CN107226795B

CN107226795B - Linezolid hapten and complete antigen as well as preparation method and application thereof

Info

Publication number: CN107226795B
Application number: CN201610177211.1A
Authority: CN
Inventors: 郭彦飞; 何扬锦; 宗伟英; 吴婉欣
Original assignee: Zhejiang Quasi Policy Biological Technology Co ltd
Current assignee: Zhejiang Quasi Policy Biological Technology Co ltd
Priority date: 2016-03-24
Filing date: 2016-03-24
Publication date: 2022-07-22
Anticipated expiration: 2036-03-24
Also published as: CN107226795A

Abstract

The invention discloses a linezolid hapten derivative, a corresponding complete antigen and application thereof in preparation of linezolid antibodies and immunoassay detection. The structure of the linezolid hapten is shown as a formula 2, and the structure of the complete antigen is shown as a formula 3. The invention also discloses an anti-linezolid monoclonal antibody prepared by the complete antigen, a hybridoma cell producing the monoclonal antibody, a detection card and a detection kit for detecting linezolid. The invention discloses an ELISA method and a fluorescence immunochromatography method for detecting linezolid, wherein the fluorescence immunochromatography method is simple to operate, short in detection time, low in cost, high in specificity and good in repeatability.

Description

Linezolid hapten and complete antigen as well as preparation method and application thereof

Technical Field

The present invention relates to the detection of antibiotics. Specifically, the invention relates to a linezolid hapten, a linezolid monoclonal antibody obtained by using the hapten and application of the linezolid monoclonal antibody in detecting linezolid by a fluorescence immunochromatography method.

Background

Since the advent of antibacterial drugs, while healing and saving the lives of countless patients, the unreasonable application of antibacterial drugs also brings many hazards to patients or society: the number of resistant bacteria increases, and the number of bacteria that produce resistance to various drugs tends to increase year by year. Unreasonable medication is a big matter about human health and is a common problem in all countries around the world. Unreasonable conditions of medicines in China are serious, according to the notice of the national food and drug administration, at least 20 million people die each year due to wrong medicines and improper medicines, and 40 percent of people die from unreasonable application of antibacterial medicines. The problem of rational medication has been a focus of attention by professionals and the public. The research and management for the reasonable use of the antibacterial drugs are early in foreign countries, and the method plays an active promoting role in the reasonable use of the antibacterial drugs in China. In order to improve the clinical reasonable application level of antibacterial drugs, ensure the medication safety of patients and reduce the drug resistance of bacteria, the ministry of health and the national administration of traditional Chinese medicine jointly make and release the "guiding principle for clinical application of antibacterial drugs" (the "guiding principle" for short) in 2004, which is the first technical guideline and the management standard of China on the clinical application of antibacterial drugs.

In recent years, the problem of drug resistance of bacteria has become more serious due to the wide application and unreasonable use of clinical antibacterial drugs. To combat the rapidly growing drug-resistant bacteria, many pharmaceutical companies are actively developing new antibacterial drugs. Oxazolidinone medicines represented by linezolid have a brand-new structure and a unique action mechanism, and have a good treatment effect on infection caused by most gram-positive bacteria. However, it has been found clinically that not all patients can achieve the desired results given the same regimen. Critically infected patients, such as cystic fibrosis patients, severe burn patients, severe renal failure patients, organ transplantation patients and the like, can cause some changes in the in-vivo process of the medicine due to the existence of various factors (such as edema, body fluid treatment, ascites and the like) which influence the in-vivo distribution and excretion of the medicine in pathophysiological conditions. Therefore, monitoring of blood drug concentration is necessary for these critical patients to ensure effective treatment of the drug, optimize clinical use of linezolid, and reduce the generation of drug-resistant strains and adverse reactions. To achieve the aim, a simple, quick, sensitive and reliable quantitative detection method for linezolid in human blood needs to be established for monitoring the blood concentration of linezolid used by critically ill patients.

At present, liquid chromatography-mass spectrometry detection methods and High Performance Liquid Chromatography (HPLC) methods are reported in foreign documents. The liquid chromatography-mass spectrometry detection has high requirements on instruments and is difficult to popularize in China. The domestic Linezolid HPLC method is reported, but an external standard method is adopted, and the recovery rate of the method is low. Although the method has strong specificity, high sensitivity and accuracy, the sample pretreatment operation steps are complicated, the cost is high, and the method is not suitable for screening and detecting mass samples due to expensive equipment, high cost, high operation skill requirement and the like. Immunoassay methods (such as an enzyme-linked immunosorbent assay, a colloidal gold method and a fluorescence immunochromatography method) have more and more important functions in the detection of small-molecule drugs because the defects of physicochemical analysis are made up by the unique advantages in the qualitative and quantitative aspects of antigen and antibody and the advantages of large analysis sample and the like due to simple operation. The ELISA can detect a plurality of samples simultaneously, has the advantages of sensitivity, strong specificity and the like, but is not suitable for on-site timely detection. The fluorescence immunochromatography method is simple and rapid to operate, does not need large-scale detection instruments and equipment, is more suitable for on-site timely detection, and is a trend of rapid detection development.

The fundamental reason influencing the quality of immunoassay is the specificity and affinity of the antibody, which depend on the molecular structure of the immune hapten, so the design and selection of the immune hapten molecules are the most critical steps for generating specific antibodies and establishing a rapid detection technology of the content of small molecule drugs.

Therefore, the hapten of linezolid is urgently needed in the field, and the complete antigen of linezolid can be obtained through the hapten, so that a specific antibody of linezolid can be obtained.

