Supplementary Materials Theoretical calculation and experimental verification demonstrated the impossibility of finding haptens to identify triphenylmethane dyes and their leuco metabolites simultaneously De-Xin Konga, b, #, *, Fang Lvc, d, # Ben Hua, b and Li-Min Caoc,* State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China;
[email protected] (B.H.) b Agricultural Bioinformatics Key Laboratory of Hubei Province, College of Informatics, Huazhong Agricultural University, Wuhan 430070, China c Food Safety Laboratory, Ocean University of China, Qingdao 266003, China;
[email protected](F.L.) d Laboratory of Quality&Safety Risk Assessment for Aquatic Product, Ministry of Agriculture, Aquatic Product Technology Promotion of Beijing, Beijing 100021, China # The first two authors contributed equally * Correspondence:
[email protected] (D.X.K);
[email protected] (L.M.C); Tel.: +86-27-8728-0877 (D.X.K); +86-532-8203-2389(L.M.C) a
Supplementary Information Figures .................................................................................................................... 2 Figure S1 Structure validation of the synthesized intermediate................................................................ 2 Figure S2 Structure validation of the synthesized hapten (N+). ................................................................ 3 Figure S3 Validation of the coating antigen. ................................................................................................. 4 Figure S4 The standard curve of antigen with -N+ structure and proteins (BSA and KLH) ................. 5 Supplementary Information Tables ...................................................................................................................... 6 Table S1 Calculated partial charge of MG’s dimethylamino atoms at different theoretical levels ....... 6 Table S2 Calculated partial charge of dimethylamino carbon atoms of five reported haptens ............ 7 Table S3 Calculated partial charge of dimethylamino carbon atoms of TDs analogues from SciFinder, ACD or Zinc database ...................................................................................................................................... 8 Table S4 Calculated partial charges of haptens’ carbon atoms for the designed haptens ................... 12 Table S5 Structure-cross reactivity relationship of the hapten with TDs ............................................... 17 Table S6 Working dilution of coating antigen and goat IgG secondary antibody by checkerboard test (n=3) .................................................................................................................................................................. 18
S1
Supplementary Figures
a
b
c
d
e
f
Figure S1. Structure validation of the synthesized intermediate. a. 1H NMR. b. 13C NMR. c. 1H-1H COSY. d. HMBC. e. HSQC. f. TOF- mass spectra.
S2
a
b
c
d
e
f
Figure S2. Structure validation of the synthesized hapten (N+). a. 1H NMR. b. 13C NMR. c. 1H-1H COSY. d. HMBC. e. HSQC. f. TOF- mass spectra.
S3
a
b
c
Figure S3. Validation of the coating antigen. a. The SDS-PAGE of BSA, cBSA and complete antigen. Marker:low molecular standard protein; 1:BSA; 2:cBSA; 3: complete antigen. b. Ultraflex II MALDI-TOF/TOF-MS of BSA. C. Ultraflex MALDI-TOF/TOF-MS of coating antigen.
S4
Absorbance (OD)
0.35 y = 0.029x - 0.0166
0.3
2 R = 0.9901
0.25 0.2 0.15 0.1 0.05 0 -0.05 0
2
4
6
8
10
12
Absorbance (OD)
Concentration of the hapten (μg/L)
1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0
y = 1.7637x + 0.0238 2
R = 0.9929
0
0.1
0.2
0.3
0.4
0.5
0.6
Absorbance (OD)
Concentration of the BSA (mg/L)
0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0
y = 1.5798x + 0.0051 R2 = 0.9957
0
0.1
0.2
0.3
0.4
0.5
0.6
Concentration of the KLH (mg/L)
Figure S4. The standard curve of antigen with -N+ structure and proteins (BSA and KLH).
