RU196919U1 - DEVICE FOR IMMUNOCHROMATOGRAPHIC SIMULTANEOUS INDIVIDUAL DETECTION OF FLUORQUINOLONE ANTIBIOTICS OFLOXACIN, ENROFLOXACIN AND CYPROFLOXACIN IN FOOD - Google Patents

Abstract

The claimed device is intended for immunochromatographic rapid laboratory and off-laboratory simultaneous individual detection of toxic contaminants - fluoroquinolone antibiotics (ofloxacin, enrofloxacin, ciprofloxacin) in food. The device is a multi-membrane composite coated with immunoreagents (test strip). A multi-membrane composite consists of a working nitrocellulose membrane on a solid polystyrene base with deposited immunoreagents, a fiberglass membrane with deposited immunoreagents, a membrane for the absorption and separation of the test sample and the final adsorbing membrane for absorption of the components of the sample after the reaction. A distinctive feature of the claimed device is that polyclonal goat antibodies specific for rabbit immunoglobulins are applied to the control zone (K.Z.). Ofloxacin conjugate with a carrier protein, a soybean trypsin inhibitor (SIT), was applied to the first test zone (T.Z. 1). Enrofloxacin conjugate with SIT carrier protein was applied to the second test zone (T.Z.2). In the third test zone (T.Z. 3), a conjugate of ciprofloxacin with an SIT carrier protein was applied. A mixture of conjugates with colloidal gold of polyclonal rabbit antibodies specific for ofloxacin, enrofloxacin and ciprofloxacin is coated on a fiberglass membrane. For the analysis does not require any additional devices. The analysis time is 10 min, excluding sample preparation. The technical task of the claimed utility model is the simultaneous individual detection of three toxic contaminants - fluoroquinolone antibiotics ofloxacin, enrofloxacin and ciprofloxacin in food. The technical result of the claimed utility model consists in the simultaneous individual detection of ofloxacin, enrofloxacin and ciprofloxacin on a single test strip using a simple one-step analysis procedure provided by applying all the necessary reagents to the test strip.

Description

The utility model relates to the determination of toxic contaminants and can be used as an express means of laboratory and off-laboratory control to ensure the safety of food consumer products (milk and dairy products, meat and meat products, eggs, honey).

The device is intended for express immunochromatographic laboratory and off-laboratory simultaneous individual detection of toxic contaminants, fluoroquinolone antibiotics ofloxacin, enrofloxacin and ciprofloxacin in food.

Today, one of the most common contaminants of animal foods is various veterinary drugs.

Fluoroquinolones, piperazine derivatives of quinolone nalidixic acid are one of the most promising groups of modern antibacterial chemotherapy drugs used to treat infections of various etiologies and localization in humans and animals. By inhibiting DNA gyrase or topoisomerase II, fluoroquinolones inhibit the activity of both gram-negative and gram-positive bacteria. The widespread veterinary use of fluoroquinolones poses significant risks to human health associated with the intake of these contaminants both through the food chains (with livestock products) and through environmental pollution. Uncontrolled ingestion of fluoroquinolones into the human body can lead to the development of resistant forms of microorganisms, as well as cause a number of pathological processes, such as dysbiosis, allergic reactions, inhibition of the activity of certain enzymes, etc. With this in mind, the maximum allowable levels of fluoroquinolone residues in milk, poultry, and beef have been established. and pork equal to 30-300 μg / kg in Europe and Russia, 100-400 μg / kg in China, 50-200 μg / kg in Japan and 200 μg / kg in the USA.

For a wide screening of food contamination with fluoroquinolones, various types of liquid chromatography and the proton nuclear magnetic resonance method are used, but they do not solve the problem of mass testing outside centralized laboratories, because require sophisticated expensive equipment, highly purified solvents, qualified personnel. Immunochemical methods are more effective in this regard: enzyme immunoassay, immunofluorescence analysis, electrochemical and optical biosensors, immunochromatographic analysis (IHA).

