A monoclonal antibody combination for NfL protein detection and its application

Abstract

This invention belongs to the field of biodetection technology, specifically relating to a monoclonal antibody combination for the detection of NfL protein and its application. The combination comprises monoclonal antibodies 9E1 and 10B4, whose complementary determinant amino acid sequences of the heavy and light chain variable regions are shown in SEQ ID NO. 1-12, respectively. The antibody combination can be used to construct a double-antibody sandwich ELISA detection system, particularly suitable for use with a biotin-avidin signal amplification system to achieve highly sensitive and specific detection of NfL protein, with a detection sensitivity of up to 20 pg / mL. This method is low-cost, simple to operate, and suitable for the quantitative analysis of NfL protein in serum, plasma, and other samples.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a combination of monoclonal antibodies for the detection of NfL protein and its application. Background Technology

[0002] Neurological disorders refer to structural or functional abnormalities of the central or peripheral nervous system, including the brain and spinal cord, caused by various factors such as genetics, infection, metabolic disorders, vascular lesions, and trauma. They are a leading cause of poor health and disability worldwide. The harm caused by neurological damage, such as stroke, Alzheimer's disease and other dementias, neonatal encephalopathy, and diabetic neuropathy, is receiving increasing public attention.

[0003] Neurofilaments (NFs) are major intermediate filaments widely distributed in mature neurons of the central and peripheral nervous systems. They are key components of the neuronal cytoskeleton of myelinated axons and play a crucial role in nerve conduction. Neurofilaments consist of four distinct subunits, commonly referred to as the "neurofilament triad": the neurofilament light chain (NfL), the neurofilament medium chain (NfM), and the neurofilament heavy chain (NfH). The neurofilament light chain (NfL) is a cylindrical protein located in the neuronal cytoplasm and is considered the core scaffold around which the other subunits assemble. NfL plays a vital role in maintaining the stability of neuronal structure. Normally, NfL is primarily found inside nerve cells, with very low concentrations in cerebrospinal fluid and blood. However, when neurons or axons are damaged or degenerate, NfL is released into the extracellular space, then into the cerebrospinal fluid, and finally into the bloodstream. Therefore, elevated NfL levels in the blood or cerebrospinal fluid directly reflect neuronal damage and axonal degeneration. Furthermore, NfL may also serve as a predictor of cognitive decline. Therefore, the detection of NfL has important guiding significance for the diagnosis, treatment and prognosis of neurological diseases, and is considered a very promising neurodegenerative marker.

[0004] Neurofilament light chain protein (NfL) detection is currently mainly used for research and clinical auxiliary diagnosis, playing an important role in the management of many neurological diseases, such as multiple sclerosis (MS), Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), brain injury, and stroke. Cerebrospinal fluid (CSF) NfL indicators have superior diagnostic performance compared to blood NfL indicators. However, blood NfL is readily available, avoiding the discomfort and procedural risks associated with lumbar puncture, and is more suitable for point-of-care testing and prognostic monitoring in future in vitro diagnostic development. Therefore, developing high-performance diagnostic reagents for neurofilament light chain protein (NfL) is essential. Currently, methods for measuring NfL include enzyme-linked immunosorbent assay (ELISA), electrochemiluminescence immunoassay (ECL), and single-molecule assay (SiMoA). Among these, the single-molecule assay (SiMoA) kit is costly, the testing instruments are expensive, and specialized training is required. Summary of the Invention

[0005] In view of the above-mentioned shortcomings and deficiencies of existing technologies, this invention provides a monoclonal antibody combination for NfL protein detection and its application, which solves the technical problems of insufficient sensitivity, high cost, or reliance on expensive specialized instruments in existing NfL detection methods. Considering sensitivity, cost, and required instruments, the ELISA method is more convenient and lower cost. This study obtained NfL-specific paired monoclonal antibodies through immunization and screening, and combined with a signal amplification system, established a biotin-avidin amplified double-antibody sandwich detection system for the ELISA method, which can specifically detect NfL protein, providing a highly sensitive, low-cost, and easily scalable solution for the clinical detection of biomarkers related to neurological diseases.

[0006] To achieve the above objectives, the main technical solutions adopted by the present invention include:

[0007] In a first aspect, the present invention provides a monoclonal antibody combination for the detection of NfL protein, the monoclonal antibody combination comprising monoclonal antibody 9E1 and monoclonal antibody 10B4, wherein the heavy chain variable region of monoclonal antibody 9E1 includes three complementarity-determining regions, and the amino acid sequences of the complementarity-determining regions are shown as SEQ ID NO.1-SEQ ID NO.3 respectively;

[0008] The light chain variable region of monoclonal antibody 9E1 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.4-SEQ ID NO.6, respectively.

[0009] The heavy chain variable region of monoclonal antibody 10B4 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.7-SEQ ID NO.9, respectively.

[0010] The light chain variable region of monoclonal antibody 10B4 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.10-SEQ ID NO.12, respectively.

[0011] In some embodiments, the amino acid sequence of the heavy chain variable region of monoclonal antibody 9E1 is shown in SEQ ID NO.13; the amino acid sequence of the light chain variable region of monoclonal antibody 9E1 is shown in SEQ ID NO.14.