S5
Supplementary Tables Table S1. Calculated partial charge of MG’s dimethylamino atoms at different theoretical levels. atoms
AM1
HF/STO-3G
HF/6-31G
B3LYP/6-31G*
1 N 2 C 3 C 4 N 5 C 6 C
-1.366955 0.414306 0.427113 -1.217105 0.344703 0.34695
-1.565772 0.338409 0.383298 -1.565786 0.338414 0.383302
-0.928144 0.359168 0.360676 -0.928144 0.359168 0.360676
-0.463849 0.228822 0.230521 -0.463849 0.228822 0.230521
S6
Table S2. Calculated partial charge of dimethylamino carbon atoms of five reported haptens. 45
45
40
40
N
N
C
C
41
CH
32
31 HOOC
N
N
36
C
C
36
N S
N
HOOC
37
31
A 31
46 N
B 0.233730
31
C
C 0.230090
32
C
0.156683
36
C
0.235630
36
C
0.228719
37
C
0.156024
40
C
0.232989
40
C
0.228749
41
C
0.157618
45
C
0.235172
45
C
0.230224
46
C
0.156384
46
41 N
46
41 N
CH CH
37
N
H2N
HOOC
37
N
O
32
32
D
E
32
C
0.151416
32
C
0.154602
37
C
0.150437
37
C
0.154195
41
C
0.150399
41
C
0.154641
46
C
0.151539
46
C
0.155146
S7
Table S3. Calculated partial charge of dimethylamino carbon atoms of TDs analogues from SciFinder, ACD or Zinc database. 42
25
37
27 46 41 25
33
28
N
N
1
30 3
10
N
32
11 8 23 13 5 6 14 21 15 17 19 18 O 51
O
50
24
33
37
4
80
55 64 45
50
26 30 34 38
40
38 36 1
11 13
25
26
21 8
39
28
1
3
53
19 17
49
0.228808 0.227721 0.227721 0.228808 60
33
7 15 6 11 13
OH
S
HO
17
9
1
43
N
63
19
NH2
57 55
67
22
31
34
O
H O N 51 S 5354 20 O
64 59
14
24
55
16
C C C C
35
62 O 64
65
31 35 40 44
26
29 27
47
21072011_141628
0.157249 0.158332 0.156130 0.155910
35 39 43 47
C C C C
0.153237 0.152950 0.157305 0.150799
43
57
F 47
54
N
42
O 53
F
51
45
68 62
4
21072011_133159
56
43
C C C C
2
59
0.211749 0.211565 0.210805 0.211220
35
8
15 16
21072011_143144
N
6
32
C C C C
21
39
N
10
30
HO 57 S 52 O 58 O
28
30
25 29 33 37
31
26
11 5 21 13 48 20 14 60 50 15
18
20O
68 72 76 80
0.156409 0.156648 0.155104 0.154922
23
24
5
4 10
N
44
59
41
N2
3N
C C C C
57
43 22
29
40 68
61
46
47
48
23
19
C 12 C 13 C 41
47
N 66
62
53
52
14 11
21072011_144235
72
76
N
18
42
0.152416 0.153087 0.152927 0.152151
67
6
52
C C C C N
7
2
21072011_133601
33 37 42 46
16
9
1
29
28
26
29
24 14 31 4 15 13 6 2 11 37 17 45 33 35 CH3 H3C 19 22 7 1 CH 20 9 CH
CH3
6
F
53
N
34
43
1
5
28 26
42
15
19 52 O 51
38 24
8 10
6
N
33 11 5 1 31 13 52O 3 O 53 14 15 S 22 51 OH 20 16 54 18
29
3
14
20
49
10 N 33
11
22
CH3
41
8
24
31 13
54 F
47
38
28 26
17
F
34
55
21072011_132958
37 41 45 49
C C C C
-0.004178 -0.004651 -0.004410 -0.004167
N 42
36
34 38 43 47
31
23
27 12 16
21
1 5 14 3 6
4
10 8
19
32
30 29
Cl 33
18
N
37
25
C C C C
0.155869 0.154652 0.155245 0.154802
42
46
38
N 30
50
46
11 15
36
20
3
4
1 2
35
7
O
O-
34
MFCD00100147
34 38 43 47
C C C C
0.153310 0.154470 0.153443 0.153351
