Nanobody for detecting cyclosporin a and application thereof

Abstract

This invention provides a nanobody for detecting cyclosporine A and its application, belonging to the field of medical detection technology. The nanobody includes complementarity-determining regions CDR1, CDR2, and CDR3; the amino acid sequence of CDR1 is shown in SEQ ID NO:1; the amino acid sequence of CDR2 is shown in SEQ ID NO:2; and the amino acid sequence of CDR3 is shown in SEQ ID NO:3. This nanobody exhibits high precision, high sensitivity, and good specificity when used to detect cyclosporine A, providing accurate and reliable detection results.

Description

Technical Field

[0001] This invention belongs to the field of medical testing technology, and in particular relates to a nanobody for detecting cyclosporine A and its application. Background Technology

[0002] Cyclosporine A (CsA) is a potent calcineurin inhibitor widely used for immunosuppressive therapy after organ transplantation and for the treatment of certain autoimmune diseases. However, it has a narrow therapeutic window, significant inter-individual pharmacokinetic variability, and its blood concentration is closely related to efficacy and toxicity; therefore, routine therapeutic drug monitoring (TDM) is essential.

[0003] Currently, the main methods for detecting cyclosporine A blood concentrations include high-performance liquid chromatography (HPLC), liquid chromatography-tandem mass spectrometry (LC-MS / MS), and immunoassay. Immunoassay is the most widely used method in clinical laboratories due to its simplicity, speed, low cost, and ease of automation. However, traditional cyclosporine A immunoassays often employ competitive methods, which have inherent drawbacks such as limited sensitivity, susceptibility to cross-reactivity with metabolites, and the need for pre-centrifugation treatment.

[0004] Sandwich immunoassays are considered a superior choice due to their high sensitivity and specificity, but they are generally suitable for large molecular antigens with multiple non-overlapping epitopes. Cyclosporine A, as a small molecule drug (molecular weight approximately 1202 Da), is difficult to provide two independent epitopes for simultaneous recognition by two antibodies, thus making its sandwich assay a persistent technical challenge.

[0005] Nanobody (also known as VHH) is a variable region fragment derived from heavy chain antibodies of camel species. It has unique advantages such as small molecular weight (about 15kDa), high stability, good solubility, and ease of genetic engineering modification.

[0006] Therefore, providing a nanobody for detecting cyclosporine A and applying it to multiple detection platforms for the detection of cyclosporine A is of significant practical importance. Summary of the Invention

[0007] Therefore, the purpose of this invention is to provide a nanobody for detecting cyclosporine A and its application, wherein the nanobody has high precision, high sensitivity and good specificity in detecting cyclosporine A, and the detection results are accurate and reliable.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0009] This invention provides a nanobody for detecting cyclosporine A, the nanobody comprising complementarity-determining regions CDR1, CDR2 and CDR3;

[0010] The amino acid sequence of CDR1 is shown in SEQ ID NO:1;

[0011] The amino acid sequence of CDR2 is shown in SEQ ID NO:2;

[0012] The amino acid sequence of CDR3 is shown in SEQ ID NO:3.

[0013] Preferably, the nanobody further includes framework regions FR1, FR2, FR3 and FR4;

[0014] The amino acid sequence of FR1 is shown in SEQ ID NO:4;

[0015] The amino acid sequence of FR2 is shown in SEQ ID NO:5;

[0016] The amino acid sequence of FR3 is shown in SEQ ID NO:6.

[0017] Preferably, the amino acid sequence of the nanobody is as shown in SEQ ID NO:8.

[0018] This invention provides the application of nanobody in the preparation of reagent for detecting cyclosporine A, wherein the nanobody is the aforementioned nanobody.

[0019] The present invention provides a test strip or kit for detecting the concentration of cyclosporine A, wherein the test strip or kit comprises the above-mentioned nanobody.

[0020] Preferably, the test strip or kit further includes a solid support coated with a first capture antibody that specifically binds to cyclosporine A; the nanobody specifically recognizes and binds to the cyclosporine A-first capture antibody complex.

[0021] This invention provides a method for detecting cyclosporine A, the method being used for non-therapeutic or diagnostic purposes, comprising the following steps:

[0022] (1) A solid support is provided, wherein a first capture antibody is immobilized on the solid support, the first capture antibody specifically binding to cyclosporine A;

[0023] (2) After the sample to be tested is brought into contact with the first capture antibody, the above-mentioned nanobody is added for detection.

[0024] Preferably, the detection platform adapted to the method is selected from enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay, electrochemiluminescence immunoassay, fluorescence immunoassay, or immunochromatographic strip method.

[0025] Preferably, the method further includes preprocessing the sample to be tested;

[0026] When using enzyme-linked immunosorbent assay (ELISA) for detection, the pretreatment uses sample release agent A, which comprises the following components: a final concentration of 40-60 mmol / L Tris-HCl, a mass-volume fraction of 0.5%-1.5% NaCl, a mass-volume fraction of 0.1%-0.5% saponin, and a mass-volume fraction of 0.5%-2% sodium salicylate.

[0027] The pH value of the sample release agent A is 7.0~8.0;

[0028] When using the immunochromatographic test strip method for detection, the pretreatment uses sample release agent B, which comprises the following components in the following amounts: a final concentration of 40-60 mM Tris-HCl, 0.5%-1.5% NaCl (w / v), 0.1%-0.5% saponin (w / v), 0.5%-2% sodium salicylate (w / v), and 0.05%-0.15% ethyl phenyl polyethylene glycol (w / v).

[0029] The pH value of the sample diluent is 7.0~8.0.

