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Heterologous immunoassay forscreening macrolide antibioticsresidues in milk based on themonoclonal antibody of tylosinJian Kang Zhangab, Yong Hua Qic, Ju Xiang Liuab & Jian Ping Wangab

a College of Veterinary Medicine, Agricultural University of Hebei,Baoding, Hebei 071000, Chinab Hebei Engineering and Technology Research Center of VeterinaryBiological Products, Agricultural University of Hebei, Baoding,Hebei 071000, Chinac College of Animal Science, Henan Institute of Science andTechnology, Xinxiang, Henan 453003, ChinaPublished online: 17 Jul 2012.

To cite this article: Jian Kang Zhang, Yong Hua Qi, Ju Xiang Liu & Jian Ping Wang (2013)Heterologous immunoassay for screening macrolide antibiotics residues in milk based onthe monoclonal antibody of tylosin, Food and Agricultural Immunology, 24:4, 419-431, DOI:10.1080/09540105.2012.705820

To link to this article: http://dx.doi.org/10.1080/09540105.2012.705820

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Heterologous immunoassay for screening macrolide antibiotics residuesin milk based on the monoclonal antibody of tylosin

Jian Kang Zhanga,b, Yong Hua Qic, Ju Xiang Liua,b and Jian Ping Wanga,b*

aCollege of Veterinary Medicine, Agricultural University of Hebei, Baoding, Hebei 071000,China; bHebei Engineering and Technology Research Center of Veterinary Biological Products,Agricultural University of Hebei, Baoding, Hebei 071000, China; cCollege of Animal Science,Henan Institute of Science and Technology, Xinxiang, Henan 453003, China

(Received 7 March 2012; final version received 20 June 2012)

In this study, tylosin (TYL) was derivatized with 4-aminophenylacetic acid tosynthesise a new hapten and the hapten was used to produce the monoclonalantibody. The obtained antibody simultaneously recognised TYL, tilmicosin,acetylisovaleryltylosin and the metabolite of TYL (desmycosin) with cross-reactivities of 100%, 62%, 97% and 93%, respectively. After evaluation of twocoating antigens, a heterologous competitive indirect enzyme linked immunosor-bent assay was developed to determine the four analytes in milk simultaneously.The limits of detection for the four analytes were in the range of 1.5�3.1 ng mL�1.The recoveries from fortified milk were in the range of 76.3�97.4% with coefficientsof variation of 5.3�15.7%.

Keywords: tylosin; monoclonal antibody; heterologous ELISA; milk

1. Introduction

Macrolide antibiotics (MACs) are a class of drugs highly active against Gram-positive

bacterial and Mycroplasma species. Therefore, MACs are widely used to treat various

diseases in swine, cattle, sheep and poultry (Christodoulopoulos, Warnick, Papaioan-

nou, & Fthenakis, 2002; Fajt et al., 2003; Laven and Andrews, 1991; Shryock, Staples,

& DeRosa, 2002; Zhang et al., 2004). The commonly used MACs include tylosin

(TYL), tilmicosin (TMC) and acetylisovaleryltylosin (ATYL; Figure 1). TYL is a

traditional MAC that is generated by Streptomyces fradiae. ATYL and TMC are both

semi-synthetic drugs derived from TYL. TYL is unstable and it can be degraded in

sugar syrup (Kochansky, Knox, & Shimanuki, 1999), honey (Kochansky, 2004) and

acidic media (Paesen, Cypers, Pauwels, Roets, & Hoogmartens, 1995) to yield its

predominant metabolite, desmycosin (DMC), also referred to as tylosin B (Figure 1).

The wide use of MACs in farm animals may produce residues in food of

animal origin and induce the resistance of bacterial strains to antimicrobials in

human use. For protection of consumer health, it is very important to monitor the

residues of these MACs in foods of animal origin. In the last decade, high-

performance liquid chromatography (Prats, Francesch, Arboix, & Perez, 2001) and

liquid chromatography�mass spectrometry (Benetti, Dainese, Biancotto, Piro, &

*Corresponding author. Email: wjpcau@yahoo.com.cnJian Kang Zhang and Yong Hua Qi contributed equally to this work.

