EXPERIMENTAL

AND

MOLECULAR

PATHOLOGY

53, 112-125 (19%)

Biochemical Characterization and Serological immunoassay of a Pancreatic Carcinoma-Associated Antigen Defined by Monoclonal Antibody LD-Bi’ FAWAZ Department

Received

HALWANI*

of Pathology, Montreal, March

McGill Quebec,

AND SERGE JOTHY University, Canada

2, 1990, and in revised

3775 University H3A 284 form

Street,

June 13, 1990

Several glycosylated macromolecules associated with normal and malignant pancreatic ductal cells have been described. We have generated a monoclonal antibody, LD-Bl, by immunizing Balb/c mice with the postmicrosomal extract of fresh human pancreatic ductal carcinoma tissue and used it in this study to characterize the nature of the target antigen. The antigen detected by LD-Bl antibody was purified to homogeneity by affinity chromatography. Enzymatic and biochemical analysis showed it to be a nonsialylated glycoprotein that on Western blotting and immunoprecipitation analyses had an apparent molecular weight of 300-400 kDa. The mobility on gels was not affected by reducing or denaturing conditions. Competitive inhibition assays with various MoAbs and lectins indicated that the epitope recognized by LD-Bl antibody involves the carbohydrate sequence Galgl + 3Galgl+ 3(or 4)GlcNAcRl -P 3Gal. Using a double determinant sandwich ELISA, elevated antigen levels were detected in the sera of 5 of 6 patients with pancreatic carcinoma, 0 of 3 patients with chronic pancreatitis, and 12 of 137 normal controls. These results suggest that patients with pancreatic carcinoma exhibit altered expression of a heavily glycosylated molecule related to a blood group precursor immunodeterminant. Q 1990Academic PESS, 1~.

INTRODUCTION The malignant cells of pancreatic carcinoma contain large amounts of mutinous substances and glycolipids, most of which are poorly defined. Some of these substances are also present in normal epithelial cells of pancreatic ducts (Kalthoff et al,, 1986). Several such substances show increased or altered glycosylation of their carbohydrate moieties. Altered glycosylation of proteins or membrane lipids has been demonstrated to be part of the oncogenic change (Santer et al., 1984; Holmes et al., 1985) and is also seen in embryonic and,fetal cellular differentiation (Gooi et al., 1981). In a previous report, we described the tissue distribution of an antigen associated with human pancreatic carcinoma defined by the monoclonal antibody, LDBl (Halwani et al., 1990). It was observed that LD-Bl strongly binds to an antigen present in the cytoplasm of pancreatic carcinoma cells. Furthermore, the study indicated that the antigen was also shed into the extracellular microenvironment of tumor cells. This led us to postulate that this antigen has access to the circulation, and therefore might be useful in a diagnostic serum immunoassay. It was however essential to first purify the antigen and characterize its biochemical nature. In this report, we identify the LD-Bl antigen as a large glycoprotein molecule containing an extended sequence of the carbohydrate epitope Gall31 -+ ’ This work was supported by the Medical Research Council of Canada. F.H. is the recipient of a James Douglas Award, Royal Victoria Hospital Research Fellowship, and Kuwait MNH Fellowship. ’ To whom requests for reprints should be addressed. 112 0014-4800/90 $3.00 Copy@ht 0 1990 by Academic Press. Inc. Au rights of reproduction in any form reserved.

LD-Bl

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ANTIBODY

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3GaQ31+ 3 (or 4) GlcNAcPl + 3Gal. In addition, we have used a double determinant sandwich ELISA3 to demonstrate increased levels of this antigen in the sera of patients with pancreatic carcinoma. MATERIALS AND METHODS Antigen Purification

LD-Bl MoAb coupled to Sepharose 4B was used to purify the target antigen from the postmicrosomal pancreatic carcinoma extract as previously described (Cuatrecasas et al., 1%8). Briefly, after washing the column with 10vol of TS, the postmicrosomal fraction of the tissue extract was applied onto the column and incubated at 4°C for 2 hr. The column was then washed with 10vol of TS, followed by 10 vol of 1 M NaCl in TS to remove nonspecific binding material. The bound antigen was then eluted with 0.05 M diethylamine, pH 11.5, into aliquots of 500 p.1 containing 100 ~1 of 1 M TS. The eluted peak was detected by measuring absorbance at 280 nm. The fractions containing the eluted peak were pooled, dialyzed immediately in PBS, then concentrated sevenfold by ultrafiltration with lO-kDa cutoff membrane (Amicon Corp., Danvers, MA). The protein concentration of the eluted fraction was determined by absorbance at 280 nm. Aliquots of the purified fraction, the void flow-through volume, and the washing buffers were assayed for LD-Bl reactivity by ELISA as described previously (Halwani et al., 1990). Purification was expressed as OD units per microgram of total protein adsorbed to the ELISA microtiter wells. Aliquots of the antigen before and after the affinity chromatography procedure were compared for purity and LD-Bl reactivity by SDS-PAGE analysis and Western blot using OD units equal to those determined by ELISA (see above). SDS-PAGE

