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研究生:殷韶禪
研究生(外文):Shao Chen Yin
論文名稱:利用單株抗體探討人類Haptoglobin之化學結構及免疫特性
論文名稱(外文):Solution properties of human plasma haptoglobin as probed by monoclonal antibodies
指導教授:毛仁淡
指導教授(外文):Simon JT Mao
學位類別:碩士
校院名稱:國立交通大學
系所名稱:生化工程研究所
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2005
畢業學年度:93
語文別:英文
論文頁數:58
中文關鍵詞:單株抗體抗原決定基免疫反應
外文關鍵詞:Haptoglobinmonoclonal antibodyantigenic mappingimmunoreactivitysite-directed mutagenesisepitope
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Haptoglobin (Hp) 在人體中有三種不同的型態,分別是Hp 1-1,Hp 2-1以及Hp 2-2。其中Hp 2-1以及Hp 2-2個體之alpha-2鏈 (142個胺基酸) 相對於alpha-1鏈 (83個胺基酸) 多了一段重複性序列 (從胺基酸12到70),故在alpha-2鏈上亦多了一個SH group,因此可藉由雙硫鍵的形成而分別連接成線形的Hp 2-1聚合物與圓圈形的Hp 2-2聚合物。為了解三種型態Hp之免疫特性與其結構的關係,我們以Hp 2-1或Hp 2-2為抗原製作出之專一性抗體來闡示。單株抗體3H8其抗原決定位經由peptide array證實位於alpha鏈之胺基酸31至39 (RYQCKNYYK),利用細菌表現之alpha-1及alpha-2重組蛋白亦可證實此抗體對alpha-2之反應較強,然而對於三種型態Hp之免疫反應卻幾近相同,可能導因於Hp 2-1及2-2之聚合物結構使得胺基酸31至39區域無法完全曝露。此外此抗體以競爭型酵素免疫連結分析法 (competitive-ELISA) 對三種型態Hp之免疫反應亦幾乎相同,故利用此抗體,即能有效快速地於人類血漿之Hp定量。另一方面,我們以Hp 2-1及2-2為抗原製作出之單株抗體,大多數僅能辨識雙硫鍵結未被破壞之Hp,且無法辨認以還原劑如beta-mercaptoethanol分離之alpha鏈或beta鏈,可能是由於Hp 2-1及2-2具有複雜程度較高之聚合物結構,而形成獨特的抗原決定位。這些單株抗體(conformational-dependent mAbs)中,我們發現有兩株(3B7與4H11)對於Hp 1-1之免疫反應明顯比Hp 2-1及2-2弱.於人類血漿中亦可區分Hp 1-1與另外兩種具有聚合物之Hp型態,只要再結合專一辨識Hp 1-1之單株抗體.即可快速區分不同型態之Hp。
Abstract in Chinease …………………………………………………………… i
Abstract……………. …………………………………………………………… ii
Acknowledement….. …………………………………………………………… iii
Content…………….. ………………………………………………………….... v
Figure content…….. …………………………………………………………… vi
Table content……… …………………………………………………………… viii
Abbreviation………. …………………………………………………………… ix
Chapter 1 Introduction……………………………………………… 1
1.1 Biological functions of Haptoglobin (Hp)……………..... 1
1.2 The structural arrangement of Hp……………………….. 2
1.3 Clinical conception with Hp phenotypes………………... 3
1.4 The purpose of this study………………………………... 4
Chapter 2 Experimental procedures………………………………… 6
2.1 Phenotyping……………………………………………… 6
2.2 Purification of Hp1-1, 2-1, and 2-2……………………… 7
2.3 Preparation of monoclonal antibodies (mAb)…………… 7
2.4 Native and SDS-PAGE………………………………….. 8
2.5 Western blot analysis……………………………………. 9
2.6 Enzyme-linked immunosorbent assay…………………… 9
2.7 Competetive ELISA……………………………………... 10
2.8 Antigenic mapping (Peptide array)……………………… 11
2.9 Expression of the recombinant Hp α1 and α2 subunits… 11
2.10 Site directed mutagenesis……………………………….. 13
vi
2.11 CD Spectrum……………………………………………. 14
2.12 Hemoglobin inhibition assay……………………………. 14
Chapter 3 Results…………………………………………………… 16
3.1 MAb prepared against Hp 2-1 or 2-2……………………. 16
3.2 MAb specific to Hp α chain of Hp that reacted equally
with Hp phenotypes………………………………………
18
3.3 Immunoreactivity of mAb 3H8 against Hp phenotypes in
solution…………………………………………………...
19
3.4 Determination of plasma levels of Hp in subject with Hp
1-1, 2-1, or 2-2 using α-chain specific mAb 3H8……….. 19
3.5 Antigenic mapping for mAb 3H8……………………….. 21
3.6 Immunoreactivity of mAb 3H8 for recombinant α1 and 2
chains……………………………………………………. 22
3.7 Conformation of recombinant α1 and 2 and Hp 2-1……. 23
Chapter 4 Discussion……………………………………………….. 24
Chapter 5 Reference………………………………………………… 32
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