٣٣
هزاﺪﻧا يﺮﻴﮔ
ﻞﻳﺎﻤﺗ ﺖﺑﺎﺛ )
ﻲﺘﻴﻨﻴﻓا (
ﻲﺘﻧآ لﺎﻧﻮﻠﻛﻮﻧﻮﻣ يدﺎﺑ IgG
اﺰﻴﻟا شور زا هدﺎﻔﺘﺳا ﺎﺑ زﺎﺗﺎﻔﺴﻓ ﻦﻴﻟﺎﻜﻟا ﻢﻳﺰﻧآ ﺪﺿ
يﺮﺻﺎﻧ ﻦﺴﺤﻣ -1
ﻲﻧذﺆﻣ ﺪﻤﺤﻣﺪﻴﺳ ﺮﺘﻛد -2
ﻲﻠﻋ ﺮﺘﻛد ﺢﺘﻓرﻮﭘ ﺮﺒﻛا
ا
2ﷲ - حﺎﺒﺼﻣ داﺰﻬﺑ
3هداز
هﺪﻴﻜﭼ
فﺪﻫ و ﻪﻨﻴﻣز ﻲﺘﻴﻨﻴﻓا:
ﻲﺘﻧآ ﻪﺑ يدﺎﺑ ﻲﺘﻧآ اﻮﻣ زا يرﺎﻴﺴﺑ رد نژ نآ ﺖﻴﻟﺎﻌﻓ رد ﻲﻤﻬﻣ ﻲﻜﻳژﻮﻟﻮﻴﺑ ﺮﻴﺛﺄﺗ در
ﻲﺘﻧآ يدﺎﺑ و
ﻲﺳاﻮﻧﻮﻤﻳا ﻮﻳدار ﺪﻨﻧﺎﻣ ﻲﻜﻳژﻮﻟﻮﻧﻮﻤﻳا ﻲﻫﺎﮕﺸﻳﺎﻣزآ يﺎﻬﺸﺠﻨﺳ مﺎﺠﻧا رد ﻦﻴﻨﭽﻤﻫ
(RIA)
دراد ﻲﻤﻴﺷﻮﺘﺴﻴﻫﻮﻧﻮﻤﻳا و .
ﻲﺘﻴﻨﻴﻓا ﺶﺠﻨﺳ فﺪﻫ ﺎﺑ ﺮﺿﺎﺣ ﻪﻌﻟﺎﻄﻣ ﻲﺘﻧآ
يﺎﻣوﺪﻳﺮﺒﻴﻫ نﻮﻠﻛ لﻮﺼﺤﻣ لﺎﻧﻮﻠﻛﻮﻧﻮﻣ يدﺎﺑ
A1G8F7
هﺪﺷ ﺪﻴﻟﻮﺗ
ﻢﻳﺰﻧآ ﻪﺑ ﺖﺒﺴﻧ آ
زﺎﺗﺎﻔﺴﻓ ﻦﻴﻟﺎﻜﻟ
(ALP)
ﺪﻳدﺮﮔ مﺎﺠﻧا .
ﻲﺳرﺮﺑ شور ﻪﻌﻟﺎﻄﻣ ﻦﻳا رد:
،ﻲﺑﺮﺠﺗ ﻲﺘﻳﺪﻳوآ و ﻲﺘﻴﻨﻴﻓا ﻦﻴﻴﻌﺗ ياﺮﺑ يدﺪﻌﺘﻣ يﺎﻬﺷور ﻲﺘﻧآ
عاﺪﺑا نﻮﻨﻛﺎﺗ يدﺎﺑ
هﺪﺷ ﺪﻧا . نآ رد ﻪﻛ هﺪﺷ هدﺎﻔﺘﺳا ﻲﻤﺘﺴﻴﺳ زا ﺎﻬﺷور ﻦﻳا ﻪﻤﻫ رد ﻲﺘﻧآ
و نژ ﻲﺘﻧآ هﺎﮕﻧآ و ﺪﻨﺳﺮﺑ لدﺎﻌﺗ ﻪﺑ ﻢﻫ ﺎﺑ يدﺎﺑ
ﺣ نداد ﺮﻴﻴﻐﺗ نوﺪﺑ لدﺎﻌﺗ ﺖﻟﺎ
، ﺮﻳدﺎﻘﻣ ﻲﺘﻧآ و دازآ نژ ﻲﺘﻧآ
ﺎﺑ هﺪﺷ هاﺮﻤﻫ نژ ﻲﺘﻧآ
هﺪﻴﺠﻨﺳ يدﺎﺑ ﻲﻣ
دﻮﺷ . ﻦﻳا رد
ﻖﻴﻘﺤﺗ زا ﻲﺘﺑﺎﻗر ياﺰﻴﻟا ﻊﻳﺮﺳ شور ﻚﻳ -
ﻪﻛ ﻲﺘﻴﻨﻴﻓا ﻲﺘﻧآ لﻮﻠﺤﻣ ﺖﻟﺎﺣ رد ار يدﺎﺑ هزاﺪﻧا
يﺮﻴﮔ ﻲﻣ ﺪﻨﻛ - ياﺮﺑ
يﺪﻧﻮﻴﭘ ﻞﻴﻣ ﺶﺠﻨﺳ ﻲﺘﻧآ
ﻣ يدﺎﺑ ﻮ لﺎﻧﻮﻠﻛﻮﻧ ﺪﺿIgG
آ ا هﺪﺷ ﺪﻴﻟﻮﺗ زﺎﺗﺎﻔﺴﻓ ﻦﻴﻟﺎﻜﻟ نﻮﻠﻛ ز
A1G8F7
هدﺎﻔﺘﺳا ﺪﺷ .
ﻪﺘﻓﺎﻳ ﺎﻫ ﺖﺑﺎﺛ ﺖﻈﻠﻏ: ﻲﺘﻧآ
ﻦﻴﻴﻌﺗ مود ﻪﻠﺣﺮﻣ ﻲﺻﺎﺼﺘﺧا ياﺰﻴﻟا رد هدﺎﻔﺘﺳا ياﺮﺑ يدﺎﺑ
Dissociation Constant
(Kd)
ﺮﺑاﺮﺑ ﻲﺘﺑﺎﻗر ياﺰﻴﻟا ﻚﻳ ﻂﺳﻮﺗ ،ﺰﺗﻼﻛ شور ﻪﺑ 1μg
ﺑ ﻪ ﺪﻣآ ﺖﺳد
؛ ﺎﺑ ﺰﺗﻼﻛ رادﻮﻤﻧ ﻢﺳر
Kd
نﻮﻠﻛ
A1G8F7
ﺮﺑاﺮﺑ
9
10 -
× 8 / 3 ﺪﺷ هدز ﻦﻴﻤﺨﺗ .
ﺠﻴﺘﻧ ﻪ يﺮﻴﮔ ﻦﻳا ﻪﺑ ﻪﺟﻮﺗ ﺎﺑ : ﻪﻛ
Kd
ﻲﺘﻧآ يدﺎﺑ ياﺮﺑ نﺪﺑ ﻲﻌﻴﺒﻃ يﺎﻫ ﺮﺘﺸﻴﺑ
ﻲﺘﻧآ نژ ﻦﻴﺑ ﺎﻫ 7 - 10 ﻲﻟا 11 - 10
ﻦﻳا ﻪﺑ ﻪﺟﻮﺗ ﺎﺑ و ﺖﺳا ﺮﻴﻐﺘﻣ ﺮﻫ ﻪﻛ
ﻪﭼ هزاﺪﻧا
Kd
ﻲﺘﻴﻨﻴﻓا ،ﺪﺷﺎﺑ ﺮﺘﻜﭼﻮﻛ ﻲﺘﻧآ
دﻮﺑ ﺪﻫاﻮﺧ ﺮﺘﺸﻴﺑ يدﺎﺑ
، ﻲﺘﻧآ يدﺎﺑ
نﻮﻠﻛ زا ﻲﺤﺷﺮﺗ
A1G8F7
ياﺮﺑ ﺮﺘﺸﻴﺑ ﺐﺳﺎﻨﻣ ﻲﻜﻳژﻮﻟﻮﻧﻮﻤﻳا يﺎﻬﺸﺠﻨﺳ ﺮﻈﻧ ﻪﺑ
ﻲﻣ ﺪﺳر . رد ًﻼﻤﻋ يﺎﻬﺸﻳﺎﻣزآ
ﻦﻳا ﺎﺑ ﻪﻛ ﻲﻤﻴﺷﻮﺘﺴﻴﻫﻮﻧﻮﻤﻳا و ﻲﻤﻴﺷﻮﺘﻴﺳﻮﻧﻮﻤﻳا ﻲﺘﻧآ
لﺎﻧﻮﻠﻛﻮﻧﻮﻣ يدﺎﺑ -
ﻢﻳﺰﻧآ ﺎﺑ هاﺮﻤﻫ مﻮﺳ ﻪﻳﻻ ردALP
- مﺎﺠﻧا
ﺪﻳدﺮﮔ
، ﮓﻧر ﻪﺠﻴﺘﻧ ﻲﺗرﺎﺠﺗ ﻪﺑﺎﺸﻣ ﻪﻧﻮﻤﻧ ﺎﺑ ﻪﺴﻳﺎﻘﻣ ﻞﺑﺎﻗ و ﻲﻟﺎﻋ رﺎﻴﺴﺑ يﺰﻴﻣآ
APAAP) ﺖﻛﺮﺷ
(Dako
دﻮﺑ .
