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additional manipulation, etc., is of greatest interest for. Immunochromatographic Technique for Express Determination of Ampicillin in Milk and Dairy Products.
ISSN 00036838, Applied Biochemistry and Microbiology, 2011, Vol. 47, No. 6, pp. 627–634. © Pleiades Publishing, Inc., 2011. Original Russian Text © N.A. Byzova, E.A. Zvereva, A.V. Zherdev, B.B. Dzantiev, 2011, published in Prikladnaya Biokhimiya i Mikrobiologiya, 2011, Vol. 47, No. 6, pp. 685–693.

Immunochromatographic Technique for Express Determination of Ampicillin in Milk and Dairy Products N. A. Byzova, E. A. Zvereva, A. V. Zherdev, and B. B. Dzantiev Bach Institute of Biochemistry, Russian Academy of Sciences, Moscow, 119071 Russia email: [email protected] Received May 16, 2011

Abstract—An immunochromatographic method for determination of βlactam antibiotic ampicillin has been developed. The method is based on the competitive interaction between antibiotic molecules contained in the sample and protein conjugate of penicillin immobilized on a membrane for binding with specific anti bodies labeled with colloidal gold, which occurs during movement of the sample to be tested and reagents along the membrane. The completion of the test system ensures control of exceeding the maximum permis sible content of the antibiotic in milk and dairy products (10 ng/mL). The possibility of testing milk, raw milk, and dairy products for 10 minutes at room temperature without sample pretreatment has been demonstrated. Keywords: ampicillin, immunoassay, immunochromatography, colloidal gold, milk, dairy products. DOI: 10.1134/S0003683811060032

Currently, antibiotics are widely used both to com bat human diseases and for the prevention and treat ment of animal diseases. A significant risk factor is antibiotic contamination of food chains and their nontherapeutic intake in the human body with food, which can cause the development of resistant forms of microorganisms, dysbacterioses, allergic reactions, suppression of the activity of certain enzymes, etc. [1– 5]. In this regard, the content of antibiotics is an important parameter in evaluating the safety of food. Microbiological and chemical methods are most extensively used for the control of antibiotics [4]. Microbiological testing is based on the suppression of antibiotics contained in the sample, the growth of the test organism (usually streptococci, micrococci, or sporeforming aerobes), or the synthesis of a certain enzyme by this organism. Adding a test organism to a sample of milk can cause a shift of pH controlled by litmus, bromocresol purple solutions, etc. and redox reactions; the process can be tested using methylene blue or triphenyltetrazolium [6–9]. Although micro biological methods provide adequate information about the content in a sample of physiologically active antibiotics molecules, they require a long time and limit the room in which you are working with microor ganisms. Alternatives to microbiological testing methods are chemical testing methods. For the detection of antibi otics of different classes, a variety of methods based generally on chromatography with mass spectrometry or capillary electrophoresis was developed [10–13]. Despite the low limits of detection (up to 0.1 ng/ml), these methods have significant drawbacks. Complex

and expensive equipment and skilled personnel are needed to implement these methods. In addition, chromatographic analysis is rather laborious, because it involves a multistep sample preparation—extrac tion, concentration, etc. These factors determine the high cost of testing and the inability to use these meth ods for wide monitoring. Based upon the above, the basic requirements to develop new methods for the determination of antibi otics are the lowest labor costs and operational ranges under the maximum permissible concentration of contaminants in food. These conditions are satisfied in the transition to immunoanalytical methods. The application of antibodies provides high selectivity of analysis, and their labeling provides signal gain and the possibility of detecting low concentrations of antibi otic antigens. Currently, immunoanalytical methods are being successfully applied for solving various prac tical problems, especially in medical and veterinary diagnostics [14–16], which indicates their potential, and for the purposes of food control. Traditional microplate enzyme immunoassay (EIA), the most intensively used immunoanalytical method [17–20], takes approximately 2 hours because of diffusion limitations. The ability to use immu nochemical approaches has led to a substantial increase in the number of alternative formats with the time of analysis 10–20 minutes. [14, 21, 22]. Immu nochromatography, in which all the specific reactions and the formation of the detected signal are initiated by contact of the test strip with the sample and which does not require auxiliary reagents, instruments, and additional manipulation, etc., is of greatest interest for

