A GFAP recombinant protein, monoclonal antibody and preparation method and application thereof

Abstract

The invention provides a GFAP recombinant protein, a monoclonal antibody and a preparation method and application thereof. The invention realizes efficient, soluble and active expression of GFAP in Escherichia coli by optimizing antigen peptide sequences and expression vectors. Mouse monoclonal antibodies specifically binding to GFAP protein are screened based on the GFAP recombinant protein, and paired monoclonal antibodies are screened using the mouse monoclonal antibodies. An ELISA detection method or kit for double antibody sandwich immunization and biotin-anti-biotin monoclonal antibody amplification applied to GFAP is further constructed based on the monoclonal antibody combination. The method or kit can specifically detect human GFAP protein, has no cross reaction with other neural markers S-100B protein and neuron-specific enolase (NSE), is consistent with the detection value of a fixed-value calibrator, and has high detection sensitivity.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and specifically relates to a GFAP recombinant protein, a monoclonal antibody and a preparation method and application thereof. Background Art

[0002] Craniocerebral injury is an important cause of death and disability worldwide, including stroke, mild traumatic brain injury (TBI), etc. These brain injuries often lead to death after progression, seriously threatening the patient's life safety. Usually, imaging methods such as cranial CT and MRI can be used to diagnose most craniocerebral injuries, but they are not sensitive enough to distinguish stroke, early concussion, microbleeding foci, diffuse axonal injury, etc. Therefore, finding biomarkers with high sensitivity and specificity for the severity, progression and prognosis evaluation of brain injury diseases such as TBI and stroke will help improve the clinical treatment effect of brain injury diseases such as TBI and stroke, improve patient prognosis, and have important value for diagnosis, treatment and prognosis evaluation.

[0003] Glial fibrillary acidic protein (GFAP) is a unique component of the cytoskeletal protein of astrocytes in the brain and is specific to the central nervous system. Most studies have shown that GFAP positivity is negatively correlated with improved neurological function and reduced infarct volume. GFAP has been widely used as a candidate biomarker for the diagnosis of brain injury. Under normal circumstances, GFAP will degrade itself inside and outside the cell and is stably expressed in the serum. It has the functions of nourishing and protecting nerves and participating in the electrophysiological activities of brain neurons. Under pathological conditions, the central nervous system is damaged to varying degrees, astrocytes are damaged, GFAP polymers break, and diffuse from the damaged site into the blood, resulting in an increase in serum GFAP.

[0004] GFAP is an acidic protein composed of 432 amino acids with a molecular weight of 50-52kDa. It is a cytoskeletal protein and a specific marker protein in the cytoplasm of astrocytes. When astrocytes are stimulated, the expression of GFAP in the cells changes significantly. Studies have found that GFAP is significantly associated with dementia, multiple sclerosis, and stroke. GFAP increases rapidly after a hemorrhagic stroke and reaches a peak within 6-12 hours. In ischemic stroke, the increase in GFAP is relatively late. Since these two types of stroke have different mechanisms of onset, the treatment strategies will be different. Therefore, GFAP is considered as a biological marker for acute cerebral hemorrhage. It is a diagnostic tool to distinguish between the two types of stroke. The serum GFAP level of patients with cerebral hemorrhage can also be used to judge the patient's condition and prognosis. In addition, there is a close positive correlation between GFAP in the serum of patients with acute cerebral hemorrhage and the volume of cerebral hemorrhage. Therefore, GFAP is also a marker for traumatic brain injury (TBI). For mild and moderate TBI, GFAP will increase significantly within 8 hours of injury, and the concentration level of GFAP can also be used to predict the prognosis after injury. However, the existing detection methods for GFAP markers have the problems of high cost and low detection accuracy. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a GFAP recombinant protein, monoclonal antibody and preparation method and application thereof. The present invention uses bioinformatics software to optimize a GFAP polypeptide segment, which can be expressed in Escherichia coli after prediction and analysis, obtains GFAP specific paired monoclonal antibody through immunization and screening, and establishes a biotin-anti-biotin monoclonal antibody amplified double antibody sandwich detection system of ELISA method in combination with a signal amplification system, providing a new approach for early diagnosis, treatment monitoring and prognosis evaluation of craniocerebral injury.

[0006] The technical solution of the present invention is:

[0007] A GFAP recombinant protein, whose amino acid sequence is shown as SEQ ID No.1.

[0008] The nucleotide sequence of the gene encoding the GFAP recombinant protein is shown in SEQ ID No.2.

[0009] A method for preparing GFAP recombinant protein, the specific operations are as follows:

[0010] (1) connecting the gene encoding the recombinant antigen to an expression vector to construct a recombinant plasmid containing the recombinant antigen gene;

[0011] (2) The recombinant plasmid is transformed into competent cells, induced for expression, and then purified to obtain the GFAP recombinant protein.

