Molecular and Biochemical Parasitology, 40 (1990) 43-52
43
Elsevier MOLBIO 01303
Production of a m o u s e monoclonal antibody against the alkaline phosphatase of adult Schistosoma mansoni Flor H. Pujol 1., Ferdinando Liprandi2, Margarita Rodriguez I and Italo M. Cesari 1 llmmunoparasitology Laboratory and 2Virus Biology Laboratory, lnstituto Venezolano de Investigaciones Cienn'ficas, Caracas, Venezuela (Received 7 August 1989; accepted 3 November 1989)
This study describes the production and characterization of a monoclonal antibody (mAb) against the alkaline phosphatase of
Schistosoma mansoni from splenocytes of chronically infected mice. Convenient selection of the mAb was achieved using the catalytic activity of the antigen in a developed enzyme-antigen immunoassay. The mAb was of the IgG1 subclass and it specifically recognized the alkaline phosphatase in adult worm sections by indirect immunofluorescence. Preincubation of the antibody with partially purified adult alkaline phosphatase did not result in inhibition of the enzyme activity and it did not mediate complementdependent cytotoxicity against mechanically transformed schistosomula in vitro. The mAb was able to immunoprecipitate under reducing conditions a polypeptide of 65 kDa, similar in size to the monomeric subunit of the schistosome enzyme. The specificity of the mAb was assessed by competitive inhibition with antibodies of infected human sera in an immunoadsorption assay. Periodate treatment of the antigen resulted in altered electrophoretic mobility of alkaline phosphatase, which confirmed the presence of carbohydrate in the molecule, but this did not prevent binding by the mAb. Although the use of the mAb in capture assays for detection of circulating alkaline phosphatase in infected host sera was unsuccessful, the production of this antibody confirmed that the enzyme is exposed by adult worms to the host and that it is immunogenic; additionally, a monoclonal probe is available for further characterization of the structure and function of this important parasite surface molecule. Key words: Schistosoma mansoni; Alkaline phosphatase; Monoclonal antibody; Affinity chromatography; Circulating antigen
Introduction
The tegument of Schistosoma mansoni adult worms is rich in alkaline phosphatase [1], an enzyme which has been widely used as a surface membrane marker [2-5]. The role of this enzyme is uncertain, but it is probably related to purine Correspondence address: Italo M. Cesari, Laboratorio de Inmunoparasitologia, Centro de Microbiologia y Biologia Celular, I.V.I.C., Apartado 21827, Caracas 1020 A, Venezuela. "Present address: Dpto. de Biotecnologia, Polar C.A., Apdo. 2331, Caracas 1010 A, Venezuela. Abbreviations: DEAE, diethylaminoethyl; ELISA, enzymelinked immunosorbent assay; IFA, indirect immunofluorescence assay; mAb, monoclonal antibody; MEM, minimum essential medium; PBS, phosphate buffered saline; PMSF, phenylmethylsulfonyl fluoride; pNPP, p-nitrophenylphosphate.
