scholarly journals Detection of Aspergillus Flavus in Wheat Grains Using Anti-Mannoprotein (MP1) and Spore Proteins Polyclonal Antibodies

2021 ◽  
Author(s):  
Ranjana Kumari ◽  
Ananta Ghosh
Keyword(s):  
Aspergillus Flavus ◽  
Polyclonal Antibodies ◽  
Protein A ◽  
Fungal Species ◽  
Cross Reactivity ◽  
Soluble Form ◽  
Minimum Concentration ◽  
Wheat Grains ◽  
Stored Wheat ◽  
Spore Proteins

Abstract Cell wall mannoprotein (MP1) gene of an aflatoxigenic strain of Aspergillus flavus, isolated from stored wheat grains, was cloned and sequenced. MP1 protein was expressed in E. coli in soluble form and purified. Polyclonal antibodies were raised against recombinant MP1 protein and inactivated spores of this fungus in rabbit, and purified by ammonium sulphate precipitation, Protein A sepharose and antigen affinity chromatography. The minimum concentration of purified mycelial or spore proteins that could be detected by ELISA was determined as 100 ng using 2 µg of these antibodies. The anti-MP1 antibody was found more sensitive than anti-spore protein antibody. Western blot and immunofluorescence analysis showed reactivity of these antibodies to various proteins (30 kDa to 200 kDa) distributed throughout the surface of mycelia and spore of A. flavus. Cross reactivity of these antibodies was detected with fungi belonging to different Aspergillus, Rhizopus and Alternaria species out of fourteen different fungal species tested. In fungal contaminated wheat grains these antibodies could detect presence of as low as 1 µg mycelia or 103 spores per gram of wheat grains using ELISA. The results suggest that the developed antibodies could be successfully applied for the detection of predominant fungal infestation in stored wheat grains.

scholarly journals Development of Murine Monoclonal Antibodies for the Immunohistochemical Diagnosis of Systemic Bovine Aspergillosis

1996 ◽  
Vol 8 (1) ◽  
pp. 68-75 ◽  
Author(s):  
H. E. Jensen ◽  
B. Aalbaek ◽  
P. Lind ◽  
H. V. Krogh ◽  
P. L. Frandsen
Keyword(s):  
Monoclonal Antibodies ◽  
Polyclonal Antibodies ◽  
Fungal Species ◽  
Cross Reactivity ◽  
Water Soluble ◽  
Enzyme Linked Immunosorbent Assay ◽  
Immunohistochemical Techniques ◽  
Immunohistochemical Diagnosis ◽  
Murine Monoclonal Antibodies

Murine monoclonal antibodies (MAbs) against water-soluble somatic antigens (WSSA) and the wall fraction (WF) from Aspergillus fumigatus were produced by fusion of splenocytes from immunized BALB/c mice with mouse myeloma X63-Ag 8.653 cells. The supernatants of in vitro cultured hybridomas were initially screened for reactivity with the WSSA and the WF from A. fumigatus and WSSA of other fungi in an enzyme-linked immunosorbent assay (ELISA). Supernatants reacting only with A. fumigatus antigens were subsequently screened for homologous and heterologous reactivity with immunohistochemical techniques using formalin-fixed, paraffin-embedded tissues from experimentally infected mice. Because of a high immunohistochemical reactivity with homologous fungi, 4 MAbs raised against A. fumigatus WSSA and WF were selected for a further evaluation of cross-reactivity (diagnostic specificity) in immunohistochemical and immunoblotting assays. In immunohistochemical assays, all MAbs raised against WSSA cross-reacted heavily with a number of other fungal species. All 4 MAbs (MAb-WF-AF-1-4) raised against the WF reacted strongly with hyphae of Aspergillus spp.; hyphae of Scedosporium apiospermum were also strongly labeled by MAb-WF-AF-3 and-4. The 2 specifically reacting MAbs (MAb-WF-AF-1 and-2) were of the IgM biotype and were precipitating, and in immunoblotting experiments both bound to a 106-kD antigen of the WF, whereas they did not bind to WSSA of A. fumigatus. One of the 2 aspergillosis-specific MAbs (MAb-WF-AF-1) was used to screen 145 mycotic lesions of cattle. The diagnoses on bovine lesions obtained by MAb-WF-AF-1 were compared with results based on reactivity with heterologously absorbed polyclonal antibodies and, for some lesions, to culture results. In the vast majority of lesions ( n = 133), the MAb-WF-AF-1 and the polyclonal anti-Aspergillus antibodies reacted in a similar pattern, i.e., positively in 41 aspergillosis lesions and negatively in 92 zygomycotic lesions. Hyphae in 3 of 12 lesions that were not stained by the polyclonal antibodies reacted with the specific MAb-WF-AF-1; i.e., aspergillosis was diagnosed. The characteristics of the 2 MAbs (MAb-WF-AF-1 and-2) raised against the WF of A. fumigatus in ELISA and immunoblotting and immunohistochemical assays justify their application for the in situ diagnosis of systemic aspergillosis of cattle.

