CN1384103A - Carbofuran half-antigen, antigen and antibody and their prepn process - Google Patents

Abstract

The present invention discloses one kind of carbofuran half-antigen. By dissolving furan phenol in toluene and leading in phosgene to react to obtain intermediate, the intermediate reacts with aminobutyric acid or aminohexyl acid in alkali dioxane solution to produce half-antigen BFNB or BFNH, the half-antigen is coupled with protein to obtain immunogen, and the immunogen is used to immunize animal to prepare antibody capable of reacting specially with carbofuran. The present invention has high specificity, high sensitivity, high accuracy, simple operation and other advantages.

Description

Carbofuran hapten, antigen and antibody and preparation method thereof

The invention relates to heterocyclic compounds and a preparation method thereof, in particular to a carbofuran hapten, antigen and antibody and a preparation method thereof.

The traditional methods of residue analysis of pesticides and their metabolites mainly rely on physical and chemical analysis methods such as gas chromatography (GC), liquid chromatography (HPLC) or mass spectrometry. Chronic and long-term effects are increasingly concerned and worried by people, and the restrictions on pesticide residues are therefore becoming more and more stringent. New requirements and higher standards are put forward for the analysis and measurement objects, types, quantities, ranges, indicators, etc. However, the traditional physical and chemical analysis methods are usually cumbersome and complicated, the sample pretreatment process is complicated, the workload is heavy, the instruments are expensive, and skilled technicians are required and the analysis period is long. Therefore, people are eager to have a simple, fast, sensitive and cheap detection technology that can carry out large-scale screening tests in the field and in the laboratory. Immunoassay has these advantages, so although immunoassay has been applied to the analysis of pesticide residues for a short time, it has been quickly used in the analysis of pesticide residues in environmental samples and food.

In 1958, Berson S.A and Yallow founded the radioimmunoassay (RIA), which was first widely used in the quantitative determination of biomedicine and clinical drugs. Due to the complexity of pesticide residue analysis objects, immunoassay was gradually applied in this field until the 1980s. Immunoassay technology can achieve selectivity and sensitivity that traditional physical and chemical analysis techniques cannot achieve, and is fast, simple, and suitable for detection Biomacromolecules can also detect trace components such as small molecules such as pesticides in complex samples.

The first report on the immunoassay of pesticides was the antiserum of the pesticide malathion prepared by Centen et al. in 1967. The reactivity of the antiserum with malathion was proved by antigen-antibody precipitation reaction. During the ten years from 1967 to the end of the 1970s, due to technical limitations, although several immunoassay methods for pesticides have been developed, they have not received widespread attention and their development has been quite slow. Since the 1980s, due to the increasing requirements for the selectivity and sensitivity of analytical methods, the requirements for the types of analysis objects and the number of environmental samples are also increasing. Highly specific, sensitive, and rapid Immunoassay technology has developed rapidly in the field of pesticide analysis. Since the 1990s, the immunoassay technology for pesticides has developed rapidly. So far, more than 60 kinds of pesticides have developed immunoassay technologies, among which there are more herbicides and insecticides, and less fungicides. Domestic research in this area has also started gradually, and there have been reports on the synthesis of haptens of pesticides such as parathion, triadimefon, gredazine, methamidophos, and atrazine-triazine.

Carbofuran (Carbofuran, 2,3,-dihydro-2,2-dimethyl-7-benzofuryl-N-methylcarbamate) was developed and produced by FMC and Mobay Chemical Company in 1969 That is, as a high-efficiency, broad-spectrum insecticide and nematicide, it is widely used in the control of pests in food, vegetables, fruits and economic crops worldwide. However, due to the high toxicity of carbofuran, it is not easy to degrade in acidic soil, and it is very easy to pollute soil and groundwater. In recent years, the environmental biotoxicity caused by carbofuran is not uncommon. Especially the large amount of use on crops and vegetables will form pesticide residues, which will cause great harm to human health, which has attracted people's attention. Therefore, it is of great practical significance to develop a simple and rapid trace analysis method suitable for on-site monitoring of pesticide residues.

Conventional methods for detecting carbofuran residues include gas chromatography (GC) and high performance liquid chromatography (HPLC). Since carbofuran is easily decomposed into phenols under normal gas chromatographic conditions, large errors are likely to occur in GC analysis. Of course, this problem can be solved by changing the operating conditions or derivatizing the pesticide. The currently designated more sensitive method for the detection of N-methylcarbamate pesticides is HPLC post-column derivatization with fluorescence detection. However, the sensitivity of these methods is greatly affected by steps such as sample purification, concentration, and derivatization; moreover, these methods require complex instruments that most laboratories do not have, and the process is cumbersome, so they are not suitable for the detection and analysis of large batches of samples. analyze. Immunoassay provides a new analytical detection method for the study of carbofuran residues.

