Selection and characterisation of membranes by means of an immunofiltration assay. Application to the rapid and sensitive determination of the insecticide carbaryl.
The characterisation and selection of membranes by means of an immunofiltration assay is described. The chemical composition of the membranes was: nitro-cellulose, polyamide, polyvinylidene difluoride, polyethersulfone, cellulose acetate, regenerated cellulose, cellulose nitrate, and glass fibre. In order to characterise the membranes according to their binding capacity, immobilisation stability, sensitivity and hydrodynamic properties, two basic immunofiltration formats were performed. In both formats, enzyme label (horseradish peroxidase, HRP) and colorimetric detection were used. In the immobilised antibody format, three monoclonal antibodies (mAb) against the insecticide carbaryl were immobilised on the membranes by passive adsorption. In the immobilised hapten format, two haptens conjugated to bovine serum albumin (BSA) were immobilised. Immobilon-P was the best membrane with regard to the characterisation criteria and permitted the filtration of large volume (5.0 ml) through the membrane without release of the receptor. The immobilisation of the receptor (antibody or haptenic conjugate) was pH dependent. Good results with regard to mAb-antigen recognition, were obtained using 50 mM carbonate/bicarbonate buffer, pH 9.6. However, the most sensitive assays were achieved using, 10 mM phosphate buffer, 137 mM NaCl, 2.7 mM KCI (PBS), pH 7.4 as immobilisation buffer. Furthermore, all these results permit the choice of the best membrane for the rapid and sensitive determination of carbaryl. This study will assist the development of dipsticks, immunoelectrodes, membrane-based immunoreactors or immunoconcentration devices that are based on the use of membranes as immunosupports.
Morais
S
S
Departamento de Química, Universidad Politécnica de Valencia, Spain.
Maquieira
A
A
Puchades
R
R
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D002021:Buffers; D012721:Carbaryl / Q000032:analysis* / Q000276:immunology; D002417:Cattle; D005374:Filtration; D006241:Haptens; D006863:Hydrogen-Ion Concentration; D008567:Membranes, Artificial; D012680:Sensitivity and Specificity; D013997:Time Factors
D000911:Antibodies, Monoclonal; D002021:Buffers; D006241:Haptens; D008567:Membranes, Artificial; D012721:Carbaryl