54 26 31 + N 37 50
22
9
N+
21072011_133231
21072011_133035
18
28 O
38
32
13
24
5 33 29 O17 S O
39 33N 35
19
51 O
7
29
2 1 17
5 15 27
47
21
9 3 4 11 13
30 23
N 34 43
25 31
O
35 34
MFCD00151208
MFCD00144834
S8
38 42 46 50
C C C C
0.158490 0.158268 0.158512 0.159161
42 46 50 54
39
43
32
11 55
33
30
9
15 27
13
21
25
31
51 47
5
48 67
34
2
11 23
N
C C C C C C
21
19
57
32 29 N
45
60 26 12 16 4 3 1Si 10 2 24 68 8
47
21
34 N 43
C C C C
30 23
51
3 1 4 11 13
2
23
18
1
3
11
46
27 31
9 15
33
39
N+ 34
49
26
4
13
2
41
72
34
77 70
20
63
5
7
17 65
19 29
58
N 38
35
32
57 59 67
61 68
56
74
O 79 S 76O 80 O 81
MFCD03931987
48 52 56 60 64 68 72 76 80
C C C C C C C C C
29 11
31
34 N
31 25
0.153389
33
36
40
N
30
11 3 P 41
51
0.189165 0.225108 0.191031 0.192583
18
20
15 27
43
C C C C
23
9
38
41 45 49 53
30
21
N 35
MFCD00068428
-0.007497 -0.022254 -0.009972 -0.010685 -0.023857 -0.011524 -0.023187 -0.010893 -0.007577 47 33 N
36
13
2
5
48
17 7 19
29 N 32
39
22
P
34 N 31 44
6 2 14 1 4 3 O 16 12 5
10 8
24
26 32 28
56 N 35
52
35
MFCD00012483
C
45
53 30 21
48
35
35
5 6
52
27 25
N
43
58
5 15
41
76 28
0.156358 0.155816 0.156696 0.157039 0.156060 0.156618
36
19
7
O
59
29
F 14
19
C C C C C C
O19
22
0.188905 0.188305 0.191707 0.244071
9
39 43 47 51 55 59
80
14 32
37 44
MFCD00166979
41 45 49 53
45
64
35
38
5
25 10F 20
31 55 35 N 38
MFCD00813906
0.163356 0.160938 0.161189 0.163395 0.166779 0.153173
30
62
17 64
7
9
30
67
5
2
3 1
41 33
36 40 44 48 52 56
O O 73 66 S 71 72 O
69
15
34
21
27 12 8 6 16 2 3 1 4
18
74 27
33
F
38
4
47
51
72
13
36
37N
49
31
23
12
MFCD02177828
N 25
P1
43
36
23
44
0.007204 0.020663 0.024351 0.007517 0.020909 0.024479 0.021025 0.007314 0.024232
53 34
0.156307 0.158011 0.153389 0.154595 N
N 34
35 14 N 33 25
63 59
C C C C C C C C C
32 3
19 8 16 27 4
MFCD02683475
35 39 43 47 51 55 59 63 67
C C C C
40
21
10
O
6
18 29
5
+ 3 S 4 1
23
N
56
2
35 39 43 47
39
31
17
7
0.194282 0.191635 0.209925 0.196617
52
35
29 19
C C C C
MFCD00014866
35
C
0.156463
MFCD00025649
36
C
0.160255 S9
39 43 47
C C C
0.154595 0.158010 0.156304
55 35
39
51
25
27
19 14
4 1 2 5 12 3 10 17 8 21
0.155951 0.156205 0.155612
36
29 N 63 16 30
6
C C C
59
40
N
39 43 47
59 63
57 19
65
36
17 8
4
23
46 39 N 21 34
66
48 11
70
12
44
68
35
53
0.216765 0.217281 0.214926 0.215507
41 45 49 53
C C C C
62
0.217800 0.218154 0.215667 0.216447
50 54 58 62
53
62
28 9 3
4
713
2 1
O
5
Cl
32 N 35 11
24
58 56
54
45
22
15
20
65
63
O
40
53 60 55
49
52
20
9
N
2
57
22
7
35 1
O
6
4
6
31
N
0.217060 0.216619 0.218236 0.218932
64
26
34
C C C C
Cl
N
33
41
58
45
MFCD00036331
36
57
54
N
49
MFCD00011878
MFCD00011876
C C C C
39
N+
9 37 3 13 28 2 6 15 1 4 7 17 19 30 32 38
