Molecular and Biochemical Parasitology, 50 (1992) 127 138
127
~) 1992 Elsevier Science Publishers B.V. All rights reserved. /0166-6851/92/$05.00 MOLBIO01639
Isolation and partial characterization of a surface glycoconjugate of
Entamoeba histolytica S a m u e l L. Stanley, Jr. 1"2, H u g h H u i z e n g a ~ a n d Ellen Li 1"3 Departments q[l Medicine, 2Molecular Microbiology and 3Molecular Biology and Biophysics, Washington University School q[ Medicine, St. Louis, MO, U.S.A. (Received 10 July 1991; accepted 7 August 1991)
To study surface molecules of Entamoeba histolytiea we produced monoclonal antibodies from mice immunized with lysates from the pathogenic amebic strain HM 1:IMSS, and screened them for the ability to inhibit E. histolytica adhesion. One monoclonal antibody, CC 8.6, was a potent inhibitor of amebic adhesion to a Chinese hamster ovary cell line, and was capable of inhibiting H M I : I M S S mediated cytotoxicity by 50%. We found that monoclonal antibody CC 8.6 bound to an amebic glycoconjugate. The glycoconjugate is present only in E. histolrtica and not in other Entamoeba sp. It migrates as a polydisperse band on SDS-PAGE, and can be metabolically radiolabeled with [14C]glucose, [~2P]phosphate, and [3H]palmitate. The glycoconjugatc can be purified by hydrophobic interaction chromatography on octyI-Sepharose: enzymatic hydrolysis with phosphatidylinositol-specific phospholipasc C alters the hydrophobic properties of the molecule. HPLC analysis of [HC]glucose-labeled glycoconjugate saccharides revealed that approximately 82% of the incorporated label was in glucose and 12% in galactose. Our studies demonstrate that one of the immunogenic surface molecules of E. histolytica is a phosphorylated, lipid-containing, glycoconjugate, and that antibodies to this antigen may have the potential to protect against E. histolytica adhesion and cytotoxicity. Key words: Entamoeba histolyti~a; Adhesion; Glycoconjugate; Lipid: Amoebiasis
histolytica disease emphasizes the need for
Introduction
The
ability
of
the protozoan parasite Entamoeba histolytica to adhere to target cells appears to be a critical component for pathogenesis. Studies of axenically cultured E. histolytica interacting with mammalian cell lines have shown that target cells in direct contact with trophozoites show extensive blebbing, morphologic changes, and trypan blue uptake, while neighboring cells not in direct contact remain intact [1]. The importance of adhesion in the pathogenesis of E. Corre~v~ondence address. S.L. Stanley, Jr., Dept. of Medicine, Washington University School of Medicine, St. Louis, MO 63110, U.S.A.
isolating surface molecules that may play a role in this critical function. To approach this problem we generated monoclonal antibodies against trophozoites from the E. histolytica HMI:IMSS strain and screened them for the ability to inhibit HMI:IMSS adhesion to a Chinese hamster ovary (CHO) cell line. We found that one monoclonal antibody, which inhibits both amebic adhesion and cytotoxicity, binds to a surface glycoconjugate. In this report we describe the isolation, and partial characterization of this E. histolytica-specific glycoconjugate.
Materials and Methods
Abbreviations." CHO, Chinese hamster ovary: ELISA, enzyme-
Cells.
linked immunosorbent assay: PBS, phosphate-buffered saline.
200:NIH
E.
