An immunoassay for terbutryn using direct hapten linkage to a glutaraldehyde network on the polystyrene surface of standard microtiter plates

Abstract

2-Aminobutylamino-4-ethylamino-6-isopropylamino-1,3,5-triazine (ABA-atrazine) has been synthesized and used as a coating hapten in an immunoassay with a monoclonal antibody against terbutryn. Coating was achieved by covalently linking ABA-atrazine to a glutaraldehyde polymer network directly bound to the polystyrene surface of a standard 96-well microtiter plate. The assay was carefully optimized. In particular, the coating hapten concentration had a strong effect on the ELISA sensitivity. By including a pre-incubation step a low test midpoint (IC50-value) of 0.130 microg L(-1) was achieved. As far as we are aware this is the most sensitive ELISA for terbutryn yet reported. The coating-hapten-format presented is proposed as generally applicable, because the glutaraldehyde-modified microtiter plate surface enables stable immobilization of a broad variety of coating haptens.

Direct hapten coated immunoassay format for the detection of atrazine and 2,4-dichlorophenoxyacetic acid herbicides

“…In case of 2,4-D, the related analogues were different due to their ester, butyric acid and sodium monohydrate groups rather than the phenoxyacetic acid group, and hence in their cross-reactivity. The detection limit of our assay was thus observed much higher (0.260 ng mL −1 for 2,4-D) than the detection limit reported in the previous studies [26,27].…”

An Immunoassay for Dibutyl Phthalate Based on Direct Hapten Linkage to the Polystyrene Surface of Microtiter Plates

“…Thus it was needed to immobilize the aminated hapten DBAP to the surface of polystyrene. Holthues et al had linked an aminated hapten ABA–atrazine to a glutaraldehyde polymer network directly bound to polystyrene surface and developed very sensitive ELISA to detect terbutryn [10]. However, it was a failure to establish hapten coated ELISA for DBP based on covalently linking DBAP to a glutaraldehyde polymer network on polystyrene surface.…”

Comparison of an Indirect Format ELISA on Modified Graphite and Polystyrene Surfaces against Triazines

“…To improve the IC50 value pre-incubation techniques are discussed in literature (Zettner and Duly, 1974;Weller, 1992). Using this sequential incubation of antibody and hapten (Holthues, 1998) could decrease the IC50 value by a factor of two, compared to an ELISA without pre-incubation, for an competitive ELISA with immobilization of atrazine derivatives to MTP. In the present report pre-incubation was checked for the antibody P6A7 and C6 as coating hapten and the IC50 value decreased by a factor of two in the case of MPT, but for graphite the IC50 value decreased markedly by nearly a factor of six.…”

“…The assay on MTPs was carried out as described previously (Holthues et al, 2001) After washing with distilled water coating with the ELISA haptens (C2 to C8, respectively) was performed through imino coupling of the ELISA haptens to the free aldehyde groups of the GA polymer network using 200 µl coating hapten solutions prepared in PBS buffer at pH 8. Reaction was over night at room temperature.…”

“…In Piehler et al (1996) glass-type surfaces modified by several hydrophilic polymers of different functional properties to immobilize aminocaproic acid atrazine are used, and in Trau et al (1997) 2,4-D (2,4-dichlorophenoxyacetic acid) was immobilized to the surface of quartz capillaries utilizing poly-l-lysine. A glutaraldehyde network was used for immobilizing several atrazine derivatives on polystyrene microtiter plates (MTP) (Holthues et al, 2001(Holthues et al, , 2005. A thiolated pyrene derivative was immobilized on a gold surface in an amperometric immunosensor for the determination of benzo[a]pyrene (Liu et al, 2000).…”