CN1710424A - Colloidal gold labeling method for rapid detection of carbaryl test strips and its production method and application - Google Patents

Abstract

A test paper for quick detecting pesticide sevin residue is nitrocellulose filter coated with two antigens of artificial antigen and sheep to rabbit antigen in pesticide sevin. The method for analyzing sevin residue includes using high specificity sevin antibody as immune detection antibody, using colloidal gold as reaction label matter , applying direct competition immune detection method with mixed liquid vertical flowing mode to finish immune reaction within short time for obtaining detection result being viewed directly .

Description

Colloidal gold labeling method for rapid detection of carbaryl test strips and its production method and application

technical field

The invention belongs to the technical field of fast immunoassay for pesticide residues. The invention relates to a colloidal gold-labeled pesticide carbaryl small-molecule compound (molecular weight less than 1000 Daltons) rapid detection test strip and its preparation method and its application to rapid detection of pesticide carbaryl technology.

Background technique

Carbaryl (also known as carbaryl) is a representative product of carbamate pesticides. In my country, carbaryl can be used for 141 kinds of crops and control 565 kinds of pests. It is currently the most widely registered insecticide. Carbaryl is widely used in vegetables, cotton, and field crops. Carbaryl is used as a flower thinning agent on fruit trees, and it is used to control harmful organisms in fish ponds in aquaculture. Because carbaryl is widely used in my country, carbaryl residues exist in many agricultural products in our country. With the continuous expansion of the scale of pesticide use, the impact of pesticide residues on the environment and the chronic and long-term effects on human health have attracted increasing attention and concern. For this reason, the restrictions on pesticide residues are becoming more and more stringent, and new requirements and higher standards are put forward for the analysis and measurement objects, types, quantities, ranges, indicators and other aspects. my country has stricter testing standards for carbaryl residues in agricultural products. According to GB 5009.21 regulations on carbaryl residues in grain, oil and vegetables: grain ≤ 5.0mg/kg, vegetables ≤ 2.0mg/kg, fruits ≤ 2.5mg/kg, edible oil ≤ 0.5mg/kg, tobacco ≤ 1.0 mg/kg.

However, due to the small molecule toxic chemicals with a molecular weight less than 1000dolton (Dalton), such as pesticides and their metabolites, the traditional residual analysis methods mainly rely on gas chromatography (GC), liquid chromatography (HPLC) or mass spectrometry, etc. means of physical analysis. The traditional physical and chemical analysis method is cumbersome and complicated, with high cost and slow analysis speed, and it is difficult to meet the needs of actual analysis. Therefore, it is urgent to develop simple, fast and sensitive analysis techniques.

In 1967, the antiserum of the pesticide malathion was prepared by Centen et al. Since then, immunoassay technology has been introduced into the field of pesticide residue analysis. Immunoassay technique has become one of the analytical techniques with the most development and application potential because of its fast, simple and sensitive characteristics, and has been widely valued.

Based on the analysis technology of antigen-antibody immune reaction detection of small molecules and substances, various labeling methods such as enzyme labeling, radioactive labeling, fluorescent labeling, and colloidal gold labeling can be used to display the detection results. Immunoassay techniques can be used for both quantitative and qualitative detection. The pesticide ELISA and simple enzyme immunology EIA technology developed since the 1980s have enabled the analysis of pesticide residues to gain greater vitality in the method; it is quite suitable for the rapid analysis of sample matrices that are too complex and difficult to analyze with ordinary physical and chemical methods. Value.

At present, various pesticide residue laboratory immunological rapid detection techniques and rapid detection kits widely used are basically quantitative detection, while test strip technology belongs to qualitative detection technology. Compared with the test kit, the test strip is easier to carry and detects more quickly. In the actual detection process, especially on-site rapid detection, it is not necessarily necessary to obtain quantitative data for each sample, but only to qualitatively determine whether a certain sample contains a certain pesticide, and whether the content exceeds the standard. Therefore, the rapid detection test strip that only takes a few minutes or more than ten minutes to obtain the result is the most suitable detection tool.