and Western Blotting

Electrophoresis was performed in duplicate in acrylamide gradient gels at a constant current of 2.5 mA/cm under reducing (Zmercaptoethanol) and nonreducing conditions (Laemmli et al., 1970). One gel was stained with Bio-Rad silver stain (Merril et al., 1981). The duplicate gel was blotted onto nitrocellulose paper (70 V, 18 hr, 4°C) in 25 mM Tris, 192mA4 Glycine, 20% methanol buffer, pH 8.3 (Towbin et al., 1979). For immunostaining, the nitrocellulose paper was then incubated in TS containing 5% BSA and 0.02% sodium azide (blocking solution) to saturate additional protein binding sites (30 min at 37”C), followed by affinitypurified LD-Bl MoAb ascites (or mouse myeloma ascites as negative control) diluted to 2 @rl in blocking solution (1 hr at 37°C). Excess antibody was washed with TS once, then TS plus 0.05% Nonidet P-40 (Sigma, St. Louis, MO) twice, then TS once. The blot was then incubated with excess peroxidase-conjugated F(Ab)‘, sheep anti-mouse IgG (Amersham, Arlington Heights, IL) diluted 500fold in blocking solution (1 hr at 37°C). The nitrocellulose paper was washed again as above and developed in diaminobenzidine (50 mg% in TS, 0.015% H,O&. 3 Abbreviations used: BSA, bovine serum albumin; CEA, carcinoembryonic antigen; EDC, N-ethyl-N’-(3-dimethylaminopropyl)~carbodiimide; ELISA, enzyme-linked immunosorbent assay; Fuc, fucose; Gal, galactose; GalNAc, N-acetyl galactose; Glc, glucose; GlcNAc, N-acetyl glucose; Mes, 2-(N-morpholino)ethane sulfonic acid; MoAb, monoclonal antibody; PBS, 0.01 M sodium phosphate, 0.15 M NaCl, pH 7.4; POA, pancreatic oncofetal antigen; SDS, sodium dodecyl sulfate; TS, 0.01 M Tris-HCl, 0.15 M NaCl, pH 7.4.

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Zmmunoprecipitation

Cells (2 x 106)of the CAPAN- pancreatic carcinoma cell line (American Type Culture Collection, Rockville, MD) were cultured for 4 hr in methionine-free RPM1 medium (GIBCO, Grand Island, NY) containing 10% dialyzed FBS, followed by the addition of [35S]methionine (1.25 mCi, Amersham) in 10 ml of methionine-free medium, and a further incubation for 16 hr at 37°C. Cells were collected by trypsinization and washed in complete medium. Radiolabeled cells were solubjlized with ice-cold buffer containing 10 m&f Tris-HCl, 140mM NaCl, 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS, 1 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride, pH 7.5 (Linsley et al., 1988). The lysate was centrifuged at 8000 g for 15 min. Aliquots of the supematant (100 ~1) were incubated with 5 pg of LD-Bl MoAb (1 hr at 4”C), followed by 10 ug of rabbit anti-mouse IgG (Miles Inc., Kankakee, IL) for 30 min at 4°C. Immune complexes were then precipitated with 100~1of a 10% suspension of Staphylococcus aureus coupled to Sepharose 4B (Pharmacia, Uppsala, Sweden) for 1 hr at 4°C with constant agitation. The precipitates were washed in TS once, 1 M NaCl in TS once, then TS once again. Radiolabeled precipitates were then separated by SDS-PAGE as above and visualized by autoradiography. Enzymatic

and Biochemical

Characterization

Affinity purified antigen was incubated with a range of concentrations of SDS (Bio-Rad) with 2-mercaptoethanol (Sigma), pepsin (Sigma), trypsin (Sigma), proteinase K (Beohringer-Mannheim, W. Germany), neuraminidase from Arthrobacter ureafaciens (Calbiochem, San Diego, CA), lysozyme (Sigma), collagenase (Sigma), elastase (Sigma), RNase (Sigma), or PBS alone as a negative control. In each instance, digestion was carried overnight at 37°C in PBS buffer except pepsin (acetate buffer, pH 4). Aliquots of the digested antigen were then tested for residual activity by ELISA and dot-immunoblots (Herbink et al., 1982). The antigen was also exposed to mild periodate oxidation as previously described (Woodward et al., 1985). Lipid extraction from fresh human pancreatic carcinoma tissues and the CAPAN- cell line was also performed, followed by thin-layer chromatography as previously described (Brockhaus et al., 1981). Sections from frozen pancreatic carcinoma tissues were also exposed to lipid extraction by immersion in chlorofotmmethanol (2:l) or methanol alone for 5 mitt, washed in PBS, then reacted with LD-Bl MoAb using immunoperoxidase histochemistry as previously described (Halwani et al., 1990). Zmmunochemical

