Development of ELISA and immunochromatographic assay for the detection of neomycin

Clinica Chimica Acta 364 (2006) 260 – 266 www.elsevier.com/locate/clinchim

Development of ELISA and immunochromatographic assay for the detection of neomycin Yong Jin a, Jin-Wook Jang a, Mun-Han Lee a, Chang-Hoon Han b,* a

Department of Biochemistry, College of Veterinary Medicine, Seoul National University, 151-742 Seoul, South Korea b Brain Korea 21, School of Agricultural Biotechnology, Seoul National University, 151-742 Seoul, South Korea Received 9 April 2005; received in revised form 18 July 2005; accepted 19 July 2005 Available online 31 August 2005

Abstract Background: Reliable analytical methods are required to monitor neomycin residue levels in the livestock products. In particular, a more simple and rapid detection method is required in the veterinary fields. Methods: Competitive direct ELISA and immunochromatographic assay were developed using monoclonal antibody to detect neomycin in the animal plasma and milk. Results: No cross-reactivity of the antibody was observed with other aminoglycosides based on competitive direct ELISA methods, indicating that the antibody is highly specific for neomycin. Based on the standard curves, the detection limits were determined to be 6.85 ng/ ml in PBS, 3.61 ng/ml in plasma, and 2.73 ng/ml in milk, respectively. Recoveries of neomycin from spiked plasma and milk at levels of 50 – 200 ng/ml ranged from 87% to 108%. Concentration of intramuscularly injected neomycin was successfully monitored in the rabbit plasma through competitive direct ELISA. Immunochromatographic method was also developed using colloidal gold-conjugated monoclonal antibody. Through this method, the detection limits were estimated to be about 10 ng/ml of neomycin in PBS, plasma, and milk. Conclusions: Immunochromatographic assay developed in this study is suitable for the simple screening of neomycin residues in the veterinary field. Observed positives can be confirmed using a more sensitive laboratory method such as competitive direct ELISA. D 2005 Elsevier B.V. All rights reserved. Keywords: Neomycin; Monoclonal antibody; Competitive direct ELISA; Immunochromatographic assay; Plasma; Milk

1. Introduction Neomycin, an aminoglycoside antibiotics produced by Streptomyces fradiae, is widely used in veterinary medicine to treat bacterial infections in animals [1]. It is classified as a broad-spectrum antibiotic due to its growth inhibition of Gram-positive bacteria such as Staphylococcus aureus and Mycobacterium tuberculosis, and Gram-negative bacteria such as E. coli, Enterobacter aerogenes, Klebsiella pneumoniae, and Proteus vulgaris [2]. Neomycin is known to perturb protein synthesis in bacteria by binding the 30 S subunit of ribosomal RNA, which causes misreading of the genetic code and inhibits translation [3,4].

* Corresponding author. Tel.: +82 2 880 1240; fax: +82 2 888 2754. E-mail address: [email protected] (C.-H. Han). 0009-8981/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2005.07.024

However, despite its impressive clinical successes, neomycin is potentially ototoxic and nephrotoxic to human and animals [5,6]; thus monitoring of its residues in foods is essential for the maintenance of public health. For consumer protection, the European Union (EU) established maximum residue limits (MRL) for edible tissues, fat, milk, and eggs: 500 Ag/kg for meat, fat, liver, eggs, 1500 Ag/kg for milk, and 5000 Ag/kg for kidney [7]. Therefore, simple and reliable analytical methods are required to monitor neomycin residue levels in the livestock products. Various techniques have been developed for the detection of aminoglycoside residues in milk, urine, blood, and tissue including microbioassay [8,9], gas chromatography (GC) [10], high-performance liquid chromatography (HPLC) [11 –14], and enzyme-linked immunosorbent assay (ELISA) [15,16], among which ELISA has become the most popular method for the detection of chemicals in foods due

Y. Jin et al. / Clinica Chimica Acta 364 (2006) 260 – 266

to its high sensitivity, simplicity, and ability to screen large number of small-volume samples. In the veterinary fields, however, a more simple and rapid detection method is required. Several studies on immunochromatographic assay, relatively sensitive and simple for the detection of antibiotics, were reported [17 – 20]. In addition, recent studies have reported on the colloidal gold-based immunochromatographic assay. Using this method, Shyu et al. [21] developed a simple and reliable immunochromatographic assay for the detection of ricin, and Putalun et al. [22] developed a one-step immunochromatographic strip test for the detection of sennosides A and B. In this study, we developed a competitive direct ELISA using neomycin monoclonal antibody for the detection of neomycin in the animal plasma and milk. In addition, an immunochromatographic method, which can be used as a rapid and simple screening test kit for the detection of neomycin in veterinary fields, was developed using colloidal gold-conjugated antibody.