Disclosure of Invention

The invention aims to provide a hapten of linezolid and a complete antigen of linezolid obtained by the hapten.

Another object of the present invention is to provide an antibody specific to linezolid, a detection kit comprising the antibody and a method for detecting linezolid using the antibody.

In a first aspect, the present invention provides a hapten having a structure represented by formula 2:

in the formula, R includes but is not limited to the following groups: -CH₂-、

-ο-、

Or a combination thereof;

n is an integer of 2 to 8.

In a preferred embodiment, R is-CH₂-。

In a preferred embodiment, n is an integer from 4 to 6; most preferably 4.

In a second aspect, the present invention provides a complete antigen having the structure of formula 3:

wherein the Protein is a Protein carrier; and

r and n are as described in the first aspect of the invention.

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).

In a preferred embodiment, the protein carrier is Bovine Serum Albumin (BSA), or Ovalbumin (OVA).

In a third aspect, the present invention provides a method of preparing a hapten according to the first aspect of the invention, the method comprising the steps of:

(a) deacetylating linezolid to give deacetylated linezolid

(b) Reacting the deacetyllinezolid with a diacid compound or an ester-group-containing cycloalkane dione compound to obtain the hapten of the first aspect of the invention.

In a preferred embodiment, the conditions of step (b) are as follows: the reaction temperature is 16-30 ℃, preferably 25-28 ℃, and more preferably 25 ℃; the reaction time is 20-50 minutes, preferably 30-40 minutes, and more preferably 35 minutes; the reaction solvent is methanol, ethanol, THF, DMF, dichloromethane, tetrahydrofuran, preferably methanol, dichloromethane, tetrahydrofuran, more preferably dichloromethane/tetrahydrofuran.

In a fourth aspect, the present invention provides a method of preparing a complete antigen according to the second aspect of the invention, the method comprising the steps of:

linking a hapten as described in the first aspect of the invention to a protein carrier to produce a complete antigen as described in the second aspect of the invention.

In a preferred embodiment, the conditions for linking the resulting hapten to a protein carrier are as follows: the reaction temperature is 20-28 ℃, preferably 23-28 ℃, and more preferably 25 ℃; the reaction pH is 7.0 to 8.0, preferably 7.2 to 7.6, more preferably 7.5; the reaction time is 1 to 6 hours, preferably 3 to 4 hours, more preferably 3 hours.

In a fifth aspect, the present invention provides a use of the hapten of the first aspect or the complete antigen of the second aspect of the invention for the preparation of a monoclonal antibody specific for linezolid.

In a sixth aspect, the present invention provides a monoclonal antibody that specifically binds to linezolid.

In specific embodiments, the monoclonal antibody is produced by a mouse hybridoma cell line that is deposited with the China center for type culture Collection (CCTCC, China, Wuhan, university of Wuhan) with a preservation number of CCTCC NO: C201614; the classification is named as: hybridoma cell line LH3A8C 2.

In a preferred embodiment, the monoclonal antibody detects linezolid with a sensitivity of 0.28 ug/ml.

In another preferred embodiment, the monoclonal antibody does not bind to other antibiotics.

In another preferred embodiment, the other antibiotic is dobutamine hydrochloride, dopamine hydrochloride, vancomycin, teicoplanin, midazolam, fluconazole, furosemide, dexamethasone phosphate or omeprazole.

In a seventh aspect, the present invention provides a hybridoma cell line producing the monoclonal antibody of the sixth aspect of the invention, wherein the hybridoma cell line is a mouse hybridoma cell line deposited by China center for type culture Collection (CCTCC, China, Wuhan, university of Wuhan) with a preservation number of CCTCC NO: C201614; the classification is named as: hybridoma cell line LH3A8C 2.

In an eighth aspect, the present invention provides a use of the monoclonal antibody according to the sixth aspect of the present invention for preparing a reagent, a detection card or a kit for detecting linezolid in a sample.

In a preferred embodiment, the sample is a biological sample, preferably a blood sample.

In a ninth aspect, the present invention provides a method of detecting the presence or absence of linezolid in a biological sample, the method comprising the steps of:

(a) contacting said biological sample with a monoclonal antibody according to the sixth aspect of the invention;

(b) detecting the formation of an antigen-antibody complex, wherein the formation of the complex indicates the presence of linezolid in the sample.

In a preferred embodiment, the monoclonal antibody carries a detectable label; preferably, the marker is selected from the group consisting of: a colloidal gold label, a colored label, or a fluorescent label.

In a preferred embodiment, the detection method is a fluorescence detection method.

In a preferred embodiment, the detection method is non-diagnostic in vitro.

In a tenth aspect, the present invention provides a fluorescence immunochromatographic detection card for detecting linezolid, the detection card comprising a substrate; a liquid absorbing member; a detection section; the sample adding component is characterized in that the detection component is fixed on the substrate, the middle part of the detection component is provided with a quality control band and a detection band, the two ends of the detection component are fixed with the imbibing component and the sample adding component in a partially overlapped mode, wherein the detection band is coated with the complete antigen of the second aspect of the invention, and the quality control band is coated with rabbit antibody IgG.

In a preferred embodiment, the linezolid fluorescence immunochromatography detection card further comprises a card box, wherein the card box consists of a lower cover and an upper cover, the upper cover is provided with a sample adding window and a detection window, the linezolid fluorescence immunochromatography detection card is completely arranged in the lower cover, and the detection window and the sample adding window respectively correspond to a sample adding component, a quality control band and a detection band on the linezolid fluorescence immunochromatography detection card.

In a preferred embodiment, the upper cover is further provided with a product number area; a bar code identification area.

