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 PBS-T, 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 / total / In those with data on atopy at 12 months / In those with data on atopy at 7 years / In those with data on asthma at 7 years / In those with data on BHR at 7 yearsCCot / 279 / 276 / 268 / 279 / 258
UCot / 370 / 366 / 360 / 370 / 338
Gas stove / 380 / 376 / 367 / 380 / 348
NO2 / 155 / 151 / 150 / 155 / 145
Dog allergen / 379 / 375 / 366 / 379 / 347
Dog ownership / 377 / 373 / 364 / 377 / 345
TOTAL / Control / Intervention
Environmental tobacco smoke:
CCot (ng/ml)
UCot (ng/mg) / 279
9.2 ±38.2
370
89.5 ±280.0 / 130
9.9 ±39.4
173
75.5 ±231.1 / 149
8.5 ±37.2
197
101.9 ±316.9
NO2: (parts per billion) / 155
11.3 ±5.1 / 72
12.1 ±6.0 / 83
10.6 ±4.0
Dog allergen: (µg/g of dust) / 379
9.0 ±21.0 / 177
8.4 ±21.2 / 202
9.6±20.9
Presence of gas stove / 380
37 (9.7) / 178
21 (11.8) / 202
16 (7.9)
Dog ownership / 377
60 (15.9) / 177
26 (14.7) / 200
34 (17.0)
Atopy / 376
90 (23.9) / 175
42 (24.0) / 201
48 (23.9)
Supplemental Table 2. Sample size and mean ±standard deviation or frequency (percent) of early exposures.
Supplemental Table 3.Frequency (%) of asthma and BHR by dog allergen and other (non-allergen) exposure status; associated ORs
Exposures^ / Asthma / No asthma / Adjusted* OR** / BHR / No BHR / Adjusted* OR**--dog allergen AND NO2
--only dog allergen
--only NO2
--neither / 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
--dog allergen AND gas stove
--only dog allergen
--only gas stove
-- neither / 4 (5.6)
26 (36.6)
4 (5.6)
37 (52.1) / 8 (2.6)
94 (30.5)
21 (6.8)
185 (60.1) / 3.0 (0.7-12.5)
1.2 (0.6-2.3)
1.0 (0.3-3.4)
1 / 4 (2.8)
37 (26.2)
11 (7.8)
89 (63.1) / 6 (2.9)
71 (34.5)
13 (6.3)
116 (56.3) / 1.0 (0.2-3.8)
0.7 (0.4-1.1)
1.1 (0.5-2.6)
1
--dog allergen AND UCot
--only dog allergen
--only UCot
-- neither / 22 (31.0)
8 (11.3)
21 (29.6)
20 (28.2) / 62 (20.8)
34 (11.4)
86 (28.9)
116 (38.9) / 1.8 (0.8-4.0)
1.1 (0.4-3.0)
1.1 (0.5-2.5)
1 / 24 (17.6)
15 (11.0)
47 (34.6)
50 (36.8) / 49 (24.4)
24 (11.9)
52 (25.9)
76 (37.8) / 0.8 (0.4-1.5)
0.8 (0.4-1.8)
1.3 (0.8-2.3)
1
--dog allergen AND CCot
--only dog allergen
--only CCot
-- neither / 13 (26.0)
7 (14.0)
18 (36.0)
12 (24.0) / 37 (16.2)
39 (17.1)
68 (29.8)
84 (36.8) / 2.7 (1.1-7.1)
1.3 (0.4-4.1)
1.4 (0.6-3.5)
1 / 15 (14.3)
14 (13.3)
36 (34.3)
40 (38.1) / 29 (19.1)
29 (19.1)
44 (28.9)
50 (32.9) / 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^ / Asthma / No asthma / Adjusted* OR** / BHR / No BHR / Adjusted* OR**--atopy AND NO2
--only atopy
--only NO2
--neither / 5 (25.0)
5 (25.0)
7 (35.0)
3 (15.0) / 16 (12.2)
17 (13.0)
48 (36.6)
50 (38.2) / 5.9 (1.2-28.1)
4.4 (0.9-20.8)
2.3 (0.6-9.5)
1 / 9 (15.5)
11 (19.0)
18 (31.0)
20 (34.5) / 12 (14.5)
9 (10.8)
31 (37.3)
31 (37.3) / 1.2 (0.4-3.3)
1.9 (0.7-5.4)
0.9 (0.4-2.0)
1