recovery and/or regulation and transport of phosphate ions across the tegument [6,7]. The enzymatic activity has been purified from the tegumental double surface membrane as a 260-kDa glycoprotein, composed of 4 65-kDa subunits [8]. It is found as a particulated membrane complex in excretion and secretion products of adult worms incubated in vitro [9]. It has been shown that the alkaline phosphatase activity from adult worm tegumental membranes is partially inhibited by the IgG fraction of sera from chronically infected mice, suggesting that the enzyme is immunogenic in a natural infection [7]. The presence of circulating antibodies against the enzyme in human and experimental infection has recently been studied, and its application in an alkaline phosphatase immunoassay has shown that the enzyme may serve as an antigen for immunodiagnosis [9,10]. In the present study, we describe the produc-
0166-6851/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
44
tion of specific monoclonal antibody (mAb) against the alkaline phosphatase of S. mansoni adult worms, taking advantage of the catalytic activity of this protein to select it. An enzyme capture immunoassay based on this antibody was not able to detect alkaline phosphatase as circulating antigen during an experimental infection. The adult S. mansoni enzyme was easily purified in a two-step procedure by butanolic extraction and affinity chromatography on the anti-alkaline phosphatase mAb conjugated to Sepharose. Materials and Methods
Materials. Alkaline phosphatase from human placenta, a-naphthyl acid phosphate, aprotinin, goat anti-mouse specific immunoglobulin isotypes, rabbit ant-mouse Ig conjugated to peroxidase or to fluorescein isothiocyanate, EDTA, molecular weight markers, phenylmethylsulfonyl fluoride (PMSF), p-nitrophenylphosphate (pNPP), pristane and RPMI-1640 were from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Polyethylene glycol was from Dupont New Research Products (MA, U.S.A.). D E A E cellulose was from Whatman (Maidstone, U.K.). Protein A-Sepharose and cyanogen bromide activated Sepharose 4B were from Pharmacia (Uppsala, Sweden). Fast blue RR from Michrome (Edward Gurr, London, U.K.). Polyvinyl microtiter plates MIC-2001 were from Dynatech Laboratories (Alexandria, VA, U.S.A.). Tissue culture plates, Minimum Essential Medium with Earle's salts (MEM) and fetal bovine serum were from Flow Laboratories (McLean, VA, U.S.A.), Iodo-Gen from Pierce Chemical Co. (Rockford, IL, USA). NaX25I (carrier-free, 16.9 Ci mg -1 of iodine) was from Amersham Laboratories (Aylesbury, U.K.). Tissue-Teck (O.C.T. compound) resine was from Ames Co., Miles Laboratories (Naperville, IL, U.S.A.) and an ELISA amplifying substrate system from Bethesda Research Laboratories (Gaithersburg, MD, U.S.A.). Parasites. Adult S. mansoni worms from the Venezuelan strains JL and YT (the latter kindly provided by R.N. Incani, Departamento de Parasitologla, Universidad de Carabobo, Valencia, Venezuela) and the Brazilian strain BH (kindly
given to us by W. Lobato Paraense, Instituto Oswaldo Cruz, Rio de Janeiro, Brazil) were obtained by perfusion from hamsters infected 6 weeks previously with 400 cercariae [11]. Except where otherwise indicated, the JL strain was used in all experiments. Parasite fractions. Tegument membranes were obtained by incubation of adult worms in the presence of phosphate ions for 5 min at 37°C; the worm-free medium was then centrifuged at 65 000 x g for 1 h at 4°C and the membranous fraction recuperated in the pellet [12]. An adult worm homogenate was washed four times with cold distilled water, and the final pellet was extracted twice with two volumes of n-butanol saturated with water. The aqueous phases were pooled, incubated with 1% Triton X-100 for 30 min, and centrifugated at 65 000 x g for 1 h at 4°C. The supernatant was recovered as the solubilized butanolic extract. Eggs were obtained from the granulomatous livers of infected hamsters [13], and soluble egg antigens were obtained by homogenization and ultracentrifugation [14]. Protein and enzyme assays. Protein determinations were performed according to Peterson [15]. Alkaline phosphatase activity was determined with pNPP as substrate [7]. Sera. Human sera from coprologically positive S. mansoni-infected patients were kindly provided by B. Alarc6n de