Can f1 ELISA detailed methods
The ELISA kit for Can f1 was purchased from Indoor Biotechnologies , Charlottesville, VA. The protocol for the assay was
provided by the company. Briefly, 100 ul monoclonal antibody (against Can f1) in bicarbonate buffer, pH 9.6, was added to
a microtiter plate (96 wells). The plate was incubated at 4 deg C overnight. The plate was washed 3X in PBS-tween (PBS-T)
on the next day. 100 ul PBS-T containing 1% BSA (bovine serum albumen) was added to each well and was incubated at
room temperature for 30 minutes. The plate was washed 3X in PBS-T. 100 ul of Can f1 reference standard ( 1000 ng/ml to 2
ng/ml) in BSA PBS-T; 100 ul of dust samples (both in duplicates) were added to each well and the plate was incubated at
room temperature for 1 hour. After 5X washing with PBS-T, 100 ul rabbit anti-Can f1 was added to each well and the plate
was left at room temperature for 1 hour. The plate was washed again 5 times with PBS-T. 100 ul peroxidased goat anti-rabbit
IgG was added to each well and the plate was incubated at room temperature for 1 hour. After washing for 5 times with PBST, 100ul ABTS/hydrogen peroxide was added to each well. OD was read at 405nm after the top of curve reached 2.0. A
standard curve was plotted with the absorbance vs concentration of the standard. The value of the sample was obtained from
the standard curve.
ONLINE REPOSITORY Supplemental TABLES
Supplemental Table 1. Number of children for which there was data on given exposure variable and outcome
Variable
CCot
UCot
Gas stove
NO2
Dog allergen
Dog ownership
total
279
370
380
155
379
377
In those
In those
with data
with data
on atopy at on atopy at
12 months
7 years
276
268
366
360
376
367
151
150
375
366
373
364
In those
with data
on asthma
at 7 years
279
370
380
155
379
377
In those
with data
on BHR at
7 years
258
338
348
145
347
345
Supplemental Table 2. Sample size and mean ± standard deviation or frequency (percent) of early exposures.
Environmental tobacco
smoke:
CCot (ng/ml)
UCot (ng/mg)
NO2: (parts per billion)
Dog allergen: (µg/g of dust)
Presence of gas stove
Dog ownership
Atopy
TOTAL
Control
Intervention
279
9.2 ± 38.2
130
9.9 ± 39.4
149
8.5 ± 37.2
370
89.5 ± 280.0
155
11.3 ± 5.1
379
9.0 ± 21.0
380
37 (9.7)
377
60 (15.9)
376
90 (23.9)
173
75.5 ± 231.1
72
12.1 ± 6.0
177
8.4 ± 21.2
178
21 (11.8)
177
26 (14.7)
175
42 (24.0)
197
101.9 ± 316.9
83
10.6 ± 4.0
202
9.6 ± 20.9
202
16 (7.9)
200
34 (17.0)
201
48 (23.9)
Supplemental Table 3. Frequency (%) of asthma and BHR by dog allergen and other (non-allergen) exposure
status; associated ORs
Exposures^
--dog allergen AND NO2
--only dog allergen
--only NO2
--neither
--dog allergen AND gas
stove
--only dog allergen
--only gas stove
-- neither
--dog allergen AND UCot
--only dog allergen
--only UCot
-- neither
--dog allergen AND CCot
--only dog allergen
--only CCot
-- neither
Asthma
No asthma
Adjusted*
OR**
BHR
No BHR
Adjusted*
OR**
5 (25.0)
3 (15.0)
7 (35.0)
5 (25.0)
14 (10.4)
18 (13.3)
51 (37.8)
52 (38.5)
4.8 (1.1-21.5)
1.0 (0.2-5.2)
1.3 (0.4-4.5)
1
5 (8.5)
9 (15.3)
22 (37.3)
23 (39.0)
13 (15.1)
11 (12.8)
31 (36.0)
31 (36.0)
0.6 (0.2-1.8)
1.1 (0.4-3.1)
0.9 (0.4-2.0)
1
4 (5.6)
26 (36.6)
4 (5.6)
37 (52.1)
22 (31.0)
8 (11.3)
21 (29.6)
20 (28.2)
13 (26.0)
7 (14.0)
18 (36.0)
12 (24.0)
8 (2.6)
94 (30.5)
21 (6.8)
185 (60.1)
62 (20.8)
34 (11.4)
86 (28.9)
116 (38.9)
37 (16.2)
39 (17.1)
68 (29.8)
84 (36.8)
