毒物分析 免疫檢驗
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Transcript of 毒物分析 免疫檢驗
毒物分析 免疫檢驗
賴滄海教授
09-29-2008
Characteristics of the Screening and Confirmation Assays Screening Assays
Easy to Perform (automation) Low cost Sensitivity Precision Accuracy
Confirmation Assays Accuracy Precision Sensitivity Low Cost Easy to Perform
濫用藥物免疫檢驗試劑之原理 ( 一 )
1. Homogenous Immunoassay
1) Enzyme immunoassay
Enzyme Multiplied Immunoassay Technique (Syva EMIT), DRI.
Cloned Enzyme Donor Immuno Assay (CEDIA).
2) Fluorescence immunoassay
Fluorescence Polarization Immunoassay (FPIA). Abbot TDx.
3) Microparticle agglutination immunoassay
Roche Abuscreen Online
2. Heterogeneous Immunoassay
1) Radio immunoassay (Roche, DPC)
2) Enzyme Linked Immunoassay (DCB)
濫用藥物免疫檢驗試劑之原理 ( 二 )
EMIT 的原理
G6PD - * (Full Enzyme activity) + G6PD- * :Ab (↓Enzyme activity) + * :Ab
G6P + NAD+ 6-phosphogluconate + NADH (↑A 340)G6PD - *
+ * in sample
G6PD - * + Ab → G6PD - *: Ab (↓Enzyme activity)
Schematic Illustration of CEDIA
ED * : Ab + EA
No enzyme activity
Active enzyme–– *
EA + ED * + Ab →Ab : * + (EA-ED * )4
Fluorescence Polarization Immunoassay (FPIA)
Ab : Ag + Ab : Ag -Fl + Ag - Fl
(Decreased Polarization)
Ag
Ab + Ag - Fl → Ab : Ag -Fl
(Polarized Light)
Schematic Illustration of RIA
DRUG * :Ab1
DRUG :Ab1
DRUG *DRUG
DRUG * :Ab1 :Ab2
DRUG :Ab1 :Ab2
Ab2
++ Ab1DRUG *DRUG
(ppt)
Schematic illustration of DCB EIA
No enzyme activity
wash
+ *
Plate -Ab + * -Enz wash Enzyme Activity
+ substrate
+ substrate- *
Amphetamine analogs
Percent cross reactivity of amphetamine analogs with various immunoassays (1)
ReagentsAnalyte
ug/mL
Cross reactivity (%)
Beckman (DRI)
Cobas I400
Syva Emit p
Syva Emit II
CEDIA (microgenic
s)
AxSYM (Abbot)
D-Amphetamine 1 151.4 88.8 202.7 97.9 97.9 135.5
BDB 10 20.6 4.4 3.9 4.8 94.5 137.5
4-Bromo-2,5-dimethoxyphenethylamine
10 0.0 0.0 1.3 0.0 2.8 0.0
Bupropion 10 0.0 0.8 0.0 0.3 13.9 0.1
Cathinone 10 0.0 0.1 0.1 0.0 2.4 0.0
Chlorpheniramine 10 0.0 0.0 0.4 0.0 1.4 0.0
N-Desmethylselegiline 10 1.7 2.6 4.1 21.5 49.9 2.1
ethylamphetamine 10 8.8 2.9 5.2 20.4 221.0 45.1
Fenfluramine 10 9.2 3.2 6.7 5.0 152.1 16.2
Ketamine 10 0.0 0.0 0.1 0.0 0.4 0.0
MBDB 1 135.3 28.4 127.8 12.5 861.0 26.6
Percent cross reactivity of amphetamine analogs with various immunoassays (2)
ReagentsAnalyte
ug/mL
Cross reactivity (%)
Beckman (DRI)
Cobas I400
Syva Emit p
Syva Emit II
CEDIA (microgenic
s)
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 1 101.8 28.1 110.1 27.7 978.0 132.2
Mescaline 10 0.3 0.0 0.0 0.0 0.1 0.1
Methadone 10 0.2 0.0 0.7 0.0 0.9 0.0
Methaqualone 10 0.1 0.0 5.6 0.0 0.8 0.1
Methcathinone 10 0.9 0.0 4.4 0.0 2.3 0.0
Methylephedrine 10 0.9 0.0 1.5 0.0 1.1 0.1
Methylphenidate 10 0.1 0.0 0.1 0.0 1.0 0.0
Methylpseudoephedrine 10 0.1 0.0 1.3 0.0 1.6 0.0
Norketamine 10 0.1 0.0 0.7 0.0 2.4 0.0
Percent cross reactivity of amphetamine analogs with various immunoassays (3)
ReagentsAnalyte
ug/mL
Cross reactivity (%)
Beckman (DRI)
Cobas I400
Syva Emit p
Syva Emit II
CEDIA (microgenic
s)
AxSYM (Abbot)
Norpseudoephedrine (cathine) 10 7.4 1.2 2.7 1.4 3.0 0.0