2. Materials and methods 2.1. Materials Neomycin sulfate, kanamycin sulfate, gentamicin sulfate, streptomycin sulfate, amikacin sulfate, tobramycin sulfate, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC), horseradish peroxidase (HRP), goat anti-mouse IgG – horseradish peroxidase conjugate, o-phenylenediamine dihydrochloride (OPD), hydrogen peroxide, Freund’s complete adjuvant, Freund’s incomplete adjuvant, polyoxyethylene-sorbitan monolaurate (Tween 20), and colloidal gold particle were from Sigma-Aldrich (St. Louis, MO). Dulbecco’s modification of Eagle’s medium (DMEM), fetal bovine serum (FBS), antibiotic –antimycotic, polyethylene glycol 1500 (PEG 1500), hyphoxanthine –aminopterine – thymidine (HAT) medium, hyphoxanthine – thymidine (HT) medium, microtiter plates, and microculture plates (96- and 24-well plates) were from Gibco BRL (Rockville, MD). BALB/c mice and rabbits were from Charles River Technology (Seoul, Korea). Nitrocellulose membrane was from Millipore (Billerica, MA). 2.2. Preparation of KLH, BSA, and HRP conjugates Neomycin –KLH and neomycin – BSA conjugates were prepared by the method of Mahon et al. [23]. Neomycin was conjugated with HRP according to the procedure described by Haasnoot et al. [15] using EDC. 2.3. Immunization of mice Immunization was performed as described previously [24]. Four BALB/c female mice, 8– 10 weeks of age,

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weighing approximately 18 g, were given four intraperitoneal injections with neomycin –KLH conjugate (100 Ag/ injection). The first injection consisted of 0.2 ml of conjugate in a 1 : 1 ratio of saline and Freund’s complete adjuvant. The second (day 14) and the third (day 28) injection consisted of 0.2 ml of conjugate in saline and Freund’s incomplete adjuvant (1 : 1). Ten days following the third injection, serum was collected from the retrobulbar plexus of each mouse and the highest sensitivity antiserum was determined by competitive indirect ELISA. The spleen from the mouse with serum showing optimum relative inhibition was used for the subsequent fusion. Three weeks after the third injection and 4 days prior to fusion, this mouse was given a fourth injection of conjugate in PBS (0.1 ml). 2.4. Monoclonal antibody production Hybridoma cell lines were produced through the fusion of myeloma cells (Sp2/0) and spleen cells obtained from immunized mice using PEG 1500 as described previously [25]. Twelve days after the fusion, competitive indirect ELISA was performed to screen for antibody-producing cells using the culture supernatant. A stable hybridoma cell producing antibody with the highest binding capacity and sensitivity to neomycin was selected, and cloned to 0.5 cells/well by limit dilution. The cultured hybridoma cells (5  106 cell) were injected intraperitoneally into the mice to produce monoclonal antibody in the ascites fluid. Immunoglobulin was prepared from the ascites fluid of each mouse as described previously [25]. 2.5. Cross-reactivity with other aminoglycosides Cross-reactivity of the antibody with other aminoglycosides (kanamycin, gentamicin, streptomycin, amikacin, and tobramycin) was determined by competitive direct ELISA as described below. B / B 0 value of 50% (CR50) was calculated as described previously [26]. 2.6. Competitive direct ELISA Competitive direct ELISA was developed using monoclonal antibody and neomycin –HRP conjugate. Each well of the microtiter plates was coated with 100 Al aliquots of neomycin antibody (diluted 1 / 2000 in PBS) and incubated for 3 h at 37 -C. Unbound antibody was removed from the plate with the washing solution (0.02% Tween 20 in PBS), and each well was blocked with 200 Al blocking solution (1% skim milk in PBS) at 37 -C for 1 h. Neomycin standards (50 Al each; 1 to 1000 ng/ml) were added to each well, and incubated with 50 Al diluted neomycin – HRP conjugate (diluted 1 / 2000 in PBS) for 1 h at 37 -C. After removing the unbound neomycin and neomycin – HRP conjugate with the washing solution, 100 Al substrate solution was added to each well, which was then incubated

