Series Of Haptens Research Articles

Development of Cu(II)/Cu(I)-induced quantum dot-mediated fluorescence immunoassay for the sensitive determination of ethyl carbamate.

A series of haptens were rationally designed for producing monoclonal antibodies specific for EC and a simple fluorescence immunoassay platform was developed for the sensitive determination of EC based on alkaline phosphatase (ALP)-triggered Cu+ quenching of CdSe quantum dots (QDs). It was noted that Cd as a fluorescence substrate in CdSe QDs can be selectively substituted by Cu+ that resulted in a more significant fluorescence quenching in comparison with Cu2+. Meanwhile, because ALP catalyzed ascorbic acid phosphate and then assisted the transformation of Cu2+ to Cu+, the change in fluorescence intensity was found to be proportional to ALP concentration. After simple magnetic separation, the sensitivity and linear range of the established assay were improved approximately 53-fold and an order of magnitude, respectively, when compared with the conventional ELISA. The proposed platform was able to both amplify the signal and eliminate matrix interferences, making it a promising to determine EC as well as other contaminants in complex food matrix in a highly sensitive and simple manner. Graphical abstract.

Highly sensitive monoclonal antibody-based immunoassays for boscalid analysis in strawberries

Boscalid is an agrochemical recently developed for crop protection and the most significant member of the succinate dehydrogenase inhibitor group of fungicides. In this study, a collection of high-affinity monoclonal antibodies was generated to boscalid. By using a series of haptens with a linker at alternative tethering sites of the boscalid framework, specific antibodies were isolated as well as antibodies that also recognized the main boscalid metabolite. Two immunoassays were developed using different ELISA formats. Optimized assays displayed very high sensitivities (limits of detection were near 0.01µg/L). Trueness and precision for the determination of the target analyte in strawberry samples was evaluated. Moreover, immunoassay performance was validated with a reference chromatographic method using QuEChERS extracts of fruits from fungicide-treated crops. A monitoring study with strawberry samples from local markets was carried out by immunoassay, showing an occurrence of boscalid of 15% with a maximum residue concentration of 43µg/kg.

Synthesis of derivatives and production of antiserum for class specific detection of pyrethroids by indirect ELISA

Two series of haptens including 3-phenoxybenzoic acid (PBA) and 3-(2-chloro-3, 3,3-trifluoroprop-1-enyl)-2,2-dimethylcyclo-propanecarboxylic acid (CF3MPA) were used to prepare immunogens through attachment of 4-C or 6-C handles. Class selective antibodies were produced by immunising rabbits. Ab502 showed the highest reactivity towards tau-fluvalinate (IC50 1.3 ng mL−1), λ-cyhalothrin (IC50 2.3 ng mL−1), cyfluthrin (IC50 2.2 ng mL−1) and fenpropathrin (IC50 18.5 ng mL−1) among the antibodies in a competitive ELISA. The effects of methanol, pH and salt concentration were optimised for maximum efficiency of the ELISA (Enzyme-Linked ImmunoSorbent Assay). Ab502 (1:80000)/2-OVA-1(0.2 µg mL−1) was chosen for ELISA optimisation. Finally, 0.05 M phosphate buffered saline (PBS) at pH 6.5 containing 30% methanol (v/v) was used to dilute the standards. Target analytes in honey samples were extracted with ethyl acetate by sonication. The samples were spiked with three different concentrations of each compound (tau-fluvalinate, 0.5 ng g−1, 3 ng g−1, 12 ng g−1; λ-cyhalothrin and cyfluthrin 1 ng g−1, 5 ng g−1, 65 ng g−1). The recoveries were 36–59% at the lowest spiking concentration and 61–81% at the higher concentration. This assay might be useful to screen pyrethroid residues in honey or other matrix.

Development of Immunoassays for Type II Synthetic Pyrethroids. 1. Hapten Design and Application to Heterologous and Homologous Assays.

Immunoassays differing in selectivities for pyrethroid insecticides have been developed for the detection of type II pyrethroids, including deltamethrin, cypermethrin, and lambda-cyhalothrin. Two approaches were employed in hapten synthesis to raise antibodies with different cross-reactions: (1) use of three spacer attachment points to offset different parts of molecules from the points of attachment and (2) use of linkers with and without bulky groups in the enzyme conjugate to reduce antibody affinities for the spacer arm in the immunoassay. The first approach resulted in the preparation of three series of haptens with a spacer attached (1) at the aromatic moiety of pyrethroid, (2) through the middle of the molecule, and (3) at the cyclopropane moiety. Haptens based on the derivatives of the pyrethroid metabolites were also prepared. The second approach involved the use of a linker with a bulky (cyclohexane ring) functionality for preparation of an enzyme conjugate. While most combinations of antibody and conjugate could be used in immunoassays for detection of deltamethrin in the 10-100 µg/L range, in most cases the limits of detection of the assays (for total isomers of a particular target pyrethroid) were lowered 10-50 fold by treatment of the pyrethroid standards with dilute alkali to produce a different isomer mix. Fifteen antisera prepared using 8 haptens were each screened with 14 peroxidase conjugates, and 26 antibody/conjugate combinations were selected for further study on the basis of the assay sensitivity, dynamic behavior, and specificity for deltamethrin, cypermethrin, and cyhalothrin. These immunoassays provided 50% inhibition of antibody binding (IC(50)) values between 1.5 and 4.2 µg/L of isomerized total deltamethrin and limits of detection of 0.2-0.7 µg/L. The most sensitive immunoassay for total deltamethrin was obtained using cypermethric acid-KLH as the immunogen and a conjugate based on a derivative of cypermethrin coupled through the middle of the molecule to peroxidase. These provided an IC(50) of 2 µg/L and a limit of detection of 0.2 µg/L of isomerized total deltamethrin. However, no particular hapten design produced antisera of clearly superior sensitivity or specificity for deltamethrin. Differing cross-reactions with the closely related pyrethroids, deltamethrin, cypermethrin, and cyhalothrin, were obtained, and for several antibodies the cross-reaction as well as the limits of detection could be altered by varying the conjugate combinations. Each of the 12 antibody/enzyme conjugate combinations that sensitively detected deltamethrin were very stereospecific, detecting the alphaS, 1R cis, (DM1), and alphaR, 1R cis (DM2) isomers only; the assay sensitivity was greater for the latter isomer.

Patch testing in children.

Over a 3-year period, 533 children aged between 4 months and 14 years were patch-tested with a “pediatric” series of haptens. The children were affected with various types of eczema or with psoriasis of the hands (n = 31). In 78 positive children, there were 112 positive test reactions to various allergens. Nickel, cobalt, ethylenediamine, and potassium dichromate were the most frequent sensitizers. Positive reactions were relevant in 52 (67%) of 78 positive children.

Patch testing in children.

Over a 3-year period, 533 children aged between 4 months and 14 years were patch-tested with a “pediatric” series of haptens. The children were affected with various types of eczema or with psoriasis of the hands (n = 31). In 78 positive children, there were 112 positive test reactions to various allergens. Nickel, cobalt, ethylenediamine, and potassium dichromate were the most frequent sensitizers. Positive reactions were relevant in 52 (67%) of 78 positive children.