Quantifying Aß(1-40) and Aß (1-42) Using Sandwich-ELISA.

The role of Aß accumulation in the pathogenesis of Alzheimer's disease (AD) is supported by genetic studies showing that mutations in the amyloid-ß precursor protein (APP) that alter Aß production are linked to a subset of familial AD (FAD) cases with autosomal penetrance (reviewed in ref. 1). Several of these FAD-associated APP mutations, as well as FAD-associated mutations in the presenilin 1 (PS1) and presenilin 2 (PS2) genes, lead to an increase in the production of Aß(1)-(42) relative to Aß(1)_(40). This, combined with the observation that these peptides are differentially deposited in senile plaques (SPs) in vivo, suggests that differential production of Aß(1)-(40) and Aß(1)_(42) may be crucially important in the pathogenesis of AD. Thus, it is important to use techniques that not only quantitate Aß production, but also specifically differentiate between these two peptides in a variety of experimental paradigms. Here we describe the use of a highly sensitive sandwich-ELISA (enzyme-linked immunosorbent assay) to quantitate both Aß(1)-(40) and Aß(1)-(42) in soluble pools, after secretion by cultured cells into the medium or in human cerebrospinal fluid (CSF) samples, as well as in insoluble pools, as found intracellularly in cultured cells, or deposited in the brain parenchyma.

A Cell-Cell Fusion Assay to Monitor HIV-1 Env Interactions with Chemokine Receptors.

Biological membrane fusion is an important part of many cellular processes and is a critical step in the entry of enveloped viruses, such as HIV-1, into cells. For HIV-1 to infect cells, the virus must bind to the cell surface, after which the viral Env protein must be triggered to undergo a conformational change that mediates membrane fusion. Cell-surface binding has long been known to occur via a high-affinity interaction between Env and CD4. However, the cell-surface molecules responsible for triggering the fusion-inducing conformational change in the Env protein have been only recently identified, permitting the study of HIV-1 Env-mediated membrane fusion in much greater detail (for review, see 1). These molecules, termed coreceptors, have been shown to be members of the nine-transmembrane domain receptor family. The most important HIV-1 coreceptors are the chemokine receptors CCR5 and CXCR4 (2-7), although at least nine other chemokine receptors or orphan receptors have been shown to support cellular entry for subsets of HIV-1 or SIV strains (3,5,8-11). The ability of a given virus strain to utilize particular chemokine receptors is a major determinant of cellular tropism. Thus, it is desirable to identify the receptors used by virus strains in a rapid, quantitative, and reproducible manner.