In a preferred embodiment, the substrate is a dark hard substrate; a black PVC substrate is preferred.

In a preferred embodiment, the detection member is a nitrocellulose membrane.

In a preferred embodiment, the sample addition member is glass fiber.

In a preferred embodiment, the absorbent member is absorbent paper.

In an eleventh aspect, the present invention provides a test kit for detecting linezolid, the kit comprising:

(a) the linezolid fluorescence immunochromatography detection card according to the tenth aspect of the present invention; and

(b) linezolid detection analysis solution matched with the linezolid fluorescence immunochromatographic detection card in the tenth aspect of the invention; and

(c) instructions for using the linezolid detection kit to detect linezolid;

wherein the detection analysis solution comprises the monoclonal antibody and the anti-rabbit IgG antibody of the fluorescence labeling of the sixth aspect of the invention.

In a preferred embodiment, the fluorescent dyes for the detection of the label in the assay solution include, but are not limited to, FITC (Fluorescein), Alexa Fluor 647, CFTM647, TRITC (Rhodamine), CAL Fluor (R) Red 610, and the like.

In a preferred embodiment, the solvent portion of the assay solution is a phosphate buffer containing BSA.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows the preparation of linezolid derivatives¹H-NMR identification results;

figure 2 shows uv scan spectra of linezolid immunogen and coatingen;

FIG. 3 shows a schematic diagram of the structure of linezolid fluorescence immunochromatographic detection card; wherein:

FIG. 3A shows a test strip card configuration without a plastic card; 1: a black PVC substrate; 2: water absorption paper: 3: a nitrocellulose membrane; 4: glass fibers; 5: a quality control line (C line); 6: a detection line strip (T line);

figure 3B shows a linezolid detection card structure with a plastic cartridge; 1': a lower cover; 2': an upper cover; 3': a sample application window; 4': glass fibers; 5': detecting a window; 6': a quality control line (C line); 7': detecting a line tape (T line); 8': a nitrocellulose membrane; 9': linezolid project designation (LIN); 10': a barcode identification area;

FIG. 4 shows a standard curve spectrum of linezolid fluorescence immunochromatography assay;

figure 5 shows a standard curve profile for linezolid ELISA assay.

Detailed Description

The inventor synthesizes linezolid derivatives serving as haptens through long-term and intensive research, connects the linezolid derivatives with a proper protein carrier to generate complete antigens, immunizes a Balb/C mouse with the complete antigens serving as immunogens, fuses spleen cells of the Balb/C mouse with mouse myeloma SP20 cells to obtain a hybridoma cell strain which specifically secretes an anti-linezolid monoclonal antibody, further prepares and purifies the linezolid monoclonal antibody, and then prepares the linezolid immunodetection card with high sensitivity and specificity by using the complete antigens and the linezolid antibody. On the basis of this, the present invention has been completed.

Haptens

Certain small molecule substances, such as linezolid, are not of large molecular weight and are not capable of inducing an immune response alone, i.e. are not immunogenic, but can gain immunogenicity and induce an immune response when cross-linked or conjugated to a carrier such as a macromolecular protein or non-antigenic polylysine. These small molecule substances can bind to response effector products, have antigenicity, are immunoreactive only and are not immunogenic, and are also called incomplete antigens.

Therefore, in order to prepare a complete antigen of linezolid, the present inventors derivatized linezolid to prepare the hapten of the present invention. As used herein, the terms "hapten", "linezolid hapten" or "linezolid derivative" all refer to a derivative of linezolid having the following structural formula 3 when derivatized:

wherein R includes, but is not limited to, the following groups: -CH₂-、

-ο-、

Or a combination thereof;

n is an integer of 2 to 8.

In a preferred embodiment, R is-CH_2-. In a preferred embodiment, n is an integer from 4 to 6; most preferably, n-4.

The linezolid hapten of the invention can be prepared by the following method:

(a) deacetylating linezolid to give deacetylated linezolid

(b) Reacting the deacetylated linezolid with diacid compound or esterate-bearing cycloalkane dione compound to obtain the hapten of the invention.

In a specific embodiment, the preparation of linezolid hapten is shown in the following reaction scheme:

complete antigens

Substances with immunogenicity and immunoreactivity, called complete antigens (complete antigens), such as most proteins, bacteria, viruses, bacterial exotoxins, animal sera, etc. The complete antigen can stimulate body to produce antibody or sensitized lymphocyte and produce specific combination reaction with the antibody in vivo and in vitro.

Typically, haptens need to be conjugated to macromolecules such as Bovine Serum Albumin (BSA), Ovalbumin (OVA) or hemocyanin (KLH) or covalently bound to become complete antigens that are both immunoreactive and immunogenic.

As used herein, the term "complete antigen" refers to the product of the linezolid hapten of the invention when combined with a suitable protein carrier. As used herein, the term "protein carrier" refers to any immunologically acceptable protein for forming a complete antigen, including, but not limited to: bovine Serum Albumin (BSA) or Ovalbumin (OVA) is preferred, among them, Keyhole Limpet Hemocyanin (KLH), Human Serum Albumin (HSA), and artificially synthesized Polylysine (PLL).

The structure of the linezolid complete antigen is shown as a formula 3:

wherein, Protein is a Protein carrier, and Bovine Serum Albumin (BSA) or Ovalbumin (OVA) is preferred in the invention; the part covalently crosslinked with the protein carrier is a linezolid derivative shown as a formula 2; r and n are as described above.

On the basis of the linezolid hapten prepared as described above, the hapten obtained can be further linked to a protein carrier to prepare the complete antigen of the invention.

