GFAP recombinant protein, monoclonal antibody as well as preparation method and application of monoclonal antibody

Abstract

The invention discloses a GFAP recombinant protein, a monoclonal antibody as well as a preparation method and application of the monoclonal antibody, and belongs to the technical fields of immunology, peptide chemistry and photochemistry. The monoclonal antibody comprises a monoclonal antibody 1 and a monoclonal antibody 2, the monoclonal antibody 1 is used as a coating antibody, and amino acid sequences of a heavy chain and a light chain of the monoclonal antibody 1 are shown as SEQ ID NO.3 and 2; the monoclonal antibody 2 is used as a labeled antibody, and amino acid sequences of a heavy chain and a light chain of the monoclonal antibody 2 are shown as SEQ ID NO.5 and 4. The obtained GFAP antibody is high in affinity, the detection sensitivity and accuracy are ensured, and the CLIA kit prepared from the GFAP antibody is suitable for early diagnosis of various nervous system diseases and has the advantages of convenience and rapidness in detection.

Description

Technical Field

[0001] The present invention belongs to the technical fields of immunology, peptide chemistry and photochemistry, and in particular relates to a GFAP recombinant protein, a monoclonal antibody and a preparation method and application thereof. Background Art

[0002] GFAP (Glial Fibrillary Acidic Protein) is composed of 432 amino acids with a molecular weight of approximately 50 kDa. GFAP is a major structural protein in the central nervous system (CNS), primarily expressed in astrocytes. As one of the intermediate filament proteins, GFAP plays an important role in maintaining astrocyte morphology, stability, and function. Astrocytes are the most numerous type of glial cell in the brain and spinal cord, supporting neuronal function, maintaining the integrity of the blood-brain barrier, participating in neuronal metabolism, and regulating the immune response of the nervous system (Emsley et al., 2003). Under normal physiological conditions, GFAP is primarily located within astrocytes, where it provides support and stress resistance. However, in various neurological diseases or injuries, GFAP expression and release are altered, making it a potential biomarker for these conditions (Hol & Pekny, 2015).

[0003] As a biomarker for neurological diseases, GFAP plays an important role in the following diseases:

[0004] (1) Traumatic brain injury (TBI)

[0005] In traumatic brain injury, astrocytes may be directly damaged or activated, leading to the release of GFAP into the blood. Elevated GFAP levels are closely associated with the severity of brain injury and have been shown to be an important biomarker for the diagnosis of TBI (Papa et al., 2012). GFAP blood concentrations are generally proportional to the extent of injury. Therefore, after acute brain injury, GFAP measurement can help assess injury severity and guide clinical treatment (Jung et al., 2015).

[0006] (2) Alzheimer's disease (AD)

[0007] Alzheimer's disease is a progressive neurodegenerative disorder characterized by neuronal loss, accumulation of amyloid plaques, and tau protein tangles. GFAP, a marker of neuroinflammation, is closely associated with the neuroinflammatory response in Alzheimer's disease (Akiyama et al., 2000). Studies have shown that GFAP levels are elevated in patients with Alzheimer's disease, particularly in the early stages of the disease, and changes in GFAP levels can reflect disease progression (Koch et al., 2019).

[0008] (3) Neuroinflammatory diseases

[0009] In neuroinflammatory diseases such as multiple sclerosis (MS) and encephalitis, astrocyte activation leads to the release of GFAP. Changes in GFAP levels can reflect neuroinflammation and help assess disease severity and treatment efficacy (Lee et al., 2015). Studies have found that GFAP levels are closely correlated with neuroinflammatory activity, thus providing a potential monitoring tool for these diseases (Frohman et al., 2016).

[0010] (4) Glioma and other brain tumors

[0011] Gliomas are a type of brain tumor that originates from glial cells. GFAP is highly expressed in gliomas, making it a potential biomarker for this type of tumor (Aldape et al., 2015). Elevated GFAP levels not only aid in the early diagnosis of gliomas but can also be used for tumor monitoring during treatment (Wang et al., 2017).

