- Th^nh phan hoa hgc va tmh khong thuan nhat: Chi kich thuoc vd tinh la chua du dl xac dinh
1.2. Khdng huyet thanh
Cdc khdng huyet thanh (antibody) hay cdn ggi Id cdc globulin mien dich (immunoglobulin - dm chi thdnh phin cung cdp khd ndng mien dich nam d phan globuUn ciia huyet thanh khi phdn tich bdng difn di) Id nhOng phdn hi protein hoat dgng nhu nhimg thy thd tren be mat te bao lympho B dl nh$n difn khdng nguyen hodc nhu nhihig sdn phdm tiet ciia te bao plasmạ Ve ngudn goc, ti bdo
plasma chinh la te bdo lympho B sau khi da nhan dien khdng nguyen, hoat hod va bift hod thdnh. Nhihig khang huyet thanh do te bao plasma tiet ra cd tinh đc hieu vdi khang nguyen gidng het nhu tinh dac hieu cua phan tir khdng huyit thanh tren bd mat tl bao B đng vai trd Id thy thi đnh cho khang nguyen đ nhan dien khar^ nguyen ban diụ Cdc khdng huylt thanh ndy se Imi hanh trong mdu vd bach huyet, chiing hoat đng nhu nhimg thdnh phin thuc hien ciia đp ihig miin dich dich the bdng each phdt hien vd trung hod hodc loai bd cac khang nguyen. Cdc khdng huyet thanh thyc hien hai chiic ndng chinh: chiing gan mgt cdch dac hieu vao mgt khang nguyen vd chiing tham gia trong mgt sd chirc nang sinh hgc khac. Chuong nay chimg ta se tap trung vao van de cau tnic cua cdc phan tir khdng huyet thanh. DEIC diem cdu tnic ndo da tao ra tinh d^c hieu vd chiic ndng cua nhftng phan tir ndỵ
1.2.1.Ciu triic ccfban ciia khdng huyit thanh
Tir dau thi k^ XX ngudi ta da biet rdng cdc khdng huylt thanh Id cdc phan tir thuc hien cua đp ling miin dich dich thi xuit hifn trong huyit thanh. Ndm 1939 Tiselius .A vd Kabat .E .A da tiln hdnh phdt hien phin protein huylt thanh chiia cac khdng huylt thanh. Cdc tdc gid da gay miln dich cho thd bdng ovalbumin sau đ chia huylt thanh ciia thd da dugc gay miin djch thanh hai phan bdng nhaụ Phan thii nhat dugc difn di de phan tach thdnh bdn thanh phin: albumin va cac a-, b- vd g- globulin; Phan thii hai dugc cho phan iing vdi khdng nguyen dl hinh thdnh chit ket tua, sau đ loai bd chit tua ndy r^i sir dyng phdn huyet thanh cdn lai de dien dị So sdnh hinh dnh dien di cua hai miu huylt thanh ndy, cdc tdc gid phdt hifn thiy trong mau dien di thir hai cac thdnh phin trong phin g-globulin gidm di đng kẹ Nhu v^y phan g-globulin cd chiia cdc khdng huydt thanh
cua huylt thanh vd vi vdy chiing dugc d^t ten Id globulin miln dich dl phdn biet vdi cdc protem
khde cdn lai trong phan g-globulm.