[0030] Preferably, the solid support is selected from perforated plates, magnetic beads, nitrocellulose membranes, glass fiber membranes, or microfluidic chips;

[0031] When the nanobody is used for detection, the nanobody is coupled to a detection marker; the detection marker is selected from enzymes, chemiluminescent substances, fluorescent substances, biotin, radioactive isotopes, or colloidal gold.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] This invention provides a nanobody for detecting cyclosporine A. The nanobody has unique CDR1, CDR2, and CDR3 region sequences, and has the ability to specifically recognize and bind to cyclosporine A. When used to detect cyclosporine A, it has high precision and high sensitivity, and shows extremely low cross-reactivity rates (all <0.1%) with 21 other common clinical drugs, indicating that the nanobody has good specificity for detecting cyclosporine A, and the results are accurate and reliable.

[0034] This invention also provides a method for detecting cyclosporine A. Compared with conventional cyclosporine A detection kits that use a competitive method to detect cyclosporine A, the method using the nanobody of this invention to detect cyclosporine A using a sandwich method has a better signal-to-noise ratio, better precision and sensitivity, higher specificity, and more accurate and reliable detection results. Attached Figure Description

[0035] Figure 1 Calibration curve for the detection of cyclosporine A in blood using the magnetic microparticle chemiluminescence sandwich method;

[0036] Figure 2 Calibration curve for the detection of cyclosporine A in blood by magnetic microparticle chemiluminescence competitive assay;

[0037] Figure 3 The correlation between sandwich method and competitive method mass spectrometry for detecting cyclosporine A concentration in blood. Detailed Implementation

[0038] Unless otherwise defined, all technical terms used herein have the same meaning as understood by one of ordinary skill in the art.

[0039] This invention provides a nanobody for detecting cyclosporine A, the nanobody comprising complementarity-determining regions CDR1, CDR2 and CDR3;

[0040] The amino acid sequence of CDR1 is as shown in SEQ ID NO:1, or an amino acid sequence having more than 80% identity with the amino acid sequence shown in SEQ ID NO:1.

[0041] The amino acid sequence of CDR2 is as shown in SEQ ID NO:2, or an amino acid sequence having more than 80% identity with the amino acid sequence shown in SEQ ID NO:2.

[0042] The amino acid sequence of CDR3 is as shown in SEQ ID NO:3, or an amino acid sequence having more than 80% identity with the amino acid sequence shown in SEQ ID NO:3.

[0043] In this invention, the CDR (complementarity-determining region) of the nanobody is also called the HCDR (heavy chain complementarity-determining region), that is, CDR1, CDR2 and CDR3 can be called HCDR1, HCDR2 and HCDR3 respectively.

[0044] In this invention, the nanobody comprises an amino acid sequence obtained by modifying, deleting, substituting, and / or adding amino acid sequences shown in SEQ ID NO:1-3, wherein the amino acid sequence has the ability to specifically recognize and bind cyclosporine A. Further, the amino acid sequence of the nanobody differs from the amino acid sequence shown in SEQ ID NO:1-3 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 amino acids.

[0045] In this invention, SEQ ID NO:1 is: EFTFDDSD. SEQ ID NO:2 is: ISSDGST. SEQ ID NO:3 is: AAGAPNNNPILCGAPSGY.

[0046] In this invention, the nanobody further includes framework regions FR1, FR2, FR3, and FR4; the amino acid sequence of FR1 is preferably as shown in SEQ ID NO:4, or an amino acid sequence having more than 80% identity with the amino acid sequence shown in SEQ ID NO:4; the amino acid sequence of FR2 is preferably as shown in SEQ ID NO:5, or an amino acid sequence having more than 80% identity with the amino acid sequence shown in SEQ ID NO:5; the amino acid sequence of FR3 is preferably as shown in SEQ ID NO:6, or an amino acid sequence having more than 80% identity with the amino acid sequence shown in SEQ ID NO:6.

[0047] In this invention, the amino acid sequence of the nanobody comprises an amino acid sequence obtained by modifying, deleting, substituting, and / or adding amino acids to the amino acid sequence shown in SEQ ID NO:4-7, and the amino acid sequence is functionally identical or similar to the amino acid sequence shown in SEQ ID NO:4-7. Further, the amino acid sequence in the nanobody differs from the amino acid sequence shown in SEQ ID NO:4-7 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, or 38 amino acids.

[0048] In this invention, SEQ ID NO:4 is: QVTLMESGGGSVQAGGSLRLSCTAS. SEQ ID NO:5 is: MGWYRQAPGNECELVST. SEQ ID NO:6 is: YYEDSVKGRFTISQDNAKNTVYLQMNSLKPEDTAVYYC. SEQ ID NO:7 is: WGQGTQVTISS.

[0049] In this invention, the amino acid sequence of the nanobody comprises: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4; the amino acid sequences of CDR1, CDR2, and CDR3 are as shown in SEQ ID NO:1-3, or functional active variants of the amino acid sequences differing from the amino acid sequences shown in SEQ ID NO:1-3 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 amino acids. The amino acid sequences of FR1, FR2, FR3, and FR4 are as shown in SEQ ID NO:4-7, or functional active variants of the amino acid sequences having 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequences shown in SEQ ID NO:4-7. As a preferred embodiment, the amino acid sequence of the nanobody is as shown in SEQ ID NO:8, or an amino acid sequence having more than 80% identity with the amino acid sequence shown in SEQ ID NO:8. SEQ ID NO:8 is: QVTLMESGGGSVQAGGSLRLSCTASEFTFDDSDMGWYRQAPGNECELVSTISSDGSTYYEDSVKGRFTISQDNAKNTVYLQMNSLKPEDTAVYYCAAGAPNNPILCGAPSGYWGQGTQVTISS.

[0050] Furthermore, the nanobody is an antibody that specifically recognizes the "cyclosporine A-antibody complex".

