Development Of Immunoassays For Small Molecule Organic Contaminants In The Environment

Based on the affinity of antigens and antibodies, the enzyme-linked immunoassay was considered as a sensitive and selective method in environmental and biomedical analysis. Antibody, including polyclonal antibody (Pab), monoclonal antibody (Mab) and recombinant antibody, is one of the most important steps in immunoassay. It is also one of the hot topics in biomedical analysis. In this study, take low molecular compounds existed in environment and food as examples:Para red, sudan red Ⅰ,2,2’,4,4’-tetrabromodiphenyl ether and tetrabromobisphenol A, Pab, Mab and camel id single domain antibody (VHH) were introduced and their immunoassays were developed. The details were showed as follows:(1) Development of immunoassay with high sensitivity and specificity for Para red and Sudan red I. Haptens P1-P9of Para red were synthetized, and anti-para red Pab was produced with P1-BSA immunization. Then a sensitive and selective ELISA based on Pab was developed for the detection of Para red in food samples, with the limit of detection (LOD) and inhibition half-maximum concentrations (IC50) of0.06and2.2ng mL-1, respectively. The linear detection ranged from0.24ng mL-1to19ng mL-1. Negligible cross-reactivities were obtained with the structural analogues including Sudan red Ⅰ,Ⅱ,Ⅲ,Ⅳ, and G, sunset yellow,2-naphthol, and4-nitroaniline. The spiked recoveries in tomato sauce, chilli sauce, chilli powder and sausage samples were in a range of90-108%, showing good correlation with high-performance liquid chromatographic method; Monoclonal antibody for sudan red I was produced and its cross-reactivity for para red was18%. The Mab labeled with nanocolloidal gold, was used to detect sudan red I and para red simultaneously by a semiquantitative dip strip assay. The visual limit of detection10ng g-1of sudan red I and50ng g-1of para red in tomato sauce and chilli powder samples were obtained, with the detection time of10min. The strip was stable for at least2months at4℃.(2) Development of competitive and non-competitive enzyme-linked immunosorbent assay for2,2’,4,4’-tetrabromodiphenyl ether (BDE-47). In this study, Mab for BDE-47was produced and a sensitive direct ELISA for BDE-47in environmental samples was developed, with IC50of1.4±0.05ng mL-1. This assay was used to determine BDE-47in soil, sediment and house dust samples, showing a statistically significant correlation with those of a gas chromatography-mass spectrometry method (R2=0.79-0.85); And then, phages selectively bind to Mab1H2specific to BDE-47and "BDE-47-Mab" complex have been selected from phage display libraries. Competitive and noncompetitive phage ELISA for BDE-47were developed, with slightly differences on assay sensitivities, IC50of the competitive phage ELISA and the half-maximum signal enhancement concentration (EC50) of the noncompetitive phage ELISA for BDE-47being6.8ng mL-1and4.2ng mL-1, respectively. The noncompetitive phage ELISA showed higher cross-reactivities with BDE-28, BDE-99and BDE-100(1.3-6.5%) than those of the competitive one. The average recoveries of competitive phage ELISA and noncompetitive phage ELISA in sewage sludge and fish fillet samples were in a range of96-124%and 97-120%, respectively. The results in sewage sludge samples showed good correlation (0.99and0.99) with a gas chromatograph/electron capture detector-ion trap mass spectrometer method, while in fish fillet samples lower correlations (0.57and0.75) were obtained.(3) Development of enzyme linked immunosorbent assay for tetrabromobisphenol A (TBBPA) based on camiled single domain antibodies. In this study, naturally occurring antibodies in camelids are devoid of light chain, named single domain antibodies (nanobodies, VHH), which were employed for TBBPA detection. An alpaca was immunized with a TBBPA hapten T5coupled to thyroglobulin and a phage display VHH library was constructed. VHH T3-15highly selective for TBBPA was isolated from the phage display VHH library using heterologous coating antigen T3-BSA. An indirect immunoassay was developed for TBBPA by purified VHH, with IC50of0.40ng mL-1. The recoveries of TBBPA in soil and fetal bovine serum samples were102%-111%and90%-96%, respectively, by ELISA and agreed well with a liquid chromatography-tandem mass spectrometry (HPLC-MS) method (R2=0.98); Then T3-15was fused with alkaline phosphatase (AP), showing both an integrated TBBPA-binding capacity and enzymatic activity. A one-step immunoassay based on T3-15-AP was developed for TBBPA in5%dimethyl sulfoxide (DMSO)/phosphate buffered saline (PBS, pH7.4), with IC5o of0.20ng mL-1. A simple pretreatment method of diluting urine samples with DMSO (60%) was developed for TBBPA in a one-step assay, with average recoveries of97-110%.Compared with the instrumental analysis, immunoassays, which have proved to be ease of operation, time-saving, sensitive, selective and capable of high throughput, is a useful tool in environmental analysis.