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Y. Jin et al. / Clinica Chimica Acta 364 (2006) 260 – 266 Table 1 Background levels determined in PBS, plasma, and milk (n = 4)

100

80 PBS Plasma Milk

B/B0

60

Mean background (ng/ml)

Standard deviation (ng/ml)

Limit of detectiona (ng/ml)

2.59 2.26 2.22

1.42 0.45 0.17

6.85 3.61 2.73

a Limit of detection: the mean background levels plus three times standard deviation (B 0 + 3s).

40

20

0 1

10

100

1000

10000

ng/ml, then diluting 10-fold in PBS. The recoveries of neomycin from the spiked rabbit plasma or bovine milk were calculated based on the standard curves constructed by competitive direct ELISA. The detection limits were determined as the mean background levels +3  S.D. (n = 4).

Aminoglycosides concentration (ng/ml) Fig. 1. Cross-reactivities of monoclonal antibody against neomycin (n), to gentamicin (?), kanamycin (4), streptomycin (r), amikacin (>), and tobramycin (g), where B is the absorbance of the well containing aminoglycosides and B 0 is the absorbance of the well without aminoglycosides. Each value shows the mean (TS.D.) of B / B 0 (n = 4).

for 20 min at 37 -C. Absorbance was measured at 490 nm using an ELISA reader (Emax, Molecular Devices, CA). To prepare the calibration curves of neomycin in the plasma and milk, neomycin stock standard solutions (1000 Ag/ml) were prepared by dissolving neomycin in the rabbit plasma or bovine milk. The solutions were further diluted with the plasma or milk to 0, 10, 20, 50, 100, 250, 500, 1000, 2500, 5000, and 10,000 ng/ml, which were then diluted 10-fold in PBS. The standard curves of neomycin in the plasma and milk were established by competitive direct ELISA. For recovery test, neomycin-spiked solutions were prepared by dissolving neomycin in the rabbit plasma or bovine milk to give final concentrations of 50, 100, and 200

2.7. Monitoring of blood neomycin concentration Neomycin was administered intramuscularly to rabbits at 20 mg/kg/day for 3 consecutive days. Blood samples were collected from the ear vein of each rabbit 2, 4, 6, 8, 10, and 12 h after the last injection of neomycin, and were centrifuged (2000 g) for 10 min to obtain plasma. Plasma samples were diluted 10-fold in PBS and subjected to competitive direct ELISA to determine the neomycin concentration in the blood. The slope of the plasma concentration – time curve was estimated based on the linear-regression of the depletion curve, under the assumption that tissue neomycin is eliminated by the first-order kinetics. The elimination half-life (t 1/2), defined as the time required for the body to eliminate 50% of the remaining drug, was calculated using the following equation [27]: t 1/2 = 0.693 / k, where k, the overall elimination constant, is the slope of the plasma concentration – time curve. 2.8. Immunochromatographic assay Colloidal gold (40 nm in diameter) was conjugated with neomycin monoclonal antibody as described previously [21]. Briefly, 20 Ag of purified monoclonal antibody was added to 1 ml colloidal gold solution (pH 8.0). After incubation at room temperature for 10 min, the conjugates were blocked with 1% BSA solution for 30 min. The goldlabeled antibody was supplied, ready to use, in 0.2 M Tris – HCl buffer (pH 8.7) containing 1% Tirton X-100 at an optical density of 10 at 540 nm.

100

80

B/B0

60

40

20

Table 2 Recoveries of neomycin from spiked plasma and milk (n = 4)

0 1

10

100

1000

Neomycin concentration (ng/ml) Fig. 2. Standard curve of neomycin in PBS (n), rabbit plasma (?), and bovine milk (r) constructed by competitive direct ELISA. B and B 0 are the absorbances of the sample with/without neomycin, respectively. Each value shows the mean (TS.D.) of B / B 0 (n = 4).

