Immunoblotting identifies and antigen recognized by anti gp63 in the immune complexes of Indian kala-azar patient sera

In SDS-PAGE the immune complexes (IC) of kala-azar patient sera showed intense bands at 55 kDa and 20 kDa corresponding to heavy and light chains of immunoglobulins. In immunoblot experiment, kala-azar and normal IC after treatment with patient sera showed multiple bands of which the band at 55 kDa was most prominent in kala-azar IC. It is known that in kala-azar sera antihuman IgG is present, so the heavy band at 55 kDa region may be due to higher amount of IgG and/or other antigen(s) present at that region. Immunoblot experiments of kala-azar IC with anti gp63 also developed a major band at 55 kDa. It suggests that the antigen (55 kDa) and gp63 have common antigenic epitope (s). Normal IC did not react with anti gp63 indicating absence of this antigen in normal IC. Antigenic similarity between the IC antigen (55 kDa) and gp63 indicated that the former antigen may have been processed from gp63. In summary, identification of a parasite antigen (55 kDa) in IC of kala-azar patients sera may be useful in developing a serodiagnostic assay for visceral leishmaniasis. (Mol Cell Biochem130: 11–17, 1994)Abbreviations IC Immune Complexes - PEG Polyethylene Glycol (Mol wt 8000) - PBS Phosphate Buffer Saline - VL Visceral Leishmaniasis - AVL American Visceral Leishmaniasis - IgG Immunoglobulin G - TBS Tris Buffer Saline - SDS-PAGE Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis - gp63 A leishmanial surface glycoprotein of molecular mass 63,000 - TEMED N,N,N,N-Tetramethylethylenediamine     

Serologic Analyses of Cell-Surface Antigens of Acanthamoeba spp. with Plasma Membrane Antisera*†

SYNOPSIS. Antisera were raised against plasma membrane-enriched fractions of the species Acanthamoeba castellanii and Acanthamoeba culbertsoni to determine whether cell-surface antigens would facilitate species identification of Acanthamoeba isolated from the environment or in human infections. Acanthamoeba castellanii and A. culbertsoni plasma membranes were purified, after homogenization, by differential and isopycnic centrifugation. Electron microscopic examination of purified membrane samples showed an enrichment of membranes with a typical trilaminar structure. Occasionally, mitochondria were recognized in the electron microscope preparations. 5′-Nucleotidase, Mg²⁺-ATPase, and alkaline phosphatase were enriched 11-fold, 2-fold, and 7-fold, respectively, in the A. castellanii membranes, as determined from analyses of the enzyme activities in whole cell homogenates and membrane preparations. 5′-Nucleotidase was not detected in A. culbertsoni, but the activities of Mg²⁺-ATPase and alkaline phosphatase were increased 2- to 3-fold. Both membrane preparations showed no glucose-6-phosphatase activity and less than 5% contamination with succinic dehydrogenase. From assays of acid phosphatase activity, the most apparent contamination of the plasma membrane preparations was with membranes of phagocytic vacuoles. Acanthamoeba castellanii membrane antisera produced significant agglutination and fluorescence of homologous cells to titers of 1:8192 and 1:1024, respectively. Acanthamoeba polyphaga and Acanthamoeba rhysodes gave the most cross-reactions in heterologous tests. They were agglutinated to a titer of 1:128 and positively fluoresced to titers of 1:32 and 1:64, respectively. Antisera of A. culbertsoni membrane agglutinated homologous cells at a dilution up to 1:4096 and produced homologous fluorescent titers up to 1:512. Other than agglutination of A. polyphaga to a titer of 1:128, these antisera did not cross-react significantly with any remaining heterologous species. Three new isolates were identified with these plasma membrane antisera: 2 of them, contaminants from tumor tissue cultures, were identified as A. culbertsoni. Preliminary information is also given on the use of the membrane antisera for species identification of Acanthamoeba in several new cases of amebic encephalitis.     