histolytica and
Rahman,
HMI:IMSS, HK-9, were all obtained
128
from the American Type Culture Collection (ATCC), (Rockville, MD). Entamoeba invadens, Entamoeba moshkovskii, and the E. histolytica-like Laredo strain were also obtained from the ATCC. All amebae were grown in TYI-33 media as previously described [2]. All E. histolytica species were grown at 35~'C, and E. hTvadens, E. moshkovskii and the Laredo strain at 25"C. The lectin-resistant C H O cell line 1021 was graciously provided by S. Kornfeld and grown as described previously [3]. Antigen preparation and #nmunizations. E. histoO, tica lysates were used to immunize BALB/c mice. 2 x 10:' E. histoO'tica H M I : I M S S trophozoites were harvested by chilling 48-h cultures followed by low-speed centrifugation. The trophozoites were washed three times with phosphate-buffered saline (PBS) and suspended in 1 ml of PBS. The trophozoites were lysed by sonication (three 10-s bursts). The homogenate was centrifuged at 1100 x g f o r 3 0 m i n a t 4 ' ~ C , and the pellet was discarded. 100 #1 of the E. histoh'tica lysate combined with 100 i~1 of complete Freund's adjuvant was used to immunize each mouse. Mice received two additional boosts with 80 #1 E. histoh'tica lysate and 80 Id incomplete Freund's adjuvant at weeks 2 and 6, and received 15 /~1 of soluble antigen intravenously on week 8, 3 days before fusion. An identical protocol was used to prepare lysates from all other ameba species tested. Monoclonal antibodies. Monoclonal antibodies were prepared using previously described methods [4]. Antibodies were screened using an enzyme-linked immunoabsorbent assay (ELISA) against E. histolytica lysates, and underwent a second screening against bovine serum albumin (BSA), and Laredo lysates. The monoclonal antibody CC8.6 was isotyped as previously described [5]. The origin and characteristics of monoclonal antibodies HPC/~2 (routine IgM, tc-phosphorylcholine binding antibody), M58.6, and 187.1 have been described [5]. Both M58.6 and 187.1
were coupled to alkaline phosphatase as previously described [6]. EL1SAs were performed according to the protocol in [5] using a 10 l~g ml I solution of the target antigen in PBS. For immunoprecipitation studies monoclonal antibodies CC8.6, and HPC/12 were coupled to Affi-Gel beads (Biorad, Richmond, CA) using the manufacturer's protocol. Adherence oJ ' [-~H]thymldme-laheled " " E. histoh'tica trophozoites to monolayers q / C H O 1021 cells. These experiments were performed as described in [7], except varying concentrations of monoclonal antibody CC8.6, media alone, or control monoclonal antibody HPC/~2 were added to the monolayers after the addition of the labeled trophozoites. Subsequent steps were performed as previously described [7]. Ameba-mediated cytoloxici O' assays. These assays were performed as described in [8], except varying concentrations of monoclonal antibody CC 8.6, media alone, or control monoclonal antibody HPC/t2 were added to the monolayers of 5tCr-labeled C HO 1021 cells immediately before the addition of the trophozoites. 1 x 105 H M l : l M S S t r o p h o z o i t e s w e r e added to each monolayer, and cytotoxicity was measured at two hours. Subsequent steps were performed as previously described [8]. Western hlottinlz. Western blotting of trophozoite lysates was performed using CC 8.6 ascites diluted 1:1000 in blocking buffer with 1251-1abeled 187.1 (rat anti-mouse ~,) as the secondary antibody using the protocol described in [9]. The same protocol was utilized for dot immunoblots. h7 vivo labeling. For labeling experiments a standard protocol was used. The standard media of 48 h cultures of ameba, containing 4 x 10(' trophozoites, was replaced with 1 ml of appropriately modified labeling media (~minimal essential medium containing 0.5% heat inactivated bovine serum, 0.5% BSA, and 5.7 mM cysteine), and the label to be tested. Cultures were incubated for 4 h at 3 5 C then processed as described below. For glucose
129
labeling, 250 /~Ci [U-14C]glucose 2 mCi mmol 1 (American Radiolabeled Chemicals Inc. (ARC), St. Louis, MO) was added to 1 ml of glucose-free labeling medium (glucose free c~-minimal essential medium (~-M EM) containing 0.5% heat-inactivated bovine serum, 0.5% BSA, 5 m M sodium pyruvate and 5.7 m M cysteine). For phosphate labeling 250 #Ci of carrier-free [32p]phosphate (Amersham, Arlington Heights, IL) was added to phosphatefree labeling medium (phosphate free ~-MEM media containing 0.5% heat-inactivated bovine serum, 0.5% BSA, and 5.7 m M cysteine). For palmitate labeling, 1 mCi of [9,10-3H]palmitate (NEN, Boston, MA) was added to c~-MEM medium containing 1 mg ml 1 defatted BSA (Sigma, St. Louis, MO), and 5.7 m M cysteine, and incubated for 4 h at 35°C. The labeled trophozoites were harvested and washed 3 times in PBS. Pellets were lysed by vortexing for 1 rain in a lysis buffer of 1.2 ml 0.02 M Tris-phosphate/0.14 M sodium chloride/0.005 M EDTA/0.002 M benzamidine/ 0.001 M phenylmethylsulfonyl fluoride ( P M S F ) / I % Triton X-100/l% deoxycholate/ 10% BSA/0.02% sodium azide, then spun at 10000 x g at 4°C for 10 min. The supernatants were used immediately for immunoprecipitation as described below.
Immunoprecipitation.
