An antibody kit for detecting brain glioma and application thereof

Abstract

This invention discloses an antibody kit for detecting glioma and its application. The kit includes: a solid-phase carrier coated with the anti-GFAP-N monoclonal antibody Cap-ab, recombinant GFAP protein standards, and enzyme-labeled anti-GFAP-C monoclonal antibody T-ab. The variable regions of the Cap-ab heavy and light chains are shown in SEQ ID NO.11 and SEQ ID NO.10, respectively; the variable regions of the T-ab heavy and light chains are shown in SEQ ID NO.19 and SEQ ID NO.18, respectively. This kit, based on a double-antibody sandwich ELISA method, can specifically and sensitively detect the level of glial fibrillary acidic protein (GFAP) in human peripheral blood, and can be used for the auxiliary diagnosis of glioma. It has advantages such as high sensitivity, strong specificity, simple operation, and small blood sample volume.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to an antibody kit for detecting gliomas and its application. Background Technology

[0002] Gliomas are the most common primary malignant tumors of the central nervous system, originating from glial cells. They account for approximately 40%-50% of all intracranial tumors and are characterized by high incidence, high recurrence rate, and high mortality. According to the World Health Organization's classification system, gliomas are divided into grades I-IV, with high-grade gliomas (grades III-IV) being extremely malignant, and the median survival for patients is only 12-15 months. Currently, the diagnosis of gliomas mainly relies on imaging examinations (such as MRI and CT) and histopathological biopsy. However, imaging examinations have limited ability to identify early, small lesions and are difficult to differentiate between tumor types and grades; while histopathological biopsy is the gold standard, it is an invasive procedure with risks of bleeding, infection, and neurological damage, making it unsuitable for dynamic monitoring and early screening. Therefore, developing a non-invasive, convenient, and sensitive serological detection method is of great significance for the early diagnosis, treatment evaluation, and prognosis of gliomas.

[0003] Glial fibrillary acidic protein (GFAP) is a specific intermediate filament protein of astrocytes, primarily found in astrocytes of the central nervous system. When brain tissue is damaged or tumors develop, astrocytes are activated and release GFAP into the bloodstream, leading to elevated GFAP levels in peripheral blood. Studies have shown that serum GFAP levels in glioma patients are significantly higher than in healthy individuals, and these levels are closely related to tumor grade and prognosis. Therefore, GFAP is considered one of the most promising serum biomarkers for gliomas. However, existing GFAP detection methods are mostly based on polyclonal antibodies or single monoclonal antibodies, which suffer from insufficient specificity, low sensitivity, and large batch-to-batch variability, making it difficult to meet the needs of precise clinical diagnosis.

[0004] In recent years, the double-antibody sandwich ELISA method has been widely used for the quantitative detection of biomarkers due to its high specificity and sensitivity. This method requires a pair of paired antibodies targeting different epitopes of the target protein. One antibody is immobilized on a solid-phase carrier as a capture antibody, while the other acts as a detection antibody coupled with a signal amplification system. Therefore, developing high-affinity, high-specificity paired monoclonal antibodies is crucial for constructing high-quality ELISA kits. Based on this technical approach, this invention prepared high-affinity monoclonal antibodies Cap-ab and T-ab targeting the N-terminal conserved region (amino acids 1-120) and C-terminal conserved region (amino acids 351-432) of the GFAP protein, respectively. A double-antibody sandwich ELISA detection system was successfully constructed, achieving highly sensitive and specific quantitative detection of GFAP in peripheral blood, providing a new technical means for the non-invasive auxiliary diagnosis of gliomas. Summary of the Invention

[0005] To address the aforementioned problems, the present invention first provides an antibody kit for detecting glioma, comprising: a solid-phase carrier coated with anti-GFAP-N monoclonal antibody, recombinant GFAP protein standard, and enzyme-labeled anti-GFAP-C monoclonal antibody.

[0006] In some embodiments, the amino acid sequence of the heavy chain variable region of the anti-GFAP-N monoclonal antibody is shown in SEQ ID NO. 11, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 10.

[0007] In some embodiments, the amino acid sequence of the heavy chain variable region of the anti-GFAP-C monoclonal antibody is shown in SEQ ID NO. 19, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 18.

