Functional fragment, ubiquitin carboxyl terminal hydrolase L1 antibody and application thereof

Abstract

The invention provides a functional fragment, a ubiquitin carboxyl terminal hydrolase L1 antibody and application thereof, and belongs to the technical field of antibodies. The functional fragment comprises a complementary determining region Ab1 and a complementary determining region Ab2, wherein the Ab1 comprises CDR-VL1, CDR-VL2, CDR-VL3, CDR-VH1, CDR-VH2 and CDR-VH3, and the Ab2 comprises CDR-VL1, CDR-VL2, CDR-VL3, CDR-VH1, CDR-VH2 and CDR-VH3; and the Ab2 comprises a CDR-VL1, a CDR-VL2, a CDR-VL3, a CDR-VH1, a CDR-VH2 and a CDR-VH3. The invention provides an antibody for specifically recognizing ubiquitin carboxyl terminal hydrolase L1 (Uch-L1), which has the characteristics of high specificity and high affinity, is suitable for quantitative or qualitative detection of Uch-L1, effectively reduces the detection cost of Uch-L1, shortens the detection time, improves the detection efficiency, and can realize sensitivity, stability and high sensitivity required by rapid diagnosis. And general standards such as economy and no equipment are needed.

Description

Technical Field

[0001] The present invention relates to the field of antibody technology, and in particular to a functional fragment, an antibody against ubiquitin carboxyl terminal hydrolase L1 and applications thereof. Background Art

[0002] Ubiquitin Carboxyl-Terminal Hydrolase L1 (Uch-L1) is a deubiquitinating enzyme. Studies have shown that Uch-L1 is associated with various neurological diseases, such as Parkinson's disease, Alzheimer's disease, and other forms of dementia. In these diseases, altered expression or activity of Uch-L1 affects neuronal function and survival. Furthermore, due to its high expression in brain tissue and presence in the blood, Uch-L1 is considered an important biomarker for studying neurodegenerative diseases or other brain injuries, particularly during the development and progression of TBI.

[0003] Ubiquitin Carboxyl-Terminal Hydrolase L1 (Uch-L1) is a deubiquitinating enzyme belonging to the ubiquitin-specific protease (USP) family and plays a key role in neurons.

[17] . The main functions of Uch-L1 include: 1) In terms of ubiquitin cycle, Uch-L1 can cut the end of the ubiquitin chain and remove the ubiquitin monomer from the tagged protein, thereby participating in the recycling of ubiquitin. This is crucial for maintaining the homeostasis of the ubiquitin pool in the cell. 2) In terms of protein stabilization, Uch-L1 can prevent certain proteins from being degraded by the proteasome by removing the ubiquitin tag, directly affecting the stability of the protein. 3) Uch-L1 plays an important role in synaptic function, especially in presynaptic terminals. Studies on TBI have shown that the concentration of Uch-L1 in serum and cerebrospinal fluid increases significantly after injury, and this increase lasts for at least one week, causing the concentration of Uch-L1 in serum to rise from the basal level of 2pM to 20pM or even higher. This characteristic suggests that Uch-L1 can be used as an effective biomarker for early detection of TBI and prediction of long-term prognosis. In addition, the localization of Uch-L1 in the neuronal cytoplasm makes it a key biomarker in TBI research, and its role in TBI and its value as a diagnostic and prognostic indicator have been widely explored.

[0004] Studies have shown that serum Uch-L1 levels rise rapidly after TBI and can even be detected within one hour after injury, making Uch-L1 a strong candidate for early diagnosis of TBI.

[28] Of particular note, elevated Uch-L1 levels in patients with mild to moderate TBI were not affected by drug or alcohol intake, further confirming its reliability as an independent biomarker. Furthermore, the association of Uch-L1 with multiple neurological diseases suggests its potential value in assessing neuronal health.

[0005] In summary, the development of ubiquitin carboxyl-terminal hydrolase L1 (Uch-L1) is of great significance for the diagnosis of nervous system diseases and the discovery of nervous system antibody drugs. Summary of the Invention

[0006] The present invention aims to provide functional fragments, ubiquitin carboxyl-terminal hydrolase L1 antibodies and their applications, which have the characteristics of high specificity and high affinity and are suitable for the quantitative or qualitative detection of Uch-L1, and are particularly suitable for the development of a time-resolved fluorescence immunochromatographic kit. They have the advantages of low detection limit and high sensitivity, effectively reducing the detection cost of Uch-L1, shortening the detection time, and improving the detection efficiency, and can achieve the universal standards required for rapid diagnosis, such as sensitivity, stability, economy, and no need for equipment.

