The complement components are present in serum in inactive form.

Transcription

1 Lab 3 complement A complex consisting of at least 20 serum proteins, which, once activated, Its lytic activity is destroyed when heated at 56ᵒC for 30 min. acts like a part of the innate immune defense. The complement components are present in serum in inactive form.

2 The complement is activated in a cascading manner (= each protein activates that following) and it has widespread physiologic and pathophysiologic effects. Complement proteins are synthesized mainly in the liver, but tissue macrophages and fibroblasts can synthesize some complement proteins as well.

3 Functions of the complement system: Component C3a C4a C5a Biological Activity Anaphylatoxins (= they cause release of histamine and other vasoactive compounds from basophils and mast cells, increasing capillary permeability) C3b C4b Opsonization (= Activation of phagocytic cells) C3a C5a C567 C5b6789 Chemotaxis (=attracts phagocytic cells to sites of inflammation and increases their overall activity Components of MAC (= complement activation results to the formation of a polymeric protein complex (MAC), causing osmolysis or apoptosis of microbes)

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5 Pathways of complement activation: The classical pathway of complement activation: The pathway is activated through antigen-antibody complexes. The alternative pathway of complement activation: The pathway is activated through reaction of the complement system and some substances of microbial origin(polysaccharides e.g. lipopolysaccharides of G negative bacteria, teichoic acid of G positive bacteria, zymosan from yeast cell walls, surface components of some animal parasites) or other foreign materials.

6 The lectin pathway of complement activation: The pathway is activated through binding of MBL (mannose-binding lectin) to microbial surface.

7 Pathways of complement activation CLASSICAL PATHWAY LECTIN PATHWAY ALTERNATIVE PATHWAY antibody dependent antibody independent Activation of C3 and generation of C5 convertase activation of C5 LYTIC ATTACK PATHWAY

8 THE PRACTICAL STEPS: Measurment of complement concentaration by Single radial immunodiffusion: Principal: Antibody is incorporated into agar and antigen introduced into the well. As antigen diffuses into agar precipitation rings form depending on the concentration of the antigen. Radial Immunodiffusion is used to measure IgG, IgM and complement components.

9 Measurment of Complement Activation by Heat Inactivation methods: Principal: Heat inactivation is a process in which finished serum is placed in a heated water bath and maintained at a temperature of 56 C for 30 minutes. This is the most common method and it was used in the past to inactivate the complement system for immunoassays.

10 0.6 ml serum 0.6 ml normal saline 0.6 ml heated serum 56 C for 30 minutes 0.4 ml E.coli 0.4 ml E.coli 0.4 ml E.coli

11 Incubate the tubes in 37 c for 30 minutes. Calculate number of colonies by plate count method. Calculate cmplement activation by: Complement activation = number of colony in plate heated serum number of colony in plate non heated serum. Example: number of colony in plate normal saline = 80 colony number of colony in plate heated serum = 50 colony number of colony in plate non heated serum = 30 colony Complement activation = 50-30= X 100 = 20 * 100 / 80 = 25%

12 Complement Fixation: Based on the principle that antigen and antibody reaction activates complement. Antigen and antibody, one known and the other unknown are mixed. A measured amount of complement is added. If antigen-antibody reaction has occurred it will combine fix complement.

13 An indicator system consisting of sensitized red blood cells (red blood cells plus anti-red blood cell antibody) is added. If the complement was fixed because of antigen antibody reaction red cells will not be hemolyzed i.e. the test is positive. If the antigen antibody reaction did not occur in the first step complement will not be fixed and will be available to lyse RBCs a negative test.

14 Complement Fixation Methodology Ag mixed with test serum to be assayed for Ab Standard amount of complement is added Erythrocytes coated with Abs is added Amount of erythrocyte lysis is determined Ag No Ag Ag Ag Patient s serum