هژاو يﺪﻴﻠﻛ يﺎﻫ :
دﺎﺑﻲﺘﻧآ
لﺎﻧﻮﻠﻛﻮﻧﻮﻣ ي
؛ ﻲﺘﻴﻨﻴﻓا
؛ ﻲﺘﻳﺪﻳوآ
؛ اﺰﻴﻟا
؛ زﺎﺗﺎﻔﺴﻓ ﻦﻴﻟﺎﻜﻟآ
ﻣ ﺪﻨﺟﺮﻴﺑ ﻲﻜﺷﺰﭘ مﻮﻠﻋ هﺎﮕﺸﻧاد ﻲﻤﻠﻋ ﻪﻠﺠ )
هرود 11 , هرﺎﻤﺷ 4 , لﺎﺳ 1383 (
يژﻮﻟﻮﻧﻮﻤﻳا ﺪﺷرا سﺎﻨﺷرﺎﻛ ؛لوﺆﺴﻣ هﺪﻨﺴﻳﻮﻧ 1
؛ ﻜﺷﺰﭘ هﺪﻜﺸﻧاد ﻲﻤﻠﻋ تﺄﻴﻫ ﻮﻀﻋ ﺪﻨﺟﺮﻴﺑ ﻲﻜﺷﺰﭘ مﻮﻠﻋ هﺎﮕﺸﻧاد ،ﻲ
سردآ : ﺪﻨﺟﺮﻴﺑ - يرﺎﻔﻏ نﺎﺑﺎﻴﺧ -
ﺪﻨﺟﺮﻴﺑ ﻲﻜﺷﺰﭘ مﻮﻠﻋ هﺎﮕﺸﻧاد -
ﻲﻜﺷﺰﭘ هﺪﻜﺸﻧاد -
يژﻮﻟﻮﻧﻮﻤﻳا ﺶﺨﺑ
ﻦﻔﻠﺗ : 4443041 - 0561 رﺎﮕﻧرود : 4443001 - 0561 ﻚﻴﻧوﺮﺘﻜﻟا ﺖﺴﭘ [email protected] :
رﺎﻴﺸﻧاد2
،يژﻮﻟﻮﻧﻮﻤﻳا ﻲﺷزﻮﻣآ هوﺮﮔ ﺸﻧاد ،ﻲﻜﺷﺰﭘ مﻮﻠﻋ هﺪﻜﺸﻧاد
ناﺮﻬﺗ سرﺪﻣ ﺖﻴﺑﺮﺗ هﺎﮕ
ﺪﻨﺟﺮﻴﺑ ﻲﻜﺷﺰﭘ مﻮﻠﻋ هﺎﮕﺸﻧاد ،ﻲﻜﺷﺰﭘ هﺪﻜﺸﻧاد ،يژﻮﻟﻮﻛﺎﻣرﺎﻓ و يژﻮﻟﻮﻳﺰﻴﻓ ﻲﺷزﻮﻣآ هوﺮﮔ ﻲﻤﻠﻋ تﺄﻴﻫ ﻮﻀﻋ ؛يژﻮﻟﻮﻳﺰﻴﻓ ﺪﺷرا سﺎﻨﺷرﺎﻛ3
٣٤ ﻪﻣﺪﻘﻣ يﺪﻧﻮﻴﭘ ﻞﻴﻣ ﻦﻴﻴﻌﺗ ﻲﺘﻧآ
يدﺎﺑ
ﻚﻳ ﻲﺘﻴﻨﻴﻓا ﻲﺘﻧآ
ﻪﺑ يدﺎﺑ ﻲﺘﻧآ
ﻃﻮﺑﺮﻣ نژ ﺔ
،دﻮﺧ رد
نآ ﻞﻤﻋ رد ﻲﻤﻬﻣ ﻲﻜﻳژﻮﻟﻮﻴﺑ ﺮﻴﺛﺄﺗ دراﻮﻣ يرﺎﻴﺴﺑ ﻲﺘﻧآ
دﺎﺑ ي
دراد ﻦﻴﻨﭽﻤﻫ ؛ دﺎﻳز ﺪﻴﻟﻮﺗ
ﻲﺘﻧآ يدﺎﺑ ﻪﺑ ﻦﻴﻳﺎﭘ ﻲﺘﻴﻨﻴﻓا ﺎﺑ يﺎﻫ
ﻪﺘﻓﺮﮔ ﺮﻈﻧ رد ﻲﻨﻤﻳا ﺺﻘﻧ ﻚﻳ ناﻮﻨﻋ ﻲﻣ
دﻮﺷ . ﻲﺘﻴﻨﻴﻓا
ﻲﺘﻧآ و ﻦﻤﻳادﻮﺧ يﺎﻬﻳرﺎﻤﻴﺑ يژﻮﻟﻮﺗﺎﭘﻮﻧﻮﻤﻳا رد ﻚﺷ نوﺪﺑ يدﺎﺑ
دراد ﺖﻟﺎﺧد ﺰﻴﻧ ﻲﻨﻤﻳا ﺲﻜﻠﭙﻤﻛ يﺎﻬﻳرﺎﻤﻴﺑ )
1 - 4 .(
ﻲﺘﻴﻨﻴﻓا ﺶﻘﻧ ﻲﺘﻧآ
ﺪﻨﻧﺎﻣ ﻲﺳاﻮﻧﻮﻤﻳا يﺎﻬﺷور رد يدﺎﺑ ،اﺰﻴﻟا
RIA**
، ﻲﻤﻴﺷﻮﺘﺴﻴﻫﻮﻧﻮﻤﻳا ،ﻲﻤﻴﺷﻮﺘﻴﺳﻮﻧﻮﻤﻳا
، ﺶﻳاﺰﻓا
رد و ﻲﺻﺎﺼﺘﺧا ﺮﻴﻏ تﻻﺎﺼﺗا ﺶﻫﺎﻛ و ﻲﺻﺎﺼﺘﺧا تﻻﺎﺼﺗا دﻮﺑ ﺪﻫاﻮﺧ ﺶﻳﺎﻣزآ باﻮﺟ نﺎﻨﻴﻤﻃا ﺐﻳﺮﺿ ﺶﻳاﺰﻓا ﻪﺠﻴﺘﻧ .
ﻲﺘﻳﺪﻳوآ و ﻲﺘﻴﻨﻴﻓا ﻦﻴﻴﻌﺗ ياﺮﺑ يدﺪﻌﺘﻣ يﺎﻬﺷور نﻮﻨﻛﺎﺗ ﻲﺘﻧآ هﺪﺷ عاﺪﺑا يدﺎﺑ ﺪﻧا
. ﺮﺳ زا ﺎﻬﺷور ﻦﻳا زا ﻚﻳﺮﻫ رد ﻲﻣ
ﻲﻣ هدﺎﻔﺘﺳا نآ رد ﻪﻛ دﻮﺷ
ﻲﺘﻧآ و نژ ﻲﺘﻧآ لدﺎﻌﺗ ﻪﺑ ﻢﻫ ﺎﺑ يدﺎﺑ
هﺪﻴﺳر ﺪﻧا Ab-Ag) Ab+Ag
(
؛ ﺮﻴﻴﻐﺗ نوﺪﺑ هﺎﮕﻧآ
ﺮﻳدﺎﻘﻣ ،لدﺎﻌﺗ ﺖﻟﺎﺣ نداد ﻲﺘﻧآ
و دازآ نژ ﻲﺘﻧآ
ﺎﺑ هﺪﺷ هاﺮﻤﻫ نژ
ﻲﺘﻧآ يدﺎﺑ
، هﺪﻴﺠﻨﺳ ﻲﻣ دﻮﺷ . دﻮﺟو هار ﺪﻨﭼ ،رﺎﻛ ﻦﻳا مﺎﺠﻧا ياﺮﺑ
دراد . ﺎﻘﻘﺤﻣ زا ﻲﺧﺮﺑ ﺪﻨﻧﺎﻣ ﻲﻜﻳﺰﻴﻓ يﺎﻬﺷور زا هدﺎﻔﺘﺳا ﺎﺑ ن
ژﻮﻔﻳﺮﺘﻧﺎﺳ ،لژ ﺎﺑ نﻮﻴﺳاﺮﺘﻠﻴﻓ ،ﺰﻴﻟﺎﻳد و
،بﻮﺳر ﻲﺘﻧآ ﺎﻳ يدﺎﺑ
ﻲﺘﻧآ زا ار دازآ نژ ﻲﺘﻧآ
ﻪﺑ ﻞﺼﺘﻣ نژ ﻲﺘﻧآ
اﺪﺟ يدﺎﺑ ﻲﻣ
ﺪﻨﻨﻛ
) 5 - 7 .(
ﺑ تاﺮﻴﻴﻐﺗ زا ﺰﻴﻧ ﻲﺧﺮﺑ ﻪ
يﺎﻬﻴﮔﮋﻳو رد هﺪﻣآ دﻮﺟو
ﺖﻨﺳرﻮﺌﻠﻓ ﻲﺘﻧآ
ﻪﺑ ﻞﺼﺘﻣ نژ ﻲﺘﻧآ
هدﺎﻔﺘﺳا يدﺎﺑ ﻲﻣ
ﺪﻨﻨﻛ ) 8 ( .
