: H IV. Screen ing of 1F7 Id iotyp ic An ti-gp160 An tibod ies from Phage An tibody L ibrary

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1 14 1 V o l114,n o Ch inese Jou rnal of B iochem istry and M o lecu lar B io logy Feb 1998 H IV-1 gp 160 2) 1) 1) 1) 3) (, ; 2), ; 3), ) : H IV m RNA, IgG Fd Fab, pcom b3, XL 12B lue,,, Fab g N H IV 21 IF7 : gp 160 gp 41, gp EL ISA EL ISA gp B 1D EL ISA 1F7, :, H IV, Screen ing of 1F7 Id iotyp ic An ti-gp160 An tibod ies from Phage An tibody L ibrary Zhao Yun2Feng 1) W ang H ai2t ao 2) W ang Q uan2l i 1) D u Zh i2yan 1) Yang J ing2geng 1) Sun Hong2Yan 1) M a L i2r en Y in Zhen 3) 1) (Institu te of R ad iation M ed icine, B eij ing ) 2) (Insitu te of M icrobiology and Ep id em iology, B eij ing ) 3) (U niversity of A g ricu ltu ral and A nim al S cience, Chang chun ) Abstract T he m RNA ex tracted from sp leen cells of the H IV 212infected person w as reverse tran scrip ted and am p lified by po lym erase chain reaction u sing general p rim ers scann ing Fd and ligh t chain of IgG T he am p lified fragm en ts w ere ligated in to phagem id pcom b3 and elec2 tro tran sinfected com peten t E coli XL 12B lue cells D u ring co2cu ltu re w ith helper phage, the recom b inan t phage lysis and Fab disp layed on the su rface as fu sion p ro tein w ith the N 2term i2 nal of coat p ro tein g By then, a clone lib rary w as estab lished Screen ing an tibodies bo th again st H IV 21 and recogn ized by an ti2idio typ ic an tibody 1F7 Specific an tibodies a2 gain st H IV 2gp 160 w ere en riched by 100 tim es after th ree rounds of pann ing w ith recom b inan t gp 160 and gp 41 Tw o clones nam ed 1D and 3B exh ib ited specific b inding to gp 160 and m ono2 clonal an ti2idio typ ic an tibody 1F7 iden tified by direct and petitive EL ISA m ethodṡ T he 20 : , :

2 success of iso lating hum an an ti2gp 160 w ith 1F7 idio type p roved the u sefu lness of phage disp lay system in hum an M ca b p reparation and laid a foundation fo r the screen ing of an ti2id2 io typ ic pep tides w h ich can b lock dom inan t clone of B cells in H IV 21 infected individualṡ Key words: Phage an tibody lib rary, H IV Idio typ ic an tibody,, (A ID S) H IV,, H IV, B H IV, H IV H IV, Ko β [ 1 h ler, ] 1F7 [ 2 ], 70% H IV, H IV, IgM,,, H IV 21, 1F7, pcom B 3 Scripp s XL 12B lue V CSM 13 Stratagene H IV ( rgp 160) PA STU ER 1F7 ( IgM ) 11E7 ( IgM ϑ) Ken2 tucky Ko β h ler M 132 (ho rseradish perox idase, HR P) SB (30 g, 20 g, 10 g M op s 1 L H 2O, ph 710) 112 -PCR [3 ], Fd Xhog Speg, Sacg Xbag m RNA H IV 21 PCR 94 1 m in, 45 1 m in, 72 2 m in, GCA T GT A CT A GT T T T GTC A CA A GA T T T GGG 23 CG1z CA G GT G CA G CTC GA G CA G TCT GGG 23 V H 1a 5 2 GA G GT G CA G CTC GA G GA G TCT GGG 23 V H 3a 5 2 CA G GT G CA G CT G CTC GA G TCT GGG 23 V H 1f 5 2 GA G GT G CA G CT G CTC GA G TCT GGG 23 V H 3f 5 2 CA G GT G CA G CTA CTC GA G TCG GG 23 V H 2f 5 2 CA G GT G CA G CT G CTC GA G TCG GG 23 V H 4f 5 2 CA G GTA CA G CTC GA G CA G TCA GG 23 V H 6a 5 2 CA G GTA CA G CT G CTC GA G TCT GGT CCA 23 V H 6f ϑ GCG CCG TCT A GA A CT AA C A CT CTC CCC T GT T GA 21

