Journal of Clinical LaboratoryAnalysis 494-78 (1990)
Specific Enzyme lmmunoassay for a-Fetoprotein From Hepatocellular Carcinoma Takeyuki Kohno,' Tooru Kitamura,2 Kazunori Tsuda,2 and Eiji Ishikawa' 'Department of Biochemistry and 'Second Department of Internal Medicine, Medical College of Miyazaki, Miyazaki, Japan a-Fetoprotein in sera from healthy subjects and patients with liver cirrhosis and hepatocellular carcinoma was fractionatedinto three peaks by affinity chromatography on a column of Lens culinaris agglutinin-Sepharose 48. One peak (the first peak) was found in all the serum samples, but the other two peaks (the second and third peaks) were found only in patients with hepatocellularcarcinoma. For a-fetoprotein in the second and third peaks, a specific enzyme immunoassay was developed. Lens culinaris agglutinin-coated polystyrene balls were incubated with a-fetoprotein and, after washing, with affinity-purified antia-fetoprotein Fab'-p-D-galactosidase conjugate. p-D-galactosidaseactivity bound to the polystyrene balls was assayed by fluorime-
Key words:
try. The maximal volume of serum that could be used without interferencewas 0.2 kl, and the minimal detectableserum concentrations of a-fetoprotein in the second and third peaks were 3.5 mgiliter and 0.1 mgiliter, respectively. The maximal serum concentration of afetoprotein in the first peak that did not affect the detection limits of a-fetoprotein in the second and third peaks was 10 mg/liter. It was possible to confirm the presence of a-fetoprotein in the second and third peaks by a significant difference between bound p-0galactosidase activities in the absence and presence of a-methyl-D-mannoside or Dglucose. This assay may be useful for diagnosis of hepatocellular carcinoma, although further improvements remain to be made.
Lens culinaris agglutinin, anti-a-fetoproteinantibody, p-D-galactosidase, hepatoma, tumor marker
INTRODUCTION Serum from patients with hepatocellular carcinoma has been shown to contain carbohydrate moieties bound to a-fetoprotein molecules, which are reactive with Lens culinaris agglutinin and distinct from those produced by normal hepatocytes (1,2). By these studies, the detection of a-fetoprotein carrying carbohydrate moieties reactive with Lens culinaris agglutinin has been suggested to be useful for diagnosis of hepatocellular carcinoma, since high levels of serum a-fetoprotein are observed in liver cirrhosis and do not always indicate the presence of hepatocellular carcinoma. However, no simple and specific method for this purpose is available except for immunoelectrophoresis with Lens culinaris agglutinin (1,2). This paper describes a simple enzyme imrnunoassay using Lens culinaris agglutinin-coated polystyrene balls and affinity-purified antia-fetoprotein Fab'-P-D-galactosidase conjugate, which can specifically measure a-fetoprotein produced by hepatocellular carcinoma.
albumin (fraction V, Armour Pharmaceutical Co., Kankakee, IL), 1.0 mmol/liter CaCI,, 1.0 mmol/liter MgC12, 1 . 0 mmol/liter MnCL2, and 1.O g/liter NaN3 (buffer A). Antibodies Goat anti-a-fetoprotein serum was obtained from Medical and Biological Laboratories Co., Ltd. (Nagoya, Japan). IgG was prepared from serum by fractionation with Na2S04 followed by passage through a column of DEAE-cellulose (3). F(ab')* was prepared by digestion of IgG with pepsin (3), and Fab' was prepared by reduction of F(ab'), (3). The amounts of IgG and its fragments were calculated from the absorbance at 280 nm (3).
Fractionation of Serum a-Fetoprotein Serum (0.1- 1 .O ml) was applied to a column (1 .O x 20 cm) of Lens culinaris agglutinin-Sepharose 4B (Pharmacia Fine Chemicals AB, Uppsala, Sweden) using 0.1 mol/liter
MATERIALS AND METHODS
Buffer The buffer regularly used was 10 mmol/liter Tiis HCI buffer, pH 7.0, containing 0.1 mol/liter NaCl, 1.O g/liter bovine serum 0 1990 Wiley-Liss, Inc.
Received June 2, 1989; accepted June 16, 1989. Address reprint requests to E. Ishikawa, Department of Biochemistry, Medical College of Miyazaki, Kiyotake, Miyazaki 889- 16, Japan.
Enzyme lmmunoassay for a-Fetoprotein
sodium acetate buffer, pH 7.0, containing 0.5 mol/liter NaCl, 1.0 mmol/liter CaCI2, 1.0 mmol/liter MgCI2, and 1.0 mmol/liter MnC12 (1,2). a-Fetoprotein was eluted with the same buffer and subsequently with the same buffer containing 1 .O mol/liter D-glucose (1,2). The flow rate was 15 ml/hr, and the fraction volume was 1.5 ml. The whole process was performed at 23-25°C. The amount of a-fetoprotein in each fraction was measured by sandwich enzyme immunoassay using anti-a-fetoprotein IgG-coated polystyrene balls and affinitypurified anti-a-fetoprotein Fab'-P-D-galactosidase conjugate (4). a-Fetoprotein from human placenta (Cosmo Bio Co., Ltd., Tokyo, Japan) was used as standard. The amount of afetoprotein was calculated from the absorbance at 280 nm by taking the extinction coefficient and the molecular weight to be 0.53 g - ' liter . cm-' and 70,000, respectively (5,6). a-Fetoprotein in the third peak was dialyzed against 10 mmol/liter Tris HCl buffer, pH 7.0, containing 0.1 mollliter NaCl, 1 .O mmol/liter CaCI2, I .O mmol/liter MgC12, 1 .O mmol/liter MnC12, and 1 .O g/liter NaN3, with three changes of the buffer at 4"C, for 8 hr and was used for further experiments.
