A simple method for improved assay demonstrates that HIV p24 antigen is present as immune complexes in most sera from HIV-infected individuals.

Improved detection and quantitation of p24 antigen of the human immunodeficiency virus (HIV) in sera was obtained by pH 2.5-3.0 pretreatment of samples before using a standard HIV p24 antigen ELISA. Pretreatment dissociated immune complexes and denatured antibodies with little or no compromise of p24 antigen immunoreactivity. For 652 HIV antibody-positive sera, direct comparison of the pretreatment with the conventional assay demonstrated substantial increase in both antigen positivity (50.6% vs. 12.4%) and in the level of p24 antigen in sera. Serum HIV antigen is mainly in the form of immune complexes in most individuals at all stages of HIV infection. Longitudinal study of 1 year (three measurements) on 29 seroconverters demonstrated two main patterns of p24 antigen expression in sera: 34.5% of infected individuals never express any form of detectable HIV antigen and 58.6% persistently demonstrate serum p24 antigen, usually in complex with antibody. Only 6.9% show episodic p24 antigen positivity.

Absence of infectious HIV-1 in the urine of seropositive viremic subjects.

Urine and peripheral blood samples from 48 human immunodeficiency virus type 1 (HIV-1) seropositive individuals (38 adults and 10 children) were evaluated for the presence of HIV-1 by cocultivation and for HIV-1 p24 antigen by ELISA. None of the urine samples contained replication-competent HIV-1; 41 (85%) of 48 simultaneously obtained peripheral blood mononuclear cell samples contained replication-competent HIV-1. None of 26 urine samples available for analysis contained HIV-1 p24 antigen as determined by ELISA; 12 (34%) of 35 simultaneously obtained peripheral blood samples had detectable serum HIV-1 p24 antigen. Two of the individuals studied had HIV nephropathy, three had pyuria, and five had microscopic hematuria. Culture sensitivity was maximal when mycostatin (and not amphotericin B) was used as an antifungal agent. Our findings indicate that urine from HIV-1-seropositive individuals is unlikely to contain infectious HIV-1. This would imply that the risk of transmission of HIV-1 by urine is low to nonexistent.

Halibut muscle 3-phosphoglycerate kinase. Chemical and physical properties of the enzyme and its substrate complexes.

An efficient procedure for the purification of 3-phosphoglycerate kinase (PGK) from Pacific halibut muscle is described. The molecular weight (43500) and specific activity are similar to those of other species of PGK. The isoelectric point (greater than 9.5) is more than 1.4 pH units higher than that reported for mammalian muscle PGK. The reaction of the seven thiol groups with 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2) is kinetically biphasic; reaction at a single fast-reacting thiol inactivates the enzyme. The binding of all substrates and products to PGK was observed by 31P NMR. 1,3-Diphosphoglycerate (1,3-P2G) is more tightly bound than is any of the other reaction components. Unlike 1,3-P2G in aqueous solution, the complex with PGK is protected from hydrolysis over a period of weeks. The 31P chemical shifts of this complex are insensitive to pH which suggests that solvent water is excluded from the substrate-bound cleft. As with yeast PGK, the equilibrium constant for the phosphoryl transfer reaction is near unity in the enzyme site environment in contrast to a value of approximately 10(3) (in favor of ATP) in aqueous solution. Since the ternary complex equilibrium 31P NMR spectrum can be accounted for entirely on the basis of the various binary complex spectra, there is no compelling evidence for the involvement of a stoichiometrically substantial phosphoenzyme intermediate.