Vdo nhOng ndm 1950 - 1960 Porter .R vd Edelman .G da chiing minh cdu tnic co ban ciia globulin miln dich. Cdng trinh ndy c6 mgt y nghia dang ke vd hai nhd khoa hgc da dugc trao gidi thudng Nobel vi y hpc nam 1972. Cdc cdch tiep can thyc nghiem ciia Edelman vd ciia Porter rit khde nhaụ Porter phdn cit cdc phan tii globulin mien dich bdng cac enzyme de thu dugc cac manh peptide, trong khi đ Edelman da tdch phan tii globulin miin dich bdng cdch khii cac ciu disulfide lien chuSị Kit qud cua mdi phuong phap da bd sung cho nhau vd cho phdp chiing ta hieu dugc can tnic CO ban cua phan tOr globulin miln dich. Cd Porter vd Edelman Idn dau tidn da phan tach phdn g- globulin ciia huylt tiianh bdng sidu ly tdm dl thu dugc hai phin nhd: phin thii nhat cd trgng lugng phdn tii cao cd hdng so Idng Id 19S; phin thii hai co trgng lugng phdn tir thdp va hdng sd Idng Id 7S. Cac tdc gid đ tdch rieng phin 7S xdc djnh dugc trgng lugng phdn tir la 150.000 Da vd ky hifu Id
globulin miln djch G (IgG). Porter đ phdn cdt IgG bdng enzyme papain thanh cdc mdnh khde nhaụ M$c du papam la mdt enzyme boat dgng thuy phdn protein khdng dac hieu va phan cat todn bg phdn tu protein, nhimg neu thdi gian tdc dyng ngdn thi enzyme ndy chi phan cat cdc cdu disulfide nhdy cdm nhat. Trong dilu kien nhu vdy papain da phan cat phdn tir IgG thdnh hai mdnh gidng nhau (moi manh c6 trgng lugng phan tu Id 45.000) dugc ggi la cdc mdnh Fab (antigen binding fragment), bdi vi mdnh Fab van giir nguyen khd ndng gin vdi khang nguyen ciia phan tii khdng huyet thanh nguyen ven. Ngodi hai manh Fab tdc gid cdn thu dugc mdt manh niia cd trong lugng phdn tii Id 50.000 dugc gpi Id mdnh Fc vi khi bao qudn trong lanh chung bi ket tinh hod (chu c bit ngudn tir chQ cristalliable nghia Id cd khd ndng kit tinh) (Hinh 4.2). Nisonoff .A ciing tilp can nghidn ciiu bdng cdch tuong tu nhung dimg enzyme pepsin thay cho enzyme papain. Khi cho pepsin tdc dyng ngdn vdi phan tir IgG thi thu dugc mgt mdnh cd trgng lugng phdn tir 100.000 gdm 2 manh Fab ggp 1^ vd ky hifu la F(ab')2. Mdnh F(ab')2 ciing gdy kit tua khdng nguyen. Tuy nhien khi xii ly bdng pepsin khdng thu dugc mdnh Fc ma thay vdo đ la mgt sd manh peptide nhd
c i u tnic chuoi cua IgG lin diu tien dugc nghien ciiu bdi Edelman vd sau nay dugc khdng djnh bdi Porter. Edelman da khir cdc ciu disulfide ciia phdn tii IgG bdng mercaptoethanol rdi tien hdnh difn di trdn gel tinh bgt trong mdi tnrdng ure 8M de khii cdc cau disulfide trong chuoi vd hen chu§i 1dm cho phdn tir dugc hdi ra md khdng gip. Tdc gid thu dugc 2 vf t dien di vd dilu ndy chiing td ring phdn tii IgG cd nhilu hon mgt chudi protein. Porter ciing phdt triln tin nghifm nay bdng cdch t^o ra phdn img khir nh? hon sao cho chi cit cdc cdu disulfide lien chuSi ma thdi, sau đ tien hdnh alkyl hod cdc nhdm sulfhydryl (SH) 10 ra bdn ngodi bdng iodoacetamide di ngdn cdn sy tai tao ngSu nhidn ciia cac cau disulfidẹ Ngoai ra tdc gid cdn cho them acid propionic hiiu co de ngdn can sy ngung tgp. Sau đ tiln hdnh sic ky trdn cgt di de phdn tdch cdc phan tir dya tren kich thudc cua chiing . Thi nghifm ndy da cho tiiay phdn hi IgG cd ti-gng lugng phdn tir 150.000 Da dugc hgp thdnh bdi hai chuoi peptide, mSi chuSi cd trgng lugng phdn tir 50.000 dugc ky hieu Id cdc chuoi ndng (chuoi H - heavy chain) va hai chuoi mdi chudi cd trgng lugng phan tir la 25.000 dugc ky hifu Id cdc chuli nh? (chudi L - light chain).