ﺑ تﺎﻋﻼﻃا زا هدﺎﻔﺘﺳا ﺎﺑ ﻪ
ﻦﻳا زا هﺪﻣآ ﺖﺳد
،ﺎﻬﺷور
ﻲﻣ لدﺎﻌﺗ ﺖﺑﺎﺛ ناﻮﺗ )
ﺎﻳ يﺪﻧﻮﻴﭘ ﻞﻴﻣ ﻲﻨﻌﻳ Ka
( ﺑ ار ﻪ ﺖﺳد
دروآ : [Ab]
[Ab]
Ag]
- K=[Ab
،ﻪﻟدﺎﻌﻣ ﻦﻳا رد [Ab-Ag]
ﺖﻈﻠﻏ لدﺎﻌﻣ ﻲﺘﻧآ
ﻞﺼﺘﻣ نژ
ﻪﺑ ﻲﺘﻧآ ،يدﺎﺑ ﺖﻈﻠﻏ لدﺎﻌﻣ[Ag]
آ ﻲﺘﻧ و دازآ نژ لدﺎﻌﻣ[Ab]
ﻞﺼﺘﻣ دازآ ﻲﺣاﻮﻧ ﺖﻈﻠﻏ ﻪﺑ هﺪﻧﻮﺷ
ﻲﺘﻧآ لدﺎﻌﺗ لﺎﺣ رد نژ
ﺖﺳا .
§ Affinity
** Radioimmunoassay (RIA)
†† Association Constant (Ka)
ﻦﻴﺑ ﺶﻨﻛاو ﻲﺘﻧآ
و نژ ﻲﺘﻧآ ﻪﺑ ﻲﻟدﺎﻌﺗ ﺶﻨﻛاو ﻚﻳ يدﺎﺑ
ﺖﺳا ﺮﻳز ترﻮﺻ :
[Ab] + [Ag] [x]
ﻪﭽﻧﺎﻨﭼ دازآ يﺎﻬﻫﺎﮕﻳﺎﺟ ﺖﻈﻠﻏ [Ab]
ﻲﺘﻧآ يدﺎﺑ [Ag] ،
دازآ يﺎﻬﻫﺎﮕﻳﺎﺟ ﺖﻈﻠﻏ ﻲﺘﻧآ
نژ [x] ، ﺲﻜﻠﭙﻤﻛ ﺖﻈﻠﻏ
Ag-Ab [Abt] ،
ﺖﻈﻠﻏ ﻲﺘﻧآ ﻞﻛ يدﺎﺑ [Agt] و
ﺖﻈﻠﻏ
ﻲﺘﻧآ ﺪﻧﻮﺷ ضﺮﻓ ﻞﻛ نژ
، ﻪﻟدﺎﻌﻣ قﻮﻓ ﻪﻟدﺎﻌﻣ رد ﻲﺗاﺮﻴﻴﻐﺗ ﺎﺑ
ﺑ ﺰﺗﻼﻛ ﻪ ﺖﺳد ﻲﻣ ﺪﻳآ : Klotz Equation: [Abt]/[x]= Kd/[Ag]+1
ﺖﻈﻠﻏ ﻪﭽﻧﺎﻨﭼ لﺎﺣ [Abt]
[Agt] و ،ﻢﻴﺷﺎﺑ ﻪﺘﺷاد ار
ناﺰﻴﻣ ﻦﻴﻴﻌﺗ Kd
ﺎﻳ Ka
ﻲﻜﻳ ﻦﺘﺴﻧاد مﺰﻠﺘﺴﻣ ﻪﺳ زا
ﺺﺧﺎﺷ
،[Ab]
ﺎﻳ[Ag]
دﻮﺑ ﺪﻫاﻮﺧ[x]
) 9 .(
ﻲﺘﻧآ ﻲﺘﻴﻨﻴﻓا ﺶﺠﻨﺳ فﺪﻫ ﺎﺑ ﺮﺿﺎﺣ ﻪﻌﻟﺎﻄﻣ يدﺎﺑ
يﺎﻣوﺪﻳﺮﺒﻴﻫ نﻮﻠﻛ لﻮﺼﺤﻣ لﺎﻧﻮﻠﻛﻮﻧﻮﻣ A1G8F7
هﺪﺷ ﺪﻴﻟﻮﺗ
،ناﺮﻬﺗ سرﺪﻣ ﺖﻴﺑﺮﺗ هﺎﮕﺸﻧاد رد ﻢﻳﺰﻧآ ﻪﺑ ﺖﺒﺴﻧ
آ ﻦﻴﻟﺎﻜﻟ
زﺎﺗﺎﻔﺴﻓ (ALP) ﺪﻳدﺮﮔ مﺎﺠﻧا
.
ﻲﺳرﺮﺑ شور
هزاﺪﻧا ﻲﺘﻴﻨﻴﻓا يﺮﻴﮔ ﻲﺘﻧآ
يدﺎﺑ ﺎﺑ لﻮﻠﺤﻣ ﺖﻟﺎﺣ رد
ﻲﺘﺑﺎﻗر ياﺰﻴﻟا شور زا هدﺎﻔﺘﺳا :
هزاﺪﻧا ياﺮﺑ يﺮﻴﮔ
Kd
A1G8F7 نﻮﻠﻛ ﻦﻳا رد
ﻪﻌﻟﺎﻄﻣ
ﻲﺑﺮﺠﺗ ، زا ﻲﺘﻴﻨﻴﻓا ﻪﻛ ﻲﺘﺑﺎﻗر ياﺰﻴﻟا شور ﻚﻳ ﻲﺘﻧآ
رد ار يدﺎﺑ
هزاﺪﻧا لﻮﻠﺤﻣ ﺖﻟﺎﺣ يﺮﻴﮔ
ﻲﻣ ﺪﺷ هدﺎﻔﺘﺳا ،ﺪﻨﻛ ﻦﻳا ؛
شور
ياراد ود ﻲﻣ ﻲﺳﺎﺳا ﻪﻠﺣﺮﻣ ﺪﺷﺎﺑ
:
ﻒﻟا - ﻲﺘﺴﻳﺎﺑ اﺪﺘﺑا ﻲﺘﻧآ
ﺖﺑﺎﺛ ﺖﻈﻠﻏ ﻚﻳ رد يدﺎﺑ )
ﻪﻛ
ﺖﺳا هﺪﺷ هداد ﺢﻴﺿﻮﺗ ﺖﻈﻠﻏ ﻦﻳا راﺪﻘﻣ ﻦﻴﻴﻌﺗ (
، يﺎﻬﺘﻗر ﺎﺑ
زا ﻲﻟاﻮﺘﻣ ﻲﺘﻧآ
لﻮﻠﺤﻣ زﺎﻓ ﻚﻳ رد ﺎﺗ ﺪﻧﻮﺷ روﺎﺠﻣ ﻪﻟﻮﻟ رد ،نژ
ﺪﻨﺳﺮﺑ لدﺎﻌﺗ ﻪﺑ .
ب - ناﺰﻴﻣ ﺲﭙﺳ ﻲﺘﻧآ
زا ﻚﻳ ﺮﻫ رد هﺪﻧﺎﻤﻴﻗﺎﺑ دازآ يدﺎﺑ
ﺎﻬﺘﻗر - ﺎﻌﺗ ﺖﻟﺎﺣ ﻪﺑ نﺪﻴﺳر زا ﺲﭘ ﻪﻟﻮﻟ رد لد
- شور ﺎﺑ
ﻢﻴﻘﺘﺴﻣ ﺮﻴﻏ ياﺰﻴﻟا -
نآ رد ﻪﻛ ﻲﺘﻧآ
هﺪﺷ تﻮﻛ ﺖﻴﻠﭘ ﻒﻛ نژ
ﺖﺳا - ددﺮﮔ ﻲﺑﺎﻳزرا )
11،10 ( .
٣٥
ﺖﺑﺎﺛ ﺖﻈﻠﻏ ﻦﻴﻴﻌﺗ ﻲﺘﻧآ
يدﺎﺑ :
ﻦﻳا ياﺮﺑ ﺖﻈﻠﻏ زا ﺢﻴﺤﺻ ﻦﻴﻤﺨﺗ ﻪﻛ
ﻲﺘﻧآ رد دازآ يدﺎﺑ
ﺑ لدﺎﻌﺗ لﺎﺣ ﻪ
ﺪﻳآ دﻮﺟو
، ﻪﺘﻜﻧ ﺪﻨﭼ رد ﺪﻳﺎﺑ
ﺮﮔ ﺮﻈﻧ دﻮﺷ ﻪﺘﻓ :
1 - بﺬﺟ ﺑ ﻪ ﺖﺳد ﺣﺮﻣ رد هﺪﻣآ ﻠ
ﺔ ﻢﻴﻘﺘﺴﻣﺮﻴﻏ ياﺰﻴﻟا ﺮﺧآ
- ﺖﻈﻠﻏ ﻪﻛ ﻲﺘﻧآ
ﺲﻜﻌﻨﻣ ار دازآ يدﺎﺑ ﻲﻣ
ﺪﻨﻛ - ﺎﺑ ﻲﺘﺴﻳﺎﺑ
ﺖﻈﻠﻏ ﻲﺘﻧآ هﺪﺷ ﺖﺴﺗ يدﺎﺑ
، ﺪﺷﺎﺑ ﺐﺳﺎﻨﺘﻣ .
