Annals of Tropical Medicine & Parasitology
ISSN: 0003-4983 (Print) 1364-8594 (Online) Journal homepage: http://www.tandfonline.com/loi/ypgh19
Leishmania donovani: isolation of a ConcanavalinA specific antigen and its evaluation for serodiagnosis of visceral leishmaniasis M. Bhattacharya, N. Banerjee & D. K. Ghosh To cite this article: M. Bhattacharya, N. Banerjee & D. K. Ghosh (1992) Leishmania donovani: isolation of a Concanavalin-A specific antigen and its evaluation for serodiagnosis of visceral leishmaniasis, Annals of Tropical Medicine & Parasitology, 86:4, 341-345, DOI: 10.1080/00034983.1992.11812676 To link to this article: http://dx.doi.org/10.1080/00034983.1992.11812676
Published online: 15 Nov 2016.
Submit your article to this journal
View related articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ypgh19 Download by: [Cornell University Library]
Date: 28 June 2017, At: 00:07
Annals of Tropical Medicine and Parasitology, Vol. 86, No.4, 341-345 (1992)
Leishmania donovani: isolation of a Concanavalin-A specific antigen and its evaluation for serodiagnosis of visceral leishmaniasis Bv M. BHATTACHARYA, N. BANERJEE Department of Organic Chemistry (Carbohydrate), Indian Institute of Chemical Biology, 4 Raja S.C. Mu//ick Road, Calcutta 700 032, India AND
D. K. GHOSH*
Department of Immunochemistry, Leishmania Group, Indian Institute of Chemical Biology, 4 Raja S.C. Mu//ick Road, Calcutta 700 032, India Received 14 January 1992, Revised 16 March 1992, Accepted 17 March 1992
A glycoconjugate antigen of 27-39 kDa was isolated from a cell-free extract of Leishmania do novani by affinity chromatography using a Concanavalin-A sepharose-4B column and eluted with 0·5 M a-methylmannoside. The antigen was recognized specifically by sera from kala-azar (visceral leishmaniasis) patients and did not react with sera from tuberculosis, leprosy or malaria patients. The antigen may therefore be useful in developing a serodiagnostic assay for visceral leishmaniasis.
Leishmania donovani, a digenetic protozoan parasite, is the aetiological agent of visceral leishmaniasis (kala-azar). Lectin-binding studies have demonstrated the presence of a mannose moiety on the surface of L. donovani cells (Dwyer, 1974, 1977, 1980) and other glycoconjugates (Lepay et a/., 1983; Jaffe and McMahon-Pratt, 1988), which react with Concanavilin-A (Con-A) or lentil lectin, on the surface of all Leishmania spp. tested. Such surface glycoconjugates may have a regulatory role in the parasite uptake by and the immunological response of the mammalian host (Hand man and Goding, 1985; Russell and Wilhelm, 1986). High titres of specific and non-specific antibodies are present in patients with visceral leishmaniasis (VL) and several different serological tests are in use for diagnosis of this disease. Although these tests are sensitive and "Author to whom correspondence should be addressed. 0003-4983/92/040341 +05 $08.00/0
gradually gaining importance as complementary procedures to more invasive techniques, they are not entirely specific and cross reactions occur between leishmania! antigens and sera from patients with Chagas' disease, malaria and some bacterial infections. A solution to this problem of non-specificity would be a sensitive serodiagnostic assay which uses a purified antigen specific for VL Uaffe and Zalis, 1988). In this communication we report isolation of a Con-A-specific glycoconjugate antigen from L. donovani and its evaluation for serodiagnosis ofVL. MATERIALS AND METHODS Cell-free Extract Leishmania donovani promastigotes (MHOM(
78/UR-6), grown in blood agar medium at 24 ± 1°C, were washed four times in phosphate © 1992 Liverpool School of Tropical Medicine
342
BHATTACHARYAETAL.
