CA2003316A1 - Hdl-binding proteins - Google Patents

Abstract

HDL-BINDING PROTEINS

Abstract of the Disclosure Substantially pure mammalian proteins having the following characteristics are disclosed: (a) molecular weight of approximately 110,000 Daltons; (b) bind HDL3; (c) bind apoA-I proteoliposomes and apoA-II protecliposomes; and (d) contain approximately 10% carbohydrate by weight.
The present invention also discloses a substantially pure protein having the following characteristics: (a) a molecular weight of approximately 38,000 Daltons; (b) binds HDL3; and (c) contains approximately 331 amino acid residues.
DNA sequences encoding these proteins, as well as cells transfected or transformed with an expression vector containing such a DNA sequence are also disclosed. Antibodies that bind to these proteins, methods for isolating an HDL3-binding protein and methods for detecting a genetic defect in an HDL-binding protein in a patient are also disclosed.

Description

11~15~89 17:~2 ~2~ ~82 ~031 S~d & BerrY ~ 7 2003~

HUI,-~IN~IN~ P~O'rEIN9 Tl~ p~ n~ lnv~n~lon r~lAte-~ to aQl~ular ~0 b~nding prot~n~, ~NA aequ~nc~e~ onc~o~in~ them, mathod~
~or th~l~ p~odu~t~on, an~ a~y E~y~ mploylng th~m.
MorQ ope~ifi~llv~ it rsl~ to bind~ng pro~n~ for high-densi~y l'popro~in~ t~ . ~s~ay ~y~ u~ul for l~n~lgy~ng high-densi~y l~pop~ot~ln snalo~;, and to ~he 25 ~n~log~ ~h~m~lves.
; ~
Ao~umulation of ~hol~t~rol in th~ va~aul~r endothellum i~ b~l iaved t~ b~ c~y ~v~n~ ln th~
O development o~ at~e~o~cle~ro~ or re~iew j o~ Ro~, ~ . M@d~ .88-97, lg36) . ~arly ln the developm~nt o~ Ath~roscleroti~ le~ion~, the artery walls ar~ ~ene~rated by ch~ t~rol-cont~lni~g: ath~rsgen~G
partial~ h~s~ p~r~lal~ a~ b~ ved t~ ~e d~ri~d 35 ~rom low~ n~ty lipopro~ein~ ) or ~o b@ mitlo~
~ubpo~ulation~ of ~terol-~ich lipopr~'cein~. They ~e ~ecogni~ed a3 ~ore~gn by ~ croph~ges ~ w~iah pick up the 11/15~ 17:/d3 ~2~B B82 6~31 S~ed ~ BerrY
2g~33~
~.

par~lcle~, in~ra~e th~ ~:holR~t~rol, and th~roby boaom~
~oam cells . ~ccumulatlon o~ ~o~m cello 1~ ~h~ ~lr~t ~ta~ of Yatty ~tre~c forma~ion. The~ ~atty st~e~k~
~hen ~nlarge th~ou~h th~ gr~du~ um~llati~n of lipid-S ~ontain~-n~ macroph~g~ and ~o~h mus~l~ c~ va~u-~lly developing ln~o i~ibrous pla~ lah in ~mooth r~u~cle cell~.
The s~x~Aat;uxe and aa~iv~ty o~ pl~ na lipo~
pr~tein~ hav~ b~on r~vl~w~d by ao~o ~ al. ~s~h~n 10 ~n~QLQ~ 3-41, ~ ). HlS~ en~l~y lipopr~t~in~
a~e a cla~s ~ lasm~ ~ lpoprot:~ain~ whlah aor~ t 0~ ~ole5terol t choles~e~yl ~ ter~, pho~pholipld~, lyceride~, and apollpc~prot~in~ (~rin~pally apo~I
and apoA-I~). q~o ~ain ~ubclo.~s o~ ~DL ~ I.2 an 15 HD~3 ) ~n~y b~ ~epar~d on th~ ba~i~ o~ d~n~i~y. ~ third subclas~, design~t~d }~DIIl or ~a~ cc>ntai~ apolipopro-t~in E, ~ut no~ apoA . ~ h~v~ n impl ~a~d ln th~
tran~port of aholQY~e~ol ~prin~pal~y ln th~ ~or~ o~
ohol~st~ryl es~r~) ~r~m p6~ri~h~aral t~ ssu~ to th~
~0 l~ar, wher~ th~ chole~t~r~ t~bol~z~d or ~xo~6lt~d (Glomset, ~idl~e~ g: l5~-~7, 19~ o~r ~ud1~
(~ram et al., ~Li~ ~,~ 21, 1983) h~0 shown th~t HDI~ car~ promo~ th~ aP~lux ~ ah~ erol fxom c~ltur~d ~all~, pr~a~umab~ y ~y bln~ing ~o those 25 ~lls ~nd, through a ~e~30nd ~ ng~r ~}3on~e, ~ti~
latlng ~h~ t~n~er ~ ~ n~c~llula~ ~hO~t~l AC~0$~
th~ cell m~mbr~ne. Th~ shol~t~rc~ hen ~bso~b~d by Hl)~ ox oth~ plasnla p~Ote~ nd i;ran~porte~ tç> th~
liver~ In addi~l~n, H~ hav~ b~en ~hown t~ }: ind ~o 30 st~roidogetlic tis~ue~ in r~t~ an~ ~r~ beli~Y~d to paxticlpat~ in the d~live~y a~ ::hole~terol to ~he~e t~ssues . ThU~, th~ir biolog~ c~l rol~ is compl~ac an~
app~a~s to 1 nvolvQ a ~r~r~ ~ty o~ ti~u~ e~i~ls ~f ~ct~ .
~ carry about on~-~hird o~ ~h~ tot~l s~r~ hola~t~3rol 35 in hum~n~.
HDL llav~ an ~hown to bind to exl:~hQ~atic non~eroi~og~nl~ cell~ in cultu~o ~th high a$~inity in ~i ~ ~

S/8~ 17:~4 ~06 682 6~31 Seed & BerrY Ql~9 ' 3~6 a ~atuxable mann~rr ~hi~ bin~ing i~ up-r~gul~t~d :by lo~din~ c~ wi'ch un~t~ri~ri~d ch~ t~rolr Although thl~ ~ugges~ that ~he ~IDr. lndu~ed ~lu~ o~ ahol~t~rol ~rorn aultured coll~ i~ m~diat~d th~ough bindin~ o~
5 l:o a ce~ ur~aae recept~, the ~xi~t~n~ of ~uah a reaeptor i~ dieputed ln tho lit~ nd cor~lloting dat~ hav~ b~n r~po~t~d . ~hon ~t al ~ ~l~h~
~,: 916~67, l~o) d~ onstrat~d that ra~ te~ hav~ a spec:lfia high-a~ini~y bindin~ ~lte rO~ t~DI. which i~ not 10 ~n~ltive ~o proteolytia ~nzymo~ ggo6lting th~t tha t~t~ and oth~r &t~roidog~nic ~ ues o~ ;~a~s obtain chol~te~ol ~rom HDL by a ~llulas~ proc~ no~ involv1ng r~c~pto~ pro~ein~. Gwynn~ and He~
~ 1087~-83, 19~0) ~ound ~ha~ rat ad~en~ ell~ poGses~
15 a sp~ci~ aturabl~ up~aX~ m~chanism ror HD~
~hol~sterol, bu~ ~he ~::ell birl~ln~ m~hanism o~ HDL ~a~
not ah2raot~rized. ~id~ ~t al. ~9 7 5~-65, 1g85 ) ~nd Fidg~ 2 65-~8, 1~6) i~olat~d 78,0G0 Dalton ~4h-binding protein~
ZO ~or~ ~nem~r~ne~ of ~veral cell typ~ Taba~ ~nd Tall s,~ s 13897~gO5, 1~4) ~tudied th~ ~8~
ciation of HDL3 w~th v~riou~ ~11 typQ~ ~n~ conclud~d that this as~oci~tlc)n i not, m~di~ by ~p~al~ gand and re~pt~ pro~ains, ~u~ in~olv~s lipid in~H~aa~:Lorls.
25 ~nael ~t al . (~ s l3l4n~ 8) tu~ied the e~ects o~ ~dlA~ on ~n~ativa~lon o~ HD~-blnding ~Lt~s on human ~lbroblast~ ~nd ~onclladed tnat ~hey were composed o~ p~ptide~ too ~mall ~ b~ ~onslder~d biolo~i~
~cep~ox~ .
3 ~ G~en ~he r~l~ o~ ~D~ in ~he remov~l o~
~hole~terol ~ro~ per~ p~ro.l aells, ~3J, ~ . agonists hav~ a potent~ a~ appllaa~n ln th~ tre~men'c o~
A~ero ~leros;ls~ Ind~d, ther~ ~ a n~ative ~orr~
tion between ~ v~l~ an~ tCh~ inc:~d~nce c~ cor~n~ry 3~ ar~ery dis~se. ~ goni6t~ ~ou~Ld ~ u~ed to ~tl~nul~e ~he tran~lo~a~ion o~ ~hole~ter~ on~ tra~ ular membr~nes to the cell &urf~c~e, wher~ 1~ would ba plc:kad _ , ........ .. .

. .

11~15/~ 17:~5 ~P2~6 682 ~031 Seed & BerrY ~10 ~2003~L6 d Up by ~ L or other txan~por~ protein ~ B) ~ ~hrough ~he u~e o~ ~n aBsay sys~em ~mploy~ng an R~ indln~ ~r~ln~
th~ a~nle.t~ a~n ~3~ idQnti~l~d. ~ tabl~ agonl~ts lncl~de an~ bodl~ ~o ~h~ H~-bind.in~ prot~ n and o~her 5 p~ot~ln and nonpro~n mol~ul~.
In a~dition, a ~onad D~ ~rA~ment ~ncoding an HDT~-bin~ing pre~ein aould ~ u~d ~o id~nti~y lndi~tidu-al~ ~ rl~k ~or d~veloping aths~o~c~ro~ hrough analysi~ a~ re~rl~tion fragment l~n~h ~lymorphi~
10 (~I.P~) or ~NA ~equ~n~, daic~nc~.~3s in H~L-binding p~ot~in~ aould ~e ldenti~lad. ~th~rmor~, monoclonal ~ntl~odies ao~ld bR U5ed t;o scr~an blood ~ 3 ~r ~h~
~b~nc~ o~ ~un~tiorlal HD~bindln~ pro1;~1n B~ie~ly ~t~ted, t.h~ ~r~3~n~ ~n~t~ntlon ,.
dl~clo~s ~ub3t~nt~ ally ~ur~ ma~allAn pr~teln~ haYing l;he ~oll~wing 4har~c~t~rl~1as~ ~a) molGculAr w~ht o~
approximately ~.O,OOo ~lton~ (b) ~lnd II~I~3; t) bin~
~0 ~po~~I proteolipos~m~ ~nd a~oA-I~ protooli~o~ome3t and ~d) cont~in approxlmAtoly l~$ o~r~ohy8ratR by w~i~h~.
~h~ proteins in a~rtain embo~imQn~3 ~A~y al~o b~ aharac~
t~rized as h~v~n~ ~n i~oelectric~ polr~ tw~n about 5 .
and 6.0, and may ~ d~r~ved ~om bovino o~ hum~n c~115.
~5 W~thin a relat~d a~p~t, the ~ nt in~nt~on dis¢lo3~3 arl i~olat~d ~NA ~ol~oul~ ~n~od~ng a p~o~eir~
h~lng ~h~ ~oll~wing ~haxaat~ ic~: ~ a~ c-llar we~ght o~ ~pproxir~at~ly 100, 000 D~lton exclusi~rQ o~
carbohy~rate; (b) blnds HD~3 and ~ inds ~poA I ~n~
3 O ApoA-II p~oteol ~po~om~3 . Wi~hin on~ di~n~nt, thR DNP, ~equenc~ erlc~ode~ th~ amin~ a~d sl~uenc~ ~own in F~ r~
3 ~rom glycin~, am~no aaid num~r 2 ~ tO glu'camio ac~d, ~mino acid num~sr l~O ~
Within ano~e~ ~bo~ nt, the ~NA G~quence 3 5 encode~ ~h~ amint) ~ ;:id ~ena~ 5h~n in ~igure~ 4 ~r~m m~thionine, am~no ac::id nutnb~r l, ~o g~ut~m~ acld, ~nino as;:id n~m~r 119~ ithlrl y~ ~no~h~r e~od~mel~, th~

`'. ~, ~

. ~:

11/15~ 17:~6 ~20~ ~8~ ~31 Seed ~ B~rry 2003~
.~

~N~ secauen~e ~ncod~ the amino ~c~d ~e~anae 3hown ln Fi~ur~ 5 ~r~m methionin~, ~mlno acid num~ar 1, to i~oleuc~n~, amino aaid numbf.~r 12~2.
Th~ pr~on~ v~ntion ~ di~los~ ~ethod~
~or ;1 ~olating ~n HD~3~binding pXo~in, ¢omprising olubilizing mamm~l.lan cell m~brana pro~ln~ t~
prod~ce an ~nrich~ r~otion; ~b) oX~ing ~h~ richQd ~rac~ion to an antlbody that birld~ ~o 8 prot~in ~hat blnd~ ~DI.3, to p~oduce ~ bo~nd rr3.ctlon and a~ ~mbourld lO ~a~tio~s ~c) r~coYe~ g ~h~ ~und ~raG~on: and td) ls~latin~ ~h~ ~DI-3 bindi~g p~o~in ~om thQ bound f~a~tion. ~h~ m~thod may ~l~o inc~ ho ~p ~ ad-ing the ~ lian C~118 wi~h ~hol~ ol p~:ic,r to the g~gp o~ 3~lubillzlng.
Xn anothar asp~ o~ t.he pr~n~ in~ren~i~n, an isola~ed D~a~ molecul~a ~n~:o~lng a pro~in hav~n~ t~e followin~ cha~ateri~ic~ is d~clo~e~ ) a mol~qul~r w~ ht ~X approximately 3B, 000 ~alton~ S (b) bind~ ~D~
and ~c) ~ontaln~ app~oxim~toly 331 ~mino a~id rosidu~
20 With~n on~ ~mbodim~nt, th~ prot~i~ oompr~ th~ amino seqll~na~ ~hown in Figure 1 f~om glycin~ ~mlno ~c~d 2, to glut~m~c aal~, a~ino a~id 331.
Wi~in a r~l~t~d ao~o~ of ~he proson~
inven~ion, a ~u~s~an~l~lly ~rs prote~n i~; d~lo~2d 2 5 h~ving t~ ~ollowirlg charaot~ a ~ oular ~eigh~ o~ approximat~ly 3~,000 ~alt~n~J (13) ~inds ~ 3;
and (a) c~r~tain~ appxoxima~ly 3~1 a~ino ~cid rt~s~du8~;.
Wi~hin ~rt~in ~bodi~n~nt~/ ~he prot~in re~ w~th poly~lon~1 ~n~i~era to ~ 110, 00~-D~lton }~D~3 ~aindlng 30 ~o~ein i~rom bovln~ aox~c endo~heli~
C~ ran~fect~d o~ ~r~n~ormed with a~
~xpre~aion ~rec~o~ ~om~ lng a ~omote~ operably linked to a DNA ~equ~nc~ ~nc:o~ing 2 pro~in a~ de~ib~d above ~r~ 50 di~clo~d.
~n addi~ion, ~nt~30~ tha~ hind to t~e protein~ d~s~ e~l ~bove are ~l~o di~clo~ed~

11/15~8g 17 ~7 ~ B82 G~31 Seed ~ BerrY _ _ 00~2 26~03~

In yet ~noth~r fl~p~Clt o~? the pre3~nt invention, mQthod~ ~o~ do~cting a g~n~ic de~a~ ;ln ~n HD~ ind~ng protoin in ~ pati~nt ~re di~c:lo~3d. The mathod~ ~fln~r~lly ::o~pris~: ~a) i~olating DN~ ~rom a 5 l?~tlQnt J tb) cl ~ving ~h-3 ~NP ~ith a r~t~lotion endo nu~lR~8~ to p~odu~e DN~ ~raç~ment~ ~ ~a) hybrldl~in~ ~h~
~NA ~r~ nts ~o a ONA m~leaul~ com~p~rinlng a portlon Or ~hs nucloo~id~ n~q o~ ~igu~ 1, th~ nucl~okl~e se~uonoe o~ Flgure 3, t~ nu~l~ot~qQ ~ ce o~ u~
10 4, the nual~otide ~;e;~uenc~ o~ F1~ 9, 5~ 0~ ~ompl~montary t4 a poxti~n OI on~ o~ th~s~ ~e~uenc~, whereln ~h~ 1: N~
molecule iB ~ lea~ a~out ~8 nu~l~8tlde~ i~ length5 and ~d) deteoting ~ p~t~rn ~ h~bridi~ti~n ~ the DNP~
~r~gment~ and th~ D~A molacul~, whar~in th~ p~tt~n i~ ~.
15 dia~no~ti~ ~or ~h~ pr~aence or ~senc~ o~ a gene~i~
~e~e~t ln ~n ~ bindlng pro~ain .
Th~ e a~d oth~r a~peat~ o~ th~4 p~e~en~
nvon~lon will become ~vident upon re~Qrf3no~ ~o ~he ~llowing de~ailed deEwrlp~ion and att~ch~d drawing~. These drawings are not to be construed as limiting the scope of the invention.
Figure 1 illust~te~ thR nucl ~o~ quence~
of the co~ing ~rand~ o$ ~wo ol)NA ~lc7ne~ en~oqlng ~DL-bir~ding prot~in, toge~h~r wi~h ~h~ ~n~rred ~mino 2~ ~cld ~RqUenC~ of on~ o~ the t:l4r~ Nu~ber~ ~ove the lines re~r t~ nu~leo~ Ide po~itlonæ~ ~un~ers b~low ~h~
;L lne~ re~e~ ~o amino a~id po~itlon~ .
~l~ure ~ illu~t~a~es th~ con~t~c ~on of~ the pl~mid Zem217.

3 0 Fig~lre 3 lllu~1:rat~ tka~ nu~leotid~ s~quen~
o~ a ~loned cDNA ~noo~ing ;~ po~tic7n o~ ~ h~an t~D~
blnding pro~ein, together w~th ~h~ d~dUC~d ~ino a~id se~uen~e. ¢~rt~in ~ iC~i4n æ~y~e git~ a~e ~own.
The tQrmin~l Eco RI ~lt~ a~e th~ re~ult o~ ~he ~lo~in~
: 35 pro~edure. Numb~r~ ~t the end~ o~ linos ~ 0 nucleo~l~e po5~tion~ Number~ ~lo~ the lin~ ~e~er to , ,,__. . .. .. .. . _. _ .. _ _ _ . ~ . .. . .....

