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A 6-benzyladenine hapten, artificial antigen and its application in immunoassay

A benzyl adenine and hapten technology, applied in the field of immunoassay, can solve the problems of long detection time, expensive equipment and instruments, and inability to realize rapid on-site detection, and achieve strong specificity

Active Publication Date: 2022-04-19
GUANGDONG DAYUAN OASIS FOOD SAFETY TECH CO LTD +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the methods used to detect 6-BA are mainly traditional detection methods, such as spectrophotometry, high performance liquid chromatography and its combination technology, etc. Although the above methods can be accurately quantified, due to expensive equipment and instruments, the detection time is long , and requires professional operation, it is impossible to achieve rapid on-site detection in the true sense; another detection method is immunological detection technology, which has the characteristics of high specificity and high selectivity, and is very suitable for trace components in complex matrices With the interpenetration of disciplines, the detection methods emerge in endlessly, and the scope of application is also expanding. Compared with traditional detection methods, immunological detection technology has the characteristics of economy, rapidity, low technical points, and easy operation.

Method used

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  • A 6-benzyladenine hapten, artificial antigen and its application in immunoassay
  • A 6-benzyladenine hapten, artificial antigen and its application in immunoassay
  • A 6-benzyladenine hapten, artificial antigen and its application in immunoassay

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Experimental program
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Embodiment 1

[0058] The preparation of embodiment 1 6-BA hapten

[0059] A 6-benzyl adenine hapten, the 6-benzyl adenine hapten has a structural formula shown in formula I:

[0060]

[0061] Such as figure 1 As shown, the 6-BA hapten of the present invention is prepared by the following method, and the steps are as follows:

[0062] (1) Take 2.0g (14.8mmol) of compound a, add 10ml of toluene and 4.9g (29.6mmol) of compound b in turn, and react at 100-120°C for more than 5h. After the reaction was completed, the solvent was removed under reduced pressure, and 20ml of absolute ethanol was added to dissolve the residue. After cooling in an ice bath, 1.1g (29.6mmol) of NaBH was added. 4 , After no gas is produced, the temperature is raised to 60-70°C for more than 2 hours. After the reaction is complete, remove the solvent under reduced pressure, add 20ml of purified water, adjust the pH value of the aqueous phase to 1-2 with 1M dilute hydrochloric acid, extract twice with ethyl acetate,...

Embodiment 2

[0066] Example 2 Preparation of 6-BA artificial antigen

[0067] 6-BA artificial antigen among the present invention, its structural formula is:

[0068]

[0069] Wherein, protein is a protein carrier, and the protein carrier is any one of bovine serum albumin, ovalbumin, human serum albumin or hemocyanin.

[0070] The synthetic route of the artificial antigen is as follows: image 3 shown.

[0071] 1. The carrier protein is the 6-BA artificial antigen of bovine serum albumin, and the synthesis method is as follows:

[0072] 1) Take 10 mg of the 6-BA hapten prepared in Example 1, dissolve it in 0.2 mL of dimethylformamide (DMF), stir well, add 10 mg of 1-(3-dimethylaminopropyl)-3- Ethylcarbodiimide (EDC) and 10 mg N-hydroxysuccinimide (NHS), stirred at room temperature for 4 hours, can obtain the hapten-activated ester;

[0073] 2) Weigh 40 mg of bovine serum albumin (BSA), fully dissolve it in 4 mL of 0.01 mol / L PBS solution to form a carrier protein solution, slowly a...

Embodiment 3

[0081] Example 3 Application of 6-BA artificial antigen in the preparation of anti-6-BA monoclonal antibody

[0082] The preparation method of anti-6-BA monoclonal antibody is as follows:

[0083] The 6-BA artificial antigen whose carrier protein is hemocyanin in Example 2 was used as the immunogen, emulsified with an equal volume of Freund's adjuvant, and then immunized BALB / C mice. The immunization dose of each mouse was 100 μg, and the immunization interval was 2 weeks. After 3 times of immunization, the blood of the tail vein of the mice was collected to detect the serum titer. If the antibody titer does not meet the requirements, a booster immunization is required. After the antibody titer no longer rises, subcutaneous booster immunization is performed with 100 μg of the whole antigen. After 5 days, mouse splenocytes are taken to fuse with SP20 cells (mouse myeloma cells). The fused cells were selected in HAT medium, and after 5 days, the complete medium was replaced wit...

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Abstract

The invention discloses a 6-benzyl adenine hapten, an artificial antigen and an application thereof in immunoassay. The present invention obtains the 6-benzyladenine hapten through the multi-step reaction of compound a and compound b, and the arm introduced by the obtained hapten not only has active groups, but also completely retains all the amine groups of the target to be tested; and uses the hapten The obtained artificial antigen and monoclonal antibody IC50 value is 1.6 μ g / L, can realize the detection of 6-BA quickly and conveniently, and the fluorescence quantitative immunochromatography test strip prepared in the present invention is 2 μ g to the detection sensitivity of 6-BA / L.

Description

technical field [0001] The invention belongs to the field of immune detection, in particular to a 6-benzyl adenine hapten, an artificial antigen and the application thereof in immune detection. Background technique [0002] 6-benzyl adenine (6-benzylarninopuring, 6-BA) is the first synthetic cytokinin, white or off-white crystal, insoluble in water, slightly soluble in ethanol, stable in acid and alkali, It has various effects such as inhibiting the decomposition of chlorophyll, nucleic acid and protein in plant leaves, keeping green and anti-aging; transporting amino acids, auxin, inorganic salts, etc. to the treatment site, etc., and is widely used in agriculture, fruit trees and gardening crops from germination to harvest. stage. Because of its good efficacy and low price, 6-BA is widely used as a growth regulator in the cultivation of rootless bean sprouts. [0003] Excessive intake of 6-BA by the human body will stimulate the skin and mucous membranes, causing damage ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07D473/34C07K14/765C07K14/77C07K14/795C07K1/107C07K16/44G01N33/577G01N33/58G01N33/558G01N33/545
CPCC07D473/34C07K14/765C07K14/77C07K14/795C07K16/44G01N33/577G01N33/582G01N33/558G01N33/545
Inventor 李斌梁科梁巧雯李韵婷石松
Owner GUANGDONG DAYUAN OASIS FOOD SAFETY TECH CO LTD