Development of enzyme-linked immunosorbent assay (ELISA) for the detection of neomycin residues in pig muscle, chicken muscle, egg, fish, milk and kidney

Meat Science 82 (2009) 53–58

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Development of enzyme-linked immunosorbent assay (ELISA) for the detection of neomycin residues in pig muscle, chicken muscle, egg, fish, milk and kidney S. Wang *, B. Xu, Y. Zhang, J.X. He Key Laboratory of Food Nutrition and Safety, Ministry of Education of China, Tianjin University of Science and Technology, 29 The Thirteenth Road, Tianjin 300457, PR China

a r t i c l e

i n f o

Article history: Received 21 July 2008 Received in revised form 3 December 2008 Accepted 4 December 2008

Keywords: Neomycin Polyclonal antibody Competitive direct ELISA Animal-derived food products Residues

a b s t r a c t A colorimetric competitive direct enzyme-linked immunosorbent assay (ELISA) method was developed using polyclonal antibody to determine neomycin residues in food of animal origin. No cross-reactivity of the antibody was observed with other aminoglycosides. The limit of detection of the method was 0.1 lg/kg. A simple and efficient sample extraction method was established with recoveries of neomycin ranged from 75% to 105%. The detection limits were 5 lg/kg(l) in pig muscle, chicken muscle, fish and milk, 10 lg/kg in kidney and 20 lg/kg in egg, respectively. Chemiluminescence assay was developed for detecting neomycin residues in pig muscle and chicken muscle. The limit of detection of the method was 0.015 lg/kg, and the detection limits were 1.5 lg/kg in pig muscle and 6 lg/kg in chicken muscle. The ELISA tests were validated by HPLC, and the results showed a good correlation (r2) which was greater than 0.9. Ó 2008 Elsevier Ltd. All rights reserved.

1. Introduction Neomycin is an aminoglycoside antibiotics produced by Streptomyces fradiae and widely used in veterinary medicine to treat bacterial infections in animals (Waksman & Lechevalier, 1949). Neomycin has a broad-spectrum antibiotic due to its growth inhibition of Gram-positive bacteria and Gram-negative bacteria (Waksman, Katz, Lechevalier, & Brunswick, 1950). It is known that neomycin can disturb protein synthesis in bacteria by binding the 30S subunit of rRNA, so it can cause misreading of the genetic code and inhibits translation (Fourmy, Yoshizawa, & Puglisi, 1998; Ren, Martinez, Kirsebom, & Virtanen, 2002). Neomycin is used to treat gastrointestinal infections of cattle, sheep, pigs, goats and poultry by the oral route and to treat mastitis by intramammary administration. However, neomycin presents well-known potentially ototoxic and nephrotoxic to human and animals (Cleveland et al., 1990; Waisbren & Spink, 1950). Moreover, neomycin, like other aminoglycosides, is poorly absorbed after oral administration. Despite the minor fraction absorbed, which accumulates mainly in the kidney, a short withdrawal time (<3 weeks) is recommended. After intra-muscular (IM) application of neomycin, a long withdrawal period (60–90 d) before slaughter should be exercised for kidney (Vlietstra, Masman, & Steijger, 1993), and a much shorter withdrawal time is required for muscle tissue (<5 d). For milk, intravenous or intra-muscular injections of aminoglycosides normally produce few residues in milk if the withdrawal time is respected. However, intramammary * Corresponding author. Tel.: +86 22 6060 1456; fax: +86 22 6060 1332. E-mail address: [email protected] (S. Wang). 0309-1740/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.meatsci.2008.12.003

