1. 毒物分析 免疫檢驗
賴滄海教授

2. Characteristics of the Screening and Confirmation Assays
Screening Assays
Easy to Perform (automation)
Low cost
Sensitivity
Precision
Accuracy
Confirmation Assays
Low Cost
Easy to Perform

3. 濫用藥物免疫檢驗試劑之原理 (一) 1. Homogenous Immunoassay Enzyme immunoassay
Enzyme Multiplied Immunoassay Technique (Syva EMIT), DRI.
Cloned Enzyme Donor Immuno Assay (CEDIA).
Fluorescence immunoassay
Fluorescence Polarization Immunoassay (FPIA). Abbot TDx.
Microparticle agglutination immunoassay
Roche Abuscreen Online

4. 濫用藥物免疫檢驗試劑之原理 (二) 2. Heterogeneous Immunoassay
Radio immunoassay (Roche, DPC)
Enzyme Linked Immunoassay (DCB)

5. EMIT的原理 G6PD -＊ + Ab → G6PD -＊：Ab (↓Enzyme activity) + ＊ in sample
G6PD -＊ (Full Enzyme activity) + G6PD-＊:Ab (↓Enzyme activity) + ＊:Ab
G6P + NAD phosphogluconate + NADH (↑A 340)
G6PD -＊
+ ＊ in sample
G6PD -＊ + Ab → G6PD -＊：Ab (↓Enzyme activity)

7. Schematic Illustration of CEDIA
ED＊: Ab + EA
No enzyme activity
Active enzyme
– ＊
EA + ED＊ + Ab →Ab :＊+ (EA-ED＊)4

8. Fluorescence Polarization Immunoassay (FPIA)
Ab：Ag + Ab：Ag -Fl + Ag - Fl
(Decreased Polarization) Ag
Ab + Ag - Fl → Ab：Ag -Fl
(Polarized Light)

9. Schematic Illustration of RIA
DRUG ＊ :Ab1
DRUG :Ab1
DRUG ＊
DRUG
DRUG ＊:Ab1 :Ab2
DRUG :Ab1 :Ab2
Ab2 +
+ Ab1
(ppt)

10. Schematic illustration of DCB EIA
No enzyme activity
wash +＊
Plate -Ab + ＊-Enz wash Enzyme Activity
+ substrate -＊

11. Amphetamine analogs

12. Percent cross reactivity of amphetamine analogs with various immunoassays (1)
Reagents
Analyte
ug/mL
Cross reactivity (%)
Beckman
(DRI)
Cobas
I400
Syva
Emit p
Emit II
CEDIA
(microgenics)
AxSYM
(Abbot)
D-Amphetamine 1
151.4
88.8
202.7
97.9
135.5
BDB 10
20.6
4.4
3.9
4.8
94.5
137.5
4-Bromo-2,5-
dimethoxyphenethylamine
0.0
1.3
2.8
Bupropion
0.8
0.3
13.9
0.1
Cathinone
2.4
Chlorpheniramine
0.4
1.4
N-Desmethylselegiline
1.7
2.6
4.1
21.5
49.9
2.1
ethylamphetamine
8.8
2.9
5.2
20.4
221.0
45.1
Fenfluramine
9.2
3.2
6.7
5.0
152.1
16.2
Ketamine
MBDB
135.3
28.4
127.8
12.5
861.0
26.6

13. Percent cross reactivity of amphetamine analogs with various immunoassays (2)
Reagents
Analyte
ug/mL
Cross reactivity (%)
Beckman
(DRI)
Cobas
I400
Syva
Emit p
Emit II
CEDIA
(microgenics)
AxSYM
(Abbot)
MDA 1
85.6
40.2
122.2
68.9
86.1
119.6
MDEA 10
12.4
3.4
8.6
15.2
793.6
57.0
MDMA
101.8
28.1
110.1
27.7
978.0
132.2
Mescaline
0.3
0.0
0.1
Methadone
0.2
0.7
0.9
Methaqualone
5.6
0.8
Methcathinone
4.4
2.3
Methylephedrine
1.5
1.1
Methylphenidate
1.0
Methylpseudoephedrine
1.3
1.6
Norketamine
2.4

14. Percent cross reactivity of amphetamine analogs with various immunoassays (3)
Reagents
Analyte
ug/mL
Cross reactivity (%)
Beckman
(DRI)
Cobas
I400
Syva
Emit p
Emit II
CEDIA
(microgenics)
AxSYM
(Abbot)
Norpseudoephedrine (cathine) 10
7.4
1.2
2.7
1.4
3.0
0.0
Pheniramine
0.1
1.8
Phentermine
4.0
85.5
17.8
51.6
13.8
Phenopropanolamine
(Norephedrine, PPA)
0.3
0.6
2.5
PMA
(p-methoxyamphetamine)
27.7
70.6
59.6
12.1
85.3
18.1
PMMA
(p-methoxymethamphetamine) 1
49.3
25.4
52.0
11.0
936.8
146.1
Propoxyphene
6.1
1.5
Pseudoephedrine
0.7
Selegiline
1.9