[0012] Chemiluminescence immunoassay (CLIA) is a technique that combines highly sensitive chemiluminescence measurement technology with highly specific immune reactions and is used for the detection and analysis of various antigens, haptens, antibodies, hormones, enzymes, fatty acids, vitamins, and drugs. Summary of the Invention

[0013] One of the objects of the present invention is to provide a GFAP monoclonal antibody, wherein the monoclonal antibody is monoclonal antibody 1 and / or monoclonal antibody 2, wherein the amino acid sequences of the heavy chain and light chain of monoclonal antibody 1 are shown in SEQ ID NOs. 3 and 2; and the amino acid sequences of the heavy chain and light chain of monoclonal antibody 2 are shown in SEQ ID NOs. 5 and 4.

[0014] A second object of the present invention is to provide the use of the above-mentioned GFAP monoclonal antibody in the preparation of products for detecting GFAP.

[0015] In one embodiment of the present invention, the product is a reagent or a kit.

[0016] In one embodiment of the present invention, the kit is a chemiluminescent immunoassay kit.

[0017] In one embodiment of the present invention, in the chemiluminescent immunoassay kit, monoclonal antibody 1 is used as the coating antibody; and monoclonal antibody 2 is used as the labeling antibody.

[0018] A third object of the present invention is to provide a chemiluminescent immunoassay kit for detecting GFAP, wherein the kit contains monoclonal antibody 1 and monoclonal antibody 2, wherein monoclonal antibody 1 serves as a coating antibody and monoclonal antibody 2 serves as a labeling antibody.

[0019] In one embodiment of the present invention, the monoclonal antibody 1 is coupled to magnetic beads; and the monoclonal antibody 2 is labeled with an acridinium ester.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] (1) The GFAP antibody obtained by the present invention has a strong affinity, which ensures the sensitivity and accuracy of the detection.

[0022] (2) The CLIA kit provided by the present invention is not only suitable for the early diagnosis of various neurological diseases, but also has the advantages of convenient and rapid detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the SDS-PAGE detection result of GFAP protein in Example 1.

[0024] Figure 2 The SDS-PAGE detection results of the G151-coated antibody and the G167-labeled antibody in Example 3, wherein M refers to Marker, NR refers to Non-reduced sample, and R refers to Reduced sample.

[0025] Figure 3 This is the GFAP standard curve in Example 5.

[0026] Figure 4 This is the GFAP linear graph in Example 5.

[0027] Figure 5 This is the AUC curve diagram in Example 6. DETAILED DESCRIPTION

[0028] Example 1 Preparation of recombinant GFAP antigen

[0029] Antigen preparation:

[0030] Cloning and Transformation: Clone the full-length GFAP gene (corresponding amino acid sequence is SEQ ID NO. 1) into an appropriate expression vector (between NcoI and EcoRI of the pET28a vector) containing a His tag for subsequent purification. Transform the expression vector into competent E. coli (BL21 DE3 expression strain) and select colonies on plates containing kanamycin.

[0031] SEQ ID NO.1 is as follows:

[0032] MHHHHHHGGSGGSGGMERRRITSAARRSYVSSGEMMVGGLAPGRRLGPGT

[0033] RLSLARMPPPLPTRVDFSLAGALNAGFKETRASERAEMMELNDRFASYIEKVR

[0034] FLEQQNKALAAELNQLRAKEPTKLADVYQAELRELRLRLDQLTANSARLEVE

[0035] RDNLAQDLATVRQKLQDETNLRLEAENNLAAYRQEADEATLARLDLERKIES

[0036] LEEEIRFLRKIHEEEVRELQEQLARQQVHVELDVAKPDLTAALKEIRTQYEAM

[0037] ASSNMHEAEEWYRSKFADLTDAAARNAELLRQAKHEANDYRRQLQSLTCDL

[0038] ESLRGTNESLERQMREQEERHVREAASYQEALARLEEEGQSLKDEMARHLQ

[0039] EYQDLLNVKLALDIEIATYRKLLEGEENRITIPVQTFSNLQIRETSLDTKSVSEG

[0040] HLKRNIVVKTVEMRDGEVIKESKQEHKDVM

[0041] Protein expression: Inoculate a single transformed colony into LB medium containing kanamycin, culture with shaking at 37°C overnight, transfer the culture to fresh medium, and grow to an OD600 of 0.6–0.8. Add IPTG (e.g., 0.5–1 mM) to induce protein expression and continue incubation at 18–25°C for 4–6 hours.

