Counterimmunoelectrophoresis in Determination of Prostatic Acid ...

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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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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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