[0051] Furthermore, the nanobody comprises a heavy chain framework region or a variant thereof selected from human, mouse, primate, or camelid sources.

[0052] This invention provides a nucleic acid molecule that encodes the aforementioned nanobody. Specifically, the sequence of the nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO:8 is shown in SEQ ID NO:9.

[0053] SEQ ID NO:9 is:

[0054] CAGGTGACCCTGATGGAATCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTACAGCCTCTGAATTCACTTTTGATGATTCTGACATGGGCTGGTACCGCCAGGCTCCAGGGAATGAGTGCGAGTTGGTCTCAACTATTAGTAGTGATGGTAGCACATACTATGAAGACTC CGTGAAGGGCCGATTCACCATCTCCCAAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTGTATTACTGTGCGGCCGGCGCTCCTAATAACCCAATACTATGCGGCGCTCCCTCTGGTTACTGGGGCCAGGGGGACCCAGGTCACCATCTCCTCA.

[0055] In this invention, the nanobody has the ability to specifically recognize and bind to cyclosporine A, and has high precision, high sensitivity and good specificity when detecting cyclosporine A, and the detection results are accurate and reliable.

[0056] This invention provides the application of nanobody in the preparation of reagent for detecting cyclosporine A, wherein the nanobody is the aforementioned nanobody.

[0057] The present invention provides a test strip or kit for detecting the concentration of cyclosporine A, wherein the test strip or kit comprises the above-mentioned nanobody.

[0058] In this invention, the test strip or kit further includes a solid support coated with a first capture antibody that specifically binds to cyclosporine A; the nanobody specifically recognizes and binds to the cyclosporine A-first capture antibody complex.

[0059] In this invention, the kit is an enzyme-linked immunosorbent assay (ELISA) kit, which further includes a well plate, cyclosporine A standard solution, buffer, washing solution, substrate solution, stop solution, and detection substrate. The well plate can be a 96-well plate, a 6-well plate, a 12-well plate, a 24-well plate, or a 48-well plate. Whole blood samples tested by the kit undergo pretreatment. The pretreatment uses pretreatment reagents, which include a dissociating agent and a hemolysin. The dissociating agent is preferably selected from sodium salicylate, salicylic acid, or 8-aniline-1-naphthalenesulfonic acid (8-ANS). The hemolysin is preferably selected from saponins, polyethylene glycol octylphenyl ether (Triton X-100), ethylphenyl polyethylene glycol (NP-40), cholamidopropyl dimethylaminopropanesulfonate (CHAPS), or sodium dodecyl sulfate (SDS). More preferably, the hemolytic agent is sample release agent A, which comprises the following components in the following amounts: a final concentration of 40-60 mmol / L Tris-HCl, a mass-volume fraction of 0.5%-1.5% NaCl, a mass-volume fraction of 0.1%-0.5% saponin, and a mass-volume fraction of 0.5%-2% sodium salicylate; the pH value of sample release agent A is 7.0-8.0. Even more preferably, sample release agent A comprises the following components in the following amounts: a final concentration of 45-55 mmol / L Tris-HCl, a mass-volume fraction of 0.7%-1.2% NaCl, a mass-volume fraction of 0.2%-0.4% saponin, and a mass-volume fraction of 0.7%-1.5% sodium salicylate; the pH value of sample release agent A is 7.2-7.8. The sample release agent A comprises the following components in the following amounts: a final concentration of 40-60 mmol / L Tris-HCl, a mass-volume fraction of 0.5%-1.5% NaCl, a mass-volume fraction of 0.1%-0.5% saponin, and a mass-volume fraction of 0.5%-2% sodium salicylate, with a pH value of 7.0-8.0. More specifically, the sample release agent A comprises the following components in the following amounts: a final concentration of 50 mmol / L Tris-HCl, a mass-volume fraction of 0.9% NaCl, a mass-volume fraction of 0.2% saponin, and a mass-volume fraction of 1% sodium salicylate; the pH value of the sample release agent A is 7.4. The mass-volume fractions are expressed in g / mL.

[0060] In this invention, the test strip is a fluorescent immunochromatographic test strip, which further includes a base plate and a sample pad, a fluorescent microsphere binding pad, a nitrocellulose membrane, and absorbent paper sequentially overlapped and adhered to the base plate. The whole blood sample tested by the kit undergoes pretreatment. The pretreatment uses pretreatment reagents, which include a dissociating agent and a hemolysin. The dissociating agent is preferably selected from sodium salicylate, salicylic acid, or 8-aniline-1-naphthalenesulfonic acid (8-ANS). The hemolysin is preferably selected from saponins, polyethylene glycol octylphenyl ether (Triton X-100), ethylphenyl polyethylene glycol (NP-40), cholamidopropyl dimethylaminopropanesulfonate (CHAPS), or sodium dodecyl sulfate (SDS). More preferably, the hemolytic agent is sample release agent B, which preferably includes the following components in the following amounts: a final concentration of 40-60 mM Tris-HCl, 0.5%-1.5% NaCl (by mass / volume), 0.1%-0.5% saponin (by mass / volume), 0.5%-2% sodium salicylate (by mass / volume), and 0.05%-0.15% ethylphenyl polyethylene glycol (by mass / volume); the pH value of the sample diluent B is 7.0-8.0. More preferably, the sample release agent B comprises the following components in the following amounts: a final concentration of 45-55 mM Tris-HCl, 0.7%-1.2% NaCl (by mass / volume), 0.2%-0.4% saponin (by mass / volume), 0.7%-1.5% sodium salicylate (by mass / volume), and 0.05%-0.15% ethylphenyl polyethylene glycol (by mass / volume); the pH value of the sample diluent is 7.2-7.8. The sample release agent B is composed of the following components in the following amounts: a final concentration of 40-60 mM Tris-HCl, 0.5%-1.5% NaCl (by mass / volume), 0.1%-0.5% saponin (by mass / volume), 0.5%-2% sodium salicylate (by mass / volume), and 0.05%-0.15% ethylphenyl polyethylene glycol (by mass / volume), with a pH value of 7.0-8.0. Furthermore, the sample release agent B comprises the following components in the following amounts: a final concentration of 50 mmol / L Tris-HCl, 0.9% NaCl (w / v), 0.2% saponin (w / v), 1% sodium salicylate (w / v), and 0.1% ethylphenyl polyethylene glycol (w / v); the pH value of the sample release agent B is 7.4. The units for the w / v fractions are g / mL.

