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1
Research Division, R & D Center, BML, Inc., 1361-1 Matoba, Kawagoe, Saitama 350-1101, Japan.
2
Department of Cardiovascular Biochemistry, St.
Bartholomew’s & The Royal London School of Medicine & Dentistry,
Charterhouse Square, London EC1M 6BQ, United Kingdom.
a Author for correspondence. Fax 44-20-7882-6169; e-mail n.e.miller{at}mds.qmw.ac.uk
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: A sandwich ELISA for PLTP has been developed, using two monoclonal antibodies against recombinant human PLTP (rhPLTP) expressed in Chinese hamster ovary cells. The ELISA allows for the quantification of PLTP in the range 0.625–15.0 ng/assay (1.2–30.0 mg/L). Intra- and interassay CVs were <3.0% and <4.2% respectively. The assay was used to quantify plasma PLTP concentrations in 132 Japanese subjects (75 males and 57 females).
Results: PLTP concentrations were 12.0 ± 3.0 mg/L (mean ± SD; range, 4.9–20.5 mg/L). No sex difference was observed. Plasma PLTP concentration was positively correlated with HDL-cholesterol (r = 0.72; P <0.001), apolipoprotein (apo) A-I (r = 0.62; P <0.001) and HDL2-cholesterol (r = 0.72; P <0.001), and was negatively correlated with triacylglycerol (r = -0.45; P <0.001). There was no correlation with plasma apo A-II. These results agree with other evidence that plasma PLTP is associated with large apo A-I-containing lipoproteins. There was no correlation (r = -0.01) between plasma PLTP and plasma phosphatidylcholine transfer activity (range, 3.5–10.5 µmol · mL-1 · h-1), suggesting that PLTP may exist in active and inactive forms.
Conclusion: This new ELISA will be of value for further studies of PLTP in health and disease.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cholesteryl ester transfer protein (CETP) also catalyzes the transfer of phospholipids between plasma lipoproteins (10), and PLTP has been found to increase CETP-mediated transfer of cholesteryl esters between VLDLs and HDLs (11). Nevertheless, PLTP and CETP show neither cooperativity nor competition in the transfer of phospholipids between HDLs and LDLs (11), suggesting that the two proteins transfer phospholipids by different mechanisms.
The apparent molecular mass of PLTP purified from human or pig plasma is between 69 and 81 kDa (4)(11)(12)(13)(14). Human PLTP cDNA has been cloned and is 1750 bp in length, coding a signal sequence of 17 amino acids and a mature protein of 476 residues (13).
Plasma PLTP activity has been reported to be increased in patients with non-insulin-dependent diabetes mellitus (15)(16) and obesity (17), and to be increased by cigarette smoking (18) and alcohol consumption (19). In view of the likely importance of PLTP in health and disease, reliable and accurate methods for measuring plasma PLTP concentrations are required. We have raised monoclonal antibodies (mAbs) against recombinant human PLTP (rhPLTP) expressed in Chinese hamster ovary (CHO) cells. Two of the mAbs reacted against purified human plasma PLTP and were used to develop a new sandwich ELISA.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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subjects
Blood samples were collected into EDTA-containing glass tubes
(final concentration, 1 g/L; Terumo), and samples were immediately
centrifuged at 2500g at 4 °C for 10 min. Plasma was
obtained from 132 apparently healthy subjects (75 males and 57
females). Their lipid profiles are summarized in Table 1
. Plasma samples were stored at -80 °C. No subject was
taking any medication known to affect plasma lipoproteins.
