CN105367645A - Preparation method of tramadol artificial antigen - Google Patents

Abstract

The invention provides a preparation method of a tramadol artificial antigen. The method comprises the following steps: preparing and detecting a semiantigen: chemically modifying tramadol and ethyl 5-bromovalerate used as raw materials, linking arms, and reacting to obtain the semiantigen containing a carboxyl group; and 2, preparing and detecting the artificial antigen: combining the semiantigen containing a carboxyl group with bovine gamma globulin (BGG) through a carbodiimide technology to prepare the tramadol artificial antigen which is tramadol-bovine gamma globulin. The tramadol artificial antigen prepared in the invention can undergo animal immunization to obtain a corresponding tramadol antibody, can be used in researches of various tramadol immunoassays, and provides a convenient, fast and accurate approach for detecting tramadol.

Description

The preparation method of a kind of U-26225A artificial antigen

Technical field

The invention belongs to technical field of biochemical industry, be specifically related to the preparation method of a kind of U-26225A artificial antigen.

Background technology

U-26225A, English name: Tramadol(INN), be a kind of non-opium central analgesics, though also can be combined with opiate receptor, its avidity is very weak, is mainly used as anodyne, can alleviate common to serious pain.This medicine is synthetic, acts on μ-opioid receptor and norepinephrine and serum tension force prime system and unites.U-26225A can be used for treating medium extremely serious pain.There are some researches show, U-26225A is to the effect of norepinephrine and serum tension force prime system system and alleviate the effect of pain, can alleviate the misery of dysthymia disorders and anxiety disorder.

U-26225A mainly acts on central nervous system, and its effect is wide, effective to Acute or chronic pain, to the similar morphine of the effect of human body and heroine.As unique a kind of central analgesics, because its analgesic effect is powerful and additive low so be used widely clinically.But along with widely using of U-26225A, the problem of U-26225A habituation causes concern.According to pertinent data display, normal people, as taken U-26225A 200 milligrams every day, can produce pharmacological dependence after about half a year, and if to take 300 to 400 milligrams (6 to 8 medicines) every day even more, can be addicted in a short time.Long-term, high-dose is taken and can be caused central nervous excitation, respiration inhibition, and can produce tolerance and additive and other untoward reactions.The phenomenon of tramadol hydrochloride habituation, is worldwide all found, and it is the fifth-largest by the medicine abused that therefore this medicine has been listed in the world by the World Health Organization.2008, tramadol hydrochloride was carried out control as psychotropic substances by China.

Its structural formula is:

At present, high performance liquid chromatography (HPLC) is mainly relied on to the detection of U-26225A, gas-chromatography (GC), thin-layer chromatography (TLC), simple (MS) etc., but there is expensive equipment, during check fee, and need professional and technical personnel to operate, modern measure can not be reached to fast, requirement accurately.And immunoassay can make up above all shortcomings, immunoassay is that one utilizes antigen and antibody specific association reaction to detect the analytical procedure of various material (medicine, hormone, protein, microorganism etc.), and the prerequisite of the method needs to provide specific antigen and antibody exactly.Therefore be necessary the preparation method providing a kind of effective U-26225A artificial antigen, the U-26225A artificial antigen of preparation can be used for immunity preparation and has specific U-26225A antibody, is further used for detecting.

Summary of the invention

The object of the invention is to overcome the shortcomings and deficiencies existed in prior art, the preparation method of a kind of U-26225A artificial antigen is provided, prepared U-26225A artificial antigen can carry out animal immune, obtain corresponding U-26225A antibody, can be used for the research of various U-26225A para-immunity analytical method, the detection for U-26225A class provides convenient approach fast and accurately.

A preparation method for U-26225A artificial antigen, is characterized in that, comprises the following steps:

(1) artificial semiantigen is prepared:

A tramadol hydrochloride is dissolved in purified water by (), drip 25% sodium hydroxide, dissociates and makes it pH ≈ 10; Add 5-bromine Valeric acid ethylester 330ul, react 3 hours in the oil bath of 80 ~ 90 DEG C.Developping agent: 95% ethanol: ammoniacal liquor: methylene dichloride: Isosorbide-5-Nitrae-dioxane=8:1:10:1(v/v), Rf=0.6-0.7.

B () reaction terminates after, adjust pH=5.5 with 1N hydrochloric acid, evaporated under reduced pressure, obtains oil product I.

C () product I thin-layer chromatographic analysis (TLC) method is separated, developping agent: 95% ethanol: ammoniacal liquor: methylene dichloride: Isosorbide-5-Nitrae-dioxane=8:1:10:1(v/v), Rf=0.6-0.7.Collect required part silicone gel, by dehydrated alcohol extraction, evaporated under reduced pressure, obtain colorless oil as product II.

D () product II adds purified water in proportion after, adjust pH=9 with DAS, evaporated under reduced pressure.Add DMF in proportion, filter, filtrate evaporate to dryness, obtains U-26225A haptens.Reaction ratio: product II: purified water: DMF :=100mg:10ml:10ml.

