Anal Bioanal Chem (2011) 399:2275–2284 DOI 10.1007/s00216-010-4599-2
ORIGINAL PAPER
Development of a fluorescence polarization immunoassay for the detection of melamine in milk and milk powder Qiang Wang & Simon A. Haughey & Yuan-Ming Sun & Sergei A. Eremin & Zhen-Feng Li & Hui Liu & Zhen-Lin Xu & Yu-Dong Shen & Hong-Tao Lei
Received: 17 October 2010 / Revised: 6 December 2010 / Accepted: 12 December 2010 / Published online: 10 January 2011 # Springer-Verlag 2011
Abstract A fluorescence polarization immunoassay (FPIA) based on a polyclonal antibody was developed for the determination of melamine in milk. To obtain an antibody with improved sensitivity and specificity, 6-hydrazinyl1,3,5-triazine-2,4-diamine was coupled to bovine serum albumin and used as the immunogen for the rabbit immunization. Three fluorescein-labeled melamine tracers with different structures and spacer bridges were synthesized. The structural effect of the tracers on the assay characteristics was investigated. 6-(4,6-Diamino-1,3,5triazin-2-ylamino)-N-(2-(3-(3′,6′-dihydroxy-3-oxo-2,3dihydrospiro[indene-1,9′-xanthene]-5-yl)thioureido)ethyl) hexanamide demonstrated better sensitivity than 5-(2-(4, 6-diamino-1,3,5-triazin-2-yl)hydrazinecarbothioamido)-2(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid and 3(4,6-diamino-1,3,5-triazin-2-ylthio)-N-(2-(3-(3′,6′-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthene]-5-yl) thioureido)ethyl)propanamide. The limit of detection (10% inhibition) of the FPIA was 9.3 ng mL-1 and the IC50 (50% inhibition) value was 164.7 ng mL-1. The antibody in the FPIA showed 21.2% cross-reactivity to the Q. Wang : Y.-M. Sun : Z.-F. Li : H. Liu : Z.-L. Xu : Y.-D. Shen : H.-T. Lei (*) The Institute of Food Quality and Safety, South China Agricultural University, Guangzhou 510642, China e-mail:
[email protected] S. A. Haughey Institute of Agri-Food and Land Use, Queen’s University Belfast, Belfast BT9 5AG, UK S. A. Eremin Department of Chemical Enzymology, Faculty of Chemistry, M.V. Lomonosov Moscow State University, Moscow 119998, Russia
fly-killing insecticide cyromazine, but had no crossreactivity to other natural structurally related compounds. Recoveries, measured in spiked milk and milk powder samples, ranged from 79.4 to 119.0%. Milk samples fortified with melamine were analyzed by this method and confirmed by high-performance liquid chromatography– mass spectrometry. Excellent recoveries and correlation with spiked levels were observed, suggesting that this immunoassay could be applied to the screening of melamine residues in milk and milk powder after a simple dilution procedure. Keywords Melamine . Fluorescence polarization immunoassay . Polyclonal antibody . Milk Abbreviations BSA Bovine serum albumin CAAT 2-chloro-4,6-diamino-1,3,5-triazine CAC Codex Alimentarius Commission CR Cross-reactivity DCC N,N′-dicyclohexylcarbodiimide DMF N,N-dimethylformamide EDA Ethylenediamine EDC 1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide hydrochloride EDF 1-(2-aminoethyl)-3-(3′,6′-dihydroxy-3-oxo-3Hspiro[isobenzofuran-1,9′-xanthene]-5-yl)thiourea ELISA Enzyme-linked immunosorbent assay FITC Fluorescein isothiocyanate isomer I FPIA Fluorescence polarization immunoassay HPLC High-performance liquid chromatography LOD Limit of detection MS Mass spectrometry NHS N-hydroxysuccinimide TLC Thin-layer chromatography
