Teicoplanin complete antigen and antibody and preparation method and application thereof

Abstract

The present invention discloses a complete teicoplanin antigen and its use in the preparation of teicoplanin antibodies and immunoassay detection. The structure of the complete teicoplanin antigen is shown in Formula 1. The present invention also discloses anti-teicoplanin monoclonal antibodies prepared using the complete antigen, hybridoma cells producing the monoclonal antibodies, and detection cards and kits for detecting teicoplanin analogs. The present invention discloses an ELISA method and a fluorescent immunochromatographic assay for detecting teicoplanin analogs. The fluorescent immunochromatographic assay offers simple operation, short detection time, low cost, high specificity, and good reproducibility.

Description

Technical Field

[0001] The present invention relates to the detection of antibiotic drugs, in particular to a complete teicoplanin antigen, a teicoplanin monoclonal antibody obtained by using the complete antigen, and its application in the detection of teicoplanin analogs by fluorescent immunochromatography. Background Art

[0002] Teicoplanin is a novel glycopeptide antibacterial drug produced by Actinomycetes motile. It is a complex composed of five glycopeptides with similar structures, similar to vancomycin. Due to its unique acetyl substituent, it is 30-100 times more lipophilic than vancomycin, allowing for easier tissue and cell penetration, a long half-life, and the ability to be administered intravenously or intramuscularly. Teicoplanin's mechanism of action is similar to that of other glycopeptide antibacterial drugs. Teicoplanin binds to the tail of the last two amino acids of the pentapeptide attached to N-acetylmuramic acid (D-alanyl-D-alanine), forming a complex that prevents the essential disaccharide decapeptide, the essential structural component of the cell wall mucopeptide, from being transported to the synthesis site. This blocks the addition of new units, disrupting the integrity of the bacterial cell wall and membrane, leading to bacterial death. Overdose may cause injection site pain, thrombophlebitis, rash, itching, bronchospasm, drug fever, allergic reactions, nausea, vomiting, diarrhea, lethargy, headache, eosinophilia, leukopenia, neutropenia, thrombocytopenia, and thrombocytosis. The "Expert Consensus on Clinical Dosage of Teicoplanin" states that trough blood concentration is an important indicator for adjusting the initial loading dose of teicoplanin. Glycopeptide antimicrobial drugs are water-soluble and primarily distributed in extracellular fluid. The strength of teicoplanin's antimicrobial effect is directly proportional to the drug concentration in extracellular fluid, which in turn is parallel to the blood concentration. Therefore, the blood concentration indirectly reflects the drug concentration at the site of infection. Teicoplanin has a high plasma protein binding rate and a low free concentration. The optimal teicoplanin dose for early efficacy in treating MRSA infections is a trough concentration >10 ng / mL, but not higher than 60 ng / mL. Physicians should adjust the dosing interval based on the patient's renal function and the dose based on the trough concentration.

[0003] Currently, teicoplanin detection primarily involves liquid chromatography coupled with mass spectrometry (HPLC-MS), high-performance liquid chromatography, and chemiluminescence methods. These methods are expensive, complex, and time-consuming. Fluorescence quantitative immunochromatography is a novel quantitative detection technology that combines the advantages of immunofluorescence and traditional immunochromatography. This technology is flexible and simple to operate, low-cost, and has a short reaction time, enabling timely quantitative detection. This provides physicians with data support for adjusting patient medications, enabling personalized dosing and ensuring both efficacy and safety. Summary of the Invention

[0004] In order to solve the above technical problems, the present invention provides a complete teicoplanin antigen, a teicoplanin monoclonal antibody obtained by using the complete antigen, and its use in detecting teicoplanin analogs by fluorescent immunochromatography.

[0005] Teicoplanin complete antigen, the complete antigen having the structure shown in Formula 1:

[0006]

[0007] Formula 1

[0008] Among them, Protein is a protein carrier.

[0009] In a preferred embodiment, the protein carrier is any one protein selected from the group consisting of bovine serum albumin (BSA), ovalbumin (OVA), keyhole limpet hemocyanin (KLH), human serum albumin (HSA) and artificially synthesized polylysine (PLL).

