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Folic acid-aminocaproic acid-hemocyanin conjugate and preparation method thereof

An aminocaproic acid, hemocyanin technology, applied in the field of bioengineering, can solve the problems of long time, low production efficiency, insufficient sensitivity, etc.

Inactive Publication Date: 2020-05-15
上海领潮生物新材料有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0012] In the prior art, the immunogens used to prepare the above-mentioned specific antibodies include FA (folate)-BSA (bovine serum albumin), FA (folate)-ACA (aminocaproic acid)-BSA (bovine serum albumin), etc., and The methods for preparing the above immunogen mainly include the following: 1. In the presence of EDC / NHS, mix FA and BSA according to a certain molar ratio, react overnight at 4 degrees, and dialyze 10mmol / LpH7.4 PBS to obtain FA-BSA; 2. FA is activated with glutaraldehyde and coupled to an inert protein (BSA) to obtain FA-BSA; 3. The document "Antibody to folic acid" reports: first prepare ACA-BSA, dissolve 500mgBSA in 10ml purified water, add 1gACA and 300mgEDC, reaction at room temperature for 4 hours, 10mmol / L pH7.4 PBS dialyzed to obtain ACA-BSA; preparation of FA-ACA-BSA, 25mgFA, 10mgNHS, 23mgDCC, direct reaction with 100mg ACA-BSA, 4 overnight, 10mmol / LpH7 .4 PBS dialysis to obtain FA-ACA-BSA; however, when the immunogens prepared by these methods are used to prepare antibodies, especially monoclonal antibodies, they have poor immunogenicity, and at the same time, the prepared antibodies, especially monoclonal antibodies, have specificity when used to detect folic acid. However, there are defects such as poor performance and insufficient sensitivity, and there are also defects such as low production efficiency, low yield, and long time-consuming

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  • Folic acid-aminocaproic acid-hemocyanin conjugate and preparation method thereof
  • Folic acid-aminocaproic acid-hemocyanin conjugate and preparation method thereof
  • Folic acid-aminocaproic acid-hemocyanin conjugate and preparation method thereof

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Embodiment 1

[0047] This embodiment provides a folic acid-aminocaproic acid-hemocyanin conjugate, the preparation method of which specifically includes the following steps:

[0048] (1) 1 g of aminocaproic acid and 500 mg of hemocyanin were reacted in 10 mL of water at room temperature in the presence of 300 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide for 4 hours, Then, it was dialyzed with PBS with a pH value of 7.5, centrifuged at 5000 rpm for 3 min, and the supernatant containing aminocaproic acid-hemocyanin conjugate was obtained, and the measured OD concentration was OD280=12.5 mg / ml;

[0049] (2) 23 mg of folic acid was dissolved in carbonate buffer solution, and the control concentration was 22 mg / mL; 8 mg of N-hydroxysuccinimide (NHS) was dissolved in dimethyl sulfoxide, and the control concentration was 7.6 mg / mL; Dissolve 24 mg of dicyclohexylcarbodiimide (DCC) in dimethyl sulfoxide, and control the concentration to 23 mg / mL; then mix the three and react at room temperatu...

Embodiment 2

[0051] This embodiment provides a folic acid-aminocaproic acid-hemocyanin conjugate, the preparation method of which specifically includes the following steps:

[0052] (1) 1 g of aminocaproic acid and 500 mg of hemocyanin were reacted in 10 mL of water at room temperature in the presence of 300 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide for 4 hours, Then, it was dialyzed with PBS with a pH value of 7.5, centrifuged at 5000 rpm for 3 min, and the supernatant containing aminocaproic acid-hemocyanin conjugate was obtained, and the measured OD concentration was OD280=12.5 mg / ml;

[0053] (2) Dissolve 25mg of folic acid in carbonate buffer, the control concentration is 17mg / mL; 10mgN-hydroxysuccinimide (NHS) is dissolved in dimethyl sulfoxide, the control concentration is 7mg / mL; 23mg of dicyclohexylcarbodiimide (DCC) was dissolved in dimethyl sulfoxide, and the control concentration was 16mg / mL; then the three were mixed, and reacted at room temperature for 2 hours to ob...

Embodiment 3

[0055] This embodiment provides a folic acid-aminocaproic acid-hemocyanin conjugate, the preparation method of which specifically includes the following steps:

[0056] (1) 1 g of aminocaproic acid and 500 mg of hemocyanin were reacted in 10 mL of water at room temperature in the presence of 300 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide for 4 hours, Then, it was dialyzed with PBS with a pH value of 7.5, centrifuged at 5000 rpm for 3 min, and the supernatant containing aminocaproic acid-hemocyanin conjugate was obtained, and the measured OD concentration was OD280=12.5 mg / ml;

[0057] (2) 30 mg of folic acid was dissolved in carbonate buffer, and the control concentration was 10 mg / mL; 10 mg of N-hydroxysuccinimide (NHS) was dissolved in dimethyl sulfoxide, and the control concentration was 3.5 mg / mL; Dissolve 35 mg of dicyclohexylcarbodiimide (DCC) in dimethyl sulfoxide, and control the concentration to be 12 mg / mL; then mix the three and react at room temperature fo...

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Abstract

The invention discloses a folic acid-aminocaproic acid-hemocyanin conjugate and a preparation method thereof. The preparation method includes the following steps that (1) aminocaproic acid and hemocyanin are reacted in water under the existence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, dialysis separation is carried out to obtain supernatant containing an aminocaproic acid-hemocyanin conjugate; and (2) folic acid dissolved in a carbonate buffer solution and N-hydroxysuccinimide and dicyclohexylcarbodiimide separately dissolved in organic solvents are mixed and reacted to obtain a reaction mixture, the supernatant is added, and stirring is carried out overnight at room temperature to prepare the folic acid-aminocaproic acid-hemocyanin conjugate. The novel immunogenic folic acid-aminocaproic acid-hemocyanin conjugate can be rapidly and efficiently prepared with high-quality(good activity), time is consumed less, the yield of the target conjugate is high, the purity is high, and antibodies, especially single cloned antibodies, prepared by the folic acid-aminocaproic acid-hemocyanin conjugate are highly sensitive and specific in folic acid detection.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, in particular to a folic acid conjugate that can be used as an immunogen, in particular to a folic acid-aminocaproic acid-hemocyanin conjugate (FA-ACA-KLH) and a preparation method thereof. Background technique [0002] Folic acid (Folate) was extracted and purified from spinach by H.K. Mitchell in 1941. It is a water-soluble vitamin. It is named after its rich content in green leaves. There are several forms of existence in nature, and its parent compound is a combination of three components: pteridine, p-aminobenzoic acid and glutamic acid, and its molecular formula is C 19 h 19 N 7 o 6 , the chemical structure is as follows: [0003] [0004] Folic acid is one of the essential vitamins for cell growth and reproduction in the body, and its deficiency will affect the normal physiological activities of the human body. Many literatures have reported that the lack of folic acid is di...

Claims

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Application Information

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IPC IPC(8): C07K14/795
CPCC07K14/795
Inventor 罗朝领茅柳娟曹雪姣尹文偲倪伟青
Owner 上海领潮生物新材料有限公司