Food and Agricultural Immunology, 2013

Vol. 24, No. 4, 419�431, http://dx.doi.org/10.1080/09540105.2012.705820

# 2012 Taylor & Francis

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Mutinelli, 2004; Bogialli, Ciampanella, Curini, Di Corcia, & Lagana, 2009; Cherlet,

De Baere, Croubels, & De Backer, 2002; Draisci, Palleschi, Ferretti, Achene, &

Cecilia, 2001; Heller and Nochetto, 2004; Lucchetti et al., 2005; Martos, Lehotay, &

Shurmer, 2008; Msagati and Nindi, 2004; Wang & Leung, 2007; Wang, Leung,

& Butterworth, 2005) were used to determine the residues of MACs in various

animal original foods. For detection of TYL residues, most reported methods were

focused on the parent compound, TYL. In consideration of the structural instability

of TYL, animal derived foods for human consumption should be analysed for both

TYL and DMC rather than parent TYL alone. However, there has been only one

article reporting the simultaneous determination of TYL and DMC, and that study

was in honey (Thompson, Pernal, Noot, Melathopoulos, & van den Heever, 2007).

These analytical methods are time-consuming, and sophisticated and expensive

instruments are required. In comparison, enzyme linked immunosorbent assay

(ELISA) is a low cost and sensitive method capable of screening large numbers

of samples in a single test. The core reagent of an ELISA method is antibody.

By now, there have been several articles reporting the production of different anti-

bodies to these MACs. Yao and Mahoney (1989) produced a polyclonal antibody

against 23-deoxy-23-amino-O-mycaminosyl-tylonolide. The antibody showed cross-

reactivity (CR) to the 12-, 14- and 16-membered macrolides that contain amino

sugar moieties. Wicker et al. (1994) prepared a polyclonal antibody against TYL

that cross-reacted with TMC. Jackman, Spencer, Silverlight, Marsh, and Bellerby

(1997) produced a polyclonal antibody against DMC and Silverlight, Brown, and

Jackman (1999) showed the anti-DMC antibody exhibited high CR (96%) with TYL.

Creemer, Beier, and Kiehl (2003) synthesised a hapten of TYL and a hapten of

TMC. Beier, Creemer, Ziprin, and Nisbet (2005) used the hapten of TMC to produce

a monoclonal antibody that was specific to TMC and showed no CR with TYL

Figure 1. Chemical structures of the four macrolide antibiotics.

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and other MACs that do not contain the structure of 3,5-dimethylpiperidine (the

D ring in TMC molecule, Figure 1). The authors did not determine the antibody

reactivity to ATYL and DMC. Peng et al. (2012) produced a monoclonal antibody

against TYL that showed CR to TMC.Recently, we have produced a monoclonal antibody against TYL that showed

the following cross-reactivities: TYL (100%), ATYL (91%), DMC (76%) and TMC

(49%) (Zhang, Liu, Wang, Chai, & Wang, 2012). Therefore, TYL can be regarded as

a generic hapten of the four analytes to generate a broad-specific antibody. In this

study, TYL was used to synthesise a new hapten with the aims to enhance the anti-

body CR and to develop a multi-determination immunoassay for the four analytes.

2. Materials and methods

2.1. Reagents and chemicals

Tylosin tartrate, ATYL, TMC and DMC, bovine serum albumin (BSA), Ovalbumin

(OA), and Freund’s adjuvant were all from Sigma-Aldrich (St. Louis, MO, USA).

3, 3?, 5, 5?-tetramethylbenzidine (TMB) was purchased from Serva (Heidelberg,Germany). Other chemical reagents were all analytical grade from Beijing Chemical

Company (Beijing, China). The stock solutions of the four analytes were prepared by

dissolving each compound in methanol to obtain a concentration of 100 mg mL�1.

These solutions were stored at �20 8C in amber glass bottles. Working solutions

of the four drugs with series concentrations were prepared by dilution of the

stock solutions with PBS (1, 2, 5, 10, 20, 50, 100 and 200 ng mL�1). PBS (pH 7.2)

was prepared by dissolving 0.2 g KH2PO4, 0.2 g KCl, 1.15 g Na2HPO4, and 8.0 g

NaCl in 1000 mL water. Coating buffer was prepared with sodium carbonate andsodium hydrocarbonate (0.1 M, pH 9.6). Washing buffer was PBS containing 0.05%

Tween (PBST). Substrate buffer was prepared with sodium hydrogen phosphate

and citrate (0.1 M, pH 5.5). The substrate system was prepared by adding 200 mL

1% (w/v) TMB in DMSO (dimethyl sulfoxide) and 64 mL 0.75% (w/v) H2O2 to

20 mL substrate buffer.