[0061] This invention provides a method for detecting cyclosporine A, the method being used for non-therapeutic or diagnostic purposes, comprising the following steps:

[0062] (1) A solid support is provided, wherein a first capture antibody is immobilized on the solid support, the first capture antibody specifically binding to cyclosporine A;

[0063] (2) After the sample to be tested is brought into contact with the first capture antibody, the above-mentioned nanobody is added for detection.

[0064] In this invention, the detection platform adapted to the method is selected from enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay, electrochemiluminescence immunoassay, fluorescence immunoassay, or immunochromatographic strip method. The method also includes pretreatment of the sample to be tested. When using ELISA, the pretreatment uses sample release agent A, the composition of which is described above and will not be repeated here.

[0065] In this invention, when using the immunochromatographic test strip method for detection, the pretreatment uses sample release agent B. The composition of sample release agent B can be found in the previous description and will not be repeated here.

[0066] In this invention, the solid support is selected from perforated plates, magnetic beads, nitrocellulose membranes, glass fiber membranes, or microfluidic chips.

[0067] In this invention, when the nanobody is used for detection, the nanobody is coupled to a detection marker. The detection marker is selected from enzymes, chemiluminescent substances, fluorescent substances, biotin, radioactive isotopes, or colloidal gold. The enzyme is preferably selected from horseradish peroxidase or alkaline phosphatase. The chemiluminescent substance is preferably selected from acridine ester, isoluminol derivatives, or ruthenium tripyridine.

[0068] In this invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art.

[0069] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0070] In the following examples, cyclosporine A (Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number C106893), anti-cyclosporine A mouse monoclonal antibody (Beijing Deoping Biotechnology Co., Ltd., catalog number E001M13), cDNA synthesis kit (product name SuperScript™ III Reverse Transcriptase, Thermo Fisher Scientific, catalog number 18080044), and Expi293 kit (product name Expi293) were used. TMThermo Fisher Scientific, catalog number A14635, saponin (China National Pharmaceutical Group Chemical Reagent Co., Ltd., catalog number XW018047152020), AMPPD luminescent solution (Beijing Danda Biotechnology Co., Ltd., catalog number DY001-50), sample release agent (Beijing Danda Biotechnology Co., Ltd., catalog number SF004-100), cyclosporine A detection kit (Beijing Danda Biotechnology Co., Ltd., catalog number F11409), time-resolved latex microspheres (1% solid content, Suzhou Weidu Biotechnology Co., Ltd., catalog number FT0200CA), sulfo-NHS (Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number H109337), EDC (Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number E106172-5g), nitrocellulose membrane (NC membrane, Sartorius, model CN140), goat anti-mouse IgG (Feipeng Biotechnology Co., Ltd., catalog number goat anti-mouse IgG), fluorescence detector (Beijing Danda Biotechnology Co., Ltd., model number D10).

[0071] The present invention will be further illustrated below with reference to the embodiments:

[0072] Example 1: Preparation and Screening of Nanobodies

[0073] 1. Immunogen preparation and unicorn immunization

[0074] Cyclosporine A (CsA) and anti-cyclosporine A mouse monoclonal antibody (as the first capture antibody) were mixed in PBS at an equimolar ratio and incubated at room temperature (20-25°C) for 1 hour to form a "cyclosporine A-antibody complex". Using this cyclosporine A-antibody complex as an immunogen, Ankavo Biotechnology (Beijing) Co., Ltd. was commissioned to perform subcutaneous multi-site immunization on a 3-year-old dromedary camel. The immunization protocol is shown in Table 1.

[0075] Table 1. Immigration Schedule for Dromedary Camels

[0076]

[0077] Serum test results: The 409600 dilution well was positive, with an antibody titer >1:102400, which meets the requirements for library construction.

[0078] 2. Construction of a nanobody phage library

[0079] (1) Blood collection: 100 mL of blood was collected.

[0080] (2) Isolation of peripheral blood lymphocytes (PBMCs)

[0081] Approximately 100 mL of whole blood was collected from each camel, diluted with an equal volume of DPBS, and mixed thoroughly, totaling 200 mL. This mixture was then aliquoted into 50 mL centrifuge tubes and centrifuged to separate lymphocytes. After centrifugation, the cells separated into four layers from top to bottom: a plasma layer, a PBMC layer, a lymphocyte separation medium layer, and a erythrocyte layer. 40 mL of the plasma layer was transferred to a clean container and frozen at -80°C for later use. Approximately 5 mL of the PBMC (peripheral blood mononuclear cell) layer was transferred to a new 15 mL centrifuge tube, and the cells were washed twice with PBS. Quantitative analysis was performed using a cell counter, yielding a result of 3.5 × 10⁻⁶ cells / mL. 8 PBMCs / mL.

[0082] (3) RNA was extracted from PBMCs using the Trizol method. The extracted RNA concentration was 455.8 ng / µL and the volume was 40 µL.