[0012] In some embodiments, the amino acid sequence of the heavy chain variable region of monoclonal antibody 10B4 is shown in SEQ ID NO.15; the amino acid sequence of the light chain variable region of monoclonal antibody 10B4 is shown in SEQ ID NO.16.

[0013] In some embodiments, the nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 9E1 is shown in SEQ ID NO. 17; the nucleotide sequence encoding the light chain variable region of monoclonal antibody 9E1 is shown in SEQ ID NO. 18.

[0014] In some embodiments, the nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 10B4 is shown in SEQ ID NO. 19; the nucleotide sequence encoding the light chain variable region of monoclonal antibody 10B4 is shown in SEQ ID NO. 20.

[0015] Secondly, the present invention provides the application of the above-mentioned monoclonal antibody combination (monoclonal antibody 9E1 and monoclonal antibody 10B4) in the preparation of tools for detecting NfL protein.

[0016] In some embodiments, the tools include reagents, kits, test strips, and antibody chips.

[0017] In some embodiments, the kit includes a double-antibody sandwich ELISA kit.

[0018] In some embodiments, the kit includes a double-antibody sandwich ELISA kit with a biotin-avidin amplification system.

[0019] In some embodiments, the kit is coated and labeled with monoclonal antibodies 9E1 and 10B4.

[0020] Beneficial effects:

[0021] The monoclonal antibody combination for NfL protein detection provided by this invention is based on the complementarity-determining region (CDR) sequences of monoclonal antibodies 9E1 and 10B4. Specifically, the heavy chain of monoclonal antibody 9E1 is shown in SEQ ID NO. 1-3, and the light chain is shown in SEQ ID NO. 4-6; the heavy chain of monoclonal antibody 10B4 is shown in SEQ ID NO. 7-9, and the light chain is shown in SEQ ID NO. 10-12.

[0022] Monoclonal antibodies 9E1 and 10B4, possessing the specific CDR sequences described above, can be used to construct a highly sensitive immunoassay platform. This antibody combination is particularly useful in the detection of NfL protein using a double-antibody sandwich assay. This invention successfully prepared immunologically active recombinant NfL protein via a prokaryotic expression system and screened for highly efficient binding monoclonal antibodies. The established detection method achieves a sensitivity of up to 20 pg / mL and exhibits high specificity for NfL protein. Compared to existing technologies that rely on expensive instruments or costly detection methods, the solution provided by this invention offers significant advantages such as high sensitivity, strong specificity, low cost, ease of operation, and easy promotion. It can be widely applied to the detection of NfL in serum and plasma samples, providing reliable technical support for early screening, disease assessment, and prognostic monitoring of neurodegenerative diseases such as Alzheimer's disease, multiple sclerosis, and stroke, and has significant clinical application value. Attached Figure Description

[0023] Figure 1 This is an SDS-PAGE protein identification image;

[0024] Figure 2 The image shows the results of identifying NfL recombinant protein with purchased antibodies.

[0025] Figure 3 This is a diagram illustrating the binding activity of paired monoclonal antibodies.

[0026] Figure 4 This is a sensitivity identification diagram for the biotin-avidin amplified ELISA.

[0027] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Detailed Implementation

[0028] The embodiments of this application will be further described in detail below with reference to the accompanying drawings and examples. The detailed description of the following embodiments and the accompanying drawings are used to illustrate the principles of this application by way of example, but should not be used to limit the scope of this application. This application can be implemented in many different forms and is not limited to the specific embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.

[0029] These embodiments are provided to make the application thorough and complete, and to fully express the scope of the application to those skilled in the art. It should be noted that, unless otherwise specifically stated, the relative arrangement of components and steps, material composition, numerical expressions, and values ​​illustrated in these embodiments should be interpreted as merely exemplary and not as limiting.

[0030] Example 1.

[0031] 1. Preparation of recombinant proteins

[0032] The NfL gene sequence was downloaded from NCBI. After codon optimization for the E. coli expression system, the gene was synthesized and cloned into the pET28a expression vector. Gene synthesis was performed by Anhui General Biotechnology. The recombinant plasmid pET28a-NfL expressing NfL was transformed into BL21(DE3) competent cells using standard methods (Molecular Cloning). The transformed cells were plated on LB agar plates (containing 50 μg / mL kanamycin) and incubated overnight at 37°C. A single colony was picked and inoculated into 5 mL of LB medium (containing 50 μg / mL kanamycin) and incubated overnight at 37°C with shaking at 220 rpm. 1% of the total culture volume was inoculated into LB medium (containing 50 μg / mL kanamycin) and incubated at 37°C with shaking at 220 rpm for approximately 3 hours, until OD (outlet count) was reached. 600 The concentration was 0.6-0.9, and the final concentration was 0.1 mM IPTG. The cells were collected after induction at 30℃ and 200 rpm for 4 hours.

[0033] The NfL nucleotide sequence is shown in SEQ ID NO.21:

[0034]

[0035] The NfL amino acid sequence is shown in SEQ ID NO.22:

[0036] *

[0037] The asterisk (*) at the end of the amino acid sequence represents the stop codon of the protein sequence, which is not shown in the sequence listing.

