Apr 17, 1995 - Key words : Lipoparticles, ELISA, Atherosclerosis, Diabetes. Studies on the fractionation of LDL by immunoprecipita- tion procedures have ...
45 Journal
of Atherosclerosis
Original
Article
and
A Monoclonal
Antibody-based
Measurement
of Native
B-containing
Carlos Calvo, Verdugo, and
Vol. 4, No. 1
Thrombosis
Enzyme
and
Glycated
Immunoassay
for the
Apolipoprotein
Particles
Paulina Lorena
Bustos, Toledo
Paolo
Giraudo,
Natalia
UIIoa,
Jorge
Sepulveda,
Cecilia
Departamento de Bioquimica Clinicae lnmunologia, Facultad de Farmacia, Universidad de Concepcion, Concepcion, Chile.
We describe the development of sandwich enzyme-linked immunosorbent assays designed to measure native and glycated apolipoprotein B-containing particles in plasma. The assays utilize monoclonal antibodies anti native or glycated apo B-LDL for coating and a polyclonal anti apoB-LDL-peroxidase conjugate as the detecting antibody. The method is specific, sensitive and precise. The intra-assay coefficient of variation for the plasma native and glycated apolipoprotein B-containing particles was determine to be 7.8% and 7.5%, respectively. The method described can provide specific and reproducible determinations of apoB and glycated-apoB containing particles in plasma it will be of great interest in the evaluation of atherosclerotic risk in dyslipoproteinemic states in diabetic and nondiabetic subjects. J Atheroscler Thromb, 1997; 4 45-49. Key words : Lipoparticles,
ELISA, Atherosclerosis,
Diabetes
Studies on the fractionation of LDL by immunoprecipitation procedures have shown the occurrence of two types of apoB-containing lipoproteins. The major subspecies of lipoprotein particles contains apoB as the sole protein constituent (LpB), while the minor types of lipoprotein particles contain apoB in association with apoC and/or apoE (1). A study designed to investigate the relation of LpB particles to coronary artery disease in middle-aged men indicates, that apoB particle determination might be a more efficient screening test for coronary artery disease high-risk subjects than is total apoB determination (2). Lipoprotein abnormalities are related to the development of atherosclerosis and, therefore, non-enzymatic glycation of the protein moiety of lipoproteins (apolipoproteins) has been the subject of intensive study (3-8). Glycation of apoB-LDL was found significantly in-
creased in diabetic patients compared with normal subjects, even in the presence of good glycemic control (9). Metabolic abnormalities associated with glycation of LDL induce diminished recognition of LDL by the classic LDLreceptor, increased covalent binding of LDL in vessel walls and enhanced uptake of LDL by a low-affinity, highcapacity receptor pathway on macrophages, thus stimulating foam cell formation, an early feature of atherosclerosis (10). We have developed a sandwich enzyme-linked immunosorbent assay (ELISA) for the quantitation of native and glycated apoB-containing particles in human plasma using monoclonal antibodies against native apoB-LDL and glycated apoB-LDL.
Address for correspondence : Carlos Calvo, Ph. D., Departmento Bioquimica clinica, e Inmunologia, Facultad de Farmacia, Universidad de Concepcibn, Casilla 237, Concepcibn, Chile. Received April 17, 1995. Accepted for publication June 27, 1996.
Isolation of apoB-LDL Low density lipoproteins (LDL) were isolated as described by Havel et al. (11). Fresh human plasma was obtained from healthy donors and adjusted to 0.01% EDTA.
Materials and Methods
46
A Monoclonal
Protein using
was
Glycation
serum
of
Two
mg
37•Ž
as
bituric
pH
described
et
al.
100
mM
in
glucose
0.5
penicillin
50
M
and
Isolation
phosphate
U/ml
for
of
7 days
method
determined
by
the
Mab
activated
thiobar-
was
column
were 50 ƒÊg
15
30,
the
in
55,
St.
same
Freund's MO)
booster of
50 ƒÊg
50
mice
of apoB-LDL
by or
adjuvant
intraperitoneal-
in incomplete
consisted
and
BALB/c
pg
Louis,
doses A
which
intraperitoneally
1, 100
complete
intraperitoneally.
day
female
: day
Company,
and
adjuvant,
follows
apoB-LDL
Chemical
day
on
as
glycated
(Sigma ly
of two-month-old
immunized
Freund's
isolate
2.5•~107
NSO/2
Milstein
from
Cambridge,
wells
for
7.4.