11/15/89 17 ~8 ~B B82 B~31 Seed & Berry 1~ 3 2~

amino ~cld po~itior~. ,Th~ ar~ow india~ th~ poln~ oP
div~rgen~ ln th~ Eleclu-anaola o~ olon~ ~2a-1 and ~IEI. 13.
El~ur~ 4 illu~t~r~ tho nuolooti~ qu~n~
o~ ~ ~lon~cl DNA, de~i~nated H~ 13, ~3naoding a human 5 ~ inding p.ro~ n ~ogeth~r wi~h the dedu~d amno a~ld ~quenc~ . Carta~n re~r;l ~ n ~t~ ar~ ~hown. Number~
at th~ ~nd~ o~ the~ lln~ r~ o nucl~o~lda po3~ion.
N~mb~rj below ~h~ lin~ r~fe~r to ~mino ~d po~ition.
Flgure 5 illustrate~ the nualoot~de ~quanc~
10 oS a cloned DNA, ~ nated H~I. 13/8 ~pllc~, ~n~ding a human ~ blndiny ~ro~ in, ~og~h~ wlth the d~duc~d a~4lno aald ~quenc~ C~rt~in r~stric~lon ~ite~ are ~hown. Numbe~ a~ th~ en~ th~ lin~ reS~r ~o nucl~o~ld~ po~ition. Num~ers ~elow ~he line~ re~3r to 15 amlrlo aaid po~ition~
Figur~ G ~hows p~;rti~l r~t~lotion map~ o~
human H~L-~inding prot~in cDN~ clon~s. ~s~rlotior ~it~ ar~ d~ign~ted a~; RI, E~c~ RI ~ P~ P~t Bam HI; G, Bg1 IIS N, Nar IS A, APa IJ ~, H1nd II~7 V, 20 ~vU II~ S, S~t I7 X, Xma I- Th~ min~1 E~ it~5 ar~ th~ re~U1t O~ the O1Oni~ Pr~ Ur~.
F1gUXe 7 ~ Btr~te~ ~e ~n~ruatlon o~ ~he v~cto~ Z~m22~F~ and ZMB4. SylTbols u~ed are: DHFR, mouse dlhydrofol~t~ r~du~ e g~n~s SV40p, S~140 p~o~t~5 ~5 sv40~, sv4~ te~lna~; MT~l, mou~e met~ iorlein pr~mo~er; MII~, adeno~irus 2 ~or l~ke p~omo~e~5 and S~;, ~pli~ng ~i~n~

3~ Pri~ to ~et~in~ for~h the lnYen~e:ion~ {~ m~y l~e h~lprul to an und~rs~anding ther~o~ to sot forth de~initions o~ certain i:~3~15 ~Q b~:u~e~ ~r~ln.
~ t~ A DNA molecule or s~quenc~ which h~s ~en en~ymatlo~lly ~yn~h2~ized fr~m 35 the se~u~nc~s present in ~n m~NA ~ernplat~, or a clone o~
such a molecule.

........... _ ,.... ~ ......... ;

11/15/~ 17 ~9 2~2~)6 6~2 6~31 S~ed ~ ~errY 1~1~14 3~6 Ex~e~ion Y~ A ONA molec:u~ or a clon~3 o~ ~u~h a molecul~ whlch ha~ b~en mo~l~led th~u~h human ~s~terv~ntlon ~o con~in ~egments o~ D~A whl~h ~r~
~m~in~d and ~uxtapo~d in a mann~r whiah ~ould not 5 o~ho~ e ~x1~t in n~t.~re and which aontAin~ at l~a~t on~ D~A ~equanc~ to be exp~ d in ~ ho~t aall and s~quence~ whlch :~aailitat~a f3uah expr~iont ln~ludin~ a t~anscription p~omot~r and t~minator. Repli~At~orl ~
an e7cpre~sion v~:at~r in a ho~ oell ~o~nonly oacs~rs 10 autonomously, du~ to ~he prQsence ~ a repll~a~ion origin on ~he veator, or may occur ~y in~g~ on into ~h~ ho~t ahromo~ome.
The p~ent ln~ten~ion pro~rides HR~-binding prot~ins and methods o~ m~Xln~ thase prot~ins, a~ well 15 as u~e~ul polypeptide ~ragment~ o~ thelse ~rot~ ~s . Th~
inding ~ro~ein~ and p~lype~t~es ~re i~olElted ~rom cell~ which naturally produce ~h~m, o~, pr~er~bly, ~re ~nade ~y eXpre~ing cl~n~d ~ ~quenaes ~n recoml~in~nt ~ell~. ~h~ ~nven1:lc)n provid~ t~e3~ protein~ ~n 20 substan~ially p~re ~orm, that i~, ~t lea~ ~Q~6 ~ur~, and in one embodimen~, free of other pro~aine o~ ma~nalian origin. lrh~ L-~indin~ prot~ and polyp~p~de~ ~hu~
prod~ ad are ~s~ul 1YI te~t ~y~tem~ ~or ~c:resn~n~ poten-tlal HD~ analogs, $ncludlng a~oni~ts and ~nt~goni3tæ O:e 25 H~h~ In anothex embodimen~ o~ the pre3~nt inVen~ion~
th~ binding pro~eirss and polyp~p~iào ~r~m~n~ ~hereo~
ar~ u~ed as an~1 g~ns ~o generat~3 ~n~b~di~e~ whic:h may be u~e~ D~ analog~ analog~, in~ludin~ ~ti1~ot~ e~, other pro~ine ~ peptide~ an~ nonpr~t~in c~ompound~ y 3 0 ~e u~ed thex~peutl¢;~lly in th~ ea~men~ hy~erc:hol~-~ero~emia snd atherosc~ ~rosls, HDI. a~ g~ may ~1BO be U~d ~o ~reen pa~ent~ ~o~ da~e~ or defi~i~ncies in HDI.-bin~llng protein~ and. t~ I. rec~pkor pathway.
Th~ pre ~nt invsn~lor~ al~ p~ovid~s ~NA
35 sequence~ enco~lng H~L-b;indin~ pro~ein~. The3~
sequen~:e~ are u~ ul in producing ~e HD~ binding prot~ns as not~d a~ov~, and aXe ~lso uY~ul in the '~.

11/15/g~ 17 1~ ~2~6 ~82 ~31 S~J & Berry ~ 15 26~0~31~i diagnosls o~ ~n~tla d~fa~s in ~L-b~ndln~ prot~in produ~tion, s~ch d~ t~, whlOh m~y ~ ocl~d with a hi~h risk ~or ~h~ de~elopm~nk o~ atheroocl~ro~i~, may ~a d~tect~d by using tho ~NA ~que~aes ae probe~ ~or the pr~ence ~f r~tr~c~ion ~ragm~nt length polymorphi~ms ~F~Ps). Thn~e polymorphi~m0 ar~ th~ r~sult o~ ~hange~
ln DNA ~e~uehae~ and c~n b~ detoct~d as di~erena~ in the 8~ZRS of ~ra~m~nt~ gen~ra~d ~fter d~ge~ion of D~A
wit~ re~t~i~tion ~ndonucl~ase~. ThU~ a c~NA ~ncoding an HDL-bin~in~ pro~cin or a ~ragmen~ o~ ~uch a c~NA aan u~e~ to probe ~or RFLP~ in dige~t~ o~ genomic DNA
i~ola~ed ~ro~n, for ex~lnpl~, bloo~ l~uXocyteu. ~:~ner-ally, ~rob~ w~ll be at least a~ou~ 14, pr~fe~ably at 16~Ast 18, ~lUC180~ n length. Hulnan ~ub~eat~ wi'ch 15 variou~ ~orm~ o~ hyp~r- arld hypo-lipideml~ are ~ar~ened to iden~i~y particular }~LP~ ~hat are dia~n~tio ~or sorders in lipoprot~in ~e~aboli~m An~ predi~p~sition ~o ~th~ro~ol~roslR . ~F~,Ps ahow Mend~l lAn lnheritano~
pa~ternF~ and ~an be u~ed t~ study the segr~g~iorl o~
2 0 genes: ln :Ea~nily E;tudi~s . ~ethods ~or carrying out ~FI,P
analyses are generally }~own in th~3 art and are desoribed, ~or ~xample, by ~3 et al. (~_9~t.
76:10~0-g5, 1~5). Al~c~rn~ively, the ~NA ~an~s provlded by the pre~nt inven~on ca~ be us~d a~
25 ~pecl~i~ probes or ~rime~s in ~thl3r co~lY~ntional sc:rsening ~skem~.
~ rie~ly, H~ bin~ing pro~ins ~re i~olat~d î~om mammalian cell ~y ~o~iliz tion o~ membr~n~
Pollowed by a combination of ~nvention~l ~olumn 2nd 30 sla}: ~el chr~mato~ap~y techni~uQs, ln~ ding ar~l nity chroma~ogr~phy ~Qing H~L apoprote~ ovalently }30und to aL sui~al~le in~c:lu~ ma~rix (e.g~, Sepha~o~e, Pharma~ia, Pisc~ataway, ~ he ~ ln~ns~ p~cot~ns m~y also b~
purl~ied by immunoa~f~ni~ ~hronl~to~r~phy ~ing an~i-3 5 bodi~ ~r iln~isera px~par~d a~ ~eE;cr~ ~ed ~ereina~te~.Monoclonal anti~odle~ ar~ parti~:ul~rly ~r~ferr~.
~r~ red ~ourc:~s o~ ~L~binding pxo~eins Ln~lud~ ao~t ~ c ,. .: '' ''''` ~ ' ' , : ~

1~1 01 11/15/~ 17 11 ~2~B 6f~ 31 See~ & Berry ~033~6 ~o endc)thelial ~ells and fibroblas~3. c~ells are pre~erably lo~ded wlth c}~ol~trol by incubdtion in c2l01e3t~rol~ or I:DL-con~ainin~ dium prior ~o protq~in i~olatiorl.
~lt~rnati~e~ly, ~h~3 ~n~ indin~ protQirl~ Ihay be 5 p~odu~ed by expres~ing olon~d DNA ~eS~Qnce~s ln reco~nbi-nant cell~ pr~arrecl ~o u~ loned ~DNA, because ~he u~e o~ intron~cont~ining gerl~mia ~NA o~n result in ~h~rant expr~s~ n, particul~rly in ~ roorgan~ s~ oe~t cells~ Suit~ DNA ~qu~n~3s m~y ~ o be ~yn~h~izad 10 aacording tc) con~ention~l m~thod~ (t3Q~, ~0~ exampl~, Caruth~s et al~, U.S. Patent No. 4,458,06~ Ita~cura, in ~ ~, E1B~ r Bio~hemi~al P~e~ 82) ba~ed on th~ amine aaid ~e~uence 4~ an i~ola~ed ~DL-bin~ln~ protein, Two ~uch amlno ~cid sequRnce~ ar~ disclosed her~in.
Im~unog~nlc polypept~d~ Pr~an~ may b~
prep~red ~rom H~L-bin~lng protein~ ~y ~ ion ~ith proteolytic enzym~s or ~her ~g~n~ .g~, ~yan~æn bromide~. Alternatively, p~p~id~s m~y ~ pr~du~ed ~y ~o ex~res~ion o~ ~loned ~NA ~eqUQna~ or by ~tandard ch~mical syn~he~i~ t~chnique3.
For expressl4n ln ~ecombinant a~ , a DNA
~q~enae encoding ~n H~ inding prot~in 1~ a~ted into a ~ull;able ~xpre~aien v~ator, whia~ ih ~urn i~
25 in~e~t:ed ~ n~o appropria~e ho~ he m~thod o~
~nsertion Will depen~ upon ~h~ p~t~culax }~oE~I: c~
~hos~ll. M~thods fo~ tr~n~ect~ng 2n~mrnal1an c~alls ~nd tran~orming bacteria ancl ~ungi wit:h ol~d DN~ ~e well known in the art. ~ t~ble eXpxqa~io~ ve~t~
3~ co~p~ise ~ ~romo~e~F whic~h i~ ~ap~ble of dir~c~iny the t~ansa~ipt~.on o~ ~ cloned ~NA ~quon~3 in a ~30~ cell and ~ runational ~r~ns~;:ription termination ~taO In ~m~ in~t~nces, i~ i~ pr~er~d t~at ~xpr~ ion ve~tor~
~urth6~r co~nprise an ~rigin ~ reF)li~ion, ~uenaes 35 ~hi~h regula~e ~d/o~ ~nh~nc~ ~3xpre~3sion l~vels, and one or more sel~c~ble n~ark~r3, ~ep~n~ing on th~ ho~t c~ll selected. Suitable ~xpr~ ion v~tor~ y b~ d~riv~d ' , 11/15/~g 17 12 ~B 682 6031 .',e~d ~ Berry ~17 2~ 3~6 ~rom plA~mi~, RNA and DNA vl~u~, or c~311ular DNA
sequence3, or may contain ~l~m~n~ o~ 6~ac~h.
Pr~err~d prokaryotlo ho~t~ ~or u~e in ~arryiny out the present lnv~n~lon ~r~ ~train~ o~ the 5 bActeria ~Dh~1~ S~ , althou~h ~L~I~ an~ other gonera are also w~ul. ~hn~ or tran~ortnln~
the~e ho~t~ and for eXpr~Sing ~or~iyn PNA a~quenc-3~
aloned in thelP ~ro wel 1 ~cnown in th~ ar~ ( s~e, ~or 2xample, Maniatis et al. (ad~. ~, ~1 10 l ~o~at~rv ~n~ , Cold Spring Har):~or ~a~ora~or~ Cold Spri~g ~ar~or, N.Y., 1~2). tra~tor~ ~ad ror e~xpre~ing ~eign ~NA in ba¢terial hos~ wlll g~n~rally contaln a ~eleo~abl~ m~rke~, ~u~h ~a a ~eno ~or ~n~i10lo~1~ ra~
tance, a~d a promote~ whlch fun~tlon~ ~n th~ ho~t cell~.
15 Appr~pria~ prc~mo~er~ include ~ ~ tNiahol~ and Yano~cy, ~q~ ~ , 155~ 3) ~
(~asadab~n e~ al ~, J BAQ~ 71~0 , 1980~, ~AC
(Ru~all et al ,, Ç~ ~Q : 2~1~4~ , 198~ n~ ph~e pr4mot~r~. Pla~mid~ usef~l for trAn~f~r~ning baateria ~0 include pBR~22 ~Boli~ar et al., ~a a:~5-113, 1~77) ~
the ptJC pl~sm~d ~M~s1n~, M~h~in ~ SLY LQ~ ~~
q7, 1~83~ and V~eixa 2nd ~ sing, ~ 25~-~8, 1~3~), p~Q~t2 (~ue~ lo , 1983 ), ~nd der~va~ives t~reo~.
Eukaryot;i~ roorge~ ms, s~:h ~ ~he yeast 5Z~Ç~!i~ or ~ilam~n~ou~ fungl, ~nclud~ng Asp~gillu~, m~y Alsc7 be used ~s ho~t a~lls. Partic~-larly prefe~ed ~pecie i; of A~per~illus include 30 Teohnlqu~ r trarls~orming y~a~st ~r~ d~aribe~ Py Begg~
~ 27~:~U4-0~, ls7~) and Hlnn~n e~ al. ~Q~
Ag~S~ 1740~ 84). Expres~lon v~t~r~
~or u~e in yea6t inclllde Y~p7 ~Btruhl ~t al~, 1 ~ Z~,S 103~-3~ 7~), YEpl3 (Broa~h 35 ~t ~1~, ~ 8 s 121-33 J lg7~), p3~B2~ at~d pJDB21g ~B~gg~, ibid~ ), and ~riva~iv~ t~n~reof~ Slloh vec~ors w~ 11 ge~nerally compris~ a ~el~ a~le Darker, su~h as ~he 11/15/8~ 17 14 ~06 682 ~031 _ Se~d & B~rrY
~01~33~6 ~3erectlve ~le~table Inark~r E~ X~wJ~ ki nnd hell EP 171,14~ MUrray ~t dl~,~, U.S. P~tant ~0.4,7~,O7~), or a mltritlonal mark~r, ~uoh a~ the I,~ ~ he, P~a~erred pr~motar~ ~or u~ ln yda3t eXp~eO~Oh v~c~or~ ln~lude 5 ~prolnoter~ ~rom y~ col~lo S~on~a ~tltoanlAn e~
ao7~-~o~ l9~0J Albar ~nd K~waoaki, ;L~L~o~ 41~-34, l~na~ X~w~aki, U S
pa~eht No. 4,59~,311~ o~ als~ohol d~hyqro~ana~pe gen~s ~Y~uny ~ al ., ln Jiollaolld~ ot al . (n~. ~, E~l~
lo ~ ~ ~, Now Yorlt: Ploh~4nl, lg~, p, 33~ ~nd ~m~Qr, ~y~ ~U.sl92-~ol, ~3~ a~ilit4t~ pllrl~ica~lon ol~ r~ign prDtoin ~roduc~d in A ye~t trans~ormant ~nd to obt~ln, ~hon neoa~a~a~y, prop~r di~ul~ orl~ ~or~natlon, ~ signal 15 ~e~u~nao ~rom a y~A~t gene enooding ~ ~oa~st~d protoin i~ ~olnetl in thQ propo~ reading rr~ o tho ~eqUan~Q
on~odlng tl~e ror~n prot~$ll. Par~ ula~ prer~rr~d oi~n~l ~e~eh~s ihqlude tho~3 onoocling ~h6~ pr~-pro re~ n oP ~he ~E~l ge~n~ ~Xur~n ~n~ Horoxowltz~ SQ;~.
~0~933-q~, lsaa; slngh~ EP 123,54~t and Xurl~n a~ al., U,~, Patorlt No. 4~s4~oa2) Ahd th~ ~oor~ry p~ptld~
portlon o~ tho Oh~ ~MaoRay et al., U. ~ . P~!ltelht llo ~ ,572~ ~aoKay, EP 2~0~68Dt ~aaK~y ~t Rl., U.S
P~ten~ ~pplla~tion 07/Z70,93~.
~rah~orm~d coll~ ~r~ a~l~urad ln appropriate ~edla contalnlng oar~on ~nd ni~rog~h ~o~o~ 3 w~ll a~
oth~r nu~ nt~ or s~lea~lve a~entd whl~h ~ay b~
r~guit~ by th~ partlaul~r ~train. y~s~ ho~t a~
tran~or~d ~i~h pla~ld~ aontaihing th~ 1 o~lea~able marker m~y be ~41tured i~ ao~pl~x ~qi~ aOh~ining gl4coo~ a~ a ~a~on ~oura~. ~
H~gher ~uX~ryotlq aell~ ~y alr~o s~r~ a~ ~or~t qell~ ln oar~ying out the preseht ~hve~ion.~ ~ultu~
mAmr~ an ~lla, sUah A~3 th~ ~X, CJI~, Ns-l~ sp~/o~ H~60 an~ J658L o~ ine~, ~re prè~e~r~d. Th~se an~ othe~`
~itabl~ 0911 lIne~ a~e ~v~ ro~ a ~ar~ety of aou~a~ u~h a~ tha Am~3rla~n ~ypq Cu~ 3 c~l~ w ~lon, a : .

