10 Rapid Microbial Detection Assays Flashcards
What do immunodiagnostic assays do? And what are they good for?
Detect and identify pathogens
Good for clinical/pharmaceutical samples
What do immunodiagnostic assays require?
An antibody to recognise the antigen (pathogen)
Immunodiagnostic assays don’t require what?
The cultivation of the microbes
What do immunodiagnostic assays rely on?
The quality of antigen detection, higher sensitivity means better assay
In agglutination assays, how does clumping occur?
The interaction between antibodies and antigens due to multiple sites on the outside of the pathogen and antigen recognition sites on the antibody
What are the benefits of agglutination assays?
Cheap to perform, highly specific, very quick and useful for many pathogens
Explain the latex bead agglutination test.
Latex beads (0.8 um) are coated with the antibody. The sample is incubated with antibody-latex mix. Serial dilutions are performed with saline and all dilutions are checked for agglutination
What happens if the antigen (ie organism) is present?
The latex suspension agglutinates and clumps indicate a positive reaction
What is the detection limit of a latex bead agglutination test?
0.1-0.2 um / ml
What does ELISA stand for?
Enzyme Linked Immunosorbent Assay
Explain ELISA and the detection limit.
ELISA uses enzymes attached to antibodies which are indirect forms of detection.
Detection limit is about 1 ng/ml of antigen
What enzymes are most commonly used?
Peroxidase or β-galactosidase
What is TMD and what does it act as?
Tetramethylbenzidine and acts as a donor of hydrogen
What reaction does peroxidase catalyse?
Reduction of hydrogen peroxidase (H2O2) to water by peroxidase enzymes
What colour does the reaction change to?
Blue
How is an ELISA quantified.
Can be dejtected by eye but better to use a spectophotometer
What is a direct sandwich ELISA?
Initially, the reaction vessel is coated with primary antibody, specific for the organism being detected. Excess antibody is washed away. The sample is then added and then washed then the second antibody is added and washed again. Then assay changes colour
Antigen is sandwiched between 2 different antibodies (primary and secondary)
What is an indirect ELISA?
Antigen is attached to the reaction vessel and then the serum is added. It is then incubated and washed. Then an antihuman antibody is added and then detected by enzyme
What does immunocytochemistry use?
Uses tissue sections or fixed cells
How is immunocytochemistry carried out?
Incubate sample with antibody against antigen. Incubate with a second antibody - which has a label
How many antigens is it possible to detect by immunocytochemistry?
More than 1 antigen