Characterization

LD-Bl MoAb was tested for reactivity with highly purified CEA (Dr A. Fuks, McGill Cancer Center) and pancreatic oncofetal antigen (Gelder et al., 1978) (Calbiochem) by competitive ELISA. Each antigen was coated to a microtiter plate, and incubated with a range of concentrations of LD-Bl MoAb (0.1-10 p,g) overnight at 4°C in PBS. Affinity-purified LD-Bl antigen and BSA were used as controls. Aliquots from each microtiter well were then tested by ELISA for residual LD-Bl antibody activity with its affinity-purified antigen coated on a different microtiter plate. In a similar assay, LD-Bl MoAb was tested for reactivity with erythrocytes from individuals of 0, A, B, and AB blood types. Serial dilutions of LD-Bl MoAb

LD-Bl

PANCREATIC

CANCER-ASSOCIATED

ANTIBODY

115

were added to 1 x lo4 of the washed erythrocytes. After overnight incubation at 4°C the cells were pelleted by centrifugation at 1000g for 15min, and aliquots of the supernatant were tested for residual reactivity with at&my-purified antigen by ELISA. Specific carbohydrate sequences were detected on the antigen defined by LDBl antibody by its reactivity with other MoAbs and lectins using competitive inhibition of biotinylated LD-Bl MoAb. MoAb B47 (Biomira Inc., Edmonton, Canada) binds to sialyl Lea antigen (Skoczenski et al., 1988). MoAb B72.3 (Dr. J. Schlom, NCI, Bethesda, MD), 47DlO (DuPont, North Billerica, MA), B27.1, and B43.13 (Biomira Inc.) bind to as yet incompletely characterized glycoproteins (TAG-72 (Thor et al., 1986), 47DlO (Ho et al., 1987), and CA 125 (Krantz et al., 1988), respectively). MoAb AH6 (Dr. S. Hakomori, University of Washington, Seattle, WA) binds to Fucol14 2GalB1--* 4[Fucal --f 3lGlcNAc (simple LeY), and KHI (Dr. S. Hakomori) binds to extended LeY antigen (Kim et al., 1988). Lectins (Sigma) with known carbohydrate specificities were also tested for their ability to block LD-Bl MoAb reactivity with affinity-purified LD-Bl antigen. Arachis hypogaea (PNA) binds to terminal Gall31 + 3GalNAc, Bandeiraea simplicifoliu I (BS-I) binds to terminal a-D-Gal or cx-D-GalNac, Bandeiruea simplicifoliu II (BS-II) binds to D-GlcNAc, Dolichos biflorus (DBA) binds to terminal ol-D-GalNAc, Euonymus europaeus (EEA) binds to Gall31 --, 3GalBl + (3 or 4)GlcNAc, Glycine max (SBA) binds to D-GalNAc, Phytolacca americana (PWM) binds to P-D-G~cNAc, Ulex europaeus (UEA) binds to a-L-Fuc, and Viciu villosa isotype B4 (VVA) binds to D-GalNAc. Five micrograms of affinity-purified LD-B 1 antigen in PBS were added to each well of a microtiter plate and incubated overnight at 4°C. The plates were washed and incubated with PBS containing 1% BSA for 1 hr at 37°C. This was followed by incubation of serial dilutions of the test antibodies or lectins in TSBSA for 2 hr at 37°C. After washing the unbound material in PBS, 5 pg of biotinylated LD-Bl MoAb in PBS:BSA was added to each well and incubated for 1 hr at 37°C. Binding of the biotinylated LD-Bl MoAb was detected using a streptavidinbiotin-peroxidase complex (Amersham) diluted lOOO-foldin PBS:BSA for 1 hr at 37°C. Unlabeled LD-Bl MoAb and mouse myeloma ascites were used as positive and negative controls, respectively. Another negative control was carried out by testing the ability of these lectins to block the reactivity of the biotinylated B18 anti-CEA MoAb (reacts with the polypeptide moiety of CEA) under the same conditions. Serological

Assay

The amount of LD-B 1 antigen present in the sera of various patient populations was quantified using a double antibody sandwich ELISA (Butler et al., 1987)with some modification. The test was performed on the assumption that LD-Bl antibody reacts with a multideterminant epitope. Each serum sample was tested in triplicate. MoAb LD-Bl diluted to 5 p&ml in 0.1% EDC in 25 mM Mes buffer (Rotmans and Scheven, 1984; Calbiochem), pH 6.0, was added to the wells of polyvinyl microtiter plates and incubated overnight at 22°C covered with Parafilm to avoid drying. After washing with PBS, the microtiter wells were treated with 250 p,l of 5% BSA in PBS for 1 hr at 37°C to minimize nonspecific protein absorption. This was followed by incubating 200 ~1of the serum samples to be tested for 3 hr at 37°C. In each assay, normal human serum was used as negative control,