Samples

Level added (ng/ml)

Level found (ng/ml)

Recovery (%)

Plasma

50 100 200 50 100 200

53.8 T 6.9 95.8 T 6.8 187.0 T 6.3 48.3 T 5.7 100.9 T 11.4 174.7 T 13.9

108 96 94 97 101 87

Milk

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Neomycin concentration (ng/ml)

7000

263

the membrane, the intensities of red color band on the membranes were monitored.

6000 5000

3. Results and discussion

4000

The purified neomycin monoclonal antibody did not show cross-reactivity with other aminoglycosides, indicating that the monoclonal antibody is highly specific for neomycin (Fig. 1). 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) [28]; the aminoglycoside families are distinguished by the amino sugars attached to the nucleus. In neomycin three amino sugars are attached to its nucleus, whereas gentamicin has 2 amino sugars attached. In addition, the molecular structure of amino sugars in neomycin is different from that of kanamycin family (kanamycin, tobramycin, and amikacin) [15]. These structural differences enable each antibody to recognize its own specific antigen. To determine the detection limits of neomycin in the plasma and milk, standard curves of neomycin in PBS, rabbit plasma, and bovine milk were constructed by competitive direct ELISA (Fig. 2). Based on these curves, 50% of B / B 0 ratio was about 50 ng/ml for both rabbit plasma and bovine milk. Background levels determined in PBS, plasma, and milk are summarized in Table 1. The detection limits were 6.85 ng/ml in PBS,

3000 2000 1000 0

0

2

4

6

8

10

12

Withdrawal time (hours) Fig. 3. Plasma depletion profile of neomycin after intramuscular administration of neomycin. Neomycin was administered intramuscularly to rabbits at 20 mg/kg/day for 3 consecutive days. Blood samples were collected from rabbits 2, 4, 6, 8, 10, and 12 h after the last injection of neomycin. Plasma samples were diluted 10-fold in PBS and subjected to the competitive direct ELISA. Each value shows the mean (TS.D.) of neomycin concentration (n = 4).

One microliter (3 Ag neomycin – BSA) of neomycin – BSA (3 mg/ml) conjugate was applied to one end of the nitrocellulose membrane strip (HF 135, 25  4.5 mm). After drying, the lower edge of the test strip was dipped into the well containing each concentration of neomycin in 50 Al of buffer (0.2 mol/l Tris –HCl, pH 8.7, 1% Triton X-100) and 2 Al colloidal gold-conjugated monoclonal antibody. After the mixture of sample and the gold-labeled antibody rising up

Fig. 4. A schematic description of the immunochromatographic assay. In the absence of neomycin in the sample, the gold-labeled monoclonal antibody can bind to the immobilized neomycin – BSA conjugate on the membrane, and develop red color band. In the presence of neomycin in the sample, however, free neomycin molecules compete with the immobilized neomycin – BSA conjugate for the binding to gold-labeled antibody. Thus, negative and positive results are judged by the appearance and disappearance of red color band on the strip, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 5. Cross-reactivity of aminoglycosides with the monoclonal antibody of neomycin in immunochromatographic assay. Three microgram of neomycin – BSA, gentamicin – BSA, kanamycin – BSA, streptomycin – BSA, amikacin – BSA, or tobramycin – BSA conjugate was applied to each strip of nitrocellulose membrane. After the gold-labeled antibody rising up the membrane, the intensities of the red color band on each membrane strip were observed. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

3.61 ng/ml in plasma, and 2.73 ng/ml in milk, respectively (Table 1). Recoveries of neomycin from the plasma and milk spiked with different concentrations of neomycin (50, 100, and 200 ng/ml), as determined by competitive direct ELISA based on the values obtained from the standard curves, were 108%, 96%, and 94%, and 97%, 101%, and 87%, respectively (Table 2). The neomycin concentration sharply increased to 5700 ng/ml after the intramuscular administration up to 2 h, then rapidly decreased to < 2300 ng/ml after 4 h of withdrawal (Fig. 3), during which the elimination half-life (t 1/2) of neomycin in the rabbit plasma was estimated to be 3.2 h of withdrawal. When aminoglycosides are administered into the body cavities, which contain serosal surfaces, extremely rapid and complete absorption takes place, whereas slow absorption can be observed when administered orally or rectally [29]. In addition, Isoherranen and Soback [30] reported that aminoglycosides bind readily to tissue proteins and macromolecules via ionic bounds, while less to the plasma proteins. They also showed that aminoglycoside accumulation in the

