Dot enzyme-linked immunosorbent assay (Dot-ELISA): a micro technique for the rapid diagnosis of visceral leishmaniasis.

A micro enzyme-linked immunosorbent assay utilizing antigen dotted onto nitrocellulose filter discs (Dot-ELISA) was developed for the rapid diagnosis of visceral leishmaniasis. Leishmania donovani promastigotes applied to filter discs in volumes of 1 microliter were placed in 96-well microtiter plates, blocked with bovine serum albumin, then incubated with 4-fold dilutions of patient sera. After incubation with peroxidase-conjugated anti-human antibody, washing and addition of precipitable substrate, positive reactions appeared as blue dots on a white background which were easily read by eye. The procedure is performed at room temperature, takes about 2 h and is economical. At a reciprocal diagnostic titer of greater than or equal to 32, 41 of 42 (98%) leishmaniasis patients were positive, and positive titers ranged from 512 to 524,288. Control sera from healthy individuals showed 1 of 50 (2%) false positive reactions. Sera from patients with African trypanosomiasis, Chagas' disease, and lupus erythematosus were cross-reactive in the Dot-ELISA. No cross-reactivity was noted with sera from patients with amebiasis, coccidioidomycosis, cutaneous leishmaniasis, viral hepatitis, hydatidosis, malaria, schistosomiasis, syphilis, toxoplasmosis or trichinosis. In replicate experiments, 90% of 167 sera tested did not vary in titer. This rapid and inexpensive test should prove to be an important field diagnostic technique for visceral leishmaniasis.

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.

Evaluation of promastigote and amastigote antigens in the indirect fluorescent antibody test for American cutaneous leishmaniasis.

Leishmania braziliensis panamensis promastigotes, temperature-induced in vitro-cultivated amastigotes, Vero cell-derived amastigotes, and rodent lesion-derived amastigotes were evaluated as antigens in the indirect immunofluorescent antibody (IFA) test for American cutaneous leishmaniasis. Test sensitivity was determined using sera from 34 U.S. soldiers with leishmaniasis diagnosed by demonstrating parasites in their skin lesions. Sera were collected from 3 to 24 months after exposure to Leishmania. Positive IFA reactions among patient sera were 82% with promastigotes or lesion amastigotes, 79% with in vitro amastigotes, and 76% with Vero cell amastigotes (P = N.S.). Positive titers ranged from 1:8 to 1:128 using all antigens. Test specificity was determined with 30 sera from healthy individuals. False positive reactions ranged from 0-5% depending on the antigen and all titers were less than or equal to 1:8. Test cross-reactivity was assessed with 47 sera from patients with other diseases. Depending on the antigen, cross-reactions occurred with sera from patients with Chagas' disease, lupus erythematosus, malaria, toxoplasmosis and amebiasis. None of the antigens cross-reacted with sera from patients with viral hepatitis, coccidioidomycosis, syphilis, schistosomiasis, and trichinosis. In replicate experiments, 99-100% of the sera varied no more than +/- 1 titer dilution. As sensitivity, specificity, cross-reactivity, and reproducibility of the four antigens were statistically similar, promastigotes, which can be easily and economically cultured in large numbers in vitro are recommended for use in the IFA test for American cutaneous leishmaniasis.

Determination of IgM and IgG antibodies to Toxoplasma using the IFA test, ELISA, and Dot-ELISA procedures.

