CN114685648B - Theophylline complete antigen and preparation method and application thereof - Google Patents

Theophylline complete antigen and preparation method and application thereof Download PDF

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CN114685648B
CN114685648B CN202011563628.4A CN202011563628A CN114685648B CN 114685648 B CN114685648 B CN 114685648B CN 202011563628 A CN202011563628 A CN 202011563628A CN 114685648 B CN114685648 B CN 114685648B
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theophylline
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monoclonal antibody
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antigen
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CN114685648A (en
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李忠鹏
何扬锦
陈颖
费晓旭
周晓静
张盛朝
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Zhejiang Quasi Policy Biological Technology Co ltd
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Abstract

The invention discloses a theophylline complete antigen and application thereof in preparation of a theophylline antibody and detection by an immunoassay method. The theophylline complete antigen has a structure shown in a formula I, wherein R is optionally substituted C1-C3 alkyl; preferably optionally substituted methyl; protein is a Protein carrier. The invention also discloses an anti-theophylline monoclonal antibody prepared by the complete antigen, a hybridoma cell for producing the monoclonal antibody, and a detection card and a detection kit for detecting the theophylline analogue. The invention discloses an ELISA method and a fluorescence immunochromatography method for detecting theophylline analogues, wherein the fluorescence immunochromatography method has the advantages of simple operation, short detection time, low cost, high specificity and good repeatability.

Description

Theophylline complete antigen and preparation method and application thereof
Technical Field
The invention relates to the field of detection. In particular, the invention relates to a theophylline complete antigen, a theophylline monoclonal antibody obtained by using the complete antigen and application of the theophylline monoclonal antibody in detecting theophylline by using a fluorescent immunochromatography method.
Background
Theophylline is applied to clinic for half a year, but has a narrow safety treatment window due to a plurality of adverse reactions, and after inhaled glucocorticoids (ICS) and beta 2 receptor agonists are widely applied to clinical treatment of asthma and have better curative effects, the position of the theophylline in the treatment of asthma gradually declines to the third and fourth positions. The advent of the theophylline sustained-release preparation effectively improves the clinical application safety of the theophylline medicament, leads the status of the theophylline in the asthma therapeutic medicament to rise, formally lists the sustained-release theophylline medicament as a medicament for controlling asthma symptoms in the global asthma control initiative (GINA) in 2006, and indicates by the Chinese bronchial asthma control guidelines in 2008 that ICS+the sustained-release theophylline medicament can be used as a first-line therapeutic scheme of chronic asthma which cannot be controlled by ICS alone in the poor regions of China. Currently, more than 300 theophyllines and derivatives thereof are clinically known, and the most commonly used theophyllines are: theophylline, aminophylline, doxofylline, and the like.
According to the statistical results of the literature, various factors such as age, individual difference, dosage form, combined medication, concurrent pathological conditions (liver insufficiency) and the like can influence the pharmacokinetic parameters of theophylline, so that the blood concentration of the theophylline has large inter-individual difference. The safe and effective range of the theophylline is narrow, the optimal concentration is 10-20 mug/ml, when the optimal concentration exceeds 20 mug/ml, the toxic and side effects are increased, the individual difference of the theophylline metabolism is large, and the theophylline is extremely easily influenced by diseases and other medicines, so that the medicine monitoring of the theophylline becomes an auxiliary means for safe medicine application. In summary, in order to further optimize and standardize the use of theophylline in different populations, it is necessary to monitor the blood concentration of theophylline (therapeutic drug monitoring, TDM) in a specific population by adjusting the dosing regimen according to the pathophysiological condition of the patient.
At present, the detection of theophylline mainly comprises ultraviolet spectrophotometry, high performance liquid chromatography, gas chromatography, enzyme-free method and the like, and the instrument is expensive, and the operation is complex and time-consuming. The fluorescent quantitative immunochromatography technology is a novel quantitative detection technology combining the advantages of the immunofluorescence technology and the traditional immunochromatography technology. The technology has flexible and simple operation, lower cost and short reaction time, and can realize timely quantitative detection. The medicine taking device can adjust the medicine taking of a patient in time for improving the data support, achieves the aim of individual medicine taking, ensures the curative effect and ensures the medicine taking safety.
Disclosure of Invention
The invention aims to provide a complete antigen of theophylline, by which a specific antibody of the theophylline can be obtained.
It is another object of the present invention to provide a detection reagent or detection kit comprising the theophylline-specific antibody of the present invention, and a method for quantitatively determining theophylline using the antibody or detection kit.