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and affinity-purified LD-B 1antigen diluted to known concentrations in the control serum was used as positive control. The material bound to the capture antibody was detected with biotinylated LD-Bl MoAb diluted to 3.5 p&ml in PBS and incubated at 37°C for 1 hr. Binding of the biotinylated antibody was detected with the streptavidin-biotin-peroxidase complex (Amersham) diluted lOOO-fold in PBS:BSA for 1 hr at 37°C. The reaction was developed with o-phenylenediamine (0.2 g% in 0.017 M sodium citrate, 0.065 M sodium phosphate buffer, pH 6.3, 0.015% HzOz).After 15 min, the reaction was blocked with 4 N sulfuric acid, and the absorbance measured at a wavelength of 492 nm in a single beam microplate reader (Titertek, Finland). The concentrations of LD-Bl antigen in the unknown samples were calculated using the linear portion of the absorbance curve generated with the positive serum standard. When absorbance was at the upper limit of the ELISA reading, the assay was repeated using serial dilutions of the test serum in PBS. Immunoglobulin

Subclass Determination

Immunoglobulin subclass was determined by ELISA by means of isotypespecific rabbit anti-mouse antibodies (Zymed, San Francisco, CA). RESULTS Antigen Purification

The LD-Bl antibody, which has an IgGZb isotype, was used to purify the corresponding antigen by affinity chromatography from the postmicrosomal extract of pancreatic carcinoma. Both the flowthrough and the washing buffers showed no residual reactivity with LD-Bl antibody by ELISA. Essentially all LD-Bl reactivity was retained in the bound fraction of the column. Approximately 80 pg of antigen was extracted per gram of pancreatic carcinoma wet tissue. When equal amounts of ELISA reactivity units of the postmicrosomal extract and the purified LD-Bl antigen were compared by Western blotting, similar patterns of reactivity were observed before and after purification (Fig. 1, lanes C and E). Although this caused certain overloading of the postmicrosomal gel, we,found that this was the minimum amount that can be used and still be able to demonstrate the antigen in the unpurified, crude state. The antigen consisted of two closely associated bands of apparent molecular weight of 300 to 400 kDa. The gel mobility was similar under reducing and nom-educing conditions, suggesting the absence of intrachain or interchain disulfide bonds. The purification procedure did not appear to cause dispersion of the band by shearing of the antigen into lower molecular weight components, since immunoblotting of the unpurified postmicrosomal fraction showed a similar staining pattern (Fig. 1, lane C). Silver stain of SDS-PAGE did not reveal any staining bands in the purified fraction (Fig. 1, lane D), nor in the same molecular weight region of the postmicrosomal extract (lane B). Yet, the presence of antigen was confIrmed on a duplicate gel blotted to nitrocellulose paper and immunostained with LD-Bl antibody (lane C and E). This has been described in heavily glycosylated glycoproteins due to masking of the protein core by carbohydrate chains (Johnson et al., 1986) (see below under antigen characterization). Silver stain was however valuable in confirming the purity of LD-Bl antigen after affinity chromatography.

LD-Bl

PANCREATIC A

CANCER-ASSOCIATED B

ANTIBODY

117

CDEF

FIG. 1. SDS-polyacrylamide gel and immunoblot profiles of the postmicrosomal fraction and aflinity purified fraction of human pancreatic carcinoma tissues. Gels were loaded with equal amounts of ELISA reactivity units of the postmicrosomal extract and the purified LD-Blantigen then compared by silver stain and Western blotting. (B) Silver stain of the postmicrosomal fraction, (C) immunoblot of the postmicrosomal fraction stained with LD-Bl MoAb, (D) silver stain of affinity-purified fraction, (E) immunoblot of aflinity-purified fraction stained with LD-Bl antibody, (F) same as E stained with control ascites, (A) molecular weight standards (Sigma: B-galactosidase, M, 116 kDa, phosphorylase B, M, 97.4 kDa, albumin, M, 66 kDa, ovalbumin, M, 45 kDa, and carbonic anhydrase, M, 29 KDa).

Antigen

Characterization

To characterize the epitope recognized by LD-Bl MoAb, the affinity-purified antigen was subjected to the action of various enzymatic treatments. Among the proteolytic enzymes, only trypsin caused some reduction in antigenicity (58% of control) after overnight incubation at a high enzyme concentration of 4 mg/ml. Lowering the trypsin concentration to 1 mg/ml or less did not result in significant antigenic loss (Table I). Enzymatic digestion with pepsin, lysozyme, proteinase K, collagenase, elastase, and RNase did not alter the antigenic immunoreactivity as measured by ELISA. To determine whether the complete LD-Bl antigen was cleaved by trypsin digestion into smaller peptide chains that retained immunoreactivity but were unable to adsorb to polyvinyl microtiter plates, enzymatic digestion was carried on the purified antigen after adsorption to nitrocellulose paper, and immunoreactivity was tested using a dot-immunoblot assay. The results obtained under these conditions were identical to the ELISA method (data not shown). The differential sensitivity of LD-Bl antigen to trypsin digestion among other proteolytic enzymes is not clear. It appears that the antibody binding site either contains protein or requires the protein moiety for its antigenic integrity. Yet, we cannot exclude the possibility that the other proteolytic enzymes have resulted in proteolytic digestion without affecting the LD-Bl antigenic site. Denaturing and reducing the antigen by 2-mercaptoethanol and SDS, or by

