Evaluation of antibody responses in American visceral leishmaniasis by ELISA and immunoblot

American visceral leishmaniasis (AVL) is an important disease among children of northeast Brazil. In order to characterize antibody responses during AVL, sera of hospitalized patients were analyzed by ELISA and Western blot using a Leishmania chagasi antigen preparation. The ELISA was positive (absorbance greater than or equal to 0.196) at a serum dilution of 1:1024 in all patients at presentation, and fell to ward control levels over the following year. Only one of 72 control subjects tested positive, and that donor had a sibling with AVL. Immunoblots of the patients' sera recognized multiple bands, the most frequent of which were at approximately 116 kDa, 70 kDa, and 26 kDa. Less frequently observed were bands at approximately 93 kDa, 74 kDa, 62 kDa, 46 kDa and 32 kDa. The ELISA responses and patterns of banding were distinctive for AVL, and could be used to differentiate patients with AVL from those with Chagas' disease or cutaneous leishmaniasis. Sera from six AVL patients followed for up to six weeks after treatment identified no new bands. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of surface iodinated parasite proteins showed one major band and four minor bands, whereas SDS-PAGE of biotinylated parasite proteins revealed a banding pattern similar to those of patient sera. AVL appears to produce characteristic immunoblot patterns which can be used along with a sensitive screening ELISA to diagnose AVL.     

Comparative antigen analysis of Trichomonas vaginalis by enzyme-linked immunoelectrotransfer blot technique]

Analysis of six isolates of Trichomonas vaginalis was carried out with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and enzyme-linked immunoelectrotransfer blot (EITB). Trichloroacetic acid-treated antigens of the 6 isolates revealed 25 protein profiles ranging 12-170 kDa of molecular weight in SDS-PAGE. In EITB, the specific immunogenic bands were visualized at 51 kDa and 96 kDa when HY-1 antigen was probed with different mice sera immunized with 6 isolates of T. vaginalis. The banding patterns with different sera showed isolate-to-isolate variability. In EITB, homologous antigen (HY-1) did not show any enhanced response in reacting to homologous antiserum (HY-1) when 6 isolates of T. vaginalis were probed with a single serum (HY-1). It is assumed that the different banding patterns of six isolates show isolate-to-isolate variability and immunogenic common bands in 41, 47, 74 and 94 kDa on EITB may connote the important significance on immune response in T. vaginalis infection.     

Identification of an antigen specific to Trypanosoma congolense by using monoclonal antibodies

Using indirect immunofluorescence assays on acetone-fixed smears of a series of different parasites, we have shown that two monoclonal antibodies bind specifically to Trypanosoma congolense organisms. The antibodies bind to both bloodstream trypomastigotes and procyclic culture forms of the parasite and are thus not stage specific. Immunoprecipitation and immunoblot analysis showed that both monoclonal reagents bound a protein of approximately 31,000 m.w. This antigen appeared to be located on the plasma membrane of T. congolense, but the epitope was not exposed on the surface of living bloodstream or procyclic organisms. The antigen was detectable on acetone-fixed organisms or in trypanosome lysates in enzyme-linked immunosorbent assays and may therefore by useful as a species-specific marker in field assays for epidemiologic and clinical investigations.     

Soluble interferon-alpha receptor molecules are present in body fluids.

Soluble forms of the interferon-alpha receptor (sIFN-alpha R) were identified in human serum and urine by Western blotting with monoclonal antibodies (MAb) directed against IFN-alpha R, and by immunoprecipitation (Iptn) of a covalently cross-linked complex of IFN-alpha R and [125I]IFN-alpha with anti IFN-alpha MAb. Elevated levels of sIFN-alpha R were found in sera of hairy cell leukemia patients. The soluble receptor from serum migrated as a 55 kDa protein in SDS-PAGE, and, as expected, the cross-linked product migrated as a 75 kDa protein. The soluble receptor from urine was found to be a protein of mol. wt. 45 kDa and its cross-linked complex migrated as a 65 kDa protein. The calculated mol. wt. of the entire extracellular domain of the IFN-alpha R prior to post-translational modifications is 47,000. Since there are 12 potential glycosylation points in this extracellular domain, its actual mol. wt. may be as high as 70,000 Da. It is therefore concluded that sIFN-alpha R molecules, corresponding to truncated forms of the extracellular domain of the cell surface IFN-alpha R, are present in human serum and in normal human urine.     

The kidney form of Trypanosoma musculi: A distinct stage in the life cycle?

