United States: (12) Patent Application Publication (10) Pub. No.: US 2011/0318847 A1

US 20110318847A1
( 19 ) United Sta tes

( 12) Pa tent Applica tion Publica tion ( 10) Pub. N o. : US 2011/0318847 A1
SUN DREHAGEN ( 43) Pub. Da te: Dec. 29 , 201 1
( 5 4) CVD ASSAY ( 30) F or eig n Applica tion Pr ior ity Da ta
( 75 ) I nv entor ; Er ling SUN DREHAGEN Os lo Dec. 20, 2002 ( GB) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0229 747. 1
( N O) Publica tion Cla s s i? ca tion
( 73) As s ig nee: Ax is - Sh ield ASA, 05 10 ( N O) ( 5 1) I nt- Cl
G01N 33/82 ( 2006 . 01)
( 21) Appl. N o. : 13/170, 387 3821, 15 /00 ( 201101)
( 5 2) US. Cl. . . . . . . . . . . . . . . . . . . . . . . . . . . 436 /5 01; 9 77/773; 9 77/9 20
( 22) F iled: J un. 28, 2011 ( 5 7) ABSTRACT
. . An a s s a y meth od a nd k it f or th e detection of potentia l f or
Rela ted U' s ' Apph ca tlon Da ta CVD or pr opens ity to CVD in a h uma n or non- h uma n a nima l
( 6 2) Div is ion of a pplica tion N o, 10/ 5 39 , 7 9 7, ? led on Dec, s ubj ect, s a id meth od compr is ing a s s es s ing th e concentr a tion
19 , 2005 , ? led a s a pplica tion N o. PCT/GB03/05 6 07
on Dec. 22, 2003.
of ca lpr otectin in a ca lpr otectin- conta ining s a mple ta k en
f r om s a id s ubj ect.
Pa tent Applica tion Publica tion Dec. 29 , 2011 Sh eet 1 0f 7 US 2011/0318847 A1
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CVD ASSAY
[ 0001] Th e pr es ent inv ention r ela tes to a n a s s a y meth od f or
detecting potentia l f or or pr opens ity to ca r diov a s cula r dis ea s e
( cv D) in a s ubj ect, e. g . a h uma n or non- h uma n a nima l, es pe
cia lly a ma mma l, a nd in pa r ticula r to a n a s s a y meth od Wh ich
ma y be us ed to detect a potentia l f or CVD or a pr opens ity to
CVD bef or e th e ons et of CVD s y mptoms noticea ble by th e
s ubj ect.
[ 0002] CVD is a ma j or s our ce of ill h ea lth a mong th e
h uma n popula tion. I n 19 9 a ppr ox ima tely 40% of a ll dea th s in
th e Wes ter n Wor ld Wa s a r es ult of CVD ( i. e. 1 in ev er y 2. 5
dea th s ) . F or 2002, it is es tima ted th a t, in th e USA, ov er one
million people Will s uf f er f r om a neW or r ecur r ent cor ona r y
a tta ck , a nd mor e th a n 40% of th e people s uf f er ing f r om th es e
a tta ck s Will die. M a ny of th es e people Will die s uddenly
With out ev er h a v ing been h os pita lis ed or tr ea ted. M a ny Will
not h a v e r ea lis ed th a t th ey Wer e s us ceptible to CVD.
[ 0003] Ea r ly or pr e- emptiv e tr ea tment s uch a s a ch a ng e of
diet, r eduction or ces s a tion of s mok ing , incr ea s e in ex er cis e,
r eduction of body Weig h t, etc. , h a s , h oWev er , a h ig h s ucces s
r a te of pr ev enting CVD or r educing th e pr opens ity to CVD.
Th us if CVD or potentia l f or or pr opens ity to CVD ca n be
detected, ef f ectiv e tr ea tment is a v a ila ble.
[ 0004] Th er e is a ccor ding ly a need f or meth ods Wh ich ca n
be us ed to detect CVD a nd es pecia lly , th e potentia l f or or
pr opens ity to CVD, bef or e th e dis ea s e h a s pr og r es s ed bey ond
th e s ta g e Wh er e tr ea tment ( eg ch a ng e of lif e s ty le a nd/or
h a bit) is r outinely s ucces s f ul. I n pa r ticula r , th er e is a need f or
meth ods Wh ich ca n be us ed to detect CVD a t th e ea r ly s ta g es
Wh en th e s y mptoms a r e not a ppa r ent to a s ubj ect or to a th ir d
pa r ty , eg a ph y s icia n, i. e. meth ods f or tes ting s y mptom
f r ee s ubj ects a r e r eq uir ed.
[ 0005 ] Such meth ods ma y be us ed to s cr een th e g ener a l
popula tion ( i. e. in ma s s s cr eening ) or a t- r is k g r oups With in
th e popula tion, e. g . ma les ov er 40, Wor k er s in h ig h s tr es s j obs ,
indiv idua ls With unh ea lth y diets , indiv idua ls s uf f er ing f r om
clinica l obes ity , s mok er s , etc. I n ca s es Wh er e potentia l f or
CVD or pr opens ity to CVD is dia g nos ed, pr e- emptiv e tr ea t
ment ma y be g iv en a nd/ or th e pa tient ma y be encour a g ed to
ma k e a dj us tments to lif es ty le a nd h a bit.
[ 0006 ] L ik eWis e, Wh er e potentia l f or CVD or pr opens ity to
CVD is detected, a pa tient ma y be s ubmitted to f ur th er tes t
ing , eg us ing mor e ex pens iv e or time cons uming tech niq ues ,
s uch a s ECG, With a nd With out ph y s ica l a ctiv ity , r a diois otope
ima g ing of my oca r dia l per f us ion, X - r a y ( e. g . CT) my oca r dia l
a ng iog r a ph y , M R my oca r dia l a ng iog r a ph y or per f us ion
ima g ing , etc. Th us by con? r ming th e pos s ible pr es ence of , or
potentia l f or , or pr opens ity to, CVD by us ing th e ch ea p a nd
f a cile a s s a y meth od of th e inv ention in a n initia l s cr een ( eg
in a ma s s s cr een of s y mptom- f r ee h ea lth y s ubj ects ) th e
lik elih ood of detecting or identif y ing undis cov er ed CVD, or
potentia l f or CVD, bef or e h ea lth da ma g e becomes ir r ev er s
ible is incr ea s ed Wh ils t, a t th e s a me time, unneces s a r y us e of
ex pens iv e a nd time- cons uming tes ts is limited.
[ 0007] By CVD is mea nt a ny condition of th e h ea r t,
a r ter ies , or v eins Wh ich dis r upts th e s upply of ox y g en to
lif e- s us ta ining a r ea s of th e body s uch a s th e br a in, th e h ea r t
etc. Ex a mples of CVDs a r e a r ter ios cler os is , a cute my oca r dia l
inf a r ction, a ng ina pector is , is ch emic h ea r t dis ea s e, cer e
br ov a s cula r dis ea s e, s tr ok e, s uba r a ch noid h a emor r h a g e,
Dec. 29 , 2011
intr a - cer ebr a l h a emor r h a g e, cer ebr a l inf a r ction, cong es tiv e
h ea r t f a ilur e, a ng ina , h ea r t a tta ck , ca r dia c a r r es t a nd a r r h y th
mia .
[ 0008] Th e pr es ent inv ention is ba s ed on th e s ur pr is ing
? nding th a t th e pr otein ca lpr otectin is a us ef ul ma r k er or
indica tor of potentia l f or CVD or pr opens ity to CVD bef or e
th e ons et of CVD s y mptoms ( i. e. in s y mptom- f r ee s ubj ects ) .
I n pa r ticula r it h a s been s ur pr is ing ly f ound th a t a bnor ma lly
h ig h ca lpr otectin lev els in v a r ious body ? uids is indica tiv e of
s us ceptibility to CVD bef or e th e ons et of CVD s y mptoms is
a ppa r ent to a s ubj ect or to a th ir d pa r ty ( eg a ph y s icia n) .
[ 0009 ] F or th e a v oida nce of doubt, th e ter m ca lpr otectin is
us ed h er ein s y nony mous ly With L I pr otein , M RP 8/14",
cy s tic ? br os is ( a s s ocia ted) a ntig en ( CF A) a ndca lg r a nulin .
[ 0010] Ca ipr otectin ex is ts in both dimer ic a nd tr imer ic
f or ms . As a dimer , ca lpr otectin compr is es th e poly peptide
ch a ins S100A8 a nd S100A9 . As a tr imer , ca lpr otectin is a 36
k Da h eter otr imer ic pr otein With tWo h ea v y ( 14 k D) a nd one
lig h t ch a in ( 8 k D) non- cov a lently link ed.
[ 0011] Ca ipr otectin is a ca lcium binding pr otein a nd Wh en
bound to ca lcium, ca lpr otectin is r es is ta nt to h ea t a nd to
pr oteoly s is . Th is ma y a lloW f or a Wide r a ng e of a s s a y tech
niq ues a nd conditions to be employ ed.
[ 0012] Epitope ma pping of ca lpr otectin s h oWs th a t a nti
bodies With s peci? city f or th e complex a nd/or its s ing le pr o
tein ch a ins ma y be pr oduced. At lea s t f our s epa r a te immuno
g enic s ites h a v e been s h oWn to ex is t on th e ca lpr otectin
complex . Some a ntibodies r ecog nis e eith er th e h ea v y or th e
lig h t ch a in, Wh ils t oth er s r ecog nis e both .
[ 0013] Ca lpr otectin is f ound in cells , tis s ues a nd ? uids in a ll
pa r ts of th e h uma n body a nd is der iv ed pr edomina ntly f r om
neutr oph ils a nd monocy tes . Ca lpr otectin is pr oba bly pr es ent
in a ll indiv idua ls s ince a mong s t mor e th a n 5 , 000 indiv idua ls
tes ted, no ca lpr otectin f r ee indiv idua l Wa s f ound. Ca ipr otec
tin is a ls o f ound in r a ts , mice, r a bbits , s h eep, ca ttle a nd pig s .
I t is th er ef or e a n a bunda nt ubiq uitous molecule.
[ 0014] I n v iv o, ca lpr otectin is inv olv ed in numer ous bio
log ica l f unctions including intr a cellula r s ig na l tr a ns duction,
neutr oph il a ctiv a tion, inh ibition of intr a cellula r enZ y mes
inv olv ed in cell pr olif er a tion, a ntimicr obia l a ctiv ity a nd in
neutr oph il def ence. Ca lpr otectin is a ls o a r eg ula tor y pr otein
in in? a mma tor y r ea ctions a nd in th is r ole ma y f unction to
s timula te immunog lobulin pr oduction, ch emota ctic f a ctor
a ctiv ity a nd neutr oph il immobilis ing f a ctor .
[ 0015 ] Wh ils t body ? uids pr oba bly a lWa y s compr is e
ca lpr otectin, th e concentr a tion of ca lpr otectin in v a r ious body
? uids h a s been f ound to ch a ng e, f or ex a mple, to incr ea s e, in
a number of dis ea s e conditions ( e. g . in? a mma tor y , inf ectious
a nd ma lig na nt dis ea s es ) . Th us mea s ur ement of th e concen
tr a tion of ca lpr otectin in body ? uid f r om pa tients s uf f er ing
f r om s uch dis ea s e conditions ( i. e. in indiv idua ls s h oWing
s y mptoms noticea ble to th e s ubj ect a nd/or to a th ir d pa r ty )
a nd compa r ing th e ca lpr otectin concentr a tion deter mined to
th a t in body ? uid f r om, f or ex a mple, a h ea lth y ( i. e. a non
dis ea s ed) s ubj ect ma y be us ed a s a mea ns of dia g nos ing s uch
dis ea s es .
[ 0016 ] F or ex a mple, Wh ils t th e s y mptoms of ba cter ia l a nd
v ir a l inf ections a r e v er y s imila r a nd dia g nos is f r om th eir
s y mptoms a lone ma y be di? icult, th e concentr a tion of ca lpr o
tectin in th e pla s ma / s er um of th e inf ected s ubj ect incr ea s es
a ppr ox ima tely 1 to 2 times With v ir a l inf ections but a r ound 1
to 18 times With ba cter ia l inf ections . Th us th e s ubj ect h a v ing
noticed th e s y mptoms of inf ection, ca n h a v e th e concentr a
US 2011/0318847 A1
tion of ca lpr otectin in th eir body ? uid mea s ur ed a nd th eir
inf ection dia g nos ed a nd tr ea ted a ccor ding ly .
[ 0017] Oth er dis ea s es in Wh ich ca lpr otectin ma y be us ed a s
a dia g nos tic tes t include: r h euma tic dis ea s es ( eg r h euma toid
a r th r itis , j uv enile r h euma toid a r th r itis , s y s temic lupus er y th e
ma tos us ) , Sj og r ens s y ndr ome, intr a ocula r in? a mma tor y con
ditions , cy s tic ? br os is , a cute a nd ch r onic lung dis ea s e, lung
ca r cinoma ( s q ua mous cells ) , pulmona r y ca ncer s , color ecta l
ca ncer , in? a mma tor y boWel dis ea s e, g a s tr ic ca ncer , color ec
ta l a denoma or ca ncer , Cr oh ns dis ea s e, ulcer a tiv e colitis ,
g a s tr ointes tina l mucos a l in? a mma tion, ur ina r y s tones , a lco
h olic liv er dis ea s e, or a l in? a mma tor y mucos a l dis ea s e, CN S
in? a mma tor y dis ea s e ( eg multiple s cler os is a nd a cute
enceph a litis ) , HI V inf ection, s econda r y CN S inf ections in
HI V inf ected pa tients , ur ina r y tr a ct inf ections , cy s titis , py elo
neph r itis , endog enous pos ter ior uv eitis , h a ema tolog ica l con
ditions ( e. g . leuk a emia ) , f ebr ile conditions ( inf ectious a nd
non- inf ectious ) , a cute my oca r dia l inf a r ction a nd a ph er es is .
[ 0018] Th e pla s ma concentr a tion of ca lpr otectin h a s a ls o
been f ound to incr ea s e dur ing open h ea r t s ur g er y ( Semb, A.
G. et a l, Eur . J . Ca r dio- th or a c Sur g . ( 19 9 1) 5 136 3- 36 7,
Sa a tv edt, K . et a l. , Sca nd, J . Th or . Ca r diov a s c. Sur g . ( 19 9 6 )
30: 5 3- 6 0, M oen, O. et a l. , Per f us ion ( 19 9 4) 9 : 109 - 117) .
