Food Chemistry 202 (2016) 141–148
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Development a monoclonal antibody-based enzyme-linked immunosorbent assay for screening carotenoids in eggs Dapeng Peng, Feng Liao 1, Yuanhu Pan, Dongmei Chen, Zhenli Liu, Yulian Wang ⇑, Zonghui Yuan ⇑ National Reference Laboratory of Veterinary Drug Residues (HZAU) and MAO Key Laboratory for Detection of Veterinary Drug Residues, Hubei Collaborative Innovation Center for Animal Nutrition and Feed Safety, Huazhong Agricultural University, Wuhan, Hubei 430070, China
a r t i c l e
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Article history: Received 15 June 2015 Received in revised form 5 January 2016 Accepted 27 January 2016 Available online 28 January 2016 Keywords: Carotenoids Monoclonal antibody Indirect competitive enzyme linked immunosorbent assay Eggs
a b s t r a c t In this study, a monoclonal antibody (mAb) with broad-specificity against several carotenoid analogs with equal or similar efficacy was prepared. The obtained mAb C11, with the IgG1 isotype, showed cross-reactivity (CR) with canthaxanthin (100%), b-ionone acid (140.4%), b-carotene (92.9%), capsanthin (90.1%), b-apo-80 -carotenal (92.7%), and xanthophyll (95.8%). Using the mAb C11, a highly sensitive and inexpensive indirect competitive enzyme linked immunosorbent assay (ic-ELISA) was developed with a simple sample preparation procedure for the simultaneous detection of these carotenoid compounds in eggs. The limit of detection of the various carotenoids ranged from 1.31 mg kg1 to 1.48 mg kg1. Recoveries from egg yolks spiked with the above carotenoids ranged from 91.8% to 113.3%, with coefficients of variation (CVs) of less than 14.8%. These results suggest that the developed ic-ELISA is a sensitive, specific, accurate, and inexpensive method that is suitable for the screening of carotenoid residues in routine monitoring. Ó 2016 Published by Elsevier Ltd.
1. Introduction Color is a key factor for the consumer acceptance of many foods. The red fillet color caused by the deposition of carotenoid pigments (e.g., astaxanthin and canthaxanthin) in the muscular tissue is an important quality criteria for consumer acceptance and willingness to pay in salmonid fishes (Caballo, Costi, Sicilia, & Rubio, 2012; Dissing, Nielsen, Ersbøll, & Frosch, 2011; Folkestad et al., 2008). In eggs, a golden yellow yolk color, which is also caused by the deposition of carotenoid pigments (e.g., canthaxanthin, b-carotene, b-apo-80 -carotenal, and capsanthin) in the yolk, is associated with health and quality (Furusawa, 2011; Grashorn & Steinberg, 2002; Ren & Zhang, 2008). However, neither salmonid fishes nor egg-laying hens can produce carotenoids, and they have to obtain these pigments from dietary sources. Although more than 600 carotenoids have been defined in nature, only a few of them are used in animal feed, pharmaceuticals, cosmetics and food coloring (Kop & Durmaz, 2008; Ong & Tee, 1992). In fish and poultry farming and food processing, several carotenoids, including canthaxanthin, b-carotene, b-apo-80 carotenal, capsanthin, and xanthophyll (Fig. 1A), are frequently ⇑ Corresponding authors. E-mail addresses: [email protected]
(Y. Wang), yuan5802@mail. hzau.edu.cn (Z. Yuan). 1 Co-first author. http://dx.doi.org/10.1016/j.foodchem.2016.01.123 0308-8146/Ó 2016 Published by Elsevier Ltd.
