a cumbersome dialysis step, which is needed to remove perchloric acid and equilibrate the sample ..... similar when saving in technician time ... program. Proper sample collection must also be appreciated. The effects of patient posture, length.
CLIN. CHEM.
24/1, 135-137 (1978)
Buffer-Exchange
for Rapid Separation
Column
Antigen from Perchioric Darrow
E. Haagensen,
Acid
Jr.,’ Richard
L. Easterday,2
A Sephadex 0-50 medium (Pharmacia) buffer-exchange column has been developed for rapidly changing the medium for carcinoembryonic antigen from perchioric acid to acetate buffer. Analytical recovery of the extracted antigen exceeded 95%. Plasma for analysis can be so prepared and samples ready for analysis within an hour. The technique also results in uniform assay volume, thus eliminating
this
variable
from
the
assay.
The commercially available carcinoembryonic antigen (CEA) assay marketed by Hoffmann-La Roche (“RocheCEA”) contains a cumbersome dialysis step, which is needed to remove perchloric acid and equilibrate the sample in the final assay buffer, ammonium acetate (10 mmol/liter, pH 6.8 ± 0.2). The dialysis requires a minimum of four buffer changes over 12 h and the resulting sample volume may vary from 4 to 6 ml. A potential alternative method for removing perchioric acid and equilibrating the sample in the assay buffer is to use buffer-exchange column chromatography for this step. We demonstrate in this paper the usefulness of this procedure. A perchloric acid-extracted sample can be prepared for analysis in about 20 mm with a constant volume for all samples.
Materials
and Methods
Prepacked Sephadex G-50 medium columns (Pharmacia) were designed to meet the following specifications: (a) elution stops automatically when the applied sample has all entered the top of the gel bed; (b) complete buffer exchange for a 4.3-4.8 ml sample volume with a 6.5-mi elution of the column; (c) a minimum of 95% analytical recovery or greater for CEA in the 6.5-ml eiution volume. Column preparation. Polypropylene columns (10 X 2.1 cm i.d.) had polyethylene frita (2 mm X 2.1 cm diameter, 70-120 ism
pore
size)
inserted
into
the
bottom
of
each
column.
The
1 Department of Surgery (Box 3236), Duke University Medical Center, Durham, N. C. 27710. 2 Research Department, Pharmacia Fine Chemicals, Piscataway, N. J. 08854. Received
Sept.
2, 1977;
accepted
Oct.
11,
of Carcinoembryonic
1977.
Catherine
A. Stolle,2
columns
and Samuel
A. Wells,
Jr.1
filled with 1.9 g of Sephadex G-50 M, swollen acetate solution (10 mmol/liter, pH 6.75) to give a 5.5-5.8 cm settled bed volume before compression. A top polyethylene frit (4 mm X 2.1 cm diameter, 70120 m pore size) was inserted and the gel bed was compressed to 4.8 cm bed height. The columns were then washed with the ammonium acetate solution to equilibrate the column with the CEA assay buffer. Sample recovery determination. We evaluated recovery of sample added to the Sephadex G-50 M columns by determining sample content in three fractions: (a) the eluate from a 4.5-mi added sample; (b) the eluate from the 6.5-mi buffer exchange sample; and (c) the eluate from 5 ml of sample column washings (the total volume of these three eluent fractions is equivalent to the liquid bed volume of the column). The sample content of the 6.5-mi buffer exchange volume (fraction b) divided by the total sample content of all three fractions and multiplied by 100 was the percentage recovery. The amount of sample present was determined by measuring the absorbance at 280 nm for the various proteins tested. Technique of sample preparation. The Hoffmann-La Roche procedure for plasma perchloric acid extraction (1) was followed precisely. This procedure resulted in approximately a 4.5-mi volume of perchloric acid extract after centrifugation. Each patient’s plasma was extracted in quadruplicate. We dialyzed two of the samples from each patient after the Hoffmann-La Roche procedure. The perchioric acid supernates for the other two samples from each patient were carefully poured onto the surface of Sephadex G-50 M columns that had been pre-equilibrated in the ammonium acetate in
were
ammonium
buffer
and
the
columns
were
allowed
to
drain,
stopping
au-
tomatically when the meniscus reached the gel surface. This effluent was discarded. Next, exactly 6.5 ml of water was added to each column and 6.5 ml of effluent was collected in assay tubes. This 6.5-mi eluate contained the sample, now present in the buffer with which the column was pre-equilibrated. Column sample elution automatically stopped when the 6.5 ml of wash reached the gel-surface. The flow rate was about 1 mi/mm. Sample addition and elution required about 20 mm. The eluted samples were analyzed by the Roche-CEA assay, the only modification being the use of a 6.5-mi volume of Roches’ ethylenediaminetetraacetate buffer for the CEA standard curve, to match the sample volume.