8
N
43 38 47
43 47 51 55
41
26
25
27
N+
50 24
22
40
23
3 10
14
34
38
34
21
6
61
32
0.163242 0.162739 0.159910 0.162778 0.159986 35
N+
21 5
C C C C C
45
32
30 67 16 N 29 40
40 44 48 52 56
11
13
14
O
37
29
17
33
36
17
15
24
31
18
N
19
28
26
N
MFCD00070611
37 41 45 49 53 57
C C C C C C
56
0.170124 0.160380 0.154495 0.156577 0.158690 0.157325
36 40 44 48
60 54
60
55
N
29 8
14
HOOC
11
74 72
35 26
2
5 19
1N
17
18
S
32
34
11
70 68
54 38
14
73
41 40
HOOC
68
N
8
2
11
74 72
5
1 18
N 17
S
23
22
64
1
5
N
S
19 20
18
26
46 24 48
21
28
23
31
55 49
47
64
43 41
34 36
N
68
31
52
1C
2N
58
56
C C
0.172212 0.176510
56 60
C C
0.173441 0.181802
52 56
C C
0.154600 0.155000
64
C
0.173922
64
C
0.175572
60
C
0.154124
68
C
0.172447
68
C
0.175278
64
C
0.154542
8
14
68
11
2
5
17 1
S
20
N 19
52 27 25 41
18
20 22
2C
43
24
28
31
45
HOOC
N 50
30
14
73
46
5 6
74
72
2
8 11
5
38 27 36
1
S
18
24
25
31
48
32
39
34
37
64 51
1CC 60
32
35
40
73
N
54
HOOC 64
74
72
14
2
8 11
5
64 27
52 50
20
24
32
35
34
N
40
N
54
68
38 36
52
45 47
49
41
25 43
22
47
55
19
17
41 43
45
56
N18 1
S
34
36
N
28
68
17N 19 29
N
32
37
N
45
51 60
30
39
50
26 45
24
17
2
56 60
69
70
HOOC
41
43 35 52 29 34 28 31 32
1N
HOOC
64 8
14
36
43
24 36 23
48
0.154451 0.153844 0.154876 0.153702
69
38
45
52
34
56
40
47
50
73
C C C C
N
49
27
44
2Cl
49
50
N
60
2CC
56 55
60
S10
52 56 60 64
C C C C
0.154503 0.154926 0.154619 0.155231
56 60 64 68
C C C C
0.174610 0.174031 0.172120 0.177219
56 60 64 68
C C C C
0.172658 0.172186 0.173224 0.171255
S11
Table S4. Calculated partial charges of haptens’ carbon atoms for the designed haptens. a) with simple modification 45
46
40
H3C
CH N 44 3
H3C 25
23
26
24
21
28
22
12 67
59
HOOC 66
53
62
C
3
2
8 35
4 6
16
21
11
10 31
18
49
50
56
20
1
45
H3C
30
CH3
N CH3
5
31
N CH3
19
15 60
54
19
7
50
17
57
51
66 68
COOH 63
67
17
66
60
H2N
21
1
54
63
57
50
51
24 22
28
2
37
36
A1
36
H3C
30
26
13
CH 1
4
9
32
CH3
N
30
27 14
41
46
CH3
N
29
65
41
45
CH
13
11
4
2
9
7
15
31
C1
B1
C
0.226104
32
C
0.152674
32
C
0.151659
36
C
0.227602
37
C
0.152005
37
C
0.152183
40
C
0.225762
41
C
0.151952
41
C
0.151901
C
0.227075
46
C
0.153184
46
57 52N
53
33
32
28
8
1
23 13
30
6
3
6
12 21
44 34
11
7 14
CH 1 9
33
N
16
5 21
19 32 36 18 CH
48
40
55 50N
16
0.030755 0.030767 0.030755 0.030767 0.241280 0.241280
25
8
3
6 5 31 11 13
19
N41
O47
15
29 28
24
17
O42
26
46
4N
33 37 42 46 51 55
42
1
14
20
27
14 23
51
22
28
13 12
18
CVC
C C C C C C
30
N11
19
0.152705 38
10
10
25
4
36 40 44 48 53 57
37
N 27
2
C 34
CH3 N CH3
37
31
45
32
36
5
48
C C C C C C
0.147758 0.148349 0.147731 0.148262 0.147875 0.148460
43
2O
34 38 43 48
C C C C
0.153216 0.153742 0.254175 0.253940