[0042] Cell lysis: Collect the cell pellet by centrifugation (4°C, 5000×g, 15 minutes), resuspend the cell pellet in lysis buffer (such as PBS at pH 7.4, containing protease inhibitors), and lyse the cells by sonication.

[0043] Protein purification: Load the clarified lysate onto a Ni-NTA affinity chromatography column. Wash the column with PBS containing 20–50 mM imidazole to remove nonspecifically bound proteins. Elute the target protein with elution buffer (PBS containing 500 mM imidazole). Analyze the eluted fractions by SDS-PAGE to confirm protein content.

[0044] Quality control: SDS-PAGE was used to verify the molecular weight and purity >90%, e.g. Figure 1 The protein was concentrated to a concentration of 1–2 mg / mL, sterile filtered, and aliquoted into sterile 1.5 mL centrifuge tubes. Long-term storage was performed at -80°C to avoid repeated freezing and thawing.

[0045] Example 2 Rabbit Immunization to Obtain Monoclonal

[0046] 1. Adjuvant preparation: Mix recombinant GFAP antigen and Freund's complete adjuvant in a 1:1 ratio to form a stable emulsion.

[0047] 2. Rabbit immunization:

[0048] Initial injection (day 0): 50–200 μg of full-length GFAP protein was mixed with Freund's complete adjuvant and injected into the rabbit subcutaneously at multiple points with a total injection volume of 0.5–1.0 mL.

[0049] Booster injections: Booster injections are given every 14–21 days using the same dose of antigen but mixed with Freund's incomplete adjuvant.

[0050] Antibody response monitoring: 7–10 days after each booster injection, collect 0.5–1.0 mL of blood from the marginal ear vein and use ELISA to detect the anti-GFAP antibody titer in the serum. The ELISA plate should be coated with full-length GFAP protein for detection specificity. The coating concentration is 1 μg / mL, 50 μl / well.

[0051] Final boost injection: 3–5 days before spleen removal, a single subcutaneous injection of full-length GFAP protein diluted in PBS (without adjuvant) is administered to maximize the number of circulating antibody-producing B cells.

[0052] 3. Collection of splenocytes:

[0053] The rabbits were euthanized according to ethical regulations, and the spleens were removed aseptically and placed in ice-cold sterile PBS containing 1% fetal bovine serum (FBS) in RPMI-1640 medium.

[0054] Prepare a single-cell suspension: squeeze spleen tissue through a 70 μm cell strainer into RPMI-1640 medium in a culture dish. Centrifuge at 300 g for 5 minutes at 4°C. Discard the supernatant and resuspend the pellet in fresh RPMI-1640 medium. Count the number of viable cells using a hemocytometer and trypan blue staining.

[0055] 4. Hybridoma cell line screening:

[0056] a) Fusion to generate hybridoma cells: Immortalized rabbit B cell lines were used, grown in the sensitive environment of HAT medium, and spleen cells were mixed at a ratio of 2:1 or 3:1 to form fusion cells. Polyethylene glycol (PEG 1500 or PEG 3350) was slowly added dropwise while gently stirring to avoid cell damage. Within 5 minutes, PEG was gradually diluted with RPMI-1640 medium to neutralize the PEG. After centrifugation, the cells were resuspended in RPMI-1640 containing HAT medium to indicate successful fusion. The fused cells were plated at a low density (1×10 3 to 1×10 4 cells / well) were seeded in a 96-well plate, and 200 μL of culture medium was added to each well.

[0057] b) When the fused cells have grown to cover approximately 1 / 2 of the well bottom, screen for positive clones using ELISA. Subsequently, increase the number of positive clones through 2-3 rounds of limiting dilution cloning until the positive rate reaches 100%. Finally, expand the screened cell lines and cryopreserve them.