CLINICAL CHEMISTRY, Vol. 24, No. 1, 1978
135
0.8
a
Table 1. Carcinoembryonic Antigen Concentration in 19 Specimens of Plasma as Measured by the Two Techniques of Sample Preparation
0.0
0.7
-
0.6
-
1.0
2.0
--
Dialysis
0
0.5
Assay 1
-
0.4
6.0
-
0.1
7.0
0
2
4
6
8
10 2
4 16 18 20 22 24 26 28 30 3234
Column
Eiuonl
Volume
Assay 2
0.2
4.0
3.0
1.0
1.8
1.8
0.0
2.8
3.2
0.4
1.9
2.2
0.3
1.4
2.4
1.0
0.9
1.6
0.7
4.2
4.0
0.2
4.4
3.0
1.4
2.2
2.2
0.0
2.3
2.2
0.1
0.7
1.2
0.5
0.4
1.5
1.1
1.7
1.8
0.1
1.7
1.8
0.1
4.7
4.6
0.1
3.5
3.2
0.3
4.5
3.8
0.7
5.0
4.6
0.4
2.4
2.4
0.0
2.0
2.8
0.8
3.0
2.2
0.8
3.0
3.8
3.8
20.8
21.6
0.8
16.2
2.2
16.0
12.2
16.0
13.4
2.6
14.0
5.4
5.4
0.0
5.0
of the figure
14.8
14.8
0.0
16.0
respectively,
the 4.8- and
11.3-mi
elution
points
=
jg/uiter
2.1
Fig. 1. Elution profile on Sephadex 0-50 M buffer-exchange column to which was applied 4.8 ml of 0.6 mol/liter perchloric acid containing 1 mg of Dextran Blue 2000 The elution profile of Dextran Blue 2000 is depicted as absorbances of 1 ml fractions at 660 nm (#{149}). The elution profile of perchioric acid is similaly depicted from measurements of pH (A). The shaded portion of the figure depicts the 6.5-mI buffer-exchange volume we used in the CEA assay. Arrow at the bottom depict,
Difference,
2.3
36
in ml
Assay 1
1.3
.
0.2
Difference, ig/IIter
chromatography
2.4
5.0
-
Column
3.7
4.0
0.3
0
Assay 2
0.8
3.6
1.4 1.2
17.2
7.8
5.8
2.0
4.4
2.4
2.0
22.2
15.0
7.2
21.0
18.8
2.2
2.5
1.4
1.1
0.7
0.0
0.7
4, 0
4,
0 2
. c.,
00
0
4.8-mi application sample, measured in duplicate, was 97 ± 1% for either applied volume. Perchioric acid was retained
m
until at least 12 ml had emerged from the column. We collected 6.5 ml of effluent after applying 4.8 ml of 0.6 mol/liter
80
perchloric G-50 M
0
60
to 40 different No
Elution of protein ples of components -
vidually
0
tested
-
applied
0
Fig. 2. Percentage
recovery
0-50
weight
of five separate
M columns
in a 6.5-mI
volume
Each protein solution, 4.5 ml, was applied to the column; 6.5-mi buffer-exchange volume was collected, and the protein content of this fraction was compared to the total eluted protein content. The ribonuclease A, chymotrypsinogen A, ovalbumin, and pyruvate kinase were from Worthington albumin (Pentex) was from Miles Laboratories
Biochemicals,
the bovine
were
ml
ammonium
of the
that
of
these
ef-
had
in the
a molecular was
greater
for
dialyzed
CEA
each
Sephadex samples,
G-50 M column the CEA standard
used
prescribed
we applied
0.6 moi/liter (Pharmacia)
perchloric
acid
Blue 2000 in a volume of ml and washed the column, monitoring the absorbance and pH of each milliliter of eluate (see Figure 1), the Dextran Blue being monitored at 660 nm. Percentage recovery of Dextran Blue in the 6.5-ml assay volume from a 4.3- or a 4.3-4.8
CLINICAL CHEMISTRY, Vol. 24, No.