v i n dl cdn lai Id di xdc dinh xem cdc sdn phim phan cdt bdi enzyme, Fab, F(ab')2 vd Fc cd lidn quan nhu thi ndo vdi cdc sdn phim phan ra cua chudi ndng va chuoi nhẻ Dl trd Idi caii hdi ndy Porter da sir dyng khdng huylt thanh de dugc gdy miin dich bdi cdc manh Fab va Fc cua phdn tir IgG ciia thd. Ong đ phdt hifn thiy ring khdng huylt thanh khdng Fab cd thi tuong tac vdi cd chuSi n$ng vd chuSi nh?, trong khi đ khdng huylt thanh khdng Fc thi chi phdn iing vdi chudi ndng md thdị Dilu ndy chimg td Fab c6 chira cdc thdnh phin cua cd chudi nang vd chudi nhe cdn Fc thi
chi chiia cac thanh phin cua chuoi ndng. Cac kit qua nay da khdng dinh md hinh diu tien ve cau tnic phan tii IgG ma Porter da dua rạ Theo md hinh nay thi pMn tir IgG bao gdm hai chudi nang gidng het nhau vd hai chudi nhe gidng het nhau dugc lien ket vdi nhau bang cdc cdu disulfliạ Enzyme papain phan cat ngay phia tren cdu disulfiia Uen chudi ndi giiia hai chudi nang cdn enzyme pepsin thi phan cdt ngay phia dudi can disutftia nay vd vi the hai enzyme thuy phdn protein tao ra cdc sdn phan phdn rd khde nhaụ Khi sir dyng mercaptoethanol dl khir va akyl hod se cho phep ta tdch rieng dugc cdc chuoi nang vd chuoi nhẹ
1.2.2. Cdc quyit ^inh khdng nguyen tren phdn tic globulin miin dich
Do cdc khdng huyet thanh la cdc phan tir glycoprotein nen bdn thdn chiing cQng cd the boat đng nhu mgt chit smh miln dich di kich thich sinh ra mdt đp iing tao khdng huyit thanh. Ngudi ta chia cdc quylt dinh khdng nguyen (hodc cdc epitop) tren phdn tii globulm miin dich thanh ba loai chinh: cdc quyet dinh isotyp, cdc quyet dinh allotype vd cdc quylt dinh idiotyp. Cac quylt dinh ndy dugc dinh vj tren cac vi tri đc tnmg cua phdn tii khang huyit thanh.
Cac quylt djnh isotype
Cdc quyet dinh isotype ndm tren vung hdng dinh cho phep phdn biet cdc chuoi ndng cua cac Idp vd cdc 16p nhd khdng huyet thanh khde nhau, va phan biet cac typ vd cdc typ nhd ciia cdc chudi nhe trong m^t lodi nhdt dinh. Mdi m^t quylt dinh isotype dugc md hod bdi mpt gene cua vimg hdng djnh ridng bift vd tit cd cdc cd the trong cimg mdt lodi deu cd cimg cdc gene vimg hdng dinh ma hod cdc quyit djnh ndỵ Trong cimg m^t lodi, timg ca the se bilu hifn tat cd cdc quyet dinh isotype trong huylt thanh cua chiing. Cdc lodi khde nhau dugc di truyin cdc gene ciia vimg hdng dinh khde nhau vd bdi vdy chiing cd cdc isotype khde nhaụ Do do khi Idy globulin miln djch ciia lodi ndy tidm vdo lodi khde thi cdc quyet djnh isotype se dugc nhan dien nhu Id la vd kich thich sinh ra đp ling t^o khdng huyit tharUi chdng lai cdc quyet dinh isotype cua phan tir globulin miln dich lạ Cac khdng huyit thanh khdng isotype thudng dugc sii dyng de xac dinh cdc Idp hodc cdc lap nhd cua globulin miln dich trong huyit thanh hodc xac dinh Idp khang huylt thanh gin tren bi mat te bdo B (Slg).
