Characterization of Entamoeba histolytica Antigens in Circulating Immune Complexes in Sera of Patients with Amoebiasis

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1 J HEALTH POPUL NUTR 2002 Sep;20(3): ICDDR,B: Centre for Health and Population Research ISSN Circulating immune $ complexes in amoebiasis 215 Characterization of Entamoeba histolytica Antigens in Circulating Immune Complexes in Sera of Patients with Amoebiasis K. Sengupta 1, P.K. Ghosh 1, S. Ganguly 2, P. Das 2, T.K. Maitra 1, and K.N. Jalan 1 1 Department of Immunology, Kothari Medical Centre and Research Institute, 8/3, Alipore Road, Calcutta , and 2 National Institute of Cholera and Enteric Diseases, P-33, C.I.T. Road, Beliaghata, Calcutta , India ABSTRACT Isolated circulating immune complexes (CICs) from sera of patients with amoebiasis were characterized to determine Entamoeba histolytica antigens that participate in the disease process. In total, 116 serum samples were collected before starting anti-amoebic therapy, and their CICs were isolated by differential polyethylene glycol precipitation. The presence of amoeba-specific antigens in CICs was detected by antigen capture enzyme-linked immunosorbent assay (ELISA) and by immunoblot assay. Antigen capture ELISA showed significantly higher optical density (p<0.001) in all patients with amoebiasis than in the normal healthy controls and patients of non-amoebic hepatic disorder. Immunoblot assay detected amoeba-specific CICs in all 18 patients (100%) with confirmed amoebic liver abscess, 28 (80%) of 35 patients with clinically-suspected amoebic liver abscess, and 18 (78.26%) of 23 patients with amoebic colitis. No patients with non-amoebic hepatic disorders and healthy control subjects had any detectable level of amoebic antigens in CICs. Immunoblot assay revealed E. histolytica antigens of relative molecular masses of 35, 56, 70, and 90 kda present in CICs of 64 of 76 patients with amoebiasis. The 35-kDa polypeptide was observed in 52 patients (81.25%). The results of the study suggest that the 35-kDa polypeptide antigen can be a diagnostic marker in active amoebiasis. Key words: Entamoeba histolytica; Amoebiasis; Circulating immune complexes; Antigens; Antibodies INTRODUCTION Infection due to Entamoeba histolytica is one of the major health problems in developing countries. Demonstration of circulating antibodies to E. histolytica is widely used as a marker of immunodiagnosis for amoebiasis (1-3). However, the presence of anti-amoebic antibodies has no correlation with clinical intensity of the infection (3-5). In endemic areas, anti-amoebic antibodies persist for Correspondence and reprint requests should be addressed to: Dr. T.K. Maitra Kothari Medical Centre and Research Institute 8/3, Alipore Road Calcutta India ghoshprabirk@hotmail.com Fax: years even after complete recovery from the disease (1). The antibody response to E. histolytica Gal/GalNAC adherence lectin has been observed in patients with amoebiasis (6). The presence of anti-amoebic antibodies does not differentiate present from past infection (5-7). Therefore, the demonstration of amoeba-specific antigens in blood and pus would provide a better and more reliable means of diagnosis of invasive amoebiasis (8-10). Several test methods, such as counterimmunoelectrophoresis (9), enzyme-linked immunosorbent assay (ELISA) (11), and solid-phase radioimmunoassay (12), have been used for detecting E. histolytica antigens in circulating immune complexes (CICs) from sera of patients with amoebiasis. Subsequently, the presence of E. histolytica-specific JHPN-0109:118-OP

2 216 J Health Popul Nutr Sep 2002 Sengupta K et al. Gal/GalNAC lectin antigen in serum of amoebic liver abscess (ALA) and patients with amoebic colitis has also been reported (13-15 ). Elevated levels of CICs have been reported in other parasitic diseases, such as bancroftian filariasis (16), Indian kala-azar (17), and onchocerciasis (18-19). Amoeba-specific antigens in CICs were demonstrated by solid-phase sandwich radioimmunoassay (12,20), anti-amoebic antibody-based micro-elisa test (11,21), and a spot test on antibody-coated strips of nitrocellulose membrane (22). However, until now, no report is available on the characterization of