The object of the present invention is to provide a carbofuran hapten, antigen and antibody with high specificity, sensitivity, high accuracy, good precision and simple and fast operation method and a preparation method thereof.

In order to achieve the above object, the present invention takes the following measures:

The molecular structure of carbofuran hapten is:

n is any natural number from 1 to 20

The molecular structure of carbofuran antigen is:

Carbofuran antibody is an immunoglobulin that can specifically react with carbofuran.

A kind of preparation method step of carbofuran hapten is:

1) Dissolve 1 part of furanol in 1 to 4 parts of toluene, pass through 1 to 2 parts of phosgene, add sodium hydroxide until the solution is alkaline, and the reaction temperature is -10 to 10°C to obtain a mixture;

2) The above mixture was subjected to liquid-liquid distribution, and then concentrated under reduced pressure to obtain an oily mixture of furanol and the product. After cooling, the oily mixture precipitated a colorless crystal, which was 2,3-dihydro-2,2-di Methyl-7-benzofuryl chloroformate;

3) Mix 1 part of 2,3-dihydro-2,2-dimethyl-7-benzofuryl chloroformate with 1-2 parts of aminobutyric acid or aminocaproic acid in alkaline dioxane solution The reaction can give 4-[[(2,3-dihydro-2,2-dimethyl-7-benzofuryloxy)carbonyl]amino]butanoic acid (BFNB) or 6-[[(2,3- Dihydro-2,2-dimethyl-7-benzofuryloxy)carbonyl]amino]hexanoic acid (BFNH).

The steps of the preparation method of another kind of carbofuran hapten are:

1) 1 part of furanol is dissolved in 4 to 9 parts of toluene, a constant amount of phosgene is passed through, a catalyst is added, and the reaction temperature is 80 to 150°C to obtain a mixture;

2) The above mixture was subjected to liquid-liquid distribution, and then concentrated under reduced pressure to obtain an oily mixture of furanol and the product. After cooling, the oily mixture precipitated a colorless crystal, which was 2,3-dihydro-2,2-di Methyl-7-benzofuryl chloroformate;

3) Mix 1 part of 2,3-dihydro-2,2-dimethyl-7-benzofuryl chloroformate with 1-2 parts of aminobutyric acid or aminocaproic acid in alkaline dioxane solution The reaction can give 4-[[(2,3-dihydro-2,2-dimethyl-7-benzofuryloxy)carbonyl]amino] (BFNB) or butyric acid 6-[[(2,3- Dihydro-2,2-dimethyl-7-benzofuryloxy)carbonyl]amino]hexanoic acid (BFNH).

Adopting the present invention in the carbofuran residue detection technology can make the pesticide residue analysis obtain greater vitality in the method. The hapten is coupled with the protein to prepare the immunogen and then the animals are immunized to obtain carbofuran-specific antibodies. Enzyme-linked immunoassay and radioimmunoassay can be established by using antigen-antibody immune reaction and enzymatic reaction or isotope labeling. It has high specificity and sensitivity when detecting carbofuran residues in samples, the minimum detection limit can reach 0.005ppm, the accuracy is high, the recovery rate can reach 96.12%, the accuracy is good, and the intra-assay coefficient of variation CV%=7.32%, The batch-to-batch coefficient CV%=10.90%. At the same time, the operation method is simple and fast, no complicated pretreatment process is required, a large number of samples can be tested at one time, the cost is low, and the requirements for operators are low. It can be operated through simple training. This technology has considerable application value for the rapid analysis of pesticide residues that lack detection activity or the sample matrix is too complex, so it is difficult to analyze with ordinary physical and chemical methods. Therefore, the residual amount of carbofuran in the sample can be quickly and accurately analyzed and detected, which is convenient for the detection of a large number of samples and on-site sample analysis.

The present invention is described in detail below in conjunction with embodiment.

