A multicenter performance evaluation of the DPP(®) HIV-1/2 assay for the detection of HIV antibodies in various HIV testing algorithms.

BACKGROUND: Multicenter studies were conducted to evaluate the DPP(®) HIV 1/2 Assay using oral fluid (OF) and fingerstick (FS) specimens in two different countries at the point of care (POC). OBJECTIVE: To evaluate the DPP(®) HIV 1/2 Assay using OF and FS specimens when compared to various worldwide algorithms for the detection of HIV. METHODS: At each testing center, each participant was tested using the DPP HIV 1/2 Assay using OF and FS specimens. Each sample was dispersed into a premeasured buffer in a dropper bottle (DPP(®) SampleTainer™ bottle) and added to the sample well of the device followed by the addition of running buffer to the buffer well of the device. Reference testing was performed according to the National testing algorithm of each Country. RESULTS: Assay sensitivity resulted in ranges of 98.9-100% for OF specimens and 99.8-100% for FS specimens. Assay specificity resulted in ranges of 99.9-100% for OF specimens and 99.5-100% for FS specimens. CONCLUSIONS: Assay sensitivity and specificity obtained for both FS and OF were similar. The DPP HIV 1/2 Assay is highly accurate in detecting antibodies to HIV-1/2 with OF and FS specimens when compared to nationally accepted algorithms. The assay is especially advantageous in that the original sample is collected in a closed vial, eliminating the need for recollection of samples at the POC in the event of an invalid result or assay error upon testing.

Evolution of the allosteric ligand sites of mammalian phosphofructo-1-kinase.

Mammalian phosphofructokinase (PFK) has evolved by a process of tandem gene duplication and fusion to yield a protein that is more than double the size of prokaryotic PFKs. On the basis of complete conservation of active site residues in the N-terminal half of the eukaryotic enzyme with those of the bacterial PFKs, one assumes that the active site of the eukaryotic PFK is located in the N-terminal half. Again using sequence comparisons, the four allosteric ligand sites of mammalian PFK have been thought to arise from the duplicated catalytic and regulatory sites of the ancestral PFK. Previous site-directed mutagenesis studies [Li et al. (1999) Biochemistry 38, 16407-16412; Chang and Kemp (2002) Biochem. Biophys. Res. Commun. 290, 670-675] have identified the origins of the citrate and fructose 2,6-bisphosphate sites. Here, site-directed mutagenesis of two arginine residues (Arg-433 and Arg-429) of mouse phosphofructokinase is used to identify the ATP inhibitory site, and, by inference, the AMP/ADP site. Mutation of the residues to alanine reduced ATP inhibition in the case of Arg-429 and eliminated ATP inhibition in the instance of Arg-433. The Arg-433 mutant could be inhibited by citrate, and that inhibition could be reversed by fructose 2,6-bisphosphate and cyclic AMP, a high-affinity ligand for the AMP/ADP binding site. It is concluded that the two inhibitors, ATP and citrate, of mammalian PFK interact with sites that have evolved from the duplicated phosphoenolpyruvate/ADP allosteric site of the ancestral PFK. The two sites for activators, fructose 2,6-bisphosphate and AMP or ADP, have evolved from the catalytic site of the ancestral precursor.