(2) U-26225A artificial antigen is prepared:

E U-26225A haptens is dissolved in N by (), in dinethylformamide, add N-hydroxy-succinamide, cyclohexyl phosphinylidyne diimine, reaction ratio: U-26225A haptens: N, dinethylformamide: N-hydroxy-succinamide: cyclohexyl phosphinylidyne diimine=20mg:1ml:10mg:17mg, stirring at room temperature reacts more than 18 hours, and reaction terminates rear centrifuging and taking supernatant liquor and is designated as A liquid;

F sodium-chlor and disodium hydrogen phosphate, sodium dihydrogen phosphate dihydrate are that 78.3:4.2:1 is dissolved in distilled water with mol ratio by (), prepare the PBS damping fluid that Na ion concentration is 0.1mol/L, pH is 7.2 ~ 7.4;

G BGG albumen is dissolved in PBS damping fluid by (), obtain the B liquid that concentration is 5mg/ml;

H A liquid is slowly added drop-wise to B liquid by (), the volume ratio of A liquid and B liquid is 1:5, and the mixed solution obtained leaves standstill preservation and spends the night under 2-8 DEG C of condition, obtains artificial antigen mixed solution;

I artificial antigen mixed solution is dialysed by () in PBS damping fluid, dialysis terminates rear centrifuging and taking supernatant liquor and namely obtains artificial antigen: U-26225A-bovine gamma globulin(BGG).

Due to the molecular weight of U-26225A, during independent role, not there is immunogenicity or less immunogenic, after therefore itself and macromolecular carrier such as bovine gamma globulin(BGG) must being connected to form U-26225A antigen, body could be stimulated to produce corresponding U-26225A antibody.The present invention is being prepared in U-26225A artificial antigen process, and selected site and cross-linking method all obviously do not change its structure, remain antigenic determinant.Between U-26225A haptens and bovine gamma globulin(BGG), introduce bridge construction, expose antigenic determinant, the U-26225A artificial antigen obtained maintains the structural specificity of U-26225A, is conducive to the generation of corresponding U-26225A antibody.

Technical scheme of the present invention is divided into two steps, and the first step is haptenic preparation and detection: be that raw material passes through chemically modified with U-26225A, and 5-bromine Valeric acid ethylester is obtained by reacting carboxylic haptens; Second step is preparation and the detection of artificial antigen: made it by carbodlimide method to combine the artificial antigen and U-26225A-bovine gamma globulin(BGG) of preparing U-26225A with bovine gamma globulin(BGG) (BGG).Its reaction equation is as follows:

The U-26225A artificial antigen that the present invention prepares is identified by following methods:

The mensuration of conjugate protein concentration: accurate measuring Proteinstandard(BGG) to be mixed with concentration be respectively 0.2,0.4,0.6,, 0.8,1.0,1.2, the standard series concentration of 1.6mg/ml, mixing, gets 50ul each concentration standard protein solution and joins in disposable cuvette, add 250ulreagentA, mixing, then add 2.0mlreagentB, react and record absorbancy with spectrophotometer after 10 minutes.Blank solution is that reagentA250ul adds reagentB2.0ml.With OD value for X-coordinate, concentration is that ordinate zou makes typical curve, draws typical curve linear equation.Accurate measuring sample size is 50ul, joins in disposable cuvette, adds 250ulreagentA, shake up, then add 2.0mlreagentB, reacts and records absorbancy with spectrophotometer after 10 minutes.Sample concentration is tried to achieve by the typical curve drawn.The protein concentration that the present invention calculates U-26225A antigen is 2.35mg/ml.

Embodiment

The preparation of U-26225A artificial antigen is divided into two steps, and the first step is haptenic preparation and detection: be that raw material passes through chemically modified with U-26225A, and Succinic anhydried is obtained by reacting carboxylic haptens; Second step is preparation and the detection of artificial antigen: made it by carbodlimide method to combine the artificial antigen and U-26225A-bovine gamma globulin(BGG) of preparing U-26225A with bovine gamma globulin(BGG) (BGG).

embodiment 1

(1) artificial semiantigen is prepared:

A 200mg tramadol hydrochloride is dissolved in 4ml purified water by (), drip 4 25% sodium hydroxide, dissociates and makes it pH ≈ 10; Add 5-bromine Valeric acid ethylester 330ul, react 3 hours in the oil bath of 80 ~ 90 DEG C.Developping agent: 95% ethanol: ammoniacal liquor: methylene dichloride: Isosorbide-5-Nitrae-dioxane=8:1:10:1(v/v), Rf=0.64.

B () reaction terminates after, adjust pH=5.5 with 1N hydrochloric acid, evaporated under reduced pressure, obtains oil product I.

C () product I thin-layer chromatographic analysis (TLC) method is separated, developping agent: 95% ethanol: ammoniacal liquor: methylene dichloride: Isosorbide-5-Nitrae-dioxane=8:1:10:1(v/v), Rf=0.6-0.7.Collect required part silicone gel, by dehydrated alcohol extraction, evaporated under reduced pressure, obtain 124mg colorless oil as product II.

D () product II adjusts pH=9 with DAS after adding 12.4ml purified water, evaporated under reduced pressure.Add 12.4mlN, dinethylformamide, filter, filtrate evaporate to dryness, obtains U-26225A haptens 110mg.