Cdc quylt djnh allotyp
M$c dii tit cd cdc cd thi trong cimg mdt loai thira hudng cimg mgt loai bd gene isotype nhung 1^ cd m^t so gene allele khde nhau giira cdc ca thẹ Nhihig gene allele nay ma hod mdt sd dogji peptide cd trinh ty cdc acid amine khde nhau, ngudi ta gpi do Id cdc quyet dinh allotypẹ Cdc quylt dinh ndy chl xuit hifn d mgt sd chii khdng phai la tdt cd cac thdnh vien ciia mdt lodị d ngudi
da xdc dinh dugc allotype cua 4 Idp nhd IgG, 1 Idp nhd IgA vd chudi nhe (. Cac quylt dinh allotype ciia chuoi (dugc ky hieu Id Gm. Cho din nay da nhdn dang dugc 25 loai quyit dinh allotype Gm, chiing dugc ky hieu bing chii G (viit tit cua chii gamma) sau đ Id sd thii tu Idp nhd rdi din sd tiiii ty allotype [vi du Glm(l), G2m(23), G3m(l 1), G4m(4a)]. Ngudi ta chi mdi xdc dinh dugc allotype cua Idp nhd IgA2 dugc ky hieu la A2m(l) vd A2m(2). Chudi nhe (cd 3 quylt dinh allotype dugc ky hieu la (m(l), (m(2), vd (m(3). Moi allotype nay cd su khac nhau tir 1 din 4 acid amine dugc ma hod bdi cdc gene allele khac nhaụ
Cdc khdng huyet thanh chdng cdc quyet dinh allotype cd thi thu dugc bdng cdch tiem cdc globulin miln dich cua mdt cd thi ndy cho mdt cd thi khde trong cimg loai khdng cd cdc quylt djnh allotype tuong iing. Khdng huylt thanh khang allotype ciing cd thi xuit hien d ngudi phy nii trong qua trinh mang thai khi đp iing vdi quylt dinh allotype cua ngudi chong cd trSn phan tu globulin mien dich ciia ngudi con. Khdng huylt thanh khang allotype cung cd thi xudt hifn sau khi truyin mdụ
Cdc quyet d}nh idiotype
Trinh ty cdc acid amine cd duy nhat d vimg VH vd VL ciia mpt phdn tir khdng huyit thanh biet trudc cd the hoat dOng khdng chl nhu mpt vi tri ket hgp khang nguyen md cdn hoat đng nhu m0t quyit djnh khdng nguyen. Cdc quyit dinh idiotype dugc sinh ra do cac vimg thay dii cua chuoi n$ng vd chuSi nh? hinh flianh mgt ciu tnic đc bift vl khdng gian. Mil m^t khang nguySn rieng le ciia vimg thay doi dugc xem nhu Id mgt quyit dinh idiotype hay cdn ggi Id idiotopẹ Trong mdt s6 tnrdng hgp idiotope cd the Id mOt vi tri ket hgp khdng nguydn thuc sy, nhung trong mgt sd trudng hgp mgt idiotope ciing cd the Id mpt dogn cua vimg thay đi ndm ngodi vi tri kit hgp khdng nguyen. Moi phan tii khdng huyet thanh se cd nhieu idiotopẹ Tat ca cdc idiotop cua mdt phan tir khdng huyet thanh se tao ra hinh dnh idiotype ciia khdng huyet thanh dọ
Bdi vi cdc khdng huyit thanh do cac te bao B ndm trong cimg mgt clone sdn xuit ra cd vimg thay đi gidng hft nhau nen chiing cd cimg mgt hinh dnh idiotypẹ Cdc khdng huyit thanh khdng cimg mpt clone td bdo B sdn xudt ra ciing cd the cd quyet dinh idiotj^e gidng nhaụ Quyit dinh idiotype ndy dugc ggi Id quydt dinh idiotype chung gidng nhau, chiing phdn anh sy huy đng phd biln cung raOt gene vimg thay đi cua td bdo mdm bdi cdc te bao B khac nhau ciia cung mpt đng thuan chung. Cdc khdng huyet thanh khdng idiotype dugc tao ra bang cdch thu hep din miic tdi thilu sy khde bift isotype hodc allotype de den miic cd the nhdn dang dugc su khac bift idiotypẹ Thdng thudng cd the sir dyng m^t khdng huyet thanh thuan khiet nhu protein u tuy hoac mdt khdng
huylt thanh don clonẹ Tiem khdng huylt thanh nay cho mdt co the nhan đng gene thi se hinh thanh dugc khdng huyet thanh khdng i d i o ^ e .
