ABSTRACT
In addition to tests for the group-specific hexon antigen of adenoviruses, adenoviruses can be detected in clinical specimens by hybridization assays utilizing the widely shared base sequences of the region of the hexon gene that codes for the group-reactive determinants. We have developed a liquid-phase hybridization system with biotin- and europium-labeled probes which are reacted after DNA amplification of a 161-bp region of the hexon gene and which are quantitated by time-resolved (TR) fluorometry in streptavidin-coated microtiter wells. Polymerase chain reaction (PCR)-TR fluorometry is not a rapid test in the usual sense, but it is highly useful for specimens with inherent toxicity or with low virus yield, such as organ minces and specimens obtained late in the course of an illness. In a survey of 103 specimens tested by this method, including urine, stool, and tissue suspensions, the agreement with the hexon-specific TR fluoroimmunoassay antigen test for positive specimens was 100% and the sensitivity compared with that of virus culture was 91%. The PCR-TR fluorometry system was also shown to be advantageous as a quantitative measure of PCR products.
Subject(s)
Adenoviruses, Human/genetics , Adenoviruses, Human/isolation & purification , Capsid Proteins , Polymerase Chain Reaction/methods , Adenoviruses, Human/immunology , Antigens, Viral/genetics , Base Sequence , Capsid/genetics , Capsid/immunology , DNA Probes , DNA, Viral/genetics , Evaluation Studies as Topic , Fluorometry , Humans , Molecular Sequence Data , Nucleic Acid HybridizationABSTRACT
The platelet membrane glycoproteins IIb (GpIIb) and GpIIIa form calcium-dependent heterodimers containing binding sites for fibrinogen, von Willebrand factor, and fibronectin. Although GpIIb and GpIIIa are distinct proteins, both GpIIb and GpIIIa are deficient in platelets from individuals with the recessive disorder Glanzmann's thrombasthenia. To gain a better understanding of the genetic basis for GpIIb and GpIIIa synthesis, we studied their synthesis by two human leukemia cell lines, HEL and K562. HEL cells contained complexes of GpIIb and GpIIIa, and K562 cells expressed GpIIIa, but not GpIIb, when stimulated with phorbol-12-myristate-13-acetate (PMA). RNA from HEL cells directed the in vitro synthesis of a 110,000-Mr precursor for GpIIb and a 92,000-Mr precursor for GpIIIa, which indicates that the synthesis of GpIIb and GpIIIa by HEL cells is directed by separate mRNAs. In contrast, RNA from PMA-stimulated K562 cells only directed the synthesis of a 92,000-Mr precursor for GpIIIa. The dissociation of GpIIb and GpIIIa synthesis in K562 cells suggests that GpIIb and GpIIIa may be the products of separate genes.