Brain-specific protein product 9.5 determination kit
By developing a single-domain antibody against brain-specific protein product 9.5 combined with magnetic microparticles and chemiluminescence detection technology, a reagent kit for the assay of brain-specific protein product 9.5 was prepared. This kit achieves highly sensitive, highly specific, and highly accurate quantitative detection of brain-specific protein product 9.5 in human serum, overcoming the shortcomings of existing technologies and meeting the needs of early diagnosis and dynamic monitoring.
Patent Information
- Application Number
- CN202511791954.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-01
- Publication Date
- 2026-03-20
AI Technical Summary
There is currently no chemiluminescent detection kit for brain-specific protein product 9.5 based on single-domain antibodies, which cannot achieve highly sensitive, highly specific, and highly accurate quantitative detection of brain-specific protein product 9.5 in human serum.
A single-domain antibody against brain-specific protein product 9.5 was developed and combined with magnetic microparticles and chemiluminescence detection technology to prepare a kit for the assay of brain-specific protein product 9.5. By using this antibody to couple with magnetic microparticles and combining it with an acridinium ester-labeled detection antibody, high sensitivity and high specificity of detection can be achieved.
It exhibits good linearity in the range of 25-25000 pg/mL, with good repeatability and high accuracy, meeting the needs of early diagnosis and dynamic monitoring, avoiding false positives, simplifying operation procedures, and improving detection efficiency.
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Abstract
Description
Technical Field
[0001] This application relates to the field of biomedical detection technology, specifically to a reagent kit for the determination of brain-specific protein product 9.5. Background Technology
[0002] Brain-specific protein product 9.5 (PGP9.5) (also known as ubiquitin C-terminal hydrolase L1, UCH-L1) is a deubiquitinating enzyme and a nervous system-specific protein. As one of the most abundant proteins in the brain, PGP9.5 accounts for up to 2% of brain soluble proteins. PGP9.5 regulates cell proliferation, differentiation, and apoptosis via the ubiquitin-related pathway. Studies have shown that PGP9.5 is involved in maintaining axonal stability, neurogenesis in embryonic brain tissue, and stress-mediated apoptosis, thereby affecting the nervous system.
[0003] In traumatic brain injury (TBI), PGP9.5 crosses the blood-brain barrier and enters the bloodstream within one hour, leading to a significant increase in serum PGP9.5 levels. This is of great significance for the early diagnosis, differential diagnosis, and prognosis of TBI, and is mainly used clinically as an adjunct to the diagnosis of brain injury. Chemiluminescence immunoassay (CLIA) combines highly sensitive chemiluminescence assays with highly specific immunoreactions for the detection and analysis of various antigens, haptens, antibodies, hormones, enzymes, fatty acids, vitamins, and drugs. In recent years, single-domain antibodies have shown great potential in the field of immunoassay due to their small molecular weight, high stability, ease of genetic engineering, and conjugation. However, there are currently no reports on chemiluminescence detection kits for brain-specific protein products PGP9.5 based on single-domain antibodies.
[0004] Therefore, there is an urgent need in this field to develop a reagent kit for the assay of brain-specific protein product 9.5, which can achieve highly sensitive, highly specific and highly accurate quantitative detection of brain-specific protein product 9.5 in human serum, and has important clinical application prospects. Summary of the Invention
[0005] In view of this, this application provides a single-domain antibody against brain-specific protein product 9.5 (PGP9.5) and a kit for the assay of brain-specific protein product 9.5 prepared based on the antibody.
[0006] In a first aspect, this application provides a single-domain antibody against brain-specific protein product 9.5, comprising VHH, wherein the VHH comprises CDR1, CDR2 and CDR3 in the amino acid sequence shown in SEQ ID NO:1.
[0007] Optionally, CDR1 to CDR3 are determined according to the Kabat numbering system, AbM numbering system, IMGT numbering system, Chothia numbering system, or Contact numbering system.
[0008] Optionally, the amino acid sequence of CDR1 is shown in SEQ ID NO:2, the amino acid sequence of CDR2 is shown in SEQ ID NO:3, and the amino acid sequence of CDR3 is shown in SEQ ID NO:4.
[0009] Optionally, the VHH comprises an amino acid sequence with more than 90% homology obtained by substituting, deleting, and / or adding one or more amino acids and / or terminal modification of any one or more amino acids in the amino acid sequence shown in SEQ ID NO:1.
