BM214 Immunology - Lab 3 Study Notes

Context: Investigating a potential outbreak of Mycobacterium tuberculosis (TB) among students volunteering in KwaZulu-Natal, South Africa.
Objective:
- Students are not responding to conventional treatments - possibly a virulent strain.
- Aim is to understand the immune response and produce a report on the findings.

Purpose of ELISAs:
- Measure the quantity of a specific protein (antigen) using an antibody that binds to the antigen.
- Quantitative Application: Measuring the amount of an antigen directly.
- Qualitative Application: Detecting antibodies against an antigen, useful for diagnosing infections.

Testing Setup:
- Compare previous lab work (e.g., immune cell counts, antimicrobial activity) with outputs from this ELISA.

Antigen Coating: Coating the plate with TB antigen (complete initially).
Blocking: Blocking of non-specific binding sites (complete initially).
Sample Addition: Introduce human serum samples (identified as P1-P4 for patients and H1-H4 for healthy controls).
Conjugate Addition: Add anti-human enzyme antibody conjugate.
Substrate Addition: Introduce substrate to initiate color change.
Stopping Reaction: Add acid to halt the enzyme-substrate reaction.
Reading Results: Measure results via ELISA reader.

Recognition: The ELISA detects antibodies that recognize a specific antigen.
Coating Ingredients: Antigen coated on ELISA plate in a coating buffer at pH 9 for optimal binding.
Specific Antigen used: MB1 (TB-specific).

Definition: Serum from a patient contains various antibodies, potentially cross-reacting with antigens from different pathogens.
Antibody Structure:
- Fab Portion: Binds specifically to an antigen.
- Fc Portion: Interacts with cell surface receptors.

Negative Control: Serum from healthy individuals that should not react, preventing false positives.
Positive Controls: Serum known to produce a positive reaction to ensure the assay is functioning correctly.

Measurement Objective: Read results at 450 nm, which indicates the absorbance of samples.
Data Analysis Method: Plot absorbance against the dilution reciprocal to find the endpoint—the highest dilution producing a positive result indicates the titre.

Initial Setup: Coat wells with 100 μL of M. tuberculosis antigen MB1 (10 μg/mL in PBS, pH 9) and incubate overnight at 4°C.
Washing: Wash plates thrice with PBS/Tween (0.05 % vol/vol Tween 20, pH 7.4).
Blocking: Add 150 μL of blocking solution (5 % non-fat dried milk in PBS/Tween) and incubate for 20 minutes at 37°C.
Final Wash: Again wash plates three times with PBS/Tween.
ELISA Reader: Follow the lab manual and results will be posted on MyPlace.

Each student receives an individual plate; reagents are shared.
Pipetting Technique:
- First stop to draw liquid, second stop to expel.
- Use incubation times efficiently to collect required materials for the next step.

Dilution Scheme:
- Series of serial dilutions: 1:1, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, 1:512, 1:1024.
Plate Configuration:
- Each position labeled A - H, with different samples (P, H) assigned to respective spots starting at a dilution of 1:100.

Collect PBS in a labelled universal container.
Add 100 µL of PBS to all highlighted wells.
Add 200 µL of serum sample (from patient and healthy control) to designated wells in column 1.
Conduct serial dilutions across the plate.

Competency Check: After the 20-minute incubation period, request a lab team member to verify and sign off on lab techniques.
Buffer Collection: Acquire wash buffer for washing wells from designated areas.

Sample Arrangement:
- Rows A-C: Patient Sample
- Rows D-F: Healthy Control Sample
Assays for Antibodies: Add 100 µL of specific antibodies to respective rows:
- IgA: Rows A and D
- IgE: Rows B and E
- IgG: Rows C and F

Determine if a patient is infected by evaluating endpoint titres of antibodies.
Calculate the mean endpoint titre with standard error of the mean (SEM) across all samples for different Ig classes.
Plot absorbance versus dilution for each antibody class (graphs not included in reports).
Analyze and compare antibody levels in infected versus healthy controls using the Mann Whitney U test via Minitab software; significance set at p < 0.05.
Present results visually in bar graphs with corresponding legends in the lab report.

Definition: An endpoint titre of 1:8 implies that antibodies can still be detected in a sample diluted by 8 parts saline while not detectable at 1:16.
Reporting: Drop the leading 1 for presented results (e.g., reported as 8). A higher titre indicates a greater abundance of antibodies.

Visual Representation: Graphical analysis displayed via absorbance at 450 nm against serum dilutions across defined intervals (e.g., 100, 200, etc.) showing responses from negative, positive, and unknown samples.

Further Help: Refer to the ELISA analysis section on MyPlace for further guidance and insights regarding the experiment procedures and analysis techniques.