II 
Figure 4.5-A. Cotton rat serum anti-adenovirus antibody titers following 
respiratory tract delivery of Ad-CFTR, a replication deficient, recombinant 
adenovirus vector containing the human CFTR cDNA. Controls are presented as 
open symbols (□ untreated; A intratracheal administration of vehicle on day 
0; o intratracheal administration of vehicle on day 0 and day 7). Animals 
receiving Ad-CFTR are presented as closed symbols (a intratracheal adminis- 
tration of Ad-CFTR on day 0; • intratracheal administration of Ad CFTR on 
day 0 and vehicle on day 7) . Animals were sacrificed and serum obtained at 
the indicated time points. Anti -adenovirus antibodies were determined by 
enzyme linked immunosorbent assay (ELISA). Briefly, 96 well ELISA plates 
were coated overnight with Ad-CFTR at 4°; after washing [with phosphate 
buffered saline containing 0.05% Tween-20 (PBS -Tween) ] , serial, 4-fold 
dilutions of serum [in phosphate buffered saline containing 1% fetal bovine 
serum (PBS-1%FBS)] were added and the plates incubated overnight at 4°. 
Plates were washed with PBS -tween and incubated with a rabbit anti-cotton 
rat IgG serum (gift of Dr. B. Murphy, NIAID) for 5 hr at 4°. Plates were 
washed with PBS-tween and incubated with goat anti-rabbit alkaline phospha- 
tase conjugate overnight at 4°. Plates were then washed with PBS-tween, 
developed for one hour with p-nitrophenyl phosphate, disodium- 6 -H 2 0 , and 
optical density was determined at 405 nm in an ELISA plate reader. Back- 
ground, determined from samples assayed as above on wells left uncoated 
with virus, was subtracted and titers determined. Standardization was 
insured by the evaluation of standard positive and negative controls on 
each plate. Data is expressed as the inverse maximum dilution at which 
detectable signal over negative control was detected. All data points 
indicate the mean of two independent determinations of serum from a single 
cotton rat. 
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Recombinant DNA Research, Volume 16 