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11/15~89 17 14 ~2~6 ~8~ B~31 Se~d ~ Berry E~
~ 33~6 ~3 ~oc:kvlll~, Md. ~hH ~k~ K a~ll lln~ tk-t~ Wa~ t~ar ~nd Ba~rg~, prQ~ . _Natl . A~ad . S~ 7~ ~1106-lO, 19~2) i~ ~ pa~xtla~ rly Fr~r~rr~d. ndhe~nt ~ell line.
Expr~lon v~a~or~ r~r ~t~ m~mn~ian c~ wlll 5 ~omlprl~ a promot~r a~p~bl~ OL~ dlr~a~ln~ th~ tran5arlp-tlon oP a oloned gen~ or cD~tA. Pu~icUlarly pr~er~d promot~r~ inalud~ ths SV40 promotor ~ubram~ni Gt ~
~5Ql,5~1~ ;L: 354-~4 ~ 19~), thO m~tnllothlon~in qqn~ -l) promot6~ tPal~nit~r ~t al. . ~QlQn~a iL~L~8o9 10 14~ 1933J Palt~lt61r ~t nl.~ U.S. P~tl~nt No. 4,57~,~21)~
the 1llDU5El ketpp~t ~ens p;ro~otsr ~o~an o N~:l. ~c~d. ~c~ U~ ~,:70~1-4~, 1984), and t~.te ad~noviru~ ta~or lat~ promot~r ~B~rkn~r ~tn~ Aharp, ~, ~ a4l~57, lga5~ 0 aDntain~d is~ tha 15 oxpr~ts~iort v~or~t io a tran~a~pti~n te~ntlnator, l~oat~d down~ aT~ Or ~ho lnoe~tio~ ~lt~ ~or tho 1: NA
~t~qu~no~ to b~ exp~o~d ~ A p~ore~r~d tqrltt~ nator i~ the hu~ttan growth ho~non~a ~hGH~ ~ene t~r~lna~or ~D~Noto ~t al., ~u~ Aq~a~ 1~ 9~3719-30~ 19el) . Sn ~ddition, 2D veotor~ will pr~:erably ont~in ~nh~na~r ~equenaa~
approp~iat~ to th~ particular ho~t c~ll lin~.
For ~xpras~tlon in ~ultured mnmn~llan aE~ t, expra~olon v~to~ aon~aining clonRd ~ quanc~ ~r0 int~odua~d into t~ cRlls by ~ppro~riate t~ns~c~ion 25 t~chnlque~ ~Uoh as ~al~Lum phoelpha~o~ t~ t~ s~-tl3n l~or ex~ plo, a~ m an~ n dcr ~b~ ~y ~s456-67, 1~73, a~ modi~led by Wigl~ ~t ~
,, ~, BCi,~ 3~67-70, 1~8~) or ~l~atropora-tion ~M~umann e~ al. ~Q~. 1:84L-45~ 1932). ;5a~ ~
30 typical tr~n~ e~tic~ a D~lA-oal~iu~ phC~ph~t~ p~oalpi~
~a~ lz i~or~ and ~hen appl i~d ~¢o the 0~ 9 ln ~h~
pre~no~ o~ mAdluEn contalning ahloroquin~ ~100 ,~1). The ro lrlcub~te~ 3eor g~r hour~ ~rith t~ ~ro~ipi~te, rollow~d l~y a ~wo~inute, l5% gly~e~ol ~ioolc, ~ por~i~n 35 o~ tho 01~113 ~ake ~p t;he ~N~ ~n~ nt:~ln it in~ the c~ or sev~ral day~ m~ll r~a~ion Or ~h~ ~ell~
lnt~qrat~ th~ U~A lnt~ ~he g~no=~ ma~7~t~in 'c~ NP. in ~,. :
`, ' ~ , ~

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il~l5/8~ 17 15 ~2~6 682 6~31 Seed ~ Berr~ !~02~
~33~

~4 nonshromo~o~ ucl~Ar ~truo~ur~- The~e tr~n~otonts can ba 1~nti~1o~ by cotran~oction wlth ~ ~ane that confor~ a ~alectable phonotype ~a ~Hl~a~ablo mark~r).
Pro~orrod ~loot~lo marker~ inalud~ tho DHF~ ~eno, 5 whlqh l~np~rt~ oollul~r r~a~tano~ to h~othotrexa~ tMTX~
An lnhibitor o~ nucloo~ yntha31~1 or thn b~at~riAl nuo~oin r~ noc ~no, ~hlOh o~nr~ro rooi~kanco ~o ~ho dru~ a~4~ n i~hlbitor o~ prot~in ~yntho~
A~tqr the hoet ooll~ h~v~ ta~en up th~ ~NA, dru~ ~el~a-tion i~ appll~d to ~fll~ct ~ar a pop~lation o~ c~ that are expre~lng 'che ~ o~ablc ma~X~r ~ 3ve~18 hi~h ~nough to ~on~r r~31~t~noe.
Trnn~ t~d mammall~n oelle are ~ùl~u~ed ~n ~orum Qontainlng or ~ru~-~re~ m~di~ ~on~lning appro-p~latn nutri~nt and antibiotlG s~ppl~nts. sult~bl~mod~a ara ~vail~la ~ro~ aomm~ralal ~ppl~er6 or m~y b~
prepare~ aqoo~dln~ te publi~hed ~oai~D (50H, catalo~ o~ th~ Am~rican ~yp~ ~ul~ur~ ~oll~otion).
Coampllfl~atlon can b~ u~d to inar~a~a ~0 nXP~ iQn 10Y~1~ ln eell5 tran~oot~d wi~h thn ~IF~
n~ar~er. ~lgh ~ona~n~tlon~ 0~ M~X are add~d t~ the ~ultur~ medium at the ti~n Or lnltl~l ~olQc~lon~ ~ m~y ~e added by s~uenti~lly inGr~a~in~ th~ ~onc~ntration of M$X in ~he mQdlum, ~ollowe~ by ~apeated olonlny by dilu tian o~ th~ d~ug-r~ tan~ ~ell lln~ a~te~ ~aah ampli-floatlon ~tep. ~ wh~ah expr~3~ th~ PHPR m~ke~
~hen 3elected and ~¢reened ~or pro~u~tion o~ tha ~L-binding pro~oLn.
~r~ening o~ n~Pa~ted oells may b~ don~ ~y the liyand blott~ng teahniq~4 ~G~h~ ~nd Ora~ ~. Biol.
Çh~m~ 7439-42, l98~ ia~ly~ o~ atlons or a~lture mèdia ar~ ~p~q~d ~y ~ rophor~ on polya~rylam~da ~lab gel~. ~ho ~ r~tod pro~ln~ are thRn tran~es~ed to nl~ro~e~lulo~e ~y ~lectrop~or~
~he nitroaellu1o~o blo~ ockad ts prev~nt non~peol~1~ blndlng, then in~ubat~ ~it~ ~h~ la~ d lig~n~ o~ Ln~ere~t (~.g., HDL~. Alc~rnativ~ly, ~

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llr15/~9 17 16 ~p206 632 6031 Seed ~ BerrY ~ ~21 ~33~

~y be &a~e~ned by a ~unct.ion~ ~s~y ~ha~ mes~ure~
~lux o~ la~ d chol~t~rol ~ro~ o~ in the pr0~na~
o~ ~D~. ~rl~ly, tr~n~eot~d o~lla a~ lncubat~ in ot~cl-rloh m~dl~m to le~ o~ with ~holo~rol, an~
intrAohllul~ pool~ o~ uxoes~ ohnlentorol ar~ radio-l~eled with ~ ~ynthutla proaur~or ~o.g., 3H-mev~lono-la~tone). Hor,-mod~tod t~n~loca~ion ot intraaoll~l~r 3H-~hol~s~rol ~o tho a~ u~o~ ~nd l~to t~ m~lum i~ ln~iaatlv~ o~ HDL roaeptor ~tl~ity ~lu~e e~
~ ol~ Çh~m. æ~ ago4-so7~ 1~87).
~ not~d abov~, ~ntlbodie~ to HDL-binding protein~ ~ay b~ u~ed ~ gonist~ ~nd a~tagonl~ts and rO~ tn~ ldRn~ifl~lon and purifl~a~lon o~ ~$-~indlng p~at~in~, Nonoolonal an~i~odi~ Ar# ~n~ally pre~err~d 15 ~or th~s~ purposo~ . HD~-bindlng p~o~ ar r~ n~
th~reo~ A~o ua~d to make mo~oclon~l ~ntlbodle~ acco~lng to m~thod~ g~er~lly d~s~ribqd ~n tho ll~e~ature.
P~ein ~r~gment~ u8~ul ln lmmunlz~tlon wlll ~ o~
su~lalent ~iZ~ ~ be immunogenlo, pr~orA~ly at le~t ~lx ~ino aold~ in length, moro p~ ra~ly about fi~t~on Amino aoido ln length. Pre~e~od ~ub~ct ~nim~l~ ar6!
~ic~ o~ rat~, with ~A~B~4 mlca bqi~ ularly pro~rrod~ ~h~ appropriAt~ anlm~l~ aro lmmuniz~d wl~h A
pr~paratlon o~ the HD~-blnding pr~ln, pr~r~bly a pu~ or p~rtially pu~ prepA~lon. ~r~rab~y, th~
anlmale a~o lmmunlz~d wlth ae lea~t 140 n~ ~oh o~ ~he prot~in pr~pa~a~lon, mo~t p~er~bly gre~tor than 500 n~
e~oh. For immuniz~tien, th~ teln 1~ pre~era~ly adsorb~d to a ~olld-phasa ~trlx, p~e~ably to nitro-o~llulo~e pape~. The matrl~ h~ lnt~odua~d lnto ~hoa~mal. ~ahni~ue# ~or $ntroduatlon ~ t~ ~djo~4ed prot~in include lmpl~ntatihn ~U.8. ~Aten~ No. 4,6s~2~0 or ~ billzation ~r the ~olid ph~ a,nd lnjeckion o~
~h~ ~olubiliz~d m~ ial ~Xnuda~n, An~l" Ri~dh~.
3$ ~:z:2as ~8~ l9a~ h~ a~lld-phJ~e m~trlx may be ~olu-bill~ed in An appropria~ organiG ffolv~n~ ~o.~., hM5s) and sither ~lxed with ad~u~nt or ~lln~, or in~eoted ' ~22 11/15/8~ 17:17 ~2~6 G~2 ~31 ______See~ ~ BerrY
20~33~6 lt ~lroctly. Altcrnatlv~ly, ~e protein m~y be ln~eatad ln the d~na~ o~ ~ soli~ matrix and/or a~uvah~. ~n~nc tlon or implanta~ion may b~ lntrAporlton~Dl, ~ub~u~-noou~, inkramu~cul~r or lntrn~cno-~, pro~ra~ly 5 lntr~p0~itono~1. Th~ anl~ln may al~o b0 ~n~oat~d wlth ~uv~nt, ~uo~ ~s ~reund'~ ~diuvan~. 31ng.lo or m~ltipl~
boont~ i~nuniz~tionD ar~ U3~ a~an ono And 4~v~n d~y~ prlo~ to kh~ ~u~ion d~to, pro~rAbly on day~ on~
bhrough rour, lntraveno~ in~ctlonH o~ th~ HDL-blnding 1~ prot~ln may b~ ql~n dally. W~9~ u~lng ~m~ll p~ptld~
a~ l~nmunogen~ it 1~ Pre~or~ed to qon~u~a~o th~ p~ptide~
to ~ hapten, ~u~h a~ koyhol~ limpat h~mooyanln~ Mathod~
~r ao~u~atlon a~a well ~no~n in tho art.
Betwa~n Oh~ ~hd ~o~n d~y~, pr~rer~bly ~aur d~y~, a~a~ the adminl~tratlQn a~ the flnal b~o~t~r ~ nlz~tion, ~pl~Qns or po~tion~ th~oo~ ara ha~Y~t~d rrOm ~ha lmmunlzod ~nlm~l~. A~ thl~ tim~, th~ lymph nad~0 ~y al~a be haxva~t~d ~nd ino~udod in th~ o~ll ~rap~tion. ~ho harves~d org~no ~3 mlnc~d u~in~
tdc~ni~uo~ whicb di~up~ the s~ructuro o~ tb~ or~n but ~lch ~r~ not dot~lm~ntal ~o the lymphocy~a~. ~h~
o~an~ Are p~o~ably minaed ~ th B~isdo~s ~ pr~s~ed through A me~h ~roun, and ~lx~d ~ith growth m~ql~m te enxi~h thU pr~paration ~or lymphoayt~s~ ThR ~inc~d ~nd ~train~d tia~ue ls h~rves~d by ~an~rl~u~ation, th~n mix~d ~ith ~ro~th ~qdlu~ to ~o~m a c~Il su~pen~lon. ~he red bloo~ a~ may ~e ly~d ~y ~d~lng ~ ~ypo~nlo or hyp~rtanl~ ~olu~lon to tb~ uq~n~lon. ~ pref~r~ed m~thad ~er aell ly~ 5 to fl~d di~llRd ~ter to tho ~usp~n~ion~ ~nd quiaXly return thQ ~u ~nuio~ to an 190toni~ ~tat~ ~ith ~odiu~ ahlorid~. Any rem~lnlng:
ti~u~ may b~ r~oYed by ~lt~tloa through ~u2e.
~ ha harv~t~ oell s~pe~slQn ls th~n mix~d wlt~ ~ my~loma ~ell; line, pr~ra~ly ~n~ ~hiuh Is ~yngenela wit~ tha immunl2~d anl~ y~lo~ a~ll lln ~o~ vn~iouo spe~le~ a~ wid~ly ~v&ila~l~ throu~h, ~or ~xampl~, Ame~ n Type Culture C~ t~on, Rc~kvll1e, , . .........
_........... . .... ~

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11/15/8~ 17 18 ~2~6 ff82 6~31 Seed ~ ~err~ 23 33~

Mcl. Mya~om~ o~ll lin~6 oommonly ut~od inalude Po563Ag8 (A~CC TIB 9), 8Pa/O-A~14 ~A~C~ C~ 15Ql)~ PO (ATCC CR~
~646), ~nd 2l0-RCY-A~l ~Galrre at ~l., ~ ~,1 ~31, 1~7i). A pr~err~ o~ll llne i~ p3~91~ g4 1 ~A~CC
5 ~rI:~ la), hR~RlnA~er r~rretl to l~a N~-l. 'rhe N~-~~Qll~ ~r~ pra~rably tont-d to dt~tormlno the olonln~
e~lo~onoy o~ ~ho ~tr~in. ~hi~ may te don~ by olonlng ~u~ the N9-1 ~t~ln by llmltLng dll~tion and carrying out tu~t fu~lon~ wi~h the lndlviduAl N8-1 olones to ~lnd 1~ cAndid~te~ wlth tho hlyhest ~usion ~ n41~- The myolomn cell~ ~re oultur~ in An ~pprcprlate mAmmallAn c~ ow~h m~dlum~ A vArloty 0~ w~oh ~r~ y~nar~
known ln the urt nn~ .avnil~bl~ ~rom oomnor~ urc~.
Mammall~n ~ell llnos are routlnoly ~rown b~tw~en 36 c and 40'~ und~r aondltlon~ wb~ch maintaln ~n optlmal p~
b~w~en 6.0 nnd ~.0, pre~erably a~out p~ 7.Z. ~he pH o~
the cul~u~e m~y be main~lnud throu~h ~h~ ~8~ o~ ~
~arlety Or bur~0~ ~ys~m~ known ln ~ha ~rt. A pr~ d bu~r oy~t~m lnvolvu~ ~rowln~ th~ ~lls ln ~ bl~a~bon-ate bu~ ln A humldi~l~d lno~ba~or ~ont~inlng ~2pre~rably about 74 C02, Th~ ~u~ion bs~w~n ~h3 lymphO~yt~ rrO~ t~
lmmunlzed ~nlm~l ~hd tho myeloma oell~ mAy b~ carrled out by a varioty o~ m2thod~ de~ri~o~ in the lltq~tu~e.
The~e methods includ~ th~ us~ o~ ~oly~hylens gly~ol Ea) (~rown ~t ~1., JL~ L_ a~ 9ao~83, 1980) and ~l~otro~u~lon (Zlmmerman nnd Vl~X~n, L~ UOhL-~Q1L *7~ Zt an ale~tro~u~lon g~n~ o~
mh~r¢lal~y avail~ rom B~ot~chnologles ~n~
3~ Exp~rlmen~al R~earoh, ~no" ~n ~le~o, C~lir.~.
Followlng th~ fu~ion, th~ o~ are platedl~o multi-well aulture pla~, pra~erA~ly ~6-Well . A reagent which ~el~o~lvely ~llows ~e~ th~
~growth o~ the ~u~d myoloma ~ell~ ov~r th~ un~U~e~ oell~
-:3s i~ a~e~ to th~ cultux~ mod~um. ~ pr~rr~d ~el~tlon t~hnlg~e u~e~ HA~ ~hypoxanthin-, a~inop~erin, , ~ I
', ' ~`~' ' .~ ~ . ~. .
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ll/15r8g 17 19 ~20~ 6~2 ~)31 Seed ~ BerrY ~ 024 ;~0~33~
1~ , thymldln~) ~el~c~lon. Othe~ ~la~tlon ~chnLque~ may Also bo us~d, d~ponding on tho my~lom~ aoll lLna uqed.
Alt~rnAtive ~q~hod~ Or ~rod~oln~ mo~oelon~l ~tibodio~ u~llLz~ invit~ l~uni~tlon toohn1quH~.
Lympha~yta~ m~y be harva~tod ~rom ly~phold or~ns, ouoh Ae ~pl~un~ or lymph noda~, or ~rom whole blood aB
p~riphor~l blood lymphooyto~. ~ho ly~pb~oyto~ a~a p~t lnto aultur~ in ~h~ pr~a~nac o~ the ~D~-b~ndin~ Protoln.
O~cn immunoSt1mulatory polyp4~tld~ will bo add~d to ~he culture ~dium oonou~r~ntly. At v3r~0U3 ~lmo~
followlng the oultu~in~ o~ tho lym~hooyt2~ In vi~, th~
lymphoayt0~ ar~ hArve~tod and PU~e~ wlth th~ my~loma o~ll lins a~ d~arib~ abovo.
Othe~ ~eohnlqu~ ~or pr~dualn~ ~nd maintalnln~
~ntibody-searstln~ lymph~ay~e c~ aul~ure lnalude viral tran3~tlon o~ th~ ly~phoa~t~s to prcduoo tranfl~ox~d a~ll line~ WhiQh will do~tinu~ to graW in cultura. Ep~eln-B~rr vir~ ~E~V) hA~ ~-nn used ~or thi~ teohniqu~ tr~n~orm~d a~ do not r~quire fuslon wi~h myeloma oollff to allow 4Qntinuad ~row~h ln c~lturo.
Thymocyt~ ~tay bo u~ed a~ A ~eeder l~y~r to &ondltion th~ mediu~ ~or th~ ~uc~d ~ t. Th~,tncyt~
m~y be prep~red from ~uvenlla mLo~ lo~ ~han sigh~ wo~s old. ~ha thymus ~land~ ~r~ ~rvest~d, and ~ u~p~n-~ions aro pr~parcd a~ d~crl~eP a~ov~t. Al~h~1v~ly, periton~al ~a~ropha~os or ncttlm~une sple~n ~Ctl~ may used as ~ ~eAer layer. ~nothnr #~orn~lv~ 1~ to us~
~ondltion~d modium ~ro~ thymooytus o~ ~orophaqe~.
~t ~n ~ppr~pria~ tl m~ fDllo~ing the ~u~on dayl the fu~d ~ells (by~rldomas~ ~o t~en an~lyz~d ~o~
thç produotion Or ~nti~ody ag~ eha ~n~ en of ~hol~e, Thl~ ean ~q don~ by ~ wl~e ~Ariety o~ t~oh n~qu~s, l~olu~ln~ We~t~rn ~l~t, EL~AI lmmunopre~lPl~a~
tion, lnflu~no~ on ~lolo~le~l aa~ity ~ays, and lmmuno~ytoch~ l atainin~ h~s~ teohni~u~s And others arra well de~3ribed in t~ tcra~ure. ~s~, fo~