Example 1

The steps of the cold method preparation method of carbofuran hapten (n=3) are:

1) In 120ml of toluene, 75 grams of phosgene was introduced, and then 63 grams of furanol was added for reaction. At the same time, sodium hydroxide was slowly added until the solution was slightly alkaline, and the reaction was stopped to obtain a mixture. The reaction temperature was -8°C;

2) The above mixture is subjected to liquid-liquid distribution, phosgene is removed, and then concentrated under reduced pressure to obtain an oily mixture of furanol and the product. After cooling, the oily mixture precipitates a colorless crystal. This substance is 2,3-dihydro-2 , 2-Dimethyl-7-benzofuryl chloroformate;

3) React 5 grams of 2,3-dihydro-2,2-dimethyl-7-benzofuryl chloroformate with 8 grams of aminobutyric acid in a dioxane solution containing sodium hydroxide to obtain 4-[[(2,3-Dihydro-2,2-dimethyl-7-benzofuryloxy)carbonyl]amino]butanoic acid, ¹ H HMR(acetone-d _s )δ1.43(s, 6H, CH ₃ ), 1.86 (m, 2H, CH ₂ ), 2.42 (t, 2H, CH ₂ COOH), 3.05 (s, 2H, CH ₂ ), 3.26 (q, 2H, CH ₂ -NH), 6.72 -6.99 (m, 3H, aromatic).

Example 2

The steps of the thermal method preparation method of carbofuran hapten (n=3) are:

1) Weigh 50 grams of furanol and dissolve it in 300 mL of toluene, pass through phosgene at a flow rate of 50 ml/min, add 1 gram of catalyst, and react at a reaction temperature of 90 ° C to obtain a mixture;

2) The above mixture is subjected to liquid-liquid distribution, phosgene is removed, and then concentrated under reduced pressure to obtain an oily mixture of furanol and the product. After cooling, the oily mixture precipitates a colorless crystal. This substance is 2,3-dihydro-2 , 2-Dimethyl-7-benzofuryl chloroformate;

3) React 5 grams of 2,3-dihydro-2,2-dimethyl-7-benzofuryl chloroformate with 8 grams of aminobutyric acid in a dioxane solution containing sodium hydroxide to obtain 4-[[(2,3-Dihydro-2,2-dimethyl-7-benzofuryloxy)carbonyl]amino]butanoic acid, ¹ H HMR(acetone-d _s )δ1.43(s, 6H, 2CH ₃ ), 1.86 (m, 2H, CH ₂ ), 2.42 (t, 2H, CH ₂ COOH), 3.05 (s, 2H, CH ₂ ), 3.26 (q, 2H, CH ₂ -NH), 6.72 -6.99 (m, 3H, aromatic).

Example 3

The steps of the cold method preparation method of carbofuran hapten (n=5) are:

1) In 180ml of toluene, pass 90 grams of phosgene, then add 75 grams of furanol for reaction, and at the same time slowly add sodium hydroxide until the solution is slightly alkaline, stop the reaction, and obtain an intermediate product, the reaction temperature is 6 °C;

2) The above-mentioned intermediate product is first liquid-liquid partitioned with water to remove phosgene, and then the toluene is evaporated with a rotary evaporator to obtain an oily mixture of furanol and the product. After cooling, this oily mixture precipitates a colorless crystal. This substance is 2,3-dihydro-2,2-dimethyl-7-benzofuryl chloroformate;

3) React 5 grams of 2,3-dihydro-2,2-dimethyl-7-benzofuryl chloroformate with 8 grams of aminobutyric acid in a dioxane solution containing sodium hydroxide to obtain 4-[[(2,3-Dihydro-2,2-dimethyl-7-benzofuryloxy)carbonyl]amino]hexanoic acid, ¹ H HMR(acetone-d _s )δ1.43(s, 6H, 2CH ₃ ), 1.41-1.68 (m, 6H, CH ₂ CH ₂ CH ₂ ), 2.38 (t, 2H, CH ₂ COOH), 3.05 (s, 2H, CH ₂ ), 3.20 (q, 2H, CH ₂ NH), 6.72-6.99 (m, 3H, aromatic).

Example 4

The steps of the thermal method preparation method of carbofuran hapten (n=5) are:

1) Weigh 50 grams of furanol and dissolve it in 400 mL of toluene, pass through phosgene at a flow rate of 50 ml/min, add 1.2 grams of catalyst (quaternary ammonium salt), and react to obtain an intermediate product. The reaction temperature is 120 ° C, 2,3- Dihydro-2,2-dimethyl-7-benzofuryl chloroformate;

2) The above-mentioned intermediate product is first liquid-liquid partitioned with water to remove phosgene, and then the toluene is evaporated with a rotary evaporator to obtain an oily mixture of furanol and the product. After cooling, this oily mixture precipitates a colorless crystal. This substance is 2,3-dihydro-2,2-dimethyl-7-benzofuryl chloroformate;

