Serono Laboratories, Inc., Braintree, MA 02184; and âPAP-. Checkâ prostatic acid phosphatase from NMS. All these RIA procedures are competitive-binding.
OLIN. CFEM. 27/10, 1747-1752 (1981)
Comparison of CountercurrentImmunoelectrophoreticAssaywith CommercialRadioimmunoassayKitsfor MeasuringProstaticAcid Phosphatase G. L Wright, Jr., P. F. Scheilhammer, D. N. Brassli, S. M. Sieg, and M. S. Leffeil We evaluatedand compared five commercial radioimmunoassay kits with a standard counter-immunoelectrophoretic assay for the measurement of prostatic acid phosphatase in serum. Four ofthe five radioimmunoassays performed as described by the supplier with respect to sensitivity, stability, precision, linearity, analytical recovery, and expected values for the normal male population. None of the radioimmunoassays was more clinically sensitive then the counter-Immunoelectrophoretic assay for detecting increased prostatic acid phosphatase In serum.
Results obtained by counter-immunoelectrophoretic assay ageed with results obtained by radloimmunoassay in 96% of the tests. The proportion of positive results in patients with confirmed prostatic adenocarcinoma Increased with disease proession. The fewer positive tests in localized adenocarcinoma (Stages A and B) suggests that neither the counter-immunoelectrophoretic assay nor the radioimmunoassay procedures are useful for screening unselected populations for adenocarcinoma of the prostate. The high percentage of normal values found in those patientsclinically free of disease after treatment is encouraging and supports the use of the prostatic acid phosphatase Immunoassays in prospectively monitoring the treatment of prostatlc cancer patients. cancer #{149} prostatic nlfinitorlng Ulrapy . cutoff vafres screening
Additional Koyphrases screening Interval
.
disease refevnce
.
Measurement of serum acid phosphatase has been used for more than 40 years in the clinical staging of prostatic adenocarcinoma. Most of these studies involved enzymic colonmetric assays to detect this enzyme. These assays have some inherent problems, the major one being a lack of specificity for the isoenzyme that is specific for prostatic acid phosphatase (PAP) (1).’ In recent years, several immunoassays have been developed and shown to have greater sensitivity and specificity than the enzyme assays (2-10). Countercurrent immunoelectrophoresis (CIE) and radioimmunoassay (RIA) have been the two immunoassay techniques most extensively evaluated
(2-19).
Recently, several RIA kits for specific measurement of PAP have become commercially available. We evaluated these kits and compared results with those obtained with an established CIE assar for measuring serum PAP. Departments of Microbiology/Immunology and Urology and the Immunology Program, Eastern Virginia Medical School, P.O. Box 1980, Norfolk, VA 23501. ‘Nonstandard abbreviations use& PAP, prostate acid phosphatase; CIE, countercurrent immunoelectrophoretic assay; RIA, radioimmunoassay; NEN, New England Nuclear, Boston, MA 02118; CA, Clinical Assays, Cambridge, MA 02139; MAL, Mallinckrodt Diagnostics, St. Louis, MO 53134; NMS, Nuclear Medical Systems, Newport Beach, CA 92863. Received April 15, 1981; accepted June 9, 1981.
Materials and Methods Human subjects.
The study population
consisted of 25
apparently healthy men (ages 43-67) who had not previously any form of urinary difficultyor urogenital disease; 22 patients with histologically proven benign prostatic hyperplasia; and 265 patients with various stagesof prostatic adenocarcinoma. The prostatic carcinoma patients were clinically staged by digital rectal palpation, cystoscopic examination, bone scan, and, where appropriate, skeletal survey and chest roentgenograms. CAT scan and lymphangiograms were not routinely used. Of these patients, 68% were also surgically staged by lymphadenectomy in conjunction with ‘I implant for definitive treatment of the disease. In the case of 60 of the prostatic cancer patients, PAP tests were done before and periodically after therapy; 205 had received definitive therapy either by im1 implantation or external beam radiation, or by hormonal manipulation. These patients were followed up every three to six months with rectal examination and PAP tests, and often with bone scan, cystocopy, and prostate biopsy between 12 and 30 months after treatment. Serum samples. Ten mililiters of blood was drawn into a Corvac evacuated tube (Corning Glass Works, Corning, NY 14830) from the antecubital vein before rectal examination. Blood samples were allowed to clot at room