Sera from paraneoplastic pemphigus (PNP) immunoprecipitate multiple antigens from human epidermal protein extract. In this study, we further characterized the autoantibodies in 12 PNP sera. Immunoblotting using recombinant linker subdomains of envoplakin, periplakin, desmoplakin, and bullous pemphigoid antigen I found that 11 of the 12 sera recognized linker subdomains of envoplakin and periplakin. We then synthesized 12 peptides covering the linker subdomain of envoplakin for ELISA. One of the peptides, peptide no. 8, was recognized by nine out of the 12 sera with a higher affinity. A method of ligand-receptor binding assay was designed and performed using this peptide labeled with fluorescence as the ligand. Peptide no. 8 bound to CD20(+) cells in Castleman's tumors from the patients whose sera were positive to this peptide by ELISA. Our data suggest that the linker subdomain of plakin proteins may be one of the major areas recognized by PNP autoantibodies, and epitopes in the linker subdomain of envoplakin recognized by PNP autoantibodies with a high affinity are dispersed in several areas and are variable among PNP patients. We also demonstrate that B-lymphocyte clones specifically reacting to epidermal proteins exist in Castleman's tumors from PNP.

Up-converting phosphor technology (UPT)-based lateral-flow immunoassay has been developed for quantitative detection of Yersinia pestis rapidly and specifically. In this assay, 400 nm up-converting phosphor particles were used as the reporter. A sandwich immumoassay was employed by using a polyclonal antibody against F1 antigen of Y. pestis immobilized on the nitrocellulose membrane and the same antibody conjugated to the UPT particles. The signal detection of the strips was performed by the UPT-based biosensor that could provide a 980 nm IR laser to excite the phosphor particles, then collect the visible luminescence emitted by the UPT particles and finally convert it to the voltage as a signal. V-T and V-c stand for the multiplied voltage units for the test and the control line, respectively, and the ratio V-T/V-C is directly proportional to the number of Y pestis in a sample. We observed a good linearity between the ratio and log CFU/ml of Y pestis above the detection limit, which was approximately 10(4) CFU/mI. The precision of the intra- and inter-assay was below 15% (coefficient of variation, CV). Cross-reactivity with related Gram-negative enteric bacteria was not found. The UPT-LF immunoassay system presented here takes less than 30 min to perform from the sample treatment to the data analysis. The current paper includes only preliminary data concerning the biomedical aspects of the assay, but is more concentrated on the technical details of establishing a rapid manual assay using a state-of-the-art label chemistry. (c) 2006 Elsevier B.V. All rights reserved.