Specificity of antigens from pathogenic Aspergillus species. I. Studies with ELISA and immunofluorescence

Studies were made by enzyme linked immunosorbent assay (ELISA) and indirect fluorescent antibody (IFA) tests on the reactivities and specificities of 13 antigens prepared from four species of Aspergillus against antisera from immunized rabbits and 64 sera from patients with aspergillosis, other systemic mycoses and nocardiosis. Although reactions in both serological tests were invariably strongest with homologous antigen: antibody systems, antisera from rabbits immunized with A. fumigatus, Blastomyces dermatitidis, Candida albicans and Paracoccidioides brasiliensis reacted in the ELISA test with all of the Aspergillus antigens. In contrast, cross-reactivity was virtually non-existent with antiserum to Histoplasma capsulatum. Of five antigens prepared from A fumigatus tested by ELISA against human sera from patients with aspergillosis and other nocardial and systemic fungal infections, sensitivities varied from 81 to 100% for sera from 32 patients with aspergillosis, and specificities from 20 to 97% for sera from 30 patients with nocardiosis and other systemic mycoses. Purified A. fumigatus C antigen reacted weakly with sera from eight of these 30 patients, but the reactions were readily distinguishable from those obtained with sera from patients with aspergillosis. At optimal serum dilutions, cross-reactivities of A. fumigatus in the IFA studies were non-existent in the sera from 28 patients with candidosis, coccidioidomycosis, cryptococcosis, histoplasmosis, paracoccidioidomycosis and nocardiosis. Sensitivities of IFA were 94% for patients with aspergilloma and 83% for patients with allergic bronchopulmonary aspergillosis.     

Specificity of cytoplasmic and cell-wall antigens from four species of Phytophthora.

Cytoplasmic and cell-wall antigens and antisera were prepared from four Phytophthora species, and cell-wall antigens were prepared from two Pythium species. Immunodiffusion of the Pythium and Phytophthora cell-wall antigens showed that the two Pythium species did not cross-react with the Phytophthora cell-wall antisera. Immunodiffusion analysis of both cell-wall and cytoplasmic antigens of Phytophthora revealed some degree of specificity between species but not between A1 and A2 mating types in Phytophthora cinnamomi. Species specificity was improved by using indirect fluorescent antibody techniques and by the use of cross-absorbed sera. Agglutination and quantitative precipitation techniques did not significantly improve specificity. It was possible to distinguish serologically between Phytophthora cinnamomi and Phytophthora cambivora and the Phytophthora cryptogea-Phytophthora drechsleri group. The absence of consistent serological variation between P. cryptogea and P dreschsleri is consistent with the suggestion (Bumbieris, 1974) that P. cryptogea and P. drechsleri should be considered as one species.     

Specificity of the S1 nuclease from Aspergillus oryzae.

Conditions are described for digesting single-stranded DNA by S1 nuclease without introducing breaks in double-stranded DNA. The enzyme is inhibited by low concentrations of various compounds of phosphate. Under certain conditions S1 nuclease cleaves the strand opposite a nick in bacteriophage T5 DNA; under other conditions, the enzyme cleaves a loop in one strand of heteroduplex lambdaDNA while leaving the opposite strand intact. S1 nuclease makes many single strand breaks in ultraviolet-irradiated duplex lambdaDNA. Superhelical DNA of phiX174 (Form I) is converted first to a relaxed circular molecule (Form II), and then to a linear molecule (Form III) by cleavage at one site per molecule. Since the cleavage occurs at many sites in the population of molecules, the partially single-stranded regions in phiX174 superhelical DNA are not determined by specific nucleotide sequences.     

Substrate Specificity of Alkaline Proteinases from Aspergillus sydowi and Aspergillus sulphureus

l-Fucose (l-galactose) dehydrogenase was isolated to homogeneity from a cell-free extract of Pseudomonas sp. No 1143 and purified about 380-fold with a yield of 23 %. The purification procedures were: treatment with polyethyleneimine, ammonium sulfate fractionation, chromatographies on phenyl-Sepharose and DEAE-Sephadex, preparative polyacrylamide gel electrophoresis, and gel filtration on Sephadex G-100. The enzyme had a molecular weight of about 34,000. The optimum pH was at 9 — 10.5 and the isoelectric point was at pH 5.1. l-Fucose and l-galactose were effective substrates for the enzyme reaction, but d-arabinose was not so much. The anomeric requirement of the enzyme to l-fucose was the β-pyranose form, and the reaction product from l-fucose was l-fucono- lactone. The hydrogen acceptor for the enzyme reaction wasNADP⁺, and NAD ⁺ could be substituted for it to a very small degree. Km values were 1.9mm, 19mm, 0.016mm, and 5.6mm for l-fucose, l- galactose, NADP⁺, and NAD⁺, respectively. The enzyme activity was strongly inhibited by Hg^{2 +}, Cd^{2 +}, and PCMB, but metal-chelating reagents had almost no effect. In a preliminary experiment, it was indicated that the enzyme may be usable for the measurement of l-fucose.     

Studies in Xyridaceae II. two new species of Xyris from Brazil

Two new species of Xyridaceae, Xyris jolyi and X. coutensis, from Minas Gerais, Grazil, are distinguished from close relatives on the basis of morphological features including bracts, sepals, and leaves.     

Separation of three class II antigens from a homozygous human B cell line

Three class II molecules were isolated from a homozygous DRw6 human B lymphoblastoid cell line using the monoclonal antibodies L243 (L203), L227, LKT 111, and Genox 3.53. Two of the antigens appeared to employ the same heavy chain but expressed different light chains. The two light chains were separated after denaturation using L227 and LKT 111. One or both of these two molecules carried the DRw6 and MT2 determinants. The third class II antigen expressed the DC1 determinant. It was composed of a heavy and light chain different from the DR-like antigen subunits. The antibodies L243, L227, and LKT 111 did not preclear the cell lysate of the DC1 antigen recognized by Genox 3.53. However, a xenoanti-DR serum immunoprecipitated both the DR-like and the DC1 antigens. Thus, in total, one cell line can express at least two class II heavy chains and three class II light chains. This observation was not unique to this cell line.     

The demonstration of transmissible gastroenteritis viral antigens by immunoelectrophoresis and counterimmunoelectrophoresis.

Immunoelectrophoresis of alkaline intestinal extracts, or the supernatants after precipitation of these extracts with 60% ammonium sulfate, prepared from piglets experimentally infected with the DL or Purdue strains of transmissible gastroenteritis virus, revealed up to three antigens. Two antigens migrated towards the anode, and the third migrated towards the cathode. Antigens with anodal or cathodal mobility were also demonstrated in the same materials by counterimmunoelectrophoresis, and the procedure for counterimmunoelectrophoresis was modified to detect both antigens in a single test.     

Identification of the pathogenic Aspergillus species by nested PCR using a mixture of specific primers to DNA topoisomerase II gene

For PCR-based identification of Aspergillus species, a common primer of the DNA topoisomerase II genes of Candida, Aspergillus and Penicillium, and species-specific primers of the genomic sequences of DNA topoisomerase II of A. fumigatus, A. niger, A. flavus (A. oryzae), A. nidulans and A. terreus were tested for their specificities in PCR amplifications. The method consisted of amplification of the genomic DNA topoisomerase II gene by a common primer set, followed by a second PCR with a primer mix consisting of 5 species-specific primer pairs for each Aspergillus species. By using the common primer pair, a DNA fragment of approximately 1,200 bp was amplified from the Aspergillus and Penicillium genomic DNAs. Using each species-specific primer pair, unique sizes of PCR products were amplified, all of which corresponded to a species of Aspergillus even in the presence of DNAs of several fungal species. The sensitivity of A. fumigatus to the nested PCR was found to be 100 fg of DNA in the reaction mixture. In the nested PCR obtained by using the primer mix (PsIV), the specific DNA fragment of A. fumigatus was amplified from clinical specimens. These results suggest that this nested PCR method is rapid, simple and available as a tool for identification of pathogenic Aspergillus to a species level.     

Identification of lipoglycan antigens from the Acholeplasma laidlawii cell membrane in crossed immunoelectrophoresis

Membranes from the wall-less prokaryote Acholeplasma laidlawii contain a component termed lipoglycan or lipopolysaccharide (LPS). The lipoglycan has extraction properties, which are similar to those of LPS of gram-negative bacteria, but it is chemically distinct from bacterial LPS. The membrane-bound lipoglycan of A. laidlawii did not seem to be particularly immunogenic and antibodies against it could not always be detected by rocket immunoelectrophoresis (RIE) or crossed immunoelectrophoresis (CIE) in hyperimmune sera raised against membranes. The immunoprecipitate corresponding to the lipoglycan, obtained by CIE of Tween 20-solubilized A. laidlawii membranes, has been identified and shown to be both a cathodically and anodically migrating component at pH 8.6. The shape of the immunoprecipitate in both RIE and CIE showed that the lipoglycan antigen is composed of at least two components, which are immunologically related.