Analytical Biochemistry 462 (2014) 51–59
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
A heterogeneous biotin–streptavidin-amplified enzyme-linked immunosorbent assay for detecting tris(2,3-dibromopropyl) isocyanurate in natural samples Dan Bu a, Huisheng Zhuang a,⇑, Xinchu Zhou b, Guangxin Yang a a b
School of Environment Science and Technology, Shanghai Jiao Tong University, Minghang District, Shanghai 200240, People’s Republic of China School of Agriculture and Biology, Shanghai Jiao Tong University, Minghang District, Shanghai 201101, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 6 March 2014 Received in revised form 14 May 2014 Accepted 3 June 2014 Available online 12 June 2014 Keywords: Tris(2,3-dibromopropyl) isocyanurate Biotin–streptavidin-amplified system Enzyme-linked immunosorbent assay Environmental monitoring
a b s t r a c t Tris(2,3-dibromopropyl) isocyanurate (TBC) is a novel brominated flame retardant (BFR) that is widely used to substitute the prohibited BFRs throughout the world. With the development of research, the potential environmental and ecological harms of TBC have been revealed. For sensitive and selective detecting TBC, an indirect competitive biotin–streptavidin-amplified enzyme-linked immunosorbent assay (BA–ELISA) has been established in this study. The small molecular TBC–hapten was synthesized first; it mimicked the chemical structure of TBC and possessed a secondary amine group. The as-obtained hapten was then conjugated with carrier proteins to prepare artificial antigen. After immunization, the anti-TBC polyclonal antibody was obtained from separating rabbit serum. The procedures of this BA– ELISA were optimized. Under the optimal conditions, the limit of detection (IC10) was 0.0067 ng/ml and the median inhibitory concentration (IC50) was 0.66 ng/ml. Cross-reactivity values of the BA–ELISA with the tested TBC analogues were 65%. This immunoassay was successfully applied to determine the TBC residue in river water samples that were collected near a BFR manufacturing plant. Satisfactory recoveries (92.1–109.2%) were obtained. The results indicated that this proposed BA–ELISA is suitable for the rapid and sensitive determining of TBC in environmental monitoring. Ó 2014 Elsevier Inc. All rights reserved.
With the increasing application of polymeric materials in electronic, construction, and household products, brominated flame retardants (BFRs)1 have been used largely for the reason of fire safety. Global market demand for BFRs continues to increase, growing from 145,000 tonnes in 1990 [1] to 310,000 tonnes in 2000 [2].
⇑ Corresponding author. Fax: +86 21 54740825/62419587. E-mail addresses: [email protected], [email protected] (H. Zhuang). 1 Abbreviations used: BFR, brominated flame retardant; PBDE, polybrominated diphenyl ether; PBB, polybrominated biphenyl; TBBPA, tetrabromobisphenol A; HBCD, hexabromocyclododecane; TBC, tris(2,3-dibromopropyl) isocyanurate; ELISA, enzyme-linked immunosorbent assay; BA–ELISA, biotin–streptavidin-amplified ELISA; SA–HRP, streptavidin–horseradish peroxidase; BSA, bovine serum albumin; OVA, egg albumin; BNHS, biotinylated N-hydroxysuccinimide ester; DMF, N,Ndimethylformamide; EDCHCl, 1-ethyl-(3-dimethyllaminopropyl) carbodiimide hydrochloride; DMSO, dimethyl sulfoxide; TMB, 3,30 ,5,50 -tetramethylbenzidine; NMR, nuclear magnetic resonance; PBS, phosphate-buffered saline; CBS, carbonate buffer solution; PBST, PBS with 0.05% Tween 20; pAb–TBC, anti-TBC polyclonal antibody; Bi–pAb–TBC, biotinylated TBC antibody; LOD, limit of detection; SPE, solid phase extraction; HPLC, high-performance liquid chromatography; UV, ultraviolet; PEG, polyethylene glycol; PVA, polyvinyl alcohol; CV, coefficient of variation; CR, cross-reactivity; PBB15, 4,40 -dibrominated biphenyl. http://dx.doi.org/10.1016/j.ab.2014.06.003 0003-2697/Ó 2014 Elsevier Inc. All rights reserved.
BFRs can be mainly divided into two subgroups according to the way of incorporating into polymeric materials: reactive and additive flame retardants. The most commonly used BFRs include polybrominated diphenyl ethers (PBDEs), polybrominated biphenyls (PBBs), tetrabromobisphenol A (TBBPA), hexabromocyclododecanes (HBCDs), and tris(2,3-dibromopropyl) isocyanurate (TBC). Recently, concerns about the potential environmental and ecological problems caused by BFRs have been raised. Studies have proved that many BFR materials could potentially harm ecosystems and human health [3–5]. Many types of BFRs have been found to exist in various environmental or biological samples such as breast milk [6,7], serum [8,9], living body [10,11], food [12,13], water [14,15], and sediment [16–18]. Currently, two types of polybrominated diphenyl ethers, penta- and octa-BDEs, are banned in Europe [19,20] and listed in the Stockholm Convention on persistent organic pollutants (POPs) [21], In 2013, HBCD was included in the Stockholm Convention as well. Meanwhile, TBBPA is listed in the convention on the protection of the marine environment of the North-East Atlantic as a hazardous substance. With growing stringent regulations on BFRs, other substitutes have been produced to fill the market vacancy of those obsolete BFRs. However,
52
BA–ELISA for tris(2,3-dibromopropyl) isocyanurate / D. Bu et al. / Anal. Biochem. 462 (2014) 51–59
these substitutes have similar semi-volatile properties and may also harm the environment. TBC is one of these alternative BFRs that had been identified in the environment first in 2009 [22]. TBC (Fig. 1) is a novel BFR that has a hexabrominated heterocyclic s-triazine structure. As an alternative BFR, TBC has the features of high thermal stability, low viscosity, and low tendency of photodegradation. In China, production and use of TBC were begun in 1980 [23]. The production amount of TBC in China is unknown, but judging from the usage and output information, its production quantity is relatively large [22]. So, it can be assumed that the potential for TBC to be released into the environment should not be ignored [22,24]. Recent studies have proved that TBC is a potential environmental hazard. High Kow and bioaccumulation factor calculated results indicated that TBC has the characteristics of semi-volatility and bio-accumulation [25]. A study on zebrafish embryos showed that TBC could remarkably inhibit the expression of vitellogenin genes in liver and further affect gonadal development [26]. Therefore, it proved that TBC is a potential endocrine disruptor. For TBC determination, the liquid chromatography tandem mass spectrometry method was commonly used [24,27]. It is well known that instrument methods are effective, accurate, and reliable. Nonetheless, some drawbacks exist. These methods are generally expensive, time-consuming, and labor-intensive and also require complex pretreatment procedures that restrict their widespread application for rapid detection in environmental studies. So, it makes sense to develop a more highly sensitive, selective, highthroughput, and simpler analytical method. Immunoassay, which is based on the principle of molecular biology, has the above advantages. Compared with instrumental methods, enzyme-linked immunosorbent assay (ELISA) is more suitable for detecting trace organic pollutants in the environment. Meanwhile, some ELISA methods have been established for detecting PBDEs [28–32] and other BFRs [33,34]. To further improve sensitivity, conventional ELISAs have been modified with a combination of chemiluminescence, fluorescence, or biotin–streptavidin system. Among these strategies, the biotin– streptavidin system is an effective technique and has been widely used for sensitivity improvement [35–39]. In biotin–streptavidinamplified ELISA (BA–ELISA), several biotins are conjugated to one immunoglobulin without altering the biological activity of immunoglobulin. Then, streptavidin–horseradish peroxidase (SA–HRP) combines with biotins in the following step. The affinity of biotin and avidin is very strong, with an affinity constant of 1015 L/mol [40]. The signal intensity can be increased due to more enzyme molecules catalyzing the substrate.