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Introduction Melamine, with an IUPAC name of 1,3,5-triazine-2,4,6triamine, is an organic base. In its chainlike “polymerized” form, melamine is an industrial chemical and has been used for decades in the manufacturing of dishes, plastic resins, flame-retardant fibers, components of paper and paperboard, and industrial coatings [1, 2]. Melamine is not approved for addition to foods or feeds, nor is it permitted for use as a fertilizer anywhere in the world. Ingestion of melamine may lead to reproductive damage and bladder or kidney stones, which in turn may lead to bladder carcinogenesis [3]. The maximum amount of melamine allowed is 1 mg kg-1 in powdered infant formula and 2.5 mg kg-1 in other foods and animal feed, according to the rulings of the Codex Alimentarius Commission (CAC) and the European Commission [4, 5]. Melamine is usually analyzed by physicochemical methods based on gas or liquid chromatography. Highperformance liquid chromatography (HPLC) has been proposed for quantitative determination of melamine; the limit of detection (LOD) was 0.1 mg kg-1 [6]. Ion-pair liquid chromatography coupled with electrospray tandem mass spectrometry (MS) was used to determine residues of melamine in chard samples using a C18 reverse-phase column; the LOD was 10 μg kg-1 [7]. Gas chromatography–MS was also used to detect melamine; the LOD proposed was 10 μg kg-1 [8]. The instrumental methods meet the requirement of the CAC and are accurate and reliable but expensive, laborious, and time-consuming as well as requiring professional personnel. To date, immunoassay technology is increasingly used for screening food contaminants owing to its sensitivity, selectivity, time efficiency, and cost-effectiveness. Over the past 20 years the development of immunochemical methods and their potential applications, especially the enzyme-linked immunosorbent assay (ELISA), have increased significantly. In general, ELISA has many advantages over the other techniques and allows direct analysis of a large number of samples; however, ELISA is multistep procedure and is time-consuming. Simplifying the assay and minimizing the analysis time are the primary goals in developing screening methods for high-throughput sample analysis. The shift from the use of heterogeneous methods (with separation) to homogeneous methods (without separation) for assay simplification for routine applications has shown great potential [9]. Fluorescence polarization immunoassay (FPIA) is one of the most extensively used homogeneous techniques, and meets the requirements of a simple, reliable, fast, and costeffective analysis. FPIA is a competitive immunoassay method based on the increase in the polarization of the fluorescence of a small fluorescein-labeled hapten (tracer)
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when it is bound by a specific antibody. Recently, the use of FPIA for the determination of pesticides [10, 11], biological toxins [12, 13], mycotoxins [14, 15], and veterinary drugs [16, 17] in agricultural products and environmental samples has been reported. In a previous study, an ELISA method for the detection of melamine was developed [18]. Yin et al. [19] synthesized a hapten, 4,6-diamino-1,6-dihydro-1,3,5-triazin-2-ylaminoacetic acid, and developed an ELISA for melamine. A competitive 1,1′-oxalyldiimidazole chemiluminescent enzyme immunoassay based on commercial immunoreagents for the screening of melamine in milk was also developed [20]. Several commercial ELISA kits for melamine have become available, such as the Abraxis melamine plate kit (Abraxis, Warminster, PA, USA), the Romer AgraQuant melamine sensitive test kit (Romer Labs, Union, MO, USA), and the MaxSignal melamine enzymatic assay kit (Bioo Scientific, Austin, TX, USA). The LODs for melamine in liquid infant formula and wheat products were usually less than 1 mg L-1 and less than 2.5 mg kg-1, respectively [2]. Immunoassays for the detection of melamine have also been reported by other researchers [21–23]. However, no FPIA method for the detection of melamine has been published thus far. In the present study, the first development of an FPIA for the detection of melamine is described. The assay is based on an improved polyclonal antibody produced by a new melamine hapten (6-hydrazinyl-1,3,5-triazine-2,4-diamine, hapten A) that has not previously been reported in the literature and its application for detection of melamine in artificially spiked milk and milk powder samples.
