CN107219216A - A kind of method of aflatoxin B1 in chemiluminescence immunoassay detection baby rice powder - Google Patents

Abstract

A method for chemiluminescent immunodetection of aflatoxin B1 in infant rice flour, comprising the following steps: (1) sample pretreatment: dilute infant rice flour and water at a rate of 5% to obtain a sample solution, moisturize and seal at 37°C, and set aside; (2) Preparation of aflatoxin B1 antigen AFB1-BSA; (3) preparation of acridinium ester-labeled aflatoxin antigen AFB1-BSA-AE; (4) establishment of a standard curve; (5) adding samples for immunoreaction determination; (6) Quantitative readings. The method of the present invention has low detection limit, high sensitivity, quickness, convenience and low cost, and is very suitable for the detection of aflatoxin B1 in baby food, such as rice noodles.

Description

A method for chemiluminescent immunodetection of aflatoxin B1 in infant rice flour

technical field

The invention relates to the field of medical immunological in vitro diagnosis, and more specifically relates to a method for chemiluminescence immunological detection of aflatoxin B1 in infant rice flour.

Background technique

Aflatoxin B1 (Aflatoxin B1 is abbreviated as AFB1) is a derivative of dihydrofuran oxinone, which contains a difuran ring and an oxinone (coumarin). Aflatoxin B1 is one of the most carcinogenic chemicals known. Aflatoxin B1 is highly toxic to humans and some animals, and its toxic effect is mainly damage to the liver.

Aflatoxin B1 is the most common and most harmful in natural food. AQSIQ stipulates that aflatoxin B1 is one of the mandatory inspection items for most foods; it is listed in the key research poisons determined by WTO for the first place. When food and feed products are not dried in time or stored improperly, they are easily contaminated by aflatoxin B1. Aflatoxin B1 can directly or indirectly enter the human food chain, interfere with the synthesis of information RNA and DNA in the body, and then affect cells. The synthesis of protein leads to systemic damage to the body, and its main target organ is the liver. In severe cases, it can cause cirrhosis, liver cancer and even death. Moreover, aflatoxin B1 is very heat-resistant, with a decomposition temperature of 268°C, so it is difficult to destroy it during cooking. Therefore, it is very important to establish a simple, rapid and sensitive detection method for aflatoxin B1 by controlling the entrance of food and feed.

The commonly used methods for the early detection of aflatoxin in China include thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC), but both of them have the disadvantages of long detection time, expensive equipment, complicated operation and not suitable for large quantities of samples. Detection, the need for professionals to operate and other shortcomings. The application of immunochemical analysis has opened up a new field of detection of aflatoxin B1. Currently, commonly used methods include radioimmunoassay, fluorescence immunoassay and enzyme-linked immunoassay. Among them, although radioimmunoassay has high sensitivity and specificity, it has the disadvantages of short validity period and radioactive contamination; although fluorescence immunoassay has strong specificity, high sensitivity and fast speed, the problem of non-specific staining has not been completely solved, and the result judgment The objectivity of the method is insufficient, and the procedure is relatively complicated; enzyme-linked immunoassay is a new immunoassay technology that appeared in the 1970s, and its principle is that the antigen (or antibody) is adsorbed on the immunosorbent on the carrier and the antibody (or antibody) labeled with enzyme antigen) and the analyte (antigen or antibody) in the specimen have a specific immunological reaction, and then add an enzyme substrate for color reaction, and judge the content of the analyte in the sample by the depth of the color, although the sensitivity is high and the specificity However, there are disadvantages such as endogenous enzyme interference, corrosive sulfuric acid as the stop solution, most of the substrates are toxic or carcinogenic, and the detection of absorbance is easily affected by various external factors such as scratches.

Chemiluminescence immunoassay is based on the specific combination of antigen and antibody, and according to the principle that the concentration of the analyte in the chemiluminescence detection system and the chemiluminescence intensity of the system have a linear quantitative relationship under certain conditions. A trace analysis method for detection to determine the content of the analyte. Acridinium esters are good fluorescent reagents with high luminous efficiency, small photo-quenching effect, mild reaction conditions, and are not affected by the length of the connecting arm. They have received extensive attention from immunochemists in recent years. Acridine esters can be used to realize chemiluminescence immunoassay by labeling biological macromolecules such as antigens, antibodies and DNA. Due to the advantages of high sensitivity, wide linear dynamic range, long light signal duration, simple and fast analysis method, stable results, small error, good safety and long service life, chemiluminescence immunoassay has been widely used in food, clinical and veterinary medicine in recent years. It plays an important role in the fields of life sciences, environment, etc., and is an important means of rapid detection of trace toxin residues in food and feed.