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isolation of lipoproteins
The HDL3 fraction (d =
1.125–1.21 kg/L) was isolated from fresh human plasma by
sequential preparative ultracentrifugation in a Beckman Ti 50.2 rotor,
using solid KBr to adjust the density (20). The washed
HDL3 was refloated at d = 1.21
kg/L (14); dialyzed against phosphate-buffered saline (PBS),
pH 7.4, containing 10 mmol/L Tris-HCl, 150 mmol/L NaCl, and 1 mmol/L
EDTA; and stored at 4 °C. Plasma
HDL2-cholesterol (d =
1.063–1.125 kg/L) and HDL3-cholesterol were
quantified after preparative ultracentrifugation as described
previously (20).
preparation of rhPLTP
rhPLTP was prepared from CHO cell culture medium and purified
using phenyl-Sepharose, Ni-NTA agarose, and heparin-Sepharose
chromatography, as described previously
(21)(22). As a primary calibrator,
heparin-purified rhPLTP was used. The purity of purified rhPLTP,
subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) and visualized by silver staining, was determined by gel
scanning using the Intelligent Quantifier system (BioImage). This
showed one 80-kDa protein band, representing >90% of total protein
(Fig. 1A
). Purified rhPLTP has been shown to be similar to plasma PLTP
in PC transfer activity, remodeling of HDLs, and apo A-I cleavage
activity (22).
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preparation of mAbs AGAINST rhPLTP
Balb/c mice were immunized with 25 µg of purified rhPLTP
(22), and spleen cells from the mice were fused with Sp2/0
cells (23). The supernatants of hybridoma cells were
screened by ELISA using plates coated with purified rhPLTP (100
ng/well) and by immunoblotting. Positive hybridoma cells were cloned
three times by limiting dilution and injected intraperitoneally into
pristane-primed Balb/c mice. The IgG fraction was isolated from ascitic
fluid using protein A-Sepharose CL-4B according to the manufacturer’s
instructions, dialyzed at 4 °C against PBS, and stored at
-80 °C. The specificities of mAb113 and mAb114 were confirmed by
immunoblotting against purified human plasma PLTP and human plasma. mAb
isotype was characterized using the Mouse mAb Isotyping kit (Boehringer
Mannheim) and was IgG2b and IgG1 for mAb113 and mAb114, respectively.
measurement of phospholipid transfer activity
Phospholipid transfer activity was measured as described by Damen
et al. (24), using [14C]PC liposomes
prepared as described by Cheung et al. (25). A diluted
plasma sample (100 µL of a 1:50 dilution) was added to 400 µL of
reaction mixture containing washed HDL3 (250 µg
of protein) and liposomes (75 nmol of PC) and incubated at 37 °C for
30 min. Liposomes were then precipitated by the addition of 300 µL of
a solution containing 230 mmol/L NaCl, 92 mmol/L
MnCl2, 150 IU/L heparin (24), and the
radioactivity in the supernatant was measured. Phospholipid transfer
activity was expressed as µmol of PC transferred to
HDL3 per milliliter of plasma per hour. All
assays were performed using the same batches of liposomes and
HDL3. The intra- and interassay CVs (n = 8)
were 5.9% and 7.7%, respectively. The PC transfer activity of rhPLTP
was a linear function of concentration up to 13.0
µmol · mL-1 · h-1.
Polyclonal antibody raised against the rhPLTP in rabbits almost
completely inhibited the PC transfer activities of both rhPLTP and
normal human plasma (data not shown).
The PC transfer assay was tested by adding to each of three plasma samples of differing PC transfer activities (6.3, 9.5, and 11.0 µmol · mL-1 · h-1) increasing amounts of rhPLTP of known transfer activities (0.7, 1.8, and 3.2 µmol · mL-1 · h-1), and then measuring the PC transfer activities of the mixtures. The final measured PC transfer activities increased in a dose-dependent manner and were within 4–20% of the predicted values (mean, 8.4%).