Trypanosoma musculi is a parasite specific to mice, which resides in the blood and lacks intracellular stages. After immune clearance of the flagellates from the general circulation, mice are resistant to reinfection. Yet, long after parasites are no longer detected in the peripheral blood, they persist in the vasa recta of the kidneys and it has been proposed that this is an immunologically privileged site for T. musculi. This relationship provides a useful model for studies of latent or chronic infections in immune hosts. Here, Fernando Monroy and Donald Dusanic consider the immune responses of mice to T. musculi and compare characteristics of the parasites from the vasa recta (kidney forms, KFs) of mice with latent infections to trypanosomes from the peripheral blood (bloodstream forms, BSFs) of animals during active infections. They consider how KFs evade immune destruction and suggest that these sequestered parasites represent a distinct stage in the life cycle.     

草鱼Ⅲ型呼肠孤病毒NS66抗体制备及应用

[背景]草鱼Ⅲ型呼肠孤病毒(grass carp reovirus,GCRV genotypeⅢ) 104株可导致典型性草鱼出血病,对其编码片段的分析有望为临床免疫学检测提供依据。[目的]研究GCRV104株s6基因节段编码蛋白NS66的可能功能,制备良好的GCRV104株NS66蛋白多克隆抗体并分析其特异性。[方法]PCR方法扩增GCRV104株s6基因片段,并克隆至表达载体pGEX-4T-3,转化到大肠杆菌BL21后用IPTG诱导表达,其产物经SDS-PAGE鉴定分析后,通过纯化获得目的蛋白。然后用纯化的pGEX-4T-3-NS66重组蛋白免疫小鼠,获得Anti pGEX-4T-3-NS66多克隆抗体,Western blot测定抗体效价,Western blotting和间接免疫荧光试验(indirect immunofluorescence assay,IFA)鉴定抗体特异性。[结果]SDS-PAGE分析显示表达的重组蛋白约66 kD,大小与预期相符,主要存在于包涵体中;Western blotting测得制备的多克隆抗体效价大于1:50 000,Western blotting和IFA结果表明,制备的多克隆抗体能特异性识别GCRV104病毒。[结论]GCRV104病毒编码的非结构蛋白NS66可能参与了复制和组装过程,形成病毒包涵体,这为建立GCRV104免疫诊断方法及研究GCRV编码的NS66蛋白的功能奠定了前期基础。     

Monoclonal antibody to human 66,000 molecular weight plasminogen activator from melanoma cells. Specific enzyme inhibition and one-step affinity purification

Hybridomas producing a monoclonal IgG1 antibody to a human plasminogen-activating enzyme with an apparent mol. wt. of 66,000 (66 K, HPA66) from human melanoma cells were obtained by fusion of NSI-Ag 4/1 mouse myeloma cells with spleen cells from a mouse immunized with a partially purified preparation of the enzyme. Screening for clones of hybridomas producing antibodies to HPA66 was performed with the impure enzyme preparation. A preliminary screening included enzyme-linked immunosorbent assay and SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting; the final identification was based on inhibition of the enzymatic activity of HPA66 which was complete at high antibody concentrations. No inhibition of three other human and murine plasminogen activators or of plasmin was observed. Employing a one-step affinity procedure with the antibody coupled to Sepharose, HPA66 was purified approximately 200-fold from conditioned medium from the melanoma cells with a yield of 79%. The purified HPA66 was homogeneous as evaluated by SDS-PAGE. Electrophoresis under reducing conditions indicated that it consisted of one polypeptide chain. The binding constant between the antibody and 125I-labelled HPA66 was approximately 2.5 x 10(9) l/mol. The antibody did not bind to a variety of other plasminogen activators, including 52-K and 36-K human enzymes and 48-K and 75-K murine enzymes. Previously, a monoclonal antibody against another enzyme was derived by the sole use of enzyme inhibition for screening. The present study represents a modification of this procedure that can be used when antibody-unrelated inhibitors of the enzyme are present in hybridoma culture fluid.     

