prostatic acid phosphatase in detection of prostatic cancer. After staining for acid phosphatase, we could detect as little as 0.3 ng of purified enzyme standard.
CLIN.
Cl-EM.
24/1,
140-142(1978)
Counterimmunoelectrophoresis Phosphatase Andras
We
G. Foti,’
evaluated
in Determination
in Human Serum
J. Fenimore
Cooper,2
and Harvey
counterimmunoelectrophoresis
for use
Herschman3
in
measuring prostatic acid phosphatase in detection of prostatic cancer. After staining for acid phosphatase, we could detect as little as 0.3 ng of purified enzyme standard complexed with antibody by this technique. However, when serum samples were used as antigen, the method was less sensitive (1.5-2.0 ng) because some of the serum proteins migrate with the phosphatase and decrease the intensity of the stain for acid phosphatase. For this reason we could not detect the phosphatase in serum samples of normal persons; only patients with moderately (or greater) increased activity in their serum showed positive results. In contrast, by radioimmunoassay as little as 1.0 ng of the phosphatase can be detected in serum. Keyphrases: intermethod comparison . enzyme radioimmunoassay prostatic cancer . diagnostic screening procedure for small laboratories
Addftlonal
assay
.
aids
.
Prostatic
carcinoma
men after of prostatic disease
is one
considered
it is still PAP)
the
to
Increased activity
leading
detection of this and
phosphatase
is a potent
assay) it can
for
capsule
acid
serum
(1-6). (enzyme (7-13). However,
PAP
prostatic
prostatic
in the
malignancy techniques
serum
malignancies
screening test for the lead to early recognition
confined
operable.
(EC 3.1.3.2, of prostatic Biochemical
measure
of the
age 60. A feasible carcinoma could
when
are also
indicator
now used to be measured
by the use of serologic assays. We have previously described in detail a radioimmunoassay for serum PAP (14, 15), which has several advantages: (a) the immunological activity of the enzyme
is more
specificity the
and
stable
the
conventional
results
than
sensitivity enzyme
in substantially
prostatic
cancer,
enzyme activity (16); (b) the of this assay are far better than assay (16), which consequently (c) its
improved
in all
stages
detection
of the
disease,
of untreated as compared
with
enzyme
assay.4 However, the need to radioiodinate the antigen and the need for an expensive gamma counter led us to investigate the use of alternative serologic procedures for detection of PAP. Counterimmunoelectrophoresis has been extensively used
for the
detection
Antigen
and
quantitation
precipitated
is
of Prostatic Acid
with
of many
the
antigens
appropriate
(17-20).
antibody
after
migration and
in an electric
does
not
field.
require
use
dioimmunoassay.
The
It is faster
of
than
radioactive
technique
immunodiffusion
materials
can
be very
as does
useful
laboratories However,
where only a few samples are tested routinely. it must be borne in mind that the sensitivity of the radioimmunoassay far exceeds that of counterimmunoelectrophoresis (19). We describe here
Materials
our
findings.
and Methods
Materials: Disodium p-nitrophenyi Violet LB salt, and naphthol AS-MX obtained from Sigma Chemical Co., agarose from Kallestad Laboratories 55318; and barbital from Maliinckrodt 63147.
All
other
obtained
from
chemicals
were
J. T. Baker
phosphate, Fast Red phosphoric acid were St. Louis, Mo. 63178; Inc., Chaska, Minn. Inc., St. Louis, Mo.
of analytical
Chemical
grade
Co.,
Medical
of
Group,
2 Department
Group
and
Research,
Los Angeles, of Urology,
Kaiser
Foundation
Southern
Calif.
California
90027.
Southern
California
Hospital,
cancer.
of Biological Chemistry UCLA School of Medicine, A. G., Herschman, H., Cooper, randomized clinical study N. Engi. J. Med., in press.
Received
140
Aug.
12, 1977;
accepted
Permanente
Los Angeles,
Department Medicine, Foti, comparative
Permanente
Los
Medical
Calif.
and Laboratory Angeles, Calif.
J. F., and for the
Oct. 4, 1977.
CLINICAL CHEMISTRY, Vol. 24. No. 1, 1978
of Nuclear 90024.
Malvaez,
detection
90027.
R. R., A
of prostatic
and
were
Phillipsburg,
N. J.
08865.
Prostatic acid prostatic fluid and
previously Collection
phosphatase the antiserum
was purified raised in rabbits
from human as described
(15).
of serum samples: The normal subjects were healthy men who were undergoing routine physical examinations. In all prostatic cancer patients, the stage of the disease was confirmed by rectal examination, prostatic needle biopsy, and bone scan. In all patients, the blood was obtained at least 48 h after rectal examination was stored at -25 #{176}C until use. Counterimmunoelectrophoresis: cm, Kodak 1402130) barbital buffer (0.05
and
separated.
