Phenol artificial antigen synthetic method

Abstract

The invention relates to a preparation method of phenol artificial antigen. Ferulic acid hapten is used as a phenol structure analogue, and ferulic acid and carrier protein (bovine serum albumin BSA) are coupled by adopting a mixed anhydride method to prepare the phenol artificial antigen. The method is simple in synthetic step, reaction conditions are easy to realize, operation is convenient, aftertreatment separation method is simple and convenient, product coupling ratio n is between 20 and 35, BSA artificial antigen with the coupling ratio in the range has favorable immunocompetence and is suitable for being used as an immunizing antigen to prepare the antibody, the prepared antibody has favorable valence and has favorable recognition detection performance to phenol, and the level of sensitivity of the antibody for detecting the phenol reaches 0.15ppm.

Description

technical field

[0001] The invention relates to a method for synthesizing an artificial antigen, in particular to a method for synthesizing a phenol artificial antigen with high immune activity. Background technique

[0002] Phenol (chemical formula: C 6 h 5 OH, PhOH), also known as carbolic acid and hydroxybenzene, is an important organic chemical raw material. The phenol in the environment mainly comes from coking, oil refining, petrochemical, fertilizer, pesticide, plastic manufacturing, combustion and other processes. During the burning of cigarettes This compound is also produced and is one of the seven harmful components of cigarette smoke. Phenol mainly enters the human body through the respiratory tract and skin to cause poisoning. It is a highly toxic substance that directly damages cells and can corrode, damage and inhibit mucous membranes, cardiovascular and central nervous systems. It is very harmful to organisms. The environment will cause serious pollution. ...

Examples

Embodiment 1

[0051] Preparation of phenol antigen:

[0052] Weigh 0.0511g of ferulic acid, add 2ml of dioxane, 30kHz, 200W power for 5 minutes to dissolve, add 65ul tri-n-butylamine, 45ul isobutyl chloroformate at room temperature, and stir for 1h at room temperature. After the reaction, stand at 4° C. for 1 hour, then centrifuge at 4000 rpm for 10 minutes, and take the supernatant. Take by weighing BSA0.105g, dissolve with 10mlPBS damping fluid (the pH=7.25 of PBS damping fluid, comprise the component of following concentration: NaCl is 137mmol / L, KCl is 2.7mmol / L, NaCl 2 HPO 4 10mmol / L, KH 2 PO 4 2mmol / L), the mixed anhydride supernatant obtained by centrifugation was added to the BSA reaction solution under ice-water bath conditions at a speed of 2 drops / s, and the reaction was stirred for 6h. The reaction solution system is put into the treated semi-permeable membrane dialysis bag (take a dialysis bag with a width of 10-15 cm, place it in a mixed aqueous solution containing 2 wt% s...

Embodiment 2

[0054] Preparation of phenol antigen:

[0055] Weigh 0.0985g of ferulic acid, add 4ml of dioxane, 45kHz, 300W power ultrasonic for 10 minutes to dissolve, add 130ul tri-n-butylamine, 190ul isobutyl chloroformate at room temperature, and stir at room temperature for 3h. After the reaction, stand at 25° C. for 1 hour, then centrifuge at 10,000 rpm for 20 minutes, and take the supernatant. Take by weighing BSA0.208g, dissolve with 15mlPBS damping fluid (the pH=7.25 of PBS damping fluid, comprise the component of following concentration: NaCl is 137mmol / L, KCl is 2.7mmol / L, NaCl 2 HPO 4 10mmol / L, KH 2 PO 4 2mmol / L), the mixed anhydride supernatant obtained by centrifugation was added to the BSA reaction solution under ice-water bath conditions at a speed of 4 drops / s, and the reaction was stirred for 10h. The reaction solution system is put into the treated semi-permeable membrane dialysis bag (take a dialysis bag with a width of 10-15 cm, place it in a mixed aqueous solution ...

Embodiment 3

[0057] Preparation of phenol antigen:

[0058] Weigh 0.0752g of ferulic acid, add 3ml of dioxane, dissolve by ultrasonication at 40kHz, power 200W for 5 minutes, add 88ul tri-n-butylamine and 113ul isobutyl chloroformate at room temperature, and stir for 2 hours at room temperature. After the reaction, stand at 4°C for 1 hour, then centrifuge at 8000 rpm for 10 minutes, and take the supernatant. Take by weighing BSA0.162g, dissolve with 13mlPBS damping fluid (the pH=7.25 of PBS damping fluid, comprise the component of following concentration: NaCl is 137mmol / L, KCl is 2.7mmol / L, NaCl 2 HPO 4 10mmol / L, KH 2 PO 4 2mmol / L), at a speed of 3 drops / s, the mixed anhydride supernatant obtained by centrifugation was added to the BSA reaction solution under ice-water bath conditions, and the reaction was stirred for 8h. The reaction solution system is put into the treated semi-permeable membrane dialysis bag (take a dialysis bag with a width of 10-15 cm, place it in a mixed aqueous ...

