Immunoassay of chemical contaminants in milk: A review

Journal of Integrative Agriculture 2015, 14(11): 2282–2295 Available online at www.sciencedirect.com

ScienceDirect

REVIEW

Immunoassay of chemical contaminants in milk: A review XU Fei1, REN Kang2, YANG Yu-ze2, GUO Jiang-peng2, MA Guang-peng3, LIU Yi-ming1, LU Yong-qiang2, LI Xiu-bo1 1

National Feed Drug Reference Laboratories, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R.China 2 Beijing General Station of Animal Husbandry, Beijing 100107, P.R.China 3 China Rural Technology Development Center, Ministry of Science and Technology of China, Beijing 100045, P.R.China

Abstract The detection of chemical contaminants is critical to ensure dairy safety. These contaminants include veterinary medicines, antibiotics, pesticides, heavy metals, mycotoxins, and persistent organic pollutants (POPs). Immunoassays have recently been used to detect contaminants in milk because of their simple operation, high speed, and low cost. This article describes the latest developments in the most important component of immunoassays — antibodies, and then reviews the four major substrates used for immunoassays (i.e., microplates, membranes, gels, and chips) as well as their use in the detection of milk contaminants. The paper concludes with prospects for further applications of these immunoassays. Keywords: milk, chemical contaminants, immunoassay, antibodies

1. Introduction Milk is an indispensable source of high quality protein and is especially important to infants, children, the elderly, and the sick. Global milk production reached 782 million tones or 109.6 kg per capita in 2013 (International Dairy Federation 2014). In addition, milk is a raw material for many dairy products such as yogurt, butter, cheese, ice cream, candy, etc. Before getting to consumers, milk goes through production, processing, and circulation. Each step involves

Received 16 April, 2015 Accepted 25 June, 2015 XU Fei, E-mail: [email protected]; Correspondence LI Xiu-bo, Tel: +86-10-82106059, E-mail: [email protected]; LU Yong-qiang, Tel: +86-10-84929001, E-mail: [email protected] © 2015, CAAS. All rights reserved. Published by Elsevier Ltd. doi: 10.1016/S2095-3119(15)61121-2

potentially unsafe factors such as chemical contamination that can affect milk quality. The potential chemical contaminants in milk products include veterinary medicines, antibiotics, pesticides, heavy metals, mycotoxins, persistent organic pollutants (POPs), radionuclides, nitrates or nitrites, as well as packaging contaminants or adulterants (Griffiths 2010). The source of this problem of these main problems are farmland contaminated with medication, forage pollution, drinking water contamination, illegal additions as well as transportation and processing pollution. Table 1 lists some of the major milk chemical contaminants and their maximum residue limits in the EU and China (MOA 2002; Council Regulation (EU) 2010). Researchers have focused on the detection of chemical contaminants in milk. The EU, the U.S., China, and others have enacted many validated methods for monitoring milk safety including gas chromatography (GC) (Hernandes et al. 2014; Meneghini et al. 2014), high-performance liquid chromatography (HPLC) (Karami-Osboo et al. 2014), gas

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Table 1 Primary chemical containments in milk Category Veterinary drugs

Pesticides Heavy metal

Mycotoxin Others

Contamination Ampicillin Cefalexin Enrofloxacin Gentamicin Streptomycin Sulfonamides Tetracyclines DDT HCH Pb Cd As Hg Aflatoxin M1 Melamine

Pathway Drug abuse in the process of breeding or failure to follow milk withdrawal time

Feed stuff (forage or cereal) pollution Environment (soil or water) pollution

Feed stuff stored improperly and added illegally

chromatography-mass spectrometry (GC/MS) (Liu et al. 2014; Zheng et al. 2014), liquid chromatography tandem mass spectrometry (LC-MS/MS) (Freitas et al. 2014; Sniegocki et al. 2014; Young et al. 2014), differential pulse anodic stripping voltammetry (DPASV) (Sadeghi et al. 2014), etc. These technologies are highly sensitive, specific, and reliable, but their application was limited by costly instrumentation and complicated sample preparation. In contrast, immunoassays based on specific antibody recognition are a rapid method of screening samples (Wild 2013). Unlike chromatography-based methods, they are simple, specific, easy to use, high-throughput, and do not require costly instrumentation. This makes them suitable for on-site detection. Therefore, a combination strategy with fast immunoassay screening followed by instrument-based confirmation has been widely considered. Moreover, immunoassay is also suitable for the analysis of milk. Because milk is a fluid, milk samples can be analyzed after only simple pretreatment such as dilution or protein precipitation. They can even be analyzed without sample preparation. For this reason, immunoassays have become the subject of increasing research attention in the detection of chemical contaminants in milk. Immunoassays include three major elements: (1) preparation of the target-specific antibody; (2) antigen-antibody recognition based on a specific carrier; and (3) acquisition of the detection signals. This review describes recent developments in antibodies and then details the four major carriers widely used in milk contaminant immunoassays.

2. Antibodies and antibody substitutes Antibodies are a key factor for a successful immunoassay. In addition to the continuous improvements in traditional

MRLs (μg L–1) EU 4 100 100 100 200 100 100 40 Forbidden 20 Forbidden Forbidden Forbidden 0.05 2 500

China 10 100 100 200 200 100 100 20 20 50 300 50 10 0.5 2 500

antibody production, the discovery and production of novel antibodies or antibody substitutes with specific properties has been reported including single-chain antibody fragments (Ahmad et al. 2012), aptamers (Toh et al. 2015), receptor proteins (Beltrán et al. 2014), and molecularly-imprinted polymers (Song et al. 2014) (Fig. 1).

2.1. Traditional antibodies Polyclonal and monoclonal antibodies are generated from immunized animals (rabbits, mice, or goats). Polyclonal antibodies can recognize multiple epitopes on any one antigen, and are inexpensive to produce. However, they are not as popular as monoclonal antibodies because they have high batch-to-batch variability and non-specificity. Monoclonal antibody technology is based on secreted antibodies from a hybridoma amplified in vitro. It was invented by British scientists Köhler and Milstein (1975) and was a major breakthrough in the field of immunology. In recent decades, monoclonal antibody preparation technology has gradually matured and mass production is now possible. Several companies, such as Biopharm (Germany), IDEXX (U.S.), and Randox (U.K.), have produced rapid screening products (kits and strips) for the assessment of milk contamination using monoclonal antibodies. Traditional antibodies are inferior in stability to many of their alternatives considered below because of the long time required for preparation and ethical concerns associated with the use of animals. Another term of “nanoantibody” or “nanobody” was given more attention these years. They are single-domain variable fragments of special type of antibodies, which naturally exist in blood of Camelidae family animals and in some chondrichthyan fishes (Tillib 2011). It is interesting that only a single variable domain of this antibody (without the first СН1

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Fig. 1 Types of antibody and antibody substitutes. A, IgG1 antibody. B, fragments of antibody such as Fab, single chain antibody fragments (scFV). C, schematic diagram of penicillin-binding proteins (PBPs) recognizing and binding on beta-lactam antibiotics. D, schematic overview of DNA aptamer selection, reprinted with permission from Sefah et al. (2010). E, schematic overview of the imprinted polymer preparation, reprinted with permission from Song et al. (2014).

domain) is necessary and sufficient in order to specifically recognize an antigen and bind to it. Because these camel mini-antibodies are more easily to be “humanized”, they have a very potential prospect as a passive immunisation treatment or antibody drugs (Tillib et al. 2010).

