week 1 elisa practical

Detection of IgE and IgG against Aspergillus fumigatus

• Detection of $IgE$ and/or $IgG$ against major Aspergillus fumigatus antigen Asp f1 in human serum using Indirect binding ELISA and Sandwich ELISA.

• Asp f1 is a $18$ kDa ribotoxin and a major allergen recognized by the immune system in over $85\%$ of patients with Allergic Bronchopulmonary Aspergillosis (ABPA).

Practical Sessions

• Schedule Details:

  • The practical will occur during week $18$ (Thursday & Friday).

  • Sign-up for sessions on Brightspace; registration closes at $12$ noon on Wednesday, $15^{th}$ January $2025$ (week $16$).

• Arrival Instructions:

  • Attend the practical on time (arrive by $09:45$).

  • Late arrivals will not be admitted and are unlikely to repeat the practical due to strict biosafety and timing protocols.

Basic Instructions for Practical

• Safety Briefing:

  • A mandatory safety briefing will be conducted at the start of each practical session.

• Necessary Items:

  • Bring lab coats, a copy of the practical schedule, and lab books.

  • A video will be posted on Brightspace to guide preparation; it is essential to watch this before attending.

• Lecture Content:

  • Background on Aspergillus, allergic aspergillosis, and ELISA mechanisms will be covered to provide theoretical context.

Aspergillus and Aspergillosis

• Aspergillus Overview:

  • Member of the Ascomycota phylum.

  • The name is derived from "aspergere," meaning ‘to sprinkle’, referencing the shape of the asexual spore-forming structure.

• Classification:

  • Aspergillus is a genus of around $200$ species, with approximately $18$ known to be pathogenic to humans. A. fumigatus is the most common clinical isolate.

• Habitat and Characteristics:

  • Filamentous fungi (molds) that are globally distributed.

  • Spores (conidia) are small ($2-3 \mu m$), allowing them to reach deep into the pulmonary alveoli.

  • Found in decaying organic matter, soil, air vents, and hospital environments (showerheads, water storage, potted plants).

Life Cycle of Aspergillus

• Phases of Life Cycle:

  1. Inhalation: Conidia reach the lungs.

  2. Germination: Under favorable conditions, spores swell and break dormancy.

  3. Hyphal Growth: Development of a hyphal mass through elongation and dichotomous branching ($45^{\circ}$ angle).

  4. Sporulation: Formation of conidiophores and release of new conidia.

• Cell Structure:

  • Universal cell wall components include $\beta$-(1,3)-glucan, chitin, and galactomannan. These act as primary Pathogen-Associated Molecular Patterns (PAMPs).

Immune Response to Aspergillus

• Antibody Responses:

  • PAMPs are recognized by Pattern Recognition Receptors (PRRs) like Dectin-1 (which recognizes $\beta$-glucan) and TLRs.

  • Recognition can induce apoptosis in neutrophils and trigger pro-inflammatory cytokine release.

• Variability in Immune Response:

  • Innate: Macrophages and neutrophils attempt to clear conidia and hyphae.

  • Adaptive: Involves Th2-mediated responses (leading to $IgE$ production and allergies) or Th1/Th17 responses for fungal clearance.

Clinical Manifestations of Aspergillosis

• Chronic Conditions:

  • CPA: Chronic Pulmonary Aspergillosis.

  • CNPA: Chronic Necrotizing Pulmonary Aspergillosis (subacute).

• Allergic Conditions:

  • SAFS: Severe Asthma with Fungal Sensitization.

  • ABPA: Allergic Bronchopulmonary Aspergillosis (typically involving high $IgE$ levels).

• Acute/Invasive Conditions:

  • CAPA: COVID-19 Associated Invasive Pulmonary Aspergillosis.

  • IPA: Invasive Pulmonary Aspergillosis (primarily in immunocompromised hosts).

Allergic Aspergillosis Details

• Pathophysiology:

  • Asp f1 is the primary allergen targeted in diagnostics. It is a ribotoxin that inhibits protein synthesis.

  • Aspergillus contributes to asthma in $10-15\%$ of patients.

  • Ungerminated conidia stimulate the production of specific $IgE$, leading to mast cell degranulation and bronchoconstriction.

  • ABPA hallmarks include elevated total and specific $IgE$, specific $IgG$, and peripheral eosinophilia.

Diagnostic Techniques: ELISA Methods

• ELISA Overview:

  • Enzyme-Linked Immunosorbent Assay uses an enzymatic color change (quantified by optical density) to measure target molecules.

• Indirect ELISA (Detecting Antibodies):

  1. Coating: Asp f1 peptide antigen is bound to the well.

  2. Blocking: Non-specific sites are blocked.

  3. Primary Antibody: Patient serum (containing $IgG$ or $IgE$) is added.

  4. Secondary Antibody: HRP-conjugated anti-human $IgG$ or $IgE$ is added.

  5. Detection: OPD substrate ($o$-phenylenediamine) is added; Horseradish Peroxidase (HRP) catalyzes a color change.

• Sandwich ELISA (Detecting Antigens):

  • Measures the concentration of antigen ($Asp f1$) between two layers of antibodies (capture and detection antibodies).

Conceptual and Ethical Considerations

• ELISA Rationale: Why choose Sandwich over Indirect? Sandwich ELISA typically offers higher sensitivity and specificity by using two antibodies that recognize different epitopes.

• Markers: Advantages of using purified peptide markers (like Asp f1) include reduced cross-reactivity compared to crude full-length fungal extracts.

• Detection Sensitivity: Methods like signal amplification or using chemiluminescence can improve sensitivity but may increase the risk of false positives.

• Clinical Correlation: The necessity of combining microscopic identification (gold standard) with antigen/antibody detection to ensure diagnostic accuracy.