[0010] In a preferred embodiment, the protein carrier is bovine serum albumin (BSA) or ovalbumin (OVA).

[0011] A method for producing a complete teicoplanin antigen, the method comprising the steps of:

[0012] (1) linking the teicoplanin hapten to bovine serum albumin (BSA) or ovalbumin (OVA) to prepare the complete antigen;

[0013] (c) Dissolving teicoplanin hapten and BSA / OVA in 0.01 M PBS to form solution A;

[0014] (d) Liquid A obtained in step (a) was added dropwise to a 20-30% glutaraldehyde solution and reacted at room temperature for 2.5-3.5 hours to obtain a teicoplanin-BSA / OVA conjugate, i.e., a complete teicoplanin antigen.

[0015] A use of the complete teicoplanin antigen is for preparing a specific monoclonal antibody against teicoplanin.

[0016] A monoclonal antibody that specifically binds to the complete teicoplanin antigen.

[0017] Preferably, the monoclonal antibody is produced by a mouse hybridoma cell line; the hybridoma cell line is deposited by the China Center for Type Culture Collection (CCTCC, Wuhan University, Wuhan, China) with the accession number CCTCC NO: C2020226.

[0018] A hybridoma cell line producing the monoclonal antibody, wherein the monoclonal antibody has a sensitivity of 7-8 ng / mL for detecting teicoplanin analogs;

[0019] The monoclonal antibody does not bind to other antibiotic therapeutic drugs;

[0020] The other antibiotic therapeutic drugs are levofloxacin, rifampicin, vancomycin, linezolid and fosfomycin.

[0021] A use of the monoclonal antibody is to prepare a reagent, a detection card or a kit for detecting teicoplanin in a sample.

[0022] A method for detecting the presence of teicoplanin in a biological sample, the method comprising the steps of:

[0023] (a) contacting the biological sample with the monoclonal antibody;

[0024] (b) detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of teicoplanin in the sample;

[0025] Wherein, the monoclonal antibody carries a detectable marker; and the detection method is fluorescence detection.

[0026] Preferably, the label is selected from the group consisting of colloidal gold labels, colored labels, and fluorescent labels.

[0027] A fluorescent immunochromatographic test card for detecting teicoplanin analogs, comprising a substrate; a liquid aspirating component; a detection component; and a sample loading component, characterized in that the detection component is fixed on the substrate, a quality control strip and a detection strip are provided in the middle of the detection component, and the liquid aspirating component and the sample loading component are fixed at both ends of the detection component in a partially overlapping manner, wherein the detection strip is coated with the complete teicoplanin antigen, and the quality control strip is coated with rabbit antigen IgG.

[0028] In a preferred example, the teicoplanin fluorescent immunochromatographic test card also includes a card box, which consists of a lower cover and an upper cover, the upper cover is provided with a sample loading window and a detection window, and the teicoplanin fluorescent immunochromatographic test card is completely arranged in the lower cover, and the detection window and the sample loading window correspond to the sample loading component, quality control strip and detection strip on the teicoplanin fluorescent immunochromatographic test card respectively.

[0029] In a preferred embodiment, the upper cover is further provided with a product number area and a bar code identification area.

[0030] In a preferred embodiment, the substrate is a dark hard substrate, preferably a black PVC substrate.

[0031] In a preferred example, the detection component is a nitrocellulose membrane.

[0032] In a preferred example, the sample adding component is glass fiber.

[0033] In a preferred embodiment, the liquid-absorbing component is absorbent paper.

[0034] A detection kit for detecting teicoplanin analogs, the kit comprising:

[0035] (a) the teicoplanin fluorescent immunochromatographic test card;

[0036] (b) a teicoplanin detection analysis solution compatible with the teicoplanin fluorescence immunochromatographic test card;

[0037] (c) instructions for use of the teicoplanin detection kit for detecting teicoplanin analogs;

[0038] Wherein, the detection and analysis liquid is a detection and analysis liquid containing the fluorescently labeled monoclonal antibody and anti-rabbit IgG antibody;

[0039] The fluorescent dyes used for labeling in the detection analysis solution include but are not limited to FITC (Fluorescein), AlexaFluor 647, CF TM 647, TRITC (Rhodamine).