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, ll/15/$~ 17 2~ ~S2~6 682 6~31 Seed & BerrY ~ ~25 2~ 3~6 example, ~. G. R. Hurroll (ed.~, A~i~Qsli~: ,"T~ DO~ }~aton, ~la.:C~C P~ o., 1~8a.) Uybrldoma~ w~h ~eo~
~ntibodles ~ in~a~t ~r~ maintainoa ~n oultur~. Tho ~all~ ~ro axp~nd~d ln cul~ur~ und, ~t th~ Dam~ tlm~, inaybe clona~ ln su~h n mann~r ~ to obtain oolonlo~
orl~lnAtin~ ~rai~ ~lngla oall~. ~hi~ ~rovld~ ~o~ ~h~
monoalonallty or tho nntlbcdla~ obtaln~d ~rom ~ho hybr,idonua. A wl~ loty o~ t~hhlquo3 oxl~t ~or ~lonlng ~ , lnoluding llmltlng dllu~on, BD~t n~a~
olDnin~, and ~luore~c~no~-~otlvated o~ iorting.
ono~ olanuo o~ o~ ara ~btain~d, they ar~
r~a~yed ~or th~ produ~tion o~ th~ ~ntlbody ~
intere~. Th~aa cell~ ar~ th~n ~xpand~d in aultur~ to ~illow ~or th~ produa~ion Or larger ~ount~ o~ the ~ntlbody. M~thods for ~xpan~lon o~ th~ ludo m~intaining th~ c~113 ln ou~tu~a, plaoo~nt o~ th~ ~all~
in a biorRaotor or o~ha~ typ~ Or lurge-scal~
aul~uro environmont, or oulturln~ the o~ u3in~
~ari~u~ a~r cr g~latin o~rrl~ ma~rio~. Antibodl~
ar~ th~n isolat~d from ~ho cell cultute modla.
A pre~err~d method ~or p~odu~in~ l~r~ amount6 o~ ~ntib~lo~ involva~ ~rowln~ ~ha hy~rid~m~ cRll~ ln a~oit~ flul~. ~he hybridomas aro pre~r~bly i~ol~ad from tha ~ultur~ media by o~ntri~ga~lon ~n~ wa~hed wl~h an i30-03mo~ic aol~tion, pr~fQrably ph~p~a~o-bu~r~d salina. ~h~ o~ are th~n ~a~u~penfl~d ln an i~c-o~otlo ~olution and in~otad into the ~orieon~al cAvi~y Qf ~n ~pproprl~te hoet ~imal, pr~r~bly ~ mo~o~, ~nd allowed to q~ow wlthln tho h~st Anim~ h~ ~o~ animal ~ay r~c~ivo a prein~e~ion ~ p~iJt~n~ l~,6,10,14~
tetr~m2~hylp~nt~deoane) prior to the i~e~tlon oP ~h0 hybrldoma 4O11e, pre~r~ly ~eysn to thl~y day3 pri~r to the lnt~o~uotlon o~:th~ hyb~idom~ ollowing ~r~wth oP th~ oells in th~ peritoneal c~vity, a~aite~ flUi~, ~ontain~ng the antlbody o~ int~r4~ 15 coll~oted.

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., 11~15w29 17 2~ B82 G~31 Seed ~ ~errY . ~
133~6 Ahtlbodi~s m~y be puri~l~d ~rom conditlonad m~dla or ~olt~ fluid ~y ~ ~siaty Or ~o~hod~ known ln th~ art- ~h~ m~tbada lncluda ~mmJnlu~ ~ul~Ato praQ
ltation, ion-oxchnngo ehramato~n~by ~a~ ~urrQ
lbicl~), nnd hlgh-pr~a~ur~ llquld ~h~o~q~ogrAph~ uBing ~
hy~roxylap~tit~ ~upport tytan~or ~t ~1. . Ll_;U~Ui~L
MQ~Q~a ~157, lg85~. A pro~errad ma~hod ~r pu~1ryin7 antibodi~ Pro~ ~anditionqd m~dla or a~aita~ ~luld U~lliz~ ~ oommorQially nvAllA~l~ Prot~ln ~-8epha~os~
CL-4B aolu~n ~Ph~rmac1a, ~ t~way, N.~.~ Sl~ma~ ~t.
Loul~, Mo.). An~lbod~a~ may be puri~iH~ with th~Q
calumns u31ng condition~ ~ugg~tæd ~y ~h~ manu~aotUrar, ~yplo~lly, th~ aon~ltloned mec~lum or agcit~ ~lUld i~
m~x~d w~th ~n ~u~l volum~ Or ~NEN ~0 WM ~rl~-~a~ l pH
8.0, lO0 ~M ~aCl, 1 .nN ED~A, 0.94 NP-40) a~d ~pplled to th~ aolumn. ~he an~lbo~ ara ~luto~ uaing ~ p~
~radie~. Pr~rably, th~ ~lutlon ~ or comp~i~os 0~1 N sodiu~ aLtr~to.
A~tib~Les may al~ ~e pro~ua~d by ~0combin~n~
~thod~. Antibody g~ne o~qu~na4~ o~ ln~r~st aro l~olat~d ~om antlbody-pr~d~alng coll~ ~nd ar~ aloned and ~xpre~ed aca~dln~ to Xnawn ~r3codure~. Recombl-n~nt m~thad~ ~acllltata tho produa~lon o~ human mono-~lonal antibodie~ ~hd ~h~m~ri ~ntibodioa aontninLng human con3tant rogi4n~. M~thod~ ~or produain~ r~com~1-~ant ~ntlbodl~ a~e dl~olo~od by~ ~o~ ex~pla, No~b~r~ar ~t al. (WO 86/01533~ and Win~er ~publi~hed E~rop~n pat0nt appllo~tion ~P 23~,400).
Antibodi~s and oth~r pot~ntl~ goni~t~
ar~ ~a~ned in a ~ul~i~tap ~o~ay smploying tho i~olate~
bLndlng protein and/or recombinan~ ~oll8 whiah expre~
the proto~h~ Po~en~lal a~onlsts aro rL~t or~ened for t~e~r a~lllty to bind to th~ ~D~,blh~ing prot~ih by the llgand bl~tlng ~ech~ique. m o~ ao~psund~ whl~h a~e ~ount to bind are ~h~n oareen~d ~o~ thglr abll~ty ~0 ~tl~ulato ~ran&port ~ aholes~erol r~Om ~all~ e~en-tially A~ desorlbed ab~v~. B~ ly, ~b~ ar oholu~-: ~

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11/~5/~9 17 21 2~2~6 682 6~31 Seed ~ Ber~y~ 7 33~

Zl ~erol pool6 ~r~ labeled and th~ c~ ro inoub~ted ln the pr~nc~ Or tho o~mpou~d to b~ tootc~ Total llpld~
~r~ th~n ~xtr~otod ~rom the ~dium, ~ap~r~t~d by thln-~ r ohromato~r~phy, and A~sayed ~or th~ preoen~ o~
5 laboled oholo~tarol.
Prat~ln~, inolu~in~ antlbodl~, th~t bind ta I~DL-~in~iny protoln~ Ar~ uoe~ul wlthln din~nostla aompo-~itlono tor ld~nt.l~ying d~ato in the ~yntho~ nd praa~ooinq Or HDL-~lnding proteino ln a p~tlent ~nd ~o~
lo d~t~otin~ struatural deieot~ in ~D~-blndln~ protein~.
Given the rolo th~t ~DL 1~ b~ vad ta play ln aholoa-terol ~ransport ~nd the preventlon o~ ~tharosclerosl~.
dlagno~ie oi ouah de~eot~ ~ay b~ use~ tQ identlf~
i~dl~idu~16 at rlok ~or ath~rcr~ol~ro~i~ or to ~lagno~e tho p~rticular oondltion rosponsible ~or ~th~rogone~
To æ4reen for gunet~c de~at~ in HDL-bindlng p~otein~, c~lls n~e ~tained irom ~ p4tiont ~nd ~naly~ed ~or th~ pre~ena~ and 9trUCtUrH or th~ p~oteln~-Pre~xred aell~ in thi~ ~eg~d inolu~e ~lbro~la~t~ ~nd leukocy~e~. Fib~obl~ot~ may b~ o~tnl~ from ~kln ~bmples nnd a~ reAdily GUltU~d in V~VO. ~ko~yte~ may be obtaln~d ~rom ~lood campl~ by ~tRn~d p~ocodur~.
Analyoi~ o~ cell membr~n~ oo~pan~nt~ by ~o~ern blot~iny (~owbin e~ al.~ Pr~ 1. AQ~dl Sci~ a ~4350~4, ~5 1~) with antlbodi~ to ~D~-hlnding p~otoin~ i~ u~d to datect ~tructur~l dqfe~t~ or d~iclena~o~. Tb~ use e~ ~
panel o~ ~n~lbo~lo~ thAt reoognlz~ dl~er~nt ~pl~op~ on a protein i8 parti~ularly ~dv~ntdg~ous ~r det~atih~
~trua~ural d~ects~ Q~netla de~eots ~ay al~ b~
dotucte~ by pr~lng o~ lAr D~A rro~ 8 ~tient ~ith ~oned NA ~eguenoe~ ~naoding H~ indln~ p~ot~ins or fragmon~ ~her&of ~nd an~ in~ tho hybrldis~tl~n pattern, whi~h i~ lndlca~l~e o~ th~ ~re~e~ er ~b~ena~
~ a ~utatlon.
~L agoni~t-~ ~ay ~e u~d to ~tl~ula~ ole~r-anoe Or chole~t~rol lh p~ti~nt~ wlth ~QW l~vel~ L
or defect~ in apopr~toin ~tr~atur~. P~pt~d~ R~o~l~t~

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11/15~ag 17 22 ~2~6 682 ~1 Seed & aerrY ~ 2~
33~

wlll g~nerally b~ oombin~d wlth a phy~ologlc~lly aaa~ptabl~ o~rrl~ or dllu~nt and admlni~t~rud by ln~oa-tlon, although small peptlde~ may also bo ~dmini~tored by '1ngQ~tl~n o~ inhal~ n u~ aonvuntional ~ormu~a-tlon~. chemio~ onia~o wll~ pr~orably b~ ~dmini~-tar~d by ln~o~tlon, ~n~L
iLQ
LDL and H~L3 wr~ od e~om tho pl~sma 0 h~althy ~ting ~ubj~at~ by ~qu~tl~l qontrl~ùg~tlon, ~o d~orlb~d by deL~ and GO~m~n ( ~ nnl~ 1:45~-7B, ~5~ h~ N~3 ~r~ction wa~ OUb~t~d to h~p~in~Rpharo~e a~inlty chrom~tQ~rn~hy t~ x~movo apo~-~o~t~lning partlol~ ~B1~sbrOe~k Qt ~1 " a~ LLnL
rnvQQ~L 711525-39 1983). Llpoprotain~ wa~o 1~dln~tod by ~ od1~ication o~ th~ ~ono~h10rldo proo~dur~ ~ilhel~or ~t ~ 15S~U~L ~ E~Y~h~ a~Qs21~21, 1~7~).
~rot~olipoD~m~ w~ro prhpared u~ln~ ~g~ loo1t~in hnd puri~led apopro~oln ~-X, ~ or ~ b~ tho ~ol~t~
dlalyql~ m~hod ~Ch~n and Alber~ 0-gl, 1~8a) ~t ~ lnolar ratio o~ lth~ to apopro~eln 300:1, ~ultur~d h~an a~ln fibrobla~t~, bovlnq Aortio cndoth~ l c~ , swi~ 3~3 ~ibrobla~t~, ænd tran6~0rm~d maarophag~-llk~ 401l linea (~g- ! m~u~e J~74 ~acropha~a~ and human ~EL aell~ e plat~d intD 1,~0 ~m oultu~e di$hes ~n Dul~e~ao'~ modl~i~d Eagle~ meilum ~o auppl~ented with 10~ ~Qt~l bov~n~ oe~ nd wer~ g~own tc con~lu~ncy. C~ re loa~ed wi~h a4Ole~erol by inoubatlng them ~or z4 to 4S hour~ With ~e~um rr~e medlum c~n~ainin~ 0.2~ b~vine ~erum ~lbu~i~ p~u~ elther nonlipoproteln ahole~terol ~ ~dded ln ~th~n~l ~r aao~y~ated LDL ~Or~ 45-59,~

11/15/8g 17~3 ~20~ 6~2 ~31 Seod & BerrY 1~l~2~
2~)~3~6 Cell membr~ne~ we~ p~par~d ~om oh~le~l;erol-tro~d a~lls e~ntl~lly a~ da~orlbad by aa~u ot ~1.
lol. ~hq~. ;~.: 38~2-5~, 1978) . t~ll monolayer~
w~ra w~hed three tima~ Wi~h Pa~ ~lU~ O.~t ~ d~ room 5 tomp~r~tux~ nd th~n chlll~d ~o 4'C ~n~ ~arap~d with A
nylon pollc1~m~n lnt~ ~.0 nM Triu-NCl, 3~H 7.4, oc;ntAin~ng 150 ~ tA~ banzar41dln~ 0~9 mM ~DTA~ And 1 DM
ph~nylrao~hyl-~ul~ortyl ~luoridq di~olYua in di~thyl-~ul~oxido ~ ell~ w~ra ~entri~uy~l at 200 x g ~or 10 S ~inut~3 at 4'~, re~u~p~nded in T85, pooled, homog~nl~ed with two 5-~eaond p~ of ~ Polytron homog~nlzer ~T~km~r ~o " cln~lnn~t~ Ohlo), ~nd ~entri~u~ed a~ ~00 x ~ ~e~ 10 minutos at 4'C. ~h0 ~00 x g ~up~rnat~nt W~s ~entrl~uyod ~ ~00.000 X g ~or 60 ~inute~ at 4'C, and th~ mumbrano ~llut wa~ ~tored at -70'C. In ~ ~ploa~ prepar~tlon, th~ total mem~r~n~
~rotein yi~ qu~ntlfled ~y tho mo~hod o~ ~,owry Qt al. ~J. ~iQl. chQ~ a~-73, 1~ om 3~ 130-m~
di~h~ WaH approxima~æly ~ M~
M~mbran~ p~llue~ Werc ~olublll~e~ by ne~dl~
~plratlon in ~letrophore~ia 4~mpl~ bu~er ~nd ~ollod ~or 3 ~lnu~ ln th~ pr~n~ o~ 104 (v~ 0-moraAp-to~hAnol~ ~r~lons ~ontainlng ~lAl amount~ o~
pro~oin ~ner~ 500 ~9) p~r lan~ ~r~ ~leot~ophor~d on 7~ polyA~rylaml~o ~ g~l~ aocor~lng ~o Laem~l~
~n~ 2~680-85~ 1970). Soparat~l prot~ns w~3r~
tran~orre~ onto nl~rooellulo~e ~Soh1~1Ohe~ ~nd 9chuell, 5 ~m~ ~y ~leotxopho~e~ Towbln et a~ 5YJ ~lt ~ CA~ . US~ 4350-54, aQ79), 0~1~ we~e ~ t~d u~in~ pro~tain~ or iodinat~d ~Dl~oular w~ight ndDrd8, To a~say protein bindin~ ~at~vity, nitro~llu-.~o e ~trlp~ we~e lnoub~t~ f~ 2 ~our~ wlth blockin~
bu~rer (1~ ~M ~ris-~Cl/ p~ 7~4, o~n~lning lSo ~M~Cl~
35 1~ w~v h9~ w/v Carn~tion n~n~ iRd ~ilk, 0,Dl~ i v/v antl~oam A, 50 ~g/ml LPL~, ~ollo~d by a further ~- !
hour inoubatiDn in t~ ~a=e bu~r~r oon~ai~n~ ~ ~gJml I

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. 1 11~15~8g 17 24 g~206 682 6~31 Seed ~ herry ~ 030 ~33~L~

l~5I-~DL3. stri~ wer~ wa~hed once rapidly and than flv~ tlm~3 ~or lS minut~ with wa~hing bu~P~r ~blc~kin~
buf~r withaut ~ lottin~ Inouba~ion~ ~nd wa~A~ w~r3 a~ roo~P ~au~parn~u~e, ~f~r drying, proteln b~nd~ w~ vl~uallzed Py æutoradlogr~hy~ ~u~orndlo graphs ruv~Al~d ~h~ tho 125I-HD~3 bound to a m~or m~mb~no protein h~vin~ ~n appar~nt m~looular mn~ o~
approxlmat~ly 110,000 Dalton~ in all th~ ~o~omention~d ~qll typ~ The relatlv~ migr~tlon ~nd bin~ing actlvity of thi~ protoin worR not ~lt~rhd by bollln~ oP the ~olubill~d m~mbr~nu3 o~ by pr~reatmont Wlth 1~ v) ~-~erc~ptoathanol. A~ oo~p3red to chol~t~rol-lo~de~
~113, 0~116 incubated with ~hol~st~rol-~ra~ m~dlum ha~
low~r or undotea~Able ~mount~ o~ the 110 kDA ~rot~in, ~u~ ting that it~ binding ~tlvl~y w~ ~p-reg~l~t~d by chol~tero~ lo~ding o~ o~ . Pr~in~u~Atlon o~ hu~an arteri~l ~mooth m~s~le c~ wlth ino~u~ aona~ntra-tl~ns of ~hol~tqrol pr~uc~d B doae-do~nd~nt ln~rease in th~ binding ~otlvlty o~ ~h~ 110 ~Pa pro~eln.
ao ~ryp~l~ treatment ~f tho ~n~th~lial aell ~onol~y~r a~olish~d th~ a~tiVity o~ tho HDL~blnd~n~
pr4teln, indloating ~hat 1~ o~ted in the pla~a m~mbran~ ~nd i~ ~ho~fore avail~ h~ o~ ur~a~
for l~t~a4~ion ~lth ~DL. Und~r th~e a~ndl~ion~, ~hc aell monalayer r~main~d intao~i ~nd ~bll ~orphology, a~
a~s~ d by llght ~iaroa~opy, wa~ ~nA~r~ot~d.
Troat~ont o~ the 110 k~a HP~blndlng protei~
wlth endo~lyoo~l~as~ H ~0114~ed ~y ~ lea~opho~
o~ th~ tr~a~o~ prot~ln lndlaat~ ~ o~rbo~ydrato oo~tent o~ appraxl~atoly 1~ by w~ight.
~x~pl~ 2 - Purifiçati~n ~ L~
Th~ HD~-binding ~rot~in d~ribod 1~ ~x~mple 1 i3 pr~p~red Pro~ mi~o~o~al m~F~n~ pr~p~ed ~rom ~ul~ured bo~ine endo~h311al ~Rll~ by ~oluhili~ation in 1~ Tri~on X-100. gol~bili~d prot~ina ar~ p~s~æ~
throu~h a wh~at g~rm ~glutti~in ool~mn. ~he ad~or~ed I