[0008] In some embodiments, the amino acid sequences of the heavy chains CDR1, CDR2, and CDR3 of the anti-GFAP-N monoclonal antibody are shown in SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6, respectively, and the amino acid sequences of the light chains CDR1, CDR2, and CDR3 are shown in SEQ ID NO.7, SEQ ID NO.8, and SEQ ID NO.9, respectively.

[0009] In some embodiments, the amino acid sequences of the heavy chains CDR1, CDR2, and CDR3 of the anti-GFAP-C monoclonal antibody are shown in SEQ ID NO.12, SEQ ID NO.13, and SEQ ID NO.14, respectively, and the amino acid sequences of the light chains CDR1, CDR2, and CDR3 are shown in SEQ ID NO.15, SEQ ID NO.16, and SEQ ID NO.17, respectively.

[0010] In some embodiments, the solid support is an enzyme-labeled plate, and the enzyme label is horseradish peroxidase label.

[0011] In some embodiments, the recombinant GFAP protein standard is the full-length GFAP protein or its immunologically active fragment.

[0012] The present invention also provides a method for detecting glioma, comprising the following steps: adding the sample to be tested into a pre-coated ELISA plate of the above kit for incubation, washing, adding enzyme-labeled anti-GFAP-C monoclonal antibody for incubation, washing, adding a chromogenic substrate for color development, measuring the absorbance value, and calculating the GFAP content in the sample according to the standard curve.

[0013] In some embodiments, the sample to be tested is human peripheral blood serum.

[0014] The present invention also provides the application of the above-described kit in the preparation of products for the auxiliary diagnosis of glioma.

[0015] Compared with the prior art, the present invention has at least the following beneficial effects:

[0016] (1) The antibody kit provided by the present invention has prepared capture antibody and detection antibody for the conserved regions of the N-terminus and C-terminus of GFAP, a serum marker of glioma, respectively, forming a highly specific double-antibody sandwich detection system, which significantly improves the specificity of detection and avoids cross-reaction.

[0017] (2) The sensitivity of the kit of the present invention reaches 10 pg / mL, which is much higher than that of the conventional ELISA detection method. It can effectively distinguish the peripheral blood GFAP level of glioma patients and healthy people, and provide a reliable means for early screening and auxiliary diagnosis of glioma.

[0018] (3) The kit of the present invention is easy to operate, requires no complicated equipment, and has a short detection time (about 1 hour), making it suitable for promotion and use in medical institutions at all levels, especially for large-scale population screening and postoperative recurrence monitoring.

[0019] (4) The antibody used in this invention is a monoclonal antibody, which has good batch-to-batch stability, ensuring the repeatability and reliability of the kit and reducing the risk of false positives and false negatives. Attached Figure Description

[0020] Figure 1 The image shows the SDS-PAGE identification results of GFAP-N and GFAP-C proteins in this invention, where M is the protein molecular weight marker, lane 1 is the non-reduced GFAP-N protein, and lane 2 is the non-reduced GFAP-C protein.

[0021] Figure 2The image shows the ELISA results of the anti-GFAP-N antibody titer in mouse serum according to the present invention.

[0022] Figure 3 This is a graph showing the ELISA results of the binding activity of hybridoma cell supernatant to GFAP-N according to the present invention.

[0023] Figure 4 This is a graph showing the ELISA results of the binding activity of monoclonal hybridoma cell supernatant to GFAP-N according to the present invention.

[0024] Figure 5 This is a graph showing the ELISA results of the binding activity of monoclonal hybridoma cell supernatant to GFAP-C according to the present invention.

[0025] Figure 6 The image shows the SDS-PAGE identification results of the purified Cap-ab and T-ab antibodies of this invention, where M is the protein molecular weight marker, lane 1 is the reduced Cap-ab, lane 2 is the non-reduced Cap-ab, lane 3 is the reduced T-ab, and lane 4 is the non-reduced T-ab. Detailed Implementation

[0026] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0027] Example 1: Design and preparation of antigen fragments

[0028] Glial fibrillary acidic protein (GFAP), derived from astrocytes, is an important serum biomarker for gliomas. According to UniProt (P14136), this protein consists of 432 amino acids with a molecular weight of 49.88 kDa. The N-terminal amino acids 1-120 (SEQ ID NO: 1) were selected.