[0083] (4) Reverse transcription to synthesize cDNA: cDNA was synthesized by reverse transcription using a cDNA synthesis kit in accordance with the instructions.

[0084] (5) Nested PCR amplification of VHH gene fragment. After the first round of PCR and the second round of PCR amplification, the second round of PCR products were detected by electrophoresis to obtain the target band. The target band was extracted to obtain the purified second round of PCR products.

[0085] (6) Ligation and transformation: The purified second-round PCR product and the phage vector pAKW-SCNB were double-digested with NcoI and NotI, ligated overnight, and then electroporated to transform freshly prepared TG1 competent bacteria.

[0086] (7) Counting and identification: After electroporation, the colony count of the immunobank was calculated by titration plate to be 3.85 × 10⁻⁶. 8 One colony was selected, and 40 single clones were chosen for PCR identification. All were confirmed to be correct clones, indicating a library capacity >10. 8 .

[0087] (8) Phage library presentation: The above bacterial colonies were collected, amplified, and packaged and displayed using helper phage M13KO7. The phage library was then purified and its titer was determined, yielding a phage library titer of 7 × 10⁻⁶. 13 cfu / mL.

[0088] 3. Biological panning

[0089] Solid-phase affinity panning was used. Anti-cyclosporine A mouse monoclonal antibody was diluted to 20 µg / mL with carbonate buffer (first round) and coated onto ELISA plates. After blocking, cyclosporine A solution (5 µg / mL) was added and incubated to form a "cyclosporine A-antibody complex" target in situ on the plate. A pre-blocked phage library was added to the wells and incubated overnight at 4°C. Non-specifically bound phages were removed using a progressively stronger washing protocol (PBS and PBST containing 0.1% Tween-20). Specifically bound phages were eluted with 0.1 M glycine-HCl (pH 2.2) and immediately neutralized with Tris-HCl (pH 8.0). The eluted phages were used to infect TG1 cells for amplification and rescued with helper phage M13KO7 for the next round of panning or analysis. Three rounds of panning were performed, with the capture antibody coating concentration decreasing progressively (20→15→10 µg / mL) and the washing intensity increasing progressively. The input and output data of the three rounds of screening are shown in Table 2. The significant increase in output rate indicates that the specific phages were effectively enriched.

[0090] Table 2 Data from the Three Rounds of Biological Selection

[0091]

[0092] 4. Screening and identification of positive clones (phage-ELISA)

[0093] Hundreds of monoclonal antibodies were randomly selected from the output plates after the third round of screening and cultured in 96-well deep-well plates. A helper phage was used for rescue, and monoclonal phage supernatant was prepared. Monoclonal phage supernatant was added to two types of ELISA plates: one coated with cyclosporine A-antibody complex and the other coated only with capture antibody (control group). Detection was performed using HRP-labeled anti-M13 secondary antibody. Result interpretation: Phase transition ratio = (experimental group - control group) / control group; data with a phase transition ratio > 0.3 were interpreted as positive clones. Based on the results in Table 3, a total of 5 positive clones were obtained.

[0094] Table 3. Identification of positive clones by phage-ELISA

[0095]

[0096] 5. Sequence analysis and antibody expression

[0097] Phagemid Af14-4, a strongly positive clone, was selected for sequencing (using primer SEQ ID NO:10:5'-gattagcggatcctacctg-3') to obtain the VHH gene sequence. The unique VHH gene subcloning was expressed in the eukaryotic expression vector pcDNA3.4, with a His tag at the C-terminus, using the Expi293 kit to obtain soluble nanobody protein.

[0098] Af14-4 base sequence (SEQ ID NO:9):

[0099] CAGGTGACCCTGATGGAATCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTACAGCCTCTGAATTCACTTTTGATGATTCTGACATGGGCTGGTACCGCCAGGCTCCAGGGAATGAGTGCGAGTTGGTCTCAACTATTAGTAGTGATGGTAGCACATACTATGAAGACTC CGTGAAGGGCCGATTCACCATCTCCCAAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTGTATTACTGTGCGGCCGGCGCTCCTAATAACCCAATACTATGCGGCGCTCCCTCTGGTTACTGGGGCCAGGGGGACCCAGGTCACCATCTCCTCA.

[0100] Af14-4 amino acid sequence (SEQ ID NO:8):

[0101] QVTLMESGGGSVQAGGSLRLSCTASEFTFDDSDMGWYRQAPGNECELVSTISSDGSTYYEDSVKGRFTISQDNAKNTVYLQMNSLKPEDTAVYYCAAGAPNNPILCGAPSGYWGQGTQVTISS.

[0102] Example 2: Magnetic microparticle chemiluminescence method for detecting cyclosporine A blood concentration (sandwich method reagent)

[0103] (1) Take 50 mg of tosylactivated magnetic beads (Dynalbeads M280 Tosylactivated), dilute them in 2.5 mL of 50 mM borate buffer (BB, pH 8.0), add 0.25 mg of anti-cyclosporine A mouse monoclonal antibody, mix well, and shake at 37 °C for 8 h to obtain the reaction solution.

[0104] (2) The supernatant in the solution after the reaction was removed by magnetic adsorption. 50 mL of TBST (50 mM Tris, 0.9% NaCl and 0.1% Tween-20, pH 7.4) was added to the modified magnetic beads and the mixture was shaken at 42 °C for 16 h to block the unreacted Tosyl groups on the surface of the magnetic beads, thus obtaining the blocked magnetic beads.

[0105] (3) Remove the supernatant of the sealed magnetic beads with magnetic adsorption, and wash the sealed magnetic beads three times with the above-mentioned TBST. Finally, resuspend the sealed magnetic beads with TBST to make the final concentration 0.3 mg / mL, which is the magnetic microparticle working solution, and store it at 2-8℃ for later use.

