毒物分析 免疫檢驗 賴滄海教授 03-018-2010

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

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

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)

EMIT的原理 G6PD -＊ + Ab → G6PD -＊：Ab (↓Enzyme activity) + ＊ in sample 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 + + Ab1 (ppt)

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

Amphetamine analogs

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

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

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

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

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

Opiate

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

Benzodiazepine

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

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

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.

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）

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