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the express monitoring of immunoassay formats [23, 24]. However, some methodological sophistication is often required for real sample analysis, because the native sample may flow with difficulty along a test strip or interfere with immunochemical interactions. For example, when testing milk samples, placing the test strip in a thermostat with temperature ranging from 45 to 56°С is usually recommended [www.charm.com]. Previously, we implemented immunochromato graphic assay (ICA) for chloramphenicol [25] and streptomycin [26] without pretreatment and incuba tion, which was achieved through a combination of the parameters of antibodies, colloidal particles, and membranes. The purposes of this work are development and characterization of express ICA for ampicillin—a rep resentative of a widespread class of antibiotics, beta lactam antibiotics. METHODS Monoclonal antibodies against βlactam antibiot ics and penicillin–bovine serum albumin conjugate (BSA) (DCN, United States) were used in the devel opment of test systems. Goat (GAMIss), rabbit (RAMIss), and sheep (SAMIss) antimouse IgG anti bodies; sheep (SARIss) antirabbit IgG antibodies (Imtek, Russia); goat (GAMI) anti mouse IgG anti bodies (Arista Biologicals, USA); bovine antimouse IgG antibodies conjugated with peroxidase (Medga mal, Russia); ampicillin, penicillins G and V, amox icillin, cloxacillin, chloramphenicol, streptomycin, gentamicin, kanamycin, tetracycline, rifampicin, sul fonylamide, Tris, Triton X100, dihydrochloride of 3,3',5,5'tetramethylbenzidine, sodium azide (Sigma, United States), neomycin, cephalexin, ciprofloxacin, sulfaqvinoxamine, sulfamethoxipyridazine, gold hydrochloric acid (Fluka, Germany), Tween 20, BSA, sodium citrate, dimethylsulfoxide (DMSO, MP Bio medicals, United Kingdom), glycerol, NaCl, K2CO3 (DiaeM, Russia), Na2CO3, NaHCO3, KH2PO4, and KOH (Himmed, Russia) were also used. All ancillary reagents (salts, acids, alkalis, organic solvents) were of analytical or chemical purity. The solutions for obtaining colloidal gold (CG) and its conjugates were prepared in water deionized using a MilliQ purification system (Millipore, United States). The stock solutions of antibiotics (1–5 mg/ml in 50 mM citrate buffer, pH 6.4) were prepared on the day of the experiment, except chloramphenicol, rifampicin, sulfonylamide, and sulfaqvinoxamine. Stock solutions of chloramphenicol in ethanol, rifampicin in DMSO, and sulfonylamide and sulfaqvinoxamine in methanol were used as both freshly prepared and stored at 4°С for a month. EIA was performed in a Costar 9018 96well trans parent polystyrene microplate (Corning Costar,

United States). Immunochromatographic test strips were prepared with the use of membranes from the mdi Easypack membrane kit (Advanced Microde vices, India) consisting of a membrane attached to a working membrane, a colloidal gold conjugate pad, a sample pad, a final adsorbent pad, and a protective laminate film. Competitive EIA of ampicillin. Penicillin–BSA conjugate in the concentration of 2 µg/ml in 50 mM Kphosphate buffer (pH 7.4) with 0.1 M NaCl (PBS) was immobilized from a volume of 100 µl in wells of a microplate for a night at 4°С. Then, PBS microplate was washed four times with 0.05% of Triton X100 (PBST). Further, 50 µl of ampicillin solution (con centration range from 1 µg/ml to 10 pg/ml) were placed in wells of the microplate, and 50 µl of specific antibodies in the concentration of 0.02 µg/ml were added in PBST. The microplate was incubated for 1 h at 37°С, then PBS was washed four times, and 100 µl of immunoperoxidase conjugate (dilution of 1 : 6,000 in PBST) was added into the wells and it was again incubated for 1 h at 37°С. Peroxidase activity of the enzyme label associated with the carrier was deter mined after washing (three times by PBST and once by distilled water). For this, 100 µl of 0.4 mM 3,3',5,5' tetramethylbenzidine solution in 40 mM Nacitrate buffer (pH 4.0) with 3 mM Н2О2 was placed into wells of the microplate and it was incubated for 15 min at room temperature. The reaction was stopped by the addition of 50 µl of 1 M H2SO4, and D450 was mea sured. Instead of ampicillin, penicillins G and V, amox icillin, and cloxacillin were used in immunoenzyme experiment of characteristic of antibodies’ specificity in concentrations from 10 µg/ml to 1 ng/ml. The dependence of optical density (y) versus the antigen concentration in the samples (x) was approxi mated with the 4parametric sigmoid function y = (A – D)/(1 + (x/C)B) + D using Origin 7.5 software (OriginLab, United States). С parameter value corre sponds to antigen concentration inhibiting binding of antibodies by 50% (IC50). Antigen concentration causing 10% of inhibition (IC10) was calculated using the same function and it was considered as the detec tion limit of the analysis. Obtainment of CG by the citrate method [25, 27]. One milliliter of 1% HAuCl4 solution was added to 97.5 ml of deionized water and it was boiled. When mixing, 1.5 ml of 1% sodium citrate was added. The mixture was boiled for 25 min, then it was cooled and stored at 4–6°С. Transmission electron microscopy. CG prepara tions were applied on grids (300 mesh, Pelco Interna tional, United States) coated with a support film of polyvinylformal dissolved in chloroform. Images were obtained using a CX100 electron microscope (Jeol, Japan) at accelerating voltage of 80 kV and an increase