Affer
20,
a blocking
administered
several
fug
administered
particles
on
the
same
mouse
using
Milstein
(15).
myeloma
MRC
the
determined
Screening
by
of
During
the
the
to
by
measuring
supernatants
native
cyanogen
bromide
by
the
anti
Pharmacia. apoB
affinity
particles.
120
reactivities
of
glycated
at
37•Ž,
100 ƒÊl
of
the
were washings,
and
5.0,
containing
tion
was
were
the
25
readings nm.
cate
to
for
of
50
to for
90
min.
,ƒÊ1 of
20%
H2SO4.
an
ELISA
glycated
5E5
apoB-LDL
conditions
min
and
and
The
reader
were
as
The
30
apoB-containing MAb
Affer
substrate
added. for
on
The
(10
buffer,
37°C
20 ƒÊg/ml
similar
apoBwell.
(OPD)
was
90
similarly
each
at
taken
for
washed
37•Ž
F1202)
glycated
with
under
30%
of
incubating
M citrate-phosphate
proceed
was
washing,
anti-human
added at
in PBS
apoB-containing
again
diluted
pH
Tween
Affer
or
were
was
0.15
were
ELISA
centrations by
of
addition
at
450
plates
5 Jul of
bance
(OVA)
Affer
incubated
ml
saline, 0.05%
37•Ž.
apoB-LDL
corresponding
allowed by
at
in duplicate. the
microtiter
buffered
containing
min
performed
plastic
ovalbumin
60 of
was
onto
o-phenylenediamine
standardized
culture
apoB-LDL
of 3% for
again
in
stopped
PBS
conjugate
further
2D9
phosphate
with
added
min
OPD
.
with
solution
were
particles
MAb
concentrations
The
hybridomas
4•Ž
incubated
mg
kit (Sigma)
at
washing
plates
(MAbs)
MAbs,
and
C.
obtained
a commercial
2 ƒÊg/ml
18 h
4000
kohler
antibodies
the
the
against
by
antibodies of
Dr.
glycol
described
monoclonal
preparation
on
apoB-containing
LDL-peroxidase
Biology,
poliethylene
using
with
by
Molecular
monoclonal
ELISA
the
selected
50%
fused
(provided
of
procedure
of
were
cells
Laboratory in
subclasses
were
mice
for
and
was
intravenously
immunized
England)
(Merck)
The
from
to
described
apoB-containing
immobilizing
and cells
ELISA
added
day. Spleen
as
ELISA
The
groups
linked
4B
(14).
production
Different
was
chromatographed to
Sandwich Hybridoma
2D9
Sepharose
LDL was
lipoparticles
Purified
(13).
(TBA)
Immunoassay
BRL). The antibodies were eluted with 0.1 M glycine hydrochloride, pH 2.6, and neutralized immediately with 1 M Tris buffer, pH 8.0.
(12)
standard.
with
7.4,
of glycation
acid
Lowry
Enzyme
vitro
incubated
(PBS),
degree
to a
cyanoborohydride
saline
The
as
in
were
sodium
buffered
according
albumin
apoB-LDL
LDL
12 mg/ml
at
measured
bovine
Antibody-based
pH reacwas
absor-
(BIO-TEK)
particles
was
different
con-
added
described,
in
dupli-
previously
.
ELISA.
Specificity
of
The and
the
monoclonal
specificity
of
each
immunoblotting.
LDL)
and
sferred
and
slab to
determined
sheets
incubated
with
or
Proteins (0.45 ƒÊm
pH 8.3.
The
the
ELISA (HDL
onto
in agarose
electrophoresis.
described
by
lipoproteins immobilized
separated
gel
buffer,
then
previously
was
were
nitrocellulose
in Tris-glycine were
albumin
plates
acrylamide
MAb
Apolipoproteins,
human
microtiter
Results
antibodies
ELISA
size,
nitrocellulose
monoclonal
, a
SDS-polywere
pore
Native
and
obtained
single
band
The
tran-
furfural
sheets
of
per
a
A
Monoclonal
was
with antiserum
produced
in rabbits.
Sepharose-Protein
A
idase
by
the
periodate
to The (Sigma)
human
plasma
antibodies and
method
were
conjugated
apoB-LDL purified to
of
Monoclonal
and by
perox-
(17).
affinity
chromatography
purified
One
the
on
Protein
from
ascites
G-agarose
glycated.
measured
nmoles
using
5-hydroxymethyl-
indicates
that
about
70%
fluid (Gibco,
by
production
the
MAbs
other
a
apoB
hybridoma,
as
1 and
glycated
belonging
Figs.