The simplest form of immunochemical analysis to use is the ICA, which detects the presence of an antigen (toxic contaminant) in a liquid sample in 10-15 minutes. ICA is based on the movement of a liquid sample along the membrane, which leads to the formation of specific immune complexes in different parts of the membrane. A stained marker (colloidal gold) conjugated with specific antibodies is distributed across the membrane, and its presence or absence in certain sections of the membrane at the end of the analysis is the basis for the conclusion about the results. The use of IHA for the detection of toxic contaminants provides a number of advantages - control in laboratory and off-laboratory conditions, express analysis with minimal sample preparation, analysis in one step without the need for additional reagents and manipulations and ease of detection and interpretation of the results.

In the scientific literature there are descriptions of the development of immunochromatographic tests to determine individual fluoroquinolone antibiotics - ciprofloxacin (1-3), enrofloxacin (4-6), danofloxacin (7), ofloxacin (8), which use antibodies with individual specificity, and for simultaneous determination several (up to 12) fluoroquinolone antibiotics (9), which use antibodies specific for the group of fluoroquinolone antibiotics. In commercial immunochromatographic test systems, group-specific antibodies are mainly used, for example, in the designs of AE Solution (South Africa), Unisensor (Belgium), Elabscience (USA), Shenzhen Lvshiyuan Biotechnology), Ballya and Beijing Meizheng Bio-Tech (all from China).

The closest analogue of the claimed utility model is the commercial immunochromatographic test system QuinoSensor. Rapid test detecting all quinolones and fluoroquinolones in milk ”, KIT038, Unisensor (Belgium), based on antigen-antibody reaction.

The test system is a set consisting of:

-8 test strips with reagents applied, packed in an aluminum container with a lid in which a desiccant is placed;

-plastic 8-hole strip, in the wells of which a conjugate specific for fluoroquinolone antibiotics (norfloxacin, enrofloxacin, danofloxacin, difloxacin, sarafloxacin, marbofloxacin, ciprofloxacin, cololofexacolofolefolacin, cololofecoline, cololofecin, cololofecoline, olofolecino, cololofecin, cololofinacoline, cololofinacoline, cololofinacoline, cololofinacoline, cololine, cololofinacoline, cololine, cololofinacoline, cololine

-instructions for using the kit;

cardboard box with labeling for packing set.

The test strip is a multi-membrane composite, which consists of:

- a solid-base nitrocellulose working membrane with anti-virus antibodies applied to the control zone and the hapten-carrier protein conjugate applied to the test zone (the hapten-carrier-protein conjugate design is the know-how of the manufacturer);

- membranes for the absorption and separation of the test sample;

- the final absorbent membrane to absorb the components of the sample after the reaction;

- a covering protective film with marking.

The analysis is carried out as follows:

1. Milk and liquid dairy products are analyzed without prior sample preparation.

2. From the strip, 1 well with a dried conjugate of colloidal gold-specific fluoroquinolone-specific antibodies is separated.

3. Pipette 200 μl of milk into the well.

4. The hole with the sample is placed in the cell of the heating unit, close the lid and automatically mix the sample for 5-10 seconds.

5. Turn on heating at 37 ° C and incubate the sample well for 6 minutes.

6. Open the block cover and dip the test strip vertically with the lower end of the membrane to absorb the sample into the liquid sample for 4 minutes.

7. After 10 minutes from the start of the analysis, the test strip is removed. The result of the analysis is recorded visually or using a READSENSOR APP038 detector.

The analysis result is interpreted as follows:

1. If after 10 minutes two lines appear on the working membrane of the test strip (control and test), painted in dark red, the analysis result is considered negative, ie the sample does not contain fluoroquinolone antibiotics or their concentration is below the detection limit.

2. If after 10 minutes two dark red lines (control and very pale test) or one dark red line (control) appear on the working membrane of the test strip, then the analysis result is considered positive, ie The sample contains fluoroquinolone antibiotics at concentrations equal to or above the detection limit.

3. If after 10 minutes no dyed lines are formed on the working membrane of the test strip or only one dyed colored line is formed, the analysis result is considered invalid.