[0012] In step (1), the coding gene is codon-optimized by the E. coli expression system, and the expression vector is any one of pET28a, pET32a, pET40b, pSmart1, and pCold-TF.

[0013] In step (2), the competent cells are any one of BL21 (DE3), Rosetta, and OrigamiB (DE3).

[0014] A monoclonal antibody that specifically reacts with the GFAP recombinant protein is used to screen a monoclonal antibody that can be paired with the monoclonal antibody, and the two monoclonal antibodies are combined to construct a double antibody sandwich immunization and biotin-anti-biotin monoclonal antibody amplified ELISA detection method that can specifically detect human GFAP protein.

[0015] The method for preparing the monoclonal antibody comprises the following specific steps:

[0016] S1) adding adjuvant to the purified GFAP recombinant protein, mixing, and immunizing female BALB / c mice;

[0017] S2) taking all spleen cells of the immunized mice and fusing them with SP2 / 0 myeloma cells in the logarithmic growth phase, screening and culturing them to obtain hybridoma cells;

[0018] S3) The hybridoma cells obtained by screening are expanded and cultured, and injected intraperitoneally into female BALB / c mice. The ascites is collected, and the ascites is purified after centrifugation to obtain monoclonal antibodies.

[0019] In step (S1), the adjuvant is any one of Freund's complete adjuvant, MF59 aqueous adjuvant, and aluminum hydroxide adjuvant;

[0020] The volume ratio of the purified recombinant antigen to the adjuvant is 1:1.

[0021] The ELISA detection method is constructed based on the monoclonal antibody, specifically: based on the monoclonal antibody, a monoclonal antibody that can be paired with it is screened, and the two monoclonal antibodies are further used to construct a double antibody sandwich immunization and biotin-anti-biotin monoclonal antibody amplified ELISA detection method that can specifically detect human GFAP protein.

[0022] The application of the ELISA detection method in detecting human GFAP protein. Specifically, the ELISA detection method is an ELISA detection method based on double antibody sandwich immunity and biotin-anti-biotin monoclonal antibody amplification, which can specifically detect human GFAP protein, has no cross reaction with other neural markers S-100B protein and neuron-specific enolase (NSE), is consistent with the detection value of the fixed value calibrator, and has high detection sensitivity.

[0023] The beneficial effects of the present invention are:

[0024] (1) The present invention provides a GFAP recombinant protein, which achieves efficient, soluble and active expression of GFAP in Escherichia coli by optimizing the antigen peptide sequence and expression vector;

[0025] (2) Based on the GFAP recombinant protein, the present invention screened out mouse monoclonal antibodies that specifically bind to the GFAP protein, and used the mouse monoclonal antibodies to screen paired monoclonal antibodies. Further, based on the monoclonal antibody combination, an ELISA detection method or kit for GFAP using double antibody sandwich immunization and biotin-anti-biotin monoclonal antibody amplification was constructed, which can specifically detect human GFAP protein, and has no cross-reaction with other neural markers S-100B protein and neuron-specific enolase (NSE). The detection value is consistent with that of the fixed value calibrator, indicating that the ELISA detection method or kit of the present invention has high detection sensitivity and has certain clinical application value in the early diagnosis of craniocerebral injury and the prediction of disease progression. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0027] Figure 1 Shown is the SDS-PAGE electrophoresis identification result of the GFAP recombinant protein of the present invention;

[0028] Figure 2 It shows the ELISA reaction result between the GFAP recombinant protein of the present invention and the specific monoclonal antibody. DETAILED DESCRIPTION

[0029] To make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0030] The above technical solution is described in detail below in conjunction with specific embodiments.

[0031] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0032] Example 1 Preparation of recombinant antigen

[0033] This embodiment provides a GFAP recombinant protein, whose amino acid sequence is shown in SEQ ID No.1, and the nucleotide sequence of the gene encoding the recombinant antigen is shown in SEQ ID No.2.

[0034] SEQ ID No. 1 is as follows:

[0035] MNQLRAKEPTKLADVYQAELRELRLRLDQLTANSARLEVERDNLAQDLATVRQKLQDETNLRLEAENNLAAYRQEADEATLARLDLERKIESLEEEIRFLRKIHEEEVRELQEQLARQQVHVELDVAKP DLTAALKEIRTQYEAMASSNMHEAEEWYRSKFADLTDAAARNAELLRQAKHEANDYRRQLQSLTCDLESLRGTNESLERQMREQEERHVREAASYQEALARLEEEGQSLKDEMARHLQEYQDLLNVKLA.

