The determination of acetylcholine receptor antibody (AChR Ab) titer by an enzyme-linked immunosorbent assay (ELISA) in patients with myasthenia gravis was introduced. The optimal conditions were determined by chequerboard determination. The specificity was confirmed by inhibition tests. The sensitivity is 9 p mole. The comparison of AChR Ab titers among 49 myasthenic patients, 19 non-myasthenic neurological patients and 20 healthy blood donors has shown that it is a highly sensitive, specific, reproducible, rapid, simple and inexpensive method for determining AChR Ab and that it is highly valuable for the diagnosis of myasthenia gravis.
The zot gene encoding Zonula occludens toxin was amplified from classic Vibrio cholerae genomic DNA by PCR. The result of sequencing indicated that zot gene encodes 399 amino acid residues. The sequence of zot gene was a little bit different from that of reported including 14 nucleotides and four amino acid residues. The expression plasmid pET-ZOT was constructed by inserting zot gene into plasmid pET-28a(+) containing the T7 promoter. The expression plasmid was induced into E. coli BL21 (DE3) and expression strain BLZOT was selected. SDS-PAGE analysis revealed that the ZOT protein was expressed and accumulated up to above 15% of bacterial soluble protein after induced by IPTG. A protein of 47 kD was expressed as including body. Western blot analysis revealed that the expressed protein was ZOT.
Tumor necrosis factor alpha (TNF alpha) mRNA expression in bronchoalveolar lavage (BAL) cells from patients with lung fibrosis was studied by a reverse transcription-DNA polymerase chain reaction (RT-PCR). The results indicated that two thirds of the tested samples were TNF alpha mRNA positive. On the other hand, TNF alpha activity was detected in a big part of the BAL supernatants from the patients. Furthermore, recombinant TNF alpha was found to be mitogenic to lung fibroblasts. These data suggest that TNF alpha expression in BAL cells may play a role in the development of lung fibrosis in human.
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