The dot enzyme-linked immunosorbent assay (Dot-ELISA) and the enzyme-linked immunosorbent assay (ELISA) were compared with the immunofluorescent antibody test (IFA) for detection of IgM- and IgG-specific antibodies to human toxoplasmosis. Reciprocal titers were determined in all three assays using sera from 56 patients with suspected toxoplasmosis or with symptoms and diseases requiring exclusion of toxoplasmosis and control sera from 56 healthy persons. Using the Dot-ELISA, six patient sera (10.7%) were positive at titers of greater than equal to 1024 for IgM antibodies (titer range 1024-16 384) and 47 sera (84%) were positive for IgG antibodies (titer range 16-262 144) at a titer of greater than or equal to 16. One control serum was reactive for IgM (titer 1024) and 10 control sera (18%) were positive for IgG in the Dot-ELISA. In the ELISA, at titers of greater than or equal to 128, five sera (9%) were reactive for IgM (titer range 128-512) and 52 sera (92.8%) were reactive for IgG (titer range 32-8192) at a titer of greater than or equal to 32; no control sera gave positive reactions for IgM while 10 sera (18%) were positive for IgG in the ELISA. Using the IFA test at reciprocal titers of greater than or equal to 16, four sera (7.1%) were positive for IgM (titer range 32-512), and 51 sera (91%) were positive for IgG (titer range 16-8192). None was reactive for IgM, and eight sera (14%) were positive for IgG (titer range 32-128) in the IFA test. The Dot-ELISA correlated well with the IFA test (correlation coefficient = 0.895) and the ELISA correlated slightly higher with the IFA test (correlation coefficient = 0.910) for detection of IgG antibodies to Toxoplasma gondii.

Dot enzyme-linked immunosorbent assay (Dot-ELISA): comparison with standard ELISA and complement fixation assays for the diagnosis of human visceral leishmaniasis.

The dot enzyme-linked immunosorbent assay (Dot-ELISA), standard ELISA and the complement fixation (CF) tests were compared in the serodiagnosis of African visceral leishmaniasis (kala-azar). Assay sensitivity was determined using sera from 44 patients with parasitologically confirmed kala-azar. Using the Dot-ELISA, 42 of 44 patients (95%) were positive at a reciprocal titer of greater than or equal to 32 (titer range 512-524 288). In the standard ELISA technique, 43 of 44 patients (98%) were positive (titer range 32-32 768). At a reciprocal titer of greater than or equal to 8 in the CF test, 35 patients (80%) were positive, 1 (2%) was negative and 8 patients (18%) showed anticomplementary (AC) activity (titer range 8-2048). Specificity, determined using 33 sera from healthy individuals not living in endemic areas, was 97% in both the Dot-ELISA and the standard ELISA (32 of 33 sera); in he CF test, all sera were negative except 1 (3%) which showed AC activity. Sera from patients with Chagas' disease cross-reacted in the dot-ELISA up to a titer of 512. In the standard ELISA, cross-reactions occurred mainly using sera from patients with Chagas' disease, malaria and syphilis, and to a lesser extent with sera from amebiasis, schistosomiasis and trichinosis patients. Overall titer agreement in replicate experiments was highest in the Dot-ELISA (89%), followed by the standard ELISA (80%) and the CF test (72%).

Reduced false positive reactions in the Dot-enzyme-linked immunosorbent assay for human visceral leishmaniasis.

Specificity of the Dot-enzyme-linked immunosorbent assay (Dot-ELISA) for visceral leishmaniasis was significantly improved through the use of enzyme-conjugated antisera specific for IgG heavy chains. Of sera from Kenyans with visceral leishmaniasis, 97% (29/30) were positive using horseradish peroxidase (HRP)-conjugated anti-IgG (heavy and light chain specific) which detected bound IgG and IgM. False positive reactions occurred in 80% of sera from both trypanosomiasis-infected patients (8/10) and apparently healthy Africans (24/30). HRP-conjugated anti-IgG (heavy chain specific), which detected only bound IgG, significantly reduced false positive reactions among trypanosomiasis-infected (2/10, P less than 0.02) and healthy Africans (6/30, P less than 0.001), without reducing test sensitivity in leishmaniasis patients. No false positives occurred when either HRP-conjugated antiserum was used to assay sera from 30 North Americans. Application of enzyme-conjugated antisera specific for IgG improves the serodiagnostic value of the Dot-ELISA for individual patient evaluation and epidemiologic investigations.