Preparation of monoclonal antibodies

The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible small numbers of possible spontaneous mutations. Thus, the modifier "monoclonal" indicates that the antibody is not a mixture of discrete antibodies in nature.

The antibodies of the invention can be prepared by a variety of techniques known to those skilled in the art. For example, a complete antigen of the invention may be administered to an animal to induce the production of monoclonal antibodies. For Monoclonal Antibodies, they 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 hybrids, Elsevier, N.Y.,1981) or can be prepared using recombinant DNA methods (U.S. Pat. No. 4,816,567).

Representative myeloma cells are those that fuse efficiently, support stable high-level production of antibody by selected antibody-producing cells, and are sensitive to Culture medium (HAT medium matrix), 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 Salk Institute Cell Distribution Center, san diego, california, usa), and SP-2, NZ0, or X63-Ag8-653 cells (available from American Type Culture Collection, rockwell, maryland, usa). Human myeloma and mouse-human hybrid myeloma 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 (Monoclonal Antibodies Production Techniques and Applications), pp 51-63 (Marcel Dekker, Inc., New York, 1987).

The medium in which the hybridoma cells are grown is assayed to detect the production of monoclonal antibodies of the desired specificity, e.g., by in vitro binding assays such as enzyme-linked immunosorbent assay (ELISA) or Radioimmunoassay (RIA). The location of the antibody-expressing cells can be detected by FACS. The hybridoma clones can then be subcloned by limiting dilution procedures (subcloned) and grown by standard methods (Goding, Monoclonal Antibodies): Principles and Practice (Principles and Practice), Academic Press (1986) pp 59-103). Suitable media for use in this purpose include, for example, DMEM or RPMI-1640 medium. In addition, hybridoma cells can grow in animals as ascites tumors.

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.

The monoclonal antibody of the invention is produced by a mouse hybridoma cell line which is preserved in China center for type culture Collection (CCTCC, China, Wuhan university) at 2016, 02, 25 days, and has a preservation number of CCTCC NO: C201614; the classification is named as: hybridoma cell line LH3A8C 2.

In particular embodiments, the monoclonal antibodies of the invention carry a detectable label. More preferably, the marker is selected from the group consisting of: a colloidal gold label, a colored label, or a fluorescent label.

In a specific embodiment, the monoclonal antibody of the invention detects linezolid with a sensitivity of 0.28 ug/ml. The monoclonal antibodies of the invention do not cross-react with carrier proteins of the linezolid complete antigen, such as BSA or OVA. Further, the monoclonal antibodies of the invention also do not bind to other antibiotics, including but not limited to dobutamine hydrochloride, dopamine hydrochloride, vancomycin, teicoplanin, midazolam, fluconazole, furosemide, dexamethasone phosphate, or omeprazole.

Detection kit

The detection kit of the invention refers to a kit which contains the monoclonal antibody of the invention and can be used for detecting linezolid. The kit can comprise a container, an instruction for use, a buffering agent, an immune auxiliary agent and the like according to needs.

The detection kit of the present invention may take various forms such as a detection card, a test kit containing various reagents required for the test, and the like. In the examples, the kit of the present invention is described by taking a test card as an example, but it should not be construed that the kit of the present invention is limited to the test card.

In a specific embodiment, the fluorescence immunochromatographic assay card for detecting linezolid of the present invention comprises a substrate; a liquid absorbing member; a detection section; and a sample addition member; the detection component is fixed on the substrate, the middle part of the detection component is provided with a quality control band and a detection band, the two ends of the detection component are fixed with the liquid absorption component and the sample adding component in a partially overlapped mode, wherein the detection band is coated with the complete antigen of the invention, and the quality control band is coated with rabbit antibody IgG.

In a preferred embodiment, the linezolid fluorescence immunochromatographic detection card further comprises a card box, wherein the card box is composed of a lower cover and an upper cover, the upper cover is provided with a sample adding window and a detection window, the linezolid fluorescence immunochromatographic detection card is completely arranged in the lower cover, and the detection window and the sample adding window respectively correspond to a sample adding component, a quality control band and a detection band on the linezolid fluorescence immunochromatographic detection card. The upper cover can also be provided with a product number area; a bar code identification area. The substrate may be a dark hard substrate; a black PVC substrate is preferred. The detection member may be a nitrocellulose membrane. The sample addition component can be glass fiber. The wicking component may be absorbent paper.

As used herein, "secured in a partially overlapping manner" means that two adjacent components form an overlapping region, rather than a complete overlap where one component is completely contained within the other, and the two components are secured by the overlapping region. The manner of fixing can be chosen at the discretion of the person skilled in the art, for example by gluing or the like.

On the basis of the detection card, the invention also provides a detection kit for detecting linezolid, which is filled with:

(a) the linezolid fluorescence immunochromatography detection card;

(b) linezolid detection assay solution matched with the linezolid fluorescence immunochromatography detection card; and

(c) instructions for using the linezolid detection kit to detect linezolid;

wherein the detection analysis solution comprises the monoclonal antibody of the invention and an anti-rabbit IgG antibody which are fluorescently labeled.

Fluorescent labels can be selected autonomously by one skilled in the art as desired, including but not limited to FITC (Fluorescein), Alexa Fluor 647, CFTM647, TRITC (Rhodamine), CAL Fluor (R) Red 610, and the like.

Immunoassay application of linezolid hapten and artificial antigen

The linezolid hapten and the artificial antigen are applied to antibody preparation, and the antibody is a monoclonal antibody or a polyclonal antibody; the linezolid hapten and the artificial antigen application thereof and the corresponding antibody prepared by using the linezolid artificial antigen are applied to various immunological detection fields for detecting the content of linezolid, including but not limited to the immunological detection fields such as ELISA, chemiluminescence method, colloidal gold method, fluorescence immunochromatography and the like.

