dard dilution series was present in duplicate on every single ELISA plate.
2
.
5
. Other methods Total cholesterol and HDL cholesterol after precipi-
tation of b-lipoproteins with dextran sulphate-magne- sium
chloride were
determined enzymatically
CHOD-PAP method
using the
reagents from
Boehringer Mannheim, Mannheim, Germany [24]. Tri- acylglycerol concentration was determined with the
fully enzymatic method of Wahlefeld [25]. Apolipo- proteins apo A-I, A-II and B were quantified by
immunoturbidometry [26]. LDL cholesterol was calcu- lated according to the formula of Friedewald [27].
Concentrations of LpA-I and LpA-IA-II particles were measured by rocket immunoelectrophoresis using
commercial reagents Sebia, Paris, France. In Western blot analysis, human plasma samples 1 ml of 1:10
diluted plasma per well were subjected to SDS – PAGE analysis on 12.5 homogeneous polyacrylamide gels
under reducing conditions, whereafter the proteins were transferred onto nitrocellulose sheets. After anti-PLTP
antibody treatment, the blots were visualized using the Enhanced
ChemiLuminescence detection
system Amersham.
2
.
6
. The study population The study subjects, n = 159, were from a large
Finnish cross-sectional study FINRISK [28]. They represented men and women in four age groups from
25 to 65 years in 10-year intervals. The participants of the FINRISK study were randomly selected from the
inhabitants of North Karelia, the Kuopio province, and Southwestern Finland, thus closely representing the
Finnish population. Among the study subjects, 86.2 were moderate drinkers, 8.8 drank so much they felt
intoxicated at least once per week and 5 did not use alcohol. Of the subjects, 51 were regular tobacco
users, 15 were occasional smokers, and 34 had never smoked. Among women, 12.3 used oral contra-
ceptives, 1.4 used intrauterine devices delivering hor- mones, 19.2 used estrogen for menopause symptoms,
and 1.4 were gravid. Diabetes type 1 or 2 had been diagnosed in 2.5 of the participants. Blood samples
from the study subjects were collected in non-fasting state. All participants have given informed consent and
the study has been approved by an ethics committee.
2
.
7
. Statistical analyses Statistical testing was performed using the Statistical
Package for Social Sciences SPSS version 7.5 SPSS, Chicago, USA. The dependence of serum PLTP mass
or phospholipid transfer activity on gender was tested by analysis of variance ANOVA. The correlation of
PLTP mass, phospholipid transfer activity and specific activity with age, body mass index BMI, serum
cholesterol, triacylglycerol, HDL cholesterol, apoB, apoA-I, apoA-II, apoA-I in LpA-I or LpA-IA-II
particles, and serum gamma glutamyl transferase S- GT was analyzed with unadjusted values.
3. Results
3
.
1
. De6elopment of an ELISA for determination of PLTP mass
The two antibodies selected for the PLTP ELISA, the monoclonal antibody JH59 used as capture antibody
and the rabbit polyclonal antibody R176 used as detec- tion antibody, have been characterized previously using
PLTP derived from a stably transfected HeLa cell line [29,30]. The immunoreactivity of the capture mAb
JH59 as well as the polyclonal detection antibody R176 was further tested with human plasma samples.
In Western blots visualized using the sensitive En- hanced Chemiluminescence method, both antibodies
detected a major 80-kDa PLTP band and only minor immunoreactivity with more slowly migrating protein
bands was observed, probably representing aggregated PLTP Fig. 1.
For calibration of the ELISA, PLTP was purified about 6000-fold from human plasma. The purified
PLTP, when subjected to SDS – PAGE and visualized by silver staining, showed one main 80-kDa protein
band Fig. 2. The 80-kDa band represented 90.1 of the total protein in the preparation as determined by
gel scanning using the Bioimage System Millipore. The protein concentration of this primary PLTP stan-
dard was assayed using the Lowry method with BSA as standard. Typically, purified PLTP preparations con-
tained from 40 to 60 mgml PLTP protein.
To obtain a standard curve for the PLTP-ELISA, dilutions of the primary standard were made in PBS-
0.1 Tween 20 and used to create standard curves in the range of 25 – 250 ng PLTP protein per well. As
shown in Fig. 3, the ELISA is linear over the entire range used and is suitable for the quantification of
PLTP levels as low as 0.125 mgl. The reproducibility of the ELISA was good. Between runs n = 3 each
primary standard point differed on average 4.3 from the mean value.
Because the assay was developed for measuring PLTP from a variety of sources the interference of
plasma was evaluated. Horse plasma was used as ma- trix since it did not give absorbance values over back-
ground when analyzed with the ELISA assay. The purified human plasma PLTP was added to horse
plasma and then diluted to exactly match the PLTP
Fig. 2. Electrophoretic analysis of purified plasma PLTP used as primary standard in ELISA. PLTP was purified from plasma as
described in Section 2. To characterize the preparation and assess purity, 0.63-mg lane 2 and 0.23-mg lane 3 aliquots were subjected to
SDS – PAGE using 12.5 gels. The protein bands were visualized either by silver staining lane 2 or by immunoblotting using the
monoclonal antibody JH59 lane 3. Lane 1 shows molecular weight standards.
Fig. 1. Western blot analysis of human plasma with anti-PLTP antibodies. Plasma samples 1 mlwell of 1:10 diluted plasma and
purified PLTP were subjected to SDS – PAGE analysis on 12.5 polyacrylamide gels under reductive conditions whereafter the
proteins were transferred onto nitrocellulose sheets. After antibody treatment, the blots were visualized using the Enhanced Chemilu-
minescence method Amersham. Lane A: immunoblot with the monoclonal antibody JH59 the ELISA capture antibody; lane B:
immunoblot with the polyclonal antibody R176 the ELISA detection antibody; lane C: immunoblot of a purified plasma PLTP with JH59
as a control.
to minimize a possible bias caused by differences in the lipidlipoprotein composition of the specimens.