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AND JOTHY

TABLE I Sensitivity of LD-B 1 Antigen to Enzymatic and Chemical Treatment Treatment Control Trypsin Trypsin Pepsin Proteinase K Lysozyme Collagenase Eiastase RNase Neuraminidase Periodate

Concentration 4mg/ml 1 &ml 4 mg/ml 4 mg/ml 4 mg/ml 4 mg/ml 4 ms/ml 4 mg/ml 4 mg/ml 20 mM 1mM 0.1 mM

Absorbance 1.210 0.705 1.160 1.184 1.115 1.118 1.202 1.142 1.110 1.205 0.308 0.680 0.916

(100) (58) (%) (98) (92) (92) (99) (94) (92) (100) (25) (56) (76)

Note. Effect of enzymatic and chemical treatment on the binding of LDBl MoAb to its antigen. Numbers in parenthesis represent percentage of reactivity compared to control sample.

boiling for 30 min, did not alter its immunoreactivity with LD-Bl antibody, implying that the antibody recognizes a nonconformational determinant. However, the epitope was sensitive to mild periodate treatment at acid pH, which cleaves the carbohydrate vicinal hydroxyl groups without altering the structure of polypeptide chains (Woodward et al., 1985), implying that the epitope is associated with the carbohydrate moiety. Neuraminidase digestion, on the other hand, did not alter the antibody reactivity, indicating the absence of sialic acid residues on the epitope recognized by LD-Bl antibody. LD-Bl reactivity was not affected by lipid extraction of pancreatic carcinoma tissue. LD-Bl antibody did not bind to the glycolipid fraction extracted from pancreatic carcinoma tissue or the CAPANcell line, as determined by immunostaining of thin-layer chromatograms (data not shown). Furthermore, when sections of pancreatic carcinoma tissue were immersed in chloroform and methanol or methanol alone, the antibody binding property showed no reduction in immunostaining compared to controls. To confirm the protein nature of the antigen, we metabolically labeled CAPAN2 cells, a pancreatic carcinoma cell line rich in LD-Bl antigen, with [35S]methionine. The radiolabeled cells were detergent solubilized and incubated with LD-Bl MoAb, and the immune complexes were precipitated with rabbit anti-mouse Ig and S. aureu~ coupled to Sepharose 4B. LD-Bl antigen appeared as a broad band with a molecular weight of 300 to 400 kDa (Fig. 2, lanes A and B), supporting its identity with the affinity-purified antigen from human tissue. The ability to metabolically incorporate [35S]methionine into LD-Bl antigen confirms that is contains a protein core. To test if LD-Bl antibody was reacting with other known antigens, it was incubated with serial dilutions of purified CEA and POA antigens, using affinitypurified LD-Bl antigen and BSA as controls. After overnight incubation, the antibody was tested for residual activity on pancreatic carcinoma by ELISA. No blocking with either CEA or POA was observed, and the reaction was similar to the antibody incubated with BSA. In another test, using a dot-immunoblot assay,

LD-Bl

PANCREATIC

CANCER-ASSOCIATED

il

ANTIBODY

119

t *

FIG. 2. Autoradiograms of (A) gradient gel from whole cell lysate of [35S]methionine-labeled CAPAN- pancreatic carcinoma cell line, (B) immunoprecipitate with LDBl MoAb, (C) same as B, but with control ascites. Arrows represent same molecular weight standards as in Fig. 1.

no significant binding of LD-Bl MoAb occurred over a wide range of CEA concentrations. Similarly, no cross-reactivity with POA was noted. To explore the carbohydrate moiety recognized by LD-Bl antibody, human erythrocytes of blood types A, B, AB, and 0 were incubated overnight with LD-Bl antibody, and the supematant was then tested for residual reactivity with its antigen by ELISA. The concentration of LD-Bl MoAb used was half the saturation level in order to be a limiting step for this assay. Under these conditions, there was no decreased reactivity of LD-Bl antibody with its purified antigen, indicating that the antibody does not bind to A, B, or H blood group determinants. Similarly, LD-Bl binding was not blocked by several MoAbs known to react with carbohydrate antigens present on glycoproteins or glycolipids of pancreatic carcinoma tissue (sialyl Lea, TAG-72, CA 125, 47D10, simple and extended LeY). In another assay, the same MoAbs were used in a double determinant sandwich ELISA, whereby they were coated on a microtiter plate and incubated with the postmicrosomal extract of pancreatic carcinoma tissue. After being washed, biotinylated LD-Bl antibody was added to test its reactivity with the bound material. However, no reactivity with LD-Bl MoAb was detected (data not shown), indicating that the LD-Bl glycoprotein has antigenic determinants distinct from those previously identified antigens. To further explore the nature of the carbohydrate determinants recognized by