Noya, Instituto de Medicina Tropical, Universidad Central de Venezuela. Chronically infected mouse sera were obtained from C57BL/6 infected 15 weeks previously with 200 cercariae. Production of monoclonal antibodies. A chronically infected BALB/c mouse was used as the splenocyte source; this mouse received an i.p. injection 15 days before fusion and an i.v. injection 4 days before fusion, of a mixture of 75 txg of butanolic extract and 75 Ixg of soluble egg antigen. The fusion of spleen cells with non-Ig-secreting myeloma cell line P3X63Ag8.653 was done in the presence of 38% (v/v) polyethylene glycol, according to standard techniques [16]. Hybridoma
45 supernatants were tested by: (1) ELISA [17] to detect antibodies against butanolic extracted antigens or soluble egg antigens, using 8 ~g of butanolic extract or 1 g,g of soluble egg antigen per well. (2) Alkaline phosphatase immunoassay [10] for the selection of anti-alkaline phosphatase hybridoma; briefly, rabbit anti-mouse Ig serum (1:1000) was adsorbed to polyvinyl microtiter plates. The plates were then saturated with 1% casein and the hybridoma supernatant added. The Triton X-100 solubilized butanolic extract (0.3 mU m1-1 = 0.3 ~mol p-nitrophenol liberated min -1 at 37°C, pH 9.5) was the source of antigen. The pNPP substrate was then added for 1 h and the enzyme activity determined at 405 nm in a Multiskan II spectrophotometer (Flow Laboratories, U.S.A.). Cloning of selected hybridomas was performed by limiting dilution, using a splenocyte feeder layer. Expansion of hybridoma cell lines was performed in vitro on 24-well culture plates and in vivo by inoculation of about 106 hybridoma cells to a BALB/c mouse injected 2 weeks previously with 0.2 ml of pristane [16]. The limit of detection of the anti-alkaline phosphatase mAb was assessed in the alkaline phosphatase immunoassay by diluting the butanolic extract from 0.3 mU m1-1 to 0.003 mU m1-1. A 1:100 dilution of normal or chronically infected mouse sera were also tested in this assay as controis. Besides pNPP, an ELISA amplifying substrate system for detection of alkaline phosphatase was used. For the latter system, the wells were incubated for 15 min with the NADP substrate and the amplifying solution subsequently added for another 15 min. The reaction was stopped with 2 M H2SO4 and the absorbance read at 492 nm. Inhibition of the alkaline phosphatase activity by the mAb was assessed by incubation of 5 I~1 of butanolic extract (0.5 mU m1-1) with 5, 10, 20 or 40 Ixl of inactivated (30 min, 56°C) ascitic fluid rich in anti-alkaline phosphatase mAb or with an irrelevant ascitic fluid. The alkaline phosphatase activity of a butanolic extract not incubated with antibody was used as control.
anti-mouse IgG1, IgG2a, IgG2b and IgM antisera [18].
Immunofluorescence.
Indirect immunofluorescent assay (IFA) was performed on adult worms [19]. These were included in resin and cryostat sections (10 Ixm) prepared at -20°C. Sections were fixed with cold acetone in 0.7% (w/v) gelatin-treated slides for 5 min, and washed with 0.075 M NaCl, 0.075 M phosphate, pH 7.4 (PBS). Antialkaline phosphatase mAb in the ascitic fluid or normal mouse serum (each 1:100 in MEM with 20% fetal bovine serum) was then added for 30 min at 37°C. Slides were washed three times with PBS and then incubated with fluorescein isothiocyanate-conjugated goat anti-mouse Ig (1:100 in PBS with 1% Evans blue and 0.1% Tween 20). Slides were washed four times with PBS and mounted in alkaline glycerol. IFA studies were also performed with whole glutaraldehyde-fixed mechanically transformed [20] 3-h-old schistosomula, cercariae, eggs and miracidia [21].
Immunoprecipitation. Tegument membranes (25 ixg) were radioiodinated with 1251for 5 min at 4°C using Iodo-Gen [22]. Excess iodine was removed by PBS washing and four centrifugations at 100 000 x g for 1 h at 4°C in a Beckman L8-55 M ultracentrifuge (Beckman Instruments, Inc., Palo Alto, CA, U.S.A.). The preparation was solubilized in 50 mM Tris-HC1, pH 8.0 with 0.15 M NaCI, 1% (v/v) Triton X-100, 1 mM EDTA, 1 mM PMSF, 1 mM aprotinin, and centrifugated at 2000 x g for 5 min. After incubation with the an: tibodies, immune complexes were precipitated with protein A-Sepharose and identified by electrophoresis in 10% polyacrylamide gel with SDS [23] and autoradiography [24].
