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Radioimmunoassay of Human Serum Antibody Specific for Adenovirus Type 5-Purified Fiber¹

From the Department of Microbiology and Immunology and Department of Virology and Epidemiology, Baylor College of Medicine, Houston, Texas 77025

Abstract

A radioimmunoassay (RIA), utilizing a second antibody to separate immune complexes, was developed to provide a sensitive and specific measure of serum antibody to adenovirus type 5 (Ad 5) fiber. Purity of fiber antigen was ascertained by sodium dodecyl sulfate urea-polyacrylamide gel electrophoresis and isoelectric focusing in ampholyte pH gradients. After labeling with ¹²⁵I to high specific activity, the iodinated fiber did not exhibit loss of antigenic reactivity and remained stable for 3 weeks when stored at -20°C with supplemental protein. Rabbit anti-Ad 5 serum with a neutralization titer of 1:320 precipitated 50% of the labeled fiber at a serum dilution of 1:50,000 when tested by the RIA. In competition assays as little as 0.5 ng of unlabeled fiber per milliliter was sufficient to inhibit the ¹²⁵I fiber-antibody reaction. Serum specimens from 20 volunteers, obtained before and after vaccination with purified Ad 5 fiber or hexon subunit vaccine, were tested by RIA, hemagglutination-inhibition (HI), and neutralization tests. A comparison of mean antibody titers of post-inoculation sera showed that the RIA was 300 and 1000 times more sensitive than the HI and neutralization tests, respectively. Moreover, 19 of the men who were negative by the standard serologic tests before vaccination were shown to have anti-fiber antibody, with a mean RIA titer of 1:1028. Specificity of the RIA was demonstrated by the lack of an increase in antibody to Ad 5 fiber among those individuals vaccinated with the hexon subunit. Thus, the development of a highly sensitive and reproducible RIA allows for the detection of antibody specific for the Ad 5 fiber in serum which contains antibodies to the different virion antigenic determinants associated with Ad 5.

Footnotes

¹ This work supported in part by Contract N01 AI 32506 from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20014 and Grant Q-435 from the Robert A. Welch Foundation, Houston, Texas.

² Present address: Department of Virology, Hadassah Medical School, Hebrew University, Jerusalem, Israel.