-
Lens culinaris Agglutinin-Coated Polystyrene Balls Polystyrene balls (3.2 mm in diameter, Precision Plastic Ball Co., Chicago, IL) were coated by physical adsorption with Lens culinaris agglutinin (0.1 g/liter, Vector Laboratories Inc., Burlingame, CA) in 0.1 mol/liter Tris HCl buffer, pH 7.5, containing 1.O mmol/liter CaC12, 1.O mmoliliter MgC12, 1 .O mmol/liter MnCI2, and 1 .O giliter NaN3 (7). The amount of Lens culinuris agglutinin was calculated from the absorbance at 280 nm by taking the extinction coefficient and the molecular weight to be 1.26 g - liter cm- and 49,000, respectively (8). Lens culinaris agglutinin-coated polystyrene balls were stored in buffer A at 4°C.
'
1
'
a-Fetoprotein-Sepharose 4 6 a-Fetoprotein from human placenta (0.1 mg; Cosmo Bio Co., Ltd.) was coupled to CNBr-activated Sepharose 4B (0.1 g, Pharmacia Fine Chemicals AB) according to the instructions of Pharmacia. a-Fetoprotein-Sepharose 4B was stored in 10 mmol/liter sodium phosphate buffer, pH 7.0, containing 0.1 mol/liter NaCI, 1.O g/liter bovine serum albumin, and 1 .O g/liter NaN3 at 4°C.
Affinity-Purified Anti-a-Fetoprotein Fab'-P-DGalactosidase Conjugate Anti-a-fetoprotein F(ab')* was affinity purified by elution from a column of a-fetoprotein-Sepharose 4B at pH 2.5 (4). Affinity-purified anti-a-fetoprotein Fab' was conjugated to P-D-galactosidase (EC.3.2.1.23) from Escherichia coli (lyophilized for enzyme immunoassay, Boehringer Mannheim GmbH, Mannheim, FRG) using N,N'-o-phenylene dimalei-
75
mide (3). The average number of Fab' molecules conjugated per P-D-galactosidase molecule was I .7. Affnity-purified antia-fetoprotein Fab'-P-D-galactosidase conjugate (60 pmol) in buffer A (1 .O ml) was passed through a column (1 .O x 7.0 cm) of Lens culinaris agglutinin-Sepharose 4B (Pharmacia) using buffer A at a flow rate of 1 .O ml/hr. The amount of the conjugate was calculated from the enzyme activity by taking the specific activity to be 52 U/nmol (9).
Enzyme lmmunoassay for a-Fetoprotein a-Fetoprotein, serum, and other additions were diluted with buffer A and mixed to a total volume of 100 ~ 1 The . diluted mixture (100 ~ 1 was ) mixed with 50 ~1 of 10 mmol/liter Tris HCl buffer, pH 7.0, containing 1.O mol/liter NaCl, 1.O g/liter bovine serum albumin, 1.O mmol/liter CaC12, 1.O mmol/liter MgCI2, 1 .O mmol/liter MnC12, and 1 .O g/liter NaN3 and was incubated with two Lens culinaris agglutinin-coated polystyrene balls at 20°C for 3 hr and at 4°C overnight. The polystyrene balls were washed twice by addition and aspiration of 2 ml of buffer A and incubated with 10 fmol of affinity-purified anti-a-fetoprotein Fab'-P-D-galactosidase conjugate in 150 pl of buffer A at 20°C for 3 hr. Finally, the polystyrene balls were washed as described above, and P-D-galactosidase activity bound to the polystyrene balls was assayed by fluorimetry at 30°C for 30 min using 4-methylumbelliferyl-~-D-galactoside as substrate (4). The fluorescence intensity was measured relative to lo-' mol/liter 4-methylumbelliferone (4).
Expression of the Detection Limit of a-Fetoprotein The detection limit of a-fetoprotein by enzyme immunoassay was taken as the minimal amount of a-fetoprotein that gave a bound P-D-galactosidase activity significantly in excess of that nonspecifically bound in the absence of a-fetoprotein (background). The existence of a significant difference from the background was confirmed by t test ( P < 0.001, n = 5 ) .
Patient Specimens Serum was collected from healthy subjects, liver cirrhosis patients, and 19 patients with hepatocellular carcinoma. The liver disease patients were diagnosed by clinical and histologic criteria.
RESULTS AND DISCUSSION Fractionation of Serum a-Fetoprotein a-Fetoprotein in sera from healthy subjects and patients with liver cirrhosis and hepatocellular carcinoma was fractionated into three peaks by affinity chromatography on a column of Lens culinaris agglutinin-Sepharose 4B (Fig. 1). The recovery of a-fetoprotein was 85-94%. The first peak contained a-fetoprotein that passed through the column without adsorption. The second broad peak contained a-fetoprotein that was
Kohno et al.