Leishmania species: evidence for transglutaminase activity and its role in parasite proliferation

Albeit transglutaminase (TGase) activity has been reported to play crucial physiological roles in several organisms including parasites; however, there was no previous report(s) whether Leishmania parasites exhibit this activity. We demonstrate herein that TGase is functionally active in Leishmania parasites by using labeled polyamine that becomes conjugated into protein substrates. The parasite enzyme was about 2- to 4-fold more abundant in Old World species than in New World ones. In L. amazonensis, comparable TGase activity was found in both promastigotes and amastigotes. TGase activity in either parasite stage was optimal at the basic pH, but the enzyme in amastigote lysates was more stable at higher temperatures (37-55 degrees C) than that in promastigote lysates. Leishmania TGase differs from mouse macrophage (M Phi) TGase in two ways: (1) the parasite enzyme is Ca(2+)-independent, whereas the mammalian TGase depends on the cation for activity, and (2) major protein substrates for L. amazonensis TGase were found within the 50-75 kDa region, while those for the M Phi TGase were located within 37-50 kDa. The potential contribution of TGase-catalyzed reactions in promastigote proliferation was supported by findings that standard inhibitors of TGase [e.g., monodansylcadaverine (MDC), cystamine (CS), and iodoacetamide (IodoA)], but not didansylcadaverine (DDC), a close analogue of MDC, had a profound dose-dependent inhibition on parasite growth. Myo-inositol-1-phosphate synthase and leishmanolysin (gp63) were identified as possible endogenous substrates for L. amazonensis TGase, implying a role for TGase in parasite growth, development, and survival.     

Antigenic analysis of excretory-secretory products of Wuchereria bancrofti and Brugia malayi infective larval forms by SDS-PAGE

Excretory-secretory (ES) products of W. bancrofti and the closely related B. malayi infective larval forms were analysed for their antigenic activity by SDS-PAGE followed by Western blotting as well as by gel elution-sandwich ELISA using filarial serum immunoglobulin-G (FSIgG) as a capture antibody. In W. bancrofti infective larval ES products, the protein molecules of 66, 46, 35, 33, 30 and 14 kDa molecular wt. showed antigenic activity by immuno blotting technique. In sandwich ELISA technique eventhough all SDS-PAGE fractions except ESA 6 (55-47 kDa) showed antigenic positivity, the fractions ESA 8 (37-31 kDa) and ESA 9 (31-25 kDa) showed high reciprocal antigen titre of 262144 and 32768 respectively. In B. malayi infective larval ES products, the protein molecules of 109, 102, 97 and 77 kDa molecular wt. showed reactivity with FSIgG by blotting technique, where as in sandwich ELISA except ESA 7 (47-37kDa), all fractions showed antigenic positivity. However, these fractions failed to show high antigen titre similar to W. bancrofti ES products with FSIgG.     

Differentiation between Trichinella spiralis and T. pseudospiralis infective larvae by a monoclonal antibody

Crude saline extracts of Trichinella spiralis and T. pseudospiralis infective larvae were studied by Western blot analysis using a monoclonal antibody, named ES/TA2 and produced against T. spiralis larvae. This monoclonal antibody recognized seven major antigenic components in T. spiralis larvae with apparent Mr: 45, 48, 50, 68, 70, 92 and 105 kDa and five in T. pseudospiralis larvae: 38, 50, 70, 72 and 92 kDa. SDS-PAGE of both extracts did not reveal appreciable differences in the range of molecular weights recognized by ES/TA2. These facts show the existence of immunological differences among proteins with apparently identical molecular weights.     

Component proteins in cystic fluid of Taenia solium metacestodes collected surgically from neurocysticercosis patients.

Surgically collected cystic fluid of Taenia solium metacestodes from patients of intracranial cystic lesion were compared in their protein composition with those from naturally infected pigs in Cheju Do, Korea and Ecuador. In non-denaturing discontinuous-polyacrylamide gel electrophoresis (disc-PAGE), no discernible differences were recognized in banding patterns between the cystic fluids from Cheju Do and Ecuador, and between the cystic fluids from pigs and human lesions except wider bands that corresponded to human albumin and gamma-globulin (in 4 of 9 patients). In reducing SDS-PAGE, bands in the cystic fluid from Ecuador showed the same banding pattern with that from Cheju Do but two bands of 21 and 17 kDa were stained darker. Cystic fluids from patients revealed the same protein compositions of the major protein bands of 94, 64, 15, 10 and 7 kDa as in the cystic fluid of pig origin, but human albumin (66 kDa), heavy and light chains of gamma globulin (55 and 22.5 kDa) were contaminated in 4 of 9 cystic fluids. Human CSF proteins seem to have been contaminated during cystic fluid collection. In any cystic fluid from patients, the major protein component was 150 kDa which was subdivided into 15, 10 and 7 kDa in reducing SDS-PAGE.     

Purification and characterization of an alpha 1-antichymotrypsin-like 66 kDa protein from the human breast cancer cell line, MCF-7.

