58 



Analysis of Antigenic Properties of PPO 



The competitive ELISA adopted from Seymour et al . (1991) was 

 employed to study whether PPO from mushroom, potato, white shrimp, and 

 brown shrimp possessed similar antigenic determinants as the Florida spiny 

 lobster. After the microplate was coated with lobster PPO at 10 ng/well 

 for one hour at room temperature, 100 /xL aliquot of antibody-competitive 

 PPO mixture was added. The antibody-PPO mixture was prepared by mixing 

 the competitive PPO (either mushroom, potato, or lobster, white shrimp, 

 and brown shrimp) (0.2 - 2.0 iig/ml) with an equal volume of primary 

 antibody solution (10 or 20 /zg/mL of IgY) for 1 hr at ambient temperature. 

 The assay procedures were conducted as previously described. 



Immunoblottinq 



Purified mushroom, potato, lobster, white shrimp, and brown shrimp 

 PPO and a crude lobster PPO preparation were subjected to SDS-PAGE under 

 reduction condition (Laemmli, 1970) and then electro-transferred to 

 nitrocellulose membrane (Bio-Rad). Slab gels (16 cm x 20 cm) at 1.0 mm 

 thickness, consisting of stacking gel (4% acrylamide/ 0.1% bisacryl amide) 

 and separating gel (7.5% acrylamide/ 0.2% bisacryl amide), were prepared 

 according to the Protean^" II Slab Cell Instruction Manual (Bio-Rad, 

 1985a). A constant current of 13 mA/gel was applied during stacking and 

 18 mA/gel during separation. Fifty-/ig aliquots of test samples were 

 applied to each well and run with the protein standards (SDS-6H Molecular 

 Weight Marker Kit). 



Following electrophoresis, the gel was equilibrated in 500 mL of 

 Towbin transfer buffer (25 mM Tris, 192 mM glycine, 0.1% SDS, and 20% 