1 ml of trophozoite lysate supernatant was added to 50 /~1 antibody coupled (CC8.6 or HPC/~2) Affi-Gel beads. The mixture was incubated at 4°C with end-over-end mixing for 20 h. The supernatant was removed, and the beads washed twice in lysis buffer, twice in 0.5 M NaC1/0.1 M NaHCO3, and once in 0.1 M sodium phosphate/0.001 M benzamidine/0.0005 M P M S F / 0.001 M EDTA. The beads were boiled in 50/tl of SDS-PAGE sample buffer containing 2mercaptoethanol. Samples were analyzed using 10% and 12.5% acrylamide gels. For analysis of 3H- and 14C-labeled immunoprecipitates, the gels were soaked in E N 3 H A N C E (NEN, Boston, MA), dried, and exposed to Kodak X A R film (Eastman Kodak, Rochester NY) at 70°C. Prestained molecular weight standards -
(Amersham, Arlington Heights, IL) were used for each gel.
Octyl-Sepharose chromatography. 72 h cultures of H M I : I M S S trophozoites (28 x 106) were labeled with 750/xCi [U-14C]glucose under the conditions described above, harvested, washed twice in 50 mM sodium phosphate buffer/0.17 M NaC1, pH 7.3, and then extracted with 20 vols. of chloroform/methanol/water (4:8:3, v/v/v) [10]. The delipidated residue was extracted twice with water saturated with 1butanol with continuous stirring for 18 h at 4°C [11], then insoluble material removed by centrifugation (10000 x g, 30 min) [12]. The supernatants were lyophilized and washed with chloroform/methanol (2:1 v/v) [12]. The residue was dissolved in 0.1 M 2-{[2-hydroxy-l,l-bis(hydroxymethyl)ethyl]amino}ethanesulfonate (Tes) buffer, pH 7.0, containing 5% (v/v) lpropanol, and loaded onto an equilibrated octyl-Sepharose (Pharmacia) column at 4 ml h 1 [12]. The column was eluted by addition of a linear gradient of 0.1 M Tes buffer containing 5 65% 1-propanol with 3 ml fractions collected. The radioactivity of each fraction was determined by counting 20 /xl in 10 ml ScintiVerse (Fisher Scientific, Pittsburgh, PA). Immunoreactivity with CC 8.6 was determined by spotting 2/tl of a fraction on nitrocellulose paper and immunoblotting with CC 8.6 as described above. Phosphatidylinositol phospholipase C hydrolysis. Purified [14C]glucose-labeled glycoconjugate (80000 cpm) was incubated with 2 units phosphatidylinositol-specific phopholipase C (PI-PLC) derived from culture supernatants of Bacillus subtilus transfected with the PI-PLC gene from Bacillus thuringensis [13] (graciously provided by M. Low, Columbia University) in 25 mM Hepes, pH 7.4, at 37°C for 2 h. Cleavage of the hydrophobic moiety was determined by passing the sample through the octyl-Sepharose column as described above, and measuring the label in bound and unbound fractions by liquid scintillation spectrometry [12].
130
Carbohydrate analysis. The [14C]glucose-labeled fraction obtained from octyl-Sepharose chromatography (fraction 59) was hydrolyzed in 2 N trifluoracetic acid for 2 h at 10WC. The trifluoracetic acid was removed by evaporation at reduced pressure. The acid hydrolysate was analyzed directly by borate anion exchange H P L C to identify labeled aldoses as described by Barr and Nordin [14]. Alternatively, the acid hydrolysate was reduced with sodium borohydride, N-acetylated, and deionized on a mixed bed resin (Amberlite M B-3) prior to analysis on a borate anion exchange column to identify the labeled alditols [14]. [14C]Aldoses and alditols were detected by monitoring the H P L C effluent mixed RPI 3a70 liquid scintillation cocktail using a Berthold H P L C radioactivity monitor.