or intrauterine use is a potential cause of the presence of residues in milk (Manners & Stewart, 1982; Ziv & Sulman, 1974). Thus monitoring of its residues in animal-derived foods is essential for public health. In order to protect the consumers, the European Union (EU) established maximum residue limits (MRLs) for animal edible tissues, 500 lg/kg for meat, fat, liver, eggs, 1500 lg/kg for milk and 5000 lg/kg for kidney (EC. No. 1183/2002) Therefore, establishment of a simple, quick and reliable analytical method is required to monitor neomycin residue levels in the livestock products. Various techniques have been developed for the detection of neomycin residues in milk, urine, blood, plasma and tissues including the microbial inhibition screening tests (Lantz, Lawrie, Witebsky, & Maclowry, 1980; Posyniak, Zmudzki, & Niedzielska, 2001; Rosner & Aviv, 1980; Shaikh, Jackson, & Guyer, 1991), high-performance liquid chromatography (HPLC) with either pre- or post-column derivatization (Binns & Tsuji, 1984; Tsuji & Jenkins, 1986) and thin layer chromatography (TLC) (Bossuyt, Renterghem, & Waes, 1976). Microbiological methods are preferred for a large scale first screening because of their convenience, low costs and broad-spectrum characteristics. For microbiological methods, long experiment period, poor sensitivity and the high limit of detection are its disadvantages. While, HPLC might be the technique for the confirmation of positive screening results. HPLC requires costly samples extraction and clean-up procedures, highly qualified personnel and expensive equipment, and the number of samples that can be processed daily is small. But ELISA methods can detect tens of samples at one time in a shorter time. In recent years, biosensor assay is developed to detection neomycin in milk (Valerie, Nathalie, & Pascal, 2005; Willem, Geert, Marjo, & Amerongen, 2003), and the colloidal gold-based

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immunochromatographic assay is studied to detection neomycin in plasma and milk. Till now, enzyme-linked immunosorbent assay (ELISA) has become the most popular method for the detection of veterinary medicine and pesticide residues in foods due to its high sensitivity, simplicity and ability to screen large number of small-volume samples. Several groups have developed immunoassays for neomycin detection, most of the assays relied on the direct ELISA to determine neomycin residues in milk and kidney. Haasnoot et al. (1999) developed a direct competitive ELISA for screening neomycin in milk and kidney using commercially available antiserum of neomycin with the detection limit of 6.3 ng/ml and 42 ng/g in samples. Loomans, Wiltenburg, Koets, and Amerongen (2003) developed an indirect competitive ELISA using neamin as an immunogen for the development of a generic ELISA detecting neomycin residues in milk with 50% inhibition levels at 113 ng/ml in samples. Yong, Jin-Wook, Mun-Han, and Chang-Hoon (2006) detected neomycin in plasma and milk by direct competitive ELISA using monoclonal antibody with detection limit of 3.61 ng/ml and 2.73 ng/ml in samples. However, samples preparations of most of these reports were comparatively laborious, and most of them tested less than two kinds of foods samples. Nowadays, the chemiluminescence assay has been developed quickly for high sensitivity. However, there are few reports on detecting neomycin in sample with chemiluminescence assay. In this study, a direct competitive ELISA was developed by producing neomycin polyclonal antibody for detection of neomycin in six kinds of animal-derived food products. Optimization of buffer solution used in direct ELISA assay was carried out. In addition, a simple and rapid extraction procedure that could evidently reduce matrix effects was conducted. At the same time, chemiluminescence assay was developed to detect neomycin in two livestock samples, and the comparison of effects of colorimetric assay and chemiluminescence assay on the ELISA detective sensitivity of the standard curve and the limits of detection of neomycin in samples was carried out. Besides, the ELISA results were compared with a traditional HPLC method. 2. Materials and methods 2.1. Reagents Neomycin B sulfate was obtained from Shymax Chem. Neamin sulfate, kanamycin sulfate, gentamycin sulfate, streptomycin sulfate, amikacin sulfate, tobramycin sulfate, sisomicin sulfate, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), ovalbumin (OA), horseradish peroxidase (HRP), 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride) (EDC), o-phthaldialdehyde (OPA), fish skin gelation (FG) and Freund’s complete and incomplete adjutants were purchased from Sigma–Aldrich (St. Louis, MO, USA). Protein A-Sepharose 4B was purchased from Amersham Biosciences (Uppsala, Sweden). HPLC grade methanol was obtained from Merck (Germany). Reagent grade 3,3’,5,5’-tetramethylbenzidine (TMB), hydrogen peroxide, luminol and p-iodophenol were from Sigma–Aldrich (St. Louis, MO, USA). Polystyrene 96-well microwell plates were from Nunc (Roskilde, Denmark), and microplate washer was from Bio-Rad (Hercules, CA). Immunoassay absorbance was read with a Multiskan Spectrum purchased from Thermo (Labsystems, Vantaa, Finland) in dual-wavelength mode (450–650 nm). All solutions were prepared with water purified with a Milli-Q system (Millipore, Bedford, MA). 2.2. Solutions Phosphate-buffered saline (PBS, 10 mmol/l sodium phosphate, 137 mmol/l NaCl, pH 7.5), phosphate-buffered saline with 0.05%