Using colloidal gold-labeled antibodies to display the quantitative binding of antigens and antibodies is simple to operate and has a high sensitivity. It has developed rapidly in recent years and is currently the most widely used rapid detection technology. It is used in the detection of macromolecular substances such as proteins. It has been widely used, and the vast majority of commercial test strips currently available on the market use colloidal gold technology. But this kind of test strips can not be well applied in the detection of small molecules, and needs to be improved in terms of detection accuracy and stability. Therefore, the development of colloidal gold-labeled pesticide carbaryl rapid detection test strips has a wide market need.

Contents of the invention

issues that need resolving:

In view of the above problems, the purpose of the present invention is to fill the gap that there is currently no rapid detection test strip for the colloidal gold-labeled pesticide carbaryl. Provide a simple, rapid, on-site qualitative detection test strip and method with good antibody specificity and high detection sensitivity.

Technical solutions:

The present invention utilizes nitrocellulose membrane as a solid phase carrier, coats carbaryl artificial antigen and secondary antibody on the membrane and carries out immobilization treatment, adopts colloidal gold as reaction marker and carries out labeling to carbaryl antibody to display antigen, The results of the specific immunological reaction of the antibody, so as to qualitatively detect the ultra-trace amount of carbaryl in the sample. The key to this technology research is the preparation of colloidal gold-labeled pesticide carbaryl antibody, immobilization treatment technology and solid-phase surface immunoreaction technology.

Colloidal gold labeling method for rapid detection of carbaryl test strip, which is coated with pesticide carbaryl hapten and artificial antigen linked with OVA as the detection line area A and goat anti-rabbit secondary antibody as the test strip effectiveness standard line area The strip-shaped solid-phase support membrane of B, wherein the solid-phase support membrane adopts nitrocellulose membrane.

It should be noted:

The colloidal gold labeling method quickly detects carbaryl test strips. The pesticide carbaryl hapten uses the hapten in the patent application No. 200410020332.2. The artificial antigen concentration of the hapten and OVA is 10 ^-3 mg/L. The specification is 1.0-2.2cm×4.5-9.0cm, the widths of A and B are equal, both are 3-5mm, and the distance between A and B is 10-15mm.

The colloidal gold labeling method for rapid detection of carbaryl test strips is to be fixed on the solid-phase carrier membrane through the steps of spotting, fixing, sealing and washing. The specific method is as follows:

· Coated

Use a pen to mark two regions A and B on the solid-phase carrier film. Region A is the detection line region, and region B is the standard line region for the effectiveness of the test strip. The carbaryl artificial antigen synthesized by carbaryl hapten and OVA is coated in the area, and the goat anti-rabbit secondary antibody phosphate buffer solution (PBS) dilution is coated in the standard line area, and the solid phase carrier membrane adopts nitrocellulose membrane;

·fixed

After coating, first fix at room temperature for 5-10 minutes, and then fix in a 30°C incubator for 40-60 minutes, so that the artificial antigen and secondary antibody can be fully fixed on the nitrocellulose membrane;

·Closed

Soak the nitrocellulose membrane of the fixed antibody in 1-2% gelatin phosphate buffer solution for 5-10 minutes, and block it in a thermostat at 30°C for 20-40 minutes;

·dry

Rinse the sealed nitrocellulose membrane three times with phosphate buffer solution containing 0.5ml/L Tween-20, namely PBST washing solution, gently shake off excess water, and then place the rinsed membrane at 37°C for 30 Minute drying or freeze-drying, and airtight storage at 4°C;

Through the above process, the production of the colloidal gold-labeled pesticide carbaryl rapid detection test strip is completed.

Among them, the hapten used for coating is the carbaryl hapten in the patent application No. 200410020332.2. The concentration of the carbaryl artificial antigen synthesized by linking the carbaryl hapten with OVA is 10 ^-3 mg/L, and the nitrocellulose membrane adopts the specification It is 1.8-2.2cm×4.5-9.0cm. When coating, use a pen on the solid-phase carrier film at a distance of 5-10mm from one end of the carrier film, and mark the two regions A and B in turn, and the distance between the two regions 10-15mm, and the width is 3-5 mm, and then use an automatic laying machine to coat the carbaryl hapten and OVA artificial antigen 0.5- 2 μl, spread 0.5-2 μl of 1:50 PBS dilution of goat anti-rabbit secondary antibody on the standard line area.

