Monoclonal antibody composition for resisting neurofilament light chain protein and application

Abstract

The invention discloses a monoclonal antibody composition for resisting neurofilament light chain protein and application, the monoclonal antibody composition comprises NfL-Clone 1 and NfL-Clone 2, the amino acid sequence of a heavy chain variable region of the NfL-Clone 1 is as shown in SEQ ID NO.1, and the amino acid sequence of a light chain variable region of the NfL-Clone 1 is as shown in SEQ ID NO.2; the amino acid sequence of a heavy chain variable region of the NfL-Clone 2 is as shown in SEQ ID NO.3, and the amino acid sequence of a light chain variable region of the NfL-Clone 2 is as shown in SEQ ID NO.4. The antibody combination has the characteristics of specificity and high affinity, the NfL can be used as a brain injury biomarker, and meanwhile, the invention also provides a diagnostic detection kit of the antibody for early diagnosis of brain injury of a patient, so that the specificity and sensitivity of a diagnostic reagent are improved.

Description

Technical Field

[0001] The invention belongs to the field of antibody preparation and sequence determination, and specifically relates to a monoclonal antibody composition against neurofilament light chain protein and its application. Background Art

[0002] Neurofilament light chain protein (NfL) is a 68 KD neuronal cytoplasmic protein. It is the smallest and most abundant neurofilament subunit, responsible for maintaining the function of the nervous system. Since NfL is a neuronal protein, it can better reflect the loss of connection between nerve cells and axons. The NfL content in the blood is low, generally <10 pg / mL, while it is relatively high in the cerebrospinal fluid, <1800 pg / mL. When axonal damage or neuronal degeneration-related diseases occur, the NfL levels in both the cerebrospinal fluid (CSF) and the blood will increase, so NfL can be used as a biomarker for brain damage.

[0003] Several studies have indicated that neurofilament light chain protein (NfL) can be used as a diagnostic, prognostic and monitoring marker for various neurological diseases, such as Alzheimer's disease (AD), Parkinson's disease / syndrome (PD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), etc.

[0004] The core raw material for developing excellent neurofilament light chain protein (NfL) immunodiagnostic reagents is anti-neurofilament light chain protein (NfL) monoclonal antibody. The specificity and sensitivity of general manufacturers are not high, and most of the core raw materials are difficult to obtain, so it is very necessary to develop excellent neurofilament light chain protein (NfL) diagnostic reagents. Summary of the invention

[0005] In order to solve the problems of sensitivity and specificity of antibodies on the market, the present invention provides a monoclonal antibody composition against neurofilament light chain protein. The antibody composition has excellent performance and high accuracy.

[0006] At the same time, a chemiluminescence kit based on the above-mentioned neurofilament light chain protein (NfL) antibody is provided, which provides a basis for early screening of brain damage by detecting NfL in the blood.

[0007] The present invention provides the following technical solutions: A monoclonal antibody composition against neurofilament light chain protein, including NfL-Clone1 and NfL-Clone2, The amino acid sequence of the heavy chain variable region of NfL-Clone1: QVQLQESGPELVKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYINPYNDGSKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARGGFYYAMDYWGQGTSVTVSS The amino acid sequence of the light chain variable region of NfL-Clone1: DIVLTQSPALMAASPGEKVTITCSVSSSISSSSLHWYQQKSETSPKPWIYGTSNLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSRYPLTFGAGTKLELK The amino acid sequence of the heavy chain variable region of NfL-Clone2: QVQLEQSGPDLVKPSQSLSLTCTVTGYSITSGYSWHWIRQFPGNKLEWVGYIHFSGTTNYNPSLKSRISITRDTSRNQFFLQLNSVTTADTATYYCASVIRYALDYWGQGTSVTVSS The amino acid sequence of the light chain variable region of NfL-Clone2: DIVLTQSPSYLAASPGETITINCRASKSISKYLAWYQEKPGKTNKLLIYSGSTLKSGIPSRFSGSGSGTDFTLTISSLEPEDFAMYYCQQHNEYPWTFGGGTKLEIK Preparation method of monoclonal antibody composition against neurofilament light chain protein (NfL): Expression and production of neurofilament light chain protein (NfL): After pTT5-NfL expression plasmid was mixed with transfection reagent PEI, HEK293 cells were transfected. At the same time, enhancers and auxiliary materials were added to OPM-293 CD03 Medium cell culture medium for culture. When the cell viability was <70%, the cell culture was collected, centrifuged and the cell supernatant was collected.

