毒物分析 免疫檢驗 賴滄海教授 03-04-2009. Characteristics of the Screening and...

毒物分析 免疫檢驗

賴滄海教授

03-04-2009

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

）

Table 3. Effects of Adulterants in Urine on Immunoassay Results

Immunoassay

Adulterant Amp Barb Bzd Coc THC Opi PCP

NaCl E E E E E E E

Base Buffer EF

Lime-A-Way or Liquid Plumber R F ER F

Vinegar ERK EK EK EK EFRK ERK EK

Bleach C F C EFCKR R K

Drano EFCK R ECK R ECK EFCK R EFCK R EFCK R EFCK RKDetergent/Soap EC R EFC R EC EC R EFC R EC R FC RVisine (benzalkonium Cl) E F EK F E CE ECK R E E

Golden Seal C F EFCKR

Urinaid (glutaraldehyde) E KR CE E CE R EFC R E R CEF KRKlear (KNO2) E RK E RK E RK E R E RK E RK E RK

DETOX EFRK

TEST CLEAN EFR EFR EFR EFR EFR EFR EFR

E=EMIT, F=FPIA, C=CEDI, R=RIA, K=KIMS

Underlined abbreviations indicate a false positive result.

Abbreviations without underlines indicate a false negative result.

Sone of data are from a 1998 personal communication from Sal Salamone, OraSure Technologies, Inc., Bethlehem, PA and Yale Caplan, Baltimore, MD.

Thanks for you attentions!Thanks for you attentions!