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Fig. 1. Determination of ampicillin in buffer by competi tive EIA method. The parameters of the approximating curve: А = 1.00996; D = 0.07089; С = 0.43601; B = 1.23197.

Fig. 2. Determination of specific antibody concentration (µg/ml) used for conjugation with CG. Zero level D580 corresponds to CG solution without antibodies and with out addition of 10% NaCl. The selected concentration of antibodies (12 µg/ml) is marked by an arrow.

of 3300000. Photos in digital form were analyzed in the Image Tool program. Synthesis of CGantibodies conjugates. The pre liminary characterization of the conjugation of anti bodies with CG was carried out according to [28]. For this purpose, 1.0 ml of CG solution (D520 = 1.0) was added to 0.1 ml of aqueous solution of antibodies (concentration varied from 5 to 250 µg/ml) and it was mixed and incubated for 10 min at room temperature. Then, 0.1 ml of 10% NaCl was added into each probe and it was mixed. D580 was measured in 10 min. Before conjugation with CG, the antibodies were dialyzed against 1,000fold volume of 10 mM trisHCl buffer (pH 8.5) for 2 h at 4°C. To CG solution, 0.2 M К2СО3 was added (D520 = 1.0) until pH 8.5, and a solu tion of antibodies at a certain concentration was spiked. The mixture was incubated for 30 min at room temperature and with mixing; following this, BSA was carried up to a final concentration of 0.25%. CG par ticles with antibodies immobilized on them were sep arated from noninteracting antibodies by centrifuga tion at 8000 g for 30 min. After removal of superna tant, the precipitate was resuspended in PBS containing 0.25% BSA. If longterm storage was nec essary, NaN3 was added to the received product up to final concentration of 0.02%. Preparation of the immunochromatographic test system. Application of reagents on membrane in the test system was conducted using an IsoFlow (Imagene Technology, United States) automatic dispenser. The colloidal goldantibody conjugate was applied on a pad at a dilution corresponding to D520 = 2.0 (32 µl for 1 cm of substrate width). For formation of analytic

zone, the penicillin–BSA conjugate (0.2 mg/ml in 0.2 M carbonate buffer, pH 9.6) was used; for forma tion of control zone, goat antimouse IgG antibodies (GAMI, 0.25 mg/ml in PBS) were used. Both solu tions were stabilized and applied in 2.0 µl for 1 cm of working membrane width. The resulting membranes and pads were air dried at 20–22°C no less than 20 h. Multimembrane composite was obtained from which strips with the width of 3.5 mm were obtained using an Index Cutter1 (APoint Technologies, United States) automatic guillotine cutter. These test strips with silica gel as the desiccant were vacuumpacked in bags from laminated aluminum foil using a FR900 (Wenzhou dingli packing machinery, China) solder with a mini conveyor. Cutting and packing were conducted at 20– 22°C in a special room with relative air humidity no more than 30%. Preparation of the milk, raw milk, and dairy prod ucts. Cow’s milk with a fat content from 0.5% to 6.0%, kefir with a fat content of 1.0%, sour clotted milk with a fat content of 3.2%, and drinking frugurt with a fat con tent of 1.0% were bought in a retail distribution network. Full cream raw cow milk was given by E.A. Yurova (All Russian Research Institute of Dairy Industry of the Russian Academy of Agricultural Sciences). Samples of these products were spiked with different amounts of antibiotics and stirred. Probes of finished milk and dairy product were analyzed by the immunochro matographic method without sample pretreatment, and full cream milk before testing was diluted by dis tilled water in ratio of 3 : 1 (vol./vol.) Immunochromatographic assay and results. Analy sis was carried out at room temperature. The bag was

