Novel microneutralization assay for HCMV using automated data collection and analysis

Abstract

In addition to being sensitive and specific, an assay for the assessment of neutralizing antibody activity from clinical trial samples must be amenable to automation for use in high-volume screening.To that effect, we developed a 96-well microplate assay for the measurement of HCMV-neutralizing activity in human sera using the HCMV-permissive human cell line HEL-299 and the laboratory strain of HCMV AD169. The degree to which neutralizing antibodies diminish HCMV infection of cells in the assay is determined by quantifying the nuclei of infected cells based on expression of the 72 kDa IE1 viral protein. Nuclear IE1 is visualized using a highly sensitive immunoperoxidase staining and the stained nuclei are counted using an automated ELISPOT analyzer. The use of Half Area 96-well microplates, with wells in which the surface area of the well bottom is half the area of a standard 96-well microplate plate, improves signal detection compared with standard microplates and economizes on the usage of indicator cells, virus, and reagents. The staining process was also streamlined by using a microplate washer and data analysis was simplified and accelerated by employing a software program that automatically plots neutralization curves and determines NT 50 values using 4-PL curve fitting.The optimized assay is not only fast and convenient, but also specific, sensitive, precise and reproducible and thus has the characteristics necessary for use in measuring HCMV neutralizing activity in the sera of vaccine trial subjects such as the recipients of Vical's HCMV pDNA vaccine candidates.

A cytomegalovirus DNA vaccine induces antibodies that block viral entry into fibroblasts and epithelial cells

“…After incubation for 1 h at 37°C, reactions were transferred to wells of clear 96-well plates containing confluent ARPE-19 or MRC-5 cells. Foci of immediate early (IE) antigen-positive cells were detected by the method of Abai et al [36] 5 days post infection of MRC-5 cultures or 10 days post infection of ARPE-19 cultures. The amounts of each virus stock used in neutralizing assays were predetermined to result in 10-20 foci per well by titration on MRC-5 or ARPE-19 cultures and staining for IE antigen-positive cells as described above.…”

Development of an Enzyme-Linked Immunosorbent Spot Assay To Measure Serum-Neutralizing Antibodies against Coxsackievirus B3

“…However, this method is not suitable for high-throughput screening. Abai et al (17) developed a fast neutralization assay for human cytomegalovirus based on the ELISPOT assay. This method quantifies the infected cells based on the expression of the immediate-early 1 (IE1) viral protein through immunoperoxidase staining and the use of an automated ELISPOT analyzer.…”

“…However, the ELISPOT assay is seldom used to assess humoral immunity, even though it was originally developed to analyze antibody-secreting cells (15,16). Exploiting the highly sensitive and high-throughput properties of the ELISPOT assay, some studies in the last few years have attempted to apply it to measure neutralizing antibodies (17,18). This type of ELISPOT-based neutralization test (Nt-ELISPOT) has properties similar to those of the ELISPOT assay, which makes Nt-ELISPOT an ideal alternative method for the measurement of the neutralizing capacities of serum on a large scale.…”

In vitro affinity maturation of a natural human antibody overcomes a barrier to in vivo affinity maturation

“…Neutralization assay Neutralization assays were performed as previously described 19 but with infected nuclei detected using anti-IE antibody Mab810 (Millipore, Cat. MAB810-500UG) followed by Alexa Fluor 488 conjugated secondary (Invitrogen, Cat.…”