2 - لﻮﻜﻟﻮﻣ زا ﻲﻤﻛ ﺪﺻرد ﻂﻘﻓ يﺎﻫ
ﻲﺘﻧآ دازآ يدﺎﺑ )
ﺮﺘﻤﻛ
زا 10
% ( ﻪﺑ ﻲﺘﺴﻳﺎﺑ ، ﻲﺘﻧآ
نژ تﻮﻛ يﺎﻫ اﺰﻴﻟا ﺖﻴﻠﭘ ﻒﻛ رد هﺪﺷ
ﺪﻧﻮﺷ ﻞﺼﺘﻣ اﺮﻳز ؛
ﺮﻫ زا ﻪﻧﻮﮔ رد لﻼﺘﺧا ﻊﻳﺎﻣ زﺎﻓ لدﺎﻌﺗ
يﺮﻴﮔﻮﻠﺟ دﻮﺷ
.
3 - نﻮﭼ Kd
تراﺮﺣ ﻪﺑ ًﻻﻮﻤﻌﻣ ﻪﺘﺴﺑاو
ﻲﺘﺴﻳﺎﺑ ﺎﻣد ،ﺖﺳا
دﻮﺷ ﻪﺘﺷاد هﺎﮕﻧ ﺖﺑﺎﺛ ﻞﺣاﺮﻣ مﺎﻤﺗ رد .
تﺎﻜﻧ ﺖﻳﺎﻋر ياﺮﺑ 1
و 2 ﺖﻈﻠﻏ ﻪﻨﻣاد ﻦﻴﻴﻌﺗ ﻪﺑ زﺎﻴﻧ ،
ﻲﺘﻧآ تﻮﻛ نژ نﺎﻣز و هﺪﺷ
بﻮﻠﻄﻣ لﻮﻠﺤﻣ نﻮﻴﺳﺎﺑﻮﻜﻧا
ﻲﺘﻧآ تﻮﻛ ﻚﻫﺎﭼ رد يدﺎﺑ ﺖﺳا هﺪﺷ
. ﺑ ياﺮﺑ ﻪ ا ندروآ ﺖﺳد ﻦﻳ
ﻪﻨﻣاد زا ﺖﻈﻠﻏ ﻲﺘﻧآ ﻪﺑ ﻢﻴﻘﺘﺴﻣﺮﻴﻏ ياﺰﻴﻟا شور ﻚﻳ زا يدﺎﺑ
هدﺎﻔﺘﺳا ﺮﻳز حﺮﺷ ﺪﻳدﺮﮔ
:
1 - ﺪﺷ هدﺮﺑ رﺎﻛ ﻪﺑ اﺰﻴﻟا ﺖﻴﻠﭘ ود
؛ ﺖﻴﻠﭘ رد هرﺎﻤﺷ
1
، ﻪﺑ
3 هرﺎﻤﺷ ﺖﻴﻠﭘ رد و ﻒﻳدر 2
ﻪﺑ 2 ،ﺎﻬﻜﻫﺎﭼ زا ﻒﻳدر
μg/ml 1 آ زﺎﺗﺎﻔﺴﻓ ﻦﻴﻟﺎﻜﻟ -
ناﻮﻨﻋ ﻪﺑ ﻲﺘﻧآ
نژ - ﺪﺷ ﻪﻓﺎﺿا و
ﺖﻴﻠﭘ ﺎﻣد رد ﺐﺷ ﻚﻳ ﺎﻫ ي
4 راﺮﻗ داﺮﮕﻴﺘﻧﺎﺳ ﻪﺟرد ﺪﻨﺘﻓﺮﮔ
.
2 - هرﺎﻤﺷ ﺖﻴﻠﭘ ﻪﺑ 1
، زا ﺲﭘ 3 ﻮﺸﺘﺴﺷ رﺎﺑ
، يﺎﻬﺘﻗر /2
1
ﻲﻟا /64
زا1
ﻲﺘﻧآ ﺖﻈﻠﻏ ﺎﺑ هﺪﺷ جرﺎﺧ يدﺎﺑ μg/ml
2 ترﻮﺻ ﻪﺑ
ﺖﻴﻜﻴﻠﭙﻳﺮﺗ (Triplicate)
ﺪﺷ ﻪﻓﺎﺿا .
PBS -T زا ناﻮﻨﻋ ﻪﺑ
ﺪﻫﺎﺷ و ﻲﻔﻨﻣ ﺖﻗر ﺎﺑ شﻮﻣ مﺮﺳ زا /1000
ناﻮﻨﻋ ﻪﺑ 1
ﺪﻫﺎﺷ
ﺪﺷ هدﺎﻔﺘﺳا ﺖﺒﺜﻣ .
يﺎﻣد رد ﺖﻋﺎﺳ ﻢﻴﻧ ياﺮﺑ 37
ﻪﺟرد
ﺪﺷ مﺎﺠﻧا نﻮﻴﺳﺎﺑﻮﻜﻧا ﻞﻤﻋ داﺮﮕﻴﺘﻧﺎﺳ .
3 - ﺖﻴﻠﭘ ﻦﻳا تﺎﻳﻮﺘﺤﻣ -
ﻪﺳ ﻒﻳدر ﺮﻫ ﻚﻳ رد ﻲﻳﺎﺗ
ﻪﻧﺎﮔاﺪﺟ فرﺪﻨﭘا -
ﺑ ﺎ ﻊﻤﺟ ﺖﻗد ﺪﺷ يروآ
) زا ﺮﺘﻤﻛ ﻲﻤﻛ ﻪﻛ
300 ﺪﺷ ﺪﻫاﻮﺧ فرﺪﻨﭘا ﺮﺘﻴﻟوﺮﻜﻴﻣ (
ترﻮﺻ ﻪﺑ مود ﺖﻴﻠﭘ ﻪﺑ و
‡‡ Optical Density (OD)
§§ Phosphate Buffered Saline (PBS)
ياﺮﺑ و ﺪﺷ ﻪﻓﺎﺿا ﻲﻠﺒﻗ ﺐﻴﺗﺮﺗ نﺎﻤﻫ ﻪﺑ ﻲﻳﺎﺗود رد ﺖﻋﺎﺳ ﻢﻴﻧ
يﺎﻣد 37 ﺪﺷ ﻪﺑﻮﻜﻧا داﺮﮕﻴﺘﻧﺎﺳ ﻪﺟرد .
4 - ﻞﺣاﺮﻣ نﻮﻴﺳﺎﺑﻮﻜﻧا نﺎﻳﺎﭘ زا ﺲﭘ ﺖﻴﻠﭘ ﺮﻫ ياﺮﺑ
ﻪﻣادا اﺰﻴﻟا ﺶﻳﺎﻣزآ ﺖﻓﺎﻳ
.
5 - ود زا هﺪﻣآ ﺖﺳد ﻪﺑ يرﻮﻧ بﺬﺟ ﺐﺴﺣ ﺮﺑ يرادﻮﻤﻧ
ﻒﻠﺘﺨﻣ يﺎﻬﺘﻗر ﻞﺑﺎﻘﻣ رد ﺖﻴﻠﭘ ﻲﺘﻧآ
ﺪﺷ ﻢﺳر يدﺎﺑ )
رادﻮﻤﻧ 1 .(
ﻲﺘﻧآ زا ﺖﻗر ﻦﻳﺮﺗﻻﺎﺑ يدﺎﺑ
) ﻦﻳﺮﺘﻤﻛ ﻲﻨﻌﻳ ﻦﻜﻤﻣ ﺖﻈﻠﻏ
(
ﺖﻴﻠﭘ زا ﻞﺻﺎﺣ رادﻮﻤﻧ ﻪﻛ يﺎﻫ
1 و 2 ،ﺪﻨﺷﺎﺑ ﻖﺒﻄﻨﻣ ﻢﻫ ﺮﺑ
ﺪﺷ بﺎﺨﺘﻧا )
ﺖﻗر 4 / 1 ﺖﻈﻠﻏ ﺎﺑ ﺮﺑاﺮﺑ ﻪﻛ 2μg
/ 1 ﻲﻣ ﺪﺷﺎﺑ .(
ﻦﻴﻴﻌﺗ Dissociation Constant (Kd)
1 - زﺎﺗﺎﻔﺴﻓ ﻦﻴﻟﺎﻜﻟآ اﺰﻴﻟا ﺖﻴﻠﭘ ﻪﺑ )
ناﻮﻨﻋ ﻪﺑ (Ag
ﺎﺑ
μg/ml ﺖﻈﻠﻏ 1
يﺎﻣد رد ﮓﻨﻴﺗﻮﻛ ﻞﻤﻋ و ﺪﺷ هدوﺰﻓا 4
ﻴﺘﻧﺎﺳ ﻪﺟرد ﺪﺷ مﺎﺠﻧا ﺐﺷ ﻚﻳ ياﺮﺑ داﺮﮕ
.