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11/15/~g 17:25 ~2~ 6~2 6~31 Ssed ~ B~rry 1~1~31 ~0~33~6 ylycop~ot~in i~ alutad ln th~ p~e~or~oo 0~ exae~
N-~c~tylgluc:o~mlnu, dlaly~d, ~nd ~u1b~Qtad t~ l~o~l~c-tric focuDln~ ct~Dphor~si.~ ln tll~ pr~noe O 4 ure~ . Protalh~ havlng th~ iH~oloctrla point o~ th~
blndln~ protoln tpH 5 . o to pH 6 . o~ ar~ l~olatHd nnd puri~iHd by ~D~-polya~ryla~ a ~al tlla~ophor~oi~. Th~
po~ition o~ the ~DL-bln~ing ~rc'c~in on tho ~ol 1SD
ldantlrled by ~llver atAlnlng and ~on~irmod ~ the r,d blot ~a~ay. ~h~ H~-binding p~ot~ln 1~ ~han 10 ~lHtro~lut~d ~rDm thH gHl. Pro~e~n pllrJ rlod by thi~
mathod 1~ a~ l~R~t ~09~ pure~ Al~ detu~qnln~d by ~llv~r staining ~ poly~a~.~yl~mld~ 51o~ h~ prot~in me~y b~
~urth~r puri~i~d by UPL~ ale n~a~ssa~y. Al~erna'cl~ol~t, the HDL-bindlng protuin msy bH puri~ied by lmmuno-lS ~lnlty ohrom~ogr~phy o~ ~lubili~ed m~mb~an~ ~slng n3noclonal ~ntib~die~ pro~uoed ~ de~o~ib~d h~r~in. ~ho purl~led ~re~eln i~ us~d for antib~dy ~ropar~tlon ~nd prot~ln oharac~erizatlon~

~g~
. Pr~para~l.on 0~ m~ 9~LL_~ItL olL L 2 -~Y
Fi~rQ~L~t ~ RNA
~otal ~NA wae pr~p~x~d fr~ b~man flb~obla~t ~SK-5) c~ cultu~d 1~ the ~r~nce o~ lo~ a~ru~ by ~h~ gu~nldlne l~othlooyanat~ m~thod t~hir~win et al., t5Z-94, 197~ POly tA)~ RNA WA~
obtain~d by two round~ o~ a~init~ chromat~raphy on ~ o (~ U10B~ ~Manlatl~ et ~ " lbid~ t and ~econd ~trAnd ~N~ wa~ ~yn~h~lzad ~ ~ith~r ollqo ~d~) or random pri~in~ riF~ rand ~ynthssl~
lFri8~h~r ~t ~1., S~ .771017-28, 138~) ~rom 5 ug o~ 2 X ~elected poly ~A)~ 2N~ de~orlbed by aubler ~nd ~D~m~n ~Ç~n~ ~s263-69, 1~83) a~d ~a~n b~ X~
Natl. A~d. ~ d ~s241~-16, 19~6~, Th~ ~dou~le-strAnd~d a~A w~s m~hylAted With E~0 X~ ~ethyl~e '~
.~

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11/15/$9 17 25 ~2~ 632 6~31 Se~d & BerrY 1~1~32 ;~o~363~gi ~M~niat1q et nl ., ibid. ), Paaa~ Ver a S~Ph2~rC~ CL-¢3 ~PhaL~ P1DC~UW~Y~ N3. ) oolu~ luntlld wlth 1'4 ~A pol~r~o ~Mnn1~t1~ ~t A1~ ih~d.), li~7~ted to ~Sco'~I lln~cc2r~, fli~ d W1t~h ECO R~ pUI!~3/~S OV~
5 S~PI]~r~ CI,_2~ tYh~aC1n) ~O1U~I~ Th~ Cl:)NA Waa ll~tt~d tc~ ~n E~o F~-dig~tod, phoaph~ta~d l~mbd~ a v~ctor ~YoUn~ and ~avlEI~ ~"~;lq~
.QQ~1194-~, 1983) and p~cXnqad in l~mbda p~ak~ln~
~xtr~ot ~S~r~tag~n~, ~nc., ~a ,Joll~, CA11~ ~ . Plht~
10 lysat~ libl~rle~ w~re propar M~ni~atl~ et ~ ibld. 3 u~ing ~ i. YlO~ a~ t hu ho4~ ~train. Th~s~ plate ly~at~ llbrari~a werfl ~torod ov~r ~hl~ro~o~n ~ 4'C
un~ll usod ~or l:Lgand ~or~n~ng. ~h~ ra~om-prim~d oDNA
ll~rary was oompo~d of approx~ 'co~y 1~, millioJ;
15 i~olltes, and th~ ollgo (dT)-pr~mud ll~r~ry w~ aompo~ed af approxlmat~ly ~3 r~llllon l~ola~

Thlrty platoa (lS0 mm) s~ N2~ r waro e~h 2~ ovorl~y~d with 10 r~l o~ NZY 0. 7~ a~aro~ aon~alning nll YlOP0 c~ ad~or~d wlth app~oxl~a~ly SC~00 ~ll that aontalnod ollgo ~r)-pr~ nd random~prlm~d human ~l~robla~t ~NA in~er~d at th~ E~o RI ~ rh~
platos were incubated at 37'~ ~or 3 ho~lro, overl~yed a5 wlth 137-mm nltrooellule~e ~l~t~r~ ~chlelohe~ ~nd S~uell~ pravlou~ly sonk~3d in 1~ m~ I~G ti~pl~opy~ D~
thioqRlaCltOpyr~lnO:~Ildli~ lh c:homical 00" ~t. LouiF~, Mo. ), ~nd ln-~ub~ed at ~7'C overnight. me fllt~rs W~
w~ hea twi~e fo~ 15 mlnu~ ch ~t roo~ t~mpor~tur~ ln 30 ~0 mM ~ris-~l, p~ 8.5, lgO mM NaCl, and w~hod ~or a hour~ ht rooln t~mp~r~tUre in li~and~^~lottin~ l; loc:king bu~er. l~he ~ilters wo~e trans~e~r~d 'e~ ~re~h ~locking b~f~r oont~lnlng ~ ~y~ BI-HDL3 and i~cub~od for 2 2~4U~Y ht ~04m temp~tu~e. ~e ~i1t~ ere ~hoa thr~o ~ ror 15 mlnutlss ebch at. 10-~ in wa~hing b~rzffr, two t;im~ fo~ lS ~ninut~ ~aa~ ~ lo~ in 50 ~M
Tri3-~SCl, p~ 8.S, lS0 m~S N~l, 0~2~ ~$A, and or,~ ~or 5 ,~ .
'~ ~
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" , ~ . , 11/15/~g~ 17:~6 ~2~6 ~ 31 Seed ~ BerrY ~33 Z~ 391 6 minu~s at lO'C ln 50 mM ~rls-llCl, pll B.5, l50 mM II~Cl.
Th~ ~ilt~r~ we~ air drlod and oxp~od to X-r~y ~ilm at roo~n tomp6lr~tur~D for lO dsys. Po~ lv~ ~glons ~n th~
plal~ ro plok~d, t:lt~r~d, An~ r~plat~d ~ qlva lo, ooo S and 50,000 pla~uo~ ~aoh on ~ 5~ , YlO~o cnlls ~Ic~;Jrdlng ~o Atandar~ proaod~ (M~nla~i~ et ~1., lbld. ~, ~a-~o~d ~ qrs w~r~ ov~rl~yqd and ~c~e~ned a~ d~orib~d abovo . Po~itiv~ pl~ w~ro plok~d and r~plat~d to glva 500 plag,ue~ e~oh on ~ L Y1~9C~ aell~ G-~oakqd $11ter~ wRr~ ov~rla~ed And slo~e~nad 8:}~ de~rlb~dabov~. Pl~te lys~te ~took~ War~ pro~nred a~ d~o~ribod by Naniati~ et al. ~ibld.). ~hag~ ~N~ Wa~ prep~r~ by ln~u~nting ~ ODbo~ unlt~ o~ i YlO~0 o~ in~cted with 5 x 1a6 phhg~ in 500 ml c~ N2Y broth flt 37-C ~or 3 15 hou~ he phag~ were r~aov0r~d by pr~alpitn~ion with ~
NaCl and la~ ~wfv) polyethyl~ne glyaol 3000 at 4'C and aentrl~ug~ion at ~000 rpm in a ~orvall ~3 rot~r or 15 mlnute~. ~he pha~e pr~olpltnt~ w~re ea~h dl~olv~d ~n 5 ml Or p~l.50 ~mfml ~3Cl in lQ mM ~rl~-~el, pH 7.4~ ~00 2a ~ NaCl, 10 ~M MgsO4, snd ~ntrl~g~ ln ~ 8~vall 8W55 roto~ ~ 35~ ooo rpm ov~rnlgh~. Th~ O~Cl-bnnd~d phage w~r~ reoev~r~d hy 4th~01 prealyltAtlon~ ~n4 DNA wa~
uxt~aot~d by r~su~pending th~ ~ p~llet in ~50 ~l o~ 50 mM
Tris-HC~, pH ~ adding 100 ~1 o~ lO0 m~ ~rl~ ~Cl, pl~
~5 8.0, 100 mM E~TA, 0.1~ 9DS and ~0 ~1 o~ 1 mg~ml prot~ina~e X ~ollow~d by inaubat10n ~or 1 ho~r ~ ~7'C.
~he DNA W~s ~xt.raot~d twico with phonolJc~l3:l~on~
al~ohol (~4~ PCI), once ~lth CRal3tl~oa~yl alaoho~
(24~ , and pre~lpitate~ wl~h A~OniUm ~tate ~nd eth~nol. Th~ puri~led a~N~ W~ u~p~n~e~ ln 75 ~1 o~ 10 mN Trl~ p~ ~.0, 1 mM EDTA, ~ig~ d with ~co R~
at 37-~, and the oDNA ins~t~ per~ re~ove~e~ by eleatrop~ore~is ~n O.a% ag~o~ gels. The o~ fraq~nt ~rom a posi~l~e phag~ alone ~ro~ tho ran~om-prlmed 3S libra~y (designa~e~ 17~1~3) wa~ ~aovered on ~A45 (~ohlaiahs~ an~ S~huell~ ~n~.~ X~n~, N.~.~, elut~d ln 1,5 ~ N~l, 10 ~M ~ria-~l, pH ~.0, 1 ~ TA ~t ~5C

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il/15/~ 17:27 ~206 6~2 6031 Seed ~ ~errY ~034 2(~33~

~or ~O minut~, PC~ and CI ~xtract~d ~nd ~th~nol pr9cip-ita'~ed. Th~ oDNA ~ag~Qnt wa~ ln~rt~d at the ~oo RI
slt~ of M13mpl~ ~n~ pUCl~, and th~ V~tA soq~lenoe wa~
d~te~lr~d by ~h~ thod ~ sange~ k al . ~L
5 ~ggl s~ .U~54~3-67, 1~77). P~ult~ d$a'ce~
tha~ thi~ ~lon~ ~on~inod nuol~ctldo~ 257-8a8 o~ th~
~quon~e ~hown in ~igur~ h~a pU~ a~N~ cl~no wa~
d~ n~te~ pM~O-1.
A ~ kb human ~lb~obla~t ctD~tA w~ ol~t~d by ~r~enln~ thR human ~ibrobla~t ~gtll DN~ llbxsry With th~ p~tlal cDNA ~om pl~mid pM2~0 l. All bne~crln ph~g~ platln~, plaquo ~url~icatlonG, and DNA p~ri~ica-tion~ were p~r~ormed n~ described abo~o. ~hR 2 kb c~NA
wa~ ~xoi~sd ~ro~ ~ cl~no 1~ and lneo~ed in~o th~
E~o Rl ~lte o~ pUCls to constru~ umid pM253-3. Tho aDNA WA6 ~l~o in~ert~d into Ml~m~18 a~d Ml~mpl~, nnd the DNA ~uenc~ Was d~termlned by the me~he~ o~ sanq~r c~ lb~d.~. Th~ nucleotide s~queno~ o~ th~ olon~d ~
kb cDNA and th~ inferred amino a~ld ooquuno~ are ~hown ~0 in Flgur~ 1. Whlle ~he amino ~ld s~qu~noo ln ~l~uro 1~ ~hown With an ~mlno~rmin~l methl~nine ~id~e, oellul~r proce~lng ~ay re~ult in ~ ~rot~ln hnvin~ an ~mlno-t~rmlnal ~lyain~.
C. ~onst~u~ o~L_p~_g_~an C~mid ~14r~x~
The Ye~tor 8~217 wan pr~p~d ~ro~ pJB8 ~ Lau 8nd X~n, _~v~ 5l~ ~ ~L~ C~ Q:~22~-2~ 3) ~n~ pgV2~n~ ~Sou~hR~n and BRr9, L _ILaL.~ eL ~en ~53a7-4~ 8~) by oomblnln~ th~ 3.4 kb ~ind III
~r~gment o~ p~B~, wh~h c~nta~ne th~ ~V~O prom~*ex and ~OS reglon, wlth th~ 2.65 kb ~ind III~Eoo ~ ~ra~m~n~ o~
pSV2-nso, ~hich cun~ins ~h~ neomyo~n r~ tan~e ~en~
~nd SV40 t~rmln~tor, lnto t~e ~l~nd ~X~ and E~O ~I ~itH~
o~ pUCl3 ~ee Figu~ 2).
Hlgh mole~ul~r woigh~ ~NA w~ olatea ~om ~he hu~n acut~ lympho~l~Yti~ leu~eml~ c~ n~ MO~T-4 M4nowa~a ~t al., I ~U~ =U~ 91~5, 19~;

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11/15/89 17:2$ ~2~ ~82 ~1 Seed & BerrY
)33~
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as A~CC ~R~ 1582). ~he 4N~ waA ~arti~lly d1~e~ted with ~a~
3A and ~1zo-~r4at10rl~t~q on ar~ NaCl gr~cl1~t ~3 desc~ibod by Dill~ln And Wo~ ,a, 1 4, lg~
DNA rraatlorl~ oont~inin~ 30 ~o ~0 k~ g~n~s wore 5 11g~t:o~i .tnto t~o ~am ~ o~ Zo~21~, ~nd th~ lig~tion ron~t~ion wa~ fn vl'~o paokas~od into ph~ ptlrtlolo~ ~u~lng paok~ing Xi~ obtnln~d ~rom 5t~at~geno, ~no., ~4 ~olla, ~ali~.) wh10h were u~o~ to ~rAn~oct ~",~, 1~1.
~ ho proc~urR ~ave rloo ~o ~pproxl~tely ~.~ x 10 lO6 ~mplc~illih-ritsl~tant ~oloni~ whioll wore hAn,~eeted ~ro~n plate~, ~lxed tog6~the~, allquoti~d, and ~ro~esl ~t -70-C. StAphyloa~acal g~ow~h wa~ prev~nt~ by th~ adcll-tior of v~noomyain-HCl tLilly) at Approxim~tely 3 ~g~ er to A~ploillin~ con~ainin~ madla .
15 ~h~ h~man cos~nld llbr~ a~ ~c~re~n3d witb 3~P~ eled pM253-3 2-Xb C~N~. ~wen~ pl~t~ ~150 ~m) of ~B ~edla con~a~,nlng 50 ~g per ~ r o~ neomyaln woro lnoaul~ted with approxllR~tely S0, 000 aoloniql~ oAah Or th~ hu~h~n ~oslnld 114 a~y and inaub~t~d o~nlght ~t ~0 37'C. Nitroacllulo~e ~ er llft~ or ~he platn~ wq~s~u pr~par~d and hybridl~ed wlth the p~53-3 oDl~A, and po~l-tlve oolonie~ wero pu~lfiod a~oordlns~ to at~nd~d ~nathod~ (~7anlatls et al . ~ ibld . ) . q~h~ thrae po~itivo ~lone~ o~tain~d WH~ signathd pM~0-16, pM380-~7 ~nd ~5 pM:~80-20.

Polyalonal ~ntl~tra weri3 pooled ~rc~ w~ H.
whioh had boen immunized witb a pa~tially p~2rl~i~d 30 pxop~tion ~ tl~o bo~rinR llO,000 Dall~an ~lrJL~ di~g p~otoin. ~ha posled ~r~ wbr~ d~ ted o~ aht anti~odieR b~ ~roma~4gr~ph~ on ~N~ ati~at~ ~eph~lros~
~pharmAci~ Pi~ taw~f, N.~. ~ oonju~ted t~ ar~ extraot o~ a. 5~ Y10~0~ ~wunty plate~ ~130 mln~ O~ NZY a~r 35 ~ re oaoh o~erl~y~ with 10 wl ~ N2Y 0. 7~ ig~ros~
~ntaining ~., çQ;Li ~lO90 Cly~ d~o~d with npproxi-matel5~ 50, QOO ~gtll ph~ o~n~aining r~ndoh-p~lm~d .
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', [~ ~3ff 5~9 17 2g ~2~6 ~2 6~31 Seed ~ ~e~
)33~

human fi4roblaRt oDN~ in5ertQd at t~ Eao ~I sit~. The platea w~re inoub~t~d at 37'~ ~or ~ hour3, thRn over l~yHd With 137 ~m nitro~llulo~ ~llt~r~, whinh h~d b3fln pr~vlou~ly ~oA~fl in 10 mM IPTG, a"d inOUbntUd ~t 37'C
ovornlqht. The filtura wqr~ W~h~d at room tbmp~r~t~r~
ln 50 mN ~r ~ C1, P}~ 8.5, 1~0 h~M NQ~1 ~er 1~ m1nUt~8~
1n 50 m~ C1, PH B,5, 130 ~t Na~1, 3~ 83A ~O~ 1 heur; ~nd ov~rnight lh So m~ Trl~-~Cl~ p}~ 8.5~ 1~0 ~M
Na~1, 3g ~SA nontAlninu th~ pool~d, d~ t~d an~is~
o dllut~d approxi~na~ly 1 ~ ~oo . ~h~ ~llter6 w~r~ then W~shafl ~ ro~m tomp~r~tur~ in 50 mN ~ris-HCl, pH 0.5, 150 m~ NaCl ~or lo ~inute~ ln 50 m~ ~rls-~Cl, pH 8.5, 150 mM ~aCl, 0.1~ NP~40 ~or lQ mln~t~s~ ~wl~a In 50 ~Y
~rls-~Cl, p~ 8.5, 150 m~ NaCl ~or 10 ml~u~es e~ah~ ~nd in 50 mM Tr1S-~C1, Pll 8.~ 0 mM NaCl, 3~ ~8~ ~o~ lO
mlnute~. T~o Yllt~r~ wer6l th~n inoubated ~02~ a . S hou~o ln 50 mM ~ri~-N~l, pH ~.5, 1~0 ~M NRC1, 3~ P~A With app~ox1~atQly 1,000,000 op~ per ~ilt~r o~ 125I-l~b~led aYflnlty-pu~i~ied rAb~i~ anti-~ou~ Ig~ ~O~anon ~0 T~nik~-CAp~ll, Malv~rn~ P~ he ~ ers w~re th~n ~ashed at r~om te)nper~ture in 30 m~ Trl~-H~l~ p~ 8.5, 150 m~ Na~ r 10 mlnutess in 50 ~M ~rl3-HOl, pH ~
150 ~ N~Cl, D.l~ NP-40 ~or 10 mlnute~) and twl~ ln 50 rnM ~rl~-HCl, p~ a.5, 150 n~ NA~1 ~0~ 10 mlnute~ ~ah.
~h~ ~ilt~rs Wer~ Air drl~d an~ a~tor~d~gr~phed at -70'C
with ~n lnten~l~ying ~ar~nu PosltiVe r~glon~ on th~
pl~t~ wG~r~ plaque-puri~led as d~a~i~od ~b~v~. ~b~ on~
positlve c:l~no obt~ln~d W~E~ de~:ign~t~d la~-l. P~ DN~
w~ puri~sd ~rorn alon~ l~B-l as d~Aarib~d abov~.
Plhgu~ la~-l wa~a u~30d to lyqo~ni~e ~
Ylo~s~ l~he lnr~cted ~a~toria W~r~ pl~te~ on ~
ampicillin media at 30'~, thah r~pllsa plo1~ed onto ~-amplcillln and lnaubat~d at 30'C an~ d2tC. ~10~ 5 th~t ~iled to grow at 42'G ~r~ lysogeniæed by ir,~a~lon 35 with IP~, and the ~0-g~lacto~ld~ rwlOn p~otQln W~5 pu~lfioà ~y ihmUno-afrihit~ ~romatogr~phy ~u~ing Proto$o~b l~a~ ~d~rb~3nt ~obtAiri~d ~roln pro~sTR Blot~

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11~15/89 17 30 ~2~ 2 6031 Se~ & ~erry, , ~ ~37 33~6 ~.