[0029] Using MERRRITSAARRSYVSSGEMMVGGLAPGRRLGPGTRLSLARMPPPLPTRVDFSLAGALNAGFKETRASERAEMMELNDRFASYIEKVRFLEQQNKALAAELNQLRAKEPTKLADVYQAEL) as the capture antigen, capture antibodies were prepared using hybridoma technology to capture GFAP in serum. Codon optimization tools were used ( https: / / www.novopro.cn / tools / codon-optimization.html The captured antigen sequence was codon-optimized for prokaryotic expression in *E. coli*, resulting in the optimized nucleotide sequence:

[0030] ATGGAACGTCGCCGTATTACTTCTGCCGCTCGCCGTTCTTATGTGTCCTCTGGTGAAATGATGGTTGGCGGTCTGGCACCGGGCCGTCGTCTGGGCCCGGGTACCCGCCTGTCTCTGGCCCGTATGCCACCTCCGCTGCCTACGCGTGTCGACTTTTCCCTGGCTGGTGCCTGAACGCTGG TTTCAAAGAAACCCGTGCTTCTGAACGTGCCGAAATGATGGAACTGAACGATCGTTTCGCTTCCTACATCGAAAAAGTGCGTTTCCTGGAACAGCAGAACAAAGCGCTGGCTGCGGAACTGAACCAACTGCGTGCAAAAGAACCGACTAAACTGGCGGATGTTTTACCAGGCGGAACTG (SEQ IDNO.2).

[0031] The target gene was synthesized by BGI Genomics Co., Ltd. in Shenzhen, using pET-28a as the vector. The resulting plasmid was transformed into BL21 competent cells, plated overnight, and then single colonies were picked and transferred into 5 mL of 2×YT liquid medium containing 50 μg / mL kanamycin. The culture was incubated at 37°C and shaken at 220 rpm until OD reached. 595 Approximately 0.5, transferred to 150 mL liquid culture medium, and incubated at 37°C until OD... 595 Approximately 0.7, add 0.5M IPTG at a 1:1000 ratio, and continue incubation overnight at 16°C. Centrifuge at 8000 rpm for 10 min at 4°C and collect the bacterial cells. Resuspend the bacterial cells in lysis buffer (50mM NaH2PO4, 300mM NaCl, 10mM imidazole, 1mM PMSF, 100×EDTA-free Protease Inhibitor Cocktail, pH=8.0), and sonicate at low temperature to lyse the cells and collect the lysate. Transfer the lysate to a new 50mL centrifuge tube, centrifuge at 12000 rpm for 30 min at 4°C, and collect the supernatant. Purify the protein using a Ni column using the His tag on the vector. The protein elution buffer is 250mM imidazole buffer. After initial purification, the protein buffer was replaced with PBS by dialysis. Finally, the obtained protein solution was concentrated using a 3kDa ultrafiltration tube and named GFAP-N. After determining the protein concentration, the protein was filtered through a 0.22μm filter membrane for sterilization. The protein concentration was adjusted to 1mg / mL using PBS and then dispensed into 0.2mL vials, which were then frozen at -80℃.

[0032] Similarly, amino acids 351-432 of the C-terminus of the GFAP protein are relatively conserved and can be used as an immunogen to prepare detection antibodies (SEQ ID No: 3: DLLNVKLALDIEIATYRKLLEGEENRITIPVQTFSNLQIRETSLDTKSVSEGHLKRNIVVKTVEMRDGEVIKESKQEHKDVM). The target sequence, after codon optimization, was cloned into the pET-28a vector. Protein expression and purification methods were the same as described above for capturing antigen fragments. The purified protein was named GFAP-C. 3 μg of both GFAP-N and GFAP-C proteins were analyzed by SDS-PAGE to confirm protein purity. The experimental results are shown below. Figure 1 As shown, lane M is the protein molecular weight marker, lane 1 is GFAP-N, and without the addition of DTT, the protein band is located at approximately 13 kD, consistent with the theoretical molecular weight. At the same time, the band is single, indicating that its purity is high. Lane 2 is GFAP-C, and the molecular weight in the non-reduced state is approximately 9.5 kD, which is consistent with the theoretical calculation value.