76
I
1 s t peak A
B
L
n
9
0
*0° 6
t InI 4
C 2nd peak
F r a c t i o n Number
Fig. 1. Fractionation of a-fetoprotein in sera from a healthy subject (A) and patients with liver cirrhosis (B) and hepatocellular carcinoma (C). Sera were collected from a healthy male subject aged 35 years, a male patient aged 41 years with liver cirrhosis, and a male patient aged 64 years with hepatocellular carcinoma and were subjected to affinity chromatography using a column ( I .O X 20 cm) of Lens culinaris agglutinin-Sepharose 4B. The volumes of sera used and the concentrations of a-fetoprotein in sera used were 1 ml, 16 pgiliter (healthy subject), 0.2 rnl, 1.4 mgiliter (liver cirrhosis patient), and 0. I ml, 200 mgiliter (hepatocellular carcinoma patient). The fraction volume was 1.5 ml.
only loosely bound to the column and eluted without sugar. The third peak contained a-fetoprotein that was bound to the column and eluted with 1.O mol/liter D-glucose. The first peak was found in both healthy subjects and patients. The second and third peaks were found in patients with hepatocellular carcinoma but not in healthy subjects or patients with liver cirrhosis. The percentages of a-fetoprotein in the first, second, and third peaks in the elution profiles obtained with sera from 20 patients with hepatocellular carcinoma were 20-24%, 71-74%, and3.1-3.7%, respectively. From theseresults, the detection of a-fetoprotein in the second and third peaks may aid diagnosis of hepatocellular carcinoma.
Detection Limit of a-Fetoprotein in Each Peak Serum was collected from 19 patients with hepatocellular carcinoma, and a-fetoprotein in the pooled serum (0.85 g/liter,
1.2 x mol/liter) was fractionated into three peaks as described above. a-Fetoprotein in the middle fractions of each peak was subjected to enzyme immunoassay (Fig. 2). Lens culinaris agglutinin-coated polystyrene balls were incubated with a-fetoprotein in each peak and, after washing, with affinitypurified anti-a-fetoprotein Fab’-P-D-galactosidase conjugate. The detection limits of a-fetoprotein in the first, second, and third peaks were 100 fmol(7 ng)/tube, 10 fmol(0.7 ng)/tube, and 0.3 fmol (0.02 ng)/tube, respectively, and 30 fmol (2 ng) of a-fetoprotein in the first peak gave no significant increase in bound P-D-galactosidase activity. These results indicated that a-fetoprotein in the second and third peaks could be detected specifically when the amount of a-fetoprotein in the first peak was 30 fmol(2 ng)/tube or less. In these experiments, there was no serum interference when the amounts of a-fetoprotein tested in the first, second, and third peaks were less than 570 fmol, 1,750 fmol. and 75 fmol, respectively. These values were the amounts of a-fetoprotein in the first, second, and third peaks in 0.2 pl of the pooled serum (the maximal serum volume that could be used without serum interference as described below), which were calculated from the concentration of a-fetoprotein in the pooled serum (0.85 g/liter, 1.2 X l o p 5 mol/liter) and the percentage of a-fetoprotein in each peak in the elution profile obtained by affinity chromatography as shown in Figure 1.
Serum Interference and Minimal Detectable Serum Concentration of a-Fetoprotein in the Second and Third Peaks To find the minimal serum concentrations of a-fetoprotein in the second and third peaks that could be detected specifically, Lens culinaris agglutinin-coated polystyrene balls were incubated with a-fetoprotein in each peak in the presence of increasing volumes of sera from four healthy subjects. When increasing volumes of sera were added, P-D-galactosidase activity specifically bound to the polystyrene balls in the presence of a-fetoprotein in the second and third peaks were lowered. Sera might have contained substance(s) reactive with Lens culinaris agglutinin-coated polystyrene balls, and, with increasing volumes of sera added, the binding of a-fetoprotein in the second and third peaks to the polystyrene balls might have been decreased. The maximal volume of serum that could be used without affecting the detection limits of a-fetoprotein in the second and third peaks was 0.2 pl. As a result, the minimal detectable serum concentrations of a-fetoprotein in the second and third peaks were 3.5 mg/liter and 0.10 mg/liter, respectively. In these experiments, there was no interference from the presence of a-fetoprotein in the sera used, which was indicated by the following calculation. The concentration of a-fetoprotein in the sera used was 30-230 pmol (2.1-16 p,g)/liter, and the volume of the sera used was 0.1-50 pl. Therefore, the amount of a-fetoprotein added with the sera
Enzyme lmmunoassay fora-Fetoprotein was 0.003- 11 f m o h b e , which was much less than the amount of a-fetoprotein in the first peak to give a significant increase in bound P-D-galactosidase activity.
300
77
1
Maximal Serum Concentration of a-Fetoprotein in the First Peak Not To Affect the Detection Limits of a-Fetoprotein in the Second and Third Peaks When 100 fmol of a-fetoprotein in the first peak was subjected to enzyme immunoassay, P-D-galactosidase activity bound to the polystyrene balls increased significantly, and no significant increase was observed with 30 fmol ( 2 ng) as described above. The maximal volume of serum that could be used without affecting the detection limits of a-fetoprotein in the second and third peaks was 0 . 2 p1as described above. Therefore, the maximal serum concentration of a-fetoprotein in the first peak, which did not affect the minimal detectable serum concentration of a-fetoprotein in the second and third peaks, was 10 mgiliter. When test serum contained higher concentrations of a-fetoprotein in the first peak, the volume of test serum used had to be less than 0.2 pl, and the detection limits of serum a-fetoprotein in the second and third peaks were raised to levels higher than 3.5 mg/liter and 0.10 mg/liter, respectively.