The colloidal gold labeling method for rapid detection of carbaryl test strips is used to detect the pesticide carbaryl in the sample to be tested. The detection method can use the vertical flow-through reaction method or the terminal immunodiffusion reaction method to detect the sample. The specific method is as follows:

·Using vertical flow-through reaction method to detect samples:

1. Prepare the sample mixture to be tested and the blank control mixture

Mixed solution of the sample to be tested: the colloidal gold-labeled carbaryl antibody and the extract of the sample to be tested are mixed at a volume ratio of 1:3-5, and a competition reaction is carried out for 5-10 minutes.

Blank control mixture: the colloidal gold-labeled carbaryl antibody was mixed with phosphate buffer solution PBST containing 5-10% methanol and 0.05-0.10% Tween at a volume ratio of 1:3-5.

2. Detection

Two test paper strips of the present invention are all closely attached on the water-absorbing filter paper, one of which is used as the detection test paper strip I, and the other is used as the contrast test paper strip II. Then drop 50-100uL of the sample mixture to be tested in the center between the detection line area and the effectiveness control line area on the test strip I, and add 50-100uL of the test sample mixture to the test line area and the validity control area on the control test strip II. Add 50-100uL of the blank control mixture to the center between the line areas. After the mixture flows completely vertically through the test strips, observe and compare the color development of the two test strips. The effective control area is red. It shows that the test strip is valid, and there is no color change in the effective control area, which shows that the test strip is invalid. Then observe and compare the color of the detection area of test paper I and test paper II, the color of the detection area of test paper I is lighter or the same as that of test paper II, indicating that the sample to be tested contains or does not contain carbaryl, and the large difference in color indicates carbaryl. High vitamin content.

·Using terminal immunodiffusion reaction method to detect samples:

1. Prepare the mixed solution of the sample to be tested and the mixed solution of the blank control with the vertical flow-through method.

2. Detection

One end of two test paper strips of the present invention is closely attached to the water-absorbing filter paper, one of which is used as the detection test paper strip I, and the other is used as the contrast test paper strip II. Then drop 50-100uL of the sample mixture to be tested at one end of the detection line area of the test strip I, and drop 50-100uL of the blank control mixture at one end of the test line area of the test strip II. Place absorbent filter paper at the other end of I and test strip II to absorb the mixed solution of the sample to be tested and the blank control mixed solution. After the mixed solution has completely spread to the entire test strip, observe and compare the color development of the two test strips, which is effective The color of the control area is red, indicating that the test strip is effective, and the effective control area has no color change, indicating that the test strip is invalid; then observe and compare the colors of the detection areas of the test strip I and the test strip II. The light or the same color of the detection area of II indicates that the sample to be tested contains or does not contain carbaryl, and the degree of light color qualitatively indicates the content of carbaryl.

The preparation method of the colloidal gold-labeled pesticide carbaryl antibody used in the above-mentioned detection of the sample to be tested is: the particle diameter obtained by the trisodium citrate reduction method is about 20-40nm, and the pH value is 9.0-10 deep red colloidal gold solution , and then take 1 mL of colloidal gold solution and add the carbaryl antibody solution dropwise under continuous stirring conditions, let it stand overnight at 4°C, and centrifuge at 15000rpm at 4°C for 30min. The supernatant was discarded and the pellet was resuspended in 2.5 mM borate stock solution, pH 7.2, containing 0.3% BSA and 0.1% _NaN3 . The carbaryl antibody in the preparation of the colloidal gold-labeled pesticide carbaryl antibody is the carbaryl antibody in the Chinese patent application No. 200410020332.2, and the antibody concentration is 1:100000-500000.

The colloidal gold labeling method for rapid detection of carbaryl test strips is used to detect the pesticide carbaryl in the sample to be tested, and the extraction solution of the sample to be tested is prepared using the method for extracting the sample to be tested in the Chinese patent application No. 200410020332.2 or 200410071941.0.

Beneficial effect:

The colloidal gold labeling method provided by the present invention quickly detects carbaryl test strips, which fills the gap that there is currently no colloidal gold-labeled pesticide carbaryl rapid detection test strips. It is specific, sensitive, accurate, fast, convenient, cheap and so on. Because the antibody used in the test strip has good specificity and high sensitivity, the detection limit reaches 1.0×10 ⁹ ng/ml, and the test strip shows good anti-interference ability and stability. Through the actual detection test, the carbaryl residues in vegetables, fruits and grains were qualitatively detected with test strips by using the vertical flow-through method and the terminal immunodiffusion method respectively, showing good stability.