2 - رد 10 ناﺰﻴﻣ ﻪﺑ ﻪﻟﻮﻟ 250
زا ﺮﺘﻴﻟوﺮﻜﻴﻣ 10
لﺎﻳﺮﺳ
ﻲﺘﻧآ ﻒﻠﺘﺨﻣ ﺖﻗر نژ
) رد هﺪﺷ ﻪﻴﻬﺗ PBS-T
ﺖﻗر زا
μg/ml 100 μg/ml ﺎﺗ 39 / 0 ( ﺎﻬﻧآ ﻪﺑ ﺲﭙﺳ ؛ﺪﺷ ﻪﻓﺎﺿا 250
ﻲﺘﻧآ زا ﻲﺘﻗر زا ﺮﺘﻴﻟوﺮﻜﻴﻣ ،ﺪﺷ ﻦﻴﻴﻌﺗ لوا ﻪﻠﺣﺮﻣ رد ﻪﻛ يدﺎﺑ
) 4 / 1 ( ﺪﺷ هدوﺰﻓا .
ﻪﻟﻮﻟ ﻚﻳ رد )
هرﺎﻤﺷ ﻪﻟﻮﻟ 11
( 500
ﻲﺘﻧآ ﺮﺘﻴﻟوﺮﻜﻴﻣ ﻲﺘﻧآ رﻮﻀﺣ نوﺪﺑ يدﺎﺑ
ﺪﺷ ﻪﻓﺎﺿا نژ .
3 - ﻪﻟﻮﻟ ﻲﻣﺎﻤﺗ يﺎﻣد رد ﺐﺷ ﻚﻳ ياﺮﺑ ﺎﻫ
37 ﻪﺟرد
ﻲﺘﻧآ ﺶﻨﻛاو ﺎﺗ ﺪﻨﺘﻓﺮﮔ راﺮﻗ داﺮﮕﻴﺘﻧﺎﺳ نژ
- ﻲﺘﻧآ لدﺎﻌﺗ ﻪﺑ يدﺎﺑ
ﺪﺳﺮﺑ .
رادﻮﻤﻧ 1 - ﺖﺑﺎﺛ ﺖﻈﻠﻏ ﻦﻴﻴﻌﺗ ﻪﺑ طﻮﺑﺮﻣ رادﻮﻤﻧ ﻲﺘﻧآ
يدﺎﺑ
ﺎﻧﻮﻠﻛﻮﻧﻮﻣ ﭙﭘ ﻞ
A1G8F7
0 0.5 1 1.5 2 2.5 3
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 RECIPROCAL OF THE DILUTION
ABSORBANCE AT 492nm
R2= 0.9474 R2= 0.6834
٣٦ 4 - ناﺰﻴﻣ ﻲﺳرﺮﺑ رﻮﻈﻨﻣ ﻪﺑ ﻲﺘﻧآ
ﺮﻫ رد دﻮﺟﻮﻣ دازآ يدﺎﺑ
ﻪﻟﻮﻟ يﻮﺘﺤﻣ زا ﻲﻳﺎﺗود ترﻮﺻ ﻪﺑ ﻪﻟﻮﻟ رد ﻪﻛ اﺰﻴﻟا ﺖﻴﻠﭘ ﻪﺑ ﺎﻫ
ﺪﺷ مﺎﺠﻧا اﺰﻴﻟا نﻮﻣزآ و ﺪﺷ ﻪﻓﺎﺿا ؛دﻮﺑ هﺪﺷ ﻪﻴﻬﺗ ﻚﻳ ﺪﻨﺑ .
5 - ﺪﺷ ﻪﺒﺳﺎﺤﻣ ﻲﺘﻴﻨﻴﻓا ﺰﺗﻼﻛ شور ﺎﺑ .
1 1]
KD[Agt A
A A
1 0
0 = +
: −
ﺰﺗﻼﻛ ﻪﻟدﺎﻌﻣ
A0
: ﻜﻫﺎﭼ يرﻮﻧ بﺬﺟ تﺎﻳﻮﺘﺤﻣ يوﺎﺣ ﻪﻛ ﺖﺳا ﻲ
ﺪﻗﺎﻓ فرﺪﻨﭘا ﻲﺘﻧآ
ﺖﺳا نژ .
A1
: يوﺎﺣ ﻪﻛ ﺖﺳا ﻲﻜﻫﺎﭼ زا هﺪﺷ هﺪﻧاﻮﺧ يرﻮﻧ بﺬﺟ
هرﺎﻤﺷ فرﺪﻨﭘا تﺎﻳﻮﺘﺤﻣ 1
- راﺪﻘﻣ ﻦﻳﺮﺗﻻﺎﺑ ياراد ﻪﻛ ﻲﺘﻧآ
نژ
μg) 100 ( ﺖﺳا - ﻲﻣ ﺪﺷﺎﺑ . راﺪﻘﻣ ﺮﺑاﺮﺑ ﺰﻴﻧx [1/Agtn] ﺪﻫاﻮﺧ
دﻮﺑ
؛ ﺖﻈﻠﻏ لﻮﻣﺮﻓ ﻦﻳا رد ﻲﺘﺴﻳﺎﺑ ﻪﺘﺒﻟا ﻲﺘﻧآ
ﻮﻣ ﺐﺴﺣﺮﺑ نژ ل
ﺪﺷﺎﺑ .
ﻪﺘﻓﺎﻳ ﺎﻫ لﻮﻠﺤﻣ ﺐﺳﺎﻨﻣ ﺖﻈﻠﻏ ﻲﺘﻧآ
ﺮﺑاﺮﺑ يدﺎﺑ μg/ml
2 / 1 ﺑ ﻪ
ﺪﻣآ ﺖﺳد
؛ نﺎﻤﻫ ﻪﺘﺒﻟا ﺖﻈﻠﻏ ،ﺪﺷ نﺎﻴﺑ ﺰﻴﻧ ًﻼﺒﻗ ﻪﻛ رﻮﻃ
لﻮﻠﺤﻣ ﻲﺘﻧآ ﻪﻟﻮﻟ رد يدﺎﺑ ﻫ
ﻪﺘﻓﺮﮔ ﺮﻈﻧ رد ﺮﺑاﺮﺑ ود ﻲﺘﺴﻳﺎﺑ ﺎ
دﻮﺷ
؛ لﻮﻠﺤﻣ ندوﺰﻓا زا ﺲﭘ اﺮﻳز ﻲﺘﻧآ
ﻢﻫ نژ ﺖﻈﻠﻏ ،نآ ﻢﺠﺣ
ﻲﺘﻧآ ﻒﺼﻧ ﻪﺑ يدﺎﺑ μg)
2 / 1 ( ﺶﻫﺎﻛ ﻲﻣ ﺪﺑﺎﻳ . پﻮﺳ زا ﻪﭽﻧﺎﻨﭼ
ﺺﻴﻠﺨﺗ ﻲﻟﻮﻠﺳ هدﺎﻔﺘﺳا ﻲﺘﻴﻨﻴﻓا ﻦﻴﻴﻌﺗ ياﺮﺑ هﺪﺸﻧ
دﻮﺷ
، ﻲﺘﺴﻳﺎﺑ
زا ﻲﺘﺑﺎﺛ ﺖﻗر ،لوا ﻪﻠﺣﺮﻣ ﻲﺻﺎﺼﺘﺧا ياﺰﻴﻟا ﻚﻤﻛ ﻪﺑ مود ﻪﻠﺣﺮﻣ ياﺮﺑ مزﻻ ﺖﻧﺎﺗﺎﻧﺮﭘﻮﺳ )
ﻦﻴﻴﻌﺗ Kd
( ﺑ ﻪ و ﺪﻳآ ﺖﺳد
ﺮﻴﻐﺘﻣ يﺎﻬﺘﻈﻠﻏ ﻲﺘﻧآ
ﺑ ﺖﻗر ﻦﻳا ﺎﺑ ﻪﻟﻮﻟ رد نژ ﻪ
رد هﺪﻣآ ﺖﺳد
دﻮﺷ روﺎﺠﻣ لوا ﻪﻠﺣﺮﻣ .
نﺎﻤﻫ لوﺪﺟ رد ﻪﻛ ﻪﻧﻮﮔ 1
هﺪﻫﺎﺸﻣ ﻲﻣ
دﻮﺷ
، ﻪﻠﺣﺮﻣ رد
ﻦﻴﻴﻌﺗ Kd
ﻢﻳﺰﻧآ ﺖﻗر لﺎﻳﺮﺳ ، زا ALP
μg/ml 100 ﺎﺗ
μg/ml 18 / 0 ) 250 ﺖﻈﻠﻏ ﺮﻫ زا ﺮﺘﻴﻟوﺮﻜﻴﻣ (
ﻪﻛ ﺪﺷ بﺎﺨﺘﻧا
ﺖﺒﺴﻧ ﺎﻬﻧآ زا ﻚﻳ ﺮﻫ ﻪﺑ 1:1
لﻮﻠﺤﻣ زا ﻲﺘﻧآ
ﺖﻈﻠﻏ ﺎﺑ يدﺎﺑ
ﺖﺑﺎﺛ μg/ml 4 / 2 ﺪﺷ ﻪﻓﺎﺿا .
