CN-122011194-A - Amitriptyline antibody and kit

Abstract

The invention belongs to the technical field of biochemical engineering, and particularly relates to an amitriptyline antibody and a kit. The structural formula of the amitriptyline hapten is shown as a formula (I) or a formula (II): . The antibody of amitriptyline prepared by the invention has high titer, strong specificity and high affinity, the LOD of the amitriptyline by using the established Elisa method is 0.013ng/mL, the IC50 is 0.865ng/mL, the quantitative detection range is 0.07-470.3ng/mL, the detection sensitivity is high, and the linear range is wide.

Inventors

• GUO CHENGXIAN
• LEI HONGTAO
• LIU YANTING
• TAN HONGYI
• YIN JIYE
• GONG YAFANG
• ZHOU BOTING

Assignees

• 中南大学

Dates

Publication Date

20260512

Application Date

20251204

Claims (6)

1. 1. An amitriptyline antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3, and the light chain variable region comprises a light chain CDR1, a light chain CDR2 and a light chain CDR3, and is characterized in that the sequence of the heavy chain CDR1 is shown in SEQ ID NO.4, the sequence of the heavy chain CDR2 is shown in SEQ ID NO.6, the sequence of the heavy chain CDR3 is shown in SEQ ID NO.8, the sequence of the light chain CDR1 is shown in SEQ ID NO.11, the sequence of the light chain CDR2 is RAN, and the sequence of the light chain CDR3 is shown in SEQ ID NO. 13.
2. 2. Amitriptyline antibody according to claim 1, wherein the heavy chain variable region is as shown in SEQ ID No.1 and/or the light chain variable region is as shown in SEQ ID No. 2.
3. 3. Amitriptyline antibody according to claim 1, which is prepared from amitriptyline hapten or amitriptyline antigen, wherein the structural formula of the amitriptyline hapten is shown as formula (I) or formula (II): ; The structural formula of the amitriptyline antigen is shown as a formula (III) or (IV): ; Wherein Z is a carrier protein.
4. 4. Amitriptyline antibody according to claim 3, wherein the carrier protein is chicken ovalbumin or bovine serum albumin.
5. 5. A kit for detecting amitriptyline, characterized in that the kit for detecting amitriptyline comprises an amitriptyline antibody according to any one of claims 1 to 4.
6. 6. The kit for detecting amitriptyline according to claim 5, which comprises an enzyme-labeled secondary antibody, a substrate chromogenic solution, a stop solution, a washing solution, a diluent, a blocking solution, an enzyme-labeled plate coated with amitriptyline antigen and the amitriptyline antibody according to any one of claims 1 to 4; The structural formula of the amitriptyline antigen is shown as a formula (III) or (IV): ; Wherein Z is a carrier protein.

Description

Amitriptyline antibody and kit The application relates to an amitriptyline hapten and a preparation method thereof, an amitriptyline antigen and a preparation method thereof, an antibody, a kit and application thereof, which are separate applications of Chinese patent applications with application numbers 2025118120330 and application dates 2025.12.04. Technical Field The invention belongs to the technical field of biochemical engineering, and particularly relates to an amitriptyline antibody and a kit. Background Amitriptyline, also known as AMITRIPTYLINE, N, N-dimethyl-3- [ 10, 11-dihydro-5H-dibenzo [ a, d ] cycloheptatrien-5-ylidene ] -1-propylamine, has the molecular formula C 20H23 N. The structural formula is as follows:。 Amitriptyline is a tricyclic antidepressant, is used for treating various depression or depression states, has good curative effects on endogenous depression and climacteric depression, and is also effective on reactive depression and depression states of neurosis. The amitriptyline has better curative effect than imipramine on patients with anxiety and depression symptoms. The combination of the above-mentioned drugs with electric shock can reduce the number of times of electric shock. And can be used for relieving chronic pain and treating infantile enuresis and childhood hyperkinetic syndrome. Amitriptyline, however, has the characteristic of treating narrow window and significant inter-individual pharmacokinetic differences. The effective blood concentration range is close to the potential poisoning concentration, and the metabolic process is easily influenced by various factors such as age, liver and kidney functions, combined medication, genetic polymorphism and the like. Therefore, it is very urgent to establish an immunoassay method for detecting amitriptyline content in serum. The key point of the establishment of the immune detection method is to design a proper amitriptyline artificial antigen and obtain an antibody with high sensitivity and strong specificity. However, no report on amitriptyline antibodies is known. Disclosure of Invention The invention aims to provide an amitriptyline antibody and a kit for solving the problem of low affinity of the existing amitriptyline antibody. In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: An amitriptyline hapten has a structural formula shown in a formula (I) or a formula (II): 。 The amitriptyline hapten shown in the formula (I) is named as 4- ((3- (10, 11-dihydro-5H-dibenzo [ a, d ] [7] annulen-5-ylidene) propyl) (methyl) amino) butanoic acid, namely 4- ((3- (10, 11-dihydro-5H-dibenzo [ a, d ] [7] benzocyclohepten-5-ylmethylene) propyl) (methyl) amino) butyric acid by adopting a systematic naming method. The amitriptyline hapten shown in the formula (II) is named as 6- ((3- (10, 11-dihydro-5H-dibenzo [ a, d ] [7] annulen-5-ylidene) propyl) (methyl) amino) hexanoic acid, namely 6- ((3- (10, 11-dihydro-5H-dibenzo [ a, d ] [7] cyclononen-5-subunit) propyl) (methyl) amino) hexanoic acid by adopting a systematic naming method. According to the embodiment of the invention, the invention can be further optimized, and the following technical scheme is formed after the optimization: Based on the same inventive concept, the invention also claims a preparation method of the amitriptyline hapten, which comprises the following steps: dissolving 3- (10, 11-dihydro-5H-dibenzo [ a, d ] cycloheptene-5-subunit) -N-methyl-1-propylamine hydrochloride, adding weak base and bromoester, stirring at 50-70 ℃ for reaction, separating and purifying the obtained product, drying, dissolving, and adding trifluoroacetic acid for reaction to obtain the amitriptyline hapten; When the structural formula of the amitriptyline hapten is shown as a formula (I), the bromo ester is tert-butyl 4-bromobutyrate; When the structural formula of the amitriptyline hapten is shown as a formula (II), the bromo ester is tert-butyl 6-bromohexanoate. In one preferred embodiment, the weak base is triethylamine or anhydrous potassium carbonate. In one of the preferred embodiments, the reaction is stirred at 50-70℃for 2-4h. In one preferred embodiment, the separation and purification steps are extraction and silica gel column chromatography of the obtained product. In one preferred embodiment, the extraction is carried out by adopting an ethyl acetate aqueous solution, and collecting an ethyl acetate phase, wherein the volume ratio of ethyl acetate to water in the ethyl acetate aqueous solution is 1-2:1-2. In one preferred embodiment, the developing agent for the silica gel column chromatography is a mixed solution of petroleum ether and ethyl acetate, wherein the volume ratio of petroleum ether to ethyl acetate in the mixed solution is 2-4:1. In the silica gel column chromatography process, a sample is added, gradient elution is carried out by using a developing agent, and a target product is collected. In one preferred embodiment,