[0008] Affinity purification of neurofilament light chain protein (NfL): The cell supernatant was fully resuspended and filtered with salt and imidazole, and then affinity purified using a pre-loaded gravity column. The protein was washed and eluted using a gradient of imidazole concentrations. The protein purity was detected by SDS-PAGE running. The protein was dialyzed into phosphate buffer, sterile filtered through a 0.22μm filter, and the protein concentration was determined by BCA.

[0009] The affinity-purified NfL protein was mixed with Freund's adjuvant in equal volumes for emulsification, with an immunization dose of 100ug / mouse. A second immunization was performed 2 weeks later, with an immunization dose of 50ug / mouse. The interval between subsequent immunizations was 3 weeks. Blood was collected from mice 7 days after immunization to separate serum, and the antibody titer was determined by enzyme-linked immunosorbent assay. Mice with better immunity were selected for hybridoma fusion experiments. After screening and culture in HAT screening medium, NfL protein was used as the coated antigen on the ELISA plate, and the supernatant was taken for enzyme-linked immunosorbent assay to screen out high-quality positive cell lines, and two rounds of cell subcloning were performed to clone monoclonal cell lines, and the cells were amplified, and the antibodies were expressed in ascites for antibody purification.

[0010] Application of monoclonal antibody composition against neurofilament light chain protein in the preparation of early diagnosis reagent for brain injury in patients.

[0011] Monoclonal antibodies are used in the NfL chemiluminescent kit. The kit uses NfL-Clone1 and NfL-Clone2 as the coating antibody and detection antibody, respectively.

[0012] Compared with the prior art, the present invention has the following beneficial effects: the present invention provides an anti-NfL protein for preparing a monoclonal antibody for diagnosis; the monoclonal antibody composition of the anti-neurofilament light chain protein of the present invention includes NfL-Clone1 and NfL-Clone2, which can recognize immunogens; this antibody combination has the characteristics of specificity and high affinity; and a chemiluminescent detection kit based on the above-mentioned antibodies is also provided, which can be used for early diagnosis and condition assessment of patients with Alzheimer's disease (AD), Parkinson's disease / syndrome (PD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), etc., which is of great significance, thereby improving the specificity and sensitivity of the diagnostic reagents; and the accurate detection results provide important medical value for guiding the use of drugs in subsequent clinical treatments. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is a correlation diagram of the test results of the kit of the present invention and other kits. DETAILED DESCRIPTION

[0014] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0015] Construction of NfL protein expression vector: The amino acid sequence of human NfL was searched on the official website of NCBI, a 6*His tag was added to the C-terminus of the NfL sequence, and the mammalian cell expression system was selected for codon optimization in the codon optimization software. The optimized gene sequence was synthesized, and the gene was subcloned into the pTT5 expression vector using the EcoRI / BamHI restriction endonuclease cutting site to construct the pTT5-NfL recombinant expression plasmid, and the gene sequence was verified by sequencing.

[0016] Expression and production of NfL protein: After the cells were revived and cultured, they were cultured at a rate of 0.8 × 10 6 / ml was inoculated in OPM-293 CD03 Medium culture medium and placed at 37°C with 5% CO 2 The cells were cultured in an incubator and transfected when the cells expanded to a certain number and the viability was greater than 95%. The pTT5-NfL recombinant expression vector was mixed with PEI and incubated at room temperature for 15-30 minutes. The cells were slowly added to the HEK293 cells, and the enhancer and excipients were added at 37°C and 5% CO. 2 Continue to culture in the incubator. When the cell viability is less than 70%, centrifuge at 5000 g for 10 min, collect the supernatant and cells, and perform WB identification.

[0017] Affinity purification of NfL protein: NaCl and imidazole were added to the cell supernatant, and the working concentrations were adjusted to 300 mM and 5 mM respectively, and then filtered with a 0.22 μm filter membrane. Ni TED-6FF beads were pre-loaded on a gravity column for affinity purification, washed with 20 mM PB, 300 mM NaCl, 10% glycerol, 50 mM imidazole, pH 8.0 solution, eluted with 20 mM PB, 300 mM NaCl, 10% glycerol, 250 mM imidazole, pH 8.0 solution, and the eluted components were collected and tested for protein purity by SDS-PAGE. The proteins that met the requirements were dialyzed into 20 mMPB, 300 mM NaCl, 10% glycerol, pH 8.0 buffer, sterile filtered with a 0.22 μm filter, and the protein concentration was determined by BCA.