[0007] The technical solution of the present invention is achieved as follows:

[0008] In a first aspect, the present invention provides an antibody or a functional fragment thereof targeting ubiquitin carboxyl-terminal hydrolase L1 (Uch-L1), comprising the following complementarity determining regions:

[0009] Ab1:

[0010] CDR-VL1: amino acid sequence is DSTM, as shown in SEQ ID NO.1;

[0011] CDR-VL2: amino acid sequence PTDWTGG, as shown in SEQ ID NO. 2;

[0012] CDR-VL3: amino acid sequence is SSVNY, as shown in SEQ ID NO. 3;

[0013] CDR-VH1: amino acid sequence is ANWDWYFD, as shown in SEQ ID NO. 4;

[0014] CDR-VH2: amino acid sequence is IDPANGNT, as shown in SEQ ID NO. 5;

[0015] CDR-VH3: The amino acid sequence is IKDTY, as shown in SEQ ID NO.6.

[0016] Ab2:

[0017] CDR-VL1: amino acid sequence is VNYMHWYQ, as shown in SEQ ID NO.7;

[0018] CDR-VL2: amino acid sequence KLASGV, as shown in SEQ ID NO. 8;

[0019] CDR-VL3: amino acid sequence is SNPLTF, as shown in SEQ ID NO.9;

[0020] CDR-VH1: amino acid sequence is DTYMH, as shown in SEQ ID NO.10;

[0021] CDR-VH2: amino acid sequence is GFNIKDTYM, as shown in SEQ ID NO. 11;

[0022] CDR-VH3: The amino acid sequence is WYFDVW, as shown in SEQ ID NO.12.

[0023] In some embodiments, the antibody further comprises a constant region;

[0024] In some embodiments, the constant region is selected from the constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD;

[0025] In some embodiments, the species of origin of the constant region is cow, horse, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose, or human.

[0026] In some embodiments, the functional fragment is selected from any one of F(ab')2, Fab', Fab, Fv, Fd, dAb, and scFv of the antibody.

[0027] The functional fragments of the above-mentioned antibodies generally have the same binding specificity as the antibody from which they are derived. Based on the disclosure herein, those skilled in the art will readily appreciate that the functional fragments of the above-mentioned antibodies can be obtained by, for example, enzymatic digestion (including pepsin or papain) and / or by chemical reduction cleavage of disulfide bonds. Based on the structure of the intact antibody disclosed herein, those skilled in the art can readily obtain the above-mentioned functional fragments.

[0028] The functional fragments of the above antibodies can also be synthesized by recombinant genetic techniques known to those skilled in the art or by, for example, an automatic peptide synthesizer, such as those sold by Applied BioSystems.

[0029] In some embodiments, a nucleic acid molecule encoding the antibody or a functional fragment thereof is introduced into a host cell to obtain a recombinant cell, which is then cultured and the antibody or a functional fragment thereof is obtained through separation and purification.

[0030] In a second aspect, the present invention provides nucleic acids encoding the antibodies or their functional fragments. Based on the amino acid sequences and codon conventions of the antibodies or their functional fragments described above, those skilled in the art can obtain the nucleotide sequences of nucleic acid molecules encoding the antibodies or their functional fragments. Due to the degeneracy of codons, the nucleotide sequences of nucleic acid molecules encoding the antibodies or their functional fragments are not unique, and all nucleic acid molecules capable of encoding the antibodies or their functional fragments are within the scope of protection of the present invention.

[0031] In a third aspect, the present invention provides a product comprising the antibody or its functional fragment, or the nucleic acid.

[0032] In some embodiments, the product is any one of an antibody conjugate, a biological material, a drug, a detection reagent or kit, and a test strip.

[0033] In some embodiments, in the product, the antibody or functional fragment thereof is labeled with a detectable marker.

[0034] In some embodiments, the product is used to detect a neurological disease (Uch-L1).

[0035] In some embodiments, the antibody conjugate is obtained by conjugating the antibody or a functional fragment thereof to a detectable label.

[0036] In some embodiments, the detection reagent or kit is a quantitative or qualitative detection reagent or kit for Uch-L1.

[0037] In some embodiments, the detection reagent or kit is used for ELISA detection, immunochemiluminescence detection, or immunofluorescence detection.

[0038] In some embodiments, the detection kit is a lateral flow immunochromatography diagnostic kit.

[0039] In some embodiments, the test strip is a time-resolved fluorescent immunochromatographic test strip.

[0040] In some embodiments, the test strip is a lateral flow immunochromatographic test strip.

[0041] In some embodiments, the detectable marker refers to a class of substances that have properties that can be directly observed by the naked eye or detected or detected by an instrument, such as luminescence, color development, radioactivity, etc., through which qualitative or quantitative detection of the corresponding target can be achieved.

[0042] In some embodiments, the detectable markers include, but are not limited to, fluorescent dyes, enzymes that catalyze substrate color development, radioactive isotopes, chemiluminescent reagents, and nanoparticle markers. In actual use, those skilled in the art can select an appropriate marker based on the detection conditions or actual needs. Regardless of the marker used, it falls within the scope of protection of the present invention.