2.2. Single-chain antibodies To overcome the bottle-neck of high-throughput screening of hybridomas, a strategy combining chemistry, cell biology, and molecular biology methods has been used to generate recombinant antibodies. This is a new way to improve the specificity and affinity of antibodies. Single chain antibody fragments (scFv) are an important class of recombinant antibodies. The antibodies are usually expressed in Escherichia coli by genetic engineering and are composed of 15–20 short peptides from the variable regions of the heavy chain and light chain of the monoclonal antibody. They are only 1/6 of the molecular weight of the whole IgG. Versus monoclonal antibodies, scFv have higher affinity for the antigen, smaller size, better tissue penetration, shorter preparation time, and easier labeling; they can also be expressed in prokaryotes. Currently, single-chain

antibodies have been made to quinolones (Gomes et al. 2010; Leivo et al. 2013), sulfonamides (Korpimäki et al. 2004; Qi et al. 2009), zearalenone (Edupuganti et al. 2013), aflatoxins (Moghaddam et al. 2001), chloramphenicol (Van Dorst et al. 2012), fenitrothion (Luo and Xia 2012), etc. Chen et al. (2014) reported the clone, expression, and purification of a double specific scFv in 2014 that showed specific affinity to 20 fluoroquinolones and 14 sulfonamides drugs. Nevertheless, some studies have indicated low stability and yield of other scFvs.

2.3. Receptor proteins Most receptor proteins are membrane-associated proteins. Studies have indicated that after some drugs enter an organism, they can function by binding to receptor proteins on the cell membrane. That is, the receptor protein acts as an “antibody”. The β-lactam antibiotics including penicillins and cephalosporins are widely used in dairy cows and are thus the main contaminants in milk. Because of the different chemical structures of penicillins and cephalosporins, broad-spectrum antibodies against β-lactam antibiotics cannot be prepared

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by traditional approaches. However, the pharmacological effects of β-lactam antibiotics are exerted through similar mechanisms by inhibiting cell wall peptidoglycan synthases, i.e., (penicillin-binding proteins (PBPs). Zeng et al. (2013) reported the preparation and multi-residue monitoring methods for PBP that can determine 7 penicillins and 8 cephalosporins with sensitivities that satisfy the EU limits. Currently, drug receptor proteins expressed in vitro include sulfonamide receptor proteins (Liang et al. 2013), and tetracycline receptor proteins (Granier and Lepage 2011). Due to the challenges of in vitro expression and preparation, the study and application of receptor proteins are limited to specific antibiotics.

2.4. Aptamers Aptamers are a single-stranded oligonucleotide sequence of DNA or RNA that consist of 20–60 nucleotides and can specifically bind to a transfer ligand. The specific affinity of the aptamers and binding ligands are based on a diverse conformation of single-stranded nucleic acids. The aptamers can fold automatically into a stable three-dimensional structure via electrostatics, hydrogen bonds, base-pairing, etc. Some aptamers have already been screened to identify specific proteins (Erdem and Congur 2014), heavy metal ions (Chung et al. 2013; Gao et al. 2014), veterinary drugs (Zhang et al. 2010; Ni et al. 2014; Pilehvar et al. 2014), nucleotides (Feng L et al. 2014), and small molecule toxins (Medibena 2014). Aptamers offer a rapid and efficient identification strategy for clinical diagnosis and food safety inspection with significant prospects in practical applications. Versus antibodies, aptamers have many advantages (Toh et al. 2015): (1) Preparation is based on simple chemical synthesis and is cost efficient; (2) good stability with highly reversible denaturation; (3) the single nucleic acids are usually composed of no more than 30 bases with relatively small molar amounts and molecular sizes. The low steric hindrance is suitable for binding with nanomaterials for the generation of high-precision sensors; (4) there is a stronger affinity and specificity between the aptamer and the target molecules but with lower dissociation coefficients (~10–9–10–12 mol L–1).

2.5. Imprinted polymer Imprinted polymers, also known as plastic antibodies, are polymers generated by molecular imprinting. They have selective adsorption or specific recognition of target molecules and their structural analogs. Imprinted polymers have been extensively studied because of their stable chemical properties and higher susceptibility to acids and bases than biological antibodies. Currently, the use of molecularly-im-

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printed polymers (MIPs) in dairy quality control has focused on pre-processing. Analytes measured with these polymers include bisphenol A (Alexiadou et al. 2008), estrogen (Lan et al. 2014), sulfonamide (De Guzmán-Vázquez Prada et al. 2006), quinolones (Zheng et al. 2010), oxytetracycline (Lv et al. 2012), amphenicols (Mohamed et al. 2007), and selenium (de Lima et al. 2013). As synthetic techniques have advanced, MIPs with specific recognition abilities as artificial antibodies have received much attention. They have been deployed to various fields including food analysis. Additional details on the use of molecularly-imprinted polymers in food analysis can be found in the literature (Song et al. 2014).

3. Solid supports for immunoassay By now, most immunoassays are based on immobilizing the antigen or antibodies on the support surface of the carrier. This section describes immunoassay methods with different assay carriers (Fig. 2).

3.1. Microplate-based carriers in immunoassays The most currently used microplates for immunoassays are polystyrene. Polystyrene is a linear polymerized styrene monomer linked via a covalent radical reaction. The weak polarity and strong hydrophobicity easily makes the antigens or antibodies adsorb to the microplate surface by hydrophobic interactions. The preparation of microplates is also cost efficient. Microplates with 96, 384, and even larger numbers of wells are available, but 96-well plates are the most common. These can be transparent plates (for enzyme-linked immunosorbent assay (ELISA)), white opaque plates (for chemiluminescence assays (CIA)), black opaque plates (for fluorescent immunoassay (FIA)), or black opaque plates with low non-specific binding surface (for fluorescence polarization analysis (FPIA)). The advantages of this method include high throughput (one plate can detect 40 to 80 samples simultaneously), semi-quantitative results, and high sensitivity and specificity. The disadvantages include the analysis time (>2 h) and the need for a microplate reader or chemiluminescence detector. ELISA is the most common assay mode and base of many commercialized assay kits. Competitive ELISA is mostly used in dairy studies because milk contaminants are mostly small-molecule compounds. The enzymes used in the assay are the horseradish peroxidase (HRP). Quantification of the target analytes is based on TMB color change. ELISA is simple and low cost, but is also limited by low sensitivity of the photometric measurement and poor stability of the labeled enzymes. To further enhance assay sensitivity, many new methods have been developed on the basis of ELISA. Most of these

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Fig. 2 Different supports for immunoassay. A, microplates. B, nitrocellulose (NC) membrane. C, gel bead. D, immunochip. The procedure for analytical microarrays includes microarray manufacture, measurement techniques for microarray readout, establishment of multi-analyte assays, and processing of multiple sets of quantitative data. Reprinted with permission from Seidel and Niessner (2014).

improve signal acquisition. CIA are efficient non-radioactive detection methods using a luminescent substrate to replace the ELISA chromogenic substrate. This method has high sensitivity, stable luminescent markers, a wide detection range and high automation. Therefore, CIA has been widely applied in the clinic and used in food safety tests (Zhao et al. 2009). Fluorescent labels can also be used to increase the sensitivity of the methods. The major methods via fluorescence include time-resolved immunoassay method (TRFIA), fluorescence polarization analysis (FPIA), liposome immunoassay (LIA) and fluorescent-linked immunosorbent assay (FLISA). Of these, it is vital to select a tracer that is efficient, highly sensitive, and easy to label. Fluorescent markers are mostly organic fluorescent dyes such as fluorescein (Wang et al. 2011), cyanine dyes (Xu et al. 2013), fluorescent marker proteins (e.g., phycoerythrin (Wang et al. 2011; Bienenmann-Ploum et al. 2012), green fluorescent proteins (Charest Morin et al. 2013, Christie et al. 2013), metal complex markers such as Eu3+, Tb3+, noble metal ion complexes, etc. (Wang et al. 2013), nanoparticles, e.g., quantum dots (Chen et al. 2009), fluorescent microsphere (Guirgis et al. 2012), carbon nanodots (Bu et al. 2014; Zhu et al. 2014), etc. Nanoparticles have been the focus of recent studies in the field of

immunoassays, and many new nanoparticles have been reported in the past few years. These fluorescent markers not only enhance the sensitivity of the current detection methods, but also provide unparalleled flexibility for labeling. Recent reports on the detection of milk contamination based on microplate carriers are summarized in Table 2. The 96-well filter plate is made of bio-inert polypropylene with chemical susceptibility. The transparent cover material is polystyrene and is compatible with automated processing devices. It is mainly used for high-throughput preparation of biological samples and LC-MS/MS analysis. Recently, 96-well filter plates have been introduced in milk analysis. Tien et al. (2013) developed a novel bead-based 96-well filtration plate competitive immunoassay by integrating 96well filtration plates and beads to perform immunoassays for gentamycin sulfate. The assay was stable, sensitive, and convenient. In the assay format above, a 96-well filter plate served as the reaction carrier, and the limit of detection (LOD) was sufficient to detect the Codex maximum residual level (MRL).

