CLINICAL

IMMUNOLOGY

AND

IMMUNOPATHOLOGY

4,

362-373

(1975)

Measurement of Carcinoembryonic Antigen in Serum of Patients by Using a Technique of Passive Hemagglutination Inhibition RONALD

L. ANTHONY

AND KENNETH

M. SOSNOWSKI

A technique of passive hemagglutination inhibition (PHI) which can detect nanogram levels of carcinoembryonic antigen (CEA) in serum of patients has been developed. Human O-negative erythrocytes were sensitized with CEA recovered from primary adenocarcinemas of the colon and rectum. Sera from patients were then examined for their capacity to inhibit the agglutination of the sensitized erythrocytes in the presence of a predetermined amount of goat anti-CEA serum. Positive sera were defined as those which produced inhibition of agglutination at a dilution of I : 8 or greater. The percentage of positive sera was 82% for primary adenocarcinomas. 55% for all other cancers, 62% for benign diseases of the gastrointestinal tract, 60% for alcoholic cirrhosis, and 17% for normal healthy controls. Results are presented for 240 patients and attempts have been made to correlate PHI titers with CEA levels determined by radioimmunoassay.

Following the identification of the carcinoembryonic antigen (CEA) in primary adenocarcinomas of the colon and rectum (I), efforts were directed toward development of in vitro tests for demonstration of CEA in serum of patients. Thomson et al. (2) developed a radioimmunoassay capable of detecting as little as 2.5 ng CEA/ml of serum. Early results of the assay appeared promising and claims were made that it was highly specific for primary adenocarcinomas of the colon and rectum when the 2.5 ng level was taken as the lower limit of positivity. More recent studies, however, tend to refute these original claims. CEA or CEA-like proteins have been demonstrated in serum of patients with adenocarcinemas of other gastrointestinal organs (3-5). adenocarcinomas of nongastrointestinal origin (6,7), nonmalignant diseases of the gastrointestinal tract (8-l 2) and in normal human plasma (13) and feces (I 4-l 6). These studies led to several extensive programs aimed at evaluating the usefulness of the radioimmunoassay of CEA as a clinical test to aid in the diagnosis of cancer ( 17-20). It was concluded that while the assay does lack specificity in its present form and it has little value as a screening test for cancer, it may be of considerable value when used to monitor possible recurrence of tumor growth and/or metastases following surgical therapy (2 I-26). The present investigation deals with the development of a technique of passive hemagglutination inhibition (PHI) capable of detecting nanogram levels of CEA in serum of patients with various types of malignancies. This test differs from the hemagglutination inhibition test described by Lange et ~1. (27) in that it utilizes 362 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved

CARCINOEMBRYONIC

ANTIGEN

IN

HUMAN

SERUM

363

25 ~1 of untreated serum. The test can be completed in an 18 hr period and it is simple enough to be performed on a routine basis in any clinical laboratory. MATERIALS

AND

METHODS

Extraction and purification oj’ CEA. Primary adenocarcinomas of the colon from ten patients were pooled and were extracted for recovery of CEA. The total weight of the tumor pool was 486 g. The extraction procedure included homogenization in deionized water (3 ml/g wet tumor) followed by solubilization in an equal volume of 2 M perchloric acid. The soluble phase was dialyzed extensively against deionized water and was lyophilized. The dry residue, which represented crude CEA, was reconstituted in phosphate buffered saline (0.02 M, pH 5.8) at a concentration of 50 mglml. Ten milliliters of the crude CEA were then eluted through a column (2.5 X 100 cm) of Sepharose 4B using the PBS as the eluent. The protein content of the effluent, after being concentrated by an Amicon on-line eluate concentrator fitted with a UM-I 0 membrane (MW cut-off level of lO,OOO), was monitored using an LKB ultraviolet absorptiometer connected to a continuous recorder plotting percentage transmittance at 280 nm. The variable tubing-constrictor valve on the concentrator was set to achieve a threefold total volume concentration. The ultrafiltrate, which represented twothirds of the total volume of the effluent, was not collected. The effluent retained by the UM-10 membrane was collected in 3 ml vol by using an automatic fraction collector. Fractions representative of each area of the elution profile were assayed for CEA by immunodiffusion. Goat anti-CEA serum, supplied by Cordis Laboratories, Miami, Florida, was used as the source of antibody. Fractions exhibiting CEA activity were pooled, dialyzed against deionized water and lyophilized. The final residue was reconstituted in PBS at a concentration of 50 mg/ml and was eluted through a column of Sephadex G-200 following the procedures described above. Fractions of the G-200 effluent which contained CEA were pooled and were used as the antigen for the development of the passive hemagglutination inhibition test and the radioimmunoassay. Anti-CEA serum. Pregnant goats were hyperimmunized with CEA recovered from the G-200 fractionation. The resultant antisera were absorbed with perchloric acid extracts of normal colons and packed human type AB-positive erythrocytes. Specificity of the antisera was confirmed by comparative immunodiffusion studies using the goat anti-CEA serum supplied by Cordis Laboratories, Miami, Florida. Clinical specimens. Serum was collected from patients who, in the opinion of their physician, could be placed into any one of the following categories: (1) primary adenocarcinomas, (2) cancers other than primary adenocarcinomas, and (3) benign disease of the gastrointestinal tract and pancreas. At the time of the initial visit the patient’s chart was abstracted for pertinent clinical data (blood type, hematocrit, etc.) and a specimen of serum was obtained. All patients were studied clinically by the Gastroenterology Service with assistance from the Departments of Radiology and Pathology. When indicated, surgical biopsy was performed by the respective surgical service in cooperation with the gastroenterologists and pathological examination was carried out by staff pathologists.