b) with electron donating group for oxidized form TDs 36 31 32
H3C
51
COOH
1
8 6
19
706968
HOOC
43
13
18
42
16
54
14
11
12
34 38 47 51
38
34 38
18
27 N 33
57
42
34
43 60
eth1
C C
16
54
62
34 38 50 54
C C C C
50
0.225739 0.229821
65
34 38
69
C C
1
10
8
5
6
0.226079 0.227357 0.227773 0.226470 46
12
23
70
28
14
25
16
54
67
27
21 30 19 COOH 77 78 79
34
38
50 N 32
3
11
28 27 N 33
0.226994 0.224763 0.225300 0.224286
13
6 30
34
phe2
14
25
28
8
25
33
38
C C C C
73
23
61 64
30
54
10
3
14 5 13 18 11 12 42 19 21 47 COOH 23 69 70 71 58
N 32
30
57
8 6
28
46
5
67
59 16
51
N
50
10
61
27
32N
3
32 N 1
65
43
25
0.225636 0.226451 0.227441 0.225166 46
32
10 5
23
41 CH 3 N CH3
phe1
C C C C
N
63
12
phe0
21
11
13
46
62 64
32 36 42 46
19
COOH
40
3
14
42
9
1
69 7071 21
7
15 63
55
16
18
44
5
3
13 11
17
50
22 4 2
42
67
47
55
43
65
24
28 20 1
18 58
59
63
30
26
50
61
CH3
N
38
18
N33
57
42 43
34
60
10
8
5
6
13
11
12
23
19 COOH 74 75 76
25 27
21 30 28
38 N33 34
63
2ETH
eth2
0.225122 0.226288
1 3
34 38
C C
0.226198 0.224792
S12
46 50
C C
0.225970 0.225331 46
46 50
C C
0.227461 0.225981
50
46
32N
21 737271 HOOC
4319 18
42 54
C C
0.226076 0.224996 46
50
50 32 N
32 N
1 10
8
1
10
8
1
10
8
3
6
3
5
6
3
5
6
5
23
13 11 12
21
25
54 19
27
14 30
58
N 33
28
16
57
34
63
42
61
23
13 11 12
38
61 67
14
18
25 27
30
62
54
57
42
34
C C C C
0.225135 0.228727 0.225199 0.224542
34 38 46 50
69
C C C C
14 45
54
0.226549 0.225338 0.227335 0.226154
34 38 46 50
60
10
8
3
5
6
11
12
23
42
73
14
25 27
28
16
54
62
37
18
N32
42
57 60
33
65
64
1 3
5
13
8 6 23
12
11
25 27
21 30 19
38 N33
28
34
69
ie2
ie1
33 C 37 C 69 C 73 C
50
10
COOH 77 78 79
43
COOH 77 78 79
51
0.281606 0.279353 0.279900 0.278811
N 32
1
19
41
48
C C C C
46
21 30
18
34
76
73
13
16
38 N 33
28
2ISO
N 68
56
16
COOH 80 81 82
43 60
72
iso2
iso1
27 14 30
18
68
25
11 12
13
19
38 N33
28
16 COOH 43 717273
23
21
64
67
63
34 38 46 50
46 50
0.223948 0.225779 -0.013546 0.004094
34 38 46 50
C C C C
0.225402 0.224145 0.225763 0.224761
c) with electron withdrawing group for reduced form TDs 54
49
50
33
14 68 66
44
HOOC 67
47
38 41
5
13
18
1
1
3
3
F
11
28
19
C C C C
66
27
HOOC
62
34
67 65 46
N
40
19 34
68
66 46
40
28
68 66 46
N
40
19 20
34
32
49 53 57 61
C C C C
25
61
0.156169 0.156026 0.156622 0.156383 33
61
49 53 57 61
C C C C
0.154421 0.153093 0.154284 0.153328
57
57
25
28
23
30
30
13 17
25 33
21 30
20
15
23
28
27
N
67 61
HOOC
43
68 66 46
37 19 40
34
20
11
21
5
6
17
8
70 68
32
3 1
10
Br
57
N
53
43
HOOC 69
46
37 40
49 53 57 61
C C C C
0.155067 0.153737 0.154972 0.154122
21 20
34
O
Br
25
23 13 15 14 6 5 11
19
67
49
65
Cl
0.155137 0.153799 0.154891 0.154142
14
61
27
27
13
33
N
N
6 11
57
F
8
14
