[Ultrastructural localization of leptospiral antigens by colloidal gold technique].

The rabbit anti-mouse antibodies were successfully labelled with colloidal gold. The distribution of leptospiral antigens in ultrastructure was researched with McAbs and labelled antibodies. It was found that colloidal gold particles were mainly distributed at the outer membrane of leptospiral strain O17 cells, which indicated that the antigens recognized by McAbs 1A7E7, and 2F9D4 were localized at the outer membrane of leptospiral cells. It was thought that colloidal technique would provide a method for research on the antigen distribution of leptospiral cells in ultrastructure.

[Comparative study of outer envelope protein and LPS of five strains of leptospires by SDS-PAGE and 2D-PAGE].

We applied SDS-PAGE, 2D-PAGE and Western blot to analyse the outer envelopes protein and LPS of five strains of leptospires. The work would lay foundations for taxonomy, the development of vaccination regimens and the elucidation of pathogenic mechanisms. The outer envelope proteins of leptospires were analyzed by SDS-PAGE and silver staining. We found that the protein profiles of the pathogenic leptospires were basically identical. A comparison of the protein profiles of the pathogenic L. with those exhibited by two nonpathogenic L. indicated that there was no obvious relationship between these organisms and any of the L. interrogans strains examined. The quantity of 21.5 kd protein of strain 017 was greater than that of strain 601 and 156. Approximately 200, 225, 238 distinct polypeptides were detected in the strain 017, 601 and 156 in 2D-PAGE by silver staining respectively. The profiles of 2D-PAGe showed obvious differences in pI. The pI of strain 017, 601 and 156 were mainly 6.68-7.4, 6.55-6.9, 5.85-7.1 respectively. The 21.5 kd protein of strain 017 was made up of six polypeptides. Our immunoblots revealed that McAb (LB1) reacted with a 41 kd antigen, which was common to the three virulent leptospires tested. SDS-PAGE profiles of silver stained outer envelope LPS of pathogenic L. differed greatly from those of the nonpathogenic L. There was a distinct differences between strain Patoc I and 3055. Our studies showed that each of the five strains of leptospires possessed characteristic outer envelope LPS, which may be used to identify the genus, species and serovars of a strain of L.

[Production, identification of four McAbs against leptospiral outer membrane antigens, and analyses of outer membrane antigens of leptospires with McAbs].

Four McAbs (1A7E7, 2F9D4, 2G3H5, 1A7H12) against outer membrane antigens of L. interrogans serovar Lai strain 017 were produced by hybridoma technique. McAb 1A7E7 was identified to be IgG2a, the rest IgG1 by immunodiffusion. Outer membrane antigens of three pathogenic leptospiral strains (017, 601, 156) and two non-pathogenic leptospiral strains (Patoc I, 3055) were analysed by SDS- polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting with McAbs. It was found that McAb 1A7E7 recognized specifically the 42kd antigenic band of strain 017; McAb 2F9D4 the 42kd antigenic bands of strain 017, 601, 156, and McAbs 2G3H5 and 1A7H12 the 31 kd antigenic bands of 017, 601, 156. Further study of antigenic properties possessed by pathogenic leptospira may provide some new clues to look for specific diagnostic and protective antigens.

[The ultrastructure of leptospires after freeze-etching].

The ultrastructure of three strains of leptospires, i.e. L. interrogans serovar Lai strain 017, L. biflexa serovar patoc strain Patoc I and L. illini strain 3055, were studied with the technique of freeze-etching replica. The results showed that (1) the ultrastructure of leptospires after freeze-etching are similar to that observed by means of negative staining and ultrasectioning. There are no dramatic differences among strains of leptospires studied. (2) Globular particles exposed on the resulting two inner membrane faces are asymmetrically distributed. Large areas on EF face studied with numerous globular particles, whereas the PF face had few of them. The density of globular particles varies in different parts of leptospires, from 776/microns2 to 2303/microns2. The globular particles, 14.25 +/- 2.25 nm in diameter, represent membrane proteins in outer envelope of leptospires. (3) Axial filament, 20 nm in diameter, were seen closely surrounded by a 7 nm sheath-like structure. (4) There are subcellular structures in cytoplasm of leptospires. They appear to be ribosomes, chromatins and inclusions.

[Endocytosis and cytotoxin effect induced by the attachment of the glycolipoprotein of leptospires to vascular endothelial cell in culture].

The Glycolipoprotein (GLP) of various virulent leptospires was extracted, purified and identified. The GLP was combined with fluorescence rhodamine B and the colloidal gold as the probe. Meanwhile, the bovine aortic endothelial cell (EN) was isolated and cultured in the medium 199 "Nissui". The GLP-rhodamine B and GLP-colloidal gold probe were added to cultured EN and incubated at 4 degrees C for 2 h then at 37 degrees C for 2 h. The interaction between GLP-rhodamine B, GLP-CG probe and EN were observed and photography by fluorescence microscopy (Leitz) and scanning electronmicroscopy. The releasing rate of lactic acid dehydrogenase (LDH), acid phosphatase (AcP) and protein from the EN were detected. The results indicated that the GLP of Leptospira interrogans serovar Lai, strain 017 attached to EN and a endocytosis was happened subsequently. The LDH and AcP releasing rate were significantly higher than control and the cell protein was much less than control. The results showed that the GLP of L. interrogans strain 017 had the cytotoxin effect through a specific attachment, endocytosis in EN. The endocytosis probably is a receptor-mediated endocytosis.

[Purification and SDS-page analysis of axial filaments from leptospires].

A modified method, differential centrifugation followed by sucrose density centrifugation, was used to purify axial filaments from three strains of Leptospires. Ultrastructure of the axial filaments was studied and profiles of the axial filaments were characterized and compared. The results have shown that all the three strains of Leptospires, i.e., L. interrogsans serovar Lai strain 017, L. biflexa serovar patoc strain Patoc I and L. illini strain 3055, have two axial filaments in one cell. The axial filament is 20 nm in diameter. It is the first observation that the end which inserts the cytoplasms cylinder is wider in diameter than the free one. An insertion pore structure is observed. The new method yields 1.5mg axial filaments from 12 g leptospires cells. SDS-PAGE was first employed in the analysis of axial filaments of leptospires. The results have also shown that there are 6 proteins in the axial filaments of strain 017, MW 26,000-50,000 while 7 proteins in the axial filaments of strain Patoc I and strain 3055. MW 29,000-80,000 and 28,500-80,000 respectively. Interestingly, all the axial filaments of the three strains have a common protein band of MW 31,500. The possibility of using axial filament proteins as a new criterion for typing and a serodiagnosis antigen is discussed.