In this study, a highly sensitive and selective indirect competitive ELISA, using a biotin–streptavidin amplification system, was developed for detecting TBC. For establishing this proposed BA– ELISA, TBC–hapten and immunogens were prepared primarily. Based on the optimal immunization, polyclonal anti-TBC antibodies were produced. Procedures for BA–ELISA were optimized, and some influencing factors were also discussed. Subsequently, this BA–ELISA was implemented to detect TBC residues in environmental samples. The accuracy and sensitivity of the testing results were good. We believe that this proposed BA–ELISA will be useful for environmental studies. Materials and methods Reagents and apparatus The TBC standard and organic materials for hapten synthesis, including 2,4,6-triallyloxy-1,3,5-triazine and liquid bromine, were purchased from J&K Chemical (Beijing, China). Hapten was purified through column chromatography using silica gel (40 lm average particle size) acquired from Shanghai Sanpont (China). Bovine serum albumin (BSA), egg albumin (OVA), biotinylated N-hydroxysuccinimide ester (BNHS), N,N-dimethylformamide (DMF), 1-ethyl(3-dimethyllaminopropyl) carbodiimide hydrochloride (EDCHCl), glutaraldehyde, dimethyl sulfoxide (DMSO), hydrogen peroxide, Coomassie brilliant blue G250, Tween 20, complete and incomplete Freund’s adjuvant, and 3,30 ,5,50 -tetramethylbenzidine (TMB) were purchased from Sinopharm (China). SA–HRP was acquired from Sangon Biotech (Shanghai, China). All reagents were of analytic grade unless specified otherwise. The 1H nuclear magnetic resonance (NMR) spectrometer was an Avance III 400-MHz instrument (Bruker, Switzerland) with CDCl3 solution. Fourier transform infrared spectrometry was performed on a Nicolet 6700 instrument (Thermo, USA). A Multiskan MK3 ELISA reader (Thermo) was used to determine absorbance in dual-wavelength mode (450/650 nm) with polystyrene 96-well microplates purchased from Sangon Biotech. TBC–protein conjugates were characterized on a UV-2012 PC spectrophotometer (UNICO, USA). Ultrapure water was prepared using a Milli-Q system (Millipore, Bedford, MA, USA). Buffers and solutions Phosphate-buffered saline (PBS: 137 mmol/L NaCl and 10 mmol/L sodium phosphate, pH 7.4), carbonate buffer solution (CBS: 15 mmol/L Na2CO3 and 34.9 mmol/L NaHCO3, pH 9.6), PBST (PBS with 0.05% Tween 20), phosphate–citrate buffer (0.1 mol/L citric acid and 0.2 mol/L Na2HPO4, pH 4.3), TMB substrate solution (0.4 ml of 2.5 g/L TMB ethanol solution, 10 ml of phosphate–citrate buffer, and 10 ll of 30% H2O2) were used. Synthesis of TBC–hapten
Fig.1. Molecular structure of tris(2,3-dibromopropyl) isocyanurate (TBC). CAS: 52434-90-9.
Compound 1 was synthesized as Likhterov’s method [41] and is shown in Fig. 2A. Here, 12.46 g (0.05 mol) of 2,4,6-triallyloxy-1,3,5triazine, 0.9 g (0.05 mol) of water, and 0.38 g (2.25 mol) of CuCl22H2O were mixed in toluene. The mixture was sustained by stirring in a round-bottom flask and heating at 95 °C for 5 h. The reaction was monitored by thin-layer chromatography (TLC) in developing solvent (n-hexane/ethyl acetate = 5:1). After cooling to room temperature, the reaction solvent was evaporated to obtain the crude product. Then, the crude product was recrystallized from petroleum ether and dichloromethane. Finally, the pure compound 1 was obtained after being washed by diluted hydrochloric acid. M.P.: 146–148 °C; name: 1,3-diallyl isocyanurate; yield: 63.6%.
BA–ELISA for tris(2,3-dibromopropyl) isocyanurate / D. Bu et al. / Anal. Biochem. 462 (2014) 51–59
53
Fig.2. The synthetic route of TBC–hapten, immunogen, and coating antigen.