Antibody competition assay. Alkaline phosphatase immunoassay was carried out using different concentrations of butanolic extract preincubated for 10 min at 37°C in the presence of 1:2 dilution of pooled sera from either S. mansoni-infected patients or healthy people.
Specificity. The specificity of the mAb was asIsotype analysis. This was done by Ouchterlony immunodiffusion of ascitic fluid and specific goat
sessed in the alkaline phosphatase immunoassay by testing it against the enzyme from human pla-
46
centa, butanolic extract from mouse liver or intestine, or butanolic extract from adult worms of three South American S. mansoni strains. All enzyme sources were solubilized with 1% (w/v) Triton X-100 and adjusted to an enzymatic concentration of 0.3 mU m1-1 for the assay.
Periodate treatment. Butanolic extract was treated with different concentrations of sodium metaperiodate for 1 h at room temperature in the dark; the reaction was stopped by adding 50 mM glycerol and by dialysis overnight at 4°C [25]. The periodate-treated fractions (all adjusted to 0.3 mU ml-t) were used as enzyme source in the alkaline phosphatase immunoassay. SDS-PAGE of the different fractions was done according to Laemmli [23]; alkaline phosphatase activity was detected by incubation of the gel with tx-naphthyl-sodium phosphate (2 mg m1-1) and Fast Blue RR (1 mg m1-1) in 1 mM MgC12 [1]. Search for circulating S. mansoni alkaline phosphatase in the serum of infected hamsters. The antialkaline phosphatase mAb ascitic fluid (1:1000) was adsorbed onto microtiter plates. After saturation with casein either plasma or sera from infected patients, or sera from hamsters infected with 1000 cercariae 6 weeks previously, was added diluted 1:10 or undiluted. After incubation for 90 min at 37°C and washing, substrate (pNPP or amplifying substrate) was added.
Results
Production of monoclonal antibodies. Various mAbs against S. mansoni butanolic extracted antigens and soluble egg antigens were obtained using as splenocyte source a chronically infected BALB/c mouse; one mAb was against the alkaline phosphatase of adult worms. The characteristics of the non anti-alkaline phosphatase mAbs will be described in a separate communication. The IgG1 anti-alkaline phosphatase mAb (2B5C7), could be detected only by the described alkaline phosphatase immunoassay; by standard ELISA, the reaction of this antibody with alkaline phosphatase in butanolic extracts could not be detected. Characterization of the anti-alkaline phosphatase monoclonal antibody. The ascitic fluid of the 2B5C7 hybridoma cell line could immunoadsorb the worm alkaline phosphatase activity even if diluted up to 10 -8 . Non specific adsorption was excluded using a normal mouse serum as control; on the other hand, the immunoadsorption shown by the mAb was 6 to 10 times higher (in terms of absorbance) than that shown by the chronically infected mouse serum (Fig. 1). The limit of alkaline phosphatase detection by the mAb in this immunoassay was approximately 0.01 mU m1-1 >. 2.5
o
>
Purification of the alkaline phosphatase by affinity chromatography. Immunoglobulins from the mAb ascitic fluid, purified by ammonium sulfate precipitation and chromatography on DEAE-cellulose, were coupled to cyanogen bromide-activated Sepharose [18]. 1 mg of butanolic extract was applied to a 0.5 ml of the anti-alkaline phosphatase-Sepharose column, and chromatography performed with 0.005 M Tris-HCl, pH 7.4, containing 0.1% (w/v) Triton X-100. Elution of the bound proteins was performed with 2 M MgCI2 pH 8.0 with 0.1% (w/v) Triton X-100. The different fractions were analyzed by SDS-PAGE [23] and proteins detected by silver staining [26]; alkaline phosphatase activity was detected as described above.