temperature for 30 mm and the serum was collected by centrifugation (1500 X g, 15 mm) and divided into two aliquots, one for CIE assay and one for RIA. To the sample for CIE, 10 g of disodium citrate was added per milliliter of serum. Both samples were stored at -70 #{176}C until analyzed. Radioimmunoassays. We examined the following RIA kits for PAP: “RIANEN” prostatic acid phosphatase, from NEN; “Gamma Dab” prostatic acid phosphatase from CA; “RIAQuant” P.A.P. test from MAL; “P.A.P. Check RIA” from Serono Laboratories, Inc., Braintree, MA 02184; and “PAPCheck” prostatic acid phosphatase from NMS. All these RIA procedures are competitive-binding assays and were used according to the manufacturers’ instructions. Countercurrent immunoelectrophoresis (CIE). The CIE procedure was used as described by Chu and associates (11, 19). The assay was performed on 13 X 18 cm Mylar sheets covered with 35 mL of phosphate buffer (50 mmolfL, pH 6.5) containing 7.5 g of agarose. Parallel rows of wells, 4 mm in diameter, were cut 5mm apart in the agarose. Ten microliters of serum or PAP standards (source of standard PAP was prostate adenocarcinoma tissue) was placed in each cathodic well and an equal volume of a 32-fold diluted rabbit anti-PAP antiserum was added to each anodic well, (The PAP standards and rabbit anti-PAP antiserum were kindly supplied by Dr. T. M. Chu, Roswell Park Memorial Institute.) The agarose sheet was placed in the electrophoresis chamber and electrophoresed at 40 mA for 2 h at 4#{176}C. The agarose plate was then stained for 3 to 5 h at room temperature in 0.1 molfL ammonium acetate buffer (0.1 mol/L, pH 5.0) containing, per liter,! g each of a-naphthyl phosphate and Fast Garnet GBC experienced
CLINICALCHEMISTRY, Vol. 27, No. 10, 1981
1747
Table 1. Characteristics of the Five PAP Kits HEN
Sample volume, Total counts/mm
CA
Serono
MAL
NMS
100 30 000 40 5 SA SD,ON Yes 1-50
100 29 000 62 4 SA SD,ON No 1-30
200 35 000 42 4 SA ON No 0.5-30
200 86 000 30 4 SA, PEG SD,ON No 1-40
100 15 000 35 5 SA SD Yes 2.5-40
Controls included Sensitivity, sg/L
2
2 0.4
3 0.5
NO 0.15
2 0.10
Expected normal range, j.gIL PAP antigen source Anti-PAP antibody source
0.6-3.3 Seminal fluid
0-2.0
0-4.0
0-2.0
0.5-3
Seminal fluid Goat
Seminal fluid Rabbit
Seminal fluid Rabbit or goat
Seminal fluid C Rabbit
LL
% of total counts found, zero standard No. pipetting steps Separation Incubation
technique8 tlmeb
Addition of buffer Standards, range, .ig/L
0.6
Rabbit
a SA, second antibody; PEG, polyethylene glycol. tissue as soszce of PAP for ‘25I abeled trace.
b
S1),sa
C
day ,
salt. The plate was rinsed in distilled water and the plates were evaluated independently when wet and again after having been air dried, by two laboratory personnel.
Results Comparison
of methods.
characteristics
of
Table 1 summarizes the principal the five RIA procedures; all in-
0.999); 0.36 to 30 zg/L with the CA assay (r = 0.999); = 0.999); 0.83 to 30 ,ug/L for the Serono assay (r = 0.998); and 1.83 to 40 figfL for the NMS =
0.28 to 40 sg/L for the MAL kit (r assay (r
=
0.973). Various serum samples were diluted with zero or with a pool of normal-female serum and assayed
Linearity. standard
assayed. All kits demonstrated dilution and gave no indication
acceptable parallelism on of nonspecific interfering
in serum.
recovery. Known amounts of PAP (up to 100 tg/L) were added to aliquots of a pool of normal serum from women, containing 1.5 tg of PAP per liter, and assayed by four of the five methods. Each sample was assayed in four replicates in four separate assays, yielding 16 values for each sample. The analytical recovery for NEN averaged 92% (range 92-95%); for CA, 94% (range 90-95%); for Serono, 104% (range 93-114%); for MAL, 99% (range 93-105%); and for NMS, 97% (range 94-101%). Sensitivity. The sensitivity or detection limits of the five methods are shown in Table 1. The sensitivity of the standard Analytical
volve very similar operations. Standard curves. Logit-log transformation of the standard curve data showed linearity from 0.94 to 50 g/L for the NEN
assay(r
by all five methods. The PAP concentration in these samples was found to be linearly proportional to the amount of sample
substances
Performance Characteristics of the RlAs analytical
Seminal fluid used as sowce of PAPfor standards,prostatic adenocarclnoma
curve is defined as the smallest concentration that can be distinguished from zero. These values were calculated from
Table 2. Quality Control Studies NEN (30)8
CA
(22)
S.rono (16)
MAL
2.34 ± 2 2.43 0.21 8.60 10.18 ± 2 9.43 0.43 4.60