amoebaspecific antigen(s) in CICs from sera of patients with amoebiasis. This lacunae of information promoted us to characterize amoeba antigens in CICs. In the present investigation, attempts were made to characterize E. histolytica-specific antigenic polypeptides present in CICs in sera of patients with amoebiasis. MATERIALS AND METHODS Clinical samples Serum samples were collected from patients attending the Outpatients Department and/or Inpatients Department of Kothari Medical Centre, Calcutta, India. In total, 116 subjects who were not given any treatment prior to our collection of blood samples were included in the study. Consents were obtained from the patients and from the institutional review board for this study. Patients were categorized into the following five groups: Confirmed amoebic liver abscess (18 patients): Amoebic liver abscess (ALA) was diagnosed based on the following criteria: (i) patients had enlarged tender liver, febrile-associated toxaemia, and abscess demonstrated on ultrasound; (ii) patients had fever and pain in the epigastrium; (iii) bacteriologically sterile abscess aspirate; (iv) a positive serology for antibody to amoeba determined by an ELISA; E. histolytica antigen was also detected in aspirate by ELISA; and (v) improvement after treatment with an anti-amoebic drug. Suspected cases of amoebic liver abscess (35 patients): All these patients had enlarged palpable liver, toxaemia, fever, and pain in the epigastrium. Ultrasonographically, no abscess was demonstrated, and no aspiration was made in any of these cases. Patients showed high anti-amoebic antibody titre. Of the 35 patients given anti-amoebic treatment, 28 recovered. Amoebic colitis (23 patients): These patients were confirmed by sigmoidoscopy, stool examination, and biopsy examination. Smear of stool was prepared in 0.85% saline and Lugol s iodine, and was examined for presence of red blood cells within the trophozoite under microscope. The stool samples were inoculated in Robinson s medium (23) and examined after hours for growth of E. histolytica/e. dispar complex trophozoites. E. histolytica cysts were also concentrated by the formal ether method and inoculated in Robinson s medium, and were observed under microscope for E. histolytica/e. dispar complex trophozoites after hours of culture. In these patients, high anti-amoebic antibody titre in serum was observed. E. histolytica antigen in stool was detected by doubleantibody sandwich ELISA (24). Patients with non-amoebic hepatic disorder (20 patients): This group comprised four patients with hepatocellular carcinoma confirmed by fine needle aspirate cytology (FNAC) and seven patients with pyogenic liver abscess confirmed by bacteriological examination of pus. In this group, one patient had acute cholecystitis, and eight patients had acute viral hepatitis. Although no E. histolytica antigen was detected in liver abscess aspirate, poor anti-amoebic antibody titre in sera was observed. Control group (20 subjects): These patients were aged years, and their sex and profession matched those of the patients group. They had no recent history of diarrhoea and dysentery, and stool samples were negative for E. histolytica/e. dispar complex trophozoites under microscope, which was also confirmed by culture in Robinson s medium. No E. histolytica/e. dispar-specific stool antigens were detected by double-antibody sandwich ELISA (24). Microscopy and culture Direct microscopic observation of collected liver abscess pus from patients with amoebic liver abscess revealed that 1 of 18 (5.87%) liver abscess specimens was positive for E. histolytica trophozoites, and their growth in culture medium was also observed (23). In the aspirate of patients with non-amoebic hepatic disorder, we did not observe any E. histolytica trophozoites either by direct microscopic examination or by their growth in culture medium (23). Smears of stool samples prepared in 0.85% saline and Lugol s iodine obtained from 18 ALA patients, 35 suspected ALA, and 20 patients of non-amoebic hepatic disorder did not show any E. histolytica/e. dispar complex