Aflatoxins are highly toxic and carcinogenic. Aflatoxin is produced by microorganisms, unlike melamine, which is maliciously added. Aflatoxins are fat soluble and if present in cattle feed, the levels in beef will not be high. However, the content in internal organs, especially the liver, will be high. Aflatoxin is very heat-resistant and will not be destroyed when heated to 280°, so as long as it is contained in rice noodles, it will not be removed. Ultraviolet rays can only damage a small amount. As a result, rice flour may contain aflatoxin. Since babies will almost always choose a certain amount of rice flour to feed during their growth period, it is necessary to carry out aflatoxin B1 detection on infant rice flour (or other foods), according to " GB 10765-2010 National Food Safety Standard for Infant Formula Foods stipulates: aflatoxin M1 or aflatoxin B1≤0.5μg/kg.

At present, although there are many detection methods and detection kits for aflatoxin B1, these detection products have high detection limits and are used in routine detection. It is difficult to detect aflatoxin B1 in baby food. To achieve the effect, even if there are some more sophisticated detection products, the detection cost is high, generally speaking, therefore, it is necessary to provide a method that is convenient, fast and has a relatively low detection limit (meeting the detection of aflatoxin B1 in baby food) .

Contents of the invention

In order to solve the above defects in the prior art, the present invention provides a faster and more sensitive method for detecting the content of AFB1.

The technical scheme of the present invention is: a method for chemiluminescent immunodetection of aflatoxin B1 in infant rice flour, comprising the following steps:

(1) Sample pretreatment:

Dilute baby rice flour and water at 5% to make a sample solution, keep it moisturized and sealed at 37°C, and set aside;

(2) Preparation of aflatoxin B1 antigen AFB1-BSA:

Aflatoxin B1 (AFB1) is reacted with carboxymethylhydroxylamine hemihydrochloride in pyridine to generate aflatoxin B1 oxime (AFB1O), and then the active ester method is used to couple AFB1O with carrier protein to form a complete antigen. Using this method, the oximation rate of AFB1 and the utilization rate of AFB1O are both high.

(3) Preparation of aflatoxin antigen AFB1-BSA-AE labeled with acridinium ester:

Weigh 1mg of acridinic acid and dissolve it in 200ul N,N-dimethylformamide, take 50μL of this solution and slowly drop it into 250μL of 3.5mg/mL AFB1-BSA solution, and add 40μL of 0.1mol/L PBS. React overnight at 25°C in the dark at 150r/min. After the reaction, dialyze with distilled water at 4°C for 2 days and set aside.

(4) Establish a standard curve:

Coat two chemiluminescence plates with secondary antibody, incubate at 37°C for 2 h; wash the plate three times, add 350 μL of distilled water to each well, and seal with moisture at 37°C for 1.5 h; wash the plate three times, add an equal amount of AFB1 monoclonal antibody to each well, Incubate at 37°C for 45 min; add 50 μL each of AFB1 standard substance and 50 μL each of diluted AFB1-BSA-AE at concentrations of 0, 0.02, 0.1, 0.5, 1, 5, 10, 50, and 100 ng/mL, and mix well. Add 100 μL of PBS to the blank well, keep the temperature at 37°C for 45 minutes; wash the plate 4 times, add 100 μL of Triton X-100 acridinium ester initiator to each well, and then add 100 μL of initiator CTAB through the chemiluminescence analyzer, and measure while adding, a total of 10 times , take the average value; calculate the binding rate of each concentration, and make a standard curve.

(5) adding samples for immunoreaction determination:

Coat with secondary antibody, incubate at 37°C for 2 hours; wash the plate 3 times, add 350 μL sample solution to each well, and seal with moisture at 37°C for 1.5 hours; wash the plate 3 times, add AFB1 monoclonal antibody 4 μg/mL for competitive chemiluminescence detection.

(6) Quantitative reading:

The content of aflatoxin B1 was obtained by comparing the detection results with the standard curve.

Further, when making a standard curve, binding rate (%)=B/B0×100%, where B0 is the luminescence value without AFB1, and B is the luminescence value with AFB1.

Further, when making a standard curve, 100 μL of AFB1 monoclonal antibody was added to each well at a ratio of 1:1800, and AFB1-BSA-AE was diluted at a ratio of 1:500.