measurement of pltp concentration
mAb114 (100 µL of a 5 mg/L solution in PBS) was coated on a
microtiter plate (Nunc Immunoplate II) by incubation at 4 °C
overnight. The wells were then blocked with 200 µL of PBS containing
40 g/L Block Ace (Snow Brand Milk Products) for 2 h at
room temperature. After the plate was washed with 200 µL of PBS
containing 1 mL/L Tween 20, 100 µL of the calibrator solution
and diluted plasma samples (1:200) was added and incubated for 2 h
at room temperature. After the plate was washed five times, 100 µL of
1 mg/L biotinylated mAb113 was added to each well, and the mixture was
incubated for 2 h at room temperature. After the plate was washed
five times, 100 µL of 1 mg/L horseradish peroxidase-conjugated
streptavidin (Vector Laboratories) was added, and the mixture was
incubated for 1 h. After the plate was washed, 100 µL of
substrate solution containing 0.25 g/L o-phenylenediamine
and 0.15 mL/L H2O2
was added to each well. After 30 min, the reaction was stopped by
addition of 100 µL of 4 mol/L
H2SO4. The absorbance was
measured immediately at 492 nm by a microplate reader. Pooled culture
medium from CHO cells expressing rhPLTP served as a secondary
calibrator (1.2–40 mg/L), which was calibrated against the
heparin-purified rhPLTP primary calibrator.
other analytical methods
Measurements of plasma total cholesterol, triacylglycerol, and
HDL-cholesterol concentrations were performed in a Hitachi 7450
automated analyzer using commercial kits. Total HDL-cholesterol was
measured after precipitation of apo B-containing lipoproteins with
dextran sulfate and magnesium chloride. The LDL-cholesterol
concentration was calculated according to Friedewald et al.
(26). The protein content of purified rhPLTP was determined
with the BCA protein assay kit (Pierce), using bovine serum albumin as
the calibrator. SDS-PAGE was performed by the Laemmli method
(27) and immunoblotting as described by Towbin et al.
(28). SDS-PAGE for analysis of purified rhPLTP and plasma
PLTP was performed using 5–20% polyacrylamide gels; proteins were
detected with a silver staining kit (Daiichi Pure Chemical Co.) or by
immunoblotting using a chemiluminescent reagent (NEN Life
Science Products).
statistical analysis
Results are expressed as means ± SD. Analysis of variance
was used for group comparisons. Correlations were assessed by
least-squares regression analysis. P <0.05 was considered
statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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), the molecular mass of which (
80
kDa) was similar to that previously reported for human PLTP
(12). Neither mAb inhibited PC transfer activity (data not
shown). A sandwich ELISA for PLTP was established using mAb114 for
capture and biotinylated mAb113 for detection. This system showed a
dose-dependent response to heparin-purified rhPLTP (1:64 000 to
1:200), rhPLTP culture medium (1:2560 to 1:20), and plasma (1:2560 to
1:40; Fig. 2
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standardization of elisa for pltp concentration
For calibration of the ELISA, rhPLTP was purified from the culture
medium of transfected CHO cells (22). When subjected to
SDS-PAGE and visualized by silver staining, the heparin-purified rhPLTP
showed a single major 80-kDa band (Fig. 1A
), which represented >90%
of the total protein in the preparation (as determined by gel scanning
using the Intelligent Quantifier system). The protein concentration of
this primary rhPLTP calibrator, assayed using a BCA protein kit with
bovine serum albumin as calibrator, was typically 80–90 mg/L.
To obtain a calibration curve for the ELISA, dilutions of the primary
calibrator were made in PBS containing 1 mL/L Tween 20 to provide
0.156–20.0 ng of rhPLTP protein per well (0.31–40.0 mg/L). As shown
in Fig. 3
, the ELISA was linear up to 30 mg/L and suitable for
quantifying PLTP concentrations as low as 1.2 mg/L. When the rhPLTP
culture medium, as a secondary calibrator (1:2560 to 1:20), was diluted
in PBS containing 1 mL/L Tween 20 to cover the PLTP concentration range
1.2–40.0 mg/L, the curve was identical to that attained with the
primary calibrator (Fig. 3
). To avoid potential nonlinearity caused by
very low or high absorbance, the PLTP concentrations in plasma samples
were measured using several dilutions (1:2560 to 1:40), and the least
diluted aliquot that gave an absorbance value between 0.8 and 1.2 was
chosen.