In a first aspect, the present invention provides a theophylline complete antigen having the structure shown in formula I:
Wherein R is optionally substituted C1-C3 alkyl; preferably optionally substituted methyl;
protein is a Protein carrier.
In a specific embodiment, the complete antigen has a structure represented by formula II:
wherein R and Protein are as defined in claim 1.
In a preferred embodiment, the "optionally substituted" means substituted with one or more lower alkyl, hydroxy or halogen.
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), etc.
In specific embodiments, the protein carrier is Bovine Serum Albumin (BSA), or Ovalbumin (OVA).
In a second aspect, the present invention provides a method of preparing the theophylline complete antigen of the first aspect, the method comprising the steps of:
Linking theophylline to a protein carrier via a linking agent to produce the complete antigen of any one of claims 1-3.
In a preferred embodiment, the linking agent is glutaraldehyde.
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), etc.
In a preferred embodiment, the protein carrier is Bovine Serum Albumin (BSA), or Ovalbumin (OVA).
In a preferred embodiment, the conditions for the attachment of theophylline to the protein carrier are as follows: the reaction temperature is 20-35 ℃, preferably 25-32 ℃, more preferably 30 ℃; the reaction pH is from 6.0 to 9.0, preferably from 7.2 to 9.0, more preferably 8.4; the reaction time is 4 to 9 hours, preferably 7 to 9 hours, more preferably 8 hours.
In a third aspect, the present invention provides the use of the theophylline complete antigen of the first aspect for the preparation of a specific monoclonal antibody to theophylline.
In a fourth aspect, the invention provides a monoclonal antibody that specifically binds theophylline.
In a preferred embodiment, the monoclonal antibody is produced by a mouse hybridoma cell line which is deposited with the China center for type culture Collection (CCTCC, china, university of Wuhan, wuhan) under accession number C202081; the classification is named: hybridoma C214238.
In a preferred embodiment, the monoclonal antibody detects theophylline with a sensitivity lower than 2ng/mL; preferably about 1.88ng/mL.
In a preferred embodiment, the monoclonal antibody does not bind to other asthma therapeutic agents; such other asthma treatment drugs include, but are not limited to, budesonide, terbutaline, cefoperazone.
In a fifth aspect, the present invention provides a hybridoma cell line producing the monoclonal antibody of the fourth aspect, which is deposited with the China center for type culture collection (CCTCC, china, the university of Wuhan, and the university of Wuhan) under accession number C202081; the classification is named: hybridoma C214238.
In a sixth aspect, the present invention provides the use of a monoclonal antibody according to the fourth aspect for the preparation of a reagent, test card or kit for the detection of theophylline in a sample.
In a preferred embodiment, the sample is a biological sample, preferably a plasma sample.
In a seventh aspect, the present invention provides a method of detecting the presence or absence of theophylline in a biological sample, the method comprising the steps of:
(a) Contacting the biological sample with the monoclonal antibody of the fourth aspect;
(b) Detecting whether an antigen-antibody complex is formed, wherein the formation of a complex indicates the presence of theophylline in the sample.
In a preferred embodiment, the monoclonal antibody carries a detectable label; preferably, the marker is selected from the group consisting of: colloidal gold labels, colored labels or fluorescent labels.
In a preferred embodiment, the detection method is a fluorescent detection method.
In a preferred embodiment, the detection method is non-diagnostic in vitro.
In an eighth aspect, the present invention provides a fluorescent immunochromatographic assay card for detecting theophylline, said assay card comprising a substrate; a liquid-absorbing member; a detection section; the sample adding component is characterized in that the detecting component is fixed on the substrate, a quality control belt and a detecting belt are arranged in the middle of the detecting component, the liquid absorbing component and the sample adding component are fixed at two ends of the detecting component in a partially overlapped mode, the detecting belt is coated with the theophylline complete antigen of the first aspect, and the quality control belt is coated with rabbit antigen IgG.
In a preferred embodiment, the theophylline fluorescence immunochromatography detection card further comprises a card box, 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 theophylline 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 part, a quality control belt and a detection belt on the theophylline fluorescence immunochromatography detection card.
In a preferred embodiment, the upper cover is further provided with a product numbering zone; a bar code identification area.
In a preferred embodiment, the substrate is a dark colored rigid substrate; black PVC substrates are preferred.
In a preferred embodiment, the detection member is a nitrocellulose membrane.
