one-stepcompetitive lmmunochromatographic ... · then lp(a) is>300 mgfl, the relative risk...
TRANSCRIPT
CLIN. CHEM. 39/4, 619-624 (1993)
CLINICAL CHEMISTRY, Vol. 39, No. 4, 1993 619
One-Step Competitive lmmunochromatographic Assay for SemiquantitativeDetermination of Lipoprotein(a) in Plasma
Sheng C. Lou, Chandu Patel, ShanFun Ching, and Julian Gordon
Numerous studies have associated high concentrations oflipoprotein(a) [Lp(a)] with atherosclerosis. We developeda rapid, one-step competitive immunochromatographicassay to measure Lp(a) in plasma. The assay is per-formed on a nitrocellulose membrane strip and the resultis determined by a visual readout of rust-colored colloidalselenium. The assay is based on the principle that Lp(a)in the sample will compete with Lp(a)-coated colloidalselenium for binding to the anti-Lp(a) monoclonal anti-body immobilized on the assay strip in the format of fourladder bars. The number of capture bars that appear as aresult of the formation of colloidal selenium color isproportional to the concentration of the Lp(a) protein in thesamples. The strip assay semiquantitatively measuresLp(a) concentrations ranging from 0 to 180 mg/L of Lp(a)protein in serum, plasma, or fingerstick whole-blood sam-ples. This assay appears very useful for quick identifica-tion of individuals with above-normal concentrations ofplasma Lp(a) protein (>70 mg/L), and has potential formonitoring a patient’s response to treatment with Lp(a)-lowering drugs.
Indexing Terms: lipoproteins . immunochromatographynitrocellulose reagent strip . dipstick methods colloidal sele-nium monoclonal antibodies
In 1963, Berg (1) described lipoprotein(a) [Lp(a)] as a
genetic variant of low-density lipoprotein (LDL).’ Later,
the protein composition, electrophoretic mobility, parti-cle size, and buoyant density of Lp(a) were found to
differ from those of LDL (2). After a simple disulfidereduction, Lp(a) dissociates into LDL-like and apoli-poprotein(a) [apo(a)1 molecules, which suggests that the
apo(a) molecule is covalently linked to ape B-100 by a
disulfide bond (3-5). The apo(a) molecule on the Lp(a)particle was determined to have a cDNA sequence
similar to that of human plasminogen (6, 7).
The Lp(a) particle is more atherogenic than LDL (8,
9), probably because, in part, Lp(a) disturbs the balanceetween thrombogenesis and fibrinolysis. High concen-rations of Lp(a) may favor atherosclerotic plaque for-aation by inhibiting plasminogen activation by tissue�lasminogen activator (7, 10, 11). Numerous studies
rave indicated that high concentrations of plasma Lp(a)
re strongly associated with atherosclerosis (12-16).Then Lp(a) is >300 mgfL, the relative risk of coronary
therosclerosis is about doubled. When LDL and Lp(a)
Abbott Diagnostics Division, Abbott Laboratories, North Chi-go, IL 60064-3500.‘Nonstandard abbreviations: Lp(a), lipoprotein(a); LDL, low-nsity lipoprotein; and apo, apolipoprotein.Received April 20, 1992; accepted November 6, 1992.
are both above normal, the relative risk is increased
about fivefold (16). Given reports that patients withheterozygous familial hypercholesterolemia with high
plasma Lp(a) may develop atherosclerosis earlier thanthose with low Lp(a) (17-19), the measurement ofplasma Lp(a) is considered a very strong predictor of
premature coronary artery disease.
Lp(a) has been measured quantitatively by radioim-
munoassay (20), radial immunodiffusion (21), rocketimmunoelectrophoresis (22), and more recently by
ELISA (23-25). All of these methods require eithermultiple-step procedures or a long assay time. Here, we
report on a rapid, one-step, noninstrumented, competi-
tive immunochromatographic method that measures
plasma Lp(a) semiquantitatively to identify individuals
with higher risk for coronary artery disease.
Materials and Methods
Animal and Human Plasma
Female BALB/c mice 8-10 weeks old were purchased
from Charles River Labs., Portage, MI. Human plasma
used for Lp(a) isolation and testing was ordered from
Interstate Blood Bank, Milwaukee, WI. Purified Lp(a),
apo(a), and 29 clinical plasma samples were obtainedfrom A. M. Scanu and G. M. Fleas, University of Chi-
cago, Chicago, IL.