2.2. Synthesis of the hapten

The hapten synthesis route is shown in Figure 2. About 470 mg tylosin tartrate

(0.5 mmol) and 75 mg 4-aminophenylacetic acid (0.5 mmol) were added to a mixture

of 20 mL of water and 0.5 mL of acetic acid. The mixture was refluxed under heating

until the solution turned yellow. Then the solvent was dried in drying box at 40 8C and

the dry residue was dissolved in 10 mL of ethanol. After 10 mL of ethyl acetatewas added into the ethanol solution, some lemon yellow sediment appeared. The

mixture was filtered under vacuum and the residue was washed with 50 mL of water,

and subsequently dried to yield the hapten PTYL (melt point of 220 8C;

IR (KBr) Vmax 3426, 3100, 2971, 2933, 1704, 1594, 1375, 1155, 1266, 713, 680 cm�1).

2.3. Preparation of the conjugates

The conjugates were prepared as shown in Figure 2. About 4 mL of N,N-

dimethylformamide dissolving 53 mg hapten and 25 mL triethylamine were added

Food and Agricultural Immunology 421

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Figure 2. Synthetic route of the hapten PTYL and the three conjugates.

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into a glass jar. Then 20 mL of isobutyl chloroformate was added and the mixture

was stirred for 60 min at 4 8C. Then, the mixture was added dropwise into 2 mL

of sodium bicarbonate solution containing 74 mg BSA or 30 mg OA and stirred

for 12 h at 4 8C. The resulting immunogen (PTYL-BSA) and coating antigen(PTYL-OA) were dialysed against three changes of PBS for 3 days and stored

at �20 8C until used. TYL, PTYL, BSA and the conjugates were all scanned on a

UV-Vis spectrophotometer to verify the conjugation. The hapten/protein coupling

ratios were determined according to the previous 2,4,6-trinitrobenzene sulphonic

acid method (Sashidhar, Capoor, & Ramana, 1994).

2.4. Production of the monoclonal antibody

Six 8-week-old female BALB/c mice were immunised subcutaneously with an

emulsion of the immunogen (50 mg protein per mouse) in Freund’s complete

adjuvant. Beginning two weeks later, mice were boosted at 2-week intervals. The

serum of each mouse was collected and the antibody titre was monitored. The spleenfrom the mouse with the highest titre after six boosters was removed and the

splenocytes were fused with SP2/O myeloma cells and cultured in 96-well plates.

Following a first screening by indirect ELISA, the positive hybridomas were

rescreened using the competitive indirect ELISA described later with TYL as the

competitor. Hybridomas producing the specific monoclonal antibody to TYL were

sub-cloned twice by the limiting dilution method and the sub-cloned hybridoma

cells were collected, centrifuged and frozen in liquid nitrogen. The ascites from

hybridoma-induced mice were purified using saturated ammonium sulphate pre-cipitation and used for development of the competitive indirect ELISA.

2.5. Competitive indirect ELISA

In this study, coating antigen PTYL-OA (coating antigen 1, Figure 2) and a

coating antigen previously prepared in our laboratory (coating antigen 2, Figure 2)

(Zhang et al., 2012) were used to develop the homologous and heterologous ELISA.

The optimal dilutions of coating antigen and antibody were determined by using the

checkerboard procedure, in which the well with an absorbance of 1.0 was defined as

the optimal dilutions of the coating antigen and the antibody. After that, each well of

a microtitre plate was coated with 100 mL of coating antigen, incubated overnight at

4 8C, and then blocked with 1% foetal calf serum. The plate was washed three timeswith PBST, and then, 50 mL of the optimal antibody dilution and 50 mL of TYL

standard with series concentrations were added to the wells (in triplicate) for

incubation for 1 h at 37 8C. The plate was washed as aforementioned. About 100 mL

of horseradish peroxidase labelled goat anti-mouse IgG was added before incubation

for 30 min at 37 8C. After washes, 100 mL of TMB substrate system was added prior

to 15 min incubation at 37 8C. Finally, the reaction was stopped by the addition of 50

mL of 2 M H2SO4, and the plate was read on an ELISA plate reader at 450 nm to

obtain the absorbance (B) of each well.The other three analytes shown in Figure 1 and several other drugs (erythromycin,

spiramycin and avermectin) were all determined by the ELISA. The limits of

detection (LOD) for these analytes were defined as the concentrations showing

10% of inhibition, respectively. The competitive inhibition curves were developed