-66
-
45
-
36
-
29
-
24
-
20·1
-
14·2
et al., 1979) and the cell-free extract loaded on top of the column. After thorough washing with PBS containing 500 mM NaCI, the Con-A reactive glycoconjugate was eluted with 0· 5 M a-methylmannoside in PBS containing 100 mM NaCI. The eluents containing the glycoconjugate were pooled, dialysed, concentrated and kept at - 20°C for further study. SDS-PAGE Polyacrylamide gel electrophoresis was carried out under reducing conditions, using the Laemmli (1970) buffer system. After electrophoresis, the gels were fixed and stained for proteins and carbohydrates using I% Coomassie Brilliant Blue R-250 and periodic acid-Schiffs reagent (PAS), respectively. Dot-Radio Immunosorbent Assay (Dot-RIA) ANTIGEN DISC PREPARATION
{A)
{B)
{C)
Fig. 1. (A) SDS-PAGE (11%) separated Leishmania donovani cell-free extract, stained for proteins with Coomassie blue; (B) Coomassie blue and (C) periodic acid Schiff's stained SDS-PAGE (11%) of the isolated antigen (glycoconjugate). The molecular weight standards are indicated at the right.
Nitrocellulose filter discs (5 mm, 0·22 f.Lm pore size; Millipore) were dotted with the isolated glycoconjugate in 1 f.Ll volumes (900 ng), stabilized by drying for 15 minute at 56°C and placed individually into the wells of a microtitre plate. Non-specific binding sites were blocked by incubating each disc with 150 f.Ll 3% (w/v) bovine serum albumin in PBS (3% BSA-PBS) for 15 minutes at room temperature (r.t.). ANTIBODY INCUBATION
buffered saline, pH 7·1 (PBS). The final pellet was resuspended in PBS containing 1 mM CaCl 2, 1 mM MgC12 and 0·1% (w/v) phenyl methyl sulphonic fluoride, sonicated on ice (10, 30-second pulses; Labsonic 2000 sonicator) and centrifuged at 10 000 g for 20 minutes at 4°C and stored at -70°C. Protein and carbohydrate were measured by the Folin-Ciacaltau (Lowry et al., 1951) and phenolsulphuric acid methods (Dubois et al., 1956), respectively. Affinity Chromatography Cyanogen bromide activated Sepharose-4B coupled with Con-A (Cuatracasas and Anfinsen, 197la,b) was used to prepare the affinity column (5 x 1 em). The column was equilibrated with glycyl-glycine buffer, pH 7·1 (Lo
The blocking solution was aspirated off each antigen disc and replaced with 150 f.Ll of serum sample diluted 1:100 with 1% BSA-PBS in each well. The plate was shaken and then incubated at r.t. for 90 minutes. The sera were then. aspirated off and the discs washed three times with PBS containing 0·05% Tween 20 (Sol-A). INCUBATION WITH
125
1-LABELLED PROTEIN-A
After aspirating off the wash solution, 150 f.Ll of 1251-labelled protein-A (Markwell, 1982) (50 000 counts per minute) in BSA-PBS solution were pipetted into each well and the plate incubated for 90 minutes at r.t. The discs were again washed three times in Sol-A and then the radioactivity of each of the discs was measured with a gamma counter (ECIL, India).
SERODIAGNOSIS OF KALA-AZAR
343
4
10 . - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - ,
X X
" I
Normal
I
Malaria Kala-czar Leprosy Tuberculosis Patient-sera
Fig. 2. Dot-RIA. The purified antigen (glycoconjugate) was coated on nitrocellulose discs and incubated with a I: 100 dilution of human sera from normal control ( \7 ); patients with leprosy ( T ), malaria ( 0 ), tuberculosis (e) and kala-azar (x). The binding of the antibody with the leishmania! antigen was measured using radiolabelled protein-A.
Antisera Production Antisera were prepared by injecting rabbits with promastigotes (UR-6) with Freund's adjuvant (Rassam and Al-Mudhaffar, 1981). Patient Sera Sera were collected from patients with kalaazar, leprosy, tuberculosis or malaria, who had been admitted to the School of Tropical Medicine, Calcutta, India. Sera from healthy research workers were used as controls from an endemic area.
Reactivity of the Antigen with Normal and Patient Sera All the kala-azar patient sera were positive against the glycoconjugate in gel diffusion, counter-current and immunoelectrophoresis tests but sera from the other patients and control sera were negative (data not shown). The binding of the glycoconjugate antigen with the different sera was also examined by dot-RIA. The antigen was specifically recognized by the sera from patients with kala-azar (4-9 x 103 counts per minute); reaction with the other sera was at a very low level (0·09--0·19 x 103 counts per minute) (Fig. 2).