The application of the linezolid hapten and the artificial antigen in antibody preparation refers to the preparation of the artificial antigen by using the linezolid hapten derivative, and the preparation of an anti-linezolid polyclonal antibody and an anti-linezolid monoclonal antibody by using the artificial antigen to immunize an experimental animal; the experimental animal should not be understood as a simple mouse in the specific embodiment, and should include but not be limited to: experimental animals such as mice, rats, rabbits, goats, sheep, horses, donkeys, chickens, dogs, etc.

The application of the linezolid hapten and the artificial antigen in the field of linezolid immunoassay determination refers to the establishment of various immunoassay methods for detecting the linezolid content by using the artificial antigen prepared from linezolid hapten and the corresponding antibody prepared from the artificial antigen of linezolid as immunoassay raw materials. The immunoassay field includes but is not limited to immunological detection methods such as ELISA, chemiluminescence method, colloidal gold method and fluorescence immunochromatography; the immunoassay method for detecting linezolid not only specifies quantitative detection, but also comprises semi-quantitative and qualitative detection methods based on immunological detection in each.

In the specific embodiment, the invention takes the preparation of a specific monoclonal antibody by an immunized mouse as an example, and takes ELISA and fluorescence immunochromatography as specific examples to illustrate the application of linezolid hapten and artificial antigen in linezolid immunological detection.

The invention has the advantages that:

1. the invention discloses a general formula structure of a linezolid hapten derivative and a derivatization method of the hapten derivative for the first time, wherein the method comprises the following steps: the existing deacetylated linezolid is used as a raw material, and a polycarbochain carboxyl group is added at the amino terminal of the deacetylated linezolid to complete derivatization, so that the core structure of linezolid is retained to the maximum extent, and the core structure is used for preparing a specific linezolid antibody;

2. the invention discloses a structure of a linezolid complete (artificial) antigen and a preparation method thereof for the first time;

3. the invention discloses the application of linezolid hapten and artificial antigen in the fields of linezolid antibody preparation and immunological detection for the first time, and provides a reliable method for promoting clinical linezolid blood concentration detection;

4. the monoclonal antibody can detect linezolid with high sensitivity and is not combined with other antibiotics; and

5. the linezolid detection kit can simply and quickly detect linezolid on site.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations.

Examples are given.

Example 1 preparation of Linezolid hapten

The compound, namely, the deacetyllinezolid (CAS: 168828-90-8) (compound 1), is prepared by a one-step reaction, and the preparation route is shown as follows:

5- (aminomethyl) -3- (3-fluoro-4-morpholinophenyl) oxazolidinone compound (1) (800mg, 2.71mmol) was dissolved in dichloromethane/tetrahydrofuran (30ml/15ml) at room temperature, and cyclohexane-2, 7-dione compound (2) dissolved in dichloromethane/tetrahydrofuran (5ml/2ml) was added thereto. The mixture was stirred at room temperature for thirty minutes, and then 1mol/L sodium hydroxide solution (20ml) was added to the mixture, and stirring was continued for five minutes. After the reaction mixture was washed with dichloromethane (15 ml. times.3), the organic phase was removed and the pH of the aqueous phase was adjusted to 5 with concentrated hydrochloric acid. The precipitate was collected by filtration, washed with ice water and finally dried under vacuum to give 6- ((3-fluoro-4-morpholinophenyl) -2-oxazolidinyl) aminomethyl) -6-oxo-n-hexanoic acid (i.e.: linezolid derivative) (490mg, yield: 47%) white solid. Through a process¹The results of H-NMR identification are shown in FIG. 1.

Example 2 preparation of Linezolid complete antigen (immunogen and coatingen)

The linezolid derivative (hapten) obtained in example 1 was coupled with Bovine Serum Albumin (BSA) and Ovalbumin (OVA) by the carbodiimide method, respectively. The specific coupling method is as follows:

weighing 40mg of linezolid derivative compound, dissolving in 2ml of DMF to form a final concentration of 20mg/ml, mixing 90 mu L of linezolid derivative compound with 6 mu L of EDC (100mg/ml), adding 10ul of NHS (50mg/ml), uniformly mixing, and reacting for 15-60 mins under the stirring condition.

The reaction mixture was centrifuged (1600rmp), added to 1ml of 6mg/ml BSA solution (or 1ml of 4.5mg/ml OVA solution) and reacted at room temperature with stirring for 1-5 h, and then dialyzed 4 times against phosphate buffer, and the solution was changed once for 12 h. The dialysate was collected and the concentrations of immunogen and coating antigen were 5.1mg/ml and 4.3mg/ml, respectively, as determined using the Quick Start Bradford Protein Assay Kit of Burle, USA (BIO-RAD). The structural formula of the obtained linezolid artificial antigen (immunogen and coating antigen) is shown as follows, wherein the Protein is BSA (bovine serum albumin) or OVA (ovalbumin)

Comparison of the ultraviolet scanning peak results of linezolid artificial antigens (immunogen and coatingen) is shown in fig. 2, and the peak of the conjugate is distinguished from the peak of BSA and OVA, which indicates the success of coupling.

Example 3 preparation of monoclonal antibody Using Linezolid complete antigen

1. Immunising an animal

The linezolid immunogen obtained in example 2 was diluted to 0.2mg/ml, 500ul of the immunogen was mixed with an equal volume of Freund's complete adjuvant, emulsified completely, and immunized against BALB/c mice (Shanghai Si Laike laboratory animals, Ltd.) by subcutaneous injection on the back and foot of the mice. The first immunization was with Freund's complete adjuvant, followed by incomplete Freund's incomplete adjuvant. Blood was drawn from the orbit one week after the fourth immunization, serum was separated, and the titer of anti-linezolid antibody was measured. The antibody titer of the mice after four times of immunization is 1:256,000 by ELISA detection.