3.2. Membrane-based carrier in immunoassays Immunoassays based on membrane carriers are common

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Table 2 Representative examples of microplate-immunoassay for milk analysis Species Antibiotics

Veterinary drug

Pesticides POPs Others Toxin Heavy metal 1)

2)

Analyte Cephalexin/Cefadroxil Tetracyclinesn Macrolide antibiotics Gentamicin Fluoroquinolones/Sulfonamides Sulfonamides Sulfonamides/Melamine/ Quinolones Fluoroquinolone Orbifloxacin Chloramphenicol Atrazine Avermectins Tris-(2,3-dibromopropyl) isocyanurate Melamine 5-Hydroxymethyl-2-furfural Aflatoxin B-1 and aflatoxin M-1 Abrin Mercury(II) ion

Method1) FPIA ELISA ELISA CIA FPIA ELISA/FLISA FLISA

Signal2) FITC TMB TMB Luminol FITC TMB Quantum dot

Reference Zhang et al. (2014) Wang et al. (2014) Zhang et al. (2013) Li et al. (2012) Chen et al. (2014) Shen et al. (2007); Liang et al. (2014) Zhu et al. (2011)

ELISA/TRFIA

TMB/Europium

FPIA ELISA/CIA ELISA ELISA ELISA

FITC TMB/Luminol TMB TMB TMB

Suryoprabowo et al. (2014); Leivo et al. (2013) Mi et al. (2014) Tao et al. (2013); Liu et al. (2014) Barchanska et al. (2012) Wang et al. (2012) Feng H Y et al. (2014)

ELISA/FLISA ELISA ELISA ELISA ELISA

Quantum dot TMB TMB TMB TMB

Wu et al. (2013); Gong et al. (2014) Guan et al. (2013) Jiang et al. (2013) Zhou et al. (2012) Wang et al. (2012)

FPIA, fluorescence polarization immunoassay; ELISA, enzyme-linked immunosorbent assay; CIA, chemiluminescence assays; FLISA, fluorescent-linked immunosorbent assay; TRFIA, time-resolved fluorescent immunoassay. FITC, fluorescein isothiocyanate; TMB, tetramethylbenzidine.

format for the rapid detection. Its initial development is flow-through immunoassay, which is known as immuno-filtration assay. The test principle involves a flow of fluid containing the analyte through a porous membrane and into an absorbent pad. It is a rapid (within 5 min) and simple immunoassay for qualitative screening, with the advantage of large volumes of samples, but now its application is often restricted by relatively low sensitivity, especially for the analysis of food contaminants such as chemical pesticides and antibiotic residues (Sui et al. 2009). Lateral flow tests are also known as lateral flow immunochromatographic assays. When a sample solution is applied to the end of a test strip, it moves forward by capillary effects; when it reaches the conjugation pad, it dissolves and reacts with the colloidal gold-labeled reagents on the conjugation pad. It then further moves to an area on the test strip with antigens or antibodies attached. If the compound formed between samples and the gold-labeled reagents specifically reacts with the antigens or antibodies, they will accumulate in this zone. This causes a color change visible to the naked eye. Immunochromatographic methods are widely applied in dairy quality control. It is most commonly used with a nitrocellulose membrane (NC) because of the following reasons (Mansfield 2009): (1) They have high affinity to target across a wide range and at low ion strength buffer. Here, most negatively charged proteins will be associated with the nitrocellulose membrane due to hydrophobicity; (2)

the binding site is easily blocked, which reduces nonspecific binding; (3) it is suitable for mass production and is stable with low cost. The label most commonly used in immunochromatographic assay is colloidal gold (Hayat 2012). The production process is relatively easy. Chloroauric acid (HAuCl4) can be reduced into gold nanoparticles (colloidal gold) in the presence of reducing agent. The colloidal gold is negative under weak basic conditions - this is attractive to positively charged protein molecules. They bind strongly without changing the biological properties of the proteins. Many studies use colloidal gold as a label because it is easy to prepare, inexpensive, stable, and yields a bright color. Reports using it in milk include aminoglycoside antibiotics (Watanabe et al. 2002; Jin et al. 2006; Wu et al. 2010), cephalosporins (Chen et al. 2009; Guo et al. 2015), quinolones (Sheng et al. 2011; Liu et al. 2014), sulfonamides (Chen et al. 2009), and aflatoxin (Wang et al. 2011; Zhang et al. 2012). Many of these have been commercialized. Latex particles are another common type of labeling molecule in immunoassay. Versus colloidal gold, latex particles bind antibodies with strong covalent bonds. By mixing with the dye molecules, the latex particles show a variety of colors. However, the preparation of latex particles is relatively complicated and is not as extensively studied as colloidal gold. Currently, the US companies IDEXX Laboratories (IDEXX 2015) and Charm Sciences (Charm 2015) use latex particles for dairy assays including β-lactam antibiotics,

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tetracyclines, aminoglycosides, and sulfonamides with many latex color choices available depending on the analyte. Although colloidal gold and latex particles have been studied and applied widely, both of them rely on observation with the naked eye. There are limitations to the sensitivity of this method. Yang et al. (2011) reported that colloidal gold with silver enhancement in an immunochromatographic assay can detect abrin in milk with a 100-fold increase in sensitivity vs. gold only. Other reports have studied novel immunochromatographic assays combining colloidal gold with immunomagnetic nanobeads for separation and enrichment (Huang et al. 2014; Liu et al. 2015). Other studies have focused on fluorescent labeling materials including nanosilver (Anfossi et al. 2013), fluorescent silica particles (Zhang et al. 2013), liposomes (Anfossi et al. 2013), colloidal carbon (Blažková et al. 2009), upconverting phosphors (Guo and Sun 2012), quantum dots (Garcia-Fernandez et al. 2014), fluorescent biological markers (Liotta et al. 1999), and low molecular weight (LMW) fluorescent dyes (Goryacheva et al. 2013). Fluorescent microspheres and quantum dots have gained the most attention. The others have not been used broadly due to difficult synthetic steps or limitations in application. Most quantum dot- or fluorescent microsphere-based immunochromatography techniques in the dairy field were reported in the past 3 years. Xie et al. (2014) developed two sandwich immunochromatography assays for detecting E. coli O157:H7 using colloidal gold (CG-ICTS) and fluorescent microspheres (FM-ICTS) as labels. The results showed that the sensitivity of FM-ICTS is 8-fold higher than that of CG-ICTS under optimum conditions. Bian et al. (2013) detected cephalexin in milk, serum, and animal tissues based on fluorescent microsphere strips in 2013; Chen et al. (2013) measured sulfamethazine in milk with a fluorescent microsphere strip method in 2013, and Zhou et al. (2014) detected lincomycin in milk, honey, cattle, and swine urine with a fluorescent microsphere strip method in 2014. Berlina et al. (2013) detected chloramphenicol in milk with a quantum dot-based immunochromatographic method in 2013. The use of quantum dots enable highly sensitive analysis and multiplexing, and the limit of CAP detection is 0.2 ng mL–1 - the limit of quantitation is 0.3 ng mL–1. In 2015, Taranova et al. (2015) reported a novel “traffic light” immunochromatographic test based on multicolor quantum dots for the simultaneous detection of chloramphenicol, streptomycin, and ofloxacin in milk. Fig. 3 illustrates that the system was designed in a traffic light format with three lines in different colors (red, yellow, and green). The test system exhibited high sensitivity with LOD values that are 80–200 times lower than those achievable with ELISA using the same antibodies (Taranova et al. 2015).