technical field

[0001] The invention relates to a method for synthesizing an artificial antigen, in particular to a method for synthesizing a phenol artificial antigen with high immune activity. Background technique

[0002] Phenol (chemical formula: C 6 h 5 OH, PhOH), also known as carbolic acid and hydroxybenzene, is an important organic chemical raw material. The phenol in the environment mainly comes from coking, oil refining, petrochemical, fertilizer, pesticide, plastic manufacturing, combustion and other processes. During the burning of cigarettes This compound is also produced and is one of the seven harmful components of cigarette smoke. Phenol mainly enters the human body through the respiratory tract and skin to cause poisoning. It is a highly toxic substance that directly damages cells and can corrode, damage and inhibit mucous membranes, cardiovascular and central nervous systems. It is very harmful to organisms. The environment will cause serious pollution. ...

Examples

Embodiment 1

[0051] Preparation of phenol antigen:

[0052] Weigh 0.0511g of ferulic acid, add 2ml of dioxane, 30kHz, 200W power for 5 minutes to dissolve, add 65ul tri-n-butylamine, 45ul isobutyl chloroformate at room temperature, and stir for 1h at room temperature. After the reaction, stand at 4° C. for 1 hour, then centrifuge at 4000 rpm for 10 minutes, and take the supernatant. Take by weighing BSA0.105g, dissolve with 10mlPBS damping fluid (the pH=7.25 of PBS damping fluid, comprise the component of following concentration: NaCl is 137mmol / L, KCl is 2.7mmol / L, NaCl 2 HPO 4 10mmol / L, KH 2 PO 4 2mmol / L), the mixed anhydride supernatant obtained by centrifugation was added to the BSA reaction solution under ice-water bath conditions at a speed of 2 drops / s, and the reaction was stirred for 6h. The reaction solution system is put into the treated semi-permeable membrane dialysis bag (take a dialysis bag with a width of 10-15 cm, place it in a mixed aqueous solution containing 2 wt% s...

Embodiment 2

[0054] Preparation of phenol antigen:

[0055] Weigh 0.0985g of ferulic acid, add 4ml of dioxane, 45kHz, 300W power ultrasonic for 10 minutes to dissolve, add 130ul tri-n-butylamine, 190ul isobutyl chloroformate at room temperature, and stir at room temperature for 3h. After the reaction, stand at 25° C. for 1 hour, then centrifuge at 10,000 rpm for 20 minutes, and take the supernatant. Take by weighing BSA0.208g, dissolve with 15mlPBS damping fluid (the pH=7.25 of PBS damping fluid, comprise the component of following concentration: NaCl is 137mmol / L, KCl is 2.7mmol / L, NaCl 2 HPO 4 10mmol / L, KH 2 PO 4 2mmol / L), the mixed anhydride supernatant obtained by centrifugation was added to the BSA reaction solution under ice-water bath conditions at a speed of 4 drops / s, and the reaction was stirred for 10h. The reaction solution system is put into the treated semi-permeable membrane dialysis bag (take a dialysis bag with a width of 10-15 cm, place it in a mixed aqueous solution ...

Embodiment 3

[0057] Preparation of phenol antigen:

[0058] Weigh 0.0752g of ferulic acid, add 3ml of dioxane, dissolve by ultrasonication at 40kHz, power 200W for 5 minutes, add 88ul tri-n-butylamine and 113ul isobutyl chloroformate at room temperature, and stir for 2 hours at room temperature. After the reaction, stand at 4°C for 1 hour, then centrifuge at 8000 rpm for 10 minutes, and take the supernatant. Take by weighing BSA0.162g, dissolve with 13mlPBS damping fluid (the pH=7.25 of PBS damping fluid, comprise the component of following concentration: NaCl is 137mmol / L, KCl is 2.7mmol / L, NaCl 2 HPO 4 10mmol / L, KH 2 PO 4 2mmol / L), at a speed of 3 drops / s, the mixed anhydride supernatant obtained by centrifugation was added to the BSA reaction solution under ice-water bath conditions, and the reaction was stirred for 8h. The reaction solution system is put into the treated semi-permeable membrane dialysis bag (take a dialysis bag with a width of 10-15 cm, place it in a mixed aqueous ...

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