120

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LD-Bl antibody, various lectins to specific carbohydrates or blood group oligosaccharides (see Materials and Methods) were tested in a competitive inhibition ELISA for their ability to block the reactivity of biotinylated LD-B 1 antibody with its antigen (Fig. 3). The lectins were selected for their known reactivity with pancreatic carcinoma tissue (Ching et al., 1988), or on the basis of their carbohydrate specificities. Under these assay conditions, the lectins PNA, DBA, SBA, PWM, UEA, and VVA did not show any significant blocking of LD-Bl reactivity (Fig. 3). On the other hand, the reactivity of LD-Bl MoAb with its purified antigen was inhibited by the lectins EEA (45%), BS-II (670/o), and BS-I (72%). BS-I binds to terminal a-~-Gal, BS-II binds to D-GlcNAc, while EEA binds to GalBl + 3GalBl + (3 or 4)GlcNAc (Petryniak et al., 1977). However, inhibition required high concentrations of lectins up to a molar ratio of 32: 1 (see Discussion below). Another finding was that low concentrations of EEA and BS-I, or high concentrations of PNA and PWM, resulted in a significant enhancement of LD-Bl antibody binding. This was confirmed by repeating the test twice on different occasions . As a negative control, the same experiment was carried out to test the ability of these lectins to block the reactivity of biotinylated B18 MoAb with the heavily glycosylated molecule CEA. B18 MoAb reacts with the polypeptide moiety of CEA (Haggarty et al., 1986). As expected, no blocking or enhanced reactivity was noted by any of the lectins on the reactivity of B18 MoAb with CEA. Serological Assay

Sera from various patient populations were assayed for the presence of LD-B 1 antigen by ELISA using LD-Bl MoAb immobilized on ELISA plates as catching antibody, and biolinylated LD-Bl MoAb as detecting antibody. Adsorption of capture antibody to the solid phase was carried in EDC/Mes buffer to provide increased assay sensitivity (Rotmans and Scheven, 1984). A standard curve was O-O l -•BS-1

140120100

PNA

A-AK-2 q -ODBA n --ItSA V-V EEA

-

:z::j!j O-OVVA l -e

LD-81

40 20 1 0 I 1E-2

.

. .

. ..., 1 E-l

,

,

, , ,,,,,

, 1

Lectin/MoAb

, , ,,,,

I 10

,

, . , . ..1 100

Ratio

FIG. 3. Competitive inhibition of various lectins to LD-Bl MoAb. Unlabeled MoAb (LD-Bl) was used as control. Concentrations are expressed in molar ratios of lectins to labeled antibody (see text). Abscissa represents the relative absorbance compared to the control sample incubated with buffer alone.

LD-Bl

PANCREATIC

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121

constructed using known concentrations of purified LD-Bl antigen diluted in a negative serum. The assay was standardized in sensitivity and reproducibility by determining the optimum concentration of capture antibody and labeled antibody to be used. The standard curve was essentially linear for antigen concentrations above 80 rig/ml (Fig. 4). The standard deviation of the curve carried on six separate occasions ranged from 5 to 12% for different antigen concentrations, demonstrating that the assay could be used for screening set-aaccurately and reproducibly over that range of antigen concentrations. A pilot study was conducted on serum samples from 137 healthy controls, 3 patients with chronic pancreatitis, and 6 patients with histologically diagnosed pancreatic carcinoma (Fig. 5). The assay revealed antigen concentration below 100 rig/ml in 118 control sera (86%) with a mean value of 50 + 40 rig/ml. Using a cut-off value of 300 &ml, 12 of 137 normal controls were considered to have elevated serum antigen levels. Sera from 3 patients with chronic pancreatitis also showed low antigen concentrations with a mean value of 80 rig/ml. On the other hand, 5 of 6 samples from patients with pancreatic carcinoma had elevated serum values of LD-Bl antigen. The mean value for 5 sera was 650 rig/ml, with a range of 350 to 1300 &ml. One sample had a value of 65 &ml. Review of that case revealed no obvious differences in tumor characteristics. DISCUSSION Biochemical characterization of the pancreas carcinoma-associated LD-B 1 antigen was based on affinity chromatography which allowed purification of the crude postmicrosomal extract to homogeneity in a single step. Biochemical analysis of the eluted fraction from the affinity column indicates that LD-Bl MoAb

Antigen

Concentration

trig/ml)

4. LD-Bl standard antigen titration curve expressed as the concentration of affinity puritied LD-BI antigen in rig/ml of serum vs absorbance units in a double determinant sandwich ELISA. Bars indicate the standard deviation of six different measurements on separate occasions. Line represents linear regression to the fmt order calculated on an IBM microcomputer with Sigmaplot software. FIG.