[0036] SEQ ID No. 2 is as follows:

[0037] ATGAATCAGCTGCGCGCCAAAGAACCGACCAAACTGGCAGATGTGTATCAGGCAGAACTGCGTGAACTGCGCCTGCGTCTGGATCAGCTGACCGCCAATAGTGCCCGTCTGGAAGTTGAACGCGATAATCTGGCACAGGATCTGGCCACCGTGCGTCAGAAACTGCAGGATGAAACCAATCTGCGTCTGGAAGCAGAAAATAATCTGGCCGCATATCGCCAGGAAGCCGATGAAGCCACCCTGGCACGTCTGGATCTGGAACGCAAAATTGAAAGCCTGGAAGAAGAAATTCGCTTTCTGCGCAAAATTCATGAAGAAGAAGTTCGTGAACTGCAGGAACAGCTGGCACGTCAGCAGGTGCATGTTGAACTGGATGTTGCAAAACCGGATCTGACCGCAGCACTGAAAGAAATTCGTACCCAGTATGAAGCCATGGCAAGTAGCAATATGCATGAAGCCGAAGAATGGTATCGCAGCAAATTTGCAGATCTGACCGATGCCGCAGCACGCAATGCCGAACTGCTGCGTCAGGCAAAACATGAAGCAAATGATTATCGTCGCCAGCTGCAGAGCCTGACCTGCGATCTGGAAAGCCTGCGTGGCACCAATGAAAGTCTGGAACGTCAGATGCGCGAACAGGAAGAACGCCATGTTCGTGAAGCAGCAAGCTATCAGGAAGCCCTGGCACGCCTGGAAGAAGAGGGTCAGAGTCTGAAAGATGAAATGGCACGCCATCTGCAGGAATATCAGGATCTGCTGAATGTGAAACTGGCC。

[0038] This embodiment provides a method for preparing a GFAP recombinant protein, comprising the following steps:

[0039] (1) Plasmid construction:

[0040] The 102aa-358aa containing the main antigenic epitope of GFAP protein was predicted and selected for expression using computational software, as shown in SEQ ID No. 2. This segment of polypeptide can be expressed in the E. coli prokaryotic expression system. The gene was codon-optimized in the E. coli expression system (adding the start codon ATG at the N-terminus) and synthesized by Anhui General Biotechnology, and cloned into the pET28a vector to obtain the recombinant plasmid pET28a-GFAP expressing GFAP protein;

[0041] (2) Inducible expression:

[0042] The recombinant plasmid pET28a-GFAP was transformed into BL21 (DE3) competent cells, and the transformed bacteria were spread on LB agar plates containing 50 μg / mL kanamycin and cultured at 37°C overnight. A single colony was picked and inoculated into 4 mL of LB medium containing 50 μg / mL kanamycin, and cultured at 37°C and 230 rpm overnight. 1% of the total volume of the culture medium was inoculated into LB medium containing 50 μg / mL kanamycin, and cultured at 37°C and 220 rpm for about 3 hours, until the OD600 was 0.6, and a final concentration of 0.1 mM IPTG was added, and the bacteria were collected after induction at 16°C and 180 rpm for 15 hours.

[0043] (3) Purification of recombinant protein:

[0044] Because the expressed recombinant protein has a histidine tag, we used ACHROM Base protein purification instrument and HisTrap TM HP chromatography column was used for preliminary crude purification, and then ion exchange chromatography was used to obtain the purified recombinant protein, which is the GFAP recombinant protein, and the amino acid sequence is shown in SEQ ID No.1.

[0045] Specifically, buffer A is 10mM phosphate, 500mM NaCl, pH 8.0, buffer B is 10mM phosphate, 500mM NaCl, 0.5M imidazole, pH 8.0, the chromatography column is balanced with buffer A, then the fermented bacterial solution is centrifuged at 8000rpm for 5min, the precipitate is resuspended with liquid A, ultrasonically broken in an ice water bath for 20min, ultrasonicated for 3 seconds at intervals of 4 seconds, centrifuged at 10000rpm for 30min, the supernatant is filtered with a 0.22 micron filter, loaded, the chromatography column is washed with buffer A, and finally gradient eluted with buffer B, the target protein collection peak is selected and dialyzed with 10mM phosphate pH 8.0, and then DEAE ion exchange chromatography is performed, and the target protein peak is collected by gradient elution of different NaCl concentrations, and the purification is observed by SDS-PAGE protein gel electrophoresis. The protein concentration is determined by Thermo's ultra-micro spectrophotometer Nanodrop and stored at -20℃.

[0046] (4) Identify the purity of the recombinant protein by SDS-PAGE electrophoresis:

[0047] Protein sample pretreatment: add an equal volume of 2×SDS loading buffer to each sample, boil in a boiling water bath for 10 min, centrifuge at 12000 rpm for 3 min, and add the supernatant to the sample well.

