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Journal of Immunoassay Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ljii19

Development of Monoclonal Antibodies Reactive With Methamphetamine Raised Against A new Antigen a

a

a

Koici Terazawa , Liying Ji , Takehiko Takatori , b

b

Kimiko Aoki , Yasuko Hirose & Yukio Kuroiwa

b

a

Department of Legal Medicine , Hokkaido University School of Medicine , Sapporo, 060, Japan b

Department of Biochemical Toxicology , Pharmaceutical Sciences, Showa University , Tokyo, 142, Japan Published online: 23 Oct 2006.

To cite this article: Koici Terazawa , Liying Ji , Takehiko Takatori , Kimiko Aoki , Yasuko Hirose & Yukio Kuroiwa (1991) Development of Monoclonal Antibodies Reactive With Methamphetamine Raised Against A new Antigen, Journal of Immunoassay, 12:2, 277-291, DOI: 10.1080/01971529108055072 To link to this article: http://dx.doi.org/10.1080/01971529108055072

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JOURNAL OF IMMUNOASSAY, 1 2 ( 2 ) ,

277-292

(1991)

Downloaded by [University of Calgary] at 11:52 24 March 2015

DEVELOPMENT OF MONOCLONAL ANTIBODIES REACTIVE WITH METHAMPHETAMINE RAISED AGAINST A NEW ANTIGEN

Koici Terazawa, Liying Ji, Takehiko Takatori, Kimiko Aoki*, Yasuko Hirose* and Yukio Kuroiwa* Department of Legal Medicine, Hokkaido University School of Medicine, Sapporo 060, Japan. *Department of Biochemical Toxicology, Pharmaceutical Sciences, Showa University, Tokyo 142, Japan.

ABSTRACT Monoclonal antibodies (McAbs) specific to methamphetamine (MA) were produced using p-amino MA coupled to bovine serum albumin (BSA) with glutaraldehyde (GA) as an immunogen and with conventional hybridoma techniques. Hybridoma clones secreting the McAbs were selected by an enzyme-linked immunosorbent assay (ELISA) system using both the above conjugate and BSA modified with GA as screening antigens. In the ELISA system were used avidin and biotinyl-alkaline phosphatase which converts nicotinamide adenine dinucleotide phosphate (NADP) into NAD. The final enzyme activity was determined using diformazan of nitroblue tetrazolium formed together with the NAD produced, alcohol dehydrogenase and phenazine methosulfate. The McAbs from 9 clones were characterized by a crossreactity test using the ELISA. The McAbs recognized MA (100 % ) , methoxyphenarnine (8.0 %), ephedrine (2.3 % ) , but did not react with metylephedrine, amphetamine, OH-amphetamine, dimethylamphetamine, 8-phenylethylamine, norephedrine, phentermine and ranitidine. An inhibition curve for MA was obtained in the range of 0 . 7 5 to 50 ng. (KEY WORDS: p-Amino-methamphetamine, Monoclonal Methamphetamine, antibdy, Immunoassay, Enzyme-linked immunosorbent assay. ) 277 Copyright 0 1991 by Marcel Dekker, Inc.

TERAZAWA ET AL.

278

INTRODUCTION

Immunoassays

for

methamphetamine (MA) have

been

established by preparing antibodies raised against hapten-carrier

antigens

The immunogens

(1-8).

the have

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been synthesized by modifying two parts of MA molecule, i.e., amino group and p-position of the aromatic ring. To were

amino group of MA the

the

-CHI COOH ( I ) ,

attached:

-(CH2 )4NH2(4-7,9): duced

spacers

CH2) 3-COOH

( 3 ) and

the carboxy or amino group

was combined with carrier

polyclonal

-(

following

molecules.

However,

antibodies (PcAbs) raised against the above

immunogens crossreacted with not only MA-related but

intro-

drugs

their metabolites (2-6);methylephedrine (2-20.8

crossreactivity), (0.6-6.6

%),

ephedrine

(0.5-13

%),

amphetamine

norephedrine (0.1 and 2 % ) and

phenamine ( 0 . 0 5 - 2 . 2

%).