Tween 20 (PBS-T), coating buffer (CB, 50 mmol/l sodium carbonate buffer pH 9.6), TMB substrate solution (prepared by adding 3.3 mg TMB in 250 ll DMSO to 25 ml of phosphate-citrate buffer (0.1 mol/ l citric acid + 0.2 mol/l Na2HPO4, pH 4.3) containing 3.25 ll of a 30% H2O2 solution), luminol-H2O2- p-indophenol substrate solution (prepared by adding 4 mg luminol, 2 mg p-indophenol dissolved in DMSO and 4 ll H2O2 to 20 ml 0.1 M Tris buffer pH 8.6) and OPA derivative solution (1 mg OPA in 1 ml methanol and 10 ll mercapto-ethanol to 10 ml boric acid buffer(pH 10.5), and diluted 100-folds before used)were used. 2.3. Preparation of KLH and HRP conjugates The immunogen was made by coupling neomycin to keyhole limpet hemocyanin (KLH) according to the method described by (Mahon, Ezer, and Wilson (1973)). 10 mg KLH was dissolved in 1 ml 0.01 mol/l pH 7.4 PBS which contained 7  10 2 mmol neomycin. And then 400 mg 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide hydrochloride freshly prepared in 0.5 ml water was added drop-wise to this solution. The reaction was allowed to take place at room temperature with gentle stirring for 2 h and overnight at 4 °C. Then the solution was dialyzed against PBS for 3 d. Neomycin was conjugated with HRP according to the procedure described by Haasnoot et al. (1999) using EDC. 2.4. Polyclonal antibody production Antibodies were produced in rabbits as described by Wang, Allan, Skerritt, and Kennedy (1998). White rabbits were immunized by intradermal and intra-muscular injections of the neomycin conjugated to KLH. IgG from antisera was purified by Sepharose 4Bprotein A affinity chromatography. The IgG fraction was dialyzed, and the antibodies were concentrated and then used for the competitive ELISA format described below. 2.4.1. Direct competitive ELISA Direct competitive ELISA was developed using polyclonal antibody and neomycin–HRP conjugate. The microtiter plates were coated with purified anti-neomycin IgG at 0.5 lg per well in 100 ll coating buffer and incubated overnight at room temperature. Unbound antibodies were removed by washing plates three times with the washing solution (10 mmol/l PBST) and unbound active sites were blocked with 200 ll of 1% BSA/PBS per well for 1 h. After the plates were washed four times, 100 ll neomycin standards in PBS (or diluted sample solution) and 100 ll neomycin–HRP conjugate (diluted 1/100000 in PBS) were then added to each well, and incubated for 1 h at room temperature. After washed five times to remove the unbound neomycin standards and neomycin–HRP conjugate, the HRP tracer activity was then measured by adding 150 ll of TMB substrate solution to each well. The enzymatic reaction was stopped after 30 min by adding 2.5 mol/l H2SO4 (50 ll per well) and the absorbance was then read in dual-wavelength mode (450 nm as test and 650 nm as reference). 2.4.2. The optimization of neomycin ELISA In this assay, the effects of pH value and ionic strength were studied to improve the performances of the selected immunoassay format. The optimized buffer condition was used in following ELISA assay. pH value: Using optimized PBS buffer concentration, the effect of different pH value ranging from 5.5 to 8.5 were tested. Ionic strength: Different concentrations of PBS, including 0, 10, 20, 30, 40, 50 mmol/l concentrations of PBS were tested.

