Screening and optimisation of an ELISA method for the quantitative detection of enterovirus specific RT-PCR products by means of a two-level experimental design.
In a previous paper, optimal reaction conditions were determined for the RT-PCR part of a quantitative enterovirus specific RT-PCR ELISA method (J. Pharm. Biomed. Anal., 25 (2001) 131-142). In order to obtain a detection limit as low as possible, the ELISA part of the procedure was optimised as well. This was done by investigating the influence of seven factors at three levels in a multivariate approach. A reflected two-level screening design, derived from a Plackett-Burman design, was used. Optimal reaction conditions were established by calculation and by evaluation of the effects of the factors on the measured absorbance of the ELISA detection. Under these conditions, the linear range and detection limit of the test were determined and compared with the ELISA conditions before optimisation. The optimised RT-PCR ELISA will be used to study a possible longitudinal relationship between enteroviruses and the development of multiple sclerosis and juvenile diabetes.
Lauwers
S
S
Department of Microbiology and Hygiene, Vrije Universiteit Brussel, Laarbeeklaan 103, B-1090, Brussels, Belgium.
Vander Heyden
Y
Y
Rombaut
B
B
D004770:Enterovirus / Q000235:genetics / Q000302:isolation & purification*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D016679:Genome, Viral; D012367:RNA, Viral / Q000032:analysis*; D012107:Research Design; D020133:Reverse Transcriptase Polymerase Chain Reaction
D012367:RNA, Viral