In-cell assembly of single-chain Fv from peripheral blood lymphocytes from vitiligo patients / 中华皮肤科杂志

ABSTRACT

Objective To obtain in situ pairing of the variable region genes of the immunoglobulin heavy and light chains of B cells from vitiligo patients. Methods Fifty vitiligo patients were screened by a live melanocyte enzyme-linked immunoabsorbent assay. The sera from seven patients were proved to be strongly positive. Then CD19+ B cells were isolated from the peripheral blood lymphocytes of the 7 patients, then fixed in 10% buffered formaldehyde and permeabilised by Proteinase K. The mRNA was amplified within the cells by reverse transcription-polymerase chain reaction (RT-PCR) with specific primers. The immunoglobulin VH and VL DNA assembled within the same cells using the Cre/loxP system. Nested primers were designed to amplify the known major human VH and VL gene familes. Result A unique 800bp band was obtained corresponding in size to single chain Fv fragments. Conclusion The in situ pairing of the variable region genes of the immunoglobulin heavy and light chains of B cells is obtained from vitiligo patients by in-cell PCR.