33 27 N
F 65
57
53
28
21 23
3
65
C C C C
27
37
43
HOOC
61
30
13
11
CN
Cl
49 53 57 61
33
67
1
17 19 34
17
25
20
5
HOOC
15 14
23
28
15 37
8 6
5
21 30
10
43
3
13
11
N
67
1
6
CN
0.156452 0.155477 0.155636 0.156204 49
37
43
58
F0
50 54 58 62
15 14
17
25
21 30
35
8 5
23
12
53
N 10
10
10
16
32
49
N
8 6
53
32
N
N 65
8 32
3 1
10
N
53
49
O 66 NO2
49 53 57 61
C C C C
0.156698 0.156531 0.156784 0.156766 S13
33
42
38
N 19
20
17
22
15 9
13
8
1 37
O
24 26 34
31
6
3
35 N
14
11
28
29
4
50
N
O
46
36
NOO
38 42 46 50
C C C C
0.157667 0.158686 0.157551 0.157857
d) with N+ electron withdraw groups for oxidized form TDs 33
37
32
H3C
37
CH3
N
H3C
31 27
HOOC 71
18 51
10
4
21
42
6 8
19
66
41
47
CH3
N 43 CH3
69 67
57
HOOC 68
16
63
27
25
27
25
23
29
23
60
14 1
2
18 51
33 37 43 47
C C C C
8
19
co1
0.171242 0.166441 0.167129 0.168775 33
H3C
33 37 43 47
C C C C
33
CH3
72 70
56
O2N 71
51
21
42
6 8
19
41
47
CH3
N 43 CH3
32
N
16
69 67 68
18
NH
60
51
55
33 37 43 47
69
33 37 43 47
18 51
no1
CH3
N 43 CH3
66 64
37
H3C
CH3
32
N
58 55 53
O2N 65
18
N H2
61
51
33 37 43 47
C C C C
6
33
37
CH3
H3C
25
27
23
29
8
2 4
42
21 12 10
41
43
CH3
N 47 CH3
0.170494 0.165265 0.166478 0.167635
63
H2N
16 57 60 55
14 1
18
NH
51
5
CH
6 8
2 4
53 19
42
21 12 10
66
41
43
CH3
N 47 CH3
16
64
58
H2N 61
C C C C
0.168521 0.167558 0.171216 0.166289
14 1
32
N
47
CH3 N 43 CH3
33
CH3
N 55 H2
18 51
25 23 5
CH
6 8
2 4
53 19
42
21 12 10
43
CH3 N 47 CH3
41
nh2
nh1
33 37 43 47
42
31
29
6
10 41
0.170340 0.169414 0.168430 0.173488
27
19
21
8
23
CH
4
31
5
12
2
19
25
14 1
29
CH
no2
0.169794 0.169081 0.172819 0.167485
33
CH3 27
14 1
29
nh0
C C C C
8
41
47
16
27
53
N55
59
42
21 6
C C C C
47
CH3 N 43 CH3
37
32
N
5
10
2 4
19
42
31
12
CH
63
31
H2N
14 1
53
57
O2N
0.166087 0.171436 0.167404 0.168415
16
33
H3C
23
37
65
37
25
10
10 41
0.169345 0.167934 0.172271 0.167207
29
H3C
56
C C C C
27
no0
62
33 37 43 47
23
12
6
co2
25
CH
4
21
8
CH3
5
2
19
29 2 4
C C C C
51
27
66
33 37 43 47
N
61
18
N 55 H2
23 14 1
18
N 43 CH3
65
25
53
N 55
59
41
CH3
12
CH
53
58
HOOC
31
5 16
32
H3C
31
62
47
66 64
0.168745 0.167911 0.171481 0.166406
37
32
N
42
6
14 1
16
10
4
21
co0
5
12
CH
53
N55 H
CH3
31
5
12
CH 2
53
N55
59
14 1
16
62
56
32
N
29
25 23 5
33
37
H3C
CH3
31
29
72 70
33 32
N
33 37 43 47
C C C C
0.169093 0.167576 0.171874 0.166925 S14
37
32
H3C
33
37
CH3
N
31
29
25
27
23
29
5 62
56
HS
53 18
N55
59
14 1
16
51
12
CH 2
4
21
6
10 42
57
HS
CH3
N 43 CH3
53 18
NH
60
51
55
2
4
21
6
C C C C
0.171480 0.166369 0.167937 0.168928 33
33 37 43 47
C C C C
3
HOOC
81 79
73
37 19
N 55
67
5 11
13
58 31 34
35
26
20
28
63
34 47
N
39 43 47 51
C C C C
HOOC
32
16
62 66 70 74
80 78
43
HOOC 79
46
37 19 40
13