[0040] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 UV scans of the teicoplanin immunogen and coating are shown;

[0042] FIG2 shows a schematic diagram of the structure of a Teicoplanin fluorescent immunochromatographic test card; wherein:

[0043] Figure 2A Shows the structure of the test card without a plastic card; 1: black PVC substrate; 2: absorbent paper; 3: nitrocellulose membrane; 4: glass fiber; 5: quality control line (C line); 6: test line (T line);

[0044] Figure 2B The structure of the teicoplanin test card with a plastic card box is shown; 1': lower cover; 2': upper cover; 3': sample loading window; 4': glass fiber; 5': detection window; 6': quality control line (C line); 7': detection line (T line); 8': nitrocellulose membrane; 9': teicoplanin project logo; 10': barcode identification area;

[0045] Figure 3The standard curve profile of the teicoplanin fluorescence immunochromatographic assay is shown;

[0046] Figure 4 The standard curve profile of the teicoplanin ELISA assay is shown. DETAILED DESCRIPTION

[0047] After extensive and in-depth research, the inventors synthesized a complete teicoplanin antigen. Using this antigen as an immunogen, they immunized Balb / C mice, fused their spleen cells with mouse myeloma SP20 cells, and obtained a hybridoma cell line that specifically secreted anti-teicoplanin monoclonal antibodies. They then prepared and purified teicoplanin monoclonal antibodies. Subsequently, they used this complete antigen and teicoplanin antibodies to prepare a highly sensitive and specific teicoplanin immunoassay card. This work led to the completion of the present invention.

[0048] Complete antigen

[0049] Substances that are both immunogenic and immunoreactive are called complete antigens, such as most proteins, bacteria, viruses, bacterial exotoxins, and animal sera. Complete antigens can stimulate the body to produce antibodies or sensitize lymphocytes and can also undergo specific binding reactions with them in vivo and in vitro.

[0050] Usually, haptens need to be coupled to macromolecules such as bovine serum albumin (BSA), ovalbumin (OVA) or hemocyanin (KLH) or covalently bonded to become complete antigens that are both immunoreactive and immunogenic.

[0051] As used herein, the term "complete antigen" refers to the product of the teicoplanin hapten of the present invention conjugated to an appropriate protein carrier. As used herein, the term "protein carrier" refers to any immunologically acceptable protein used to form a complete antigen, including but not limited to bovine serum albumin, ovalbumin, keyhole limpet hemocyanin (KLH), human serum albumin (HSA), and synthetic polylysine (PLL), preferably bovine serum albumin (BSA) or ovalbumin (OVA).

[0052] The structure of the complete teicoplanin antigen of the present invention is shown in Formula 1:

[0053]

[0054] Formula 1

[0055] Here, Protein is a protein carrier, and in the present invention, preferably bovine serum albumin (BSA) or ovalbumin (OVA).

[0056] The conditions for linking the teicoplanin hapten to the protein carrier are as follows: the teicoplanin hapten is linked to bovine serum albumin (BSA) or ovalbumin (OVA) to prepare a complete antigen.

[0057] (a) Teicoplanin hapten and BSA / OVA were dissolved in 0.01 M PBS to prepare solution A.

[0058] (b) Liquid A obtained in step (a) was added dropwise to a 25% glutaraldehyde solution and reacted at room temperature for 3 h to obtain a teicoplanin-BSA / OVA conjugate, i.e., a complete teicoplanin antigen.

[0059] Preparation of monoclonal antibodies

[0060] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible spontaneous mutations that may be present in minor amounts. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete antibodies.

[0061] The antibodies of the present invention can be prepared by various techniques known to those skilled in the art. For example, the complete antigens of the present invention can be administered to animals to induce the production of monoclonal antibodies. Monoclonal antibodies can be prepared using hybridoma technology (see Kohler et al., Nature 256:495, 1975; Kohler et al., Eur. J. Immunol. 6:511, 1976; Kohler et al., Eur. J. Immunol. 6:292, 1976; Hammerling et al., In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, NY, 1981) or can be prepared using recombinant DNA methods (U.S. Patent No. 4,816,567).