1
H NMR (CdCl3) d (ppm): 8.44 (1H ArH non-benzene ring), 5.89 (1H, AC@CAH conjugated), 5.23–5.34 (2H, AC@CH2), 4.45–4.47 (2H, ArACH2AR). IR (KBr) m/cm–1: 3430.65 (N-H, stretching vibration), 1582.13 (NAH, in-plane bending vibration), 1137.33 (secondary amine CAN, stretching vibration), 1723.99 (C@O, stretching vibration), and 875.55, 778.72, 734.95 (CAH, flexural vibrations). TBC–hapten (compound 2) was synthesized as follows. First, 0.5 g (0.94 mmol) of compound 1 and 120 ll (2.36 mmol) of liquid bromine was mixed in 20 ml of dichloromethane. The red reaction solution was maintained at reflux temperature for 1 h and then cooled to room temperature. After the reaction was completed, saturated Na2S2O3 solution was added to stop the reaction. The mixture was extracted with CH2Cl2 three times, dried over anhydrous Na2SO4, and concentrated in vacuum. TBC–hapten was obtained after purification by recrystallization in the mixture of ethyl acetate and n-hexane. M.P.: 128–131 °C; name: 1,3-bis-(2,3dibromo-propyl)-1,3,5-triazine-2,4,6-trione; yield: 47.1%. 1 H NMR (CdCl3) d (ppm): 9.22 (1H NAH); 4.42 (1H RACHABr); 3.77, 3.88 (2H ACH2ABr); 1.44, 1.28 (2H ACH2A); IR (KBr) m/cm–1: 3293.82 (OAH stretching vibration); 1671.98 (C@O stretching vibration); 1430.92 (OAH, in-plane bending vibration); 3320.82 (secondary amine NAH, stretching vibration); 1295.93, 1315.21 (aromatics CAN stretching vibration); 2989.12 (CAH stretching vibration); 1648.84–1432.85 (benzene C@C, frame vibration); 910–665 (CAH out-of-plane bending vibration), 621.08 (CABr, stretching vibration). Preparation of immunogen and coating antigen For preparing immunogen and coating antigen for TBC, the glutaraldehyde method and the carbodiimide method were employed to couple hapten to the carrier proteins using the terminal
secondary amine group on the TBC–hapten molecule. Here, different coupling methods were used to reduce the specific binding caused by linking arms. Linking arm [42] is a contact portion between hapten and carrier protein that could become an antigenic determinant and specifically combine with antibody. Detailed coupling procedures were as follows. For the glutaraldehyde method shown in Fig. 2B, 1 ml of 0.5 mol/L TBC–hapten DMF solution was dropwise added into 10 ml of 12 mg/ml BSA solution. Then, 200 ll of 25% glutaraldehyde was dropwise added into the above solution. The reaction was sustained by magnetic stirring at 4 °C for 24 h. After the reaction was accomplished, the suspension was dialyzed against PBS (0.01 M, pH 7.4) for 3 days. For the carbodiimide method shown in Fig. 2C, equimolar TBC– hapten and EDC were dissolved in 1 ml of DMF. The obtained supernate was dropwise added into OVA solution (10 mg/ml in NaAc–HAc buffer, 0.01 M, pH 5.0) and then stirred at 4 °C for 6 h. After that, another 10 mg of EDC was added into the solution. The reaction was sustained by stirring in the dark at 4 °C for 24 h. After the reaction was completed, the suspension was dialyzed against PBS for 3 days. Prepared hapten–protein conjugates BSA–TBC and OVA–TBC were used as the immunogen and coating antigen, respectively. Preparation of anti-TBC polyclonal antibody TBC–immunogen (BSA–TBC) was injected into three male New Zealand white rabbits (2.0–2.5 kg). Then, 1 mg/ml TBC–immunogen, emulsified with Freund’s complete adjuvant (1:1, v/v), was hypodermic injected at multiple sites on the neck or back. The second immunization, emulsified with Freund’s incomplete adjuvant, was the same as that of the first after 2 weeks. The rest of the booster injection was given at intervals of 2 weeks. The serum titer was determined by ELISA. When the antiserum titer reached 60,000,
54
BA–ELISA for tris(2,3-dibromopropyl) isocyanurate / D. Bu et al. / Anal. Biochem. 462 (2014) 51–59
the rabbit blood (containing anti-TBC antibody) was collected. This collected blood was rested at 4 °C overnight, and then the antiserum was separated by centrifugation and purified by octanoic acid and ammonium sulfate. After that, antiserum (containing anti-TBC polyclonal antibody [pAb–TBC]) was stored at 20 °C until use. Biotinylated TBC antibodies (Bi–pAb–TBCs) were prepared as follows. Purified pAb–TBC was diluted by sodium carbonate buffer (0.1 M, pH 9.6) to 1–2 mg/ml. Then, 1 mg/ml BNHS solution (in DMSO) was dropwise added into the dilute antibody solution with stirring for 4 h. The mass ratio of pAb/BNHS was 1:10. After that, the mixture was dialyzed against PBS for 3 days and then stored at 4 °C before use.
the specificity of the proposed immunoassay. So, in this study, the above parameters are discussed. In addition, to evaluate the interference of different matrices on proposed BA–ELISA performance, four common water miscible organic solvents—DMSO, methanol, acetone, and acetonitrile— were selected and added during the competitive procedure. The reason for choosing these organic solvents was that they are commonly used in sample extraction. All determinations were performed in triplicate, and the mean absorbance values were calculated. For data analysis, the inhibition (%) is an important parameter and is calculated as Eq. (1):
Inhibition ð%Þ ¼ Procedures of heterologous indirect competitive BA–ELISA All indirect competitive BA–ELISAs were performed in 96-well microplates. The wells in each microplate were coated with 100 ll/well coating antigen (OVA–TBC) at 4 °C overnight. After washing, unbound active sites were blocked with 200 ll/well blocking reagent and then incubated at 37 °C for 60 min. The wells were washed with PBST and then incubated for 1 h at 37 °C with biotinylated antibody diluents (50 ll/well) as well as different concentrations of TBC standard solution or practical sample extract (50 ll/well). After washing, 100 ll/well SA–HRP conjugate (dilution 1:1000) was added and incubated for 60 min. After a final washing step, 100 ll/well TMB substrate solution was added to each well, and the developing reaction was stopped after 15 min by 50 ll/well 2 M H2SO4. The absorbance of each well was read in dual-wavelength mode at 450 nm as the test and 650 nm as the reference.
ðAmax Amin Þ ðAs Amin Þ 100%; Amax Amin
ð1Þ
where Amax is the absorbance in the absence of TBC, Amin is the absorbance of the blank sample, and As is the absorbance of TBC at the standard concentration. Under optimal conditions, a dose–response curve of BA–ELISA was determined by plotting inhibition (%) versus the logarithm of TBC concentration. The sensitivity of the assay was evaluated by IC50, the concentration at which a compound inhibits a particular phenomenon by 50%. Analogously, the limit of detection (LOD) was assessed in terms of IC10. Sample preparation
In general, the specificity of ELISAs was mainly based on the efficient combination of antibody and antigen. However, several parameters—for example, the concentration of biotinylated antibody and immobilized antigen, the blocking reagent, the incubation time of the competition reaction, and some factors (pH and ionic strength) in assay buffer—were deeply affected by the efficient combination of antibody and antigen, further influencing
All water samples were collected from a tributary of the Huangpu River near a TBC manufacturing plant located southwest of Shanghai in eastern China. The sampling map and sites are illustrated in Fig. 3. Four sampling sites were placed downstream of the outlet of a TBC manufacturing plant except for two control sites in the upstream location. River water samples were collected using 1-L glass bottles that had been precleaned with acetone. After collection, water samples were directly carried to the laboratory and stored at 4 °C until analysis. The procedures of sample pretreatment were similar to the description in the literature [22]. Briefly, 1 L of river sample was filtrated through a 0.45-lm filter and then loaded onto a solid phase extraction (SPE) cartridge at the rate of 2 ml/min. First, the SPE cartridge needed to be preconditioned with 3 ml of methanol and distilled water. Then, 3 ml of 40% methanol
Fig.3. Location of sampling sites.