Materials and methods Reagents and instrumentation General reagents and organic solvents were of analytical grade unless otherwise specified. Melamine, bovine serum albumin (BSA), 1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), N,N′-dicyclohexylcarbodiimide (DCC), N,N-dimethylformamide (DMF), fluorescein isothiocyanate isomer I (FITC), and complete and incomplete Freund’s adjuvants were purchased from Sigma (St. Louis, MO, USA). Ethylenediamine (EDA) was obtained from Jingke Company (Guangzhou, China). Silica gel G glass sheets (type GF254, layer thickness 0.2 mm) for thin-layer chromatography (TLC) were obtained from Taizhou Shenghua Material Corporation (Zhejiang, China). Microplates (96 wells) were obtained from Jinchanhua Corporation (Shenzhen, China). 2-Chloro-4,6-diamino-1,3,5-triazine (CAAT) and 6hydrazinyl-1,3,5-triazine-2,4-diamine (hapten A) were obtained from Shanghai Chaoyan Biotechnology Company
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(Shanghai, China). Cyanuric chloride and cyanuric acid were obtained from Accela Chembio Company (Shanghai, China). Atrazine was a gift from Shandong Zhongke Qiaochang Chemical Company (Shandong, China). Cyromazine was bought from Changzhou Zhineng Animal Pharmaceutical Company (Changzhou, China). 3-(4,6-Diamino-1, 6-dihydro-1,3,5-triazin-2-ylthio)propanoic acid (hapten B) and 6-(4,6-diamino-1,6-dihydro-1,3,5-triazin-2-ylamino) hexanoic acid (hapten C) were prepared previously [18]. Fluorescence polarization values were recorded using a Wallac 1420 VICTOR3 multilabel counter (PerkinElmer, USA). Ultraviolet spectra were recorded with a UV-3010 spectrophotometer (Hitachi, Japan). Synthesis of fluorescein-labeled tracers Tracer A Hapten A (5 mg, 45 μmol) was dissolved in 0.5 mL of absolute DMF. Triethylamine (50 μL) and FITC (5 mg, 12.8 μmol) were added and the solution was stirred overnight at room temperature. A sample of reaction mixture (50 μL) was isolated by TLC using chloroform and methanol (4:0.4, v/v) as the eluent. The main yellow band at Rf =0.5 was scraped and extracted with 1 mL of methanol, thus giving 5-(2-(4,6-diamino-1,3,5-triazin-2-yl)hydrazinecarbothioamido)-2-(6-hydroxy-3-oxo-3H-xanthen9-yl)benzoic acid (tracer A) (Fig. 1). The tracer concentration was estimated spectrophotometrically at 492 nm, assuming the absorbance in sodium borate buffer (50 mM, pH 8.0) to be the same as for fluorescein (ε=8.78×104 M-1 cm-1) [11]. Tracers B and C FITC (11.7 mg, 0.03 mmol) was dissolved in methanol (1 mL), containing triethylamine (10 μL). EDA (20 mg, 0.3 mmol) was added and the mixture was stirred for 1 h at room temperature. After concentration, the reaction mixture was subjected to silica gel column chromatography using ethyl acetate and methanol (3:1, v/v) as an eluent. The yellow band of the column was collected until no fluorescein was eluted. Following this, the column was treated with anhydrous methanol, and the eluent was collected and concentrated to dryness to obtain the linker 1-(2-aminoethyl)-3-(3′,6′-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthene]-5-yl)-thiourea (EDF). Tracers B and C were synthesized using the active ester method [10, 11]. Briefly, hapten B (8.6 mg, 40 μmol) or hapten C (10 mg, 40 μmol) was mixed and stirred with DCC (16.0 mg, 80 μmol) and NHS (9.7 mg, 80 μmol) in absolute DMF (0.4 mL) and the reaction was allowed to proceed overnight. The precipitate was removed by centrifugation, and EDF (4.5 mg, 10 μmol) was added to
Fig. 1 Structures of melamine haptens, fluorescein-labeled tracers, and immunogens. BSA bovine serum albumin, FITC fluorescein isothiocyanate isomer I
the supernatant. After the color of the solution had changed to yellow, a small portion of the reaction mixture (approximately 50 μL) was subjected to TLC using chloroform and methanol (4:1, v/v) as the eluent for 3(4,6-diamino-1,3,5-triazin-2-ylthio)-N-(2-(3-(3′,6′-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthene]-5-yl) thioureido)ethyl)propanamide (tracer B) and chloroform and methanol (5:1, v/v) as the eluent for 6-(4,6-diamino1,3,5-triazin-2-ylamino)-N-(2-(3-(3′,6′-dihydroxy-3-oxo2,3-dihydrospiro[indene-1,9′-xanthene]-5-yl)thioureido)ethyl)hexanamide (tracer C) (Fig. 1). The main yellow band at Rf =0.8 for tracer B and at Rf = 0.9 for tracer C was isolated and the concentration was estimated spectrophotometrically at 492 nm as described above. Antibody production Hapten A was coupled to BSA to produce an immunogen for the immunization. Briefly, 3 mg (20 μmol) hapten A was dissolved in 100 μL of absolute DMF. Then, hapten A solution was added dropwise with stirring to BSA (10 mg) in 1.9 mL of 0.9% (m/v) NaCl, followed by the addition of 5 mg (25 μmol) EDC, mixed thoroughly and kept at room temperature overnight. The conjugates were purified by dialysis against phosphate-buffered saline (0.05 M, pH 7.4) for 3 days with three changes per