3 Circulating immune complexes in amoebiasis 217 trophozoites or cyst under direct microscopic examination or by their growth in Robinson s medium after hours of culture. Direct microscopic examination of stool samples showed E. histolytica/ E. dispar complex trophozoites and cyst in 19 of the 23 patients with amoebic colitis. Four patients showed only E. histolytica/e. dispar complex cyst. Stool culture was positive for E. histolytica/e. dispar complex trophozoites in 13 patients. Detection of stool antigen A monoclonal antibody-based sandwich ELISA was performed to detect stool antigen as described earlier (24). Briefly, a polystyrene 96-well ELISA plate (Nunc, Denmark) was coated with 3 µg/well rabbit anti-e. histolytica IgG (found optimal as determined in checker board titration) in carbonate-bicarbonate buffer ph 9.6 (sodium carbonate 1.59 g/l, sodium hydrogen carbonate 2.93 g/l in distilled water) and kept overnight at 4 o C. Excess antibody was removed by washing the plate twice with phosphate-buffered saline ph 7.2 containing 0.05% Tween-20 (PBST). Non-specific sites were blocked with 3% gelatin for two hours. The wells were then washed with PBST, and 100 µl of 1:50 dilution of faecal extract (1 g of faecal sample homogenized in 2 ml of PBS ph 7.2 centrifuged and collected supernatant) from which antigen was to be detected was added and incubated at 37 o C for one hour. The wells were washed thoroughly for four times with PBST and then incubated with 3 µg/100 µl/well (optimal concentration determined by checker board titration) of MAb NICED 11 for one hour at 37 o C. After thorough washings (5 times with PBST), goat anti-mouse IgG labelled with HRP was added and incubated for one hour. The plate was washed thoroughly, and substrate containing 10 mg of o-phenylenediamine dihydrochloride and 10 µl of 30% H 2 O 2 per 25 µl was added. This reaction was developed in the dark for 15 minutes, and then optical density was measured at 492 nm. E. histolytica/e. dispar antigen was not present in the stool samples of 18 ALA patients, 35 suspected ALA patients, and 20 patients of non-amoebic hepatic disorder, and showed optical density equal to a normal healthy individual. However, patients with amoebic colitis showed the presence of E. histolytica/e. dispar-specific stool antigens 5-10 times higher optical density (p<0.001) than normal healthy individuals. Parasite antigen and anti-serum Amoeba antigen was prepared from axenic E. histolytica (strain HM1: IMSS) grown in TYI-S-33 medium (25). Briefly, E. histolytica trophozoites from 48-hour old culture were harvested and suspended in hypotonic buffer (sodium phosphate buffer, ph 7.4 containing 1 mm CaCl 2, 1 mm MgCl 2, 1 mm PMSF, 3 mm NEM, and 2 µm leupeptin). The cell suspension was homogenized using Potter-Elvejhem homogenizer, and the homogenate was then centrifuged at 10,000 g for 30 minutes. The clear supernatant was saved as the soluble antigen, and the yield of total protein was quantitated by the method of Bradford (26) using bovine serum albumin as the standard. Antibodies to soluble antigen of E. histolytica were raised in an albino rabbit (weighing kg) by giving four subcutaneous injections, at an interval of seven days, containing 2 mg of soluble antigen emulsified in an equal volume of Freund s adjuvant and two intravenous doses with antigen alone. Seven days after the last dose, the rabbit was bled, and serum was separated. Immune sera were pooled, and the antibody titre was determined by ELISA. The immunoglobulin G fraction was purified from the pooled immune serum by affinity column chromatography using protein-a Sepharose CL-4B matrix (24). Enzyme-linked immunosorbent assay ELISA was performed to determine anti-amoebic antibody titres in clinical samples (3). Briefly, 96-well microtitre ELISA plates (Nunc, Denmark) were coated with 2 µg/well E. histolytica soluble antigen in 50 mm carbonate-bicarbonate buffer ph 9.6 (sodium carbonate 1.59 g/l, sodium hydrogen carbonate 2.93 g/l in distilled water) and kept at 4 o C overnight. The plates were washed twice with PBS ph 7.2 containing 0.05% Tween- 20 (PBST), and 200 µl of bovine serum albumin (BSA) was then added to 1% in PBST for one hour. After incubation, the plates were washed twice, and test sera were then added to the first well of each row at 1:50 dilution in PBST containing 1% BSA and applied serial dilutions. The plates were incubated at room temperature for one hour. The wells were then washed four times with PBST, and 100 µl of peroxidase-conjugated antihuman IgG (Jackson Immuno Research Laboratories, USA) was added and incubated for one hour. After thorough washings with PBST, citrate buffer containing 10 µg of o-phenylenediamine dihydrochloride (OPD) and 10 µl of 30% H 2 O 2 per 25 ml was then added. The