used as colorings and feed additives to pigment the eggs or meat of hens, broilers, salmon, and trout to make food more attractive and appetizing (Caballo et al., 2012; Fujii, Shimizu, & Nakamura, 2001; Furusawa, 2011; Ong & Tee, 1992). However, some of these substances pose a potential risk to human health, especially if they are excessively consumed. For example, canthaxanthin has been reported to cause liver injury and an eye disorder called canthaxanthin retinopathy, the formation of yellow deposits on the retina (FDA, 2003). Although no adverse effect of high-dose oral b-carotene supplementation was observed in several standard toxicological studies in various experimental animals (rat, mice, rabbits) (Grenfell-Lee, Zeller, Cardoso, & Pucaj, 2014; Woutersen, Wolterbeek, Appel & Berg, 1999), intervention trials with large doses of b-carotene found adverse effect on the incidence of lung cancer in smokers and workers exposed to asbestos (Omenn et al., 1996). In addition, there is evidence that both b-apo-80 -carotenal and b-carotene at high doses could significantly enhance DNA strand breaks and lipid peroxidation and impair mitochondrial functions (Siems et al., 2005; Yeh & Wu, 2006). For these reasons, safety data, such as acceptable daily intakes, based on toxicological studies with experimental animals and human clinical studies have been repeatedly determined and evaluated by the Food and Agricultural Organization (FAO) and World Health Organization (WHO) (Minioti, Sakellariou, & Thomaidis,
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Fig. 1. The structure of some b-carotenoid pigment: A, the structure of some b-carotenoid pigment and haptens; B, stereochemical structure of some b-carotenoid pigment, 1, b-apo-80 -carotenal; 2, retinoic acid; 3, canthaxanthin; 4, b-carotene; 5, b-ionone acid; 6, b-ionone; 7, 4-keto-b-ionone acid; 8, retinol; 9, capsanthin; 10, xanthophyl.
2007). The European Commission adopted a directive in 2003 to reduce the authorized level of canthaxanthin in animal feed. Recently, the maximum residue limits (MRLs) of canthaxanthin in animal tissues and feeds were set by the European Food Safety Authority (EFSA) and the Japanese Ministry of Health, Labour and Welfare, respectively (Furusawa, 2011). Therefore, monitoring the presence of such carotenoids in edible animal tissues and products is necessary to ensure the safety and appropriateness of products for human consumption, satisfactory product quality and adequate production costs. Numerous analytical methods, such as spectrophotometry (Schoefs, 2002), thin layer chromatography (Hayashi et al., 2003) and high performance liquid chromatography (HPLC) (Akhtar & Bryan, 2008; Barba, Hurtado, Mata, Ruiz, & Tejada, 2006; Breithaupt, 2004; Caballo et al., 2012; Hu, Lin, Lu, Chou, & Yang, 2008; Minioti et al., 2007; Ren & Zhang, 2008), have been used for the determination of carotenoids in a variety of food matrices. These
methods, however, are not suitable for the routine monitoring of carotenoids because they involve expensive instrumentation and are time consuming. This has in turn created a demand for better analytical methods that are inexpensive, rapid, and robust for the detection and quantification of such carotenoids in a variety of food matrices. Indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) is the most popular method for detecting drugs in animal tissues due to its high sensitivity, low cost, and ability to screen large numbers of samples. Production of antibodies against carotenoids or development of ic-ELISA methods for the analysis of carotenoids in edible animal tissues have not been reported. Therefore, this study aimed to prepare a monoclonal antibody (mAb) with broad-specificity against several carotenoid analogs and to develop a highly sensitive and low cost ic-ELISA for the simultaneous detection of these carotenoid compounds in edible animal tissues and products with a simple sample preparation procedure.