Fig.4. UV spectrogram of TBC–hapten, protein, and conjugates. Absorbance value at characteristic peak, 256 nm: ODTBC–BSA = 0.940, ODhapten = 0.396, ODBSA = 0.117; 253 nm: ODTBC–OVA = 0.938, ODhapten = 0.379, ODOVA = 0.663; CBSA: 0.23 g/L; COVA: 0.15 g/L; Chapten: 0.03 g/L. Protein and conjugate were dissolved in 0.01 M PBS buffer (pH 7.4). Hapten was dissolved in DMSO.
Immunoassay optimization
55
BA–ELISA for tris(2,3-dibromopropyl) isocyanurate / D. Bu et al. / Anal. Biochem. 462 (2014) 51–59
was used to wash the cartridge. Subsequently, the cartridge was eluted with 9 ml of methanol. Finally, the eluent was evaporated by a gentle nitrogen flow to 200 ll.
HPLC analysis Detection results of BA–ELISA were verified by the high-performance liquid chromatography (HPLC) method. HPLC analysis was performed on a Waters 1515-2489 HPLC system equipped with an Athena C18-Wp column (250 4.6 mm, 5 lm). The mobile phase was methanol/acetonitrile (70:30, v/v) at a flow rate of 0.3 ml/min. The injection volume was 20 ll, and detection was at 267 nm.
Results and discussion Characterization of immunogen, coating antigen, and antibody TBC is a small molecule organic that has no immunogenicity unless being conjugated with carrier proteins. For conjugating with carrier proteins, TBC–hapten has been synthesized as with the above method. The results of NMR and infrared spectrophotometric analyses proved that the synthesis was successful. The secondary amine on the TBC–hapten molecular structure could be a feasible group to attach a linker. Different cross-linking agents, glutaraldehyde and EDC, were used for preparing the immunogen and coating antigen. The conjugates were analyzed by an ultraviolet–visible (UV–Vis) spectrophotometer, and the results are shown in Fig. 4.
Table 1 Optimal concentrations of biotinylated antibody and coating antigen in terms of absorbance. Dilution of biotinylated antibodya
100 200 500 1000 1500 2000 3000 Blank a
TBC–OVA concentration (lg/ml) 30.10
15.05
6.02
3.01
1.51
0.75
2.281 1.599 1.114 1.020 0.894 0.566 0.346 0.146
1.719 1.315 1.268 1.126 1.053 0.924 0.694 0.131
1.690 1.446 1.354 1.173 1.060 0.946 0.856 0.180
1.644 1.538 1.394 1.139 0.893 0.742 0.441 0.172
1.855 1.553 1.252 0.998 0.740 0.594 0.320 0.158
2.057 1.926 1.666 1.156 0.887 0.763 0.585 0.177
The concentration of biotinylated anti-TBC antibody was 0.862 mg/ml.
Fig.5. Suitable operating conditions of immunoassay method; (A) blocking reagent; (B) incubation time; (C) pH of buffer; (D) ionic strength (NaCl solution, 0–2.0 mol/L) in PBS buffer.
56
BA–ELISA for tris(2,3-dibromopropyl) isocyanurate / D. Bu et al. / Anal. Biochem. 462 (2014) 51–59
Suitable operating conditions for immunoassay method
Fig.6. Effects of common solvents on BA–ELISA performance. A stands for the absorption value with a certain amount of solvent, and A0 stands for the absorption value without any solvent. The concentrations of coating antigen and biotinylated anti-TBC antibody were 6.02 and 0.57 lg/ml, respectively. The solvent percentage was a final concentration in the assay system.
Fig.7. Calibration curve of BA–ELISA for detecting TBC. The inhibition was calculated in different TBC concentrations (10–4 to 104 ng/ml in 1% DMSO). The plate was coated with 57 ng of TBC–OVA per well, and the biotinylated anti-TBC antibody was diluted 1500-fold in PBS. The data were averages of eight replicates.
The characteristic absorption peaks of TBC–hapten and proteins appeared at 281 nm (for hapten), 227 and 273 nm (for BSA), and 219, 224, and 268 nm (for OVA). Dissimilar to carrier proteins, new characteristic peaks of TBC–BSA and TBC–OVA were exhibited at 256 and 253 nm, respectively. The blue shift of absorption peaks indicated that the hapten was successfully conjugated with the protein. The coupling ratio [43,44] was 35 for BSA–TBC and was 12 for OVA–TBC. The concentrations of BSA–TBC and OVA–TBC were 5.34 and 3.01 mg/ml, respectively, which were measured by the Coomassie blue staining method.
Experimentally, the concentrations of coating antigen and biotinylated antibody were very important to improve the sensitivity of immunoassay. According to checkerboard titration, the optimal reagent concentrations were the ones that resulted in the maximum absorbance (A0max) of approximately 1.0 and lower concentrations of antibody and coating antigen. The results are shown in Table 1. The optimal concentration of the coated TBC–OVA was 6.02 lg/ml, and the Bi–pAb–TBC was at 1:1500 dilution. Blocking is required in immunoassay procedures to avoid nonspecific absorption. Otherwise, unoccupied sites may absorb components such as Bi–pAb–TBC and SA–HRP during subsequent steps. Different common blocking solutions were compared, and the results are exhibited in Fig. 5A. As a result, the 1% gelatin blocking agent achieved a lower background value (0.094) than those of 0.1% gelatin (0.107), 0.5% gelatin (0.120), 0.5% OVA (0.113), 3% milk powder (0.217), 2% glucan (0.361), 2% PEG (polyethylene glycol) 20000 (0.244), and 1% polyvinyl alcohol (PVA, 0.129). Therefore, 1% gelatin was selected in the succeeding experiments. The incubation time of competitive reaction was tested to enhance the sensitivity. Different incubation times of 15, 30, 45, 60, 75, and 90 min were investigated. The results are exhibited in Fig. 5B. Although the A0max value increased with increasing incubation times, the lowest IC50 (0.69 ng/ml) was obtained at 60 min. Hence, an incubation time of 60 min was selected for the competitive reaction between antigen and antibody. In addition, assay buffer-related factors, pH and ionic strength in buffer, were analogously examined as possible means to enhance the analytical sensitivity. As buffer pH values changed between 5.0 and 10.0, the IC50 and A0max (in Fig. 5C) varied in the ranges of 0.74 to 32.78 ng/ml and 0.585 to 1.581 AU, respectively. The best combination of IC50 and A0max (IC50 = 0.742, A0max = 1.026) was obtained at pH 7.4. Therefore, pH 7.4 was selected in further optimization assays. Similarly, the influence of ionic strength was tested in the range of 0.05 to 2 mol/L (in Fig. 5D). As a result, 0.1 mol/L NaCl was selected for the immunoassay buffer preparation because the best combination of IC50 and A0max (IC50 = 1.02, A0max = 1.053) was received at this concentration. To evaluate the influence of different matrices on BA–ELISA performance, four water miscible organic solvents were added into the immunoassay system, and the results are shown in Fig. 6. These results indicated that lower amounts of organic solvent (6600
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
A heterogeneous biotin–streptavidin-amplified enzyme-linked immunosorbent assay for detecting tris(2,3-dibromopropyl) isocyanurate in natural samples Dan Bu a, Huisheng Zhuang a,⇑, Xinchu Zhou b, Guangxin Yang a a b
School of Environment Science and Technology, Shanghai Jiao Tong University, Minghang District, Shanghai 200240, People’s Republic of China School of Agriculture and Biology, Shanghai Jiao Tong University, Minghang District, Shanghai 201101, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 6 March 2014 Received in revised form 14 May 2014 Accepted 3 June 2014 Available online 12 June 2014 Keywords: Tris(2,3-dibromopropyl) isocyanurate Biotin–streptavidin-amplified system Enzyme-linked immunosorbent assay Environmental monitoring