The QuinoSensor test system provides information only on the total contamination of a sample with fluoroquinolone antibiotics, while the claimed utility model allows the identification of a toxic contaminant. Analysis using the QuinoSensor test system involves an intermediate stage of incubation of the sample with a conjugate of specific antibodies with colloidal gold. The analysis using the claimed utility model is one-step, because all necessary reagents are applied to the test strip. Also, for analysis using the QuinoSensor test system, additional equipment is required - a heating unit with an integrated mixing device, which complicates the analysis compared to analysis using the inventive utility model.

The technical task of the claimed utility model is the simultaneous individual detection of three toxic contaminants - ofloxacin, enrofloxacin and ciprofloxacin in food.

The technical result of the claimed utility model consists in the simultaneous individual detection on the same test strip of three toxic contaminants - fluoroquinolone antibiotics ofloxacin, enrofloxacin and ciprofloxacin using a simple one-step analysis procedure provided by applying all the necessary reagents to the test strip.

The proposed device is a multi-membrane composite coated with immunoreagents (test strip). A multi-membrane composite consists of a working nitrocellulose membrane on a solid polystyrene base with deposited immunoreagents, a fiberglass membrane with deposited immunoreagents, a membrane for the absorption and separation of the test sample and the final adsorbing membrane for absorption of the components of the sample after the reaction.

The specified technical result is achieved by the fact that:

- in K.Z. polyclonal goat antibodies specific for rabbit immunoglobulins are applied;

- in TZ.1, ofloxacin conjugate with the carrier protein SIT is applied;

- in TZ.2 the conjugate of enrofloxacin with the carrier protein SIT is applied;

- in T.Z.3 a conjugate of ciprofloxacin with a protein carrier SIT is applied;

- a mixture of conjugates with colloidal gold of polyclonal rabbit antibodies specific for ofloxacin, enrofloxacin and ciprofloxacin is applied to the glass fiber membrane.

The attached drawing (Fig. 1) shows the inventive device, where 1 is a solid base of a working nitrocellulose membrane made of polystyrene, 2 is a working nitrocellulose membrane with deposited and dried immunoreagents, 3 is a final adsorbing membrane for absorption of the components of the sample after the reaction, 4-glass fiber membrane with deposited and dried immunoreagents, 5 - membrane for the absorption and separation of the test sample, A - K.Z. with applied polyclonal goat antibodies specific for rabbit immunoglobulins, B - T.Z.3 with a ciprofloxacin conjugated protein with a carrier protein SIT, C - T.Z.2 with a coated enrofloxacin conjugate with a protein carrier SIT, G - T.Z. .1 coated with ofloxacin conjugate with SIT carrier protein.

Table 1 shows the characteristics of the materials from which the elements of the claimed device are made.

Analysis using the inventive device is as follows:

1. 100-120 μl of the analyzed sample (milk, liquid dairy product, meat or egg extract, diluted honey, water) is added to a plastic tube with a capacity of 1.5 ml.

2. The test strip is immersed vertically with the lower end of the membrane to absorb the sample to a depth of 0.5 cm in liquid and incubated at room temperature for 3 minutes.

3. Take out the test strip and place it on a dry horizontal surface for 7 minutes.

4. 10 minutes after the start of fluid movement along the test strip, the analysis result is recorded visually or using a detector with video-digital registration.

The inventive device operates as follows (see Fig. 1):

If ofloxacin, enrofloxacin and / or ciprofloxacin are present in the sample, then they move with a fluid stream along the absorbent membrane (5), reach the glass fiber membrane (4) and react with conjugates of colloidal gold with specific antibodies to form double complexes (dissolved antigen - antibodies labeled with colloidal gold). Complexes under the action of capillary forces move along the working nitrocellulose membrane (2) and interact with conjugates ofloxacin antigens with protein immobilized in TZ.1 (D), TZ.2 (B) and TZ.3 (B) carrier of SIT (T.Z. 1, G), enrofloxacin with a protein carrier of SIT (T.Z.2, B) and ciprofloxacin with a protein carrier of SIT (T.Z.3, B) with the formation of triple complexes (immobilized on membrane through a carrier protein antigen - antibodies labeled with colloidal gold - antigen from solution). The excess of unbound binary complexes continues to move along the working nitrocellulose membrane (2) and interacts with anti-species antibodies immobilized in K.Z. (A), with the formation of binary complexes (anti-species antibodies immobilized on the membrane - antibodies labeled with colloidal gold).