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The main procedures of the chemiluminescence assay are same to the procedures of direct competitive ELISA. The difference between colorimetric assay and chemiluminescence assay is different substrates. After removing the unbound neomycin standards and neomycin–HRP conjugate from microplate, the HRP tracer activity was then measured by adding 150 ll of luminal-H2O2-p-indophenol substrate solution to each well, and then read the value of chemiluminescence. 2.5.1. Sample preparation Six different matrices obtained from local supermarkets such as pig hind leg muscle, chicken breast muscle, pig kidney, egg, cod fish and milk were chosen to evaluate the method. Before spiked and recovery studies, each test sample was verified neomycin free (less than 5 lg/kg) by high-performance liquid chromatography (HPLC). Each sample was spiked three different concentrations of neomycin dissolved in water. Besides milk, all the other sample tissues should be chopped before extraction. The extraction and dilution procedures of these six samples are described as follows: Milk: one millilitre of milk was centrifuged at 4500 g for 10 min at 4 °C, and then removed the fat, the liquid layer was diluted in 50-folds with 0.5%BSA/PBS (pH 9.0–9.5) for analysis. Pig muscle, chicken muscle and fish: one gram samples and 10 ml PBS (pH 8.5) were thoroughly mixed for 1 min and centrifuged at 4500 g for 10 min at 4 °C. The supernatant liquid layer was diluted 5-folds with 0.5%BSA/PBS (pH 9.0–9.5) for colorimetric analysis. For chemiluminescence assay, the supernatant liquid layer was diluted 10-folds for pig muscle and 40-folds for chicken muscle. Egg: Yolk and egg white were mixed adequately and stored at 20 °C, then defreezed before experiments. 1 g samples and 10 ml PBS (pH 8.5) were thoroughly mixed for 1 min and centrifuged at and 4500 g for 10 min at 4 °C. The supernatant liquid layer was diluted 20-folds with 0.5%BSA/PBS (pH 9.0–9.5) for analysis. Kidney: one gram samples and 10 ml PBS (pH 8.5) were thoroughly mixed for 1 min and centrifuged at 4500 g, for 10 min at 4 °C. The supernatant liquid layer was diluted 10-folds with 0.5%BSA/PBS (pH 9.0–9.5) for analysis. 2.5.2. Instrumentation for HPLC analysis The ELISA results were verified using a Shimadzu HPLC equipped with a LC-10AT vp. pump with HAMILTON injector (25 ll loop), and a DGU-12A online degasser, a CTO-10AS vp. column oven and system controller SCL-10A utilized RS-232C interface for communication with CLASS-VP chromatography workstation. A fluorescence detector RF-10AXL was used to measure signals at excitation and emission wavelengths of 345 and 445 nm, respectively. The separation was performed on a C18 reversed-phase column (15 cm  4.6 mm I.D., 5 lm, Alltech, USA) with methanol–water (50:50, v/v) as the mobile phase at a flowrate of 1.0 ml/min. The temperature of column oven was 35 °C Before HPLC analysis, 1 g pig muscle or chicken muscle samples obtained from homogenized sample previously (at 0, 250, 500, 1000 ng/g different concentrations of neomycin) were added 10 ml PBS (pH 8.5) and mixed for 1 min thoroughly, then heated at 70–80 °C for 10 min and centrifuged at 4 °C and 4500 g for

3. Results and discussion 3.1. Optimization of neomycin ELISA The optimization of pH and ionic strength was the preliminary study that should be used in further determination of neomycin residues. Immunoassay for neomycin is more sensitive at pH 8.5 than other pH tested (Fig. 1). IC50 was very low at pH 8.5 because neomycin as an aminoglycoside antibiotics produced by S. fradiae is an alkaline antibiotics. It was confirmed that the stability of neomycin in slightly alkaline solution is preferable. Furthermore, the absorbance (A0) did not decrease at pH 8.5 and it was suitable for ELISA. Therefore, pH 8.5 was selected for further study. The effect of ionic strength on the assay performance is shown in Fig. 2. The values of IC50 increased and the values of absorbance (A0) decreased as salt concentration increased. The increased ionic strength has a detrimental effect on interactions where ionic driving forces prevail. The buffer will crystallize in high concentration of PBS. In this assay, 10 mmol/l PBS was chosen to achieve the maximum sensitivity. Taking account of these results, the best performances were achieved with 10 mmol/l PBS, pH 8.5. The standard curve of neomycin is showed in Fig. 3. A six-point standard curve was performed in each ELISA plates with the average result of IC50 1.0 ng/ml, IC15 0.1 ng/ml, which is lower than results of some previous reports. 3.2. Cross-reactivity determination The specificity of the antibody for neomycin was determined by testing seven aminoglycoside antibiotics, and the results were listed in Table 1. It can be seen that most tested aminoglycoside antibiotics showed low cross-reactivity (<0.01%). So the purified neomycin polyclonal antibody did not have cross-reactivity with