4 218 J Health Popul Nutr Sep 2002 Sengupta K et al. reaction was developed in the dark for 15 minutes, and optical density was measured in an ELISA reader at 492 nm. Polyethylene glycol precipitation of circulating immune complexes CICs of clinical samples were isolated by precipitation of sera with polyethylene glycol (PEG MW 8000, Sigma, USA) as described earlier (27). Briefly, 2 ml of serum was mixed with an equal volume of 5% PEG (final concentration 2.5%) in 0.1 M borate buffer, ph 8.5 and incubated at 4 C overnight. The precipitates were separated by centrifugation at 1,500 g for 45 minutes at 4 C. Thereafter, the precipitates were washed six times with 2.5% PEG in borate buffer and solubilized in 0.2 ml PBS, ph 7.2. The suspension containing immune complexes was dialyzed against PBS, ph 7.2 and stored at -80 C in aliquots. The protein content was measured by the method of Bradford (26). Antigen capture ELISA The method used was a modification of double-antibody sandwich ELISA described earlier (24). Briefly, polystyrene 96-well ELISA plates (Nunc, Denmark) were coated with rabbit anti-e. histolytica IgG (2 µg/ well affinity-purified IgG was found optimal as determined by checker board titration) in 50 mm carbonate-bicarbonate buffer ph 9.6. After incubation at 4 C overnight, the wells were washed with PBST, and the non-specific sites were blocked with 2% gelatin (Sigma Chemical, USA) in PBST for one hour at room temperature. To each well, 100 µl of CICs containing 50 µg of immune complexes after thorough washings with PBST was added and incubated for two hours at 37 C. The wells were washed four times with PBST, and 100 µl of peroxidase-conjugated anti-human IgG (Jackson Immuno Research Laboratories, USA) was added for one hour. After thorough washings with PBST, substrate buffer containing 10 mg of o-phenylenediamine dihydrochloride and 10 µl of 30% H 2 O 2 per 25 ml was added. The colour reaction was developed in the dark and measured an in ELISA reader at 492 nm. Sodium dodecyl sulphate polyacrylamide gel electrophoresis and immunoblot assay The protein profiles of CICs were analyzed by 10% polyacrylamide gel under denaturing condition using Laemmli s discontinuous buffer system (28). Briefly, 100 µg of protein of each CIC sample was boiled in sodium dodecyl sulphate sample buffer. Following electrophoresis, the protein bands were visualized by staining the gel with 0.2% Coomassie blue. The electrophoretically-separated proteins of CICs were transferred onto nitrocellulose strips (29) and incubated with 3% gelatin to block the non-specific binding sites. The washed strips were incubated with 50 µg/ml of purified IgG of immune rabbit serum for two hours at room temperature. The antigen-antibody reaction was probed by anti-rabbit IgG-HRP (Jackson Immuno Research Laboratories, USA). The polypeptide bands were visualized by colour reaction developed in 0.05% 3-3' diamino benzedene dihydrochloride (Sigma, USA) in 20 mm Tris HCl buffer, ph 7.4 containing 0.03% H 2 O 2. RESULTS Determination of anti-amoebic antibody titre in sera The serum anti-amoebic antibody titres were detected in the patients group by ELISA and were plotted in Figure 1. Sera of all patients from confirmed ALA, suspected ALA, and amoebic colitis had an anti-amoebic Fig. 1. Antibody titres (x10 3 ) Anti-amoebic antibody titres in sera of patients and healthy control subjects. Each point represents serum antibody titre of patients with confirmed amoebic liver abscess (Panel A), suspected amoebic liver abscess (B), amoebic colitis (C), non-amoebic hepatic disorder (D), and healthy control (E) n=18 n=35 n=23 n=20 n=20 antibody titre of 2000, whereas all the sera from control subjects and patients with non-amoebic hepatic disorders had a titre of Therefore, in our assay, a titre of 2000 was considered as the cut-off limit.