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The detergent Tween 20 was included in the diluent to avoid any effects of differences between samples in lipid/apolipoprotein composition. We examined several detergent for sample dilution, including Triton X-100, CHAPS, CHAPSO, BIGCHAP, deoxy-BIGCHAP, n-octyl-ß-D-glucoside, n-heptyl-ß-D-thioglucoside, n-octyl-ß-D-maltoside, MEGA-9, sucrose monocaprate, and sodium cholate (Detergent Starter Kit II; Wako Pure Chemicals). Plasma samples diluted with buffer containing other detergents, such as n-dodecyl-ß-D-maltoside, gave higher absorbance, but the values for PLTP concentration did not differ from those obtained with PBS containing 1 mL/L Tween 20 (data not shown). Incubation of plasma samples at 56 °C for 2 h, which as found by others (12)(25) produced complete loss of PC transfer activity, had no effect on the values obtained for PLTP concentration (data not shown).
When purified rhPLTP was added to samples of plasma in sufficient
amounts to raise the total PLTP concentration by 2.5–13.2 mg/L, the
final concentrations given by the ELISA averaged 102% of those
predicted (Table 2
). The intra- and interassay CVs of the ELISA were <3.0% and
4.2%, respectively (n = 8). No interference with the ELISA was
observed with hemoglobin (10 g/L), bilirubin (0.2 g/L), or
triacylglycerol (4.25 g/L).
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plasma pltp concentrations in healthy subjects
PLTP concentrations and activities were measured in plasma samples
from 132 healthy Japanese subjects (Table 3
). Concentrations averaged 12.0 ± 3.0 mg/L (mean ±
SD), with a range of 4.9–20.5 mg/L. PC transfer activity averaged
6.3 ± 1.3
µmol · mL-1 · h-1,
with a range of 3.5–10.5
µmol · mL-1 · h-1.
Mean PLTP-specific activity, calculated from these data, was 0.56
± 0.20
µmol · h-1 · µg-1.
There were no sex differences in PLTP concentration, PC transfer
activity, or PLTP specific activity. There was no significant
correlation between plasma PLTP concentration and plasma PC transfer
activity (Table 4
and Fig. 4
). In both sexes, plasma PLTP concentration was positively
correlated with the concentrations of HDL-cholesterol, apo A-I, and
HDL2-cholesterol (Fig. 5
and Table 4
) and was negatively correlated with triacylglycerol
concentration (Table 4
). In females but not in males, PLTP
concentration was also negatively correlated with plasma apo B
concentration. Values for PC transfer activity in plasma were not
significantly correlated with any of the lipoprotein measurements in
either sex (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-tocopherol (40) between lipoproteins.
To date there have been few studies of the function of PLTP in the
regulation of lipid transport in humans in vivo. In the absence of a
reliable of immunoassay of plasma PLTP concentration, most clinical
studies have been limited to measurements of phospholipid transfer
activity (15)(16)(17)(18)(19). For a better understanding of PLTP
function, there is a need for reliable and sensitive methods for
measuring PLTP concentration in plasma and other biological fluids. To
this end, we prepared a series of mAbs against rhPLTP and used two of
them to develop the sandwich ELISA. By SDS-PAGE and immunoblotting,
both mAbs recognized a single protein of 80 kDa under both reducing
and nonreducing conditions, indicating that they react with a linear
epitope of PLTP protein. We found that mAb113 cross-reacted with pig
plasma PLTP, the amino acid sequence of which has 93% homology with
human PLTP (41). However, mAb114 did not cross-react with
pig PLTP, demonstrating that the two mAbs recognize different epitopes
on the PLTP molecule. The ELISA was found to be suitable for assaying
plasma PLTP concentrations in the range 1.2–30.0 mg/L. No differences
in dilution curves were observed between purified rhPLTP (primary
calibrator), rhPLTP culture medium (secondary calibrator), and human
plasma. Intra- and interassay CVs were <5%. These results indicate
that our ELISA is specific, accurate, reproducible, and sufficiently
sensitive for a wide range of applications.
