Leishmania major: common antigen responsible for induction of delayed-type hypersensitivity response in guinea pigs.

The cellular response to Leishmania major (L. major) was evaluated in vivo by delayed-type hypersensitivity (DTH) skin test reaction using leishmanin as antigen. Our previous study had shown the development of species-specific DTH reaction in sensitized guinea pigs by application of a single purified antigen from promastigotes and filtered culture supernatants of L. major. This study has shown that purified antigen is common in both stages of the life cycle and filtered culture supernatant of L. major. The common antigen was purified and analyzed from soluble Leishmania antigen (SLA) of amastigotes, promastigotes, and filtered culture supernatant of L. major by specific monoclonal antibody coupled to sepharose-4B. The purified antigen, which gave a single band of 56 kDa on sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) electrophoresis, elicited DTH response in guinea pigs sensitized with L. major. It was almost of the same degree as that produced by whole SLA. These results show that DTH inducer antigen is present in both stages of the life cycle and filtered culture supernatant of L. major.

Analysis of specific IgE and IgG subclass antibodies for diagnosis of Echinococcus granulosus.

The potential roles of specific antibodies of different immunoglobulin G (IgG) subclasses and IgE in serological diagnosis of cystic echinococcosis (CE) were investigated by an enzyme linked immunosorbent assay (ELISA) based on Antigen 5 (Ag5). Presence of IgG1 was demonstrated in all sera from 58 patients with CE. The most discriminatory and specific antibodies found in this study belonged to IgG4 and IgE. Only one false-positive reaction was observed with IgG4 and no IgE cross-reactivity occurred with 40 sera from healthy controls. In 36 sera from patients infected with parasites other than CE two false-positive reactions with IgG4 were observed but none occurred with IgE. In immunoblotting, it was shown that IgG1 subclass was responsible for cross-reactivity of human antibodies that reacted with a 38 kDa subunit of Ag5. IgG4 and IgE antibodies could not recognize the 38 kDa subunit and under non-reducing conditions reacted with the 57 kDa subunit without any cross-reactivity to other parasites. The results demonstrated that IgG4 and IgE are the most important antibodies for serological diagnosis of hydatid cyst in an Ag5 based immunoassay system.

Leishmania major: species specific delayed hypersensitivity reaction induced by exogenous secreted antigen in the guinea pig.

The cellular response to Leishmania major (L. major) is usually evaluated in vivo by the delayed-type-hypersensitivity (DTH) test using leishmanin. Leishmanin can give false-positive reactions in areas where there is a background of leishmaniasis. In a previous study, it was shown that a 56 kDa antigen purified from promastigote and culture supernatant of L. major induce strong DTH reactions in sensitized guinea pigs. In this study, the species-specificity of this antigen was further investigated. Three groups of guinea pigs were sensitized with L. major, L. tropica, and L. infantum and both flanks of sensitized animal were injected intradermally with purified 56 kDa antigen or soluble leishmania antigen (SLA). The extent of indurations were measured after 24, 48, and 72 h. In animals which were sensitized with three species of leishmania, only those immunized with L. major showed skin reactions to purified antigen by an increase in skin thickness. Since complex antigen mixtures such as SLA and leishmanin show cross-reactivity and can be non-specific, the result obtained here suggest that 56 kDa antigen may be a useful diagnostic tool for species specific diagnosis in field studies of leishmaniasis.

Leishmanin skin test in guinea pig with a single purified protein of Leishmania major.

A series of hybridomas was produced by fusion of SP2/0 myeloma cells with spleen cells of mice immunized with Leishmania major (L. major). The reactivity of secreted monoclonal antibodies (mAbs) was evaluated against available leishmanin antigen by enzyme linked immunosorbent assay. Only one hybridoma designated as 7F9 secreted IgG1 mAb which was shown to be reactive with leishmanin. This mAb was further tested against four species of Leishmania (L. donovani, L. tropica, L. infantum, L. major) and a recombinant gp63. Among the four species tested it was shown to be only reactive with promastigotes of L. major. The antigen recognized by this mAb was purified and analyzed from both sonicated and supernatant cultures of L. major by immunoaffinity chromatography and reverse phase high performance liquid chromatography. The purified antigen, which gave a single band of 56kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis elicited a strong delayed-type hypersensitivity (DTH) reaction in guinea pigs sensitized with L. major. It was almost of the same degree as that produced by leishmanin. These results suggest that an L. major-specific antigen is an alternative as a specific diagnostic skin test reagent, which could lead to a better understanding of the mechanism of DTH in L. major.

Antiepileptogenic and anticonvulsant activity of interleukin-1 beta in amygdala-kindled rats.

Ischaemic, excitotoxic and traumatic brain injuries have been associated with the occurrence of epileptic seizures. Microglia, the principal immune cells in the brain, produce a variety of proinflammatory and cytotoxic factors especially interleukin-1 (IL-1) early after an acute insult. We studied the effect of intracerebroventricularly administered IL-1beta on seizure acquisition and on fully kindled seizures in amygdala kindling model of epilepsy. IL-1beta (0.01 ng/rat) retarded acquisition of kindled behavioral seizures and growth of afterdischarges (AD). IL-1beta (0.01-10 ng/rat) also exhibited significant anticonvulsant effect on established kindled seizures and AD duration. This effect began 0.5 h after administration and was continued up to 72 h. Pretreatment of the kindled animals with nitric oxide synthase inhibitor, N(G)-nitro-L-arginine methyl ester, or cyclooxygenase inhibitor, piroxicam, reversed the anticonvulsant effect of IL-1beta at early time points. Although most of the previous studies indicate a proconvulsant or convulsant property of IL-1, our results support a protective and antiepileptogenic role of IL-1beta.

Production of monoclonal antibody against alkaline phosphatase.

BACKGROUND: Monoclonal antibody against alkaline phosphatase (Alp) has many applications which Alp-anti Alp complex (APAAP) formation is one of the most important ones. This complex is applicable in many immunohistochemical and immunocytochemical techniques such as diagnosis of various kinds of leukemias, lymphomas, skin diseases, kidney dysfunctions, etc. OBJECTIVE: Production of anti-Alp monoclonal antibody for utilization in APAAP complex. METHODS: After several arranged injections of Alp to Balb/c mice and determining the specific antibody titer by ELISA test, the spleen lymphocytes of immunized mice and Sp2/0 myeloma cells were fused using polyethylene glycol as fusing agent and hybridoma cells were cultured in HAT medium. Identification and selection of anti-Alp producing clones were done by performing ELISA test on supernatants of all resulting clones. Limiting dilution method was used to attain monoclones and the effect of obtained antibodies on enzyme activity was investigated by a specific ELISA test. For production of concentrated Ab, the hybridoma cells were injected to peritoneal cavity of mice and the produced ascetic fluids were collected. Finally class and subclass of the obtained antibodies were determined by Isostrip kit. RESULTS: After six rounds of fusion, 104 Hybridoma clones were obtained and two Anti-Alp producing clones (A_1G_8 and A_1G_9) were selected and subcloned. Both antibodies were IgG_1 with kappa (kappa) light chains. These antibodies did not affect the enzyme activity and the electrophoresis of ascetic fluids showed an obvious band in gamma (gamma) position. CONCLUSION: Because these antibodies are from IgG class and don't affect the enzyme activity, it seems that they are suitable for APAAP complex formation.