3.0 (0.7-12.5)
1.2 (0.6-2.3)
1.0 (0.3-3.4)
1
1.8 (0.8-4.0)
1.1 (0.4-3.0)
1.1 (0.5-2.5)
1
2.7 (1.1-7.1)
1.3 (0.4-4.1)
1.4 (0.6-3.5)
1
4 (2.8)
37 (26.2)
11 (7.8)
89 (63.1)
24 (17.6)
15 (11.0)
47 (34.6)
50 (36.8)
15 (14.3)
14 (13.3)
36 (34.3)
40 (38.1)
6 (2.9)
71 (34.5)
13 (6.3)
116 (56.3)
49 (24.4)
24 (11.9)
52 (25.9)
76 (37.8)
29 (19.1)
29 (19.1)
44 (28.9)
50 (32.9)
1.0 (0.2-3.8)
0.7 (0.4-1.1)
1.1 (0.5-2.6)
1
0.8 (0.4-1.5)
0.8 (0.4-1.8)
1.3 (0.8-2.3)
1
0.7 (0.3-1.4)
0.6 (0.3-1.3)
1.0 (0.5-1.8)
1
^ Exposures defined as per methods
*Models are adjusted for group allocation (“intervention”), which was forced in, and for any baseline characteristic (race,
gender, family history of asthma, maternal education, residence city, season, child’s atopic status) with p<0.05 upon being
entered in a stepwise manner. The specific variables retained in model after stepwise process are noted as follows for each
of the four models, by co-exposures respectively: for NO2 = intervention, gender, atopic status; for gas stove = intervention,
gender, history of asthma, residence city, atopic status; for UCot = intervention, gender, history of asthma, residence city,
atopic status; for CCot = intervention, gender, history of asthma, atopic status.
** odds of outcome given elevated dog allergen, other exposure or both relative to having neither (95% CI); bold indicates
p<0.05
Supplemental Table 4. Frequency (%) of asthma and BHR by atopy and non-allergen exposure status;
associated ORs
Atopy status/exposures^
--atopy AND NO2
--only atopy
--only NO2
--neither
--atopy AND gas stove
--only atopy
--only gas stove
-- neither
--atopy AND UCot
--only atopy
--only UCot
-- neither
--atopy AND CCot
--only atopy
--only CCot
-- neither
Asthma
No asthma
Adjusted*
OR**
BHR
No BHR
Adjusted*
OR**
5 (25.0)
5 (25.0)
7 (35.0)
3 (15.0)
4 (5.6)
30 (42.3)
4 (5.6)
33 (46.5)
20 (28.2)
14 (19.7)
23 (32.4)
14 (19.7)
15 (30.0)
10 (20.0)
16 (32.0)
9 (18.0)
16 (12.2)
17 (13.0)
48 (36.6)
50 (38.2)
6 (2.0)
50 (16.4)
22 (7.2)
227 (74.4)
21 (7.1)
33 (11.2)
126 (42.7)
115 (39.0)
20 (8.8)
18 (8.0)
86 (38.1)
102 (45.1)
5.9 (1.2-28.1)
4.4 (0.9-20.8)
2.3 (0.6-9.5)
1
8.6 (2.0-37.1)
5.5 (2.9-10.3)
1.3 (0.4-4.4)
1
8.0 (3.3-19.5)
4.2 (1.71-10.0)
1.0 (0.5-2.2)
1
9.3 (3.4-25.5)
9.4 (3.1-28.3)
2.2 (0.9-5.3)
1
9 (15.5)
11 (19.0)
18 (31.0)
20 (34.5)
6 (4.3)
41 (29.3)
9 (6.4)
84 (60.0)
25 (18.5)
22 (16.3)
45 (33.3)
43 (31.9)
16 (15.4)
17 (16.3)
35 (33.7)
36 (34.6)
12 (14.5)
9 (10.8)
31 (37.3)
31 (37.3)
3 (1.5)
32 (15.7)
15 (7.4)
154 (75.5)
11 (5.5)
22 (11.1)
90 (45.2)
76 (38.2)
15 (9.9)
10 (6.6)
59 (39.1)
67 (44.4)
1.2 (0.4-3.3)
1.9 (0.7-5.4)
0.9 (0.4-2.0)
1
3.7 (0.9-15.1)
2.4 (1.4-4.0)
1.1 (0.5-2.6)
1
4.1 (1.8-9.2)
1.8 (0.9-3.6)
0.9 (0.5-1.5)
1
2.0 (0.9-4.5)
3.2 (1.3-7.6)
1.1 (0.6-2.0)
1
^ Exposures defined as per methods
*Models are adjusted for group allocation (“intervention”), which was forced in, and for any baseline characteristic (race,
gender, family history of asthma, maternal education, residence city, season) with p<0.05 upon being entered in a stepwise
manner. The specific variables retained in model after stepwise process are noted as follows for each of the four models, by
co-exposures respectively: for NO2 = intervention; for gas stove = intervention, gender, history of asthma, residence city; for
UCot = intervention, gender, history of asthma, residence city; for CCot = intervention, gender, history of asthma.