ﺪﻌﺑ ﻪﻠﺣﺮﻣ رد -
ﺶﻨﻛاو مﺎﺠﻧا زا ﺲﭘ ﻲﺘﻧآ
و نژ ﻲﺘﻧآ يدﺎﺑ
ﻪﻟﻮﻟ رد - ﻪﻟﻮﻟ يﻮﺘﺤﻣ تﻮﻛ ياﺰﻴﻟا ﺖﻴﻠﭘ ﻪﺑ ﺎﻫ
ﺎﺑ هﺪﺷ 1μg
ﻲﺘﻧآ ALP نژ بﺬﺟ اﺰﻴﻟا نﻮﻣزآ مﺎﺠﻧا زا ﺲﭘ و ﺪﺷ ﻪﻓﺎﺿا
رد ﺎﻬﻧآ يرﻮﻧ 492
ﺪﻳدﺮﮔ ﺖﺋاﺮﻗ ﺮﺘﻣﻮﻧﺎﻧ .
هﺎﮕﺘﺳد زا هﺪﻣآ ﺖﺳد ﻪﺑ داﺪﻋا ﺮﮔا ،ﺰﺗﻼﻛ ﻪﻟدﺎﻌﻣ ﻖﺒﻃ ،ﻢﻴﻫد راﺮﻗ ﻪﻟدﺎﻌﻣ ﻦﻳا رد ار اﺰﻴﻟا ﺮﮕﺘﺋاﺮﻗ نﺎﻤﻫ ﻪﻛ ﻪﻟدﺎﻌﻣ y
n 0
0
A A
A
ﻲﻣ −
،ﺪﺷﺎﺑ ﻲﻣ ﺖﺳد ﻪﺑ راﺪﻘﻣ و ﺪﻳآ
ﺰﻴﻧ ﻪﻟدﺎﻌﻣ x
[1/Agtn] ﻲﻣ
ﺪﺷﺎﺑ
؛ ﻢﻳﺰﻧآ ﺖﻈﻠﻏ راﺪﻘﻣ - ALP
ترﻮﺻ ﻪﺑ ﻪﻛ
ﻦﻴﻴﻌﺗ مود ﻪﻠﺣﺮﻣ رد ﺖﻗر لﺎﻳﺮﺳ Kd
دﻮﺑ هﺪﺷ ﻪﺘﻓﺮﮔ رﺎﻛ ﻪﺑ -
هدﻮﻤﻧ بﺎﺴﺣ لﻮﻣ ﺐﺴﺣﺮﺑ ار
، يﺎﺟ ﻪﺑ Agtn
ﻲﻣ راﺮﻗ ﻢﻴﻫد
) رادﻮﻤﻧ 2 ( .
ﺚﺤﺑ ﻪﺠﻴﺘﻧ و يﺮﻴﮔ
هزاﺪﻧا ﻲﺘﻧآ ﻲﺘﻴﻨﻴﻓا ﻖﻴﻗد يﺮﻴﮔ يدﺎﺑ
ًﻼﻤﻋ ﻲﻤﻧ نﺎﺳآ ﺪﺷﺎﺑ
.
ﻲﺘﻧآ ياﺮﺑ نژ
ﻲﻣ ﺺﻟﺎﺧ ﻲﻧﺎﺳآ ﻪﺑ ﻪﻛ لﻮﻠﺤﻣ يﺎﻫ ،ﺪﻧﻮﺷ
ﻲﻣ ﻲﺘﻧآ مزﻻ راﺪﻘﻣ ناﻮﺗ راﺪﻧﺎﺸﻧ و دروآ ﺖﺳد ﻪﺑ ار ﺺﻟﺎﺧ نژ
دﻮﻤﻧ . شور ،ترﻮﺻ ﻦﻳا رد نﺎﻜﻣا ﻪﻛ ،درﺎﭼﺎﻜﺳا ﺰﻴﻟﺎﻧآ ﺎﺑRIA
ار ﻲﺘﻴﻨﻴﻓا ﺖﺑﺎﺛ ﻪﺒﺳﺎﺤﻣ ﻲﻣ
ﺖﺳا ﺐﺳﺎﻨﻣ ،ﺪﻫد .
لوﺪﺟ 1 - ﻦﻴﻴﻌﺗ ﻪﺑ طﻮﺑﺮﻣ ﺞﻳﺎﺘﻧ Kd
نﻮﻠﻛ A1G8F7
بﺬﺟ ﻢﻳﺰﻧآ ﺮﺘﻴﻟوﺮﻜﻴﻣ250 زﺎﺗﺎﻔﺴﻓ ﻦﻴﻟﺎﻜﻟآ μg/ml)
(
250 ﺮﺘﻴﻟوﺮﻜﻴﻣ
ﻲﺘﻧآ يدﺎﺑ μg/ml) (
هرﺎﻤﺷ ﻪﻟﻮﻟ 123
/
0 25 2/4 1
133 /
0 15 2/4 2
145 /
0 12/5 2/4 3
135 /
0 10 2/4 4
156 /
0 8 2/4 5
157 /
0 6/25 2/4 6
227 /
0 4 2/4 7
215 /
0 3/25 2/4 8
327 /
0 1/56 2/4 9
032 /
1 0/78 2/4 10
231 /
1 0/39 2/4 11
321 /
1 0/19 2/4 12
351 /
1 0/09 2/4 13
353 /
1 0/00 2/4 14
08 /
0 (PBS) ﻲﻔﻨﻣﺪﻫﺎﺷ 15
٣٧
رادﻮﻤﻧ 2 - لﺎﺼﺗا زا ﻞﺻﺎﺣ ﺰﺗﻼﻛ رادﻮﻤﻧ ﻪﺑALP
ﻲﺘﻧآ يدﺎﺑ
لﺎﻧﻮﻠﻛﻮﻧﻮﻣ A1G8F7
نﻮﭼ رد يﺮﻴﻴﻐﺗ ﻦﻳﺮﺘﻜﭼﻮﻛ ،ﻦﻴﺌﺗوﺮﭘ ندﺮﻛراﺪﻧﺎﺸﻧ ﻞﺣاﺮﻣ
لﻮﻜﻟﻮﻣ رد ﻲﺘﻧآ
ﻲﭘا ﺖﺳا ﻦﻜﻤﻣ نژ ﻲﺻﺎﺼﺘﺧا پﻮﺗ
ﻲﺘﻧآ يدﺎﺑ
لﺎﺼﺗا ﻲﺘﻴﻨﻴﻓا ﻞﻗاﺪﺣ ﺎﻳ دﺮﺒﺑ ﻦﻴﺑ زا ار ﻲﺘﻧآ
ﺪﻫد ﺮﻴﻴﻐﺗ ار يدﺎﺑ ،
ﺑ زا ﻪﻛ ﻲﺠﻳﺎﺘﻧ ﻪ
ﻲﺘﻧآ ندﺮﺑرﺎﻛ ﺑ راﺪﻧﺎﺸﻧ نژ
ﻪ ﺖﺳد ﻲﻣ ﺪﻳﺎﺑ ﺪﻳآ
دﻮﺷ ﺮﻴﺴﻔﺗ طﺎﻴﺘﺣا ﺎﺑ .
ﺑ مﺎﮕﻨﻫ ﻪ ندﺮﺑرﺎﻛ ﻲﺘﻧآ
دازآ يدﺎﺑ -
ﻲﺘﻗو ﻲﺘﻧآ رد راﺪﻧﺎﺸﻧ يدﺎﺑ نﻮﻣزآ
ﺑ ﻪ هدﺮﺑ رﺎﻛ ﻲﻣ
دﻮﺷ - ﺖﺑﺎﺛ
ﺑ ﻲﺘﻴﻨﻴﻓا ﻪ هﺪﻣآ ﺖﺳد
، ﺪﻳﺎﻤﻧ ﺮﻴﻴﻐﺗ ﺖﺳا ﻦﻜﻤﻣ )
13،12 .(
ﺐﻠﻏا ﻪﻛ ﺖﺷاد ﻪﺟﻮﺗ ﻲﺘﺴﻳﺎﺑ ﻦﻴﻨﭽﻤﻫ ﻲﺘﻧآ
مﺮﺳ يﺎﻫ
ﻲﻠﭘ زا ﻲﻧﻮﮔﺎﻧﻮﮔ ﺖﻴﻌﻤﺟ يوﺎﺣ لﺎﻨﻠﻛ ﻲﺘﻧآ
يﺎﻬﺘﻤﺴﻗ ﻪﻴﻠﻋ يدﺎﺑ
ﻚﻳ ﻒﻠﺘﺨﻣ ﻲﺘﻧآ
ﺮﻫ و ﺖﺳا نژ ﻧآ
ﻲﺘ ﺮﻳدﺎﻘﻣ ياراد يدﺎﺑ K
ﺖﺳا دﻮﺧ صﺎﺧ
؛ درﺎﭼﺎﻜﺳا رادﻮﻤﻧ ﺮﮕﻳد ﻂﻳاﺮﺷ ﻦﻳا ﺖﺤﺗ
رد ﻲﻨﺤﻨﻣ ﻚﻳ ترﻮﺻ ﻪﺑ و دﻮﺑ ﺪﻫاﻮﺨﻧ ﻲﻄﺧ ﻲﻣ
ﻪﻛ ﺪﻳآ
ﻲﺘﻧآ ﺮﻫ زا ﺎﻬﻄﺧ نﺪﺷ ﺐﻴﻛﺮﺗ ﺮﮕﻧﺎﺸﻧ ﺖﺳا يدﺎﺑ
.