5 Circulating immune complexes in amoebiasis 219 Determination of E. histolytica antigen in circulating immune complex Antigen capture ELISA revealed that all 18 (100%) patients with confirmed amoebic liver abscess had amoebic antigens in CICs with a mean optical density of 2.2; 23 (65.71%) of the 35 suspected ALA patients had amoebic antigen-specific CICs with a mean optical density of 1.20; and 17 (73.91%) of the 23 patients with amoebic colitis showed amoebic antigen in CICs with a mean optical density of Amoeba antigen-specific CICs were detected neither in 20 patients with nonamoebic hepatic disorders nor in 20 healthy individuals, the observed mean optical densities of whom were 0.55 and 0.25 respectively. Immunoblot assay Immunoblot assay revealed E. histolytica antigenspecific major immunoreactive polypeptides at 35, 56, The presence of amoeba antigen-specific polypeptides was observed in CICs isolated from all 18 patients (100%) of confirmed ALA cases, 28 (80%) of the 35 suspected ALA patients, and 18 (78.26%) of the 23 patients of amoebic colitis. None of the patients with non-amoebic hepatic disorders nor the healthy control subjects showed amoebic antigens in CICs (Table). Immunoblot reactions showing amoeba-specific antigen polypeptides present in CICs in sera of patients of confirmed ALA, suspected ALA, amoebic colitis, nonamoebic hepatic disorder, and control group respectively are presented in Figure 2. The electrophoreticallyseparated CIC proteins of ALA subjects transferred onto nitrocellulose strips were also incubated with anti-human IgG HRP (Jackson Immuno Research Laboratories, USA) in a similar manner. However, no polypeptide band was seen (Fig. 3). Table. Recognition of amoebic antigen-specific circulating immune complexes in different groups of patient sera Patient group No. of sera tested No. (%) of sera positive in antigen capture ELISA No. (%) of sera positive in immunoblot assay Confirmed amoebic liver abscess (100) 18 (100) Suspected amoebic liver abscess (65.71) 28 (80) Amoebic colitis (73.91) 18 (78.26) Non-amoebic hepatic disorders Healthy control Fig. 2. Immunoblot assay represents E. histolyticaspecific polypeptide antigens in CIC obtained from patients with confirmed amoebic liver abscess (Panel A); suspected amoebic liver abscess (B); amoebic colitis (C); non-amoebic hepatic disorder (D); and healthy control (E). Positions of protein molecular weight markers are shown in the left (M). The antigen antibody reaction was probed by anti-rabbit IgG-HRP kda Fig. 3. Immunoblot assay represents CIC proteins of ALA patients incubated with anti-human IgG-HRP, molecular marker (M), and ALA patients (C) kda M A B C E F 29 70, and 90 kda in CICs obtained from the patients of confirmed ALA, suspected ALA, and amoebic colitis. M C

6 220 J Health Popul Nutr Sep 2002 Sengupta K et al. DISCUSSION The elevated level of CICs in sera of patients and their disappearance after anti-amoebic therapy during infection due to E. histolytica were reported (21). These findings clearly suggest the importance of CICs in diagnosis of current amoebic infection and also relationship of CICs with pathogenesis of amoebiasis. In the present study, an attempt was made to characterize antigenic polypeptides of E. histolytica that participate in CICs in sera of patients with amoebiasis. It was observed that all symptomatic amoebic patients contained an anti-amoebic antibody titre of 2000, but sera of non-amoebic patients never showed a titre of more than Thus, this value can be used as the cut-off limit from amoebic to non-amoebic cases. The presence of serum antibodies to amoebae means tissue invision by E. histolytica in the past or present. The prevalence of such antibodies in asymptomatic individuals of an endemic area can differentiate from symptomatic to asymptomatic amoebiasis. Antigen capture ELISA showed significantly high (p<0.001) E. histolytica-specific circulating antigen in CICs of ALA patients, patients of suspected amoebic liver abscess, and patients with amoebic colitis compared to patients of non-amoebic hepatic disorders and normal healthy controls. Thus, antigen capture ELISA was useful in diagnosis of invasive amoebiasis. In consequence to our study, Mohimen et al. reported the presence of CICs in patients of asymptomatic E. histolytica carriers by sandwich radioimmunoassay and solid-phase antibodyspecific RIA method (27). They showed a positivity of 61.53% and 76.92% by sandwich RIA and solid-phase