Isolation of Lp(a) from Human Plasma
One unit of human plasma was collected from 1 L ofwhole blood. To minimize proteolysis, we added 30 mL of
protease inhibitor stock solution (containing, per liter,
0.2 mol of sodium EDTA, 200 g of chioramphenicol, 25 g
of sodium aside, 10 g of gentaniicin sulfate, 20 millionunits of kallikrein inactivator, 1 mol of benzamidine, 0.2
mol of phenylmethylsulfonyl fluoride, and 0.3 mol ofNaCl, pH 7.4) to the plasma. Total lipoproteins were
isolated by adjusting the plasma to density 1.21 kg/L
with solid NaBr and centrifuging in a Beckman 60Ti
rotor (Beckman Instruments, Palo Alto, CA) at 477 000
x g (59 000 rpm) for 20 h at 15#{176}C.Total lipoprotein
fractions were then dialyzed against NaC1 (0.15 mol/L)plus sodium EDTA (0.1 g/L, pH 7) for 24 h with three
changes to remove the extra salt. Total lipoproteins
were further passed through an apo(a)-specific monocle-nal antibody-affinity column, prepared by coupling an-tibody 4F2 (see next sections) to Affl-Gel 10 (Bio-Rad,
Richmond, CA). The bound Lp(a) was eluted from the4F2-affinity column with glycine . HCI (0.1 mol/L, pH2.8), and the eluted fractions were immediately adjusted
to neutral pH by titration with Tris-base buffer (1 mol/L,
pH 10.5). The concentration of Lp(a) protein was deter-
mined by using a modified Lowry method (26). Purity of
MeasurementRegion/
SampleLoading
Area\Conjugate
Pad
NitrocelluloseMembrane
Strip
\\End of Assay Holder
Indicator//Cover Tape
the isolated Lp(a) was evaluated by sodium dodecyl
sulfate-.polyacrylamide gel (66 g/L) electrophoresis un-
der reducing conditions and by immunoblotting to seewhether human plasminogen contaminated the Lp(a)
purification (27, 28). We observed only ape B-100 and
apo(a) bands on the stained gels. This isolated Lp(a) wasused to prepare the Lp(a)-selenium colloid conjugate for
the Lp(a) strip assay.
The Lp(a) standard was obtained from A. M. Scanu’slaboratory at the University of Chicago. Isolation and
characterization of Lp(a) were described by Fless et al.(5,25). The Lp(a) standard had the following chemical
composition based on weight fraction (g/kg): protein236, phospholipid 218, free cholesterol 80, cholesteryl
ester 376, and triglyceride 90. The factor for convertingLp(a) protein to lipoprotein is 4.2 (5). Therefore, a 70mg/L Lp(a) protein standard is equivalent to 300 mgfL
for total Lp(a) lipoprotein mass, the concentration rec-
ommended as the cutoff point for higher risk of athero-
sclerosis (16).
Anti-Lp(a) Monoclonal Antibody
Production. Female BALB/c mice were immunized
four times at 2- to 3-week intervals with 50 �g of Lp(a)protein emulsified with Ribi adjuvant (Ribi Immuno-
Chem Research, Inc., Hamilton, MT). Four days after
the last boosting, the mice were killed and their im-
mune spleen cells were fused with myeloma cells SP2/0,
according to the procedure reported by Gefter et al. (29).After 2 to 3 weeks, spent tissue culture media were
collected from hybrid-growing wells of microtiter plates
and tested for Lp(a)-binding monoclonal antibodies. Thescreening procedure was carried out with an ELISA
method, in which spent culture media were first incu-bated on an Lp(a) or apo(a)-coated microtiter plate andthen with horseradish peroxidase-goat anti-mouse Ig(G
+ M) conjugate. An enzyme substrate solution, o-phe-nylenediamine, was added to each well for signal devel-
opment, and absorbances were read at 492 nm with aCLS microtiter plate reader (Cambridge Life Sciences,
Ltd., Cambridgeshire, UK).Characterization. Initially, 29 Lp(a)-binding monocle-
nal antibodies were selected from four cell fusions. Tenof the antibodies reacted with apo B-100 on the LDLparticle but not with the isolated apo(a) molecule. Four
monoclonal antibodies (8B4, 4D2, 1E1, and 4F2) withhigh affinity for Lp(a) and apo(a) were purified from
mouse ascites fluid with a Protein A-agarose column,
and antibodies were eluted from the column with 0.1moIJL citric acid, pH 3.0. The purified monoclonal anti-
bodies were further characterized in terms of cross-reactivity with human plasminogen and the ability to
bind to different Lp(a) isoforms. As much as 1 g/L ofhuman plasminogen did not significantly inhibit the
binding of these four monoclonal antibodies to Lp(a)-coated plates in the ELISA. Also, each of these four
Lp(a)-specific monoclonal antibodies recognized all ofthe different isoforms of Lp(a) that were previouslyseparated by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis under reducing conditions.