[0048] Preparation of SDS-PAGE electrophoresis gel: Wash and dry the vertical electrophoresis glass plate, place it vertically on the gel preparation rack, prepare the separation gel system as required, add it to the gap between the glass plates, and add anhydrous ethanol to seal it after the gel surface reaches the required height; after solidifying at room temperature for 30min-1h, discard the sealing liquid on the top of the gel and dry it with absorbent paper, prepare the concentrated gel in proportion, insert a suitable comb, and solidify it at room temperature for 30min-1h.

[0049] SDS-PAGE electrophoresis: dilute 5× glycine buffer to the working concentration, add it to the electrophoresis tank to the appropriate liquid level, and gently pull the comb out of the solidified gel. Add protein marker and 10μL of treated protein sample to the sample well. Turn on the power supply, adjust the voltage to 90V constant voltage electrophoresis to the separation gel, and then change to 130V until bromophenol blue reaches the bottom of the gel. Staining and decolorization: cut the gel from the glass plate, place it in Coomassie brilliant blue dye, shake and dye for 3 hours, and then decolorize it with decolorizing solution until the bands are clear.

[0050] like Figure 1 As shown, "M" in the figure represents protein marker, and "1" represents lane 1, which is the purified recombinant protein GFAP. It can be seen from SDS-PAGE that the recombinant GFAP protein has a very high purity, the estimated molecular weight of the fusion protein is 34.7 kDa, and the band position is consistent with the expected size.

[0051] ELISA to identify the activity of recombinant protein

[0052] The purified recombinant GFAP protein was coated (1 μg / mL), and the reaction with the commercially available HyTest GFAP mouse monoclonal antibody (GFAP81cc) was identified by ELISA. First, the recombinant protein was coated in the microplate: the coating buffer was carbonate buffer (1.59 g sodium carbonate, 2.93 g sodium bicarbonate, fixed to 1 L pure water), the coating concentration was 1 μg / mL, 50 μl / well, overnight at 4°C; 3% sucrose, 1% BSA blocking, 150 μl per well, 37°C for 2 hours, wash the plate once with washing buffer PBST (PBS containing 0.05% Tween-20), and pat dry; GFAP81cc was diluted with PBS according to the gradient of 10 μg / mL, 1 μg / mL, 100 ng / mL, and 10 ng / mL, and 50 μL was added to the microplate coated with the antigen. At the same time, neurofilament light chain protein NfL mouse monoclonal antibody was used as a negative control, and the reaction was carried out at 37°C for 30 minutes. Shake out the liquid in the wells, wash the plate 4 times with PBST washing solution, pat dry, add 50 μL / well of HRP-labeled goat anti-mouse (diluted 1:5000 in PBS), react at 37℃ for 30 min, wash the plate 4 times again, pat dry, add 50 μL / well of TMB color developing solution, develop at room temperature for 10 min, finally add 0.5 M sulfuric acid to terminate the reaction, and measure the OD450nm value with an enzyme reader.

[0053] like Figure 2 As shown, "Ctrl" represents the control NfL monoclonal antibody. It can be seen from the figure that indirect ELISA shows that the recombinant GFAP protein can specifically bind to the commercial GFAP monoclonal antibody, indicating that the protein has good biological activity and can be used for subsequent immune and screening tests.

[0054] Example 2 Screening of monoclonal antibodies

[0055] This embodiment provides a method for obtaining monoclonal antibodies based on the recombinant protein GFAP screening, and the specific operations are as follows:

[0056] S1) Mouse immunization

[0057] 50 μg of recombinant protein GFAP was mixed with an equal volume of Freund's complete adjuvant to obtain a mixture, which was then injected subcutaneously at multiple points to immunize 6-week-old female BALB / c mice. At week 0 and week 2, Freund's incomplete adjuvant was mixed with the recombinant protein in equal proportions and emulsified according to the above method, and then immunized again. At week 6, mouse serum was collected to test the antibody titer. The mouse with the highest titer was selected and boosted with 20 μg of recombinant protein GFAP through the tail vein. Three days later, the mouse spleen was taken for the preparation of hybridoma cells;

[0058] S2) Screening of hybridoma cell lines

[0059] All spleen cells of immunized mice were fused with SP2 / 0 myeloma cells in the logarithmic growth phase and then placed in HAT medium for screening and culture. When the fused cells grew to 1 / 2 of the bottom of the well, positive clones were screened by GFAP protein indirect ELISA method, and the positive rate was 100% through 2-3 consecutive limiting dilution cloning methods, and the cell lines were expanded and frozen;

[0060] Among them, the method of indirect ELISA method for screening positive clones is as follows:

[0061] First, the recombinant protein was coated in the microplate: the coating buffer was carbonate buffer (1.59 g sodium carbonate, 2.93 g sodium bicarbonate, fixed to 1 L pure water), the coating concentration was 1 μg / mL, 50 μl / well, 4°C overnight; 3% sucrose, 1% BSA blocking, 150 μL per well, 37°C for 2 hours, wash the plate once with washing buffer PBST (PBS containing 0.05% Tween-20), pat dry; add 50 μL cell culture supernatant, react at 37°C for 30 minutes. Shake out the liquid in the wells, wash the plate 4 times with PBST washing solution, pat dry, add 50 μL / well of HRP-labeled goat anti-mouse (diluted 1:5000 in PBS), react at 37℃ for 30 min, wash the plate 4 times again, pat dry, add 50 μL / well of TMB color developing solution, develop at room temperature for 10 min, finally add 0.5 M sulfuric acid as a terminator to terminate the reaction, and measure the OD450nm value with an enzyme reader.

[0062] S4) Preparation of monoclonal antibody ascites

[0063] After the selected monoclonal cell lines were expanded and cultured, 0.2 mL (containing 2.5×10 6 Cells) were pretreated with Freund's incomplete adjuvant in female BALB / c mice. About 10 days later, when the abdomen of the mice was obviously swollen, ascites was collected using a sterile syringe needle. The collected ascites was centrifuged at 3000r / min for 10 minutes to collect the middle layer.

[0064] S5) Purification of monoclonal antibodies

[0065] Centrifuge the ascites at 12000r / min for 5 minutes, take 5mL of the supernatant, add 20ml of acetate buffer (68mmol / L, pH 4.5), mix well, slowly add 50μL of octanoic acid, stir while adding, continue stirring for 30 minutes after adding, centrifuge at 12000r / min for 30 minutes at 2-8℃, and take the supernatant. Filter the supernatant with absorbent cotton, add saturated ammonium sulfate at a ratio of 50% (V / V) of the final volume, stir while adding, continue stirring for 30 minutes after adding, let it precipitate at 2-8℃ for 4-5 hours, centrifuge at 12000r / min for 30 minutes at 2-8℃, and take the precipitate. The precipitate was resuspended with 3 ml Tris-HCl (10 mmol / L, pH 9.0), placed in a dialysis bag (MW: 8000-14000), and dialyzed in 2 L Tris-HCl (10 mmol / L, pH 9.0) solution at 2-8 ° C for 14 hours. The liquid in the dialysis bag was transferred to a centrifuge tube and centrifuged at 12000 r / min for 5 minutes. The supernatant was the purified monoclonal antibody. The concentration of the purified monoclonal antibody was measured with an ultra-micro spectrophotometer and stored at -20 ° C to avoid repeated freezing and thawing.

[0066] S6) Identification of binding activity of monoclonal antibodies

[0067] The recombinant protein GFAP antigen and the control carrier protein (pET28a-Tau recombinant protein) were coated with the indirect ELISA method, and the purified antibodies were tested by ELISA to identify the affinity of the monoclonal antibody. The antibody was diluted to 1 μg / ml. The results are shown in Table 1. "Ctrl" in Table 1 represents the negative control NfL mouse monoclonal antibody.

[0068] Table 1 - Binding activity identification of monoclonal antibodies

[0069]

[0070] As can be seen from Table 1, the binding activity of the 32 screened GFAP monoclonal antibodies to GFAP protein and control carrier protein was detected by indirect ELISA. The data showed that the monoclonal antibodies could specifically bind to GFAP protein.

[0071] Example 3 Screening and application of paired monoclonal antibodies

[0072] 1. HRP labeling of antibodies:

[0073] The screened monoclonal antibodies were labeled according to the instructions of G-Biosciences' HOOK™ HRP PLUS Labeling Kit (Cat#:786-313). Finally, the labeled antibodies were dialyzed overnight in 0.01M PBS, pH 7.4 buffer, glycerol was added at a volume of 1:1, and the cells were stored at -20°C.

[0074] Specifically: dilute the antibody to be labeled with coupling buffer to a final concentration of 2 mg / mL, then add the diluted antibody solution to the tube containing HRP (provided with the kit), mix it by pipetting, and incubate it at room temperature for 1 hour. Mix it regularly during the incubation period. Add 50 μL of stop solution, mix it for 15 minutes to terminate the labeling reaction, dialyze it in PBS buffer overnight, and add an equal volume of glycerol for freezing.