21 23 20
34
N 66 65
46 41 49
47
N 60 70 78
HOOC 79
9
22
16
74
28 33 25 27 N 61
70
22
77
HOOC 78
76
2
16 19
13
9
40 50 47 39 37 49 33 32 57 35 30
52 54
69
N 60
55
73
27 28
1
N NH 25
5
NC
NNH
61 65 69 73
C C C C
0.168287 0.171517 0.170615 0.166524 67
76
80
61
31
40
45 44 42
60
64
N 58
40
64
32
27
85
1N O 25
59
62
49
47 38 39 36
26 34
HOOC
86 84
8
14
11
2
5
22
42 30
N 1
34
35
47 39
44
37
57 46 48
55 53 66 50
17 26
52
N
68
72
18
5
CN1
NO
N
42
N
29
57
65
2 13
19
77
47
CH3 N 43 CH3
N
53 51
56
30
42
0.172449 0.167207 0.168682 0.169421
68
N
6
5 11
C C C C
25
54
3
10 41
8
74
0.170394 0.166587 0.168363 0.171802
8
1
6
19
59 72
10
15 14
44
31
C C C C
53
21
45
28 29
1N
4
44
5
N
17
2 13
19
80 78
0.170105 0.164528 0.167038 0.166533 49
22
79
51
N2
9
2
sh2
33 37 43 47
51 40 42 33
36
12
CH
59
38 29
22 24
51
53
56
15 14
17
59
61
61
55
1
6
18
N H2
62
N
10
56
HS
61 66
8
70
58 55 53
0.169049 0.168174 0.171839 0.166811
39
23 5
14 1
16
64
CH3
N 43 CH3
65
N
76
41
47
sh1
43
80
42 8
sh0
25
29 10
19
CH3
27
12
CH
33
N
31
23 5
32
H3C
25
14 1
16
63
47
41
8
19
68
33 37 43 47
37
CH3
N
31
27
64
33
32
H3C
49 53 57 61
C C C C
0.172261 0.177335 0.174581 0.174055
60 64 68 72
C C C C
0.199318 0.200997 0.194599 0.193333
68 72 76 80
C C C C
0.170571 0.169572 0.168176 0.173469
59
C
0.171082
59
C
0.170810
62
C
0.172528
63
C
0.169480
63
C
0.170822
66
C
0.167533
67
C
0.172691
67
C
0.168898
70
C
0.168038
71
C
0.168395
71
C
0.175269
74
C
0.169410
S15
79 68
N 51 22
86
HOOC
87 85
16 19
5
2 13
1
N 9
26 25 33
28
36 30 35 31 43
49 38 48 40 41
54 56 45 65
47 58 60
63 62
N
67
NPH
81
53
69
73
69
C
0.165927
65
C
0.173222
0.175581
73
C
0.161498
69
C
0.168625
0.170265
77
C
0.162523
73
C
0.169289
0.171459
81
C
0.164074
77
C
0.170825
0.172705
S16
Table S5. Structure-cross reactivity relationship of the hapten with TDs.
Compounds
Average charge
Absolute charge
Percentage
Cross-
Cross-
of C atoms
difference*
difference %**
reactivity
reactivity
(rabbit)
(mice)
MG
0.229671
0.057
33.14
24.50
20.56
LMG
0.152528
0.020
11.58
51.22
37.79
CV
0.216933
0.044
25.75
29.24
23.49
LCV
0.150946
0.021
12.50
68.73
55.64
-N+
0.172503
0
0
100%
100%
* Charge difference of the indicative C atoms with hapten (–N+). ** The charge difference in percentage by dividing the charge of hapten (0.172503).
S17
Table S6. Working dilution of coating antigen and secondary antibody by checkerboard test (n=3). Dilution of IgG secondary antibody anti rabbit IgG anti mouse IgG
Concentration of the coating antigen(µg•mL-1) 0.1
0.2
0.5
1
2
5000
0.813
0.813
0.814
0.991
0.757
10000
0.506
0.542
0.566
0.598
0.476
5000
0.493
0.535
0.594
0.691
0.657
10000
0.706
0.782
0.866
0.998
0.776
S18