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Fig. 3. Dependence of ampicillin calibration curve ICA on the buffer type used at the immobilization of penicillin– BSA conjugate in analytic zone of test strip. 1—0.2 M car bonate buffer, pH 9.6; 2—50 mM phosphate buffer, pH 7.4, with 0.1 M NaCl; 3—0.1 M citrate buffer, pH 6.4

Fig. 4. Dependence of ampicillin calibration curve ICA on the penicillin–BSA conjugate concentration used for the immobilized on the test zone of test strip. 1–4—concen trations of penicillin–BSA conjugate solution are 0.4, 0.3, 0.2, and 0.1 mg/ml, respectively.

opened, the test strip was pulled, and its lower end was vertically submerged into an aliquot of the sample (50 µl) for 1 min following which the test strip was placed on level surface. The effect was controlled 10 min after the beginning of the analysis getting a dig ital image of the test strip on a Bear Paw 4800TA pro scanner (Mustek, Taiwan) and calculating the integral color intensity in the test and control zones as written in [29].

plateau correspond to practically equal concentrations of the analyte [30]. Antibodies were characterized by specificity to ampicillin (100%) and cephalexin (150%) and low crossreactivity with respect to other betalactams: penicillin G—0.05%; penicillin V—0.04%; amoxicil lin—0.07%; cloxacillin—0.03%. CG was obtained to the Frens method [27]. Elec tron microscopy shows a high degree of homogeneity of particles by sizing feature [25]. Mean value of max imal axis was 37 ± 8 nm, of minimal axis was 30 ± 5 nm. Thus, particles were characterized by a mean diameter of 34 nm, which corresponds to generally applicable recommendations on the optimal colloidal gold particle size for immunochromatography (3040 nm) [31]. For particles obtained, the adsorption immobilization allows formation of a monolayer con taining up to 180 antibody molecules for one CG par ticle (the average surface area of the contact is suppos edly 20 nm2). The conditions for the conjugation of antibodies with CG were chosen based on the photometric data reflecting the aggregation of the product of this reac tion at a high ionic strength of the solution. The con centration dependence obtained (Fig. 2) was charac terized by increase in optical density up to 2.5 µg/ml, abrupt decrease up to 6.0 µg/ml, and slow decrease with outlet on the plateau up to 12 µg/ml. We note that molar ratio antibody : CG is 190 : 1 at an antibodies concentration equal to 6 µg/ml, i.e., this ratio practi cally coincides with the theoretical limit of surface sat uration of particles at the monolayer immobilization of antibodies. Antibody concentration exceeding exit point D580 on plateau by 10–15% was chosen for con jugation as recommended in [28], which allows stabi

RESULTS AND DISCUSSION Preparation and characterization of immunoreac tants used in ICA. Analytical possibilities of commer cial immunoreagents was preliminarily characterized by EIA. As follows from the calibration curve of EIA obtained under optimized conditions (Fig. 1), mono clonal antibodies combined with hapten–protein con jugate allowed us to determine the ampicillin in con centration up to 0.05 ng/ml. However, we note that, during visual inspection of the results of competitive immunochromatographic assay the recorded disap pearance of coloration in the analytical zone corre sponds to the output of the calibration curve at the bottom not on the upper plateau. With regard to microplate EIA, this effect was achieved at ampicillin concentration equal to 10 ng/ml. This value corre sponds to maximum allowable concentration of ampi cillin in milk and dairy products [SanPiN 2.3.2.1078 01] and allows us to recommend the reagents for development of express analytic system. Actually, in our previous experiment the difference threshold of positive and negative samples in immunochromatog raphy and outlet of calibration curve of EIA on lower

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Fig. 5. Immunochromatographic detection of ampicillin in (a, b) a buffer and (c, d) milk with fat content of 3.5%: a, c—external view of test strips after analysis (I—analytic zone; II—control zone); 1–7—concentrations of ampicillin are 0, 0.3, 1, 3, 10, 30, and 100 ng/ml, respectively; b, d – the dependence of the color intensity in the test zone (rel. un.) on the ampicillin concentration (ng/ml).

lization of surface of colloidal particle by antibodies and prevention of the formation of aggregates. Thus, antibodies in a concentration of 12 µg/ml were in syn thesis. The excess of unreacted antibodies was removed in the step of the precipitation of the conju gate. Development of competitive ICA of ampicillin. First of all, we checked ability of synthetic CGantibodies conjugate to interaction in control and analytic zones of the test strip and chose optimal reagents for applica tion in these zones. A series of preparations of antimouse IgG anti bodies were correlated with binding of CGantibodies conjugate in the course of immunochromatography. At saturating concentrations, the color intensity of the control line was 115 and 133 rel. un. for goat antibod ies (GAMIss and GAMI, respectively), 80 rel. un. for rabbit antibodies (RAMIss), and 95 rel. un. for sheep APPLIED BIOCHEMISTRY AND MICROBIOLOGY

antibodies (SAMIss). Although the differences are not big, goat antibodies GAMI (Arista Biologicals) pro viding maximal binding of CG were chosen for further application. Optimal concentration of antispecies antibodies at immobilization in control zone was 0.25 mg/ml corresponding to outlet on the plateau concentration dependence of binding of colloidal conjugate. PenicillinBSA conjugate was used for formation of an analytic zone of the test strip. The buffer in which conjugate was applied in analytic zone was chosen on the first step. As follows from Fig. 3, the color intensity in the test zone in the absence of the analyte in the sample substantially differs from that observed at a sat urating (0.5 mg/mL) concentration of the penicillin– BSA conjugate: 155 rel. units in the case of immobili zation from a carbonate buffer, pH 9.6, 83 rel. units from PBS, pH 7.4; and 17 rel. units from a citrate buffer, ðÍ 6.4. Obviously, there is an advantage of the

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the assay (disappearance of coloration in the analytical area) corresponds to the controlled level of compound content (its maximum allowable concentration) and, therefore, does not require the additional dilution of the sample by a particular factor or its concentration before the assay. A timeconsuming solution to this problem consists in screening a large number of anti bodies that differ in affinity, which is not always possi ble. Changing the concentration of the hapten–pro tein (penicillin–BSA) conjugate was used to control the threshold of differentiation between positive and negative samples for immobilization.

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Fig. 6. Coloration dynamics of analytic zone in immuno chromatographic system of determination of ampicillin in milk with fat content of 1.5% in the absence of analyte.

carbonate buffer, which was used in further prepara tion of test strips. Based on data from our previous work [25, 26, 30], CGantibody conjugates were applied from a solution, the concentration of which corresponded to D520 = 2.0, which ensured the formation of intensely colored areas in the analysis combined with the completeness of washing of reagent from the starting area and the lack of nonspecific coloration of the working mem brane. For a productive immunochromatographic test, it is important that the change in the qualitative result of

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Figure 4 shows the dependences of the intensity of color of the analytical zone obtained on the content of ampicillin in the sample at different concentrations of penicillinBSA conjugate. As can be seen from Fig. 4, a decrease in the concentration of the conjugate upon immobilization from 0.4 to 0.2 mg/mL resulted in a decrease in the limit of detection of ampicillin by a factor of 10 (from 100 to 10 ng/mL). Further decrease of the concentration of penicillin–BSA conjugate to 0.1 mg/ml led to a significant (2.5 times) reduction in the amplitude of the calibration curve of ICA. There fore, the concentration of penicillin–BSA conjugate equal to 0.2 mg/ml was chosen for immobilization, which provides the necessary threshold of detection (10 ng/ml in accordance with [SanPiN 2.3.2.1078 01]) at a sufficient intensity of coloration of analytical zone and the low consumption of the reactant. The results suggest the possibility to shift the threshold of differentiation between positive and neg ative samples on the order with minimal loss in signal amplitude (and, thus, in the accuracy and reliability of measurements) in the optimization of immunoassay systems.

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Fig. 7. Immunochromatographic determination of ampicillin in milk, raw milk, and dairy products. 1—milk product with fat content of 0.5%; 2—milk product with fat content of 3.2%; 3—milk product with fat content of 6.0%; 4—full cream raw milk diluted before testing by distilled water in a ratio of 3 : 1 (vol./vol.); 5—kefir with a fat content of 1.0%; 6—sour clotted milk with a fat content of 3.2%;7—drinking frugurt with a fat content of 1.5%. Left strip for each matrix corresponds to the absence of ampicillin in probe; right strip corresponds to the presence of ampicillin in 10 ng/ml. APPLIED BIOCHEMISTRY AND MICROBIOLOGY

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Characteristics of the immunochromatographic test system developed. On the basis of optimization, test strips were prepared and experiments for immuno chromatographic control of presence of ampicillin immunoassay in standard solutions and milk and dairy products were carried out. Figure 5 shows the results of sample analysis (FBS and milk with fat content 3.5%) with various concen trations of ampicillin. In the ICA, disappearance of color in the analytical zone corresponded to exceed the maximum permissible content of ampicillin (10 ng/ml) in a test sample. The accuracy of determi nation was 10%. After 5 min after the start of the front of the liquid (milk), the intensity of coloration of analytical zone was 50% of the maximum; it was 70% after 7 min and 85% after 10 minutes (Fig. 6). With this in mind, the recommended duration of the analysis is 10 min. In the immunochromatographic system, no cross reactions with other groups of antibiotics (chloram phenicol, streptomycin, tetracycline, gentamicin, neomycin, kanamycin, ciprofloxacin, and rifampicin) and bacteriostatics (sulfamethoxypyridazine, sulfanil amide, and sulfaquinoxamine) at concentrations up to 10 μg/mL were observed. The developed test system was used for analysis of full cream raw milk, milk products (fat content of 0.5– 6.0%), and dairy products (kefir with fat content 1%, sour clotted milk with fat content of 3.2%, drinking frugurt with fat content of 1.5%). The results are shown in Fig. 7. For all tested samples, the possibility of reliable detection of ampicillin with the same minimum detect able concentration as in buffer—10 ng/ml—was dem onstrated. We note that, for immunochromatographic testing of full cream raw milk, the liquid level did not rise along the test strip until the end of the working mem brane, which did not allow the carrying out of a reli able assessment of the results of the analysis. Predilu tion of tests with distilled water in a ratio of 3 : 1 (vol/vol.) exclude these problems (Fig. 8), little affect ing on the complexity and the laboriousness of the analysis as a whole. The coloration in the test zone was observed in the dilute sample containing ampicillin at concentrations lower than 10 ng/mL (in the starting sample, at a concentration lower than 13.3 ng/mL). An important advantage of this method is the abil ity to implement it at room temperature. Currently available membrane tests to determine ampicillin require preincubation at elevated temperature or sam ples of milk with special receptors (Twinsensor BT, Twinsensor, Belgium; Beta star, USBBioproducts, United States; SNAP, IDEXX, USA) or test strips inscribed with the milk samples applied (Charm SL Betalactam Test, Charm, United States). A number of immunochromatographic tests for the determina tion of βlactam antibiotics, such as PENs204R4 (NKBIO, China), Penicillin G Rapid Test (Quicking APPLIED BIOCHEMISTRY AND MICROBIOLOGY

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Fig. 8. Immunochromatographic determination of ampi cillin in whole raw milk (probes 1 and 2) and raw milk (probe 3) diluted before testing by distilled water in ratio of 3 : 1 (vol./vol.). Left strip for each probe corresponds to the absence of ampicillin; right strip corresponds to the pres ence of ampicillin in 10 ng/ml.

Biotech, China) involve the multistep milk sample preparation. In contrast to the above tests, analysis of milk using the developed test system can be carried out at room temperature without sample pretreatment. An important advantage of the test systems is their suit ability for security control of dairy products, since they provide the same thresholds for contamination, while suitability for this purpose of commercially available test systems is not characterized. Analysis results can be monitored visually or using portable photometric detector software [32]. Due to the rapidity and methodological simplicity, the devel oped test system can be considered as an efficient tool for the mass screening of raw milk, milk and cultured dairy products for the ampicillin content. ACKNOWLEDGMENTS We thank I.V. Safenkova (Bach Institute of Bio chemistry, Russian Academy of Sciences) for the elec tron microscopic measurements of CG preparation. This work was financially supported by government contracts no. 02.740.11.0868 from 28.06.2010, no. 14.740.11.0615 from 05.10.2010, and no. 16.512.11.2125 from 25.02.2011, by the Russian Foundation for Basic Research, project nos. 090801209 and 110491189, and by the Program of Basic Research of the Presid ium of the Russian Academy of Sciences no. 8 “Cre ation and Improvement of Chemical Analysis Meth ods and Investigation of Substances and Materials Structure.”

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APPLIED BIOCHEMISTRY AND MICROBIOLOGY

Vol. 47

No. 6

2011