A Highly Sensitive Indirect Competitive Enzyme-Linked Immunosorbent Assay (ic-ELISA) by Antigen Coating for Diethyl Phth
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A Highly Sensitive Indirect Competitive Enzyme-Linked Immunosorbent Assay (ic-ELISA) by Antigen Coating for Diethyl Phthalate Analysis in Foods Mingcui Zhang & Xiaona Yu & Yue Wang & Yurong Hu & Shaohui Liu
Received: 10 September 2012 / Accepted: 5 November 2012 / Published online: 17 November 2012 # Springer Science+Business Media New York 2012
Abstract As we have known, with the plasticizer disturbance in 2011 in Taiwan, long-term exposure to diethyl phthalate (DEP), one of the widely used phthalate esters, can lead to serious health problems. Therefore, a highly sensitive indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) by antigen-coated plate format for DEP in foods was proposed in this paper. The polyclonal antibodies were raised against diethyl 4-aminophthalate (4-DEAP) conjugated to bovine serum albumin by the amino diazotization linkage method. Coating antigen was prepared with 4-DEAP conjugated to ovalbumin using the same procedure. Under the optimal experimental conditions, the ic-ELISA has a linear working range of 0.005–18.6 ng/mL (R2 00.9921), with a limit of detection of 0.0049 ng/mL. Low cross-reactivity (99.5 %) were supplied by Shanghai Chemical Reagent, Co. (Shanghai, China). Freud’s incomplete adjuvants (lanoline/paraffin liquid, 1:2, concussed by ultrasonic cleaner for 3 h) and complete adjuvants (Freud’s incomplete adjuvants with Bacille Calmette–Guerin vaccine) were prepared in our laboratory. Buffers and Solutions The coating buffer (CB) was 0.05 mol/L, pH 9.6, carbonate buffer. The assay buffer phosphate-buffered saline (PBS) was 0.01 mol/L (pH 7.9), 0.296 g NaH2PO4, 2.964 g Na2HPO4, 8.0 g NaCl, and 0.2 g KCl. The washing buffer (PBST) was PBS containing 0.05 % Tween-20. Blocking solution was 1 % OVA dissolved in PBS. The substrate solution is composed of 1.02 g citric acid, 3.68 g Na2HPO4 per 100 mL, 4 mg OPD, and 15 μL of 30 % H2O2 per 10 mL before using. The stop solution is 2 mol/L sulfuric acid (H2SO4) used to stop the enzymatic reaction. In this work, all reagents were of analytical reagent grade, unless specified; tridistilled water was used for the preparation of solutions in the whole procedure. Preparation of Protein–Hapten Conjugation Because the target DEP is of a small molecular weight (MW0222.24), this must be presented to the immune system covalently linked to a carrier, customarily a protein, to be immunogenic. Hence, for the DEP, we synthesized diethyl 4-nitrophthalate and diethyl 4-aminophthalate (DEAP) in our laboratory (Zhang et al. 2007). Then, the conjugation of DEAP coupled to BSA via amino diazotization linkage was used as the immunogen (Zhang et al. 2006). Simultaneously, we prepared the conjugation of DEAP with OVA as the coating antigen in the same way. Both the protein–hapten conjugations were stored at −20 °C. Obtaining the Specific Polyclonal Anti-DEP Antibodies The polyclonal antibody was prepared by immunization with DEAP-BSA to rabbits and purified by a modified saturated ammonium sulfate method in our previous work (Zhang et al. 2010). The a
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