[0043] In some embodiments, the fluorescent dyes include but are not limited to fluorescein dyes and their derivatives (for example, including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (for example, including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (for example, including but not limited to Cy2, Cy3, Cy3B, Cy3.5, C y5, Cy5.5, Cy3, etc. or their analogs), Alexa series dyes and their derivatives (for example, including but not limited to AlexaFluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or their analogs) and protein dyes and their derivatives (for example, including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), peridinin-chlorophyll protein (preCP), etc.).

[0044] In some embodiments, the enzyme that catalyzes substrate color development includes but is not limited to horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphate glucose dehydrogenase.

[0045] In some embodiments, the chemiluminescent reagent includes, but is not limited to, luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridinium esters and their derivatives, dioxetanes and their derivatives, lophanine and its derivatives, and peroxyoxalates and their derivatives.

[0046] In some embodiments, the nanoparticle-based labels include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0047] In a fourth aspect, the present invention provides the use of the antibody or functional fragment thereof, or the nucleic acid, or the product, or the Uch-L1 time-resolved fluorescent immunochromatographic test strip in at least one of the following aspects:

[0048] (1) Application in the preparation and detection of Uch-L1 products;

[0049] (2) Application in the preparation of products for diagnosing neurological diseases;

[0050] (3) Application in detecting Uch-L1 antigen content;

[0051] In some embodiments, the product is any one of an antibody conjugate, a biological material, a drug, a detection reagent or kit, and a test strip.

[0052] In the application, the sample can be a sample from a living human or animal (including blood, excrement, oral and nasal secretions, etc.), or can be a sample not from a living human or animal, such as in vitro cultured cells or cell culture fluid.

[0053] The application can be achieved using enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay, radioimmunoassay, fluorescent immunoassay, immunochromatography, and the like.

[0054] In a fifth aspect, the present invention provides a Uch-L1 time-resolved fluorescent immunochromatographic test strip, comprising a sample pad, a conjugation pad, a reaction membrane, a water-absorbing pad and a base plate; the reaction membrane is provided with a detection line and a quality control line, and the sample pad, conjugation pad, reaction membrane and water-absorbing pad are sequentially pasted on the base plate; the conjugation pad or the detection line contains the antibody or its functional fragment.

[0055] In some embodiments, the conjugate pad is coated with time-resolved immunoblot-labeled Uch-L1-targeting antibody 9D5.

[0056] In some embodiments, the detection line is coated with antibody 5C3;

[0057] In some embodiments, the quality control line is coated with anti-DNP polyclonal antibody.

[0058] In some embodiments, the concentration of the antibody coated on the colloidal gold conjugate pad is 20-25 μg / mL.

[0059] In some embodiments, the concentration of the antibody coated on the test line is 1-2 mg / mL.

[0060] The Uch-L1 time-resolved fluorescence immunochromatographic test strip of the present invention is a lateral flow immunochromatographic test strip, typically in a narrow strip shape, typically 4-6 mm wide and 6-7 cm long. Preferably, the sample pad is made of cellulose; the colloidal gold binding pad is made of glass fiber; the reaction membrane is made of nitrocellulose membrane (NC membrane); and the absorbent pad is made of cellulose.

[0061] The present invention adopts the following steps to perform the detection operation of the immunochromatographic test strip:

[0062] (1) Sample preparation: Use a pipette to pipette 48 μL of the test solution, 1 μL of the TRFM-9-D5-B8 probe, and 1 μL of the TRFM-BSA-DNP probe into a centrifuge tube. Mix thoroughly by shaking and incubate at room temperature for 5 minutes.

[0063] (2) Sample addition: After incubation, pipette 35 μL of the mixture into the slot of the time-resolved fluorescence immunoassay test strip to start the reaction. After 15 minutes of reaction, place the test strip into the fluorescence analyzer for reading.

[0064] (3) Data analysis: By measuring the fluorescence intensity on the T line (test line) and the C line (control line), the T value is calculated and inserted into the pre-established standard curve to determine the concentration of human Uch-L1 protein in the test solution.

[0065] Detection principle Figure 1 When the test sample contains Uch-L1, the fluorescent probe (TRFM-9-D5-B8) specifically binds to it. These complexes are then captured by the capture antibody on the T line, forming a stable signal peak. Simultaneously, the probe containing DNP-BSA reacts with the anti-DNP antibody on the C line, ensuring the effectiveness of the control line. When reading on an immunofluorescence analyzer, two signal peaks are displayed, corresponding to the T line and the C line.

[0066] On the contrary, if the sample to be tested does not contain Uch-L1, the fluorescent probe will not undergo a specific binding reaction with Uch-L1, so only a fluorescence peak is observed on the C line. In this case, no fluorescent signal appears on the T line, indicating that the target protein is not detected in the sample.

