A novel exo-antigen-based ELISA for the detection of canine leishmaniasis

Dogs which are infected with leishmania parasites serve as major reservoir hosts for zoonotic visceral leishmaniasis. The incidence of zoonotic visceral leishmaniasis is rising in many countries. This may be associated with the continuing drift of people and their pets from rural areas into peri-urban settings, particularly at the fringe of large cities. At the same time, there is evidence of adaptation of sand fly vectors to these urban settings. This has created an alarming situation because, even though domestic and stray dogs may be infected, many remain asymptomatic but are still highly infectious to the sand fly vectors and thus pose a serious threat to human health. Over half of the infected dogs have asymptomatic infections and current assays are not sensitive enough under field conditions to distinguish asymptomatic from symptomatic dogs. There is an urgent need for a specific and sensitive screening tool for use in the field. We have previously demonstrated that promastigote exo-antigen-based ELISAs can be used in the specific diagnosis of human visceral leishmaniasis (HVL). A cocktail of exo-antigens prepared from three species (L. infantum, L. donovani, and L. major) was used to develop and optimize a canine ELISA assay. Serum samples from dogs with a variety of pathological conditions but living in a non-leishmania endemic area were used as negative controls and their reactivity was used to determine a cut-off value for the ELISA. Samples from dogs residing in a leishmania endemic area were tested in parallel using direct agglutination (DAT), immunofluorescence (IFAT), and ELISA. The ELISA results correlated closely (100%) with the clinical symptoms, and were elevated in one asymptomatic dog. This sample was also found to be positive by IFAT. Based on its sensitivity and specificity, the cocktail exo-antigen-based ELISA may prove useful, even at 1:2,000 serum dilutions, for screening dogs in different geographical regions of the world.     

Standardization of the dot enzyme-linked immunosorbent assay (Dot-ELISA) for human visceral leishmaniasis

The Dot-ELISA, a rapid, visually read micro enzyme immunoassay for visceral leishmaniasis utilizing minute volumes of antigen "dotted" on nitrocellulose filter discs and precipitable chromogenic substrate, was analyzed under a variety of experimental parameters. Raising assay incubation temperatures from 23 degrees C to 28 degrees C resulted in titer increases in three of five leishmaniasis patient sera; at 37 degrees C, all five patient sera and one of five normal human sera showed titer increases. The amount of antigen used could be reduced 50% by incubating patient serum overnight at 4 degrees C. Antigen discs stored at - 20 degrees C were optimally reactive with leishmaniasis sera over a 270-day period. Antigen discs stored at 4 degrees C and 23 degrees C showed reproducible titer decreases at 90 days. Aging either peroxidase-conjugated antibody or substrate for up to 28 days at 4 degrees C did not adversely affect titers of positive and negative control sera and reagent controls. Activated substrate stored at 23 degrees C was optimally reactive in the assay for at least 24 hours. No changes in titers of positive and negative control sera or nonspecific reactions in reagent controls occurred when using different brands of microtiter plates. The long shelf lives and stabilities of Dot-ELISA antigen and reagents indicate this test should prove useful both in the laboratory and in the field.     

A new 'total' activin B enzyme-linked immunosorbent assay (ELISA): development and validation for human samples

Background and objective  There are currently no sensitive and specific assays for activin B that could be utilized to study human biological fluids. The aim of this project was to develop and validate a 'total' activin B ELISA for use with human biological fluids and establish concentrations of activin B in the circulation and fluids from the reproductive organs.
Design  The new ELISA was validated and then used to measure activin B levels in the circulation of healthy participants, IVF patients, pregnant women and in ovarian follicular fluid and seminal plasma.
Patients and measurements  Healthy adult subjects ( n  = 143), subjects from an IVF clinic ( n  = 27) and pregnancy groups ( n  = 29) were sampled.
Results  The sensitivity of the assay was 0·019 ng/ml. Validation of the activin B ELISA showed good recovery (90·7 ± 9·8%) and linearity in biological fluid and cell culture media and low cross-reactivity with related analytes (inhibin B = 0·077% and activin A = 0·0034%). There was a negative correlation between activin B concentration ( r  = −0·281, P  < 0·011) and females with increasing age. Patients attending IVF clinics had significantly lower levels of activin B compared with gender-matched control subjects. Ovarian follicular fluid and seminal plasma had 50–80 fold higher levels of activin B (mean = 5·35 and 3·66 ng/ml respectively) than sera (mean = 0·071 ng/ml).
Conclusions  This fully validated ELISA for activin B offers a tremendous utility for measuring this protein in a variety of normal physiological processes and in various clinical pathologies.     