I.~
•~ 0.~
43
Elsevier MOLBIO 01303
Production of a m o u s e monoclonal antibody against the alkaline phosphatase of adult Schistosoma mansoni Flor H. Pujol 1., Ferdinando Liprandi2, Margarita Rodriguez I and Italo M. Cesari 1 llmmunoparasitology Laboratory and 2Virus Biology Laboratory, lnstituto Venezolano de Investigaciones Cienn'ficas, Caracas, Venezuela (Received 7 August 1989; accepted 3 November 1989)
This study describes the production and characterization of a monoclonal antibody (mAb) against the alkaline phosphatase of
Schistosoma mansoni from splenocytes of chronically infected mice. Convenient selection of the mAb was achieved using the catalytic activity of the antigen in a developed enzyme-antigen immunoassay. The mAb was of the IgG1 subclass and it specifically recognized the alkaline phosphatase in adult worm sections by indirect immunofluorescence. Preincubation of the antibody with partially purified adult alkaline phosphatase did not result in inhibition of the enzyme activity and it did not mediate complementdependent cytotoxicity against mechanically transformed schistosomula in vitro. The mAb was able to immunoprecipitate under reducing conditions a polypeptide of 65 kDa, similar in size to the monomeric subunit of the schistosome enzyme. The specificity of the mAb was assessed by competitive inhibition with antibodies of infected human sera in an immunoadsorption assay. Periodate treatment of the antigen resulted in altered electrophoretic mobility of alkaline phosphatase, which confirmed the presence of carbohydrate in the molecule, but this did not prevent binding by the mAb. Although the use of the mAb in capture assays for detection of circulating alkaline phosphatase in infected host sera was unsuccessful, the production of this antibody confirmed that the enzyme is exposed by adult worms to the host and that it is immunogenic; additionally, a monoclonal probe is available for further characterization of the structure and function of this important parasite surface molecule. Key words: Schistosoma mansoni; Alkaline phosphatase; Monoclonal antibody; Affinity chromatography; Circulating antigen
Introduction
The tegument of Schistosoma mansoni adult worms is rich in alkaline phosphatase [1], an enzyme which has been widely used as a surface membrane marker [2-5]. The role of this enzyme is uncertain, but it is probably related to purine Correspondence address: Italo M. Cesari, Laboratorio de Inmunoparasitologia, Centro de Microbiologia y Biologia Celular, I.V.I.C., Apartado 21827, Caracas 1020 A, Venezuela. "Present address: Dpto. de Biotecnologia, Polar C.A., Apdo. 2331, Caracas 1010 A, Venezuela. Abbreviations: DEAE, diethylaminoethyl; ELISA, enzymelinked immunosorbent assay; IFA, indirect immunofluorescence assay; mAb, monoclonal antibody; MEM, minimum essential medium; PBS, phosphate buffered saline; PMSF, phenylmethylsulfonyl fluoride; pNPP, p-nitrophenylphosphate.