Interpretation of the analysis results is carried out in accordance with the scheme presented in Fig. 2:

1. If after 10 minutes four red lines appear on the working membrane of the test strip (KZ, T.Z. 1, T.Z.2 and T.Z.3), then the result of the analysis is considered negative, i.e. . the sample does not contain ofloxacin, enrofloxacin and ciprofloxacin or their concentrations are below the detection limits (Fig. 2a).

2. If after 10 minutes two red lines appear on the working membrane of the test strip (K.Z. and the first along the liquid TZ.1), then the analysis result is considered negative for ofloxacin and positive for enrofloxacin and ciprofloxacin, etc. e. the sample does not contain ofloxacin (or its concentration is below the detection limit) and contains enrofloxacin and ciprofloxacin at concentrations equal to or higher than the detection limits (Fig. 2b).

3. If after 10 minutes two red lines appear on the working membrane of the test strip (K.Z. and the second along the liquid T.Z.2), then the analysis result is considered negative for enrofloxacin and positive for ofloxacin and ciprofloxacin, t. e. the sample does not contain enrofloxacin (or its concentration is below the detection limit) and contains ofloxacin and ciprofloxacin at concentrations equal to or higher than the detection limits (Fig. 2c).

4. If after 10 minutes two red lines appear on the working membrane of the test strip (K.Z. and the third in the direction of the liquid T.Z.3), the analysis result is considered negative for ciprofloxacin and positive for ofloxacin and enrofloxacin, etc. e. the sample does not contain ciprofloxacin (or its concentration is lower than the detection limit) and contains ofloxacin and enrofloxacin at concentrations equal to or higher than the detection limits (Fig. 2d).

5. If after 10 minutes three red lines appear on the working membrane of the test strip (K.Z., second and third along the liquid, T.Z.2 and T.Z.3), then the result of the analysis is considered to be positive for ofloxacin and negative for enrofloxacin and ciprofloxacin, i.e. the sample contains ofloxacin at a concentration equal to or higher than the detection limit, and does not contain enrofloxacin and ciprofloxacin (or their concentration below the detection limit) (Fig. 2e).

6. If, after 10 minutes, three red lines appear on the working membrane of the test strip (K.Z., first and third along the liquid, T.Z. 1 and T.Z.3), then the result of the analysis is considered positive for enrofloxacin and negative for ofloxacin and ciprofloxacin, i.e. the sample contains enrofloxacin at a concentration equal to or higher than the detection limit, and does not contain ofloxacin and ciprofloxacin (or their concentration below the detection limit) (Fig. 2f).

7. If after 10 minutes three red lines appear on the working membrane of the test strip (K.Z., first and second along the liquid, T.Z.1 and T.Z.2), then the analysis result is considered positive for ciprofloxacin and negative for ofloxacin and enrofloxacin, i.e. the sample contains ciprofloxacin at a concentration equal to or higher than the detection limit, and does not contain ofloxacin and enrofloxacin (or their concentration below the detection limit) (Fig. 2g).

8. If after 10 minutes one red line appears on the working membrane of the test strip (KZ), then the result of the analysis is considered positive for ofloxacin, enrofloxacin and ciprofloxacin, ie the sample contains ofloxacin, enrofloxacin and ciprofloxacin at concentrations equal to or higher than the detection limits (Fig. 2c).

9. If after 10 minutes no colored lines are formed on the working membrane of the test strip or only test lines are formed (one, two or three in any combination), then the result of the analysis is considered invalid (Fig. 2i).

The effectiveness of this approach is confirmed by the following examples:

Example 1 (study of the time of receiving negative ofloxacin, enrofloxacin and ciprofloxacin analysis results using the inventive device):

Using the inventive device, an analysis of milk with a fat content of 3.2% that does not contain ofloxacin, enrofloxacin and ciprofloxacin according to ELISA is performed. 120 μl of sample is added to the tube. The inventive device (test strip) is immersed vertically with the lower end of the membrane to absorb the sample to a depth of 0.5 cm in the sample and incubated at room temperature for 3 minutes Take out the test strip and place it in the groove of the retractable carriage of the detecting device for video-digital recording. The result of the analysis is evaluated after 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15 minutes using the video-digital detector software. Analysis time is controlled using a stopwatch. The analysis is carried out in five repetitions using test strips of five different series (strips No. 1-5).