** odds of outcome given atopy, non-allergen exposure or both relative to having neither (95% CI); bold indicates p<0.05
Supplemental Table 5. OR (95% CI) for “intercondition” comparisons in main text figures 1 and 2.
Note that the ORs presented here differ from those in figures 1 and 2, as they represent distinct
comparisons, as described within this table
Asthma
BHR
Supplemental Table 5a
(reflects Figure 1 “intercondition” comparisons)
Combined exposure, relative to elevated dog
allergen only:
Dog allergen and NO2: Dog allergen
Dog allergen and gas stove: Dog allergen
Dog allergen and UCot: Dog allergen
Dog allergen and CCot: Dog allergen
2.1 (0.4-10.3)
1.9 (0.5-7.0)
1.5 (0.6-3.7)
2.0 (0.7-5.6)
0.3 (0.1-1.7)
1.7 (0.4-7.5)
0.8 (0.3-1.9)
0.8 (0.3-2.0)
Single exposure, relative to elevated dog allergen
only:
NO2: Dog allergen
Gas stove: Dog allergen
UCot: Dog allergen
CCot: Dog allergen
2.9 (0.8-10.9)
2.9 (0.6-14.9)
1.2 (0.6-2.5)
1.4 (0.6-3.3)
0.6 (0.2-1.8)
0.9 (0.2-4.2)
0.5 (0.3-1.0)
0.6 (0.3-1.4)
Elevated dog exposure alone, relative to single
elevated exposure only:
Dog allergen: NO2
Dog allergen: gas stove
Dog allergen: UCot
Dog allergen: CCot
0.8 (0.2-3.6)
0.7 (0.2-2.2)
0.8 (0.3-2.4)
1.2 (0.4-3.2)
0.9 (0.3-2.4)
2.3 (0.8-6.1)
1.4 (0.7-3.1)
1.6 (0.7-3.5)
Supplemental Table 5b
(reflects Figure 2 “intercondition” comparisons)
Atopy and exposure, relative to atopy only:
Atopy and NO2: Atopy
Atopy and gas stove: Atopy
Atopy and UCot: Atopy
Atopy and CCot: Atopy
1.0 (0.2-4.4)
2.4 (0.5-11.4)
1.8 (0.7-4.6)
0.5 (0.1-2.0)
0.2 (0.0-1.1)
1.7 (0.4-7.8)
3.1 (1.1-8.6)*
0.7 (0.2-2.2)
Atopy and exposure, relative to exposure only:
Atopy and NO2:NO2
Atopy and gas stove:gas stove
Atopy and UCot:UCot
Atopy and CCot: CCot
2.7 (0.7-10.4)
3.6 (0.7-19.0)
6.6 (2.9-15.1)*
5.0 (2.0-12.8)*
1.4 (0.4-4.1)
3.5 (0.7-17.9)
4.6 (2.1-10.1)*
1.9 (0.8-4.3)
1.9 (0.5-6.8)
3.7 (1.1-12.5)*
4.3 (1.7-10.6)*
3.4 (1.3-9.1)*
2.0 (0.7-5.8)
2.1 (0.8-5.4)
2.0 (1.0-4.0)*
2.9 (1.2-7.0)*
Atopy only, relative to exposure only:
Atopy:NO2
Atopy:gas stove
Atopy:UCot
Atopy:CCot
Models are adjusted for group allocation (“intervention”), which was forced in, and for any baseline characteristic (race,
gender, family history of asthma, maternal education, residence city, season, child’s atopic status) with p<0.05 upon being
entered in a stepwise manner.
* indicates p<0.05