The synthesis of a 66 kDa protein immunoreactive with antibodies to human alpha 1-antichymotrypsin (alpha 1-ACT) is induced by estradiol (E2) in the human breast cancer cell line MCF-7. We have purified this alpha 1-ACT-like 66 kDa protein from medium conditioned by MCF-7 cells, performed a comparative physico-chemical characterization with serum alpha 1-ACT, and analysed its presumed positive regulatory effect on growth of MCF-7 cells. The 66 kDa protein is a functional antiproteinase which is antigenically identical to serum alpha 1-ACT. The 66 kDa protein does however deviate from serum alpha 1-ACT with respect to mol. wt. and pattern of microheterogeneity, the molecular mechanism for this is probably an incomplete glycoprotein processing in the MCF-7 cells. The results of our growth experiments suggest that the 66 kDa protein is a minor positive growth regulatory factor, which may contribute to breast carcinoma cell proliferation in a cooperative manner.     

Trypanosoma musculi co-express several receptors binding rodent IgM, IgE, and IgG subclasses

The present work demonstrates the expression of receptors for the Fc portion of rodent Ig by the murine parasite Trypanosoma musculi. By using a rosette assay adapted to the parasite morphology and by flow cytometry analysis, three distinct receptors were identified. A receptor binding rabbit or rat polyclonal IgG and mouse monoclonal IgG1, IgG2a, and IgG2b was found on parasites purified from the blood and the peritoneal cavity of infected mice and on parasites maintained in culture conditions. This IgG receptor was degraded by pepsin. A separate receptor, binding only mouse monoclonal IgG3 was observed on cultured parasites. A receptor binding rabbit, rat, and mouse IgM was found on cultured and peritoneal parasites, but not on blood parasites. This receptor did not bind IgG or IgA but it bound mouse and rat IgE as well as IgM. It was degraded by trypsin. IgG and IgM/IgE receptors were co-expressed on single parasites. They were not of host origin but synthesized by trypanosomes as shown by reexpression in vitro after proteolytic degradation. Their expression was variable with the development of trypanosomes both in vitro and in vivo.     

Purification of cross-reacting protein antigen shared by Yersinia enterocolitica and other gram-negative bacteria with monoclonal antibody

A monoclonal antibody against the Yersinia enterocolitica 60-kilodalton (kDa) antigen, designated cross-reacting protein antigen (CRPA), was obtained by cell fusion. The CRPA common to gram-negative bacteria was purified from Y. enterocolitica by the affinity chromatography with the monoclonal antibody (IgG1) thus obtained. The purified CRPA showed a single band of 60 kDa in SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and reacted with rabbit antisera against Y. enterocolitica, Vibrio cholerae, Escherichia coli, Pseudomonas aeruginosa, and Shigella sonnei in Western blot analysis. The monoclonal antibody, however, reacted with a 60 kDa peptide from Y. enterocolitica, but not with the antigens from other gram-negative bacteria such as V. cholerae, E. coli, S. sonnei, Salmonella enteritidis, Serratia marcescens, Klebsiella pneumoniae, Proteus mirabilis, and P. aeruginosa. The results suggested that both species-specific and cross-reactive epitopes were present on a CRPA molecule.     

Production and characterization of a monoclonal antibody to dopamine D2 receptor: comparison with a polyclonal antibody to a different epitope.

A monoclonal antibody (Mab) that recognizes the rat dopamine D2 receptor (DAR) has been generated using DAR specific peptide. The Mab, IgM isotype recognizes five proteins (Mr 220, 145, 95, 66 and 47 kDa) in striatal membrane on Western blot. Preincubation of Mab with free peptide blocked the labeling of all five bands. A polyclonal antibody against peptide from a different region of the DAR, reacted with three out of five proteins (220, 66, and 47 kDa) in these membranes. The DAR antagonist NAPS-biotinyl binds to a 220 kDa protein in striatal membrane on ligand blotts; the labeling can be blocked by the addition of 2 microM sulpride. The 220 kDa Mab reactive protein was less in cerebellum and was absent in the liver. Neither the Mab nor polyclonal antibody inhibited binding of a DAR antagonist, [3H]YM09151-2, to the striatal membranes. These antibodies will enable us to study the structure/function and regulation of the synthesis of DAR protein.     