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2. Materials and methods 2.1. Chemicals b-ionone, urea hydrogen peroxide, Freund’s adjuvant (complete and incomplete), polyethylene glycol (PEG), dimethylsulfoxide (DMSO), RPMI-1640, N-hydroxysuccinimide (NHS), dicyclohexylcarbodiimide (DCC), bovine serum albumin (BSA), ovalbumin (OVA), goat anti-mouse IgG horseradish peroxidase conjugate (HRP-IgG), and b-apo-80 -carotenal were purchased from Sigma– Aldrich (St. Louis, MO, USA). Canthaxanthin, b-carotene, 4-ketobeta-ionone acid, capsanthin, b-ionone acid, retinoic, retinol and xanthophyll were obtained from Xi’an Sino-herb Bio-technology Co., Ltd (Xian, China). Fetal calf serum was purchased from Hangzhou Sijiqing Biological Engineering Materials Co., Ltd. (Hangzhou, China). All other chemicals were purchased from the Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China) and were of analytical grade. 2.2. Synthesis of Haptens The four Haptens, b-ionone-O-carboxymethoxylamine (b-ionone-CMO, Hapten A), b-ionone acid (Hapten B), 4-keto-bionone acid (Hapten C) and retinoic acid (Hapten D), used for the immunization and coating of antigens are presented in Fig. 1A. All of the obtained products were confirmed by LC/MS-IT-TOF (Shimadzu Corp., Kyoto, Japan). The detailed procedure for the synthesis of Haptens A–C is as follows. 2.2.1. Synthesis of Hapten A Hapten A was synthesized according to previously described procedures (Banerjee & Dureja, 2005; Peng et al., 2012). Briefly, O-carboxymethoxylamine hemihydrochloride (109.3 mg dissolved in 1 mL saturated solution of NaHCO3) was added to a stirred solution of b-ionone (1 mL) dissolved in dry and distilled ethyl alcohol (5 mL). Stirring continued for another 2 h. After completion of the reaction, the reaction mixture was washed with a saturated solution of NaHCO3 and extracted with diethyl ether. After discarding the diethyl ether phase, the water phase was adjusted to pH 3.0 with HCl (4.0 mol L1) and then extracted with dichloromethane (3 10 mL). The water phase was dried over anhydrous sodium sulfate and concentrated in vacuo to obtain the product Hapten A. MS m/z calculated for C15H21O3N [M H] 264.1602, found 264.1594. 2.2.2. Synthesis of Hapten B Synthesis of Hapten B was based on a procedure described by He and Wu (1988). Briefly, one gram of Br2 was added to a NaOH solution (3.54 g NaOH dissolved in 14 mL of water) and incubated for 1 h at room temperature. One gram of b-ionone (dissolved in 6 mL of 1,4-dioxane) was added to this solution, which was then incubated for 4 h at room temperature. Then, 10% NaHSO3 was added to the mixture with simultaneous monitoring using potassium iodide starch test paper. After completion of the reaction, the reaction mixture was adjusted to pH 3.5 with HCl (12 mol L1). After the solvent was removed with a vacuum suction filter, the residue was recrystallized twice with absolute methanol and evaporated in vacuo to obtain the product Hapten B. MS m/z calculated for C12H18O2 [M H] 193.1229, found 193.1302. 2.2.3. Synthesis of Hapten C Synthesis of Hapten C was based on modified procedures (Della & G., 2007; He & Wu, 1988). Briefly, 10 g of b-ionone, 50 mL of methylene chloride, 1.01 g of KI (dissolved in 6 mL of water), and 1.76 g of NaHSO4 (dissolved in 4 mL of water) were added to a