620 CLINICAL CHEMISTRY, Vol. 39, No. 4, 1993
Preparation of Assay Strip Components
Lp(a)-coated colloidal selenium. Colloidal selenium
was prepared essentially as described by Devereaux etal. (30). After adjusting 1 mL of colloidal selenium
solution (A� = 15) to pH 8.0 by adding 50 �L of borate
buffer (100 mmol/L, pH 8.5), we added 10 pg of Lp(a)protein, gently vortex-mixed the colloidal mixture for 2
mm, and added 10 �LL of 100 g/L polyethylene glycol (Mr
20000) to block and stabilize the Lp(a)-coated colloidalselenium. This conjugate was stable in liquid form for 2
months, and in lyophilized form for �8 months at roomtemperature under low humidity.
Assay strip. As illustrated in Figure 1, the floppyassay strip consisted of a sample loading area, a conju-gate pad, a measurement region, and an end-of-assay
indicator.Conjugate pad. The assay strip’s conjugate pad is a
piece of glass-fiber material (0.8 x 0.3 cm) called Lydall
(Lydall, Inc., Hamptonville, NC), which contains dried
Lp(a)-coated colloidal selenium, sheep anti-humanerythrocyte antiserum, and casein. The anti-erythrocyte
antiserum traps erythrocytes in the pad when whole-
blood samples are tested; thus, only plasma flows intothe membrane strip. The casein helps the colloidalselenium migrate through the membrane strip. The
conjugate pad was prepared by saturating a piece ofLydall (18 x 0.8 x 0.01 cm) with 1.5 mL of the conjugate
mixture [Lp(a)-coated colloidal selenium at A� = 10
and a 50-fold dilution of sheep anti-erythrocyte antise-rum in (final concentration) 20 g/L casein, 20 mmol/L
Ti-is buffer, pH 7.21. The conjugate pad was frozen at
-40 #{176}Cfor 30 miii and then dried at 10#{176}Cfor 16 h in a
shelf lyophilizer (VirTis, Inc., Gardiner, NY).Immobilization of antibody onto nitrocellulose mem-
brane. The measurement region of the assay strip is apiece of nitrocellulose membrane (5 X 0.3 cm), to which
was immobilized the Lp(a)-speciflc monoclonal antibody
8B4 in a four ladder-bar format. A computer-controlledreagent jet was used to dispense the anti-Lp(a) mono-
clonal antibody reagent, 4 gIL, in a straight line (18 x
I �1 H N � �
__ __Fig. 1. Schematic of assay strip design
The assay strip consists of a sample loading area, conjugate pad, measuiment region, end-of-assay indicator, and a holder. The conjugate pad contaldried Lp(a)-coated colloidal selenium, which provides an easily visible reeawing to its rust color; anti-erythrocyte antiserum for whole-blood samples; acasein. In the measurement region, a nftrocellulose membrane strip Is usedchromatographic support and is impregnated with antl-Lp(a) monocloiantibody in a four ladder-bar format. The first capture bar contaIns 2 �uganti-Lp(a) antibody; the remaining capture bars contaIn 1 �ug of antibody. Tschematic assembly of assay strip is shown at the bottom of the panel
Holder
EOAIndicator
MeasurementRegion
ConjugatePad
SampleLoading Area
Assay Apply <40 40 to 70 to 120 to Lp(a) Protein
Strip Sample <70 <120 150 mg/L
Fig. 2. Illustration of Lp(a) strip assay performance and assaydynamic rangesThe sample Is applied to the sample loadIng area. When the end-of-assay(EOA) Indicator has developed, the assay Is complete. The number of capturebars that appear Is a function of the concentration of the Lp(a) protein In thesample. Each successive capture bar that Is stained by the Lp(a)-coatedcolloIdal selenIum color Is defined by a range of Lp(a) proteIn concentrationsas shown under each strip
CLINICAL CHEMISTRY, Vol. 39, No. 4, 1993 621
0.1 cm) onto a piece of 5-pm pore size nitrocellulose
membrane (20 x 15 cm) mounted on a mobile platform(31). The reagent jetting rate was 2 pL per inch. Two
jetted lines without spacing formed the first capture bar
(18 x 0.2 cm). The second, third, and fourth capture bars
were printed in a single jetted line (18 x 0.1 cm) with
0.2-cm spacing between each bar.