[0010] Optionally, the VHH comprises an amino acid sequence as shown in SEQ ID NO:1.
[0011] Secondly, this application provides the use of the above-mentioned anti-brain-specific protein product 9.5 single-domain antibody in the preparation of a detection kit.
[0012] Optionally, the kit is a chemiluminescence assay kit for brain-specific protein products 9.5.
[0013] Thirdly, this application provides a brain-specific protein product 9.5 assay kit, which includes the aforementioned anti-brain-specific protein product 9.5 single-domain antibody.
[0014] Optionally, the kit further includes magnetic microparticles; wherein the magnetic microparticles are conjugated to the anti-brain-specific protein product 9.5 single-domain antibody.
[0015] Optionally, the kit may also include an acridinium-labeled antibody against brain-specific protein product 9.5.
[0016] In summary, this application includes at least one of the following beneficial technical effects: 1. The single-domain antibody screened in this application (such as Lib218-0-16) can specifically recognize brain-specific protein product 9.5 and has no cross-reactivity with ubiquitin C-terminal hydrolase L3 (UCH-L3) and ubiquitin C-terminal hydrolase L5 (UCH-L5), which can effectively avoid false positives; 2. Combining magnetic microparticle enrichment with chemiluminescence detection technology, this kit exhibits good linearity in the range of 25-25000 pg / mL, with good repeatability and high accuracy, meeting the needs of early diagnosis and dynamic monitoring. Detailed Implementation
[0017] In this disclosure, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the laboratory procedures for cell culture, molecular genetics, nucleic acid chemistry, and immunology used herein are all standard procedures widely used in their respective fields. To better understand this disclosure, definitions and explanations of relevant terms are provided below.
[0018] The term “antibody” is used in the broadest sense in this article to refer to a protein that contains an antigen-binding site, encompassing natural and artificial antibodies of various structures, including but not limited to polyclonal antibodies, monoclonal antibodies, single-chain antibodies, intact antibodies, and antibody fragments.
[0019] As used herein, “sdAb” refers to a single monomeric variable antibody domain capable of binding to an antigen (e.g., a single-domain antibody that binds to brain-specific protein product 9.5). Single-domain antibodies include the VHH domain described herein. Examples of single-domain antibodies include, but are not limited to, antibodies naturally lacking the light chain, such as antibodies from camelid species (e.g., camels), single-domain antibodies derived from conventional 4-chain antibodies, engineered antibodies, and single-domain antibodies other than single-domain scaffolds derived from antibodies. Single-domain antibodies can be derived from any species, including but not limited to mice, humans, camels, goats, rabbits, and cattle. For example, single-domain antibodies can be derived from antibodies produced in camelid species (e.g., camels, dromedary camels, alpacas, and guanacos), as described herein. Other species besides camelids may also produce heavy-chain antibodies naturally lacking the light chain; VHHs derived from these other species are also within the scope of this disclosure. The single-domain antibody (e.g., VHH) provided in this article can have the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
[0020] The term "complementarity-determining region" or "CDR region" or "CDR" refers to a region within the variable domain of an antibody that is highly variable in sequence and forms a structurally defined loop ("hypervariant loop") and / or contains antigen contact residues ("antigen contact sites"). CDRs are primarily responsible for binding to antigen epitopes and are sequentially numbered from the N-terminus as CDR1, CDR2, and CDR3. In a given variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any of a number of known antibody CDR assignment systems or combinations thereof, including, for example: Chothia (Chothia et al. (1989) Nature 342: 877-883, Al-Lazikani et al., “Standard conformations for the canonical structures of immunoglobulins”, Journal of Molecular Biology, 273, 927-948 (1997)) based on antibody sequence variability; Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, US Department of Health and Human Services, National Institutes of Health (1987)) based on antibody sequence variability; AbM (University of Bath); Contact (University College London); and the International ImMunoGeneTics database (IMGT). (http: / / imgt.cines.fr / ), and the North CDR definition based on affinity propagation clustering utilizing a large number of crystal structures. Unless otherwise stated, in this invention, the term "CDR" or "CDR sequence" encompasses a CDR sequence determined in any of the foregoing methods. A CDR may also be determined based on having the same AbM numbering position as a reference CDR sequence (e.g., any of the CDR sequences in the examples of this invention). In one embodiment, the CDR of the single-domain antibody of this invention is positioned according to the AbM numbering scheme. Unless otherwise stated, in this invention, when referring to the positions of residues (including heavy chain variable region residues) in the antibody variable region and CDR, it refers to the numbering position according to the AbM numbering system.
