Delayed Hypersensitivity Foot-Pad Test
Objective: Measurement of delayed-type hypersensitivity response in mouse foot-pad to Listeria antigens by measuring swelling at 24 hours post-injection
Gather these items before starting the experiment. Check off items as you prepare.
As an Amazon Associate, we earn from qualifying purchases. Product links help support this free resource.
Protocol Steps
Prepare protein-free medium for antigen production
Prepare protein-free medium by dialyzing a concentrated solution of BHI medium against distilled water
Note: This removes proteins from the medium
View evidence from paper
“The organism was grown in a protein-free medium prepared by dialysing a concentrated solution of BHI medium against distilled water”
Sterilize dialyzed medium
Sterilize the dialysate by autoclaving or Seitz filtration
Note: Two sterilization methods are acceptable
View evidence from paper
“The dialysate was sterilised by autoclaving or by Seitz filtration”
Culture Listeria monocytogenes in protein-free medium
Seed a bulk culture with organisms grown in the same protein-free medium and incubate with continuous aeration
Note: Continuous aeration is required
View evidence from paper
“A bulk culture, seeded with organisms grown in the same medium, was incubated with continuous aeration for 24 hours”
Remove bacteria and prepare antigen filtrate
Remove bacteria by centrifuging and filtering through sintered glass to obtain culture filtrate containing antigens
Note: The filtrate contains the antigens for injection
View evidence from paper
“The bacteria were removed by centrifuging and filtering through sintered glass”
Inject antigen into right hind foot-pad
Inject the culture filtrate antigen into the right hind foot-pad of sensitized mice
Note: Volume is critical for standardization
View evidence from paper
“The filtrate was injected in a volume of 50 µl into the right hind foot-pad”
Inspect injection site for immediate reaction
Inspect the injection site after 6 to 8 hours to exclude an immediate (non-delayed) reaction
Note: This confirms the response is delayed-type hypersensitivity, not immediate
View evidence from paper
“The injection site was inspected after 6 to 8 hours to exclude an immediate reaction”
Measure foot-pad thickness at 24 hours
Measure the thickness of each hind foot using dial-gauge calipers to quantify swelling response
Note: Measurement precision is 0.1 mm
View evidence from paper
“after 24 hours the thickness of each hind foot was measured to 0.1 mm with dial-gauge calipers”