15. Percent cross reactivity of amphetamine analogs with various immunoassays (4)
Reagents
Analyte
ug/mL
Cross reactivity (%)
Beckman
(DRI)
Cobas
I400
Syva
Emit p
Emit II
CEDIA
(microgenics)
AxSYM
(Abbot)
2C-H (2,5-dimethoxy- phenethylamine) 10
0.9
0.0
2.8
0.3
2.4
2C-I (4-Iodo-2,5-
Dimethoxyphenethylamine)
0.2
2.1
2.3
2C-T-2 (2,5-dimethyl-4- ethylthiophenethylamine)
1.9
2.0
0.1
2C-T-7 (2,5-dimethoxy-4- propylthiophenethylamine)
3,4-dimethoxyphenethylamine
6.4
0.7
1.6

16. Percent cross reactivity of amphetamine analogs with various immunoassays (5)
Reagents
Analyte
ug/mL
Cross reactivity (%)
Beckman
(DRI)
Cobas
I400
Syva
Emit p
Emit II
CEDIA
(microgenics)
AxSYM
(Abbot)
DOB (4-bromo-2,5- dimethoxyamphetamine) 10
0.1
0.0
3.5
1.1
Methamphetamine 1
168.8
31.9
401.9
186.3
288.4
147.7
HMA ( 4-hydroxy-3- methoxyamphetamine)
5.9
1.0
11.1
5.0
3.6
HHMA
1.4
0.4
4.4
8.8
1.8
MTA (4-methylthioamphetamine)
3.0
16.4
83.8
12.8
96.2
65.9
PCP
1.7
15.6
Tramadol
0.3
78.2

17. Opiate

18. Apparent cross-reactivity (%) of various opiate immunoassays
Analyte
Roche RIA
DPC RIA
Syva EMIT
Abbott FPIA
CEDIA
DRI
O6-Acetylmorphine
14.9
15.7
92.8 81 ?
Codeine
198
0.06
330
111
125
100
Hydrocodone
24.5
138.3
100.5 59 30
Hydromorphine
9.9
0.7
69.2
105.4 66
Morphine
84.7
83.5
86.3
90.0
Morphine-3-Glucuronide
52.4
0.9
45.3
64.2 94 50
Oxycodone
0.1
3.4
18.4
3.1 3
Oxymorphine
0.01
1.2
10.8
1.9
0.6

19. Benzodiazepine

20. Cross Reactivity of Benzodiazepine Immunoassay (1)
Benzodiazepines
Cross Reactivity(%)
DRI
Syva Emit d.a.u.
Roche RIA
CEDIA
(microgenics)
AxSYM
(Abbot)
Alprazolam
1832
1916
158
309
107
α-Hydroxy Alprazolam
916
1610 99
239 89
Chlordiazepoxide 23 31 45 55
Clobazam 52
150 77 92 49
Clonazepam 27 39 35
134
7-Amino Clonazepam 17 21 9 63 24
Diazepam
2294
2190
166
294
140
Flunitrazepam
110 32 53
7-Amino Flunitrazepam 33 13 78 40
N-Desalkyl Flunitrazepam 37
7-Hydroxy Flurazepam
250
1399 82
160

21. Cross Reactivity of Benzodiazepine Immunoassay (2)
Benzodiazepines
Cross Reactivity(%)
DRI
Syva Emit d.a.u.
Roche RIA
CEDIA
(microgenics)
AxSYM
(Abbot)
N-Desalkyl Flurazepam
709
691 77
289 85
Lometazepam 42 97 59
169 54
Lorazepam 38 36 57
167 40
Midazolam
203
888 73
120 65
Nitrazepam 90
119 18
96** 68
7-Amino Nitrazepam 32 66
Nordiazepam
1538
1345
92**
260
102**
4’-Hydroxy Nordiazepam 24 55 53 86
Oxazepam
106**
86** 94 62
Prazepam
811
1955
108
150 76
Triazolam
115
847 98
234
α-Hydroxy Triazolam 49
295 92
205 56

22. Adulterants that Affects the Immunoassays
1. In Vivo Adulteration.
2. In Vitro Adulteration.
(1) Detecting Adulteration.
(2) EIA are more likely to be affected.
(3) Substances used as adulterants.
Alcohols, Ammonia, Ascorbic acid, Bleach, Blood,
Detergent, Drano, Lemon Juice, Peroxide, Salt,
Soap, Visine, Urinaid.

23. 單一步驟免疫層析法 （One-Step Immunochromatography ）
水分吸收區 C T
反應區：
抗體-色素結合物
樣品吸收區
C：品管線（陽性反應線）
T：藥物結合線（陰性反應線） 陽性 陰性
(二條藍色線條）
(一條藍色C線）

26. Thanks for you attentions!