recovery and/or regulation and transport of phosphate ions across the tegument [6,7]. The enzymatic activity has been purified from the tegumental double surface membrane as a 260-kDa glycoprotein, composed of 4 65-kDa subunits [8]. It is found as a particulated membrane complex in excretion and secretion products of adult worms incubated in vitro [9]. It has been shown that the alkaline phosphatase activity from adult worm tegumental membranes is partially inhibited by the IgG fraction of sera from chronically infected mice, suggesting that the enzyme is immunogenic in a natural infection [7]. The presence of circulating antibodies against the enzyme in human and experimental infection has recently been studied, and its application in an alkaline phosphatase immunoassay has shown that the enzyme may serve as an antigen for immunodiagnosis [9,10]. In the present study, we describe the produc-
0166-6851/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
44
tion of specific monoclonal antibody (mAb) against the alkaline phosphatase of S. mansoni adult worms, taking advantage of the catalytic activity of this protein to select it. An enzyme capture immunoassay based on this antibody was not able to detect alkaline phosphatase as circulating antigen during an experimental infection. The adult S. mansoni enzyme was easily purified in a two-step procedure by butanolic extraction and affinity chromatography on the anti-alkaline phosphatase mAb conjugated to Sepharose. Materials and Methods
Materials. Alkaline phosphatase from human placenta, a-naphthyl acid phosphate, aprotinin, goat anti-mouse specific immunoglobulin isotypes, rabbit ant-mouse Ig conjugated to peroxidase or to fluorescein isothiocyanate, EDTA, molecular weight markers, phenylmethylsulfonyl fluoride (PMSF), p-nitrophenylphosphate (pNPP), pristane and RPMI-1640 were from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Polyethylene glycol was from Dupont New Research Products (MA, U.S.A.). D E A E cellulose was from Whatman (Maidstone, U.K.). Protein A-Sepharose and cyanogen bromide activated Sepharose 4B were from Pharmacia (Uppsala, Sweden). Fast blue RR from Michrome (Edward Gurr, London, U.K.). Polyvinyl microtiter plates MIC-2001 were from Dynatech Laboratories (Alexandria, VA, U.S.A.). Tissue culture plates, Minimum Essential Medium with Earle's salts (MEM) and fetal bovine serum were from Flow Laboratories (McLean, VA, U.S.A.), Iodo-Gen from Pierce Chemical Co. (Rockford, IL, USA). NaX25I (carrier-free, 16.9 Ci mg -1 of iodine) was from Amersham Laboratories (Aylesbury, U.K.). Tissue-Teck (O.C.T. compound) resine was from Ames Co., Miles Laboratories (Naperville, IL, U.S.A.) and an ELISA amplifying substrate system from Bethesda Research Laboratories (Gaithersburg, MD, U.S.A.). Parasites. Adult S. mansoni worms from the Venezuelan strains JL and YT (the latter kindly provided by R.N. Incani, Departamento de Parasitologla, Universidad de Carabobo, Valencia, Venezuela) and the Brazilian strain BH (kindly
given to us by W. Lobato Paraense, Instituto Oswaldo Cruz, Rio de Janeiro, Brazil) were obtained by perfusion from hamsters infected 6 weeks previously with 400 cercariae [11]. Except where otherwise indicated, the JL strain was used in all experiments. Parasite fractions. Tegument membranes were obtained by incubation of adult worms in the presence of phosphate ions for 5 min at 37°C; the worm-free medium was then centrifuged at 65 000 x g for 1 h at 4°C and the membranous fraction recuperated in the pellet [12]. An adult worm homogenate was washed four times with cold distilled water, and the final pellet was extracted twice with two volumes of n-butanol saturated with water. The aqueous phases were pooled, incubated with 1% Triton X-100 for 30 min, and centrifugated at 65 000 x g for 1 h at 4°C. The supernatant was recovered as the solubilized butanolic extract. Eggs were obtained from the granulomatous livers of infected hamsters [13], and soluble egg antigens were obtained by homogenization and ultracentrifugation [14]. Protein and enzyme assays. Protein determinations were performed according to Peterson [15]. Alkaline phosphatase activity was determined with pNPP as substrate [7]. Sera. Human sera from coprologically positive S. mansoni-infected patients were kindly provided by B. Alarc6n de