Cryptic species of Paracoccidioides brasiliensis: impact on paracoccidioidomycosis immunodiagnosis

We aimed to evaluate whether the occurrence of cryptic species of Paracoccidioides brasiliensis, S1, PS2, PS3 and Paracoccidioides lutzii, has implications in the immunodiagnosis of paracoccidioidomycosis (PCM). Small quantities of the antigen gp43 were found in culture filtrates of P. lutzii strains and this molecule appeared to be more variable within P. lutzii because the synonymous-nonsynonymous mutation rate was lower, indicating an evolutionary process different from that of the remaining genotypes. The production of gp43 also varied between isolates belonging to the same species, indicating that speciation events are important, but not sufficient to fully explain the diversity in the production of this antigen. The culture filtrate antigen AgEpm83, which was obtained from a PS3 isolate, showed large quantities of gp43 and reactivity by immunodiffusion assays, similar to the standard antigen (AgB-339) from an S1 isolate. Furthermore, AgEpm83 was capable of serologically differentiating five serum samples from patients from the Botucatu and Jundiaí regions. These patients had confirmed PCM but, were non-reactive to the standard antigen, thus demonstrating an alternative for serological diagnosis in regions in which S1 and PS2 occur. We also emphasise that it is not advisable to use a single antigen preparation to diagnose PCM, a disease that is caused by highly diverse pathogens.     

Immunoblotting of soluble antigens in Paracoccidioides brasiliensis culture

This study investigated the major soluble antigens produced by Paracoccidioides brasiliensis (Pb339) cultured in solid Sabouraud (pH 5.6 and 8.5), Sabouraud plus brain heart infusion and liquid tomato juice‐enriched complex medium media at intervals of 3 days over 30 days by immunoblotting and concluded that, to optimize the source of each antigen, both time and growth conditions should be considered.     

Detection of antibody responses and delayed dermal hypersensitivity with Blastomyces dermatitidis yeast and mycelial lysate antigens

Yeast cell lysate and mycelial lysate antigens prepared from one strain (T-58) of Blastomyces dermatitidis were evaluated with respect to the detection of antibodies and delayed dermal hypersensitivity. Comparable ELISA sensitivity values were evidenced with the two antigens when assayed against serum specimens from dogs with blastomycosis, sera from non-infected dogs residing in endemic and nonendemic areas for blastomycosis and sera from rabbits that were hyperimmunized with B. dermatitidis antigens. Specificity determinations with anti -Histoplasma capsulatum rabbit sera indicated that both reagents exhibited only minimal cross-reactivity; the mycelial antigen was slightly more specific than the yeast phase reagent. Similar sensitivity and specificity results were experienced when the two antigens were used to detect delayed dermal hypersensitivity in guinea pigs previously sensitized with B. dermatitidis or H. capsulatum.     

Detection of IgG and IgM in Sera from Canines with Blastomycosis using Eight Blastomyces dermatitidis Yeast Phase Lysate Antigens

The objective of this study was to compare the efficacy of eight Blastomyces dermatitidis yeast phase lysate antigens (T-58: dog, Tennessee; T-27: polar bear, Tennessee; ERC-2: dog, Wisconsin; B5894: human, Minnesota; SOIL: soil, Canada; B5896: human, Minnesota; 48089: human, Zaire; 48938: bat, India) in the detection of the immunoglobulins IgG and IgM in serum specimens from canines with blastomycosis. An indirect enzyme-linked immunosorbent assay (ELISA, peroxidase system) was used to analyze sera collected during four different intervals post-infection. The yeast lysate antigen 48938 was a reactive antigen for the detection of both IgG (mean absorbance value range: 1.198–2.934) and IgM (mean absorbance value range: 0.505–0.845). For the same sera, antigen T-27 was also effective in the detection of IgG (mean absorbance value range: 0.904–3.356) and antigen 48089 was useful for the detection of IgM (mean absorbance value range: 0.377–0.554). The yeast lysate antigen B5894 proved to be a poor antigen for the detection of both IgG and IgM (mean absorbance value ranges: 0.310–0.744 for IgG, 0.025–0.069 for IgM). Inherent variations in yeast lysate antigens such as these may be utilized to develop improved immunoassay procedures for the specific detection of IgG or IgM in cases of blastomycosis.     