122

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AND JOTHY

..*.I 1

I NV

. CA

.. LEL CP

FIG. 5. Distribution of serum LD-Bl antigen levels in normal volunteers (NV, n = 137), patients with chronic pancreatitis (CP, n = 3), and those with carcinoma of pancreas (CA, n = 6). Each symbol (m) represents the mean value of triplicate determinations for each serum sample. Samples from normal volunteers with antigen concentrations less than 100 rig/ml (n = 118) are shown as a solid rectangle. The mean value of each study group is shown as a short solid line.

recognizes a nonconformational determinant. The epitope is contained in a glycoprotein on the basis of its sensitivity to mild periodate oxidation at acid pH, its decreased activity after trypsin digestion, and the ability to label it with radioactive methionine. Its mobility pattern on polyacrylamide gel, and the inability of silver and Coomassie blue stains to react with it, suggest that it consists of a heavily glycosylated molecule (Allen et al., 1984). The insensitivity to various proteolytic enzymes is consistent with the presence of a protein core that is shielded by multiple glycosylated side chains. Lipid extraction on the other hand resulted in no appreciable loss of antigenicity, and the antibody showed no reactivity with the extracted lipid fraction of pancreatic carcinoma tissues. Carbohydrate antigens have assumed a significant value as markers associated with gastrointestinal neoplasias (Itzkowitz et al., 1986). CA 19-9 is a sialylated Lewisa antigen expressed in the tissue and sera of patients with pancreatic carcinoma (Steinberg et al., 1986). DU-PAN-2 is a high molecular weight sialylated mucin-like glycoprotein (Lan et al., 1985; Borowitz et al., 1984) defined by a MoAb prepared against a human pancreatic carcinoma cell line. Competitive assays using a MoAb reactive with sialylated Lewisa molecule indicate that the molecules detected by anti-sialylated Lewisa and LD-Bl MoAbs are different. DU-PAN-2 MoAb was not available to use for competitive studies, yet certain physical, chemical, and immunologic properties indicate that it is also different from LD-Bl. Both antigens consist of heavily glycosylated proteins and show similar, but not identical, electrophoretic properties. Yet, DU-PAN-2 is a sialylated molecule that is susceptible to neuraminidase, pepsin, proteinase K, and papain digestion (Lan et al., 1987). LD-Bl antigen is resistant to all of these enzymesincluding neuraminidase. The tissue reactivities of the two antibodies are different and indicate that they do not recognize the same antigen. In normal

LD-Bl

PANCREATIC

CANCER-ASSOCIATED

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pancreatic tissue, LD-Bl shares with both CA 19-9 and DU-PAN-2 antigens their expression in the ductal epithelium and centroacinar cells. The fact that LD-Bl MoAb reacts with a carbohydrate determinant, and its variable expression on normal gastric and colonic tissue suggested that the antigen may be related to a blood group-related determinant (Mollicone et al., 1985). On the other hand, the antibody showed no reactivity with human ABO erythrocytes. To clarify this observation, various monoclonal antibodies and lectins of known carbohydrate and blood group specificity were tested for reactivity with LD-Bl antigen. The reactivity of LD-Bl antibody with its antigen was decreased by the lectins EEA, BS-II, and BS-I to 45, 67, and 72% of control, respectively. The carbohydrate epitope common to these lectins is present in both type I and type II blood group determinants. Since EEA showed the highest degree of LD-Bl blocking, and since the carbohydrate sequence recognized by EEA also contains the carbohydrates recognized by BS-I and BS-II, the epitope recognized by LDBl probably shares the same epitope recognized by EEA. Therefore, it appears that the LD-Bl antigen contains the epitope Gall31 + 3GalPl + 3 (or 4) GlcNAcPl * 3Ga1,which is present in the precursor chain of both type I and type II blood group-related antigens (Lloyd et al., 1987). However, competitive blocking was partial and required high lectin concentrations, up to a molar ratio of 32: 1. This may be due to a lower affinity of the lectins to the antigen compared to LD-Bl antibody, but this is unlikely considering the ability of most affinitypurified lectins to agglutinate erythrocytes at a much lower concentration than that used in this assay. Another possibility is that the lectins are limited in their binding to part of, and not the entire, epitope recognized by LD-Bl MoAb. Therefore, it may well be that the epitope recognized by LD-B 1 antibody is an extended carbohydrate moiety formed by repeat sequences of the epitope recognized by these lectins. In support of the latter possibility is the ability of EEA and BS-I at low molar ratios to potentiate the reactivity of the antibody. It may well be that the lectin binding to their reactive sites causes changes in the steric configuration of LD-B 1epitope, making it more accessible to antibody binding. The inability of UEA I to block LD-Bl reactivity indicate that ol+fucose does not contribute to the epitope recognized by LD-Bl antibody. It is not possible with the present work to predict whether LD-Bl MoAb can differentiate between the two precursor chains, type I and type II. The sandwich ELISA, as described in this report, is a sensitive and rapid method for detecting and quantifying LD-Bl antigen in body fluids. The use of ECD/Mes buffer (Rotmans et al., 1984) to covalently link the capture antibody resulted in a sevenfold increased sensitivity compared to PBS buffer. Although the antigen was detectable in low concentrations in the serum of most (91%) normal individuals, 12of 137controls had elevated levels and therefore specificity for carcinoma of the pancreas was not present. More significant was the notable differences in serum concentration, and therefore antigen accessibility to the circulation, between patients with pancreatic carcinoma and those with chronic pancreatitis. LD-Bl antigen levels were elevated in the sera of 5 of 6 pancreatic carcinoma patients (mean 650 rig/ml; range 350-1300 m&l), whereas the serum samples of 3 patients with chronic pancreatitis had low LD-Bl antigen content (mean 80 rig/ml; range 50-100 &ml). This is in contrast to the tissue studies where we found similar antigen expression by benign and malignant pancreatic cells (Halwani et al., 1990). The mechanism of this phenomenon is not clear. It is

