Laboratory Class

IMMUNOLOGY
• Tran Thi Hai Yen/ PhD.

2023-2024
 Lab 5. Enzyme-Linked Immunosorbent Assay
for Detection of Hepatitis B surface Antigen

Objective:

- Understand ELISA principle

- Detect Hepatitis B surface Antigen (HBsAg) in human
serum sample
Lab 5. Enzyme-Linked Immunosorbent Assay
for Detection of Hepatitis B surface Antigen
Lab 5. Enzyme-Linked Immunosorbent Assay
for Detection of Hepatitis B surface Antigen
Lab 5. Enzyme-Linked Immunosorbent Assay
for Detection of Hepatitis B surface Antigen
Lab 5. Enzyme-Linked Immunosorbent Assay
for Detection of Hepatitis B surface Antigen
Lab 5. Enzyme-Linked Immunosorbent Assay
for Detection of Hepatitis B surface Antigen
Anti-HBsAg monoclonal antibody - coated strips
↓
Dispense 50µL of Controls (Negative, Positive) or 100µL of samples into each well
↓
Dispense 100µL of Peroxidase-conjugated anti-HBsAg antibody into wells
↓
Incubate plate at 37oC | 60mins (Cover the plate with aluminum foil)
↓
Remove liquid from wells by “flicking” the plate → Blot the plate of a sheet of paper towel
to remove remaining liquid
↓
Fill all wells with 150µL washing reagent → Flick off and blot plate (x4 times)
↓
Quickly add 100µL of TMB-H2O2 substrate to each well
↓
Incubate at tor | 30 minutes (in the dark condition)
↓
Add 100µL of stopping reagent (H2SO4) to each well to stop the reaction
↓
Wait at least 4 minutes
↓
Observe and record the color change in each well
↓
Read your plate on the plate reader at 450nm (OD450)
Lab 5. Enzyme-Linked Immunosorbent Assay
for Detection of Hepatitis B surface Antigen
Lab 5. Enzyme-Linked Immunosorbent Assay
for Detection of Hepatitis B surface Antigen
 Lab 5. Enzyme-Linked Immunosorbent Assay
for Detection of Hepatitis B surface Antigen

❑ Data interpretation

Cut-off OD450 (Co) = Average OD450 of Negative control + 0.050

S/Co Interpretation
< Co Negative
≥ Co Positive
 Lab 5. Enzyme-Linked Immunosorbent Assay
for Detection of Hepatitis B surface Antigen

Report:

➢ Keynotes of today’s class

➢ Labwork report
➢ Feedback for Instructors