renal proximal tubules were several-fold higher than in the plasma or other tissues, and half-lives of aminoglycosides were 2– 3 and 30– 700 h in the plasma and tissues, respectively. Therefore, due to the longer and more variable half-lives of aminoglycosides in the tissues than in the plasma, our future study will focus on the estimation of the time-dependent concentration of neomycin in tissues using monoclonal antibodies. A schematic description of the immunochromatographic assay is illustrated in Fig. 4; negative and positive results are judged by the appearance or disappearance of red color band on the strip, respectively. Monoclonal antibody of neomycin did not show any cross-reactivity with the other aminoglycosides tested as revealed through immunochromatographic assay (Fig. 5). PBS, rabbit plasma, and bovine milk spiked with neomycin (0, 2, 4, 6, 8, and 10 ng/ml) were tested by immunochromatographic assay (Fig. 6). The color intensity gradually decreased with increasing concentration of neomycin, and disappeared completely at 10 ng/ml of neomycin in the samples. Through immunochromatographic assay, there-

Fig. 6. Immunochromatographic assay for the detection of neomycin. A series of dilutions (0, 2, 4, 6, 8, and 10 ng/ml) of neomycin were prepared in PBS (A), rabbit plasma (B), and bovine milk (C). After the mixture of sample and gold-labeled antibody rising up the membrane, the intensities of the red color band on each membrane strip were observed.

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Fig. 7. Comparison of competitive direct ELISA and immunochromatographic assay. Neomycin was administered intramuscularly to rabbits at 20 mg/kg/day for 3 consecutive days. Blood samples were collected from rabbits 2, 4, 6, 8, 10, and 12 h after the last injection of neomycin. Plasma samples were subjected to immunochromatographic assay. Subsequent competitive direct ELISA determined the neomycin concentration in the blood.

fore, the detection limits were estimated to be about 10 ng/ml of neomycin in PBS, plasma, and milk. Plasma samples collected from rabbits 2, 4, 6, 8, 10, and 12 h after intramuscular injection of neomycin (20 mg/kg/ day for 3 consecutive days) were subjected to immunochromatographic assay (Fig. 7). No color development was observed in all strips, which suggests that plasma samples contain higher than 10 ng/ml of neomycin. Subsequent ELISA gave the accurate numbers of neomycin concentration (Fig. 7). Therefore, the immunochromatographic assay is suitable for the screening of neomycin residues in the veterinary field. Observed positives can be confirmed using competitive direct ELISA. Recently, several studies have focused on the colloidal gold-based immunochromatographic assays [21,22]. The application of immunogold detection has several advantages. Firstly, the nanoparticles of colloidal gold show better mobility than other materials in the porous nitrocellulose membrane. The colloidal gold particles are also less susceptible to aggregation during the preparation of the test device. Finally, the gold-labeled antibody improves the assay sensitivity [21]. In the present study, a compromise was made between the sensitivity and nonspecific binding of antigen – antibody reaction in the immunochromatographic assay. Based on the findings that the concentration of the gold-labeled antibody and the pH of the developing solution were important for better resolution, we optimized the assay conditions for better sensitivity without any cross-reactivity or non-specific bindings. In conclusion, immunochromatographic assay could be applied to the detection of aminoglycosides in veterinary fields due to its rapid and simple procedure. For greater accuracy, however, the detection should be supported by a more sensitive laboratory method such as competitive direct ELISA method. The assays developed in this study could complement each other as well as the veterinary field and laboratory findings. Moreover, instead of slaughtering the animals to obtain test samples, the

methods developed in the present study could be applied to determine aminoglycoside concentration in the plasma of live animals.

Acknowledgements This study was supported by Brain Korea 21 project from the Ministry of Education, and by Research Institute for Veterinary Science (RIVS) of College of Veterinary Medicine in Seoul National University, Republic of Korea.

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