Reactivity

Compared with aqueous-based immunoassays, lateral-flow tests require that the capture reagents remain biologically active after being desiccated on the nitrocellulose for months to years. Many capture reagents meet this criterion, but not without consideration of the chemistry of the buffers used to apply them (10). In general, the reagent buffers should be kept as simple as possible: (1) buffer molarity <10 mM, (2) sodium chloride and other salts eliminated, (3) surfactants and detergents eliminated, and (4) other additives eliminated. Evaporation of the water used in the carrier buffer concentrates other solutes around and onto the capture reagent. The concentration of the buffer and other salts increases as a larger proportion of the water is evaporated, potentially denaturing proteins. The buffer and salts eventually crystallize and may mask the capture reagent. Larger crystals can also clog the pores of the membrane, retarding sample flow. In either case, the net effect is reduction of the ability of the capture reagent to participate in the immunoreaction. Thus, the reagent buffer should contain only those constituents necessary and sufficient to retain biological activity.

Although the application buffer should be kept as simple as possible, the lability of a particular reagent may require a more complex buffer for retaining biological activity or solubility during its preparation. In this case, factors that may compromise adsorption to the membrane must be considered, with the recognition that some reagents may not be usable in a lateral-flow format.

Relative to drug-of-abuse assays, reactivity can be problematic. Drug-of-abuse assays are modeled after competitive and inhibition assays, and often there is only a single antibody available. If the antibody is unreactive when adsorbed to the membrane, it will have to be conjugated to the detector particle. The drug conjugate then has to be used as the capture reagent on the membrane. Because a drug's structure is not subject to denaturation like a protein molecule, the carrier would only need to have sufficient mass to adsorb effectively and prevent steric hindrance by the nitrocellulose polymer. The reagent buffer will have to be tailored to the specific chemical properties of the drug conjugate.

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