End-of-assay indicator. The end-of-assay indicatorbar was located at the end of the assay strip. A pH-
sensitive dye, qumaldine red, was used as the end-of-assay indicator. At pH <1.4, the quinaldine red solution
is colorless. When the pH is >3.2, the indicator turns
red. The quinaldine red solution (0.2 g/L, pH 0.9) wasdispensed with the reagent jet onto the nitrocellulose
membrane in a 1-mm-thick line located 2.5 cm abovethe fourth capture bar. When a plasma sample (pH >5)
flows through the end-of-assay indicator bar on theassay strip, the bar turns from colorless to red, indicat-
ing that the assay is completed.
Assembly. The schematic assembly of the assay strip
is shown in the bottom panel of Figure 1. The conjugate
pad (15 x 0.8 cm) is attached to the top side of theantibody-immobilized nitrocellulose membrane (15 x 5cm) 0.5 cm below the first capture bar, with a 0.1-cm
junctional area. The assay strip is then laminated onboth sides of the conjugate pad and membrane with8-cm-wide adhesive tape (Adhesive Research, Inc.,
Glenrock, PA). The assembled conjugate pad and mem-brane are then attached to the top of a clear, transpar-
ent plastic film (3M, St. Paul, MN) with double-sided
adhesive tape. A 1-cm length of plastic ifim extends
beyond the conjugate pad to be used as the sampleloading area. This whole assembly is cut into 6 x 0.3 cm
assay strips (Figure 1) with a slicing cutter (Schleicher
& Schuell, Keene, NH).
Comparison Methods
The Lp(a) ELISA developed by A. M. Scanu’s labora-tory was described in detail by Fless et al. (25). Briefly,
microtiter plates were coated with 100 p.L of affinity-
purified rabbit anti-human apo(a) antibody for 2 h at
37#{176}C.After incubating the samples and then washing
away the unbound fraction, we incubated each well ofthe microtiter plates with 100 �tL of goat anti-humanapo B antibody for 1 h at 37 #{176}C.After washing theplates, we added 100 �tL of rabbit anti-goat IgG-alka-line phosphatase conjugate per well and incubated the
plates for 1 h at 37#{176}C.After washing again, we added100 �tL of p-mtrophenyl phosphate substrate solution
r well and incubated the plates in a dark chamber for
0 mm at room temperature to develop color. The
bsorbance of the plates was read at A410.
The Terumo (Elkton, MD) Lp(a) sandwich assay was�ed out according to the manufacturer’s instructions.
e added 10 pL of the samples, calibrators, or controls to
mL of dilution buffer to make a 201-fold dilution, thenipetted 100 pL of the diluted samples into Lp(a) mono-
lonal antibody-precoated wells of the Terumo microtiter
late. The plate was incubated at room temperature for 1
on a rotating platform. After washing away the unbound
portion, we pipetted 100 pL of anti-Lp(a)-horseradish
peroxidase conjugate into each well. The plate was incu-
bated as above for 20 miii and then washed. We then
added 100 pL of o-phenylenediamine substrate solution
and incubated the plate for 20 mm to develop color. Theenzyme reaction was stopped and the absorbance of the
plate was read at A4�. The concentration of Lp(a) in the
samples was determined from a standard calibration
curve of total Lp(a) mass (sum of lipid and protein) rang-
ing from 0 to 800 mg/L.