2D9, epitope
namely
5E5, apoB-LDL.
to
by their
reactivity
apolipoproteins
designated
native
with
examined
and
(Table
hybridoma,
only
was
lipoproteins
immunoblotting
identified were
which
were
showed
electrophoresis.
apoB-LDL 40
apoB-LDL
of
recognized
MAbs IgGs
of was
antibody
different
reacted Purification
gel
glycation
standard
mg
ultracentrifugation
agarose
as
(16).
antibody
polyclonal
sequential
1%
residues
Specificity Polyclonal
on of
as
lysine
apoB-LDL by
degree
fructose
Sigma)
antibodies
glycated
LDL
the lgG1
by
la
and
produced in the
LDL
produced Both subclass.
ELISA
lb). a
MAb
complex a
MAb
MAbs
that and that were
Calvo Table
1.
Specificity
of the monoclonal
antibodies
ELISA of the culture supernatants
et al.
47
by ELISA
were done in duplicate.
Absorbances
higher than 0.5 were considered
as positive (+).
2
1
1
apoB -
2
LDLn apoB - LDLgly
Fig. 1b. Immunoblotting of lipoprotein with MAID (5E5) anti glycated apoB-LDL. Purified glycated apoB-LDL was electrophoresed in agarose gel, transfer to nitrocellulose and identified with MAb against glycated apoB-LDL : lane 1, Fig. 1a. Immunoblotting of lipoprotein with MAID (2D9) anti apoB-LDL. Purified apoB-LDL was electrophoresed in agarose gel, transfer to nitrocellulose and identified with MAb against apoB-LDL : lane 1, apoB-LDL ; lane 2, glycated apoB - LDL.
Isolation
of
An linked
to
rose
4B
from the
apoB-containing
affinity
column 2-3
ml
allowed
a
LDL
particles
with of
cyanogen
the
isolation
preparation
of purified activated
2).
column
ApoB-containing
10 mg bromide
MAb
The
was
lipoparticles
were
capacity
mg
per
used
ml
to
of
of
gel.
standardize
ELISA.
Standardization
of
Different blocking LDL of
solution
apoB-LDL curve
0.99
and
2D9 or
particles lation
was
was
5
with
were
the
to
of
0.98
0.2 of
to
curve and
with
from absorbance
detect
a stand-
coefficient
of
0.9).
20 ƒÊg/m1
glycated
2 ƒÊg/ml to
(correlation from
determine
anti-apoB-
Thus,
concentration
50 ƒÊg/ml
a standard
the
lipoparticles
concentration to
concentration, of
assayed.
optimal
range
a
coefficient
antibody dilution
apoB-containing
from
used
sandwich the
working
absorbance
Furthermore, 5E5
like
conjugate
MAb
ard
ELISA
and
peroxidase
the
the
conditions
of
the
MAb
apoB-containing 3 to
30 ƒÊg/m1 range
apoB-LDL.
to 1.0). The variation coefficient intra-assay for the plasma native and glycated apoB-containing lipoparticles was determined to be 7.8% and 7.5%, respectively (Table 2). Discussion
particles
binding
0.2
lane2, glycated
2D9
Sepha-
of apoB-containing
(Fig.
chromatographic
the
about
apoB-LDL;
correfrom
(0.1
A number of assays for apoB-containing particles have been developed in recent years, mostly ELISA. The development and use of anti apoB monoclonal antibodies which recognize apoB-containing atherogenic lipoprotein particles in plasma might permit a more accurate assessment of coronary artery disease risk (2). In addition, a number of case-controlled studies have shown that high levels of plasma apoB-containing particles are associated with risk of myocardial infarction (18, 19). Analysis of serum glycated apoB-LDL has been performed by others methods including immunoassays with polyclonal antibodies against glycated-LDL (20). The use of specific monoclonal 'antibodies to evaluate glycated apoB-LDL yield better results than those obtained for those methods. Recently, it has been reported the use of monoclonal antibodies that recognize glycated apoB in a competitive
48
A Monoclonal
Antibody-based
Enzyme
(2)
(3)
Immunoassay
Fi6vet C, Nuttens MC, Ducimetiére P, Fruchart JC, Bertrand M, and Salomez JL : Relation of arteriographically defined coronary artery disease to serum lipoprotein particles mapped with monoclonal antibodies. Circulation, 84 : 153-159, 1991 Curtis LK and Witzum JL : Plasma apolibobrotein A-I, AII,B, C-I and E are glycosilated in hyperglycemic
(4)
(5)
Fig. 2. Elution profile of apoB-containing munoaffinity chromatography. The arrow of the particles.
particles by imdenotes the elution
(6)
(7) Table
2.
glycated
Standardization apo
of
B-containing
the
ELISA
for
native
and
particle.
