f
AQOBLNVIWNOTOMIOU AX @ a
ASNOW-LLNY (i)- Sample-diiting solution (0.1%-BSA-contsining PAS
solution}e
-<6> Reaction-trminating liquid (0.5 M H.SO, solution)
«<7» Standand preparation (CD14 (1-307) S286C)
‘Measuring Instruments for Perorming an Assay Using the
Above Assay Kit inthis order.
-<7> Housing ease (OEM case available from NIPPN Tech-
socluster, Inc)
By the way, the outlines of <1> to <5> are represented in
FIG. 10).
84(16) t0 (19) Example of Immonochromtographie Assay
Kit
Table 8 shows, in addition to 8-15), examples ofan assay
kit fora sandwich EIA system utilizing the scond specific
binding between binding of biotin and streptavidin, and
‘examples of an assay kit fora sandwich FIA system utilizing
the fragment ofan antibody that binds to a peptide having 16
amino acid residues described in SEQ TD NO: 2. repre-
sents a labeled binding substance. The constituent elements
‘> to <7> are identical. Asa substance to be applied on <>,
-<3>-{i) represents binding substance to be immobilized on
an immobilized membrane andl <3>-(i) represents a binding
‘substance to be immobilized on acontrol line. <> represents
‘an antibody bound with a second specific binding substance,
the substance being a reagent to be applied on <2> oF as
in the ease of , of o be added o-a specimen or simul
neously added togethor with the spocimen,
By the way, the outlines of (16)<1> to ate shown in
FIG. 1(3),and (17) to 20) ate similarly understood.
old
TABLES
as Sisoed” sti
19) Godson ‘Savas winston
ay Rabjyefge—ruuoni3a. Amie
By the way, F(ab), of S68 antibody labeled with gold
colloid of (20)<1> is prepared as follows. The preparation ofUS 7,465,547 B2
45
F(ab), from S68 antibody is performed a follows using
Immobilized Pepsin PIERCE). That is, $68 antibody is is
solved in a20 mM acetate ber (pH 4.5} to be prepared to $
‘mg/ML. 0.25 mal. of immobilized Pepsin is prepared by suse
pension aovonding to the protocol of PIERCE and mised with
‘mi. of the above antibody. Next, the mixture is tired foe 4
hours in an incubator at 37° C., and then the reaction is
terminated by the addition of 1.5 mL of 10 mM Tris-HCl (pH
7.5). The reaction solution is centei fuged (1000) 10 separate
‘gel and a superntaat. Then, the separated supernatant is
faded to | mL of prosep-A (Millipore) allow the binding of
Peptides including Fe portion such as Fe fragment and undi
iBeved, Likewise, the mixture is centrifged to collect the
Supernatant, and then the supernatants dialyzed against PBS
(pH16.4).Theabsorbanceof F(ab) at 280m ismeasuredand
then the concentration of Faby. is calculated from the
absorption coustant (0.533 mp/mnLiem!). The resulting
F(ab), is labeled wit gold colloid as inthe case of Example
4, resting in F(ab’) of the gold collo-labeled S68 anti
body.
84(21) Configuration Fxample of Flow-Through System
Dye-lbeled antibody: RED VIOLET dye-labeled S68
antibody
-<2> Conjugate pad: Filter paper (No. 63, manufactured by
‘Advantec Toyo Co. [4 impregnated with the above (I)<
-<3>-Antibody-immobilized membrane: Nitrocellulose mem-
‘brane (Advantec Toyo) on which S68 antibody i immobi
lized
‘<4> Absorbing pad: Filter paper (No. 28, Advantec Tokyo)
laminated with polypropylene
<5 Housing ease: Case described in JP 06-273419 A (mame
Tctured by Mochida Pharmaceutical), <2> 10 <8 are
assembled in <5> such that liguid dropped in <2> is
allowed to flow through <2>, <3, and <4 in this onder.