Use of the enzyme-linked immunosorbent assay (ELISA) for detection of circulating antibodies to human leishmaniasis

In an attempt to lay the background work of immunodiagnosis of human leishmaniasis in Taiwan, the usefulness of enzyme-linked immunosorbent assay (ELISA) for detection serum antibodies in large regional groups was studied. Antigens were prepared from promastigotes of Leishmania tropica major recently isolated in our laboratory by Chao. One hundred and sixty-five serum samples were collected from medical students of National Yang-Ming Medical College (YM), Taipei, 61 from residents of San-hsing District (SH), I-lan County, 9 from residents of Wu-lai District (WL), Taipei County, 2 from patients with cutaneous leishmaniasis, 6 from laboratory workers in the Department of Parasitology (LW), National Yang-Ming Medical College, and 1 from a patient with the history of hypersensitive to mosquito bites. The result of the ELISA were analyzed statically. YM students had the lowest serum antibody levels to this parasite. The laboratory workers and the SH residents had a significantly higher antibody prevalence rate than the YM students, but a significantly lower rate than positive patients. Positive/pseudopositive rate in non-patient groups was 0.82%. Tracing of these two suspected cases are in progress. The regional high antibody levels in the SH residents suggest that a risk of human leishmaniasis may be significant in the population tested. Using our antigen preparations we demonstrated that the ELISA is sensitive, rapid, and easy to perform and is therefore useful in screening and detecting of circulating anti-leishmania antibodies in serosurvey of large sample size.     

ELISA system for detection of immune responses to FVIII: a study of 246 samples and correlation with the Bethesda assay

Inhibitors of FVIII are usually IgG polyclonal antibodies that develop as alloimmune responses in patients with congenital haemophilia A or as autoimmune responses resulting in acquired haemophilia. Their recognition can be difficult, especially when the titre is low. Furthermore, results from a Bethesda assay often require several days as samples are referred to a specialty laboratory. The aim of this study is to assess the utility of an ELISA system for detecting immune responses to FVIII. A total of 246 plasma samples submitted from 176 individuals with immune responses to FVIII, as verified with the Bethesda assay, and samples from 50 control subjects were tested for the presence of FVIII-specific IgG using an ELISA-based assay. Paired sera from 18 of the patients were also tested by the ELISA. Of the 246 samples that were positive for a FVIII inhibitor by the Bethesda assay, 235 (95.5%) were also positive by ELISA. The regression coefficient, using Log BU was r = 0.82. The correlation data were strengthened when 27 inhibitor samples were diluted further. There was a strong correlation between ELISA results for the 18-paired serum and plasma samples (r = 0.99). There is a strong correlation between the ELISA and Bethesda methods in detecting immune responses to FVIII. The ELISA provides rapid screening that could be available well in advance of confirmation by the Bethesda assay.     

Micro enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of New World leishmaniasis

Of 21 confirmed cases of New World leishmaniasis, 16 exhibited antibody to antigens of the promastigote of Leishmania braziliensis panamensis by the enzyme-linked immunosorbent assay (ELISA). Comparison of antibody titers obtained by ELISA with titers obtained by indirect immunofluorescence using an amastigote substrate confirmed that the sensitivities of the two techniques were within the same range (r = 0.80). Although sera from patients with New World leishmaniasis failed to react with antigens extracted from epimastigotes of Trypanosoma cruzi, sera from 39 cases of Chagas' disease were reactive with promastigotes of L. braziliensis panamensis. This apparent unidirectional cross-reactivity has been attributed to differences in potency of the antigenic stimulus presented in the two diseases.     

A rapid and simple procedure for dissociation of tumor tissue from the human colon

Summary A rapid procedure for dissociation of colon tumor tissue which combines enzymatic and mechanical methods is described. Using this method a cell suspension almost free of cell aggregates is obtained which is further characterized by high cell yield and excellent cell viability.Presented in part at the fourth symposium of the section of experimental cancer research (SEK) of the German Cancer Society (1987), J Cancer Res Clin Oncol 113:S8Dedicated to Professor Fritz Linder on the occasion of his 75^th birthday     

Impact of canine control on the epidemiology of canine and human visceral leishmaniasis in Brazil.

Brazil is the only country endemic for zoonotic visceral leishmaniasis (ZVL) that regularly conducts epidemiologic and prophylactic control programs that involve the treatment of human cases, insect vector control, and the removal of seropositive infected dogs. This report reviews 60 studies reporting data on the efficacy of these recommended control tools and concludes that in Brazil 1) eradication of the disease in Minas Gerais was achieved by the concomitant use of the three control methods, 2) although seropositivity by an immunofluorescent assay is not completely related to infectiousness, the removal of seropositive dogs leads to a significant reduction of canine and human incidence, 3) improvement of the sensitivity of the diagnostic tool used for canine control should optimize the efficacy of control, and 4) although difficult and expensive, the public health dog control campaigns performed in Brazil reduced the incidence of ZVL and should be maintained since treatment of dogs is an unrealistic intervention, both because of its prohibitive cost and relatively poor effectiveness.     

Standardization of dot-ELISA for the serological diagnosis of toxocariasis and comparison of the assay with ELISA.

The dot-enzyme-linked immunosorbent assay (dot-ELISA) was standardized using somatic (S) and excretory-secretory (ES) antigens of Toxocara-canis for the detection of specific antibodies in 22 serum samples from children aged 1 to 15 years, with clinical signs of toxocariasis. Fourteen serum samples from apparently normal individuals and 28 sera from patients with other pathologies were used as controls. All samples were used before and after absorption with Ascaris suum extract. When the results were evaluated in comparison with ELISA, the two tests were found to have similar sensitivity, but dot-ELISA was found to be more specific in the presence of two antigens studied. Dot-ELISA proved to be effective for the diagnosis of human toxocariasis, presenting advantages in terms of yield, stability, time and ease of execution and low cost.     

A rapid and simple assay for growth hormone-binding protein activity in human plasma

The newly discovered circulating growth hormone binding proteins dictate a re-evaluation of the state of GH in plasma in health and disease as the binding proteins are known to affect GH metabolism and action. We describe a rapid and simple GH-binding assay that allows determination of free and complexed plasma GH, as well as GH-binding protein activity as an index of GH-binding protein levels, with relative ease. The method is based on incubation of plasma with 125I-GH and separation of bound from free GH on small DEAE-cellulose columns; it can be used on a large scale for routine determinations. The results obtained by this method are comparable to those obtained with the previously used slow and more cumbersome gel filtration technique. Initial data obtained in normal subjects and certain disease states show that the bound fraction of plasma GH is similar in men, women and children, is unaffected by pregnancy or acute infection, but is marginally decreased in liver cirrhosis. In acromegaly, binding protein activity also appears normal when allowance is made for partial saturation of the binding proteins by the high prevailing GH levels. The technique we describe should facilitate investigations of normal and abnormal regulation of the GH binding proteins.     

A simple and practical agglutination assay for human leucocyte antigen-B27 typing

BACKGROUND AND OBJECTIVES: The human leucocyte antigen (HLA) B27 is the most frequently typed single antigen that is associated with diseases. Here, we describe a simple and rapid particle agglutination assay (PaGIA) for HLA-B27 typing. MATERIALS AND METHODS: Superparamagnetic particles were coated with a monoclonal antibody to HLA-B27 and subsequently used for testing. Anticoagulated whole-blood samples were obtained from healthy blood donors (n = 194) with known HLA patterns and from patients (n = 51) who had been typed positive for HLA-B27 by flow cytometry. RESULTS: The particles agglutinated only after incubation with HLA-27-positive blood samples, using the ID-microtyping system. Positive reactions were clearly distinguishable from negative reactions in all samples tested. Flow cytometric HLA-B27 typing revealed an indeterminate result in one patient. CONCLUSIONS: The new HLA-B27 PaGIA is suitable for rapid typing of HLA-B27. The assay is simple and easy to perform, and can be implemented in any routine laboratory.     

Gentamicin-attenuated Leishmania infantum: cellular immunity production and protection of dogs against experimental canine leishmaniasis

An attenuated line of Leishmania infantum (L. infantum H-line) has been established by culturing promastigotes in vitro under gentamicin pressure. Here, we show that L. infantum H-line induced significantly higher levels of IFN-γ and lower levels of IL-10 compared with those in dogs infected with L. infantum wild type (WT). Anti-Leishmania-specific total IgG, IgG1, and IgG2 antibodies were present in the serum of all infected dogs, with levels of IgG2 subclass highest in the sera of dogs inoculated with L. infantum H-line. Relatively high levels of IgG1 were found in the sera of dogs infected with L. infantum WT. Six of seven dogs immunized intradermally (i.d.) with the attenuated line later showed a positive skin test to leishmanin, whereas the dogs infected with L. infantum WT did not. No clinical abnormalities were observed, and no parasites found in the visceral organs of the dogs inoculated intravenously (i.v.) with L. infantum H-line over 24 months post-inoculation. Dogs which had been immunized with L. infantum H-line i.d. 12 months previously were protected against challenge with L. infantum WT. These data suggest that the L. infantum H-line was safe and induced a protection which is correlated with cellular immunity in dogs.     

Immune complex decomplementation of canine sera for use in a complement-fixation test for diagnosis of visceral leishmaniasis

Canine sera frequently become anti-complementary when heat-inactivated at 56 degrees C for 30 min, and generally cannot be used in standard complement-fixation (CF) assays. Therefore, a procedure was developed for decomplementing canine sera by absorption with particulate immune complexes consisting of sheep erythrocyte stroma optimally sensitized with anti-sheep erythrocyte antibody (hemolysin). Canine sera incubated for 20 min at 30 degrees C with sensitized stroma consistently showed less than 10% residual complement and were not anti-complementary. This decomplementation procedure was applied in a complement-fixation (CF) test for detection of serum antibodies during canine visceral leishmaniasis. Two groups of German shepherd dogs were injected intravenously with Leishmania donovani or L. donovani chagasi amastigotes, and the course of infections was followed for 12 weeks. Using freeze-thaw sonicate preparations of L. donovani parasites as antigen, reciprocal CF antibody titers above 24 were detectable in sera 7 weeks after infection and gradually increased to a maximum titer of 775 at 12 weeks. Sera from control dogs had mean titers of 24. This improved methodology enhances the potential of the CF test in the serodiagnosis of canine leishmaniasis.     

Evaluation of the micro enzyme-linked immunosorbent assay (ELISA) for antibodies in American visceral leishmaniasis: antigen selection for detection of infection-specific responses