[0075] 2. Establishment of double antibody sandwich immunoassay method:

[0076] The purified monoclonal antibody was diluted to 0.5μg / mL, 1μg / mL, and 2μg / mL concentrations with coating buffer (1.59g sodium carbonate and 2.93g sodium bicarbonate, fixed to 1L pure water, pH 9.6) and added to the ELISA plate at 50μL / well for overnight coating at 4°C. The coating solution was discarded the next day, and the plate was washed once with washing solution (PBST, PBS containing 0.05% Tween-20), patted dry, and blocked with 3% sucrose and 1% BSA at 150μL / well. Incubated at 37°C for 2h, the blocking solution was discarded, and patted dry. The GFAP recombinant protein of the sample to be tested and the S100B protein of the control sample were diluted with PBS at 10μg / mL, 1μg / mL, 100ng / mL, and 10ng / mL, and then added to the ELISA plate, 50μL / well, incubated at 37℃ for 35min, washed 4 times with PBST washing solution, patted dry, and added 50μL / well of enzyme-labeled monoclonal antibody diluted to 500, 1000, and 2000 times with PBS, incubated at 37℃ for 35min, washed 4 times, patted dry, and added 50μL / well of TMB color development solution, color developed at room temperature for 10min, and finally added 0.5M sulfuric acid, 50μL / well, terminated the reaction, and measured the OD450nm value with an ELISA reader. The condition pairing with the most obvious positive and negative differences was selected as the best monoclonal antibody combination for optimization of the biotin amplification system.

[0077] 3. ELISA detection method based on double antibody sandwich immunity and biotin-anti-biotin monoclonal antibody amplification:

[0078] The above-screened monoclonal antibody combinations were used for coating and biotin coupling, respectively. The specific operation of biotin-antibody coupling was as follows: the coupling molar ratio was 20:1. First, 2.0 mg of activated biotin was dissolved in 360 μL ultrapure water to prepare a 10 mM biotin solution. 2 mg of antibody and 26.6 μL of 10 mM biotin were shaken and reacted at room temperature for 3 hours (the reaction system was controlled at about 2 mL). The biotin-antibody mixture was then dialyzed with 0.01 M PBS to remove excess biotin. After dialysis, the antibody concentration was determined, and then an equal volume of glycerol was added and stored at -20°C.

[0079] Then, the most suitable reaction conditions for the amplification system were determined by adjusting the concentration of the coated antibody, biotin antibody and the dilution of the HRP-labeled anti-biotin monoclonal antibody. Specifically, the monoclonal antibody was coated in the microplate (coating buffer carbonate buffer: 1.59g sodium carbonate, 2.93g sodium bicarbonate, fixed to 1L pure water), the coating concentration gradient was 0.5μg / mL, 1μg / mL, 2μg / mL, 50μL / well, 4℃ overnight; the plate was washed once with washing solution (PBST, PBS containing 0.05% Tween-20), patted dry, blocked with 3% sucrose, 1% BSA, 150μL per well, 37℃ for 2 hours, patted dry and stored for later use; the recombinant protein GFAP was diluted to a concentration of 10ng / ml with PBS, and 50μL was added to the microplate coated with the monoclonal antibody, and the S100B protein was diluted to 10ng / ml as a negative control, and reacted at 37℃ for 30min. The liquid in the wells was shaken off, the plate was washed 4 times with PBST solution, and patted dry; the biotin-conjugated monoclonal antibody was diluted with PBS to a concentration of 1μg / mL, 2μg / mL, and 4μg / mL, and 50μL / well was added to the microplate, and reacted at 37°C for 30min. The liquid in the wells was shaken off, the plate was washed 4 times with PBST solution, and patted dry, and 50μL / well of HRP-labeled anti-biotin monoclonal antibody (diluted 1000, 2000, and 4000 times with PBS) was added, and reacted at 37°C for 30min, and the plate was washed 4 times, and patted dry, and 50μL / well of TMB colorimetric solution was added to develop at room temperature for 10min, and finally the stop solution was added to terminate the reaction, and the OD450nm value was measured with an enzyme reader. The coating conditions, biotin monoclonal antibody concentration, and HRP-labeled anti-biotin monoclonal antibody dilution with the most obvious positive and negative differences were selected as the optimal reaction conditions.

[0080] Through the above-mentioned coating and HRP labeling of monoclonal antibodies, the optimal pairing combination with the most obvious difference in positive and negative detection values ​​was screened out according to the detection of positive and negative samples, and then the biotin-anti-biotin monoclonal antibody amplification system was debugged. It was finally determined that the concentration of coated monoclonal antibody GFAP-8# was 1μg / mL, the concentration of biotin-coupled monoclonal antibody GFAP-23# was 2μg / mL, and the dilution of HRP-labeled anti-biotin monoclonal antibody was 1000 times. Under this condition, the background of the detection system was the lowest, the detection value of positive protein was the highest, the detection was the most sensitive, and the detection limit of GFAP standard protein was 13.2pg / mL.

[0081] The full-length amino acid sequence of the heavy chain (H chain) variable region of the monoclonal antibody GFAP-8# is shown in SEQ ID No.3, and the nucleotide sequence is shown in SEQ ID No.4.