[0067] The present invention has the following beneficial effects: the present invention provides an antibody that specifically recognizes ubiquitin carboxyl-terminal hydrolase L1 (Uch-L1), the antibody having the characteristics of high specificity and high affinity, and is suitable for the quantitative or qualitative detection of Uch-L1, and is particularly suitable for the development of a time-resolved fluorescence immunochromatographic kit, having the advantages of a low detection limit and high sensitivity, effectively reducing the detection cost of Uch-L1, shortening the detection time, and improving the detection efficiency, and being able to achieve the general standards required for rapid diagnosis, such as sensitivity, stability, economy, and no need for equipment. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0069] Figure 1 This is the detection principle diagram;

[0070] Figure 2 The results of Coomassie Brilliant Blue staining to identify Uch-L1 protein expression, where lane M: protein marker; lane 1: 0.1mM IPTG-induced empty cells; lane 2: uninduced empty cell supernatant; lane 3: uninduced empty cell inclusion bodies; lane 4: 0.1mM IPTG-induced whole cells; lane 5: 0.1mM IPTG-induced supernatant; lane 6: 0.1mM IPTG-induced inclusion bodies;

[0071] Figure 3 This is the ELISA test result diagram;

[0072] Figure 4 The figure shows the purification results of Uch-L1 monoclonal antibody identified by Coomassie Brilliant Blue;

[0073] Figure 5 This is the result diagram of Uch-L1 antibody pairing;

[0074] Figure 6 This is the standard curve of TRFIS;

[0075] Figure 7 The figure shows the detection results of TRFIS under 365nm ultraviolet light;

[0076] Figure 8 The figure shows the results of using TRFIS to detect 29 real serum samples. DETAILED DESCRIPTION

[0077] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0078] Example 1

[0079] 1. Design of Uch-L1 Antigen

[0080] The mRNA sequence of human Uch-L1 gene was obtained according to the Uch-L1 gene on NCBI (NM_004181.5), optimized for Escherichia coli codon preference to ensure efficient expression of the gene in Escherichia coli, and cloned into the pColdII prokaryotic expression vector (containing a 6×His tag).

[0081] 2. Preparation of Uch-L1 Protein

[0082] The Uch-L1 protein was expressed by E. coli expression system to obtain recombinant Uch-L1 protein, which was used as an antigen and verified by SDS-PAGE electrophoresis. The results are as follows: compared with the blank control group, lanes 4-6 showed obvious bands at 25KDa ( Figure 2 )

[0083] 3. Mouse Immunization

[0084] Four 6-8 week old Balb / c mice were used for this experiment, three of which were immunized and one served as the experimental control group. The control group mice received no treatment to ensure the accuracy of subsequent experimental results. For the first immunization, 50 μg of Uch-L1 protein (purity >95%) was emulsified with Freund's complete adjuvant and injected subcutaneously or intradermally at multiple sites in the nape of the neck (100 μL / mouse). Booster immunizations were performed on days 14 and 28 with Freund's incomplete adjuvant (same dosage as above).

[0085] 4. Serum titer detection of immune mice

[0086] Seven days after the third immunization, blood was collected from the tail vein of mice, and the immune serum titer was determined by indirect ELISA. 50 μg of Uch-L1 protein was dissolved in 10 mL of 0.05 M pH 9.6 phosphate buffer and coated onto a polystyrene 96-well plate at 100 μL / well at 4°C overnight. The plate was washed three times with PBST (0.02 M PBS containing 0.05% v / v Tween-20), blocked with 100 μL / well of 10 mM PBS containing 1% BSA blocking solution at 37°C for 2 h, and washed three times with PBST (0.02 M PBS containing 0.05% v / v Tween-20) before use. 96-well plates were blocked with 5% skim milk (100 μL / well) and incubated at 37°C for 1 hour. The plates were then washed three times with PBST (0.02 M PBS containing 0.05% v / v Tween-20). Serum samples were serially diluted in PBS (1:1000, 1:10,000, and 1:320,000). Unimmunized mouse serum (same dilution) was used as a negative control, and PBS was used as a blank control. 100 μL of serum or PBS was added to each well and incubated at 37°C for 1 hour. A 1:10,000-diluted horseradish peroxidase-conjugated goat anti-mouse IgG was added at 100 μL / well and incubated at 37°C for 30 minutes. The plates were washed as above. TMB (100 μL / well) was then added to each well for color development and incubated at 37°C in the dark for 10 minutes. The reaction was terminated by adding 50 μL / well of 2 M H₂SO₄.