Monoehmal antibody CC 8.6 inhibits HM1.'IMSS trophozoite adhesion and cytotoxicity. We examined the ability of monoclonal antibody CC8.6 to inhibit the adhesion of [3H]thymidine-labeled HM I:IMSS trophozoites to monolayers of the 1021 C H O cell line. We found that CC8.6 was a potent inhibitor of amebic adhesion, reducing adhesion to 2% + 2% of control values (media alone) (Table I). Equivalent concentrations of the isotype and allotype matched control antibody HPClz2 showed a moderate effect, reducing adhesion to 81 _+ 4% of the values seen with a media control. The difference between the inhibitory effect of CC 8.6 and the control antibody was significant at the P
127
~) 1992 Elsevier Science Publishers B.V. All rights reserved. /0166-6851/92/$05.00 MOLBIO01639
Isolation and partial characterization of a surface glycoconjugate of
Entamoeba histolytica S a m u e l L. Stanley, Jr. 1"2, H u g h H u i z e n g a ~ a n d Ellen Li 1"3 Departments q[l Medicine, 2Molecular Microbiology and 3Molecular Biology and Biophysics, Washington University School q[ Medicine, St. Louis, MO, U.S.A. (Received 10 July 1991; accepted 7 August 1991)
To study surface molecules of Entamoeba histolytiea we produced monoclonal antibodies from mice immunized with lysates from the pathogenic amebic strain HM 1:IMSS, and screened them for the ability to inhibit E. histolytica adhesion. One monoclonal antibody, CC 8.6, was a potent inhibitor of amebic adhesion to a Chinese hamster ovary cell line, and was capable of inhibiting H M I : I M S S mediated cytotoxicity by 50%. We found that monoclonal antibody CC 8.6 bound to an amebic glycoconjugate. The glycoconjugate is present only in E. histolrtica and not in other Entamoeba sp. It migrates as a polydisperse band on SDS-PAGE, and can be metabolically radiolabeled with [14C]glucose, [~2P]phosphate, and [3H]palmitate. The glycoconjugatc can be purified by hydrophobic interaction chromatography on octyI-Sepharose: enzymatic hydrolysis with phosphatidylinositol-specific phospholipasc C alters the hydrophobic properties of the molecule. HPLC analysis of [HC]glucose-labeled glycoconjugate saccharides revealed that approximately 82% of the incorporated label was in glucose and 12% in galactose. Our studies demonstrate that one of the immunogenic surface molecules of E. histolytica is a phosphorylated, lipid-containing, glycoconjugate, and that antibodies to this antigen may have the potential to protect against E. histolytica adhesion and cytotoxicity. Key words: Entamoeba histolyti~a; Adhesion; Glycoconjugate; Lipid: Amoebiasis
histolytica disease emphasizes the need for
Introduction
The
ability
of
the protozoan parasite Entamoeba histolytica to adhere to target cells appears to be a critical component for pathogenesis. Studies of axenically cultured E. histolytica interacting with mammalian cell lines have shown that target cells in direct contact with trophozoites show extensive blebbing, morphologic changes, and trypan blue uptake, while neighboring cells not in direct contact remain intact [1]. The importance of adhesion in the pathogenesis of E. Corre~v~ondence address. S.L. Stanley, Jr., Dept. of Medicine, Washington University School of Medicine, St. Louis, MO 63110, U.S.A.
isolating surface molecules that may play a role in this critical function. To approach this problem we generated monoclonal antibodies against trophozoites from the E. histolytica HMI:IMSS strain and screened them for the ability to inhibit HMI:IMSS adhesion to a Chinese hamster ovary (CHO) cell line. We found that one monoclonal antibody, which inhibits both amebic adhesion and cytotoxicity, binds to a surface glycoconjugate. In this report we describe the isolation, and partial characterization of this E. histolytica-specific glycoconjugate.
Materials and Methods
Abbreviations." CHO, Chinese hamster ovary: ELISA, enzyme-
Cells.
linked immunosorbent assay: PBS, phosphate-buffered saline.
200:NIH
E.
