The aperture-defined microvolume (ADM) method: automated measurements of enzyme activity using an inverted fluorescence microscope
- 17 Downloads
The aperture-defined microvolume (ADM) method is based on the relatively constant absorbance or fluorescence of a microvolume of homogeneously coloured material, which is defined by the numerical aperture of the objective.
This paper describes the princile of the method and discusses the equipment needed. The main applications reported so far for the measurement of enzyme activity are reviewed. Among these are the quantification of ELISA and DASS tests used in immunology, kinetic studies of enzymes in solution using fluorogenic substrates, and the measurement of enzyme activity in single cells or cell fractions that have been isolated by flow sorting.
Typical characteristics of automated ADM measurements include a coefficient of variation of less than 3%, a lower detection limit of a few nanogrammes of fluorescing dye (e.g. 4-methylumbelliferone) and a linear relationship between fluorescence yield and fluorophore concentration over a range of 0.01 to 2.5 nmol. The scanning of Terasaki-type trays and 96-well microtitration plates can be completely automated and requires approximately one minute.
KeywordsEnzyme Activity Fluorescence Microscope Kinetic Study Microtitration Plate Typical Characteristic
Unable to display preview. Download preview PDF.
- Deelder, A. M., Tanke, H. J. &Ploem, J. S. (1978) Automated quantitative immunofluorescence using the aperture defined microvolume (ADM) method. InImmunofluorescence and Related Staining Techniques (edited byKnapp, W., Holubar, K. andWick, G.), pp. 31–44, Amsterdam: Elsevier, North Holland Biomedical Press.Google Scholar
- Deelder, A. M., Koper, G., De Water, R., Tanke, H. J., Rotmans, J. P. &Ploem, J. S. (1980) Automated measurements of immunogalactosidase reactions with a fluorogenic substrate by the aperture defined microvolume measurement method and its potential application toSchistosoma mansoni immunodiagnosis.J. Immun. Meth. 36, 269–83.Google Scholar
- Dresden, M. H., Rotmans, J. P., Deelder, A. M., Koper, G. &Ploem, J. S. (1982) Automated measurements of proteinase activity with a fluorogenic substrate using an inverted fluorescence microscope.Analyt. Biochem. 126, 170–3.Google Scholar
- Haayman, J. J. &Wijnants, F. A. C. (1975) Inexpensive automation of the Leitz Orthoplan microfluorometer using pneumatic microcomponents.J. Immun. Meth. 7, 255–70.Google Scholar
- Jongsma, A. P. M., Hijmans, W. &Ploem, J. S. (1971) Quantitative immunofluorescence Standardization and calibration in microfluorometry.Histochemie 25, 329–43.Google Scholar
- Jongkind, J. F., Ploem, J. S., Reuser, A. J. J. &Galjaard, H. (1974) Enzyme assays at the single cell level using a new type of microfluorimeter.Histochemistry 40, 221–29.Google Scholar
- Jongkind, J. F. &Verkerk, A. (1984) Cell sorting and microchemistry of cultured human fibroblasts: applications in genetics and aging research.Cytometry 5, 182–7.Google Scholar
- Dejosselin de Jong, J. E., Jongkind, J. F. &Ywema, H. R. (1980) A scanning inverted microfluorimeter with electronic shutter control for automatic measurements in micro-testplates.Analyt. Biochem. 102, 120–5.Google Scholar
- Ploem, J. S., Tanke, H. J., Al, I. &Deelder, A. M. (1978) Recent developments in immunofluorescence microscopy and microfluorometry. InImmunofluorescence and Related Staining Techniques (edited byKnapp, W., Holubar, K. andWick, G.), pp. 3–10. Amsterdam: Elsevier, North Holland Biomedical Press.Google Scholar
- Van Dalen, J. P. R., Knapp, W. &Ploem, J. S. (1973) Microfluorometry on antigen-antibody interaction in immunofluorescence using antigens covalently bound to agarose beads.J. Immun. Meth. 2, 383–92.Google Scholar