3
.
2
. Use of the ELISA for determination of PLTP concentration in plasma
Having established the ELISA method, PLTP con- centrations were determined in human plasma samples
obtained from 159 Finnish normolipidemic individuals. PLTP mass and phospholipid transfer activity values as
well as calculated specific activities for different age groups of women and men are shown in Table 1. The
mean PLTP mass in the study subjects was 15.6 9 5.1 9 S.D. mgl range 2.3 – 33.4 mgl. No difference in
the phospholipid transfer activity, mass or specific ac- tivity in plasma could be observed between genders.
We have previously reported the plasma phospho- lipid transfer activity level of the subjects and the
correlation of phospholipid transfer activity with vari- ous lipoprotein parameters [20]. The present ELISA
method allowed us to extend the study to the relation- ships of PLTP mass and specific activity with plasma
lipid parameters Table 2. PLTP mass correlated posi- tively
with HDL-cholesterol
r = 0.36, P B 0.001,
apoA-I r = 0.37, P B 0.001, apoA-II r = 0.20, P B 0.05, LpA-I r = 0.26, P = 0.001 and LpA-IA-II
particles r = 0.34, P B 0.001, and negatively with BMI r = − 0.28, P B 0.001 and serum triacylglycerol TG
concentration r = − 0.34, P B 0.001. The specific ac- concentrations in specimens applied on ELISA plates
without matrix. The results obtained with and without matrix did not differ significantly from each other data
not shown. Having calibrated the ELISA we estab- lished a secondary standard using a normolipidemic
plasma sample from a healthy male volunteer. When analyzed from three different plasma dilutions 1:10,
1:15, and 1:20, the mean PLTP mass in our secondary standard was 12.24 mgl. To obtain a standard curve
the secondary standard was diluted in PBS-0.1 Tween 20 to cover the PLTP mass range from 25 to 250 ng
PLTP proteinwell. The slope of the secondary stan- dard curve did not differ significantly from the slope of
the curve attained with the primary standard data not shown. Between runs n = 8, each secondary standard
point differed on average 8.6 from the mean value. To avoid the potential non-linearity caused by very low
or high absorbances, the plasma PLTP concentrations of the study subjects were measured using three dilu-
tions 1:90, 1:30 and 1:10, from which the absorbance value of the least diluted specimen which did not exceed
absorbance 1.5 was chosen. The intra- and inter-assay variations were 4.3 and 8.6, respectively. The ELISA
standards used and the specimens under study were diluted in buffer containing detergent 0.1 Tween 20
Fig. 3. Calibration of the ELISA for determination of PLTP mass. The ELISA was performed as described in Section 2. The standard curve was formed by serial dilution of purified PLTP. For the ELISA the monoclonal antibody JH59 was used as a capture antibody and the polyclonal
R176 antibody as detection antibody. The standard points shown are the mean of three independent measurements after subtracting the background signals. Each standard point differed on average by B 5 between different ELISA runs.
tivity of PLTP, i.e. phospholipid transfer activity di- vided by PLTP mass, correlated positively with plasma
TG concentration r = 0.568, P B 0.001, BMI r = 0.45, P B 0.001, apoB r = 0.45, P B 0.001, total cholesterol
r = 0.42, P B 0.001, LDL-cholesterol r = 0.34, P B 0.001 and age r = 0.36, P B 0.001, and negatively
with HDL-cholesterol r = − 0.33, P B 0.001, LpA-I r = − 0.21, P B 0.01 as well as LpA-IA-II particles
r = − 0.32, P B 0.001. Phospholipid transfer activity but not mass correlated positively with plasma gamma-
GT activity r = 0.35, P B 0.001. There were no statis- tically
significant differences
in PLTP
mass or
phospholipid transfer activity due to alcohol, tobacco, or hormone usage, or between individuals having dia-
betes and healthy subjects. Surprisingly, there was no significant correlation be-
tween plasma PLTP mass and phospholipid transfer activity values measured in sera from the population
sample correlation coefficient, r = 0.06, P = 0.453; Fig. 4A. However, both phospholipid transfer activity and
PLTP mass correlated significantly with the plasma triacylglycerol concentration: phospholipid transfer ac-
tivity displayed a positive correlation while PLTP mass showed a negative correlation with TG levels. When
plasma TG concentration was included as a covariate in the analysis, a significant positive correlation be-
tween phospholipid transfer activity and mass was re- vealed
partial correlation
coefficient, r = 0.31,
P B 0.001; Fig. 4B. To exclude the possibility that differences in plasma TG concentration could cause a
bias in either the PL transfer assay or the mass determi- nation, purified VLDL was added in different amounts
in the plasma the secondary standard to obtain final TG concentrations from 0.43 to 7.0 mmoll. No signifi-
cant effect of the TG addition was observed on the plasma PL transfer activity Fig. 5A or the PLTP mass
quantitated with the present ELISA method Fig. 5B. Also Intralipid was used to study the effect of TG on
both assays; TG concentrations up to 20 mmoll did not affect the activity or mass determinations. Other
lipoprotein classes LDL, HDL, when added to the plasma in different amounts the highest final concen-
tration per incubation, 15 mmoll, showed no signifi- cant effect on these assays data not shown.
4. Discussion