Mgt trong nhung thi nghiem sdm nhit đ chiing minh sa cd mat ciia cac quyet dinh idiotype
Id thi nghifm ciia Oudin .J vd Michel .M tai vien Pasteur Paris nam 1963. Cdc tac gid nay da tiem khdng huylt thanh ciia thd khang Samonella vdo mdt con thd khac cimg nhdm allotypẹ Thd dugc tiem da sdn sinh ra khdng huydt thanh cd khd nang kit hgp dugc vdi khang huyet thanh da dimg de gdy miln dich ma khdng ket hgp vdi nhimg khdng huyit thanh cua chinh con thd đ khang lai cac khdng nguyen khde va ciing khdng ket hgp vdi khang huyit thanh ciia nhiing con thd khde khdng 1^ Samonellạ Khang huylt thanh khang idiotype cdn dugc smh ra bdi đng vat trong qud trinh đp iing miin djch vd đng vai trd quan trgng trong vif c dieu hod dap iing miln djch.
1.2.3, Cdc l&p khdng huyit thanh
Moi Idp khdng huyet thanh dugc phdn bift bdi sy khde nhau vi trinh ty cac acid amine trong viing hdng dinh chuoi ndng. Ciu tnic cua 5 Idp khdng huyet thanh chinh da dugc xdc dinh (hinh 5.12), vd tinh chdt sinh hpc ciia chiing dugc lift ke d bang dudi day:
Bang 1.2. Tdm tit tinh chit ciia cdc lap (isotype) immunoglobulin khde nhaụ Ddc diem vd boat tinh sinh hgc cua cdc Idp vd phdn Idp khdng huyet thanh
„ . . , , i B B Tyle "Hoa tri"' , IgG , Man 70% din 75% 2 IgA . 1 IgM Niem nhav Cdc dich tiet Lvmpho B Mau 15% din 20% 1 cac khang huylt ^^^^ thanh trong huyet i thanh j 2 - 4 2-10 -IgE IgD Bach cau ai kiem Te bao mast < 1 % 2 Lvmpho B < 1 % 2 13
Vai tro dpc tọ
Trung hoa cac Ngung tu, Ngung tụ
trung hoa con duong co Hoat hoa cac . , , , . , . , , , ^-. . , . truns hoa cac te bao lvmpho Vl khuan va cac vi khuan. dien cua bo , , r • ,
, : ! —. , ; — i kv sum trung B ;
I virus I virus , the , Khang huy^t thanh IgG
IgG CO nong do cao nhdt trong huylt thanh, chiem khoang 80% t6ng luong globulin miln
dich trong huylt thanh. Phan til IgG la m6t monomer gim c6 hai chudi nang (vk hai chuli nhe hoac
(hô (. Trong luong phan tii cua IgG khoang 150.000 Da, hang s6 ling 7S, vi vay IgG co the thay ca 6 trong long mach va o ngoai long mach. Co 4 lop nho IgG 6 nguoi duoc danh diu theo nong do giam din cua chiing trong huylt thanh: IgGl (9 mg/ml), IgG2 (3 mg/ml) IgG3 (1 mg/ml), va IgG4 (0,5 mg/ml). B6n lop nho nay dupe ma hoa boi 4 gene vung htog dinh chuoi nang (gene CH) khac
nhau m^ co 90 - 95% trinh ty ADN gidng nhaụ Dac trung ciu tnic dh phan biet lop nho nay vdi lop nho khac \k kich thuoc cua vung ban le, so lu(?ng cung nhu vi tri cua cac cau disulfide lien
chuSi nli cic chuoi nang . S\r khac bỉt vl acid amine giua cac lop nho cua IgG da lam cho boat tinh sinh hpc cua phan tii co nhirng khac nhaụ IgGl, IgG3 va IgG4 co thi chuyin van de dang qua
nhau thai vk d6ng vai trd trong viec bio v6 thai phat trien. M5t s6 lop nho IgG co the boat hoa bo
thi mac du hipu qua cua chiing khac nhaụ Lop nho IgG3 hoat hoa b6 the hieu qua nhit, tiep theo la IgGl r6i din IgG2 c6n IgG4 thi khdng cd kha nang hoat hoa bd thị IgG cung boat đng nhu mpt khing huylt thanh opsonin do chiing cd thi gin vio thy the danh cho Fc c6 tren be mat dai thuc