RESULTS DISCUSSION SDS-PAGE A single glycoconjugate antigen was eluted by a-methylmannoside from the Con-A column and revealed, by SDS-P AGE and Coomassie Blue staining, to have a molecular weight around 29-30 kDa [Fig. l(B)]. The antigen gave a broad band, ranging from 27-39 kDa [(Fig. 1(C)] after PAS staining. A few more antigens were eluted from the affinity matrix when it was treated with 0· 5 M a-methylmannoside containing SDS.
Affinity separation occupies a unique place amongst separation techniques. The present study was undertaken to isolate Con-A reactive antigens of an Indian strain of L. donovani and it was surprising that only a single antigen was found in the eluate. The antigen has a molecular weight of 27-39 kDa and the major portion of the antigen contains a carbohydrate moiety, as it could be stained with PAS. The protein content of the antigen appears to be very low, as only a
344
BHATTACHARYAETAL.
small portion of the antigen (29-30 kDa) was stained with Coomassie Blue. A few antigens of relatively low molecular weight (14-19 kDa) could be eluted with a-methylmannoside containing SDS, which indicates these antigens may have a higher affinity for Con-A than the one isolated. Dwyer ( 1981) indicated that the pellicular membrane of L. donovani contains at least 24 glycopeptidesfglycoproteins, of15-90·5 kDa, of which 15 contain mannose along with other sugar components; the only mannose-containing glycoprotein had a molecular weight of 60 kDa. It has also been reported that Con-A and lentillectin reacted, respectively, with 41-52 and 160-185 kDa components present in eight different species of Leishmania 0 affe and McMahon-Pratt, 1988). Besides these antigens, two other bands, in the region of 34 and 39 kDa were apparent in L. donovani infantum and Con-A reacted with two additional proteins of 76 and 92 kDa in L. major. Glycoproteins of 65 and 23-25 kDa were identified in L. donovani (IS) in a binding experiment with Con-A sepharose (Lepay eta!., 1983). It appears that the major glycoconjugate revealed in the present study of L. donovani
has not been reported before. However, an amphipathic glycoconjugate of 20-67 kDa, which migrates as a polydisperse band in SDS-PAGE, has been reported to be present in L. tropica (Handman et al., 1984). The exact identity of the glycoconjugate isolated from Indian L. donovani cannot be ascertained from the data available but it is surface-oriented, as it is labelled when isolated from surface-iodinated promastigotes (data not shown). Periodic acid oxidation of the antigen inhibits its antibodybinding activity. This Con-A specific antigen can be used, at very low concentrations, to distinguish sera from kala-azar patients from all other sera. No cross reaction was found with the other sera from patients with diseases prevalent in India. The isolated antigen appears to have good serodiagnostic potential, as tests with it were both sensitive and specific. M. Bhattacharya is a junior research fellow with the Indian Council of Medical Research. This investigation received partial financial support from the UNDP. The authors wish to thank Professor A. N. Bhaduri, Director, IICB, for his constant encouragement of this work. ACKNOWLEDGEMENTS.
REFERENCES CuATRECASAS, P. & ANFINSEN, C. B. (1971a). Affinity chromatography. Annual Review of Biochemistry, 40,
259-278. CuATRECASAS, P. & ANFINSEN, C. B. (1971b). Affinity chromatogrpahy. Methods in Enzymology, 22, 345-378. DUBOIS, M. K., CILLS, K. A., HAMILTON, J. K., BEBRS, P. A. & SMITH, F. (1956). Colorimetric method for determination of sugar. Analytical Chemistry, 28, 350. DwYER, D. M. (1974). Lectin binding saccharides on a parasitic protozoa. Science, 184,471-473. DWYER, D. M. ( 1977). Leishmania donovani, surface membrane carbohydrate of promastigotes. Experimental
Parasitology, 41, 341-358. DWYER, D. M. (1980). Isolation and partial characterization of surface membrane from Leishmania donovani promastigotes. Journal of Parasitology, 27, 17 5-182. DWYER, D. M. (1981). Structural, chemical and antigenic properties of surface membranes isolated from Leishmania donovani. In Biochemistry of Parasites, ed. Slutzky, G. M. pp. 10-28. Oxford: Pergamon Press. HANDMAN, E. & GODING, J. M. (1985). The Leishmania receptor for macrophage is a lipid containing glycoconjugate. EMBO Journal, 4, 329-336. HANDMAN, E., GREENBLATT, C. L. & GoDING, J. W. (1984). An amphipathic sulfated glycoconjugate of Leishmania: characterization with monoclonal antibodies. EMBO Journal, 3, 2301-2306. }AFFE, C. L. & McMAHoN-PRATT, D. (1988). The identification of membrane glycoconjugates in Leishmania species. Journal ofParasitology, 74, 548-561. }AFFE, C. L. & ZALIS, M. (1988). Use of purified parasite proteins from Leishmania donovani for the rapid serodiagnosis of visceral leishmaniasis. Journal of Infectious Diseases, 157, 1212-1220.