Food and Agricultural Immunology 423

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by plotting the B/B0 values (mean absorbance of the standards divided by the

mean absorbance of zero standards) verse the concentrations (Log C). The CR

among these competitors was calculated from the half-maximal inhibition concen-

tration (IC50) as follows: CR (%)�100�IC50 TYL/IC50 competitor.

2.6. Sample preparation

The extraction of MACs from milk sample was modified from the previous reports

(Bogialli et al., 2009; Wang & Leung, 2007). A milk sample (5 mL) and 30 mL of

acetonitrile were added into a 50 mL polypropylene centrifuge tube and the tube

was shaken vigorously on a variable speed reciprocal shaker for 10 min. Themixture was centrifuged at 10,000 rpm for 10 min and the acetonitrile phase was

collected and evaporated to dryness. The dry residue was dissolved in 5 mL of PBS

and filtered through a 0.45 mm Millipore filter for ELISA analysis.

Blank milk samples were obtained from several controlled cows. In order to

evaluate the matrix interference, matrix-matched standards prepared with the

extracts of blank milk sample were used to develop the matrix-matched competitive

curves. The accuracy was evaluated by determination of the recoveries from the

four analytes fortified blank milk at concentrations of 20, 50, 100 ng mL�1. A totalof 35 unknown milk samples from China (20 commercial packaged milk samples

from several supermarkets and 15 raw milk samples from several dairy farms)

were analysed by the developed ELISA method.

3. Results and discussions

3.1. Hapten and immunogen

As shown in Figure 1, the three MAC drugs and DMC all contain a 16-atom

macrocyclic lactone ring (A ring), a 5-O-mycaminosyl ring (B ring), and a

neutral sugar (C ring). The antibody specific to the three rings should recognise

the four analytes simultaneously. In a previous report, B ring and D ring

(3,5-dimethylpiperidin at C20 position in the molecule of TMC) in the hapten of

TMC were presented to the immune system and the resulting antibody only

recognised TMC (Beier et al., 2005). This result is because among the four analytes

only TMC contains the D ring (Figure 1). In other reports, A ring and C ring in themolecule of DMC were presented to the immune system, resulting in an antibody

that simultaneously recognised DMC, TYL and TMC (Jackman et al., 1997;

Silverlight et al., 1999), analytes that contain the two rings. In our recent study, TYL

was derivatized with 6-aminohexanoic acid linker at the C20 position to synthesise

the hapten. The immunogen had A ring and C ring spaced far from the carrier

(similar to the illustration of coating antigen 2, Figure 2) and resulted in an antibody

that simultaneously recognised TYL, ATYL, DMC, and TMC (Zhang et al., 2012).

In the present study, a new hapten of TYL was synthesised by derivatizationof TYL with 4-aminophenylacetic acid utilising the C20 aldehyde group in TYL

molecule (Figure 2). This was equivalent to introduction of a phenylacetic acid

at C20 position and the free carboxyl group was used to couple with the carrier.

This hapten contained the common structures of the four analytes (A ring, B ring

and C ring) to elicit antibodies that recognise all four analytes.

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The successful synthesis of the hapten PTYL was proven by following data.

First, the melting point of the hapten (2208C) was higher than that of TYL (200 8C),

indicating a new compound was obtained. Second, the infrared (IR) data showed the

main chemical groups of parent TYLwere still present and a carboxyl group, a benzene

ring and a C�N bond were obtained. Third, the UV absorbance spectra of PTYL and

TYL were similar (Figure 3), indicating the general structure of TYL was untouched.

Fourth, the UV absorbance spectra of the immunogen contained the characteristic

peaks of PTYL and BSA (Figure 3), indicating PTYLwas coupled to BSA. The hapten

density was 14 mol/mol in the immunogen and 11 in the coating antigen.

Figure 3. UV absorbance spectra of tylosin, tylosin hapten, BSA and the immunnogen.

