Development and validation of dot-ELISA on modified cellulose filter paper: a simplified novel approach

Abstract

Graft polymerization of glycidyl methacrylate (GMA) onto cellulose filter paper (CFP) was carried out by a free-radical polymerization process and grafted surfaces were characterized by standard polymer characterization techniques. Reactional profiles of GMA and cellulose were elucidated using molecular mechanics energy relationships by exploring the spatial disposition of molecular entities. CFP-g-GMA surfaces with different graft levels of GMA were evaluated and standardized for their application in dot-ELISA in two steps. In the first step, sensitivity, specificity and reproducibility of the assay on the GMA grafted cellulose surface were evaluated through a model system using rabbit anti-goat IgG, goat anti-rabbit IgG and the goat anti-rabbit IgG–HRP-conjugate. A variety of blocking agents and different levels of conjugate dilutions were screened to standardize the assay. The rabbit-anti-goat IgG antibody at a concentration as low as 6 ng mL⁻¹ was efficiently detected on CFP with a 70% GMA graft level using 5% skimmed milk as a blocking agent and the antispecies–IgG–peroxidase conjugate diluted 2000 times. In the second step, sensitivity and specificity of the developed system were established with human blood and finally used to identify the source of mosquito blood meal, an important parameter in epidemiological studies, particularly in determining the role of mosquitoes in malaria transmission. The time duration of standardized assay reduced to 90 min compared to 3–4 h of usual dot-ELISA. For mass screening, as for epidemiological studies under field conditions, sheets of higher dimensions – instead of small strips as utilized by the researchers for laboratory studies – can be used for multiple spotting.