[0087] 5. Cell Fusion

[0088] Recovery of SP2 / 0 cells: First, disinfect the surface of RPMI-1640 culture medium (containing 10% fetal bovine serum, FBS) with 75% ethanol and place it in a biosafety cabinet for ultraviolet sterilization for 30 minutes. Then take out the SP2 / 0 cells from the liquid nitrogen tank, quickly place them in a 37°C water bath for rapid thawing, and immediately disinfect the outer wall of the cryopreservation tube with 75% ethanol after thawing and transfer them to a biosafety cabinet. Slowly add the cell suspension to preheated 10mL complete culture medium (RPMI-1640 + 10% FBS), gently pipet and mix, then inoculate into a 100mm culture dish and place it in a CO2 incubator for culture. After 24 hours, replace with fresh culture medium to remove the cryoprotectant, and subculture when the cells adhere to the wall and grow to 90% confluence.

[0089] Extraction of peritoneal macrophages: 8-week-old non-immunized Balb / c female mice were selected 2-3 days in advance, and 2 mL of sterile 3% thioglycolate broth was injected intraperitoneally to stimulate the proliferation of peritoneal macrophages in the mice. 48 hours after the injection, the mice were euthanized by cervical dislocation, and the whole body was immediately disinfected with 75% ethanol and fixed in a clean bench. Under sterile conditions, pre-cooled PBS was used for peritoneal lavage. The collected lavage fluid was centrifuged at 1000 rpm for 10 minutes, and the supernatant was discarded. The precipitate was resuspended and washed twice with PBS (1000 rpm × 5 min). Finally, the cell density was adjusted to 2 × 10 using RPMI-1640 complete medium containing 10% heat-inactivated FBS. 5 cells / mL, 100 μL / well was inoculated into pre-treated 96-well plates (10 plates in total) and placed in a CO2 incubator until ready for use. All operations were performed in a biosafety cabinet.

[0090] Preparation of immune spleen cells: Balb / c mice (n=3) were selected on the 5th day after the sprint immunization. After blood was collected from the eyeballs, the mice were euthanized by cervical dislocation. The mice were immediately immersed in 75% ethanol for 5 minutes for disinfection. The spleen was aseptically removed in a biosafety cabinet and rinsed three times in culture medium to remove connective tissue. The spleen was cut into 1-2 mm pieces using sterile scissors. 3 Grind and filter tissue blocks through a 200-mesh cell sieve to prepare a single-cell suspension. Centrifuge at 1000 rpm for 10 minutes, discard the supernatant, add 3 mL of red blood cell lysis buffer, gently tap the tube to mix, and let stand for 5 minutes. Then, add culture medium to terminate the reaction and centrifuge again. Finally, determine the number and viability of spleen cells using a hemocytometer or automated cell counter to ensure cell viability is greater than 90%.

[0091] Harvest SP2 / 0 cells: Collect SP2 / 0 cells in the logarithmic growth phase from ten 100 mm culture dishes, combine them into a 50 mL sterile centrifuge tube, and centrifuge at 1000 rpm for 10 min (centrifuge radius 10 cm). Discard the supernatant and gently resuspend the cells in 10 mL of culture medium. Wash the cells by centrifugation twice under the same conditions to completely remove residual culture medium. After the final centrifugation, resuspend the cells in 5 mL of culture medium for cell counting.

[0092] Fusion: The pretreated spleen lymphocytes (1×10 8 cells) and SP2 / 0 cells (1×10 7cells) were mixed in a 50 mL centrifuge tube at a ratio of 10:1, and the cells were resuspended in 10 mL of serum-free RPMI-1640 medium (preheated at 37°C). After centrifugation at 1000 rpm for 10 minutes, the supernatant was discarded. After the cell pellet was balanced in a 37°C water bath for 5 minutes, preheated PEG1450 was slowly added dropwise using a syringe, strictly controlling the drop rate to 1 mL / min, and gently rotating the centrifuge tube to ensure uniform contact between the cells and the fusion agent. After the fusion reaction lasted for 1 minute, 15 mL of preheated serum-free RPMI-1640 medium was added dropwise and allowed to stand for 10 minutes to terminate the reaction. Finally, the fused cells were transferred to a 50 mL centrifuge tube and centrifuged at 1000 rpm for 5 minutes to collect the cells for subsequent HAT selective culture.

[0093] Dilution and plating: Add 50 mL of RPMI-1640 complete medium containing 10% FBS containing HAT selective medium to a centrifuge tube and gently pipette to mix. Distribute the cell suspension evenly into a 96-well plate pre-seeded with feeder cells, adding 100 μL of cell suspension to each well. Place the cell culture plate in a CO2 cell culture incubator.

[0094] 6. Screening of hybridoma cells secreting Uch-L1 monoclonal antibodies

[0095] The cell culture supernatant was screened by indirect ELISA, and the positive clone hybridoma cells with higher titers were selected for subcloning. The cells were cloned 2-3 times continuously by limiting dilution method until the positive rate of cells reached 100%. Figure 3 It can be seen that we have successfully obtained 9 hybridoma monoclonal cell lines specific for Uch-L1 protein.