This study was designed to evaluate the ELISA for diagnosis of American visceral leishmaniasis (AVL) using antigen prepared from different Leishmania isolates and from a strain of Trypanosoma cruzi. Two Leishmania donovani chagasi isolates from Bahia and Maranh?o (both states of northern Brazil), one L. donovani from Sudan, one L. mexicana amazonensis isolate, and one T. cruzi isolate were used. A total of 375 sera were tested, including 119 from AVL patients, 96 from nonleishmaniasis hospitalized patients, 20 from healthy persons, 30 from patients with mucocutaneous leishmaniasis, 28 from patients with Chagas' disease, 20 from patients with tuberculosis, 21 from leprosy patients, 27 from schistosomiasis patients and 14 from patients with systemic mycoses. The antigens prepared from L. d. chagasi (Bahia) and L. m. amazonensis showed the highest sensitivity (98% and 99%, respectively) for detecting antibodies in sera from AVL patients. However, the specificity of L. d. chagasi (Bahia) antigen was better than that of L. m. amazonensis (96% vs. 86%). Comparison among the three L. donovani isolates demonstrated that the antigen prepared with the isolate from the same area where the sera originated yielded higher mean absorbance than the others. By using spectrophotometric absorbance values it was possible to use a single dilution of serum (between 1/100-1/400) since a clear separation was seen between AVL patients and controls. No patients with the other diseases who were tested gave positive results. We suggest that ELISA can be a very convenient, sensitive, and specific test for diagnosis of AVL when soluble antigen, preferably from an isolate from the test area, is used.     

A new cytokine release assay: a simple approach to monitor the immune status of HIV-infected patients

Purpose

To test a new assay based on an ex vivo cytokine release from whole blood for the monitoring of immune changes in human immunodeficiency virus (HIV)-infected patients.

Methods

A pilot study of outpatients with HIV infection (n = 9) at a large academic hospital who were divided into three groups: HIV-infected patients on highly active antiretroviral therapy (HAART) with a CD4⁺ cell count >350/μL (group I) or a CD4⁺ cell count <350/μL (group II) and HIV-infected HAART-naïve subjects with a CD4⁺ cell count >350/μL (group III). All groups were compared with healthy volunteers (n = 3). The ex vivo cytokine release assay was performed in a three-step process: (1) blood collection, (2) whole-blood ex vivo incubation over 48 h without or with a standard set of well-defined recall antigens as comparable to those used formerly in the skin delayed-type hypersensitivity (DTH) test, (3) cytokine determination from the assay supernatant.

Results

Under stimulated conditions, untreated HIV-infected patients with a CD4⁺ count >350/μL had similar interleukin-2 (IL-2) levels in the supernatant of the whole-blood incubation to HIV-infected patients on HAART with a low CD4⁺ count. Both groups revealed lower IL-2 levels in the supernatant than HIV-infected patients on HAART and with a CD4⁺ count >350/μL or healthy volunteers. The determination of interferon-γ and tumour necrosis factor-α in the supernatant showed a similar arrangement of cytokines between groups.

Conclusions

Our results suggest that this cytokine release assay could be a suitable tool to mirror the immunological responsiveness of patients with HIV infection in a gradual manner; further studies are required in order to assess its value in HAART monitoring.     

Cytomegaloviral infections in the guinea pig: experimental models for human disease

The salivary gland virus of guinea pigs, or guinea pig cytomegalovirus (CMV), was first described in The Journal of Infectious Diseases in 1920. Early studies were concerned with the nature of intranuclear and intracytoplasmic inclusions, viral pathogenicity, transmissibility in animals, and similarity to human viral infections. Subsequently, tissue culture techniques and electron microscopy of guinea pig CMV-infected salivary gland and tissue culture cells demonstrated the morphogenesis which closely resembles that of human CMV. By use of tissue culture cocultivation techniques, generalized viremic infection, transplacental transmission of virus, congenital infection, CMV-associated mononucleosis, interstitial pneumonia, and transmission of virus by blood transfusion have been demonstrated. In studies of live attenuated and noninfectious envelope antigen vaccines for the prevention of transplacental transmission of virus and of interstitial pneumonia, the guinea pig model has demonstrated efficacy of such vaccines under experimental conditions. Investigations of polymorphonuclear leukocyte function during primary CMV infections may elucidate mechanisms for bacterial, parasitic, and fungal superinfections in human disease.