[0058] c) Specific steps for ELISA screening of positive clones:

[0059] Microplate coating with recombinant GFAP antigen: Use carbonate buffer (formula: 1.59 g sodium carbonate, 2.93 g sodium bicarbonate, add purified water to 1 L) to dilute the recombinant GFAP antigen to 1 μg / mL, add 50 μL to each well, and place the plate at 4°C overnight to complete protein coating.

[0060] Blocking treatment: add 150 μL of blocking solution (3% sucrose and 1% bovine serum albumin) to each well, incubate at 37° C. for 2 hours, wash the microplate once with PBS containing 0.05% Tween-20 (PBST), and pat dry.

[0061] Add culture supernatant: Add 50 μL of culture supernatant from fused cells to each well and incubate at 37°C for 30 minutes. Then, pour off the liquid in the wells, wash the plate four times with PBST, and pat dry.

[0062] Add secondary antibody: Add 50 μL of HRP-labeled goat anti-mouse antibody (diluted to 1:5000 in PBS) to each well, incubate at 37°C for 30 minutes, wash the plate again with PBST 4 times, and pat dry.

[0063] Color development and termination: Add 50 μL of TMB color development solution to each well, incubate at room temperature for 10 minutes, and then add 50 μL of 0.5 M sulfuric acid to terminate the reaction.

[0064] Results: Absorbance at 450 nm (OD450) was measured using a microplate reader to determine if the cell clones were positive. At a 1:10 dilution, the antibody's absorbance was greater than 2.3, significantly higher than the negative control's 0.081, indicating strong affinity between the antibody and recombinant GFAP protein. The results are shown in Table 1.

[0065] Table 1

[0066]

[0067]

[0068] Example 3 Antibody Purification and Sequencing

[0069] Cell culture supernatants containing G151 and G167 cell lines were directly loaded onto a Protein A / G affinity chromatography column to allow specific binding of the antibody to the filler. After binding, the column was washed with elution buffer (20mM Sodium phosphate, pH 7.0) to remove nonspecifically bound proteins. The antibody was then eluted from the column with elution buffer (0.1M Lycine-HCl, pH 2.7). The eluted antibody was immediately neutralized with neutralization buffer (1M Tris-HCl, pH 8.5) to prevent inactivation. The eluate was collected and the antibody concentration was determined using a spectrophotometer. The purified antibody was aliquoted as needed and stored at -80°C until use.

[0070] To determine the antibody's gene sequence, total RNA was extracted from hybridoma cell lines G151 and G167 in logarithmic growth phase using a Trizol Total RNA Extraction Kit (Sangon, Cat. No. B5113110). cDNA was then synthesized by reverse transcription. A third-party sequencing company was commissioned to sequence this cDNA and obtain the gene sequence information for the monoclonal antibody.

[0071] G151-coating antibody:

[0072] Light chain (SEQ ID NO. 2):

[0073] MPRGWAAPLLLLLLQGGWGDVLMTQTIGSLPVALGDQVSISHESQSIVPSKGL

[0074] SGEWYLEKNGRGPIHELKNEFSGVPERYSVADVETDFALKVSKVGEKEDLIFY

[0075] CFQGSHVPVAYTEGGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY

[0076] PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY

[0077] ACEVTHQGLSSPVTKSFNRGEC

[0078] Heavy chain (SEQ ID NO.3):

[0079] MPRGWAAPLLLLLLQGGWGQVKRLESVSGLAKWEGSDGRLSCVASLPTFED

[0080] RSNSWIRQEPGSGHEYVFTISYHGTIKHLADSIKGWFGISKENTVNSITLYMWT

[0081] LLAEGTAVNYCATRILGYLGFRDYWGNPGLVTVAEASTKGPSVFPLAPSSKST

[0082] SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV

[0083] PSSSLGTQTYICNVNHKPSNTKVDDKVEPKSCDKTHTCPPCPAPELLGGPSVFL

[0084] FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE

[0085] QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIQKTISKAKGQPREP

[0086] QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD

[0087] SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0088] G167-labeled antibody:

[0089] Light chain (SEQ ID NO.4):

