We report a protocol that employs direct coating of smaller hapten on microtiter ELISA plates for the detection of low molecular weight analytes such as pesticides in an immunoassay format. In this method, the polystyrene surface of microtiter plates was functionalize with amino groups using 3-aminoprpyltriethoxysilane (APTES) for the covalent linkage to small molecular hapten with carboxyl groups. The developed immunoassay format could be used as convenient quantitative tool for the sensitive and rapid screening of pesticides and other low molecular weight analytes in samples.
Immunological methods such as enzyme linked immunosorbent assay (ELISA) are increasingly becoming important for pesticides residual analysis due to the high inherent selectivity of detecting molecules, i.e., antibodies. The fact that antibodies could be made virtually against any substance, and its usages in developing highly sensitive assay makes this approach quite useful for the analysis of these toxic molecules1-4. These assays apart from being highly specific, exhibit the desired sensitivity and accuracy for the detection of low molecular weight contaminants present in our environment5-8.
An ELISA for small molecules, in general, needs conjugates of the hapten with large carrier protein for coating the wells of microtiter plates. The formation of such conjugates is not always reproducible. This makes it difficult to evaluate hapten-protein stoichiometry and to understand the precise orientation of the hapten on the protein9. It has been observed that protein molecules immobilized on a hydrophobic polystyrene surface by passive adsorption lose their activity and suffer considerable denaturation10. These macromolecules are found to better retain their functional activity when immobilized through extended hydrophilic spacer arms, since sorption on the surface is substantially reduced11.
A polystyrene surface can be modified to improve its hydrophilicity by incorporating various functional groups such as hydroxyl, amino, carbonyl, carboxyl etc. on its surface12-13. However, the direct attachment of hapten molecules to a polystyrene surface is not possible due to the lack of available functional groups on polystyrene. In order to avoid these drawbacks, a method for direct attachment of carboxylated hapten on polystyrene support for binding the biomolecules on modified polystyrene surface was recently reported by our group14-15. In this assay format, we describe a method for generating amino groups on polystyrene microtiter wells using simple one-step aqueous silanization method for binding carboxylated hapten to develop a highly sensitive immunoassay format for hapten (Fig. 1). This method allowed us to link carboxylated hapten to amine grafted polystyrene microtiter plates for the quantification of 2,4-D pesticide. Hapten specific antibodies against 2,4-D was used in the present assay format, which shows high degree of assay sensitivity.