M or e s peci? ca lly , Sa a tv edt et a l. r epor t th a t ca lpr otectin con
centr a tion r is es a f ter th e s ta r t of ca r diopulmona r y by pa s s a nd
pea k s 48 h our s pos t- oper a tiv ely .
[ 0019 ] I t h a s noW s ur pr is ing ly been f ound th a t th e potentia l
f or CVD or pr opens ity to CVD in a s ubj ect ca n be a s s es s ed by
deter mining th e concentr a tion of ca lpr otectin in a ca lpr otec
tin- conta ining s a mple ta k en f r om s a id s ubj ect. I n oth er
Wor ds , it h a s been f ound th a t deter mina tion of th e concentr a
tion of ca lpr otectin in a ca lpr otectin- conta ining s a mple ta k en
f r om a s ubj ect ca n be us ed to pr edict, pr ior to th e ons et of
s y mptoms Wh ich a r e noticea ble to th e s ubj ect or to a th ir d
pa r ty ( eg a ph y s icia n) Wh eth er or h oW lik ely th e s ubj ect is to
s uf f er CVD.
[ 0020] By potentia l f or or pr opens ity to is mea nt th e
lik elih ood or pr oba bility th a t th e cur r ently s y mptom- f r ee s ub
j ect being tes ted Will s uf f er CVD in th e f utur e. Th is mig h t
ta k e th e f or m of a n index , r a tio, per centa g e or s imila r number
r e? ectiv e of th e r ela tiv e r is k of CVD in th e f utur e ( e. g . in th e
f olloWing 1- 2 y ea r s , a t lea s t in th e f olloWing 6 month s ) .
[ 0021] Th us v ieWed f r om one a s pect th e inv ention pr ov ides
a n a s s a y meth od f or th e detection of potentia l f or CVD or
pr opens ity to CVD in a h uma n or non- h uma n a nima l s ubj ect,
s a id meth od compr is ing a s s es s ing th e concentr a tion of
ca lpr otectin in a ca lpr otectin- conta ining s a mple ta k en f r om
s a id s ubj ect, eg a s a mple of blood, pla s ma , s er um, cer e
br os pina l ? uid, or a l ? uid, ur ine, f a eces , s y nov ia l or empy ema
? uid.
[ 0022] By a s s eds ing it is mea nt th a t a q ua ntita tiv e or
s emi- q ua ntita tiv e v a lue f or th e concentr a tion of ca lpr otectin
is deter mined. Th is ma y be th e v a lue f or th e Concentr a tion of
th e s a mple a s tes ted, e. g . a f ter tr ea tment to r emov e th e cells or
oth er s a mple components not being a s s a y ed f or , or to con
centr a te or dilute th e s a mple or to tr a ns f er th e ca lpr otectin to
a s epa r a te medium, e. g . s olid s ubs tr a te.
[ 0023] Alter na tiv ely , th e a s s es s ment ma y s imply be q ua li
ta tiv e, i. e. to indica te Wh eth er th e ca lpr otectin is a bov e or
beloW one or mor e pr e- s elected th r es h old v a lues , eg v a lues
indica tiv e of a bs ence of potentia l f or CVD or pr opens ity to
CVD a s detecta ble by th e a s s a y . Th e pr ecis e v a lues f or th es e
th r es h old v a lues or oth er r ef er ence v a lues f or ca lpr otectin
ma y depend on th e na tur e of th e s a mple, th e a g e, Weig h t, s ex
Dec. 29 , 2011
a nd s pecies of a s ubj ect a nd ma y be deter mined in a r outine
ma nner by mea s ur ing th e ca lpr otectin concentr a tion of th e
r elev a nt body ? uid of eq uiv a lent s ubj ects With out CVD or
With CVD a t v a r ious s ta g es of dev elopment.
[ 0024] A v a lue indica tiv e of ca lpr otectin concentr a tion
deter mined or a s s es s ed in a ccor da nce With th e meth od of
th e inv ention ma y be a n a bs olute concentr a tion of ca lpr otec
tin or ma y a lter na tiv ely be a n index , r a tio, per centa g e or
s imila r number r e? ectiv e of th e concentr a tion of ca lpr otectin.
[ 0025 ] A body s a mple us ed in th e a s s a y meth od of th e
inv ention ma y be a ny ca lpr otectin- conta ining s a mple, eg a
body ? uid or tis s ue s a mple, or a s us pens ion etc. Pr ef er a bly ,
th e s a mple Will be a body ? uid, e. g . ur ine, cer ebr os pina l ? uid,
or a l ? uid, s y nov ia l ? uid or empy ema ? uid, or mor e pr ef er
a bly , blood or a blood der iv ed s a mple. Wh en th is is th e ca s e
( i. e. Wh en blood or a blood der iv ed s a mple is us ed) , th e
s a mple us ed f or a na ly s is Will pr ef er a bly be cell- f r ee ( e. g .
s er um or pla s ma ) . Alter na tiv ely f a eces ma y be us ed.
[ 0026 ] Th e s a mple ma y be tr ea ted pr ior to being us ed in th e
a s s a y meth od of th e inv ention. Th us th e s a mple ma y be
tr ea ted to r emov e a ny cells a nd/or a ny s a mple components
not being a s s a y ed f or . Th e s a mple ma y a ls o be tr ea ted to
concentr a te or dilute th e s a mple or to tr a ns f er th e ca lpr otectin
to a s epa r a te medium, e. g . s olid s ubs tr a te. F or ex a mple th e
s a mple ma y be diluted by a dding a buf f er or oth er a q ueous
medium. Alter na tiv ely , a s a mple, pa r ticula r ly a pla s ma or
s er um s a mple, ma y be us ed dir ectly .
[ 0027] Th e s a mple is optiona lly tr ea ted With ca lcium or a
ca lcium mimic ( e. g . ions of a noth er a lk a line ea r th meta l) ,
pr ior to being us ed in th e a s s a y meth od of th e inv ention. Th e
ca lcium or ca lcium mimic ma y be a ny f or m Wh ich pr ov ides
Ca 2+ ions ( e. g . Ca Cl2) I f ca lcium is us ed th en pr ef er a bly
s uf ? cient ca lcium or ca lcium mimic is a dded to th e s a mple to
s a tur a te th e ca lcium binding s ites of ca lpr otectin. F or
ex a mple, a ten mola r ex ces s of ca lcium s our ce ma y be a dded,
mor e pr ef er a bly , a ? v e mola r ex ces s or es pecia lly pr ef er a bly
a th r ee mola r ex ces s .
[ 0028] Wh ile a s s a y s f or ca lpr otectin a r e k noWn a nd ma y be
us ed in th e meth od of th e inv ention, th er e h a s not pr ev ious ly
been a ny s ug g es tion th a t ca lpr otectin is a ma r k er or indica tor
of potentia l f or CVD or pr opens ity to CVD. I n oth er Wor ds ,
th er e h a s not pr ev ious ly been a ny s ug g es tion th a t th e ca lpr o
tectin concentr a tion of s y mptom- f r ee s ubj ects mig h t be us ed
a s a ma r k er or indica tor of potentia l f or or pr opens ity to CVD
a nd, in pa r ticula r , th er e h a s not been a ny s ug g es tion th a t
ca lpr otectin concentr a tion mig h t be us ed a s th e ma r k er or
indica tor in a n a s s a y meth od s uita ble f or ma s s s cr eening of
h ea lth y ( i. e. s y mptom- f r ee) s ubj ects .
[ 0029 ] Any k noWn a s s a y meth od f or ca lpr otectin ma y be
us ed in th e a s s a y meth od of th e inv ention. Th us , f or ex a mple,
th e meth od dis clos ed in Us . Pa t. N o. 4, 833, 074 ( F a g er h ol et
a l. ) f or th e is ola tion of ca lpr otectin a nd f or th e s ubs eq uent
pr oduction of monos peci? c a nti- s er a th er eto ma y be us ed to
pr oduce a ntica lpr otectin a ntibodies f or us e in a ny conv en
tiona l a s s a y meth od. Th e a ntica lpr otectin a ntibodies pr o
duced ma y be us ed, f or ins ta nce, in enZ y me link ed- a nd
r a dio- immunoa s s a y s .
[ 0030] A N y coCa r d ( Ax is - Sh ield PoC, Os lo, N or Wa y )
immunoa s s a y f or ma t f or ca lpr otectin ma y a ls o, f or ex a mple,
be us ed. Th is a s s a y us es a s olid ph a s e, s a ndw ich - f or ma t in
Wh ich th e tes t dev ice compr is es a membr a ne coa ted With
immobilis ed a ntica lpr otectin a ntibodies . Th us th e s a mple
( optiona lly diluted) is a pplied to th e dev ice a nd Wh en th e
s a mple ? oWs th r oug h th e membr a ne, a ny ca lpr otectin pr es ent
US 2011/0318847 A1
is ca ptur ed. Th e ca lpr otectin, immobilis ed on th e membr a ne
is th en tr ea ted With a g old- a ntibody conj ug a te Wh ich binds to
th e ca lpr otectin- a ntibody complex a nd th e intens ity of colour
( due to th e g old bea ds ) , a s deter mined by a bs or ba nce of r ed
lig h t, is pr opor tiona l to th e a mount of ca lpr otectin. Th e con
centr a tion of ca lpr otectin ca n th er ef or e be ca lcula ted f r om a
ca libr a tion cur v e pr epa r ed in th e conv entiona l ma nner .
[ 0031] Alter na tiv ely , th e commer cia l tes t ( Ca lpr es t ) f or
ca lpr otectin, f or ex a mple, in f a eces ( a v a ila ble f r om Eur os pi
ta l ) ma y be us ed. Th is a s s a y us es a poly clona l a ntibody
a g a ins t ca lpr otectin in a n enZ y me link ed immuno- s or bent
a s s a y s y s tem. Th us ca lpr otectin pr es ent in a s a mple ta k en
f r om a s ubj ect becomes bound to a ntibody , Wh ich is a ds or bed
to th e s ur f a ce of a pla s tic Well. A s ubs tr a te f or th e enZ y me is
th en a dded a nd th e intens ity of th e colour ed pr oduct pr oduced
is pr opor tiona l to th e a mount of enZ y me a nd th er ef or e to th e
a mount of ca lpr otectin. Th e concentr a tion of ca lpr otectin ca n
th er ef or e be ca lcula ted f r om a ca libr a tion cur v e pr epa r ed in
th e conv entiona l ma nner .
[ 0032] I ndeed, both mono- a ndpoly clona l a ntica lpr otectin
a ntibodies a r e a v a ila ble commer cia lly . Eg g a nd r a bbit poly
clona ls a r e, f or ex a mple, a v a ila ble f r om N or Weg ia n Antibod
ies AS a nd Ax is - Sh ield Dia g nos tics r es pectiv ely , Wh ils t
mous e monoclona l a ntibody ma y be obta ined f r om Da k o A/ S,
Denma r k . Any a ntica lpr otectin a ntibody obta ined, f or
ex a mple, by a ny conv entiona l tech niq ue f or ma k ing a ntibod
ies , ma y be us ed in th e meth od of th e inv ention. F or ins ta nce,
r a bbit a ntica lpr otectin a ntibody a s Well a s monoclona l a nti
bodies ca n be pr oduced a ccor ding to th e pr otocol des cr ibed in
Ha r loW a nd L a ne ( 19 88) , Antibodies , A L a bor a tor y M a nua l,
Cold Spr ing Ha r bor L a bor a tor y , N eW Yor k , N Y.
[ 0033] Alter na tiv ely , a ntica lpr otectin a ntibodies ma y be
pr epa r ed by r eg ula r ly inj ecting a ca lpr otectin- conta ining
s olution into ch ick ens , a nd th en collecting th e y olk s of th e
ch ick ens eg g s . Ch ick en eg g poly clona l a ntibody ca n th en be
is ola ted a ccor ding to conv entiona l tech niq ues a nd pur i? ed by
a ? inity ch r oma tog r a ph y .
[ 0034] Pr ef er a bly , th e a s s a y meth od of th e inv ention is us ed
f or ma s s s cr eening of h ea lth y ( e. g . CVD s y mptom- f r ee) s ub
j ects . Wh er e potentia l f or CVD or pr opens ity to CVD is
detected, th e s ubj ect ma y be s ubj ected to f ur th er tes ting ( eg
us ing mor e ex pens iv e tech niq ues s peci? c to CVD) to con? r m
th e pr es ence or a bs ence of CVD.
[ 0035 ] I n g ener a l, bes ides th e s a mple under ev a lua tion,
ca libr a tion s a mples With k noWn ca lpr otectin content Will a ls o
be a s s es s ed in th e per f or ma nce of th e a s s a y meth od. Such
deter mina tions ca n be us ed to plot a ca libr a tion cur v e f r om
Wh ich th e ca lpr otectin content of th e s a mple under inv es tig a
tion ma y be deter mined. Th e na tur e of th e ca libr a tion s a mples
a nd s election of conv er s ion or a dj us tment f a ctor s us ed in th e
deter mina tion of th e ca lpr otectin ma y v a r y depending , f or
ex a mple, on th e ma nner in Wh ich th e ca lpr otectin is detected
in th e a s s a y tech niq ue a ctua lly us ed a nd on oth er a s pects of
th e meth od Wh ich a f f ect th e a s s a y r es ult, f or ex a mple, buf f er
compos ition, a s s a y conditions etc.
[ 0036 ] Ty pica lly ca libr a tion s a mples h a v ing ca lpr otectin
contents of 0 to 5 000 mg / L Will be us ed. Th e r ef er ence r a ng e
With in Wh ich th e v a lue f or ca lpr otectin concentr a tion Will
g ener a lly be f ound is 0. 1 to 10 mg /L .
[ 0037] I n g ener a l, th e concentr a tion of ca lpr otectin in th e
s er um a nd pla s ma of h uma ns With little or no potentia l f or
CVD or pr opens ity to CVD Will be in th e r a ng e 0. 01- 0. 75
mg /L . M or e s peci? ca lly , th e concentr a tion of ca lpr otectin in
th e s er um a nd pla s ma of s uch h uma ns Will be in th e r a ng e
Dec. 29 , 2011
0. 05 - 0. 70 mg /L , ev en mor e s peci? ca lly 0. 10- 0. 6 6 mg /L , f or
ins ta nce, in th e r a ng e 0. 15 - 0. 45 mg /L . F or ex a mple, th e con
centr a tion of ca lpr otectin in th e s er um or pla s ma of a f ema le
With little or no potentia l f or CVD or pr opens ity to CVD Will
be in th e r a ng e 0. 09 - 0. 5 3 mg /L , f or ex a mple, a bout 0. 31 mg /L
or 0. 30 mg / L . Th e concentr a tion of ca lpr otectin in th e s er um
or pla s ma of a ma le With little or no potentia l f or CVD or
pr opens ity to CVD Will be in th e r a ng e 0. 12- 0. 6 6 mg /L , f or
ex a mple, 0. 30- 0. 39 mg /L , es pecia lly 0. 31 mg /L .