--atopy AND gas stove
--only atopy
--only gas stove
-- neither / 4 (5.6)
30 (42.3)
4 (5.6)
33 (46.5) / 6 (2.0)
50 (16.4)
22 (7.2)
227 (74.4) / 8.6 (2.0-37.1)
5.5 (2.9-10.3)
1.3 (0.4-4.4)
1 / 6 (4.3)
41 (29.3)
9 (6.4)
84 (60.0) / 3 (1.5)
32 (15.7)
15 (7.4)
154 (75.5) / 3.7 (0.9-15.1)
2.4 (1.4-4.0)
1.1 (0.5-2.6)
1
--atopy AND UCot
--only atopy
--only UCot
-- neither / 20 (28.2)
14 (19.7)
23 (32.4)
14 (19.7) / 21 (7.1)
33 (11.2)
126 (42.7)
115 (39.0) / 8.0 (3.3-19.5)
4.2 (1.71-10.0)
1.0 (0.5-2.2)
1 / 25 (18.5)
22 (16.3)
45 (33.3)
43 (31.9) / 11 (5.5)
22 (11.1)
90 (45.2)
76 (38.2) / 4.1 (1.8-9.2)
1.8 (0.9-3.6)
0.9 (0.5-1.5)
1
--atopy AND CCot
--only atopy
--only CCot
-- neither / 15 (30.0)
10 (20.0)
16 (32.0)
9 (18.0) / 20 (8.8)
18 (8.0)
86 (38.1)
102 (45.1) / 9.3 (3.4-25.5)
9.4 (3.1-28.3)
2.2 (0.9-5.3)
1 / 16 (15.4)
17 (16.3)
35 (33.7)
36 (34.6) / 15 (9.9)
10 (6.6)
59 (39.1)
67 (44.4) / 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 1and 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 / BHRSupplemental Table 5a
(reflectsFigure 1 “intercondition” comparisons)
Combinedexposure, relative to elevated dog allergen only:
Dog allergen and NO2: Dog allergen / 2.1 (0.4-10.3) / 0.3 (0.1-1.7)
Dog allergen and gas stove: Dog allergen / 1.9 (0.5-7.0) / 1.7 (0.4-7.5)
Dog allergen and UCot: Dog allergen / 1.5 (0.6-3.7) / 0.8 (0.3-1.9)
Dog allergen and CCot: Dog allergen / 2.0 (0.7-5.6) / 0.8 (0.3-2.0)
Single exposure, relative to elevated dog allergen only:
NO2: Dog allergen / 2.9 (0.8-10.9) / 0.6 (0.2-1.8)
Gas stove: Dog allergen / 2.9 (0.6-14.9) / 0.9 (0.2-4.2)
UCot: Dog allergen / 1.2 (0.6-2.5) / 0.5 (0.3-1.0)
CCot: Dog allergen / 1.4 (0.6-3.3) / 0.6 (0.3-1.4)
Elevated dog exposure alone, relative to single elevated exposure only:
Dog allergen: NO2 / 0.8 (0.2-3.6) / 0.9 (0.3-2.4)
Dog allergen: gas stove / 0.7 (0.2-2.2) / 2.3 (0.8-6.1)
Dog allergen: UCot / 0.8 (0.3-2.4) / 1.4 (0.7-3.1)
Dog allergen: CCot / 1.2 (0.4-3.2) / 1.6 (0.7-3.5)
Supplemental Table 5b
(reflectsFigure 2 “intercondition” comparisons)
Atopy and exposure, relative to atopy only:
Atopy and NO2: Atopy / 1.0 (0.2-4.4) / 0.2 (0.0-1.1)
Atopy and gas stove: Atopy / 2.4 (0.5-11.4) / 1.7 (0.4-7.8)
Atopy and UCot: Atopy / 1.8 (0.7-4.6) / 3.1 (1.1-8.6)*
Atopy and CCot: Atopy / 0.5 (0.1-2.0) / 0.7 (0.2-2.2)
Atopy and exposure, relative to exposure only:
Atopy and NO2:NO2 / 2.7 (0.7-10.4) / 1.4 (0.4-4.1)
Atopy and gas stove:gas stove / 3.6 (0.7-19.0) / 3.5 (0.7-17.9)
Atopy and UCot:UCot / 6.6 (2.9-15.1)* / 4.6 (2.1-10.1)*
Atopy and CCot: CCot / 5.0 (2.0-12.8)* / 1.9 (0.8-4.3)
Atopy only, relative to exposure only:
Atopy:NO2 / 1.9 (0.5-6.8) / 2.0 (0.7-5.8)
Atopy:gas stove / 3.7 (1.1-12.5)* / 2.1 (0.8-5.4)
Atopy:UCot / 4.3 (1.7-10.6)* / 2.0 (1.0-4.0)*
Atopy:CCot / 3.4 (1.3-9.1)* / 2.9 (1.2-7.0)*
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