a b s t r a c t Tris(2,3-dibromopropyl) isocyanurate (TBC) is a novel brominated flame retardant (BFR) that is widely used to substitute the prohibited BFRs throughout the world. With the development of research, the potential environmental and ecological harms of TBC have been revealed. For sensitive and selective detecting TBC, an indirect competitive biotin–streptavidin-amplified enzyme-linked immunosorbent assay (BA–ELISA) has been established in this study. The small molecular TBC–hapten was synthesized first; it mimicked the chemical structure of TBC and possessed a secondary amine group. The as-obtained hapten was then conjugated with carrier proteins to prepare artificial antigen. After immunization, the anti-TBC polyclonal antibody was obtained from separating rabbit serum. The procedures of this BA– ELISA were optimized. Under the optimal conditions, the limit of detection (IC10) was 0.0067 ng/ml and the median inhibitory concentration (IC50) was 0.66 ng/ml. Cross-reactivity values of the BA–ELISA with the tested TBC analogues were 65%. This immunoassay was successfully applied to determine the TBC residue in river water samples that were collected near a BFR manufacturing plant. Satisfactory recoveries (92.1–109.2%) were obtained. The results indicated that this proposed BA–ELISA is suitable for the rapid and sensitive determining of TBC in environmental monitoring. Ó 2014 Elsevier Inc. All rights reserved.
With the increasing application of polymeric materials in electronic, construction, and household products, brominated flame retardants (BFRs)1 have been used largely for the reason of fire safety. Global market demand for BFRs continues to increase, growing from 145,000 tonnes in 1990 [1] to 310,000 tonnes in 2000 [2].
⇑ Corresponding author. Fax: +86 21 54740825/62419587. E-mail addresses: [email protected], [email protected] (H. Zhuang). 1 Abbreviations used: BFR, brominated flame retardant; PBDE, polybrominated diphenyl ether; PBB, polybrominated biphenyl; TBBPA, tetrabromobisphenol A; HBCD, hexabromocyclododecane; TBC, tris(2,3-dibromopropyl) isocyanurate; ELISA, enzyme-linked immunosorbent assay; BA–ELISA, biotin–streptavidin-amplified ELISA; SA–HRP, streptavidin–horseradish peroxidase; BSA, bovine serum albumin; OVA, egg albumin; BNHS, biotinylated N-hydroxysuccinimide ester; DMF, N,Ndimethylformamide; EDCHCl, 1-ethyl-(3-dimethyllaminopropyl) carbodiimide hydrochloride; DMSO, dimethyl sulfoxide; TMB, 3,30 ,5,50 -tetramethylbenzidine; NMR, nuclear magnetic resonance; PBS, phosphate-buffered saline; CBS, carbonate buffer solution; PBST, PBS with 0.05% Tween 20; pAb–TBC, anti-TBC polyclonal antibody; Bi–pAb–TBC, biotinylated TBC antibody; LOD, limit of detection; SPE, solid phase extraction; HPLC, high-performance liquid chromatography; UV, ultraviolet; PEG, polyethylene glycol; PVA, polyvinyl alcohol; CV, coefficient of variation; CR, cross-reactivity; PBB15, 4,40 -dibrominated biphenyl. http://dx.doi.org/10.1016/j.ab.2014.06.003 0003-2697/Ó 2014 Elsevier Inc. All rights reserved.
BFRs can be mainly divided into two subgroups according to the way of incorporating into polymeric materials: reactive and additive flame retardants. The most commonly used BFRs include polybrominated diphenyl ethers (PBDEs), polybrominated biphenyls (PBBs), tetrabromobisphenol A (TBBPA), hexabromocyclododecanes (HBCDs), and tris(2,3-dibromopropyl) isocyanurate (TBC). Recently, concerns about the potential environmental and ecological problems caused by BFRs have been raised. Studies have proved that many BFR materials could potentially harm ecosystems and human health [3–5]. Many types of BFRs have been found to exist in various environmental or biological samples such as breast milk [6,7], serum [8,9], living body [10,11], food [12,13], water [14,15], and sediment [16–18]. Currently, two types of polybrominated diphenyl ethers, penta- and octa-BDEs, are banned in Europe [19,20] and listed in the Stockholm Convention on persistent organic pollutants (POPs) [21], In 2013, HBCD was included in the Stockholm Convention as well. Meanwhile, TBBPA is listed in the convention on the protection of the marine environment of the North-East Atlantic as a hazardous substance. With growing stringent regulations on BFRs, other substitutes have been produced to fill the market vacancy of those obsolete BFRs. However,
52
BA–ELISA for tris(2,3-dibromopropyl) isocyanurate / D. Bu et al. / Anal. Biochem. 462 (2014) 51–59
these substitutes have similar semi-volatile properties and may also harm the environment. TBC is one of these alternative BFRs that had been identified in the environment first in 2009 [22]. TBC (Fig. 1) is a novel BFR that has a hexabrominated heterocyclic s-triazine structure. As an alternative BFR, TBC has the features of high thermal stability, low viscosity, and low tendency of photodegradation. In China, production and use of TBC were begun in 1980 [23]. The production amount of TBC in China is unknown, but judging from the usage and output information, its production quantity is relatively large [22]. So, it can be assumed that the potential for TBC to be released into the environment should not be ignored [22,24]. Recent studies have proved that TBC is a potential environmental hazard. High Kow and bioaccumulation factor calculated results indicated that TBC has the characteristics of semi-volatility and bio-accumulation [25]. A study on zebrafish embryos showed that TBC could remarkably inhibit the expression of vitellogenin genes in liver and further affect gonadal development [26]. Therefore, it proved that TBC is a potential endocrine disruptor. For TBC determination, the liquid chromatography tandem mass spectrometry method was commonly used [24,27]. It is well known that instrument methods are effective, accurate, and reliable. Nonetheless, some drawbacks exist. These methods are generally expensive, time-consuming, and labor-intensive and also require complex pretreatment procedures that restrict their widespread application for rapid detection in environmental studies. So, it makes sense to develop a more highly sensitive, selective, highthroughput, and simpler analytical method. Immunoassay, which is based on the principle of molecular biology, has the above advantages. Compared with instrumental methods, enzyme-linked immunosorbent assay (ELISA) is more suitable for detecting trace organic pollutants in the environment. Meanwhile, some ELISA methods have been established for detecting PBDEs [28–32] and other BFRs [33,34]. To further improve sensitivity, conventional ELISAs have been modified with a combination of chemiluminescence, fluorescence, or biotin–streptavidin system. Among these strategies, the biotin– streptavidin system is an effective technique and has been widely used for sensitivity improvement [35–39]. In biotin–streptavidinamplified ELISA (BA–ELISA), several biotins are conjugated to one immunoglobulin without altering the biological activity of immunoglobulin. Then, streptavidin–horseradish peroxidase (SA–HRP) combines with biotins in the following step. The affinity of biotin and avidin is very strong, with an affinity constant of 1015 L/mol [40]. The signal intensity can be increased due to more enzyme molecules catalyzing the substrate.
