Immunochromatographic technique for express determination of ampicillin in milk and dairy products

ISSN 0003�6838, 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.

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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 isantibiotic contamination of food chains and theirnontherapeutic intake in the human body with food,which can cause the development of resistant forms ofmicroorganisms, dysbacterioses, allergic reactions,suppression of the activity of certain enzymes, etc. [1–5]. In this regard, the content of antibiotics is animportant parameter in evaluating the safety of food.Microbiological and chemical methods are mostextensively used for the control of antibiotics [4].

Microbiological testing is based on the suppressionof antibiotics contained in the sample, the growth ofthe test organism (usually streptococci, micrococci, orspore�forming aerobes), or the synthesis of a certainenzyme by this organism. Adding a test organism to asample of milk can cause a shift of pH controlled bylitmus, bromocresol purple solutions, etc. and redoxreactions; the process can be tested using methyleneblue or triphenyltetrazolium [6–9]. Although micro�biological methods provide adequate informationabout the content in a sample of physiologically activeantibiotics molecules, they require a long time andlimit the room in which you are working with microor�ganisms.

Alternatives to microbiological testing methods arechemical testing methods. For the detection of antibi�otics of different classes, a variety of methods basedgenerally on chromatography with mass spectrometryor 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 areneeded to implement these methods. In addition,chromatographic analysis is rather laborious, becauseit involves a multistep sample preparation—extrac�tion, concentration, etc. These factors determine thehigh cost of testing and the inability to use these meth�ods for wide monitoring.

Based upon the above, the basic requirements todevelop new methods for the determination of antibi�otics are the lowest labor costs and operational rangesunder the maximum permissible concentration ofcontaminants in food. These conditions are satisfiedin the transition to immunoanalytical methods. Theapplication of antibodies provides high selectivity ofanalysis, and their labeling provides signal gain and thepossibility of detecting low concentrations of antibi�otic antigens. Currently, immunoanalytical methodsare being successfully applied for solving various prac�tical problems, especially in medical and veterinarydiagnostics [14–16], which indicates their potential,and for the purposes of food control.

Traditional microplate enzyme immunoassay(EIA), the most intensively used immunoanalyticalmethod [17–20], takes approximately 2 hours becauseof diffusion limitations. The ability to use immu�nochemical approaches has led to a substantialincrease in the number of alternative formats with thetime of analysis 10–20 minutes. [14, 21, 22]. Immu�nochromatography, in which all the specific reactionsand the formation of the detected signal are initiatedby contact of the test strip with the sample and whichdoes not require auxiliary reagents, instruments, andadditional manipulation, etc., is of greatest interest for

Immunochromatographic Technique for Express Determinationof Ampicillin in Milk and Dairy Products

N. A. Byzova, E. A. Zvereva, A. V. Zherdev, and B. B. DzantievBach Institute of Biochemistry, Russian Academy of Sciences, Moscow, 119071 Russia

e�mail: [email protected] May 16, 2011

Abstract—An immunochromatographic method for determination of β�lactam antibiotic ampicillin hasbeen developed. The method is based on the competitive interaction between antibiotic molecules containedin 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 reagentsalong 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

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the express monitoring of immunoassay formats [23,24]. However, some methodological sophistication isoften required for real sample analysis, because thenative sample may flow with difficulty along a test stripor interfere with immunochemical interactions. Forexample, when testing milk samples, placing the teststrip in a thermostat with temperature ranging from 45to 56°С is usually recommended [www.charm.com].

Previously, we implemented immunochromato�graphic assay (ICA) for chloramphenicol [25] andstreptomycin [26] without pretreatment and incuba�tion, which was achieved through a combination of theparameters of antibodies, colloidal particles, andmembranes.

The purposes of this work are development andcharacterization of express ICA for ampicillin—a rep�resentative of a widespread class of antibiotics, beta�lactam antibiotics.

METHODS

Monoclonal antibodies against β�lactam 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) anti�mouse IgG anti�bodies; sheep (SARIss) anti�rabbit IgG antibodies(Imtek, Russia); goat (GAMI) anti� mouse IgG anti�bodies (Arista Biologicals, USA); bovine anti�mouseIgG 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 X�100, dihydrochloride of3,3',5,5'�tetramethylbenzidine, sodium azide (Sigma,United States), neomycin, cephalexin, ciprofloxacin,sulfaqvinoxamine, sulfamethoxipyridazine, goldhydrochloric acid (Fluka, Germany), Tween 20, BSA,sodium citrate, dimethylsulfoxide (DMSO, MP Bio�medicals, United Kingdom), glycerol, NaCl, K2CO3(DiaeM, Russia), Na2CO3, NaHCO3, KH2PO4, andKOH (Himmed, Russia) were also used. All ancillaryreagents (salts, acids, alkalis, organic solvents) were ofanalytical or chemical purity.