ﻚﻳ ﻦﻴﻴﻌﺗ ﺺﺧﺎﺷ
ﻲﻤﻛ
، ﻚﻳ ﻲﻟدﺎﻌﺗ ﻪﻳﺰﺠﺗ ﺖﺑﺎﺛ ﻪﻴﺒﺷ
ﺲﻜﻠﭙﻤﻛ Ab-Ag
، ﻨﺌﻤﻄﻣ و دﺎﻤﺘﻋا ﻞﺑﺎﻗ شور ﻪﺑ ﻲ
اد زﺎﻴﻧ در .
ﻦﺘﭘﺎﻫ ياﺮﺑ ﻲﺑﺎﺨﺘﻧا شور ،ﻲﻟدﺎﻌﺗ ﺰﻴﻟﺎﻳد ﻲﺘﻧآ و ﺎﻫ
نژ يﺎﻫ
ﺰﻴﻟﺎﻳد ﻞﺑﺎﻗ ﻲﻣ
ﺪﺷﺎﺑ
؛ ياﺮﺑ ﻪﻛ ﻲﺗرﻮﺻ رد ﻲﺘﻧآ
نژ يﺎﻫ
ﻲﻤﻧ مﺎﺠﻧا ﻞﺑﺎﻗ ﻲﻟﻮﻜﻟﻮﻣوﺮﻛﺎﻣ ﺪﺷﺎﺑ
.
زا ،اﺰﻴﻟا ﻞﻴﺒﻗ زا يرﺎﻴﺴﺑ يﺎﻬﺷور Surface Plasmon
Resonance ﻲﺘﻧآ ﺎﺑ
نژ لﺎﻧﻮﻠﻛﻮﻧﻮﻣ ﺎﻳ ﻲﺘﻧآ
ﺖﺑﺎﺛ يدﺎﺑ هﺪﺷ
ﺖﻴﻠﭘ يور ﻫ
يور ﺎﻳ ﺎ Sensor Chip
) ﺎﺑ هﺪﻴﺷﻮﭘ يﻼﻃ ﺪﻨﻧﺎﻣ
ﻪﻠﻴﺘﻣ ﻲﺴﻛﻮﺑﺮﻛ ناﺮﺘﺴﻛد لژورﺪﻴﻫ هﺪﺷ
( ياﺮﺑ ﺐﻠﻏا ،
هزاﺪﻧا يﺮﻴﮔ هدﺎﻔﺘﺳا ﻲﺘﻴﻨﻴﻓا ﻲﻣ
دﻮﺷ ) 15،14 (.
ترﺪﻨﺑ ﻲﻳﺎﻬﺷور ﻦﻴﻨﭼ Kd
نﺎﺸﻧ ار ﻲﻌﻗاو ﻲﻣ
ﺪﻨﻫد
؛ اﺮﻳز ﺖﺑﺎﺛ ندﻮﻤﻧ ﻲﺘﻧآ نژ ) ﺖﻴﻠﭘ ﻒﻛ رد (
هرﻮﺗﺎﻧد ﺚﻋﺎﺑ ﺐﻠﻏا نﺪﺷ
ﻦﻴﺌﺗوﺮﭘ ﻲﻣ دﻮﺷ
؛ ﺎﻨﺑ ﺮﻴﻴﻐﺗ ﺰﻴﻧ نآ ﻲﻟﺎﺼﺗا صاﻮﺧ ﻦﻳاﺮﺑ ﻲﻣ
ﺪﻨﻛ
؛
ﻦﻳاﺮﺑ هوﻼﻋ Kd
ﺖﺳا هﺪﺷ ﻒﻳﺮﻌﺗ لﻮﻠﺤﻣ ﺖﻟﺎﺣ رد
؛ رد ﻲﻨﻌﻳ
ﻪﻛ ﻲﻌﻳﺎﻣ زﺎﻓ ﻲﺘﻧآ
و نژ ﻲﺘﻧآ يدﺎﺑ ود ﺮﻫ لﻮﻠﺤﻣ ترﻮﺻ ﻪﺑ
ﻲﻣ ﺪﻨﺷﺎﺑ
؛ ﺸﻳﺎﻣزآ رد ﻪﻛ ﻲﻟﺎﺣ رد تﺎ
ﺮﻴﻏ ﺎﻬﻧآ زا ﻲﻜﻳ ﺪﻣﺎﺟ زﺎﻓ
ﺖﺳا كﺮﺤﺘﻣ
؛ ﻲﺘﻴﻨﻴﻓا ﻦﻴﻤﺨﺗ ﻦﻳاﺮﺑﺎﻨﺑ (Ka)
ﻚﻤﻛ ﻪﺑ
ﻓ يﺎﻬﺷور ﺪﺷﺎﺑ نآ ﻲﻌﻗاو راﺪﻘﻣ زا رود ﺖﺳا ﻦﻜﻤﻣ ،ﺪﻣﺎﺟ زﺎ
) 16 (.
ﺖﻈﻠﻏ ﺮﮔا ﻲﻓﺮﻃ زا ﻲﺘﻧآ
ﺸﻴﺑ لﺎﺗﻮﺗ يدﺎﺑ ﺮﺘ
زا Kd
،ﺪﺷﺎﺑ
ﺖﻳﺎﺿر ﻲﺘﻴﻨﻴﻓا ﻦﻴﻴﻌﺗ ﺞﻳﺎﺘﻧ دﻮﺑ ﺪﻨﻫاﻮﺨﻧ ﺶﺨﺑ
. ﺖﻴﺳﺎﺴﺣ
ﻚﻴﺗﺎﻤﻳﺰﻧآﻮﻧﻮﻤﻳا شور (EIA)
ﻚﻴﻧژﻮﻣوﺮﻛ ياﺮﺘﺴﺑﻮﺳ ﺎﺑ
ﺖﻈﻠﻏ ﻞﻗاﺪﺣ ﻲﺘﻧآ يدﺎﺑ لﺎﺗﻮﺗ دوﺪﺣ ﻪﺑ ار
10 M 10-
دوﺪﺤﻣ
ﻲﻣ ﺪﻨﻛ ) 17 ( ؛ ﻚﻳ ياﺮﺑ ﻦﻳاﺮﺑﺎﻨﺑ ﺪﻳﺎﺑ ،ﻻﺎﺑ ﻲﻠﻴﺧ ﻲﺘﻴﻨﻴﻓا ﺎﺑAb
ﻪﮔوﮋﻧﻮﻛ ﺎﺑ ﺶﻳﺎﻣزآ ﺎﺘﺑ ﺎﻳ زﺎﺗﺎﻔﺴﻓ ﻦﻴﻟﺎﻜﻟآ يﺎﻫ
- ﻮﺘﻛﻻﺎﮔ ز زاﺪﻳ
دﻮﺷ مﺎﺠﻧا
؛ ﻢﻳﺰﻧآ ﻦﻳا اﺮﻳز ﺖﻴﺳﺎﺴﺣ ﺎﻫ
ﺶﻳﺎﻣزآ ﺶﻳاﺰﻓا ار
ﻲﻣ ﻦﻴﻳﺎﭘ يﺎﻬﺘﻈﻠﻏ ﺎﺑ ندﺮﻛرﺎﻛ نﺎﻜﻣا و ﺪﻨﻫد ﻲﺘﻧآ ﺮﺗ
ار يدﺎﺑ
ﻲﻣ ﻢﻫاﺮﻓ ﺪﻧزﺎﺳ
) 19،18 .(
Kd
ﻲﺘﻧآ يدﺎﺑ زا ﺎﻫ
7
10- 11 ﺎﺗ 10 -
ﺮﻴﻐﺘﻣ ا ﻪﭼ ﺮﻫ ﻪﻛ ﺖﺳ
دﺪﻋ Kd
ﺪﺷﺎﺑ ﺮﺘﻜﭼﻮﻛ
، نآ ﻲﺘﻴﻨﻴﻓا ﻲﺘﻧآ
دﻮﺑ ﺪﻫاﻮﺧ ﺮﺘﺸﻴﺑ يدﺎﺑ
؛
ﻲﻜﻳژﻮﻟﻮﻧﻮﻤﻳا يﺎﻬﺸﺠﻨﺳ ﻲﻣﺎﻤﺗ رد ﻪﺘﺒﻟا
، ﻲﺘﻴﻨﻴﻓا ندﻮﺑ ﻻﺎﺑ
دﻮﺑ ﺪﻫاﻮﺨﻧ ﺖﻳﺰﻣ Ab
؛ لﺎﻧﻮﻠﻛﻮﻧﻮﻣ زا ﻪﭽﻧﺎﻨﭼ لﺎﺜﻣ ناﻮﻨﻋ ﻪﺑ
ﻲﺘﻧآ ﺑ يدﺎﺑ ﻪ هﺪﻣآ ﺖﺳد
، ﺺﻴﻠﺨﺗ رد ﻢﻴﻫاﻮﺨﺑ آ
ﻲﺘﻧ رد نژ
ﻢﻴﻳﺎﻤﻧ هدﺎﻔﺘﺳا ﻲﻠﻳﺎﻤﺗ ﻲﻓاﺮﮔﻮﺗﺎﻣوﺮﻛ نﻮﺘﺳ شور
11 ، 10 -
Kd=
ياﺮﺑ ﻲﺘﻧآ دﻮﺑ ﺪﻫاﻮﺨﻧ ﺐﺳﺎﻨﻣ ﺮﮕﻳد يدﺎﺑ
؛ مﺎﮕﻨﻫ رد اﺮﻳز
يزﺎﺳاﺪﺟ ﻲﺘﻧآ
نﻮﺘﺳ زا نژ Allocution
ﺪﻳﺪﺷ ﺶﻫﺎﻛ ﻪﺑ زﺎﻴﻧ
ﺮﻓﺎﺑ pH ﻲﻣ ﺪﺷﺎﺑ
؛ ﺖﺳا ﺮﻛذ ﻪﺑ مزﻻ ﻲﻠﻴﺧ ﺮﻓﺎﺑ ﻪﻛ
يﺪﻴﺳا
،
ﻲﻌﻴﺒﻃ رﺎﺘﺧﺎﺳ ندز ﻢﻫﺮﺑ ﻲﻳﺎﻧاﻮﺗ ﻲﺘﻧآ
نﻮﺘﺳ ﺮﻤﻋ ﺶﻫﺎﻛ و نژ
دراد ار ﻲﻠﻳﺎﻤﺗ ﻲﻓاﺮﮔﻮﺗﺎﻣوﺮﻛ
؛ لﺎﺜﻣ ياﺮﺑ ﻖﻴﻘﺤﺗ رد
Sakharov
،نارﺎﻜﻤﻫ و يﺎﻣوﺪﻳﺮﺒﻴﻫ نﻮﻠﻛ
App.1 ﺪﻟﻮﻣ
لﺎﻧﻮﻠﻛﻮﻧﻮﻣ ﻲﺘﻧآ
IgG1 يدﺎﺑ ﻢﻳﺰﻧآ ﺪﺿ ﺖﺑﺎﺛ ياراد و ALP
ﻪﻳﺰﺠﺗ
10
10-
* 5 / 8 نﻮﺘﺳ رد زرﺎﻔﺳ ﻪﺑ لﺎﺼﺗا زا ﺲﭘ ﻪﻛ دﻮﺑ
ا ﻲﺒﺴﻧ ندﻮﺑ ﻻﺎﺑ ﻞﻴﻟد ﻪﺑ ﻲﻓاﺮﮔﻮﺗﺎﻣوﺮﻛ ﻲﺘﻴﻨﻴﻓ
ﻲﺘﻧآ ياﺮﺑ يدﺎﺑ
0 1 2 3 4 5 6 7 8
0 10 20 40 80 175
1/[Ag]
[Abt]/[x]
y= 0.0388+1.097 R2= 0.9907
٣٨ ﺑ نﻮﺘﺳ زا ﻲﺑﻮﺧ ﻲﻠﻴﺧ ﺞﻳﺎﺘﻧ ﻢﻳﺰﻧآ ﻪ
ﺪﻣﺎﻴﻧ ﺖﺳد
؛ ﻲﺗرﻮﺻ رد
ﮓﻧر رد ﻪﻛ ﺰﻴﻣآ
ﺳﻮﻧﻮﻤﻳا يﺎﻬﻳ ﻴ
ﻲﻤﻴﺷﻮﺘﺴﻴﻫﻮﻧﻮﻤﻳا و ﻲﻤﻴﺷﻮﺘ
داد نﺎﺸﻧ ﻲﻟﺎﻋ رﺎﻴﺴﺑ ﺞﻳﺎﺘﻧ )
20 (.
ﻖﻴﻘﺤﺗ رد نارﺎﻜﻤﻫ وWu
ﺰﻴﻧ
، ﻲﺘﻧآ يدﺎﺑ لﺎﻧﻮﻠﻛﻮﻧﻮﻣ يﺎﻫ
ﺑ ﻪ نﻮﻠﻛ زا هﺪﻣآ ﺖﺳد
،4G10 ﻔﺳ ﻪﺑ لﺎﺼﺗا زا ﺲﭘ رد زرﺎ
ﺑ ﺎﻔﻟآ نوﺮﻓﺮﺘﻨﻳا ﺺﻴﻠﺨﺗ رﻮﻈﻨﻣ ﻪﺑ ﻲﻓاﺮﮔﻮﺗﺎﻣوﺮﻛ نﻮﺘﺳ ﻪ
رﺎﻛ
ﻲﻟﺎﻋ رﺎﻴﺴﺑ نآ هدزﺎﺑ ﻪﻛ ﺪﻧﺪﺷ هدﺮﺑ ﺪﺷ شراﺰﮔ
) ﻪﺟرد ﺎﺑ
زا ﺶﻴﺑ صﻮﻠﺧ 95
% ) ( 21 (.
ﻪﻌﻟﺎﻄﻣ رد
،ﺮﮕﻳد يا لﺎﻧﻮﻠﻛﻮﻧﻮﻣ ﺪﻟﻮﻣ
ﻲﺘﻧآ ﻲﺘﻴﻨﻴﻓا ﺎﺑ يدﺎﺑ
5
10-