In a preferred embodiment, the loading member is a glass fiber.
In a preferred embodiment, the absorbent member is absorbent paper.
In a ninth aspect, the present invention provides a test kit for detecting theophylline, said kit comprising:
(a) The theophylline fluorescence immunochromatography detection card of the eighth aspect;
(b) A theophylline detection assay solution mated with the theophylline fluorescent immunochromatographic assay card of the eighth aspect;
(c) The theophylline detection kit detects the instruction manual of theophylline;
wherein the detection analyte is a detection analyte comprising a fluorescent-labeled monoclonal antibody according to the fourth aspect and an anti-rabbit IgG antibody.
In a preferred embodiment, the fluorescent dye for labeling in the detection assay includes, but is 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 detection assay is a phosphate buffer containing BSA.
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Drawings
FIG. 1 shows UV scan patterns of theophylline immunogens and coating immunogens;
FIG. 2 shows a schematic diagram of the structure of a theophylline fluorescence immunochromatographic assay card; wherein:
FIG. 2A shows a test card configuration without a plastic card; 1: a black PVC substrate; 2: absorbent paper: 3: a nitrocellulose membrane; 4: glass fibers; 5: a quality control line (C line); 6: a detection line strip (T line);
FIG. 2B shows a theophylline test card configuration with a plastic cartridge; 1': a lower cover; 2': an upper cover; 3': a sample application window; 4': glass fibers; 5': a detection window; 6': a quality control line (C line); 7': a detection line strip (T line); 8': a nitrocellulose membrane; 9': theophylline project mark; 10': a bar code identification area;
FIG. 3 shows a standard curve pattern of theophylline fluorescence immunochromatographic assay;
Figure 4 shows a standard curve profile of theophylline ELISA assay.
Detailed Description
The inventor synthesizes a complete antigen of theophylline through long-term and intensive research, uses the complete antigen as an immunogen to immunize a Balb/C mouse, fuses spleen cells of the Balb/C mouse with myeloma SP20 cells of the mouse to obtain a hybridoma cell strain capable of specifically secreting an anti-theophylline monoclonal antibody, further prepares and purifies the anti-theophylline monoclonal antibody to obtain the theophylline monoclonal antibody, and then prepares a theophylline immunodetection card with high sensitivity and specificity by using the complete antigen and the theophylline antibody. On this basis, the present invention has been completed.
Complete antigen
Substances having immunogenicity and immunoreactivity, called complete antigens (complete antigens), such as most proteins, bacteria, viruses, bacterial exotoxins, animal serum, etc. The complete antigen can stimulate the organism to produce antibody or sensitized lymphocyte and can also generate specific binding reaction with the complete antigen in vivo and in vitro.
Typically, haptens need to be coupled covalently or with macromolecules such as Bovine Serum Albumin (BSA), ovalbumin (OVA) or hemocyanin (KLH) to become complete antigens that are both immunoreactive and immunogenic.
As used herein, the term "complete antigen" refers to the product of the theophylline hapten of the present invention in combination with a suitable protein carrier. As used herein, the term "protein carrier" refers to any immunologically acceptable protein for forming complete antigens, including but not limited to: bovine Serum Albumin (BSA), or Ovalbumin (OVA) is preferred in the present invention, as are Bovine Serum Albumin (BSA), ovalbumin (KLH), human Serum Albumin (HSA), and synthetic Polylysine (PLL).
The theophylline complete antigen has a structure shown in a formula I:
Wherein R is optionally substituted C1-C3 alkyl; preferably optionally substituted methyl;
protein is a Protein carrier.
Herein, the term "optionally substituted" means substituted with one or more lower alkyl, hydroxy or halogen, unless such substitution is clearly not feasible.
In a preferred embodiment, theophylline is bound to a suitable protein carrier using glutaraldehyde as a linking agent. Therefore, the complete antigen of theophylline of the present invention has the structure shown in formula II:
wherein R and Protein are as described above.
In a specific embodiment, the conditions for the attachment of the theophylline hapten to the protein carrier are as follows: the reaction temperature is 20-35 ℃, preferably 25-32 ℃, more preferably 30 ℃; the reaction pH is from 6.0 to 9.0, preferably from 7.2 to 9.0, more preferably 8.4; the reaction time is 4 to 9 hours, preferably 7 to 9 hours, more preferably 8 hours.
Preparation of monoclonal antibodies
The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homologous antibodies, i.e., the individual antibodies comprising the population are identical except for the possible presence of a small number of spontaneous mutations. Thus, the modifier "monoclonal" refers to a mixture of antibodies whose properties are not discrete.
