Substrate
and enzyme control wells were included on each plate.
The plates were incubated for 30 min at 37 C, and subsequently
washed three times with 200 ll of PBS solution containing 0.05%
Tween-20. One hundred milli liter of a 1:3000 dilution of peroxidase-
labeled goat anti-cat IgG (gamma) (Kirkegaard and Perry Laboratories)
in PBS–Tween solution was pipetted into appropriate
wells. The plate was incubated for 30 min at 37 C and after another
wash step, 100 ll of substrate (TMB; SureBlue Reserve TMB
Substrate, Kirkegaard and Perry Laboratories) was pipetted into
each well. The enzyme reaction was stopped after 10 min. The
optical density of each well was read at 450 nm with an automated
micro-ELISA reader. A sample was considered positive for B. henselae
IgG if the mean OD value was greater than the mean OD value
plus 3SD of negative samples at the titer P64. Titers were estimated
by comparison to a standard curve generated from the positive
and negative control sample results.