In this study, a highly sensitive and selective indirect competitive ELISA, using a biotin–streptavidin amplification system, was developed for detecting TBC. For establishing this proposed BA– ELISA, TBC–hapten and immunogens were prepared primarily. Based on the optimal immunization, polyclonal anti-TBC antibodies were produced. Procedures for BA–ELISA were optimized, and some influencing factors were also discussed. Subsequently, this BA–ELISA was implemented to detect TBC residues in environmental samples. The accuracy and sensitivity of the testing results were good. We believe that this proposed BA–ELISA will be useful for environmental studies. Materials and methods Reagents and apparatus The TBC standard and organic materials for hapten synthesis, including 2,4,6-triallyloxy-1,3,5-triazine and liquid bromine, were purchased from J&K Chemical (Beijing, China). Hapten was purified through column chromatography using silica gel (40 lm average particle size) acquired from Shanghai Sanpont (China). Bovine serum albumin (BSA), egg albumin (OVA), biotinylated N-hydroxysuccinimide ester (BNHS), N,N-dimethylformamide (DMF), 1-ethyl(3-dimethyllaminopropyl) carbodiimide hydrochloride (EDCHCl), glutaraldehyde, dimethyl sulfoxide (DMSO), hydrogen peroxide, Coomassie brilliant blue G250, Tween 20, complete and incomplete Freund’s adjuvant, and 3,30 ,5,50 -tetramethylbenzidine (TMB) were purchased from Sinopharm (China). SA–HRP was acquired from Sangon Biotech (Shanghai, China). All reagents were of analytic grade unless specified otherwise. The 1H nuclear magnetic resonance (NMR) spectrometer was an Avance III 400-MHz instrument (Bruker, Switzerland) with CDCl3 solution. Fourier transform infrared spectrometry was performed on a Nicolet 6700 instrument (Thermo, USA). A Multiskan MK3 ELISA reader (Thermo) was used to determine absorbance in dual-wavelength mode (450/650 nm) with polystyrene 96-well microplates purchased from Sangon Biotech. TBC–protein conjugates were characterized on a UV-2012 PC spectrophotometer (UNICO, USA). Ultrapure water was prepared using a Milli-Q system (Millipore, Bedford, MA, USA). Buffers and solutions Phosphate-buffered saline (PBS: 137 mmol/L NaCl and 10 mmol/L sodium phosphate, pH 7.4), carbonate buffer solution (CBS: 15 mmol/L Na2CO3 and 34.9 mmol/L NaHCO3, pH 9.6), PBST (PBS with 0.05% Tween 20), phosphate–citrate buffer (0.1 mol/L citric acid and 0.2 mol/L Na2HPO4, pH 4.3), TMB substrate solution (0.4 ml of 2.5 g/L TMB ethanol solution, 10 ml of phosphate–citrate buffer, and 10 ll of 30% H2O2) were used. Synthesis of TBC–hapten
Fig.1. Molecular structure of tris(2,3-dibromopropyl) isocyanurate (TBC). CAS: 52434-90-9.
Compound 1 was synthesized as Likhterov’s method [41] and is shown in Fig. 2A. Here, 12.46 g (0.05 mol) of 2,4,6-triallyloxy-1,3,5triazine, 0.9 g (0.05 mol) of water, and 0.38 g (2.25 mol) of CuCl22H2O were mixed in toluene. The mixture was sustained by stirring in a round-bottom flask and heating at 95 °C for 5 h. The reaction was monitored by thin-layer chromatography (TLC) in developing solvent (n-hexane/ethyl acetate = 5:1). After cooling to room temperature, the reaction solvent was evaporated to obtain the crude product. Then, the crude product was recrystallized from petroleum ether and dichloromethane. Finally, the pure compound 1 was obtained after being washed by diluted hydrochloric acid. M.P.: 146–148 °C; name: 1,3-diallyl isocyanurate; yield: 63.6%.
BA–ELISA for tris(2,3-dibromopropyl) isocyanurate / D. Bu et al. / Anal. Biochem. 462 (2014) 51–59
53
Fig.2. The synthetic route of TBC–hapten, immunogen, and coating antigen.
