Measuring Hormone Disruption

1
Measuring Hormone Disruption

• in vivo Rat Uterus Growth
• in vitro E-Screen (MCF-7 breast cancer cell
  proliferation)
• in vitro Yeast-Estrogen Response System (YES)
• in vivo Male Yolk Production (Vitellogenin)
• in vivo Tadpole Fetal Development (FETAX)

2
African Clawed Frog

• Xenopus strangefoot
• Development model
• Sensitive to estrogen
• First pregnancy test

3
Enzyme-Linked Immunosorbant Assay (ELISA)
Low VTG
High VTG

• Stick Xenopus serum to microtiter plate
• Add primary antibody (Rabbit anti-Xenopus)
• Add secondary antibody (goat anti-rabbit linked
  to HRP enzyme)
• Add peroxide substrate

Dk Blue
Lt Blue
4
ELISA Plate
Darker blue indicates higher serum vitellogenin
5
Data Analysis
1. Plot dilutions to locate linear range
2. Choose one dilution in linear range to analyze
statistically
6
Xenopus Serum Collection

• Get frogs, place in baggie weigh
• Inject each frog with 300ul of Ketamine/Rompum
• Wait 15 min enter weight data while waiting
• Cut off one leg through femur
• Drip blood into labeled microcentrifuge tube
• Squeeze both ends of cut leg to get maximal blood
• Post-mortem
• Open abdomen of frog
• Remove testes weigh individually enter data