It has been verified by experiments that the above-mentioned colloidal gold preparation method, carbaryl artificial antigen and secondary antibody coating and immobilization treatment technical measures are not only simple and convenient, with few reaction steps, but also simple instruments and equipment, and the unqualified test strips are less than 1 %, provides a good technical basis for large-scale production of test strips, can effectively reduce production costs, and is conducive to product promotion and popularization.

The rapid detection method provided by the present invention is easy and fast to operate, and the operation performed by the user only uses the extract solution (methanol) to simply extract the sample to be tested, and can add colloidal gold-labeled antibodies without further processing using test paper It takes only 5-10 minutes to complete the entire detection process. The detection result can be judged by the naked eye through the color depth of the detection line, and due to the good anti-interference ability of the test strip, the detection accuracy can reach 90%. The above are very suitable for the needs of on-site testing.

Compared with the current domestic and foreign similar technologies, the present invention is a rapid detection tool for immunity with high detection efficiency and easy operation, and has good application prospects; it has both economic and social benefits.

Description of drawings

Fig. 1: the detection result of the test strip of the present invention adopting the vertical flow-through reaction mode;

Figure 2: The detection result of the test strip of the present invention using the terminal immunodiffusion reaction method.

Detailed ways

Embodiment 1: (colloidal gold labeling method rapidly detects the preparation of carbaryl test strip)

· Coated

Use a pen to mark two areas A and B on the nitrocellulose membrane with a specification of 1.0 × 4.5 cm. Area A is the detection line area, and area B is the standard line area for the validity of the test strip. On the phase carrier membrane, carbaryl hapten and OVA-linked synthetic carbaryl artificial antigen (using the mixed anhydride method) were coated on the detection line area, respectively, and goat anti-rabbit secondary antibody PBS dilution was coated on the standard line area, solid-phase Carrier membrane adopts nitrocellulose membrane; On the nitrocellulose membrane membrane of automatic plate-laying machine, it is the carbaryl hapten of ^10-3 mg/L to be coated with the carbaryl artificial antigen (i.e. Add 0.75 μl of ^10-3 mg of the above-mentioned artificial antigen to each liter of PBS, and spread 0.75 μl of goat anti-rabbit secondary antibody diluted 1:50 (that is, 1 ml of goat anti-rabbit secondary antibody added to 50 ml of PBS) on the control line area . It should be noted that we use the carbaryl hapten in patent application No. 200410020332.2. The widths of the two regions A and B are equal, both are 3mm, and the distance between the two regions A and B is 10mm.

·fixed:

After coating, first fix at room temperature for 10 minutes, and then fix in a 30°C incubator for 40 minutes to fully fix the antibody on the solid-phase carrier membrane.

· Closed:

Soak the immobilized antibody solid-phase carrier membrane in 2% gelatin phosphate buffer solution for 5 minutes, and block it in a thermostat at 30°C for 20 minutes;

·dry

Rinse the sealed solid-phase carrier membrane three times with phosphate buffered solution (PBST) buffer containing 0.5ml/L Tween-20 (PBST washing solution), gently shake off excess water, and place it at 37°C for 30 minutes to dry , Store in airtight at 4°C.

Embodiment 2: (colloidal gold labeling method rapid detection carbaryl test strip preparation)

· Coated

Use an automatic plate-plating machine to coat carbaryl hapten with a concentration of ^10-3 mg/L on the 2.0 cm × 9.0 cm above-mentioned treated strip solid-phase carrier membrane in the detection line area and synthesize carbaryl linked with OVA. Artificial antigen (using mixed anhydride method) 2μl, 2μl of goat anti-rabbit secondary antibody diluted in PBS with a volume ratio of 1:50 was applied to the control line area. It should be noted that we used the carbaryl hapten in patent application No. 200410020332.2. The widths of the two regions A and B are equal, both are 5mm, and the distance between the two regions A and B is 15mm.

·fixed

After coating, fix at room temperature for 5 minutes, and then fix in a 30°C incubator for 60 minutes to fully fix the antibody on the solid-phase carrier membrane.

