Synthesis and identification of artificial antigen for dibutyl phthalate
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摘要:目的
制备并鉴定1种新型邻苯二甲酸二丁酯人工抗原。
方法以4-硝基邻苯二甲酸为原料，经过酯化、还原生成4-氨基邻苯二甲酸二丁酯(DBAP)，DBAP再与戊二酸酐反应，衍生生成4-戊二酸单酰邻苯二甲酸二丁酯(DBCP)。将合成的半抗原衍生物DBCP通过活性酯法与牛血清蛋白(BSA)或卵清蛋白(OVA)偶联，得到人工抗原。通过免疫新西兰大白兔获得抗血清，经间接竞争酶联免疫法(icELISA)检测抗体特异性。
结果经质谱、氢核磁共振谱图鉴定，半抗原衍生物合成成功，经紫外光谱、SDS-聚丙烯酰胺凝胶电泳和免疫试验证实偶联成功，并计算得出DBCP与载体蛋白BSA和OVA的结合比分别为11:1和17:1。经icELISA法检测，兔1和兔2的血清效价均达到1:128 000，IC50分别为301和308 ng·mL-1，反应特异性较好。
结论半抗原衍生物的合成是方便、安全的。邻苯二甲酸人工抗原的成功制备使获得DBP多克隆抗体以及建立DBP免疫分析方法成为可能。
Abstract:ObjectiveThis study was aimed to prepare and identify a new kind of artificial antigen for dibutyl phthalate.
MethodDibutyl 4-aminophthalate (DBAP) was obtained via esterification and reduction of 4-nitrophthalic acid. The hapten derivative monoglutaryl 4-amino-dibutyl phthalate (DBCP) was prepared through DBAP reacting with glutaric anhydride. DBCP was conjugated to bovine serum albumin (BSA) or ovalbumin (OVA) to make artificial immunogen via activated ester method. Then the antiserum of two female New Zealand white rabbits was obtained after injecting with artificial immunogens which were successfully coupled. Indirect competitive enzyme-linked immunosorbent assay (icELISA) was used to analyze the sensitivity and specificity of the antiserum.
ResultThe successful synthesis of hapten derivative was identified by mass spectrometry (MS) and 1H nuclear magnetic resonance (1H NMR). The successful coupling was proved by ultraviolet spectroscopy (UV) and SDS polyacrylamide gels electrophoresis (SDS-PAGE). Furthermore, the numbers of DBCP molecules per BSA/OVA molecules for two conjugates were 11:1 and 17:1 respectively, estimated by ultraviolet spectrophotometry. The icELISA method indicated high specificity for the antiserum with the highest titer being 1:128 000 for samples from both rabbits, and the IC50 being 301 and 308 ng·mL-1 respectively.
ConclusionThe hapten derivative can be synthesized in a convenient and secure way. The successful synthesis of the artificial antigen for dibutyl phthalate makes it possible to get polyclonal antibodies and establish immunoassay for DBP.
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Keywords:
- dibutyl phthalate /
- artificial antigen /
- spectrum /
- immunity identification
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图 1 半抗原衍生物DBAP的1H NMR(a)和MS(b)图谱
Figure 1. 1H NMR (a)and MS(b) spectrums of hapten derivative DBAP
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图 2 半抗原衍生物DBCP的1H NMR(a)和MS(b)图谱
Figure 2. 1H NMR(a) and MS(b) spectrums of hapten derivative DBCP
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图 4 BSA和DBCP-BSA以及OVA和DBCP-OVA的SDS-PAGE电泳图
M1:重组蛋白Marker 1; 1：BSA；2:DBCP-BSA; M2:重组蛋白Marker 2; 3：OVA; 4:DBCP-OVA。
Figure 4. SDS-PAGE gel of BSA, DBCP-BSA, OVA and DBCP-OVA
表 2 多克隆抗体与DBCP结构类似物的交叉反应
Table 2 Cross-reactivity of the polyclonal antibody against DBCP related compounds
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