Fig. 3 Principle of the competitive detection of 3 antibiotics using a “traffic light” immunochromatographic test (1, test zone for STM; 2, test zone for CAP; 3, test zone for OFL; 4, conjugate QD-mAb/STM; 5, conjugate QD-mAb/CAP; 6, conjugate QDmAb/OFL; 7, control line). A, test strip before the assay. B, assay results for the sample containing STM. C, assay results for the sample containing CAP and OFL. Reprinted with permission from Taranova et al. (2015).

3.3. Gel-based carriers in immunoassays Sephrose 4B gel is commonly used as a carrier in protein purification. It can bind antibodies through covalent bonds after being activated by cyanogen bromide (CNBr). Conventionally, sepharose 4B gel has been used for sample cleaning in pre-processing, i.e., an immunoaffinity column. Recently, researchers have introduced it into immunoaffinity assays. The CN-Br activated Sepharose 4B gel is a solid support to immobilize goat anti-mouse IgG. Monoclonal antibodies were added to the immobilized IgG and bound to the column through specific affinity - excess monoclonal antibody flowed through the column by gravity. Then drugs and enzyme-labeled antigens were added to the column for competitive binding to the monoclonal antibody. The final step was addition of TMB chromogenic substrate. This method is called visualized gel-based ELISA (Gel-ELISA) because the TMB chromogenic substrate appears blue in the column bed. Gel-ELISA was firstly reported in 2008 (Goryacheva et al. 2008). Subsequent applications have been reported in the detection of 2,4,6-trichlorophenol (Beloglazova et al. 2010) and ochratoxin A (Rusanova et al. 2009) in red wine,

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and benzopyrene (Beloglazova et al. 2011) and zearalenone-4-glucoside in food (Beloglazova et al. 2013). Gel-ELISA is mostly used to detect toxins and organic molecules in liquid samples (Speranskaya et al. 2014). There are also milk analysis papers via Gel-ELISA. In 2012, Yuan et al. (2012) used Gel-ELISA to detect chloramphenicol in animal tissues, milk, honey, fish, and shrimp with a LOD of 1.0 μg L–1. Versus the lateral flow strip test of CAP with the same antibody, the Gel-ELISA provided a reliable assay with 100-fold sensitivity improvement. Xu et al. (2014) reported the use of Gel-ELISA to detect apramycin in milk, muscle, and liver. Compared with dcELISA, the detection time of IATC is shortened to 20 min whereas a similar sensitivity for various samples was observed. In 2015, Jiang and coworkers used Gel-ELISA to measure 14 sulfonamides and 13 quinolones in milk. The use of liposome-encapsulated quantum dots with the Gel-ELISA method had the best results with limits of detection (LODs) of 1 and 0.5 ng mL–1 for the sulfonamides (SAs) and quinolones (QNs), respectively (Jiang et al. 2015). As a novel test strategy, Gel-ELISA has shown its unique advantages and brought application potentials. First, the range of sample volumes is large. In Gel-ELISA, the pore size governs sample size in contrast to microplate approaches. The applied sample volume is usually 50 μL, and the total reaction volume is less than 100 μL. In a gel column, up to five column bed volumes can be used and eluted with gravity. Practically, sample volumes of 1–3 mL are usually applied, but that may theoretically contain up to 20-fold the amount of target actually as in DcELISA, which effectively enhances sensitivity. Second, the matrix interferences are small and sample pre-treatment is minimal. Gel-ELISA has strong tolerance to sample matrix effects. After protein precipitation, the supernatant can be completely transferred to the column— there is no sample dilution. In contrast, to eliminate sample matrix interferences in DcELISA, the supernatant sample from protein precipitation still requires dilution to reduce the matrix background. This reduces detection limits. Third, Gel-ELISA offers rapid assay times. Because it uses natural gravity flow, the method does not need any incubation or washing steps with total assay times of 15–20 min, which saves up to 80% of the time vs. the 90–120 min of ELISA.

3.4. Chip-based carriers in immunoassays Chip is another carrier for the detection of different antibiotic classes in milk. Biochips technologies use various strategies to immobilize biological molecules (oligonucleotides, DNA, cDNA, polypeptides, antigens, antibodies, etc.) on solid supports such as silicon slides, glass slides (beads), plastic

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sheets (beads), and nylon films. According to the different coating compounds, common biochips could be divided into three categories: DNA microassay, protein-chip, and microfluid. The system can capture targets from the sample with signal capture facilitated by charge-coupled-device camera (CCD) cameras or laser scanner. Digital processing offers qualitative and quantitative analyses. Compared to other immunoassays, the biochip method has huge potential in the detection of antibiotic residues in milk, which uses low sample volumes, requires no sample pre-treatment, and is high-throughput and rapid. In these methods, the analytes (chemical contaminants in milk) were coupled to the chip surface with or without linking agents, and competitive immunoassay format was used to implement the detection. Milk studies using bio-chip technologies to detect chemical contaminants in milk mainly focus on antibiotics and drug residues. In 2008, Ye et al. (2008) reported a microplate array-based SMM-FIA for the simultaneous detection of sulfamethazine, streptomycin, and tylosin in milk. In 2009, Raz et al. (2009) described a label-free multiplex detection system for streptomycin, gentamicin, neomycin, kanamycin, sulfamethazine, enrofloxacin, and chloramphenicol residues in milk using a surface plasmon resonance-based immunosensor. The detection of multiplex residues can be used to measure all the target compounds at µg L–1 levels, which were sensitive enough for milk control at maximum residue levels as established by the European Union (Raz et al. 2009). In 2009, Kloth et al. (2009) reported a hapten microarray designed for the parallel analysis of 13 different antibiotics in milk. The assay could be performed in as little as 6 min using an indirect competitive chemiluminescence microarray immunoassay (CL-MIA). This method showed high sensitivity, and the chip could be reused 50 times after antigen activation (Kloth et al. 2009). Porter et al. (2012) developed a biochip array kit for multiplex screening of residues for more than 20 anthelmintic drugs in milk and animal tissues in 2012. The results showed that biochip array technology is suitable for the simultaneous determination of multiple analytes from a single sample. Biochip-based immunoassays are still being improved. The synthesis of the biochip base involves complicated chemical processing, and the capture of chemiluminescent signals requires extremely high sensitivity CCD detectors for one-time acquisition. On the other hand, this strategy uses small sample volumes and requires specialists for such sophisticated operation. However, the high cost will likely limit its utility.

4. Conclusion and prospects Immunoassay technology has been increasingly applied to

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dairy quality control in the last ten years due to its simple operation, short analysis times, and low cost. The most mature method is microplate-based ELISA that uses small sample volumes and simple pretreatment steps. It has a high capacity without the need for complex instrumentation. It can achieve detection limits similar to HPLC or LC-MS/ MS (ng g–1 or pg g–1). The test efficiency is 10-fold better than instrument-dependent measurements. The test strip method using a NC membrane as the carrier can complete a test in 5–10 min, and the results are visible to the naked eyes. This method has developed rapidly, and its utility has extended from the field of medical diagnostics to food safety. There are multiple commercialized products in use on dairy farms and milk processing stations. Recently, the Chinese government amended the Food Safety Law of China. Article 88 of the law stressed that “rapid methods should be used in the check test of edible agricultural products”. This will greatly promote the research and development of immunoassay in the future from the aspect of policy. Moreover, new nano-materials, such as fluorescent microspheres, quantum dots, magnetic beads, and graphene have their own advantages in terms of sample separation, sample enrichment, signal amplification, and sensitivity. In addition, high-throughput, rapid analysis technologies such as biochips are increasingly applied to diary assays. In summary, immunoassays are increasingly important in the detection of chemical contaminants in milk because of developments in material techniques, antibody techniques, bioconjugate techniques, and signal acquisition techniques.

Acknowledgements This work was financially supported by the Beijing Dairy Industry Innovation Team and Feed Quality and Safety Control Innovation Team of Chinese Academy of Agricultural Sciences.