round-bottomed flask under an atmosphere of nitrogen. Then, 7.97 g of NaBrO4 (dissolved in 25 mL of water) was slowly added (drop by drop with shaking for 2.5 h) to the mixture, and then incubated for 4 h at 37 °C. After the end of the reaction, the organic phase was washed in turn with an NaOH solution (4 mol L1, 20 mL), acetic acid (2 mol L1, 10 mL), an NaHSO3 solution (1.5%, 10 mL), and water (10 mL), dried over anhydrous magnesium sulfate, and concentrated in vacuo. The residue was recrystallized twice with petroleum ether and evaporated in vacuo to obtain the product 4-keto-b-ionone. MS m/z calculated for C13H18O2 [M + H]+ 207.1385, found 207.1414. Using this product as raw material, Hapten C was synthesized essentially as described for Hapten A. MS m/z calculated for C12H16O3 [M H] 207.1021, found 207.0960. 2.3. Synthesis of conjugates All of the haptens were conjugated to BSA to prepare immunizing conjugates according to previously described procedures (Peng et al., 2012). Briefly, 23.0 mg of NHS and 43.4 mg of DCC were added sequentially to solutions of 27 mg of Hapten A, 39 mg of Hapten B, 43 mg of Hapten C, and 20 mg of Hapten D dissolved in 2.0 mL of 1,4-dioxane at 4 °C in the dark. After the mixture was stirred overnight, the activated hapten solution was centrifuged for 10 min at 5000 rpm. Next, 1.5 mL of the supernatant was added dropwise with stirring to a solution in which 70 mg of BSA had been dissolved in 1:10 (v/v) in DMF: phosphate buffered saline (PBS, 0.1 mol L1, pH 8.0). The conjugated mixture was stirred at 4 °C overnight and then centrifuged for 10 min at 5000g. The supernatant was purified by exhaustive dialysis against PBS (0.01 mol L1, pH 7.4) and then stored at –20 °C. All of the haptens were conjugated to OVA to prepare the coating conjugates according to the mixed anhydride method (Degand & Bernes-Duyckaerts, 1993). Briefly, after dissolution of 27 mg of Hapten A, 39 mg of Hapten B, 43 mg of Hapten C, and 20 mg of Hapten D respectively in a mixture of dioxane/DMF/triethylamine (1/2/0.03, v/v/v), the solution was stirred for 30 min at room temperature and then isobutyl chloroformate (30 lL) was added dropwise. After stirring for 2 h, this solution was added dropwise to an OVA solution (90 mg, in 20 mL of TBS (0.02 mol L1, pH 7.5)). The resultant solution was stirred overnight at 4 °C and subsequently centrifuged at 3,000 g for 10 min. The supernatant was purified by exhaustive dialysis against PBS (0.01 mol L1, pH 7.4) and stored at –20 °C. Verification of conjugate synthesis and estimation of the hapten/protein ratio was performed on an 8453 UV–Visible spectrophotometer. The numbers of Hapten A, Hapten B, Hapten C, and Hapten D conjugated to the carrier was estimated as follows: [e (conjugation) e (protein)]/e (hapten), where e is the absorbance coefficient of analytes. 2.4. Preparation of monoclonal antibodies All animal experiments in this study adhered to Huazhong Agricultural University Animal Experiment Centre guidelines and were approved by the Animal Ethics Committee. Eight female Balb/c mice (6–8 weeks old), purchased from Hubei Center for Disease Control and Prevention (Wuhan, China), were inoculated with the immunizing conjugates (two mice of each). The immunization schemes are shown in Table 1. First, the immunogens were prepared for injection by emulsification of the conjugates in 500 lL of sterile isotonic saline and 500 lL of Freund’s adjuvant. This cocktail was mixed vigorously until a homogeneous suspension was obtained. Complete adjuvant was used for the first injection, and incomplete adjuvant was used for the subsequent injections. The immunogen emulsion was injected subcutaneously
D. Peng et al. / Food Chemistry 202 (2016) 141–148 Table 1 The immunization schemes, titer and specificity. Immunogen
Mouse Mouse Mouse Mouse Mouse Mouse Mouse Mouse
Hapten B-BSA Hapten C-BSA Hapten D-BSA
1 2 3 4 5 6 7 8
Titera (1:X 102)
The inhibition ratiob (%) of the sera
75 75 125 125 125 125 150 150
3 3 2 2 3 3 2 2
8 16 64 64 1024 1024 8 8
85.7 85.4 64.9 89.3 30.2 6.5 67.8 36.0
Note: a The highest dilution factor that still yields a positive result. b The specificity of each sear was primarily assessed by the (B0 – B)/B0 values of 25 mg L1 canthaxanthin which was measured by ic-ELISA method.