[0038] Recombinant NfL protein is an artificially expressed NfL protein obtained through genetic engineering in a prokaryotic expression system. Typically, the NfL gene sequence is codon-optimized, cloned into the pET28a expression vector, and then induced to express. In this application, recombinant NfL protein is used as an antigen to immunize mice to prepare hybridoma cells, screen and identify specific monoclonal antibodies against NfL protein (such as 9E1 and 10B4). The obtained monoclonal antibodies can effectively recognize both native NfL protein and recombinant NfL protein, thereby achieving highly sensitive and specific detection of NfL protein in clinical samples such as serum, plasma, or cerebrospinal fluid.

[0039] 2. Purification of recombinant proteins

[0040] 2.1 Purification of recombinant proteins

[0041] Because the expressed recombinant protein carries a histidine tag, it was purified using a protein purification instrument and a nickel-ion affinity chromatography column from Taidu Biotechnology. Buffer A consisted of 50 mM PB, 300 mM NaCl, pH 8.0; Buffer B consisted of 50 mM PB, 300 mM NaCl, 0.5 M imidazole, pH 8.0. The chromatography column was equilibrated with buffer A. The fermented bacterial culture was then centrifuged at 8000 rpm for 10 min. The precipitate was resuspended in buffer A and sonicated in ice water for 30 min, with 5-second intervals between sonications. The mixture was then centrifuged at 12000 rpm for 30 min. The supernatant was filtered through a 0.22 μm filter, loaded onto the column, washed with buffer A, and finally eluted with a gradient of buffer B. The purification status was observed by SDS-PAGE protein gel electrophoresis. The target protein peak was selected for collection, dialyzed to buffer A, and the protein concentration was measured using a micro-spectrophotometer. The collected protein was aliquoted and stored at -20℃.

[0042] For SDS-PAGE protein gel electrophoresis to observe purification results, please refer to [link to SDS-PAGE protein gel electrophoresis]. Figure 1 . Figure 1 In the text, M stands for Protein Marker, and number 1 indicates pET28a-NfL recombinant protein. Figure 1 The results showed a distinct main band between 55-70 kDa, with a protein purity of approximately 90% or higher, consistent with the estimated antigen size (62.6 kDa). The purified recombinant protein can be used for further downstream experiments.

[0043] 2.2 Indirect ELISA identification of NfL recombinant protein

[0044] The purified NfL recombinant protein was coated (1 μg / ml), and its reaction with the positive monoclonal antibody was identified by indirect ELISA. The positive monoclonal antibody was a commercially available monoclonal antibody (HyTest, NF71). First, the recombinant protein was coated in microplates (coating buffer: carbonate buffer: 1.59 g sodium carbonate, 2.93 g sodium bicarbonate, diluted to 1 L of pure water, pH 9.6), with a coating concentration of 1 μg / mL, 50 μl / well, and incubated overnight at 4°C; then blocked with 1% gelatin, 150 μl per well, at 37°C for 2 hours, and washed once with washing buffer (PBST, PBS containing 0.05% Tween-20), and blotted dry; commercially available monoclonal antibody (HyTest, NF71) was diluted in PBS at gradients of 10 μg / ml, 1 μg / ml, 100 ng / ml, 10 ng / ml, and 1 ng / ml, and 50 μl was added to the coated microplates. HPV18 / E6 monoclonal antibody (Santa Cruz, G-7) was used as a negative control, and the reaction was carried out at 37°C for 30 min. Discard the liquid from the wells, wash the plate four times with PBST, pat dry, and add 50 μL / well of HRP-labeled goat anti-mouse secondary antibody (diluted 1:5000 with PBS). Incubate at 37°C for 30 min, wash four more times, pat dry, add 50 μL / well of TMB chromogenic buffer, and incubate at room temperature for 10 min. Finally, add 0.5 M sulfuric acid to stop the reaction and measure the OD using a microplate reader. 450 nm value. Results are as follows: Figure 2 , Figure 2 NF71 is a commercially available monoclonal antibody (HyTest), and Ctrl is an HPV18 / E6 monoclonal antibody (Santa Cruz, G-7).

[0045] The purified NfL recombinant protein was coated onto an ELISA plate at a concentration of 1 μg / ml. It still showed a weak positive reaction with the commercially available NfL monoclonal antibody (HyTest, NF71) at a concentration of 10 ng / ml, indicating that the purified NfL recombinant protein was active and could be used for the next step of the experiment.

[0046] 3. Mouse immunization

[0047] The purified recombinant protein was mixed with an equal volume of Freund's complete adjuvant (200 μL) and subcutaneously injected at multiple sites into 6-week-old female BALB / c mice at a dose of 30 μg / mouse. Then, at weeks 2 and 4, the same dose was mixed with an equal volume of MF59 adjuvant and injected intramuscularly. At week 5, mouse serum was collected to detect antibody titers. Mice with the highest titers were selected for a booster immunization with 20 μg NfL of recombinant protein via intraperitoneal pulse. Three days later, the spleens of these mice were harvested for hybridoma cell preparation.