1.4

35

Absorbance IC50

1.2

30

1

25

0.8

20

0.6

15

0.4

10

0.2

5

IC50(µg/L)

2.5. Chemiluminescence assay

10 min. The upper liquid was added OPA derivative solution equally and mixed for 1 min, and then filtered through a 0.22 lm filter before injected into the HPLC system.

A0

2.4.3. Cross-reactivities with other aminoglycosides Cross-reactivities of the antibody with other aminoglycosides (neamin, kanamycin, gentamycin, streptomycin, amikacin, sisomicin and tobramycin) were determined by direct competitive ELISA as described above. The sensitivity of each aminoglycoside relative to neomycin was calculated using the formula relative sensitivity = (IC50 of neomycin)/(IC50 of other aminoglycoside)

0

0

5.5

6.5

7.5

8.5

pH Fig. 1. Effect of different pH values of dilution on the direct competitive ELISA.

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1.4

Absorbance IC50

1.2

9

Table 2 Recovery results of microwell immunoassay of neomycin from spiked samples (n = 4).

8

Samples

Level added (lg/ kg(L))

Level found (lg/kg(L)) Mean ± SD

Recoveries (%)

CV (%)

7

Milk

25 50 500 25 100 500 25 100 500 25 100 500 50 100 500 100 250 500

26.4 ± 1.0 45.2 ± 1.2 523.5 ± 1.3 22.6 ± 2.5 90.9 ± 9.5 477.0 ± 6.4 18.8 ± 1.3 83.3 ± 2.9 433.5 ± 11.5 21.8 ± 1.1 85.0 ± 5.0 427.5 ± 5.1 41.6 ± 2.9 79.8 ± 3.2 438.0 ± 5.5 75.8 ± 6.0 196.2 ± 8.2 421.0 ± 10.8

105.8 90.5 104.7 90.5 90.9 95.4 75.1 83.3 86.7 87.1 85.0 85.5 83.3 79.8 87.6 75.8 78.5 84.2

3.8 2.6 0.5 11.1 10.5 1.3 6.9 3.5 2.6 5.0 5.9 1.2 7.0 4.0 1.2 7.9 4.2 2.6

1 6

0.6

4

Pig muscle

IC50(µg/L)

5

A0

0.8

3

Chicken muscle Fish

0.4 2 0.2

1

0 10

20 30 PBS(mmol/L)

40

50

Fig. 2. Effect of different ion concentration of dilution on the direct competitive ELISA.

The standard curve of neomycin

amino sugars are attached to its nucleus, whereas gentamycin has two amino sugars attached and sisomicin is the ramification of gentamycin. In addition, the molecular structure of amino sugars in neomycin is different from that of kanamycin family (kanamycin, tobramycin, and amikacin), (Loomans et al., 2003). These structural differences enable each antibody to recognize its own specific antigen. 3.3. Matrix effects and their removal

0.1 1 10 Concentration of neomycin µg/L

100

Fig. 3. Neomycin standard curve of direct competitive ELISA by using colorimetric assay.

other aminoglycosides except neamin which is the part of the structure of the neomycin, and indicated that the polyclonal antibody was highly specific for neomycin. The specificity of the antibody can be explained by the differences in the molecular structure of the aminoglycosides. All aminoglycosides consist of two or more amino sugars joined through a glycosidic linkage to a hexose nucleus, which is either streptose (found in streptomycin) or 2-deoxystreptamin (characteristic of all other aminoglycosides), (Yong et al., 2006); the aminoglycoside families are distinguished by the amino sugars attached to the nucleus. In neomycin three

Table 1 Cross-reactivity of neomycin antibody with other aminoglycoside antibiotics. Aminoglycoside antibiotics

Cross-reactivity (%)