%

methoxy-

On the other hand, monoclonal

antibodies (McAbs) were prepared recently by immunizing a conjugate of MA-(CH2)4-NHCO-human IgG ( 7 ) , but one of

15

clones

obtained

had

lower

crossreactivity

with

almost all of the above compounds than PcAbs, and still had

undesirable

reactivity with methylephedrine

(1.6

90).

To solve the problem, o u r coworkers prepared a new PcAb (8). They introduced amino group at p-position of

279

MONOCLONAL ANTIBODIES REACTIVE WITH METHAMPHETAMINE

the

aromatic ring of MA (p-arninoMA) and coupled it

to

bovine serum albumin by glutaraldehyde method (p-aminoMA-GA-BSA).

The produced PcAb did not crossreact with

methylephedrine

(7.2

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Since

are

with

methoxyphenamine

ephedrine (0.8 % ) and amphetamine (0.3 % ) .

%),

higher

but did weakly

it

is generally known that McAbs

us

give

specificity than PcAbs and that the former also

superior

latter,

in an immunohistochemical study

to

the

in this paper we deal with the production

and

characterization of McAbs raised against the p-aminoMAGA-BSA antigen.

MATERIALS

Preparation

of

AND

METHODS

Antigen Conjugates

(i) An immunogen,

2-aminoMA-GA-BSA, was prepared

to

coupling

p-aminoMA

followed

by reduction with sodium borohydride

BSA by

condensation

by

with

GA

(FIGURE

1) as previously described ( 8 , l O ) .

(ii) BSA

was modified with GA (BSA-GA) for

screening

antigen

of an

enzyme-linked

assay (ELISA) as described above.

the

McAb

immunosorbent

TERAZAWA ET A L .

280

CH, CH, I

H Z~ ~

c

H

I

, CH- NH

+ BSA-NH2 + O=CH(CH2)3CH=0 Downloaded by [University of Calgary] at 11:52 24 March 2015

1

CH3 CH3

I I BSA- N-CH (CH , ) , c H = N ~ CH,- CH-NH

10

NaBH4

IH (CH ,) HN

BSA-

CH3 CH, I

CH ,-CH

I

-N H

FIGURE 1. Synthesis of an antigen by conjugation of paminomethamphetamine and bovine serum albumin through glutaraldehyde followed by reduction.

Production of McAbs

A

female

six-week

old BALB/C

mouse

was

first

immunized i.p. with 0 . 2 5 mg of 2-aminoMA-GA-BSA in 0 . 2 5 ml of emulsion of saline and Freund's complete adjuvant ( 1 : l by v o l ) .

the

immunogen

months. cells

Thereafter i.p. injections of 0 . 2 5 mg of in

saline were given

monthly

over

Three days after the final injection, were

fused

with

P 3 U 1 myeloma

cells

4

spleen

(5:l

by

281

MONOCLONAL ANTIBODIES REACTIVE WITH METHAMPHETAMINE

number) using polyethylene glycol 1500 and cultured the

Iscove's

modified

hypoxanthine, Hybridoma

Dulbecco's

aminopterin

clones

medium

and

in

containing

thymidine

(11).

secreting McAbs were selected by the

ELISA system described below using the 2-aminoMA-GA-BSA

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and

BSA-GA

contained positive were

as screening

antigens.

Hybridoma

cells

in wells in which culture supernatants

to

p-aminoMA-GA-BSA but negative to

were

BSA-GA,

subcloned twice by a limiting dilution method

to

ensure their monoclonal origins. Isotype

analyses

were

performed

monoclonal typing kit (MMT-RC 1, Culture

a

using

U.K.).

Serotec Ltd.,

supernatant was used as a source of

mouse

antibody.

ELISA All ture mM

procedures were carried out at room

except otherwise mentioned.

tempera-

O n e hundred 1.11 of 10

sodium carbonate buffer (pH 9.6) containing 1 p g of

p-arninoMA-GA-BSA were added to each well of

-

bottomed Co.,

w e l l plate (Falcon 3915,

96

flat-

Becton Dickinson

U.S.A.) and incubated overnight at 4 ° C .