1
H NMR (CdCl3) d (ppm): 8.44 (1H ArH non-benzene ring), 5.89 (1H, AC@CAH conjugated), 5.23–5.34 (2H, AC@CH2), 4.45–4.47 (2H, ArACH2AR). IR (KBr) m/cm–1: 3430.65 (N-H, stretching vibration), 1582.13 (NAH, in-plane bending vibration), 1137.33 (secondary amine CAN, stretching vibration), 1723.99 (C@O, stretching vibration), and 875.55, 778.72, 734.95 (CAH, flexural vibrations). TBC–hapten (compound 2) was synthesized as follows. First, 0.5 g (0.94 mmol) of compound 1 and 120 ll (2.36 mmol) of liquid bromine was mixed in 20 ml of dichloromethane. The red reaction solution was maintained at reflux temperature for 1 h and then cooled to room temperature. After the reaction was completed, saturated Na2S2O3 solution was added to stop the reaction. The mixture was extracted with CH2Cl2 three times, dried over anhydrous Na2SO4, and concentrated in vacuum. TBC–hapten was obtained after purification by recrystallization in the mixture of ethyl acetate and n-hexane. M.P.: 128–131 °C; name: 1,3-bis-(2,3dibromo-propyl)-1,3,5-triazine-2,4,6-trione; yield: 47.1%. 1 H NMR (CdCl3) d (ppm): 9.22 (1H NAH); 4.42 (1H RACHABr); 3.77, 3.88 (2H ACH2ABr); 1.44, 1.28 (2H ACH2A); IR (KBr) m/cm–1: 3293.82 (OAH stretching vibration); 1671.98 (C@O stretching vibration); 1430.92 (OAH, in-plane bending vibration); 3320.82 (secondary amine NAH, stretching vibration); 1295.93, 1315.21 (aromatics CAN stretching vibration); 2989.12 (CAH stretching vibration); 1648.84–1432.85 (benzene C@C, frame vibration); 910–665 (CAH out-of-plane bending vibration), 621.08 (CABr, stretching vibration). Preparation of immunogen and coating antigen For preparing immunogen and coating antigen for TBC, the glutaraldehyde method and the carbodiimide method were employed to couple hapten to the carrier proteins using the terminal
secondary amine group on the TBC–hapten molecule. Here, different coupling methods were used to reduce the specific binding caused by linking arms. Linking arm [42] is a contact portion between hapten and carrier protein that could become an antigenic determinant and specifically combine with antibody. Detailed coupling procedures were as follows. For the glutaraldehyde method shown in Fig. 2B, 1 ml of 0.5 mol/L TBC–hapten DMF solution was dropwise added into 10 ml of 12 mg/ml BSA solution. Then, 200 ll of 25% glutaraldehyde was dropwise added into the above solution. The reaction was sustained by magnetic stirring at 4 °C for 24 h. After the reaction was accomplished, the suspension was dialyzed against PBS (0.01 M, pH 7.4) for 3 days. For the carbodiimide method shown in Fig. 2C, equimolar TBC– hapten and EDC were dissolved in 1 ml of DMF. The obtained supernate was dropwise added into OVA solution (10 mg/ml in NaAc–HAc buffer, 0.01 M, pH 5.0) and then stirred at 4 °C for 6 h. After that, another 10 mg of EDC was added into the solution. The reaction was sustained by stirring in the dark at 4 °C for 24 h. After the reaction was completed, the suspension was dialyzed against PBS for 3 days. Prepared hapten–protein conjugates BSA–TBC and OVA–TBC were used as the immunogen and coating antigen, respectively. Preparation of anti-TBC polyclonal antibody TBC–immunogen (BSA–TBC) was injected into three male New Zealand white rabbits (2.0–2.5 kg). Then, 1 mg/ml TBC–immunogen, emulsified with Freund’s complete adjuvant (1:1, v/v), was hypodermic injected at multiple sites on the neck or back. The second immunization, emulsified with Freund’s incomplete adjuvant, was the same as that of the first after 2 weeks. The rest of the booster injection was given at intervals of 2 weeks. The serum titer was determined by ELISA. When the antiserum titer reached 60,000,
54
BA–ELISA for tris(2,3-dibromopropyl) isocyanurate / D. Bu et al. / Anal. Biochem. 462 (2014) 51–59
the rabbit blood (containing anti-TBC antibody) was collected. This collected blood was rested at 4 °C overnight, and then the antiserum was separated by centrifugation and purified by octanoic acid and ammonium sulfate. After that, antiserum (containing anti-TBC polyclonal antibody [pAb–TBC]) was stored at 20 °C until use. Biotinylated TBC antibodies (Bi–pAb–TBCs) were prepared as follows. Purified pAb–TBC was diluted by sodium carbonate buffer (0.1 M, pH 9.6) to 1–2 mg/ml. Then, 1 mg/ml BNHS solution (in DMSO) was dropwise added into the dilute antibody solution with stirring for 4 h. The mass ratio of pAb/BNHS was 1:10. After that, the mixture was dialyzed against PBS for 3 days and then stored at 4 °C before use.
the specificity of the proposed immunoassay. So, in this study, the above parameters are discussed. In addition, to evaluate the interference of different matrices on proposed BA–ELISA performance, four common water miscible organic solvents—DMSO, methanol, acetone, and acetonitrile— were selected and added during the competitive procedure. The reason for choosing these organic solvents was that they are commonly used in sample extraction. All determinations were performed in triplicate, and the mean absorbance values were calculated. For data analysis, the inhibition (%) is an important parameter and is calculated as Eq. (1):
Inhibition ð%Þ ¼ Procedures of heterologous indirect competitive BA–ELISA All indirect competitive BA–ELISAs were performed in 96-well microplates. The wells in each microplate were coated with 100 ll/well coating antigen (OVA–TBC) at 4 °C overnight. After washing, unbound active sites were blocked with 200 ll/well blocking reagent and then incubated at 37 °C for 60 min. The wells were washed with PBST and then incubated for 1 h at 37 °C with biotinylated antibody diluents (50 ll/well) as well as different concentrations of TBC standard solution or practical sample extract (50 ll/well). After washing, 100 ll/well SA–HRP conjugate (dilution 1:1000) was added and incubated for 60 min. After a final washing step, 100 ll/well TMB substrate solution was added to each well, and the developing reaction was stopped after 15 min by 50 ll/well 2 M H2SO4. The absorbance of each well was read in dual-wavelength mode at 450 nm as the test and 650 nm as the reference.
ðAmax Amin Þ ðAs Amin Þ 100%; Amax Amin
ð1Þ
where Amax is the absorbance in the absence of TBC, Amin is the absorbance of the blank sample, and As is the absorbance of TBC at the standard concentration. Under optimal conditions, a dose–response curve of BA–ELISA was determined by plotting inhibition (%) versus the logarithm of TBC concentration. The sensitivity of the assay was evaluated by IC50, the concentration at which a compound inhibits a particular phenomenon by 50%. Analogously, the limit of detection (LOD) was assessed in terms of IC10. Sample preparation
In general, the specificity of ELISAs was mainly based on the efficient combination of antibody and antigen. However, several parameters—for example, the concentration of biotinylated antibody and immobilized antigen, the blocking reagent, the incubation time of the competition reaction, and some factors (pH and ionic strength) in assay buffer—were deeply affected by the efficient combination of antibody and antigen, further influencing
All water samples were collected from a tributary of the Huangpu River near a TBC manufacturing plant located southwest of Shanghai in eastern China. The sampling map and sites are illustrated in Fig. 3. Four sampling sites were placed downstream of the outlet of a TBC manufacturing plant except for two control sites in the upstream location. River water samples were collected using 1-L glass bottles that had been precleaned with acetone. After collection, water samples were directly carried to the laboratory and stored at 4 °C until analysis. The procedures of sample pretreatment were similar to the description in the literature [22]. Briefly, 1 L of river sample was filtrated through a 0.45-lm filter and then loaded onto a solid phase extraction (SPE) cartridge at the rate of 2 ml/min. First, the SPE cartridge needed to be preconditioned with 3 ml of methanol and distilled water. Then, 3 ml of 40% methanol
Fig.3. Location of sampling sites.