[0062] Representative myeloma cells are those that fuse efficiently, support stable high-level production of the antibody by the selected antibody-producing cells, and are sensitive to a culture medium (HAT medium medium), including myeloma cell lines, such as murine myeloma cell lines, including myeloma cell lines derived from MOPC-21 and MPC-11 mouse tumors (available from the Salk Institute Cell Distribution Center, San Diego, California, USA), and SP-2, NZ0, or X63-Ag8-653 cells (available from the American Type Culture Collection, Rockville, Maryland, USA). Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies [Kozbor, J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibodies Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)].

[0063] The culture medium in which the hybridoma cells are grown is analyzed to detect the production of monoclonal antibodies with the desired specificity, such as, for example, by in vitro binding analysis, enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA). The location of cells expressing the antibody can be detected using FACS. The hybridoma clones can then be subcloned by limiting dilution steps and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986) 59-103 pages). Suitable culture medium used to achieve this purpose includes, for example, DMEM or RPMI-1640 culture medium. In addition, hybridoma cells can be grown as ascites tumors in animals.

[0064] The monoclonal antibodies secreted by the subclones are suitably isolated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0065] The monoclonal antibody of the present invention is produced by a mouse hybridoma cell line, which was deposited in the China Center for Type Culture Collection (CCTCC, Wuhan University, Wuhan, China) on November 27, 2020, with the deposit number CCTCCNO: C2020226; the classification name is: hybridoma cell line C214242.

[0066] In a specific embodiment, the monoclonal antibody of the present invention carries a detectable label. More preferably, the label is selected from the group consisting of colloidal gold labels, colored labels, and fluorescent labels.

[0067] In a specific embodiment, the monoclonal antibody of the present invention has a sensitivity of 7.4 ng / ml for detecting a teicoplanin analog. The monoclonal antibody of the present invention does not cross-react with carrier proteins of the complete teicoplanin antigen, such as BSA or OVA. Furthermore, the monoclonal antibody of the present invention does not bind to other antibiotic therapeutic agents, including but not limited to levofloxacin, rifampicin, vancomycin, linezolid, and fosfomycin.

[0068] Detection kit

[0069] The detection kit of the present invention refers to a kit containing the monoclonal antibody of the present invention and can be used for the detection of teicoplanin analogs. The kit may include a container, instructions for use, a buffer, an immunomodulator, etc. as needed.

[0070] The detection kit of the present invention can be in various forms, such as a detection card, a test box containing various test reagents, etc. In the examples, the detection card is used as an example to illustrate the kit of the present invention, but it should not be understood that the kit of the present invention is limited to the detection card.

[0071] In a specific embodiment, the fluorescent immunochromatographic test card for detecting teicoplanin analogs of the present invention includes a substrate; a liquid aspiration component; a detection component; and a sample loading component; wherein the detection component is fixed on the substrate, a quality control band and a detection band are arranged in the middle of the detection component, and the liquid aspiration component and the sample loading component are fixed at both ends of the detection component in a partially overlapping manner, wherein the detection band is coated with the complete antigen of the present invention, and the quality control band is coated with rabbit antigen IgG.

[0072] In a preferred embodiment, the teicoplanin fluorescent immunochromatographic test card of the present invention further includes a card cartridge, which comprises a lower cover and an upper cover. The upper cover is provided with a sample loading window and a detection window. The teicoplanin fluorescent immunochromatographic test card is completely disposed within the lower cover. The detection window and sample loading window correspond to the sample loading component, quality control strip, and test strip on the teicoplanin fluorescent immunochromatographic test card, respectively. The upper cover may also be provided with a product number area and a barcode identification area. The substrate may be a dark hard substrate, preferably a black PVC substrate. The detection component may be a nitrocellulose membrane. The sample loading component may be fiberglass. The liquid absorbing component may be absorbent paper.