·Closed

Soak the immobilized antibody solid-phase carrier membrane in 1% gelatin phosphate buffer solution for 10 minutes, and block it in a thermostat at 30°C for 40 minutes;

·dry

Rinse the sealed solid-phase carrier membrane three times with phosphate buffer buffer solution containing 0.5ml/L Tween-20, that is, PBST washing solution, gently shake off excess water, freeze-dry the membrane after washing, and then Store airtight at 4°C.

All the other steps are the same as in Example 1.

Embodiment 3: (colloidal gold labeling method rapidly detects the preparation of carbaryl test strip)

· Coated

Use an automatic plate-plating machine to apply 1.2 μl of carbaryl hapten and OVA-linked synthetic carbaryl artificial antigen with a concentration of ^10-3 mg/L on the above-mentioned strip solid-phase carrier membrane. 1.2 μl of goat anti-rabbit secondary antibody diluted in PBS at a volume ratio of 1:50 was applied to the area of the control line. It should be noted that we recommend using the carbaryl hapten in patent application No. 200410020332.2.

·fixed

After coating, first fix at room temperature for 8 minutes, and then fix in a 30°C incubator for 50 minutes to allow the antibody to be fully fixed on the solid-phase carrier membrane.

·Closed

Soak the immobilized antibody solid-phase carrier membrane in 1.5% gelatin phosphate buffer solution for 10 minutes, and block it in a thermostat at 37°C for 30 minutes;

·dry

Rinse the sealed solid-phase carrier membrane three times with phosphate buffer buffer solution containing 0.5ml/L Tween-20, that is, PBST washing solution, gently shake off excess water, freeze-dry the membrane after washing, and then Store airtight at 4°C.

All the other steps and methods are the same as in Example 1.

Application Example 1

This embodiment is a method for detecting the content of carbaryl in rapeseed by using a vertical flow-through reaction method and using a colloidal gold-labeled rapid detection test strip. The specific method is:

Preparation of colloidal gold-labeled carbaryl antibody: the particle diameter obtained by the trisodium citrate reduction method is about 20-40nm, the pH value is 9.2 dark red colloidal gold solution, and then take 1mL of colloidal gold solution under continuous stirring conditions. Carbaryl antibody (carbaryl antibody in China Patent Application No. 200410020332.2, the antibody concentration is 1:100000.) solution was added dropwise, left standing at 4°C overnight, centrifuged at 15000rpm, 4°C for 30min. The supernatant was discarded and the pellet was resuspended into a sol with 2.5 mM borate stock solution containing 0.3% BSA and 0.1% NaN ₃ , pH 7.2.

· Preparation of blank control mixed solution: mix gold-labeled carbaryl antibody with phosphate buffered solution (PBST) containing 5% methanol and 0.05% Tween at a volume ratio of 1:3.

·sampling:

Randomly buy 2.5 kg of rapeseed in the vegetable market, cut the leaves and stems of rapeseed into 0.25 cm × 0.2 cm pieces with scissors, and weigh 5 grams of rapeseed stem and leaf samples respectively.

·extract:

The samples were mixed with 100% methanol according to the mass ratio of 1:5 (that is, take 1g of the sample and add 5g of methanol), and pulverize the homogenate with a homogenizer (that is, the extraction method of the sample to be tested adopts the method in No. 200410020332.2 patent application) . The homogenate was allowed to stand for 20 minutes, and 1ml of the supernatant was carefully aspirated, diluted 20 times with 0.05% Tween phosphate buffer (20ml PBST), and the final concentration of methanol was 5%.

Detection: Wet the filter paper with double distilled water, shake off the excess water, stick it on a smooth plastic plate, use a smooth glass test tube to gently roll back and forth on the filter paper, so that there are no air bubbles between the filter paper and the plastic plate. All two test paper strips of the present invention are closely attached to the water-absorbing filter paper, one of which is used as the detection test paper strip I, and the other is used as the contrast test paper strip II. bubble. If the filter paper has too much moisture, dry filter paper can be placed around it to absorb excess moisture.

Mix the colloidal gold-labeled carbaryl antibody sol with the above-mentioned sample extract at a ratio of 1:3 by volume, and after 5 minutes of competitive reaction, draw 50uL of the reaction mixture and add it dropwise to the pre-marked detection area on the test strip I. line area and validity control line area. Drop 50uL of the blank control mixture in the center between the detection line area and the effectiveness control line area on the control test strip II.