Fig.4. UV spectrogram of TBC–hapten, protein, and conjugates. Absorbance value at characteristic peak, 256 nm: ODTBC–BSA = 0.940, ODhapten = 0.396, ODBSA = 0.117; 253 nm: ODTBC–OVA = 0.938, ODhapten = 0.379, ODOVA = 0.663; CBSA: 0.23 g/L; COVA: 0.15 g/L; Chapten: 0.03 g/L. Protein and conjugate were dissolved in 0.01 M PBS buffer (pH 7.4). Hapten was dissolved in DMSO.
Immunoassay optimization
55
BA–ELISA for tris(2,3-dibromopropyl) isocyanurate / D. Bu et al. / Anal. Biochem. 462 (2014) 51–59
was used to wash the cartridge. Subsequently, the cartridge was eluted with 9 ml of methanol. Finally, the eluent was evaporated by a gentle nitrogen flow to 200 ll.
HPLC analysis Detection results of BA–ELISA were verified by the high-performance liquid chromatography (HPLC) method. HPLC analysis was performed on a Waters 1515-2489 HPLC system equipped with an Athena C18-Wp column (250 4.6 mm, 5 lm). The mobile phase was methanol/acetonitrile (70:30, v/v) at a flow rate of 0.3 ml/min. The injection volume was 20 ll, and detection was at 267 nm.
Results and discussion Characterization of immunogen, coating antigen, and antibody TBC is a small molecule organic that has no immunogenicity unless being conjugated with carrier proteins. For conjugating with carrier proteins, TBC–hapten has been synthesized as with the above method. The results of NMR and infrared spectrophotometric analyses proved that the synthesis was successful. The secondary amine on the TBC–hapten molecular structure could be a feasible group to attach a linker. Different cross-linking agents, glutaraldehyde and EDC, were used for preparing the immunogen and coating antigen. The conjugates were analyzed by an ultraviolet–visible (UV–Vis) spectrophotometer, and the results are shown in Fig. 4.
Table 1 Optimal concentrations of biotinylated antibody and coating antigen in terms of absorbance. Dilution of biotinylated antibodya
100 200 500 1000 1500 2000 3000 Blank a
TBC–OVA concentration (lg/ml) 30.10
15.05
6.02
3.01
1.51
0.75
2.281 1.599 1.114 1.020 0.894 0.566 0.346 0.146
1.719 1.315 1.268 1.126 1.053 0.924 0.694 0.131
1.690 1.446 1.354 1.173 1.060 0.946 0.856 0.180
1.644 1.538 1.394 1.139 0.893 0.742 0.441 0.172
1.855 1.553 1.252 0.998 0.740 0.594 0.320 0.158
2.057 1.926 1.666 1.156 0.887 0.763 0.585 0.177
The concentration of biotinylated anti-TBC antibody was 0.862 mg/ml.
Fig.5. Suitable operating conditions of immunoassay method; (A) blocking reagent; (B) incubation time; (C) pH of buffer; (D) ionic strength (NaCl solution, 0–2.0 mol/L) in PBS buffer.
56
BA–ELISA for tris(2,3-dibromopropyl) isocyanurate / D. Bu et al. / Anal. Biochem. 462 (2014) 51–59
Suitable operating conditions for immunoassay method
Fig.6. Effects of common solvents on BA–ELISA performance. A stands for the absorption value with a certain amount of solvent, and A0 stands for the absorption value without any solvent. The concentrations of coating antigen and biotinylated anti-TBC antibody were 6.02 and 0.57 lg/ml, respectively. The solvent percentage was a final concentration in the assay system.
Fig.7. Calibration curve of BA–ELISA for detecting TBC. The inhibition was calculated in different TBC concentrations (10–4 to 104 ng/ml in 1% DMSO). The plate was coated with 57 ng of TBC–OVA per well, and the biotinylated anti-TBC antibody was diluted 1500-fold in PBS. The data were averages of eight replicates.
The characteristic absorption peaks of TBC–hapten and proteins appeared at 281 nm (for hapten), 227 and 273 nm (for BSA), and 219, 224, and 268 nm (for OVA). Dissimilar to carrier proteins, new characteristic peaks of TBC–BSA and TBC–OVA were exhibited at 256 and 253 nm, respectively. The blue shift of absorption peaks indicated that the hapten was successfully conjugated with the protein. The coupling ratio [43,44] was 35 for BSA–TBC and was 12 for OVA–TBC. The concentrations of BSA–TBC and OVA–TBC were 5.34 and 3.01 mg/ml, respectively, which were measured by the Coomassie blue staining method.
Experimentally, the concentrations of coating antigen and biotinylated antibody were very important to improve the sensitivity of immunoassay. According to checkerboard titration, the optimal reagent concentrations were the ones that resulted in the maximum absorbance (A0max) of approximately 1.0 and lower concentrations of antibody and coating antigen. The results are shown in Table 1. The optimal concentration of the coated TBC–OVA was 6.02 lg/ml, and the Bi–pAb–TBC was at 1:1500 dilution. Blocking is required in immunoassay procedures to avoid nonspecific absorption. Otherwise, unoccupied sites may absorb components such as Bi–pAb–TBC and SA–HRP during subsequent steps. Different common blocking solutions were compared, and the results are exhibited in Fig. 5A. As a result, the 1% gelatin blocking agent achieved a lower background value (0.094) than those of 0.1% gelatin (0.107), 0.5% gelatin (0.120), 0.5% OVA (0.113), 3% milk powder (0.217), 2% glucan (0.361), 2% PEG (polyethylene glycol) 20000 (0.244), and 1% polyvinyl alcohol (PVA, 0.129). Therefore, 1% gelatin was selected in the succeeding experiments. The incubation time of competitive reaction was tested to enhance the sensitivity. Different incubation times of 15, 30, 45, 60, 75, and 90 min were investigated. The results are exhibited in Fig. 5B. Although the A0max value increased with increasing incubation times, the lowest IC50 (0.69 ng/ml) was obtained at 60 min. Hence, an incubation time of 60 min was selected for the competitive reaction between antigen and antibody. In addition, assay buffer-related factors, pH and ionic strength in buffer, were analogously examined as possible means to enhance the analytical sensitivity. As buffer pH values changed between 5.0 and 10.0, the IC50 and A0max (in Fig. 5C) varied in the ranges of 0.74 to 32.78 ng/ml and 0.585 to 1.581 AU, respectively. The best combination of IC50 and A0max (IC50 = 0.742, A0max = 1.026) was obtained at pH 7.4. Therefore, pH 7.4 was selected in further optimization assays. Similarly, the influence of ionic strength was tested in the range of 0.05 to 2 mol/L (in Fig. 5D). As a result, 0.1 mol/L NaCl was selected for the immunoassay buffer preparation because the best combination of IC50 and A0max (IC50 = 1.02, A0max = 1.053) was received at this concentration. To evaluate the influence of different matrices on BA–ELISA performance, four water miscible organic solvents were added into the immunoassay system, and the results are shown in Fig. 6. These results indicated that lower amounts of organic solvent (6600