Antibodies of the invention may be prepared by various techniques known to those skilled in the art. For example, the complete antigens of the invention may be administered to animals to induce monoclonal antibody production. For monoclonal antibodies, hybridoma technology can be used to prepare (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, N.Y., 1981) or can be prepared using recombinant DNA methods (U.S. Pat. No. 4,816,567).
Representative myeloma cells are those which fuse efficiently, support stable high levels of antibody production by the antibody-producing cell of choice, and are sensitive to the medium (HAT medium matrix), including myeloma cell lines, e.g., murine myeloma cell lines, including those 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 C μ Lture Collection, rocyveromyces, 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 antibody production techniques and uses (Monoclonal Antibodies Production Techniques and Applications), pages 51-63 (MARCEL DEKKER, inc., new york, 1987).
The culture medium in which the hybridoma cells are grown is analyzed to detect the production of monoclonal antibodies having the desired specificity, such as by an in vitro binding assay, e.g., an enzyme-linked immunosorbent assay (ELISA) or a Radioimmunoassay (RIA). The location of cells expressing the antibody can be detected by FACS. The hybridoma clones can then be subcloned by limiting dilution steps (subcloned) and grown by standard methods (Goding, monoclonal antibody (Monoclonal Antibodies): principles and practices (PRINCIPLES AND PRACTICE), ACADEMIC PRESS (1986) pages 59-103). Suitable media for this purpose include, for example, DMEM or RPMI-1640 medium. In addition, hybridoma cells can grow as ascites tumors in animals.
Monoclonal antibodies secreted by the subclones are suitably isolated from culture medium, ascites fluid or serum by conventional immunoglobulin purification procedures such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography.
The monoclonal antibody is produced by a mouse hybridoma cell line which is preserved in China center for type culture collection (CCTCC, china, the university of Wuhan, and the university of Wuhan) for 3 months and 4 days in 2020, and the preservation number is C202081; the classification is named: hybridoma C214238.
In a specific embodiment, the monoclonal antibodies of the invention bear a detectable label. More preferably, the marker is selected from the group consisting of: colloidal gold labels, colored labels or fluorescent labels.
In a specific embodiment, the monoclonal antibody of the invention has a theophylline detection sensitivity of 1.88 μg/mL. The monoclonal antibodies of the invention do not cross-react with the carrier protein of the theophylline complete antigen, e.g., BSA or OVA. Further, the monoclonal antibodies of the invention also do not bind to other asthma treatment drugs including, but not limited to, budesonide, terbutaline, cefoperazone.
Detection kit
The detection kit of the present invention refers to a kit containing the monoclonal antibody of the present invention and useful for detection of theophylline analogues. The kit can comprise a container, instructions for use, buffers, immunoassays, etc., as needed.
The test kit of the present invention may take various forms, such as a test card, a test kit containing various reagents required for the test, and the like. The kit of the present invention is described using a test card as an example in the examples, but it should not be construed that the kit of the present invention is limited to the test card.
In a specific embodiment, the fluorescent immunochromatographic assay card for detecting theophylline of the present invention comprises a substrate; a liquid-absorbing member; a detection section; and a loading component; the detection part is fixed on the substrate, the quality control belt and the detection belt are arranged in the middle of the detection part, the liquid absorbing part and the sample adding part are fixed at two ends of the detection part in a partially overlapped mode, wherein the detection belt is coated with the complete antigen of the invention, and the quality control belt is coated with rabbit antigen IgG.
In a preferred embodiment, the theophylline fluorescence immunochromatography detection card further comprises a card box, 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 theophylline fluorescence immunochromatography detection card is completely arranged in the lower cover, and the detection window and the sample adding window correspond to a sample adding part, a quality control belt and a detection belt on the theophylline fluorescence immunochromatography detection card respectively. The upper cover can be also provided with a product numbering area; a bar code identification area. The substrate may be a dark colored rigid substrate; black PVC substrates are preferred. The detection member may be a nitrocellulose membrane. The loading member may be a glass fiber. The absorbent member may be absorbent paper.
By "partially overlapping secured" as used herein is meant that two adjacent components form a certain overlap region, rather than a complete overlap in which one component is fully contained within the other component, and the two components are secured by the overlap region. The manner of fixation may be selected autonomously by the person skilled in the art, for example by means of gluing or the like.