Example 9
Detection of Human Low-Molecular-Weight CD14
(1) Ge Filtation Chromatography
Por analyzing the substance in serum of a patient sufering
from sepsis detected by the assay kit deserbed in Example
8.(1), the serum of the patient suffering from sepsis was
fractionated through a gel itration chromatography column
Superdex 200PC 3.2130 (Amersham Bioscience). with
SMART SYSTEM (Amersham Bioscience) using D-PBS as
a elution buile. Then, each fraction was assayed using the
assay kit described in Example &(1) and the commercially
available CDIELA kit (BL-Hamburg). The molecular
‘weight thereof was etleulated by calibrating the column
using aldolase (18 kDa), BSA (67 kDa), ovalbumin (43
Da), and chymotrypsin 25 kDa) from the LMW calibration
Kitand FMW calibration kit (Amersham Bioscience),
Asa result, a8 shown in FIG. 6, the commercially avilable
CDIGEIA kit detected soluble CDI having a molecular
weight of about $7 KDa, which was defined as high-molecu-
Jar-weight soluble CDI4 of 49 to 35 kDa conventionally
reported, On the other hand, in the kt described in Example
S-(1), a peak derived from human jow-moleculat-weight
‘CD1d detected inpatient sutfering from sepsis was detected
around a molecular weight of 35 to 45 kDa but no peak was
‘detected around S7 kDa. Thus, the result confiemed thatthe
kit deseribed in Example 8-(1) specifically detects only a
soluble protein present in blood.
46
2) Gel Filtration Chromatography <2>
‘As in the case of 2}-e1>, $0 yl of serum from a patient
sulTering from sepsis was ractionated through gelation
chromatography colimn Superdex 75 10/30) GL, (Amer
sham Bioscience) using 200 mM ammonium acetate (pH 68)
fsa elution bufler and was subjected ta the assay sing each
it The molecular weight thereof was calculated by calibrat-
ing the columa sing BSA (67 kDa), ovalbumin (43 kD),
ciymotrypsinogen (25 kDa), and ribonuclease A (13.7 kDa}
tom the MW calibrationkit and HMW calibration kit (Am-
ersham Bioscience),
"The results are shown in FIG. 7, In the kit desribed ia
Example 8-(1), a peak derived from human low-molecular-
weight CD14 was detected around a molecular Weight of 25
1035 kDa,
(8) F1025-3.1 Antibody Affinity Column Chromatography
‘When a peaked fraction (¢. faction 12) derived from
human Jow-molecular-weight CD14 obtained in (2)-<2> is
applied to P1025-3-1 antibody affinity column chromatogr-
phy; peak derived from human low-molocular-weight CD14
Js eluted inanafisity column aon-absorbing fraction. By the
way, te adjustment and operation ofthe F1025-3-1 antibody
affinity column can be performed on the basis ofthe method
described in Example 10 of WO 01/22085.
“These results show that the human low-molecular-weight
CD14 is. soluble protein in blood that specitieally binds to
antibodies against a specifi peptide described in SEQ ID
NO: 2 having a sequence detected only in human CD14 and
also binds to an ant-CD14 unibody recognizing an amino
‘acid sequence at positions 17 to 26 from the N-terminal of
hhuman CD14, The gel filtration determines the molecular
‘weight thereof to be 25 to 45 kD. Thus, itean be defined that
‘he lhuman low-moleeular-weight CD14 smallerin molecu-
CD14 does not bind o F1025-3-1 antibody that specifically
binds vo high-molecular-weight CDI.
Example 10
Assay of Low-Molecular-Weight CD14 in Sera of
Patients Suffering from Various Kinds of Diseases
10 examples, from which isolates were identified, were
sed (Table 9) asthe ser of patients suffering from sepsis. ln
addition, the assay was conducted using the assuy it
described in Fxample 8-1) on 52 examples of normal ind
Viduals (male 31 examples and female 21 examples), and
patients suffering from various kinds of diseases (20 diseases,
60 examples.
TABLES
T Wale al Couple arate bce
a
4 "Mile $2 Soro basen
3 Male 37 Exhonohocoll
& Femi Eich col
; Mae 4)—_‘Suppforwt eas
‘ Male $1 Fame axtomerne
3 Fammle, MLB
Mle Exerc
‘The level of low-molecular-weight CD14 in secum of a
‘normal individual was inthe range of 0.008 to 0.100 g/m.