[0096] 7. Preparation and Purification of Ascites

[0097] Hybridoma cell lines were cultured at 1×10 6 The antibody was injected into the peritoneal cavity of 8-10 week old female BALB / c mice pretreated with liquid paraffin at a dose of 100 mg / day. After 10-14 days of observation, ascites was extracted when the abdomen of the mice became distended. The monoclonal antibody was purified by affinity chromatography and its purity was determined by SDS-PAGE, which showed a purity of over 90%. The results were verified by SDS-PAGE electrophoresis as follows: Figure 4 .

[0098] Example 2 Antibody pairing

[0099] This experiment uses the simple sodium periodate method to prepare enzyme-labeled antibodies. The specific coupling steps are as follows:

[0100] (1) Dissolution: Weigh 5 mg of horseradish peroxidase and dissolve it in 1 mL of double-distilled water.

[0101] (2) Oxidation: Add 200 μL of freshly prepared 0.1 M NaIO4 solution to the horseradish peroxidase solution and react in the dark for 20 minutes.

[0102] (3) Dialysis: The oxidized solution was transferred to a dialysis bag with a molecular weight cutoff of 50 kDa and dialyzed against 1 mM NaAc buffer at 4 °C overnight to remove unreacted sodium periodate.

[0103] (4) Coupling: Adjust the pH value of the solution to 9.0-9.5 by adding 20 μL of 0.2 M CBS (pH = 9.5). Then, add 1 mL of 0.2 M CBS (pH = 9.5) containing 10 mg of monoclonal antibody IgG, and stir the solution at room temperature in the dark for 2 hours.

[0104] (5) Reduction: Add 100 μL of NaBH4 (4 mg / mL) solution to the above solution, mix thoroughly, and react at 4°C for 2 hours to stabilize the formed Schiff base bond.

[0105] (6) Dialysis: The reduced solution was re-loaded into a dialysis bag with a molecular weight cutoff of 10 KDa and dialyzed overnight at 4°C in a buffer solution containing 0.15 M PBS (pH = 7.4) to remove unbound components.

[0106] (7) Purification: Add an equal volume of saturated ammonium sulfate solution to the dialyzed solution, mix thoroughly, and let stand at 4°C for 1 hour to promote antibody precipitation.

[0107] (8) Washing: Centrifuge (3000 rpm, 30 minutes), wash the precipitate twice with half-saturated ammonium sulfate solution, and finally dissolve the precipitate in a small amount of PBS.

[0108] (9) Final dialysis and separation: The dissolved solution was placed back into a 50 kDa molecular weight cutoff dialysis bag and dialyzed overnight at 4°C against a buffer solution containing 0.01 M PBS (pH = 7.4). The supernatant was then collected by centrifugation (10,000 rpm, 30 minutes) to obtain the desired enzyme-labeled antibody conjugate.

[0109] Then use Elisa to pair antibodies, the results are as follows Figure 5 shown.

[0110] Example 2 Preparation of Immunochromatographic Test Strips

[0111] 1. Sample Pad and Conjugate Pad Pretreatment: Lay the glass cellulose membrane flat on a clean plastic tray and add the sample pad or conjugate pad pretreatment solution. Use a roller to evenly spread the solution across the membrane surface to ensure it is flat and fully absorbed. Then, dry the membrane at 37°C for 6 hours. After drying, cut the membrane into strips according to the following dimensions and seal them in a moisture-proof cabinet until ready for use: Sample Pad: 300 mm × 14 mm; Conjugate Pad: 300 mm × 11 mm.

[0112] 2. Construction of an independent C-line quality control system: In view of the fact that human metabolites may produce non-specific reactions with interfering substances in the sample (such as heterophilic antibodies or human anti-animal antibodies), thereby affecting the specificity of the immunochromatography control line (C-line), the present invention introduces an independent quality control system based on dinitrophenol (DNP) to enhance the stability and specificity of the C-line. As a small molecule compound (molecular weight of 184.11), DNP is not a human metabolite and therefore will not undergo non-specific binding with the above-mentioned interfering substances, thereby significantly improving the specificity of the C-line. In a specific implementation, an anti-DNP antibody is coated on the C-line, and a conjugate of DNP-BSA and TRFM is used as a C-line-specific probe. This strategy not only improves the reliability of the detection process, but also ensures the validity of the immunochromatography results.

[0113] 3. Nitrocellulose membrane processing: The capture antibody is coated on the test line (T line) of the nitrocellulose membrane, while the anti-DNP monoclonal antibody is coated on the C line. First, fix the nitrocellulose membrane to a PVC base plate, and then dilute the anti-DNP antibody and capture antibody to a concentration of 1 mg / mL and 2.0 mg / mL, respectively. Use a film spray gun to evenly distribute the two antibodies on the membrane at a speed of 1 μL / cm, so that the distance between the T line and the C line is maintained at 3mm. Finally, place it in a 37°C environment to dry overnight and prepare for test strip assembly.