[0048] 4. Screening, preparation, and antibody purification of hybridoma cell lines

[0049] 4.1 Screening of hybridoma cell lines

[0050] All spleen cells from immunized mice were fused with SP2 / 0 myeloma cells in logarithmic growth phase and then cultured in HAT medium for selection. When the fused cells reached halfway to the bottom of the well, clones positive for NfL recombinant protein were selected by indirect ELISA. Since the immunogen was of prokaryotic origin and contained a His tag, background components needed to be screened to identify specific cell lines targeting NfL protein. Positive cells were cloned to monoclonal state using limiting dilution, and then the cell lines were expanded and cryopreserved.

[0051] Indirect ELISA method for screening positive clones:

[0052] NfL recombinant protein and other recombinant proteins of the pET28a vector (HIS tag) pET28a-HPV8 / E6 were coated in microplates (coating buffer: carbonate buffer: sodium carbonate 1.59 g, sodium bicarbonate 2.93 g, diluted to 1 L of pure water, pH 9.6), with a coating concentration of 1 μg / mL, and incubated overnight at 4 °C; blocked with 1% BSA, 150 μL per well, at 37 °C for 2 hours, washed once with washing buffer, and patted dry; 50 μL of cell culture supernatant was added, and the reaction was carried out at 37 °C for 30 min. Discard the liquid from the wells, wash the plate four times with PBST, pat dry, and add 50 μL / well of HRP-labeled goat anti-mouse secondary antibody (diluted 1:5000 with PBS). Incubate at 37°C for 30 min, wash four more times, pat dry, add 50 μL / well of TMB chromogenic buffer, and incubate at room temperature for 10 min. Finally, add 0.5 M sulfuric acid to stop the reaction and measure the OD using a microplate reader. 450 nm value. Positive cell lines that reacted only with the NfL recombinant protein and not with the control antigen were selected for subsequent experiments. The screening results are shown in Table 1.

[0053] Table 1: Screening results of NfL monoclonal antibodies

[0054]

[0055] 4.2 Preparation of Monoclonal Antibody Ascites

[0056] After the selected monoclonal cell lines were expanded and cultured, 0.2 ml (containing 2.5 × 10⁻⁶ cells) was injected intraperitoneally. 6 Female BALB / c mice (cells) were pretreated with Freund's incomplete adjuvant. After about 10 days, when the mice's abdomens were significantly swollen, ascites was collected using a sterile syringe needle.

[0057] 4.3 Affinity chromatography purification of monoclonal antibodies

[0058] Centrifuge the ascites fluid at 12000 rpm for 5 minutes. Collect the supernatant and dilute it 10-fold with binding buffer (20 mM PB, 150 mM NaCl, pH 7.4). Filter the supernatant through a 0.22 μm filter. Pump the filtered sample slowly into a Protein L purification column equilibrated with binding buffer using a peristaltic pump. Connect the column to a protein purification instrument and wash with binding buffer for 5-10 column volumes until the UV absorption peak flattens. Then elute with elution buffer (0.1 M glycine, pH 2.7). Collect the elution peak and adjust the collected sample to neutral with 1 M Tris-HCl (pH 9). Transfer the solution to a dialysis bag (MW: 8000~14000) and dialyze for 14 hours at 2~8°C in 0.01 M PBS (pH 7.4). Transfer the liquid from the dialysis bag to a centrifuge tube and centrifuge at 12000 rpm for 5 minutes. The supernatant is the purified monoclonal antibody. The purified monoclonal antibody was analyzed for concentration using a micro-spectrophotometer and then aliquoted and stored.

[0059] 4.4 Identification of the binding activity of monoclonal antibodies

[0060] Using an indirect ELISA method, NfL recombinant protein antigen and control vector protein (pET28a-HPV18 / E6 recombinant protein, see patent CN120329424B) were coated separately. The purified antibody was then subjected to ELISA to identify the affinity of the monoclonal antibody. The antibody was diluted to 1 μg / ml. The results are shown in Table 2. In Table 2, Ctrl represents the negative control GFAP mouse monoclonal antibody (see patent CN118930634B).

[0061] Table 2: Identification of the binding activity of monoclonal antibodies.

[0062]

[0063] Table 2 shows that the binding activity of the 22 selected NfL monoclonal antibodies to recombinant NfL protein and control vector protein was detected by indirect ELISA. The data indicate that the selected monoclonal antibodies specifically bind to recombinant NfL protein, and some monoclonal antibodies, including monoclonal antibody 9E1 and monoclonal antibody 10B4, exhibited high affinity activity.

[0064] 5. Screening of paired antibodies in double-antibody sandwich ELISA

[0065] To improve the efficiency of screening paired monoclonal antibodies, monoclonal antibodies with high binding activity were selected for coating and labeling to screen for paired combinations that can specifically detect NfL recombinant proteins.