Food and Agricultural Immunology 425

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3.2. Antibody performance

Six hybridomas producing specific monoclonal antibody to TYL were obtained

and the antibodies were named TY01, TY02, TY07, TY13, TY25 and TY56. Their

IC50 and CRs to the four analytes are shown in Table 1 (coating antigen 1). Antibody

TY01 and TY13 showed high specificity for TYL and showed low CRs to ATYL

(6.4% and 8.1%, respectively) and negligible CRs to TMC and DMC (B 2%). This

pattern was possibly because the two antibodies mainly bind the E ring in the

molecule of TYL (Figure 1). The four other antibodies simultaneously recognised

the four analytes. Antibody TY56 showed the best performance with IC50 in the

range of 14.2�22.9 ng mL�1 and CRs in the range of 62�100% (Table 1). Based

on these results, we speculated that the four antibodies mainly bind A ring and

C ring, because they showed negligible cross-reactivity (CRsB1%) to the MACs that

do not contain the two rings (erythromycin, spiramycin and avermectin, data not

shown). The CRs of these antibodies to the four analytes were better than those

of the previously reported antibodies (Beier et al., 2005; Jackman et al., 1997; Peng

et al., 2012; Silverlight et al., 1999; Wicker et al., 1994; Yao and Mahoney, 1989).

The CRs of the four antibodies to ATYL and DMC (48�97%) were similar to our

previous anti-TYL antibody (37�94%), but the CRs to TMC (29�62%) were higher

than that antibody (16�49%) (Zhang et al., 2012). This was because of the different

molecular structure of the two haptens. In the molecule of the previously described

TYL hapten, there is a simple straight chain at C20 position and the structure at

this position is different from that in TMC molecule (Figures 1 and 2), so the CRs

were low. In the new hapten, PTYL, there is a benzene ring at C20 position and

the general structure near this position is similar to that in the TMC molecule.

The general structure of TMC could be regarded as a part of PTYL, so the CRs

to TMC were high. Therefore, the new hapten of TYL improved the antibody

selectivity, resulting in the antibody that could be used for multi-analyte immu-

noassay of the four analytes.

Table 1. Performances of the six antibodies for the four analytes with different coating

antigens.

TY01 TY13 TY02 TY07 TY25 TY56

IC50a CRb IC50 CR IC50 CR IC50 CR IC50 CR IC50 CR LODa

Homologous format (coating antigen 1)

TYL 13 100 21 100 9.6 100 38.0 100 19.5 100 14.2 100 2.1

ATYL 162 8.1 328 6.4 15.2 63 48.7 78 20.7 94 14.6 97 2.6

DMC �1000 B1.2 �1000 B2 21.3 48 66.5 57 21.9 89 15.3 93 3.0

TMC �1000 B1.2 �1000 B2 27.4 35 131 29 31.9 61 22.9 62 5.4

Heterologous format (coating antigen 2)

TYL NEc NE NE NE 8.4 100 27.3 100 15.0 100 12.6 100 1.8

ATYL NE NE NE NE 11.8 71 33.2 82 15.8 95 11.9 106 1.5

DMC NE NE NE NE 17.0 49 43.3 63 16.5 91 13.4 94 2.7

TMC NE NE NE NE 20.5 41 71.8 38 27.7 54 18.8 67 3.1

aThe unit of IC50 and LOD is ng mL�1.bThe CR is expressed as %.cNE means ‘not evaluated’.

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3.3. ELISA method

Nowadays, heterology in the coating antigen has been commonly used to improve

the sensitivity and specificity of an immunoassay (Franek, Diblikova, Cernoch, Vass,

& Hruska, 2006). Therefore, the four antibodies (TY02, TY07, TY25 and TY56),

PTYL-OA (coating antigen 1) and the previously prepared coating antigen TYL-OA

(coating antigen 2, Figure 2) were arranged into homologous and heterologous

format to optimise the reagent combinations.The performances of these antibodies in homologous format were described

earlier. As shown in Table 1, the specificities and sensitivities of the four antibodies in

heterologous format (using coating antigen 2) were generally better than that in

homologous format (using coating antigen 1). For example, the CRs of antibody

TY56 to the four analytes were in the range of 67�106% and IC50 were in the range

of 11.9�18.8 ng mL�1. In addition, the CRs differences among these competitors in

heterologous format were lower than that in homologous format. In heterologous

format, the coating antigen 2 (containing a long straight chain) eliminated theinfluence of the antibody from the spacer arm in PTYL-BSA to show low

competitive binding to the antibodies; thus increases the antibody binding for the

competitors to achieve broad specificity and high sensitivity. Among these reagent

combinations, antibody TY56 and coating antigen 2 produced the highest sensitivity,

with LODs in the range of 1.5�3.1 ng mL�1 (Table 1). Therefore, this combination

was used for the subsequent experiments. The competitive inhibition standard curves

for the four analytes in the range of 1�200 ng mL�1 are shown in Figure 4.