Glass
slides
Serum (8.3
X 10.2 solution in and dried.
were precoated with agarose mol/iiter, pH 8.6), 10 g/iiter,
9.0 ml of the agarose solution was layered onto the slides. The gel-covered slides were placed in a moist chamber at 4#{176}C for at least 20 mm. Wells 3 mm in diameter were cut 6 mm apart. A second row of wells was cut 10mm from the first row. Then
One
row
or serum
of wells
was
samples.
filled
The
with
other
10 sl of purified
row of wells
PAP
standard
was filled
with
di-
luted antiserum. The buffer (0.05 moi/liter,
electrode vessels were filled with barbital pH 8.6). The slide was placed in the electrophoretic chamber in such a way that the wells filled with antibody were on the anodic side, the wells with PAP on the cathodic side. The slide was connected to the electrolyte with a filter-paper bridge. A constant current of 10 mA was for 1.5 hat 4 #{176}C. After the electrophoresis, stained by the Burstone technique (21). Histochemical staining of acid phosphatase: applied
Department
ra-
for small
Violet sodium
LB salt, acetate
50
mg/di
buffer
of
water,
was
mixed
(2.5 mol/liter,
pH
5.2)
the
Fast with
(15)
and
in all
enzyme
serum
samples
assay
(22).
by
both
10
containing
of naphthoi AS-MX phosphate. The electrophoresis incubated in this mixture at 37 #{176}C for 5 h. Determination of prostatic acid phosphatase: determined
gel
radioimmunoassay
was
Red ml
of
25 mg
slide This
was was
Table
1. PAP Content
of Sera of Patients
RIA
PatIent
ng/0.1
with Prostatic
Enzyme assay Sigma unlts/1.0 ml
ml
AE AL SP FR SL
3.0 3.2 3.2 3.3 3.4 7.2
0.07 0.06 0.06 0.05 0.07 0.06
LA
7.2
EE
Cancer, CIE 10 izl
N
Untreated
OG KE AP
9.2 10.0 16.5 10.3 12.8 21.5
0.18 0.11 0.31 0.13 0.14 0.30
I I I II Il II
Untreated Untreated Untreated Untreated
Dl
22.4
II
Untreated
TC
28.4
0.23 0.75
CK KJ BM HH BM
22.0 27.0 32.0 79.6 20.8 30.0 36.0 38.0 202.0
II Ill
DES RAD, DES
III III
RAD Untreated
III
RAD
#{149} Abbreviations
used are same
0.45 0.40 0.74 1.89 0.29 0.34
#{149} #{149}
a variety
Untreated
#{149}
1.10
IV IV
DES DES, RAD
1.12 7.60
IV IV
DES
we
examined,
of conditions
the
of PAP the anode
its antibody gen precipitate
meet
most
used
for
able
counterimmuno-
is
suitable
was
sodium
barbital
at 10 mA. Under these of the antibody is equal
condito the
in the 10 g/liter gel. The PAP migrates at pH 8.6. Consequently, the enzyme and between
the
two
made visible with phosphatase/antibody
wells.
acid
The
antibody/anti-
phosphatase
stain,
1.75
and
3.5 ng of PAP
were
used
against
dilutions of 10-, 50-, 100-, 500-, 1000-, 1500-, and 3000-fold, a visible precipitate was detected 2000-fold dilution. For subsequent studies a 100-fold antiserum was used.
2000-,
antisera
to our results
to detect
PAP
for purified
enzyme,
we were
un-
of normal patients, even when more serum (20 uI) was used in the wells. Similarly, we could not detect PAP in most cases in sera from patients with in-
in serum
tracapsular prostatic tumors (Stage I or II). Detection improved when serum proteins other than immune tates were washed out of the gels after electrophoresis, the volume of the gel was increased to accommodate crease volume of sample (25-50 pI), or when antibody of 10- or 25-fold were used. The
mean
persons
because the acid complex retains the ability to hydrolyze phosphate groups from the substrate (23, 24). Figure 1 shows the results of counterimmunoelectrophoresis when purified enzyme standards were used. The limit of detection is between 0.21 and 0.43 ng with a 25-fold diluted When
DES
as in Fig. 2.
electrophoresis (the buffer system and its pH, the current, duration of electrophoresis, and the amount of gel used to coat the slides), to maximize the measurement of PAP. Of those mol/liter, pH 8.6), the electroendosmosis
Untreated
IV
In contrast
altered
Untreated
#{149}
Results
antiserum.
N
Untreated
JA LM BL
migration towards
-
I
IG
(0.05 tions,
N
a
Treatment
Stag.