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day. The dialyzed product was spun at 5,000 rpm for 15 min, and the supernatant was collected as immunogen A and stored at -20 °C until use. Two New Zealand rabbits weighing 1.5–2.0 kg were immunized five times using immunogen A at intervals of 14 days by the Guangdong Medical Laboratory Animal Center. Bleeds were taken from the rabbits on the eighth day after immunization, starting 40 days after the first injection, and tested for the presence of melamine antibodies using an indirect ELISA [18]. The serum obtained was divided into aliquots (1 mL), labeled as antibody A and stored at –20 °C until use. Antibody B was obtained from immunogen B, which was prepared by coupling hapten C to carrier protein BSA using the active ester method [18]. Fluorescence polarization immunoassay Sodium borate buffer (0.05 M, pH 8.0) with 0.01% sodium azide was used for all FPIA experiments. Standard solutions of melamine and cross-reactants were prepared by dilution of stock solutions of these compounds. Antibody dilution curves were constructed by mixing the fluorescein-labeled tracer (100 μL, 1 nM solution in sodium borate buffer) with the serially diluted antibody (100 μL). THe fluorescence polarization signal (mP) was measured using a Wallac Victor3. To achieve the optimal incubation time, a competitive kinetic curve was plotted. Ninety microliters of the tracer solution (0.8 nM) in sodium borate buffer and 20 μL of standard solution (0, 50, 100, and 500 ng mL -1 , respectively) were mixed, then 90 μL of working antibody was added, and the mP value was recorded successively versus time beginning at 1 min to obtain the kinetic curves To construct the competitive calibration curves, the melamine standard (20 μL) at concentrations of 0.001–2,000 μg mL-1 was mixed with the labeled tracer (90 μL) and diluted antibody (90 μL). The mP was measured after an appropriate incubation time, based on the kinetics of the curve. The mP values were plotted against the analyte concentration, and a h i four-parameter equation, y ¼ ðADÞ= 1 þ ðx=CÞB þ D, was used to fit the experimental sigmoidal curve in Origin 7.5 for Windows. A and D are the maximal and minimal mP, B corresponds to the slope of the sigmoidal curve, and C is the melamine concentration at 50% of tracer binding (IC50). The LOD was defined as the standard concentration that inhibited 10% of tracer binding, and the detectable concentration range was defined as the standard concentration that inhibited 20– 80% of tracer binding [24]. Cross-reactivity (CR) was calculated according to the following equation: CR% ¼ ½IC50 ðmelamineÞ=IC50 ðstructurally related compoundsÞ 100%.
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Analysis of spiked samples To evaluate the recovery of the FPIA developed, melaminefree milk and milk powder were spiked with melamine and recoveries were determined by FPIA. For milk samples (1 mL), 0.5, 1, 5, and 50 μg of melamine standard were added, respectively. Each milk solution was centrifuged for 10 min (12,000 rpm, 4 °C) to remove the fat in milk. The lower liquid nonfat phase was diluted 1:30 in sodium borate buffer before being used in FPIA. For milk powder samples (1 g), 1, 2, 10, and 50 μg of melamine standard were added, respectively. Methanol–H2O (1:1, v/v, 5 mL) solution was added to each sample. The mixture was vortexed for 10 min, and centrifuged for 10 min (10,000 rpm, 4 °C). The middle clear liquid nonfat phase was diluted 1:12 in sodium borate buffer before being used in FPIA. Blank samples were prepared as described above but were not spiked with melamine. The melamine concentration in spiked samples was calculated after fitting of the standard curve using the four-parameter logistic model. To validate the FPIA, correlation studies between methods were performed on 0.5, 1.0, 5.0, 10.0, and 50.0 mg L-1 melamine-spiked milk samples. Each sample was divided two portions; one was analyzed using FPIA, and the other was analyzed by HPLC–MS. The HPLC–MS analysis was carried out by Guangdong Testing Institute for Product Quality (Shunde, Guangdong Province, China) using the procedure described by Yin et al. [19].
