2010-0003669). a relative migration speed. We demonstrated the applicability of this proposal by using it to select an appropriate membrane for the development of an ICA of the pesticide diazinon. strong class=”kwd-title” Keywords: Immunochromatographic assay, Low-molecular weight compounds, Membrane, Diazinon 1. Introduction Immunochromatographic assays (ICAs) have recently begun to gain acceptance as simple, rapid, and inexpensive tools for detecting trace amounts of chemicals and biomolecules [1C3]. The key advantage of an ICA using colloidal gold as the label is that this technique involves only one step unlike other immunoassays that usually require three or four steps. ICAs specific for monovalent low-molecular weight compounds such as pesticides must be competitive in contrast to ICAs for high-molecular compounds that usually involve a noncompetitive sandwich procedure [4C11]. Compared to the large number of other types of immunoassays developed for small molecules, the number of ICAs for small molecules is rather small. This is probably due to difficulties in establishing effective competition in a competitive ICA format. Pre-incubation of sample with AbCCG before performing an ICA described in some papers appears to be evidence of such a difficulty [12C14]. In competitive ICAs, competition is between the migrating analyte and immobilized analyte hapten (capture antigen) Tivozanib (AV-951) for the binding to the migrating AbCCG. The degree of inhibition of AbCCG binding to the capture antigen would be proportional to the frequency of collision between AbCCG and analyte before completion of AbCCG binding to the capture Rabbit polyclonal to KLK7 antigen. Meanwhile, the collision frequency would depend on the concentration of analyte around the migrating AbCCG and the time required for AbCCG to reach the capture antigen. Concentration of the analyte around the migrating AbCCG, in turn, would depend on the relative migration speed of the analyte and AbCCG on the test strip. Therefore, we suggest in the present study that the relative migration speed of the two migrating substances is critically important for sensitive detection by a competitive ICA. We also propose that a suitable Tivozanib (AV-951) relative migration speed of the two migrating substances depends on the type of ICA. We previously discussed the topic of suitable relative migration speed in lateral ICAs [10,11] and we further explore this issue in the current study. Here, we also present a discussion about suitable relative migration speeds for dipstick type ICA. The proposal on Tivozanib (AV-951) suitable relative migration speeds for two types of ICAs was also tested in this study by using the proposal to select an appropriate membrane for detecting the organophosphorus pesticide diazinon. Using the selected membrane and a monoclonal antibody to diazinon, an ICA for the pesticide was developed and validated. 2. Experimental 2.1. Materials and chemicals Pesticides including diazinon were purchased from Dr. Ehrenstorfer (Augsburg, Germany). Gold (III) chloride trihydrate, sodium citrate, BSA, ovalbumin (OVA), polyethylene sorbitan monolaurate (Tween 20), phosphate buffered saline (PBS), indophenyl acetate (IPA), and anti-mouse IgG were purchased from Sigma (St. Louis, USA). Cellulose (Rapid 24) for sample pad and NC membranes (nitrocellulose 8.0, Immunopore RP, FP and SP) for sample pad were obtained from Whatman (Maidstone, UK). Cellulose (Millipore SA3H645H9) for the absorption pad was acquired from Millipore (Billerica, MA). 2.2. Capture antigen and antibody The monoclonal anti-diazinon antibody and capture antigen (diazinon hapten-OVA) used for this study were ones previously prepared in the laboratory of one of the authors (Y.T.L.) [15]. 2.3. Preparation of the AbCCG complexes Colloidal gold was prepared using the method developed by Frens [16]. The procedure was as follows. Fifty mL of 0.01% tetra-chloroauric acid solution was boiled and 1 mL of 1% sodium citrate solution was added under constant stirring. Stirring was continued until the color changes from purple to reddish-orange and, then, the solution was cooled. The cooled solution was adjusted to pH 9.0 with 0.1 M K2CO3. Conjugation of the anti-diazinon antibody to colloidal gold was carried out according to the method by Roth Tivozanib (AV-951) [17]. The optimal ratio of the antibody to colloidal gold was determined using the procedure by Beasley [18]. Before conjugation, the optimal concentration of antibody for conjugation was determined. One milliliter of colloidal gold solution was distributed into each of a series of vial. The antibody solution (0C15 L) was.