(8)
(9)
enzyme-linked immunosorbent assay to measure glycated-LDL concentration in plasma from non-diabetic and diabetic subjects (21). In the present report we described the development of a precise, specific and sensitive enzyme-linked immunosorbent assay for apoB and glycated-apoB containing particles determinations in plasma that agree well with those reported early. However, little information is available on the quantification of these lipoparticles in plasma. Finally, the system described here can provide specific and reproducible determinations of native and glycated apoB-lipoparticles in plasma that will be of great interest in the evaluation of atherosclerotic risk in dyslipoproteinemic
states
in diabetic
and non diabetic
Acknowledgments : This work was supported 92-0218 from FONDECYT and grant 91.31.42-1 University of ConcepciOn, Chile.
(10) (11)
(12)
(13)
(14)
subjects. by grant from the
(15)
(16)
References (17) (1)
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(18)
diabetic
subjects. Diabetes, 34 : 452-461, 1985 Calvo C, Ponsin G, and Berthezene F : Characterization of the non-enzymatic glycation of high density lipoprotein in diabetic patients. Diabete Metab, 14 : 264269, 1988 Calvo C, Talussot C, Ponsin C, and Berthezene F : Nonenzymatic glycation of apolipoprotein A-I. Effects on its self-association and lipid binding properties. Biochem Biophys Res Commun, 153 : 1060-1067, 1988 Calvb C and Verdugo C : Association in vivo of glycated apolipoprotein A-I with high density lipoproteins. Eur J Clin Chem Clin Biochem, 30 : 3-5, 1992 Calvo C, Ulloa N, Del Pozo R, and Verdugo C : Decreased activation of lecithin : Cholesterol acyltransferase by glycated apolipoprotein A-I. Eur J Clin Chem Clin Biochem, 31 : 217-220, 1993 Calvo C, Ulloa N, Campos M, Verdugo C, and AyraultJarrier M : The preferential site of non-enzymatic glycation of human apolipoprotein A-I in vivo. Clin Chim Acta, 217 : 193-198, 1993 Lyons TJ, Baynes JW, Patrick JS, Colwell JA, and LopezVirella MF : Glycosilation of low density lipoprotein in patient with type I (insulin dependent) diabetes : Correlations with other parameters of glycaemic control. Diabetologia, 29 685-689, 1986 Lyons T : Lipoprotein glycation and its metabolic consequences. Diabetes, 41 : 67-73, 1992 Havel RJ, Eder HA, and Bragdon JH : The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J Clin Invest, 34 : 13451353, 1955 Lowry OH, Rosebrough AL, Farr AL, and Rondall RJ : Protein measurement with the Folin phenol reagent. J Biol Chem, 193 : 265-275, 1951 Witzum JL, Mahoney EM, Branks MJ, Fisher M, Elam R, and Steinberg D : Non-enzymatic glycosilation of low density lipoproteins alters its biological activity. Diabetes, 31 : 283-291, 1982 Mehl TD, Wenzel SE, Russell B, Gardner D, and Merimee TJ : Comparison of two indices of glycemic control in diabetic subjects : Glycosilated serum protein and hemoglobin. Diabetes Care, 6 : 34-39, 1983 KOhler G and Milstein C : Continuous cultures of fused cells secreting antibody of predefined specificity. Nature, 256 : 495-497, 1975 Towbin H, Staehlin T, and Gordon J : Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets : Procedure and some applications. Proc Natl Acad Sci USA, 76 : 4350-4354, 1979 Catty D and Raykundalia C : ELISA and related enzyme immunoassays. In : Antibodies. Vol II, ed by Catty D, IRL Press Limited, Oxford, 97-154, 1988 Genest JJ, Bard JM, Fruchart JC, Ordovas JM, Wilson PFW, and Schaeffer EJ : Plasma apolipoprotein A-I, A-II, B, E and C-III containing particles in men with premature coronary artery disease. Atherosclerosis, 90 : 149-157,
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munogenic in the guinea pig. Proc Natl Acad Sci USA, 80 : 2757-2761, 1983 (21) Cohen MP, Lautenslager G, and Slua E : Glycated LDL concentrations in non-diabetic and diabetic subjects measured with monoclonal antibodies reactive with glycated apolipoprotein B epitopes. Eur J Clin Chem Clin Biochem,
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