Hybridoma cell strain secreting modafinil monoclonal antibody and application of hybridoma cell strain

A technology of hybridoma cell lines and cloning antibodies, which is applied in the direction of analytical materials, microbial-based methods, biochemical equipment and methods, etc., can solve the problems of cumbersome steps, high cost, and time-consuming, and achieve the effect of good detection sensitivity

Inactive Publication Date: 2021-11-30
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Modafinil content analysis methods include high-performance liquid chromatography (HPLC), HPLC-UV and other instrumental methods. These detection methods have the disadvantages of time-consuming, cumbersome steps, inability to perform rapid on-site detection, and high cost. Therefore, a quick and easy method is established. The detection method of modafinil is of great significance

Method used

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  • Hybridoma cell strain secreting modafinil monoclonal antibody and application of hybridoma cell strain
  • Hybridoma cell strain secreting modafinil monoclonal antibody and application of hybridoma cell strain
  • Hybridoma cell strain secreting modafinil monoclonal antibody and application of hybridoma cell strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Preparation of hybridoma cell line 4F5

[0035] (1) Preparation of immunogen:

[0036] Weigh 3.64mg of modafinil hapten, 7.58mg of 1-ethylcarbodiimide hydrochloride, 4.57mg of N-hydroxysuccinimide, and dissolve in 800μL of anhydrous N,N-dimethylformamide, A1 solution was obtained, and the reaction was stirred at room temperature for 6 h. Take bovine serum albumin BSA 10mg, dissolve it with 2mL 0.1M borate buffer solution to obtain B1 solution, add A1 solution dropwise to B1 solution at room temperature, and react at room temperature for 8 hours to obtain the conjugate modafinil -BSA mixture solution, the complete antigen and unconjugated small molecule hapten are separated by dialysis to obtain the conjugate modafinil-BSA.

[0037] The structural formula of the modafinil hapten is:

[0038] (2) Preparation of coated original modafinil-OVA:

[0039] Weigh 1.82 mg of modafinil hapten, 3.84 mg of 1-ethylcarbodiimide hydrochloride, and 2.30 mg of N-hydroxysu...

Embodiment 2

[0048] Example 2 IC of Modafinil Monoclonal Antibody 50 Determination of

[0049] The measurement steps are:

[0050] (1) Coating: The coated original modafinil-OVA was diluted with 0.05M pH 9.6 carbonate buffer starting from 1 μg / mL, 100 μL / well, and reacted at 37°C for 2 hours.

[0051] (2) Washing: Pour off the solution in the plate, and wash 3 times with washing liquid, each time for 3 minutes.

[0052] (3) Blocking: After patting dry, add 200 μL / well blocking solution and react at 37°C for 2 hours. Wash and tumble dry for later use.

[0053] (4) Adding samples: Dilute the antiserum (after blood collection from the tail of the mouse, dilute the corresponding multiple with antibody diluent) from 1:1000, and add it to the coated wells of each dilution, 100 μL / well, react at 37°C for 30 min; after washing thoroughly, add 1:3000 diluted HRP-goat anti-mouse IgG, 100 μL / well, react at 37°C for 30 min.

[0054] (5) Color development: Take out the ELISA plate, wash it thoroug...

Embodiment 3

[0057] Embodiment 3: Specificity experiment-cross reaction

[0058] Adopt ELISA detection method to test the cross-reactivity of several drugs similar to modafinil effect, the cross-reactivity value is calculated as follows:

[0059] (IC of Modafinil 50 / Compound IC 50 )×100;

[0060] Interactions: [(S)-(diphenylmethyl)sulfinyl]acetic acid, Ritalin, amphetamine.

[0061] The experimental results are shown in Table 1:

[0062] Table 1

[0063]

[0064] As can be seen from the experimental results in Table 1, the obtained modafinil monoclonal antibody of the present invention only has inhibition to modafinil, IC 50 The value is 0.54ng / mL, and the crossover to analogs is less than 1%, indicating that the monoclonal antibody has high sensitivity and specificity.

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Abstract

The invention discloses a hybridoma cell strain capable of secreting a modafinil monoclonal antibody and application of the hybridoma cell strain and belongs to the field of food safety immunodetection. Two modafinil haptens are provided, a modafinil complete antigen is prepared, the modafinil complete antigen and an equal amount of a Freund's adjuvant are mixed and emulsified completely, a mouse immunization experiment is carried out, high-titer and low-IC50 mouse splenocytes are taken and fused with myeloma cells through a PEG method, and the hybridoma cell strain is obtained through screening by an indirect competitive enzyme-linked immunosorbent assay and three times of subcloning. The monoclonal antibody secreted by the cell strain has relatively good specificity and detection sensitivity (IC50 is 0.54ng / mL) to modafinil. The result of the invention can be used for establishing an immunodetection method for modafinil content in human plasma, and thus, the hybridoma cell strain has a practical application value.

Description

technical field [0001] The invention belongs to the technical field of immunoassay in human plasma, in particular to a hybridoma cell line secreting modafinil monoclonal antibody and application thereof. Background technique [0002] Modafinil is a wakefulness-promoting drug used to treat excessive sleep and improve mental clarity in narcolepsy and spontaneous narcolepsy, as well as obstructive sleep apnea syndrome and sleep disorders caused by shift work. Spend. Abuse of modafinil may lead to impaired cognitive function, especially in attention, working memory, and response inhibition. Therefore, it is necessary to establish a rapid and efficient method for the detection of modafinil in human plasma. [0003] Modafinil content analysis methods include high-performance liquid chromatography (HPLC), HPLC-UV and other instrumental methods. These detection methods have the disadvantages of time-consuming, cumbersome steps, inability to perform rapid on-site detection, and hig...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/44G01N33/577G01N33/53C12R1/91
Inventor 胥传来晁梦佳匡华徐丽广孙茂忠刘丽强吴晓玲宋珊珊胡拥明郝昌龙高巍马伟吴爱红
Owner JIANGNAN UNIV
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