2. Cell fusion and screening

Four immunizations were performed and mice were re-boosted by intraperitoneal injection of approximately 100 μ g of immunogen, 3 days later, and the spleens of the mice were harvested for fusion. SP2/0 cells (Nanjing military medical science college) were mixed with splenocytes, serum-free culture medium (Hyclone SH30022.018 DMEM (high glucose)) was added, centrifugation (1500rpm, 3min) was carried out, precipitated cells were taken, 1ml of 50% polyethylene glycol 4000 was added dropwise, and the mixture was allowed to stand for 90 seconds. Then 10ml of serum-free culture solution pre-warmed at 37 ℃ is added dropwise and kept stand for 5 min. After the fusion, the cell suspension was centrifuged (1000rpm, 3min), and the complete culture medium was used to inoculate the cell suspension to the feeder layerCells in 96-well plates, 2X 10 per well⁴Myeloma cells/ml. Standing at 37 deg.C for 5% CO₂After two days of culture in the incubator, 2 XHAT of complete culture medium was added to the wells to a final concentration of 1 XHAT. When the hybridoma colonies grow to the bottom of the wells in the area of 1/10-1/5, wells positive for antibody from the fused cells are screened by ELISA.

3. Ascites preparation and antibody purification

Injecting BALB/c mouse with 0.5ml paraffin oil into abdominal cavity, and injecting 0.5ml 1 × 10 into abdominal cavity 7 days later⁶Positive hybridoma cells. The growth condition of the mouse is observed, the abdominal bulge can be seen in about 7 days, and the ascites is collected in time. The monoclonal antibody with high purity is obtained by purification by utilizing affinity chromatography (Protein G Resin affinity purification), and the Protein amount is 4 mg.

Example 4 immunoassay with Linezolid complete antigen

1. Fluorescence immunochromatography assay

1) Preparation of detection analysis solution

a. The monoclonal antibody obtained in example 3 and an anti-rabbit IgG antibody (Kitty, Hangzhou, Biotech Co., Ltd.) were each fluorescently labeled;

b. the antibody after fluorescent labeling is diluted with phosphate buffer containing BSA to prepare a detection analysis solution.

2) Preparation of linezolid fluorescence immunochromatographic test paper card

a. The prepared linezolid-coated antigen (Lin-OVA) and rabbit antibody IgG are respectively diluted to an appropriate concentration (0.4-3.0mg/ml) by using a coating buffer (phosphate buffer). Uniformly spraying the diluted Lin-OVA and rabbit antibody IgG on a nitrocellulose membrane (a detection line and a quality control line are respectively formed) at the temperature of 25 +/-5 ℃, drying for about 5-8 hours under the humidity condition of 12-30%, and drying and storing for later use;

b. and (b) respectively and sequentially sticking the coated nitrocellulose membrane, the glass fiber paper and the absorbent paper obtained in the step (a) on a black PVC substrate to form a detection card (as shown in figure 3A), and cutting the detection card into proper widths as required.

c. And c, putting the detection card obtained in the step B into a lower cover of the card box, and covering an upper cover to form a complete detection card with the card box (as shown in figure 3B).

3) Detection

And (3) uniformly mixing 10 mu L of diluted sample with 100 mu L of detection analysis solution, adding 100 mu L of the diluted sample into a sample adding window of the detection card, reacting for 5-10 min, detecting in an FCR fluorescence immunoassay analyzer (Huzhou HaichongBiotechnology Co., Ltd.), and comparing the ratio (T/C value) of the T line signal value and the C line signal value of the sample with a built-in standard curve to display the detection result.

4) Linezolid fluorescence immunochromatography detection test paper card detection principle

And (3) detecting by adopting a competition method, wherein linezolid antigen in the sample and linezolid antigen (coating antigen) on a detection line (T line) compete and combine with a fluorescence-labeled anti-linezolid antibody in the detection analysis solution. When the concentration of the antigen in the sample is low, the fluorescent antibody combined on the detection line is increased, and further the fluorescent signal on the detection line is strong, so that the ratio (T/C value) of the fluorescent signal on the detection line (T line) to the fluorescent signal on the quality control line (C line) is large; on the contrary, when the concentration of linezolid antigen in the sample is higher, the T/C value is very small. Therefore, the higher the linezolid content in the sample, the lower the T/C value. And comparing the T/C value with a built-in standard curve and displaying the detection result.

5) Sensitivity and standard curve for detecting linezolid by fluorescence immunochromatography

The blank serum is added with linezolid standard substance to prepare samples with the series concentration of 0ug/ml, 0.39ug/ml, 0.78ug/ml, 1.56ug/ml, 3.125ug/ml, 6.25ug/ml, 12.5ug/ml, 25ug/ml and 50 ug/ml. The samples with the series of concentrations are detected according to the detection steps, each sample is repeated for 3 times, the detection experimental result is shown in table 1, and a standard curve (Logit-log linear regression) is drawn according to the data in table 1 by taking the concentration as the abscissa and the T/C value as the ordinate, and is shown in figure 4. The equation corresponding to the curve in FIG. 4 is shown in Table 2, and IC is calculated₅₀＝4.59ug/ml。

TABLE 1 fluorescence immunochromatography assay for detecting linezolid samples of different concentrations

TABLE 2 equation for inhibition curves (Logit-log linear regression)

The test was repeated 10 times for 0ug/ml samples, and the mean (X), Standard Deviation (SD), and precision (CV) of the T/C values were calculated. The sensitivity was calculated as the T/C value of X-2 × SD versus the concentration value of linezolid in the standard curve of figure 4.