[0066] 5.1 HRP labeling of antibodies

[0067] The selected labeled antibody was diluted to a final concentration of 2 mg / mL using carbonate coupling buffer (1.59 g sodium carbonate, 2.93 g sodium bicarbonate, diluted to 1 L of pure water, pH 9.6). 2 mg HRP was dissolved in 0.5 mL of ultrapure water and thoroughly mixed with 0.5 mL of 0.06 M sodium periodate solution. Then, 0.5 mL (1 mg antibody) of the diluted antibody solution was added to the matching tube containing HRP, and the mixture was pipetted and incubated at room temperature for 1 h, with periodic mixing during incubation. The labeling reaction was terminated by adding 50 μL of 5 mg / mL sodium borohydride and mixing for 15 min. Finally, the labeled antibody was dialyzed overnight in 0.01 M PBS, pH 7.4 buffer. Glycerol was added at a 1:1 volume ratio, and the mixture was aliquoted and stored at -20 °C.

[0068] 5.2 Establishment of the double-antibody sandwich method

[0069] The purified monoclonal antibody was diluted to a concentration of 1 μg / mL with 50 μL / well of coating buffer (1.59 g sodium carbonate and 2.93 g sodium bicarbonate to 1 L of pure water, pH 9.6) and coated overnight at 4°C. The coating buffer was discarded the next day, and the plate was blocked with 1% BSA at 150 μL / well. The plate was incubated at 37°C for 2 h, and the blocking buffer was discarded. The test antigen NfL recombinant protein and the control antigen HPV18 / E6 were diluted with PBS at 200 ng / mL and added to the microplate at 50 μL / well. The plate was incubated at 37°C for 35 min, washed 4 times with PBST, and then coated with PBS. 50 μL of 1000-fold diluted enzyme-labeled monoclonal antibody was added to each well and incubated at 37°C for 35 min. After washing the plate four times and patting it dry, 50 μL of TMB chromogenic buffer was added to each well. The plate was incubated at room temperature for 10 min. Finally, 50 μL of 0.5 M sulfuric acid was added to each well to stop the reaction. The OD was measured using a microplate reader. 450 nm value. Based on the pairing results, the combination with the highest P / N value was selected for biotin amplification system testing. The results are shown in Table 3. According to the data in the table, monoclonal antibody 9E1 for coating and monoclonal antibody 10B4 for HRP labeling showed the best pairing effect.

[0070] Table 3. Results of paired monoclonal antibody screening.

[0071]

[0072] In the table, "*" indicates the dilution factor.

[0073] The table only lists the screening results of some of the better-performing monoclonal antibody pairs; data for the remaining antibody combinations are not shown.

[0074] 6. Identification of binding activity of paired monoclonal antibodies

[0075] Based on the screening of potential paired antibodies using a double-antibody sandwich ELISA, and referring to the aforementioned indirect ELISA method, the paired monoclonal antibodies screened by the double-antibody sandwich ELISA and other murine unrelated monoclonal antibodies (HPV18 / E6 monoclonal antibody G-7) were serially diluted at 10 μg / ml, 1 μg / ml, 100 ng / ml, 10 ng / ml, 1 ng / ml, and 100 pg / ml. Other murine unrelated monoclonal antibodies (G-7) were used as negative controls. The binding activity of NfL monoclonal antibodies was measured. Other murine unrelated monoclonal antibodies were used as negative controls in the experiment to exclude the influence of non-specific binding. The results are as follows: Figure 3 , Figure 3 9E1 is monoclonal antibody 9E1, 10B4 is monoclonal antibody 10B4, and Ctrl is HPV18 / E6 monoclonal antibody G-7. The binding activity of the screened monoclonal antibodies 9E1 and 10B4 to the NfL recombinant protein was identified using indirect ELISA. Both monoclonal antibodies reacted with the NfL recombinant protein at a concentration of 1 ng / ml, indicating that 9E1 and 10B4 have high binding titers.

[0076] 7. Application of paired monoclonal antibodies in biotin amplification systems

[0077] 7.1 Biotin-antibody conjugation

[0078] Biotin can specifically bind to avidin or biotin monoclonal antibodies, exhibiting multi-stage amplification. The binding is highly stable and specific, significantly improving the sensitivity of detection methods and minimizing non-specific binding of reagents in practical applications. Therefore, utilizing a biotin-avidin amplification system can effectively enhance the detection performance of antibody pairs.

[0079] The molar ratio of biotin (Thermo, EZ-Link NHS Biotin, 20217) to antibody was 20:1. First, 2.0 mg of activated biotin was dissolved in 360 μL of ultrapure water to prepare a 10 mM biotin solution. Then, 2 mg of antibody and 26.6 μL of 10 mM biotin were reacted at room temperature with shaking for 3 h (the reaction volume was controlled at approximately 2 mL). Afterward, the biotin-antibody mixture was dialyzed against 0.01 M PBS to remove excess free biotin. The antibody concentration was measured after dialyzing, and an equal volume of glycerol was added. The mixture was then stored at -20 °C.

[0080] The final concentration of the labeled antibody was approximately 0.5 mg / ml. The biotin-conjugated monoclonal antibody used in this experiment was the optimal monoclonal antibody 10B4, which was selected in the previous screening. This antibody was biotin-labeled and used to construct a biotin-avidin signal amplification system.