[0073] The term "partially overlapping" as used herein means that two adjacent components form a certain overlapping area, rather than a complete overlap where one component is completely contained within the other, and that the two components are secured via this overlapping area. The securing method may be selected by those skilled in the art, such as by bonding.

[0074] On the basis of the above-mentioned detection card, the present invention also provides a detection kit for detecting teicoplanin analogs, which contains:

[0075] (a) the aforementioned Teicoplanin Fluorescent Immunochromatographic Test Card;

[0076] (b) a teicoplanin analogue detection analysis solution compatible with the teicoplanin fluorescence immunochromatographic test card;

[0077] (c) instructions for use of the teicoplanin analog detection kit for detecting teicoplanin analogs;

[0078] Wherein, the detection and analysis liquid is a detection and analysis liquid containing the fluorescently labeled monoclonal antibody of the present invention and anti-rabbit IgG antibody.

[0079] Those skilled in the art can select fluorescent labels according to their needs, including but not limited to FITC (Fluorescein), Alexa Fluor 647, CF TM 647, TRITC (Rhodamine), etc.

[0080] In a preferred embodiment, the solvent portion of the detection analysis solution is a phosphate buffer containing BSA.

[0081] Application of Immunoassay of Teicoplanin Complete Antigen

[0082] The complete teicoplanin antigen of the present invention is used for antibody preparation, and the antibody should be a monoclonal antibody or a polyclonal antibody; the corresponding antibodies prepared from the complete teicoplanin antigen of the present invention are used in various immunological detection fields for detecting the content of teicoplanin analogs, including but not limited to ELISA, chemiluminescence, colloidal gold method and fluorescent immunochromatography.

[0083] The above-mentioned "application of complete teicoplanin antigen for antibody preparation" refers to the use of the complete teicoplanin antigen of the present invention to immunize experimental animals with the complete antigen to prepare anti-teicoplanin polyclonal antibodies and monoclonal antibodies; the experimental animals should not be understood as simple mice in the specific embodiment, and should include but are not limited to: mice, rats, rabbits, goats, sheep, horses, donkeys, chickens, dogs and other experimental animals.

[0084] The aforementioned "application of complete teicoplanin antigen in teicoplanin immunoassays" refers to the use of antibodies prepared from complete teicoplanin antigen as immunoassay raw materials to establish various immunoassay methods for detecting the content of teicoplanin analogs. This immunoassay field includes, but is not limited to, immunological assays such as ELISA, chemiluminescence, colloidal gold, and fluorescent immunochromatography. These immunoassays for teicoplanin analogs encompass not only quantitative assays but also semi-quantitative and various qualitative immunoassay-based methods.

[0085] In a specific embodiment, the present invention takes the preparation of specific monoclonal antibodies by immunizing mice as an example, and uses ELISA and fluorescent immunochromatography as specific examples to illustrate the application of complete teicoplanin antigen in the immunological detection of teicoplanin.

[0086] Advantages of the present invention:

[0087] 1. This invention discloses for the first time the structure of a complete (artificial) teicoplanin antigen and its preparation method;

[0088] 2. This invention discloses for the first time the application of a complete teicoplanin antigen in the preparation of teicoplanin antibodies and immunological detection, providing a reliable method for clinical teicoplanin blood concentration detection.

[0089] 3. The monoclonal antibodies of the present invention can detect teicoplanin analogs with high sensitivity and do not bind to other antibiotic therapeutic drugs;

[0090] 4. The teicoplanin analog detection kit of the present invention can simply and quickly detect teicoplanin analogs on site.

[0091] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, were generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer.

[0092] Example 1: Preparation of Teicoplanin Complete Antigen (Immunogen and Coating Gen)

[0093] Teicoplanin (hapten) was conjugated to bovine serum albumin (BSA) and ovalbumin (OVA) respectively by EDC method. The specific conjugation method is as follows:

[0094] Dissolve 18.0 mg of teicoplanin and 36 mg of OVA / 57.2 mg of BSA in 6 mL of 0.01 M PBS. Add 20 μL of 25% glutaraldehyde dropwise and incubate at room temperature in the dark for 3 h. Dialyze the solution against phosphate buffered saline for 2-3 days, changing the dialysate 2-3 times daily. Collect the dialysate by centrifugation to prepare teicoplanin-BSA / OVA. Maintain a teicoplanin to BSA / OVA molar ratio of 30:1.