[0018] Preparation of monoclonal antibodies: NfL protein and Freund's adjuvant were mixed and emulsified in equal volumes, with an immunization dose of 100ug / mouse, and multiple subcutaneous injections. The second immunization was performed 2 weeks later, with an immunization dose of 50ug / mouse. The interval between subsequent immunizations was 3 weeks. The mouse blood was collected 7 days after immunization to separate serum, and the antibody titer was determined by enzyme-linked immunosorbent assay (ELISA). Mice with serum ELISA titers that met the requirements after immunization were subjected to cell fusion. The final immunization was performed 3 days before fusion, and 100ug of antigen was injected intraperitoneally. On the day of fusion, the mice to be fused were euthanized by cervical dislocation. Soaked in 75% alcohol for 5 min. The spleen was sterilely removed and placed in a culture dish containing 10 mL DMEM basal culture. The sieve was placed in another plate, the spleen was transferred to the sieve, and the spleen was ground with a syringe. DMEM was added to the sieve and the sieve was rinsed to collect more spleen cells in the plate. The cells were transferred to a 50 mL centrifuge tube, and the spleen cells were washed once with serum-free DMEM and centrifuged at 1500 rpm for 5 min. Collect spleen cells and remove red blood cells with red blood cell lysis buffer, wash spleen cells once with serum-free DMEM, and centrifuge at 1500 rpm for 5 min. Collect spleen cells and count. Mix myeloma cells and spleen cells to make the number of myeloma cells and spleen cells reach an appropriate ratio. Put the cells in a 50 mL centrifuge tube, dilute with DMEM basal medium, and then centrifuge at 1500 rpm for 5 min. Discard the supernatant. Shake the centrifuge tube to make the cells uniform, wash the cells once with electrofusion solution, and then centrifuge at 1500 rpm for 5 min. Discard the supernatant. Dilute the cells to 2×107 / mL with electrofusion solution, then add 2mL of cell suspension to the fusion pool for electrofusion experiment, and let it stand for 10 min after electroporation. Then add HAT DMEM medium. Spread the fused cells into a 96-well plate, 100 μL per well. Then put the cell culture plate in a CO2 incubator for culture. Check 6 days after fusion, and start screening detection 10 days after fusion. On the day before the test, the antigen was coated on the ELISA plate with PBS overnight. The next day, 100 μL / well of the cell supernatant was aspirated for ELISA test. According to the ELISA results, the positive wells were judged (the sample well OD value / negative well OD value ≥ 2.1 was judged as a positive well). Use a single-channel pipette to pick the positive wells detected on the whole plate, and perform a second confirmation test to further confirm the positive wells. The cells in the confirmed positive wells were subcloned. Blow up the cells in the positive wells, count them, add N / 4 mL DMEM culture medium to the centrifuge tube (N is the cell count result), take 100 μL of cell suspension into the centrifuge tube, blow it evenly and leave 1 mL, add DMEM to 4 mL, blow it evenly, and leave 100 μL (about 2 drops) at the bottom of the tube.Add DMEM to 5 mL in a centrifuge tube, mix well and drop it to the first three rows of a 96-well plate, leaving about 1.8-2 mL at the bottom of each well, add DMEM to 5 mL, blow well and drop it to the D, E, and F rows of the 96-well plate, one drop per well, leaving about 1.5-1.8 mL at the bottom of the tube, add DMEM to about 2.8-3 mL, blow well and drop it to the G and H rows of the 96-well plate, one drop per well, observe under a microscope after 7-10 days, detect the wells with clone growth, mark the wells with monoclonal clones, pick out positive monoclonal clones as much as possible for re-subcloning, and pick out the monoclonal clones to expand and culture the cells after 100% positive detection. Amplify the cells, express antibodies through ascites, purify the antibodies, load the ProteinG agarose gel medium into the affinity purification column, slowly load the ascites, elute with glycine elution buffer after the antibody binds, and obtain the desired purified antibody. The purified antibodies were analyzed for diagnostic properties.

[0019] Monoclonal Antibody Performance Analysis: The kit was prepared based on the double antibody sandwich method and acridinium ester luminescence method.

[0020] 1. Carboxyl magnetic beads coated with antibodies Mix the carboxyl magnetic beads thoroughly, take out the corresponding amount of magnetic beads and place them in a centrifuge tube, use a magnetic rack to remove the supernatant, add the coating activation solution after washing, add EDC and NHS dissolved in the coating activation solution after mixing, and activate them in a water bath at 37°C and 220 rpm for 0.5-0.7h after mixing thoroughly; Ultrasonic dispersion of magnetic beads, washing with coating activation solution for three times, adding the coating antibody of the present invention, the mass ratio of magnetic beads to antibodies being 100:1, mixing thoroughly, reacting in a water bath at 37° C. and 220 rpm for 2.5-3 h; Use a magnetic rack to remove the supernatant, wash three times, disperse the magnetic beads by ultrasonication, add the blocking agent, mix thoroughly, and block in a water bath at 37°C, 220 rpm for 1-1.2h; Ultrasonic dispersion of magnetic beads was performed, and the supernatant was removed using a magnetic stand. The beads were washed three times, and magnetic bead storage solution was added to dilute the beads to a concentration of 1 mg / mL for later use.

