Abstract
BackgroundRespiratory syncytial virus (RSV) is the major cause of lower respiratory tract infection in infants and young children. Although several experimental RSV vaccines are under investigation, immuno therapy is the only treatment currently available. In assessing the immunogenicity of various vaccine formulations, a plaque reduction neutralization assay for the evaluation of RSV neutralizing antibody has been widely used. The method produces reliable results, but it is tedious and labor intensive as it relies on manual counting by laboratory personnel. To facilitate evaluation of phase II and phase III vaccine clinical trials, a more rapid, reliable and efficient neutralization assay is needed.ResultsAn improved microneutralization assay for quantifying RSV neutralizing antibodies was developed using an ImmunoSpot® Series I Analyzer (Cellular Technology Ltd., Cleveland, OH) for automated plaque counting. The method is an improvement of the established classical microneutralization assay in which immunostained plaques on transparent tissue culture plates are counted manually under a dissecting microscope. Image analyzer technology allows for fully automated counting of plaques distributed throughout an entire well. Adjustments, such as the use of opaque tissue culture plates and the TMB substrate, True Blue™ (KPL, Gaithersburg, MD), were required to adapt the assay for optimal detection of plaques by the image analyzer. The suitability and the accuracy of the method for counting RSV plaques were determined by comparative testing of a reference serum and two control sera by manual and automated counting methods. The results showed that the two methods were highly correlated (R = 0.9580) and the titers generated by them were within two-fold.ConclusionOur results demonstrate that the semi-automated assay is rapid and reliable. It provides results within two fold to the classical plaque microneutralization assay and is readily applied to the evaluation of neutralizing antibody titers in sera obtained from epidemiology or vaccine clinical trials.
Highlights
Respiratory syncytial virus (RSV) is the major cause of lower respiratory tract infection in infants and young children
Respiratory syncytial virus (RSV) is the most common form of lower respiratory viral infection affecting infants, the elderly, and immunocompromised individuals [1]. It may cause complications ranging from pneumonia and bronchiolitis to death [2]
Our results show good agreement between the visual and automated high throughput counting methods for determining RSV serum neutralization antibody titers
Summary
Respiratory syncytial virus (RSV) is the major cause of lower respiratory tract infection in infants and young children. In assessing the immunogenicity of various vaccine formulations, a plaque reduction neutralization assay for the evaluation of RSV neutralizing antibody has been widely used. Respiratory syncytial virus (RSV) is the most common form of lower respiratory viral infection affecting infants, the elderly, and immunocompromised individuals [1]. In severe cases, it may cause complications ranging from pneumonia and bronchiolitis to death [2]. We demonstrate the utility of a method that automates the most laborious and subjective part of the serum neutralization assay – the determination of plaque number. Our results show good agreement between the visual and automated high throughput counting methods for determining RSV serum neutralization antibody titers
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