ED: 6-year course Class 9 General properties of viruses. Methods of

Katedra i Zakład Mikrobiologii Lekarskiej
Warszawski Uniwersytet Medyczny
ED: 6-year course
Class 9
General properties of viruses. Methods of culture.
Laboratory diagnosis of viral infections.
General properties of viruses
Viral taxonomy
Replication cycles of the viruses
Methods of virus cultivation
Collection, transport and preparation of clinical material for virological diagnostic procedures
Isolation of viruses from material taken from the patient, initial identification of the isolated virus
based on cytopathogenic effect
Laboratory methods of virus detection and identification
Serological methods in diagnosis of viral infections
Demonstrations:
1. Labware and media used for cell cultures
2. Growing cell culture
3. Complement fixation assay: paired sera
4. Immunochromatographic assays for the detection of viral diarrhoeas
To perform:
1. ELISA: antibodies detection in IgM and IgG class
2. ELISA: antibodies detection, quantitative method
Maciej Przybylski
Copyright © 2013/2014 Warszawski Uniwersytet Medyczny/Medical University of Warsaw
Katedra i Zakład Mikrobiologii Lekarskiej
Warszawski Uniwersytet Medyczny
1. ELISA: antibodies detection (IgM and IgG), qualitative method
Surface of the wells was covered with viral antigens specific for given antibodies.
- High titre of specific standard antibodies was added to „C+” well (positive control).
- Border amount of specific standard antibodies was added to „C/O” well (cut-off control). Results with
lower values are treated as negative.
- Well labelled „C-” constitutes a negative control (no antibodies added)
- Patients’ sera were added to the wells „P1” to „P4”.
Strips were incubated (1h, 37°C) and unbound antibodies were washed away.
Next, enzyme-labelled antibodies against human IgGs and (respectively) IgMs were added to each well in
adequate strips. Strips were incubated for 0,5 hour at 37°C with subsequent washing.
(above steps are already done, see lower section „to be performed”)
2. ELISA: antibodies detection, quantitative method
Surface of the wells was covered with viral antigens specific for given antibodies.
- Wells marked as „1IU”, „10 IU”, „50 IU” and „200 IU” are set of standard antibodies titres calibrated
in international units (IU).
- Patients’ sera were added to the wells „P1” to „P4”.
Strip was incubated (1h, 37°C) and unbound antibodies were washed away.
Next, enzyme-labelled antibodies against human IgGs were added to each well.
Strip was incubated for 0,5 hour at 37°C with subsequent washing.
(above steps are already done, see lower section „to be performed”)
ELISA ASSAYS: TO BE PERFORMED
To each well add 50 µl of ELISA substrate (tetramethylbenzidin – TMB).
Incubate the strip at room temperature for 10 minutes in the darkness.
After incubation stop enzymatic reaction by adding 50 µl of 0,5N H2SO4 (stop solution).
Copyright © 2013/2014 Warszawski Uniwersytet Medyczny/Medical University of Warsaw