into multiple sites on the back of each mouse. Blood was collected, and titers of antisera were determined by indirect ELISA. The mice producing the best dose-response curves were selected for fusion. Spleen cells of the immunized mice were fused with myeloma cells Sp2/0 at a ratio of 10:1 according to a previous procedure (Peng et al., 2012). Hybridoma from wells having a positive response in the ELISA described below were cloned twice by limiting dilution and expanded to guarantee monoclonality. After cell culture, the cultured hybridoma was intraperitoneally injected into mice to produce ascites. The class and subclass of the isotypes of the secreted antibody were determined using a mouse monoclonal antibody isotyping kit (Proteintech Group, Inc., Chicago, IL, USA). The mAbs raised against each of the immunizing conjugates were screened against each of these corresponding coating antigens in a checkerboard for the best dilution of the coating conjugate and ascites. The extent of cross-reactivity (CR) was assessed by determining the IC50 values in the ic-ELISA. Several carotenoid compounds, canthaxanthin, b-carotene, b-apo-80 -carotenal, 4-keto-beta-ionone acid, xanthophyll, capsanthin, b-ionone acid, b-ionone, retinoic, and retinol, were selected to test for CR. The concentrations of the standard solutions of the compounds ranged from 0.001 to 100 mg L1. The CR values were calculated as follows: CR = (IC50 of canthaxanthin/IC50 of other carotenoid compounds) 100%. The detailed ELISA procedure is described in the section on indirect ELISA and ic-ELISA. The antibody with the lowest IC50 was selected for the remainder of this study. 2.5. Indirect ELISA and ic-ELISA The indirect ELISA protocol was similar to that described by Peng et al. (2012). Briefly, 96-well Maxisorp microtitre plates (Nunc, Roskilde, Denmark) were coated with coating conjugates in 100 lL of coating buffer (0.05 mol L1 carbonate buffers, pH 9.6) overnight at 4 °C. The plates were washed three times with PBS containing 0.1% Tween-20 (PBST) and incubated with 200 lL of 1% OVA in PBS at 37 °C for 0.5 h. The plates were then washed with PBST, followed by addition of 100 lL of antiserum in each well. After 0.5 h of incubation at 37 °C, the plates were washed with PBST and then incubated with 100 lL of HRP-IgG (1:5000) at 37 °C for 0.5 h. After washing the plate with PBST, 100 lL of TMB substrate solution was added to each well. The samples were incubated for 15 min at room temperature in the dark, followed by addition of the stop solution (2 mol L1 H2SO4). The absorbance at 450 nm was measured on a Tecan Sunrise 2.5 Microplate Reader (Sunrise, Austria). The ic-ELISA protocol was similar to that described by Peng et al. (2012). Briefly, 96-well Maxisorp microtitre plates were coated with 100 lL of coating conjugates. After being washed and blocked, 50 lL of the antibody and 50 lL of varying concentra-
tions of standard analyte or the samples were added to each well. The plates were then incubated, washed and measured on a microplate reader as described above. A standard dose response curve was obtained according to the OD values and concentrations of standards. 2.6. Sample preparation The samples were prepared according to a previous procedure (Furusawa, 2011) with slight modification. Briefly, 10 Single Comb White Leghorn (SCWL) laying hens (approximate weight 1.5 kg) were purchased from the Breeding Poultry Testing Centre (Huazhong Agriculture University, Wuhan, China). These hens were held in individual cages, and feed and water were given ad libitum. Eggs from seven laying hens that were continuously fed basal layer diets free of carotenoid compounds were collected and used as blank eggs. To validate the present method for routine monitoring, eggs with residual canthaxanthin from three laying hens that were fed a diet containing 10 mg kg1 canthaxanthin for 3 weeks were collected and used as real eggs. The egg yolks were separated from their albumen, homogenously pooled and used as the blank and real egg yolk samples, respectively, and stored at 20 °C until analysis. One gram of each homogenized sample was weighed into 50 mL polypropylene centrifuge tubes. A total of 14 mL of extract solvent (ethyl acetate:2% NaOH solution = 2:1) was added, and the mixture was agitated on a shaker for 5 min. After centrifuging at 4000 g for 10 min at 4°C, 5 mL of the supernatants was transferred into another 10 mL tube and dried using nitrogen gas at 40–50 °C. The residue was re-dissolved in 500 lL acetdimethylamide and then diluted with PBS (0.01 mol L1, pH 7.4) for ic-ELISA. 2.7. Validation of the ic-ELISA method The standard solution (canthaxanthin, b-carotene, b-apo-80 carotenal, xanthophyll, capsanthin) was diluted in PBS (0.01 mol L1, pH 7.4) to obtain a five-point standard curve. Immunoassay validation was carried out using 20 different blank egg yolk samples, which were prepared according to the section on sample preparation and were previously confirmed using HPLC analysis, to be free of carotenoid compounds. Each sample was assayed using ic-ELISA to demonstrate the range of blank matrix effects and determine the LOD. The LOD determination was based on 20 blank samples, accepting no false positive rates, with an average +3 standard deviation (SD). The accuracy and precision of the method were represented by the recovery and coefficient of variation (CV), respectively. The recoveries (%) of spiked canthaxanthin, b-carotene, xanthophyll, capsanthin, and b-apo-80 -carotenal were determined using 5 spiked
