RU2361203C1 - Method of determining quality of milk and dairy products - Google Patents

Abstract

FIELD: chemistry. ^ SUBSTANCE: invention relates to control over quality of milk and dairy products for contamination with mycotoxins. Method includes carrying out immunoaffinity concentration and immunoenzymatic detecting of aflatoxin M1 in volume of gel with inoculated antibodies. ^ EFFECT: method simplification, reduction of its duration and cost, as well as increase of sensitivity of detection. ^ 2 dwg

Description

The invention relates to analytical chemistry and can be used to determine aflatoxin M1 in milk and dairy products while controlling the quality of products for mycotoxin contamination.

In modern practice, various enzyme-linked immunosorbent assays are widely used for the semiquantitative and qualitative determination of aflatoxin M1.

The most common method is enzyme-linked immunosorbent immunoassay based on a heterogeneous reaction of immunoreagents bound to a solid surface with immunoreagents and analytes in solution. Usually performed in a 96-well microplate format (Rosi P., Borsari A., Lasi G., Lodi S., Galanti A., Fava A., Girotti S., Ferri E. Aflatoxin M1 in milk: Reliability of the immunoenzymatic assay International Dairy Journal 17 (2007) 429-435; Velasco MLR, Delso MMC, Escudero DO ELISA and HPLC determination of the occurrence of Aflatoxin M1 in row cow's milk. Food Addit. Contam. 20 (2003) 276-280).

The disadvantage of this method is the duration of its implementation (2-48 hours), a large number of stages of analysis and incubation, the need for sample preparation and the use of special equipment for recording analysis results.

A known method for the determination of mycotoxins, based on the use of membranes with covalently bound antibodies (see EP patent No. 0893690, IPC G01N 33 / 543K; G01N 33 / 569F).

This method, as well as other immunofiltration tests based on membranes, does not allow to achieve the necessary sensitivity.

Closest to the proposed invention is a method for determining aflatoxin M1 in milk (Sibanda L., De Saeger S., Van Peteghem C. Development of a portable field immunoassay for the detection of aflatoxin M1 in milk. Intern. J. Food Microbiology 48 (1999 ) 203-209). The method consists in the following: liquid milk was heated on a magnetic heated stirrer and filtered through a Whatman No. 4 filter to remove fats. Aflatoxin M1 was extracted from milk using Aflaprep ® M immunoaffinity columns (Rhone-Diagnostics Technologies, Glasgow, UK). 50 ml of filtered milk was slowly passed through the column, then the column was washed with distilled water. Aflatoxin M1 was eluted with 1.25 ml of methanol and 1.25 ml of phosphate-buffered saline. The resulting solution (2.5 ml, 50% methanol) was diluted with phosphate-buffered saline to a volume of 8.33 ml to reduce the methanol content to 15%. Secondary rabbit anti-mouse antibodies were applied to the membrane, dried, then blocked with casein solution and dried again. The membrane was fixed over a piece of cotton wool, primary antibodies were applied, washed with phosphate-buffered saline, an aliquot of the analyzed milk was added, then the aflatoxin B1 conjugate with horseradish peroxidase was washed again and the substrate was added. The intensity of the developing color was controlled visually.

The disadvantage of this method is the use of immunoaffinity columns to increase sensitivity and reduce the matrix effect, which complicates the analysis procedure, increases its duration and cost.

The technical result of the claimed invention is to simplify, reduce the duration and cost of the enzyme-linked immunosorbent assay for the determination of aflatoxin M1 in milk while increasing the sensitivity of the determination.

This technical result is achieved by combining the stages of purification, concentration and determination directly in the immunoaffinity carrier.

The essence of the invention lies in the fact that in the method of enzyme-linked immunosorbent determination of aflatoxin M1 in milk and dairy products, including immunoaffinity concentration and enzyme-linked immunosorbent determination of aflatoxin M1, the membrane is replaced by a gel with grafted antibodies, while the immuno-affinity concentration and the enzyme-linked immunosorbent determination of aflatoxin M directly , which allows to reduce the time and economic costs of the analysis, as well as to increase the sensitivity of the determination.

The invention is illustrated by drawings, where in Fig.1 shows a detecting column, in Fig.2 is a diagram of the immunochemical determination of aflatoxin M1. The positions in the figures indicate: 1 - column: 2 immunoaffinity gel; 3 - porous filters (frits); 4 - sample; 5 - conjugate; 6 - flushing; 7 - substrate; 8 - negative sample; 9 is a positive sample.

The method is implemented as follows. A porous filter 3 is placed at the bottom of column 1, over which an immunoaffinity gel 2 with grafted specific antibodies is poured, and another porous filter 3 is placed on top to fix the gel. Sample 4 is passed through column 1 from top to bottom, then conjugate 5 is introduced from bottom to top. Washing step 6 is used to remove excess conjugate 5. Substrate 7 is then added to column 1 and color development is observed. In the case of color, the result is considered a negative sample 8, in the absence of color - a positive sample 9.

Consider an example of the implementation of the claimed method of enzyme-linked immunosorbent assay for aflatoxin M1 in milk and dairy products (immunochromatographic analysis with sample preparation and concentration).

To determine aflatoxin M1, a 1 ml column was used in which a detecting immunolayer was placed, which was a mixture of a gel with grafted specific antibodies (for affinity purification and binding of the analyte) and a gel with blocked binding centers (for sample preparation). The gel was prepared on the basis of sepharose with CNBr activated groups, diluted with a 1: 3 solution of phosphate-buffered saline. The prepared gel (200-400 μl) was placed on a porous filter (frit) at the base of the column; another porous filter was placed on top of the immunoaffinity gel for column fixation.

Sample preparation of whole, homogenized milk, lactic acid products consisted in the separation of fats by centrifugation for 5-10 minutes at 5000-10000 rpm. For analysis, a skimmed water portion was used. To determine the aliquot (10 ml) of the analyzed milk was passed through a column with immunoaffinity gel using a syringe, then the conjugate of the analyte with horseradish peroxidase. The conjugate was left in the column for 3-5 minutes, then the column was washed with 5-10 ml of phosphate-buffered saline with Tween 20. The last step in the determination was to add tetramethylbenzidine-based substrate to the column, after which a blue color developed over several minutes ( in the absence of aflatoxin M1 in the sample). In the presence of aflatoxin M1 above the detection limit, the immunoaffinity gel remains white. Color development was recorded visually. The absence of color during the selected time was interpreted as a positive sample (aflatoxin M1 concentration is above the maximum permissible). The development of blue color was interpreted as a negative sample (the concentration of aflatoxin M1 is below the maximum permissible).

Using this method allowed to achieve a sensitivity determination of aflatoxin M1 at the level of 0.05 ng / ml, which meets the requirements of the EU for food quality control.

Claims (1)

A method for determining the quality of milk and dairy products, characterized in that it includes immunoaffinity concentration and enzyme-linked immunosorbent assay of aflatoxin M1 in a sample in a gel volume with grafted antibodies by color when adding a substrate based on tetramethylbenzidine for a selected time, while at a concentration of aflatoxin M1 in the sample above the maximum allowable gel remains white, and at a concentration of aflatoxin M1 in the sample below the maximum allowable gel turns blue.