[0081] 7.2 Establishment of the Biotin-Avidin Amplified ELISA System

[0082] The monoclonal antibody combinations selected above were coated and conjugated with biotin, and the most suitable reaction conditions for the scale-up system were determined by exploring the concentrations of coating antibody, biotin antibody, and the dilution of HRP-labeled streptavidin. The monoclonal antibody was coated in microplates (coating buffer: carbonate buffer: 1.59 g sodium carbonate, 2.93 g sodium bicarbonate, diluted to 1 L of pure water) at concentration gradients of 0.5 μg / mL, 1 μg / mL, and 2 μg / mL, 50 μL / well, and incubated overnight at 4 °C. The plates were washed once with washing buffer (PBST, PBS containing 0.05% Tween-20), blotted dry, and blocked with 3% BSA, 150 μL per well, incubated at 37 °C for 2 hours, blotted dry, and then dried for later use. The NfL recombinant protein was diluted to a concentration of 10 ng / mL with PBS and added to 50 μL of the monoclonal antibody-coated microplates. At the same time, the recombinant protein GFAP was diluted to 10 ng / mL as a negative control, and the reaction was carried out at 37 °C for 30 min. Discard the liquid from the wells, wash the plate four times with PBST, and blot dry. Dilute the biotin-conjugated monoclonal antibody with PBS to concentrations of 1 μg / mL, 2 μg / mL, and 4 μg / mL, and add 50 μL / well to each well. Incubate at 37°C for 30 min. Discard the liquid from the wells, wash the plate four times with PBST, blot dry, and add 50 μL / well of HRP-polymerized streptavidin (BIOSYNTH, 65R-S105PHRP, diluted 10000, 20000, and 40000 times with PBS). Incubate at 37°C for 30 min, wash four more times, blot dry, and add 50 μL / well of TMB chromogenic buffer. Incubate at room temperature for 10 min. Finally, add stop solution to terminate the reaction and measure the OD using a microplate reader. 450 nm value. The optimal reaction conditions were selected based on the coating concentration, biotin-conjugated monoclonal antibody concentration, and HRP-labeled avidin dilution, which showed the most significant differences between positive and negative results. The preparation process of recombinant protein GFAP is described in patent CN118930634B and will not be repeated here for brevity.

[0083] By coating and HRP-labeling monoclonal antibodies separately, and based on the detection of positive and negative samples, the optimal pairing combination was selected based on the most significant difference in positive and negative detection values. The biotin-avidin amplification system was then adjusted. Ultimately, the following conditions were determined: the coating monoclonal antibody was monoclonal antibody 9E1 at a concentration of 1 μg / mL; the biotin-conjugated monoclonal antibody was monoclonal antibody 10B4 at a concentration of 1 μg / mL; and the HRP-labeled streptavidin was diluted 20,000 times. Under these conditions, the detection system exhibited the lowest background and the highest detection value for positive proteins. The detection method in this application does not include disease diagnosis.

[0084] 7.3 Sensitivity determination of ELISA detection method based on double antibody sandwich immunoassay and biotin-avidin amplification

[0085] To determine the optimal coating concentration, biotin-conjugated monoclonal antibody concentration, HRP-avidin dilution, and other reaction conditions, following the above detection steps, the NfL recombinant protein was first serially diluted with PBS buffer to obtain concentrations of 2000 pg / mL, 200 pg / mL, 20 pg / mL, 2 pg / mL, and 0 pg / mL (blank control). Simultaneously, GFAP recombinant protein was used as a negative control at the same dilution. 50 μL of each diluted protein concentration was then used for detection.

[0086] See Figure 4 , Figure 4 NfL refers to the recombinant NfL protein, and GFAP refers to the recombinant GFAP protein. The selected paired monoclonal antibodies were applied and optimized in a biotin-streptavidin amplified ELISA system. The optimal reaction conditions were determined as follows: 9E1 monoclonal antibody coating concentration of 1 μg / ml, biotin-labeled monoclonal antibody 10B4 dilution of 1 μg / ml, and HRP-streptavidin dilution of 20,000-fold. This system showed a positive reaction for NfL recombinant protein diluted to 20 pg / ml and no reaction with GFAP recombinant protein, demonstrating good specificity and high sensitivity. It can be used for the detection of NfL protein and NfL recombinant protein, providing technical support for biomarker research in neurological diseases and showing significant application potential in the early screening and diagnosis of neurological diseases.

[0087] 8. Antibody variable region gene sequence

[0088] Total RNA was extracted from hybridoma cells using the RNeasy Mini Kit (Cat. No. 74104), and cDNA was synthesized by reverse transcription using RandomPrimers. Universal primers for the variable region of mouse antibodies were designed, and the VH and VL genes were amplified by two rounds of PCR. Age1 and Bsiw1 restriction sites were introduced into the primers for the third round of PCR. The PCR products were purified by gel extraction and ligated into the pUC19 vector, transformed into TOP10 strain, and single colonies were picked and sequenced after culturing at 37°C for 14 h to obtain the gene sequences of the light and heavy chains of the monoclonal antibody.