[0106] (4) Dissolve 1 mg of alkaline phosphatase (ALP) in 1 mL of phosphate-buffered saline (PBS), add 0.2 mg of the Af14-4 antibody prepared in Example 1, mix well, add 10 µL of 50% glutaraldehyde aqueous solution to the resulting Af14-4 antibody solution, and react at room temperature (20~25℃) in the dark for 2 h. After the reaction, dialyze into PBS to completely remove free glutaraldehyde, collect the dialysis bag solution, and obtain the dialysis product. After dialysis, dilute the dialysis product to 1 µg / mL (based on antibody amount) with MES diluent (50 mM MES, 0.9% NaCl, 5 mg / mL BSA, 1 mM MgCl2, pH 6.7) to obtain the enzyme-labeled working solution.

[0107] (5) Weigh 6.06g of Tris, 9.0g of sodium chloride, 2.0g of saponin, and 10.0g of sodium salicylate, dissolve them in 900mL of purified water, and stir at room temperature (20~25℃) until the saponin is completely dissolved. Adjust the pH to 7.4 with 1M hydrochloric acid, then transfer the solution to a 1L volumetric flask, add purified water to make up to 1L, mix well, and you will get sample release agent A (containing 50mM Tris-HCl, 0.9% NaCl, 0.2% saponin, 1% sodium salicylate, pH 7.4).

[0108] (6) Sample pretreatment method: Take 100µL of EDTA anticoagulated whole blood and add it to 200µL of sample release agent A. Vortex mix for 10 seconds to fully lyse the whole blood and obtain the sample solution A to be tested.

[0109] (7) Reaction procedure: Take 10µL of the above sample solution A and add it to the reaction well. Add 50µL TBST, 50µL magnetic microparticle working solution and 50µL enzyme-labeled working solution, incubate at 37℃ for 10min, wash, and add AMPPD luminescent solution for color development.

[0110] Calibrators of cyclosporine A at different concentrations (0, 25, 50, 100, 200, 400, 800, and 1600 ng / mL) were prepared using negative EDTA-anticoagulated whole blood. The concentration of cyclosporine A was detected using the method described in this embodiment, and a standard curve was established as follows: Figure 1 The results are shown in Table 4, and the precision and limit of detection were determined.

[0111] Comparative Example 1: Magnetic Particle Chemiluminescence Method for the Determination of Cyclosporine A Blood Drug Concentration (Competitive Reagent Method)

[0112] Take 200µL of EDTA-anticoagulated whole blood and place it in a 1.5mL centrifuge tube. Add 200µL of sample release agent and vortex to mix for 10 seconds. Place the mixed sample in a centrifuge and centrifuge at 10,000 rpm for 5 minutes.

[0113] After centrifugation, remove the centrifuge tube and carefully aspirate the supernatant. Follow the instructions for the cyclosporine A assay kit to perform the subsequent measurements.

[0114] Calibrators of cyclosporine A at different concentrations (0, 25, 50, 100, 200, 400, 800, and 1600 ng / mL) were prepared using negative EDTA-anticoagulated whole blood. The concentration of cyclosporine A was determined using the method described in Comparative Example 1, and a standard curve was established as follows: Figure 2 The results are shown in Table 4, and the precision and limit of detection were determined.

[0115] Table 4. Calibration curve data of cyclosporine A by magnetic particle luminescence method

[0116]

[0117] Figures 1-2 The results in Table 4 show that, compared with the conventional cyclosporine A detection kit which uses a competitive method to detect cyclosporine A, the sandwich method using the Af14-4 antibody of the present invention has a better signal-to-noise ratio (S1 / S0) and a better dynamic range (S7 / S0) for detecting cyclosporine A.

[0118] Table 5 Precision and Sensitivity

[0119]

[0120] The results in Table 5 show that, compared with the conventional cyclosporine A detection kit which uses a competitive method to detect cyclosporine A, the sandwich method using the Af14-4 antibody of this invention has better precision and sensitivity for detecting cyclosporine A.

[0121] Further evaluation was conducted using the methods of Example 2 and Comparative Example 1 to assess the cross-reactivity of rapamycin, digoxin, imatinib, voriconazole, phenytoin, rifampin, valproic acid, paclitaxel, tacrolimus, acetaminophen, everolimus, furosemide, prednisolone, chloramphenicol, linezolid, erythromycin, mycophenolate mofetil, carbamazepine, cephalosporin C zinc salt, gentamicin, and vancomycin. Different concentrations of the cross-test agent were added to cyclosporine A samples at concentrations of 100 ng / mL and 400 ng / mL, respectively, and the cross-reactivity rates were calculated according to the cross-reactivity rate formula, as shown in Table 6.

[0122] Cross-reactivity rate (%) = (Cyclosporine A concentration measured with cross-reactive agent - Cyclosporine A concentration without cross-reactive agent) ÷ Concentration of cross-reactive agent × 100

[0123] Table 6 Cross-reactivity

[0124]

[0125] The results in Table 6 show that the sandwich method for detecting cyclosporine A using the Af14-4 antibody of the present invention exhibits extremely low cross-reactivity rates (all <0.1%) against 21 common clinical drugs. In contrast, conventional cyclosporine A detection kits using a competitive method show varying degrees of cross-reactivity (up to 9.28%) against some structural analogs (such as digoxin, rapamycin, carbamazepine, etc.). Therefore, the sandwich method for detecting cyclosporine A using the Af14-4 antibody of the present invention has superior specificity.