On the basis of the detection card, the invention also provides a detection kit for detecting theophylline, which is provided with:
(a) The theophylline fluorescence immunochromatography detection card;
(b) The theophylline detection analysis liquid is matched with the theophylline fluorescence immunochromatography detection card;
(c) Instructions for detecting a theophylline analog using the theophylline analog detection kit;
wherein the detection analyte is a detection analyte containing the fluorescent-labeled monoclonal antibody and the anti-rabbit IgG antibody.
The fluorescent markers may be selected autonomously by those skilled in the art as desired, including but not limited to FITC (Fluorescein), alexa Fluor 647, CFTM647, TRITC (Rhodamine), CAL Fluor (R) Red610, and the like.
In a preferred embodiment, the solvent portion of the detection assay is a phosphate buffer containing BSA.
Immunodetection application of theophylline complete antigen
The theophylline complete antigen is applied to antibody preparation, and the antibody is a monoclonal antibody or a polyclonal antibody; the corresponding antibody prepared by the theophylline complete antigen is applied to various immunological detection fields for detecting the content of the theophylline analogue, including but not limited to the immunological detection fields such as ELISA, a chemiluminescence method, a colloidal gold method, a fluorescence immunochromatography method and the like.
The application of the theophylline complete antigen in antibody preparation refers to the preparation of anti-theophylline polyclonal antibodies and monoclonal antibodies by utilizing the theophylline complete antigen and utilizing the complete antigen to immunize experimental animals; the laboratory animals should not be construed as pure mice in the embodiments, and should include, but are not limited to: mice, rats, rabbits, goats, sheep, horses, donkeys, chickens, dogs, and other laboratory animals.
The application of the theophylline complete antigen in the field of determination of theophylline immunodetection refers to the establishment of various immunodetection methods for detecting the content of theophylline analogues by using corresponding antibodies prepared from the theophylline complete antigen as immunodetection raw materials. The immunodetection field comprises, but is not limited to, ELISA, chemiluminescence, colloidal gold, fluorescence immunochromatography and other immunological detection methods; the immune detection method for detecting the theophylline analogue not only carries out specified quantity detection, but also comprises semi-quantitative detection and various qualitative detection methods based on immunological detection.
In specific embodiments, the invention takes the preparation of specific monoclonal antibodies by immunized mice as an example, and uses ELISA and fluorescent immunochromatography as specific examples to illustrate the application of theophylline complete antigen in the detection of theophylline immunology.
The invention has the advantages that:
1. The invention discloses a structure of theophylline complete (artificial) antigen and a preparation method thereof for the first time;
2. the invention discloses the application of the theophylline complete antigen in the fields of theophylline antibody preparation and immunological detection for the first time, and provides a reliable method for promoting clinical blood concentration detection of theophylline and analogues thereof;
3. the monoclonal antibody can detect theophylline and analogues thereof with high sensitivity, and is not combined with other medicines;
4. The theophylline analogue detection kit can simply and rapidly detect theophylline and analogues thereof on site.
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer.
Examples.
EXAMPLE 1 preparation of theophylline complete antigen (immunogen and coating antigen)
Theophylline (hapten) was coupled to Bovine Serum Albumin (BSA) and Ovalbumin (OVA), respectively, by EDC. The specific coupling method is as follows:
10mg of theophylline is weighed and dissolved in 2ml of DMF to form a final concentration of 10mg/ml, 400 μl of theophylline (10 mg/ml) is taken and mixed with 2 μl of 20% glutaraldehyde, then added to 1.5ml of 10mg/ml BSA solution (or added to 1.5ml of 7.5mg/ml OVA solution), and after uniform mixing, reacted for 3 hours under stirring at room temperature in the absence of light. Dialysis was performed 4 times with phosphate buffer and the solution was changed once for 12 hours. The dialysate was collected and the concentrations of immunogen and coating antigen were determined to be 8.3mg/ml and 4.6mg/ml using Quick Start Bradford Protein Assay Kit from BIO-RAD, inc. The structural formula of the obtained theophylline artificial antigen (immunogen and coating antigen) is shown as follows, wherein "Protein" is BSA (bovine serum albumin) or OVA (ovalbumin)
Comparison of the results of UV scanning peaks for theophylline artificial antigen (immunogen and coating) as shown in FIG. 1, the peaks of the conjugate were distinguished from the peaks of BSA and OVA, indicating successful conjugation.