References Ahmad Z A, Yeap S K, Ali A M, Ho W Y, Alitheen N B M, Hamid M. 2012. ScFv antibody: Principles and clinical application. Clinical and Developmental Immunology, 2012, 1–15. Alexiadou D K, Maragou N C, Thomaidis N S, Theodoridis G A, Koupparis M A. 2008. Molecularly imprinted polymers for bisphenol A for HPLC and SPE from water and milk. Journal of Separation Science, 31, 2272–2282. Anfossi L, Di Nardo F, Giovannoli C, Passini C, Baggiani C. 2013. Increased sensitivity of lateral flow immunoassay for ochratoxin A through silver enhancement. Analytical and Bioanalytical Chemistry, 405, 9859–9867. Barchanska H, Jodo E, Price R G, Baranowska I, Abuknesha R. 2012. Monitoring of atrazine in milk using a rapid tube-

based ELISA and validation with HPLC. Chemosphere, 87, 1330–1334. Beloglazova N V, De Boevre M, Goryacheva I Y, Werbrouck S, Guo Y, De Saeger S. 2013. Immunochemical approach for zearalenone-4-glucoside determination. Talanta, 106, 422–430. Beloglazova N V, Goryacheva I Y, Rusanova T Y, Yurasov N A, Galve R, Marco M P, De Saeger S. 2010. Gelbased immunotest for simultaneous detection of 2,4,6-trichlorophenol and ochratoxin A in red wine. Analytica Chimica Acta, 672, 3–8. Beloglazova N V, Goryacheva I Y, de Saeger S, Scippo M L, Niessner R, Knopp D. 2011. New approach to quantitative analysis of benzo[a]pyrene in food supplements by an immunochemical column test. Talanta, 85, 151–156. Beltrán M C, Borràs M, Nagel O, Althaus R L, Molina M P. 2014. Validation of receptor-binding assays to detect antibiotics in goat’s milk. Journal of Food Protection, 77, 308–313. Berlina A N, Taranova N A, Zherdev A V, Vengerov Y Y, Dzantiev B B. 2013. Quantum dot-based lateral flow immunoassay for detection of chloramphenicol in milk. Analytical and Bioanalytical Chemistry, 405, 4997–5000. Bian S M, Chu X G, Jin Y, Xing S G, Zhang Y, Hu H B. 2013. A novel microsphere-based fluorescence immunochromatographic assay for monitoring cefalexin residues in plasma, milk, muscle and liver. Analytical Methods, 5, 6441–6448. Bienenmann-Ploum M E, Huet A C, Campbell K, Fodey T L, Vincent U, Haasnoot W, Delahaut P, Elliott C, Nielen M, Schilt R. 2012. Fiveplex flow cytometric immunoassay for the simultaneous detection of six coccidiostats in feed and eggs. Conference poster. In: Residues of Veterinary Drugs in Food. Proceedings of the EuroResidue VII Conference. 14–16 May, 2012. Volume 1, 2 and 3. Egmond Aan Zee, Netherlands. pp. 899–902. Blažková M, Mičková-Holubová B, Rauch P, Fukal L. 2009. Immunochromatographic colloidal carbon-based assay for detection of methiocarb in surface water. Biosensors and Bioelectronics, 25, 753–758. Bu D, Zhuang H, Yang G, Ping X. 2014. An immunosensor designed for polybrominated biphenyl detection based on fluorescence resonance energy transfer (FRET) between carbon dots and gold nanoparticles. Sensors and Actuators (B: Chemical), 195, 540–548. Charest Morin X, Fortin J P, Bawolak M T, Lodge R, Marceau F. 2013. Green fluorescent protein fused to peptide agonists of two dissimilar G protein-coupled receptors: Novel ligands of the bradykinin B2 (rhodopsin family) receptor and parathyroid hormone PTH1 (secretin family) receptor. Pharmacology Research & Perspectives, 1, 1–13. Charm. 2015. Charm II antiboitics kit. [2015-03-15]. http://www. charm.com/products Chen J, Xu F, Jiang H, Hou Y, Rao Q, Guo P, Ding S. 2009. A novel quantum dot-based fluoroimmunoassay method for detection of Enrofloxacin residue in chicken muscle tissue.

XU Fei et al. Journal of Integrative Agriculture 2015, 14(11): 2282–2295

Food Chemistry, 113, 1197–1201. Chen L, Wang Z, Ferreri M, Su J, Han B. 2009. Cephalexin residue detection in milk and beef by ELISA and colloidal gold based one-step strip assay. Journal of Agricultural and Food Chemistry, 57, 4674–4679. Chen M, Wen K, Tao X Q, Ding S Y, Xie J, Yu X Z, Li J C, Xia X, Wang Y, Xie S L, Jiang H Y. 2014. A novel multiplexed fluorescence polarisation immunoassay based on a recombinant bi-specific single-chain diabody for simultaneous detection of fluoroquinolones and sulfonamides in milk. Food Additives and Contaminants (Part A-Chemistry Analysis Control Exposure & Risk Assessment), 31, 1959–1967. Chen M, Wen K, Tao X Q, Xie J, Wang L M, Li Y, Ding S Y, Jiang H Y. 2014. Cloning, expression, purification and characterization of a bispecific single-chain diabody against fluoroquinolones and sulfonamides in Escherichia coli. Protein Expression and Purification, 100, 19–25. Chen R, Li H, Zhang H, Zhang S X, Shi W M, Shen J Z, Wang Z H. 2013. Development of a lateral flow fluorescent microsphere immunoassay for the determination of sulfamethazine in milk. Analytical and Bioanalytical Chemistry, 405, 6783–6789. Christie M, Boland A, Huntzinger E, Weichenrieder O, Izaurralde E. 2013. Structure of the PAN3 pseudokinase reveals the basis for interactions with the PAN2 deadenylase and the GW182 proteins. Molecular Cell, 51, 360–373. Chung C H, Kim J H, Jung J, Chung B H. 2013. Nucleaseresistant DNA aptamer on gold nanoparticles for the simultaneous detection of Pb2+ and Hg2+ in human serum. Biosensors and Bioelectronics, 41, 827–832. Council Regulation (EU). 2010. Council Regulation (EU) N 37/2010, on Pharmacologically Active Substances and Their Classification Regarding Maximum Residue Limits in Foodstuffs of Animal Origin. pp. 1–72. Van Dorst B, Mehta J, Rouah Martin E, Backeljau J, De Coen W, Eeckhout D, De Jaeger G, Blust R, Robbens J. 2012. Selection of scFv phages specific for chloramphenicol acetyl transferase (CAT), as alternatives for antibodies in CAT detection assays. Journal of Applied Toxicology, 32, 783–789. Edupuganti S R, Edupuganti O P, O’Kennedy R. 2013. Generation of anti-zearalenone scFv and its incorporation into surface plasmon resonance-based assay for the detection of zearalenone in sorghum. Food Control, 34, 668–674. Erdem A, Congur G. 2014. Voltammetric aptasensor combined with magnetic beads assay developed for detection of human activated protein C. Talanta, 128, 428–433. Feng H Y, Tong X, Li W L, Zhou L P, Shi L, Cai Q Y. 2014. Indirect competitive enzyme-linked immunosorbent assay of tris-(2,3-dibromopropyl) isocyanurate with monoclonal antibody. Talanta, 128, 434–444. Feng L, Zhang Z, Ren J, Qu X. 2014. Functionalized graphene as sensitive electrochemical label in target-dependent

