The Preparation Of Four Phthalate Plasticizer Artificial Antigens And Their Identification By Immunoassay

Phthalates are plasticizers which were widely used in industry process and other consumer products to make them flexible. These products are that we use every day such as polyvinyl chloride plastics, food packing bags, children’s toys, spices, cosmetics and so on. At the same time, phthalates are environmental hormones contamination. They can be released and diverted into ecological environment under some condition. Furthermore, they can move into human body to threaten human health through skin, respiratory system and digestive system. Therefore, it is important to establish an effective and sensitive method to determine phthalates.In this study, four hapten derivatives were obtained from 4-nitrophthalic acid throμgh esterification and reduction with hydrogen. The derivatives have liner structμre, branched chain structμre, stretch arm and rings respectively. In order to make artificial antigens, hapten derivatives were coupled with carrier proteins like bovine serum albumin (BSA) and ovalbumin (OVA) via diazotization method and activated ester method. Then eight female New Zealand White rabbits were injected in subcutaneous and intramuscular to get polyclonal antibodies. Each artificial antigen inject two rabbits. Antibodies were obtained by venous blood. Some reaction conditions of the indirect competitive enzyme-linked immunosorbent assay (icELISA) were optimized to make the method more sensitive. Then it was used to detect the sensitivity and specificity of the antibodies.The successful preparation of the main derivatives were detected by 1H nuclear magnetic resonance (1H NMR), mass spectrometry (MS) and infrared spectra (IR). The successful synthesis of artificial antigens were detected and identified by Mltraviolet spectroscopies (MV) and SDS-polyacrylamide gels electrophoresis (SDS-PAGE). The sensitivity and specificity of antibodies were determined by indirect competitive enzyme-linked immunosorbent assay (icELISA). The result showed that the highest titer of DBAP-BSA antibody was 1:64000 and the other three were 1:128000. The limit of detection were DBAP-BSA (52.8ng/mL), DBCP-BSA (35.5ng/mL), DiBAP-BSA (8.7ng/mL) and DCHAP-BSA (11.9ng/mL). The liner range were DBAP-BSA (81.96-1138.037ng/mL), DBCP-BSA (55.72-832.945ng/mL), DiBAP-BSA (16.753-857.328ng/mL) and DCHAP-BSA (63.997-1222.221ng/mL). The recoveries from spiked oil samples all within the range from 86% to 105%. Therefore, we can get following conclusions: 1) The four hapten derivatives were synthesized successfully.2) The antibodies had high sensitivity and specificity.3) The hapten derivative which has branched chain structure (DiBAP-BSA) is easier to be identified and produce antibodies. The next were rings (DCHAP-BSA) and stretch arm (DBCP-BSA). Branched chain and ring structure are high immune activity groups.