SERODIAGNOSIS OF KALA-AZAR
345
LAEMMLI, U.K. (1970). Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature, 227,680-685. LEPAY, D. A., NOGOCIRA, N. & CoHN, Z. (1983). Surface antigens of Leishmania donovani promastigotes. Journal ofExperimental Medicine, 157, 1562-1572. Lo, J.-T., MuKERJI, K., AWASTHI, Y. C., HANADA, K., SuzuKI, K. & SRIBASTAVA, S. K. (1979). Purification and properties of sphingolipid P-galactosidases from human placenta. Journal of Biological Chemistry, 254, 6710-6715. LOWRY, 0. H., RosENBROUGH, M., FARR, A. & RANDALL, R. (1951). Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry, 193, 265-275. MARKWELL, M.A. K. (1982). A new solid state reagent to iodinate proteins. 1. Conditions for the efficient labelling of antiserum. Analytical Biochemistry, 125, 427-432. RASSAM, M. B. & AL-MUDHAFFAR, S. A. (1981). Immunochemical characteristics of an Iraqi Leishmania donovani. Annals of Tropical Medicine and Parasitology, 75, 145-155. RussELL, D. G. & WILHELM, H. (1986). The involvement of the major surface glycoprotein (gp-63) of Leishmania promastigotes in attachment to macrophages. Journal of Immunology, 136, 2613-2620.
ISSN: 0003-4983 (Print) 1364-8594 (Online) Journal homepage: http://www.tandfonline.com/loi/ypgh19
Leishmania donovani: isolation of a ConcanavalinA specific antigen and its evaluation for serodiagnosis of visceral leishmaniasis M. Bhattacharya, N. Banerjee & D. K. Ghosh To cite this article: M. Bhattacharya, N. Banerjee & D. K. Ghosh (1992) Leishmania donovani: isolation of a Concanavalin-A specific antigen and its evaluation for serodiagnosis of visceral leishmaniasis, Annals of Tropical Medicine & Parasitology, 86:4, 341-345, DOI: 10.1080/00034983.1992.11812676 To link to this article: http://dx.doi.org/10.1080/00034983.1992.11812676
Published online: 15 Nov 2016.
Submit your article to this journal
View related articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ypgh19 Download by: [Cornell University Library]
Date: 28 June 2017, At: 00:07
Annals of Tropical Medicine and Parasitology, Vol. 86, No.4, 341-345 (1992)
Leishmania donovani: isolation of a Concanavalin-A specific antigen and its evaluation for serodiagnosis of visceral leishmaniasis Bv M. BHATTACHARYA, N. BANERJEE Department of Organic Chemistry (Carbohydrate), Indian Institute of Chemical Biology, 4 Raja S.C. Mu//ick Road, Calcutta 700 032, India AND
D. K. GHOSH*
Department of Immunochemistry, Leishmania Group, Indian Institute of Chemical Biology, 4 Raja S.C. Mu//ick Road, Calcutta 700 032, India Received 14 January 1992, Revised 16 March 1992, Accepted 17 March 1992
A glycoconjugate antigen of 27-39 kDa was isolated from a cell-free extract of Leishmania do novani by affinity chromatography using a Concanavalin-A sepharose-4B column and eluted with 0·5 M a-methylmannoside. The antigen was recognized specifically by sera from kala-azar (visceral leishmaniasis) patients and did not react with sera from tuberculosis, leprosy or malaria patients. The antigen may therefore be useful in developing a serodiagnostic assay for visceral leishmaniasis.