[0021] The present application is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the application. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions, conditions described in a laboratory manual, or conditions recommended by the manufacturer.
[0022] Example 1: Screening of single-domain antibodies against brain-specific protein product 9.5
[0023] A human Fc tag linked to the C-terminus of the full-length PGP9.5 protein gene was used as an antigen to immunize alpacas, yielding high-titer antiserum. After animal immunization, 50 mL of fresh alpaca blood was collected, and peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Paque density gradient separation medium. RNA was extracted, reverse transcribed, amplified using universal primers, cloned into phage particles, transformed into TG1 strain, and a phage library was established for screening monoclonal antibodies. A single-domain antibody with high specificity and affinity was obtained and named Lib218-0-16.
[0024] Analysis of the Lib218-0-16 sequence revealed that the amino acid sequence of VHH of the single-domain antibody Lib218-0-16 is shown in SEQ ID NO:1, and the amino acid sequences of CDR1, CDR2 and CDR3 as defined by Kabat are shown in SEQ ID NO:2-4, respectively.
[0025] Example 2: Detection Method of Brain-Specific Protein Products 9.5 Based on Chemiluminescence
[0026] Main reagents: magnetic microparticles (Thermo Fisher, 1.0 μm); EDC (Sigma, catalog number E7750); acridinium ester (purchased from Maclean); capture antibody: single-domain antibody Lib218-0-16 prepared in Example 1 of this invention; detection antibody: anti-brain-specific protein product 9.5 antibody (antibody 2 derived from CN114563570B); the remaining reagents were conventional products obtained through commercial purchase.
[0027] 2.1 Magnetic microparticle-labeled anti-brain-specific protein product 9.5 single-domain antibody
[0028] Take 20 mg of magnetic microparticle solution and let it settle for 10 minutes under the action of a magnetic field. Discard the supernatant. Wash the settled magnetic microspheres three times with 2 mL of MES buffer (pH 6.0) with an activation buffer concentration of 0.05 M. Resuspend in 1 mL of MES buffer and add EDC to a final concentration of 7.5 mM. React at room temperature for 30 min. Add 1 mg of the nanobody Lib218-0-16 (concentration 1.5 mg / mL) prepared in Example 1 of this invention to the activated magnetic microparticles and react gently at 4 °C for 6.5 h. After labeling, block excess sites with glycine at a final concentration of 25 mM and react at 22 °C for 30 min. After washing three times, resuspend in 2 mL of magnetic microparticle preservation solution (TBST buffer containing 1% BSA and 0.1% ProClin300) and use directly.
[0029] 2.2 Acridin ester-labeled commercial anti-brain-specific protein product 9.5 antibody
[0030] Weigh 1.6 mg of acridinium ester and dissolve it in DMSO to prepare a 6.5 mM stock solution. Under light-protected conditions, mix 5 μl of detection antibody (1 mg / ml) with 195 μl of acridinium ester stock solution and react at room temperature for 1 h. Then add 100 μl of 10 g / L lysine and continue the reaction for 10 min to terminate the labeling reaction. Mix the labeled solution with a Sephadex G-25 desalting column and wash with PBS. Detect the protein using a chromatograph at 280 nm. When a clear protein peak appears on the chromatograph, collect the eluent. Add 1% BSA and 0.1% ProClin300 to the eluent, mix well, and store below -15°C. For detection, dilute 500 times with diluent (TBST buffer containing 1% BSA and 0.1% ProClin300) before use.
[0031] 2.3 Reagent kit detection
[0032] Add 50 μL of the magnetic microparticle-labeled single-domain antibody prepared in Example 2.1 to a reaction vessel; add 10 μL of sample, calibrator, or quality control sample to a reaction vessel; add 50 μL of the acridinium ester-labeled detection antibody prepared in Example 2.2; incubate, then wash; add substrate solution A (4% H2O2, pH adjusted to 1.05 with HCl) and substrate solution B (0.35 mol / L NaOH + 2% Triton X-100, pH=13.29); measure the luminescence value, and the system automatically calculates the PGP9.5 content in the sample.