Further, in step (4) and step (5), 15 μg/mL rabbit anti-mouse IgG secondary antibody was used for coating.

The invention provides a method for chemiluminescent immunodetection of aflatoxin B1 in infant rice flour, which has a faster and more sensitive effect than the prior art: the linear range of the detection in this application is 0.1-5.0 ng/mL , the recovery rate is 75%-115%, and the coefficient of variation is 0.36%-1.20%, which can detect the AFB1 content in the sample sensitively and accurately. The detection limit of the above method can meet the detection of aflatoxin B1 in baby food.

Description of drawings

Fig. 1 is the standard curve of the detection method of AFB1 by acridinium ester chemiluminescence method.

detailed description

The following examples are only used to explain the present invention, and are not intended to limit the protection scope of the present invention.

Example 1

A method for chemiluminescent immunodetection of aflatoxin B1 in infant rice flour, comprising the following steps:

(1) Sample pretreatment:

Dilute baby rice flour and water at 5% to form a sample solution, keep it moisturized and sealed at 37°C, and set aside;

(2) Preparation of aflatoxin B1 antigen AFB1-BSA:

Aflatoxin B1 (AFB1) is reacted with carboxymethylhydroxylamine hemihydrochloride in pyridine to generate aflatoxin B1 oxime (AFB1O), and then the active ester method is used to couple AFB1O with carrier protein to form a complete antigen. Specifically, the following processes are included:

A. Preparation of aflatoxin B1 oxime (AFB10): Take a certain amount of AFB1 and dissolve it in methanol:pyridine (1:1) solution, add carboxymethyl hydroxylamine hemihydrochloride, and stir at 70°C for 5 hours. During the reaction process, the reaction solution was taken every 30 min for thin-layer chromatography (TLC) to monitor the progress of the AFB1O synthesis reaction. Dissolve AFB1O with an appropriate amount of methanol, scan with an ultraviolet spectrophotometer, measure the light absorption value at the maximum absorption peak, and calculate the concentration of AFB- and AFB-O.

B. Linkage of AFB10 and Daikinin: Dissolve AFB1O with DMF, add DCC and NHS, and react for 4 hours under magnetic stirring at room temperature. Dissolve an appropriate amount of BSA (or HSA) in carbonate buffer, add the AFB1O solution dropwise into the protein solution under magnetic stirring, and react at room temperature for 2 hours. The reaction solution was dialyzed against PBS.

The oximation rate of AFB1 and the utilization rate of AFB1O are relatively high (after testing, the oximation rate of AFB1 and the utilization rate of AFB1O are 85% and 56%, respectively), and the synthetic complete antigen has a good immune effect after testing, and it has been stored for 360 days After the antigen titer did not change significantly. Moreover, choosing to synthesize a complete antigen with a relatively low binding ratio can not only reduce the production cost, but also help to improve the selection and affinity of antibodies.

(3) Preparation of aflatoxin antigen AFB1-BSA-AE labeled with acridinium ester:

Weigh 1mg of acridinic acid and dissolve it in 200ul N,N-dimethylformamide, take 50μL of this solution and slowly drop it into 250μL of 3.5mg/mL AFB1-BSA solution, and add 40μL of 0.1mol/L PBS. React overnight at 25°C in the dark at 150r/min. After the reaction, dialyze with distilled water at 4°C for 2 days and set aside.

(4) Establish a standard curve:

Coat 2 strips of chemiluminescence plates with 15 μg/mL rabbit anti-mouse IgG secondary antibody, incubate at 37°C for 2 hours; wash the plates three times, add 350 μL distilled water to each well, and seal for 1.5 hours at 37°C; wash the plates three times, and each well according to Add 100 μL of AFB1 monoclonal antibody at 1:1800, incubate at 37°C for 45 min; 50 μL each of the diluted AFB1-BSA-AE, mix well, add 100 μL PBS to the blank well, keep the temperature at 37°C for 45 minutes; wash the plate 4 times, add 100 μL of Triton X-100 acridinium ester promoter to each well, and then add the starting agent by chemiluminescence CTAB 100μL, add and measure, measure 10 times in total, take the average value; calculate the binding rate of each concentration, and make a standard curve. When making a standard curve, binding rate (%)=B/B0×100%, where B0 is the luminescence value without AFB1, and B is the luminescence value with AFB1. The standard curve is shown in Figure 1.