Pheniramine 10 0.1 0.0 1.8 0.0 1.4 0.0
Phentermine 10 4.0 0.0 85.5 17.8 51.6 13.8
Phenopropanolamine(Norephedrine, PPA)
10 0.3 0.6 2.7 0.0 2.5 0.0
PMA(p-methoxyamphetamine)
10 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 10 0.0 0.0 6.1 1.5 1.2 0.0
Pseudoephedrine 10 0.7 0.0 1.5 0.0 1.4 0.0
Selegiline 10 1.2 0.0 1.9 0.0 1.4 0.1
Percent cross reactivity of amphetamine analogs with various immunoassays (4)
Reagents
Analyteug/mL
Cross reactivity (%)
Beckman (DRI)
Cobas I400
Syva Emit p
Syva Emit II
CEDIA (microgenic
s)
AxSYM (Abbot)
2C-H (2,5-dimethoxy- phenethylamine)
10 0.9 0.0 2.8 0.3 2.4 0.0
2C-I (4-Iodo-2,5-Dimethoxyphenethylamine)
10 0.2 0.0 2.1 0.0 2.3 0.0
2C-T-2 (2,5-dimethyl-4- ethylthiophenethylamine)
10 0.3 0.0 1.9 0.0 2.0 0.1
2C-T-7 (2,5-dimethoxy-4- propylthiophenethylamine)
10 0.1 0.0 2.4 0.0 2.4 0.0
3,4-dimethoxyphenethylamine 10 0.0 0.0 6.4 0.7 1.6 0.1
Percent cross reactivity of amphetamine analogs with various immunoassays (5)
Reagents
Analyteug/mL
Cross reactivity (%)
Beckman (DRI)
Cobas I400
Syva Emit p
Syva Emit II
CEDIA (microgenic
s)
AxSYM (Abbot)
DOB (4-bromo-2,5- dimethoxyamphetamine)
10 0.1 0.0 3.5 0.1 0.0 1.1
Methamphetamine 1 168.8 31.9 401.9 186.3 288.4 147.7
HMA ( 4-hydroxy-3- methoxyamphetamine)
10 5.9 1.0 11.1 5.0 3.6 0.1
HHMA 10 1.4 0.4 4.4 0.0 8.8 1.8
MTA (4-methylthioamphetamine)
10 3.0 16.4 83.8 12.8 96.2 65.9
PCP 10 0.1 0.0 0.0 0.0 1.7 15.6
Tramadol 10 0.3 0.0 0.0 0.0 1.8 78.2
Opiate
Apparent cross-reactivity (%) of various opiate immunoassays
Analyte Roche RIA DPC RIA Syva EMIT Abbott FPIA CEDIA DRI
O6-Acetylmorphine 14.9 0 15.7 92.8 81 ?
Codeine 198 0.06 330 111 125 100
Hydrocodone 24.5 0 138.3 100.5 59 30
Hydromorphine 9.9 0.7 69.2 105.4 66 30
Morphine 84.7 83.5 86.3 90.0 100 100
Morphine-3-Glucuronide 52.4 0.9 45.3 64.2 94 50
Oxycodone 0.1 0 3.4 18.4 3.1 3
Oxymorphine 0.1 0.01 1.2 10.8 1.9 0.6
Benzodiazepine
Cross Reactivity of Benzodiazepine Immunoassay (1)
Benzodiazepines
Cross Reactivity(%)
DRI Syva Emit d.a.u. Roche RIACEDIA
(microgenics)
AxSYM (Abbot)
Alprazolam 1832 1916 158 309 107
α-Hydroxy Alprazolam 916 1610 99 239 89
Chlordiazepoxide 23 31 45 55 23
Clobazam 52 150 77 92 49
Clonazepam 27 39 35 134 35
7-Amino Clonazepam 17 21 9 63 24
Diazepam 2294 2190 166 294 140
Flunitrazepam 55 110 32 63 53
7-Amino Flunitrazepam 33 53 13 78 40
N-Desalkyl Flunitrazepam 32 37 21 63 40
7-Hydroxy Flurazepam 250 1399 82 160 82
Cross Reactivity of Benzodiazepine Immunoassay (2)
Benzodiazepines
Cross Reactivity(%)
DRI Syva Emit d.a.u. Roche RIACEDIA
(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 38 40 66 36
Nordiazepam 1538 1345 92** 260 102**
4’-Hydroxy Nordiazepam 24 55 53 86 24
Oxazepam 106** 86** 68 94 62
Prazepam 811 1955 108 150 76
Triazolam 115 847 98 234 76
α-Hydroxy Triazolam 49 295 92 205 56
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.
水分吸收區
CT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
C CT
(一條藍色 C線) (二條藍色線條)
單一步驟免疫層析法
(One-S
tep Imm
unochromatography
)
Thanks for you attentions!Thanks for you attentions!