[0090] MPRGWAAPLLLLLLQGGWDVDLMTETFVSLFVSEGDNVRIACYSAQSIEHSE

[0091] GNGYLKWYLQKHPQAPKANEHRDSVSWHGPDRFSGIGTYVDVSWKGGQYE

[0092] DIDFASFGCTAGWGEDYTFSVNTKRVKRTVAAPSVFAFPWSDEQLKSGTASVV

[0093] CLLNNFYKREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD

[0094] YEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0095] Heavy chain (SEQ ID NO.5):

[0096] MPRGWAAPLLLLLLQGGWGGVQLVEYGYGPVKLGHSLGLSCAVSVFYGSAH

[0097] FNSTVIPEGIKGTMTAFRSHVDTAHKILVVSLKVRFTTRDMNTAERLWMQTLT

[0098] VEDLAVFYTATCILRPLGKFKDAVGTGCTVSSASTKGPSVFPLAPSSKSTSGGT

[0099] AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL

[0100] GTQTYICNVNHKPSNTKVDDKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK

[0101] PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN

[0102] STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIQKTISKAKGQPREPQVY

[0103] TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG

[0104] SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0105] Example 4 Preparation of GFAP Kit by CLIA Method

[0106] 1. Preparation of coating buffer for GFAP capture antibody-coupled magnetic beads:

[0107] Dilute the required amount of G151-coated antibody to 1 mg / mL in dialysate and dialyze at 2-8°C for 16-24 hours, changing the solution at least four times with at least 1 hour between each change. Next, weigh the magnetic beads at a ratio of 50:1 (beads:G151-coated antibody) and wash them three times with coating buffer. Then, add 10 mg / mL NHS and 10 mg / mL EDC solution to the washed beads, mix thoroughly, and incubate at 25°C with rotation for 30 minutes. Wash the beads again. Then, add the coating antibody solution to the beads at a ratio of 50:1 (beads:G151-coated antibody), incubate at 25°C with rotation for 2 hours, and wash the beads again. Finally, add magnetic bead blocking solution and rotate to mix and incubate at 25°C for 3 hours. After incubation, wash the magnetic beads three times with a certain volume of magnetic bead washing solution. Store the washed magnetic beads in magnetic bead preservation solution with a concentration of 10 mg / mL. After labeling, store at 2-8°C until use. This completes the preparation process of the coating buffer for GFAP-coated antibodies.

[0108] 2. Preparation of labeling buffer for acridinium ester-labeled GFAP detection antibody:

[0109] Dilute the required amount of G167-labeled antibody to 0.5 mg / mL in dialysate and dialyze at 2-8°C for 16-24 hours, changing the dialysate at least four times with at least 1 hour between each change. Next, remove the 2 mg / mL acridinium ester DMSO solution dissolved in DMSO and thaw for later use. Calculate the amount of acridinium ester to be used based on a mass ratio of G167-labeled antibody to acridinium ester of 5:1 (mg / mL). Dilute the acridinium ester DMSO solution with labeling buffer to the same volume as the antibody to be labeled (use within 5 minutes of preparation; exposure to light will degrade the performance of the acridinium ester). Mix the dialyzed antibody solution with the acridinium ester solution to a final antibody concentration of 0.5 mg / mL. Incubate the reaction at 25°C with shaking for 120 minutes, protecting from light. Finally, a chromatography column filled with G-25 was used for purification, and the purification buffer was the labeling buffer. The purified acridinium ester-labeled antibody was stored in acridinium ester storage solution, and the stock solution concentration was diluted to 25ug / mL. After labeling, it was stored at 2-8°C until use.

[0110] Solution preparation:

[0111] a) Labeling buffer: PBS, pH 7.4.

[0112] b) Coating buffer: 10 mM Tris, 1 M NaCl, 0.05% Tween-20, pH 7.4.

[0113] c) Magnetic bead washing solution: PBS, 0.05% Tween-20, pH 7.4.

[0114] d) Magnetic bead storage solution: 50 mM Tris pH 7.4, 0.9% NaCl, 2% BSA, 2% sucrose, 0.05% Tween-20, 0.1% Proclin 300.