[0114] 4. Assembling and Cutting the Test Strips: Attach the conjugate pad and sample pad to the front of the nitrocellulose membrane, overlapping each other by 2mm. Attach an absorbent pad to the back of the membrane. After assembly, use a programmable cutter to cut the test strips into 3mm-wide strips. Enclose the strips in a cartridge and store them in a moisture-proof cabinet until ready for use.

[0115] Example 3 Preparation of TRFM probe

[0116] 1. Microsphere pretreatment: 1 mL of activation buffer was added to a solution containing 50 μL of 1% (w / v) microspheres, and the solution was sonicated for 10 minutes to ensure that the microspheres were fully dispersed. Subsequently, the solution was centrifuged at 20,000 g for 10 minutes at 15°C.

[0117] 2. Wash and Resuspend: Discard the supernatant and add 1 mL of activation buffer to the pellet. Ultrasonicate for 10 minutes and then centrifuge for 10 minutes under the same conditions. Repeat this step twice to completely remove unbound components.

[0118] 3. Addition of activator: Add 1 mL of activation buffer to the above microsphere precipitate, sonicate until uniformly dispersed, then add 3.5 μL of EDC and 33 μL of NHS solution in sequence. Vortex mix immediately after each addition to ensure sufficient contact between the reactants.

[0119] 4. Microsphere activation: Place the microsphere suspension containing the activator in a four-dimensional rotator and rotate it in the dark at 37°C for 30 minutes to complete the activation of the microsphere surface.

[0120] 5. Post-activation Wash: After activation, centrifuge at 20,000 g for 10 minutes at 15°C and discard the supernatant. Add 1.5 mL of activation buffer, sonicate to mix, and centrifuge again for 10 minutes under the same conditions. Discard the supernatant. Repeat this step once to completely remove any unreacted activator.

[0121] 6. Antibody Conjugation Preparation:

[0122] (1) Washing: After centrifugation, the washing solution was completely removed, 750 μL of coupling buffer was added to the microsphere pellet, and the microspheres were resuspended by ultrasonic treatment.

[0123] (2) Prepare the coupling solution separately: dissolve 50 μg of S100B antibody in 250 μL of coupling buffer and vortex to mix.

[0124] 7. Antibody conjugation:

[0125] (1) Quickly add the prepared coupling solution to the microsphere suspension and vortex to mix immediately.

[0126] (2) Place the mixture in a four-dimensional rotator and couple at 37°C in the dark for 2 hours.

[0127] 8. Block unreacted sites: Add 500 μL of microsphere blocking solution to the above mixture, vortex thoroughly to mix, and place it on the four-dimensional rotator again. Block in the dark at 37°C for 1 hour to prevent nonspecific binding.

[0128] 9. Post-Blocking Wash: Centrifuge at 15°C, 20,000 g for 10 minutes and discard the supernatant. Add 1.5 mL of Microsphere Wash Buffer, vortex to mix, and ultrasonically disperse. Centrifuge under the same conditions for 10 minutes and discard the supernatant. Repeat this step once to ensure removal of unbound antibody and other impurities.

[0129] 10. Storage: Add 500 μL of microsphere storage solution to the microsphere pellet, resuspend and mix the microspheres by ultrasonic treatment to obtain the detection probe for TRFM labeled with the detection antibody (hereinafter referred to as the detection probe). Store in a refrigerator at 4°C in the dark for subsequent use.

[0130] Example 4

[0131] The present invention adopts the following steps to perform the detection operation of the immunochromatographic test strip:

[0132] (1) Sample preparation: Use a pipette to pipette 48 μL of the test solution, 1 μL of the TRFM-9-D5-B8 probe, and 1 μL of the TRFM-BSA-DNP probe into a centrifuge tube. Mix thoroughly by shaking and incubate at room temperature for 5 minutes.

[0133] (2) Sample addition: After incubation, pipette 35 μL of the mixture into the slot of the time-resolved fluorescence immunoassay test strip to start the reaction. After 15 minutes of reaction, place the test strip into the fluorescence analyzer for reading.

[0134] (3) Data analysis: By measuring the fluorescence intensity on the T line (test line) and the C line (control line), the T value is calculated and inserted into the pre-established standard curve to determine the concentration of human Uch-L1 protein in the test solution.

[0135] When the test sample contains Uch-L1, the fluorescent probe (TRFM-9-D5-B8) specifically binds to it. These complexes are then captured by the capture antibody on the T line, forming a stable signal peak. Simultaneously, the probe containing DNP-BSA reacts with the anti-DNP antibody on the C line, ensuring the effectiveness of the control line. When reading on an immunofluorescence analyzer, two signal peaks are displayed, corresponding to the T line and the C line.

[0136] On the contrary, if the sample to be tested does not contain Uch-L1, the fluorescent probe will not undergo a specific binding reaction with Uch-L1, so only a fluorescence peak is observed on the C line. In this case, no fluorescent signal appears on the T line, indicating that the target protein is not detected in the sample.