Results and Discussion
Assay Performance
Twenty-five microliters of serum or plasma, or 50 pL of
whole blood was applied to the sample loading area. Theliquid portion of the samples rehydrated the dried Lp(a)-
coated colloidal selenium pad and flowed through thenitrocellulose membrane strip. Lp(a) in the sample com-
peted with the Lp(a)-coated colloidal selenium for binding
to the immobilized anti-Lp(a) monoclonal antibody on the
capture bars. When the end-of-assay indicator developed,
the assay was complete. The assay was completed in 8-10
miii with serum or plasma samples and in 12-15 mm withwhole-blood samples. The results of the assay were deter-
mined by the number of bars seen. The number of capture
bars that turned a rust color was a function of the concen-
tration of Lp(a) protein in the sample.
Assay Dynamic Range
The dynamic range of the assay was determined byusing the assay strips to assay a set of calibrators contain-
ing various amounts of purified Lp(a). Each successive
capture bar that was stained by Lp(a)-coated colloidal
selenium was defined by a range of Lp(a) protein concen-trations. As shown in Figure 2, when Lp(a) in the samples
622 CLINICAL CHEMISTRY, Vol. 39, No. 4, 1993
was <40 mg/L of Lp(a) protein, all the Lp(a)-coated colloi-
dal selenium color would be retained in the first capture
bar by the immobilized anti-Lp(a) monoclonal antibody.
When the samples contained 40 to <70 mg/L Lp(a) pro-
tein, the first two capture bars developed color. In individ-
uals with above-normal plasma Lp(a) concentrations-
>70 mg/L of Lp(a) protein has been suggested as thehigh-risk cutoff point for coronary atherosclerosis-the
third capture bar developed color. The dynamic ranges ofLp(a) protein for the third and fourth capture bars were 70
to <120 mgfL and 120 to 180 mg/L, respectively. When all
four capture bars had fully developed color on the assay
strip, the Lp(a) protein in the samples was �180 mgfL. In
such cases, the Lp(a) protein assay needed to be repeated
after a twofold dilution of the sample. When only a partial
area of the highest bar on each assay strip developed color,
the Lp(a) protein concentration was further estimated
proportionally within the assay range of that capture bar.
Accuracy and Reproducibility
Twenty-nine clinical samples with known lipid/lipo-
protein profiles, including Lp(a) concentrations that had
been previously determined by Fleas et al. (25) with
their ELISA, were assayed by the strip assay and theTerumo ELISA to determine Lp(a) concentrations. A setof strips run with these 29 clinical samples is shown in
Figure 3. By comparing the number of capture bars
developed on each strip with the Lp(a) protein standard
calibrated assay range of each capture bar as illustrated
in Figure 2, we determined the concentration of Lp(a)protein in these samples semiquantitatively. Sixteen of29 strips developed color only within the first capturebar, indicating that the Lp(a) protein in these sampleswas �40 mg/L. Furthermore, when only a partial areaof the first capture bar developed color, the Lp(a) proteinconcentration between 0 and 40 mg/L was estimated
proportionally by the visual ratio of the partial area and
the whole area of the first bar.This strip assay was very reproducible between runs
with the same sample. A 98% identical agreement wasobtained from the same set of assay strips read by two
researchers. The Lp(a) concentrations of these 29 sam-ples measured by our strip assay, the ELISA from Fleaset al.’s laboratory, and the Terumo ELISA are shown in
-rn � -�--� -�--���4.- � �
Fig. 3. Use of the assay strips to assay 29 clinical samplesSample numbers correspond to those In Table 1
Table 1. The linear correlation between Fless et al.’s
ELISA and the Lp(a) strip assay was y = -0.57(± 0.45)
+ 1.08(± 0.05)x. In the Terumo ELISA, Lp(a) concen-
tration was expressed as total Lp(a) lipoprotein mass. To
obtain a comparable unit, we multiplied the Lp(a) pro-tein concentrations measured by the strip assay andFleas et al.’s ELISA by a conversion factor of 4.2 to
convert to total Lp(a) lipoprotein mass. The linear
correlation of the Terumo ELISA with the strip assaywas y = 5.35(±3.58) + 1.06(±0.10)x. The linear corre-
lation of the Terumo ELISA with that of Fless et al.’swas y = 8.01(± 3.10) + 0.96(±0.08)x. Eleven of the 29
clinical samples (41%) were detected by all three meth-ods as having an above-normal concentration of Lp�a)
protein (>70 mgfL) or Lp(a) lipoproteun (>300 mg/L).