antibody-specific RIA respectively. Vinayak et al. (21) reported 55% and 20% amoebic antigen-specific CICs in suspected ALA and symptomatic non-dysenteric intestinal amoebic patients respectively by micro-elisa and suggested E. histolytica antigen-specific CICs in sera of patients with amoebiasis to be a reliable diagnostic approach in amoebiasis (10,11,30). We also observed that affinity-purified anti-e. histolytica antibodies did not cross-react with other parasitic antigens, such as Giardia lamblia, Ascaris lumbricoides, and Taenia solium as revealed by ELISA (data not shown). This suggests the specificity of the assay for amoebic antigens. In total, 64 of the 76 patients of amoebiasis (confirmed ALA, suspected ALA, and amoebic colitis) revealed positive immune reactions at 35, 56, 70, and 90 kda. However, the presence of 35-kDa polypeptide was observed in 52 patients (81.25%), which probably indicates its important role in systemic immune response during the disease. E. histolytica-specific polypeptides were detected in 28 of the 35 clinically-suspected ALA patients, whereas, by ELISA, amoeba-specific antigens were detected in 23 patients only. Thus, immunoblot analysis offered a greater sensitivity than the micro- ELISA test. Moreover, all these 28 patients, who showed amoebic polypeptides in CICs, responded well to antiamoebic therapy with metronidazole. Whereas, seven patients, who did not show any E. histolytica-specific polypeptides in CICs, failed to respond to metronidazole. Five patients with amoebic colitis did not show any amoebic polypeptides in CICs by immunoblot assay. However, E. histolytica/e. dispar complex trophozoites/ cyst were observed under direct microscopic observation and in culture medium; the reason may be the difference in tissue invision. Several major amoebic antigens, such as 29/30 kda peripheral membrane protein (31), 52 kda serine-rich surface protein (32), 125 kda variable surface antigen (33), and 170 kda Gal/GalNAC lectin, have been reported by various authors (34). These reported polypeptides were recognized by immune sera of patients with amoebic liver abscess (31-33). Ravdin et al. reported that immunoblotting of E. histolytica Gal/ GalNAC adherence lectin, purified by monoclonal antibody immunoaffinity chromatography, confirms the immunogenic specificity of preparation where only 170 kda heavy subunit of lectin was recognized by human anti-amoebic antibodies (6). Further, the presence of E. histolytica-specific Gal/GalNAC lectin antigen in the sera of patients with current amoebiasis which disappears after therapy has also been reported (13). In our study, we did not observe any of these reported major antigenic polypeptides present in CICs of patients with amoebiasis, and the reason for this is not known to us. A possible explanation could be that Calcutta is endemic for amoebiasis; so, infection and cure go side by side; antiamoebic and antibiotic drugs are used indiscriminately, making it difficult to obtain true treatment history. In summary, we have characterized amoebic polypeptides in CICs in sera of patients with amoebiasis by immunoblot assay. Interestingly, 35-kDa polypeptide antigen was observed in the majority (~81%) of patients with amoebiasis. The significance of recognition of these antigens needs further investigation.

7 Circulating immune complexes in amoebiasis 221 REFERENCES 1. Healy GR, Visvesvara GS, Kagan IG. Observations on the persistence of antibodies to E. histolytica. Arch Invest Med (Mex) 1974;5(Suppl 2): Knobloch J, Mannweiler E. Development and persistence of antibodies to Entamoeba histolytica in rat and its comparison with the serological responses of amoebic liver abscess. Am J Trop Med Hyg 1983;32: Ghosh PK, Gupta S, Leon LR, Ghosh R, Ordaz BHR, Ortiz-Ortiz L. Intestinal amoebiasis: antibody secreting cells and humoral antibodies. J Diarrhoeal Dis Res 1998;16: Vinayak VK. The specificity of indirect haemagglutination test in the diagnosis of amoebiasis. Ind J Prev Soc Med 1975;6: Thomas V, Sinniah B, Leng YP. Assessment of the sensitivity, specificity and reproducibility in the indirect immunofluorescent technique for the diagnosis of amebiasis. Am J Top Med Hyg 1981;30: Ravdin JI, Jackson TFHG, Petri WA, Murphy CF, Ungar BLP, Gathiram V et al. Association of serum antibodies to adherence lectin with invasive amoebiasis and asymptomatic infection with pathogenic Entamoeba histolytica. J Infect Dis 1990;162: Abioye AA, Lewis EA, McFarlane