TABLE 3 fluorescent immunochromatography repeated detection of linezolid sample at 0ug/ml

The T/C value of X-2 × SD in the data of table 3 was substituted as the y value into the equation corresponding to the standard curve of fig. 4 to obtain a concentration value of 0.56ug/ml, i.e., a sensitivity of 0.56 ug/ml.

6) Precision and accuracy deviation of linezolid detection by fluorescence immunochromatography

The established linezolid detection system is used for detecting linezolid standard substances with the concentrations of 3.125ug/ml and 25ug/ml respectively, the detection is repeated for 15 times respectively, and the precision (CV) of detecting the linezolid with high and low concentrations and the detection accuracy are calculated. Tables 4 and 5 show the precision and accuracy results of detecting the high and low concentrations of linezolid, respectively, and the results show that the precision of the high and low concentrations are less than 10%, and the accuracy deviation is less than 5%.

TABLE 4 repeated detection of 25ug/ml linezolid standard by fluorescence immunochromatography

TABLE 5 repeated detection of 3.125ug/ml linezolid standard by fluorescence immunochromatography

7) Cross reaction of linezolid fluorescence immunochromatography detection system

Preparing 9 common clinical related drugs into different gradient concentration samples with blank mixed serum, performing fluorescence immunochromatography detection, and calculating IC₅₀IC with linezolid₅₀The cross-reactivity was calculated by value comparison. Calculating the formula: rate of cross reaction (IC)_{50 linezolid}/IC_{50 clinically relevant medicine}) % cross-reactivity results are shown in table 6:

TABLE 6 Cross-reaction results of fluorescence immunochromatography assay for clinically relevant drugs

ELISA quantitative determination of linezolid

1) Establishment of ELISA detection standard curve

Diluting the prepared linezolid coating antigen (Lin-OVA) to 4-10ug/ml by using carbonic acid buffer solution (0.05M, pH9.6), coating a 96-well plate at 100 uL/well overnight at 4 ℃, and sealing with 5% skimmed milk powder for 3h at 200 uL/well. Washing for 3 times, 5 min/time; the blank serum is added with linezolid standard substance to prepare samples with the series concentration of 0ug/ml, 0.39ug/ml, 0.78ug/ml, 1.56ug/ml, 3.125ug/ml, 6.25ug/ml, 12.5ug/ml, 25ug/ml and 50 ug/ml. Respectively mixing 50ul of linezolid with different concentrations and 50ul of linezolid antibody (after being diluted by 200 times), adding the mixture into the micropores, and incubating for 1h at 37 ℃; after washing for 3 times, adding HRP-labeled secondary antibody, incubating for 1h (100 ul/well), washing for 3 times, adding color development solution, standing at room temperature for 15min, and adding stop solution to read (450 nm). Table 8 shows the standard curve absorbance results for the ELISA assay.

TABLE 8 ELISA for different concentrations of linezolid

Based on the data in Table 8, a Chart-log linear regression method was used to plot a Chart curve as shown in FIG. 5. The equation for the curve in fig. 5 is shown in table 9, where R2 is 0.99450.

TABLE 9 equation corresponding to ELISA assay inhibition curves (Logit-log regression)

2) Sensitivity of ELISA for detecting linezolid

The 0ug/ml samples were tested 10 times in duplicate and the mean (X), Standard Deviation (SD) and precision (CV) of the ELISA absorbance were calculated. The sensitivity was calculated as the T/C value of X-2 × SD versus the concentration of linezolid in the standard curve of figure 5.

TABLE 10 ELISA method repeated detection results of 0ug/ml linezolid sample

The absorbance values of X-2 SD in the data of Table 10 as y values were substituted into the equation corresponding to the standard curve of FIG. 5 to give a concentration value of 0.28ug/ml, i.e., a sensitivity of 0.28 ug/ml.

3) Precision and accuracy deviation of Linezolid detection by ELISA method

The established ELISA detection systems are used for detecting the linezolid standard substance with the concentration of 3.125ug/ml and 25ug/ml respectively, the detection is repeated for 15 times, and the precision (CV) of detecting the linezolid with high and low concentrations and the accuracy deviation of the detection are calculated. Tables 11 and 12 show the precision and accuracy results of detecting the concentrations of linezolid, which show that the precision of the concentrations is less than 8% and the deviation of the accuracy is less than 5%.

TABLE 11 results of ELISA method for repeated detection of 25ug/ml linezolid standard

TABLE 12 results of ELISA assay for repeated detection of 3.125ug/ml linezolid standard

4) ELISA method for detecting cross reaction of linezolid

The cross reaction result is consistent with the result of detecting linezolid by a fluorescence immunochromatography method.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. A hapten having a structure according to formula 2:

in the formula, R is-CH₂-；

n is an integer of 4 to 6.

2. The hapten of claim 1, wherein n is 4.

3. A complete antigen having the structure of formula 3:

wherein the Protein is a Protein carrier, and the Protein carrier is bovine serum albumin or ovalbumin; and

r and n are as defined in claim 1.

4. A method of preparing the hapten of claim 1 or 2, the method comprising the steps of:

(a) deacetylating linezolid to give deacetylated linezolid

Linezolid

Deacetylated linezolid;

(b) reacting deacetyllinezolid with a diacid compound or a esterifiable cycloalkandione compound to give the hapten according to claim 1 or 2.