Madlson, wi~., and used a~ ~pooi~i~d by the manufactu~r).
ThR puri~lq~ ~u~io~ protoin w~ dl~ly~od ngal~t 50 mM AmmonLum bio~rbonnte, lyophlllz~d, rr~u~pQndod in Pas ~nd ll~od t~ prnp~ro p~lyolon~l antl~rn, Tho protain ~l.0 ~g~l) Wn~ orbod ~o two nitrocallulo~ dl~oc using lO ~l po~ dl~o. ~h~ dloac were dri~d at 37'C ror 3-4 hour~, tb~n uaoh ~18~ wa~
dis~olvad ih 100 ~1 DM90. ~h~ ro~ul~iny ~olutlon WAB
aombln~d wl~h Pr3und'o ad~u~nt ~nd ln~eat~d intr~p~rl-ton~nlly into m~ nlb~c ~ioe ~ob~ln~d rrom ~imon~an ~b, Ina., Gilroy~ Call~ e miao w~ro i~munlz~d w~th an lniti~l in~ation of 2 ~ o~ protoln p~r mouss, ~oll~wad by ~ov~n boo3tos ln~otion~ o~ 2 o~ 4 ~g ~aoh ~5 at ~- to 3-WeQk int~rYal~. To obtain ~ntls~, blood was ooll~cted frcm tho miae by retro-orbltal bleQdO and a11Owqd to alot, and tho s~rum wa~ r~ov~d.
For ~e~uena~ ana1yel3, ph4g~ DNA ~ao r~oover~
from the ~2B-l r~eambinant a~d th~ aPNA w~s excla~d by ~o Eoo RI dlg~tlon. Eco ~I ~ra~ment~ a~ 261 an~ 339 bp w~rs ln~rt~d lnto the Eco ~I aitR or p~C19, and ~h~
r~ltant plasmld~ w~ dae~qnatod pM283~ n~
pN2a3-lo~ pectively. Tho ~ra~ment~ ~are al~
lnYorte~ lnto Ml3mplS ahd ~e~enaod b~ tho m~thod Of Sang2r e~ al. (ibid). Th~ DN~ ~qu~nce o~ th~ l2~-1 c~NA is ~hown in ~lgurQ 3. The 600 bp a~A hR~ ~n ~pen readlng ~ram~ ~hat ox~en~ ~or ~t~ ehtl~e l~ngth, suggestiny t~at it encode~ an int~rnal tra~m~nt O~ ~h~
HD~-blnding protoin. No slgnifioan~ matohc~ w~a ~ound 3~ to th~ ~DNA sequeh~ or th* aorrespo~dln~ ~lx pot~n~
tran~lation p~oducto using the ~AS~A and ~FA~TA ~Pe~rson an~ Llttmah~ Pr ~. N~l, Ao~d, sqi, ~A ~2~ 4~
1988) Gomputer ~2ar~has of th~ 6en~ank, EM3~ ~nd ~R
d atabP~es .
~ho in~ivid~al Eao RI fragman~ o~ the 12~-l phaga ol~ne were 32P-1abel~d u~n~ ~ndo~ p~
(A~ersham ~orp., Arlington H~ight~, I11.) *nd u~d ~o ~.
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11/15/8917 30 ~2~6 68~ ~031 S~d & BarrY ~3~

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p~b~ North~rn blot~ (Thomd~ Pxoa.,_~tl ~ ~ar~i, ~al. U~A
7.7 ~ 5~01, l~0) o.e poly t~) ~ RNA ~Chlr~wln ~It ~hr~ 52P4, 1979~ Avlv 6 L~dor, ProQ. N~tl.
~.i~ ~.2.:1408, 1~7~ om tha ~ow~ m~oropha~
5 oRll lln~ J77~ ~ whloh had bo~n oult~r~d ln tho ab6l~noo ~nd prus~no~ of oxogonou~ ao~tyl~ltod L~ ~or~m ot ~l ., ;L~l~Sh~ ~IZ~0~-lO, l~7~. ~d~n~lo~ blot pattorns w~r~3 oht~ d, lndloa~in~ thAt t~a o~N~ snoom-p~b~S ~ n~tur~l ~cv nI ~i~o. ~oth ~N~ pr~p~ratlonn aont~ln~d hybrldlzln~ m~NA~ o~ 4.4 o~ ~6 ~b, but ~ho~
~NA.s w~r~ npproxL~ately ~ou~Pold ~o~e ~bun~a~t ln th~
L~ own ¢~ . Eq~A1 ~m~unta of R~ r~ ~how~ to b~
pr~ont ln hoth ~m~ by ~t~ipplng t~o p~obo ~r~ tha blot and rehyb~idlzin~ th~ blot wlth ~ ~at gly~er~ldo-15 byd~-3-pho~phate d~hydrog~na~ ~DNA proba. ~be 12~-l fibro~la~t ~PNA prob~ yield~d ldantl~al ~NA blot pa~terh~ in humah ~p~2 li~r oar~lnoma, bl~dd~r oar~lnoma, rhabdomyo~arcom~, and nor~ mononual~ar ~118~ indl~atlng that ~ P~-bindlng p~o~ein ~on~ 1G
~xp~ cd in ~ny o~ll typ~.
ona ~illloh r~omblnant ~nmbd~ ~tll plaqu~s aontalning oll~o td~)-p~lmod hum~n e~ytbroleuX~mia ~H~) aell ~NA ~L~p4z ~t ~l., PI~Q~_ Na~lL~ ai. U~a ~t5~ l9, 19~7) w~re ~re~n~d ~o~ hybr~ atio~l ~o the 25 12~-l f ~ ~robla~ cDNA. Nitroc~llulo~e ~llt#rs, ~o~
aontainihg approximately 50,000 plngue~ ro hybridl2ed with ~h~ ~2~1ab~1~d o~NA in ~0~ forma~ido, 5x D~nh~rdt's (l mg~ml Fiaoll, 1 mg~l p~yvinylpryroli-don~, 1 mg~ml ~SA), 6x s9P~ 20X ~;5PE ~ 3 . ~ N ~lac~ 40 ~M Na~aPo4, 20 mM ~A, pH ~.~), O~lt S~S and loO ~g~ml dqnA~u~d ~lmon ~pa~ ~NA (M~nlAti~ ~t ~
Mol~~l~L _~lar!~nc~r A ~L~Çxal,t~y M~nU~l, C~.d pring Narbox L~o~atur~, C~l~ sprl~ rb~r, ~.Y., ls~a, pp.
326-2~ h~ ~ilter6 w~r~ wa~h~d~ ~w¦~ in ~x ~9C, O.lt ~DS ~t room temp~aturæ for 30 mlnu~ p~r wa~h~ ~wlae ln 0.2x 3$C~ O.l~ ~DS at 650 ~0~ 3~ teo p~ wa~h;
ln O.lx 98C, 0.1~ SDg at 55'C ~or 90 ~inu~os~ in Q.l~

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11/15/89 17 31 ~206 6~2 6031 SeetJ ~ BerrY ~ 39 2~30~3~

SSC, 0.1~ S~S, 50& ~or!n~mi~ A~ ~'C ~Qr D0 mlnuto~ ~nd in 0. lx S9C, 0.14 ~D5 at 65-C for 2 h~ur~. TWo ~o~iti~r~
plaqu~s~ d~ n~ted I~EL 13 and HEL ~ ~ w~re pur~ d, pha~ç DNA Wll~ obt~ino~, and th~ ODNAsl w~ro ~xcis~d by 5 ~oo E~I dl~tlon. Clon~ HE~ 13 ~ontAir,çd thron E~o RI
iragmHnt~, da~l~n~ d 13A, l~ dnd ~a~ who~ç approxi-m~e o~zo~ wçro do~e~min~d na 0.6~ o.a nnd 3.~ kb, Qo~iv~aly . ~ach ~ the H~L ~3 ~co RI rrag~m~nth w~3 ln~rt~d .tnto ~he ~o RI ~lte ~ pUCl~, yi~ldlng plazmld~ pMa~a (13~), p~72ag ~13B) ~Ah~l pM2~0 ~l~C~-ClOne ~EL B cont~ln~d two ~oo ~I ~ra~nt~, d~Nignat~d and 8~, whoa~ ~pproxi~ate ~lZ8~ We~ t~to~n~d as 0.~ and 1.~ kb, r~upa~ ly. ~ho ~ E~o RI
~ra~mRnt6 were oAch ina~rtod i~o ~hc Eco RI ~lt~ o~
pUC19, yi~ldlng pl~m~ds pN2~ ) and ~292 18~ h~
lntact ~EL 13 a~NA and rlanXlng L~b~R ~1 DN~ w~
~x~ d by dig~ion with Rpn I And S~t I, ahd wa~
lns~rt~d into pUCl9 to ~on~t~ua~ plas~id ~M293. DN~
hy~rldi~ti~n analy~ia demon~tr~t~d th~t th~ pN~8~-10 ~0 cDNA hybridlz~d to th~ 13A and 8~ ~raym~nts, whor~ the pM28~-11 cDN~ hybridisad t~ ~he l~C and 88 ~ragm~nt~.
Re6trla~1On andonuol~a m~pA ~re prepa~ed for the 4~NA
frag~ent~ ln pl~miq~ pM2~ to pN293 . Th~ ~pe lndl eat~d that the l~L 1~ ~DNA ha~ a mucn longe~ 5' end ~nd 25 dift`~rs ~r~m tha ItEL B And 1~-1 cDNA~ ~t th~ n~ by an ~ppar~nt al~ernatlv~ spllc:ing ~v~nt. ~h~ c~mparatlve r~riction msp~ are ohown ln FigurY ~ . Ans.l~r~ls ol~ the ~E~ 13 DN~ B~qUenc~ ~howed thR pr~enc~ o~ ~oU~teen in~erna~ r~p~4t~. Each r~peat wa~ ~hown ta oont~in an 3 O lhte~rnal ~ons~n~U~ ~e~u0nae bo~nd~d ~n ~ither ~ld~ by ~n ~mphipathic hslix. A oomp~ on betw~en the ~ 8 In~
13~ NA Y~ na~6 sug~eEIted ths~ 12B-1 repre~nt~ ~n lnt~rn~l ~ragmen~ Or REI. ~ . s~quQno~ ~na ~ r ~hs MEL 13 cl)NA tFigur~ ~) al~o lndioatod th~ pr~3~n~e oY
35 stop COdOh ~Pter nuol~otid~ 3~1 ~Fig~ra 3).
A oompari~n of the ~EL 13 ~nd ~E~ ~ DNA
~quenc~s ~q~ted th~t ~E~ 13 m~y ~ 2 vari~nt ~r HEL

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11/16/89 17:32 ~2~6 6~2 6031 _ Seed ~ BerrY
33~ ~i ~.

. A computer genorated DNA se~on4a ~howlng the RE~
~qqu~nco ~olned ~o th~ C-t~nnlnal DNA llqquor~oe or ~EL P, dAsignated ~fEL 13/8 ~plioe, i3 ~hown ln ~l~ura 5. ~ DNA
soquano~ enooding tl~o ~fEL ~a npli~ gan~r~t6~d a~
5 ~ollow~. Pl~mId pN2~0 i~ dig~s~tad tl~h Eao R~ ~nd ,~t I~ to ioolato ~ 3 . s5 Jcb fxA~ nt oompri~in~ tll- s ' oodin~ ~Hqunn~e iexom H~L 13. Pl~mid pM~05, ~ompri~ln~
th ~DN~ ~r~ment oP ~fs~ 8 ~ubolorlad ~a ~ p~rtial E~ R~
fr~ nt into pUCl~ dige~tod wlth 1~ and ~ol I
1~ to iool~t~ tl7e ~pp2~0ximataly 0. ~ kb Xrsg~hQnt. Pla~mld p~a91 ie dlge~ted wlth Bal ~ ~nd Eoo ~I to i~ol~ta the ~pproxlm~tely 0.39 k~ ~r~ament cnoodin~ the 3' oodis~
region o~ ~EL 8. q`he Eqo RI - ~t ~I pM2Y0 ~ras~nen~, the ~at 1~ - sol I pM305 ~ra~ nt ~nd ~he ~o~ ~ - Eco RI
15 pMasl ~ragm~n~ nrR joined w~t~ E~o lU-~ln~i2~d ZM~4 (Ex~mple 4) to ~onotruo~ a ~4mm~1ian axpt~s~n Iv~tor.
~he in~arts ,~ra oh~raate~lzed ~cr orldnta~loh ~th r~pRat to the pro~ote~. A plasmld ~ont~1nlng the ~EL
l3/a ~pl loa D~A ~qu~nc~el in ~he oo~r~t o~i~nt~tion ln 20 th~ otor ZN~4 i~ d~slqn~te~l H~L ?3~8 oPll~o~ZMF4.

The ve~o~ 8eh~a9R w~ con~rllotod ao ~hown ln ~5 Flgura ~ ~rom ~m2~3. Plasmi~ ~em~29 is ~ pUCl8-4~d expr~sion veotor a~ntalniny a unlqu~ E~am ~ lt~ ~a~
ins~iDn o~ olonQ~l DNA ~e~w~en t~e ~OoU~ etallo-t~llonein~l promotor and SV40 t~n~crlp~on te~mln~to~
ahd an ~xpr~lon unit oontainlrlg ~h~ SV40 ~rly 30 prom~ter, ~ou~e dibydro~olate reduat~ n~ ~nd S~740 terhinato~. Z0~229 W~B modlrl~d tO ~ ata the two Eoo it~ by par~lal d~6re~lon ~itb ~ao ~2, bltln~ing with DNA pol~ r~se S (xlon4w fraf~meh'c) ~nd ~P~, and re-li~ation. Pl~estlon o~ the re:~ultin~ pla~ml~ wlth ~2 3S tiI ollow~ b~ llgation o~ thq linsari~d pla~nid ~
~am Hl-Eoo R~ ~dapt~rs ~eeul~ce~ ln ~ un~ ~u~ Eao Rl .
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11/15/~g 17:33 ~0~ ~8~ ~31 Se~d ~_~erry ~41 33~ ~

cloning ~ h~ res~lltant pla~mld wa~ d~ nut~d z~22sr~ .
~ hfl axpreg!~lon ~,rea~or ZM~ wa~ ~on~truoted .~ro~ Z~ R ~nd pDX ~agen et nl ., U. ~ . Pat~n~ No .

4, 784, ~50~ o~n in ~lguro 7 . 2~P12~R ~ dl~e~tod witb l~lnd ~II and Foo RI, and thl~ 920 bp ~aSrm~nt ~ont~itling tho ~40 nnd M~-l promotorlt wa~ rolno~#d~ ~h~
lar~a ~rbgment Or Z~m2~ wa~ thon ~oinud te tho ^'llDO bp Hind I~-Eao RI ~r~mont olr pDX, whl~h aon~alnel the SV~O
10 p~moter~nh4no~ th~ adonovlrua ma~or late promot~r, and a ~ o~ ~plloin igt~nls.
~ he HE~ 1~ oDNA w~J ln~ert~J ln~o th~ E~o RI
~lt~s or th~ ~nammalian cell ~xpr~slon v~otor3 ZN~4 and Z~mzz~ by ligation o~ tho ~ollowin~ ~ra~cnt3~ ~o Rl -~ rom pMZ~o~ X - Xma ~ r~om pM2~4, ~mA I
- ~oo ~ ~ro~ p~2~9-s and Eco RI-diq~tod v~o~or. ThR
in~er~ w~ro ~har~atarlsod ~r orlcnt~tion Wit21 re~p~
to th~ pro~otHr~ A pl~id ~ont~inih~ th~ oPN~ in ~h~
3~n~ ori~ntatlon ln æMa~ wa~ ~uelqnat~d ~M2~6-10.
(Pl~mid pM2~6-lo h~ been d~poGlt~d wlth A~riOqn ~ypO
~ulture ~ollaation, ~ook~ , Md., as hn ~L ~Qll H~101 trahsrorm~nt. ~ A pl~mid containi~g ~e ~D~A ln th~
~nt1~enaR o~lo~ation ln Z~4 ~a~ d~signated pM2~S-7.
plasmld ¢ontAlning thc aDNA in tha oen~e ori~ntatl~n ln ~5 ~m2~R w~ dRcignat~d p~297~4, ~nd a pl~6mi~ ~ont~ining the a~NA ln th~ ~n~ ns~ ori~ntatlon w~a d~ignato~
pM2~7-2 (Ta~l~ 1).
Baby h4m~ter k$dnq~ t~X) c~ ttk-t~3~ in growth m~lum ~ulb~cc~s minimu~ entlal ~di4m ~DNE~] c~ntainlng 10~ fet~l b~vlne e~rum, 2 glutamln~, 1 mM ~sdium py~uvat~, 50 ~g~m1 ~enicil~ln, 50 ~s~ml ~trept~m~cin, And 10 ~g~ml neomyc~) w~r~ ~e~de~
into 10 c~2 tis~u~ ~ulture di~h~ and ~r~wn o~rnlght a~
37~ wqre trah~ect~d wlt2~ pl~mid ~N~ tlsih~ th 35 ~alolum p~c~ph~te preU~plta~ieh hi~thod ~ aham 211d V~
~er E~, $bid). Tbre~ huhdr~d ~1 of 10 mM Trl~-~Cl, 1 ~M
ED~A, pH ~.0, cont~lning 0.5 M c~ , lo ~g pl~8-1d DNA

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11/15/~9 17 34 ~2~G ~82 ~31 Seed & BerrY [~l~t4~
2~033~
~6 ~nd 12 . 5 ,ug ~z~lmon aparm DNA ~ dd~d dropwi~e, wlth ~or"exing, to 300 ,1~l o~ 2x HB9 ~2~0 ~ NaCl, ~0 ~M
Hepe6, 1.5 mM Na2~1P04, pH 7.15) . ~be illixhlro ~a~ t at raom tomperatu~a ~or 30 mlnut~ t~ ~lloo~ proeipltat~
~ormatlon. six hund1-~d pl o~ prQ~olpit~to U~ ~dcled to ~a~h 10 qm2 dish o~ ~HX qnll~, und tb~ dl~h~ wor-l lnoubatod ov~rnlght at 3 )'¢. Th~ aultur~ m~ium and pr~olpit~te wlare a~plrat~-d :rrc~m tha dloh~ nd 5 ml 0 15~ glyaerol in ~MEN W~ added ~or 3 mlnuto~ . ~he 10 dioh~ w~rH wa~h~d ono~ wlth DMEM, ~nd 10 ml o~ Slrowth mudium waa addod. A~tar 24 hour~ ths cul~ur0 mcclium w~
ahang~d to suleatlon medium ~DMEM aon~alning 10~ ~t~l ~ovln~ ~erum ahd 250 nM m~thotr~Xat~). Sub~Qqu~ntlY, ~ne ~luct~on mediu~ wa~ ~hahge~ eV~ry 3-4 day~.
~olonl~ o~ ~ran~atQd e~ Drmea wlthln 10-14 d~y3 a~t~r ~r~n~feation. ~rAn~pQ~ant aol~nle~ Were t~yp~lniz~d and ~eplnted ln ~el~o~ion ~edlu~ Por ~ontinued p~ag~ o~ th~ tr~n~otant p~ol~. ~nu pool~
were assign~d the de~lgnation~ ohown ~n ~b~e 1.
~1 ; 2-22A pM2~6-7 ~An~i~4n~) 2-~2~ pM~96-10 (~enae) 2-22C ZM3~ Vooto~ aontrol 3-14~ pM297-a ~n~l~o~e) 3-14~ pM297-~ ~aen~) 3-14~ 2~a~9~ veCto~ ~sntrol Cell mem~ran~s fro~ ~ul~u~ed tran~P~a~ant pool 2~22A, 2-22~, and 2-22C wer~ ~re~Ar~d ~P~ llgand blot ~n~ly~l~ (G~aham ~nd Or~, Z~7439-42, 19~7~. Me~b~A~e pro~eln~ w~re ~x~l~e~ ~h po~y~aryl&mld~ ~15 an~ transbl4t~ed to nl~roa~llulo~
~bran~. Inoubation o~ nl~roc~llulP~ ~t~ip~ Wlth 75I-~DL3 ravealed ~he pr~a~a~ of ~ 110 XDa p~tein in . ''~' ,, ~ .