[0082] SEQ ID No.3 is as follows:

[0083] QVQLKQSGGGLVQPGGSLKLSCAASGFDFTRYWMSWVRQAPGKGLEWIGEINPDSSTINYTPSLKDEFIISRDNAKNTLYLQMSKVRSEDTALYYCARSSIYDGYYWYFDVWGAGTTVTVSS.

[0084] The three CDRs sequences of the heavy chain (H chain) are as follows:

[0085] CDR-H1 amino acid sequence: GFDFTRYW (SEQ ID No. 11);

[0086] CDR-H2 amino acid sequence: INPDSSTI (SEQ ID No. 12);

[0087] CDR-H3 amino acid sequence: ARSSIYDGYYWYFDV (SEQ ID No. 13).

[0088] SEQ ID No. 4 is as follows:

[0089] caggtgcagctgaagcagtctggaggtggcctggtgcagcctggaggatccctgaaactctcctgtgcagcctcaggattcgattttactagatactggatgagttgggtccggcaggctccagggaaagggctagaatggattggagaaattaatccagatagcagtacgataaactatacg ccatctctaaaggatgaattcatcatctccagagacaacgccaaaaatacgctttacctgcaaatgagcaaagtgagatctgaggacacagccctttattactgtgcaagatcttcgatctatgatggttactattggtatttcgatgtctggggcgcagggaccacggtcaccgtctcctca.

[0090] The full-length amino acid sequence of the light chain (L chain) variable region of the monoclonal antibody GFAP-8# is shown in SEQ ID No.5, and the nucleotide sequence is shown in SEQ ID No.6.

[0091] SEQ ID No.5 is as follows:

[0092] DVVMTQSPSSLSASLGERVSLTCRASLEINRYLSWLQQKPDGTIKRLIYAASTLDSGVPKRFSGSRSGSDYSLTISSLESEDFADYYCQQFTVYPPTFGGGTKLEIKRTV.

[0093] The three CDRs sequences of the light chain (L chain) are as follows:

[0094] CDR-L1 amino acid sequence: LEINRY (SEQ ID No. 14);

[0095] CDR-L2 amino acid sequence: AAS;

[0096] CDR-L3 amino acid sequence: QQFTVYPPT (SEQ ID No. 15).

[0097] SEQ ID No.6 is as follows:

[0098] gatgttgtgatgacccaatctccatcctccttatctgcctctctgggagaaagagtcagtctcacttgtcgggcaagtctggaaattaatcgttacttaagctggcttcagcagaaaccagatggaactattaaacgcctgatctacgccgcatccactttagat tctggtgtcccaaaaaggttcagtggcagtaggtctgggtcagattattctctcaccatcagcagccttgagtctgaagattttgcagactattactgtcaacaatttactgtttatcctcccacgttcggaggggggaccaagctggaaataaaacgtacggtg.

[0099] The full-length amino acid sequence of the heavy chain (H chain) variable region of the monoclonal antibody GFAP-23# is shown in SEQ ID No.7, and the nucleotide sequence is shown in SEQ ID No.8.

[0100] SEQ ID No.7 is as follows:

[0101] QVQLQQSGPGILQPSQTLSLTCTFSGFSLSTSGMGVSWIRQPSGKGLEWLAHIYWDDDKRYNPSLKSRLTISKDTSRNQVFLKITSVDTADTATYYCARCLYDGSYDYWGQGTTLTVSS.

[0102] The three CDRs sequences of the heavy chain (H chain) are as follows:

[0103] CDR-H1 amino acid sequence: GFSLSTSGMG (SEQ ID No. 16);

[0104] CDR-H2 amino acid sequence: IYWDDDK (SEQ ID No. 17);

[0105] CDR-H3 amino acid sequence: ARCLYDGSYDY (SEQ ID No. 18).

[0106] SEQ ID No. 8 is as follows:

[0107] caggttcagctgcaacagtctggccctgggatattgcagccctcccagaccctcagtctgacttgtactttctctgggttttcactgagcacttctggtatgggtgtgagttggattcgtcagccttcaggaaagggtctggagtggctggcacacatttactgggatgatgacaagcg ctataacccatccctgaagagccggctcacaatttccaaggatacctccagaaaccaggtattcctcaagatcaccagtgtggacactgcagatactgccacatactactgtgctcgatgtctctatgatggttcatatgactactggggccaaggcaccactctcacagtctcctca.

[0108] The full-length amino acid sequence of the light chain (L chain) variable region of the monoclonal antibody GFAP-23# is shown in SEQ ID No.9, and the nucleotide sequence is shown in SEQ ID No.10.

[0109] SEQ ID No.9 is as follows:

[0110] DVVVTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQHSHVPLTFGAGTKLELK.

[0111] The three CDRs sequences of the light chain (L chain) are as follows:

[0112] CDR-L1 amino acid sequence: QSIVHSNGNTY (SEQ ID No. 19);

[0113] CDR-L2 amino acid sequence: KVS;

[0114] CDR-L3 amino acid sequence: FQHSHVPLT (SEQ ID No. 20).

[0115] SEQ ID No. 10 is as follows:

[0116] gatgttgtggtgactcaaactccactctccctgcctgtcagtcttggagatcaagcctccatctcttgcagatctagtcagagcattgtacatagtaatggaaacacctatttagagtggtacctgcagaagcctggccagtctccaaagctcctgatctacaaagttt ccaaccgattttctggggtcccagacaggttcagtggcagtggatcagggacagatttcacactcaagatcagcagagtggaggctgaagatctgggagtttattactgctttcaacattcacatgttccgctcacgttcggtgctgggaccaagctggagctgaaac.

[0117] 4. Sensitivity evaluation of ELISA detection method based on double antibody sandwich immunization and biotin-anti-biotin monoclonal antibody amplification

[0118] Referring to the above detection steps, the GFAP fixed value standard protein was first diluted with PBS buffer solution, and the concentrations after dilution were 2000pg / mL, 200pg / mL, 20pg / mL, 2pg / mL, and 0pg / mL, respectively. 50μL of the diluted standard protein of different concentrations was taken for detection, and three parallel experiments were performed for each concentration to prepare a standard curve. At the same time, the GFAP serum samples fixed by Hunan Novozyme's GFAP detection kit (chemiluminescence method) were tested to compare the compliance of the test results.

[0119] The results are shown in Table 2.

[0120] Table 2 - Sensitivity evaluation results of the ELISA detection method of the present invention

[0121]

[0122] As shown in Table 2, the test results of 9 serum samples were compared and found to be completely consistent with the test results of the commercial kit.

[0123] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A monoclonal antibody specific for GFAP recombinant protein, characterized in that: The monoclonal antibody comprises a VH domain and a VL domain; the VH domain is the amino acid sequence shown in SEQ ID No.3, and the VL domain is the amino acid sequence shown in SEQ ID No.5; The VH domain includes CDR-H1 with an amino acid sequence as shown in SEQ ID No.11, CDR-H2 with an amino acid sequence as shown in SEQ ID No.12, and CDR-H3 with an amino acid sequence as shown in SEQ ID No.13; The VL domain includes a CDR-L1 having an amino acid sequence as shown in SEQ ID No. 14, a CDR-L2 having an amino acid sequence of AAS, and a CDR-L3 having an amino acid sequence as shown in SEQ ID No. 15; Alternatively, the monoclonal antibody comprises a VH domain and a VL domain; the VH domain is the amino acid sequence shown in SEQ ID No.7, and the VL domain is the amino acid sequence shown in SEQ ID No.9; The VH domain includes CDR-H1 with an amino acid sequence as shown in SEQ ID No.16, CDR-H2 with an amino acid sequence as shown in SEQ ID No.17, and CDR-H3 with an amino acid sequence as shown in SEQ ID No.18; The VL domain includes CDR-L1 with an amino acid sequence as shown in SEQ ID No.19, CDR-L2 with an amino acid sequence of KVS and CDR-L3 with an amino acid sequence as shown in SEQ ID No.

20.

2. The monoclonal antibody specific for GFAP recombinant protein according to claim 1, characterized in that: The amino acid sequence of the GFAP recombinant protein is shown in SEQ ID No.1, and the nucleotide sequence of the gene encoding the GFAP recombinant protein is shown in SEQ ID No.

2.

3. A method for preparing the monoclonal antibody according to claim 1 or 2, characterized in that: The specific operations are: S1) adding adjuvant to the purified GFAP recombinant protein, mixing, and immunizing female BALB / c mice; S2) taking all spleen cells of the immunized mice and fusing them with SP2 / 0 myeloma cells in the logarithmic growth phase, screening and culturing them to obtain hybridoma cells; S3) The hybridoma cells obtained by screening are expanded and cultured, and injected intraperitoneally into female BALB / c mice. The ascites is collected, and the ascites is purified after centrifugation to obtain monoclonal antibodies.

4. The method for preparing a monoclonal antibody according to claim 3, characterized in that: In step (S1), the adjuvant is any one of Freund's complete adjuvant, MF59 aqueous adjuvant, and aluminum hydroxide adjuvant; The volume ratio of the purified recombinant antigen to the adjuvant is 1:

1.

5. An ELISA detection method constructed based on the monoclonal antibody according to claim 1 or 2 or the monoclonal antibody prepared by the method according to any one of claims 3 to 4, wherein the ELISA detection method does not include a method for diagnosing and treating a disease.

6. Use of the ELISA detection method according to claim 5 in detecting human GFAP protein, wherein the application does not include diagnosis and treatment methods of diseases.

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