RU191660U1 - IMMUNOCHROMATOGRAPHIC TEST STRIP FOR THE EXPRESS METHOD OF DETERMINING FOUR GROUPS OF ANTIBIOTICS IN MILK WITH EXCLUSION OF POSSIBLE FORMATION OF THE SAMPLE - Google Patents

Abstract

The utility model relates to biotechnology, analytical biochemistry and biochemistry of food products, specifically to a test strip for immunoassay of antibiotics in milk or dairy products and can be used to control the quality of livestock products. The proposed test strip for the determination of antibiotics and casein in milk and dairy products allows for reliable express analysis (within 5-10 minutes) at room temperature without any preliminary sample preparation of the test sample for analysis or the use of any additional equipment in off-laboratory conditions. Thus, this test strip allows you to implement a wide screening of milk or dairy products directly at the sampling site with minimal labor.

Description

Technical field

The utility model relates to biotechnology, analytical biochemistry and biochemistry of food products, specifically to a test strip for immunoassay of antibiotics in milk or dairy products and can be used to control the quality of livestock products.

State of the art

Currently, antibiotics are widely used not only to combat human infectious diseases, but also as a means of preventing and treating animal diseases. In this regard, the intake of antibiotics into the human body with food, which can cause a number of undesirable effects, is dysbacteriosis, the development of resistant forms of microorganisms, allergic reactions, suppression of the activity of certain enzymes, etc., becomes a significant risk factor.

Various analytical methods, both microbiological and chemical, immunochemical, have been proposed and used to control the antibiotic content in food products. So, in particular, immunochromatographic analysis is widely used for quality control of food and feed products (test systems for mycotoxins, antibiotics, pesticides), including for the analysis of antibiotics in milk and dairy products. Since the speed and productivity of testing is of primary importance for mass examinations, in this situation immunochromatographic analysis has undoubted advantages, for which all the necessary reagents are preliminarily applied to the membrane components of the test strip and its contact with the test sample directly initiates the movement of the liquid front across the membranes, the flow of specific reactions and the formation of immune complexes, which, due to the inclusion of a colored marker in their composition, can detected visually or by an optical detector.

The prior art methods for determining individual groups of antibiotics in milk (Berlina AN et al / Quantum dot-based lateral flow immunoassay for detection of chloramphenicol in milk / Anal Bioanal Chem. 2013.405 (14): 4997-5000. Doi: 10.1007 / s00216- 013-6876-3; NA Taranova et al / Highly Sensitive Immunochromatographic Identification of Tetracycline Antibiotics in Milk / Int J Anal Chem. 2015; 2015: 347621. doi: 10.1155 / 2015/347621; Byzova NA / Immunochromatographic technique for express determination of ampicillin in milk and dairy products / Prikl Biokhim Mikrobiol. 2011.47 (6): 685-93.).

The prior art test "4sensor" (rapid test "4SENSOR KIT060", Belgium) to determine the content of four groups of antibiotics in milk. This rapid test allows you to determine the presence of chloramphenicol, streptomycin, tetracyclines and beta-lactams in milk. Each test line corresponds to one of the antibiotics. One package contains 96 test strips. The measurement procedure takes 10 minutes. The main disadvantages of the test are the need for preliminary preparation of milk samples and a mixture of antibodies, incubation in a thermostat, the need for consumables and special conditions for the study.

The prior art also knows a test strip for determining antibiotics and a method for immunochromatographic determination of antibiotics in milk and dairy products RU2406090 (publication date 10/12/2010). The test strip according to this invention allows to determine only one type of antibiotic in one experiment, and the method described in this document is intended to determine only three groups of antibiotics of their four regulated by GOST for dairy products, and one test strip determines only one group of antibiotics, those. three different antibiotic groups require three different test strips.

Utility Model Disclosure

The objective of the claimed utility model is the development of an immunochromatographic test strip for the simultaneous determination of four groups of antibiotics, as well as allowing the determination of the presence or absence of casein in the analyzed sample of milk or dairy products.

The technical result of the claimed utility model is to reduce the time (within 5-10 minutes) for the simultaneous determination of four groups of antibiotics - β-lactams, tetracyclines, streptomycin and chloramphenicol derivatives, as well as the detection of a specific milk protein - casein, in one rapid analysis at room temperature without any preliminary sample preparation of the test sample for analysis or the use of any additional equipment in off-laboratory conditions.