histolytica and
Rahman,
HMI:IMSS, HK-9, were all obtained
128
from the American Type Culture Collection (ATCC), (Rockville, MD). Entamoeba invadens, Entamoeba moshkovskii, and the E. histolytica-like Laredo strain were also obtained from the ATCC. All amebae were grown in TYI-33 media as previously described [2]. All E. histolytica species were grown at 35~'C, and E. hTvadens, E. moshkovskii and the Laredo strain at 25"C. The lectin-resistant C H O cell line 1021 was graciously provided by S. Kornfeld and grown as described previously [3]. Antigen preparation and #nmunizations. E. histoO, tica lysates were used to immunize BALB/c mice. 2 x 10:' E. histoO'tica H M I : I M S S trophozoites were harvested by chilling 48-h cultures followed by low-speed centrifugation. The trophozoites were washed three times with phosphate-buffered saline (PBS) and suspended in 1 ml of PBS. The trophozoites were lysed by sonication (three 10-s bursts). The homogenate was centrifuged at 1100 x g f o r 3 0 m i n a t 4 ' ~ C , and the pellet was discarded. 100 #1 of the E. histoh'tica lysate combined with 100 i~1 of complete Freund's adjuvant was used to immunize each mouse. Mice received two additional boosts with 80 #1 E. histoh'tica lysate and 80 Id incomplete Freund's adjuvant at weeks 2 and 6, and received 15 /~1 of soluble antigen intravenously on week 8, 3 days before fusion. An identical protocol was used to prepare lysates from all other ameba species tested. Monoclonal antibodies. Monoclonal antibodies were prepared using previously described methods [4]. Antibodies were screened using an enzyme-linked immunoabsorbent assay (ELISA) against E. histolytica lysates, and underwent a second screening against bovine serum albumin (BSA), and Laredo lysates. The monoclonal antibody CC8.6 was isotyped as previously described [5]. The origin and characteristics of monoclonal antibodies HPC/~2 (routine IgM, tc-phosphorylcholine binding antibody), M58.6, and 187.1 have been described [5]. Both M58.6 and 187.1
were coupled to alkaline phosphatase as previously described [6]. EL1SAs were performed according to the protocol in [5] using a 10 l~g ml I solution of the target antigen in PBS. For immunoprecipitation studies monoclonal antibodies CC8.6, and HPC/12 were coupled to Affi-Gel beads (Biorad, Richmond, CA) using the manufacturer's protocol. Adherence oJ ' [-~H]thymldme-laheled " " E. histoh'tica trophozoites to monolayers q / C H O 1021 cells. These experiments were performed as described in [7], except varying concentrations of monoclonal antibody CC8.6, media alone, or control monoclonal antibody HPC/~2 were added to the monolayers after the addition of the labeled trophozoites. Subsequent steps were performed as previously described [7]. Ameba-mediated cytoloxici O' assays. These assays were performed as described in [8], except varying concentrations of monoclonal antibody CC 8.6, media alone, or control monoclonal antibody HPC/t2 were added to the monolayers of 5tCr-labeled C HO 1021 cells immediately before the addition of the trophozoites. 1 x 105 H M l : l M S S t r o p h o z o i t e s w e r e added to each monolayer, and cytotoxicity was measured at two hours. Subsequent steps were performed as previously described [8]. Western hlottinlz. Western blotting of trophozoite lysates was performed using CC 8.6 ascites diluted 1:1000 in blocking buffer with 1251-1abeled 187.1 (rat anti-mouse ~,) as the secondary antibody using the protocol described in [9]. The same protocol was utilized for dot immunoblots. h7 vivo labeling. For labeling experiments a standard protocol was used. The standard media of 48 h cultures of ameba, containing 4 x 10(' trophozoites, was replaced with 1 ml of appropriately modified labeling media (~minimal essential medium containing 0.5% heat inactivated bovine serum, 0.5% BSA, and 5.7 mM cysteine), and the label to be tested. Cultures were incubated for 4 h at 3 5 C then processed as described below. For glucose
129
labeling, 250 /~Ci [U-14C]glucose 2 mCi mmol 1 (American Radiolabeled Chemicals Inc. (ARC), St. Louis, MO) was added to 1 ml of glucose-free labeling medium (glucose free c~-minimal essential medium (~-M EM) containing 0.5% heat-inactivated bovine serum, 0.5% BSA, 5 m M sodium pyruvate and 5.7 m M cysteine). For phosphate labeling 250 #Ci of carrier-free [32p]phosphate (Amersham, Arlington Heights, IL) was added to phosphatefree labeling medium (phosphate free ~-MEM media containing 0.5% heat-inactivated bovine serum, 0.5% BSA, and 5.7 m M cysteine). For palmitate labeling, 1 mCi of [9,10-3H]palmitate (NEN, Boston, MA) was added to c~-MEM medium containing 1 mg ml 1 defatted BSA (Sigma, St. Louis, MO), and 5.7 m M cysteine, and incubated for 4 h at 35°C. The labeled trophozoites were harvested and washed 3 times in PBS. Pellets were lysed by vortexing for 1 rain in a lysis buffer of 1.2 ml 0.02 M Tris-phosphate/0.14 M sodium chloride/0.005 M EDTA/0.002 M benzamidine/ 0.001 M phenylmethylsulfonyl fluoride ( P M S F ) / I % Triton X-100/l% deoxycholate/ 10% BSA/0.02% sodium azide, then spun at 10000 x g at 4°C for 10 min. The supernatants were used immediately for immunoprecipitation as described below.
Immunoprecipitation.
