J. of Nuclear Sci. and Tech.

No. 53, pp. 10-19, 2010

1389 10-19 53

PR81 F(ab)2
99mTc
5 4 2 3 2 1*
2
4 2 2
- 45156-58145 : -1
- 14395-836 : -2
14115-331 : -3
14115-331 : -4
19969-11151 : - -5

. :
99mTc PR81 F(ab')2
) ( 20 1 PR81 .
. A F(ab')2 .
99mTc F(ab')2 . %65/25/1
HYNIC
. %70/1 5/2
4 BALB/c .
F(ab')2 .
. PR81
99m- F(ab)2 PR81 :

Production, Quality Control and Biodistribution Study of F(ab')2 Fragment of

Antibody PR81 Labeled with 99mTc for Diagnosis of Breast Carcinoma Bearing Mice
M. Salouti*1, M.H. Babaei2, H. Rajabi3, H. Forootan2, M.J. Rasaee4, A. Bitarafan Rajabi5, F. Johari Daha2,

M. Mazidi2, J. Mohammad Nejad4, M. Shafiee2

1- Department of Biology, School of Sciences, Islamic Azad University, Zanjan Branch, Postcode: 45156-58145, Zanjan - Iran
2- Radioisotopes Department, Nuclear Science Research School, Nuclear Science and Technology Research Institute, AEOI, P.O. Box: 14395-836, Tehran - Iran

3- Department of Medical Physics, Faculty of Medical Sciences, Tarbiat Modares University, P.O. Box: 14115-331, Tehran - Iran
4- Department of Clinical Biochemistry, Faculty of Medical Sciences, Tarbiat Modares University, P.O. Box: 14115-331, Tehran - Iran
5- Department of Nuclear Medicine, Rajaei Cardiovascular, Medical and Research Center, Postcode: 19969-11151, Tehran - Iran

Abstract: Immunoglobulin fragments offer a number of advantages for intratumor penetration over the
intact antibodies. The goal of this study was to prepare F(ab')2 fragmants from antibody PR81 and 99mTc
labeling in order to produce a biological radiopharmaceutical for imaging of breast cancer in nuclear
medicine. The antibody PR81 was digested with 5% (w/w) pepsin at different times to find the best time
for a complete digestion. The prepared F(ab')2 fragments were purified by protein A column
chromatography. The immunoreactivity of the complex assessed by radioimmunoassay was determined
to be 65.2% 5.1. 99mTc radiolabeling of F(ab')2 fragment by using HYNIC as a chelator and tricine as a
co-ligand resulted in a radiochemical purity of 70.1%5.2 after 1 hr with a high stability. The complex
was purified by the gel chromatography method to increase the radiochemical purity. Biodistribution and
imaging studies in BALB/c mice with breast tumor xenograft demonstrated as a specific localization of
the compound at the site of tumors 4 h post injection with high sensitivity and minimum accumulation in
non target organs. The results showed that F(ab')2 fragment of PR81 is a promising candidate for
radioimmunoscintigraphy of the human breast carcinoma.
Keywords: Breast Carcinoma, Radioimmunoscintigraphy, Antibody PR81, F(ab)2, Fragment, 99mTc Labeling
*email: saloutim@yahoo.com

89/6/14 : 88/9/1 :

10

 1389 53

-1

MCF-7

22 .

BALB/c

%70

] .[1 PR81
MUC1

2-2

Omnifuge 2 RS/ Heraeus sepatech

EG & G/ ORTEC/ Model 4001 M

99m

] .[2 Tc

Gallenkamp

Nikon/Diaphot

Sartorius Ag Gottingen/PB 110s/ Max 110g,d=0.

] 3 .[6 PR81

752 UV/VIS Grating

) (HAMA)(1 ] .[7

NeuAir CO2/Water-Jacketed Incubator

- .

) FPLC (Fast Protein Liquid Chromatography

] 8

Pharmacia LKB Biotechnology

.[12 F(ab')2

Ependorf

PR81 99mTc

Mediso, NuclineTM SPIRIT

pH M225 CORNING

UV/VIS

.
3-2

-2

BALB/c

1-2

BALB/c .

hIgG Sigma

] .[13

20 30 8 10

FCS

RPMI 1640

222

) (SDS G-25

) (DMSO A

0/2

125I 99mTc HYNIC

11

 . . .

4-2 PR81

6-2 F(ab')2

(107cells/ml) MCF7
6

PR81

. 2 3

) PBS (BSA %1

37 . 9

1 ) 3( . F(ab')2

0/3110 0/62510 1/2510 2/510 510

125

] .[18

) (%10 FCS

) (

0/5 40ng/ml

) ( RPMI

) (10000cpm .

%20 FCS .

4 .

2000rpm 5 .

PR81

. ) Lineweaver-Burk

].[19

-20
7-2 F(ab')2

PR81 Tc

99m

5-2 PR81 F(ab')2

PR81 0/1

4C pH .

