Practical 4: Complement Assay Flashcards
What is the complement assay used for?
This assay tests the integrity of the classical pathway of activation and the terminal components which result in MAC formation
How did we carry out this assay?
(2)
Sheep rbcs have been treated with anti-sheep RBC antibodies
Serum is then added - rbc lysis will occur if serum contains functioning complement system
In your own words what is meant by C50?
How much serum did you have to add to lyse 50% of the rbcs
How would you treat a patient with C3 deficiency?
Treat the ongoing infection with antibiotics - profilactic (low dose antibiotic)
Replace C3 via fusions of C3
Give another example of a haemolysis assay
AH50 - alternative pathway
What does it indicate if both CH50 and AH50 levels are highly elevated?
It indicates there is a lack of a complex such as C3 needed in both pathways
What is nephelometry?
(3)
Add antibodies directed against the protein of interest e.g. C3
Where immune complexes form thee will be a scatter of light which will be detected
Standards can be used to calculate the exact concentration
How does formation of the MAC affect a cell?
(4)
Loss of cellular homeostasis
Disruption of proton gradient across membrane
Penetration of enzymes e.g. lysozyme into the cell
Cell death
What can the MAC kill?
(5)
Gram negative bacteria (some exceptions)
Viruses including most envelope viruses
Parasites
Erythrocytes
NOT MOST GRAM POSITIVE
What does a high C50 indicate?
A poor classical complement pathway
What are the two reasons for a high C50?
Low levels of complement
A defect in one of the complement componenets
At what nm do you measure your C50 assay?
541 nm
What is the equation used to measure the amount of lysis?
spec reading of tube/spec of positive control x 100/1
What is your positive control
Red blood cells and water
100% lysis - water lysis rbcs via osmosis
What is your negative control
Buffer and rbcs
0% lysis