[0021] The formula of the magnetic bead storage solution is 50mM Tris, 0.1% Tween-20, 0.9% NaCl, 2.5% BSA, and 0.3% Proclin-300.

[0022] 2. Acridinium ester labeled antibodies The NHS-esterified acridinium ester (NSP-SA-NHS) and the coating antibody of the present invention are reacted in a labeling buffer at a molecular molar ratio of 10:1, and the labeling reaction is carried out in a water bath at 37° C. and 220 rpm for 1-1.5 h; After the reaction is completed, add the stop solution, and react in a water bath at 37°C and 220 rpm for 0.5-0.8 h. Transfer the labeled solution to a 14 kD dialysis bag and dialyze in 1× PBS by rotation. Change the solution 4-6 times until the luminescence value of the acridinium ester in the dialysate is below the quality control line. After dialysis overnight, remove the labeling solution, test the protein content in the labeling solution, calculate and add protective solution and glycerol, mix thoroughly and store at -20℃. When testing on the machine, use the labeling preservation solution to dilute the protein concentration to 0.5ug / mL for later use.

[0023] The formula of the above-mentioned labeling preservation solution is 0.1M PBS, 0.85% NaCl, 2% BSA, and 0.3% Proclin-300.

[0024] 3. Prepare calibration and quality control products Use antigen diluent to dilute NfL antigen to 0, 2, 10, 20, 100, 200, and 1000 pg / mL, respectively, and dispense into calibrator tubes.

[0025] Use antigen diluent to dilute NfL antigen to 20 and 600 pg / mL respectively, and dispense into quality control tubes.

[0026] The above-mentioned antigen dilution solution formula is 1×PBS, 1% bovine serum albumin (BSA).

[0027] Comparison of the test kit with similar products: The NfL chemiluminescent detection kit in the embodiment was compared with the product of the control company on 100 clinical specimens. The data are as follows: Figure 1 As shown. Correlation coefficient R 2 The result shows that the kit of the present invention can accurately detect the content of NfL, provide a basis for clinical diagnosis, and fully meet the needs of clinical in vitro diagnostic testing.

[0028] Sensitivity and precision of the detection kit: The blank limit and detection limit of the kit of the present invention were studied for sensitivity, and the results showed that the blank limit and detection limit were 0.5 pg / mL and 1 pg / mL, respectively.

[0029] The precision of the kit of the present invention was studied. Three batches of reagents and calibrators were used to test two levels of quality control products. The tests were conducted twice a day, in the morning and afternoon, for 5 consecutive days. The results showed that the total indoor precision of each batch was less than 5%.

[0030] Analysis of the anti-interference ability of the detection kit: Different concentrations of interfering substances were added to two serum samples with NfL concentrations of approximately 10 pg / mL and 200 pg / mL, respectively. The group without added substances was used as the control. The relative deviation between the added group and the control group was less than 5%, which was considered the acceptable interference standard. The maximum concentration levels of different interfering substances were studied (not exceeding the highest concentration observed clinically).

[0031] Table 1 Analysis data of the anti-interference ability of the detection kit

[0032] The results show that the concentrations in Table 1 do not significantly interfere with the test results.

[0033] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A monoclonal antibody composition against neurofilament light chain protein, characterized in that: Including NfL-Clone1 and NfL-Clone2, The amino acid sequence of the heavy chain variable region of NfL-Clone1 is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region of NfL-Clone1 is shown in SEQ ID NO.2; The amino acid sequence of the heavy chain variable region of NfL-Clone2 is shown in SEQ ID NO.3, and the amino acid sequence of the light chain variable region of NfL-Clone2 is shown in SEQ ID NO.

4.

2. Use of the anti-neurofilament light chain monoclonal antibody composition according to claim 1 in the preparation of an early diagnostic reagent for brain damage in patients.

3. Use of the anti-neurofilament light chain monoclonal antibody composition according to claim 2 in the preparation of an early diagnostic reagent for brain damage in patients, characterized in that: Monoclonal antibodies are used in NfL chemiluminescent kits.

4. NfL chemiluminescent kit, characterized in that: Containing NfL-Clone1 and NfL-Clone2 according to claim 1.

5. The NfL chemiluminescent kit according to claim 4, characterized in that: The kit uses NfL-Clone1 and NfL-Clone2 as coating antibody and detection antibody, respectively.

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