[0126] Example 3: Preparation of Cyclosporine A Sandwich Method Fluorescent Immunochromatographic Detection Reagent

[0127] 1. Preparation of fluorescent microspheres

[0128] (1) Washing: Take 100µL of time-resolved latex microspheres, add 400µL of MES (25mM, pH6.5), and sonicate at 10W for 5min to obtain dispersed microspheres. Centrifuge the dispersed microspheres at 12000rpm for 5min, remove the supernatant, resuspend the precipitate with MES (25mM, pH6.5), centrifuge and wash once more, discard the supernatant, and obtain the washed microspheres.

[0129] (2) Activation: Add 500 µL of MES (25 mM, pH 6.5) to the washed microsphere precipitate, and disperse by ultrasonication at 10 W for 2 min. Then add 50 µL of 10 mg / mL sulfo-NHS (sodium N-hydroxysulfosuccinimide) solution, shake well, and then add 25 µL of 10 mg / mL EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride) solution. Shake well and react at room temperature (20~25℃) for 30 min to obtain the reaction solution. Centrifuge the reaction solution at 12000 rpm for 5 min, remove the supernatant, resuspend the precipitate with MES (25 mM, pH 6.5), centrifuge and wash once more, discard the supernatant, and obtain the activated microspheres.

[0130] (3) Antibody conjugation: Add 500µL of MES solution to the activated microspheres, disperse by sonication at 10W for 2 min, add 100µg of anti-cyclosporine A mouse monoclonal antibody, shake well, and react at room temperature (20~25℃) for 2 h to obtain the conjugated solution. After the reaction is completed, centrifuge the conjugated solution at 12000rpm for 5 min, remove the supernatant, and obtain the conjugated antibody.

[0131] (4) Blocking: Add 500µL of blocking solution (5% BSA solution) to the conjugated antibody, disperse by sonication at 10W for 2 min, react at room temperature for 1 h, centrifuge the resulting blocked solution at 12000 rpm for 5 min, remove the supernatant, wash once with blocking solution by centrifugation, and obtain the blocked microspheres.

[0132] (5) Preservation: Add 1000µL of microsphere preservation solution (50mM Tris pH8.0, 5% sucrose, 1% BSA) to the sealed microspheres, and disperse by ultrasonication at 10W for 2min to obtain fluorescent microsphere labels.

[0133] 2. Preparation of fluorescent microsphere binding pads

[0134] (1) Preparation of the bonding pad: Use a chopping machine to cut the glass fiber pad into strips with a size of 2.8cm×30cm;

[0135] (2) Spraying with gold: Using a spraying gold coating instrument, the spraying volume is set to 3µL / cm, and the fluorescent microsphere markers are uniformly sprayed onto the glass fiber pad;

[0136] (3) Drying: Place the coated bonding pad on a mesh screen and transfer it to a 45℃ drying oven to dry for 24 hours to obtain the fluorescent microsphere bonding pad.

[0137] 3. Preparation of Nitrocellulose Membrane (NC) Components

[0138] (1) NC membrane preparation: Cut the nitrocellulose membrane into 30cm lengths for later use.

[0139] (2) C-line coating solution: dilute sheep anti-mouse IgG with PBS to 1 mg / mL.

[0140] (3) T-line coating solution: Dilute the Af14-4 antibody described in Example 1 to 0.5 mg / mL with PBS.

[0141] (4) Scribing: Using a gold spraying scribing instrument, scribing the C-line or T-line with the C-line coating solution or the T-line coating solution at a liquid output of 1µL / cm.

[0142] (5) Drying: Place the coated NC film in a 50°C forced-air drying oven and dry for 24 hours.

[0143] 4. Assembly of large boards and cutting of test strips

[0144] (1) Assembly of the large plate: The blank sample pad, the fluorescent microsphere binding pad prepared in step 2, the NC membrane prepared in step 3, and the absorbent paper are assembled into a large plate in sequence.

[0145] (2) Cutting: Cut the assembled large plate into 3.8mm wide test strips and put them into the cassette for testing.

[0146] 5. Preparation of sample diluent

[0147] Weigh 7.88g Tris-HCl, 9.0g NaCl, 2.0g saponin, and 10.0g sodium salicylate. Measure 1.0mL NP40 and dissolve them in 800mL purified water. After stirring and dissolving, adjust the pH to 7.4 with 1M hydrochloric acid. Then transfer the solution to a 1L volumetric flask, add purified water to make up to the 1L mark, and shake well to obtain sample dilution solution B and sample release agent B (containing 50mM Tris-HCl, pH 7.4, 0.9% NaCl, 0.2% saponin, 1% sodium salicylate, and 0.1% NP40).

[0148] 6. Testing

[0149] (1) Sample pretreatment: Take 10µL of EDTA anticoagulated whole blood and add it to 500µL of sample release agent B. Vortex mix for 10 seconds to fully lyse the whole blood and obtain the sample solution B to be tested.

[0150] (2) Sample addition and reaction: Take 80µL of the above-mentioned sample solution B and add it vertically to the sample well of the test strip prepared in step 4. Let it react horizontally at room temperature (20~25℃) for 15 min. Add it to the reagent strip and react for 15 min.

[0151] (3) Fluorescence detection: Insert the reacted test strip into the fluorescence detector to detect the C and T line signal values ​​and calculate the T / C ratio. The results are shown in Table 7.

[0152] T / C ratio = T-line signal value / C-line signal value

[0153] 7. Methodological Performance Validation

[0154] Calibrators were prepared by adding pure cyclosporine A to negative EDTA-anticoagulated whole blood to prepare calibrators at different concentrations (0, 25, 50, 100, 200, 400, 800 and 1600 ng / mL).

[0155] Table 7. Cyclosporine A fluorescence immunochromatographic assay main curve test.