Purification of Tetragalloylglucose 4-O-Galloyltransferase and Preparation of Antibodies against This Key Enzyme in the Biosynthesis of Hydrolyzable Tannins

2000 ◽  
Vol 55 (7-8) ◽  
pp. 582-587 ◽  
Author(s):  
Petra Grundhöfer ◽  
Georg G. Gross
Keyword(s):  
Quercus Robur ◽  
Polyclonal Antibodies ◽  
Protein A ◽  
Gel Filtration ◽  
Cross Reactivity ◽  
Pedunculate Oak ◽  
Hydrolyzable Tannins ◽  
Apparent Homogeneity ◽  
Key Enzyme ◽  
Rhus Typhina

Abstract The enzyme, β-glucogallin: 1,2,3,6-tetra-O-galloyl- β-ᴅ-glucose 4-O-galloyltransferase, which catalyzes the last common step in the biosynthesis of the two subclasses of hydrolyzable tannins, i.e. gallotannins and ellagitannins, was purified 868-fold from leaves of pedunculate oak ( Quercus robur, syn. Q. pedunculata) to apparent homogeneity. Polyclonal antibodies against this pivotal enzyme were raised in rabbits and purified by protein-A chromatography, gel-filtration and affinity complexation. They were found to react specifically with acyltransferase from oak, displaying no cross-reactivity towards analogous enzymes from other plants synthesizing hydrolyzable tannins along the same biogenetic route, e.g. Rhus typhina or Tellima grandiflora.

Comparison of spore proteins among species of the cellular slime moulds Dictyostelium and Polysphondylium as examined by immunological cross-reactivity

1988 ◽  
Vol 34 (7) ◽  
pp. 891-896 ◽  
Author(s):  
Yohko Yamada ◽  
Koji Okamoto ◽  
Ikuo Takeuchi
Keyword(s):  
Polyclonal Antibodies ◽  
Polyacrylamide Gel Electrophoresis ◽  
Cross Reactivity ◽  
Developmental Changes ◽  
Coat Proteins ◽  
Slime Moulds ◽  
Cellular Slime ◽  
Spore Proteins ◽  
Spore Coat Proteins ◽  
Cellular Slime Moulds

Spore proteins of six cellular slime mould species, Dictyostelium discoideum, D. mucoroides, D. purpureum, D. lacteum, Polysphondylium violaceum, and P. pallidum were studied. The spore proteins were cross-reacted with four different polyclonal antibodies produced against D. mucoroides spores and D. discoideum major spore coat proteins SP96, SP70, and SP60 by SDS polyacrylamide gel electrophoresis and immunoblotting. The spore proteins of D. discoideum and D. mucoroides showed the strongest cross-reactivity with all the antisera and also produced many common protein bands, thus reflecting their morphological similarities. Polysphondylium violaceum, which is quite distinct in morphology from D. discoideum and D. mucoroides, produced the second strongest cross-reactivity. In contrast, the proteins of D. purpureum showed little cross-reactivity, although morphologically it closely resembles D. mucoroides. The developmental changes of these spore proteins were investigated by cross-reacting the antisera against vegetative and slug cell proteins. In the cases of D. discoideum and D. mucoroides, the band patterns of slug proteins coincided with those of spores, which suggested that most of the spore proteins had already accumulated at the slug stage. However, this was not the case with P. violaceum, which suggested that spore proteins of this species cross-reactive with the antisera were synthesized or modified at the culmination stage.

scholarly journals Purification and characterization of protein disulphide-isomerase from the unicellular green alga Chlamydomonas reinhardii. A 120 kDa dimer antigenically distinct from the vertebrate enzyme

1990 ◽  
Vol 268 (1) ◽  
pp. 63-68 ◽  
Author(s):  
D D Kaska ◽  
K I Kivirikko ◽  
R Myllylä
Keyword(s):  
Green Alga ◽  
Polyclonal Antibodies ◽  
Active Form ◽  
Protein A ◽  
Gel Filtration ◽  
Cross Reactivity ◽  
Chlamydomonas Reinhardii ◽  
Protein Disulphide Isomerase ◽  
Unicellular Green Alga ◽  
A Subunit

Protein disulphide-isomerase (PDI) has been isolated from the unicellular green alga Chlamydomonas reinhardii and purified by (NH4)2SO4 precipitation, gel filtration and DEAE-Sephacel, hydroxyapatite and f.p.l.c. chromatography. The active algal enzyme is a 120 kDa dimer with a subunit molecular mass of 60 kDa when determined by SDS/PAGE. Although similar in size to the previously isolated vertebrate PDIs, the algal enzyme is antigenically distinct, polyclonal antibodies against the algal PDI showing no cross-reactivity with the vertebrate enzyme on immunoblots, and vice versa. The anti-(algal PDI) antiserum did not inhibit algal PDI activity, and C. reinhardii PDI could be immobilized on anti-PDI-Protein A-Sepharose in active form. In contrast with the situation in vertebrates, where PDI functions as a subunit of prolyl 4-hydroxylase, the C. reinhardii PDI is not associated with the algal prolyl 4-hydroxylase.

scholarly journals Examination of the Efficacy and Cross-Reactivity of a Novel Polyclonal Antibody Targeting the Disintegrin Domain in SVMPs to Neutralize Snake Venom

Toxins ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 254
Author(s):  
Shelby S. Szteiter ◽  
Ilse N. Diego ◽  
Jonathan Ortegon ◽  
Eliana Salinas ◽  
Abcde Cirilo ◽  
...  
Keyword(s):  
Snake Venom ◽  
Polyclonal Antibody ◽  
Polyclonal Antibodies ◽  
Cross Reactivity ◽  
Snake Envenomation ◽  
Disintegrin Domain ◽  
The Common ◽  
Neutralization Ability ◽  
New Strategies

Snake envenomation can result in hemorrhage, local necrosis, swelling, and if not treated properly can lead to adverse systemic effects such as coagulopathy, nephrotoxicity, neurotoxicity, and cardiotoxicity, which can result in death. As such, snake venom metalloproteinases (SVMPs) and disintegrins are two toxic components that contribute to hemorrhage and interfere with the hemostatic system. Administration of a commercial antivenom is the common antidote to treat snake envenomation, but the high-cost, lack of efficacy, side effects, and limited availability, necessitates the development of new strategies and approaches for therapeutic treatments. Herein, we describe the neutralization ability of anti-disintegrin polyclonal antibody on the activities of isolated disintegrins, P-II/P-III SVMPs, and crude venoms. Our results show disintegrin activity on platelet aggregation in whole blood and the migration of the SK-Mel-28 cells that can be neutralized with anti-disintegrin polyclonal antibody. We characterized a SVMP and found that anti-disintegrin was also able to inhibit its activity in an in vitro proteolytic assay. Moreover, we found that anti-disintegrin could neutralize the proteolytic and hemorrhagic activities from crude Crotalus atrox venom. Our results suggest that anti-disintegrin polyclonal antibodies have the potential for a targeted approach to neutralize SVMPs in the treatment of snakebite envenomations.