(2) U-26225A artificial antigen is prepared:

E U-26225A haptens is dissolved in 5.5mlN by (), in dinethylformamide, add 55mgN-N-Hydroxysuccinimide, 94mg cyclohexyl phosphinylidyne diimine, stirring at room temperature reacts 20 hours, and reaction terminates rear centrifuging and taking supernatant liquor and is designated as A liquid;

F sodium-chlor and disodium hydrogen phosphate, sodium dihydrogen phosphate dihydrate are that 78.3:4.2:1 is dissolved in distilled water with mol ratio by (), prepare the PBS damping fluid that Na ion concentration is 0.1mol/L, pH is 7.2 ~ 7.4;

G BGG albumen is dissolved in PBS damping fluid by (), obtain the B liquid that concentration is 5mg/ml;

H A liquid is slowly added drop-wise to B liquid by (), the volume ratio of A liquid and B liquid is 1:5, and the mixed solution obtained leaves standstill preservation and spends the night under 2-8 DEG C of condition, obtains artificial antigen mixed solution;

I artificial antigen mixed solution is dialysed by () in PBS damping fluid, dialysis terminates rear centrifuging and taking supernatant liquor and namely obtains artificial antigen: U-26225A-bovine gamma globulin(BGG).

(3) qualification of U-26225A artificial antigen:

The mensuration of conjugate protein concentration: accurate measuring Proteinstandard(BGG) to be mixed with concentration be respectively 0.2,0.4,0.6,, 0.8,1.0,1.2, the standard series concentration of 1.6mg/ml, mixing, gets 50ul each concentration standard protein solution and joins in disposable cuvette, add 250ulreagentA, mixing, then add 2.0mlreagentB, react and record absorbancy with spectrophotometer after 10 minutes.Blank solution is that reagentA250ul adds reagentB2.0ml.With OD value for X-coordinate, concentration is that ordinate zou makes typical curve, draws typical curve linear equation.Accurate measuring sample size is 50ul, joins in disposable cuvette, adds 250ulreagentA, shake up, then add 2.0mlreagentB, reacts and records absorbancy with spectrophotometer after 10 minutes.Sample concentration is tried to achieve by the typical curve drawn.The protein concentration that the present invention calculates U-26225A antigen is 2.35mg/ml.

Claims (1)

1. a preparation method for U-26225A artificial antigen, is characterized in that, comprises the following steps:

(1) artificial semiantigen is prepared:

A tramadol hydrochloride is dissolved in purified water by (), drip 25% sodium hydroxide, dissociates and makes it pH ≈ 10; Add 5-bromine Valeric acid ethylester 330ul, react 3 hours in the oil bath of 80 ~ 90 DEG C; Developping agent: 95% ethanol: ammoniacal liquor: methylene dichloride: Isosorbide-5-Nitrae-dioxane=8:1:10:1(v/v), Rf=0.6-0.7;

B () reaction terminates after, adjust pH=5.5 with 1N hydrochloric acid, evaporated under reduced pressure, obtains oil product I;

C () product I thin-layer chromatographic analysis (TLC) method is separated, developping agent: 95% ethanol: ammoniacal liquor: methylene dichloride: Isosorbide-5-Nitrae-dioxane=8:1:10:1(v/v), Rf=0.6-0.7; Collect required part silicone gel, by dehydrated alcohol extraction, evaporated under reduced pressure, obtain colorless oil as product II;

D () product II adds purified water in proportion after, adjust pH=9 with DAS, evaporated under reduced pressure; Add DMF in proportion, filter, filtrate evaporate to dryness, obtains U-26225A haptens; Reaction ratio: product II: purified water: DMF :=100mg:10ml:10ml;

(2) U-26225A artificial antigen is prepared:

E U-26225A haptens is dissolved in N by (), in dinethylformamide, add N-hydroxy-succinamide, cyclohexyl phosphinylidyne diimine, reaction ratio: U-26225A haptens: N, dinethylformamide: N-hydroxy-succinamide: cyclohexyl phosphinylidyne diimine=20mg:1ml:10mg:17mg, stirring at room temperature reacts more than 18 hours, and reaction terminates rear centrifuging and taking supernatant liquor and is designated as A liquid;

F sodium-chlor and disodium hydrogen phosphate, sodium dihydrogen phosphate dihydrate are that 78.3:4.2:1 is dissolved in distilled water with mol ratio by (), prepare the PBS damping fluid that Na ion concentration is 0.1mol/L, pH is 7.2 ~ 7.4;

G BGG albumen is dissolved in PBS damping fluid by (), obtain the B liquid that concentration is 5mg/ml;

H A liquid is slowly added drop-wise to B liquid by (), the volume ratio of A liquid and B liquid is 1:5, and the mixed solution obtained leaves standstill preservation and spends the night under 2-8 DEG C of condition, obtains artificial antigen mixed solution;

I artificial antigen mixed solution is dialysed by () in PBS damping fluid, dialysis terminates rear centrifuging and taking supernatant liquor and namely obtains artificial antigen: U-26225A-bovine gamma globulin(BGG).