EXAMPLE 2 preparation of monoclonal antibodies Using theophylline complete antigen
1. Immunization of animals
The theophylline immunogen obtained in example 1 was diluted to 0.2mg/mL, 500. Mu.L of the immunogen was mixed with an equal volume of Freund's complete adjuvant, emulsified completely, and BALB/c mice (Shanghai Laek laboratory animal Co., ltd.) were immunized by injection subcutaneously on the backs and feet of the mice. The first immunization was with complete Freund's adjuvant followed by incomplete Freund's adjuvant. One week after the fourth immunization, the orbit was bled, serum was isolated, and the titers of anti-theophylline antibodies were measured. The antibody titer of the mice after four immunizations was 1:256,000 as measured by ELISA.
2. Cell fusion and screening
Four immunized mice were boosted again by intraperitoneal injection of about 100 μg of immunogen, and after 3 days, the spleens of the mice were taken for fusion. Mixing SP2/0 cells (Nanjing medical science college) with spleen cells, adding serum-free culture solution (Hyclone SH30022.018 DMEM (High Glucose)), centrifuging (1500 rpm,3 min), collecting precipitated cells, dropwise adding 1mL 50% polyethylene glycol 4000, and standing for 90 seconds. Then 10mL of serum-free culture medium preheated at 37 ℃ is added dropwise, and the mixture is kept stand for 5min. After the fusion, the cell suspension was centrifuged (1000 rpm,3 min), and the whole culture was inoculated into a 96-well plate with feeder cells, 2X 10 4/mL myeloma cells per well. Culturing in a 5% CO 2 incubator at 37deg.C for two days, and adding 2 XHAT complete culture solution to obtain final concentration of 1 XHAT in the well. When the hybridoma cell colony grows to 1/10-1/5 area of the hole bottom, the ELISA method is used for screening the fusion cell antibody positive hole.
3. Ascites production and antibody purification
BALB/c mice were given an intraperitoneal injection of 0.5mL of paraffin oil, and after 7 days, 0.5mL of 1X 10 6 positive hybridoma cells were intraperitoneally injected. And (3) observing the growth condition of the mice, wherein abdominal bulge is visible about 7 days, and collecting ascites in time. The monoclonal antibody with high purity is obtained by purification through an affinity chromatography technology (Protein G Resin affinity purification), and the Protein amount is 4mg.
EXAMPLE 3 immunoassay Using theophylline complete antigen
1. Fluorescent immunochromatographic assay
1) Preparation of detection analysis liquid
A. The monoclonal antibodies and anti-rabbit IgG antibodies obtained in example 2 were each fluorescently labeled (Hangzhou Kogyo Biotechnology Co., ltd.);
b. the fluorescence-labeled antibody was diluted with a phosphate buffer containing BSA to prepare a detection assay solution.
2) Preparation of theophylline fluorescence immunochromatography test paper card
A. The prepared theophylline coating antigen (theophylline-OVA) and rabbit antigen IgG are diluted to proper concentrations (0.6-2.5 mg/mL) respectively by using a coating buffer solution (phosphate buffer solution). Uniformly spraying diluted theophylline-OVA and rabbit antigen IgG on a nitrocellulose membrane (respectively forming a detection line and a quality control line) at 25+/-5 ℃, drying for about 1.5-2 hours under the humidity condition of 12% -30%, and drying and preserving for later use;
b. And c, respectively and sequentially pasting the coated nitrocellulose membrane, the glass fiber paper and the absorbent paper obtained in the step a on the black PVC substrate to form a detection card (shown in figure 2A), and cutting the detection card into proper widths according to requirements.
C. And c, loading the detection card obtained in the step B into a lower cover of the card box, and covering an upper cover to form the complete detection card with the card box (shown in fig. 2B).
3) Detection of
Taking 60 mu l of diluted sample and 60 mu l of detection analysis solution, taking 100 mu l of sample adding window of a detection card, reacting for 15-20 min, detecting by using a FCR fluorescence immunoassay analyzer (Suzhou and Mich precision instruments Co., ltd.), and comparing and displaying detection results according to the ratio of T line signal value to C line signal value (T/C value) of the sample and a built-in standard curve.
4) Theophylline fluorescence immunochromatography test paper card detection principle
The detection method is adopted to detect, and the theophylline antigen in the sample and the theophylline antigen (coating antigen) on the detection line (T line) compete for binding with the fluorescent-labeled anti-theophylline antibody in the detection analysis liquid. When the concentration of the antigen in the sample is lower, the more fluorescent antibodies are combined on the detection line, and further the stronger the fluorescent signal on the detection line is, so that the larger the ratio (T/C value) of the fluorescent signal of the detection line (T line) to the fluorescent signal of the quality control line (C line) is; conversely, when the concentration of theophylline antigen in the sample is high, the T/C value is small. Therefore, the higher the theophylline content in the sample, the lower the T/C value. And comparing the T/C value with a built-in standard curve and displaying a detection result.