3) React 5 grams of 2,3-dihydro-2,2-dimethyl-7-benzofuryl chloroformate with 9 grams of aminocaproic acid in a dioxane solution containing sodium hydroxide to obtain 4-[[(2,3-Dihydro-2,2-dimethyl-7-benzofuryloxy)carbonyl]amino]caproic acid, ¹ H HMR(acetone-d _s )δ1.43(s, 6H, 2CH ₃ ), 1.41-1.68 (m, 6H, CH ₂ CH ₂ CH ₂ ), 2.38 (t, 2H, CH ₂ COOH), 3.05 (s, 2H, CH ₂ ), 3.20 (q, 2H, CH ₂ NH), 6.72-6.99 (m, 3H, aromatic).

Example 5

The steps of the preparation method of immunogen are:

Dissolve 100 micromoles of hapten (BFNB or BFNH) in 1-3 mL of DMF, then add an equivalent amount of dicyclohexylcarbodiimide and N-hydroxysuccinimide to the solution, and react 8-12 Hours later, slowly add the reaction solution into 4-8 mL of 15 mg/mL bovine serum albumin carbonate buffer solution, and then react for 2-4 hours with magnetic stirring, and the reaction product is the antigen.

Example 6

The steps of the preparation method of the antibody are:

1) Dilute the antigen to the required concentration with physiological saline, then emulsify it completely with an equal volume of Freund's complete adjuvant, and immunize 2-3 kg male New Zealand white rabbits by subcutaneous multi-point injection.

2) After 4 weeks, dilute the antigen to the required concentration with normal saline, emulsify it completely with an equal volume of Freund's incomplete adjuvant, and perform booster immunization by subcutaneous multi-point injection.

3) After 2 weeks, dilute the antigen to the required concentration with normal saline, emulsify it completely with an equal volume of Freund's incomplete adjuvant, and perform a booster immunization again by subcutaneous multipoint injection, and test the antigen on the 8th day after immunization. Antibody titer.

4) After that, booster immunization was carried out every 2 weeks until the titer of the antibody reached the requirement. Whole blood was collected from the carotid artery of the rabbit, the serum was separated, and the antibody was obtained after purification.

Claims (5)

1. carbofuran half-antigen is characterized in that molecular structure is:

N is any natural number of from 1 to 20

2. the red antigen of a furans is characterized in that molecular structure is:

3. the red antibody of furans is characterized in that it is the immunoglobulin (Ig) that specific immune response can take place with the furans pellet.

4. the preparation method of a carbofuran half-antigen is characterized in that its step is:

1) benzofuranol is dissolved in 1～4 part of toluene for 1 part, feeds 1～2 part in phosgene, and hydro-oxidation sodium to solution is alkalescence, and temperature of reaction is-10～10 ℃, and reaction obtains a mixture;

2) said mixture is carried out liquid-liquid partition, get the oily mixture of benzofuranol and product then through concentrating under reduced pressure, this oily mixture is separated out a colourless crystallization after cooling, is 2,3-dihydro-2,2-dimethyl-7-benzofuryl chloro-formic ester;

3) with 1 part 2,3-dihydro-2,2-dimethyl-7-benzofuryl chloro-formic ester and 1～2 part of aminobutyric acid or hexosamine react in the Jian dioxane solution and can get 4-[[(2,3-dihydro-2,2-dimethyl-7-benzofuryl oxygen) carbonyl] amino] butyric acid or 6-[[(2,3-dihydro-2,2-dimethyl-7-benzofuryl oxygen) carbonyl] amino] caproic acid.

5. the preparation method of a carbofuran half-antigen is characterized in that its step is:

1) benzofuranol is dissolved in 4～9 parts of toluene for 1 part, and constant feeds phosgene, adds catalyzer, and temperature of reaction is 80～150 ℃, and reaction obtains a mixture;

2) said mixture is carried out liquid-liquid partition, get the oily mixture of benzofuranol and product then through concentrating under reduced pressure, this oily mixture is separated out a colourless crystallization after cooling, is 2,3-dihydro-2,2-dimethyl-7-benzofuryl chloro-formic ester;

3) with 1 part 2,3-dihydro-2,2-dimethyl-7-benzofuryl chloro-formic ester and 1～2 part of aminobutyric acid or hexosamine react in the Jian dioxane solution and can get 4-[[(2,3-dihydro-2,2-dimethyl-7-benzofuryl oxygen) carbonyl] amino] or butyric acid 6-[[(2,3-dihydro-2,2-dimethyl-7-benzofuryl oxygen) carbonyl] amino] caproic acid.