STANDARDIZATION OF A MICROPLATE IN-SITU ELISA (MISE-TEST) FOR THE SUSCEPTIBILITY TESTING OF HERPES-SIMPLEX VIRUS TO ACYCLOVIR

Viral susceptibility testing has been traditionally performed by plaqu e reduction assay (PRA) which is labour intensive, time consuming and requires subjective input by the reader. An in situ enzyme-linked immu nosorbent assay (ELISA) method has been developed with the potential t o overcome many of the limitations of PRA, and has been applied to a v ariety of viruses. Previous reports of ELISA susceptibility assays hav e shown little standardisation between these methods, or any significa nt analysis of the variable factors which may influence the outcome of the assay. This study optimised the sensitivity of a microplate in si tu ELISA (MISE-test) for the detection of viral growth, manipulated th e interaction between cells, virus and acyclovir to determine the effe ct of their relationship on susceptibility results, and established st andard assay conditions based on quality controlled parameters such as assay variability and linear ranges. 33 isolates of HSV-2 were tested for susceptibility to acyclovir by PRA, and the standardised MISE. Fa ctors which were critical to the performance of the MISE included inoc ulum size, inoculation method, duration of incubation, fixative type, immunoglobulin working strengths and choice of chromogenic substrate. Using the ELISA it was possible to separate sensitive HSV-2 isolates f rom resistant isolates applying a cutoff ID50 value of 2.0 mg/l. The c orrelation coefficient between PRA and MISE was 0.65. The standardised microplate in situ ELISA was found to be an acceptable alternative to the plaque reduction assay.