The indicated technical result is achieved due to the immunochromatographic test strip for the simultaneous determination of four groups of antibiotics and casein in milk or dairy products, which is a structure made of a substrate with four membranes successively fixed on its surface in series,

the first of which is a filter membrane for applying the test sample,

the second of which is a conjugate membrane with components deposited on its surface, specifically binding antibiotics or casein,

the third of which is a working membrane, the surface of which includes a control region, a test region for determining the antibiotic of the β-lactam group, a test region for determining the antibiotic of the tetracycline group, a test region for determining the antibiotic of the streptomycin group, a test region for determining the antibiotic of the group of chloramphenicol derivatives , a test area for determining casein,

the fourth of which is an absorbent membrane.

In private versions of the utility model, the substrate is made of nitrocellulose.

In particular versions of the utility model, specific components deposited on the conjugate membrane are characterized by an affinity of at least 10 ^-9 M for four groups of antibiotics and casein.

In particular embodiments of the utility model, the specific component is conjugated to a label.

In particular embodiments of the utility model, the label is colloidal gold particles or quantum dots with fluorescent indication, enzymatic labels.

In particular embodiments of the utility model, the specific component is a mono- or polyclonal antibody, a component of a regulated expression system, an active fragment of a bacterial ribosome, a phage display product, and DNA-based aptamers.

In particular embodiments of the utility model, the membranes are fixed to the surface of the substrate by gluing.

Thus, this test strip allows you to implement a wide screening of milk or dairy products directly at the sampling site with minimal labor.

Brief Description of the Drawings

The utility model will be more clear from the description, which is not restrictive and is given with reference to the accompanying drawings, which depict:

Figure 1. The block diagram of the test strip for immunochromatographic analysis:

1 - substrate;

2 - sample for research;

3 - test (working) membrane;

4- test areas (test lines) for determining antibiotics (4 in total);

5 - control area (control line);

6 - conjugate membrane;

7 - filtering membrane;

8 - an absorbent membrane;

9 - test area (test line) for registering casein.

Utility Model Implementation

Terms and Definitions

For a better understanding of this utility model, some of the terms used in this document are given below.

Throughout this document, the terms “ includes ” and “ including ” are interpreted to mean “includes, among other things.” These terms are not intended to be construed as “consists of only”.

The test region / line is the region / line on the surface of the working membrane within which solutions of the carrier protein conjugate (BSA (bovine serum albumin) or protein A) with a detectable antigen (antibiotics (4 groups) and casein) are immobilized.

The control region / line is the region / line on the surface of the working membrane within which sheep anti-mouse antibodies or protein A are immobilized from solution.

In this document, non-limiting examples of the marker used for the present utility model include a colored particle, an enzyme, a radioactive isotope, etc., and it is preferable to use a colored particle that can be visually detected without any special equipment. Examples of a colored particle include, but are not limited to, metallic microparticles, such as gold and platinum microparticles, non-metallic particles, latex particles, etc. The colored particle can be of any size, provided that the colored particle is of such a size that it can be moved to the lower part inside the voids of the test strip, but preferably the diameter is from 1 nm to 10 μm, more preferably from 5 nm to 1 μm , even more preferably from 10 to 100 nm.

In this document, non-limiting examples of the material from which the membrane can be made include nitrocellulose, filter paper, fiberglass, fabric, cotton, mixed cellulose nitrate ester, polyvinylidene fluoride, nylon, and others.

Utility Model Implementation Examples.

An immunochromatographic test strip according to this utility model can be obtained in a known manner using commercial materials. So, in particular, to obtain a test strip, a mdi Easypack membrane kit (Advanced Microdevices, India) can be used, including FR1 (0.6) sample application membranes, as well as a PT-R5 conjugate membrane, a CNPH90 adsorbing membrane membrane AP045. Bonding of the test strip can be done sequentially: four membranes are glued alternately on the nitrocellulose substrate.

An immunochromatographic test strip for the simultaneous determination of four groups of antibiotics and casein in milk or dairy products is a structure (figure 1) made of a substrate (1) with four membranes sequentially fixed on its surface in series (7,6,3, eight),

the first of which is a filter membrane for applying the test sample (7),

the second of which is the conjugate membrane (6) with components deposited on its surface, specifically binding antibiotics and casein,

the third of which is a working membrane (3), the surface of which includes a control region (5), a test area for determining the antibiotic of the β-lactam group, a test area for determining the antibiotic of the tetracycline group, a test area for determining the antibiotic of the streptomycin group, test area for determining the antibiotic of the group of chloramphenicol derivatives (4), a test area for determining casein (9),

the fourth of which is an absorbent membrane (8).