Neomycin Neamin Gentamycin Amikacin Kanamycin Sisomicin Streptomycin Tobramycin

100 2 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01

Immunoassays are rapid and convenient for food sample analysis, which require simple sample preconcentration and clean-up steps. However, ELISA methods often have a high potential for nonspecific binding between nontarget analytes and antibodies and are consequently prone to matrix effects. Chemical compounds present in animal product samples or sample extracts, such as protein, fat, solvents and others, might affect the binding of antibody and analytes, and they also might affect other aspects of the assay. This ‘‘matrix effect” is a common problem for the immunoassay, which could reduce the sensitivity and reliability of the competitive immunoassay and cause false positives by lowering the color development (Reynolds, 1993). To study the matrix effect in this study, pig muscle, chicken muscle, kidney, egg, fish and milk were chosen as test samples. Because neomycin can not dissolve in many organic solvents, the PBS (pH 8.5) was selected for the extract solvent according to the optimum condition of buffer solution. In our study, extracts with PBS

Inhibition %

Inhibition %

Egg

0 0

100 90 80 70 60 50 40 30 20 10 0 0.01

Kidney

100 90 80 70 60 50 40 30 20 10 0 0.01

Standard curve Egg Chicken muscle Milk Fish Pig muscle Kidney

0.1

1

10

100

Concentration of nemycin (µg/kg(L)) Fig. 4. Standard curves of neomycin in animal foods after appropriate extraction and dilution.

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3.4. Results of chemiluminescence assay

100 90 80 70 60 50 40 30 20 10 0 0.100

0.1

chicken muscle

80

pig muscle

70 60 50 40 30 20 10 0 0.001

0.01

0.1

1

Concentration of nemycin (µg/kg(L)) Fig. 6. Standard curves of neomycin in animal foods after appropriate extraction and dilution.

standard curves of neomycin in extracts of chicken muscle and pig muscle. 3.5. Comparison of the results of colorimetric assay and chemiluminescence assay The chemiluminescence assay standard curve was compared with colorimetric assay, and found that the IC50 was lower; but the sensitivity was higher. For the samples, the limit of detection for neomycin in chicken muscle was higher than that by using colorimetric assay. It could indicate that matrix effects were more difficult to be removed for the higher sensitivity of chemiluminescence assay. Moreover, the stability of the substrate and practicability of chemiluminescence assay were worse than colorimetric assay. Therefore, using colorimetric assay to detect neomycin in animal-derived food products was more suitable than using chemiluminescence assay. 3.6. Correlation studies between ELISA and HPLC analysis Although previous studies have demonstrated that ELISA was a more simple and rapid method than traditional instrumental method, the confirmatory study is still required for legal and statutory purposes because of the possibility of false positive results of ELISA. Correlation studies were performed on chicken muscle and

The standard curve of neomycin

0.10

standard curve

90

1200

HPLC values µg/kg

Inhibition(%)

Fig. 5 indicates the standard curve of neomycin by using chemiluminescence assay. A six-point standard curve was performed in each ELISA plates with the average result of IC50 0.15 ng/ml, IC15 0.015 ng/ml, which performs lower than results obtained by using colorimetric assay. However, for the chemiluminescence assay, matrix interference could be removed after 10-fold dilution with 0.5%BSA/PBS for pig muscle, 40-fold dilution for chicken muscle. The limits of detection for neomycin were 1.5 ng/ml in pig muscle and 6 ng/ml in chicken muscle. Fig. 6 shows the comparation of

100

Inhibition(%)