&

The coat-

ed wells were then washed three times with PBS containing

0.05

%

Tween 20 (PBS-Tween).

In order

to

block

vacant sites on the well surface, 400 p1 of 1 % gelatin in

PBS-Tween (1%G-PBS) were added to each well and the

282

TPRAZAWA ET A L .

plate

incubated for 30 min.

100

To each well were

p 1 of culture supernatant by the

alternatively

50

McAb

added

screening,

p1 of either MA or its related

com-

pound solution in O.l%G-PBS followed by the addition of

50 ~1 of diluted McAbs in O.I%G-PBS by preparing

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bration

curve or crossreactivity test.

allowed to stand for 2 h. three

cali-

The plate was

The wells were then

times with O.l%G-PBS.

Subsequently,

washed

100 1.11 of

biotinylated horse anti-mouse IgG (H+L) (Vector, U. S. pg protein/ml in O.l%G-PBS) were added

6

A.,

the

with 0.01 M Tris-buffered saline containing 0.05

wells

8 Tween 20, pH 7.4

ated

to

(TBS) and 100 p 1 of avidin-biotinyl-

alkaline phosphatase TBS solution containing

0.5

1.11of each of A and B reagents of a Standard Vectastain

ABC Kit (Vector, U.S.A.)

were added to the wells,

incubated

After washing with TBS

times,

for

30 min.

and

three

100 p 1 of 0.05 M diethanolamine buffer (pH 9 . 5 )

containing

0.2

mM nicotinamide

adenine

dinucleotide

phosphate (NADP, Boehringer Mannheim GmbH, F.R.G.) added to the wells.

After incubating for 20 min,

1-11of 0.05 M phosphate buffer (pH 7.0) containing

(w/v) ethanol,

were 100 3

%

1 mM phenazine methosulfate (PMS, Wako

Pure Chem. Ind. Ltd., Japan), 1 mM nitro blue tetrazolium (NBT, Wako Pure Chem. Ind. Ltd., Japan), and 17 ug of

alcohol

powder,

dehydrogenase (ADH) from yeast

Oriental yeast, Japan).

280

U/mg

After a fur her incu-

283

MONOCLONAL ANTIBODIES REACTIVE WITH METHAMPHETAMINE

bation for 10 min, the

the enzymic reaction was stopped by

addition of 50 1.11 of 0.2 M sulfuric acid.

The ab-

sorbance at 660 nm in each well was measured on an MTP-

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22 microplate photometer (Corona Electric Co., Japan).

RESULTS AND DISCUSSION

Nine clones of hybridoma, all secreting McAbs spscific to p-aminoMA-GA-BSA but not reacting with BSA-GA, were

selected.

characterized

where

A

B

showed

was

Crossreact-

is the weight of MA which

+

B ) x 100

reduced

the

at "0" (1.1 in FIGURE 2) by half (to 0.55)

absorbance and

by the crossreaction test.

of an MA analogue was calculated as (A

ivity (%),

The specificity of each antibody

is that of an MA almost

the

analogue.

same features

results are shown in TABLE 1.

The and

nine

McAbs

representative

The immunoglobulin iso-

type of each McAb was tested by a reverse passive haemagglutination Ltd., U.K.) The culture assay, of McAb.

method using a commercial

kit

(Serotec

and all were identified as I g G 1 .

clone secreting the highest level of McAb medium was used to examine sensitivity of

the

in the

culture supernatant being used as a source

The inhibition curve for MA in the range

of

0.75 to 50 ng was obtained by using a 1:lOO dilution of

TERAZAWA E T A L .

284 TABLE I

Crossreactivity of the Present Monoclonal Antibody with Methamphetamine and its Analogues % Crossreactivity

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Analogues Methamphetamine

100

OH-methamphetamine

100

Methoxyphenamine

8.0

Ephedrine

2.3

Norephedrine