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11~16/8~ 17 35 ~2~6 682 6~)31 S~ed ~ Berry ~ 043 21~033~6 me~b~ane~ from ~11 thr~a tran~Sect~ht~. ~r~n~Yeata~t 2 22B ~n~) mambr~ne~ con~Aln~d thr~a tlmas more oY
thl9 p~ot~ln than ~lth~r 2-2~A ~anti-~eh~o) o~ 2-2ac ~c~n~rol) membrane~.
Mo~olay~r~ o~ ~ran~roct~n~ 2~a~ 2-22B~ ana 2-~ac wor~ r~hlll~d ~o O'C and inaubAtad with oith~
12~I-HUL3 or 125I-LDL to te~ ~or c~ urraoe ~indlng u~ the~u lipoprotoins. Roeult~ ind10~tod th~t tran~ROtast a-2a~ ~s~n~) bcun~ two ~c ~hro~ time~ more 1~5I H~I.3 t.han el~hHr tran~roctant 2-22A or 2-22c, whor~a3 blndlng o~ 1~5I-LbL w~ tho ~me ~or ~ll thr~
trsn~c4tant~
~ult~r~d monolay~ro of trAn0~e~t~n~ 2-22A, 2-22~, and 2-a2C were sterol-load~d ~y prelncuba~lon wi~ m~lum enrlche~ with ~holssterol, th~ pulse~ ~or three hour~ wl~h 31~-mav~lonolad~one to bio~yn~h~ ally radiola~el lntra~ellul~r poGls o~ ~terol. C~ w~ro then àha~d for rour ~our~ wi~h m~dlum ~ontalning ~ith~r nAtiv~ HD~3 or HDL3 prevlo~ly tr~ated with t~yp~in to ~0 dig~t apoprot~ins~ and th~ app~ar~na~ o~ radiolabeled ~t~rol 1~ th~ m~lum wa~ m~ r~d. Trypoin~tYo~tod HDL3 was u~d a~ A aontrol ~o monlt~r r~o~pt~r indop~nd~nt ~t~rol ef~lux~ sino~ th~e p~rticles do no~ lntera~t w~tn o~ll r~ceptor~. ~e~ul~s indioat~d th~ natlve H~L3 promo~d grea~er 31~-st~rol ~lUX f~o~ tr2n~fe~tan~
2-Z2~ than dld trypeln trea~ed ~3~ wher~s b~th tr~tHd and untroated HDL3 r~moved ~hc Pame ~mount o~
3H-~te~ol rro~ tr~n~reotant~ ~-aaA snd ~22C.
Cultured ~n~fectant~ 2~ , 2-2~, and 2-~2C
were in~ub~ed with hD~ containing 3H-ohol~teryl o~t~r6 to radiolabel intrA~ellular sterol pool~ vi~ a r~o~ptor-~dl~ted ly~o~omal pathway~ C811~ w~e the~ ~h~qd ~r ~our ho~r~ ~$th medlum ~ont~inln~ ~lth~r no llpop~oteln or B~h3, And th~ app~an~ o~ 3~ h~ me~lum 3~ wa~ ~a~ure~. In tho Prese~ae ~ 3, ~rano~actant 2-228 ~cr~te~ ~wo- to th~f~ld mora 3H~t~r~l than ~$ther tran5~eotant 2-2~A or 2-22~.

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11~15/8~ 17 35 ~206 682 6~31 Seed & Be~r~ ~ 044 201D3~6 .

Cholestorol-loa~d monolay~rs o~ t~hs~eotants 2-22A! 2-22B, and ~-22C wer~ lnaubat~d wltb ~edium contRinlng ~lther no llpoprot~n or ~D~I- A~ter ~ix hour~, cell~ wer~ pul~od ono hour wlt~ l4C~ololc ~old~
~nd inoorporation o~ ro~iola~4l lnt~ c~lular ahole~teryl ~t~rA w~ dete~mln~d. ~hla ao~y m~ouro~
~h~ r~la~lva deg~ou o~ d~plRtlon o~ lntr~c~llul~r pool~
of ~ree chal~t~rol ~o~g avail~bl~ ub~t~atq ~or the ~loro~om~l ~st~rl~icatlon ~nzy~. HD~3 Ohu~od a two- ~o ~o thr~e~old great~r dopletlon of thi~ oubotr~t~ po~1 ln tran~f~ctant 2-~3 than ln ~lth~r t~an~qt~nt ~-2~A or 2-2a~.
~mP~ P~o ~ ~lon ~ A~
~n"N~- ~ r~
SQl~ yloag ~llo w~r~ lyoo~nized wi~h phage clon~ 17~I-E3 es~entially as d3sorib~d ~bDvR. The h~brld ~-~al~otosid~-HDL-bindlng p~ot~ln~ wero ~PrG-lnduo~d and rc~olv~d by paly~crylRmld~ la~rophore-ao ~ Youn~ ~nd ~avis~ ibid. t SlXel~ and ~hn, ~Qg~~n~l- A~. SQ~I US~ 303~-42~ 1387). ~he h~b~ld protein WAO partlally purlfi~d u$ing a ~-q~lact~ol~
a~inlty ~ub~trat~ column (51gma Chamlcal C~, 9t.
~oul~, ~o.) o puri~iQd ~sin~ ~n ~ Q~ g~laeto~ldasG
~S antibody col~mn (Pro~g~ Bioteoh, ~dlson, Wia.) as s~ec~iQd by tho ~up~ ho par~lal~y puri~
p~otuin w~ usod s~ an Rnti~en for ~ntibo~y produatlon.
~ alb~o mloe.were irm#niz~ ~ultiple tim~ wlth th~ hyb~ld ~-~alaato~ida~ blnding pru~oln ~ol~ule.
Th~ purl~i~d solubl3 ~ol~aul~ wa~ blot~ed ontc n~t~o-oollulo3~ (s~hleio~r ~ S~h~oll~ 0.45 Pm). The ~lot~
w~r~ dri~d at 371~, and ~ho nltrooQllulo~ w~a ~ olved With D~S0. ~e s~mpl~ wa~ then mix~d ~ith Fr~und'Y
adjuv~nt ~n~ in~eo~ed intc~ 3 p~r~tone~ vit~ o~
the ~l~e. ~o obtAin poly¢lo~ ntI~a~ blood W~
remoYod from th~ mi~e ~y retro-o~b~t~ ds ~nd allow~d to olot, and tho ~e~um w~0 rR~ov~r~d~

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llrl5/~ 17:3~ ~0~ ~82 6031 Seed & BerrY ~045 33~

A ~oond ~ro~lp ~ mic~ w~ i~munlzad with p~r~Lally puriri~d m~mb~ne~ o~ boYlna ondothali~l C~118. coll mem~r~n~ is~ln~ed ~rom bovino endotholi~l ~Hll~ wero ~QpArAtRd by 9D~-PA~, th~h oloatrophor~tl-onlly tran~Hrr~d ~o nltrooollulo~u. ~ho nitro~ollulos~
~huo~ W~rQ lnaubuta~ with ~25I-lubal~ HD~. A r~lun ~ th~ nit~OURllU10~ ~huot oor~os~on~ln~ to tho lOO ~Da rango ~ tho ~D~ ~Ql Wn~ ~ound to bind the radlolab~lod ~ h~ ro~lon or th~ nitrOQ~llUlo~o ~huet abrr~pond-10 lng ta thi~ ~r~a W218 th~n cut out ~nfi U~d r~r lmmuniz~-tlon o~ the mioe. Tho nitroaollulo~o ~h~ot WA~ oolubl-ll~ed ln DM90, ~hon mlxod with F~Rund~ ad~uv~nt. Thls mlxtur~ wa~ injea~ed lnto th~ porl~on~l oa~ 6 a~ ~ho ~ice. ~h~ procRdur~ wafi rapoated ~Ultipl4 ~i~e~.
1~ ~olyolonal antiser~ w~r~ obtained ~y bl~diD~ the ml~
a~ ~xtraatin~ th~ sorum u~ing ~onv~ntion~l m~thod~.
Ilybrldom~ ~11 line~ w~re g~no~ted by ~uoln~
ly~pho~yt~ Prom ~ mouGR imm~ni~ad Wlth th~ i~olA~ed me~or~"e~ o~ bovine an~o~h~lial aoll~ Wlth th~ N~-l mous~ my~lo~ o~ll line ~A~C~ a].
~ o p~op~r~ $or th~ Pu~ion b~tw~n the immuni~d m~u~o aHll~ s~d the mou~e my~lo~a ~11 llno~
~ho ~ploen~a and lymph nod~l; o~ th~ i~muniz~fl ml~e wor~
r~movod and mina~d wl~h ~cisl~or~ ~n top o~ rln~-me~h 25 stainleq~ ntoel ~cr~r,s. ~he minaed tis~ue~ w~rq w~h~d through 'c~e ~lne wr~n~ in~o potri ~i~hel~ with 10 :ml R~PI 1640 (~RP~SI, GIBC0, I.aw~no~, ~a~o. ) q~he~ re~aind~r o~ the minc~d ~ Ub~ wa~ pr~ed th~ough th~ ~cr~nfi w~th a ~patul~, ~n~ the ~craen# w~Pr~ wa~h~d wlth 5 ml 30 ~PMI. To remo-~a ~ny remaining o~ t~rlal, the bottom~ ~ the s~r~an~ were ~crap~d and tha m~terl~l was add~d to the p~t~i ~iohes.
~ h~ atrainad ti~Rue~ wal ~ transf~rr~d to 5~ ml oentr~fuge tubes, The p~tri di~has w~e wa~h~d with lO
35 ml RPMI to r~r~ov~ any ~m2llning mat&rl~l~. 'rhi~ ce~l:
su~p~n~l4ns w~re c~n~rl~uged ~ 10 ml~u~ at ~00 x ~
~he sup~rn~ahto w~r~ ~lacard~d, ~nd ~h~ p~ t~ we~s '^~~~~ ' ~

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i ll/15/~3 17 37 ~2~6 ~2 6~31 Seed & BerrY ~4~
331~

~su~pQndRd ln 4 ml RPMI. A~tQr resuopen~i~n, 1 ~1 f~tal eal~ ~ærum ~lo~ r~on, Ca11~.~ w~ ad~'led t~
e~h tubs.
Th~ red blood c~11 oonte~minant~ ~r~ ly~d by 5 ~dding ~ t~ril~ ditltill~d watHr ~o th3 a~ u~n-~lon~. ~ho m1xturo~ Wor~ ~wirll~d qul~kly, Dnd S ~1 4 . 25~ N~1 wa~ add~Pd to ~aoh tUb~. ~!hb ~oll eU~p~n~ n~
wb~e q~rtrlru~d ~or 10 minut~ at 200 x g. 'rho uup~r-nU~unts Werb dl~aardod, ~n~ tho ce11 ~ t9 ~a~usp~ndod 10 in 10 ml RPMI .
r~mov~ Any remainln~ tl~s~;uo ~te-rlal ~ th~
~u~p~n~ions wer~ rilt~r~d through two 1~yer~ o~ at0ri1e gnuza lnto 50 ml tu~e~ . ~h~ centrl~uq~ ~ub~ an~ ~au w~:re rin~d with an addi~iona1 10 ml R~ . Dil~tion~ o~
th~ x~3ultaht o~ u6~nelon~ w~re ~oun~d wi~h h~macy~m~t~r to d~termine the yleld Or ~ymphocyte~.
Tho prep~red c~ we~e k~pt at ~oom ~mp~r~t~re ~or appro~i~ately one hour until r~dy ~or u~u.
~he N~-l mDu~ my~loma csll llne ~os u~d ~or 20 th~ ruqlOn. ~o opt~mi~R tho ftl~lon procdur~, th~ I~S-l line wa~ ~loned out to l~ol~t~ a clone wl~h A hi~h ~u~lon e~lolency. Th~ N~-~ aoll~ wcro olonod out by llmltlnsl dilutlon ln~o ~-well microtiter plates at An ~veraye of ~lv~ and ~n a~lls p~r w~ll in N~ edlum ~abl0 2) + 2.5 x 1o6 thymooytos/ml ~p~parQd a9 de~orl~d below). ~h~ pl~Q9 w~r~ in~ub~tod ~ 37'C, 7 ~2 ~ en dayg, ~A~LE Z
3~ e,~um P~r a 590 ~1 ~olution:
5 ml 10 ~M ~on~ential amino aci~
t~IB00, Lawr~nce, Ma~
5 ~1 100 mM ~odium pyruvate 35 ~IrV1ne, Santa An~, ~A~
5 m~ 200 mM ~-glutamin~ ~O~B~0) 5 m~ x P~nl~llllnJ5~r~pto~ycin~N~omy~ln ~ O) , i~.

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11/15/~ 17:38 ~2~ B82 ~3i Seed & B~rrY ~ 47 X1~33~L~

75 ml lna~tlvat~d ~et~l c)alr ~ru~
t 91 c~C~ C`~ O~1~ C~
1 9~ N~HCo3 Add RPMI lG40 ~G~CO, :CRwr~tno4~ Ma~a. ) to a totnl volumo o~ 500 ml.
~rllls~n hy ~lltrqtion through a 0.2Z ~ llt~r.
~I~Q2L~ ,StQ~
0 38 . $ m~ th!fmi~ine 136.10 mg hypoxanth~n~ ~lgma, ~t. Lo~3, Mo.) Dl~olve ~h~ tbyra~n~ an~ hypox~ntlne in di~tillltd H;~O ~nd 1~in~ volum~ up ~o lOO nll . Wa~ tn~ ~ol~tl~n to 60'-70'C to dls~olv~ th~ lld~. Att~r ~h~ l3011~ h~ve dl~olvod, r~n~u~t t~ volum~a to 1~0 ~1. St~lllz~ by ~lltration through a ~.aa ,um ~lltor. 8tcre ~roz~n ~t -~O'C~
2 0 ~9~Q~
17.~ nç~ aminopterin ~ dd ~terll~ di~ d wnt~r t~ th~ aminopt~rln an~
bring th~ volumo to so ml. Add 1 N NaOH dro~wl~a until 25 ~ch~ amlnopt~in di~olYea. Bring tha ~lhal voluln~ to 100 ml with dl~tilled ~2~ 8t~rl~ lzo b~ ratlon t}lrouyh n o. 2a ,~ llt~. 9~:ore ~ro2~n at -20'C.

50 hl 100~
s ml 1000X A stook 4s ml di~tillod El20 stsrili~e th~ ~olu~lon by fil~ratiq~n througt~ ~ O . ~2 35 ,~m ~ilt~r. S~r~ ~oz~n ~t -~O~C.

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' : , ll/15/~ 17 39 ~208 88~ ~031 Se~ err~ 4~

7 ml N~-l m~dium 2 ml ~Htal ~alf aorulD
~ ml DMSO

Mix th~ lrlgrodiQnt~ ~nd m~co ~rc~h ~or e~ah f r~q~ln~ ~
On cl~y 10~ tho ~qllo w~ro ~x~lned ml4ro~00pi-1~ o~ and ~rQQned ~or w~ oont;~inln~ sln~l~ oolonl~o.
4n th~ s~m~ d~y~ lOO ~l o~ rr~s~ iUIl~ conta~nln~
2 . ~ x 1o6 thy~oytu~/ml W~B addod to ~a~h ~ ll . on th~
~ourt~nth day, ~ight Or ~lle mo~t vlgorou~ly ~rowing ~ingl~ ~ol~rllo~ wer~ ahoo~n to ex~and ~o~ ~usion.
~5 ~l~o ~i~ht oandidat~ a~lon~a~ ~r~ ~r~o~r~rr~d to indlvldu~l 2q-w~ll plat-as oont~lnlng l.~-2 ~l NS-l ~edlu~ + 2.5 x 10~ thymo~yt~well. ~4e~e pl~te~ w~re ~alntainad a~ 3?'~ 7% ~2 and ~h~ c~ pllt ~t ~prb-priatQ int~vAl~ by bXpandin~ t~ ooll~ to ~ra~h ~llc oP thQ plate and addln~ ~r~h NS-l me~ nia prooe-dure was ~ep~ated uht~ th~a ~a~ a ~ur~lci~nt nu~b~ o~
c~ to ~xp~nd ln~o a 7~ om2 t~o~ua oul~u~ ~la~k.
thl~ point, 1 X 107 a~ rom th~ growing aulture~ Wo~
u~ed to ino~ulat~ n 75 a~2 ti~ua oultu~fl ~la~k ~ontain-2~ lhg 50 ml N~-l modium. ~hR ~1~8X~ w0~ lnc~bat~d A~
37'C, ~ Co~, untll the c~ r~ach~d ~ d~hsity o~ at le~t ~ x 105 aR1~ h~ 0~118 wer~ ~hen harv~ot~d by ~en~rl~u~Ation and dilu~ed t~ ~ con~ntr~tlon o~
ap~roximately 5 x 10~ with ~r~eElng me~
(~a~lo 2~ ~h~ oel~s wero divL~3d into 1 ~1 ~liquot~
a~d fros~n ~epwl~ Pl~t at -~0-0 and th~n at -130'C.
~ o ~ y th4 alon~ o~ NS-~ c~ o~ ~usi~n e~ficieno~, one vial for ~oh clon~ was ~uiakly ~haw~
~n wa~r held at 37-C. ~he aell~ were lno4ul~t~d ~n~o ~ Xs c~nt~lnln~ N5-1 ~dlUm ~o ~ cona~tration o~ ~ x ~05 aells~l. The a~ wer~ g~own ~t 37-~ ~ with 7S ~2 The a~ w~re cut ~ck to 2 x 105 aell~ml ~ally. on~