[0095] The structural formula of the obtained teicoplanin artificial antigen (immunogen and coating agent) is shown below, wherein "Protein" is BSA (bovine serum albumin) or OVA (ovalbumin).

[0096]

[0097] The results of ultraviolet scanning peaks of Teicoplanin artificial antigen (immunogen and coating) are compared as follows Figure 1 As shown, the peak of the conjugate is different from the peaks of BSA and OVA, indicating successful conjugation.

[0098] Example 2: Preparation of monoclonal antibodies using teicoplanin complete antigen

[0099] 1. Immunization of animals

[0100] The teicoplanin immunogen obtained in Example 1 was diluted to 0.5 mg / mL. 500 μL of the immunogen was mixed with an equal volume of Freund's complete adjuvant, emulsified, and used to immunize BALB / c mice (Shanghai Slake Laboratory Animal Co., Ltd.) by injection in the groin. Complete Freund's adjuvant was used for the first immunization, followed by incomplete Freund's adjuvant. One week after the fourth immunization, orbital blood was collected, serum was separated, and the titer of anti-teicoplanin antibodies was measured. ELISA testing showed that the antibody titer of the mice after four immunizations was 1:256,000.

[0101] 2. Cell Fusion and Screening

[0102] After four immunizations, mice were given an intraperitoneal injection of approximately 100 μg of immunogen to boost immunization again. Three days later, the spleen of the mouse was removed for fusion. SP2 / 0 cells (Nanjing Academy of Military Medical Sciences) were mixed with spleen cells and added to serum-free culture medium (Hyclone SH30022.018 DMEM (High Glucose)). The mixture was centrifuged (1500 rpm, 3 min), and the precipitated cells were removed. 1 mL of 50% polyethylene glycol 4000 was added dropwise and allowed to stand for 90 seconds. Then, 10 mL of serum-free culture medium pre-warmed at 37°C was added dropwise and allowed to stand for 5 min. After fusion, the cell suspension was centrifuged (1000 rpm, 3 min) and inoculated with complete culture medium into a 96-well plate with feeder cells, with 2×10 cells per well. 4 / mL myeloma cells. Culture in a 37°C, 5% CO2 incubator for two days. Add 2×HAT complete culture medium to a final concentration of 1×HAT in the wells. When hybridoma colonies have grown to 1 / 10-1 / 5 of the well bottom area, screen for fusion cell antibody-positive wells using ELISA.

[0103] 3. Ascites Fluid Preparation and Antibody Purification

[0104] BALB / c mice were intraperitoneally injected with 0.5 mL of paraffin oil. Seven days later, 0.5 mL of 1×10 6 Positive hybridoma cells. Observe the growth of the mice. If abdominal swelling is visible around day 7, collect ascites promptly. Affinity chromatography (Protein G Resin affinity purification) is used to purify a highly pure monoclonal antibody, yielding 8 mg of protein.

[0105] Example 3: Immunoassay using complete teicoplanin antigen

[0106] 1. Fluorescence immunochromatographic detection

[0107] 1) Preparation of assay solution

[0108] a fluorescently labeled monoclonal antibodies and anti-rabbit IgG antibodies obtained in Example 2 (Hangzhou Qitai Biotechnology Co., Ltd.);

[0109] b. Dilute the fluorescently labeled antibody with phosphate buffer containing BSA to prepare the assay solution.

[0110] 2) Preparation of Teicoplanin Fluorescent Immunochromatographic Test Card

[0111] a. Dilute the prepared teicoplanin coating agent (teicoplanin-OVA) and rabbit antigen IgG to appropriate concentrations (0.4-3.0 mg / mL) using coating buffer (phosphate buffer). Spray the diluted teicoplanin-OVA and rabbit antigen IgG evenly onto a nitrocellulose membrane at 25±5°C (forming the test and control lines, respectively). Dry the membrane at a humidity of 12%-30% for approximately 1.5-2 hours and store dry until ready for use.