H. Clinical evaluation of serum immunoglobulins in amoebiasis. Immunology 1972;23: Davis A, Pawlowski ZS. Amoebiasis and its control. Bull World Health Organ 1985;63: Mahajan RC, Ganguly NK, Chitkara NL, Dutta DV, Chuttani PN. Detection of amoebic antigen in liver pus by counter immunoelectrophoresis a preliminary report. Ind J Med Res 1974;62: Gandhi BM, Irshad M, Acharya SK, Tandon BN. Amebic liver abscess and circulating immune complexes of Entamoeba histolytica proteins. Am J Trop Med Hyg 1988;39: Vinayak VK, Purnima S, Singh K, Venkatwswarlu K, Nair CK, Mehta SK. Specific circulating immune complexes in amoebic liver abscess. J Clin Microbiol 1986;23: Pillai S, Mohimen A. A solid-phase sandwich radioimmunoassay for Entamoeba histolytica proteins and detection of circulating antigens in amoebiasis. Gastroenterology 1982;83: Haque R, Neville LM, Hahn P, Petri WA. Rapid diagnosis of Entamoeba infection by using Entamoeba and Entamoeba histolytica stool antigen detection kits. J Clin Microbiol 1995;33: Karki BMS, Parija SC. Co-agglutination test for the detection of circulating antigen in amebic liver abscess. Am J Trop Med Hyg 1999;60: Abd-Alla MD, Jackson TFGH, Gathiram V. Differentiation of pathogenic Entamoeba histolytica infections from non-pathogenic infections by detection of galactose-inhibitable adherence protein antigen in sera and feces. J Clin Microbiol 1993; 31: Kobayashi M, Niimura M, Kanazawa T, Husky MK, Malagueno E, Santana JV. Detection of micro filarial antigen in circulating immune complex from sera of Wuchereria bancrofti infected individuals. Am J Trop Med Hyg 1997;57: Sanyal T, Ghosh DA, Sarkar D. Immunoblotting identifies an antigen recognized by anti gp63 in the immune complexes of Indian kala-azar patient sera. Mol cell Biochem 1994;130: Chandrashekar R, Ogunrinade AF, Henry RW. Onchocerca volvulus: monoclonal antibodies to immune complex-associated parasite antigens. Exp Parasitol 1993;77: Chandrashekar R, Ogunrinade AF, Alvarez RM, Kale OO, Weil GJ. Circulating immune complexassociated parasite antigens in human onchocerciasis. J Infec Dis 1990;162: Pillai S, Mohimen A, Mehra S. A radioimmunoprecipitation polyethylene glycol assay for circulating Entamoeba histolytica antigens. J Immunol Methods 1982;55: Vinayak VK, Shandil RK, Bansal V, Singh K, Bhasin DK, Kaur U. Uses and limitations in the demonstration of specific circulating immune complexes in patients with amoebiasis. J Med Microbiol 1990;32: Gandhi BM, Irshad M, Acharya SK, Tandon BN. A simple spot test for circulating Entamoeba histolytica antigen-antibody complexes in patients with amoebic liver abscess. Indian J Med Res 1989;89: Robinson GL. The laboratory diagnosis of human parasitic amoebae. Trans R Soc Trop Med Hyg 1968;62: Sengupta K, Das P, Johnson TM, Choudhury PP, Das D, Nair GB. Production and characterization of monoclonal antibodies against a highly immunogenic fraction of Entamoeba histolytica (NIH: 200) and their application in the detection of current amoebic cases. J Euk Microbiol 1993; 40:722-6.

8 222 J Health Popul Nutr Sep 2002 Sengupta K et al. 25. Diamond LS, Harlow DR, Cunnick CC. A new medium for axenic cultivation of Entamoeba histolytica and other Entamoeba. Trans R Soc Trop Med Hyg 1978;72: Bradford M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 1976;72: Mohimen A, Maitra TK, Jalan KN, Mehra S. A specific solid-phase assay for the detection of immune complexes containing Entamoeba histolytica antigens. J Immunol Methods 1989; 117: Laemmli UK. Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 1970;227: Towbin H, Straekelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 1979;76: Nuti M, D Amelio R, Seminara R, Palmisano L, Aiuti F. Circulating immune complexes in amoebiasis (letter). Trans R Soc Trop Med Hyg 1982;76: Torian BE, Flores BM, Stroeher VL Hagen FS, Stamm WE. cdna sequence analysis of a 29kDa cysteine rich surface antigen of Entamoeba histolytica. Proc Natl Acad Sci USA 1990;87: Stanley SL, Becker A, Kunz-Jenkins C, Foster L, Li E. Cloning and expression of a membrane antigen of Entamoeba histolytica possessing multiple tandem repeats. Proc Natl Acad Sci USA 1990;87: Edman U, Meraz MA, Rausser S, Agabain N, Meza I. Characterization of an immunodominant variable surface antigen from pathogenic from nonpathogenic Entamoeba histolytica. J Exp Med 1990; 172: Tannich E, Ebert F, Hortsman RD. Primary structure of 170 kda surface lectin of Entamoeba histolytica. Proc Natl Acad Sci USA 1991;88: