Sharad Vishwanath Gangal

Fusarium solani: Immunochemical Characterization of Allergens

Allergenic components of the fungus Fusarium solani were isolated using (NH4)2SO4 precipitation and ion-exchange column chromatography. The allergenicity of fractions was studied by enzyme-linked immunosorbent assay and radioallergosorbent test inhibition techniques. Proteins of culture filtrate (CF), mycelium (MY), and spore (SP) extracts of F. solani were characterized by isoelectrofocusing, sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and IgE-specific immunoblotting. CF antigen of F. solani contained more allergenic proteins than MY and SP, visible on immunoblot analysis using allergenic serum pool. A 65-kD protein component of CF was found to be a major allergen, as it was strongly visible on immunoblots of all 15 patient sera tested. Crossed immunoelectrophoresis and enzyme-linked immunosorbent assay inhibition using rabbit antibodies raised against F. solani CF demonstrated shared antigenicity between CF, MY, and SP extracts. It was observed that F. solani is a significant allergen, and most of the allergens of MY and SP extracts were found in CF extract. Therefore, CF alone can be used in the preparation of a standard extract. However, few unique allergenic proteins were observed in MY as well as in SP extracts of F. solani. Hence, the use of combined CF, MY, and SP extracts of F. solani is recommended for diagnosis and immunotherapy.

Isolation and identification of pollen allergens of Artemisia scoparia

The allergenic proteins of Artemisia scoparia pollen were separated and identified with ammonium sulfate precipitation, ion-exchange chromatography, gel filtration, and RAST-inhibition techniques. The important allergenic component Artemisia VI b that constitutes 29% of total protein in the extract was purified to homogeneity. It was found to be an acidic protein with isoelectric point 3.8 and molecular weight of 14,300. It was rich in carbohydrate, but the carbohydrate portion did not appear to be important for allergenicity. In the crossed immunoelectrophoresis reference pattern of the whole pollen extract, 37 precipitin lines could be identified on the anodic side, whereas Artemisia VI b could be observed as a single precipitin line. Immunologically, the whole pollen extract of A. scoparia demonstrated shared antigenic and allergenic determinants with Ageratum conyzoides-pollen extract. The use of fast protein liquid chromatography in partial purification of allergenic components is also discussed.

Allergen Entrapped in Liposomes Reduce Allergenicity and Induce Immunogenicity on Repeated Injections in Mice

Liposomes which are nontoxic, biodegradable and biocompatible lipid vesicles are known to act as adjuvants and can be used to formulate sustained release preparation by encapsulation. In the present study, allergen entrapped in liposomes were injected at different time intervals in Swiss mice (made responders to IgE by injecting cyclophosphamide) and Balb/C mice (high IgE responders). Tissue distribution studies after intraperitoneal injection of allergen (entrapped and untrapped) revealed that liposome-entrapped allergen was retained for a longer time in all the tissues except kidney as compared to the free allergen given similarly. It was observed that serum specific IgG antibody levels were higher and specific IgE levels were lower in animals given repeated injections of liposomes-entrapped allergen as compared to those animals injected free allergen in the same manner. The entrapment of allergen in liposomes somehow suppressed the specific IgE response on repeated injections. The immunomodulatory effect of liposomes may be useful in the immunotherapy of respiratory allergic disorders.

Characterization of Cogon Grass (Imperata cylindrica) Pollen Extract and Preliminary Analysis of Grass Group 1, 4 and 5 Homologues Using Monoclonal Antibodies to Phleum pratense

Background: Previous studies have established the role of Imperata cylindrica (Ic) pollen in type I allergic disorders. However, no systematic information is available on the allergen composition of Ic pollen extract. Objectives: To characterize the IgE–binding proteins of Ic pollen extract and to detect the presence of grass group 1, 4 and 5 allergen homologues, if any. Methods: Pollen extract of Ic was analyzed by in vivo and in vitro procedures such as intradermal tests (ID), enzyme–linked immunosorbent assay (ELISA), ELISA–inhibition, thin–layer isoelectric focusing (TLIEF), sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS–PAGE) and immunoblotting. Dot blot assay was carried out to check the presence of well–known group 1, 4, and 5 allergen homologues in Ic pollen extract. Results: Out of 303 respiratory allergies patients skin–tested, 27 showed sensitivity to Ic pollen extract. Specific IgE levels were elevated in all 15 serum samples tested. The extract prepared for this study was found to be highly potent since it required only 400 ng of homologous proteins for 50% inhibition of binding in ELISA inhibition assays. TLIEF of Ic pollen extract showed 44 silver–stained bands (pI 3.5–7.0) while SDS–PAGE resolved it into 24 Coomassie–Brilliant–Blue–stained bands (MW 100–10 kD). Immunoblotting with individual patient sera recognized 7 major IgE–binding bands (MW 85, 62, 57, 43, 40, 28 and 16 kD) in Ic pollen extract. A panel of monoclonal antibodies, specific to group 1, 4 and 5 allergens from Phleum pratense pollen extract identified group 5 and group 4 homologues in Ic pollen extract. Conclusion: Ic pollen extract was characterized for the protein profile by TLIEF and SDS–PAGE. IgE reactivity was determined by ELISA and immunoblot. Monoclonal antibodies to group 5 and group 4 allergens reacted weakly showing that this pollen contains group 5 and group 4 homologous allergens.

Application of Defined Co-Immobilized Microbial Consortium as a Ready-to-Use Seed Inoculum in Bod Analysis

A number of microorganisms were isolated from sewage. Biochemical Oxygen Demand (BOD-5 day) analysis was carried out by individual pure cultures. The cultures giving higher or equal BOD values as compared to reference GGA solution were selected for the formulation of a defined mixed microbial consortium. This microbial consortium was co-immobilized on calcium-alginate beads. Four synthetic and six industrial samples were tested for BOD by using immobilized beads as well as sewage as source of seeding materials. BOD values obtained with beads for all the synthetic as well as industrial samples were fairly comparable with those obtained with sewage. Reusability of prepared microbial beads was also checked with different synthetic and industrial samples and was compared with reference GGA solution. The same microbial beads can be reused three times for different BOD-5 day estimations. It is recommended that immobilized microbial beads can be used as a ready-to-use seeding material for BOD analysis.

Monitoring of Active Groups of Cyanogen-Bromide-Activated Paper Discs Used as Allergosorbent

The amount of active groups of cyanate esters and imidocarbamates present on cyanogen-bromide-activated paper discs were estimated. The protein-binding capacity of activated discs was found to be dependent on the amount of these active groups. The results suggested that monitoring of active groups on activated discs can help in preparing consistent allergosorbent for immunoassays.