Standardisation 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 plaque reduction assay (PRA) which is labour intensive, time consuming and requires subjective input by the reader. An in situ enzyme-linked immunosorbent assay (ELISA) method has been developed with the potential to overcome many of the limitations of PRA, and has been applied to a variety of viruses. Previous reports of ELISA susceptibility assays have shown little standardisation between these methods, or any significant analysis of the variable factors which may influence the outcome of the assay. This study optimised the sensitivity of a microplate in situ ELISA (MISE-test) for the detection of viral growth, manipulated the interaction between cells, virus and acyclovir to determine the effect of their relationship on susceptibility results, and established standard 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. Factors which were critical to the performance of the MISE included inoculum 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 from resistant isolates applying a cutoff ID50 value of 2.0 mg/l. The correlation 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.
Department of Microbiology, Royal Perth Hospital, Australia.
B J
BJ
Leahy
K J
KJ
Christiansen
G
G
Shellam
D000212:Acyclovir / Q000494:pharmacology*; D000818:Animals; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D004352:Drug Resistance, Microbial; D004797:Enzyme-Linked Immunosorbent Assay / Q000295:instrumentation / Q000592:standards*; D005347:Fibroblasts; D006558:Herpes Genitalis / Q000821:virology; D006801:Humans; D008168:Lung; D008826:Microbial Sensitivity Tests / Q000295:instrumentation / Q000592:standards*; D008907:Mink; D018139:Simplexvirus / Q000187:drug effects* / Q000254:growth & development / Q000302:isolation & purification; D014709:Vero Cells; D010948:Viral Plaque Assay
D000212:Acyclovir