364

ANTHONY

AND

SOSNOWSKI

Adenocarcinomas of the colon and rectum were classified according to Duke’s method of staging (28). Negative control sera were obtained from students and employees in the Department of Pathology. This population was comprised of both smokers and nonsmokers and included five females in the second or third trimester of pregnancy. Passive hemagglutination inhibition. Human type O-negative erythrocytes were sensitized with CEA by using the bis-diazotized benzidine (BDB) technique. The BDB was prepared according to the procedures outlined by Gordon et al. (29). Erythrocytes were collected in sterile Alsever’s solution, washed three times in 0.15 M NaCl, and standardized as a 50% suspension. One-hundred microliters of 50% erythrocytes were added to 1 ml samples of CEA which had been diluted to contain from 100 to 1000 N protein. Twohundred fifty microliters of BDB, diluted 15-fold in 0.02 M PBS (pH 7.2), were then added to the erythrocyte-CEA suspensions. Following incubation at room temperature for 10 min, the suspensions were centrifuged at 75Og for 5 min. The sensitized cells were washed in PBS containing 1% fetal calf serum (PBS-FCS) and resuspended to a final volume of 5 ml. The capacity of goat anti-CEA serum to agglutinate the sensitized cells was determined. The antiserum was diluted serially in 25 ~1 vol using the PBS-FCS as the diluent. Sensitized erythrocytes (25 ~1) were then added to each dilution of antiserum. After incubation at room temperature for 1 hr the titer of the antiserum was taken as the reciprocal of the highest dilution which produced a 3+. agglutination. This titer was considered as I unit of antibody. The capacity of a patient’s serum to inhibit the agglutination of the sensitized cells in the presence of 2 units of antibody was taken as an indirect measure of the CEA in that serum. Twenty-five microliters of the patient’s serum was diluted serially in 25 ~1 vol from 1: 2 to I : 256 using PBS-FCS as the diluent. An equal volume of goat anti-CEA serum, diluted to contain 2 units of antibody. was added to serum dilutions 1 : 4 through I : 256. The 1 : 2 dilution of serum served as the serum control. Following an incubation period of 18 hr at 4”C, 25 ~1 sensitized cells were added. After continued incubation at room temperature for I hr, the highest dilution of serum which inhibited agglutination could be read without difficulty. Sera which exhibited PHI titers of I : 8 or greater were regarded as positive. Radioimmunoassay. CEA was iodinated with lZhl by using the procedure outlined by Thomson et al. (2). The capacity of the goat anti-CEA serum to bind 35% of 1 ng labeled CEA was then determined following the methods reported by Egan et al. (30). A standard curve of inhibition was then constructed by determining the capacity of known amounts of unlabeled CEA ( 1-l 000 rig/ml) to inhibit the binding of 1 ng labeled CEA to the predetermined amount of antiserum (1 : 1280). The coprecipitation technique, as described by MacSween et al. (12) was then used to quantitate CEA levels in the sera of patients. RESULTS

The profiles of elution resulting from gel filtration of CEA on columns of Sepharose 4B and Sephadex G-200 are presented in Fig. 1A and lB, respec-

CARCINOEMBRYONIC

ANTIGEN

IA Sephroso

c

IN

HUMAN

48

Millll~tsrs

SERUM

365

I

of

Effluent

sqhodex f IB G-ZOO

*: glu 20 -B “f

80

so e

L

loo

200 Mdhliters

so0 of

400

Effluent

FIG. 1A and 1B. Elution profiles resulting from gel filtration of tumor extracts through columns of Sepharose 4B and Sephadex G-200. Milliliters of effluent represent only that portion of the effluent which was retained by the UM-10 membrane. Shaded areas of the curves represent fractions containing CEA as revealed by immunodiffusion.