alone could not reduce matrix interference from the selected test samples. Dilution is a commonly used procedure to reduce the interference from matrices. To reduce matrix interference, organic solutions could be used as dilution solution. The addition of Teleostean FG, BSA, and Tween 20 to dilute PBS extract was examined to reduce matrix effects, so PBS, PBST, 0.1–0.5%BSA/PBS and 0.1– 0.5%FG/PBS of different concentrations were tested as dilution buffer. Table 2 shows the different effects between these dilution solutions. Typically, interferences are quantified by comparing a standard curve produced in a control with a calibration curve generated in the sample matrix. If the two curves are superposable, the effect of the matrix is not significant, and then, the samples can be analyzed using the standard curve prepared in the control solution. Matrix interference could be overcome after 5-fold dilution with 0.5%BSA/PBS for pig muscle, chicken muscle and fish, 10-fold dilution for kidney, 20-fold dilution for egg and 50-fold dilution for milk. The BSA in the diluent seemed to act like a stabilizer to protect the enzyme from the interfering materials or to stabilize the antibody–antigen interaction. The limits of detection for neomycin were 5 ng/ml in pig muscle, chicken muscle, fish and milk, 10 ng/ ml in kidney and 20 ng/ml in egg. The MRLs for neomycin by EU in pig muscle, chicken muscle, egg and fish are 500 lg/kg, 1500 lg/l for milk and in kidney is 5000 lg/kg. Therefore, dilution with BSA in PBS still maintained the detection limit at legal 500 lg/ kg requirements and the developed ELISA is applicable to determination of neomycin residues. The ELISA methods established here still maintain enough sensitivity after dilution to reduce the matrix interference. Through this method, the matrix effect was eliminated and recoveries for neomycin at 25, 50, 500 ng/ml concentrations in milk; 25, 100, 500 ng/ml concentrations in chicken muscle, pig muscle and fish; 50, 100, 500 ng/ml concentrations in kidney; 100, 250, 500 ng/ml concentrations in egg these six samples were between 75% and 105%. Fig. 4 shows the comparison of standard curves of neomycin in extracts of chicken muscle, pig muscle, fish, milk, egg and kidney. Compared with other articles, the extraction procedure of this study is more rapid and simple, without any purification steps. The recovery results of six samples are shown in Table 2.

1

Neomycin concentration(µg/L) Fig. 5. Neomycin standard curve of direct competitive ELISA by using chemiluminescence assay.

1000

R2 = 0.9959

800 600 400 200 0 0

200

400

600

800

1000

ELISA values µg/kg Fig. 7. Correlations between ELISA and HPLC results for two kinds of food samples spiked with neomycin (r2 = 0.9959).

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pig muscle samples by HPLC. The extraction procedure for HPLC analysis was more complicated. Fig. 7 indicates the correlations between HPLC analysis of purified extracts and ELISA results from non-purified extracts. Good correlations (r2 = 0.9959) was obtained. The results indicate that the developed ELISA is reliable for the analysis of neomycin in animal-derived food samples. 4. Conclusion As expected, the developed ELISA assay for neomycin detection possesses the virtues of high specificity and sensitivity. Simple, rapid extraction methods of samples were obtained for determination of neomycin. The colorimetric competitive direct ELISA presented in this assay successfully determined neomycin in pig muscle, kidney, chicken muscle, egg, milk and fish far below the MRL of neomycin (500 lg/kg). The chemiluminescence assay was developed to detect neomycin residues in pig muscle and chicken muscle. The accuracy and precision of ELISA was validated by the instrumental method of HPLC and indicated the developed ELISA is reliable with good correlations (r2 = 0.9959). Acknowledgements The authors are grateful for financial supports from the Ministry of Science and Technology of the People’s Republic of China (Project No. 2006BAD05A06 and 2006AA10Z448). References Binns, R. B., & Tsuji, K. (1984). High-performance liquid chromatographic analysis of neomycin in petrolatum-based ointments and in veterinary formulations. Journal of Pharmaceutical Science, 73, 69–72. Bossuyt, R., Renterghem, R. V., & Waes, G. (1976). Identification of antibiotic residues in milk by thin-lay chromatography. Journal of Chromatography, 124, 37–42. Cleveland, C.B., Francke, D.E., Heller, W.M., Kepler, J.A., Provost, G. P., & Reilly, M.J. (1990). Anti-infective agents. AHFS drug information (pp. 51–67). Bethesda, MD: American Society of Hospital Pharmacists Press. Commission Regulation, EC (2002) No. 1183/2002 of 1 July 2002 amending Annex I of Council Regulation (EEC) No. 2377/90. Fourmy, D., Yoshizawa, S., & Puglisi, J. D. (1998). Paromomycin binding induces a local conformational change in the A-site of 16S rRNA. Journal of Molecular Biology, 277, 333–345. Haasnoot, W., Stouten, P., Cazemier, G., Lommen, A., Nouws, J. F. M., & Keukens, H. J. (1999). Immunochemical detection of aminoglycosides in milk and kidney. Aanalyst, 124, 301–305.

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