After the mixed solution completely flows through the paper strips, observe and compare the colors of the detection areas of the test strip I and the test strip II. The test results show that red lines appear in the detection areas of the test strip I and the comparison test strip II, and The color of the detection area of the test strip I is lighter than that of the control strip II, indicating that carbaryl is contained in the rapeseed sample; at the same time, the effectiveness control areas of the two test strips both appear red, indicating that the test results are valid.

Application Example 2:

This embodiment is a method for detecting the content of carbaryl in rapeseed by using a vertical flow-through reaction method and using a colloidal gold-labeled rapid detection test strip. The specific method is:

Preparation of colloidal gold-labeled carbaryl antibody: the particle diameter obtained by trisodium citrate reduction method is about 20-40nm, the pH value is 9.8 deep red colloidal gold solution, then take 1mL colloidal gold solution under continuous stirring conditions Carbaryl antibody (carbaryl antibody in China Patent Application No. 200410020332.2, the antibody concentration is 1:400000.) solution was added dropwise, left standing at 4°C overnight, centrifuged at 15,000rpm, 4°C for 30min. The supernatant was discarded and the pellet was resuspended into a sol with 2.5 mM borate stock solution containing 0.3% BSA and 0.1% NaN ₃ , pH 7.2.

Detection:

Mix the colloidal gold-labeled carbaryl antibody sol with the above-mentioned sample extract at a ratio of 1:5 by volume, and after 10 minutes of competitive reaction, draw 100uL of the reaction mixture and add it dropwise to the pre-marked detection area on the test strip I. line area and validity control line area. Add 100uL of the blank control mixture dropwise to the center between the detection line area and the effectiveness control line area on the control test strip II.

After the mixed solution completely flows through the paper strips, observe and compare the colors of the detection areas of the test strip I and the test strip II. The test results show that red lines appear in the detection areas of the test strip I and the comparison test strip II, and The color of the detection area of the test strip I is lighter than that of the control strip II, indicating that carbaryl is contained in the rapeseed sample; at the same time, the effectiveness control areas of the two test strips both appear red, indicating that the test results are valid. (Other same application

Example 1).

Application Example 3

This embodiment is a method for detecting the content of carbaryl in a rapeseed sample with a test strip using a terminal immunodiffusion reaction method. The specific method is:

Detection:

Mix the colloidal gold-labeled carbaryl antibody with the above-mentioned sample extract at a ratio of 1:5 by volume, and after 10 minutes of competitive reaction, draw 100uL of the reaction mixture and add it dropwise to the end of the detection strip I near the detection line area; At the same time, draw 100uL of the blank control mixture and add it dropwise to the end of the control test strip II near the detection line. Place absorbent filter paper at the other end of test strip I and test strip II to absorb the mixed solution of the sample to be tested and the mixed solution of the blank control. After the mixed solution has completely diffused to the entire test strip, the liquid gradually diffuses upward through the detection line area and the effectiveness control line area on the test strip by means of the chromatography of the membrane. The colloidal gold-labeled carbaryl antibody that is not combined with the carbaryl standard will combine with the carbaryl artificial antigen when passing through the detection line, and the colloidal gold-labeled carbaryl antibody that has been combined with carbaryl will not interact with the area of the test line. Carbaryl is combined with the artificial antigen, but when it passes through the area of the effective control line, it reacts with the secondary antibody and is trapped in the area of the effective control line. After the reaction is completed, observe and compare the colors of the detection line areas of the test strip I and the test strip II. The test results show that red lines appear in the detection areas of the test strip I and the control test strip II, and the test strip I detects The color of the zone is lighter than that of the control test strip II, indicating that the rapeseed sample contains carbaryl; at the same time, the effectiveness control zones of the two test strips both appear red, indicating that the test results are valid. Others are the same as the application embodiment 1.

Application Example 4

Detection:

Mix the colloidal gold-labeled carbaryl antibody with the above-mentioned sample extract at a volume ratio of 1:3, and after 5 minutes of competitive reaction, draw 50uL of the reaction mixture and add it dropwise to the end of the detection strip I near the detection line; At the same time, draw 50uL of the blank control mixture and add it dropwise to the end of the control test strip II near the detection line.