[0033] Example 2: Preparation of monoclonal antibodies against GFAP-N and GFAP-C

[0034] Monoclonal antibodies against GFAP-N were prepared using classic hybridoma technology. First, GFAP-N was emulsified with complete Freund's adjuvant, and then subcutaneously immunized with the emulsion in 6-8 week old female Balb / c mice. Two weeks after the primary immunization, a second immunization was performed using GFAP-N emulsified with incomplete Freund's adjuvant. Two weeks after the second immunization, a third immunization was performed using the same method. One week after the third immunization, a small amount of blood was collected and serum was separated. Antibody titers were determined by ELISA. The coating antigen was 1 μg / mL GFAP-N protein, added at 100 µg / well, and the secondary antibody was goat anti-mouse IgG-HRP. The experimental results of ELISA detection of serum anti-GFAP-N antibody titers are as follows: Figure 2 As shown in the figure, the negative control is the serum of unimmunized mice. The vertical axis represents the OD650 reading of the sample minus the OD650 reading of the negative control, and the horizontal axis represents the serum dilution factor (logarithmic). The results in the figure show that the serum from mouse number 2 had the highest titer and the best binding activity to the GFAP-N protein.

[0035] The mice were sacrificed, and spleen cells were isolated. Mouse myeloma cells SP2 / 0 were fused with the isolated spleen cells and cultured in vitro. After culturing in HT medium, the medium was replaced with RPMI 1640 + 10% FBS. The supernatant of the hybridoma cells was used to detect their affinity for GFAP-N using ELISA. The results are as follows: Figure 3 As shown. Figure 3The vertical axis represents the absorbance value at 650 nm in the sample wells minus the absorbance value at 650 nm in the blank wells, with the blank wells containing an equal volume of basal culture medium. The horizontal axis represents the logarithmic dilution factor of the hybridoma cell supernatant. As shown in the figure, the supernatants of hybridoma cells I and III exhibited the best GFAP-N binding activity.

[0036] Cell suspensions of hybridoma cells I and III were prepared using a limiting dilution method. Within approximately two weeks, single cells could expand to occupy about 50% of the bottom area of ​​a 96-well cell culture plate. Wells derived from hybridoma cells I were labeled I-1, I-2, I-3, etc., and so on, while wells derived from hybridoma cells III were labeled III-1, III-2, III-3, etc. The supernatant was then used to determine its affinity for GFAP-N. The results are as follows: Figure 4 As shown. Figure 4 The x-axis represents the supernatant dilution factor, expressed as Log. The supernatant of monoclonal cell number III-1 showed the best binding activity with GFAP-N and can be used for subsequent antibody expression.

[0037] Similarly, monoclonal cells with optimal GFAP-C binding activity can be screened, and the monoclonal antibodies produced by them are designated as T-ab. The results of the GFAP-C binding activity assay of cell culture supernatants from monoclonal cells C1-C3 are as follows: Figure 5 As shown, the horizontal axis represents the supernatant dilution factor taken as Log, and the vertical axis represents the OD650 absorbance value. The antibody produced by monoclonal cell C1 has the best antigen-binding activity and is suitable for use as a detection antibody.

[0038] Example 3: Purification of Monoclonal Antibodies

[0039] Expand the culture of monoclonal cells III-1 and designate the monoclonal antibody produced as Cap-ab. Collect 30 mL of cell supernatant, centrifuge at 8000 rpm at 4°C for 10 min, transfer the supernatant to a 50 mL centrifuge tube, filter through a 0.22 µm filter membrane, and measure the pH. Adjust the pH to 7.0-7.5 using neutralization buffer. Purify the collected cell culture supernatant using Protein G magnetic beads, with 0.1 M Glysin-HCl pH=2.7 as the elution buffer. Add 10× neutralization buffer to the elution buffer and mix thoroughly. Transfer the elution buffer to a 30 kDa ultrafiltration tube, centrifuge at 4000 rpm at 4°C for 10 min to concentrate the protein, adding PBS to 15 mL every 10 min and centrifuging again, for a total of 50 min. Finally, completely replace the protein buffer with PBS. Perform SDS-PAGE analysis on 3 µg Cap-ab and 3 µg T-ab proteins. The results are as follows: Figure 6As shown. Lane M is a molecular weight marker for the proteins. Lane 1 represents the reduced Cap-ab protein, lane 2 represents the unreduced Cap-ab protein, lane 3 represents the reduced T-ab protein, and lane 4 represents the unreduced T-ab protein. Both molecular weights conform to the theoretical molecular weight of mouse IgG. Under unreduced conditions, the bands for both proteins are clear, with low aggregate content. Under reduced conditions, both the 55kDa heavy chain and the 28kDa light chain are clearly visible.