[0156]

[0157] Table 7 shows that the fluorescent immunochromatographic sandwich method established using the Af14-4 nanobody of this invention exhibits good signal-to-noise ratio performance in the detection of cyclosporine A. Within the concentration range of 0–1600 ng / mL, the T / C ratio shows a good linear relationship with increasing concentration, and the lowest detectable concentration reaches 25 ng / mL (S1 / S0 = 1.3), indicating that the method has high sensitivity and low background interference.

[0158] Example 4: Clinical evaluation of cyclosporine A chemiluminescent sandwich method

[0159] 1. Sample collection

[0160] We collected EDTA-anticoagulated whole blood samples from 25 kidney transplant patients who were taking cyclosporine A.

[0161] 2. Reference Method

[0162] Liquid chromatography-tandem mass spectrometry (LC-MS / MS) was used, and the detection method was based on the following reference: Michael D, Gabriele K, Klaus R, et al. Simultaneous quantification of sirolimus, everolimus, tacrolimus and cyclosporine by liquid chromatography-mass spectrometry (LC-MS). [J]. Clinical chemistry and laboratory medicine, 2002, 40 (3): 285-92.

[0163] 3. Detection Method

[0164] Take each clinical sample and test them according to the following methods:

[0165] Sandwich method: The determination was performed according to the method described in Example 2.

[0166] Competition method: The determination was carried out according to the method described in Comparative Example 1.

[0167] LC-MS / MS method: Determined according to the above reference method.

[0168] 4. Statistical Analysis

[0169] Using LC-MS / MS measurements as reference values ​​(x-axis), and measurements from the sandwich method and the comparative competition method of this invention as test values ​​(y-axis), linear regression analysis was performed to calculate the regression equation and correlation coefficient (R²). 2 ).

[0170] 5. Results

[0171] Using mass spectrometry as the standard, the correlation between the sandwich method described in Example 2 and the competitive method described in Comparative Example 1 and mass spectrometry was analyzed. The results are as follows: Figure 3 As shown in Table 8.

[0172] Table 8 Correlation Data

[0173]

[0174] Figure 3 The results in Table 8 show that the correlation regression equation between the sandwich method described in Example 2 and LC-MS / MS is: y = 0.9405x + 19.332, R0 2 =0.9894, the correlation regression equation between the competitive method described in Comparative Example 1 and LC-MS / MS is: y=0.8873x+36.519, R 2 =0.9731. The results show that, compared with the conventional cyclosporine A detection kit which uses a competitive method to detect cyclosporine A, the detection of cyclosporine A using the Af14-4 antibody of this invention using a sandwich method has a better correlation with LC-MS / MS.

[0175] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A nanobody for the detection of cyclosporin A, characterized in that, The nanobody includes complementarity-determining regions CDR1, CDR2, and CDR3; The amino acid sequence of CDR1 is shown in SEQ ID NO:1; The amino acid sequence of CDR2 is shown in SEQ ID NO:2; The amino acid sequence of CDR3 is shown in SEQ ID NO:

3.

2. The Nanobody according to claim 1, characterized in that, The nanobody also includes framework regions FR1, FR2, FR3 and FR4; The amino acid sequence of FR1 is shown in SEQ ID NO:4; The amino acid sequence of FR2 is shown in SEQ ID NO:5; The amino acid sequence of FR3 is shown in SEQ ID NO:

6.

3. The Nanobody according to claim 1 or 2, characterized in that, The amino acid sequence of the nanobody is shown in SEQ ID NO:

8.

4. The use of the nanobody according to any one of claims 1 to 3 in the preparation of a reagent for detecting cyclosporine A.

5. A test strip or kit for detecting cyclosporin A concentration, characterized by, The test strip or kit includes the nanobody as described in any one of claims 1 to 3.

6. The test strip or kit of claim 5, wherein, The test strip or kit further includes a solid support coated with a first capture antibody that specifically binds to cyclosporine A; the nanobody specifically recognizes and binds to the cyclosporine A-first capture antibody complex.

7. A method of detecting cyclosporin A, characterized in that, The method is used for non-therapeutic or diagnostic purposes and includes the following steps: (1) A solid support is provided, wherein a first capture antibody is immobilized on the solid support, the first capture antibody specifically binding to cyclosporine A; (2) After contacting the sample to be tested with the first capture antibody, add the nanobody described in any one of claims 1 to 3 or the nanobody described in claim 5 or 6 for detection.

8. The method of claim 7, wherein, The detection platform adapted to the method is selected from enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay, electrochemiluminescence immunoassay, fluorescence immunoassay, or immunochromatographic strip method.

9. The method of claim 8, wherein, The method also includes preprocessing the sample to be tested; When using enzyme-linked immunosorbent assay (ELISA) for detection, the pretreatment uses sample release agent A, which comprises the following components: a final concentration of 40-60 mmol / L Tris-HCl, a mass-volume fraction of 0.5%-1.5% NaCl, a mass-volume fraction of 0.1%-0.5% saponin, and a mass-volume fraction of 0.5%-2% sodium salicylate. The pH value of the sample release agent A is 7.0~8.0; When using the immunochromatographic test strip method for detection, the pretreatment uses sample release agent B, which comprises the following components: a final concentration of 40-60 mM Tris-HCl, 0.5%-1.5% NaCl (w / v), 0.1%-0.5% saponin (w / v), 0.5%-2% sodium salicylate (w / v), and 0.05%-0.15% ethylphenyl polyethylene glycol (w / v). The pH value of the sample diluent is 7.0~8.

0.

10. The method of claim 7, wherein, The solid support is selected from perforated plates, magnetic beads, nitrocellulose membranes, glass fiber membranes, or microfluidic chips; When the nanobody is used for detection, the nanobody is coupled with a detection label; the detection label is selected from an enzyme, a chemiluminescent substance, a fluorescent substance, biotin, a radioisotope or colloidal gold.

Images