The solutions for obtaining colloidal gold (CG) andits conjugates were prepared in water deionized usinga Milli�Q purification system (Millipore, UnitedStates).

The stock solutions of antibiotics (1–5 mg/ml in 50mM citrate buffer, pH 6.4) were prepared on the day ofthe experiment, except chloramphenicol, rifampicin,sulfonylamide, and sulfaqvinoxamine. Stock solutionsof chloramphenicol in ethanol, rifampicin in DMSO,and sulfonylamide and sulfaqvinoxamine in methanolwere used as both freshly prepared and stored at 4°Сfor a month.

EIA was performed in a Costar 9018 96�well trans�parent polystyrene microplate (Corning Costar,

United States). Immunochromatographic test stripswere prepared with the use of membranes from themdi Easypack membrane kit (Advanced Microde�vices, India) consisting of a membrane attached to aworking membrane, a colloidal gold conjugate pad, asample pad, a final adsorbent pad, and a protectivelaminate film.

Competitive EIA of ampicillin. Penicillin–BSAconjugate in the concentration of 2 µg/ml in 50 mMK�phosphate buffer (pH 7.4) with 0.1 M NaCl (PBS)was immobilized from a volume of 100 µl in wells of amicroplate for a night at 4°С. Then, PBS microplatewas washed four times with 0.05% of Triton X�100(PBST). Further, 50 µl of ampicillin solution (con�centration range from 1 µg/ml to 10 pg/ml) wereplaced in wells of the microplate, and 50 µl of specificantibodies in the concentration of 0.02 µg/ml wereadded in PBST. The microplate was incubated for 1 hat 37°С, then PBS was washed four times, and 100 µlof immunoperoxidase conjugate (dilution of 1 : 6,000in PBST) was added into the wells and it was againincubated for 1 h at 37°С. Peroxidase activity of theenzyme label associated with the carrier was deter�mined after washing (three times by PBST and once bydistilled water). For this, 100 µl of 0.4 mM 3,3',5,5'�tetramethylbenzidine solution in 40 mM Na�citratebuffer (pH 4.0) with 3 mM Н2О2 was placed into wellsof the microplate and it was incubated for 15 min atroom temperature. The reaction was stopped by theaddition 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 immunoenzymeexperiment of characteristic of antibodies’ specificityin concentrations from 10 µg/ml to 1 ng/ml.

The dependence of optical density (y) versus theantigen concentration in the samples (x) was approxi�mated with the 4�parametric 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 ofantibodies by 50% (IC50). Antigen concentrationcausing 10% of inhibition (IC10) was calculated usingthe 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 to97.5 ml of deionized water and it was boiled. Whenmixing, 1.5 ml of 1% sodium citrate was added. Themixture was boiled for 25 min, then it was cooled andstored 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 ofpolyvinylformal dissolved in chloroform. Images wereobtained using a CX�100 electron microscope (Jeol,Japan) at accelerating voltage of 80 kV and an increase


IMMUNOCHROMATOGRAPHIC TECHNIQUE FOR AMPICILLIN DETERMINATION 629

of 3300000. Photos in digital form were analyzed inthe Image Tool program.

Synthesis of CG�antibodies conjugates. The pre�liminary characterization of the conjugation of anti�bodies with CG was carried out according to [28]. Forthis purpose, 1.0 ml of CG solution (D520 = 1.0) wasadded to 0.1 ml of aqueous solution of antibodies(concentration varied from 5 to 250 µg/ml) and it wasmixed and incubated for 10 min at room temperature.Then, 0.1 ml of 10% NaCl was added into each probeand it was mixed. D580 was measured in 10 min.

Before conjugation with CG, the antibodies weredialyzed against 1,000�fold volume of 10 mM tris�HClbuffer (pH 8.5) for 2 h at 4°C. To CG solution, 0.2 MК2СО3 was added (D520 = 1.0) until pH 8.5, and a solu�tion of antibodies at a certain concentration wasspiked. The mixture was incubated for 30 min at roomtemperature and with mixing; following this, BSA wascarried 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 PBScontaining 0.25% BSA. If long�term storage was nec�essary, NaN3 was added to the received product up tofinal concentration of 0.02%.