Noya, Instituto de Medicina Tropical, Universidad Central de Venezuela. Chronically infected mouse sera were obtained from C57BL/6 infected 15 weeks previously with 200 cercariae. Production of monoclonal antibodies. A chronically infected BALB/c mouse was used as the splenocyte source; this mouse received an i.p. injection 15 days before fusion and an i.v. injection 4 days before fusion, of a mixture of 75 txg of butanolic extract and 75 Ixg of soluble egg antigen. The fusion of spleen cells with non-Ig-secreting myeloma cell line P3X63Ag8.653 was done in the presence of 38% (v/v) polyethylene glycol, according to standard techniques [16]. Hybridoma
45 supernatants were tested by: (1) ELISA [17] to detect antibodies against butanolic extracted antigens or soluble egg antigens, using 8 ~g of butanolic extract or 1 g,g of soluble egg antigen per well. (2) Alkaline phosphatase immunoassay [10] for the selection of anti-alkaline phosphatase hybridoma; briefly, rabbit anti-mouse Ig serum (1:1000) was adsorbed to polyvinyl microtiter plates. The plates were then saturated with 1% casein and the hybridoma supernatant added. The Triton X-100 solubilized butanolic extract (0.3 mU m1-1 = 0.3 ~mol p-nitrophenol liberated min -1 at 37°C, pH 9.5) was the source of antigen. The pNPP substrate was then added for 1 h and the enzyme activity determined at 405 nm in a Multiskan II spectrophotometer (Flow Laboratories, U.S.A.). Cloning of selected hybridomas was performed by limiting dilution, using a splenocyte feeder layer. Expansion of hybridoma cell lines was performed in vitro on 24-well culture plates and in vivo by inoculation of about 106 hybridoma cells to a BALB/c mouse injected 2 weeks previously with 0.2 ml of pristane [16]. The limit of detection of the anti-alkaline phosphatase mAb was assessed in the alkaline phosphatase immunoassay by diluting the butanolic extract from 0.3 mU m1-1 to 0.003 mU m1-1. A 1:100 dilution of normal or chronically infected mouse sera were also tested in this assay as controis. Besides pNPP, an ELISA amplifying substrate system for detection of alkaline phosphatase was used. For the latter system, the wells were incubated for 15 min with the NADP substrate and the amplifying solution subsequently added for another 15 min. The reaction was stopped with 2 M H2SO4 and the absorbance read at 492 nm. Inhibition of the alkaline phosphatase activity by the mAb was assessed by incubation of 5 I~1 of butanolic extract (0.5 mU m1-1) with 5, 10, 20 or 40 Ixl of inactivated (30 min, 56°C) ascitic fluid rich in anti-alkaline phosphatase mAb or with an irrelevant ascitic fluid. The alkaline phosphatase activity of a butanolic extract not incubated with antibody was used as control.
anti-mouse IgG1, IgG2a, IgG2b and IgM antisera [18].
Immunofluorescence.
Indirect immunofluorescent assay (IFA) was performed on adult worms [19]. These were included in resin and cryostat sections (10 Ixm) prepared at -20°C. Sections were fixed with cold acetone in 0.7% (w/v) gelatin-treated slides for 5 min, and washed with 0.075 M NaCl, 0.075 M phosphate, pH 7.4 (PBS). Antialkaline phosphatase mAb in the ascitic fluid or normal mouse serum (each 1:100 in MEM with 20% fetal bovine serum) was then added for 30 min at 37°C. Slides were washed three times with PBS and then incubated with fluorescein isothiocyanate-conjugated goat anti-mouse Ig (1:100 in PBS with 1% Evans blue and 0.1% Tween 20). Slides were washed four times with PBS and mounted in alkaline glycerol. IFA studies were also performed with whole glutaraldehyde-fixed mechanically transformed [20] 3-h-old schistosomula, cercariae, eggs and miracidia [21].
Immunoprecipitation. Tegument membranes (25 ixg) were radioiodinated with 1251for 5 min at 4°C using Iodo-Gen [22]. Excess iodine was removed by PBS washing and four centrifugations at 100 000 x g for 1 h at 4°C in a Beckman L8-55 M ultracentrifuge (Beckman Instruments, Inc., Palo Alto, CA, U.S.A.). The preparation was solubilized in 50 mM Tris-HC1, pH 8.0 with 0.15 M NaCI, 1% (v/v) Triton X-100, 1 mM EDTA, 1 mM PMSF, 1 mM aprotinin, and centrifugated at 2000 x g for 5 min. After incubation with the an: tibodies, immune complexes were precipitated with protein A-Sepharose and identified by electrophoresis in 10% polyacrylamide gel with SDS [23] and autoradiography [24].