2291

linkage of split aptasensor for dual detection. Biosensors and Bioelectronics, 62, 52–58. Freitas S, Paim A, Silva P. 2014. Development of a LC-ITTOF MS procedure to quantify veterinary drug residues in milk employing a QuEChERS approach. Food Analytical Methods, 7, 39–46. Gao C, Wang Q, Gao F, Gao F. 2014. A high-performance aptasensor for mercury(ii) based on the formation of a unique ternary structure of aptamer-Hg2+-neutral red. Chemical Communications, 50, 9397–9400. Garcia-Fernandez J, Trapiella-Alfonso L, Costa-Fernandez J M, Pereiro R, Sanz-Medel A. 2014. A quantum dot-based immunoassay for screening of tetracyclines in bovine muscle. Journal of Agricultural and Food Chemistry, 62, 1733–1740. Gomes F B M B, Riedstra S, Ferreira J P M. 2010. Development of an immunoassay for ciprofloxacin based on phagedisplayed antibody fragments. Journal of Immunological Methods, 358, 17–22. Gong Y F, Zhang M Z, Wang M Z, Chen Z L, Xi X. 2014. Development of immuno-based methods for detection of melamine. Arabian Journal for Science and Engineering, 39, 5315–5324. Goryacheva I Y, Beloglazova N V, Eremin S A, Mikhirev D A, Niessner R, Knopp D. 2008. Gel-based immunoassay for non-instrumental detection of pyrene in water samples. Talanta, 75, 517–522. Goryacheva I Y, Lenain P, De Saeger S. 2013. Nanosized labels for rapid immunotests. TrAC Trends in Analytical Chemistry, 46, 30–43. Granier B, Lepage S. 2011. Kit For Use in Detection of Comprising Nuclear Receptor for Use in the Detection and Measurement of Tetracyclines and Virginiamycins. Google Patents. US 7935806 B2. Griffiths M. 2010. Improving the Safety and Quality of Milk: Improving Quality in Milk Products. Woodhead Publishing Limited, Cambridge. Guan Y G, Wu X L, Meng H C. 2013. Indirect competitive ELISA based on monoclonal antibody for the detection of 5-hydroxymethyl-2-furfural in milk, compared with HPLC. Journal of Dairy Science, 96, 4885–4890. Guirgis B S, E Cunha C S, Gomes I, Cavadas M, Silva I, Doria G, Blatch G L, Baptista P V, Pereira E, Azzazy H M. 2012. Gold nanoparticle-based fluorescence immunoassay for malaria antigen detection. Analytical and Bioanalytical Chemistry, 402, 1019–1027. Guo H, Sun S. 2012. Lanthanide-doped upconverting phosphors for bioassay and therapy. Nanoscale, 4, 6692–6706. Guo J N, Liu L Q, Xue F, Xing C R, Song S S, Kuang H, Xu C L. 2015. Development of a monoclonal antibody-based immunochromatographic strip for cephalexin. Food and Agricultural Immunology, 26, 282–292. De Guzmán-Vázquez Prada A, Reviejo A J, Pingarrón J M. 2006. A method for the quantification of low concentration sulfamethazine residues in milk based on molecularly

2292

XU Fei et al. Journal of Integrative Agriculture 2015, 14(11): 2282–2295

imprinted clean-up and surface preconcentration at a Nafion-modified glassy carbon electrode. Journal of Pharmaceutical and Biomedical Analysis, 40, 281–286. Hayat M A. 2012. Colloidal Gold: Principles, Methods, and Applications. Elsevier, U.S. Hernandes T, Dores E, Ribeiro M L, Rossignoli P A, Malm O. 2014. Simple method to determine residual cypermethrin and deltamethrin in bovine milk. Journal of the Brazilian Chemical Society, 25, 1656–1661. Huang Y, Liu D, Lai W, Xiong Y, Yang W, Liu K, Wang S. 2014. Rapid detection of aflatoxin M1 by immunochromatography combined with enrichment based on immunomagnetic nanobead. Chinese Journal of Analytical Chemistry, 42, 654–659. IDEXX. 2015. IDEXX dairy tests and accessories. [2015-03-13]. https://www.idexx.com/dairy/accessories.htm International Dairy Federation. 2014. The World Dairy Situation 2014. Bulletin of the World Dairy Federation. Jiang W X, Beloglazova N V, Wang Z H, Jiang H Y, Wen K, de Saeger S, Luo P J, Wu Y N, Shen J Z. 2015. Development of a multiplex flow-through immunoaffinity chromatography test for the on-site screening of 14 sulfonamide and 13 quinolone residues in milk. Biosensors & Bioelectronics, 66, 124–128. Jiang W X, Wang Z H, Nolke G, Zhang J, Niu L L, Shen J Z. 2013. Simultaneous determination of aflatoxin B-1 and aflatoxin M-1 in food matrices by enzyme-linked immunosorbent assay. Food Analytical Methods, 6, 767–774. Jin Y, Jang J W, Lee M H, Han C H. 2006. Development of ELISA and immunochromatographic assay for the detection of neomycin. Clinica Chimica Acta, 364, 260–266. Karami-Osboo R, Shojaee M H, Miri R, Kobarfard F, Javidnia K. 2014. Simultaneous determination of six fluoroquinolones in milk by validated QuEChERS-DLLME HPLC-FLD. Analytical Methods, 6, 5632–5638. Kloth K, Rye-Johnsen M, Didier A, Dietrich R, Märtlbauer E, Niessner R, Seidel M. 2009. A regenerable immunochip for the rapid determination of 13 different antibiotics in raw milk. Analyst, 134, 1433–1439. Köhler G, Milstein C. 1975. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature, 256, 495–497. Korpimäki T, Brockmann E, Kuronen O, Saraste M, Lamminmäki U, Tuomola M. 2004. Engineering of a broad specificity antibody for simultaneous detection of 13 sulfonamides at the maximum residue level. Journal of Agricultural and Food Chemistry, 52, 40–47. Lan H, Gan N, Pan D, Hu F, Li T, Long N, Qiao L. 2014. An automated solid-phase microextraction method based on magnetic molecularly imprinted polymer as fiber coating for detection of trace estrogens in milk powder. Journal of Chromatographya, 1331, 10–18. Leivo J, Lamminmäki U, Lövgren T, Vehniäinen M. 2013. Multiresidue detection of fluoroquinolones: Specificity engineering of a recombinant antibody with oligonucleotide-

directed mutagenesis. Journal of Agricultural and Food Chemistry, 61, 11981–11985. Li Y S, Zhang Y R, Cao X Y, Wang Z H, Shen J Z, Zhang S X. 2012. Development of a chemiluminescent competitive indirect ELISA method procedure for the determination of gentamicin in milk. Analytical Methods, 4, 2151–2155. Liang X, Ni H J, Beier R C, Dong Y N, Li J Y, Luo X S, Zhang S X, Shen J Z, Wang Z H. 2014. Highly broad-specific and sensitive enzyme-linked immunosorbent assay for screening sulfonamides: Assay optimization and application to milk samples. Food Analytical Methods, 7, 1992–2002. Liang X, Wang Z, Wang C, Wen K, Mi T, Zhang J, Zhang S. 2013. A proof-of-concept receptor-based assay for sulfonamides. Analytical Biochemistry, 438, 110–116. de Lima G C, Lago A C D, Chaves A A, Fadini P S, Luccas P O. 2013. Determination of selenium using atomically imprinted polymer (AIP) and hydride generation atomic absorption spectrometry. Analytica Chimica Acta, 768, 35–40. Liotta L A, Christiansen B C, Day A R, Harlacher T, Paweletz K. 1999. Light-Emitting Immunoassay. Google Patents. US5942407. Liu D, Huang Y, Wang S, Liu K, Chen M, Xiong Y, Yang W, Lai W. 2015. A modified lateral flow immunoassay for the detection of trace aflatoxin M1 based on immunomagnetic nanobeads with different antibody concentrations. Food Control, 51, 218–224. Liu L Q, Luo L J, Suryoprabowo S, Peng J, Kuang H, Xu C L. 2014. Development of an immunochromatographic strip test for rapid detection of ciprofloxacin in milk samples. Sensors, 14, 16785–16798. Liu N, Song S Q, Lu L, Nie D X, Han Z, Yang X L, Zhao Z H, Wu A B, Zheng X D. 2014. A rabbit monoclonal antibody-based sensitive competitive indirect enzyme-linked immunoassay for rapid detection of chloramphenicol residue. Food and Agricultural Immunology, 25, 523–534. Liu T S, Xie J, Zhao J F, Song G X, Hu Y M. 2014. Magnetic chitosan nanocomposite used as cleanup material to detect chloramphenicol in milk by GC-MS. Food Analytical Methods, 7, 814–819. Luo Y, Xia Y. 2012. Selection of single-chain variable fragment antibodies against fenitrothion by ribosome display. Analytical Biochemistry, 421, 130–137. Lv Y, Wang L, Yang L, Zhao C, Sun H. 2012. Synthesis and application of molecularly imprinted poly(methacrylic acid)silica hybrid composite material for selective solid-phase extraction and high-performance liquid chromatography determination of oxytetracycline residues in milk. Journal of Chromatographya, 1227, 48–53. Mansfield M A. 2009. Nitrocellulose membranes for lateral flow immunoassays: A technical treatise. In: Lateral Flow Immunoassayp. Humana Press, Springer, U.S. pp. 1–19. Medibena. 2014. MaxSignal® Aflatoxin B1 ELISA test kit. [201503-15]. http://www.biooscientific.com/Mycotoxin-test-kits/ MaxSignal-Aflatoxin-B1-ELISA-Test-Kit Meneghini L Z, Rubensam G, Bica V C, Ceccon A, Barreto F,