Shedding of Gs Protein (A Soluble Form of the Viral Glycoprotein) by the Rabies Virus-Infected BHK-21 Cells

Virology ◽  
1993 ◽  
Vol 195 (2) ◽  
pp. 541-549 ◽  
Author(s):  
Kinjiro Morimoto ◽  
Yasumasa Iwatani ◽  
Akihiko Kawai
Keyword(s):  
Rabies Virus ◽  
Protein A ◽  
Soluble Form ◽  
Viral Glycoprotein ◽  
Gs Protein

scholarly journals PRESENTATION PATTERN AND FUNGAL AGENTS SPECTRUM CAUSING OTOMMYCOSIS

2018 ◽  
Vol 11 (1) ◽  
pp. 408
Author(s):  
Kassim Dekhil
Keyword(s):  
Candida Albicans ◽  
Aspergillus Niger ◽  
Aspergillus Fumigatus ◽  
Influencing Factors ◽  
Aspergillus Flavus ◽  
Species Distribution ◽  
Alternaria Alternata ◽  
Otitis Externa ◽  
Fungal Species ◽  
The Public

 Objective: This study was aimed to identify the public pattern of presentation, influencing factors, and sort the fungal species, distribution of sex of patients with otomycosis.Results: The predominant complaints were pruritus and found in 76 patients (88.73%), discomfort and pain found in 62 patients (72.09%), aural fullness in 48 patients (55.81%), tinnitus in 34 patients (39.53%), hearing impairment in 50 cases (58.31%), ear discharge in 22 patients (25.58%), and most of the symptoms seen in 36 patients (68.14%). The results showed a total of eight fungal species belong to six different genera, namely, Aspergillus, Candida, Penicillium, Rhizopus, Alternaria, and Cephalosporium were isolated during this study. Among identified fungi, Aspergillus niger was found to be the most prevalent fungal species with 35.71% followed by Candida albicans (27.55%), Aspergillus flavus (10.20%), Aspergillus fumigatus (8.16), Penicillium digitatum (6.12%) and Cephalosporium species (4.08%), and Rhizopus species (5.1%), while Alternaria alternata had the lowest percentage (6.54%).Conclusion: Otomycosis/mycotic otitis externa is still a common problem and there is a rise in the occurrence of otomycosis in latest years, especially in tropical and subtropical humid climates.

Allergenic cross-reactivity ofCurvularia lunatawith other airborne fungal species

Allergy ◽  
2002 ◽  
Vol 57 (7) ◽  
pp. 636-640 ◽  
Author(s):  
R. Gupta ◽  
B. P. Singh ◽  
S. Sridhara ◽  
S. N. Gaur ◽  
R. Kumar ◽  
...  
Keyword(s):  
Fungal Species ◽  
Cross Reactivity

scholarly journals Immunochemical characterization of NADPH-cytochrome P-450 reductase from Jerusalem artichoke and other higher plants

1989 ◽  
Vol 259 (3) ◽  
pp. 847-853 ◽  
Author(s):  
I Benveniste ◽  
A Lesot ◽  
M P Hasenfratz ◽  
F Durst
Keyword(s):  
Cytochrome C ◽  
Rat Liver ◽  
Higher Plants ◽  
Polyclonal Antibodies ◽  
Reductase Activity ◽  
Jerusalem Artichoke ◽  
Cross Reactivity ◽  
Cytochrome C Reductase ◽  
Nadph Cytochrome ◽  
Cytochrome P 450

Polyclonal antibodies were prepared against NADPH-cytochrome P-450 reductase purified from Jerusalem artichoke. These antibodies inhibited efficiently the NADPH-cytochrome c reductase activity of the purified enzyme, as well as of Jerusalem artichoke microsomes. Likewise, microsomal NADPH-dependent cytochrome P-450 mono-oxygenases (cinnamate and laurate hydroxylases) were efficiently inhibited. The antibodies were only slightly inhibitory toward microsomal NADH-cytochrome c reductase activity, but lowered NADH-dependent cytochrome P-450 mono-oxygenase activities. The Jerusalem artichoke NADPH-cytochrome P-450 reductase is characterized by its high Mr (82,000) as compared with the enzyme from animals (76,000-78,000). Western blot analysis revealed cross-reactivity of the Jerusalem artichoke reductase antibodies with microsomes from plants belonging to different families (monocotyledons and dicotyledons). All of the proteins recognized by the antibodies had an Mr of approx. 82,000. No cross-reaction was observed with microsomes from rat liver or Locusta migratoria midgut. The cross-reactivity generally paralleled well the inhibition of reductase activity: the enzyme from most higher plants tested was inhibited by the antibodies; whereas Gingko biloba, Euglena gracilis, yeast, rat liver and insect midgut activities were insensitive to the antibodies. These results point to structural differences, particularly at the active site, between the reductases from higher plants and the enzymes from phylogenetically distant plants and from animals.

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