U c a 0 m
L k 0
x
4 2
.r
v)
c
aJ
-
c c,
21, 0
Assay Variation The assay variation in enzyme immunoassay for a-fetoprotein in the third peak in the presence of 0.2 p1 of pooled serum from healthy subjects was examined at three different levels over the range 0.5-7.2 fmol/tube. The variation coefficients within assay and between assay were 7.0-7.8% (n = 15) and 5.4-1 1% (n= lo), respectively.
Confirmation Test for a-Fetoprotein in the Second and Third Peaks When Lens culinaris agglutinin-coated polystyrene balls were incubated with a-fetoprotein in the second and third peaks in the presence of 0.2 moliliter a-methyl-D-mannoside, bound P-D-galactosidase activity decreased significantly as compared with that in the absence of a-methyl-D-mannoside (Fig. 2). D-glucose (1 .O mol/liter) could be substituted for 0.2 mollliter a-methyl-D-mannoside. No significant decrease was observed with a-fetoprotein in the first peak (Fig. 2 ) . Therefore, the presence of a-fetoprotein in the second and third peaks could be confirmed by a significant difference between bound P-D-galactosidase activities in the presence and absence of a-methyl-D-mannoside or D-glucose.
Improvements To Be Made There were two major limitations in the present enzyme immunoassay for a-fetoprotein in the second and third peaks. First, the maximal volume of test serum that could be used without interference was only 0.2 pl, as described above. This
'
0.3
I
1
1
10
I
100
I
1000
a-FetoDrotein Added (fmol/tube)
Fig. 2. Standard curves of a-fetoprotein by enzyme immunoassay using Lens culinaris agglutinin-coated polystyrene balls and affinity-purified antia-fetoprotein Fab'- P-D-galactosidase conjugate. a-Fetoprotein from the middle fractions of each peak was used. Circles, triangles, and squares indicate curves for a-fetoprotein in the first. second, and third peaks, respectively. Open and closed symbols indicate curves in the absence and presence of amethyl-D-mannoside, respectively.
might have been due to the presence of substance(s) in serum reactive with Lens culinaris agglutinin, as also discussed above. Second, bound P-D-galactosidase activity increased significantly with 100 fmol or larger amounts of a-fetoprotein in the first peak, and the maximal serum concentration of a-fetoprotein in the first peak that did not affect the detection limits of a-fetoprotein in the second and third peaks was not very high (10 mg/liter). The increase in bound P-D-galactosidase activity with a-fetoprotein in the first peak might have been due to the nonspecific binding of a-fetoprotein in the first peak to Lens culinaris agglutinin-coated polystyrene balls. This was supported by the finding that P-D-galactosidase activity bound in the presence of 100 fmol or more of a-fetoprotein in the first peak was not lowered by the presence of a-methylD-mannoside (Fig. 2 ) . To overcome these difficulties, methods are being developed to remove from test serum substance(s) reactive with Lens culinaris agglutinin and a-fetoprotein in the first peak.
78
Kohno et al.
REFERENCES I . Breborowicz J , Mackiewicz A , Breborowicz D: Microheterogeneity of alpha-fetoprotein in patient serum as demonstrated by lectin affinoelectrophoresis. Scand J Immunol 14:15-20, 1981. 2. Taketa K, Izumi M, Ichikawa E: Distinct molecular species of human a-fetoprotein due to differential affinities to lectins. Ann NY Acad Sci 4 17:61-68, 1983. 3 . Ishikawa E, Imagawa M, Hashida S, Yoshitake S , Hamaguchi Y, Ueno T: Enzyme-labeling of antibodies and their fragments for enzyme immunoassay and immunohistochemical staining. J Immunoassay 4:209-327, 1983. 4. Imagawa M , Hashida S , Ohta Y, Ishikawa E: Evaluation of p-Dgalactosidase from Escherichin coli and horseradish peroxidase as labels by sandwich enzyme immunoassay technique. Ann Clin Biochem 21:310-317, 1984
5 . Nishi S: Isolation and characterization of a human fetal a-globulin from the sera of fetuses and a hepatoma patient. Cancer Res 30:2507-2513, 1970 6. Al-Awqati MA, Gordon YB, Chard T: A simple and reliable method for the purification of human alphafetoprotein (AFP) from amniotic fluid and fetal livers. Clin Chim Acta 89: 173-182, 1978 7. Ishikawa E, Kato K: Ultrasensitive enzyme immunoassay. Scand J Immunol8 [Suppl7]:43-55, 1978 8. Howard IK, Sage HJ, Stein MD, Young NM, Leon MA, Dyckes DF: Studies on a phytohemagglutinin from the lentil. 11. Multiple forms of Lens culinciris hemagglutinin. J Biol Chem 246:1590-1595, 1971 9. lnoue S , Hashida S, Kohno T, Tanaka K, lshikawa E: A micro-scale method for the conjugation of affinity-purified Fab’ to P-D-galactosidase from Escherichin coli. J Biochem 98:1387-1394, 1985