" ' ' "' '. , ,, ` " ' '. '. ' ~

.` , ' ~

11/15/~g 17 39 ~2~16 G82 ~3~)31 S~ed ~ BerrY ~1~4~
2~)33~6 day b~ora fu~ion, two 75 om2 ~lnsk~ contRlnlhq 50 ~tl o~
call culture w~r~ s~t up. ~aoh o~ th~ aan~ldAto ~$-1 olon~ W4~ iXI~ wltkt lmmuni~ d mou~o ~le~n o~ nnd Yu~d a~ d~rl~od ~low.
~h~ r~auLt~ o~ the ~usion~ oh~wod tl-~t on~ af tho sln~ oolony iAolnto~ showod ar~ aren~od ~uclon Q~1aionay . Tbls alono W~o do~i~nat-d c:lono ~ and w~
~ubse~uontly 115~d ~ tho my~l~rna a~ll linc ln ~urch~r ~u~ion~.
~hymu~ ~lAnd~ ob~Pinad ~rom baby miao wor~ tho sourao of th~ ~hyrn~ayte c which w~ uood ~ P ~edar l~ye~ to aondition tho oul~uro ~din rOr th~
~u~ion~. ~hymu~ ~lnnds were obtalhod ~rom 3- to 4-w~X-old ~alb/e mioe. ~he ~hym~ glsnd~ wera rih~Dd wlth N-~-l$ ~ m~dium and mlno~d on n ~ine-mo~h, ~lnl~u~ ~tfl~l screen W~th ~ls~o~ hu min~ed ~ u~ were rln~0d throù~h thR ~cr~n with 10 ml N~-l medlum lnto a pqtri dl~h. Thq thymu3 ti~sue wa~ th~n pre~d through th~
~sr~n with a ~patul~ lnto ~h~ pbtri dl~h. Tho ~ar~n wa~ then wa~hed wlth ~0 ml N~-l m~dium. ~he ~o~ O~
thR s~e~ wn~ sGr~ped to r~mov~ any a~ho~ ti~au~, and th~ tl~u~ w~u pool~d in thu petrl di~h. Tho ~tr~lned ti~u~ was trah~ss~ed to a 50 ~1 ~entXi~u~o tube throu~h ~W0 l~yers o~ ater~le srauz~. ~h~ p~3trl ~l~h ~nd 25 gau~u wer~ rln~ed wi~h arl ~dditional 10 ~1 N9-1 m~dium.
~he oel~ were ~ntrl~uged ~c~X 10 Ininutn~ ~t 200 x g.
The eupernatan~ ~er~ dilsc~rde,d ~nd ~he p6~
re~u~p~n~ed ln lo ml Na~l m~dlum. Oilu~ion~ o~ thc oell ~u~pf~n~ion~ w~r~ count~d ~ ln~ a bomA4ytomotor~ Th~
30 y~ld ~Xom tw~ tbym~ glandsl wa~ routin#~y ~bo~t 400 mlll ion cell~ Thl3 ~ell~ Were ~to~ed &t ~o~m te~hp~r~-tur~a unt~ 1 r~ady ~or u~e.
To p~epAr~ Por fu~ion, t~0 NS~ lon~3 F
a~ r~wn a~ de~c:ribed ahclve~ wc~ ~rAn~ r~d to 50 35 ml a~ntriru~e t~lb~ nd oen~xl~uged ~or lo mlnuteo ~
200 x 9. The ~up~rn~t~nt~ w~o ~i~aa~d~, aJ~d t~ aell pt3~ let~ w~re re~usp~nded ~nd ao~ln~d $n 10 ~1 R~

' :

.

. . :
.,, .' 11/15/~g 17 4~ ~2~ 2 6~31 .SQ~ & BorrY ~ 05~
~0(1 331t^j The c~lL ~uep4n~1~n wn~ 04unt~d with a h~lRacyto~ot~3r to moni~or th~ oell vlA~llity ~E~ d~t~Min~d ~ trypan blus exclu~ion. q~he a~ llity Wa~ dot~nin2d to b~
qreata~ than ~5~.
For ~u~lon, ~.5 x 107 NS-l clon~ U11B wero addad to tha p~epared l~ nl~d moua~ ~oll~ ~pYepar~d ao de~ori4a~ ~buve) . ~rh~ mixed o~ W~rl~ aentri~ug~d ~o~
lo minuk~a at aoo x g. ~he sUperP~tant; w~o ru~oved by ~pir~tlon w1th ~ Pa~teur pipo~ attn.4hod ~o a vaouu~
~0 11n~. ~h~ ~oll pell~t w~ ru~u3p~nd~a in lO0 and warmod ln a wat~ ~ath At 37^~-one ml 50% ~olysthyl~na glycol (p~a-~s ~ulu~ion in P~P~II, W~R y~l Ad~ust~d wlthin th~ r~nge o~ p~
7.0 to pH 8~0 Wlth 20 ~ul 1% ~o~ium blcar~orlata. ~he ~G
~s solutiol~ waa added to the a~ u~penslon ovor q perl~d o~ ~no mir~ute wit~ ~ehtl~ ~tlrring. ~ho ~ ponilon w~
~tirr~d for an addltion~l minUt~. on~ ml o~ NS-l m~:dlulP
wa~ add~d oYor a pericd o;~ Ohe r~ u~ h gontl~
8tirrln~. ov~r an ~ddltle~n~l p~rl~cl o~ one ~inute, 1 ml 20 o~ l medium was added to thl3 5u~p~n~ion with g~nt~o stlr~ g. Ei~h~ ml N~-l mad~um was addod over a p~rlod o~ t~o ~inut~s. Tho ~u~p~Dsion was oentrlrug~ t 12~
x g nt ~oo~n t~mp~r~ur~ ~o~ 10 minUto~. ~ho supernatant wa~ di~onrd~d, nnd 23 ~l NS~ dium wa~ addod to gontly 25 re3uspond ~h~ pelle~. ~h~ aells war~ harve~ed ~en~ly lnto ~ plp~t qnd tran~rred ~ a 175 c~m2 rl~k~
Four hundred million thy3nooyt~ ~prepAra~ a~
~o~ w~e adaed to th~ rlask. Th~3 vol~ a w~ ad~ustqtd ta 160 n~l with N9-1 m~dium, and ~he mlx~ rra wa~
30 ln~:ub~d n~ 37'C, 7t CO;! J~or tw~ to ~ur ho~r~.
A~t~r lnaubA~lon, 3.2 ~nl of 50x ~A~ ~ab~Q 2 wa~ addad. Th~ oell ~usp~n~lon wa~ tr~n~rlar~d to ~i~h~
96-w~ll cul~ur~ pl~tes a~ 200 ~l p6~r w~,l, u8ihg plp~t tip~ with ~ch~ ehds ~ut o~ to ~vo~ rln~ ror~es.
35 ~he plat~ w~3r~ lncubated at 37'C ir~ a ~umid inoub~tor with 7~c ¢C~2~ me plate~ w~re examir~ed r~1~ro~c~piq#11y aft~ ~hr2e d~ys tv d~t~rmin~ fu~ion ef~ n~y, w~th ., :.... ~5 '~ .

,~

Ij~ 051 11/1~8~ 17 41 ~7~3~ 6~2 6~31 Seecl & Berr~
20033~L~

th~ oxpecthtlon o~ ~pproxlmately ~ive hybrldoma aolonl~o p~r well. ~he oell~ w~r~ ~3d r~ r ~eYsn d~y~ by r~P].ACing 100 111 ai! t~le mSltiiUm Wit~ r~oh N~-l m~dlus~
con~ainlng 1 x ~A~ and ~ . g x 106 ~h~ocy~e~l p~ ho 5 hy~ridom~ w~r~ te~ed betwo~n d~y~ 9 ~nd 14 ~or tha p2~0duotion of 4p~0ifio ar~tibodl~.
Thc hybridom~ o~ w~rl3 ~arc1~h~ by We~tern blot (T~w~ln et ~ ibid. ~ for tho produatian aP
an~lbodl~o to ~ha b~vlnu ~ndotheli~l ca~l moh~rane~.
10 E~ovln~ Rhdoth~ mt\r~nca w~ 0pa~atHd by 9r)S-PAaE, ela~trophor0~1~ally tr~nsrerred ~o nltroodlulose, and ~hR hi~roa~llulo~ w~ in~lbated ~n bl~klng bu~er to pr~v~nt non~p~ io blndlng l~y ~b~ ~ntibodi~.
Seot1Qn~ o~ ~h~ nltroc~ ose ~heet w~rOE thon inoub~te~
~5 witl~ ~onditian~d media P~aln ~h~ h~brldema o~ll llne~, and ~h~ prel34na~ o~ ~paoirio antibody w~u d~t~hnined u3lh~ A~C-peroxida~e ~V~to~ ~borAte~tle~, ~urll~ g~no, Call~. ~ a~ desorlhed by tho m~u~ U~Qr. 8~rer~l hybridc~mA aells ~eor~tln~ antlbody ~eo ~ndotheliAl 2 0 mQmbranas w~re det~t~d .
~ h~ ~ntlbodi~o ar~ clharactRri~e~ detectlPrl oP ~he llO )cl)a ~DL-bindinç~ prot~in ~nd ~or th~l r~co~ni-tlon o~ ~he reao~blnant ~L-blndln~ pr4t~in.
~ h0 hyb~idoma c~ll lln~ produclng ~ntlbodlos o~ lntere3t aro ~lon~d 4y limltlng ~l~utl~n to obt~ln coloni~a o~ a~ orl~inatinq ~rom sln~la ~ . Th~Ae ~olon~a a~e oxpand~d in CUl~U~ ~or ~nar~tlon o~ lar~
amoun~ of Antibo~y.
~ .6 - H ~
Antl-E~DL ankil~odie~ ~r~ ~re~ 3dl f~r ~0~9i~1e antaqoni~tia ~nd agonl~ p~opertl~ pXe~inq ~DL xeceptor in A ~on~tltutive m~nne~ o~ln~
aor~lQ endot~eli~l a~ or a~lls tr~n~reat~d wlth an ~xpr~ioh v~ator en~odin~ ~n H~L-bindinq pro~in are p~ated on miorQtlte~ pla~es ~nd~ ~xpo~d to t~e anti-bodle~. Th~ oalls ar~ theh in~u~at~ with l~5T-HD~ to ~ ' ::

~ ~ ~ :

` 11/15/~ 17~4~ 25'2~6 B82 6~31 _ Seed ~ Berry ~ ~5~
~0~3~6 ~6 lden~l~y antibodle~ that ao~pet~ r~ HDL bindlng to lntact ael 1~, Parallal cultur~ Or c~lla ~r~ u3ed ~or tho prep~atlon Or m~b~na pro~einn rOr llg~nd blot immunobl~t analy~ia. M~m4~an~ protulh~ nre bound to nl~roo~llulo~ ~trlp~, And the 8t~ipJ n~ lnoubhtod wlt~
anti~ody, th~n ~xpo~d ~o lZ~I-H~ tD det~rmlno whloh ~ntlbodie~ bloa~ HD~ binding to th~ ~aolnted ~roteln~.
Anti~odie~ thdt blnd t~ lsol~tod HD~rb~n~n~ protolno ~ithout bloc~lng M~L hind~n~ are d~l~n~ed a~ non~
blocking antibodl~ oth ~looking Anq non~blooklhg Antibodl~s ar~ u~ in ~U~4tl0nQl a~ayn to a~ th~lr ablliti~s to aat ~ ~D~ ~gonl~t~ o~ ~t~gDnlet~. For the ~unctional a~Ay, ~ultu~ed c~ [o.~ " ~lbrobla~t~, lS ~nd~t~olial cells, or ~o~opha~) hr~ ~r~Ated ~lt~ 3H-m~ onolautonR to r~diol~b~l lnt~Aacllul~ pool~ o~
oholestorol. ~bo o~lls are then e~po~d to ~edl~
cantalnlng antlbodi4~ plu~ ~lth~ nati~ or oth~r ohol~t~rol aoceptor partlcle~. O~h~r ec~ptor p~r~icle~ lnoludo ~h~ia~lly tr~at~ HD~ thst no lon~Qr lnt~r~s~ wlth HD~ rocoptor~ b~t ~o Rbl~ to ~ct chol~storol aocePtors ~rlnton ~e al., 6a:4~-503, 19~6). ~ho a~o~ o~ an~ibodi~ on th~
x o~ labolod ~erol indiaat~ ~oni~tlu and ~S ~nt~gon~tla prop~rtio~. ~bOB~ antlbo~le~ ~hat ~lo~
tbo o~lux-~timUl~tlng ~P~at~ o~ ~D~ ar~ ~DL
aAtagohlsts~ tho~o tha~ st$~ulAte ~o~ol ~ x ln th~
pre~en~o o~ ~ st~ol acaop~o~ th~t ~oa~ n4t ~i~d HD~
rea~ptors qre HDL a~ohl~t~. ~h~ 0¢~0~ o~ th~
anti~odi~s on tran~locatlon of ~Idiulab~lQ~ ~t~ro~ f~o~
ln~r~a~ it~ to the c~ urraoe ~n t~:p~e~enoe ~ ~b~en~e o~ ~ative H~ ar~ ~ea~ur~ by d~to~lni~
t~ei~ Abillti~s to ~ti~ul~t~ the a~no~ o~ ~t~rol t~om et~r~l-lad~n ma~roph~g~ roph~g~ lo~g~d wlth 3s a~etylated L~-dRriv~d ohole~e~ol ~re :$nQ~'o~t~d ~h ~di~m oontainlng ~hs te~t anti~ody plu~ eitne~ ~a~ive or che~lc~lly modl~isd ~D~ pa~ticla~, And the ~o o~

. ~, ~ ..
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.

11/15/8~ 17 43 ~208 B.~2 60~1 Se~d ~ Berry ~ ~53 20~33~L6 cJisapp~aran~c Or O~cQ~ intr~aQllUlar ~torol i~ da~t3r-mill 1d U3 U fllnotlon Or timt~. q~ogq ~tudla~ p~ovida an ln~icat:lon oP tht~ t~lernpoutla potcn~lali o~ individunl nqonlst ant~ dl~ in promotin~ th~ clourAno~ o~ exco~
sterol rnonl ~onm ooll~ o~ tho ~rtt~ry wnll.
From th~ I~oro~oin~, it will ba approt~lated tl~At, ~ltho~h apooirl~ ~m~ot~imont~ o~ ~h~ inventio hAVe b~en doaori~od horoin ror purptia~ o~' lLlu~trnt~on, v,lrlous modl~ioAtloll~ m4y bo m~do ~lthout dovi~tlng ~rom 10 tha spirit and ooopa o~ tha invontion. ~a~ordl~gly, tho inv~ntion i~ not li~ni~t~d oX~Qpt ~9 by the appended elaims.

3~5 ~tl~/v~

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, ~.

., ' ". ' .~ '.
.,, ' ~' ~

Claims (18)

1. A substantially pure mammalian protein having the following characteristics:
a) molecular weight of approximately 110,000 Daltons;
b) binds HDL3;
c) binds apoA-I proteoliposomes and apoA-II
proteoliposomes;
d) contains approximately 10% carbohydrate by weight; and e) an isoelectric point between about 5.8 and 6Ø

2. A protein according to claim 1 wherein said protein comprises a sequence of amino acids selected from the group consisting of the amino acids sequences as shown in Figure 4, from methionine, amino acid number 1, to glutamic acid, amino acid number 1195, Figure 5, from methionine, amino acid number 1, to isoleuceine, amino acid number 1292, and Figure 3, from glycine, amino acid number 2, to glutamic acid, amino acid number 125.

3. An isolated DNA molecule encoding a protein having the following characteristics:
a) molecular weight of approximately 38,000 Daltons;
b) binds HDL3; and c) contains approximately 331 amino acid residues.

4. A DNA molecule according to claim 3 wherein said protein comprises the amino acid sequence of Figure 1, from glycine, amino acid number 2, to glutamic acid, amino acid number 331.

5. A DNA molecule according to claim 3 wherein said molecule comprises a sequence of nucleotides as shown in Figure 1, from nucleotide 378 to nucleotide 1370.

6. A DNA molecule comprising a sequence of at least about 45 nucleotides of the DNA sequence shown in Figure 1, said sequence encoding an immunogenic peptide.

7. A substantially pure protein having the following characteristics:
a) molecular weight of approximately 38,000 Daltons;
b) binds HDL3; and c) comprises a sequence of amino acids as shown in Figure 1, from glycine, amino acid number 2, to glutamic acid, amino acid number 331.

8. An isolated DHA molecule encoding a protein having the following characteristics:
a) molecular weight of approximately 100,000 Daltons exclusive of carbohydrate;
b) binds HDL3; and c) binds apoA-I and apoA-II proteoliposomes.

9. A DNA molecule according to claim 8 wherein said protein has an isoelectric point between about 5.8 and 6Ø

10. A DNA molecule according to claim 8 wherein said protein comprises a sequence of amino acids selected from the group consisting of the amino acid sequence as shown in Figure 4, from methionine, amino acid number 1, to glutamic acid, amino acid number 1195, Figure 5, from methionine, amino acid number 1, to isoleuceine, amino acid number 1292, and Figure 3, from glycine, amino acid number 2, to glutamic acid, amino acid number 125.

11. A DNA molecule according to claim 8 comprising a sequence of nucleotides selected from the group consisting of the nucleotide sequences as shown in Figure 4, from nucleotide 155 to nucleotide 3739, Figure 5, from nucleotide 155 to nucleotide 4030, and Figure 3, from nucleotide 10 to nucleotide 381.

12. Cells transfected or transformed with an expression vector comprising a promoter operably linked to a DNA sequence, according to any one of claims 3-5 or 8-11.

13. The cells of claim 12 wherein said cells are yeast cells or cultured mammalian cells.

14. An antibody that binds to a protein according to any one of claims 1,2 or 7.

15. A method for detecting a genetic defect in an HOL-binding protein in a patient, comprising:
isolating DNA from a patient;
cleaving said DNA with a restriction endonuclease to produce DNA fragments;
hybridizing said DNA fragments to a DNA molecule comprising a nucleotide sequence selected from the group consisting of:
a) at least about 18 contiguous nucleotides of the sequence shown in Figure 1;
b) at least about 18 contiguous nucleotides of the sequence shown in Figure 3;
c) at least 18 contiguous nucleotides of the sequence shown in Figure 4;
d) at least 18 contiguous nucleotides of the sequence shown in Figure 5; and e) a sequence of about 18 nucleotides complemen-tary to sequence (a), sequence (b), sequence (c) or sequence (d); and detecting a pattern of hybridization of said DNA
fragments and said DNA molecule, wherein said pattern is diagnostic for the presence or absence of a genetic defect in HDL-binding protein.

16. An immunogenic polypeptide comprising a portion of an amino acid sequence selected from the group consisting of the amino acid sequence of Figure 1, the amino acid sequence of Figure 3, the amino acid sequence of Figure 4, and the amino acid sequence of Figure 5, wherein said polypeptide is at least about 6 amino acids in length.

17. An oligonucleotide which hybridizes to a nucleotide sequence selected from the group consisting of the nucleotide sequence of Figure 1, the nucleotide sequence of Figure 3, the nucleotide sequence of Figure 4, and the nucleotide sequence of Figure 5, wherein said oligonucleotide is at least about 18 nucleotides in length.

18. An oligonucleotide comprising a portion of the nucleotide sequence of Figure 1, Figure 3, Figure 4 or Figure 5, wherein said oligonucleotide is at least about 18 nucleotides in length.