HYNIC

99m

] .[20 )

4/2 .

Tc

2mg/ml 1 ) 20 (

( 0/1 pH=8/5

37C

. HYNIC 30

. 28 24 20 16 12 32

50

SDS-PAGE

4 24

. ) (

2 Tris pH=8 0/025

pH=5/2 .

.
99m

Tc

99m

F(ab')2 PR81

Tc

20

SDS-PAGE ] 14 .[17

12

Tricine- F(ab')2-HYNIC

 1389 53

8-2

3-8-2 F(ab')2
99m

F(ab')2 Tc

BALB/c

] 22 21 .[23

3 5

1-8-2

BALB/c .

1 ) 3 (
99m

ITLC-SG .

F(ab')2 Tc

1cm14cm

1/5cm )(5l

10 15 200 300

100 200( 8 4 .

. 10cm

24

1cm

...

. 4 2 1 0/5

) (%ID/g .

8 6 24 .

HYNIC

9-2

3-8-2
2-8-2 F(ab')2

8 4 24

24 37

) 20 ( .

FPLC .
) (Superose 12 HR 10/30

-3

0/01 pH=7

1-3

100 . F(ab')2

SDS-PAGE

1g/ml

PR81

1ml/min

1 ) 20 ( 28

. 1

) .(1

IgG F(ab')2

.
.
F(ab')2 SDS-PAGE %95
) .(1
13

 . . .

y = 0.3075x + 1.5333
R2 = 0.9986

6
5
3
2
1

Total activity / Bond Activity

0
20

10

15

1 / Cell * 10E6

-1

-2

PR81 . :

.MCF7

) (MW F(ab')2 : hIgG

: PR81

100

: 24 20 16 12
28 32 .

)Labeling Efficiency (%

90
80

2-3 F(ab')2

F(ab')2 PR81

70

%65/25/1 .

60

2 1/53

50

) .(2

25

20

15

10

)Time Post Labeling (h

3-3

-3 F(ab)2 PR81 Tc

ITLC-SG

HYNIC 24 .

99m

8 6 4 2 1 0/5 24
.

4-3

%70/15/2

%555

24

. 3

F(ab')2

) .(4

ITLC-SG

%935/1 .

14

 1389 53
16000

4000

Mean 8
Mean 24

T um or

B la d de r

S k in

P a nc r e a s

B o ne

M us c le

B r a in

T hy r oid

S te r nu m

C o lo n

In te s tine

H e art

S p le e n

S tom a c h

-4 Tc-HYNIC-F(ab)2

L un g

)Fraction Number (ml

99m

K idn e y

15

L iv e r

30

25

20

10

B loo d

%
ID/g
% ID
/g

8000

Mean 4

Count Per Minute

12000

20
18
16
14
12
10
8
6
4
2
0

-5 99mTc-HYNIC-F(ab)2

24 FPLC 37

) (%ID/g BALB/c

8 4 24 .

5-3 Tc-HYNIC-F(ab')2

99m

BALB/c
99m

5 Tc-HYNIC-F(ab')2

BALB/c 8 4 24

.
4
.
6-3

BALB/c

-6 ) ( ) (

BALB/c

4 Tc-HYNIC-F(ab)2

7 6 8

99m

BALB/c
8 4 24 .
-4
PR81 IgG1 ) (k


].[2
99m

PR81 Tc

-7 ) ( ) (

BALB/c

] 3 .[6

8 Tc-HYNIC-F(ab)2

15

99m

 . . .

] .[27
99m

F(ab')2 PR81 Tc

%70/15/2 .


99m

Tc

.

-8 ) ( ) (

BALB/c
24 Tc-HYNIC-F(ab)2

99m

PR81

) (HAMA

] .[7 PR81

F(ab')2

] 15 14

.[23 -

99mTc

].[24

F(ab')2

99mTc

] .[25

F(ab')2 ].[26

] .[28

BALB/c

HYNIC

. HYNIC

] .[13 F(ab')2

EDDA

PR81

8 4 24

99m

Tc

99m

Tc

99m

Tc-HYNIC-F(ab')2

16

 1389 53

-1

8 4 24 Tc-HYNIC-F(ab)2

/ 4

/ 8

/ 24

2/26

1/88
0/53

2/05

3/12

0/71

2/87

0/60

0/29

2/59

2/09

1/22

2/66

1/42

0/98

0/45

0/88

0/71

24

. 4

99m

-
99mTc 45

] 5 [6

( 15

BALB/c

24

99m

- Tc

8 4 24

99m

Tc-HYNIC-F(ab')2

) (

SPECT

Planar

. planar

Wiener Metz Butterworth

1/5 2

] .[15

2/660/45 . 1

F(ab')2 PR81

HAMA

].[29

PR81
.
17

. . .

:
1- Human Anti-Murine Antibody Response

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