The inventive utility model works as follows. In the test sample of milk or milk product (the type of container with the specified sample does not matter) immerse the test strip on the side of the filter membrane (lower edge) for 1-2 minutes no more than the length of the filter membrane (alternatively, the sample can be applied with using a pipette onto the surface of the filter membrane). Then the strip is laid horizontally on any surface. After 5 minutes, a visual or photometric (including spectrofluorimetric) indication of the test result is carried out: if 4 test areas (lines) of antibiotics and a control area (line) are stained, it is concluded that there are no antibiotics in the test sample (at a concentration acceptable by GOST ), in the absence of color of a certain test line and the presence of color of the control line - there is an admixture of a certain group of antibiotics in a concentration exceeding the permissible level by GOST, in the absence of color four x test lines for determining antibiotics and the presence of the color of the control line - there is an admixture of antibiotics of four different groups at concentrations exceeding the permissible level of GOST. While maintaining the color of the test area (line) for casein, it is possible to assert falsification of the sample (not a milk or dairy product), the disappearance of the color of this test area (line) indicates the milk character of the test sample. The control line should always appear, regardless of the presence of antibiotics and / or casein in the test sample. The control line is an internal control of the performance of the test. The absence of a control strip indicates that the test system is inoperative. To carry out the described testing using a test strip according to this utility model, the use of preliminary heating of the test sample is not required, therefore, the test can be carried out at room temperature.

A test sample (sample), potentially containing antibiotics of four groups, such as β-lactams, tetracyclines, streptomycin and chloramphenicol derivatives within the concentrations stipulated by GOST-32219-2013 and Decision of the Board of the Eurasian Economic Commission N 28 of 02/13/2018, and / or casein, under the action of capillary forces, moves along the test strip (multi-membrane composite). In this case, initially there is an interaction with marker particles (in particular, colloidal gold particles or another marker), on the surface of which a specific component is adsorbed, intended for binding with antibiotics or casein (conjugate membrane). Then the liquid front overcomes the working (test) zone, which is a working membrane with four test lines for binding antibiotics, one control line and a line for indicating casein. The degree of marker binding in the test zone and, accordingly, the staining intensity of the test line are determined by the concentration of the analyzed compounds in the sample. Specific specific components for the binding of antibiotics and casein, the dimensional characteristics of membranes and markers, as well as the type of marker are selected separately in each case (for example, based on the characteristics of the test sample) so that when they are used, the threshold concentration of the antibiotic in the sample, at which the staining of the analytical strip of the test line, consistent with the controlled maximum permissible concentration of this antibiotic in food products. The whole study takes 5-10 minutes. As a result of such a study, it is possible to simultaneously determine both the presence of antibiotics of the four groups and determine the possible falsification of milk or milk products - the identification of a specific protein of casein milk. No special sample preparation, additional equipment and special personnel training is required.

A utility model has been disclosed above with reference to specific options for its implementation. For specialists, other embodiments of the utility model that do not change its essence, as disclosed in the present description, may be obvious. Accordingly, a utility model should be considered limited in scope only below the following utility model formula.

Claims (9)

1. An immunochromatographic test strip for the simultaneous determination of the presence of four groups of antibiotics and casein in milk or dairy products, which is a structure made of a substrate with four membranes successively fixed on its surface in series, the first of which is a filter membrane for applying the test sample, the second of which is a conjugate membrane with components conjugated to the label and specifically binding antibiotics and casein applied to its surface, the third of which is a working membrane, the surface of which includes a control region, a test region for determining the antibiotic of the β-lactam group, a test region for determining the antibiotic of the tetracycline group, a test region for determining the antibiotic of the streptomycin group, a test region for determining the antibiotic of the group of chloramphenicol derivatives , a test region for determining casein, wherein the test region comprises a conjugate of a carrier protein with a detectable antigen, the fourth of which is an absorbent membrane. 2. The immunochromatographic test strip according to claim 1, in which the substrate is made of nitrocellulose. 3. The immunochromatographic test strip according to claim 1, in which the specific components deposited on the conjugate membrane are characterized by an affinity of at least 10 ^-9 M for four groups of antibiotics and casein. 4. The immunochromatographic test strip according to claim 1, in which the label is a colloidal gold particles or quantum dots with fluorescence indication, enzymatic labels. 5. The immunochromatographic test strip according to claim 3, in which the specific component is a mono- or polyclonal antibody, a component of a regulated expression system, an active fragment of a bacterial ribosome, a product resulting from phage display, aptamer based on DNA.

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