The liquid on the test strip gradually diffuses upward through the detection line area and the effectiveness control line area by means of the chromatographic action of the membrane. The colloidal gold-labeled carbaryl antibody that is not combined with the carbaryl standard will combine with the carbaryl artificial antigen when passing through the detection line, and the colloidal gold-labeled carbaryl antibody that has been combined with carbaryl will not interact with the area of the test line. Carbaryl is combined with the artificial antigen, but when it passes through the area of the effective control line, it reacts with the secondary antibody and is trapped in the area of the effective control line. After the reaction is completed, observe and compare the colors of the detection line areas of the test strip I and the test strip II. The test results show that red lines appear in the detection areas of the test strip I and the control test strip II, and the detection strip I detects a red line. The color of the test strip II is lighter than that of the control test strip II, indicating that the rapeseed sample contains carbaryl; at the same time, the effectiveness control areas of the two test strips both appear red, indicating that the test results are valid. Others are the same as the application embodiment 1.

Claims (8)

1. Colloidal gold labeling method for rapid detection of carbaryl test strip, which is characterized in that it is coated with pesticide carbaryl hapten and artificial antigen connected with OVA as the detection line area A and goat anti-rabbit secondary antibody as the test strip The strip-shaped solid-phase carrier membrane in area B of the effectiveness standard line, wherein the solid-phase carrier membrane is a nitrocellulose membrane. 2. according to the colloidal gold labeling method described in claim 1 rapid detection carbaryl test strip, it is characterized in that pesticide carbaryl hapten adopts the hapten in No. 200410020332.2 patent application, the artificial antigen concentration that hapten is connected with OVA The test strip is 10 ^-3 mg/L, the specification of the test strip is 1.0-2.2cm×4.5-9.0cm, the width of the two areas A and B are equal, both are 3-5mm, and the distance between the two areas A and B is 10-15mm . 3. the colloidal gold labeling method described in claim 1 or 2 quickly detects the manufacture method of carbaryl test strip, it is characterized in that through spotting, fixing, sealing, flushing process is fixed on the solid-phase carrier membrane, its concrete method as follows: · Coated Use a pen to mark two regions A and B on the solid-phase carrier film. Region A is the detection line region, and region B is the standard line region for the effectiveness of the test strip. Carbaryl artificial antigen synthesized by linking carbaryl hapten and OVA was applied to the area, goat anti-rabbit secondary antibody PBS dilution was applied to the standard line area, and the solid phase carrier membrane was nitrocellulose membrane; ·fixed After coating, first fix at room temperature for 5-10 minutes, and then fix in a 30°C incubator for 40-60 minutes, so that the artificial antigen and secondary antibody can be fully fixed on the nitrocellulose membrane; ·Closed Soak the nitrocellulose membrane of the fixed antibody in 1-2% gelatin phosphate buffer solution for 5-10 minutes, and block it in a thermostat at 30°C for 20-40 minutes; ·dry Rinse the sealed nitrocellulose membrane three times with phosphate buffer solution containing 0.5ml/L Tween-20, namely PBST washing solution, gently shake off excess water, and then place the rinsed membrane at 37°C for 30 Minute drying or freeze-drying, and airtight storage at 4°C; Through the above process, the production of the colloidal gold-labeled pesticide carbaryl rapid detection test strip is completed. 4. according to the manufacture method of colloidal gold labeling method rapid detection carbaryl test strip described in claim 3, it is characterized in that the used hapten of coating adopts carbaryl hapten in No. 200410020332.2 patent application, carbaryl The concentration of carbaryl artificial antigen synthesized by linking hapten and OVA is 10 ^-3 mg/L, and the size of nitrocellulose membrane is 1.8-2.2cm×4.5-9.0cm. At a distance of 5-10mm from one end of the carrier film, mark two regions A and B in turn, the distance between A and B is 10-15mm, and the width is 3-5mm, and then use an automatic laying machine on the strip solid phase On the carrier membrane, 0.5-2 μl of carbaryl artificial antigen synthesized by linking carbaryl hapten and OVA was coated on the detection line area, and 0.5-2 μl of goat anti-rabbit secondary antibody PBS dilution of 1:50 was applied on the standard line area. . 