1 ml of trophozoite lysate supernatant was added to 50 /~1 antibody coupled (CC8.6 or HPC/~2) Affi-Gel beads. The mixture was incubated at 4°C with end-over-end mixing for 20 h. The supernatant was removed, and the beads washed twice in lysis buffer, twice in 0.5 M NaC1/0.1 M NaHCO3, and once in 0.1 M sodium phosphate/0.001 M benzamidine/0.0005 M P M S F / 0.001 M EDTA. The beads were boiled in 50/tl of SDS-PAGE sample buffer containing 2mercaptoethanol. Samples were analyzed using 10% and 12.5% acrylamide gels. For analysis of 3H- and 14C-labeled immunoprecipitates, the gels were soaked in E N 3 H A N C E (NEN, Boston, MA), dried, and exposed to Kodak X A R film (Eastman Kodak, Rochester NY) at 70°C. Prestained molecular weight standards -
(Amersham, Arlington Heights, IL) were used for each gel.
Octyl-Sepharose chromatography. 72 h cultures of H M I : I M S S trophozoites (28 x 106) were labeled with 750/xCi [U-14C]glucose under the conditions described above, harvested, washed twice in 50 mM sodium phosphate buffer/0.17 M NaC1, pH 7.3, and then extracted with 20 vols. of chloroform/methanol/water (4:8:3, v/v/v) [10]. The delipidated residue was extracted twice with water saturated with 1butanol with continuous stirring for 18 h at 4°C [11], then insoluble material removed by centrifugation (10000 x g, 30 min) [12]. The supernatants were lyophilized and washed with chloroform/methanol (2:1 v/v) [12]. The residue was dissolved in 0.1 M 2-{[2-hydroxy-l,l-bis(hydroxymethyl)ethyl]amino}ethanesulfonate (Tes) buffer, pH 7.0, containing 5% (v/v) lpropanol, and loaded onto an equilibrated octyl-Sepharose (Pharmacia) column at 4 ml h 1 [12]. The column was eluted by addition of a linear gradient of 0.1 M Tes buffer containing 5 65% 1-propanol with 3 ml fractions collected. The radioactivity of each fraction was determined by counting 20 /xl in 10 ml ScintiVerse (Fisher Scientific, Pittsburgh, PA). Immunoreactivity with CC 8.6 was determined by spotting 2/tl of a fraction on nitrocellulose paper and immunoblotting with CC 8.6 as described above. Phosphatidylinositol phospholipase C hydrolysis. Purified [14C]glucose-labeled glycoconjugate (80000 cpm) was incubated with 2 units phosphatidylinositol-specific phopholipase C (PI-PLC) derived from culture supernatants of Bacillus subtilus transfected with the PI-PLC gene from Bacillus thuringensis [13] (graciously provided by M. Low, Columbia University) in 25 mM Hepes, pH 7.4, at 37°C for 2 h. Cleavage of the hydrophobic moiety was determined by passing the sample through the octyl-Sepharose column as described above, and measuring the label in bound and unbound fractions by liquid scintillation spectrometry [12].
130
Carbohydrate analysis. The [14C]glucose-labeled fraction obtained from octyl-Sepharose chromatography (fraction 59) was hydrolyzed in 2 N trifluoracetic acid for 2 h at 10WC. The trifluoracetic acid was removed by evaporation at reduced pressure. The acid hydrolysate was analyzed directly by borate anion exchange H P L C to identify labeled aldoses as described by Barr and Nordin [14]. Alternatively, the acid hydrolysate was reduced with sodium borohydride, N-acetylated, and deionized on a mixed bed resin (Amberlite M B-3) prior to analysis on a borate anion exchange column to identify the labeled alditols [14]. [14C]Aldoses and alditols were detected by monitoring the H P L C effluent mixed RPI 3a70 liquid scintillation cocktail using a Berthold H P L C radioactivity monitor.
Monoehmal antibody CC 8.6 inhibits HM1.'IMSS trophozoite adhesion and cytotoxicity. We examined the ability of monoclonal antibody CC8.6 to inhibit the adhesion of [3H]thymidine-labeled HM I:IMSS trophozoites to monolayers of the 1021 C H O cell line. We found that CC8.6 was a potent inhibitor of amebic adhesion, reducing adhesion to 2% + 2% of control values (media alone) (Table I). Equivalent concentrations of the isotype and allotype matched control antibody HPClz2 showed a moderate effect, reducing adhesion to 81 _+ 4% of the values seen with a media control. The difference between the inhibitory effect of CC 8.6 and the control antibody was significant at the P