Specific Enzyme lmmunoassay for a-Fetoprotein From Hepatocellular Carcinoma Takeyuki Kohno,' Tooru Kitamura,2 Kazunori Tsuda,2 and Eiji Ishikawa' 'Department of Biochemistry and 'Second Department of Internal Medicine, Medical College of Miyazaki, Miyazaki, Japan a-Fetoprotein in sera from healthy subjects and patients with liver cirrhosis and hepatocellular carcinoma was fractionatedinto three peaks by affinity chromatography on a column of Lens culinaris agglutinin-Sepharose 48. One peak (the first peak) was found in all the serum samples, but the other two peaks (the second and third peaks) were found only in patients with hepatocellularcarcinoma. For a-fetoprotein in the second and third peaks, a specific enzyme immunoassay was developed. Lens culinaris agglutinin-coated polystyrene balls were incubated with a-fetoprotein and, after washing, with affinity-purified antia-fetoprotein Fab'-p-D-galactosidase conjugate. p-D-galactosidaseactivity bound to the polystyrene balls was assayed by fluorime-
Key words:
try. The maximal volume of serum that could be used without interferencewas 0.2 kl, and the minimal detectableserum concentrations of a-fetoprotein in the second and third peaks were 3.5 mgiliter and 0.1 mgiliter, respectively. The maximal serum concentration of afetoprotein in the first peak that did not affect the detection limits of a-fetoprotein in the second and third peaks was 10 mg/liter. It was possible to confirm the presence of a-fetoprotein in the second and third peaks by a significant difference between bound p-0galactosidase activities in the absence and presence of a-methyl-D-mannoside or Dglucose. This assay may be useful for diagnosis of hepatocellular carcinoma, although further improvements remain to be made.
Lens culinaris agglutinin, anti-a-fetoproteinantibody, p-D-galactosidase, hepatoma, tumor marker
INTRODUCTION Serum from patients with hepatocellular carcinoma has been shown to contain carbohydrate moieties bound to a-fetoprotein molecules, which are reactive with Lens culinaris agglutinin and distinct from those produced by normal hepatocytes (1,2). By these studies, the detection of a-fetoprotein carrying carbohydrate moieties reactive with Lens culinaris agglutinin has been suggested to be useful for diagnosis of hepatocellular carcinoma, since high levels of serum a-fetoprotein are observed in liver cirrhosis and do not always indicate the presence of hepatocellular carcinoma. However, no simple and specific method for this purpose is available except for immunoelectrophoresis with Lens culinaris agglutinin (1,2). This paper describes a simple enzyme imrnunoassay using Lens culinaris agglutinin-coated polystyrene balls and affinity-purified antia-fetoprotein Fab'-P-D-galactosidase conjugate, which can specifically measure a-fetoprotein produced by hepatocellular carcinoma.
albumin (fraction V, Armour Pharmaceutical Co., Kankakee, IL), 1.0 mmol/liter CaCI,, 1.0 mmol/liter MgC12, 1 . 0 mmol/liter MnCL2, and 1.O g/liter NaN3 (buffer A). Antibodies Goat anti-a-fetoprotein serum was obtained from Medical and Biological Laboratories Co., Ltd. (Nagoya, Japan). IgG was prepared from serum by fractionation with Na2S04 followed by passage through a column of DEAE-cellulose (3). F(ab')* was prepared by digestion of IgG with pepsin (3), and Fab' was prepared by reduction of F(ab'), (3). The amounts of IgG and its fragments were calculated from the absorbance at 280 nm (3).
Fractionation of Serum a-Fetoprotein Serum (0.1- 1 .O ml) was applied to a column (1 .O x 20 cm) of Lens culinaris agglutinin-Sepharose 4B (Pharmacia Fine Chemicals AB, Uppsala, Sweden) using 0.1 mol/liter
MATERIALS AND METHODS
Buffer The buffer regularly used was 10 mmol/liter Tiis HCI buffer, pH 7.0, containing 0.1 mol/liter NaCl, 1.O g/liter bovine serum 0 1990 Wiley-Liss, Inc.
Received June 2, 1989; accepted June 16, 1989. Address reprint requests to E. Ishikawa, Department of Biochemistry, Medical College of Miyazaki, Kiyotake, Miyazaki 889- 16, Japan.
Enzyme lmmunoassay for a-Fetoprotein
sodium acetate buffer, pH 7.0, containing 0.5 mol/liter NaCl, 1.0 mmol/liter CaCI2, 1.0 mmol/liter MgCI2, and 1.0 mmol/liter MnC12 (1,2). a-Fetoprotein was eluted with the same buffer and subsequently with the same buffer containing 1 .O mol/liter D-glucose (1,2). The flow rate was 15 ml/hr, and the fraction volume was 1.5 ml. The whole process was performed at 23-25°C. The amount of a-fetoprotein in each fraction was measured by sandwich enzyme immunoassay using anti-a-fetoprotein IgG-coated polystyrene balls and affinitypurified anti-a-fetoprotein Fab'-P-D-galactosidase conjugate (4). a-Fetoprotein from human placenta (Cosmo Bio Co., Ltd., Tokyo, Japan) was used as standard. The amount of afetoprotein was calculated from the absorbance at 280 nm by taking the extinction coefficient and the molecular weight to be 0.53 g - ' liter . cm-' and 70,000, respectively (5,6). a-Fetoprotein in the third peak was dialyzed against 10 mmol/liter Tris HCl buffer, pH 7.0, containing 0.1 mollliter NaCl, 1 .O mmol/liter CaCI2, I .O mmol/liter MgC12, 1 .O mmol/liter MnC12, and 1 .O g/liter NaN3, with three changes of the buffer at 4"C, for 8 hr and was used for further experiments.