[0137] The present invention constructs a standard curve for quantitative detection of Uch-L1 through a series of concentration gradient experiments ( Figure 6 and Figure 7), three independent tests were performed at each concentration point for different concentrations of 50.000ng / mL, 25.000ng / mL, 12.500ng / mL, 6.250ng / mL, 3.125ng / mL, 1.563ng / mL, 0.781ng / mL, 0.390ng / mL and 0.195ng / mL, and the fluorescence intensity was recorded. The test data showed that in the range of 0.195-12.500ng / mL, the fluorescence signal showed a good linear relationship with the antigen concentration, and the linear equation was Y=73222X+477260, R 2 =0.9844, and the detection limit was 0.156 ng / mL.

[0138] The present invention collected 29 clinical serum samples, including 15 serum samples from patients with traumatic brain injury and 14 serum samples from healthy donors, to evaluate the clinical diagnostic value of the TRFIS established by the present invention. All samples were tested three times independently using TRFIS, and the fluorescence signal intensity was displayed as a bar graph ( Figure 8 ) display. Boxplot analysis showed that the fluorescence signal values of the TBI patient group were significantly higher than those of the healthy control group (p<0.01), but the detection values of individual patients did not differ significantly from those of healthy controls. Further receiver operating characteristic curve (ROC curve) results demonstrated that the diagnostic efficacy of this detection method was excellent, with an area under the curve (AUC) of 0.9286. These results confirm that the TRFIS established in this project has extremely high detection sensitivity and specificity, and shows good application prospects in rapid on-site testing.

[0139] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A functional fragment, characterized in that The functional fragment includes complementary determining regions Ab1 and Ab2, wherein Ab1 includes CDR-VL1, CDR-VL2, CDR-VL3, CDR-VH1, CDR-VH2, and CDR-VH3, wherein the amino acid sequence of CDR-VL1 is shown in SEQ ID NO.1, the amino acid sequence of CDR-VL2 is shown in SEQ ID NO.2, the amino acid sequence of CDR-VL3 is shown in SEQ ID NO.3, the amino acid sequence of CDR-VH1 is shown in SEQ ID NO.4, the amino acid sequence of CDR-VH2 is shown in SEQ ID NO.5, and the amino acid sequence of CDR-VH3 is shown in SEQ ID NO.6; the Ab2 includes CDR-VL1, CDR-VL2, CDR-VL3, CDR-VH1, CDR-VH2, and CDR-VH3, wherein the amino acid sequence of CDR-VL1 is shown in SEQ ID NO. The amino acid sequence of the CDR-VH1 is shown in SEQ ID NO.10, the amino acid sequence of the CDR-VH2 is shown in SEQ ID NO.11, and the amino acid sequence of the CDR-VH3 is shown in SEQ ID NO.

12.

2. The functional fragment according to claim 1, characterized in that The functional fragment is selected from any one of F(ab')2, Fab', Fab, Fv, Fd, dAb, and scFv of the antibody.

3. An antibody against ubiquitin carboxyl-terminal hydrolase L1, characterized in that: Comprising the functional fragment according to claim 1 or 2.

4. The ubiquitin carboxyl-terminal hydrolase L1 antibody according to claim 3, characterized in that The antibody further comprises a constant region; the constant region is selected from any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD.

5. The ubiquitin carboxyl-terminal hydrolase L1 antibody according to claim 4, characterized in that: The species origin of the constant region is cow, horse, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose or human.

6. Use of the functional fragment according to claim 1 or 2 and / or the ubiquitin carboxyl-terminal hydrolase L1 antibody according to any one of claims 3 to 5 in the preparation of immunoassay products such as colloidal gold, fluorescence chromatography, ELISA, and chemiluminescence.

7. The use according to claim 6, characterized in that The application includes at least one of the following applications: (1) Application in the preparation and detection of Uch-L1 products; (2) Application in the preparation of products for diagnosing neurological diseases; (3) Application in detecting Uch-L1 antigen content.

8. A product, characterized in that Comprising the functional fragment according to claim 1 or 2 and / or the ubiquitin carboxyl terminal hydrolase L1 antibody according to any one of claims 3 to 5.

9. The product according to claim 8, characterized in that The product is any one of an antibody conjugate, a biological material, a drug, a detection reagent or a kit, and a test strip.

10. A Uch-L1 time-resolved fluorescence immunochromatographic test strip, characterized in that: The invention comprises a sample pad, a binding pad, a reaction membrane, a water-absorbing pad and a bottom plate; the reaction membrane is provided with a detection line and a quality control line, and the sample pad, binding pad, reaction membrane and water-absorbing pad are sequentially adhered to the bottom plate; the colloidal gold binding pad or the detection line contains the functional fragment according to claim 1 or 2 and / or the ubiquitin carboxyl terminal hydrolase L1 antibody according to any one of claims 3 to 5.

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