Results and discussion Production of immunoreagents To obtain a specific and sensitive antibody, it was crucial to design the suitable hapten structure carefully. The following considerations have been shown to be important in determining the characteristics of the resulting antibodies: (1) the ability of the hapten to mimic the analyte; (2) the functional groups selected for the hapten coupling; (3) the length of the spacer bridge between the hapten and the carrier protein; (4) the conjugation method [18, 25, 26]. In previous work, hapten C (Fig. 1) was conjugated to BSA as an immunogen for antibody (antibody B) preparation [18]. However, antibody B strongly recognized cyromazine in ELISA, which is not expected in specific analysis of melamine. In this work, a new hapten (hapten A, Fig. 1) was conjugated to the carrier protein BSA for use as an immunogen to prepare antibody A. This hapten was commercially available and therefore complicated organic synthesis was not needed. The hydrazine group of hapten A is more reactive than the –NH2 of melamine. For this reason, a single zero-length coupling reagent, EDC, was
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Table 1 Characteristic comparison of the three tracers and two antibodies Tracer
Tracer A Tracer B Tracer C
Antibody A
Antibody B
Titer
IC50 (ng/mL)
LOD (ng/mL)
Detectable concentration range (ng/mL)
Titer
IC50 (ng/mL)
LOD (ng/mL)
Detectable concentration range (ng/mL)
1:160 1:210 1:150
174,196.7 65,909.9 444.9
29,872.3 1,615.9 36.3
55,345.1–426,973.5 6,063.9–278,987.6 89.3–2,776.7
1:300 1:230 1:150
86,0397 22,727 7,934
2,455.5 1,546.5 267.0
10,427.1–425,374.4 4,321.0–74,348.5 871.0–46,661.1
LOD limit of detection
used to produce a satisfactory conjugate (immunogen A) for the immunization. Also, without any additional coupling reagent, hapten A was linked directly to FITC for use as fluorescein-labeled tracer A. Hapten A was linked to FITC directly, but haptens B and C were covalently bound to EDF using the active ester method [10, 11]. Thus, tracers A, B, and C differed in not only the spacer length, but also in chemical structure between the hapten and the fluorophore. The spacer length between the hapten and FITC in tracer A is the shortest among the three tracers. Since FITC has a big volume, this might result in a stronger steric hindrance to the binding between tracer A and each antibody. However, the spacer length in tracer C is the longest, which might make the steric effect weaker and facilitate the binding between tracer C and each antibody as well. Besides the spacer length, tracer B had a sulfur atom, which has different electronic characteristics from the hydrazine group in tracer A or the amino group in tracer C. The electronic effect was expected to result in different ability of binding to the antibody. Since all three tracers would be used as the competing molecules in the FPIA, both the length difference and the electronic difference among spacers of tracers were proposed to result in different binding strength between the tracer and the antibody. To produce antibodies to melamine, hapten A–BSA (immunogen A) was injected into two rabbits. After five immunizations, the antibody was collected and the titer was tested by FPIA. The antibody from rabbit 1 demonstrated a higher titer and better sensitivity in FPIA performed with the three tracers than the antibody from rabbit 2 (data not shown). Therefore, the antibody from rabbit 1, named antibody A, was selected for the subsequent investigation. Comparison of tracers and antibodies Antibody B was produced previously, using immunogen B (Fig. 1), which was prepared with hapten B and BSA [18]. To obtain better sensitivity and specificity of the immunoassay, two antibodies (antibody A and antibody B) and the three tracers were initially evaluated and compared. The
specificity of an immunoassay generally depends on the antibody employed, whereas the assay sensitivity is also affected by the competitive hapten (tracer) [27]. Thus, the antibody and the tracer were two key factors to consider in the development of an FPIA. The working antibody concentration is an important parameter for FPIA development and the antibody dilution titer to show 70% or 50% binding response was usually appropriate for FPIA development [24, 28]. In this study, the antibody working concentration was assessed by FPIA using serially diluted antibody with a fixed concentration of the fluorescencelabeled tracer (1 nM). An antibody dilution titer showing 70% binding response to the tracer was chosen as the appropriate one to perform the initial evaluation with the three tracers. With the fixed tracer concentration (1 nM) to obtain 70% antibody binding, the appropriate antibody A dilution titers were 1:160, 1:210 and 1:150 for tracers A, B and C, respectively. However, the optimized antibody B dilution titers were 1:300, 1:230, and 1:150 for tracers A, B, and C, respectively. To obtain the same binding (70%), the three tracers needed different antibody dilution titers, suggesting that the three tracers