* 5 ﻲﻓاﺮﮔﻮﺗﺎﻣوﺮﻛ نﻮﺘﺳ رد هدﺎﻔﺘﺳا ياﺮﺑ ًﺎﺳﺎﺳا ﻪﻛ دﻮﺑ ﻲﺘﻴﻨﻴﻓا
ﺮﻈﻧ ﻪﺑ ﺐﺳﺎﻨﻣ رﺎﻴﺴﺑ ﻲﻣ
ﺪﺳر ) 22 .(
ﻪﺑ ﻪﺟﻮﺗ ﺎﺑ ﻦﻳا
ﻪﻛ Kd
ﻲﺘﻧآ لﺎﻧﻮﻠﻛﻮﻧﻮﻣ يدﺎﺑ نﻮﻠﻛ
A1G8F7
دوﺪﺣ
9
10-
ﺪﻳدﺮﮔ ﻪﺒﺳﺎﺤﻣ
، رﺎﻈﺘﻧا ﻲﻣ دور ﺐﻠﻏا رد
ﺶﺠﻨﺳ ﺪﺷﺎﺑ ﻪﺘﺷاد ﻲﻳارﺎﻛ و ﺐﺳﺎﻨﻣ ﻲﻜﻳژﻮﻟﻮﻧﻮﻤﻳا يﺎﻫ و
ًﻼﻤﻋ ﺰﻴﻧ رد يﺎﻬﻧﻮﻣزآ اﺰﻴﻟا
- مود ﻪﻳﻻ ناﻮﻨﻋ ﻪﺑ -
و يﺎﻬﻧﻮﻣزآ
ﻤﻴﺷﻮﺘﻴﺳﻮﻧﻮﻤﻳا ﻲﻤﻴﺷﻮﺘﺴﻴﻫﻮﻧﻮﻤﻳا و ﻲ
- ﻢﻳﺰﻧآ ﺎﺑ هاﺮﻤﻫ ALP
ﺲﻜﻠﭙﻤﻛ ترﻮﺻ ﻪﺑ APAAP
مﻮﺳ ﻪﻳﻻ ياﺮﺑ -
و ﻲﻟﺎﻋ رﺎﻴﺴﺑ
ﺲﻜﻠﭙﻤﻛ ﺎﺑ ﻪﺴﻳﺎﻘﻣ ﻞﺑﺎﻗ APAAP
ﺖﻛﺮﺷ زا هﺪﺷ يراﺪﻳﺮﺧ
ﻲﻣDako ﺪﺷﺎﺑ .
ﻊﺑﺎﻨﻣ :
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167-75.
٣٩
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٤٠
Measurement of affinity constant of Anti-human IgG Monoclonal antibodies with alkaline phosphatas
enzyme by an ELISA-based method
M. Naseri*,SM. Moazzen**, AA. Pourfathollah**, B. Mesbahzadeh***
* Instructor, Department of Immunology, Faculty of Medicine, Birjand University of Medical Sciences, Birjand, Iran
** Associate Professor, Department of Immunology, Faculty of Medical Sciences, Tarbiat Modarres University, Tehran, Iran
*** Instructor, Department of Physiology and Pharmacology, Faculty of Medicine, Birjand University of Medical Sciences, Birjand, Iran
Abstract
Background and Aim: The affinity of an antibody with its antigen is a crucial parameter in its biological activity and carrying out immunologic tests such as radioimmunoassay (RIA) and immunohistochemistry. This study was done in our laboratory at Tarbiat Modarres University to measure the affinity constant of specific monoclonal antibodies/MAB (A1G8F7) with alkaline phosphatase enzyme produced.
Material and Methods: Several Methods have been innovated to determine the affinity and avidity of antibodies. In all of them a serum is used in which antigen and antibody would reach balance and, without a change in the balance, free antigen and antigen associated with antibody are measured. Applying a rapid and simple ELISA-based method the affinity constant of specific MAB (A1G8F7) against alkaline phosphatase was measured.
Results: Constant density of antibody for use in specific ELISA used to determine dissociation constant (Kd), applying klotz methodology-by means of a competitive ELISA proved to be 1μg Kd
of A1G8F7 clone; through klotz fig, it was estimated to be 3.8×10-9
Conclusion: considering that the Kd of natural antibodies in the body for most antigens ranges from 10-7 to 10-11 and that the less Kd the more affinity of the antibody, the antibody secreted by A1G8F7
clone seems suitable. Practically, in immunocytochemistry for most immunologic measuring, histochemistry test done with mAb-ALP enzyme in three line-staining led to pretty good and comparable results with the similar trade sample of APAAP (Dako, Denmark).
Key Words: Monoclonal antibody; Affinity avidity; ELISA; Alkaline phosphatase
