Higher Throughput Quantification of Neutralizing Antibody to Herpes Simplex Viruses

Area of Science:

• Virology
• Immunology
• Assay Development

Background:

• Accurate measurement of neutralizing antibodies is crucial for understanding herpes simplex virus (HSV) immunity.
• Existing methods for quantifying HSV neutralizing antibodies can be time-consuming and lack throughput.
• The Herpevac Trial for Women provided valuable clinical isolates and sera for immunological studies.

Purpose of the Study:

• To develop and optimize a rapid, high-throughput colorimetric assay for measuring neutralizing antibodies to HSV in human sera.
• To enhance the sensitivity and reliability of HSV neutralizing antibody detection.
• To investigate the effect of assay conditions, including complement and virus source, on antibody titer measurements.

Main Methods:

• A colorimetric assay utilizing the ELVIS cell line, which expresses beta-galactosidase upon HSV infection, was employed.
• Assay conditions were optimized, including the addition of guinea pig complement and the use of virus from cell supernatant.
• Incubation times for both the assay and substrate were adjusted to achieve a standard optical density of 1.0 for reliable comparisons.

Main Results:

• The optimized assay demonstrated rapid and higher throughput measurement of HSV neutralizing antibodies.
• Addition of guinea pig complement significantly enhanced neutralizing antibody titers, particularly for HSV-2.
• Using virus from cell supernatant, rather than cell lysate, yielded higher antibody titers.
• Complement enabled sera from HSV-2 gD-vaccinated subjects to neutralize both HSV-1 and HSV-2 with equal potency.

Conclusions:

• The developed colorimetric assay provides a reliable and efficient method for quantifying HSV neutralizing antibodies.
• Complement significantly improves the detection of neutralizing antibodies, especially against HSV-2, and equalizes potency against HSV-1 and HSV-2.
• This optimized assay facilitates better comparison among different virus isolates and is valuable for immunological studies of HSV.