Leishmania donovani, a digenetic protozoan parasite, is the aetiological agent of visceral leishmaniasis (kala-azar). Lectin-binding studies have demonstrated the presence of a mannose moiety on the surface of L. donovani cells (Dwyer, 1974, 1977, 1980) and other glycoconjugates (Lepay et a/., 1983; Jaffe and McMahon-Pratt, 1988), which react with Concanavilin-A (Con-A) or lentil lectin, on the surface of all Leishmania spp. tested. Such surface glycoconjugates may have a regulatory role in the parasite uptake by and the immunological response of the mammalian host (Hand man and Goding, 1985; Russell and Wilhelm, 1986). High titres of specific and non-specific antibodies are present in patients with visceral leishmaniasis (VL) and several different serological tests are in use for diagnosis of this disease. Although these tests are sensitive and "Author to whom correspondence should be addressed. 0003-4983/92/040341 +05 $08.00/0
gradually gaining importance as complementary procedures to more invasive techniques, they are not entirely specific and cross reactions occur between leishmania! antigens and sera from patients with Chagas' disease, malaria and some bacterial infections. A solution to this problem of non-specificity would be a sensitive serodiagnostic assay which uses a purified antigen specific for VL Uaffe and Zalis, 1988). In this communication we report isolation of a Con-A-specific glycoconjugate antigen from L. donovani and its evaluation for serodiagnosis ofVL. MATERIALS AND METHODS Cell-free Extract Leishmania donovani promastigotes (MHOM(
78/UR-6), grown in blood agar medium at 24 ± 1°C, were washed four times in phosphate © 1992 Liverpool School of Tropical Medicine
342
BHATTACHARYAETAL.
-66
-
45
-
36
-
29
-
24
-
20·1
-
14·2
et al., 1979) and the cell-free extract loaded on top of the column. After thorough washing with PBS containing 500 mM NaCI, the Con-A reactive glycoconjugate was eluted with 0· 5 M a-methylmannoside in PBS containing 100 mM NaCI. The eluents containing the glycoconjugate were pooled, dialysed, concentrated and kept at - 20°C for further study. SDS-PAGE Polyacrylamide gel electrophoresis was carried out under reducing conditions, using the Laemmli (1970) buffer system. After electrophoresis, the gels were fixed and stained for proteins and carbohydrates using I% Coomassie Brilliant Blue R-250 and periodic acid-Schiffs reagent (PAS), respectively. Dot-Radio Immunosorbent Assay (Dot-RIA) ANTIGEN DISC PREPARATION
{A)
{B)
{C)
Fig. 1. (A) SDS-PAGE (11%) separated Leishmania donovani cell-free extract, stained for proteins with Coomassie blue; (B) Coomassie blue and (C) periodic acid Schiff's stained SDS-PAGE (11%) of the isolated antigen (glycoconjugate). The molecular weight standards are indicated at the right.
Nitrocellulose filter discs (5 mm, 0·22 f.Lm pore size; Millipore) were dotted with the isolated glycoconjugate in 1 f.Ll volumes (900 ng), stabilized by drying for 15 minute at 56°C and placed individually into the wells of a microtitre plate. Non-specific binding sites were blocked by incubating each disc with 150 f.Ll 3% (w/v) bovine serum albumin in PBS (3% BSA-PBS) for 15 minutes at room temperature (r.t.). ANTIBODY INCUBATION
buffered saline, pH 7·1 (PBS). The final pellet was resuspended in PBS containing 1 mM CaCl 2, 1 mM MgC12 and 0·1% (w/v) phenyl methyl sulphonic fluoride, sonicated on ice (10, 30-second pulses; Labsonic 2000 sonicator) and centrifuged at 10 000 g for 20 minutes at 4°C and stored at -70°C. Protein and carbohydrate were measured by the Folin-Ciacaltau (Lowry et al., 1951) and phenolsulphuric acid methods (Dubois et al., 1956), respectively. Affinity Chromatography Cyanogen bromide activated Sepharose-4B coupled with Con-A (Cuatracasas and Anfinsen, 197la,b) was used to prepare the affinity column (5 x 1 em). The column was equilibrated with glycyl-glycine buffer, pH 7·1 (Lo
The blocking solution was aspirated off each antigen disc and replaced with 150 f.Ll of serum sample diluted 1:100 with 1% BSA-PBS in each well. The plate was shaken and then incubated at r.t. for 90 minutes. The sera were then. aspirated off and the discs washed three times with PBS containing 0·05% Tween 20 (Sol-A). INCUBATION WITH
125
1-LABELLED PROTEIN-A
After aspirating off the wash solution, 150 f.Ll of 1251-labelled protein-A (Markwell, 1982) (50 000 counts per minute) in BSA-PBS solution were pipetted into each well and the plate incubated for 90 minutes at r.t. The discs were again washed three times in Sol-A and then the radioactivity of each of the discs was measured with a gamma counter (ECIL, India).