had different relative affinities for each antibody. At the same tracer concentration, low antibody dilution means low relative affinity between the tracer and the antibody, and low affinity may result in the analyte competing for the antibody binding sites in a competitive immunoassay, leading to better sensitivity [29]. Tracer C among the three tracers showed the lowest relative affinity for both antibody A and antibody B in this case; thus, it could be concluded that tracer C would demonstrate most sensitivity in the FPIA. To confirm this, the ability of melamine to compete with the three tracers for antibody binding was investigated by means of calibration curves. Figure 2 and Table 1 show the characteristics of the two antibodies and the three tracers. When combining tracer C with antibody A or antibody B, it was found that both IC50 values were the lowest. Comparison of the IC50 of 444.9 ng mL-1 for antibody A and 7,934.0 ng mL-1 for antibody B implied that the sensitivity (IC50) had improved by about 18 times owing to the choice of hapten used in the immuno-
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(a) Tracer A Tracer B Tracer C
220
mP
200 180 160 140 -1
0
10
10
1
2
10
10
3
10
4
5
10
10
10
6
7
10
-1
Melamine / ng mL
(b) 300
Tracer A Tracer B Tracer C
280 260
mP
240 220 200 180 160 -1
0
10
10
1
10
2
3
4
5
10 10 10 10 -1 Melamine / ng mL
10
6
10
7
Fig. 2 Initial fluorescence polarization immunoassay (FPIA) calibration curves for melamine using the three traces in combination with antibody A (a) and antibody B (b). Each point of the curve represents the mean ± the standard deviation (SD) of three assays. mP fluorescence polarization signal
gen. Similar improvements were also found with both the LOD (from 267.0 ng mL-1 with antibody B to 36.3 ng mL-1 with antibody A, approximately 8 times improvement), and the dynamic concentration range (871.0–46,661.1 ng mL-1 with antibody B and 89.3–2,776.7 ng mL-1 with antibody
A) as well. A heterogeneous immunoassay format can often result in antibodies having a relatively higher affinity for the analyte [30–33]. Tracer C, with a six-carbon spacer arm as a heterogeneous competitor, gave better assay sensitivity than tracers A and B. This indicated that a longer spacer might lead to a more sensitive assay. It was interesting to note that the combination of coated hapten B and antibody B showed better sensitivity than the combination of coated hapten C and antibody B in our previous ELISA work [18], which is not in agreement with their performance in the present FPIA. This suggests that sometimes there is a performance difference between homogeneous direct FPIA and the heterogeneous indirect ELISA. Presumably, the short spacer arms of tracers A and B resulted in steric hindrance of tracer binding when the large fluorescent dye residue is brought close to the antigen-binding site of the antibody. Since the heterologous combination of antibody A and tracer C displayed the best assay sensitivities, this combination was used for further investigation. Figure 3 shows the competitive kinetics curves. It can be seen that the kinetic reaction was nearly stable at around 4 min for 500 ng mL-1 standard, at around 10 min for 100 ng mL-1, but at around 12 min for 50 and 0 ng mL-1. Therefore, it is concluded that different concentrations of competitors need different equilibration times, and higher concentrations would equilibrate faster. However, even after 1 min, the signals showed a significant difference for different concentrations, which means that 1 min is recordable for the homogeneous immunoreactions. To minimize the interassay coefficient of variation, a longer time was usually better for manual operation because the signal at the earlier stage was successively changing. Thus, 15 min was selected in the present FPIA. Figure 4 shows a typical optimal calibration curve. The concentration of tracer C in the FPIA was 0.8 nM and the
IC50 =164.7 ng mL-1
220 0 ng/mL 50 ng/mL 100 ng/mL 500 ng/mL
230
200
mP
220 210
mP
200
180
190
160
180 170
140 160
10
150 0
5
10
15
Time / min
Fig. 3 The competitive kinetic curves of tracer C and antibody A
0
10
1
2
3
10
Melamine / ng mL
10
10
4
-1
Fig. 4 FPIA calibration curve for melamine using antibody A and tracer C. Each point of the curve represents the mean ± SD of three assays
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Table 2 Cross-reactivity (CR) of antibody to melamine and related compounds determined by fluorescence polarization immunoassay (FPIA)
IC50
CR
(nM)
(%)
Melamine
1.31
100
Cyromazine
6.17
21.2
Hapten C
8.85
14.8
CAAT
20.31
6.4
Hapten A
23.36
5.6
Hapten B
41.50
3.2
Ammeline
81.01
1.6
Ammelide
1584.76
0.1
6569.74
0.02
ND
0.01
ND
0.01
Compound
Structure
Cyanuric acid Cyanuric chloride Atrazine
CAAT 2-chloro-4,6-diamino-1,3,5-triazine, ND not determined (the IC50 was too high to be available because the calibration curve could not be fitted)
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Table 3 Recovery of melamine from spiked sample determined by FPIA Sample
Spiked level