5. The colloidal gold labeling method described in claim 1 or 2 for rapid detection of carbaryl test strip is used to detect the pesticide carbaryl in the sample to be tested, and it is characterized in that: its detection method can adopt vertical flow-through reaction mode or terminal The samples were detected by immunodiffusion reaction, the specific method is as follows: ·Using vertical flow-through reaction method to detect samples: (1). Prepare the sample mixture to be tested and the blank control mixture Mixed solution of the sample to be tested: the colloidal gold-labeled carbaryl antibody and the extract of the sample to be tested are mixed at a volume ratio of 1:3-5, and a competition reaction is carried out for 5-10 minutes. Blank control mixture: the colloidal gold-labeled carbaryl antibody was mixed with phosphate buffer solution PBST containing 5-10% methanol and 0.05-0.10% Tween at a volume ratio of 1:3-5. (2). Detection Two test paper strips of the present invention are all closely attached on the water-absorbing filter paper, one of which is used as the detection test paper strip I, and the other is used as the contrast test paper strip II. Then drop 50-100uL of the sample mixture to be tested in the center between the detection line area and the effectiveness control line area on the test strip I, and add 50-100uL of the test sample mixture to the test line area and the validity control area on the control test strip II. Add 50-100uL of the blank control mixture to the center between the line areas. After the mixture flows completely vertically through the test strips, observe and compare the color development of the two test strips. The effective control area is red. It shows that the test strip is valid, and there is no color change in the effective control area, which shows that the test strip is invalid. Then observe and compare the color of the detection area of test paper I and test paper II, the color of the detection area of test paper I is lighter or the same as that of test paper II, indicating that the sample to be tested contains or does not contain carbaryl, and the large difference in color indicates carbaryl. High vitamin content. ·Using terminal immunodiffusion reaction method to detect samples: (1). Prepare the mixed solution of the sample to be tested and the mixed solution of the blank control with the vertical flow-through method. (2). Detection One end of two test paper strips of the present invention is closely attached to the water-absorbing filter paper, one of which is used as the detection test paper strip I, and the other is used as the contrast test paper strip II. Then drop 50-100uL of the sample mixture to be tested at one end of the detection line area of the test strip I, and drop 50-100uL of the blank control mixture at one end of the test line area of the test strip II. Place absorbent filter paper at the other end of I and test strip II to absorb the mixed solution of the sample to be tested and the blank control mixed solution. After the mixed solution has completely spread to the entire test strip, observe and compare the color development of the two test strips, which is effective The color of the control area is red, indicating that the test strip is effective, and the effective control area has no color change, indicating that the test strip is invalid; then observe and compare the colors of the detection areas of the test strip I and the test strip II. The light or the same color of the detection area of II indicates that the sample to be tested contains or does not contain carbaryl, and the degree of light color qualitatively indicates the content of carbaryl. 6. The colloidal gold labeling method described in claim 5 quickly detects carbaryl test strips for detecting the pesticide carbaryl in the sample to be tested, and is characterized in that the colloidal gold labeling pesticide carbaryl used when detecting the sample to be tested The preparation method of the antibody is: adopt the trisodium citrate reduction method to obtain a particle diameter of about 20-40nm, and the pH value is 9.0-10 deep red colloidal gold solution, then take 1mL colloidal gold solution and add carbaryl Add the antibody solution drop by drop, let stand overnight at 4°C, centrifuge at 15,000rpm, 4°C for 30min, discard the supernatant, and use 2.5mM borate storage solution containing 0.3% BSA and 0.1% NaN ₃ , pH value 7.2 for precipitation Resuspend. 7. according to the colloidal gold labeling method described in claim 6 rapid detection carbaryl test strip is used to detect pesticide carbaryl in the sample to be tested, it is characterized in that the carbaryl in the preparation colloidal gold labeling pesticide carbaryl antibody The antibody adopts the carbaryl antibody in the Chinese patent application No. 200410020332.2, and the antibody concentration is 1:100000-500000. 8. According to the colloidal gold labeling method described in claim 5, the rapid detection carbaryl test strip is used to detect the pesticide carbaryl in the sample to be tested, and it is characterized in that the preparation method of the sample extract to be tested adopts China No. 200410020332.2 or 200410071941.0 The method of extracting the sample to be tested in the patent application.

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