Research: Complement assay Flashcards
Project 4
The CH50 and AP50 assays for the complement its (1) and (2) rely on their (3) ability
1 Classic pathway
2 Alternative pathway
3: hemolytic
Aim: test functionality of complement system of patients
Why is the lectin pathway not analyzed with a hemolytic assay?
There are better assays available to test this complement activation pathway
Complement results
Lysis: damage membrane pathogens with the MAC consisting of C5-9
Opsonization: with C3b, to induce phagocytosis
Inflammatory responses is induced by attracting immune cells
Three pathways complement system
- Classical pathway: activation through antibody binding
- Lectin pathway: activation through mannose binding lectins, mannose is more prevalent on bacterial cell walls, induced by immune complexes
- Alternative pathway: via spontaneous C3 hydrolysis which mimics C3b. C3b also induces self-amplification loop
C3-9 activation effects
C3a and C5a (anaphylatoxins): recruit neutrophils and monocytes
C3b: opsonization
C5b: induce MAC formation for lysis consisting of C5-C9 components
Complement hemolytic assays are based on the ability of the complement to …
Form the MAC complex which induces hemolysis, causing red color by hemoglobin release through this pore > suspension will turn red
CH50 assay components
Sheep erythrocytes + patient serum
> Sheep erythrocytes are manually opsonized using IgG (rabbit anti-sheep IgG)
> presence of IgG leads to activation classical pathway through C1q of complement
> Sheep erythrocytes contain high concentrations of sialic acid in membrane, like human RBCs. Therefore there is much Factor H that can bind on the membrane, degrading C3b and therefore inhibiting the alternative pathway, so that only the classical pathway is analyzed.
Why animal erythrocytes used in CH50 and AP50 assays?
They are immunogenic and should be lysed by the complement of the sera, however in the patients it may be impaired
When complement is inactive in patients through genetics or consumption, then the suspension will turn … in CH50/AP50
Red cell pellet
> when active complement: red suspension.
In a CH50 assay, the activity of which complement factors is analyzed?
C1, C2, C4 (involved in classical route activation)
C3, C5-C9 (involved in central step and MAC complex)
AP50 assay components and concept
Rabbit erythrocytes used
> contain low concentration of sialic acid in the membrane, so low amounts of Factor H can bind and degrade C3b
> C3b can bind the membrane and initiate the amplification cycle of the alternative pathway
> classical pathway is protected from activation: lack of IgG opsonization and the addition of Mg-EDTA in the assay
> Mg-EDTA can bind Calcium, an essential mineral in the classical pathway. Classical pathway is blocked > chelator calcium
AP50 assay measures activity of which complement factors?
Factor B, Factor D, Factor P, C3, C5-C9
CH50 and AP50 > why 50?
Dilution series of sera are made for patients
> measurement hemolysis
> when 50% hemolysis?
> in standard control: for example 8x dilution needed for 50% hemolysis
> when low complement activity in patient: lower dilution leads to 50% hemolysis
» for example when 4x > then 4/8: the complement is 0.5x active
> When high complement activity: higher dilution results in 50% hemolysis of erythrocytes. For example 16x > 16/8: 2x more active
Conclusions high and low complement activity
Low activity: genetic defect in one of the factors or immuno deficiency
High activity: possible (chronic) infection
Why must sera be kept on ice at all times during complement assay?
Complement activation is temperature-dependent so ice will prevent ex-vivo complement activation before testing.
