Nasal washes, tears and stool samples were collected as described
previously.(17 (link)) Immulon 2 plates were
coated with 4 μg/ml of lipoarabinomannan H37Ra LAM
(provided by Dr. Larry Schlesinger, Ohio State University) diluted in
100% ethanol. The plates were dried for 3–4 hours in a fume hood
then washed with PBS plus 0.05% tween 20. Plates were blocked overnight
at 4°C with 1% BSA in PBS with 0.05% tween 20. Following
blocking, the plates were washed and serum, tear, nasal wash and stool samples
were added to duplicate wells at optimal dilutions predetermined for each type
of sample. The plates were incubated overnight at 4°C. After washing and
the application of goat anti-human IgG or goat anti-human IgA horseradish
peroxidase (Southern Biotechnology Associates, Inc.), plates were incubated for
1 hour at room temperature in the dark. Then plates were washed and the ABTS
substrate (Kirkegaard & Perry, Gaithersburg, MD, USA) was added. After the
plates were incubated 20 minutes at room temperature, absorbance was read at 405
nm.
previously.(17 (link)) Immulon 2 plates were
coated with 4 μg/ml of lipoarabinomannan H37Ra LAM
(provided by Dr. Larry Schlesinger, Ohio State University) diluted in
100% ethanol. The plates were dried for 3–4 hours in a fume hood
then washed with PBS plus 0.05% tween 20. Plates were blocked overnight
at 4°C with 1% BSA in PBS with 0.05% tween 20. Following
blocking, the plates were washed and serum, tear, nasal wash and stool samples
were added to duplicate wells at optimal dilutions predetermined for each type
of sample. The plates were incubated overnight at 4°C. After washing and
the application of goat anti-human IgG or goat anti-human IgA horseradish
peroxidase (Southern Biotechnology Associates, Inc.), plates were incubated for
1 hour at room temperature in the dark. Then plates were washed and the ABTS
substrate (Kirkegaard & Perry, Gaithersburg, MD, USA) was added. After the
plates were incubated 20 minutes at room temperature, absorbance was read at 405
nm.