1,
1978
with
separate
in the
Roche-CEA
ethylenediaminete-
four separate
duplicate
one
sample
assay.
Thus,
CEA
of the duplicate
was tested
0.5-mi
0.6 moi/ liter Duplicate samples
were
G-50 M columns.
on Sephadex CEA, using
for a single
which
100 000, estimated
into
and
6.5
of per-
molecules
were
then
was
with
tested
those
separate
samples
eiuted
samples
assay
of buffer exchange, and 1 mg of Dextran
proteins For
19
in the
and the
2 is a plot
Each 0.5-mi sample was extracted with acid according to the Roche procedure.
scribed niscus
the fluid mecompleteness
and
volumes. perchloric
assays
draining stopped when of the gel-bed. To assess
of
columns,
Figure
weight exceeding than 95%. buffer exchange
Sample preparation assay. We divided each traacetate-treated plasma
duplicates
so that the top
as before
eluate.
be eluted
in 4.5 ml of water,
buffer.
6.5-mI
volume. Samweights were indi-
G-50
of the five separated
for
recovery
to drain acetate
CEA
136
any
would
sample,
of the Sephadex
Completeness of buffer exchange and automatic draining properties. Sephadex G-50 M columns were packed as deabove, reached
Sephadex
in
percentage Each
allowed
of each
buffer-exchanged each sample
Results
buffer.
columns
accounted
Weighl
vs. molecular
eluted from Sephadex
buffer-exchange
found
in the buffer-exchanged of various molecular
to the surface
centage Molecular
proteins
was
to see what
6.5 ml of effluent
20
buffer-equilibrated
acid
fluents.
-
0
40
acid columns.
We analyzed from each pair of two separate sets of curves were used to
standard
samples buffer
from
either
exchange.
For
dialysis the
or
dialyzed
curve was run in a 5-mi volume according to the Roche CEA kit instructions. As already mentioned for the buffer-exchange samples, the CEA standard curve was run in a 6.5-mi volume. For both assays, we the
netetraacetate Table 1
10-fold
buffer depicts
our
dilution
with
the
for the standard
curve.
results
of the
for
assay
ethylenediami19 plasma
sam-
pies processed either by dialysis or column chromatography. Two standard CEA curves were used, one for each member of the pairs of duplicate samples. Therefore, the variability includes the variability between separate assays for singlepoint CEA determinations. The average assay variability for duplicate samples by dialysis was 1.20 zg/iiter; the average assay variability for duplicate samples by column chromatography
was
by column 7.2 pg/liter
0.88
ag/liter.
The largest duplicate variability was 2.2 tg/1iter, as compared
chromatography for dialysis.
to
CEA”)
must
loss of a sample,
6 ml.
Each
dialysis
of leakage
dialysis
be poured
and
bags,
available
mol/iiter
perchioric
acid
enediaminetetraacetate-treated
any
protein-bound
the
plasma
assay
for
is used
plasma.
CEA
proteins;
and
thus
also
CEA
(“Roche-
to extract
ethyl-
This
extraction
precipitates
about
relatively
large
volumes
frees
90% of
bag there
has
either
replacing
with
it with in the
CLIN. CHEM.
24/1,
to remove perchloric and contributes
137-139
exchange technician
sample time
method
is taken
out
buffer.
closing
of
the ends.
more convenient, and the removal of perchloric be
arriving
more acid
in a clinical
and analyzed
processed
preparation of dialysis
preparation with sample
bag,
possibility
in the
or by knotting
Samples
can
sample Costs
into
for the
variability
staples
morning
of a dialysis
to the osmotic prescan vary from 4 to
to be inspected
is inherent
We have described a faster, consistent way of accomplishing and
and
the
time is less than vs. column buffer-
appears similar preparation
1
when saving in by the column
account.
