36 

 purified IgG. This step removed non-specific precipitant 

 bands observed in the double-diffusion experiments. Pre- 

 immune rabbit serum were purified similarly. 

 Antigen; antibody assay 



Antigens (SP-I, ADH or PAT) from crude extracts or 

 extracts from various purification steps were combined 1:1 

 (v:v) (or as stated) with antibodies. Samples were 

 incubated at 37°C for 10 min and returned to an ice bath, 

 then centrifuged (5 to 10 min) at full speed in a microfuge 

 to precipitate the complexed antigen: antibody. The 

 supernatants were assayed for enzyme activities. Controls 

 were always performed with preimmune rabbit serum. 

 Ouchterlony assay 



Ouchterlony plates were prepared with 1 . 5% agarose in 

 sodium barbital and sodium azide and wells were prepared at 

 precisely measured distances (66) . Antibodies or antigens 

 were diluted with 0.9% NaCl whenever necessary and then 

 added to the wells. Plates were incubated at 37°C 

 overnight . 

 Western blotting 



Western blotting procedures from Sambrook et al . (68) 

 were followed, and bands from either SDS or native PAGE were 

 transferred to nitrocellulose filter paper. Membranes were 

 incubated in blocking solution alone and in blocking 

 solution containing diluted SP-I antibody for one h, washed, 

 incubated further with blocking buffer containing alkaline 