[0040] Example 4: Obtaining the variable region sequence of a monoclonal antibody

[0041] Candidate hybridoma clones were lysed with Trizol and total RNA was extracted. First-strand cDNA was synthesized using this as a template. Subsequent PCR amplification was then performed using antibody variable region-specific primers with the first-strand cDNA as a template to obtain the corresponding antibody light and heavy chain variable regions. After gel recovery, the sequences were sequenced by BGI Genomics to obtain the antibody variable region sequences. The sequences of the antibody heavy chain and light chain variable regions are shown in Table 1.

[0042] Table 1. Amino acid sequence of monoclonal antibody Cap-ab

[0043]

[0044] Table 2. Amino acid sequence of monoclonal antibody T-ab

[0045]

[0046] Example 5: Preparation of Antibody Kit for Glioma Detection

[0047] A kit for detecting gliomas was designed and manufactured. The principle is a double-antibody sandwich ELISA adapted to serum. The kit consists of the following components: a pre-coated ELISA plate made with a Cap-ab-coated solid-phase carrier, recombinant GFAP protein standards, and HRP-conjugated T-ab. The kit usage is as follows: Add 100 μL of sample or standard to each well of the pre-coated ELISA plate, incubate at 37°C for 30 min, wash 5 times, add 100 μL of HRP-conjugated T-ab per well, incubate at 37°C for 30 min, wash 5 times, add 100 μL of TMB per well, and incubate in the dark for 15 min.

[0048] Serum samples were collected from 30 patients diagnosed with glioma and 30 healthy individuals (all under 55 years old). After a 1:2 dilution, peripheral blood GFAP levels were measured using the aforementioned kit. The results are shown in the table below. The kit has a linear sensitivity of 10 pg / mL, meeting the standard detection limit for clinical testing. Table 3 shows a significant difference in peripheral blood serum GFAP levels between healthy individuals and glioma patients tested using this kit. This in vitro diagnostic method requires minimal blood sample, is versatile and rapid, and can be used in conjunction with other indicators, demonstrating good application potential.

[0049] Table 3. Peripheral blood testing

[0050]

[0051] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An antibody kit for detecting glioma, characterized in that, include: A solid-phase carrier coated with anti-GFAP-N monoclonal antibody, recombinant GFAP protein standard, and enzyme-labeled anti-GFAP-C monoclonal antibody.

2. The reagent kit according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the anti-GFAP-N monoclonal antibody is shown in SEQ ID NO.11, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

10.

3. The reagent kit according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the anti-GFAP-C monoclonal antibody is shown in SEQ ID NO.19, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

18.

4. The reagent kit according to claim 1, characterized in that, The amino acid sequences of the heavy chains CDR1, CDR2, and CDR3 of the anti-GFAP-N monoclonal antibody are shown in SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6, respectively, and the amino acid sequences of the light chains CDR1, CDR2, and CDR3 are shown in SEQ ID NO.7, SEQ ID NO.8, and SEQ ID NO.9, respectively.

5. The reagent kit according to claim 1, characterized in that, The amino acid sequences of the heavy chains CDR1, CDR2, and CDR3 of the anti-GFAP-C monoclonal antibody are shown in SEQ ID NO.12, SEQ ID NO.13, and SEQ ID NO.14, respectively, and the amino acid sequences of the light chains CDR1, CDR2, and CDR3 are shown in SEQ ID NO.15, SEQ ID NO.16, and SEQ ID NO.17, respectively.

6. The reagent kit according to claim 1, characterized in that, The solid support is an enzyme-labeled plate, and the enzyme label is horseradish peroxidase label.

7. The kit according to claim 1, characterized in that, The recombinant GFAP protein standard is the full-length GFAP protein or its immunologically active fragment.

8. A method for detecting glioma, comprising the following steps: The sample to be tested is added to the pre-coated ELISA plate of the kit described in any one of claims 1-7 and incubated. After washing, enzyme-labeled anti-GFAP-C monoclonal antibody is added and incubated. After washing, chromogenic substrate is added for color development, and the absorbance value is measured. The GFAP content in the sample is calculated according to the standard curve.

9. The method according to claim 8, characterized in that, The sample to be tested is human peripheral blood serum.

10. The use of the kit according to any one of claims 1-7 in the preparation of a product for the auxiliary diagnosis of glioma.

Images