week 7 Haemolytic Assay Study Notes

Overview\n\n- Practical Session: Immunology Practical on Haemolytic Assay scheduled for Tuesday, 18th November 2025 (Week 8).\n- Attendance and Safety: Participants must arrive promptly for the mandatory safety briefing. Attendance is recorded, and students are required to possess their lab handbook and wear appropriate Personal Protective Equipment (PPE).\n\n## Background of the Complement System\n\n- Innate Immunity Overview:\n - The body's rapid, non-specific first line of defense against pathogens.\n - Phagocytes: Includes macrophages and neutrophils that utilize Pattern Recognition Receptors (PRRs) to identify Pathogen-Associated Molecular Patterns (PAMPs).\n - Activation: Recognition leads to phagocytosis and the secretion of proinflammatory cytokines and chemokines, which orchestrate the inflammatory response and recruit further immune cells.\n- The Complement Cascade:\n - A system of over 30 heat-labile plasma proteins synthesized primarily by the liver.\n - It functions as a biochemical cascade to opsonize pathogens, induce inflammation, and directly lyse target cells via the Membrane Attack Complex (MAC).\n\n## Pathways of the Complement System\n\n### 1. Classical Pathway\n- Initiation: Triggered when the $C1$ complex (consisting of $C1q$, $C1r<em>{2}$, and $C1s</em>{2}$) binds to the $Fc$ region of antigen-bound antibodies ($IgG$ or $IgM$).\n- Cascade:\n - $C1s$ cleaves $C4$ into $C4a$ and $C4b$, and $C2$ into $C2a$ and $C2b$.\n - C3 Convertase: The fragments combine to form $C4b2a$ (the classical C3 convertase).\n - This complex cleaves $C3$ into $C3a$ (anaphylatoxin) and $C3b$ (opsonin).\n\n### 2. Alternative Pathway\n- Initiation: Triggered by direct contact with pathogen surfaces (lipids, carbohydrates, or proteins).\n- Mechanism: Relies on the spontaneous hydrolysis of $C3$ (known as \"tick-over\") and the action of Factors B and D. It serves as an amplification loop for the other pathways.\n\n### 3. Lectin (MBL) Pathway\n- Initiation: Triggered by Mannose-Binding Lectin (MBL) or ficolins binding to mannose residues on microbial surfaces.\n- Mechanism: MBL-Associated Serine Proteases (MASPs) substitute for $C1r$ and $C1s$ to cleave $C4$ and $C2$.\n\n## Terminal Pathway and the MAC\n- All pathways converge at the formation of C5 convertase ($C4b2a3b$ in the classical pathway).\n- Membrane Attack Complex (MAC): Composed of $C5b$, $C6$, $C7$, $C8$, and multiple $C9$ molecules. These form a transmembrane pore (approx. $10 \text{ nm}$) in the pathogen membrane, leading to osmotic lysis.\n\n## Haemolytic Assay Methodology\n\n- Objective: To quantify the functional activity of the classical complement pathway by measuring the lysis of sensitized erythrocytes.\n- Indicator: The release of haemoglobin into the supernatant is measured spectrophotometrically; the intensity of the red color is proportional to the degree of complement-mediated lysis.\n\n### Experimental Reagents\n- Erythrocytes:\n - EA Cells (Sensitized): Sheep Red Blood Cells (SRBCs) coated with rabbit anti-SRBC antibodies (haemolysin). These are targets for classical pathway activation.\n - E Cells (Unsensitized): SRBCs without antibodies, used to control for non-specific or alternative pathway lysis.\n- Sera Proteins: Sources of complement components.\n - Normal Human Serum (NHS)\n - Guinea Pig Serum (GPS): Often used because guinea pig complement is highly efficient at lysing sensitized SRBCs.\n- DGVB Buffer (Dextrose Gelatin Veronal Buffer):\n - Contains $Ca^{2+<em>{}}$ (required for the stability of the $C1qrs$ complex) and $Mg^{2+</em>{}}$ (required for the formation of the C3 convertase complex).\n\n### Procedure and Team Tasks\n- Serial Dilution: Performed in teams of two to determine the point of 50% lysis. One student handles NHS while the other handles GPS.\n- Sample Preparation: Typically involves 40 tubes with precise volumes (e.g., $180 \mu l$ DGVB + $20 \mu l$ serum).\n- Incubation: Samples are incubated at $37^\circ C$ for 1 hour to allow the enzymatic cascade to complete.\n- Separation: After incubation, tubes are centrifuged to pellet intact cells. The supernatant containing released haemoglobin is collected.\n\n## Data Analysis and CH50\n- Spectrophotometry: The optical density (OD) of the supernatant is measured (usually at $412 \text{ nm}$ or $541 \text{ nm}$).\n- CH50 Value: Defined as the reciprocal of the serum dilution that produces haemolysis of 50% of the cells in the assay. The 50% point is used because the lysis curve is sigmoidal, and the slope is steepest (most sensitive to changes) at this midpoint.\n\n## Conclusion\n- Coursework: Results will form the basis of a detailed written report for Life Sciences students.\n- Assessment: A post-practical quiz will be administered via a scan code to evaluate understanding of the protocol and theory.