The analysis results are shown in table 2. The staining intensities of each of the binding zones are normalized to its average value obtained 15 minutes after the start of the analysis. It can be seen from the experimental data that 10 minutes after the start of the analysis of the intensity of staining reach 100.0-100.5%, ofloxacin binding zones (T.Z.1) - 100.0-100.1%, enrofloxacin binding zones (T.Z.2) - 99.7-100.1%, ciprofloxacin binding zones (T.Z. 3) - 99.2-99.8% and remain virtually unchanged over the next five minutes, i.e. the analysis time using the claimed device in the absence of ofloxacin, enrofloxacin and ciprofloxacin in the sample is 10 minutes

Example 2 (study of the time of obtaining positive for ofloxacin and negative for enrofloxacin and ciprofloxacin analysis results using the inventive device):

Using the inventive device, an analysis of milk with a fat content of 3.2% containing ofloxacin at a concentration of 30 ng / ml and not containing enrofloxacin and ciprofloxacin according to ELISA is performed. 120 μl of sample is added to the tube. The inventive device (test strip) is immersed vertically with the lower end of the membrane to absorb the sample to a depth of 0.5 cm in the sample and incubated at room temperature for 3 minutes Take out the test strip and place it in the groove of the retractable carriage of the detecting device for video-digital recording. The result of the analysis is evaluated after 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15 minutes using the video-digital detector software. Analysis time is controlled using a stopwatch. The analysis is carried out in five repetitions using test strips of five different series (strips No. 1-5).

The results of the analysis are shown in table 3. The staining intensities KZ, TZ.2 and TZ.3 are normalized to their average values obtained 15 minutes after the start of the analysis. The staining intensities of T.Z. 1 are normalized to its average value obtained in the absence of ofloxacin in the sample (see Example 1).

It can be seen from the experimental data that 10 minutes after the start of the analysis of the intensity of staining, K.Z. reach 99.8-100.5%, enrofloxacin binding zones (T.Z.2) - 99.5-100.0%, ciprofloxacin binding zones (T.Z.3) - 99.3-99.8%) and virtually unchanged over the next five minutes. The staining intensities of the test zones of ofloxacin binding (T.Z. 1) 10 minutes after the start of the analysis are from 0 to 0.3% and practically do not change over the next five minutes. Thus, the analysis time of the sample containing ofloxacin at a concentration of 30.0 ng / ml and not containing enrofloxacin and ciprofloxacin according to the ELISA, using the inventive device is 10 minutes

Example 3 (study of the time to obtain positive for enrofloxacin and negative for ofloxacin and ciprofloxacin analysis results using the inventive device):

Using the inventive device, an analysis of milk with a fat content of 3.2% containing enrofloxacin at a concentration of 30 ng / ml and not containing ofloxacin and ciprofloxacin according to ELISA is performed. 120 μl of sample is added to the tube. The inventive device (test strip) is immersed vertically with the lower end of the membrane to absorb the sample to a depth of 0.5 cm in the sample and incubated at room temperature for 3 minutes Take out the test strip and place it in the groove of the retractable carriage of the detecting device for video-digital recording. The result of the analysis is evaluated after 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15 minutes using the video-digital detector software. Analysis time is controlled using a stopwatch. The analysis is carried out in five repetitions using test strips of five different series (strips No. 1-5).

The results of the analysis are shown in table 4. The staining intensities KZ, TZ.1 and TZ.3 are normalized to their average values obtained 15 minutes after the start of the analysis. The staining intensities of T.Z. 2 are normalized to its average value obtained in the absence of enrofloxacin in the sample (see Example 1).