[0112] b. On a black PVC substrate, the coated nitrocellulose membrane, glass fiber paper and absorbent paper obtained in step a are sequentially pasted to form a test card (eg Figure 2A Cut to appropriate width as needed.

[0113] c. The test card obtained in step b is loaded into the lower cover of the cartridge and the upper cover is closed to form a complete cartridge test card (eg Figure 2B shown).

[0114] 3) Detection

[0115] Take 60 μl of the diluted sample and mix it evenly with 60 μl of the detection analysis solution. Take 100 μL and add it to the sample loading window of the detection card. After reacting for 15-20 minutes, use FCR fluorescence immunoassay analyzer (Suzhou Hemai Precision Instrument Co., Ltd.) to detect it. The test result is displayed based on the comparison of the T line signal value and C line signal ratio (T / C value) of the sample with the built-in standard curve.

[0116] 4) Teicoplanin Fluorescent Immunochromatographic Test Card Principle

[0117] Using a competitive assay, the teicoplanin antigen in the sample competes with the teicoplanin antigen (coating agent) on the test line (T line) for binding to the fluorescently labeled anti-teicoplanin antibody in the assay solution. When the antigen concentration in the sample is lower, more fluorescent antibody binds to the test line, resulting in a stronger fluorescent signal. Consequently, the ratio of the fluorescent signal on the test line (T line) to the fluorescent signal on the control line (C line) (T / C value) increases. Conversely, when the teicoplanin antigen concentration in the sample is higher, the T / C value decreases. Therefore, the higher the teicoplanin content in the sample, the lower the T / C value. The test results are displayed based on the T / C value compared with the built-in standard curve.

[0118] 5) Sensitivity and standard curve of fluorescent immunochromatographic assay for teicoplanin analogs

[0119] Teicoplanin standard was added to blank serum to prepare 8 concentration gradients of 80, 60, 40, 20, 10, 5, 2.5, and 0 ng / ml, and then diluted 20 times with 0.9% NaCl. The above series of concentration samples were tested according to the above test steps, with each sample repeated 3 times. The test results are shown in Table 1. Based on the data in Table 1, a standard curve (four parameters) was drawn with concentration as the horizontal axis and T / C value as the vertical axis. Figure 3 shown. Figure 3 The equation corresponding to the middle curve is shown in Table 2, and IC is calculated 50 =6.97ng / ml.

[0120] Table 1 Detection of different concentrations of teicoplanin samples by fluorescence immunochromatography

[0121]

[0122] Table 2 Equation corresponding to the inhibition curve (four parameters)

[0123]

[0124] Repeat the test 10 times for the 0 ng / ml sample and calculate the mean (X), standard deviation (SD) and precision (CV) of the T / C values. The sensitivity is calculated as X-2*SD T / C value. Figure 3 The corresponding teicoplanin concentration values ​​in the standard curve are shown in Table 3.

[0125] Table 3 Results of repeated detection of 0 ng / ml teicoplanin sample by fluorescence immunochromatography

[0126]

[0127] Substitute the T / C value of X-2*SD in the data of Table 3 as the y value Figure 3 The concentration value obtained in the equation corresponding to the standard curve is 7.4 ng / ml, that is, the sensitivity is 7.4 ng / ml.

[0128] 6) Precision deviation of teicoplanin detection by fluorescence immunochromatography

[0129] The established teicoplanin assay system was used to test teicoplanin standards at concentrations of 20 ng / ml, 40 ng / ml, and 60 ng / ml, with 10 replicates for each assay. The precision (CV) for the detection of low, medium, and high concentrations of teicoplanin was calculated. Table 4 shows the precision results for the detection of high and low concentrations of teicoplanin.

[0130] Table 4 Results of repeated detection of 20, 40, and 60 ng / ml teicoplanin standards by fluorescence immunochromatography

[0131]

[0132] 7) Accuracy deviation of teicoplanin detection by fluorescence immunochromatography

[0133] After diluting the standard (5 mg / mL) to ng / mL using buffer, 10 μL of each standard at different concentrations was added to 90 μL of a low-concentration in-house reference solution. Testing was performed according to the above test procedures, with each sample replicated five times. Table 5 shows the accuracy results.