SERODIAGNOSIS OF KALA-AZAR
343
4
10 . - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - ,
X X
" I
Normal
I
Malaria Kala-czar Leprosy Tuberculosis Patient-sera
Fig. 2. Dot-RIA. The purified antigen (glycoconjugate) was coated on nitrocellulose discs and incubated with a I: 100 dilution of human sera from normal control ( \7 ); patients with leprosy ( T ), malaria ( 0 ), tuberculosis (e) and kala-azar (x). The binding of the antibody with the leishmania! antigen was measured using radiolabelled protein-A.
Antisera Production Antisera were prepared by injecting rabbits with promastigotes (UR-6) with Freund's adjuvant (Rassam and Al-Mudhaffar, 1981). Patient Sera Sera were collected from patients with kalaazar, leprosy, tuberculosis or malaria, who had been admitted to the School of Tropical Medicine, Calcutta, India. Sera from healthy research workers were used as controls from an endemic area.
Reactivity of the Antigen with Normal and Patient Sera All the kala-azar patient sera were positive against the glycoconjugate in gel diffusion, counter-current and immunoelectrophoresis tests but sera from the other patients and control sera were negative (data not shown). The binding of the glycoconjugate antigen with the different sera was also examined by dot-RIA. The antigen was specifically recognized by the sera from patients with kala-azar (4-9 x 103 counts per minute); reaction with the other sera was at a very low level (0·09--0·19 x 103 counts per minute) (Fig. 2).
RESULTS DISCUSSION SDS-PAGE A single glycoconjugate antigen was eluted by a-methylmannoside from the Con-A column and revealed, by SDS-P AGE and Coomassie Blue staining, to have a molecular weight around 29-30 kDa [Fig. l(B)]. The antigen gave a broad band, ranging from 27-39 kDa [(Fig. 1(C)] after PAS staining. A few more antigens were eluted from the affinity matrix when it was treated with 0· 5 M a-methylmannoside containing SDS.
Affinity separation occupies a unique place amongst separation techniques. The present study was undertaken to isolate Con-A reactive antigens of an Indian strain of L. donovani and it was surprising that only a single antigen was found in the eluate. The antigen has a molecular weight of 27-39 kDa and the major portion of the antigen contains a carbohydrate moiety, as it could be stained with PAS. The protein content of the antigen appears to be very low, as only a
344
BHATTACHARYAETAL.
small portion of the antigen (29-30 kDa) was stained with Coomassie Blue. A few antigens of relatively low molecular weight (14-19 kDa) could be eluted with a-methylmannoside containing SDS, which indicates these antigens may have a higher affinity for Con-A than the one isolated. Dwyer ( 1981) indicated that the pellicular membrane of L. donovani contains at least 24 glycopeptidesfglycoproteins, of15-90·5 kDa, of which 15 contain mannose along with other sugar components; the only mannose-containing glycoprotein had a molecular weight of 60 kDa. It has also been reported that Con-A and lentillectin reacted, respectively, with 41-52 and 160-185 kDa components present in eight different species of Leishmania 0 affe and McMahon-Pratt, 1988). Besides these antigens, two other bands, in the region of 34 and 39 kDa were apparent in L. donovani infantum and Con-A reacted with two additional proteins of 76 and 92 kDa in L. major. Glycoproteins of 65 and 23-25 kDa were identified in L. donovani (IS) in a binding experiment with Con-A sepharose (Lepay eta!., 1983). It appears that the major glycoconjugate revealed in the present study of L. donovani
has not been reported before. However, an amphipathic glycoconjugate of 20-67 kDa, which migrates as a polydisperse band in SDS-PAGE, has been reported to be present in L. tropica (Handman et al., 1984). The exact identity of the glycoconjugate isolated from Indian L. donovani cannot be ascertained from the data available but it is surface-oriented, as it is labelled when isolated from surface-iodinated promastigotes (data not shown). Periodic acid oxidation of the antigen inhibits its antibodybinding activity. This Con-A specific antigen can be used, at very low concentrations, to distinguish sera from kala-azar patients from all other sera. No cross reaction was found with the other sera from patients with diseases prevalent in India. The isolated antigen appears to have good serodiagnostic potential, as tests with it were both sensitive and specific. M. Bhattacharya is a junior research fellow with the Indian Council of Medical Research. This investigation received partial financial support from the UNDP. The authors wish to thank Professor A. N. Bhaduri, Director, IICB, for his constant encouragement of this work. ACKNOWLEDGEMENTS.