[ 0038] A ca lpr otectin, concentr a tion in s er um or pla s ma of
g r ea ter th a n 0. 75 mg /L Will g ener a lly be v er y s tr ong ly indica
tiv e of potentia l f or CVD or pr opens ity to CVD. Th us a
th r es h old v a lue a bov e Wh ich th e a s s a y ma y be h eld to be
pr edictiv e of CVD potentia l or pr opens ity to CVD ma y g en
er a lly be in th e r a ng e 0. 32- 0. 77 mg / L , f or ex a mple, a bout 0. 6 7
mg /L , pr ef er a bly a bout 0. 70 mg /L , es pecia lly a bout 0. 76
mg /L . M or e pr ef er a bly th e th r es h old v a lue a bov e Wh ich th e
a s s a y ma y be h eld to be pr edictiv e of potentia l f or or pr open
s ity to CVD is in th e r a ng e, 0. 30- 0. 5 0 mg /L , ev en mor e
pr ef er a bly in th e r a ng e 0. 32- 0. 47 mg /L , f or ex a mple a bout
0. 45 mg /L .
[ 0039 ] I n g ener a l, th e concentr a tion of ca lpr otectin in th e
f a eces of h uma ns With little or no potentia l f or CVD or pr o
pens ity to CVD Will be in th e r a ng e 0. 01- 10 mg /L . M or e
s peci? ca lly , th e concentr a tion of ca lpr otectin in th e f a eces of
s uch h uma ns Will be in th e r a ng e 0. 05 - 9 . 0 mg /L , ev en mor e
s peci? ca lly 0. 5 0- 8. 0 mg /L .
[ 0040] A ca lpr otectin concentr a tion in f a eces of g r ea ter
th a n 10 mg / L Will g ener a lly be s tr ong ly indica tiv e of potentia l
f or CVD or pr opens ity to CVD. Th us a th r es h old v a lue a bov e
Wh ich th e a s s a y ma y be h eld to be pr edictiv e of CVD potentia l
or pr opens ity to CVD ma y g ener a lly be a bout 9 mg / L , mor e
pr ef er a bly a bout 10. 5 mg /L , es pecia lly a bout 11 mg /L .
[ 0041] HoWev er , th e th r es h old v a lues a r e better ca lcula ted
f r om ca lpr otectin deter mina tions us ing th e s a me a s s a y tech
niq ues f or th e s a me body ? uid s a mple ty pe f r om a r a ng e of
pa tients of s imila r ty pe ( a g e, s ex , Weig h t, s pecies etc. ) f r om
h ea lth y th r oug h ea r ly s ta g e CVD to s er ious CVD. Ev en mor e
pr ef er a bly , th e th r es h old v a lues Will be v a lues deter mined f or
th e s a me pa tient a t a n ea r lier , h ea lth y s ta g e. Th us , pa r ticula r ly
a t- r is k , indiv idua ls could monitor th eir ca lpr otectin lev els on
a r outine ba s is ( e. g . ev er y 6 - month s to 1 y ea r ) in ma s s s cr een
ing pr og r a mmes .
[ 0042] I n a pr ef er r ed a s s a y meth od of th e pr es ent inv ention,
s a idmeth od f ur th er compr is es a dditiona lly a s s es s ing th e con
centr a tion of a noth er ma r k er f or potentia l to CVD in th e
s a mple ta k en f r om th e s ubj ect. Ex a mples of s uita ble ma r k er s
ma y be h omocy s teine, a ctiv a ted f a ctor X I I , ch oles ter ol, ch o
les ter olz HDL r a tio, ? br inog en, tis s ue- ty pe pla s minog en a cti
v a tor , F a ctor s V, VI I a nd VI I I , lipopr otein ( a ) , v on Willebr a nd
f a ctor a ntig en, pla s min- 0t2 a ntipla s min complex , pr oth r om
bin, f r a g ment 1+ 2, th r ombin- a ntith r ombin I I I complex ,
? br inopeptide A, ? br in deg r a da tion pr oducts , D- dimer , a cti
v a ted pr otein C- r es is ta nce, f a ctor VI I c a nd VI I a , th r ombin,
s er um a my loid A, v a s cula r a dh es ion molecules a nd cor ona r y
ca lcium. Pr ef er a bly th e s econd ma r k er is s elected f r om
h omocy s teine or C- r ea ctiv e pr otein.
[ 0043] M or e pr ef er a bly th e a s s a y meth od of th e pr es ent
inv ention f ur th er compr is es a dditiona lly a s s es s ing th e con
centr a tion of C- r ea ctiv e pr otein ( CRP) in th e s a mple ta k en
f r om th e s ubj ect. Pr ef er a bly , th e concentr a tion of CRP is
a s s es s ed s imulta neous ly or s eq uentia lly to s a id ca lpr otectin
a s s a y .
US 2011/0318847 A1
[ 0044] Th e mea s ur ement of CRP ma y be ef f ected us ing a ny
s ta nda r d immunoa s s a y tech niq ue ( e. g . EL I SA, RI A etc. ) or
ma y be deter mined by N y coCa r d ( a v a ila ble f r om Ax is
Sh ield PoC, Os lo, N or w a y ) .
[ 0045 ] A ca lpr otectin concentr a tion in s er um or pla s ma of
g r ea ter th a n 0. 75 mg /L in a ddition to a CRP concentr a tion of
g r ea ter th a n 1. 75 mg /L w ill g ener a lly be v er y s tr ong ly indica
tiv e of potentia l f or CVD or pr opens ity to CVD. Pr ef er a bly ,
th e pr es ence of th e a bov e- mentioned concentr a tions is mor e
s tr ong ly indica tiv e of potentia l f or CVD or pr opens ity to
CVD th a n a ca lpr otectin or CRP concentr a tion a lone.
[ 0046 ] Th us th r es h old v a lues of ca lpr otectin a nd CRP
a bov e w h ich th e a s s a y ma y be h eld to be h ig h ly pr edictiv e of
CVD potentia l or pr opens ity to CVD ma y g ener a lly be 0. 32
0. 77 mg /L a nd 1 . 70 mg /L r es pectiv ely , mor e pr ef er a bly a bout
0. 6 7 mg /L a nd 1. 75 mg /L r es pectiv ely , s till mor e pr ef er a bly
a bout 0. 70 mg /L a nd 2. 00 mg /L r es pectiv ely , es pecia lly a bout
0. 76 mg /L a nd 2. 25 mg /L r es pectiv ely . M or e pr ef er a bly th e
th r es h old v a lues a bov e w h ich th e a s s a y ma y be h eld to be
pr edictiv e of potentia l f or or pr opens ity to CVD a r e in th e
r a ng e, 0. 30- 0. 5 0 mg /L a nd 0. 75 mg /L r es pectiv ely , ev en mor e
pr ef er a bly in th e r a ng e 0. 32- 0. 47 mg /L a nd 0. 75 mg /L r es pec
tiv ely , f or ex a mple a bout 0. 45 mg /L a nd 0. 75 mg /L r es pec
tiv ely .
[ 0047] View ed f r om a f ur th er a s pect, th e pr es ent inv ention
pr ov ides a n a s s a y k it f or us e in th e meth od of th e inv ention,
s a id k it compr is ing r ea g ents a nd ins tr uctions f or th e per f or
ma nce of th e a s s a y meth od a nd f or th e inter pr eta tion of th e
r es ults a nd, optiona lly , ca lpr otectin- conta ining r ef er ence
s a mples , a nd optiona lly , a detector . Pr ef er a bly , s a id a s s a y k it
f ur th er compr is es th e r ea g ents a nd ins tr uctions f or deter mi
na tion of CRP concentr a tion.
[ 0048] Th e ins tr uctions in th e k it ma y f or ex a mple be in th e
f or m of a la bel, a ma nua l or a n ins tr uction lea ? et; h ow ev er ,
th ey ma y ins tea d ta k e th e f or m of a computer pr og r a m or a
da ta ca r r ier , eg a computer dis k .
[ 0049 ] Th e detector ; w h er e pr es ent, w ill g ener a lly be one
ca pa ble of detecting a r epor ter s pecies , eg a s pectr ometer , a
nuclea r r a dia tion detector , a s ca tter ed lig h t detector , etc.
[ 005 0] Th e r ea g ents w ill be a g ents s uita ble f or ca lpr otectin
deter mina tion, e. g . s uita ble r ea g ents a r e s peci? ed in th e lit
er a tur e a s s ocia ted w ith th e a v a ila ble tes ts f or ca lpr otectin
s uch a s f r om Eur os pita l a nd N y coCa r d ( a v a ila ble f r omAx is
Sh ield ASA, Os lo, N or w a y ) cited h er ein. Th e r ea g ents men
tioned in US. Pa t. N o. 4, 833, 074 ma y a ls o be s uita ble.
[ 005 1] A pa r ticula r ly pr ef er r ed a s s a y meth od f or a s s es s ing
th e concentr a tion of ca lpr otectin in th e pr es ent inv ention is a
pa r ticle- ba s ed immunoa s s a y . Th is is a s ens itiv e tech niq ue
w h ich is ba s ed on tur bidimetr ic deter mina tion of th e ca lpr o
tectin concentr a tion. Th e s ens itiv ity pr ov ided by th e a s s a y
a dv a nta g eous ly a llow s f or th e r ela tiv ely low concentr a tions
of ca lpr otectin in body ? uid ( e. g . pla s ma or s er um) s a mples to
be deter mined w ith a h ig h lev el of pr ecis ion. At th e s a me time,
r ela tiv ely h ig h concentr a tions of ca lpr otectin ca n a ls o be
mea s ur ed w ith a ccur a cy .
[ 005 2] Tur bidimetr ic deter mina tion a ls o h a s th e a dv a nta g e
th a t no s olid s ur f a ce is r eq uir ed f or ph y s ica l s epa r a tion in th e
a s s a y a nd numer ous w a s h ing a nd/or s epa r a tion s teps a r e not
r eq uir ed. Th us compa r ed to pr ior a r t tech niq ues ( e. g . EL I SA) ,
th e h omog enous tur bidmimetr ic deter mina tion of ca lpr otec
tin is q uick a nd ea s y to per f or m a nd ma y , f or ins ta nce, be
a utoma ted. Compa r ed to a utoma tion of non- h omog eneous
tech niq ues inv olv ing , f or ex a mple a s olid s ur f a ce, a utoma tion
of a tur bidimetr ic ba s ed a s s a y is r ela tiv ely f a cile. Als o, th e
Dec. 29 , 2011
r es ulting a utoma ted h omog enous pr oces s is of ten mor e r eli
a ble being les s pr one, f or ex a mple, to br ea k dow n.
[ 005 3] An a utoma ted tur bidimetr ic a s s a y is a ls o f a s t, a llow
ing f or a h ig h th r oug h put of s a mples , a nd is r ela tiv ely ch ea p
to r un. Ty pica lly it ca n be per f or med us ing a commer cia lly
a v a ila ble r obot, eg th e Coba s M ir a or Hita ch i 711, both of
w h ich a r e a v a ila ble f r om Roch e Dia g nos tics . Such a n a uto
ma ted a s s a y is pa r ticula r ly a ttr a ctiv e w h en r outine tes ting of
indiv idua ls f or potentia l f or or pr opens ity to CVD is env is
a g ed.
[ 005 4] F or tur bidimetr ic deter mina tion of ca lpr otectin con
centr a tion, th e ca lpr otectin- conta ining s a mple w ill g ener a lly
be a body ? uid, e. g . ur ine, cer ebr os pina l ? uid, or a l ? uid,
s y nov ia l ? uid or empy ema ? uid, or mor e pr ef er a bly , blood or
a blood der iv ed s a mple. Wh en th is is th e ca s e, th e s a mple us ed
f or a na ly s is w ill pr ef er a bly be cell- f r ee ( e. g . s er um or pla s ma )
[ 005 5 ] Th us th e s a mple ma y be tr ea ted to r emov e a ny cells
a nd/or a ny s a mple components not being a s s a y ed f or . Th e
s a mple ma y a ls o be tr ea ted to concentr a te or dilute th e s a mple
or to tr a ns f er th e ca lpr otectin to a s epa r a te medium, e. g . s olid
s ubs tr a te. F or ins ta nce, th e s a mple ma y be diluted by a dding
w a ter , a buf f er or oth er , a q ueous medium. Alter na tiv ely , a
s a mple, pa r ticula r ly a s er um or pla s ma s a mple, ma y be us ed
dir ectly .
[ 005 6 ] Th e s a mple is optiona lly tr ea ted w ith ca lcium or a
ca lcium mimic, pr ior to being us ed in th e a s s a y met h old. Th e
ca lcium or ca lcium mimic ma y be a ny f or m w h ich pr ov ides
Ca 2+ ions ( e. g . Ca ClZ ) . I f ca lcium is us ed th en pr ef er a bly ,
s uf ? cient ca lcium or ca lcium mimic is a dded to th e s a mple to
s a tur a te th e ca lcium binding s ites of ca lpr otectin. F or
ex a mple, a ten mola r ex ces s of ca lcium s our ce ma y be a dded,
mor e pr ef er a bly , a ? v e mola r ex ces s or es pecia lly pr ef er a bly
a th r ee mola r ex ces s .
[ 005 7] Opa city , f or tur bidimetr ic deter mina tion of ca lpr o
tectin concentr a tion, w ill g ener a lly be g ener a ted by conta ct
ing th e ca lpr otectin- conta ining s a mple, dr a n a liq uot th er eof ,
w ith a n a ntica lpr otectin a ntibody , a ntibody f r a g ment or mix
tur e of a ntica lpr otectin a ntibodies ( eg a mix tur e of mono
clona l a ntibodies ) . Th e eg g poly clona l a nti- h uma n ca lpr otec
tin a ntibody commer cia lly a v a ila ble f r om N or w eg ia n
Antibodies AS ma y , f or ex a mple, be us ed to g ener a te opa city .
Any a ntica lpr otectin a ntibody obta ined, f or ex a mple, by a ny
conv entiona l tech niq ue f or ma k ing a ntibodies , ma y be us ed in
th e meth od of th e inv ention.
[ 005 8] Th e a ntibodies , or a ntibody f r a g ments , w h ich a r e
us ed f or tur bidimetr ic deter mina tion of ca lpr otectin concen
tr a tion pr ef er a bly s h ow no or little cr os s r ea ctions w ith oth er
blood pr oteins th a t ma y be pr es ent in th e elua te. Th e q ua ntity
of a ntibody , or a ntibody f r a g ment, us ed s h ould of cour s e be
optimis ed a g a ins t ca lpr otectin- conta ining s ta nda r d s a mples
a s opa ci? ca tion a r is es f r om th e h ook ef f ect w h er eby multiple
ca lpr otectin binding g ener a tes th e opa ci? ca tion centr es .
Ca lpr otectin, a s mentioned a bov e h a s numer ous a ntibody
binding s ites , a nd is pa r ticula r ly s uita ble f or detection in s uch
a n a s s a y .
[ 005 9 ] I n one pr ef er r ed embodiment, th e a ntica lpr otectin
a ntibody , or a ntibody f r a g ment, ma y be immobilis ed by bind
ing or coupling , eith er dir ectly or indir ectly , to a ny w ell
k now n s olid s uppor t or ma tr ix w h ich is commonly us ed f or
immobilis a tion. Pr ef er a bly th e s olid s uppor t or ma tr ix ta k es
th e f or m of pa r ticles , pr ef er a bly na nopa r ticles . Conv eniently
th e s olid s uppor t ma y be ma de of g la s s , s ilica , la tex , meta l
US 2011/0318847 A1
( e. g . g old) or a poly mer ic ma ter ia l ( eg poly eth y lene) . Pr ef
er a bly th e s olid s uppor t is ma de of a poly mer ic ma ter ia l s uch
a s poly eth y lene.
[ 006 0] Binding or immobilis a tion of th e a ntica lpr otectin
a ntibody or a ntibody f r a g ment ma y be a ch iev ed us ing a ny
conv entiona l tech niq ue. F or ex a mple, a v idin ( a v a ila ble f r om
Pier ce Ch emica l Compa ny ) ma y be immobilis ed on ch lor om
eth y l a ctiv a ted poly s ty r ene na nopa r ticles ( a v a ila ble f r om
I nter f a cia l Dy na mic Cor por a tion, US) by a g ita tion in buf f er
( eg a t r oom temper a tur e f or 24 h our s ) a nd th en us ed in
conj unction With biotin la belled a ntica lpr otectin a ntibodies
( pr epa r ed a ccor ding to conv entiona l tech niq ues in th e a r t) .
Th us , f or ex a mple, pla s ma ta k en f r om th e s ubj ect to be tes ted
f or potentia l f or , or pr opens ity to, CVD is a dded to a s olution
of a v idin- coa ted na nopa r ticles in a q ua r tz cuv ette of a s pec
tr oph otometer , f olloWed by th e a ddition of biotin la belled
a ntica lpr otectin a ntibody . Tur bidimetr ic r ea ding s a r e th en
ta k en.
[ 006 1] Alter na tiv ely , th e biotin la belled a ntibodies ma y be
a dded pr ior to th e a ddition of pla s ma or s er um. I n oth er
Wor ds , Wh ils t th e s a me r ea g ents a r e ty pica lly us ed r eg a r dles s
of th e ins tr ument us ed f or tur bidity detection, th e pr ecis e
s eq uence in Wh ich th e v a r ious r ea g ents a r e a dded ma y v a r y .
Gener a lly , th e s eq uence us ed s h ould be in a ccor da nce With
th e ins tr uctions a ccompa ny ing th e s pectr oph otometer us ed
( eg a Sh ima dZ u UV- 16 0 s pectr oph otometer ) .
[ 006 2] Tur bidimetr ic r ea ding s a r e ma de ( i. e. th e lig h t
a bs or ption a t a s uita ble Wa v eleng th is mea s ur ed a t r eg ula r
inter v a ls ) a nd th e lig h t a bs or ption r ela tiv e to a r ef er ence is
deter mined. Optiona lly , multiple Wa v eleng th ins tr uments
ma y be us ed to ma k e tur bidimetr ic r ea ding s a nd ma y pr ov ide
mor e pr ecis e r es ults . Suita ble ins tr uments f or ta k ing tur bidi
metr ic r ea ding s include th e Coba s M ir a , Roch e I nteg r a a nd
M er ck s Tur biq ua nt.
[ 006 3] I n a n a lter na tiv e ex per imenta l s et- up, th e a nti
ca ipr otectin a ntibody , or a ntibody f r a g ment, ma y be immo
bilis ed dir ectly on ch lor ometh y l a ctiv a ted na nopa r ticles
( a v a ila ble f r om I nter f a cia l Dy na mic Cor por a tion, US) . F or
ins ta nce, a ntica lpr otectin a ntibody ( eg th e eg g poly clona l
a ntibody a v a ila ble f r om N or w eg ia n Antibodies AS) ma y be
mix ed With th e a bov e- mentioned a ctiv a ted pa r ticles in a
buf f er ( 10 mM bor a te, 15 mM s odium ch lor ide, pH 9 . 0) a nd
a g ita ted ( eg a t r oom temper a tur e f or 24 h our s ) to f ur nis h
a ntica lpr otectin a ntibody - coa ted na nopa r ticles . Such na no
pa r ticles ma y be us ed f or tur bidimetr ic deter mina tion of
ca lpr otectin concentr a tion by a dding th em to a s a mple of
pla s ma or s er um, ta k en f r om th e s ubj ect to be tes ted f or
potentia l f or , or pr opens ity to CVD, in a buf f er a nd ta k ing
tur bidimetr ic r ea ding s in k inetic mode.
[ 006 4] Alter na tiv ely , th e pla s ma or s er um ma y be a dded to
th e a ntica lpr otectin a ntibody - coa ted na nopa r ticles . I n oth er
Wor ds , Wh ils t th e s a me r ea g ents a r e ty pica lly us ed r eg a r dles s
of th e ins tr ument us ed f or tur bidity detection, th e pr ecis e
s eq uence in Wh ich th e v a r ious r ea g ents a r e a dded ma y v a r y .
Gener a lly , th e s eq uence us ed s h ould be in a ccor da nce With
th e ins tr uctions a ccompa ny ing th e s pectr oph otometer us ed
( eg a Sh ima dZ u UV- 16 0 s pectr oph otometer ) .
[ 006 5 ] Ex a mples of a utoma ted r obots Wh ich a r e s uita ble
f or ta k ing tur bidimetr ic r ea ding s in a ccor da nce With th e a s s a y
meth od of th e inv ention include th e Coba s M ir a a nd Hita ch i
711, both of Wh ich a r e a v a ila ble f r om Roch e Dia g nos tics .
[ 006 6 ] Th e pa r ticles to Wh ich th e a ntibody , or a ntibody
f r a g ment, ma y be bound a r e ty pica lly s ph er ica l. Th e s iZ e of
th e pa r ticles us ed in th e a s s a y ma y ef f ect th e pr ecis ion With
Dec. 29 , 2011
Wh ich th e ca lpr otectin concentr a tion is mea s ur ed. Wh ils t
la r g er pa r ticles a lloW f or loWer concentr a tions of ca lpr otectin
to be deteced, th eir r educed s ur f a ce a r ea mea ns th a t th ey h a v e
a loWer binding ca pa city . F or ex a mple, doubling th e pa r ticle
dia meter , h a lv es th e binding ca pa city of a ma s s unit of pa r
ticles .
[ 006 7] Additiona lly incr ea s ing th e pa r ticle dia meter
incr ea s es th e lev el of ba ck g r ound lig h t a bs or ba nce a nd lig h t
s us pens ion a t th e Wa v eleng th s ty pica lly us ed in s uch a s s a y s
( eg 330 to 6 00 nm) . Th us Wh ils t la r g er pa r ticles incr ea s e th e
s ens itiv ity of th e a s s a y , th is ma y be a ccompa nied by s ome
los s of a ccur a cy a nd in pa r ticula r , a n incr ea s e in th e number of
f a ls e neg a tiv e r es ults obta ined. Th is is pa r ticula r ly lik ely to be
th e ca s e With s a mples conta ining r ela tiv ely h ig h ca lpr otectin
concentr a tions ( i. e. th os e s a mples obta ined f r om indiv idua ls
With a h ig h potentia l f or or pr opens ity to CVD) Wh er ein th e
na nopa r ticle- bound binding s ites ma y become s a tur a ted
With out a ll of th e ca lpr otectin becoming bound.
[ 006 8] Th es e counter - a cting ef f ects a s s ocia ted With ch a ng
ing th e pa r ticle s iz e ( eg incr ea s ing th e pa r ticle s iZ e incr ea s es
s ens itiv ity but decr ea s es a ccur a cy ) r epr es ents a s ig ni? ca nt
pr oblem to be ov er come in th e dev elopment of a s ens itiv e
a s s a y f or detecting th e r a ng e of lev els of ca lpr otectin pr es ent
in body ? uids .
[ 006 9 ] Als o it is pr ef er a ble in th e a s s a y meth od of th e
inv ention th a t th e pa r ticles us ed a lloW f or a Wide r a ng e of
ca lpr otectin concentr a tions to be deter mined With pr ecis ion.
Th is ma y mea n th a t a h ig h lev el of con? dence ca n be a ttr ib
uted to both a neg a tiv e r es ult ( i. e. a concentr a tion f a lling
beloW th e th r es h old v a lue) a s Well a s a pos itiv e r es ult. I t is
pa r ticula r ly pr ef er r ed in th e a s s a y meth od of th e inv ention
th a t s a mples h a v ing a ca lpr otectin concentr a tion in th e r a ng e
0. 5 - 5 0 mg /L ( e. g . 1- 40 mg /L ) ca n be mea s ur ed.
[ 0070] Th e pa r ticles to Wh ich th e a ntibody , or a ntibody
f r a g ment, ma y be bound a r e ty pica lly s ph er ica l With a dia m
eter of 1- 15 0 nm, f or ex a mple 10- 9 0 nm or 15 - 6 0 nm, f or
ins ta nce, 44 nm. I n a pa r ticula r ly pr ef er r ed a s s a y meth od of
th e inv ention th e pa r ticles to Wh ich th e a ntibody or a ntibody
f r a g ments a r e bound h a v e a dia meter of 5 5 - 140 nm, mor e
pr ef er a bly 6 5 - 110 nm, f or ex a mple, 70- 9 0 nm.
[ 0071] Alter na tiv ely th e dia meter of th e pa r ticles ca n be
mea s ur ed once a ntibodies or a ntibody f r a g ments a r e bound to
th eir s ur f a ce. I n th is ca s e, th e dia meter of th e a ntibody or
a ntibody f r a g ment coa ted pa r ticles is pr ef er a bly 6 5 - 140 nm,
mor e pr ef er a bly 75 - 120 nm, s till mor e pr ef er a bly 80- 100 nm.
Coa ted pa r ticles of th es e s iZ es a r e es pecia lly pr ef er r ed Wh en
th e s a mple tes ted is pla s ma .
[ 0072] Th e pa r ticles , in both th e nude a nd coa ted s ta tes ,
pr ef er a bly h a v e a dia meter Wh ich does not its elf ena ble
a bs or ption of th e Wa v eleng th of lig h t us ed f or s pectr oph oto
metr ic deter mina tion. Th us th e s us pens ion of coa ted na no
pa r ticles is a ppr ox ima tely ( e. g . s ubs ta ntia lly ) tr a ns pa r ent
until ca lpr otectin induced a g g r eg a te f or ma tion occur s r es ult
ing in th e f or ma tion of a g g r eg a tes h a v ing a la r g er dia meter .
Such a g g r eg a tes h a v e th e a bility to a bs or b th e Wa v eleng th of
lig h t us ed by th e s pectr oph otometer .
[ 0073] F ur th er , th e pa r ticles a r e pr ef er a bly s ubs ta ntia lly a ll
of th e s a me s iZ e, mor e s peci? ca lly a ll of th e s a me dia meter .
Pr ef er a bly , monodis per s e meta l ( e. g . g old) or poly mer pa r
ticles a r e us ed. M onodis per s e poly mer pa r ticles a r e a v a ila ble
f r om Dy na l Biotech AS, Os lo, N or w a y .
[ 0074] Wh ils t not Wis h ing to be bound by th eor y , it ma y be
th a t th e us e of immobilis ed a ntibody or a ntibody f r a g ments
incr ea s es th e s ens itiv ity of th e a s s a y by incr ea s ing th e s iZ e of
US 2011/0318847 A1
a ny ca lpr otectin der iv ed opa city g ener a ting s ites a nd th er e
f or e th e a mount of lig h t s ca tter ed th er ef r om. By us ing a s olid
s uppor t or ma tr ix ( e. g . na nopa r ticles ) Wh ich is s ubs ta ntia lly
a ll of th e s a me s iZ e it ma y be th a t th e s ens itiv ity of th e
tur bidimetr y a s s a y is f ur th er incr ea s ed.
[ 0075 ] As is r outine in tur bidimetr ic a s s a y s , a poly mer ic
opa ci? ca tion enh a ncer , s uch a s poly eth y leneg ly col, is pr ef
er a bly a ls o a dded to th e elua te.
[ 0076 ] Bef or e th e tur bidimetr ic deter mina tion is ma de, th e
f r a ction, a ntibody or a ntibody f r a g ment, pr ef er a bly bound to
a na nopa r ticle, a nd optiona lly enh a ncer ma y be incuba ted f or
a s h or t per iod, eg 5 minutes to a n h our , pr ef er a bly a bout 10
minutes , a t r oom temper a tur e. Optiona lly , in deter mining
ca lpr otectin concentr a tion us ing th e tur bidimetr y tech niq ue,
a k inetic r ea ding mode ma y be us ed.
[ 0077] Th e lig h t us ed in th e deter mina tion of opa ci? ca tion
s h ould h a v e a n a ppr opr ia te Wa v eleng th , f or ex a mple, 300- 6 00
nm. I n th is r eg a r d it Wa s f ound th a t us e of a 300- 45 0 nm ? lter ,
pr ef er a bly a 340 nm or a 405 mm ? lter , f umis h ed pa r ticula r ly
g ood r es ults . Us e of a 5 6 0 nm ? lter ma y a ls o y ield es pecia lly
g ood r es ults .
[ 0078] I n g ener a l, in a ddition to th e s a mple under ev a lua
tion ca libr a tion s a mples With k noWn ca lpr otectin contents
Will a ls o be a s s es s ed in th e per f or ma nce of th e a s s a y meth od.
Such deter mina tions ca n be us ed to plot a ca libr a tion cur v e
f r om Wh ich th e ca lpr otectin content of th e s a mple under
ev a lua tion ca n be deter mined. Pr ef er a bly ca libr a tion s a mples
h a v ing ca lpr otectin contents of up to 5 000 mg /L ( eg 15 00,
1000, 75 0, 25 0, 100) or up to 100 mg /L ( eg 75 , 5 0, 25 , 5 , 1. 0
a nd 0. 5 mg /L ) Will be us ed. M or e pr ef er a bly th e ca libr a tion
s a mples h a v e a ca lpr otectin content of up to 10 mg /L ( e. g . 10,
8, 6 , 4, 2 mg /L ) , s till mor e pr ef er a bly up to 5 mg /L ( 5 , 4. 5 , 4,
3. 5 , 3, 2. 5 , 2, 1. 5 , 1, 0. 5 mg /L ) .
[ 0079 ] Th e a bov e des cr ibed tur bidimetr ic a s s a y f or th e
deter mina tion of ca lpr otectin is s ur pr is ing ly r elia ble, q uick ,
ch ea p, f a cile a nd a mena ble to a utoma tion. Th is is in contr a s t
to th e cur r ently a v a ila ble a s s a y meth ods Wh ich a r e r ela tiv ely
complex a nd a r e not dir ectly a pplica ble to th e a utoma ted
multi- ta s k dia g nos tic ma ch ines commonly us ed by dia g nos
tic la bor a tor ies .
[ 0080] Automa tion is pa r ticula r ly des ir a ble Wh er e numer
ous mix ing , a ddition a nd/or dilution s teps a r e inv olv ed s ince
th es e ma y be a ch iev ed With a h ig h er deg r ee of a ccur a cy .
Robots ma y a ls o of f er a h ig h er lev el of r elia bility a nd/or
r epr oducibility . Automa tion a ls o incr ea s es th r oug h put.
[ 0081] Th e cur r ently a v a ila ble a s s a y meth ods Wh ich ma y
of f er r ea s ona ble lev els of pr ecis ion ( e. g . EL I SA) a r e, h oW
ev er , di? icult to a utoma te. Th is is a t lea s t in pa r t beca us e th ey
ty pica lly inv olv e numer ous Wa s h ing a nd s epa r a tion s teps
( e. g . a tta ch ment to a s olid s ur f a ce) a nd a utoma tion of non
h omog eneous pr oces s es is of ten pr oblema tic. Als o th es e pr o
ces s es ty pica lly inv olv e a r ela tiv ely la r g e number of s teps
Wh ich incr ea s es th e complex ity of a ny a utoma ted pr otocol.
Oth er conv entiona l tech niq ues f or deter mina tion of ca lpr o
tectin ( e. g . neph elometr y ) of f er h ig h pr ecis ion but r eq uir e
s pecia l eq uipment to ca r r y out th e neces s a r y mea s ur ements .
Specia lis ed eq uipment is not ty pica lly ea s y to incor por a te
into a n a utoma ted pr otocol.
[ 0082] I ndeed th er e is a continuing need f or ch ea p, r elia ble,
q uick a nd f a cile ca lpr otectin a s s a y s f or us e in dia g nos tic
tech niq ues .
[ 0083] Th us , a ccor ding to a f ur th er a s pect, th e pr es ent
inv ention pr ov ides a n a s s a y meth od f or th e deter mina tion of
Dec. 29 , 2011
ca lpr otectin in a ca lpr otectin- conta ining body ? uid, s a id
meth od compr is ing th e s teps of :
[ 0084] ( a ) obta ining a ca lpr otectin- conta ining liq uid
s a mple of , or der iv ed f r om, s a id ? uid;
[ 0085 ] ( b) conta cting s a id s a mple of s a id body ? uid With
a n, optiona lly na nopa r ticle- bound, a ntica lpr otectin a ntibody
or a ntibody f r a g ment, to bind s a id ca lpr otectin;
[ 0086 ] ( c) optiona lly , a dding a n opa city enh a ncer ; a nd
[ 0087] ( d) a s s es s ing th e ca lpr otectin content by tur bidim
etr y .
[ 0088] Such a s a s s a y ma y be us ef ul in th e dia g nos is of
v a r ious dis ea s e conditions Wh ich a r e ch a r a cter is ed by a bnor
ma l lev els ( e. g . h ig h lev els ) of ca lpr otectin. Such dis ea s e
conditions include: r h euma tic dis ea s es ( eg r h euma toid
a r th r itis , j uv enile r h euma toid a r th r itis , s y s temic lupus er y th e
ma to s us ) , Sj og r ens s y ndr ome, intr a ocula r in? a mma tor y con
ditions , cy s tic ? br os is , a cute a nd ch r onic lung dis ea s e, lung
ca r cinoma ( s q ua mous cells ) , pulmona r y ca ncer s , color ecta l
ca ncer , in? a mma tor y boWel dis ea s e, g a s tr ic ca ncer , color ec
ta l a denoma or ca ncer , Cr oh ns dis ea s e, ulcer a tiv e colitis ,
g a s tr ointes tina l mucos a l in? a mma tion, ur ina r y s tones , a lco
h olic liv er dis ea s e, or a l in? a mma tor y mucos a l dis ea s e, CN S
in? a mma tor y dis ea s e ( eg multiple s cler os is a nd a cute
enceph a litis ) , HI V inf ection, s econda r y CN S inf ections in
HI V inf ected pa tients , ur ina r y tr a ct inf ections , cy s titis , py elo
neph r itis , endog enous pos ter ior uv eitis , h a ema tolog ica l con
ditions ( e. g . leuk a emia ) , f ebr ile conditions ( inf ectious a nd
non- inf ectious ) , a cute my oca r dia l inf a r ction a nd a ph er es is .
[ 0089 ] Th us v ieWed f r om a f ur th er a s pect th e inv ention
pr ov ides a meth od of dia g nos is of a ny of th e a bov e- men
tioned dis ea s es , compr is ing th e meth od a s des cr ibed h er ein
bef or e f olloWed by compa r is on of s a id ca lpr otectin content
With a pr edeter mined th r es h old v a lue. Th e th r es h old v a lue
indica tiv e of a ny pa r ticula r dis ea s e s ta te ma y be deter mined
by a ny conv entiona l meth od k noWn in th e a r t. Pr ef er a bly th e
meth od is us ed f or th e dia g nos is of CVD.
[ 009 0] A body s a mple us ed in th e tur bidimetr ic a s s a y
meth od ma y be a ny ca lpr otectin- conta ining s a mple, eg a
body ? uid or tis s ue s a mple, or a s us pens ion etc. Pr ef er a bly ,
th e s a mple Will be a body ? uid, e. g . ur ine, cer ebr os pina l ? uid,
or a l ? uid, s y nov ia l ? uid or empy ema ? uid, or mor e pr ef er
a bly , blood or a blood der iv ed s a mple. Wh en th is is th e ca s e
( i. e. blood or blood der iv ed s a mple is us ed) , th e s a mple us ed
f or a na ly s is Will pr ef er a bly be cell- f r ee ( e. g . s er um or
pla s ma ) . Alter na tiv ely f a eces ma y be us ed.
[ 009 1] Pr ef er a bly , th e body s a mple is s elected to pr ov ide
th e mos t s ens itiv e indica tion of th e dis ea s e being dia g nos ed.
Th us Wh ils t blood, pla s ma or s er um mig h t be tes ted to dia g
nos e inf ections ( e. g . HI V, ba cter ia l inf ection) , r h euma tic dis
ea s e, leuk a emia etc. , f a eces mig h t be tes ted dur ing dia g nos is
of dis ea s es a s s ocia ted With th e g a s tr ointes tina l tr a ct ( e. g .
Cr oh ns dis ea s e, ulcer a tiv e colitis , color ecta l ca ncer s ) .
[ 009 2] VieWed f r om y et a f ur th er a s pect, th e inv ention pr o
v ides a k it f or a dia g nos tic tur bidimetr ic a s s a y a ccor ding to
th e inv ention, s a id k it compr is ing :
[ 009 3] pr ef er a bly , a ca lpr otectin s olution of k noWn concen
tr a tion a nd mor e pr ef er a bly a s et of s uch s olutions h a v ing a
r a ng e of ca lpr otectin concentr a tions ;
[ 009 4] one or mor e a ntica lpr otectin a ntibodies or a ntibody
f r a g ments , optiona lly immobilis ed on a s olid s uppor t ( eg
na nopa r ticles ) ;
[ 009 5 ] pr ef er a bly , a lig h t tr a ns mitting v es s el;
[ 009 6 ] pr ef er a bly , a n opa ci? ca tion enh a ncer ; a nd
[ 009 7] pr ef er a bly , a detector .
US 2011/0318847 A1
[ 009 8] I f des ir ed a n a utoma ted a ppa r a tus ma y be a r r a ng ed
to r eceiv e a ca lpr otectin- conta ining body ? uid s a mple, a pply
th e a ntica lpr otectin a ntibody or a ntibody f r a g ment, option
a lly immobilis ed on a s olid s uppor t ( e. g . na nopa r ticles ) ,
optiona lly a pply a n opa ci? ca tion enh a ncer , a nd a s s es s ca lpr o
tectin content. Such a n a ppa r a tus is a ls o deemed to f a ll With in
th e s cope of th e inv ention.
[ 009 9 ] Th e inv ention Will noW be des cr ibed f ur th er With
r ef er ence to th e f ollow ing non- limiting Ex a mples a nd th e
a ccompa ny ing ? g ur es in Wh ich :
[ 0100] F I G. 1 is th e dis tr ibution cur v e f or ca lpr otectin in th e
200 s ubj ects tes ted. Th e s umma r y s ta tis tics f or F I G. 1 a r e:
[ 0101] Ander s on- Da r ling N or ma lity Tes t
A2: 8. 16 0
P- v a lue: 0. 000
M ea n 0. 403
Sta nda r d Dev ia tion 0. 238
Va r ia nce 0. 05 7
Sk eWnes s 1. 6 79
K ur tos is 3. 302
N 19 9
M inimum 0. 070
1s t Q ua r tile 0. 240
M edia n 0. 340
3r d Q ua r tile 0. 5 00
M a x imum 1. 370
9 5 % con? dence limit f or M u 0. 370
0. 436
9 5 % con? dence limit f or Sig ma 0. 217
0. 26 4
9 5 % con? dence limit f or M edia n 0. 310
0. 370
[ 0102] F I G. 2 is th e dis tr ibution cur v e f or ca lcium s cor e in
th e 200 s ubj ects tes ted. Th e s umma r y s ta tis tics f or F I G. 2 a r e
A2: 25 . 037
P- v a lue: 0. 000
M ea n 217. 06 0
Sta nda r d Dev ia tion 39 9 . 000
Va r ia nce 15 9 201
Sk eWnes s 3. 46 2
K ur tos is 15 . 341
N 200
M inimum 0. 00
M edia n 78. 00
3r d Q ua r tile 25 9 . 25
M a x imum 279 4. 00
9 5 % con? dence limit f or M u 16 1. 42
272. 70
9 5 % con? dence limit f or Sig ma 36 3. 35
442. 46
9 5 % con? dence limit f or M edia n 0. 00
1 17. 33
[ 0104] F I G. 3 is th e dis tr ibution cur v e f or h s CRP in th e 200
s ubj ects tes ted. Th e s umma r y s ta tis tics f or F I G. 3 a r e:
[ 0105 ] Ander s on- Da r ling N or ma lity Tes t
A2: 21. 221
P- v a lue: 0. 000
M ea n 2. 331
Dec. 29 , 2011
- continued
Sta nda r d Dev ia tion 2. 9 70
Va r ia nce 8. 819
Sk eWnes s 2. 243
K ur tos is 5 . 103
N 19 7
M inimum 0. 15 0
1s t Q ua r tile 0. 5 40
M edia n 1. 15 0
3r d Q ua r tile 2. 5 80
M a x imum 16 . 30
9 5 % con? dence limit f or M u 1. 9 13
2. 748
9 5 % con? dence limit f or Sig ma 2. 703
3. 29 6
9 5 % con? dence limit f or M edia n 0. 9 33
1. 375
[ 0106 ] F I G. 4 is th e dis tr ibution cur v e f or h omocy s teine in
th e 200 s ubj ects tes ted. Th e s umma r y s ta tis tics f or F I G. 4 a r e:
A2: 2. 5 35
P- v a lue: 0. 000
M ea n 8. 39 1
Sta nda r d Dev ia tion 1. 9 6 6
Va r ia nce 3. 86 7
Sk eWnes s 0. 875
K ur tos is 0. 730
N 19 9
M inimum 5 . 100
M edia n 8. 100
3r d Q ua r tile 9 . 6 00
M a x imum 15 . 300
9 5 % con? dence limit f or M u 8. 117
8. 6 6 6
9 5 % con? dence limit f or Sig ma 1. 79 0
2. 181
9 5 % con? dence limit f or M edia n 7. 6 20
8. 400
[ 0108] F I G. 5 is a dot plot f or th e dis tr ibution of ca lpr otectin
betWeen th e ca lcium pos itiv e a nd ca lcium neg a tiv e r es ults ;
a nd
[ 0109 ] F I GS. 6 a nd 7 a r e th e ROC cur v es f or ca lpr otectin
a nd h s CRP f or a ll 200 s ubj ects tes ted a nd f or th e ma le s ub
j ects tes ted r es pectiv ely .
EX AM PL E 1
Anti- Ca lpr otectin Antibody
( a ) I s ola tion of Ca lpr otectin
[ 0110] Ca lpr otectin ma y be is ola ted a ccor ding to th e meth
ods des cr ibed in Ex a mples 1 a nd 2 of U. S. Pa t. N o. 4, 833, 074
( F a g er h ol) .
[ 0111] Ca lpr otectin ma y a lter na tiv ely be pur i? ed f r om
h uma n buf f y coa ts . A cell- s us pens ion in 2. 5 mM EDTA is
ma de by th e a ddition of EDTA ( 5 0 mM , pH 7) to cells . Th e
cells a r e th en Wa s h ed in 1 6 0 mM a mmonium ch lor ide/10 mM
s odium h y dr og en ca r bona te f or 3 minutes a nd centr if ug ed
( 16 0> < g ) f or 10 minutes a t 40 C. Th e r es ulting pellet is Wa s h ed
in EDTA ( 2. 5 mM ) /N a Cl ( 15 0 mM ) a nd centr if ug ed ( 5 5 > < g )
f or a f ur th er 10 minutes a t 40 C. Th e pellet is th en r es us pended
in 0. 6 25 mM EDTA/18. 75 mM Diema l, pH 7. 4 a nd f r oZ en a t
700 C. f or a t lea s t 24 h our s .
US 2011/0318847 A1
[ 0112] F ollow ing th a w ing , th e r es ulting ma ter ia l is centr i
f ug ed ( a t 3700 x g ) f or 30 minutes , th en th e s uper na ta nt is
r emov ed a nd ? lter ed ( w ith a 0. 45 [ mi ? lter a v a ila ble f r om
M illipor e) , th en loa ded onto a DEAE ( dieth y la minoeth y l)
Seph a r os e ion- ex ch a ng e column ( a v a ila ble f r om Ph a r ma cia ) ,
pr e- pr epa r ed us ing a binding buf f er ( e. g . 0. 6 3 mM EDTA/18.
75 mM Diema l, pH 7. 4) . Any non- binding ma ter ia l pa s s es
th r oug h th e column a nd is eluted. Once a ll of th e non- binding
ma ter ia l is eluted f r om th e column, pur e ca lpr otectin is eluted
us ing a ca lcium- conta ining elution buf f er ( eg 75 mM
Diema l buf f er / 10 mM Ca Cl2) . About 25 mg ca lpr otectin is
obta ined per buf f y coa t.
( b) Pr epa r a tion of Anti- Ca lpr otectin Antibody
[ 0113] Anti- ca lpr otectin a ntibodies ma y be pr epa r ed
a ccor ding to th e meth od des cr ibed in Ex a mple 3 of US. Pa t.
N o. 4, 833, 074 ( F a g er h ol)
[ 0114] Ch ick en eg g poly clona ls ma y a lter na tiv ely be pr e
pa r ed. A s olution compr is ing ca lpr otectin ( 0. 5 mg /ml) a nd
F r eunds a dj uv a nt is inj ected into ch ick ens ev er y 14 da y s f our
times ( or f or tw o month s ) , a nd th en once ev er y 1 month . Af ter
12 w eek s , th e eg g s of th e ca lpr otectin- inj ected ch ick en ma y
be collected a nd th eir y olk s r emov ed ( w ith out th e ? lm) . F ol
low ing dilution in HCl. ( 5 mM ) , th e y olk is centr if ug ed a nd
th e s uper na ta nt is collected. Th e s uper na ta nt is th en ? lter ed
a nd tr ea ted w ith s a tur a ted a mmonium s ulf a te to a ? na l con
centr a tion of 3. 8 M . Th e mix tur e is centr if ug ed a nd th e pr e
cipita te pr oduced is collected a nd dis s olv ed in buf f er ( 0. 11 M
s odium a ceta te, 0. 15 M N a Cl, pH 7. 4) . Th e r es ulting s olution
is ? na lly dia ly s ed w ith a membr a ne h a v ing a por e s iZ e of
10, 000 k D a nd th en pur i? ed by a ? inity ch r oma tog r a ph y .
[ 0115 ] Th e column ty pica lly us ed f or a ? inity ch r oma tog
r a ph y compr is es a n a ctiv a ted ma tr ix of s uccinimide- a ctiv a ted
s eph a r os e ( HiTr a p N HS a ctiv a ted a v a ila ble f r om Amer s h a m
Ph a r ma cia ) w h ich is s uita ble f or th e immobilis a tion of
ca lpr otectin. M or e s peci? ca lly , th e a ctiv a ted r es in r ea cts
s ponta neous ly , a t pH 7- 8, w ith f r ee a mines in th e ca lpr otectin.
F or ch r oma tog r a ph y th e dia ly s is s olution is us ua lly diluted to
a concentr a tion of a bout 3 mg /ml in PBS pr ior to its a pplica
tion to th e column. Th e a ntica lpr otectin a ntibodies a r e s ub
s eq uently eluted us ing 6 M ur ea in ice cold PBS or 0. 1 M
s odium citr a te s olution, pH 3. 0. Pr ef er a bly , 0. 1 M s odium
citr a te s olution is us ed. F ollow ing elution, th e a ntica lpr otec
tin a ntibody conta ining f r a ctions a r e immedia tely diluted a nd
dia ly s ed in PBS.
EX AM PL E 2
Tur bidimetr ic As s a y f or Ca lpr otectin
( a ) Pr epa r a tion of Av idin- Coa ted N a nopa r ticles
[ 0116 ] 6 00 [ mi of 4. 2% w /v ch lor ometh y l a ctiv a ted na no
pa r ticles ( dia meter 44 nm) a v a ila ble f r om I nter f a cia l
Dy na mic Cor por a tion, US a r e dia ly s ed a g a ins t w a ter w ith a
membr a ne h a v ing a por e s iZ e of 10, 000 k D. 0. 5 ml of a bor a te
( 10 mM ) a nd s odium ch lor ide ( 15 mM ) s olution a t pH 9 . 0 is
a dded a nd mix ed. 10 mg a v idin, dis s olv ed in 0. 5 ml of a 10
mM bor a te a nd 15 mM N a Cl s olution a t pH 9 ( a v a ila ble f r om
Pier ce Ch emica l Compa ny ) is a dded a nd th e mix tur e is a g i
ta ted a t r oom temper a tur e f or 24 h our s . 40 [ 1. 1 of g ly cine
s olution ( 2M , pH 9 . 0) is th en a dded a nd th e mix tur e is a g i
ta ted f or a f ur th er 4 h our s a t r oom temper a tur e.
[ 0117] Th e pa r ticles a r e th en diluted to a v olume of 100 ml
a nd dia ? ltr a ted, ? r s tly in 5 00 ml of a 10 mM bor a te a nd 15
Dec. 29 , 2011
mM s odium ch lor ide s olution a t pH 9 . 0 a nd s econdly in a 25
mM Tr is , 15 0 mM s odium ch lor ide a nd 0. 01% Tw een 20
s olution a t pH 7. 4 ( a v a ila ble f r om Sig ma US) us ing a Pellicon
X L F ilter ( cut of f 300, 000) a nd a la bs ca le TTF Sy s tem ( a v a il
a ble f r om M illipor e) in a ccor da nce w ith th e ins tr uctions s up
plied f r om th e ins tr uments s upplier s . Th e des ir ed concentr a
tion of a v idin- coa ted na nopa r ticles is ? na lly obta ined by
centr if ug a tion a nd r e- s us pens ion of th e pa r ticles in a 25 mM
THI S, 15 0 mM s odium ch lor ide a nd 0. 01% Tw een 20 s olu
tion. Any a g g r eg a tes f or med dur ing th is pr epa r a tion pr oce
dur e ma y be r emov ed by s low centr if ug a tion.
( b) As s a y f or Ca lpr otectin Us ing Av idin- Coa ted N a nopa r
ticles
[ 0118] A s us pens ion h a v ing a concentr a tion of a bout 0. 30
mg pa r ticles of th e a bov e- des cr ibed a v idin- coa ted na nopa r
ticles per ml is pr epa r ed by centr if ug a tion a nd r e- s us pens ion
of th e a bov e- des cr ibed pr epa r a tion in a 25 mM THI S, 15 0
mM N a C1, 0. 1% Tw een 20 a nd 2% PEG 6 000 s olution a t pH
7. 4 ( a v a ila ble f r om Sig ma ) . 5 00 [ 1. 1 of th is pa r ticle s us pens ion
is mix ed w ith a pla s ma s a mple ( a bout 20 [ 1. 1) , ta k en f r om a
s ubj ect being tes ted f or pr opens ity to CVD, in a r ea ding
q ua r tz cuv ette of a r ecor ding s pectr oph otometer ( eg a Sh i
ma dZ u UV- 16 0) . Th e a bs or ption of 340 nm monoch r oma tic
lig h t is r ecor ded a nd a f ter 6 0 s , 75 [ 1g of a ntica lpr otectin
a ntibody la belled w ith 0. 15 nmol biotin ( e. g . biotin la belled
a ? inity pur i? ed eg g poly clona l pur ch a s ed f r om N or w eg ia n
Antibodies AS, N or w a y ) , diluted in 5 0 [ 1. 1 of a 25 mM THI S,
15 0 mM N a Cl a nd 0. 1% Tw een 20 s olution a t pH 7. 4 is
a dded to th e q ua r tz cuv ette a nd mix ed. Th e a bs or ption of 340
nm monoch r oma tic lig h t is immedia tely r ecor ded us ing a
r ef er ence cuv ette conta ining a s olution of 25 mM TRI S, 15 0
mM N a Cl a nd 0. 1% Tw een 20 a t pH 7. 4, a nd a g a in a t
r eg ula r inter v a ls ( e. g . ev er y 2 minutes ) until a bout 15 minutes
h a s ela ps ed. Th e incr ea s e in a bs or ption a t ea ch time point is
ca lcula ted in a ccor da nce w ith s ta nda r d tur bidimetr ic r ea ding
in k inetic mode or end- point r ea ding s . Th a t is , th e incr ea s e
in lig h t a bs or ption a t ea ch time- point is ca lcula ted r ela tiv e to
th e r ea ding ma de pr ior to th e a ddition of a ntibody - coa ted
na nopa r ticles a nd/or a t th e end of th e r ecor ding .
[ 0119 ] A ca libr a tion cur v e is cons tr ucted by ca r r y ing out a n
identica l pr ocedur e w ith s ta nda r ds h a v ing a k now n concen
tr a tion of ca lpr otectin. Th e concentr a tion of ca lpr otectin in
th e s a mple ca n th en be ca lcula ted f r om th e ca libr a tion cur v e.
EX AM PL E 3
Alter na tiv e Tur bidimetr ic As s a y f or Ca ipr otectin
( a ) Pr epa r a tion of Anti- Ca lpr otectin Antibody Coa ted N a no
pa r ticles
[ 0120] 1 m1 of 4. 2% w /v ch lor ometh y l a ctiv a ted na nopa r
ticles ( dia meter 44 nm) a v a ila ble f r om I nter f a cia l Dy na mic
Cor por a tion, US a r e dia ly s ed a g a ins t w a ter w ith a membr a ne
h a v ing a por e s iZ e of 10, 000 k D. 0. 5 m1 of a 10 mM bor a te a nd
15 mM s odium ch lor ide s olution a t pH 9 . 0 is th en a dded. 27
mg of pur i? ed a ntica lpr otectin a ntibodies ( e. g . a ? inity pur i
? ed eg g poly clona l a ntibodies a v a ila ble f r om N or w eg ia n
Antibodies AS, N or w a y ) a r e dia ly s ed a g a ins t a 10 mM bor a te
a nd 15 mM s odium ch lor ide s olution a t pH 9 . 0.
[ 0121] F ollow ing a ddition of th e na nopa r ticles to th e pur i
? ed a ntica lpr otectin a ntibodies th e mix tur e is a g ita ted f or 24
US 2011/0318847 A1
h our s a t r oom temper a tur e. 40 ul of a g ly cine s olution ( 2 M a t
pH 9 . 0) is th en a dded a nd th e mix tur e is a g ita ted f or a f ur th er
4 h our s a t r oom temper a tur e.
[ 0122] Th e pa r ticles a r e th en diluted to tota l v olume of 100
ml a nd dia ? ltr a ted a g a ins t 1000 ml of a 10 mM bor a te a nd 15
mM s odium ch lor ide s olution a t pH 9 . 0 to Wh ich 0. 1%
Tw een@ 20 a nd 3 mg /ml eg g a lbumin is a dded us ing a Pelli
con X L ? lter ( cut of 300, 000) a nd a la bs ca le TF F s y s tem
( a v a ila ble f r om M illipor e) in a ccor da nce With th e ins tr uctions
s upplied f r om th e ins tr uments s upplier s . Th e des ir ed concen
tr a tion of a ntica lpr otectin a ntibody - coa ted na nopa r ticles is
? na lly obta ined by centr if ug a tion a nd r e- s us pens ion of th e
pa r ticles in s olution. Any a g g r eg a tes f or med dur ing th is
pr epa r a tion pr ocedur e ma y be r emov ed by s loW centr if ug a
tion.
( b) As s a y f or Ca lpr otectin Us ing Anti- Ca lpr otectin Anti
body - Coa ted N a nopa r ticles
[ 0123] A s us pens ion compr is ing 400 ug of th e a bov e- de
s cr ibed a ntibody - coa ted na nopa r ticles in 5 0 ul of a 10 mM
bor a te, 15 mM N a Cl, 0. 1% Tw een@ 20, 3 g /l eg g a lbumin
s olution a t pH 9 . 0 is pr epa r ed.
[ 0124] Simulta neous ly , 20 ul of pla s ma , ta k en f r om th e
s ubj ect being tes ted f or potentia l f or CVD, in 5 00 a s s a y buf f er
( 25 mM TRI S, 15 0 mM N a Cl, 0. 1% Tw een@ 20 a nd 2% PEG
6 000 a t pH 7. 4 ( a v a ila ble f r om Sig ma ) is put in a r ea ding
q ua r tz cuv ette of a r ecor ding s pectr oph otometer ( e. g . Sh i
ma dz u UV- 16 0) a nd th e lig h t a bs or ption of 340 nm mono
ch r oma tic lig h t is mea s ur ed. Af ter 6 0 s , th e a bov e- mentioned
s us pens ion compr is ing 400 of a ntibody - coa ted na nopa r ticles
is a dded, a nd mix ed in th e cuv ette. Th e lig h t a bs or ption
immedia tely a f ter a dding th e a ntibody - coa ted na nopa r ticles
is r ecor ded, a nd a g a in a t r eg ula r inter v a ls ( e. g . ev er y 2 min
utes ) until a bout 15 minutes h a s ela ps ed. Th e incr ea s e in lig h t
a bs or ption a t ea ch time- point is ca lcula ted r ela tiv e to th e
r ea ding ma de pr ior to th e a ddition of a ntibody - coa ted na no
pa r ticles a nd/or a t th e end of th e r ecor ding . I n oth er Wor ds ,
tur bidimetr ic r ea ding s in k inetic mode or end- point r ea d
ing s a r e ma de.
[ 0125 ] A ca libr a tion cur v e is a ls o cons tr ucted by ca r r y ing
out a n identica l pr ocedur e With s ta nda r ds h a v ing a k noWn
concentr a tion of ca lpr otectin. Th e concentr a tion of ca lpr otec
tin in th e s a mple ca n th en be ca lcula ted f r om th e cur v e.
EX AM PL E 4
Tur bidimetr ic As s a y f or Ca lpr otectin
( a ) Pr epa r a tion of Str epta v idin- Coa ted N a nopa r ticles
[ 0126 ] 6 00 pm of 4. 2% W/v ch lor ometh y l a ctiv a ted na no
pa r ticles ( dia meter 6 7 nm) a v a ila ble f r om I nter f a cia l
Dy na mic Cor por a tion, US a r e dia ly s ed a g a ins t Wa ter With a
membr a ne h a v ing a por e s iz e of 10, 000 k D. 0. 5 ml of a
ph os ph a te ( 10 mM ) a nd s odium ch lor ide ( 15 0 mM ) buf f er
s olution a t pH 7. 4 is a dded tog eth er With 10 mg s tr epta v idin,
dis s olv ed in 0. 5 ml of a 10 mM ph os ph a te a nd 15 0 mM N a Cl
buf f er s olution a t pH 7. 4 ( a v a ila ble f r om Pier ce Ch emica l
Compa ny ) a nd th e mix tur e is a g ita ted a t r oom temper a tur e f or
24 h our s . 40 pa of g ly cine s olution ( 2M , pH 9 . 0) is th en a dded
a nd th e mix tur e is a g ita ted f or a f ur th er 4 h our s a t r oom
temper a tur e.
[ 0127] Th e pa r ticles a r e th en diluted to a v olume of 100 ml
a nd dia ? ltr a ted, ? r s tly in 5 00 ml of a 10 mM bor a te a nd 15
mM s odium ch lor ide s olution a t pH 9 . 0 a nd s econdly in a 25
Dec. 29 , 2011
mM Tr is , 15 0 mM s odium ch lor ide a nd 0. 01% Tw een@ 20
s olution a t pH 7. 4 ( a v a ila ble f r om Sig ma US) us ing a Pellicon
X L F ilter ( cut of f 300, 000) a nd a la bs ca le TTF Sy s tem ( a v a il
a ble f r om M illipor e) in a ccor da nce With th e ins tr uctions s up
plied f r om th e ins tr uments s upplier s . Th e des ir ed concentr a
tion of a v idin- coa ted na nopa r ticles is ? na lly obta ined by
centr if ug a tion a nd r e- s us pens ion of th e pa r ticles in a 25 mM
TRI S, 15 0 mM s odium ch lor ide a nd 0. 010 Tw een@ 20 s olu
tion. Any a g g r eg a tes f or med dur ing th is pr epa r a tion pr oce
dur e ma y be r emov ed by s loW centr if ug a tion.
[ 0128] Th e mea n pa r ticle s iz e of th e s tr epta v idin coa ted
na nopa r ticles Wa s mea s ur ed to be 82 nm by Sintef f AS, N or
Wa y .
( b) As s a y f or Ca lpr otectin Us ing Str epta v idin- Coa ted N a no
pa r ticles
[ 0129 ] A s us pens ion h a v ing a concentr a tion of a bout 0. 6 0
mg pa r ticles of th e a bov e- des cr ibed a v idin- coa ted na nopa r
ticles per ml is pr epa r ed by centr if ug a tion a nd r e- s us pens ion
of th e a bov e- des cr ibed pr epa r a tion in a 25 mM TRI S, 15 0
mM N a Cl, 0. 1% Tw een@ 20 a nd 1% PEG 6 000 s olution a t pH
7. 4 ( a v a ila ble f r om Sig ma ) . 5 00 [ 1. 1 of th is pa r ticle s us pens ion
is mix ed With a pla s ma s a mple ( a bout 5 ul) , ta k en f r om a
s ubj ect being tes ted f or pr opens ity to CVD, in a r ea ding
q ua r tz cuv ette of a r ecor ding s pectr oph otometer ( eg a Sh i
ma dz u. UV- 16 0) . Th e a bs or ption of 5 6 0 nm monoch r oma tic
lig h t is r ecor ded a nd a f ter 6 0 s , 75 ug of a ntica lpr otectin
a ntibody la belled With 0. 15 nmol biotin ( e. g . biotin la belled
a ? inity pur i? ed eg g poly clona l pur ch a s ed f r om N or Weg ia n
Antibodies AS, N or Wa y ) , diluted in 5 0 ul of a 25 mM TRI S,
15 0 mM N a Cl a nd 0. 1% Tw een@ 20 s olution a t pH 7. 4 is
a dded to th e q ua r tz cuv ette a nd mix ed. Th e a bs or ption of 340
nm monoch r oma tic lig h t is immedia tely r ecor ded us ing a
r ef er ence cuv ette conta ining a s olution of 25 mM TRI S, 15 0
mM N a Cl a nd 0. 1% Tw een@ 20 a t pH 7. 4, a nd a g a in a t
r eg ula r inter v a ls ( e. g . ev er y 2 minutes ) until a bout 15 minutes
h a s ela ps ed. Th e incr ea s e in a bs or ption a t ea ch time point is
ca lcula ted in a ccor da nce With s ta nda r d tur bidimetr ic r ea ding
in k inetic mode or end- point r ea ding s . Th a t is , th e incr ea s e
in lig h t a bs or ption a t ea ch time- point is ca lcula ted r ela tiv e to
th e r ea ding ma de pr ior to th e a ddition of a ntibody - coa ted
na nopa r ticles a nd/or a t th e end of th e r ecor ding .
[ 0130] A ca libr a tion cur v e is cons tr ucted by ca r r y ing out a n
identica l pr ocedur e With s ta nda r ds h a v ing a k noWn concen
tr a tion of ca lpr otectin. Th e concentr a tion of ca lpr otectin in
th e s a mple ca n th en be ca lcula ted f r om th e ca libr a tion cur v e.
EX AM PL E 5
( a ) Pr epa r a tion of Anti- Ca lpr otectin Antibody
Coa ted N a nopa r ticles
[ 0131] 1 ml of 4. 2% W/v ch lor ometh y l a ctiv a ted na nopa r
ticles ( mea n dia meter 6 7 nm) a v a ila ble f r om I nter f a cia l
Dy na mic Cor por a tion, US, a r e dia ly s ed a g a ins t Wa ter With a
membr a ne h a v ing a por e s iz e of 10. 000 k D, a nd th en diluted
to 10 ml With Wa ter . 27 mg of pur i? ed eg g poly clona l a nti
bodies ( e. g . a f ? nity pur i? ed eg g poly clona l a ntibodies a v a il
a ble f r om N or Weg ia n Antibodies AS, N or Wa y ) a r e dia ly s ed
a g a ins t a 10 mM bor a te a nd 15 mM s odium ch lor ide buf f er
s olution a nd ? na lly diluted to 6 ml in th e s a me 10 mM bor a te
a nd 15 mM s odium ch lor ide s olution a t pH: 9 . 0.
[ 0132] Under a g ita tion, th e pa r ticles a r e mix ed With th e
a ntibodies , a nd a g ita tion is continued a t r oom temper a tur e f or
24 h our s . 40 ul of a g ly cine s olution ( 2 M a t pH 9 . 0) is th en
a dded a nd th e mix tur e is a g ita ted f or a f ur th er 4 h our s a t r oom
temper a tur e.
[ 0133] Th e pa r ticles a r e th en diluted to a tota l v olume of
100 ml in 10 mM bor a te, 15 mM s odium ch lor ide buf f er to
Wh ich 0. 1% Tw een@ 20 a nd 3 mg /ml eg g a lbumin is a dded
a nd dia ? ltr a ted a g a ins t 1000 ml of s a id bor a te/ s odium ch lo
r ide buf f er to Wh ich 0. 1% Tw een@ 20 a nd 3 mg /ml eg g
US 2011/0318847 A1
a lbumin is a dded, us ing a Pellicon X L ? lter ( cut of f 300. 000
D) a nd a la bs ca le TF F s y s tem ( a v a ila ble f r om M illipor e) in
a ccor da nce With th e ins tr uctions s upplied f r om th e ins tr u
ment s upplier s , a nd in th e end th e pa r ticles a r e concentr a ted to
a v olume of 40 to 100 ml.
[ 0134] Th e mea n dia meter of th e pa r ticles obta ined Wa s
mea s ur ed to be 81 nm by Sintef f AS, N or w a y .
( b) As s a y f or Ca lpr otectin Us ing Anti- ca lpr otectin
Antibody - Coa ted N a nopa r ticles
[ 0135 ] A s us pens ion of 0. 7 mg /ml of th e a bov e des cr ibed
a ntica lpr otectin a ntibody coa ted pa r ticles is ma de in 0. 25
mM TRI S, 0. 15 M N a Cl a nd 0. 1% TWeen a t pH 8. 0.
[ 0136 ] 5 [ 1. 1 of a pla s ma s a mple, ta k en f r om th e s ubj ect
being tes ted f or CVD, is dis s olv ed in a s s a y buf f er ( 46 0 [ 1. 1, 25
mM TRI S, 15 0 mM N a Cl, 0. 1% TWeen, 1. 0% poly eth y leneg
ly col 6 000, pH 7. 4) in a r ea ding q ua r tz cuv ette in a r ecor ding
s pectr oph otometer ( e. g . Sh ima dZ u UV- 16 0) a nd th e lig h t
a bs or ption of 5 6 0 nm monoch r oma tic lig h t is mea s ur ed. Af ter
6 0 s , 100 [ 1. 1 of th e a bov e mentioned s us pens ion compr is ing
0. 7 mg /ml of a ntibody coa ted na nopa r ticles is a dded, a nd
mix ed in th e cuv ette. Th e lig h t a bs or ption bef or e a nd imme
dia tely a f ter a dding th e a ntibody - coa ted na nopa r ticles is
r ecor ded, a nd a g a in a t r eg ula r inter v a ls ( eg . ev er y 20 s ) until
15 minutes h a s ela ps ed. Th e incr ea s e in lig h t a bs or ption a t
ea ch time point is ca lcula ted r ela tiv e to th e r ea ding ma de pr ior
to th e a ddition of a ntibody coa ted na nopa r ticles a nd a t th e end
of th e r ecor ding . I n oth er Wor ds , tur bidimetr ic r ea ding s in
k inetic mode a nd/or end point r ea ding s a r e ma de.
[ 0137] A ca libr a tion cur v e is a ls o cons tr ucted by ca r r y ing
out a n identica l pr ocedur e With s ta nda r ds h a v ing a k now n
concentr a tions of ca lpr otectin. Th e concentr a tion of ca lpr o
tectin ib th e s a mple ca n th en be ca lcula ted f r om th e cur v e.
EX AM PL E 6
Sta tis tica l Ana ly s is
Compa r is on of Ca lpr otectin a nd Oth er M a r k er s f or Detection
of Potentia l f or CVD or Pr opens ity to CVD
[ 0138] Cor ona r y ca lci? ca tion h a s been s h oWn to be
s tr ong ly a s s ocia ted With th e occur r ence of CVD a nd h a s a ls o
been demons tr a ted to be a us ef ul meth od f or pr edicting
potentia l f or CVD ( e. g . my coca r dia l inf a r ction or , s tr ok e) .
[ 0139 ] Th e ex tent of cor ona r y ca lci? ca tion is q ua ntita tiv ely
mea s ur ed us ed electr on- bea m computed tomog r a ph y
( EBCT) a nd is r epr es ented by a ca lcium s cor e ( CS) . A h ig h
ca lcium s cor e r epr es ents a h ig h lev el of ca lci? ca tion a nd a
h ig h r is k of dev eloping CVD.
[ 0140] I n th e f olloWing s tudy th e CS of 200 s ubj ects ( 100
contr ols h a v ing a CS < 100 a nd 100 ca s es h a v ing a CS > 100)
a g ed 45 or g r ea ter Wa s tes ted a s Well a s th eir ca lpr otectin,
CRP a nd h omocy s teine pla s ma or s er um lev els . Th e s ubj ects
Wer e eith er s elf - or ph y s icia n- r ef er r ed a s y mptoma tic indi
v idua ls a nd h a d h a d a n EBCT s ca n With in th e pr ev ious 2 y ea r s
( us ua lly With in th e pr ev ious 6 month s ) to tes ting of th eir
pla s ma or s er um f or th e concentr a tion of ca lpr otectin, CRP
a nd h omocy s teine.
M eth ods
Ca lpr otectin
[ 0141] Ca lpr otectin Wa s mea s ur ed by th e Ca lpr es t tes t
( dis tr ibuted by Eur os pita l , I ta ly ) a nd da ta Wer e s umma r is ed
f or th e pa tient a nd th e contr ol g r oup us ing s ta nda r d dev ia tion,
media n, minimum a nd ma x imum. 9 5 % con? dence inter v a ls
f or media n Wer e ca lcula ted. Th e Ander s on- Da r ling ca lcula
tion Wa s us ed a s a tes t f or nor ma lity .
[ 0142] Ca lpr otectin Wa s a ls o s umma r is ed by g ender us ing
th e s a me s umma r y s ta tis tics .
Dec. 29 , 2011
Ca lcium Scor e ( CS)
[ 0143] Ca lcium s cor e Wa s deter mined by Electr on- Bea m
Computed Tomog r a ph y ( ERCT) s ca nning . Da ta f or ca s e a nd
contr ol g r oups Wer e s umma r is ed.
Hig h Sens itiv e C- Rea ctiv e Pr otein ( h s CRP) h s CRP Wa s
deter mined by Da de Beh r ing s N Hig h Sens itiv ity CRP
a s s a y ( Rober ts et a l. , Clinica l Ch emis tr y , 2000, 46 : 4, p 46 1
46 8) . Da ta Wer e s umma r is ed f or th e pa tient a nd th e contr ol
g r oups us ing mea n, s ta nda r d dev ia tion, media n, minimum
a nd ma x imum.
Homocy s teine ( Hy c)
[ 0144] Pla s ma Homocy s teine ( Hcy ) Wa s deter mined by th e
Abbott I M x meth od ( Sh ipch a ndler , M . T. a nd M oor e E. G. ,
Clinica l Ch emis tr y , 19 9 5 , 41 : 7, p. 9 5 1- 9 9 4) . Da ta Wer e s um
ma r is ed f or th e pa tient a nd th e contr ol g r oups us ing s ta nda r d
dev ia tion, media n, minimum a nd ma x imum.
Compa r is on BetWeen Cs a nd Ca lpr otectin, h s CRP, Hcy
( i) Ch i- Sq ua r ed
[ 0145 ] Th e Ch i- s q ua r ed tes t Wa s us ed to tes t f or cov a r ia nce
betWeen pa ir s of ma r k er s .
( ii) Odds Ra tio
[ 0146 ] Th e odds r a tio us ed With th e 2x 2 cr os s - ta bula tion
( ie th e ch i- s q ua r ed ta ble) is th e r a tio of th e odds of tWo tes ts
co- v a r y ing to th e odds of tWo tes ts dis a g r eeing . Th er ef or e, th e
odds r a tio ma y be inter pr eted a s a mea s ur e bf th e ma g nitude
of a s s ocia tion betWeen th e tWo tes ts .
[ 0147] Odds r a tio Wa s ca lcula ted f or CS v er s us ca ipr otec
tin, h s CRP a nd Hcy r es pectiv ely .
Compa r is on of M edia n Va lues of th e M a r k er s
( i) M a nn- Wh itney Tes t
[ 0148] Th is is a non- pa r a metr ic ( th e da ta does not need to
be nor ma lly dis tr ibuted) Wh ich Wa s us ed to tes t if th e media n
v a lues of tWo ma r k er s a r e s ig ni? ca ntly dif f er ent. M inita b
r a nk s a ll th e da ta f r om both s ets of da ta in or der , a s s ig ning 1
to th e loWes t up to 200 f or th e h ig h es t. Th e s of tWa r e th en a dds
up th e r a nk - s cor e f or th e tWo g r oups to be compa r ed a nd
r epor ts th e P v a lue Wh ich indica tes th e ch a nce th a t r a ndom
s a mpling Would r es ult in media ns a s f a r a pa r t a s th a t obs er v ed
in th e ex per iment.
[ 0149 ] Ca lpr otectin, h s CRP a nd Hcy media n v a lues Wer e
compa r ed to CS s plit into ca s e a nd contr ol g r oups a nd th e
M a nn- Wh itney tes t Wa s us ed to tes t f or s ig ni? ca nce.
( ii) ROC- cur v es f or Ca lpr otectin a nd Ca lcium Scor e
[ 015 0] Th e a bility of a tes t to dis cr imina te dis ea s ed ca s es
f r om nor ma l ca s es ca n be ev a lua ted us ing Receiv er Oper a ting
Ch a r a cter is tic ( ROC) cur v e a na ly s is . ROC cur v e a na ly s is Wa s
us ed f or both ca lpr otectin a nd h s CRP a nd a s a compa r is on of
th e tWo ma r k er s .
[ 015 1] Th e ROC cur v es Wer e pr oduced us ing 1 1 cutof f
limits f or ca lcula tion of th e s ens itiv ity ( y - a x is ) a nd th e
1- s peci? city ( x - a x is ) . Th e da ta f r om th e contr ol a nd th e ca s e
g r oups Wer e pooled tog eth er a nd r a nk ed f r om loWes t to h ig h
es t v a lue. Ana ly s is Wa s per f or med on th e tota l popula tion a nd
on th e ma le popula tion. Th e a r ea s under th e ROC cur v es Wer e
ca lcula ted us ing th e tr a peZ oida l r ule.
Res ults
Summa r y Sta tis tics f or Ca lpr otectin
[ 015 2] F I G. 1 s h oWs th e dis tr ibution cur v e f or a ll 200 s ub
j ects tes ted.
[ 015 3] Ta ble 1 beloW s h oWs th e s umma r y s ta tis tics f or
ca lpr otectin in th e contr ol a nd th e ca s e g r oups . Th e media n
ca lpr otectin concentr a tion in th e contr ol g r oup is 0. 31 mg /L
compa r ed to 0. 38 mg /L in th e ca s e g r oup. Th e media n ca lpr o
tectin concentr a tion is 0. 31 mg /L f or th e ma les a nd 0. 30 mg /L
f or th e f ema les in th e contr ol g r oup. I n th e ca s e g r oup th e
media n ca lpr otectin concentr a tion f or ma les is 0. 39 mg /L
compa r ed to 0. 31 mg /L f or f ema les .
US 2011/0318847 A1 Dec. 29 , 2011
TABL E 1
Summa r y s ta tis tics f or Ca lpr otectin
Contr ols Ca s es
Ca lpr otectin All F ema les M a les All F ema les M a les
N 100 5 9 41 100 15 85
SD 0. 228 0. 232 0. 225 0. 5 03 0. 202 0. 5 37
9 5 % CI f or media n 0. 28- 0. 34 0. 28- 0. 33 0. 27- 0. 39 0. 34- 0. 43 0. 19 - 0. 47 0. 34- 0. 45
M edia n ( mg /L ) 0. 31 0. 30 0. 31 0. 38 0. 31 0. 39
M in ( mg /L ) 0. 09 0. 09 0. 11 0. 07 0. 08 0. 07
M a x ( mg /L ) 1. 37 1. 37 1. 36 4. 85 0. 73 4. 85
Summa r y Sta tis tics f or Ca lcium Scor e
[ 015 4] F I G. 2 s h ow s th e dis tr ibution cur v e f or a ll 200 s ub
j ects tes ted.
[ 015 5 ] Ta ble 2 below s h ow s th e s umma r y s ta tis tics f or
EBCT ca lcium s cor e in th e contr ol a nd ca s e g r oups . Th e
media n ca lcium s cor e in th e contr ol g r oup is 0 compa r ed to
25 9 in th e ca s e g r oup. Th e media n ca lcium s cor e is 0 f or both
f ema les a nd ma les in th e contr ol g r oup a nd 315 a nd 25 6 ,
r es pectiv ely , in th e ca s e g r oup.
TABL E 2
Summa r y s ta tis tics f or Ca lcium s cor e
Contr ols Ca s es
Ca lcium s cor e All F ema les M a les All F ema les M a les
N 100 5 9 41 100 15 85
SD 5 . 6 0. 7 8. 7 474. 7 36 5 . 1 49 3. 3
9 5 % CI f or media n 0- 0 0- 0 0- 0 313. 5 - 215 . 0 174. 8 5 38. 0 300. 4 207. 5
M edia n 0 0 0 25 9 315 25 6
M in 0 0 0 100 100 100
M a X 5 6 5 5 6 279 4 15 07 279 4
Summa r y Sta tis tics f or h s CRP
[ 015 6 ] F I G. 3 s h oWs th e dis tr ibution cur v e f or a ll 200 s ub
j ects tes ted.
[ 015 7] Ta ble 3 beloW s h oWs th e s umma r y s ta tis tics f or
h s CRP in th e contr ol a nd ca s e g r oups . Th e media n h s CRP in
both th e contr ol g r oup a nd th e ca s e g r oup is 1. 2 mg /L . Th e
media n h s CRP is 1. 6 mg /L f or f ema les a nd 0. 7 mg /L f or
ma les in th e contr ol g r oup a nd 1. 3 mg /L a nd 1. 1 mg /L ,
r es pectiv ely , in th e ca s e g r oup.
TABL E 3
Summa r y s ta tis tics f or h s CRP
Contr ols Ca s es
h s CRP All F ema les M a les All F ema les M a les
N 100 5 9 41 100 15 85
SD 6 . 6 3 8. 36 1. 5 3 12. 70 3. 16 13. 71
9 5 % CI f or media n 0. 79 - 1. 47 1. 36 - 2. 74 0. 5 6 - 0. 9 7 0. 9 4- 1. 5 6 0. 6 5 - 2. 5 3 0. 89 - 1. 5 9
M edia n ( mg /L ) 1. 17 1. 5 9 0. 6 8 1. 23 1. 28 1. 11
M in ( mg /L ) 0. 15 0. 15 0. 15 0. 17 0. 34 0. 17
M a x ( mg /L ) 6 2. 1 6 2. 1 9 . 29 120. 0 11. 9 120. 0
US 2011/0318847 A1
1 2
Summa r y Sta tis tics f or Hcy
[ 015 8] F I G. 4 s h ow s th e dis tr ibution cur v e f or a ll 200 s ub
j ects tes ted.
[ 015 9 ] Ta ble 4 below s h oWs th e s umma r y s ta tis tics f or Hcy
in th e contr ol a nd ca s e g r oups . Th e media n Hcy concentr a tion
in th e contr ol g r oup is 7. 5 umol/L compa r ed to 8. 5 umol/L in
th e ca s e g r oup. Th e media n Hcy concentr a tion is 7. 15 umol/L
f or f ema les a nd 8. 5 5 mol/L f or ma les in th e contr ol g r oup a nd
7. 35 umol/L a nd 8. 5 5 umol/L r es pectiv ely in th e ca s e g r oup.
TABL E 4
Summa r y s ta tis tics f or Hcv
Contr ols Ca s es
Hcy All F ema les M a les All F ema les M a les
N 100 5 9 41 100 15 85
SD 1. 9 4 1. 5 8 2. 18 6 . 03 2. 18 6 . 46
9 5 % CI 7. 7- 8. 5 7. 1- 7. 9 8. 1- 9 . 5 8. 1- 10. 6 . 8- 9 . 2 81- 109
f or media n
M edia n 7. 5 7. 1 8. 5 8. 5 7. 3 8. 5
( pI nol/L )
M in ( pI nol/L ) 5 . 3 5 . 3 5 . 3 5 . 1 5 . 1 5 . 2
M a x ( pI nol/L ) 14. 7 11. 8 14. 7 6 5 . 8 12. 4 6 5 . 8
Compa r is on betWeen ca lcium s cor e a nd ca lpr otectin, h s CRP,
Hcy
( i) Ch i- Sq ua r ed Tes t
[ 016 0] Us ing M inita b, ch i- s q ua r ed ta bles Wer e per f or med
( s ee ta ble 5 beloW) . Th is tes t compa r es ex pected dis tr ibutions
betWeen s ets of da ta ( a s s uming a r a ndom dis tr ibution) a nd
th os e obs er v ed. Th e s ig ni? ca nce v a lue is a mea s ur e of th e
deg r ee to Wh ich th e da ta is not r a ndomly dis tr ibuted. F or
ex a mple, cons ider ing th e da ta f or ca lpr otectin pos itiv e it
Would be ex pected th a t th er e Would be a n ev en s pilt betWeen
ca lcium pos itiv e a nd neg a tiv e ( i. e. 30. 5 ex pected in both
columns ) . Th e obs er v ed s plit, h oWev er , is 22 neg a tiv e a nd 39
pos itiv e indica ting a tendency f or th e ca lpr otectin pos itiv es to
co- v a r y With th e ca lcium pos itiv es .
[ 016 1] M inita b pr ov ides a n ov er a ll mea s ur e of th e s ig ni?
ca nce of th es e dif f er ences ( both a g r eement a nd dis a g r ee
ment) a nd in th is ca s e P is 0. 009 . Th us th er e is a s ig ni? ca nt
co- v a r ia tion of ca lcium With ca lpr otectin.
TABL E 5
Ch i- Sq ua r e compa r is on of Ca lpr otectin v er s us Ca lcium s cor e
Ca lcium ( co 100)
Ca lpr otectin N eg a tiv e Pos itiv e All
( co 0. 45 mg /L ) ( N ) ( N ) ( N )
N eg a tiv e Obs 78 6 1 139
Ex p 6 9 . 5 6 9 . 5 139
Pos itiv e Obs 22 39 6 1
Ex p 30. 5 30. 5 6 1
All Obs 100 100 200
Ex p 100 100 200
[ 016 2] An a na log ous compa r is on Wa s ma de betWeen
ca lpr otectin a nd h s CRP ( s ee Ta ble 6 beloW) .
Dec. 29 , 2011
TABL E 6
Ch i- Sq ua r e compa r is on of Ca lpr otectin
v er s us h s CRP ( P = 0)
h s CRP
( co 1. 6 9 mg /L )
Ca lpr otectin N eg a tiv e Pos itiv e All
( co 0. 45 mg /L ) ( N ) ( N ) ( N )
N eg a tiv e Obs 100 39 139
Ex p 88. 9 6 5 0. 04 139
Pos itiv e Obs 28 33 6 1
Ex p 39 . 04 21. 9 6 6 1
All Obs 128 72 200
Ex p 128 72 200
( ii) Odds Ra tio
[ 016 3] Odds r a tios ca n be der iv ed f r om ta bles 5 a nd 6 a nd
g iv e a mea s ur e of h oW f a r th e v a lues obs er v ed dev ia te f r om
th e ex pected. I f th e ex pected a g r eement ? g ur es a r e multiplied
tog eth er a nd div ided by th e pr oduct of th e dis a g r eement
ex pected ? g ur es a v a lue of 1 s h ould be obta ined.
[ 016 4] Ta k ing th e da ta in Ta ble 6 , f or ins ta nce, th e odds
r a tio on th e ex pected v a lues is 1: ( 88. 9 6 > < 21. 9 0/( 5 0. 04> < 39 .
04) . 19 5 3. 6 4/19 5 3. 6 : 1.
[ 016 5 ] Th e odds r a tio obs er v ed in th is ta ble is : ( l00> < 33) /
( 39 x 28) : 3300/109 2: 3. 02.
[ 016 6 ] Th e f ur th er th e odds r a tio f r om 1 th e mor e pr o
nounced th e co- v a r ia tion; a n odds r a tio of 3 is s ig ni? ca nt.
[ 016 7] Th e r es ults of a n odds r a tio a na ly s is ca r r ied out on
th e co- v a r ia nce da ta obta ined f r om th e s tudy a r e s h oWn in
Ta ble 7.
TABL E 7
Summa r y of Odds - Ra tio ? nding s
Ris k ma r k er s Odds r a tio
( N l# pos , N 2# neg ) Ca lcium Ca lpr otectin CRP
Ca lcium Scor e ( co 100) i i i
Ca lpr otectin ( co 0. 45 mg L ) 2. 27 i i
CRP ( co 1. 6 9 mg L ) 1. 00 3. 02 i
Hcy ( co 12 pI nol/L ) 1. 21 1. 32 1. 5 2
N 1 =11, N 2 = 189
( co 10 pI nol/L ) 1. 6 3 1. 5 5 1. 12
co = cutof f
[ 016 8] A h ig h odds r a tio indica tes a h ig h deg r ee of co
v a r ia tion betWeen th e tes ts . I t ca nbe s een f r om Ta ble 7 th a t th e
tes t f or ca lpr otectin g iv es th e h ig h es t odds r a tio to ca lcium
s cor e a nd th er ef or e it ca n be deduced th a t ca lpr otectin h a s th e
h ig h es t deg r ee of cov a r ia nce With ca lcium s cor e out of
ca lpr otectin, CRP a nd h omocy s teine.
[ 016 9 ] Additiona lly , ca lpr otectin g iv es a h ig h odds r a tio
With CRP, a noth er ma r k er f or CVD.
Ch i- Sq ua r ed Ana ly s is Us ing M inita b
[ 0170] A ch i- s q ua r ed tes t on th e a bov e da ta , us ing th e s a me
cutof f s a s th e odds - r a tio tes t, s h oWed s ig ni? ca nt ( P 0. 009 )
a g r eement betWeen th e ca lcium s cor e a nd ca lpr otectin

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