Wilbert J. Newhall

The use of tween 20 as a blocking agent in the immunological detection of proteins transferred to nitrocellulose membranes

The determination of the immunoreactivity of protein antigens in complex mixtures has been greatly facilitated by combining their separation via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with electrophoretic transfer to nitrocellulose membrane (NCM), and probing of bound proteins with specific antisera. Methods using various buffers and blocking agents have been published, but no studies have been published which compare these methods with each other or with others of potential merit. We have performed such a comparative study using protein antigens from Chlamydia trachomatis and Neisseria gonorrhoeae. In addition, we describe a method that blocks unoccupied protein binding sites on NCM with the nonionic detergent Tween 20, rather than proteins. This system proved to be equivalent or superior to other methods evaluated in the detection of immunoreactive proteins, and permitted staining of the NCM for protein after immunological probing. Such staining allowed precise identification of immunoreactive proteins. In addition, individual stained proteins could be excised and assessed for bound antibody in an indirect radioimmunoassay.

Transmission of Chlamydia trachomatis and Neisseria gonorrhoeae among Men with Urethritis and Their Female Sex Partners

Transmission of Chlamydia trachomatis and Neisseria gonorrhoeae among infected men and their female sex partners was examined using a design enhancing the likelihood that spread was directed from men to women. Chlamydia culture-negative specimens were examined using DNA amplification tests. Infection rates in women exposed to male sex partners with Chlamydia only were 65% (20/31) and with gonorrhea only were 73% (33/45). Infection of women by either agent was not influenced by the number of sexual exposures to or coinfection in men. There was a 98% (40/41) concordance of N. gonorrhoeae isolates among partners by auxotype and serovar. Chlamydia isolates were serotyped using ELISA and immunofluorescence testing and confirmed by nested polymerase chain reaction: 50% (6/12) of men and 57% (8/14) of women yielded mixed serovars. Sixty-four percent of pairs (9/14) were infected with identical serovars and an additional 28% shared at least one serovar. Multiple serovars of C. trachomatis, but not of N. gonorrhoeae, were common in sex partners and exchanged frequently.