We used our ELISA to measure plasma PLTP concentrations in 132 healthy Japanese subjects. No sex difference was observed. No correlation existed with the PC transfer activity of plasma, suggesting that factors other than total PLTP concentration are more important as determinants of the rate of transfer PC into HDLs. Plasma PLTP concentration was positively correlated with the plasma HDL-cholesterol, HDL2-cholesterol, and apo A-I concentrations and negatively correlated with plasma triacylglycerol concentration. The correlation with HDL2-cholesterol is consistent with a report that on gel filtration chromatography PLTP coeluted with particles of similar size to HDL2 (42). Although PLTP has been shown to bind to both apo A-I and apo A-II in vitro (9), our finding that the PLTP concentration correlated positively with the concentration of apo A-I but not with that of apo A-II suggests that binding to apo A-I-containing particles may predominate in vivo. Such particles might capture newly secreted PLTP, thereby retaining it in the plasma compartment. The apo A-I/apo A-II ratio of HDLs has been shown to be a determinant of PLTP-mediated HDL remodeling (8), and PLTP cannot mediate the conversion of particles that lack apo A-I (9)(21).
Two immunoassays for plasma PLTP concentration have been developed by other groups. Desrumaux et al. (43) described a competitive ELISA that used a polyclonal rabbit antibody to purified human plasma PLTP. As determined by this assay, the plasma PLTP concentration in 30 healthy subjects was 3.95 ± 1.04 mg/L (range, 1.98–5.71 mg/L), which is much lower than the values obtained in the present study. No significant correlation was found between plasma PLTP concentration and either the HDL-cholesterol or triacylglycerol concentration. No measurements were made of apolipoproteins. Also contrasting with our results is the fact that Desrumaux et al. (43) found a strong correlation (r = 0.79; P <0.001) between plasma PLTP concentration and plasma PC transfer activity. More recently, Huuskonen et al. (44) developed an assay that uses a mAb for capture and a polyclonal rabbit antibody for detection, both raised against rhPLTP expressed in Escherichia coli. This assay gave plasma PLTP concentrations of 15.6 ± 5.1 mg/L (range, 2.3–33.4 mg/L) in 159 Finnish subjects, which is similar to those obtained by us in Japanese subjects with our assay. As in the present study, Huuskonen et al. (44) found no correlation between plasma PLTP concentration and plasma PC transfer activity (r = -0.06).
Thus, our results are in good agreement with those obtained by
Huuskonen et al. (44) in relation to both the absolute
values of plasma PLTP concentration in healthy subjects and the absence
of any correlation with plasma PC transfer activity. The apparent
inconsistency between these results and those reported by Desrumaux et
al. (43) probably cannot be explained by differences in
sample sizes or in the genetic background, dietary habits, or
life-styles of the subjects. A theoretical possibility is that there
are catalytically active and inactive forms of PLTP in plasma and that
both our immunoassay and that of Huuskonen et al. (44)
quantify both forms, but that of Desrumaux et al. (43)
quantifies only the active form. We recently studied the distribution
of PLTP mass and PC transfer activity in human plasma fractions
separated by size exclusion chromatography. Evidence was obtained that
there are indeed active and inactive species of PLTP, that the active
form constitutes only 20% of the total, and that the two species
are associated with particles of different sizes [Oka et al. The
distribution of phospholipid transfer protein (PLTP) in human plasma:
presence of two forms of PLTP, one catalytically active and the other
inactive, submitted for publication].
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Acknowledgments |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-tocopherol between lipoproteins and cells. Biochem J 1995;305:659-667.
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), or human plasma (1:2560 to 1:40; 
).
Each point represents the mean of triplicate determinations.
, purified rhPLTP; •, rhPLTP culture medium.
, females.