XU Fei et al. Journal of Integrative Agriculture 2015, 14(11): 2282–2295

Ferrao M F, Bergold A M. 2014. Multivariate optimization for extraction of pyrethroids in milk and validation for GCECD and CG-MS/MS analysis. International Journal of Environmental Research and Public Health, 11, 11421– 11437. Mi T J, Liang X, Ding L, Zhang S X, Eremin S A, Beier R C, Shen J Z, Wang Z H. 2014. Development and optimization of a fluorescence polarization immunoassay for orbifloxacin in milk. Analytical Methods, 6, 3849–3857. MOA (Ministry of Agriculture of the People’s Republic China). 2002. Annoucement of No. 235. Maximum residue limit of veterinary drugs in animal origin food. [2015-03-15]. http:// www.moa.gov.cn/zwllm/tzgg/gg/200302/t20030226_59300. htm (in Chinese) Moghaddam A, Løbersli I, Gebhardt K, Braunagel M, Marvik O J. 2001. Selection and characterisation of recombinant singlechain antibodies to the hapten Aflatoxin-B1 from naive recombinant antibody libraries. Journal of Immunological Methods, 254, 169–181. Mohamed R, Richoz-Payot J, Gremaud E, Mottier P, Yilmaz E, Tabet J, Guy P A. 2007. Advantages of molecularly imprinted polymers LC-ESI-MS/MS for the selective extraction and quantification of chloramphenicol in milk-based matrixes. Comparison with a classical sample preparation. Analytical Chemistry, 79, 9557–9565. Ni H, Zhang S, Ding X, Mi T, Wang Z, Liu M. 2014. Determination of enrofloxacin in bovine milk by a novel single-stranded DNA aptamer chemiluminescent enzyme immunoassay. Analytical Letters, 47, 2844–2856. Pilehvar S, Dierckx T, Blust R, Breugelmans T, De Wael K. 2014. An electrochemical impedimetric aptasensing platform for sensitive and selective detection of small molecules such as chloramphenicol. Sensors, 14, 12059–12069. Porter J, O´ Loan N, Bell B, Mahoney J, Mcgarrity M, Mcconnell R I, Fitzgerald S P. 2012. Development of an Evidence biochip array kit for the multiplex screening of more than 20 anthelmintic drugs. Analytical and Bioanalytical Chemistry, 403, 3051–3056. Qi Y, Wu C, Zhang S, Wang Z, Huang S, Dai L, Wang S, Xia L, Wen K, Cao X. 2009. Selection of anti-sulfadimidine specific ScFvs from a hybridoma cell by eukaryotic ribosome display. PLoS ONE, 4, e6427. Raz R S, Bremer M G, Haasnoot W, Norde W. 2009. Label-free and multiplex detection of antibiotic residues in milk using imaging surface plasmon resonance-based immunosensor. Analytical Chemistry, 81, 7743–7749. Rusanova T Y, Beloglazova N V, Goryacheva I Y, Lobeau M, Van Peteghem C, De Saeger S. 2009. Non-instrumental immunochemical tests for rapid ochratoxin A detection in red wine. Analytica Chimica Acta, 653, 97–102. Sadeghi N, Oveisi M R, Jannat B, Hajimahmoodi M, Behfar A, Behzad M, Norouzi N, Oveisi M, Jannat B. 2014. Simultaneous measurement of zinc, copper, lead and cadmium in baby weaning food and powder milk by DPASV. Iranian Journal of Pharmaceutical Research, 13, 345–349.

2293

Sefah K, Shangguan D, Xiong X, O’Donoghue M B, Tan W. 2010. Development of DNA aptamers using Cell-SELEX, 5, 1169–1185. Seidel M, Niessner R. 2014. Chemiluminescence microarrays in analytical chemistry: A critical review. Analytical and Bioanalytical Chemistry, 406, 5589–5612. Shen J, Xu F, Jiang H, Wang Z, Tong J, Guo P, Ding S. 2007. Characterization and application of quantum dot nanocrystalmonoclonal antibody conjugates for the determination of sulfamethazine in milk by fluoroimmunoassay. Analytical and Bioanalytical Chemistry, 389, 2243–2250. Sheng W, Li Y Z, Xu X, Yuan M, Wang S. 2011. Enzymelinked immunosorbent assay and colloidal gold-based immunochromatographic assay for several (fluoro) quinolones in milk. Microchimica Acta, 173, 307–316. Sniegocki T, Posyniak A, Gbylik-Sikorska M, Zmudzki J. 2014. Determination of chloramphenicol in milk using a QuEChERS-based on liquid chromatography tandem mass spectrometry method. Analytical Letters, 47, 568–578. Song X, Xu S, Chen L, Wei Y, Xiong H. 2014. Recent advances in molecularly imprinted polymers in food analysis. Journal of Applied Polymer Science, 131, 1–18. Speranskaya E S, Beloglazova N V, Lenain P, De Saeger S, Wang Z, Zhang S, Hens Z, Knopp D, Niessner R, Potapkin D V, Goryacheva I Y. 2014. Polymer-coated fluorescent CdSe-based quantum dots for application in immunoassay. Biosensors and Bioelectronics, 53, 225–231. Sui J, Lin H, Cao L, Li Z. 2009. Dot-immunogold filtration assay for rapid screening of three fluoroquinolones. Food and Agricultural Immunology, 20, 125–137. Suryoprabowo S, Liu L Q, Peng J, Kuang H, Xu C L. 2014. Development of a broad specific monoclonal antibody for fluoroquinolone analysis. Food Analytical Methods, 7, 2163–2168. Tao X Q, Jiang H Y, Yu X Z, Zhu J H, Wang X, Wang Z H, Niu L L, Wu X P, Shen J Z. 2013. An ultrasensitive chemiluminescence immunoassay of chloramphenicol based on gold nanoparticles and magnetic beads. Drug Testing and Analysis, 5, 346–352. Taranova N A, Berlina A N, Zherdev A V, Dzantiev B B. 2015. ‘Traffic light’ immunochromatographic test based on multicolor quantum dots for the simultaneous detection of several antibiotics in milk. Biosensors and Bioelectronics, 63, 255–261. Tien Y J, Chan C, Chan K, Wang Y C, Lin J, Chang C, Chen C. 2013. Development of a novel bead-based 96-well filtration plate competitive immunoassay for the detection of gentamycin. Biosensors and Bioelectronics, 49, 126–132. Tillib S V. 2011. “Camel nanoantibody” is an efficient tool for research, diagnostics and therapy. Molecular Biology, 45, 66–73. (in Russia) Tillib S V, Ivanova T I, Vasilev L A. 2010. Fingerprint-like analysis of “Nanoantibody” selection by phage display using two helper phage variants. Acta Naturae, 2, 85–93. Toh S Y, Citartan M, Gopinath S C, Tang T. 2015. Aptamers