Antibody competition assay. Alkaline phosphatase immunoassay was carried out using different concentrations of butanolic extract preincubated for 10 min at 37°C in the presence of 1:2 dilution of pooled sera from either S. mansoni-infected patients or healthy people.
Specificity. The specificity of the mAb was asIsotype analysis. This was done by Ouchterlony immunodiffusion of ascitic fluid and specific goat
sessed in the alkaline phosphatase immunoassay by testing it against the enzyme from human pla-
46
centa, butanolic extract from mouse liver or intestine, or butanolic extract from adult worms of three South American S. mansoni strains. All enzyme sources were solubilized with 1% (w/v) Triton X-100 and adjusted to an enzymatic concentration of 0.3 mU m1-1 for the assay.
Periodate treatment. Butanolic extract was treated with different concentrations of sodium metaperiodate for 1 h at room temperature in the dark; the reaction was stopped by adding 50 mM glycerol and by dialysis overnight at 4°C [25]. The periodate-treated fractions (all adjusted to 0.3 mU ml-t) were used as enzyme source in the alkaline phosphatase immunoassay. SDS-PAGE of the different fractions was done according to Laemmli [23]; alkaline phosphatase activity was detected by incubation of the gel with tx-naphthyl-sodium phosphate (2 mg m1-1) and Fast Blue RR (1 mg m1-1) in 1 mM MgC12 [1]. Search for circulating S. mansoni alkaline phosphatase in the serum of infected hamsters. The antialkaline phosphatase mAb ascitic fluid (1:1000) was adsorbed onto microtiter plates. After saturation with casein either plasma or sera from infected patients, or sera from hamsters infected with 1000 cercariae 6 weeks previously, was added diluted 1:10 or undiluted. After incubation for 90 min at 37°C and washing, substrate (pNPP or amplifying substrate) was added.
Results
Production of monoclonal antibodies. Various mAbs against S. mansoni butanolic extracted antigens and soluble egg antigens were obtained using as splenocyte source a chronically infected BALB/c mouse; one mAb was against the alkaline phosphatase of adult worms. The characteristics of the non anti-alkaline phosphatase mAbs will be described in a separate communication. The IgG1 anti-alkaline phosphatase mAb (2B5C7), could be detected only by the described alkaline phosphatase immunoassay; by standard ELISA, the reaction of this antibody with alkaline phosphatase in butanolic extracts could not be detected. Characterization of the anti-alkaline phosphatase monoclonal antibody. The ascitic fluid of the 2B5C7 hybridoma cell line could immunoadsorb the worm alkaline phosphatase activity even if diluted up to 10 -8 . Non specific adsorption was excluded using a normal mouse serum as control; on the other hand, the immunoadsorption shown by the mAb was 6 to 10 times higher (in terms of absorbance) than that shown by the chronically infected mouse serum (Fig. 1). The limit of alkaline phosphatase detection by the mAb in this immunoassay was approximately 0.01 mU m1-1 >. 2.5
o
>
Purification of the alkaline phosphatase by affinity chromatography. Immunoglobulins from the mAb ascitic fluid, purified by ammonium sulfate precipitation and chromatography on DEAE-cellulose, were coupled to cyanogen bromide-activated Sepharose [18]. 1 mg of butanolic extract was applied to a 0.5 ml of the anti-alkaline phosphatase-Sepharose column, and chromatography performed with 0.005 M Tris-HCl, pH 7.4, containing 0.1% (w/v) Triton X-100. Elution of the bound proteins was performed with 2 M MgCI2 pH 8.0 with 0.1% (w/v) Triton X-100. The different fractions were analyzed by SDS-PAGE [23] and proteins detected by silver staining [26]; alkaline phosphatase activity was detected as described above.
I.~
•~ 0.~