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probably related to altered biosynthesis or activity of a glycosyltransferase enzyme in cancer cells resulting in accumulation of a precursor substance with backflow into the extracelhtlar space, and hence into the circulation. This phenomenon has been described in the expression of blood group antigens in pancreatic and other gastrointestinal carcinomas (Itzkowitz et al., 1987). Interestingly, this observation correlates with our immunohistochemical finding of extracellular mucus staining into the stroma adjacent to malignant pancreatic glands. In conclusion, characterization of the antigen defined by LD-B 1 MoAb reveals that it is a heavily glycosylated molecule related to a blood group precursor epitope, and that the antigen can be detected at elevated concentrations in the blood circulation. REFERENCES R. C., SARAVIS, C. A., and MAURER, H. R. (Eds.) (1984). Polyacrylamide gel elecrophoresis. In “Gel Electrophoresis and Isoelectric Focusing of Proteins.” pp. 4147. de Gruyter, Berlin/New York. BOROWITZ, M. J., TUCK, F. L., SINDELAR, W. F., FERNSTEN, P. D., and METZGAR, R. S. (1984). Monoclonal antibodies against human pancreatic adenocarcinoma: Distribution of DLJ-PAN-2 antigen on glandular epithelia and adenocarcinomas. J. Natl. Cancer Inst. 72, 999-1005. BROCKHAUS, M., MAGNANI, J. L., BLASZCZYK, M., STEPLEWSKI, Z., KOPROWSKI, H., KARLSSON, K-A., LARSON, G., and GINSBURG, V. (1981). Monoclonal antibodies directed against the human Leb blood group antigen. .I. Biol. Chem. 256, 13,223-13,225. BUTLER, J. E., PETERMAN, J. H., and DIERKS, S. E. (1987). The immunochemistry of solid-phase sandwich enzyme-linked immunosorbent assays. Fed. Proc. 46, 2548-2556. CHING, C. K., BLACK, R., HELLIWELL, T., SAVAGE, A., BARR, H., and RHODES, J. M. (1988). Use of lectin histochemistry in pancreatic cancer. .I. Clin. Pathol. 41, 324-328. CUATRECASAS, P., WILCHEK, M., and ANFINSEN, C. B. (1968). Selective enzyme purification by affinity chromatography. Proc. Natl. Acad. Sci. USA 61, 636-643. GELDER, F. B., REESE, C. J., MOOSA, A. R., HALL, T., and HUNTER, R. (1978). Purification, partial characterization, and clinical evaluation of a pancreatic oncofetal antigen. Cancer Res. 38,313-324. GOOI, H. C., FEIZI, T., KAPADIA, A., KNOWLES, B. B., SOLTER, D., and EVANS, M. J. (1981). Stage-specific embryonic antigen involves al+3 fucosylated type 2 blood group chains. Nature (London) 292, 156-158. HAGGARTY, A., LEGLER, C., KRANTZ, M. J., and FUKS, A. (1986). Epitopes of carcinoembryonic antigen defined by monoclonal antibodies prepared from mice immunized with purified carcinoembryonic antigen or HCT-8R cells. Cancer Res. 46, 300-309. HALWANI, F., CHEUNG, M., and JOTHY, S. Isolation and immunohistochemical characterization of a pancreatic carcinoma-associated monoclonal antibody. Exp. Mol. Pathol., 53, 99-111 (1990). HERBINK, P., VAN BUSSEL, F. J., and WARNAAR, S. 0. (1982). the antigen spot test (AST): A highly sensitive assay for the detection of antibodies. .I. Immunol. Methods 48, 293-298. Ho, M., KATO, K. P., DURDA, P. J., MURRAY, J. H., WOLFE, H., RABIN, H., and CARNEY, W. P. (1987). Tissue distribution, immunochemical characterization, and biosynthesis of 47D10, a tumorassociated surface glycoprotein. Cancer Res. 47, 241-250. HOLMES, E. H., OSTRANDER, G. K., and HAKOMORI, S. (1985). Enzymatic basis for the accumulation of glycolipids with X and dimeric X determinants in human lung cancer cells (NCI-H69). .I. Biol. Chem. 260, 7619-7627. ITZKOWITZ, S. H. and KIM, Y. S. (1986). New carbohydrate tumor markers. Gastroenterology 90, 491494. ALLEN,

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