57 

 phosphorylase (97,400), ^galactosidase (116,000), and myosin (205,000) 

 was used. Molecular weights of the antibody proteins were determined 

 following the methods of Weber and Osborn (1969) and Weber et al . (1972). 



Antibody Titer Determination bv FnrvmP - ljnked Immimnsorbent A^^^y ( n Tc:fl) 



One hundred-/iL lobster PPO containing 2.5 - 100 ng protein in 0.1 M 

 NaHCOg, pH 8.6, (coating buffer) was applied to the sample well of a 

 microplate (Immulon 2, Dynatech). Following overnight incubation at 4°C, 

 the well was aspirated and washed 4 times with PBS-Tween [0.01 M sodium 

 phosphate, pH 7.4, containing 0.15 M NaCl and 0.2% (v/v) Tween 20] using 

 the Nunc-Immuno Wash (A/S NUNC, Denmark). After 100 /xL primary antibody 

 (anti-lobster PPO antibody) at amounts of 0.01 - 10 ^9 in PBS-Tween was 

 added to the well, incubation was allowed to proceed at ambient 

 temperature for 1 hr. The aspirations and washings were repeated as 

 previously described before 0.1 mL secondary antibody (antichicken IgG- 

 alkaline phosphatase conjugate, Sigma) was added. Following another one- 

 hour incubation, the microplate was aspirated and washed again with PBS- 

 Tween. Following the addition of 0.1 mL p-nitrophenyl phosphate disodium 

 (1.0 mg/mL) in assay buffer (0.05 M Na^COg and 0.05 M NaHCOg containing 

 0.0005 M MgCl,) to the well, the plate was incubated at ambient temperature 

 till a yellow color developed. The absorbance of the plate at 405 nm was 

 monitored every hour using an ELISA reader (Model 2550, Bio-Rad). In this 

 study, coating buffer without antigen was used as the negative control. 