[0089] The sequence of monoclonal antibody 9E1:

[0090] The nucleotide sequence encoding the variable region of the light chain of monoclonal antibody 9E1 is shown in SEQ ID NO.18:

[0091] GACGTGCTGATGACCCAGACCCCCAGCTACCTGGCCGCCAGCCCCGGCGAGACCATCACCATCAACTGCAGGCCCAGCAAGAGCATCAGGAAGTACCTGGCCTGGTACCAGGAGAAGCCCGGCAAGACCAACAAGCTGCTGATCTACCTGGGCAACTTCCTGCAG CACGGCATCCCCAGCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGGCATGTACTACTGCCAGAGGCACACCGACTACAAGTGGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGAGGACCGTG.

[0092] The amino acid sequence of the variable region of the light chain of monoclonal antibody 9E1 is shown in SEQ ID NO.14:

[0093] DVLMTQTPSYLAASPGETITINCRPSKSIRKYLAWYQEKPGKTNKLLIYLGNFLQHGIPSRFSGSGSGTDFTLTISSLEPEDFGMYYCQRHTDYKWTFGGGTKLEIKRTV.

[0094] Light chain CDR area annotation:

[0095] The amino acid sequence of CDR-L1 in the light chain variable region of monoclonal antibody 9E1 is shown in SEQ ID NO.4: RPSKSIRKYLA;

[0096] The amino acid sequence of CDR-L2 in the light chain variable region of monoclonal antibody 9E1 is shown in SEQ ID NO.5: LGNFLQH;

[0097] The amino acid sequence of CDR-L3 in the light chain variable region of monoclonal antibody 9E1 is shown in SEQ ID NO.6: QRHTDYKWT.

[0098] The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 9E1 is shown in SEQ ID NO.17: CAGGTGCAGCTGTTGGAGACTGGGGCTGAGCTTGTGAGGCCAGGGGCCTTAGTCAAGTTGTCCTGCAAAGCTTCTGGCTTCAACATTAGAGACAATTATCTGCACTGGGTGAAGCAGAGGCCTGAACAGGGCCTGGAGTGGATTGGATGGATTGATCCTGTGTATGAGAACACTAGATATGACCCGAAGTTCCAGGGCCAGGCCAGAATAACAGCAGACACATCCTCCAATACAGCCTACCTGCAGCTCAGCAGCCTGACATCTGAGGACACTGCCGTCTATTTCTGTGCTTTCGGTAGTACCTACGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA.

[0099] The amino acid sequence of the heavy chain variable region of monoclonal antibody 9E1 is shown in SEQ ID NO.13:

[0100] QVQLLETGAELVRPGALVKLSCKASGFNIRDNYLHWVKQRPEQGLEWIGWIDPVYENTRYDPKFQGQARITADTSSNTAYLQLSSLTSEDTAVYFCAFGSTYAMDYWGQGTSVTVSS.

[0101] Heavy chain CDR region annotation:

[0102] The amino acid sequence of the CDR-H1 of the heavy chain variable region of monoclonal antibody 9E1 is shown in SEQ ID NO.1: DNYLH;

[0103] The amino acid sequence of the CDR-H2 of the heavy chain variable region of monoclonal antibody 9E1 is shown in SEQ ID NO.2: WIDPVYENTRYDPKFQG;

[0104] The amino acid sequence of the CDR-H3 of the heavy chain variable region of monoclonal antibody 9E1 is shown in SEQ ID NO.3: GSTYAMDY.

[0105] Sequence labeled with monoclonal antibody 10B4:

[0106] The nucleotide sequence encoding the variable region of the light chain of monoclonal antibody 10B4 is shown in SEQ ID NO.20:

[0107] GACATTTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCATCACCTGCAAGGCCAGTCAAAATGTGAATACTGGTGTAGCCTGGTATCAACAAAAACCAGGGCAATCTCCTAAACTACTGATTTACTGGGCATCCACCCGGCA CACTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTATACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGACACTTTATTACTGTCAGCAACATTATTACACTCCTCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGGGCT.

[0108] The amino acid sequence of the variable region of the light chain of monoclonal antibody 10B4 is shown in SEQ ID NO.16:

[0109] DIVMTQSHKFMSTSVGDRVSITCKASQNVNTGVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDYTLTISSVQAEDLTLYYCQQHYYTPLTFGAGTKLELKRA.

[0110] Light chain CDR area annotation:

[0111] The amino acid sequence of CDR-L1 in the light chain variable region of monoclonal antibody 10B4 is shown in SEQ ID NO.10: KASQNVNTGVA;

[0112] The amino acid sequence of CDR-L2 in the light chain variable region of monoclonal antibody 10B4 is shown in SEQ ID NO.11: WASTRHT;

[0113] The amino acid sequence of CDR-L3 in the light chain variable region of monoclonal antibody 10B4 is shown in SEQ ID NO.12: QQHYYTPLT.