2294

XU Fei et al. Journal of Integrative Agriculture 2015, 14(11): 2282–2295

as a replacement for antibodies in enzyme-linked immunosorbent assay. Biosensors and Bioelectronics, 64, 392–403. Wang C, Wang Z, Jiang W, Mi T, Shen J. 2012. A monoclonal antibody-based ELISA for multiresidue determination of avermectins in milk. Molecules, 17, 7401–7414. Wang J, Liu B, Hsu Y, Yu F. 2011. Sensitive competitive direct enzyme-linked immunosorbent assay and gold nanoparticle immunochromatographic strip for detecting aflatoxin M1 in milk. Food Control, 22, 964–969. Wang Q, Haughey S A, Sun Y, Eremin S A, Li Z, Liu H, Xu Z, Shen Y, Lei H. 2011. Development of a fluorescence polarization immunoassay for the detection of melamine in milk and milk powder. Analytical and Bioanalytical Chemistry, 399, 2275–2284. Wang Q, Nchimi Nono K, Syrjänpää M, Charbonnière L J, Hovinen J, Härmä H. 2013. Stable and highly fluorescent europium (iii) chelates for time-resolved immunoassays. Inorganic Chemistry, 52, 8461–8466. Wang Y, Wang D, Zou M, Jin Y, Yun C, Gao X. 2011. Application of suspension array for simultaneous detection of antibiotic residues in raw milk. Analytical Letters, 44, 2711–2720. Wang Y Z, Yang H, Pschenitza M, Niessner R, Li Y, Knopp D, Deng A P. 2012. Highly sensitive and specific determination of mercury (II) ion in water, food and cosmetic samples with an ELISA based on a novel monoclonal antibody. Analytical and Bioanalytical Chemistry, 403, 2519–2528. Wang Z H, Sheng Y J, Duan H X, Yu Q, Shi W M, Zhang S X. 2014. New haptens synthesis, antibody production and comparative molecular field analysis for tetracyclines. RSC Advances, 4, 53788–53794. Watanabe H, Satake A, Kido Y, Tsuji A. 2002. Monoclonalbased enzyme-linked immunosorbent assay and immunochromatographic rapid assay for dihydrostreptomycin in milk. Analytica Chimica Acta, 472, 45–53. Wild D. 2013. The Immunoassay Handbook: Theory and Applications of Ligand Binding, ELISA and Related Techniques. Sockton Press, New York. Wu J, Xu F, Zhu K, Wang Z H, Wang Y H, Zhao K X, Li X W, Jiang H Y, Ding S Y. 2013. Rapid and sensitive fluoroimmunoassay based on quantum dots for detection of melamine in milk. Analytical Letters, 46, 275–285. Wu J X, Zhang S E, Zhou X P. 2010. Monoclonal antibody-based ELISA and colloidal gold-based immunochromatographic assay for streptomycin residue detection in milk and swine urine. Journal of Zhejiang University (Science B), 11, 52–60. Xie Q, Wu Y, Xiong Q, Xu H, Xiong Y, Liu K, Jin Y, Lai W. 2014. Advantages of fluorescent microspheres compared with colloidal gold as a label in immunochromatographic lateral flow assays. Biosensors and Bioelectronics, 54, 262–265. Xu F, Jiang W X, Zhou J, Wen K, Wang Z H, Jiang H Y, Ding S Y. 2014. Production of monoclonal antibody and development of a new immunoassay for apramycin in food. Journal of Agricultural and Food Chemistry, 62, 3108–3113. Xu X, Draney D R, Cheung L. 2013. Cyanine Dyes and Their Conjugates. Google Patents. WO2012054749A1.

Yang W, Li X, Liu G, Zhang B, Zhang Y, Kong T, Tang J, Li D, Wang Z. 2011. A colloidal gold probe-based silver enhancement immunochromatographic assay for the rapid detection of abrin-a. Biosensors and Bioelectronics, 26, 3710–3713. Ye B, Li S, Zuo L, Li X. 2008. Simultaneous detection of sulfamethazine, streptomycin, and tylosin in milk by microplate-array based SMM-FIA. Food Chemistry, 106, 797–803. Young M S, Tran K V, Goh E, Shia J C. 2014. A rapid spebased analytical method for UPLC/MS/MS determination of aminoglycoside antibiotic residues in bovine milk, muscle, and kidney. Journal of AOAC International, 97, 1737–1741. Yuan M, Sheng W, Zhang Y, Wang J, Yang Y, Zhang S, Goryacheva I Y, Wang S. 2012. A gel-based visual immunoassay for non-instrumental detection of chloramphenicol in food samples. Analytica Chimica Acta, 751, 128–134. Zeng K, Zhang J, Wang Y, Wang Z H, Zhang S X, Wu C M, Shen J Z. 2013. Development of a rapid multi-residue assay for detecting β-lactams using penicillin binding protein 2x*. Biomedical and Environmental Sciences, 26, 100–109. Zhang D H, Li P W, Zhang Q, Yang Y, Zhang W, Guan D, Ding X X. 2012. Extract-free immunochromatographic assay for on-site tests of aflatoxin M-1 in milk. Analytical Methods, 4, 3307–3313. Zhang G P, Wang F Y, Song C M, Zhi A M, Hu X F, Zhao D. 2013. Preparation of Fluorescence Strip Based on Silica for Quantity Analysis of Gentamicin. CN103439489A. (in Chinese) Zhang J, Wang Z H, Mi T J, Wenren L Q, Wen K. 2014. A homogeneous fluorescence polarization immunoassay for the determination of cephalexin and cefadroxil in milk. Food Analytical Methods, 7, 879–886. Zhang J, Zhang B, Wu Y, Jia S, Fan T, Zhang Z, Zhang C. 2010. Fast determination of the tetracyclines in milk samples by the aptamer biosensor. Analyst, 135, 2706–2710. Zhang J K, Qi Y H, Liu J X, Wang J P. 2013. Heterologous immunoassay for screening macrolide antibiotics residues in milk based on the monoclonal antibody of tylosin. Food and Agricultural Immunology, 24, 419–431. Zhao L, Sun L, Chu X. 2009. Chemiluminescence immunoassay. Trends in Analytical Chemistry, 28, 404–415. Zheng G C, Han C, Liu Y, Wang J, Zhu M W, Wang C J, Shen Y. 2014. Multiresidue analysis of 30 organochlorine pesticides in milk and milk powder by gel permeation chromatographysolid phase extraction-gas chromatography-tandem mass spectrometry. Journal of Dairy Science, 97, 6016–6026. Zheng M, Gong R, Zhao X, Feng Y. 2010. Selective sample pretreatment by molecularly imprinted polymer monolith for the analysis of fluoroquinolones from milk samples. Journal of Chromatographya, 1217, 2075–2081. Zhou J, Zhu K, Xu F, Wang W J, Jiang H Y, Wang Z H, Ding S Y. 2014. Development of a microsphere-based fluorescence immunochromatographic assay for monitoring lincomycin in milk, honey, beef, and swine urine. Journal of Agricultural

XU Fei et al. Journal of Integrative Agriculture 2015, 14(11): 2282–2295

and Food Chemistry, 62, 12061–12066. Zhou Y, Tian X L, Li Y S, Pan F G, Zhang Y Y, Zhang J H, Wang X R, Ren H L, Lu S Y, Li Z H, Liu Z S, Chen Q J, Liu J Q. 2012. Development of a monoclonal antibodybased sandwich-type enzyme-linked immunosorbent assay (ELISA) for detection of abrin in food samples. Food Chemistry, 135, 2661–2665. Zhu K, Li J, Wang Z, Jiang H, Beier R C, Xu F, Shen J, Ding

2295

S. 2011. Simultaneous detection of multiple chemical residues in milk using broad-specificity antibodies in a hybrid immunosorbent assay. Biosensors and Bioelectronics, 26, 2716–2719. Zhu L, Cui X, Wu J, Wang Z, Wang P, Hou Y. 2014. Fluorescence immunoassay based on carbon dots as labels for the detection of human immunoglobulin G. Analytical Methods, 6, 4430-4436. (Managing editor WENG Ling-yun)