-
Lens culinaris Agglutinin-Coated Polystyrene Balls Polystyrene balls (3.2 mm in diameter, Precision Plastic Ball Co., Chicago, IL) were coated by physical adsorption with Lens culinaris agglutinin (0.1 g/liter, Vector Laboratories Inc., Burlingame, CA) in 0.1 mol/liter Tris HCl buffer, pH 7.5, containing 1.O mmol/liter CaC12, 1.O mmoliliter MgC12, 1 .O mmol/liter MnCI2, and 1 .O giliter NaN3 (7). The amount of Lens culinuris agglutinin was calculated from the absorbance at 280 nm by taking the extinction coefficient and the molecular weight to be 1.26 g - liter cm- and 49,000, respectively (8). Lens culinaris agglutinin-coated polystyrene balls were stored in buffer A at 4°C.
'
1
'
a-Fetoprotein-Sepharose 4 6 a-Fetoprotein from human placenta (0.1 mg; Cosmo Bio Co., Ltd.) was coupled to CNBr-activated Sepharose 4B (0.1 g, Pharmacia Fine Chemicals AB) according to the instructions of Pharmacia. a-Fetoprotein-Sepharose 4B was stored in 10 mmol/liter sodium phosphate buffer, pH 7.0, containing 0.1 mol/liter NaCI, 1.O g/liter bovine serum albumin, and 1 .O g/liter NaN3 at 4°C.
Affinity-Purified Anti-a-Fetoprotein Fab'-P-DGalactosidase Conjugate Anti-a-fetoprotein F(ab')* was affinity purified by elution from a column of a-fetoprotein-Sepharose 4B at pH 2.5 (4). Affinity-purified anti-a-fetoprotein Fab' was conjugated to P-D-galactosidase (EC.3.2.1.23) from Escherichia coli (lyophilized for enzyme immunoassay, Boehringer Mannheim GmbH, Mannheim, FRG) using N,N'-o-phenylene dimalei-
75
mide (3). The average number of Fab' molecules conjugated per P-D-galactosidase molecule was I .7. Affnity-purified antia-fetoprotein Fab'-P-D-galactosidase conjugate (60 pmol) in buffer A (1 .O ml) was passed through a column (1 .O x 7.0 cm) of Lens culinaris agglutinin-Sepharose 4B (Pharmacia) using buffer A at a flow rate of 1 .O ml/hr. The amount of the conjugate was calculated from the enzyme activity by taking the specific activity to be 52 U/nmol (9).
Enzyme lmmunoassay for a-Fetoprotein a-Fetoprotein, serum, and other additions were diluted with buffer A and mixed to a total volume of 100 ~ 1 The . diluted mixture (100 ~ 1 was ) mixed with 50 ~1 of 10 mmol/liter Tris HCl buffer, pH 7.0, containing 1.O mol/liter NaCl, 1.O g/liter bovine serum albumin, 1.O mmol/liter CaC12, 1.O mmol/liter MgCI2, 1 .O mmol/liter MnC12, and 1 .O g/liter NaN3 and was incubated with two Lens culinaris agglutinin-coated polystyrene balls at 20°C for 3 hr and at 4°C overnight. The polystyrene balls were washed twice by addition and aspiration of 2 ml of buffer A and incubated with 10 fmol of affinity-purified anti-a-fetoprotein Fab'-P-D-galactosidase conjugate in 150 pl of buffer A at 20°C for 3 hr. Finally, the polystyrene balls were washed as described above, and P-D-galactosidase activity bound to the polystyrene balls was assayed by fluorimetry at 30°C for 30 min using 4-methylumbelliferyl-~-D-galactoside as substrate (4). The fluorescence intensity was measured relative to lo-' mol/liter 4-methylumbelliferone (4).
Expression of the Detection Limit of a-Fetoprotein The detection limit of a-fetoprotein by enzyme immunoassay was taken as the minimal amount of a-fetoprotein that gave a bound P-D-galactosidase activity significantly in excess of that nonspecifically bound in the absence of a-fetoprotein (background). The existence of a significant difference from the background was confirmed by t test ( P < 0.001, n = 5 ) .
Patient Specimens Serum was collected from healthy subjects, liver cirrhosis patients, and 19 patients with hepatocellular carcinoma. The liver disease patients were diagnosed by clinical and histologic criteria.
RESULTS AND DISCUSSION Fractionation of Serum a-Fetoprotein a-Fetoprotein in sera from healthy subjects and patients with liver cirrhosis and hepatocellular carcinoma was fractionated into three peaks by affinity chromatography on a column of Lens culinaris agglutinin-Sepharose 4B (Fig. 1). The recovery of a-fetoprotein was 85-94%. The first peak contained a-fetoprotein that passed through the column without adsorption. The second broad peak contained a-fetoprotein that was
Kohno et al.