3.4. Sample extraction

An important step in the evaluation of an analytical procedure is to assess the matrix

effect, which should be minimised by the appropriate sample preparation. In the

Figure 4. Competitive calibration curves of TYL standard and DMC standard.

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present study, ATYL and TMC standards prepared with the extracts of blank milk

were used to develop the matrix matched competitive curves. As shown in Figure 5,

the competitive inhibitory curves of the matrix matched ATYL and TMC were

similar to that of their standards, indicating the extraction method was satisfactory.Then, recovery test was carried out by fortification of each analyte in blank milk

(20�100 ng mL�1) for ELISA analysis. Intra-assay recoveries (six duplicates in

a single day) ranged from 76.4% to 97.4% with coefficients of variation (CV) of

5.3�8.9%. Inter-assay recoveries (repeat once a day on six successive days) ranged

from 76.3% to 94.6% with CVs of 7.1�15.7% (Table 2). These data meet the

requirements for an analytical method for residue determination.

3.5. Unknown samples

The 35 unknown milk samples were analysed by the ELISA, and four samples were

determined as positive. The residue levels calculated as TYL were 12, 31, 35 and

62 ng mL�1, but the specific analyte cannot be verified due to the antibody’s high

CR. A milk sample containing any of the four analytes could be detected as positive,

but the result can only be expressed as TYL equivalents. Therefore, the ELISA

positive results need to be confirmed with an instrumental method, e.g. LC-MS/MS.

4. Conclusion

Immunoassay is a commonly used method for rapid screening the residues of

veterinary drugs in animal derived foods, but the use of immunoassay to determine

MACs in foods is rare. In this study, a new hapten of TYL was synthesised and

was used to produce a monoclonal antibody that simultaneously recognised three

Figure 5. Competitive calibration curves of ATYL standard, TMC standard and their matrix

matched standards.

428 J.K. Zhang et al.

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MACs (TYL, ATYL, TMC) and DMC, a metabolite of TYL. A heterologous

competitive indirect ELISA was developed to detect the four analytes in milk

simultaneously. This method could be used as a practical tool for routine screening

of large numbers of milk samples, with positive results confirmed by instrumental

methods.

Acknowledgements

This study was financed by Hebei Scientific and Technological Project (11221001D).

References

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Benetti, C., Dainese, N., Biancotto, G., Piro, R., & Mutinelli, F. (2004). Unauthorisedantibiotic treatments in beekeeping: Development and validation of a method to quantifyand confirm tylosin residues in honey using liquid chromatography-tandem mass spectro-metric detection. Analytica Chimica Acta, 520, 87�92.

Bogialli, S., Ciampanella, C., Curini, R., Di Corcia, A., & Lagana, A. (2009). Developmentand validation of a rapid assay based on liquid chromatogaphy-tandem mass spectrometryfor determining macrolide antibiotic residues in eggs. Journal of Chromatography A, 1216,6810�6815.

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Table 2. Recoveries of the four drugs from blank fortified milk.

Intra-assay Inter-assay

Analyte Added (ng mL�1) Recovery (%) CV (%) Recovery (%) CV (%)

TYL 20 80.2 6.6 90.3 9.6

50 89.4 8.9 94.6 9.2

100 92.6 6.2 88.5 7.4

ATYL 20 97.4 7.3 86.2 12.3

50 91.0 7.6 92.4 11.4

100 93.5 8.0 89.0 8.9

TMC 20 84.7 5.4 78.9 7.1

50 82.9 5.9 76.3 8.5

100 81.0 8.7 83.9 15.7

DMC 20 76.4 8.2 83.1 10.6

50 84.1 5.3 77.2 14.3

100 92.1 7.8 91.3 9.1

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