An immunoenzymatic dot-ELISA for the detection of Giardia lamblia antigen in stool eluates of clinical cases of giardiasis.

A dot-ELISA technique for the detection of G. lamblia specific antigen in stool eluates of clinical cases of giardiasis was developed and evaluated employing monospecific antibodies to a G. lamblia specific coproantigen with a molecular mass of 66 kDa. The assay detected 22 (91.7%) of the 24 microscopically confirmed cases of giardiasis while none of the stool eluates from 20 patients with gastrointestinal parasites other than G. lamblia and 20 apparently healthy subjects had any detectable levels of G. lamblia-specific coproantigen. Of the 25 stool eluates from clinically suspected cases of giardiasis, 13 (52%) were found to contain G. lamblia-specific coproantigen. A 3-month-follow up of five of such cases where stool eluates has antigen detected by dot-ELISA assay, revealed the presence of G. lamblia cysts on repeated stool examinations. All the clinically suspected cases with detectable levels of G. lamblia-specific coproantigen by dot-ELISA were relieved of their clinical symptoms following metronidazole therapy. Single stool eluate examination by dot-ELISA was found to be sufficient to confirm the diagnosis. The dot-ELISA is an easy, rapid, sensitive and specific procedure for confirming the diagnosis of suspected cases of giardiasis. It should be a valuable diagnostic aid under field conditions as well as in the laboratory.

Depressed humoral immune responses to surface antigens of Giardia lamblia in persistent giardiasis.

The specific anti-plasma membrane- or surface-specific serum antibodies to Mr 82,000 antigen of Giardia lamblia trophozoites in cases of persistent and nonpersistent giardiasis in a pediatric population indicated significantly low concentrations of these antibodies in patients with persistent giardiasis. There was an insignificant difference in the amount of antibodies to whole trophozoite extract in persistent and nonpersistent giardiasis or asymptomatic carriers. The acute/nonpersistent cases had predominantly IgM class antigiardial antibodies, whereas persistent and asymptomatic carriers had antigiardial antibodies of the IgG class. The inability of persistent cases of giardiasis to clear infection appears to be related to low concentrations of antiplasma membrane/anti-Mr 82,000 antigiardial antibodies.

Acid phosphatase activity of promastigotes of Leishmania donovani: a marker of virulence.

Seven cloned lines of promastigotes of Leishmania donovani (UR 6) were isolated by limiting dilution. One clone, UR6-C25, failed to multiply inside the macrophages of line J774G8 and thus was labelled as avirulent. Another, UR6-C24, multiplied inside macrophages, had a virulence index as high as 93 +/- 9.8 and was thus labelled as highly virulent. The other five clones had variable degree of virulence indices ranging from 46.4 +/- 5.8 to 67.6 +/- 3.5. No significant difference in the degrees of attachment of virulent and avirulent populations of promastigotes to macrophages was observed, suggesting no difference in the ligand utilised by these populations for attachment to the macrophages. Acid phosphatase activity of cloned promastigotes correlated with the degree of virulence. These data suggest that acid phosphatase activity could be used as a marker to differentiate avirulent from virulent populations of promastigotes of L. donovani.

Immunoprotective behaviour of plasma-membrane-associated antigens of axenic Entamoeba histolytica.

Immunisation of golden hamsters with plasma-membrane-associated antigens of a virulent subline of axenic Entamoeba histolytica strain NIH 200 V, entrapped in multilamellar phosphatidylcholine liposomes (MPL) or Freund's complete adjuvant (FCA), afforded protection against intrahepatic challenge with axenic amoebic trophozoites of the same strain. Amoebic liver abscess developed in 86% and 80% of the animals that received empty liposomes or buffer emulsified in FCA but in none of the animals that received plasma-membrane-antigen vaccines. All the immunised animals had significantly higher levels (p less than 0.001) of antibodies to plasma-membrane components and significantly higher levels (p less than 0.001) of cellular sensitisation. Antibody-dependent macrophage-mediated cytotoxicity against trophozoites was also found to be significantly greater (p less than 0.001) in immunised animals. Liposome-entrapped antigens stimulated the immune system of the host as well as, or better than, antigens administered with FCA.

Immunochemical characterisation of a 29-Kda surface-associated molecule of Entamoeba histolytica and its recognition by serum from patients with amoebiasis.

A 29-Kda cytotoxic molecule of axenically-grown pathogenic Entamoeba histolytica (strain HM1) was purified from an amoebic extract by immuno-affinity chromatography with monoclonal antibodies. Immunoreactivity of the purified 29-Kda molecule altered significantly (p less than 0.01) after exposure to heat or trypsin, but remained unaltered after treatment with sodium metaperiodate. The 29-Kda molecule was recognised by serum from each of 13 patients with amoebic liver abscess. In an ELISA system, the molecule produced significantly higher (p less than 0.01) OD readings with these serum samples than with samples from asymptomatic cyst passers. No serum from healthy subjects or from patients with idiopathic ulcerative colitis or giardiasis had antibodies that reacted with the 29-Kda molecule. The immune response to the 29-Kda amoebic protein in man may indicate a specific role for this molecule in invasive amoebiasis.

The significance of free and immune-complexed hydatid-specific antigen(s) as an immunodiagnostic tool for human hydatidosis.

Micro-enzyme-linked immunosorbent assay (Micro-ELISA) systems were developed and evaluated for the detection of circulating (free or immune-complexed) hydatid antigens in the sera of patients with hydatidosis, by employing monospecific antibodies to hydatid-specific antigens of 8-kDa and 116-kDa. Fifteen (75%) of 20 sera from patients with hydatidosis had both 8-kDa and 116-kDa antigens freely circulating in their sera while three and two samples, respectively, had only 8-kDa or 116-kDa antigen. All the surgically confirmed cases of hydatidosis had detectable levels of both 8-kDa and 116-kDa circulating immune complexes in glycine HCl-treated sera. However, none of the sera from control subjects (patients with cysticercosis, ascariasis, ancylostomiasis, hymenolepiasis, amoebic liver abscess or viral hepatitis) had any detectable level of either type of circulating specific antigen. These results suggest that the demonstration of either 8- or 116-kDa antigen(s) in free or immune-complex form could confirm the diagnosis of hydatidosis.

Serum antibodies to giardial surface antigens: lower titres in persistent than in non-persistent giardiasis.

Antisera to two antigens of Giardia lamblia--plasma membrane (PM) protein and an affinity-purified surface antigen (SA56)--were raised in rabbits, and shown to agglutinate and kill trophozoites in vitro. These antibodies were also demonstrated by ELISA in the sera of paediatric patients with giardiasis. The titres of both antibodies were significantly higher in non-persistent (acute) and asymptomatic cases than in patients with persistent infection; and the latter group did not respond to anti-giardial therapy. The inability of this group to clear G. lamblia infection, in spite of therapy, may result from the low level of antibodies which mediate the killing of trophozoites.

Enzyme-linked immunosorbent assay for copro-diagnosis of giardiasis and characterisation of a specific Giardia lamblia antigen in stools.

An enzyme-linked immunosorbent assay (ELISA) has been evaluated for copro-diagnosis of giardiasis with anti-trophozoite antibody to capture specific Giardia lamblia stool antigen (GLSA), which was then detected by specific antibody conjugated with horseradish peroxidase. GLSA was demonstrated in stool eluates from all the 24 confirmed cases of giardiasis. None of the stool eluates from apparently healthy subjects or from patients carrying intestinal parasites other than G. lamblia had GLSA. Of the 25 microscopy-negative clinically suspected cases of giardiasis, 17 (68%) patients had GLSA in their stool eluates; these patients responded to anti-giardial therapy. The specific antigen was isolated and affinity-purified by the use of specific antibody; it had a Mr of 66 Kda, and its immunoreactivity was lost after treatment with heat or trypsin but unaltered by metaperiodate. ELISA seems to be a sensitive and specific method for copro-diagnosis of giardiasis, especially in highly suspected cases.

Immunogenicity of ribonucleic acid-protein fraction of Mycobacterium tuberculosis encapsulated in liposomes.

Immunologically potent RNA-protein extracted from Mycobacterium tuberculosis strain H37Ra, when entrapped in phosphatidylcholine multilamellar liposomes and injected into mice, induced both cellular and humoral immune responses. Significant protection against infection with M. tuberculosis H37Rv was also induced in the immunised mice, as monitored by (i) higher survival rates, (ii) decreased viable counts of M. tuberculosis H37Rv in lungs, livers and spleens, (iii) lower lung density, and (iv) lower root specific lung weight, in comparison with a control group of unimmunized mice.

Antibody-dependent macrophage-mediated cytotoxicity against Entamoeba histolytica.

Interactions between trophozoites of Entamoeba histolytica and peritoneal exudate macrophages from unsensitised and antigen-sensitised animals were studied in vitro. Normal macrophages killed trophozoites to some extent. This killing capacity was enhanced by prior sensitisation of the animals with specific antigen. Incorporation of anti-amoebic antiserum in the amoeba-macrophage mixture greatly enhanced the killing capacity of macrophages. Fraction one (F-I) of a crude amoebic extract was most effective in enhancing the cytotoxicity of macrophages by prior sensitisation and anti-F-I serum was the most effective antiserum. The cytotoxicity-inducing capacity of the immune serum resided in the IgG but not in the IgM fraction.

Immune responses to the protein, carbohydrate and lipid antigens of Nocardia asteroides in experimental nocardiosis in mice.

The intravenous injection of Nocardia asteroides into mice produced systemic nocardiosis involving all the vital organs. Infection of the kidneys and adrenals was more persistent and progressive than in other organs as evidenced by increased bacterial counts and histopathological findings. During the course of the experimental infection, no humoral immune response was detected against various protein antigens up to 4 weeks after challenge, but significant cell-mediated immunity (CMI) was found. Phospholipid antigens elicited only a humoral immune response. The increased CMI responses with protein antigens correlated well with the decreasing bacterial load, which suggested that CMI against proteins was important in the pathogenesis of this disease.

Immunological responses to protein, carbohydrate and lipid fractions of Nocardia asteroides in mice.

The protein, polysaccharide and phospholipid constituents of Nocardia asteroides have been partially purified and their immunogenicity studied in mice. Humoral and cellular immune responses were demonstrated against a crude cytoplasmic protein fraction (CPF). Two fractions of CPF were prepared on a Sephadex G-200 column; a high mol. wt fraction, fraction-1(F1) was capable of eliciting both types of immune responses, whereas fraction-2(F2) behaved more like a hapten. Phosphatides elicited only humoral responses whereas polysaccharides were non-immunogenic.

Uses and limitations in the demonstration of specific circulating immune complexes in patients with amoebiasis.

A micro-enzyme linked immunosorbent assay (micro-ELISA) has been evaluated as a diagnostic test to detect amoebic antigen in polyethylene glycol (PEG) precipitated circulating immune complexes (CIC) in sera from patients with amoebiasis. The immune complexes were captured on rabbit anti-amoebic IgG-coated wells of microtitration plates and the complexed antigen was detected by enzyme linked antihuman immunoglobulins. A titre of greater than 160 for the immune complexes was considered to be of clinical significance. The immunoassay detected amoebic, antigen-specific CIC in 35 (94.5%) of 37 patients with confirmed amoebic liver abscess. Twenty (55.5%) of 36 clinically suspected cases of amoebic liver abscess had amoebic antigen-specific CIC and responded favourably to anti-amoebic chemotherapy. Only two (20%) of 10 cases of non-dysenteric symptomatic intestinal amoebic infection had amoebic antigen-specific CIC. One (10%) of 10 patients with non-amoebic intestinal disorders also had amoebic antigen in CIC. However, none of 15 cases of non-amoebic hepatic disorders that included hydatid disease, metastatic adenocarcinoma, hepatocellular carcinoma, cholecystitis and choledocal cyst, 13 cases of rheumatoid arthritis and 25 apparently healthy subjects had amoebic antigen in CIC. The levels of the amoebic antigen-specific CIC did not correlate (p greater than 0.05) with either the number of abscess(es) or lobe(s) of the liver involved. However, the levels of antigen-specific CIC were higher (p less than 0.01) in patients with a liver size of more than 5 cm below the right costal margin. Antigen-specific CIC levels tended to decline or disappear during 3-6 months following completion of therapy.(ABSTRACT TRUNCATED AT 250 WORDS)

Specific antibodies in serum of patients with hydatidosis recognised by immunoblotting.

Hydatid fluids from sheep, goat, pig and man, after resolution by sodium dodecyl sulphate-polyacrylamide gel electrophoresis under reducing conditions, revealed at least 15 discrete polypeptide bands of 8-116 Kda. By ELISA, sera from all 20 cases of hydatidosis showed anti-hydatid antibody, but so did 11 (73%) of 15 sera samples from cysticercosis patients, eight (67%) of 12 sera from patients with other parasitic infections (amoebic liver abscess or hymenolepiasis) and one (4%) of 25 sera from healthy controls. Antibody to cysticercus antigen was found in 14 (93%) of 15 sera from cysticercosis patients, 17 (85%) of 20 sera from hydatid patients, six (50%) of 12 sera from patients with other parasitic infections and one (4%) of 25 sera from healthy controls. Sera from 17 (85%) of 20 hydatid patients, 11 (73%) of 15 cysticercosis patients and five (42%) of 12 patients with other parasitic infections had antibodies to both hydatid and cysticercus antigens. Sera from 20 surgically confirmed cases of hydatidosis reacted with 12 polypeptides of 8-116 Kda in Western immunoblot with hydatid antigens. Polypeptides of 16, 24, 38, 45 and 58 Kda were recognised by all hydatidosis sera but also by many sera from patients with other infections. However, polypeptides of 8 and 116 Kda were recognised by all hydatidosis sera but not by any sera from patients with cysticercosis, other parasitic infections or viral hepatitis, or from healthy controls. Thus, recognition of 8- and 116-Kda hydatid antigens by a patient's serum appears to be a specific test confirming a clinical diagnosis in an individual case of hydatidosis.

Leishmania donovani flagellum-specific epitopes mediating host-parasite interactions.

Monoclonal antibodies were developed against flagellar components of promastigotes of Leishmania donovani. The monoclonal antibody produced by clone A11 (mAb A11) recognised epitopes in the polypeptides with molecular weights of 86, 66 and weakly 53 kDa. These epitopes were found to be distributed along the flagellum and at the anterior end of promastigotes. The mAb A11 of IgG1 isotype strongly agglutinated the promastigotes of L. donovani. The prior treatment of promastigotes of L. donovani with mAb A11 resulted in a significant (P < 0.001) reduction in the attachment of promastigotes to cultured mouse peritoneal macrophages of line J774G8. The affinity-purified epitopes identified by mAb A11 were recognised by human sera of cases of visceral leishmaniasis. The present study suggest that flagellar-specific epitopes mediate host-parasite interactions and, therefore, the role of these epitopes in the disease process is speculated.

Uses and limitations of monoclonal antibodies to Giardia lamblia-specific 66-kDa copro-antigen in copro-immunodiagnosis of giardiasis.

Antigen-capture enzyme-linked immunosorbent assay for the detection of Giardia lamblia-specific antigen in stool eluates from clinical subjects employing monoclonal antibody directed at 66-kDa G. lamblia copro-antigen has been evaluated. The G. lamblia copro-antigen was detected in 67% (31 of the 46 cases) of stool eluates from clinical cases, while none of the stool eluates from subjects with other intestinal parasites or from apparently healthy individuals, had detectable levels of G. lamblia copro-antigen. Monoclonal antibodies secreted by clones B4C5 and D3F4 recognised the periodate-sensitive and -insensitive epitopes of 66-kDa G. lamblia specific copro-antigen, respectively. Eight (73%) of the 11 symptomatic cases of giardiasis had trypsin-/periodate-sensitive epitopes of 66-kDa copro-antigen while 9 (92%) of 11 of the symptomatic cases and asymptomatic G. lamblia cyst carriers had trypsin-sensitive periodate-insensitive G. lamblia specific copro-antigen. The data tend to suggest that detection of periodate-insensitive epitopes of G. lamblia copro-antigen would indicate the presence of the parasite while the detection of periodate sensitive epitopes of G. lamblia copro-antigen would suggest symptomatic active giardial infection.

Partial characterization of a 36-kDa antigen of Entamoeba histolytica and its recognition by sera from patients with amoebiasis.

A 36-kDa antigen of axenically grown pathogenic Entamoeba histolytica (HM1-IMSS) was eluted from the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)-resolved crude amoebic extract antigens. The immunoreactivity of this partially purified 36-kDa antigen with monoclonal antibody (MoAb) 3D(10) altered significantly (P<0.01) after heat and trypsin treatment but remained unaltered after treatment with sodium metaperiodate (P0.5), thereby indicating the protein nature of the epitope recognized by MoAb 3D(10). The epitope was found to be localized on the surface as well as in the cytoplasm of the E. histolytica trophozoites with the majority of it in the cytoplasm. In addition, this epitope was also found to be present on the cyst form of the parasite. The 36-kDa molecule was recognized by the sera from 29 (85%) of the 34 patients with amoebic liver abscess and five (83%) of the six patients with amoebic colitis. No serum samples from asymptomatic cyst passers, from patients with non-amoebic hepatic or intestinal disorders and apparently healthy subjects had antibodies that reacted with this 36-kDa molecule. The immune responses in man to this 36-kDa amoebic molecule indicate a potential specific role for this molecule in invasive amoebiasis.

Significance of detection of immune-complexed 8 kDa hydatid-specific antigen for immunodiagnosis of hydatidosis.

Three micro-enzyme-linked immunosorbent assay (micro-ELISA) systems were developed and evaluated for detection of specific free circulating antigen and circulating immune-complexes (CICs) of 8 kDa antigen in the sera of patients with hydatidosis. All (100%) the sera of 30 confirmed positive cases of hydatidosis had detectable levels of antigen in the acid-treated sera. However, 23 (77%) and 26 (87%) sera of 30 confirmed cases had free as well as CICs of 8 kDa antigen in the untreated and in the polyethylene glycol (PEG) precipitated sera, respectively. None of the sera from other patients with parasitic infections or viral hepatitis had any detectable levels of 8 kDa antigen in the untreated, acid-treated or PEG-precipitated serum samples. The investigations, therefore, suggested that the demonstration of circulating antigen employing monospecific antibodies to affinity purified 8 kDa antigen in acid-treated sera is more efficient as compared to detection of free circulating antigen or CICs in the untreated or in the PEG-precipitated sera which could provide a specific immunodiagnostic tool for ongoing hydatid infection.

Immune effector responses to an excretory-secretory product of Giardia lamblia.

The prior immunisation of mice with purified excretory-secretory product (ESP) led to a complete failure of Giardia lamblia colonisation following challenge inoculation of these animals with trophozoites. The prior immunisation of mice with ESP resulted in a significant stimulation of local immunity as evidenced by a significant enhancement of T helper/inducer activity along with a significant increase in immunoglobulin A-bearing cells. Further, the presence of anti-ESP antibodies in the serum of immunised as well as immunised-challenged animals indicated the stimulation of the systemic lymphoid system. This suggests that the ESP is highly immunogenic and it could be one of the major antigens of G. lamblia responsible for protection against the infection.

Low susceptibility of trophozoites of virulent Entamoeba histolytica to cellular and antibody-dependent cellular cytotoxicity by guinea-pig effector cells.

Cytotoxic potentialities of lymphocytes and macrophages were determined against trophozoites of virulent and avirulent sublines of Entamoeba histolytica in vitro. Guinea-pigs were immunized with antigens of both sublines to obtain stimulated effector cells and antiamoebic antibodies. Trophozoites of an avirulent/attenuated subline of E. histolytica (NIH200) were significantly more susceptible to killing by lymphocytes and macrophages through cellular and antibody-dependent cellular cytotoxic mechanisms. The resistance of virulent trophozoites to killing was attributed to their higher phagocytic ability and virulence characteristics. Electron microscopic studies of the interactions showed that contact between the effector cells and the trophozoites was essential for the cytolytic event which resulted in disintegration of the trophozoites. Antigens of trophozoites of the virulence subline of E. histolytica (NIH200 V) were effective in inducing cytotoxicity against both virulent and avirulent trophozoites: however, the reverse was not true.