[0114] The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 10B4 is shown in SEQ ID NO.19: GAGGTGCAGCTGCAGCAGTCTGGGACTGTGCTGGCAAGGCCTGGGGCTTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACCTTTACCAGCTACTGGATGCACTGGGTAAAACAGAGGCCTGGACAGGGTCTGCAATGGATTGGCGCTATTTACCCTGGAAACAGTAATACTGTCTACAACCAGAAGTTCAAGGGCAAGGCCAAACTGACTGCAGTCACATCCACCAGCACTGCCTACATGGAACTCAGCAGCCTGACAAATGAGGACTCTGAGGTCTATTACTGTGCAAGATCCTCCCTCTTTGATGCTTACCCTTACTGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACAGTCTCCTCA.

[0115] The amino acid sequence of the heavy chain variable region of monoclonal antibody 10B4 is shown in SEQ ID NO.15:

[0116] EVQLQQSGTVLARPGASVKMSCKASGYTFTSYWMHWVKQRPGQGLQWIGAIYPGNSNTVYNQKFKGKAKLTAVTSTSTAYMELSSLTNEDSEVYYCARSSLFDAYPYWYFDVWGAGTTVTVSS.

[0117] Heavy chain CDR region annotation:

[0118] The amino acid sequence of the CDR-H1 of the heavy chain variable region of monoclonal antibody 10B4 is shown in SEQ ID NO.7: SYWMH;

[0119] The amino acid sequence of the CDR-H2 of the heavy chain variable region of monoclonal antibody 10B4 is shown in SEQ ID NO.8: AIYPGNSNTVYNQKFKG;

[0120] The amino acid sequence of the CDR-H3 of the heavy chain variable region of monoclonal antibody 10B4 is shown in SEQ ID NO.9: SSLFDAYPYWYFDV.

[0121] The embodiments of this application have now been described in detail. To avoid obscuring the concept of this application, some details known in the art have not been described. Those skilled in the art can fully understand how to implement the technical solutions disclosed herein based on the above description.

[0122] While specific embodiments of this application have been described in detail by way of examples, those skilled in the art should understand that the above examples are for illustrative purposes only and are not intended to limit the scope of this application. Those skilled in the art should understand that modifications can be made to the above embodiments or equivalent substitutions can be made to some technical features without departing from the scope and spirit of this application.

Claims

1. A monoclonal antibody combination for the detection of NfL protein, characterized in that, The monoclonal antibody combination includes monoclonal antibody 9E1 and monoclonal antibody 10B4. The heavy chain variable region of monoclonal antibody 9E1 includes three complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3. The amino acid sequence of CDR-H1 is shown in SEQ ID NO.1, the amino acid sequence of CDR-H2 is shown in SEQ ID NO.2, and the amino acid sequence of CDR-H3 is shown in SEQ ID NO.

3. The light chain variable region of the monoclonal antibody 9E1 includes three complementarity-determining regions CDR-L1, CDR-L2 and CDR-L3, the amino acid sequence of CDR-L1 is shown in SEQ ID NO.4, the amino acid sequence of CDR-L2 is shown in SEQ ID NO.5 and the amino acid sequence of CDR-L3 is shown in SEQ ID NO.

6. The heavy chain variable region of the monoclonal antibody 10B4 includes three complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3. The amino acid sequence of CDR-H1 is shown in SEQ ID NO.7, the amino acid sequence of CDR-H2 is shown in SEQ ID NO.8, and the amino acid sequence of CDR-H3 is shown in SEQ ID NO.

9. The light chain variable region of the monoclonal antibody 10B4 includes three complementarity-determining regions, CDR-L1, CDR-L2, and CDR-L3. The amino acid sequence of CDR-L1 is shown in SEQ ID NO.10, the amino acid sequence of CDR-L2 is shown in SEQ ID NO.11, and the amino acid sequence of CDR-L3 is shown in SEQ ID NO.

12.

2. The monoclonal antibody combination for NfL protein detection according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody 9E1 is shown in SEQ ID NO.13; the amino acid sequence of the light chain variable region of the monoclonal antibody 9E1 is shown in SEQ ID NO.

14.

3. The monoclonal antibody combination for NfL protein detection according to claim 2, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody 10B4 is shown in SEQ ID NO.15; the amino acid sequence of the light chain variable region of the monoclonal antibody 10B4 is shown in SEQ ID NO.

16.

4. The monoclonal antibody combination for NfL protein detection according to claim 3, characterized in that, The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 9E1 is shown in SEQ ID NO.17; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 9E1 is shown in SEQ ID NO.

18.

5. The monoclonal antibody combination for NfL protein detection according to claim 4, characterized in that, The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 10B4 is shown in SEQ ID NO.19; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 10B4 is shown in SEQ ID NO.

20.

6. The use of the monoclonal antibody combination according to claim 1 in the preparation of a tool for detecting NfL protein.

7. The application according to claim 6, characterized in that, The tools include reagents, kits, test strips, and antibody chips.

8. The application according to claim 7, characterized in that, The kit includes a double-antibody sandwich ELISA kit.

9. The application according to claim 8, characterized in that, The kit includes biotin. Avidin amplification system double antibody sandwich ELISA kit.

10. The application according to claim 9, characterized in that, The kit uses monoclonal antibody 9E1 as the coating antibody and monoclonal antibody 10B4 as the labeling antibody.

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