5) Sensitivity and standard curve for detecting theophylline by fluorescent immunochromatography
Theophylline standard was added to the blank serum and the serum was prepared to have 8 concentration gradients of 30, 20, 15, 10, 7.5, 3.75, 1.88, 0.94, and 0ng/ml, and diluted 10-fold with 0.9% nacl, respectively. The above series of concentration samples were tested according to the above test procedure, each sample was repeated 3 times, the test results of the test are shown in table 1, and a standard curve (four parameters) is drawn according to the data of table 1 with the concentrations on the abscissa and the T/C values on the ordinate, as shown in fig. 3. The equation corresponding to the curve in fig. 3 is shown in table 2, and IC 50 =6.10 ng/ml is calculated.
TABLE 1 fluorescent immunochromatography for detecting theophylline samples of different concentrations
TABLE 2 equation (four parameters) for inhibition curves
The 0ng/ml samples were repeatedly tested 10 times and the mean (X), standard Deviation (SD) and precision (CV) of the T/C values were calculated, respectively. The sensitivity was calculated by calculating the T/C values for X-2X sd as shown in table 3 for the theophylline concentration values in the standard curve of fig. 3.
TABLE 3 repeated detection of 0ng/ml theophylline sample by fluorescence immunochromatography
The T/C value of X-2X sd in the data of table 3 was substituted as y value into the equation corresponding to the standard curve of fig. 3 to a concentration value of 1.88ng/ml, i.e. a sensitivity of 1.88ng/ml.
6) Fluorescent immunochromatography for detecting precision deviation of theophylline
And (3) detecting theophylline standard substances with the concentrations of 5ng/ml, 15ng/ml and 25ng/ml respectively by using an established theophylline detection system, repeating detection for 10 times respectively, and calculating the precision (CV) of detecting low-medium-high concentration theophylline. Table 4 shows the results of the precision of the detection of the high and low concentrations of theophylline.
TABLE 4 repeated detection of 5, 15, 25ng/ml theophylline Standard results by fluorescence immunochromatography
7) Accuracy deviation of detecting theophylline by fluorescence immunochromatography
After the standard (5 mg/mL) was diluted to 200, 100 and 20ng/mL with buffer solution, 10. Mu.L of each standard with different concentrations was added to 100. Mu.L of the low-concentration enterprise internal reference, and the detection was performed according to the above-described detection procedure, and each sample was repeated 5 times. Table 5 is the accuracy result.
TABLE 5 repeated detection of theophylline Standard results by fluorescence immunochromatography
8) Cross reaction of theophylline fluorescence immunochromatography detection system
The 3 common clinical related medicines are respectively prepared into samples with different gradient concentrations by blank mixed serum, fluorescence immunochromatography detection is carried out, and the IC 50 of the samples is calculated and compared with the IC 50 value of theophylline to calculate the cross reaction rate. The calculation formula is as follows: cross-reactivity = (IC 50 Theophylline /IC50 clinically relevant medicine )%, and the cross-reactivity results are shown in table 6:
TABLE 6 fluorescence immunochromatography detection of Cross-reaction results of clinically relevant drugs
Clinically relevant medicine Cross reaction rate
Budesonide ≦0.1％
Terbutaline ≦0.1％
Cefoperazone ≦0.1％
9) Structural analogue detection of theophylline fluorescence immunochromatography detection system
After two theophylline analogue standards (5 mg/ml) are diluted to 200, 100 and 20ng/ml by buffer solution, 10 μl of each standard with different concentration is added into 100 μl of the low-concentration enterprise internal reference, detection is carried out according to the detection steps, and each sample is repeated 5 times. Table 7 is the accuracy result.