D. Peng et al. / Food Chemistry 202 (2016) 141–148
duplicate blanks at 5 mg kg1, 10 mg kg1, and 20 mg kg1 in 3 different analyses. The recovery (%) was calculated by the following equation: (conc. measured/conc. spiked) 100. CVs were determined from the analysis of the above samples spiked with carotenoids at 3 different levels in 5 different analyses. Analysis of each concentration level was repeated in triplicate over a time span of two months. To test the reliability of the developed ic-ELISA for incurred samples, a comparison of ic-ELISA and HPLC was carried out using the samples from the animal feeding experiment. The individual eggs were homogenized and stored at 20 °C until they were subjected to the developed ic-ELISA procedure and to HPLC analysis. All HPLC analysises in this study were performed according to Furusawa’s method (Furusawa, 2011) with slight modification. Briefly, the HPLC system consists of a Waters 2695 separations module and a 2487 dual wavelength absorbance detector (Waters, USA). Chromatographic separations were obtained under gradient condition using a Venusil C8 (250 mm 4.6 mm I.D., 5 lm) column. The column was maintained at a temperature of 30 °C and the wavelength of the UV detector was set at 450 nm. The running time was 25 min, and the injection volume was 50 lL. The mobile phase consisted of 0.1% ammonium acetate, methanol, acetonitrile and water. The limit of detection (LOD) of HPLC method for the various carotenoids ranged from 20 lg kg1 to 40 lg kg1. The mean recoveries of carotenoid analytes ranged from 79.3% to 92.8%. The coefficient of variations (CV) of the intra-day and inter-day were below 14.3%. 3. Results and discussion 3.1. Hapten design and antigen preparation Carotenoid compounds, such as canthaxanthin, b-carotene, b-apo-80 -carotenal, xanthophyll, and capsanthin, are small and simple organic molecules that are nonimmunogenic by themselves. They must be coupled to carrier proteins to induce antibody production. Therefore, according to chemical and immunological constraints, the key step in the production of a broad specific
antibody is to synthesize a generic hapten containing a structure common to all of the analytes. However, as shown in Fig. 1A, such carotenoids, consist predominantly of all-trans b-carotene-4,40 dione together with minor amounts of other groups, which lack functional groups for coupling with carrier proteins. In addition, these carotenoids are sensitive to oxygen. These factors make hapten design difficult. As shown in Fig. 1B, the three-dimensional structures of the carotenoid pigments were analyzed. These carotenoids had a long carbon chain and a b-ionone ring, as well as folding in their spatial structure. The three-dimensional structures revealed that the b-ionone ring, the three methyl groups, and the three carbon atoms linked to the carbon chain were shared by all of the carotenoid pigments. Canthaxanthin, b-carotene, b-apo-80 -carotenal, capsanthin, xanthophyll, and b-ionone acid had an upward folding angle that was formed from the 1, 2, 3, 4 and 6 carbon atoms of the b-ionone ring forming a plane and the 4, 5, 6 carbon atoms of the b-ionone forming another plane; conversely, retinoic acid and retinol had a downward angle. Therefore, our hypothesis is that the b-ionone ring is the antigenic determinant. Based on this hypothesis, we selected b-ionone and retinoic acid as the basic hapten structure, and three haptens, Hapten A, B, and C, were synthesized accordingly. Their measured molecular weights by full scan mass spectra are depicted in Fig. 2. Preparation of the haptens was successfully demonstrated. Haptens A–D were then conjugated to carrier proteins. After identification on an 8453 UV–Visible spectrophotometer, the estimated incorporation rates of Hapten A-BSA (OVA), Hapten B-BSA (OVA), Hapten C-BSA (OVA), and Hapten D-BSA (OVA) were 4.3 (6.8), 4.8 (7.2), 12.1 (16.4), and 5.4 (8.2), respectively. 3.2. Characterization of the bleeding serum and the monoclonal antibody The titer and specificity of antisera from the immunized mice are shown in Table 1. Only the bleeding serum from mice immunized with Hapten B-BSA showed high titer and specificity against
Fig. 2. Full scan mass spectra of haptens: A, Hapten A; B, Hapten B; C, 4-keto-b-ionone; D, Hapten C.
D. Peng et al. / Food Chemistry 202 (2016) 141–148
canthaxanthin. Although the bleeding serum from mice immunized with Hapten A-BSA also showed high specificity against canthaxanthin, but the titer was low. The bleeding serum from mice immunized with Hapten C-BSA and Hapten D-BSA showed either low titer or no specificity against canthaxanthin. Therefore, the spleen cells from mice immunized with Hapten B-BSA were used for the fusion experiment. Supernatants from 96-well plates were first checked by non-competitive ELISA. The positive wells were further checked by indirect competitive ELISA using canthaxanthin as competitor. Twelve clones of the positive wells gave positive results with specific binding to free canthaxanthin. Out of 12 clones, 3 stable clones were chosen to be subcloned for three cycles by limiting dilution to obtained 1 cell per well. Finally, a stable hybridroma, clone C11, which exhibited the broadest selectivity and the highest sensitivity, was selected to produce monoclonal antibody. The detailed results are given in the section on the standard curve and CR for the ic-ELISA. The obtained monoclonal antibody was of the IgG1 isotype possessing a kappa light chain. 3.3. Standard curve and CR for the ic-ELISA In some cases, heterogeneous formats can result in antibodies with a higher affinity towards the analyte compared to the coating antigen or tracer hapten. Thus, the sensitivity achievable using this format as opposed to a homologous one can improve sensitivity substantially. In the present study, coating conjugate Hapten A-OVA, Hapten B-OVA, Hapten C-OVA and Hapten D-OVA were used as homogeneous (B) and heterogeneous (A, C, D) coating antigens. The IC50 with a homogeneous (Hapten B-OVA) format was 100 mg L1, compared to 12 mg L1 with the heterogeneous formats of Hapten D-OVA and Hapten A-OVA and 9.8 mg L1 with the heterogeneous format of Hapten C-OVA. These findings agree with the previous study (Kim, Lee, Chung, & Lee, 2003) in that coating conjugate heterology may improve the sensitivity of ELISA. Therefore, the coating conjugate Hapten C-OVA was selected as the coating antigen in the present study. Finally, the optimum icELISA conditions were determined as 8.0 mg L1 of coating antigen (Hapten C-OVA) and a 1:8.0 103 antibody (C11) dilution. These were the optimal conditions and were fixed for the rest of the experiment. As shown in Fig. 3, two set of standard curves based on the canthaxanthin, b-carotene, b-apo-80 -carotenal, xanthophyll,
capsanthin, and b-ionone acid matrix calibration were obtained. The first set of standard curves (shown in Fig. 3A) ranged from 1–5 mg L1, and the second set of standard curves (shown in Fig. 3B) ranged from 1 to 16 mg L1. Compared these two sets of standard curves, there was no obvious difference in the IC50, with the IC50 value were 2.99 mg L1, 3.22 mg L1, 3.06 mg L1, 3.12 mg L1, 3.32 mg L1, and 2.13 mg L1 for canthaxanthin, b-carotene, b-apo-80 -carotenal, xanthophyll, capsanthin, and b-ionone acid, respectively. However, there was a difference to the linearity of the standard curve. As a screening method, the advantages of ic-ELISA method are rapid and low cost. Therefore, the second set of standard curves was selected for the rest study in this study at last. According to the formula CR = (IC50 of canthaxanthin/IC50 of other carotenoid compounds) 100%, the CR of the obtained mAb C11 was calculated. The antibody C11 showed CR towards canthaxanthin (100%) and exhibited measurable CR with other carotenoid compounds, b-ionone acid (140.4%), b-carotene (92.9%), capsanthin (90.1%), b-apo-80 -carotenal (92.7%), and xanthophyll (95.8%). It did not exhibit measurable CR with other analytes (