
3. Biotin-labeled antibody
NHS esterified organisms (Sieimfi, EZ-link NHS-PEG 4-biotin) were combined with the anti-S100deg.S antibodies of the invention in a molar ratio of 5:1 proportion in PBS for 1h; after the reaction is finished, a 30kDa ultrafiltration centrifuge tube is used for ultrafiltration centrifugation, PBS is added into the upper part of the ultrafiltration tube for centrifugation again after the centrifugation is finished, and the process is repeated twice; collecting biotin-antibody labeled product in the ultrafiltration tube, and detecting the concentration of the antibody using BCA method; the above product was diluted to an antibody concentration of 2. Mu.g/mL as R3 reagent using R3 formulation dilution of 20mM Tris, 50mM KCl, 2% Casein, 0.1% Tween20, 0.1% Proclin300.
4. Preparing calibration product and quality control product
The S100 beta antigen is diluted to 0, 0.005, 0.05, 0.5, 5 and 50 ng/mL respectively by using an antigen diluent, and the diluted antigen is split into calibrator tubes.
And (3) respectively releasing the S100 beta antigen diluted solution to 0.08 and 1 ng/mL by using the antigen diluted solution, and subpackaging into quality control product tubes.
The antigen diluent formulation was 1 XPBS, 1% BSA.
Detection kit and similar products are compared
The correlation between the S100.beta.chemiluminescent detection kit in the example and the product of Roche company is compared on 120 clinical specimens, and the data is shown in FIG. 1. Correlation coefficient R 2 0.9948. Description of the inventionThe clear kit can accurately detect the S100deg.S content, provides basis for clinical diagnosis, and can fully meet the clinical in-vitro diagnosis detection requirement.
Sensitivity and precision investigation of detection kit
The blank and detection limits of the kit of the present invention were studied according to the clinical and laboratory standards institute sensitivity verification analysis guideline file CLSI: EP17-A2, and the results showed that the blank and detection limits were 0.003 ng/mL and 0.005 ng/mL, respectively.
According to the precision verification analysis guideline file CLSI of the clinical and laboratory standards institute, EP5-A3 carries out precision research on the kit, 3 batches of reagents and calibrators are used for detecting 2 levels of quality control products, each time of detection is carried out twice a day in the morning and afternoon, continuous investigation is carried out for 5 days, and the result shows that the total indoor precision of each batch is less than 5%.
Anti-interference capability analysis of detection kit
Different concentrations of interferents were added to two serum samples with S100 beta concentrations of approximately 0.08 ng/mL and 1 ng/mL, and the maximum concentration levels (no more than the highest clinically visible concentration) of the different interferents were studied using the non-added group as a control, with a relative deviation of less than 5% between the added group and the control group as an interference acceptable standard.
Table 1 assay data for anti-tamper capability of test kits
The results show that: the interferents in table 1 do not interfere significantly with the detection results.
Finally, it should be noted that: the foregoing description is only a preferred embodiment of the present invention, and the present invention is not limited thereto, but it is to be understood that modifications and equivalents of some of the technical features described in the foregoing embodiments may be made by those skilled in the art, although the present invention has been described in detail with reference to the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (5)

1. A monoclonal antibody composition against s100deg.P protein, characterized in that: including S100deg.P-Clone 1 and S100deg.P-Clone 2,
the amino acid sequence of the heavy chain variable region of the S100 beta-Clone 1 is shown as SEQ ID NO.1, and the amino acid sequence of the light chain variable region of the S100 beta-Clone 1 is shown as SEQ ID NO. 2;
the amino acid sequence of the heavy chain variable region of the S100 beta-Clone 2 is shown as SEQ ID NO.3, and the amino acid sequence of the light chain variable region of the S100 beta-Clone 2 is shown as SEQ ID NO. 4.
2. Use of the anti-s100deg.P protein monoclonal antibody composition of claim 1 for preparing a diagnostic reagent for early stage of brain injury of a patient.
3. Use of a monoclonal antibody composition against S100 β protein according to claim 2 for the preparation of a diagnostic reagent for early stage of brain injury in patients, characterized in that: monoclonal antibodies were used in the s100deg.C chemiluminescent kit.
4. The S100 beta chemiluminescent kit is characterized in that: comprising S100deg.P-Clone 1 and S100deg.P-Clone 2 according to claim 1.
5. The s100deg.C chemiluminescent kit of claim 4 wherein: the kit uses S100deg.P-Clone 1 and S100deg.P-Clone 2 as coating antibody and detection antibody, respectively.
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