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毒物分析 (二)

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					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
濫用藥物免疫檢驗試劑之原理 (二)

2. Heterogeneous Immunoassay
  1)   Radio immunoassay (Roche, DPC)

  2)   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




                G6PD -*
G6P +   NAD+              6-phosphogluconate + NADH (↑A 340)
Schematic Illustration of CEDIA


    EA + ED* + Ab →Ab :*+ (EA-ED*)4

              –*
                          Active enzyme


      ED*: Ab + EA
     No enzyme activity
Fluorescence Polarization Immunoassay
(FPIA)


    Ab + Ag - Fl → Ab:Ag -Fl
                   (Polarized Light)
           Ag


    Ab:Ag + Ab:Ag -Fl + Ag - Fl
           (Decreased Polarization)
Schematic Illustration of RIA


DRUG *              DRUG * :Ab1           DRUG *
DRUG     + Ab1      DRUG :Ab1         +   DRUG


                                Ab2



                    DRUG *:Ab1 :Ab2
                                    (ppt)
                    DRUG :Ab1 :Ab2
Schematic illustration of DCB EIA

                    -*               + substrate
Plate -Ab + *-Enz            wash                  Enzyme Activity

                       +*

                wash

                       + substrate

        No enzyme activity
Amphetamine analogs
Percent cross reactivity of amphetamine
analogs with various immunoassays (1)
                                                        Cross reactivity (%)
                   Reagents
                              ug/mL
 Analyte                              Beckman   Cobas     Syva      Syva         CEDIA         AxSYM
                                       (DRI)     I400    Emit p    Emit II     (microgenics)   (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-
                               10       0.0      0.0      1.3        0.0           2.8          0.0
dimethoxyphenethylamine
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)
                                                       Cross reactivity (%)
                  Reagents
                             ug/mL   Beckman   Cobas     Syva      Syva         CEDIA         AxSYM
 Analyte
                                      (DRI)     I400    Emit p    Emit II     (microgenics)   (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)
                                                          Cross reactivity (%)
                   Reagents
                               ug/mL   Beckman   Cobas    Syva      Syva         CEDIA           AxSYM
  Analyte
                                       (DRI)     I400     Emit p    Emit II      (microgenics)   (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
                                10       0.3       0.6      2.7        0.0           2.5           0.0
(Norephedrine, PPA)
PMA
                                10      27.7       70.6     59.6      12.1           85.3         18.1
(p-methoxyamphetamine)
PMMA
                                 1      49.3       25.4     52.0      11.0          936.8        146.1
(p-methoxymethamphetamine)
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)
                                                           Cross reactivity (%)
                   Reagents
                                 ug/mL
                                         Beckman   Cobas   Syva      Syva         CEDIA           AxSYM
 Analyte
                                         (DRI)     I400    Emit p    Emit II      (microgenics)   (Abbot)

2C-H (2,5-dimethoxy-
                                  10       0.9       0.0     2.8        0.3           2.4           0.0
      phenethylamine)

2C-I (4-Iodo-2,5-
                                  10       0.2       0.0     2.1        0.0           2.3           0.0
Dimethoxyphenethylamine)

2C-T-2 (2,5-dimethyl-4-
                                  10       0.3       0.0     1.9        0.0           2.0           0.1
      ethylthiophenethylamine)

2C-T-7 (2,5-dimethoxy-4-
      propylthiophenethylami      10       0.1       0.0     2.4        0.0           2.4           0.0
      ne)

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)
                                                         Cross reactivity (%)
                  Reagents
                              ug/mL
                                      Beckman   Cobas    Syva      Syva         CEDIA           AxSYM
 Analyte                              (DRI)     I400     Emit p    Emit II      (microgenics)   (Abbot)

DOB (4-bromo-2,5-
                               10       0.1       0.0      3.5        0.1           0.0           1.1
     dimethoxyamphetamine)

Methamphetamine                 1      168.8      31.9    401.9     186.3          288.4         147.7

HMA ( 4-hydroxy-3-
                               10       5.9       1.0      11.1       5.0           3.6           0.1
    methoxyamphetamine)

HHMA                           10       1.4       0.4      4.4        0.0           8.8           1.8
MTA (4-
                               10       3.0       16.4     83.8      12.8          96.2          65.9
     methylthioamphetamine)
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)
                                               Cross Reactivity(%)
Benzodiazepines                                                     CEDIA        AxSYM
                           DRI    Syva Emit d.a.u.   Roche RIA
                                                                 (microgenics)   (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)
                                               Cross Reactivity(%)
Benzodiazepines                                                         CEDIA        AxSYM
                         DRI     Syva Emit d.a.u.   Roche RIA
                                                                     (microgenics)   (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                  (單




                                  One-Step Immunochromatography
           C 反應區:C:品管線(陽性反應線)
                                                                  一
           T     T:藥物結合線(陰性反應線)                                   步
抗體-色素結合物                                                          驟
           樣品吸收區
                                                                  免
                                                                  疫
                                                                  層
                                                                  析
                   C
                                                                  法
      C
                   T




 陽性             陰性                )
(一條藍色C線)     (二條藍色線條)
Thanks for you attentions!

				
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