This document introduces an experiment to test hemagglutination and complement-mediated lysis. Red blood cells (RBCs) are incubated with serum containing antibody and complement, or with heated serum lacking complement. Hemagglutination occurs when RBCs bind to antibody alone. Hemolysis occurs when RBCs bind antibody and complement, leading to complement activation and cell lysis. The experiment uses sheep RBCs (antigen) with rabbit antibody and serum (from immunized or normal rabbits) to test these reactions.

1. Introduction to Lab Ex. 23: Hemagglutination
Complement Mediated Lysis

2. Introduction to Lab Ex.23: Hemagglutination
Complement Mediated Lysis
Agglutination is the clumping of particulate antigens through their
binding with specific antibodies.
Hemagglutination is the clumping of red blood cells through their
binding with specific antibodies.
Thus hemagglutination is used as a observable reaction between
specific antibodies with red blood cells.
Complement is a series of proteins found in the serum with
non-specific immune function resulting in the lysis of target
cells.

3. Properties of complement include:
- non-specific immune function
- inactivated by heat (560C for 30mins)
- consists of 9 different proteins in a cascade
- activated by:
specific antigen –antibody complex formation (classical
certain chemicals – endotoxin (alternate)

5. Hemagglutination
• Hemagglutination involves agglutination of RBCs.
• Viral hemagglutination inhibition tests for antibodies by the
antibodies' ability to prevent viruses from agglutinating RBCs.
Figure 18.7

6. • Serum proteins
activated in a
cascade.
The Complement System
Figure 16.10

7. Effects of Complement Activation
• Opsonization or immune
adherence: enhanced
phagocytosis
• Membrane attack complex:
cytolysis
• Attract phagocytes
Figure 16.11

8. Effects of Complement Activation
Figure 16.12

9. Experiment: Ag=antigen; Ab= antibody; C’= complement
Premise:
Antigen used is sheep RBC (sRBC)
Antibody used is anti-sRBC - antibody to sRBC
(from immunized rabbit blood)
Normal serum is from normal rabbit (Ab absent)
Immune serum is from rabbit immunized with sRBC
(Ab present)
Normal serum (Ab absent; C’ present)
Immune serum (Ab present; C’ present)
Normal serum heated (Ab absent; C’ absent)
Immune serum heated (Ab present; C’ absent)

10. Legend: Ag = sRBC; Ab = antibody to sRBC; C’ = complement
Tube 1: sRBC + saline =
(Ag)
No reaction

11. Tube 2: sRBC + Normal serum =
(Ag) (-Ab;+C’)
No reaction
Legend: Ag = sRBC; Ab = antibody to sRBC; C’ = complement

12. Tube 3: sRBC + Immune serum =
(Ag) (+Ab;+C’)
Hemolysis
Legend: Ag = sRBC; Ab = antibody to sRBC; C’ = complement

13. Tube 4: sRBC + Normal serum (heated) =
(Ag) (-Ab;-C’)
No reaction
Legend: Ag = sRBC; Ab = antibody to sRBC; C’ = complement

14. Tube 5: sRBC + Immune serum (heated) =
(Ag) (+Ab;-C’)
Hemagglutination
Legend: Ag = sRBC; Ab = antibody to sRBC; C’ = complement

15. Tube 6: sRBC + Normal serum + Heated Immune serum =
(Ag) (-Ab;+C’) (+Ab;-C’)
Hemolysis
Legend: Ag = sRBC; Ab = antibody to sRBC; C’ = complement

16. Legend: Ag = sRBC; Ab = antibody to sRBC; C’ = complement
Tube 1: sRBC + saline = No reaction
Tube 2: sRBC + Normal serum = No reaction
Tube 3: sRBC + Immune serum = Hemolysis
Tube 4: sRBC + heated normal serum = No reaction
Tube 5: sRBC + heated Immune serum = Hemagglutination
Tube 6: sRBC + Normal serum + heated Immune serum =
hemolysis

17. Legend: Ag = sRBC; Ab = antibody to sRBC; C’ = complement
Tube 1: sRBC + saline = No reaction
Tube 2: sRBC + Normal serum = No reaction
Tube 3: sRBC + Immune serum = Hemolysis
Tube 4: sRBC + heated normal serum = No reaction
Tube 5: sRBC + heated Immune serum = Hemagglutination
Tube 6: sRBC + Normal serum + heated Immune serum =
hemolysis