Because the Lp(a) strip assay is quick, easy to use,
and reliable, we have used it routinely to screen for high
Lp(a) in blood donors from local blood banks. The Lp(a)concentrations in 25 plasma samples obtained fromInterstate Blood Bank donors were determined by the
strip assay (x) and the Terumo ELISA (y); the linear
correlation of the results was y = -2.14(±2.54) + 1.39(±0.09)x. In contrast to the clinical samples from Fleas
et al.’s laboratory, 41% of which had increased Lp(a)protein concentrations, only 16% to 20% of samples fromregular blood donors had increased Lp(a).
Overall, the results from these studies indicate that
Lp(a) measured by the semiquantitative strip assay was
comparable with that measured by the quantitativeLp(a) ELISA of Fleas et al. However, we observed some
discrepancy between the Terumo assay and the strip
assay. This may be due to the different reagents used
and the assay formats or to detection of different apo(a)
isoforms in these samples.
interfering Substances
Recently Eaton et al. (6) and McLean et al. (7)
reported that the apo(a) molecule shares a cDNA se-
quence homology with human plasminogen. Therefore,it was very important to check whether the plasminogen
present in the samples would interfere with the strip
assay. We found that adding as much as 600 mg/L of
plasmninogen to 25 plasma samples with different con-centrations of Lp(a) had no visual effect on the readings
from the bar strips when compared with the readings for
those strips run with the same samples but without
added plasminogen. This also indicated that the anti-Lp(a) monoclonal antibody used as a capture antibody in
this assay did not cross-react with human plasminogen
at physiological concentrations (120-250 mg/L). As seen
in Table 1, although some samples had total triglyceride
concentrations >2500 mg/L, there was reasonable
agreement between the ELISA of Fleas et al. and the
strip assay. We also found that plasma samples collected
with different anticoagulants, such as EDTA and so-
dium citrate, resulted in identical assay performances.
Stability of the Assay Strip
The assembled floppy strip is stable for �5 months atI�oom temperature and low humidity. The strips prepared
Ifrom the same lot were tested every week to evaluate
Itheir stability. We saw no change in assay performanceIwith these strips when compared with those originally
1run with the identical concentration of Lp(a).
In summary, this rapid, noninstrumented, one-step�mmunochromatographic assay measures Lp(a) concen-
CLINICAL CHEMISTRY, Vol. 39, No.4, 1993 623
Table 1. Lp(a) Concentrations MeasuredAssay and Two ELISAs
by the Strip
Sampleno.
Lp(a) proteIn,Total mgiL
triglycerides,mg/L EUSA� Strip
Lp(a) llpoprot.In,mg/L,
byTarumo EUSA
1 4890 6 10 162 1800 18 20 105
3 2890 45 60 361
4 2920 6 10 325 2260 146 100 6556 840 224 200 6727 1210 197 180 882
8 730 84 90 4669 1270 27 30 176
10 1680 13 20 122
11 790 94 110 605
12 1380 32 40 227
13 4640 37 60 286
14 5310 13 20 74
15 1360 116 90 84016 790 114 80 781
17 2560 28 40 185
18 1110 86 120 554
19 1310 1 0 0
20 2140 12 20 116
21 3810 5 10 2522 1900 21 20 126
23 1230 193 180 71824 1180 157 150 67625 610 2 0 1026 1320 16 20 20027 610 26 30 20628 870 20 20 5229 7820 135 150 752
Determined in the laboratory of Fless et al.; see Materials and Methodssection.
trations semiquantitatively to identify individuals with
above-normal concentrations of plasma Lp(a) who thus
have a higher risk of coronary artery disease. Once
Lp(a)-lowering drugs become established, this assay
will be very useful for monitoring the effectiveness oftreatment. This assay measures Lp(a) in serum, plasma,
or fingerstick whole blood quickly, reliably, easily, and
inexpensively; therefore, it may be suitable for use in
physicians’ offices.
We thank Angelo Scanu and Gunther Fleas at the University ofChicago for their advice and for providing Lp(a) and clinicalsamples for this study. Without their help, this project could nothave been initiated. We also thank Samar Kundu for his assis-tance in measuring Lp(a) in 25 samples with the Terumo ELISA.
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