0.5 mg L-1 1.0 mg L-1 5.0 mg L-1 50.0 mg L-1 1.0 mg kg-1 2.0 mg kg-1 10.0 mg kg-1 50.0 mg kg-1
Milk
Milk powder
Observed value
Recovery (%, n=3)
0.40±0.08 mg L-1 0.84±0.14 mg L-1 4.93±0.60 mg L-1 52.80±13.44 mg L-1 1.19±0.27 mg kg-1 1.73±0.35 mg kg-1 10.41±1.55 mg kg-1 55.39±10.80 mg kg-1
79.4±15.7 83.6±13.6 98.5±12.0 105.6±26.9 119.0±26.8 86.6±17.7 104.1±15.5 110.8±21.6
Mean recovery (%)
CV (%)
Mean CV (%)
92.9
19.72 16.32 12.18 25.45 22.52 20.40 14.93 19.49
18.4
105.4
18.9
CV coefficient of variation
reaction time was 15 min. The LOD (10% inhibition) for melamine was 9.3 ng mL-1, the IC50 value was 164.7 ng mL-1, and the working range (20–80% inhibition) was 24.1–964.4 ng mL-1. Therefore, the performance of this FPIA could meet the maximum melamine amount allowed in foods and animal feed set by the CAC and the European Commission [4, 5]. Specificity The CRs for several compounds structurally related to melamine were tested and calculated as the ratio of the IC50 value of the melamine standard to the IC50 of the compounds tested. The results are presented in Table 2. Among all the structurally related compounds tested in the present study, the CR of melamine to antibody A was highest in FPIA format assays as expected, followed by cyromazine (21.1%), hapten C (14.8%), CAAT (6.4%), hapten A (5.6%), hapten B (3.2%), ammeline (1.6%), ammelide (0.1%), and cyanuric acid (0.02%). Cyanuric chloride and atrazine showed 0.01% or less CR. Comparing the CRs of melamine and CAAT and ammeline with their structures, even when one –NH2 was changed to –Cl or –
50 40
y=0.7638x+0.7078 2
HPLC / mg L
-1
r =0.9986
30 20 10 0 0
10
20
30
40
50
60
-1
FPIA / mg L
Fig. 5 Correlation between the result from FPIA and that from highperformance liquid chromatography (HPLC) for milk samples
OH, the CR decreased from 100% for melamine to 6.4% for CAAT or 1.6% for ammeline. This suggested that the three –NH2 groups in the triazine ring are crucial structural factors to determine the affinity of the analyte for the antibody. Additionally, the CR of atrazine was very much lower than that of CAAT. Probably, the steric hindrance, resulting from two alkyl groups in triazine, should be responsible for the CR change. However, the CR of CAAT was found to be higher than that of ammeline, which could be due to the different electronic effects of –Cl and –OH on the triazine ring. The difference between the CR of cyromazine and that of hapten C can be attributed to the negative charge of the acid anion. In one case reported, the influence of the negative charge was still noticeable after even changing the spacer length from two atoms to seven atoms [34]. The difference between cyromazine and hapten A may be attributed to the positive charge of the amino cation of hydrazine. In a previous report [18], with hapten C used as the coating antigen (hapten C–ovalbumin conjugate), antibody B had an extremely high CR to hapten C (1,652.9%) and cyromazine (267.6%). On the basis of the analysis of the structure–activity relationship, it was concluded that the electronic effect resulted in more influence than the steric effect of the spacer in melamine ELISA, and that there would probably be a decrease in the CR if hapten A were used in an immunogen for antibody production [18]. To verify this, in the present work antibody from hapten A was prepared and hapten C was used as a competitor (tracer). The CR of the antibody A to cyromazine and hapten C decreased from 267.6% and 1,652.9% in ELISA to 21.2% and 14.8% in this FPIA (Table 2), respectively. This was in good agreement with our previous conclusion [18]. Interestingly, although immunogen A was prepared using hapten A, it is surprising that hapten A does not show a high CR; indeed, the CR was found to be lower than the CR to melamine, cyromazine, hapten C, and CATT in the FPIA format. The steric effect of the hydrazine group in hapten A on the antibody recognition appeared to be much
Development of a FP immunoassay for melamine
smaller than that of the cyclopropyl group in cyromazine. Owing to the hydrazine amino group of hapten A being conjugated in immunogen A it was therefore converted to a amide group by reaction with the carboxyl groups on BSA under the condensing action of EDC, leading to different electronic states between free and conjugated hapten A. Thus, we presume that the electronic effect of the hydrazine amino group plays a more important role than the steric hindrance in this case, resulting in the weaker recognition between the free hapten A and antibody A. Determining the contribution that each part of the triazine molecule makes to antibody binding is important for understanding the phenomenon of CR between related compounds. Our CR study using the FPIA shows that the three –NH2 in the triazine ring must be present for antibody binding to occur, and the differences in antibody recognition of different triazine analytes could be caused by steric or/and electronic factors. As the ELISA showed, the electronic effect still appeared to influence more the antibody recognition in this FPIA.