76
I
1 s t peak A
B
L
n
9
0
*0° 6
t InI 4
C 2nd peak
F r a c t i o n Number
Fig. 1. Fractionation of a-fetoprotein in sera from a healthy subject (A) and patients with liver cirrhosis (B) and hepatocellular carcinoma (C). Sera were collected from a healthy male subject aged 35 years, a male patient aged 41 years with liver cirrhosis, and a male patient aged 64 years with hepatocellular carcinoma and were subjected to affinity chromatography using a column ( I .O X 20 cm) of Lens culinaris agglutinin-Sepharose 4B. The volumes of sera used and the concentrations of a-fetoprotein in sera used were 1 ml, 16 pgiliter (healthy subject), 0.2 rnl, 1.4 mgiliter (liver cirrhosis patient), and 0. I ml, 200 mgiliter (hepatocellular carcinoma patient). The fraction volume was 1.5 ml.
only loosely bound to the column and eluted without sugar. The third peak contained a-fetoprotein that was bound to the column and eluted with 1.O mol/liter D-glucose. The first peak was found in both healthy subjects and patients. The second and third peaks were found in patients with hepatocellular carcinoma but not in healthy subjects or patients with liver cirrhosis. The percentages of a-fetoprotein in the first, second, and third peaks in the elution profiles obtained with sera from 20 patients with hepatocellular carcinoma were 20-24%, 71-74%, and3.1-3.7%, respectively. From theseresults, the detection of a-fetoprotein in the second and third peaks may aid diagnosis of hepatocellular carcinoma.
Detection Limit of a-Fetoprotein in Each Peak Serum was collected from 19 patients with hepatocellular carcinoma, and a-fetoprotein in the pooled serum (0.85 g/liter,
1.2 x mol/liter) was fractionated into three peaks as described above. a-Fetoprotein in the middle fractions of each peak was subjected to enzyme immunoassay (Fig. 2). Lens culinaris agglutinin-coated polystyrene balls were incubated with a-fetoprotein in each peak and, after washing, with affinitypurified anti-a-fetoprotein Fab’-P-D-galactosidase conjugate. The detection limits of a-fetoprotein in the first, second, and third peaks were 100 fmol(7 ng)/tube, 10 fmol(0.7 ng)/tube, and 0.3 fmol (0.02 ng)/tube, respectively, and 30 fmol (2 ng) of a-fetoprotein in the first peak gave no significant increase in bound P-D-galactosidase activity. These results indicated that a-fetoprotein in the second and third peaks could be detected specifically when the amount of a-fetoprotein in the first peak was 30 fmol(2 ng)/tube or less. In these experiments, there was no serum interference when the amounts of a-fetoprotein tested in the first, second, and third peaks were less than 570 fmol, 1,750 fmol. and 75 fmol, respectively. These values were the amounts of a-fetoprotein in the first, second, and third peaks in 0.2 pl of the pooled serum (the maximal serum volume that could be used without serum interference as described below), which were calculated from the concentration of a-fetoprotein in the pooled serum (0.85 g/liter, 1.2 X l o p 5 mol/liter) and the percentage of a-fetoprotein in each peak in the elution profile obtained by affinity chromatography as shown in Figure 1.
Serum Interference and Minimal Detectable Serum Concentration of a-Fetoprotein in the Second and Third Peaks To find the minimal serum concentrations of a-fetoprotein in the second and third peaks that could be detected specifically, Lens culinaris agglutinin-coated polystyrene balls were incubated with a-fetoprotein in each peak in the presence of increasing volumes of sera from four healthy subjects. When increasing volumes of sera were added, P-D-galactosidase activity specifically bound to the polystyrene balls in the presence of a-fetoprotein in the second and third peaks were lowered. Sera might have contained substance(s) reactive with Lens culinaris agglutinin-coated polystyrene balls, and, with increasing volumes of sera added, the binding of a-fetoprotein in the second and third peaks to the polystyrene balls might have been decreased. The maximal volume of serum that could be used without affecting the detection limits of a-fetoprotein in the second and third peaks was 0.2 pl. As a result, the minimal detectable serum concentrations of a-fetoprotein in the second and third peaks were 3.5 mg/liter and 0.10 mg/liter, respectively. In these experiments, there was no interference from the presence of a-fetoprotein in the sera used, which was indicated by the following calculation. The concentration of a-fetoprotein in the sera used was 30-230 pmol (2.1-16 p,g)/liter, and the volume of the sera used was 0.1-50 pl. Therefore, the amount of a-fetoprotein added with the sera
Enzyme lmmunoassay fora-Fetoprotein was 0.003- 11 f m o h b e , which was much less than the amount of a-fetoprotein in the first peak to give a significant increase in bound P-D-galactosidase activity.
300
77
1
Maximal Serum Concentration of a-Fetoprotein in the First Peak Not To Affect the Detection Limits of a-Fetoprotein in the Second and Third Peaks When 100 fmol of a-fetoprotein in the first peak was subjected to enzyme immunoassay, P-D-galactosidase activity bound to the polystyrene balls increased significantly, and no significant increase was observed with 30 fmol ( 2 ng) as described above. The maximal volume of serum that could be used without affecting the detection limits of a-fetoprotein in the second and third peaks was 0 . 2 p1as described above. Therefore, the maximal serum concentration of a-fetoprotein in the first peak, which did not affect the minimal detectable serum concentration of a-fetoprotein in the second and third peaks, was 10 mgiliter. When test serum contained higher concentrations of a-fetoprotein in the first peak, the volume of test serum used had to be less than 0.2 pl, and the detection limits of serum a-fetoprotein in the second and third peaks were raised to levels higher than 3.5 mg/liter and 0.10 mg/liter, respectively.
