63 



Antibody Production and Molecular Weight Determination 



The production of lobster PPO specific antibody did not occur until 

 after the second boosting; peak activity occurred on day 18 (Figure 11). 

 Antibody activity decreased gradually with time following the third and 

 fourth boostings. The serum eventually lost antibody activity after day 

 35. The animals apparently had become tolerant to the antigen after the 

 third boosting. 



Antibody produced on day 17 and 18 having the highest activities was 

 pooled for molecular weight determination. Three distinct bands were 

 observed on the SDS-PAGE gel (Figure 12). The molecular weight for the 

 lower band was estimated as 59,000 which was close to the value reported 

 for the heavy chain of IgY (Jensenius et al . , 1981). 



Antibody Titer Determination 



Dose-related interactions of various PPO (mushroom, potato, lobster, 

 white shrimp, and brown shrimp) at 2.5 - 10 ng with lobster PPO-specific 

 antibody at 2.5 /xg are shown in Figure 13. All test samples showed 

 affinity for the antibody. Therefore, these antigens were partially 

 cross-reactive and shared similar antigenic determinants. The appropriate 

 dose range of the interaction of the various PPOs with the antibody was 

 determined to be 2.5 - 10 ng/well . When PPO was added at more than 10 

 ng/well, the antigen-antibody reaction was saturated and the sensitivity 

 of the ELISA became dull. 





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