(5) adding samples for immunoreaction determination:

Coat with 15 μg/mL rabbit anti-mouse IgG secondary antibody, incubate at 37°C for 2 hours; wash the plate 3 times, add 350 μL sample solution to each well, and seal with moisture at 37°C for 1.5 hours; wash the plate 3 times, add 4 μg of AFB1 monoclonal antibody /mL for competitive chemiluminescent detection.

(6) Quantitative reading:

The content of aflatoxin B1 was obtained by comparing the detection results with the standard curve.

Example 2

The steps are the same as in Example 1, but the sample sources are different.

Test results:

Example Measured AFB1 content (ng/ml) Example 1 0.18 Example 2 0.32

Claims (9)

1. a method for chemiluminescent immunodetection of aflatoxin B1 in infant rice flour, is characterized in that, comprises the following steps: (1) Sample pretreatment: Dilute baby rice flour and water at 5% to make a sample solution, keep it moisturized and sealed at 37°C, and set aside; (2) preparing aflatoxin B1 antigen AFB1-BSA; (3) Preparation of aflatoxin antigen AFB1-BSA-AE labeled with acridinium ester: (4) Establish a standard curve; (5) adding samples for immunoreaction determination; (6) Quantitative reading: The content of aflatoxin B1 was obtained by comparing the detection results with the standard curve. 2. as described in claim 1, the method for chemiluminescent immunodetection of aflatoxin B1 in infant rice flour is characterized in that: in step (2), use aflatoxin B1 and carboxymethylhydroxylamine hemihydrochloride in pyridine The aflatoxin B1 oxime is generated by the reaction, and then the aflatoxin B1 oxime is coupled with a carrier protein by an active ester method to synthesize a complete antigen. 3. The method for chemiluminescent immunodetection of aflatoxin B1 in infant rice flour as described in claim 1, wherein: step (3) includes: taking acridinic acid 1mg and dissolving it in 200ul N, N-dimethyl In base formamide, take 50 μL of this solution and slowly drop it into 250 μL 3.5 mg/mL AFB1-BSA solution, and add 40 μL 0.1mol/L PBS, and react overnight at 25 °C in the dark at 150 r/min. After the reaction, use Distilled water was dialyzed at 4°C for 2 days and set aside. 4. The method for chemiluminescent immunodetection of aflatoxin B1 in infant rice flour as described in claim 1, is characterized in that: in step (4), comprising the step of: coating 2 chemiluminescent plates with secondary antibodies, warming at 37° C. Incubate for 2 hours; wash the plate three times, add 350 μL of distilled water to each well, and seal at 37°C for 1.5 hours; wash the plate three times, add the same amount of AFB1 monoclonal antibody to each well, and incubate at 37°C for 45 minutes; the added concentrations are 0, 0.02, 0.1, 0.5, 1, 5, 10, 50, 100ng/mL AFB1 standard 50μL each and diluted AFB1-BSA-AE 50μL each, mix well, add 100μL PBS to the blank well, keep the temperature at 37°C for 45min; wash the plate, Add 100 μL of acridinium ester starter to each well, then add 100 μL of starter through a chemiluminescence analyzer, and measure while adding; calculate the binding rate of each concentration, and make a standard curve. 5. as described in claim 4, the method for chemiluminescence immunodetection aflatoxin B1 in infant rice flour is characterized in that: when making standard curve, binding rate (%)=B/B0 * 100%, wherein B0 is not added The luminous value of AFB1, B is the luminous value of AFB1 added. 6. The method for chemiluminescent immunodetection of aflatoxin B1 in infant rice flour as claimed in claim 4, characterized in that: when making a standard curve, 100 μL of AFB1 monoclonal antibody is added to each well at a ratio of 1:1800. 7. The method for chemiluminescent immunodetection of aflatoxin B1 in infant rice flour as claimed in claim 4, characterized in that: when making a standard curve, AFB1-BSA-AE is diluted 1:500. 8. The method for chemiluminescent immunodetection of aflatoxin B1 in infant rice flour as claimed in claim 1, characterized in that: in step (5), coat with secondary antibody, incubate at 37°C for 2h; wash the plate, every Add 350 μL of sample solution to the wells, and seal with moisture at 37°C for 1.5 h; wash the plate, and add 4 μg/mL of AFB1 monoclonal antibody for competitive chemiluminescence detection. 9. The method for chemiluminescent immunodetection of aflatoxin B1 in infant rice flour as described in claim 4 or 8, characterized in that: 15 μg/mL rabbit anti-mouse IgG secondary antibody is used for coating.