‘and the average thereof was 0.04 igiml.. In the ease of @US 7,465,547 B2
47
patient suffering fom sepsis, the level of lowmolecular-
‘weight CD14 was in the range of 0.190 to 7.260 jim and
theaveraze thereof was 2.0m. The level of ow-molecu-
Jar-weight CD14 of the patient suffering from sepsis was
higher than those of the normal individuals and patients su
{ering from other various kinds of diseases. Among patients
suffering fom other various kinds of diseases, there Was no
patient showing a high level, compared with that of the or-
‘mal indiviial
Example 11
Comparison with Commercially Available ELISA
Kit for CD14 Soluble in Blood
11-(1) Assay of Soluble CD14 in Blood of Patients Suffering
fiom Varioas Kinds of Diseases
‘Spocimensol Example 10 were assayed using the commer-
ly available CDL4-ELISA kt (IBL-Hlamburg). The level,
‘ofsoluble CD14 inblood estimated asatotal oflow-molecu-
Jae-weight CD14 and high-molecula-weight CD14) of anor
mal individual was inthe range of 5.6 011.2 ng/mL but an
‘example of ahigh evel inthe easeof a patient suffering fom
sepsis was obsorved. However, many cases that showed high
Jevels of soluble CD14 were found in sera of patients sulfer
ing from various kinds of disease, s0 that thee was no dilfer
‘ence withthe patients sufering from sepsis
11-@2) Comparison with Kit Using S68 Antibody
‘The comparison with and investigation of the measured
levels of low-molecularweight CD14 determined ia
Example 11 were performed As showa in Table 10, the
‘commercially available CDI4-EIA kit showed an almost 17>
fold diference at maximum among the normal, various dis-
ceases, and sepris, while the assay kit of Example 9-(1)
showed a S0fold difference between the normal individuals
‘and the sepsis in spite of no difference between the nonmal
individuals and various diseases, Therefore, the result Was
‘lear that the measured level of the assay’ kit of Example
9.(1) specifically increases in sepsis
TABLE 10
(Chis ieelatend ala
Nomml seat Sop _Sepss Nomad
Astor To 008
nanple 3)
‘uiic CDA
The average level43 S.D of the tested normal individuals
was provided as acutofflovel (low-molecular-weight CD14-
EIA0.134jgiml, commercially available CDI4-E1A: 11.14
HpimL.) and then the analyses were divided into postive
‘amples (sepsis) and negative samples (normal various dis-
‘eases), The resus were shown in Table I. According to the
0
48
results, rate of identieal between both kis (he number of
‘identical for ELA positivetthe number of identical for ELA
negative) total 00), sensitivity (the numberof identical for
FIA positiveipositive examplesx100), and specificity (the
‘number of dential for ETA positive!negative examplesx100)
were calculated. As. rest, as shown in Table 12, in the case
of low-molecularsweight CDI4-BIA, the identical rate was
943%, the sensitivity was 100.0%, and the specificity was
93.89%. Thus, it was found that the low-molocular-weight
(CDI4-EIA could be usefilin diferential diagnosis on sepsis
by defining the eutofflevel, On the other band, inthe ease of
the commercially aailable CDI4ELA, there was no seas
tivity and specificity which were specific to allow diagnosis
of sepsis.
TABLE 11
Nowie anal
“ase tat vs
raphe 0)
Comercial 6 8
‘abe eDLeEIA
TABLE 12
‘Asay tot Commeraly
Example walle CDIA-E1A
Specie Sue HS
INDUSTRIAL APPLICABILITY
According tothe present invention, there is provided the
antibody prepared using a peptide as an antigen, the peptide
having 8 to 30 amino acid resides selected from an amino
acid sequence at postions 1 1o 68 of human high-molecular-
‘weight CD14, and abo provided the antibody that binds to 8
peptide having amino acid residues deseribed in SEQ ID
NOS: 20 4
“Those antibodies can be used in an assay’ kit for human
Jow-molecilar-weight CD14 and the kit is ale to quant
tively or qualittvely determine human low-molecl
‘weight CD14 with high sensitivity ina simple manner, sot
the Kits useful forthe digunosis ofa patient suffering fro
sepsis. Inthe present invention, the assay kt for human low
‘molecular-weight CD14 containing the above antibody and
the assay method are provided. Furthermore, the novel diag-
nostic method for sepsis in which human low-molecular
weight CD14 is dircelly assayed is provided. Furthermore,
the peptide useful in the preparation ofthe above antibody
and the method of preparing the above antibody are prove
segues LISTING
260» mowpea oF $09 1D nos: 18
“20> 689 19 mo 1US 7,465,547 B2
49
~continued
50
soo suqumee: x
HE Ms Pro Ol Bso Cpe GIA Leu Aap ep CLL Aap Phe Arg Cys Val
cys ton the Ser Gia Peo Gln feo Aap Esp er Glu Ala Bho Gln Cyo
Val Ger 44 Val Glu Val GlM He lke Ala Gly Gly Leu Asn Leu Olu
ro the Leu Wye Aig Val Bap Ala Rap Ala Dep fro Aug Gln TYE ALA
210 £50 10 Wo 2