REFERENCES CuATRECASAS, P. & ANFINSEN, C. B. (1971a). Affinity chromatography. Annual Review of Biochemistry, 40,
259-278. CuATRECASAS, P. & ANFINSEN, C. B. (1971b). Affinity chromatogrpahy. Methods in Enzymology, 22, 345-378. DUBOIS, M. K., CILLS, K. A., HAMILTON, J. K., BEBRS, P. A. & SMITH, F. (1956). Colorimetric method for determination of sugar. Analytical Chemistry, 28, 350. DwYER, D. M. (1974). Lectin binding saccharides on a parasitic protozoa. Science, 184,471-473. DWYER, D. M. ( 1977). Leishmania donovani, surface membrane carbohydrate of promastigotes. Experimental
Parasitology, 41, 341-358. DWYER, D. M. (1980). Isolation and partial characterization of surface membrane from Leishmania donovani promastigotes. Journal of Parasitology, 27, 17 5-182. DWYER, D. M. (1981). Structural, chemical and antigenic properties of surface membranes isolated from Leishmania donovani. In Biochemistry of Parasites, ed. Slutzky, G. M. pp. 10-28. Oxford: Pergamon Press. HANDMAN, E. & GODING, J. M. (1985). The Leishmania receptor for macrophage is a lipid containing glycoconjugate. EMBO Journal, 4, 329-336. HANDMAN, E., GREENBLATT, C. L. & GoDING, J. W. (1984). An amphipathic sulfated glycoconjugate of Leishmania: characterization with monoclonal antibodies. EMBO Journal, 3, 2301-2306. }AFFE, C. L. & McMAHoN-PRATT, D. (1988). The identification of membrane glycoconjugates in Leishmania species. Journal ofParasitology, 74, 548-561. }AFFE, C. L. & ZALIS, M. (1988). Use of purified parasite proteins from Leishmania donovani for the rapid serodiagnosis of visceral leishmaniasis. Journal of Infectious Diseases, 157, 1212-1220.
SERODIAGNOSIS OF KALA-AZAR
345
LAEMMLI, U.K. (1970). Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature, 227,680-685. LEPAY, D. A., NOGOCIRA, N. & CoHN, Z. (1983). Surface antigens of Leishmania donovani promastigotes. Journal ofExperimental Medicine, 157, 1562-1572. Lo, J.-T., MuKERJI, K., AWASTHI, Y. C., HANADA, K., SuzuKI, K. & SRIBASTAVA, S. K. (1979). Purification and properties of sphingolipid P-galactosidases from human placenta. Journal of Biological Chemistry, 254, 6710-6715. LOWRY, 0. H., RosENBROUGH, M., FARR, A. & RANDALL, R. (1951). Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry, 193, 265-275. MARKWELL, M.A. K. (1982). A new solid state reagent to iodinate proteins. 1. Conditions for the efficient labelling of antiserum. Analytical Biochemistry, 125, 427-432. RASSAM, M. B. & AL-MUDHAFFAR, S. A. (1981). Immunochemical characteristics of an Iraqi Leishmania donovani. Annals of Tropical Medicine and Parasitology, 75, 145-155. RussELL, D. G. & WILHELM, H. (1986). The involvement of the major surface glycoprotein (gp-63) of Leishmania promastigotes in attachment to macrophages. Journal of Immunology, 136, 2613-2620.
