| It has been confirmed that a number of membrane molecules exist their soluble forms either shedded from the membrane form by proteolysis or generated by direct secretion from the cell due to the alternative splicing of mRNA. These soluble forms play important roles in the regulation of immunological function and some of them are found to be related to certain diseases.LAIR-1/9.1C3 was first discovered by Burns group based on the monoclonal antibody 9.1C3 raised by immunizing mice with human natural killer (NK) cells and its function that 9.1C3 mAb could inhibit the killing of NK cells against K562 cell. The LAIR-1 gene was cloned in 1997 and was named leukocyte-associated immunoglobulin-like recepter-1 (LAIR-1). Soon after, the gene of LAIR-1 was confirmed to be located within the leukocyte Ig-like receptor complex (LRC) on human chromosome 19q13.4. LAIR-1 belongs to Ig superfamily (IgSF) with a single Ig-like domain in the extracellular region and two immunoreceptor tyrosine-based inhibitouy motifs (ITIMs) in the cytoplasmic tail. Previous study shown that LAIR-1/9.1C3 is expressed on the majority of peripheral blood mononuclear cells and thatcross-linking of LAIR-1/9.1C3 in vitro by mAb is capable of inhibiting the functions of NK cells, T cells, B cells and dendritic cell precursors indicating that LAIR-1 acts as an inhibitory receptor in hematopoietic cells.In this article, the eukaryotic cell expression vector pIg/3C-9.1C3/LAIR-1 was constructed and the fusion protein of the extracellular region of 9.1C3/LAIR-1 molecule with Fc fragment of human IgGl was expressed and purified by 9.1C3 mAb affinity chromatography. Two mAbs designated FMU8 and FMU9 were raised by immunizing mice with 9.1C3/LAIR-1-hIgFc fusion protein. The isotype both of them was IgGl and the mAb titer in ascites was over 10-6 detected by indirect ELISA. Both FMU8 and FMU9 could recognize LAIR-1-Fc and LAIR-1/myc fusion protein as well as LAIR-1 on the surface of COS7 cells transfected with pcDNA3.1- LAIR-1 vector and natural LAIR-1 expressed on HL-60 and human PBMC but not PTAl-Fc and human IgG. In order to map the mAb-recognized epitops on the 9.1C3/LAIR-1, the competitive ELISA and biosensor were employed and the results indicated that FMU8, FMU9 and 9.1C3 MAb recognized three distinct epitopes of LAIR-1. Further more, the killing of p815 cells by MLC generated effect cells was significantly blocked by the presence of FMU8 and FMU9 mAb when the redirected cytotoxicity assay (RCA) was taken.A sandwich ELISA for detecting soluble LAIR-1 (sLAIR-1) was established by using 9.1C3 MAb as coating antibody and HRP-conjugated FMU8 as sandwich antibody for the first time. The ultra-assay and inter-assay coefficient of variation (CV) were 5.3%-5.9% and 13.6%-17.8 % respectively indicating that this sandwich ELISA is reproducible and with a detection limit of approximately 1.5ug/L LAIR-1 -Fc. Moreover, a higher level of sLAIR-1 was detected in the culture supernatants of PBMC activated by PHA, CD3 mAb, LPS and especially PMA. Several protease inhibitors could down-regulate the sLAIR-1 level in Jurkat cell culture supernatant suggesting that the shedding and release of membrane LAIR-1 may be one mechanism ofthe sLAIR-1 generation. Finally, we used the sandwich ELISA for detecting the level of serum sLAIR-1 of some patients and explored the correlation of sLAIR-1 with some diseases, since many members of human leucocytes differentiation antigens (LDA) have their soluble forms which play important role in immunoregulation. sLAIR-1 could be detected in sera from either healthy person or patients. Statistical analysis showed that the serum sLAIR-1 concentration levels ware significantly higher in HFRS and allograft patients than that of healthy subjects and the level of sLAIR-1 was related with the state and stage of these diseases. Thus we suggest that the high level of serum sLAIR-1 may contribute to the progress of these diseases probably by blocking the engagement of membrane LAIR-1 with its ligand Ep-CAM and resulting in pe...
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