U c a 0 m
L k 0
x
4 2
.r
v)
c
aJ
-
c c,
21, 0
Assay Variation The assay variation in enzyme immunoassay for a-fetoprotein in the third peak in the presence of 0.2 p1 of pooled serum from healthy subjects was examined at three different levels over the range 0.5-7.2 fmol/tube. The variation coefficients within assay and between assay were 7.0-7.8% (n = 15) and 5.4-1 1% (n= lo), respectively.
Confirmation Test for a-Fetoprotein in the Second and Third Peaks When Lens culinaris agglutinin-coated polystyrene balls were incubated with a-fetoprotein in the second and third peaks in the presence of 0.2 moliliter a-methyl-D-mannoside, bound P-D-galactosidase activity decreased significantly as compared with that in the absence of a-methyl-D-mannoside (Fig. 2). D-glucose (1 .O mol/liter) could be substituted for 0.2 mollliter a-methyl-D-mannoside. No significant decrease was observed with a-fetoprotein in the first peak (Fig. 2 ) . Therefore, the presence of a-fetoprotein in the second and third peaks could be confirmed by a significant difference between bound P-D-galactosidase activities in the presence and absence of a-methyl-D-mannoside or D-glucose.
Improvements To Be Made There were two major limitations in the present enzyme immunoassay for a-fetoprotein in the second and third peaks. First, the maximal volume of test serum that could be used without interference was only 0.2 pl, as described above. This
'
0.3
I
1
1
10
I
100
I
1000
a-FetoDrotein Added (fmol/tube)
Fig. 2. Standard curves of a-fetoprotein by enzyme immunoassay using Lens culinaris agglutinin-coated polystyrene balls and affinity-purified antia-fetoprotein Fab'- P-D-galactosidase conjugate. a-Fetoprotein from the middle fractions of each peak was used. Circles, triangles, and squares indicate curves for a-fetoprotein in the first. second, and third peaks, respectively. Open and closed symbols indicate curves in the absence and presence of amethyl-D-mannoside, respectively.
might have been due to the presence of substance(s) in serum reactive with Lens culinaris agglutinin, as also discussed above. Second, bound P-D-galactosidase activity increased significantly with 100 fmol or larger amounts of a-fetoprotein in the first peak, and the maximal serum concentration of a-fetoprotein in the first peak that did not affect the detection limits of a-fetoprotein in the second and third peaks was not very high (10 mg/liter). The increase in bound P-D-galactosidase activity with a-fetoprotein in the first peak might have been due to the nonspecific binding of a-fetoprotein in the first peak to Lens culinaris agglutinin-coated polystyrene balls. This was supported by the finding that P-D-galactosidase activity bound in the presence of 100 fmol or more of a-fetoprotein in the first peak was not lowered by the presence of a-methylD-mannoside (Fig. 2 ) . To overcome these difficulties, methods are being developed to remove from test serum substance(s) reactive with Lens culinaris agglutinin and a-fetoprotein in the first peak.
78
Kohno et al.
REFERENCES I . Breborowicz J , Mackiewicz A , Breborowicz D: Microheterogeneity of alpha-fetoprotein in patient serum as demonstrated by lectin affinoelectrophoresis. Scand J Immunol 14:15-20, 1981. 2. Taketa K, Izumi M, Ichikawa E: Distinct molecular species of human a-fetoprotein due to differential affinities to lectins. Ann NY Acad Sci 4 17:61-68, 1983. 3 . Ishikawa E, Imagawa M, Hashida S, Yoshitake S , Hamaguchi Y, Ueno T: Enzyme-labeling of antibodies and their fragments for enzyme immunoassay and immunohistochemical staining. J Immunoassay 4:209-327, 1983. 4. Imagawa M , Hashida S , Ohta Y, Ishikawa E: Evaluation of p-Dgalactosidase from Escherichin coli and horseradish peroxidase as labels by sandwich enzyme immunoassay technique. Ann Clin Biochem 21:310-317, 1984
5 . Nishi S: Isolation and characterization of a human fetal a-globulin from the sera of fetuses and a hepatoma patient. Cancer Res 30:2507-2513, 1970 6. Al-Awqati MA, Gordon YB, Chard T: A simple and reliable method for the purification of human alphafetoprotein (AFP) from amniotic fluid and fetal livers. Clin Chim Acta 89: 173-182, 1978 7. Ishikawa E, Kato K: Ultrasensitive enzyme immunoassay. Scand J Immunol8 [Suppl7]:43-55, 1978 8. Howard IK, Sage HJ, Stein MD, Young NM, Leon MA, Dyckes DF: Studies on a phytohemagglutinin from the lentil. 11. Multiple forms of Lens culinciris hemagglutinin. J Biol Chem 246:1590-1595, 1971 9. lnoue S , Hashida S, Kohno T, Tanaka K, lshikawa E: A micro-scale method for the conjugation of affinity-purified Fab’ to P-D-galactosidase from Escherichin coli. J Biochem 98:1387-1394, 1985
