Western blotting has been used at one time or another to aid in the diagnosis of infectious diseases including hepatitis C virus (HCV), HIV, Lyme disease, and syphilis, as well as autoimmune disorders such as paraneoplastic disease and myositis conditions, writes Curtis Balmer, PhD, in the October issue of CLN.
In recent years, however, clinical laboratories have been moving away from Western blotting toward more sensitive and specific diagnostic assays, at least for some diseases, Balmer’s article explains.
Using confirmatory diagnosis of HCV infection as an example, Sai Patibandla, PhD, director of the immunoassay group at Siemens Healthcare Diagnostics, explained that movement away from Western blotting for confirmatory diagnosis of HCV infection began with a technical modification called Recombinant Immunoblotting Assay (RIBA). RIBA streamlines the conventional Western blot protocol by spotting recombinant antigen onto strips which are used to screen patient samples for antibodies against HCV. This approach eliminates the need to separate proteins and transfer them onto a membrane.
Since then, clinical laboratories have continued to move away from Western blot-based assays for confirming HCV in favor of the more sensitive technique of nucleic acid testing (NAT).
Confirmation of HIV infection has followed a similar path. Indeed, in 2014, the Centers for Disease Control and Prevention issued updated recommendations for HIV testing that, in part, replaced Western blotting with NAT. This change was in response to the recognition that the HIV-1 Western blot assay was producing false-negative or indeterminable results early in the course of HIV infection.
This raises the question of whether any of the many efforts to improve Western blotting currently underway will produce an assay that exceeds the sensitivity of currently employed techniques such as NAT.
Some of the most exciting and groundbreaking work in this area is being done by Amy Herr, PhD, a professor of bioengineering at University of California, Berkeley. Herr’s approach is to reduce the open aspects of Western blot. “We’ve
been developing these more closed systems that allow us at each stage of the assay to account for [protein mass] losses. We can’t do this exactly for every target of interest, but it gives us a really good handle [on protein mass losses],” she explained.
Pick up the October issue of CLN to read more about efforts to improve and modify the Western blot, and what the future looks like for its uses as a clinical tool.