[0115] e) Labeling buffer: PBS, pH 7.4.

[0116] f) Acridinium ester storage solution: 50 mM citric acid, 0.9% NaCl, 1% BSA, 0.1% Proclin 300, 0.05% Tween-20, pH 6.0.

[0117] Example 5 Kit Performance Evaluation

[0118] 1. Standard curve

[0119] The GFAP antigen was diluted with a standard diluent and prepared into calibrators of different concentrations. The GFAP calibrators were then detected using the kit in Example 4. The luminescence intensity values ​​corresponding to each calibrator were read, and a standard curve was obtained by fitting with the concentration as the horizontal axis and the luminescence intensity as the vertical axis. The GFAP standard curve data are shown in Table 2, and the standard curve is shown in the figure. Figure 3 shown.

[0120] Table 2

[0121] Concentration (pg / mL) RLU 2 265 20 1397 100 6305 1000 11320 5000 280781 10000 516505 50000 2185874

[0122] 2. Linear

[0123] The high-value sample close to the upper limit of the linear range was diluted to at least 5 concentrations according to a certain ratio. The low-value concentration sample must be close to the lower limit of the linear range. Each concentration sample was tested 3 times and the average value was calculated. The average value and the dilution ratio were fitted with a linear fit using the least squares method and the linear correlation coefficient (r) was calculated. The linear correlation coefficient r should be ≥ 0.9974. The linear graph is as follows: Figure 4 shown.

[0124] 3. Repeatability

[0125] Use two plasma samples with different concentration levels (high value and low value), and repeat the test 10 times for each concentration sample. Calculate the mean (M) and standard deviation (SD) of the 10 measurement results. Then, calculate the coefficient of variation (CV) according to the formula CV = SD / M × 100%. The CV should be within ≤8.0%.

[0126] Table 3

[0127]

[0128] 4. Accuracy

[0129] Repeat the measurement three times using an accuracy reference standard at two GFAP concentration levels. Calculate the relative deviation between the mean value (denoted as M) and the labeled value (denoted as T). Calculate the relative deviation, B, of the measured concentration using the formula B = (MT) / T x 100%. The relative deviation, B, should be ≤ 15%.

[0130] Table 4

[0131]

[0132]

[0133] Example 6 Clinical Diagnosis

[0134] Based on the clinical diagnosis results, plasma samples from 17 patients with Alzheimer's disease and 18 healthy controls were collected. The GFAP concentration in the samples was detected using the kit described in Example 4. The AUC, specificity, and sensitivity of the single marker were analyzed using the ROC curve. The results are shown in Table 2. Figure 5 .Depend on Figure 5 It can be seen that the AUC value is 0.977, the specificity is 94.4%, and the sensitivity is 94.1%.

[0135] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A GFAP monoclonal antibody, characterized in that The monoclonal antibodies are monoclonal antibody 1 and / or monoclonal antibody 2. The amino acid sequences of the heavy chain and light chain of monoclonal antibody 1 are shown in SEQ ID NOs. 3 and 2; the amino acid sequences of the heavy chain and light chain of monoclonal antibody 2 are shown in SEQ ID NOs. 5 and 4.

2. Use of the GFAP monoclonal antibody according to claim 1 in preparing a product for detecting GFAP.

3. The use according to claim 2, characterized in that The product is a reagent or a kit.

4. The use according to claim 3, characterized in that The kit is a chemiluminescence immunoassay kit.

5. The use according to claim 4, characterized in that In the chemiluminescent immunoassay kit, monoclonal antibody 1 is used as a coating antibody, and monoclonal antibody 2 is used as a labeling antibody.

6. A chemiluminescent immunoassay kit for detecting GFAP, characterized in that: The kit contains the monoclonal antibody according to claim 1, wherein the monoclonal antibody 1 is used as a coating antibody; and the monoclonal antibody 2 is used as a labeling antibody.

7. The chemiluminescent immunoassay kit according to claim 6, characterized in that The monoclonal antibody 1 is coupled to magnetic beads; the monoclonal antibody 2 is labeled with acridinium ester.

Images