[0033] Example 3: Detection results of brain-specific protein products 9.5 based on chemiluminescence method
[0034] 3.1 Specificity
[0035] The kit obtained in Example 2 was used to detect samples with added interfering agents. The blank sample used a negative matrix (serum from healthy individuals). The interfering agents were 5000 pg / ml ubiquitin C-terminal hydrolase L3 (UCH-L3) and 5000 pg / ml ubiquitin C-terminal hydrolase L5 (UCH-L5). The experimental results showed that the presence of interfering agents had no effect on the detection of the target molecule PGP9.5, and there was no cross-reaction.
[0036] 3.2 Linear Range
[0037] The self-made PGP9.5 antigen standard was serially diluted using a standard dilution buffer formulation containing TBST buffer with a final concentration of 2% BSA, 5% trehalose, and 0.1% Proclin 300. The test was performed using the kit obtained in Example 2. The test results are shown in Table 1. Within the linear range of 25-25000 pg / mL, the correlation coefficient r = 1.0000.
[0038] Table 1
[0039] 3.3 Accuracy
[0040] Self-made PGP9.5 antigen standard diluent (concentrations of 300 pg / mL and 10000 pg / mL, respectively) was added to the negative matrix (serum of healthy individuals), and the kit obtained in Example 2 was used for detection. The detection results are shown in Table 2, with relative deviations within ±10.0%.
[0041] Table 2
[0042] 3.4 Repeatability
[0043] The same concentration of standard was tested 10 times, and the results are shown in Table 3. The coefficient of variation (CV) was no greater than 10.0%.
[0044] Table 3
[0045] In summary, the brain-specific protein product 9.5 detection kit prepared based on this antibody can be used for rapid detection of the content of brain-specific protein product 9.5 in samples, exhibiting high sensitivity, strong specificity, high accuracy, and good repeatability. By combining it with a fully automated chemiluminescence analyzer, the operation steps are greatly simplified, the detection speed and throughput are increased, and the detection efficiency is improved, while avoiding errors caused by human operation.
[0046] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.
[0047] sequence list
[0048] SEQ ID NO:1 Lib218-0-16 VHH
[0049] QVQLVESGGGLVQAGGSLRLSCAASGSTNSINTIGWYRQTPGKRELVASISSSGRTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDMAVYYCNAKGGVSGFTIAAMINDYWGQGTQVTVSS
[0050] SEQ ID NO:2 Lib218-0-16 CDR1
[0051] INTIG
[0052] SEQ ID NO:3 Lib218-0-16 CDR2
[0053] SISSSGRTNYADSVKG
[0054] SEQ ID NO:4 Lib218-0-16 CDR3
[0055] KGGVSGFTIAAMINDY
Claims
1. A single-domain antibody against brain-specific protein product 9.5, comprising VHH, characterized in that, The VHH contains CDR1, CDR2, and CDR3 in the amino acid sequence shown in SEQ ID NO:
1.
2. The single-domain antibody against brain-specific protein product 9.5 according to claim 1, characterized in that, The CDR1 to CDR3 are determined according to the Kabat numbering system, AbM numbering system, IMGT numbering system, Chothia numbering system, or Contact numbering system.
3. The single-domain antibody against brain-specific protein product 9.5 according to claim 2, characterized in that, The amino acid sequence of CDR1 is shown in SEQ ID NO:2, the amino acid sequence of CDR2 is shown in SEQ ID NO:3, and the amino acid sequence of CDR3 is shown in SEQ ID NO:
4.
4. The single-domain antibody against brain-specific protein product 9.5 according to any one of claims 1 to 3, characterized in that, The VHH comprises an amino acid sequence with more than 90% homology obtained by substituting, deleting, and / or adding one or more amino acids and / or modifying the end of any one or more amino acids in the amino acid sequence shown in SEQ ID NO:
1.
5. The single-domain antibody against brain-specific protein product 9.5 according to any one of claims 1 to 3, characterized in that, The VHH contains an amino acid sequence as shown in SEQ ID NO:
1.
6. Use of the anti-brain-specific protein product 9.5 single-domain antibody as described in any one of claims 1 to 5 in the preparation of a detection kit.
7. A reagent kit for detecting brain-specific protein product 9.5, characterized in that, brain-specific protein products The 9.5 assay kit comprises the single-domain antibody against the brain-specific protein product 9.5 as described in any one of claims 1-5.
8. The reagent kit according to claim 7, characterized in that, The kit also includes magnetic microparticles; wherein the magnetic microparticles are conjugated to the anti-brain-specific protein product 9.5 single-domain antibody.
9. The reagent kit according to claim 8, characterized in that, The kit also includes an acridinium ester-labeled antibody against brain-specific protein product 9.5.