3 A GC TCT T T G T GA CGG GCG A TC TCA G 23 ϑ 5 CK1z 5 2 GA C A TC GA G CT G A CC CA G TCT CCA 23 V K1a 5 2 GAA A T T GA G CTC A CG CA G TCT CCA 23 V K3a 5 2 GA T A T T GA G CTC A CT CA G TCT CCA 23 V K2a Κ CGC CGT CTA GAA CTA T GA A CA T TC T GT A GG 23 CL 2 Κ 5 5 2AA T T T T GA G CTC A CT CA G CCC CA C 23 VL TCT GCC GA G CTC CA G CCT GCC TCC GT G 23 VL TCT GT G GA G CTC CA G CCG CCC TCA GT G 23 VL TCT GAA GA G CTC CA G GA C CCT GT T GT G TCT GT G 23 VL CA G TCT GA G CTC A CG CA G CCG CCC 23 VL CA G A CT GA G CTC A CT CA G GA G CCC 23 VL [4 ], Fd PCR Xhog Speg, pcom B 3, XL 12B lue, Sacg Xbag, XL 12B lue [5 ] 114, 1 Λl 50 Λl XL 12B lue (A 600 1), 15 m in, LB g, 37, 2 d, (cfu) 115 rgp m o lgl N ahco 3 (ph 816) 50 Λggm l, 50 Λl EL ISA, 3%BSA 37 1 h,, 50 Λl ( cfu), 37 2 h, H 2O, PBSgTw een 10, H 2O 50 Λl (011 m o lgl HC l, ph 212, 1 m ggm l BSA ), 10 m in,, 3 Λl 2 m o lgl T ris 2 m l (A 600= 1) XL 12B lue 15 m in 10 m l SB ( 20 Λggm l 10 Λggm l ), 1 Λl cfu, 37 1 h 100 m l SB ( 50 Λggm l 10 Λggm l ), 1 h 1 m l V CSM 13 (10 12 cfu) 2 h, 70 Λggm l, 37 4% PEG8000 3% N ac l, 30 m in Λl gp 160 EL ISA, 37 2 h, PBSgTw een 3, H 2O 2, M 132HR P, 37 1 h, V CSM 13 A 5 EL ISA (50 Λl PBSgTw een 2 Λggm l rgp 160, ), 50 Λl 1F7 (2 Λggm l) EL ISA, 37 2 h, PBSgTw een 3, H 2O 2, M 132HR P, 37 1 h, 11E7 ( IgM ϑ) Fd 5 Λl 22

4 F ig 1 PCR amp lication of Fd genes and ligh t chain genes A 1PCR p roduct of Fd B1PCR p roduct of ligh t chain M 1123 bp ladder F ig 2 Enrichm ent of phage antibodies by panning selection A 1Befo re panning B1A fter rounds of panning M 1ΚDNA gh indg PCR 1%, 700 bp (F ig 1, A Fd, B ) Fd pcom B 3, XL 12B lue,,, Fab, cfu 212, 100 (T ab le 10, 1%, (F ig 2), 213, 80, H IV 21 gp 160, EL ISA T ab le 2 T ab le 3 Table 1 Selective enrichm ent of phage antibodies by rgp 160 panning Round of panning Input gcfu E luted gcfu Yield g% Table 2 Competitive inh ibition EL ISA of phage an tibodies Phage w ithout antibody gp160 A 490 Inh ibition w ith % gp160 clone 3B clone 1D V CSM Table 3 B inding of phage antibodies w ith 1F7 detem ined by EL ISA Phage antibody HRP2antiM 13 1F7 A E7 clone 3B clone 1D V CSM

5 3 :,, :, PCR, :,, H IV 21 rgp 160 1F7, 1F7 70% H IV 21,, References 1 Ko β h ler H, N ara P L,M uller S D ecep tive imp rinting in the imm uno response against H IV 21 Imm unology T od ay, 1994, 15 (10) : M uller S,W ang H T, Kaveri S V, Chattopadhay S, Ko β h ler H Generation and specifity of monoclonal anti2idio typ ic anti2 bodies against hum an H IV 2specific antibodies 1 C ro ss2reacting idio topes are exp ressed in subpopulations of H IV 2infect2 ed individuals J Imm unol, 1991, 147: Kang A S, Burton D R L erner R A Com binato rial imm unoglobulin libraries in phage Κ M ethod s: A Comp anion to M eth2 od s in E nzym ology, 1991, 2 (2) : Barbas g C F, L erner R A Com binato rial imm unoglobulin libraries on the surface of phage (phabs) : rap id selection of antigen2specific Fabs M ethod s: A Comp anion to M ethod s in E nzym ology, 1991, 2 (2) : M ethod s In E lectrop oration: B acterial2t ransf orm ation and P u ls Controller Instrum ent M anual: B io2rad L ab CA : H er2 culs,