Immunodiagnosis of clonorchiasis using a recombinant antigen

A cDNA expression library of Clonorchis sinensis adult worm was constructed, and screened out immunologically. One clone, pBCs31, was selected in view of its predominant reactivity with an experimentally infected rabbit serum. Recombinant C. sinensis antigen with 28 kDa as a β-galactosidase fusion protein produced in Escherichia coli was identified by immunoblot analysis. The cloned gene was composed of 16 copies of a 30 base pair repeat and an additional 320 bases. The deduced amino acid sequence of the tandem repeat was AQPPKSGDGG. On RNA slot blot analysis. C. sinensis adult worm RNA showed a positive reaction with the cloned gene. Enzyme-linked immunosorbent assay using a purified recombinant antigen of pBCs31 showed high specificity for diagnosis of clonorchiasis.     

Field evaluation of the MM3-SERO ELISA for detection of anti-Fasciola IgG antibodies in milk samples from individual cows and bulk milk tanks

We carried out a field evaluation of the MM3-SERO ELISA for the diagnosis of Fasciola hepatica infection, by analysing serum and milk samples from individual cows and samples from bulk milk tanks. The diagnostic performance of the assay was assessed with serum samples from all 257 cows in eight fluke-free herds, and 240 cows with natural fasciolosis (diagnosed in vivo and/or post-mortem). Assay performance for individual milk samples was determined by analysis of paired serum and milk samples from 947 lactating cows from 33 F. hepatica-infected farms. The diagnostic usefulness of the assay for bulk tank milk was evaluated by analysis of bulk milk from infected (33) and non-infected (35) farms. For serum samples, the sensitivity, specificity and diagnostic accuracy of the assay were respectively 99.2% (95% CI: 97.0%–99.9%), 100% (95% CI: 98.6%–100%) and 0.997 (95% CI: 0.987–1.000). The only two infected animals in which serum antibodies were not detected had very low parasitic burdens (with only 2 and 3 flukes observed). The performance of the MM3 SERO ELISA for individual milk samples was similar to that for serum samples, and the stepwise linear regression revealed a strong correlation between the results for the milk samples and the serum samples (R² = 0.84; p < 0.001). The agreement between results obtained with pairs of serum and milk samples was very high: there was matching classification in 96% (910/947) of paired samples (kappa = 0.92; p < 0.001). Individual milk samples may therefore be used, instead of serum samples, in the MM3-SERO ELISA, for reliable detection of seropositive cows. Testing bulk tank milk samples enabled detection of infected herds, even when the within-herd prevalence of infection was as low as 12%. We conclude that the MM3-SERO ELISA is a sensitive and highly specific test for serodiagnosis of bovine fasciolosis, and can be used with individual samples of either serum or milk. Use of the assay with bulk milk samples enables estimation of the within-herd prevalence of infection.     

植物病原细菌检测和细菌病害诊断方法

植物病害诊断是通过病菌鉴定和致病性测定等步骤实现的。在过去的10年里用于细菌鉴定的基因组技术的快速发展极大地简化和促进了病菌的检测和鉴定,但是DNA分子检测方法不能完全取代传统的培养检验和表型检验方法。文中介绍了未显示病害症状的植物或植物产品中已知细菌的免疫检测和基因组DNA检测方法,以及细菌病害诊断鉴定的新方法。     

Immune complex antigens as a tool in serodiagnosis of kala-azar

The 63 kDa surface antigen of Leishmania promastigotes is one of the most important virulent factors in establishing the host parasite relationship. This glycoprotein is revealed by surface iodination study as well as by metabolic labeling and immunoblot methods. In search of this specific antigen for serodiagnosis, immune complexes (ICs) were isolated from kala-azar patient sera and analysed by SDS-PAGE and Western immunoblotting. The immunoblot of kala-azar IC with patient sera, antipromastigote sera and anti gp63 sera detected the major antigen of 55 kDa. This recognition suggests that 55 kDa antigen and gp63 have common antigenic epitope(s). Normal IC did not react with anti gp63 sera indicating absence of this antigen in normal IC. To confirm the parasitic origin of the 55 kDa antigen of kala-azar IC, in vitro IC was formed with parasite antigen and acid dissociated kala-azar IC antibody. This indicated the antigenic similarity of the 55 kDa antigen and gp63 antigen of the parasite. This also suggested that the former antigen may have been processed from gp63. In summary, identification of parasite antigen (55 kDa) in IC of kala-azar patients' sera may be useful in developing a serodiagnostic assay of visceral leishmaniasis. Several other antigens are visualized in kala-azar IC when developed with patient sera. But specificity and efficacy of these antigens have not yet been evaluated in serodiagnosis of the disease.