Preparation of the immunochromatographic testsystem. Application of reagents on membrane in thetest system was conducted using an IsoFlow (ImageneTechnology, United States) automatic dispenser. Thecolloidal gold�antibody conjugate was applied on apad at a dilution corresponding to D520 = 2.0 (32 µl for1 cm of substrate width). For formation of analytic

zone, the penicillin–BSA conjugate (0.2 mg/ml in0.2 M carbonate buffer, pH 9.6) was used; for forma�tion of control zone, goat anti�mouse 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 ofworking membrane width. The resulting membranesand pads were air dried at 20–22°C no less than 20 h.Multimembrane composite was obtained from whichstrips with the width of 3.5 mm were obtained using anIndex Cutter�1 (A�Point Technologies, United States)automatic guillotine cutter. These test strips with silicagel as the desiccant were vacuum�packed in bags fromlaminated aluminum foil using a FR�900 (Wenzhoudingli 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 nomore 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 witha 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 theRussian Academy of Agricultural Sciences). Samplesof these products were spiked with different amountsof antibiotics and stirred. Probes of finished milk anddairy 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. 1. Determination of ampicillin in buffer by competi�tive EIA method. The parameters of the approximatingcurve: 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 D580corresponds to CG solution without antibodies and with�out addition of 10% NaCl. The selected concentration ofantibodies (12 µg/ml) is marked by an arrow.

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opened, the test strip was pulled, and its lower end wasvertically submerged into an aliquot of the sample(50 µl) for 1 min following which the test strip wasplaced on level surface. The effect was controlled10 min after the beginning of the analysis getting a dig�ital image of the test strip on a Bear Paw 4800TA proscanner (Mustek, Taiwan) and calculating the integralcolor intensity in the test and control zones as writtenin [29].

RESULTS AND DISCUSSION

Preparation and characterization of immunoreac�tants used in ICA. Analytical possibilities of commer�cial immunoreagents was preliminarily characterizedby EIA. As follows from the calibration curve of EIAobtained 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 competitiveimmunochromatographic assay the recorded disap�pearance of coloration in the analytical zone corre�sponds to the output of the calibration curve at thebottom not on the upper plateau. With regard tomicroplate EIA, this effect was achieved at ampicillinconcentration 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 fordevelopment of express analytic system. Actually, inour previous experiment the difference threshold ofpositive and negative samples in immunochromatog�raphy and outlet of calibration curve of EIA on lower

plateau correspond to practically equal concentrationsof the analyte [30].

Antibodies were characterized by specificity toampicillin (100%) and cephalexin (150%) and lowcross�reactivity with respect to other beta�lactams: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 homogeneityof particles by sizing feature [25]. Mean value of max�imal axis was 37 ± 8 nm, of minimal axis was 30 ± 5nm. Thus, particles were characterized by a meandiameter of 34 nm, which corresponds to generallyapplicable recommendations on the optimal colloidalgold particle size for immunochromatography (30�40nm) [31]. For particles obtained, the adsorptionimmobilization 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 antibodieswith CG were chosen based on the photometric datareflecting 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 decreasewith outlet on the plateau up to 12 µg/ml. We note thatmolar ratio antibody : CG is 190 : 1 at an antibodiesconcentration 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 immobilizationof antibodies. Antibody concentration exceeding exitpoint D580 on plateau by 10–15% was chosen for con�jugation as recommended in [28], which allows stabi�

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Fig. 3. Dependence of ampicillin calibration curve ICA onthe 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, pH7.4, with 0.1 M NaCl; 3—0.1 M citrate buffer, pH 6.4

Fig. 4. Dependence of ampicillin calibration curve ICA onthe penicillin–BSA conjugate concentration used for theimmobilized 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.



lization of surface of colloidal particle by antibodiesand prevention of the formation of aggregates. Thus,antibodies in a concentration of 12 µg/ml were in syn�thesis. The excess of unreacted antibodies wasremoved in the step of the precipitation of the conju�gate.

Development of competitive ICA of ampicillin. Firstof all, we checked ability of synthetic CG�antibodiesconjugate to interaction in control and analytic zonesof the test strip and chose optimal reagents for applica�tion in these zones.

A series of preparations of anti�mouse IgG anti�bodies were correlated with binding of CG�antibodiesconjugate in the course of immunochromatography.At saturating concentrations, the color intensity of thecontrol line was 115 and 133 rel. un. for goat antibod�ies (GAMIss and GAMI, respectively), 80 rel. un. forrabbit antibodies (RAMIss), and 95 rel. un. for sheep

antibodies (SAMIss). Although the differences are notbig, goat antibodies GAMI (Arista Biologicals) pro�viding maximal binding of CG were chosen for furtherapplication. Optimal concentration of antispeciesantibodies at immobilization in control zone was0.25 mg/ml corresponding to outlet on the plateauconcentration dependence of binding of colloidalconjugate.

Penicillin�BSA conjugate was used for formationof an analytic zone of the test strip. The buffer in whichconjugate was applied in analytic zone was chosen onthe first step. As follows from Fig. 3, the color intensityin the test zone in the absence of the analyte in thesample 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. unitsfrom PBS, pH 7.4; and 17 rel. units from a citratebuffer, ðÍ 6.4. Obviously, there is an advantage of the

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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—externalview 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).

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carbonate buffer, which was used in further prepara�tion of test strips.

Based on data from our previous work [25, 26, 30],CG�antibody conjugates were applied from a solution,the concentration of which corresponded to D520 =2.0, which ensured the formation of intensely coloredareas in the analysis combined with the completenessof washing of reagent from the starting area and thelack of nonspecific coloration of the working mem�brane.

For a productive immunochromatographic test, itis important that the change in the qualitative result of

the assay (disappearance of coloration in the analyticalarea) corresponds to the controlled level of compoundcontent (its maximum allowable concentration) and,therefore, does not require the additional dilution ofthe sample by a particular factor or its concentrationbefore the assay. A time�consuming solution to thisproblem 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 controlthe threshold of differentiation between positive andnegative samples for immobilization.

Figure 4 shows the dependences of the intensity ofcolor of the analytical zone obtained on the content ofampicillin in the sample at different concentrations ofpenicillin�BSA conjugate. As can be seen from Fig. 4,a decrease in the concentration of the conjugate uponimmobilization from 0.4 to 0.2 mg/mL resulted in adecrease in the limit of detection of ampicillin by afactor of 10 (from 100 to 10 ng/mL). Further decreaseof the concentration of penicillin–BSA conjugate to0.1 mg/ml led to a significant (2.5 times) reduction inthe amplitude of the calibration curve of ICA. There�fore, the concentration of penicillin–BSA conjugateequal 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 analyticalzone and the low consumption of the reactant.

The results suggest the possibility to shift thethreshold of differentiation between positive and neg�ative samples on the order with minimal loss in signalamplitude (and, thus, in the accuracy and reliability ofmeasurements) in the optimization of immunoassaysystems.

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

Fig. 7. Immunochromatographic determination of ampicillin in milk, raw milk, and dairy products. 1—milk product with fatcontent 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 milkdiluted 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 witha fat content of 3.2%;7—drinking frugurt with a fat content of 1.5%. Left strip for each matrix corresponds to the absence ofampicillin in probe; right strip corresponds to the presence of ampicillin in 10 ng/ml.



Characteristics of the immunochromatographic testsystem developed. On the basis of optimization, teststrips were prepared and experiments for immuno�chromatographic control of presence of ampicillinimmunoassay in standard solutions and milk and dairyproducts were carried out.

Figure 5 shows the results of sample analysis (FBSand milk with fat content 3.5%) with various concen�trations of ampicillin. In the ICA, disappearance ofcolor in the analytical zone corresponded to exceedthe 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 zonewas 50% of the maximum; it was 70% after 7 min and85% after 10 minutes (Fig. 6). With this in mind, therecommended duration of the analysis is 10 min.

In the immunochromatographic system, no crossreactions 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 to10 μg/mL were observed.

The developed test system was used for analysis offull 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%, drinkingfrugurt with fat content of 1.5%). The results are shownin Fig. 7. For all tested samples, the possibility of reliabledetection of ampicillin with the same minimum detect�able concentration as in buffer—10 ng/ml—was dem�onstrated.

We note that, for immunochromatographic testingof full cream raw milk, the liquid level did not risealong 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 theanalysis as a whole. The coloration in the test zone wasobserved in the dilute sample containing ampicillin atconcentrations lower than 10 ng/mL (in the startingsample, 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. Currentlyavailable membrane tests to determine ampicillinrequire preincubation at elevated temperature or sam�ples of milk with special receptors (Twinsensor BT,Twinsensor, Belgium; Beta star, USB�Bioproducts,United States; SNAP, IDEXX, USA) or test stripsinscribed with the milk samples applied (Charm SLBeta�lactam Test, Charm, United States). A numberof immunochromatographic tests for the determina�tion of β�lactam antibiotics, such as PENs�204R4(NKBIO, China), Penicillin G Rapid Test (Quicking

Biotech, China) involve the multistep milk samplepreparation. In contrast to the above tests, analysis ofmilk using the developed test system can be carried outat room temperature without sample pretreatment. Animportant advantage of the test systems is their suit�ability for security control of dairy products, since theyprovide the same thresholds for contamination, whilesuitability for this purpose of commercially availabletest systems is not characterized.

Analysis results can be monitored visually or usingportable photometric detector software [32]. Due tothe rapidity and methodological simplicity, the devel�oped test system can be considered as an efficient toolfor the mass screening of raw milk, milk and cultureddairy 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 governmentcontracts no. 02.740.11.0868 from 28.06.2010, no.14.740.11.0615 from 05.10.2010, and no. 16.512.11.2125from 25.02.2011, by the Russian Foundation for BasicResearch, project nos. 09�08�01209 and 11�04�91189,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 MaterialsStructure.”

1 2 3

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 of3 : 1 (vol./vol.). Left strip for each probe corresponds to theabsence of ampicillin; right strip corresponds to the pres�ence of ampicillin in 10 ng/ml.

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Immunochromatographic technique for express determination of ampicillin in milk and dairy products

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