It can be seen from the experimental data that 10 minutes after the start of the analysis of the intensity of staining, K.Z. reach 99.8-100.5%, ofloxacin binding zones (T.Z. 1) - 99.6-100.4%, ciprofloxacin binding zones (T.Z.3) - 99.2-99.8% and practically unchanged over the next five minutes. The staining intensities of the enrofloxacin binding test zones (T.Z.2) 10 minutes after the start of the analysis are from 0 to 0.3% and practically do not change over the next five minutes. Thus, the analysis time of the sample containing enrofloxacin at a concentration of 30.0 ng / ml and not containing ofloxacin and ciprofloxacin according to the ELISA, using the inventive device is 10 minutes

Example 4 (study of the time to obtain positive for ciprofloxacin and negative for ofloxacin and enrofloxacin analysis results using the inventive device):

Using the inventive device, an analysis of milk with a fat content of 3.2% containing ciprofloxacin at a concentration of 30 ng / ml and not containing ofloxacin and enrofloxacin according to ELISA is performed. 120 μl of sample is added to the tube. The inventive device (test strip) is immersed vertically with the lower end of the membrane to absorb the sample to a depth of 0.5 cm in the sample and incubated at room temperature for 3 minutes Take out the test strip and place it in the groove of the retractable carriage of the detecting device for video-digital recording. The result of the analysis is evaluated after 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15 minutes using the video-digital detector software. Analysis time is controlled using a stopwatch. The analysis is carried out in five repetitions using test strips of five different series (strips No. 1-5).

The results of the analysis are shown in table 5. The staining intensities KZ, TZ.1 and TZ.2 are normalized to their average values obtained 15 minutes after the start of the analysis. T3.3 staining intensities are normalized to its average value obtained in the absence of ciprofloxacin in the sample (see Example 1).

It can be seen from the experimental data that 10 minutes after the start of the analysis of the intensity of staining, K.Z. reach 99.8-100.5%, ofloxacin binding zones (T.Z. 1) - 99.8-100.4%, enrofloxacin binding zones (T.Z.2) - 99.3-100.0%) and virtually unchanged over the next five minutes. The staining intensities of the test zones of ciprofloxacin binding (T.Z.3) 10 minutes after the start of the analysis are from 0.2 to 0.6% and practically do not change over the next five minutes. Thus, the analysis time of the sample containing ciprofloxacin at a concentration of 30.0 ng / ml and not containing enrofloxacin and ofloxacin according to the ELISA, using the inventive device is 10 minutes

Example 5 (study of the time of obtaining positive for ofloxacin, enrofloxacin and ciprofloxacin analysis results using the inventive device):

Using the inventive device, an analysis of milk with a fat content of 3.2% containing ofloxacin, enrofloxacin and ciprofloxacin at concentrations of 30 ng / ml according to ELISA is performed. 120 μl of sample is added to the tube. The inventive device (test strip) is immersed vertically with the lower end of the membrane to absorb the sample to a depth of 0.5 cm in the sample and incubated at room temperature for 3 minutes Take out the test strip and place it in the groove of the retractable carriage of the detecting device for video-digital recording. The result of the analysis is evaluated after 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15 minutes using the video-digital detector software. Analysis time is controlled using a stopwatch. The analysis is carried out in five repetitions using test strips of five different series (strips No. 1-5).

The results of the analysis are shown in table 6. The intensity of staining K.Z. normalized to its average value obtained 15 minutes after the start of the analysis. The staining intensities TZ.1, TZ.2 and TZ.3 are normalized to their average values obtained in the absence of ofloxacin, enrofloxacin and ciprofloxacin in the sample (see Example 1).

It can be seen from the experimental data that 10 minutes after the start of the analysis of the intensity of staining reach 99.9-100.6% and remain virtually unchanged over the next five minutes. The staining intensities of the test binding zones of ofloxacin (T.Z. 1), enrofloxacin (T.Z.2) and ciprofloxacin (T.Z.3) 10 minutes after the start of the analysis are from 0 to 0.5% and practically do not change over the next five minutes. Thus, the analysis time of the sample containing ofloxacin, enrofloxacin and ciprofloxacin at concentrations of 30.0 ng / ml according to ELISA, using the inventive device is 10 minutes

References

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3. Liu L., Luo L., Suryoprabowo S., Peng J., Kuang H., Xu C. Sensors. 2014, 14, 16785-16798.