[0134] Table 5 Results of repeated detection of teicoplanin standard by fluorescence immunochromatography

[0135]

[0136] 8) Cross-reactivity of the Teicoplanin Fluorescence Immunochromatographic Detection System

[0137] Three common clinically used drugs were prepared into samples with different gradient concentrations using blank mixed serum, and fluorescent immunochromatographic detection was performed to calculate their IC 50 IC with teicoplanin 50 The cross-reaction rate was calculated by comparing the values. Calculation formula: Cross-reaction rate = (IC 50替考拉宁 / IC 50临床用相关药物 )%, and the cross-reaction rate results are shown in Table 6:

[0138] Table 6 Results of cross-reactions of clinically relevant drugs detected by fluorescence immunochromatography

[0139]

[0140] 2. Quantitative detection of teicoplanin by ELISA

[0141] 1) Establishment of ELISA standard curve

[0142] The prepared teicoplanin coating material (teicoplanin-OVA) was diluted to 1-2 ng / ml in carbonate buffer (0.05 M, pH 9.6) and coated onto a 96-well plate at 100 μL / well. The plate was incubated overnight at 4°C, blocked with 5% BSA for 3 h, and then washed three times, 5 min each time. Teicoplanin standards were spiked into blank plasma to prepare eight concentrations of 80, 60, 40, 20, 10, 5, 2.5, and 0 ng / ml, which were then diluted 20-fold in 0.01 M PBS. A mixture of 50 μL of teicoplanin at various concentrations and 50 μL of teicoplanin antibody was added to the microwells and incubated at 37°C for 1 h. After three washes, HRP-conjugated secondary antibody was added (100 μL / well) and incubated for 1 h. After three washes, the plate was washed three times, and the colorimetric solution was added. The plate was reacted in the dark for 15 min at room temperature, and the plate was read at 450 nm after adding the stop solution. Table 7 shows the absorbance value results of the standard curve of ELISA detection. Figure 4 The equation corresponding to the middle curve is shown in Table 8

[0143] Table 7 ELISA detection of different concentrations of teicoplanin

[0144]

[0145] Table 8 Equation corresponding to the inhibition curve (four parameters)

[0146]

[0147] 2) Sensitivity of ELISA for teicoplanin detection

[0148] The negative plasma samples were tested 10 times, and the mean (X), standard deviation (SD) and precision (CV) of the ELISA absorbance values ​​were calculated. The sensitivity was calculated as X-2*SD OD value in Figure 4 The corresponding teicoplanin concentration value in the standard curve.

[0149] Table 9 Results of repeated ELISA detection of 0 ng / ml teicoplanin samples

[0150]

[0151] Substitute the absorbance value of X-2*SD in the data of Table 8 as the y value Figure 4 The concentration value obtained in the equation corresponding to the standard curve is 1.6 ng / ml, that is, the sensitivity is 1.6 ng / ml.

[0152] 3) Precision and accuracy deviation of teicoplanin detection by ELISA

[0153] Teicoplanin standards at concentrations of 20 ng / mL and 60 ng / mL were tested 10 times using an established ELISA assay system. The precision (CV) and accuracy deviation (A / D) of the assays for both high and low concentrations of teicoplanin were calculated. The results showed that both the precision and A / D were less than 15% for both high and low concentrations, and less than 15% for both high and low concentrations.

[0154] Table 10 Results of repeated ELISA testing of 20 and 60 ng / ml teicoplanin standards

[0155]

[0156] 4) ELISA to detect cross-reactivity of teicoplanin

[0157] The cross-reaction results were consistent with those of teicoplanin detection by fluorescence immunochromatography.

[0158] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A monoclonal antibody, characterized in that The monoclonal antibody specifically binds to the complete teicoplanin antigen; The monoclonal antibody is produced by a mouse hybridoma cell line; the hybridoma cell line is deposited by the China Center for Type Culture Collection (CCTCC, Wuhan University, Wuhan, China) with the accession number CCTCC NO: C2020226.

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