References
of plasma
can be analyzed without concomitant nonspecific protein inhibition of the CEA/antibody reaction. The dialysis step that follows is necessary in order to remove perchloric acid and to maximize the CEA/antibody reaction, because the CEA/ antibody reaction is optimal at low ionic strengths (2).
The use of dialysis buffer is cumbersome
into
at each step. According the final sample volumes
same day because the h with our procedure.
commercially
0.6
sample
sample
laboratory
Discussion In the
Each with sure
acid and introduce to artifactual error.
I.
Hansen,
H. J., Snyder,
antigen (CEA) assay, management of cancer.
,J. J.,
Miller,
a laboratory Hum. Pathol.
E., et al.,
Carcino-embryonic
in the (1974).
adjunct 5, 139
diagnosis
and
2. Kupchik, H. Z., Hansen, H. J., Sorokin, J. J., and Zamcheck, N., Comparison of radioimmunoassays for carcinoembryonic antigens. Conference no. 720208 in Proc. 2nd Con!. and Workshop on Embryonic and Fetal Antigens in Cancer, U. S. Government. Printing Office, Washington, D. C., 1972, pp 261-265.
(1978)
Results for Serum and Plasma Compared
in 15 Selected
Radioassays Norman
P. Kubasik
We evaluated for 15 selected
and Harrison
the results radioassay
E. Sine
for serum vs. plasma procedures, using
facturers’ kits. Blood samples were collected oxalate-fluoride, or ethylenediaminetetraacetate
samples 19 manu-
with heparin, antico-
agulants and compared with serum samples. Differences were demonstrated between serum and plasma which may be of sufficient magnitude to alter clinical interpretation of the results. Assays also demonstrated significant dif-
ferences used.
based
A precise depends
upon
on the kit manufacturer
and accurate a number
radioassay of factors,
and procedure
for a given metabolite including reagent integrity
and stability, proper tools and technique, and an adequate quality-assurance program. Proper sample collection must also be appreciated. The effects of patient posture, length of time
of tourniquet
and the type
application,
of anticoagulant
can all affect the results of iaboratory tests (1). There is little information in the literature concerning the choice of serum or plasma and possible anticoagulant effects for radioassays. We have evaluated the differences between serum and The St.,
Clinical
Rochester,
Received
May
Laboratories, N. Y. 14607. 31, 1977; accepted
The
Genesee Sept.
30,
Hospital, 1977.
224 Alexander
heparinized plasma, ethylenediaminetetraacetate
selected kits.
radioassay
Methods We
used
oxaiate-fluoride (EDTA)
procedures,
with
treated treated
plasma, plasma
for
and 15
use of 19 manufacturers’
and Materials the
following
radioassay
kits:
Cortisol (“1251 Cortisol RIA Kit”; Diagnostic Corp., 12306 Exposition Blvd., Los Angeles, Calif. Choriogonadotropin (“Biocept-G”; Wampole Cranbury, N.J. 08512). C-peptide (“251-C-peptide Kit”; Calbiochem., Torrey Pines R., LaJolia, Calif. 92037). Folate (“3H Folic Acid Assay Kit”;
Diagnostic
Products 90064).
Labs., 10933
N.
Products
Corp.). Follitropin
(“FSH RIA Kit”; Amersham/Searle, Dr., Arlington Heights, Iii. 60005. Gastrin (“RSL Gastrin Kit”; Radioassay Systems ratories, Inc., 1511 E. Del Amo Blvd., Carson, Calif. Gastrin (“Gastrin Radioimmunoassay Kit
2636
S.
Ciearbrook
SchwarzfMann,
Mountain
10962). Insulin (“Immunophase Test System”; Corning Works, Medfield, Mass. CLINICAL
View
Ave.,
Insulin
Medical 02052).
Orangeburg,
(1125)
Diagnostics,
CHEMISTRY,
Labo90746). I25J;
N.
Y.
Radioimmunoassay Corning
Vol. 24, No. 1, 1978
Glass
137