-
Methods
0.16
WR
buffers
by Various
N N I
SL CG
We
as Measured
Sigma
concentration
is 65 ug/liter units/liter
of PAP
in the
by enzyme
serum enzyme
PAP assay,
in-
of normal
150-200 2 shows for patients with For sera containing less assay.
our results by counterimmunoelectrophoresis different stages of prostatic cancer. than 200 pg/liter of PAP, counterimmunoelectrophoresis no visible stain for acid phosphatase/antibody 1 summarizes noassay, by
an
dilutions
serum
by radioimmunoassay
of serum
was not precipiwhen
and
Figure
gave
complex.
Table
measurements by radioimmuand by counterimmunoelectro-
up to diluted
‘
1044111 I
99 Fig. 1. Counterimmunoelectrophoresis of purified PAP with anti-PAP PAP was applied into the upper wells and 10 zl of antisera (25-fold dilution) Into the lower wells. The upper wells from left to riglt: 0.21, 0.43, 7.0. 14.0, and 24.0 ng of PAP per 10 uI. After electrophoresis complex was stained by the Burstone (21) method
0.87, 1.75, 3.5. the immuno-
Fig. 2. Counterimmunoelectrophoresis carcinoma patients
of sera from
prostatic
Seruni. lOl. was applied into the upper wells. Left to rit: CK, St. Ill, RAD, DES; TC, St. II, DES; LM, St. IV, DES; JA, St. IV, RAD. DES; 81, St. IV, DES; BM, St. IV, untreated; 1G. St. IV. DES; BM, St. Ill, untreated. Lower wells contained 10 Ll of antisera (100-fold dilution). The immunocomplex was stained by the 84,stone (21) method. Abbreviations are (In order given) patIents initials, stage of the cancer,
and treatment
(radiation;
diethylstlibestrol)
CLINICAL CHEMISTRY,
being
Vol. 24, No.
received
1, 1978
141
phoresis
(the
antigen
along
with
patients.
of intensity
I
Stage
Stage
of the disease
a very gave
with
few
with
positive
test
by enzyme
assay
results.
precipitate after tected corresponded
munoassay Although complete
showed
stained
antibody!
5. Gutman,
to
asterisks),
increased metastases
one
four
and the treatment II of the disease
Stage
Patients
IV disease showed positive tests. Patients with a PAP by radioimmunoassay and
phoresis mg/liter
liter
of the
is indicated
the stage Only
with or
degree
precipitate
for some and
Stage
with
Cancer
none
III
counterimmunoelectro-
concentration about 7000
the
of about
2
Sigma units! PAP/antibody
greatest
staining. The least amount of complex deto 160-300 pg/liter of PAP by radioim-
and 290-580 Sigma units/liter by enzyme assay. radioimmunoassay and enzyme assays do not show correlation, the trends are clearly seen. Serum
samples
that
trophoresis
gave
positive
always
had
both of the other showing increased
assay
noassay
show
did
tometric
not
results
an
by
increased
concentration
techniques.
activity
by
by the
or positive
by
samples radioimmu-
many
of PAP
increased
assay
of PAP
In contrast,
concentration
enzymatic
counterimmunoelec-
results
spectropho-
by counterim-
munoelectrophoresis.
7. King,
It
is fast
assay
for
and the
sensitivity
for detecting
theoretically detection
appears
or
the
the
PAP assay We evaluated
and
is more and
radioimmunoassay.
the
than
that
than of
PAP.
for
the
buffers
Different
most
measuring
specific
quantitation
to be less
of
However, enzyme
were
by counterimmunoelectrophoresis. more than 100 serum samples
its assay
studied
from
for
patients
with prostatic cancer by counterimmunoelectrophoresis, with no false-positive results. However, the technique is not sensitive enough to detect PAP from serum of normal males, and so we have no mean value of PAP for normal men by it. No prostatic cancer patients test, by this technique,
with Stage I disease and only a few patients
gave a positive with Stage II
and Scott, W. W., acid phosphatase. of serum determination.
10. Huggins, as a substrate (1945).
C., and for
11. Babson, phosphatase
A. L., and Read, P. A., A new in serum. Am. J. Clin. Pat ho!.
Seligman,
Talalay, P., Sodium phosphatase tests.
A. M.,
Chauncey,
determination 190,7 (1951).
Roy,
M.
16.
Foti,
for the
prostatic
17.
H., and prostatic
Herschman,
H.,
S. S., and
Niphadkar,
Nachlas,
and
Cooper, C!in.
Chem.
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in human
This work was supported in part by Southern California Permanente Medical Group and by Contract EY-76-03-0012 between the Energy Research and Development Administration and the Regents of the University of California.
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