FIG. 2. lmmunodiffusion patterns resulting from the diffusion of CEA against absorbed goat antiCEA serum. Wells 1 and 3 contained CEA, wells 2 and 6 contained goat anti-CEA serum (University of Maryland) well 7 contained goat anti-CEA serum supplied by Cordis Laboratories of Miami. Florida. Wells 4 and 5 contained perchloric acid extracts of normal colons.

366

ANTHONY

AND

SOSNOWSKI

tively. It must be noted, however, that since the effluent was concentrated threefold by the on-line concentrator prior to its passage through the absorptiometer, the milliliters of effluent given in the figures represents only one-third of the total volume. The ultrafiltrate, which represented two-thirds of the total volume of the effluent, did not pass through the absorptiometer and it was not collected. Thus, if we consider this concentration effect, the CEA activity, as determined by immunodiffusion, was eluted in the region representing 60-90% of the 4B column volume and 30% of the G-200 column volume. These results are in agreement with those reported by other authors (3 1,32). Effluents containing CEA, as determined by immunodiffusion, are represented by the shaded areas of the elution profiles. Results of a typical immunodiffusion test, using absorbed goat anti-CEA serum as the source of antibody, are shown in Fig. 2. The anti-CEA sera prepared at The University of Maryland (wells 2 and 6) gave a reaction of identity with the Cordis anti-CEA serum (well 7). The CEA in well 3 was the unconcentrated effluent from the G-200 fractionation while the CEA in well 1 was a pool of the G-200 effluents which had been lyophilized and reconstituted in one-tenth the original volume. Neither the Cordis antiserum nor the Maryland antisera produced a zone of precipitation when diffused against perchloric acid extracts of normal colons (wells 4 and 5). The initial phase of development of the passive hemagglutination inhibition test involved the determination of the smallest amount of CEA which could be used to sensitize human type O-negative erythrocytes in the presence of a 1 : IS dilution of BDB. The proficiency of sensitization was monitored by the capacity of goat anti-CEA serum to agglutinate the cells. It was found that agglutination was inhibited when preparations of CEA containing more than 500 pg protein/ ml were used for the sensitization. Conversely, however, the erythrocytes were agglutinated spontaneously by the BDB when the protein concentration of the antigen was less than 200 pg/ml. The passive hemagglutination titer of the antiserum was maximal (1: 600) when erythrocytes were sensitized with 1 ml of a CEA preparation containing 300 pg protein. Adenocarcinomas

The capacity of preoperative sera from 94 patients with primary adenocarcinemas to inhibit the agglutination of 25 ,ul sensitized cells in the presence of 25 ~1 of a 1 : 300 dilution of antiserum is given in Table 1. Of the five patients diagnosed as having villous adenoma with foci of in situ carcinoma (Duke’s A), two had a PHI titer of 1: 8. This titer represents the lower limit of positivity. Twenty-two patients had carcinomas of the colon or rectum which were invasive but not metastatic (Duke’s B). Sera from 81% of this group gave positive titers for CEA by the PHI procedure. Of 21 patients with colorectal carcinomas with metastases to the mesenteric lymph nodes (Duke’s C), 17 or 81% were positive. Finally, 86% of the patients with distant me&stases (Duke’s D) had a PHI titer of 1: 8 or greater. Of the 13 sera from colon-rectal adenocarcinomas which had PHI titers of less than 1 : 8, 7 were also tested by a double antibody technique of radioimmunoassay. Six of these seven sera had CEA levels of less than 2.5 r&ml. The remaining serum, which was from a patient with adenocarcinoma of the colon in the stage of Duke’s C, had a level of 6.5 ng by RIA and had a PHI

CARCINOEMBRYONIC

ANTIGEN

TABLE

PASSIVE

IN

HUMAN

367

SERUM

1

TITERS OF PREOPERATIVE SERA FROM PATIENTS WITH PRIMARY ADENOCARCINOMAS

HEMAGGLUTINATION

INHIBITION

Reciprocal of PHI titer Totals

Primary adenocarcinomas Colon-rectum Duke’s A Duke’s B Duke’s C Duke’s D Stomach Noninvassive Invasive Metastatic Liver Esophagus Lung Breast ovary Pancreas Totals

Measurement of carcinoembryonic antigen in serum of patients by using a technique of passive hemagglutination inhibition.

CLINICAL IMMUNOLOGY AND IMMUNOPATHOLOGY 4, 362-373 (1975) Measurement of Carcinoembryonic Antigen in Serum of Patients by Using a Technique of...
1MB Sizes 0 Downloads 0 Views