4. Hendrickson O.D., Zvereva E.A., Shanin LA., Zherdev A.V., Dzantiev B.B. J. Sci. Food Agric. 2019, 99, 3834-3842.

5. Chen X., Xu H., Lai W., Chen Y., Yang X., Xiong Y. Food Add. Cont .: Part A, 2012, 29 (3), 383-391.

6. Zhao Y., Zhang G., Liu Q., Teng M., Yang J., Wang J. J. Agric. Food Chem. 2008, 56, 12138-12142.

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Brief Description of the Drawings

In fig. 1 shows a diagram of the inventive device for immunochromatographic express laboratory and off-laboratory simultaneous individual detection of toxic contaminants - fluoroquinolone antibiotics (ofloxacin, enrofloxacin, ciprofloxacin), in food and environmental objects. 1 - a solid base of a working nitrocellulose membrane made of polystyrene, 2 - a working nitrocellulose membrane with deposited and dried immunoreagents, 3 - a final adsorbing membrane for absorbing sample components after the reaction, 4 - a fiberglass membrane with deposited and dried immunoreagents, 5 - an absorption membrane and separation of the test sample, A - K.Z. with applied polyclonal goat antibodies specific for rabbit immunoglobulins, B - T.Z.3 with a ciprofloxacin conjugated protein with a carrier protein SIT, B - T.Z.2 with a coated enrofloxacin conjugate with a protein carrier SIT, G - T.3.1 coated with ofloxacin conjugate with SIT carrier protein.

In fig. 2 shows an algorithm for interpreting analysis results. If four red lines appear on the working membrane of the test strip (K.Z., T.Z.1, T.Z.2 and T.Z.3), the result of the analysis is considered negative for three antibiotics (Fig. 2a). If two red lines appear on the working membrane of the test strip (K.Z. and T.Z. 1), the result of the analysis is considered negative for ofloxacin and positive for enrofloxacin and ciprofloxacin (Fig. 2b). If two red lines appear on the working membrane of the test strip (K.Z. and T.Z. 2), the result of the analysis is considered negative for enrofloxacin and positive for ofloxacin and ciprofloxacin (Fig. 2c). If two red lines appear on the working membrane of the test strip (K.Z. and T.Z. 3), the result of the analysis is considered negative for ciprofloxacin and positive for ofloxacin and enrofloxacin (Fig. 2d). If three red lines appear on the working membrane of the test strip (K.Z., T.Z.2 and T.Z.3), the result of the analysis is considered negative for ciprofloxacin and enrofloxacin and positive for ofloxacin (Fig. 2e). If three red lines appear on the working membrane of the test strip (K.Z., T.Z. 1 and T.Z.3), the result of the analysis is considered negative for ofloxacin and ciprofloxacin and positive for enrofloxacin (Fig. 2f). If three red lines appear on the working membrane of the test strip (K.Z., T.Z.1 and T.Z.2), the result of the analysis is considered negative for ofloxacin and enrofloxacin and positive for ciprofloxacin (Fig. 2g). If one red line appears on the working membrane of the test strip (KZ), the result of the analysis is considered positive for three antibiotics (Fig. 2c). If no colored lines are formed on the working membrane of the test strip or only test lines are formed (one, two or three in any combination), the result of the analysis is considered invalid (Fig. 2i).

Claims (1)

A device for immunochromatographic simultaneous individual detection of fluoroquinolone antibiotics ofloxacin, enrofloxacin and ciprofloxacin in food products, which is a test strip, which consists of a polystyrene substrate with glued membranes: a nitrocellulose membrane with deposited and dried test immunoreag membranes applied and dried immunoreagents, a membrane for the absorption and separation of the test sample and the final hell an orbiting membrane to absorb the components of the sample after the reaction, characterized in that polyclonal goat antibodies specific for rabbit immunoglobulins are applied to the control zone, ofloxacin conjugated with a carrier protein is applied to the first test zone, and enrofloxacin-protein-conjugated conjugate is loaded into the second test zone , a ciprofloxacin conjugate with a carrier protein is applied to the third test zone, a mixture of conjugates with colloidal gold of three polyclonal cro drugs is applied to the glass fiber membrane ichih specific antibodies to ofloxacin, and ciprofloxacin, enrofloxacin.

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