TABLE 7 repeated detection of theophylline analog standard substance results by fluorescence immunochromatography
ELISA quantitative determination of theophylline
1) ELISA detection standard curve establishment
The prepared theophylline-coated antigen (theophylline-OVA) was diluted to 1. Mu.g/ml with a carbonic acid buffer (0.05M, pH 9.6), coated in 96-well plates, 100. Mu.l/well, overnight at 4℃and 5% nonfat dry milk was blocked for 3h, 200. Mu.l/well. Washing for 3 times and 5 min/time; theophylline standard was added to the blank plasma and diluted 10-fold to 8 concentration gradient series concentration samples of 30, 20, 15, 10, 7.5, 3.75, 1.88, 0.94, 0 ng/mL. 50 μl of theophylline with different concentrations and 50 μl of theophylline antibody are respectively mixed and added into the microwells, and incubated for 1h at 37 ℃; after 3 washes, HRP-labeled secondary antibody was added and incubated for 1h (100. Mu.l/well), and after 3 washes, color development solution was added, and the reaction was allowed to proceed at room temperature for 15min in the dark, and stop solution was added for reading (450 nm). Table 8 shows the absorbance results of the standard curve of ELISA assay, and the equation corresponding to the curve in FIG. 4 is shown in Table 9.
TABLE 8 ELISA method for detecting theophylline with different concentrations
TABLE 9 equation (four parameters) for inhibition curves
2) Sensitivity of ELISA for detecting theophylline
The negative plasma samples were repeatedly tested 10 times, and the mean (X), standard Deviation (SD) and precision (CV) of ELISA absorbance values were calculated, respectively. The sensitivity was calculated by OD values of X-2X sd corresponding to theophylline concentration values in the standard curve of fig. 4.
TABLE 10 repeated ELISA method for 0ng/ml theophylline sample results
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The absorbance of X-2X sd in the data of table 9 was substituted as y value into the equation corresponding to the standard curve of fig. 4 to a concentration value of 0.98ng/ml, i.e., a sensitivity of 0.98ng/ml.
3) ELISA method for detecting precision and accuracy deviation of theophylline
Theophylline standard substances with the concentration of 5ng/ml and 20ng/ml are detected by using an established ELISA detection system respectively, the detection is repeated for 15 times, and the precision (CV) of detecting the low-concentration theophylline and the accuracy deviation of the detection are calculated. The results show that the precision of the high concentration and the low concentration is less than 15%, and the accuracy deviation is less than 15%.
Results of repeated ELISA assays for 5, 20ng/ml theophylline standards
4) ELISA method for detecting cross reaction of theophylline
The cross reaction results are consistent with the results of detecting theophylline by fluorescence immunochromatography.
All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

Claims (8)

1. A monoclonal antibody produced by a mouse hybridoma cell line, wherein the hybridoma cell line is preserved by a China center for type culture collection (CCTCC, china, martial arts, university of martial arts) with a preservation number of C202081; the classification is named: hybridoma C214238.
2. A hybridoma cell line producing the monoclonal antibody of claim 1, deposited by the chinese collection of classical cultures (CCTCC, chinese, wuhan, university of wuhan) under accession number C202081; the classification is named: hybridoma C214238.
3. Use of the monoclonal antibody of claim 1 for the preparation of a reagent, a test card or a kit for detecting theophylline in a sample.
4. A method of detecting the presence or absence of theophylline in a biological sample, said method comprising the steps of:
(a) Contacting the biological sample with the monoclonal antibody of claim 1;
(b) Detecting whether an antigen-antibody complex is formed, wherein the formation of a complex indicates the presence of theophylline in the sample.
5. The method of claim 4, wherein the detection method is non-diagnostic in vitro.
6. A test kit for detecting theophylline, said kit comprising:
(a) Theophylline fluorescence immunochromatography detection card;
(b) The theophylline detection analysis liquid is matched with the theophylline fluorescence immunochromatography detection card;
(c) The theophylline detection kit detects the instruction manual of theophylline;
Wherein, the theophylline fluorescence immunochromatography detection card comprises a substrate; a liquid-absorbing member; a detection section; the sample adding component is characterized in that the detection component is fixed on a substrate, a quality control belt and a detection belt are arranged in the middle of the detection component, the liquid absorbing component and the sample adding component are fixed at two ends of the detection component in a partially overlapped mode, wherein the detection belt is coated with theophylline complete antigen, and the quality control belt is coated with rabbit antigen IgG;
The detection analyte is a detection analyte comprising the fluorescent-labeled monoclonal antibody and anti-rabbit IgG antibody of claim 1;
the complete antigen has a structure represented by formula II:
wherein, protein is a Protein carrier.
7. The test kit according to claim 6, wherein 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 synthetic Polylysine (PLL).
8. The test kit of claim 7, wherein the protein carrier is Bovine Serum Albumin (BSA) or Ovalbumin (OVA).
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