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high, this immunoassay is a screening method and also has an undoubted practical advantage over methods requiring in-depth sample purification. Correlation studies between methods were carried out on five milk samples each spiked with melamine. All samples were analyzed by both FPIA and HPLC-MS. Samples were identified by a code number, so the melamine concentration was unknown to the analysts. Furthermore, FPIA analysis was performed in our laboratory according to the procedure described earlier, but HPLC-MS analysis was performed in a certificated laboratory (Guangdong Testing Institute for Product Quality, Shunde, Guangdong Province, China), and the results were compared only after the all the analyses had been completed. The analytical results obtained with these two types of samples by the two methods are shown in Fig 5. Linear regression analysis yielded an excellent correlation between methods (r2 =0.99 and slope 0.76) (Fig. 5). This suggested that the FPIA developed could be used for the accurate detection of melamine without complicated sample cleanup.
Analysis of spiked samples Conclusions Immunoassays are susceptible to interferences with different components existing in some matrices [33–35]. Matrix effects were estimated in this research using two types of matrix systems, including milk and milk powder purchased in local markets. As reported previously, the most common ways to reduce such matrix effects are selective extraction (“cleanup”) and dilution to bring the interfering substances below a concentration that would interfere with the assay [35]. Cleanup is relatively time-consuming and expensive and would counteract the advantage of the FPIA method in being a rapid and simple screening application. The dilution approach was successfully applied to extract melamine from milk and milk powder samples for the ELISA method [19]. In this study, milk was prepared by centrifugation to get the defatted solution before dilution; melamine in milk powder was extracted by methanol–H2O solution. After extraction, the optimum dilution factor of the extracts of milk and milk powder samples using sodium borate buffer was determined. It was found that with 30fold and 12-fold dilutions with sodium borate buffer for milk and milk powder, respectively, the diluted extracts showed a similar mP to the signal of sodium borate buffer (data not shown). The two matrix samples were spiked with different levels of melamine to determine the recovery and coefficient of variation. The results are summarized in Table 3. The FPIA gave satisfactory recovery that ranged from 79.4 to 119.0%. The means coefficients of variation for milk and milk powder were 18.4% and 19.3%, respectively. Although the coefficient of variation is a little
In conclusion, to improve the performance of an antibody to melamine, a new hapten strategy was utilized and the antibodies obtained were found to have high titer and better sensitivity and specificity than our previous antibodies. The FPIA developed is able to detect melamine in milk at levels of regulatory relevance, with accuracy and precision comparable to those obtained with the reference method. After the sample treatment, the immunoassay takes 15 min to performed. An issue of major importance is the ability of this FPIA to detect melamine without complicated cleanup. This FPIA may be easily included as a complementary method in melamine regulatory programs. FPIA could be considered to be a valuable method for screening purposes. Overall, this work should reasonably contribute to increase the acceptance of FPIA methods among analytical chemists involved in melamine residue analysis in foods. Acknowledgements This work was supported by the National Department Public Benefit Research Foundation (201003008-08) and China Guangdong Provincial Science and Technology Projects (cgzhzd1005, 2009B040500002).
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