Qualitative characterization of antibody responses to single and multiple Brugia pahangi infections in jirds

Antibody responses of jirds, singly and multiply inoculated with Brugia pahangi infective larvae (L3), to soluble somatic extracts of adult parasites were characterized by western blot analysis. Forty-two protein bands ranging in molecular weight from 12 to 160 kDa were recognized by sera from infected jirds. Antibody recognition of individual B. pahangi antigen bands in this assay appears to be independent of antibody enzyme-linked immunosorbent assay (ELISA) titers to crude parasite extract, severity of lymphatic lesions, levels of microfilaremia, numbers of L3 inoculated, or numbers of adult parasites in individual jirds. Antibody recognition of protein bands with molecular weights of 37 kDa, 21 kDa, and 17 kDa, however, did temporally correspond with certain parasitological and pathologic events. Antibody against the 37-kDa protein band first was identified at the onset of patency, reaching a 90% prevalence rate by 90 days postinfection (DPI). The prevalence of this antibody remained high. Antibody recognition of the 21-kDa protein band first occurred at 90 DPI and gradually increased in prevalence during the course of infection temporally similar to the increase in microfilaremia. Recognition of the 17-kDa protein band first occurred at 48 DPI, reached a maximum prevalence of 80% at 90 DPI, and decreased to a minimal prevalence by 160 DPI. Prevalence of antibody responses to the 17-kDa protein band corresponded temporally with the kinetics of the rise and fall of numbers of intralymphatic thrombi. The patterns of antibody response to these 3 bands were similar in both singly and multiply inoculated animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of tegumental surface proteins of Schistosoma mansoni primary sporocysts

Axenically transformed primary sporocysts of Schistosoma mansoni (NMRI strain) were labeled with 125I in an effort to identify sporocyst proteins exposed at the tegumental surface. Using the 125I activating reagent, 1,3,4,6-tetrachloro-3 alpha,6 alpha-diphenylglycoluril, in conjunction with SDS-PAGE and autoradiography, up to 12 bands were radiolabeled out of 60 components visualized by silver staining. Labeled proteins ranged in apparent Mr from greater than 200 to less than 12 kDa. Pronase treatment of living sporocysts after radioiodination removed all labeled material, suggesting that only surface proteins were being iodinated. Western blot analysis employing 5 monoclonal antibodies (MAB's) to sporocyst surface antigens revealed a wide range of reactivities which produced banding patterns closely reflecting autoradiograms of identical samples. The concomitant removal by Pronase of immunoreactive and radiolabeled surface proteins with identical Mr in the range of 90-130 kDa suggests that epitopes recognized by these antibodies are associated with these higher molecular weight surface proteins. However, although Pronase removes all labeled surface proteins, substantial nonradiolabeled, immunoreactive material with Mr less than 90 kDa still remains on enzyme-treated parasites. This indicates that MAB-reactive epitopes, in addition to their occurrence with surface proteins, are also associated with either unlabeled, protease-resistant surface components or internal antigens. The immunohistochemical localization of antibody-reactive material in gland-like structures within sporocysts supports an internal source for nonradiolabeled, immunoreactive components. Finally, the periodate sensitivity of the epitopes recognized by all tested MAB's suggests that carbohydrate moieties may represent a common and extremely immunogenic constituent of the sporocyst surface.(ABSTRACT TRUNCATED AT 250 WORDS)     

The structure of a juvenile hormone-binding lipophorin from the hemolymph of Diploptera punctata

The high molecular weight, high affinity juvenile hormone binding protein from the hemolymph of Diploptera punctata was identified as a lipophorin by gradient KBr ultracentrifugation and SDS gradient PAGE. This juvenile hormone binding lipophorin (JHBL) was composed of two subunits, apolipoprotein I (230 kDa mol. wt) and apolipoprotein II (80 kDa mol. wt). The density of the native protein was 1.15 g/ml. Photoaffinity labeling using the JH analog [³H]EFDA demonstrated that the JH binding site resides on apolipoprotein I. The amino acid composition of both native lipophorin and its two subunits was determined and the N-terminal sequence of the 80 kDa apolipoprotein described for 19 of the first 21 amino acids. This sequence did not have similarity to any known protein. The N-terminus of the 230 kDa apolipoprotein was blocked. The specificity of a monoclonal antibody to purified native JHBL was also demonstrated. We show that the monoclonal antibody was specific to the 230 kDa subunit and did not recognize the 80 kDa apolipoprotein.     

Overexpression and increased DNA topoisomerase II-like enzyme activity in arsenite resistant Leishmania donovani

Western immunoblot analyses of whole cell lysates probed with a human specific monoclonal anti-topoisomerase IIalpha antibody identified a 190 kDa protein over expressed in the arsenite resistant Leishmania donovani strain. The crude nuclear extract of the resistant strain showed higher topoisomerase II-like enzyme activity. suggesting a possible regulatory role of putative topoisomerase II in arsenite resistant Leishmania.