5. The method of claim 4, wherein the conditions of step (b) are as follows: the reaction temperature is 16-30 ℃; the reaction time is 20-50 minutes; the reaction solvent is methanol, ethanol, THF, DMF, dichloromethane or tetrahydrofuran.

6. The method of claim 5, wherein the conditions of step (b) are as follows: the reaction temperature is 25-28 ℃; the reaction time is 30-40 minutes; the reaction solvent is methanol, dichloromethane or tetrahydrofuran.

7. The method of claim 6, wherein the conditions of step (b) are as follows: the reaction temperature is 25 ℃; the reaction time was 35 minutes; the reaction solvent is dichloromethane/tetrahydrofuran.

8. A method of preparing the complete antigen of claim 3, the method comprising the steps of:

linking the hapten of claim 1 or 2 to a protein carrier, which is bovine serum albumin or ovalbumin, to produce the complete antigen of claim 3.

9. The method of claim 8, wherein the conditions for attaching the resulting hapten to a protein carrier are as follows: the reaction temperature is 20-28 ℃; the reaction pH is 7.0-8.0; the reaction time is 1-6 hours.

10. The process of claim 9, wherein the reaction temperature is 23-28 ℃; the reaction pH is 7.2-7.6; the reaction time is 3-4 hours.

11. The process of claim 10, wherein the reaction temperature is 25 ℃; the reaction pH was 7.5; the reaction time was 3 hours.

12. Use of the hapten of claim 1 or 2 or the complete antigen of claim 3 for the preparation of a monoclonal antibody specific for linezolid.

13. A monoclonal antibody produced using the complete antigen of claim 3 and specifically binding to linezolid, wherein the monoclonal antibody is produced by a mouse hybridoma cell line deposited with the China center for type culture Collection with the accession number of CCTCC NO: C201614.

14. The monoclonal antibody of claim 13, wherein said monoclonal antibody detects linezolid with a sensitivity of 0.28 ug/ml.

15. The monoclonal antibody of claim 13, wherein said monoclonal antibody does not bind other antibiotics; the other antibiotics are vancomycin, teicoplanin and fluconazole.

16. The monoclonal antibody of claim 13, wherein said monoclonal antibody does not bind dobutamine hydrochloride, dopamine hydrochloride, midazolam, furosemide, dexamethasone phosphate, or omeprazole.

17. A hybridoma cell line producing the monoclonal antibody of any one of claims 13-16, which is a mouse hybridoma cell line deposited with the chinese type culture collection with a collection number of CCTCC NO: C201614.

18. Use of the monoclonal antibody of any one of claims 13-16 for the preparation of a reagent, a test card or a kit for the detection of linezolid in a sample.

19. The use of claim 18, wherein the sample is a biological sample.

20. The use of claim 19, wherein the sample is a blood sample.

21. A method for the non-diagnostic in vitro detection of the presence or absence of linezolid in a biological sample comprising the steps of:

(a) contacting the biological sample with the monoclonal antibody of any one of claims 13-16;

22. The method of claim 21, wherein said monoclonal antibody carries a detectable label.

23. The method of claim 22, wherein the marker is selected from the group consisting of: a colloidal gold label, a colored label, or a fluorescent label.

24. The method of claim 22 or 23, wherein the detection method is a fluorescence detection method.

25. A fluorescence immunochromatographic assay card for detecting linezolid, the assay card comprising a substrate; a liquid absorbing member; a detection section; the sample adding component is characterized in that the detection component is fixed on the substrate, the middle part of the detection component is provided with a quality control band and a detection band, the two ends of the detection component are fixed with the imbibing component and the sample adding component in a partially overlapped mode, wherein the detection band is coated with the complete antigen of claim 3, and the quality control band is coated with rabbit antibody IgG.

26. The immunochromatographic assay card of claim 25, further comprising a card housing, wherein the card housing is composed of a lower cover and an upper cover, the upper cover is provided with a sample application window and a detection window, the linezolid immunochromatographic assay card is completely arranged in the lower cover, the detection window corresponds to the quality control zone and the detection zone on the linezolid immunochromatographic assay card, and the sample application window corresponds to the sample application part on the linezolid immunochromatographic assay card.

27. The fluorescence immunochromatographic detection card of claim 26, wherein the upper cover is further provided with a product number area; a bar code identification area.

28. The fluorescence immunochromatographic test card of claim 25, wherein said substrate is a black PVC substrate.

29. The fluorescence immunochromatographic detection card of claim 25, wherein the detection member is a nitrocellulose membrane.

30. The fluorescence immunochromatographic detection card of claim 25, wherein the sample addition member is glass fiber.

31. The fluorescence immunochromatographic detection card of claim 25, wherein the liquid absorbing member is a water absorbing paper.

32. A test kit for detecting linezolid, the kit comprising:

(a) linezolid fluorescent immunochromatographic detection card of any one of claims 25 to 31; and

(b) a linezolid detection assay that is matched with the linezolid fluorescence immunochromatographic detection card of any one of claims 25-31; and

(c) an instruction manual for detecting the linezolid by using the linezolid detection kit;

wherein the detection assay solution is a detection assay solution comprising the monoclonal antibody of any one of claims 13-16 and an anti-rabbit IgG antibody that are fluorescently labeled.

33. The test kit of claim 32, wherein the fluorescent dye for detecting the label in the analyte solution comprises: FITC, Alexa Fluor 647, CFTM647, TRITC, and CAL Fluor (R) Red 610.

34. The test kit of claim 32, wherein the solvent portion of the test assay is a phosphate buffer containing BSA.