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Adjuvant is a kind of non-specific immune activators, comprising of a wide variety of agents, such as Bacillus Calmette-Guerin vaccine(BCG), lipopolysaccharide (LPS) , aluminium hydroxide, alum, mineral oil, and monophosphoryl lipid A (MPL) and CpG ODN, etc. Their biologic activities are attributed to at least three components, that is, firstly, forming a repository or depot of antigen in tissues to produce prolonged exposure, secondly, activating monocytes and macrophages and improving their ability to ptesent antigen, and thirdly, activating lymphocytes and inducing their proliferation.Unmethylated CpG motifs (a CpG dinucleotide in particular base contexts) are prevalent in bacterial but not vertebrate genomic DNAs. The recognition of CpG motifs requires Toll-like receptor (TLR) 9, which triggers alterations in cellular redox balance and the induction of cell signaling pathways including the mitogen activated protein kinases (MAPKs) and NF-KB.Oligodeoxynucleotides (ODN) containing CpG motifs can activate plasmacytoid dendritic cells (pDCs) and B cells directely, which expressTLR-9, and secondarily activate monocytes, macrophages, NK cells, T cells and some nonimmune cells, improve the ability of neutrophils to provide effective host defense, and produce Thl-like proinflammatory cytokines, interferons, and chemokines, to activate host defense mechanisms leading to innate and acquired immune responses. Certain CpG motifs (CpG-A) are especially potent at activating NK cells and inducing IFN-a production by pDCs, while other motifs (CpG-B) are especially potent B cell activators. Therefore, CpG ODN is expected to be a novel adjuvant for prophylactic and therapeutic vaccination for cancer, allergy and infectious diseases.In this study, firstly, we compared the immunoregulation between the mouse adjuvant ImmunEasy?(CpG ODN) and traditional Freund’s adjuvant. Obeying to the statistic principles, we classified the BALB/c mice into two groups, and then, immunized them with the same dose of recombinant APRIL protein mixed with ImmunEasy?adjuvant or Freund’s adjuvant respectively. When the first immunization was made 5 weeks or 8 weeks later, the mice were bled and the liters and isotypes of the antibody against APRIL in sera from the two groups were compared. In the same time, splenocytes from immunized mice in the two groups were used to detect the frequency of splenocytes secreting specific antibodies against APRIL with different isotypes through modified ELISPOT assay. The results indicate that the ImmunEasy adjuvant (CpG ODN) can induce much stronger Thl-like response than the Freund’s adjuvant and improve the secretion of IgG2a, IgM and IgA antibodies. Moreover, we also developed a modified ELISPOT assay to detect the frequency of splenocytes secreting specific antibodies against APRIL with different isotypes.It has been well accepted that the development of the Life Science is largely depended on the improvement of the technology. The technology on preparation of mAb has become one of the cores of biotechnology. Proteomics has been one of the main development trends of the Life Science after thecompletion of the Human Genome project (HOP). Scientists believe that antibody chip would provide a new tool for the study on proteomics and the application in clinic. Obviously, the lymphocyte hybridoma technique has played a vital role regarding these aspects. We concluded that in the preparation of rnAbs, ImmunEasy can remarkably save the amount of immunogen, shorten period time for immunization, and obtain mAbs of IgG2a, IgM and IgA preferentially, when compared with Freund’s adjuvant.The immunogen used in this experiment is APRIL that is a novel member of TNFSF as well as one of the new CD nomination candidates announced by the 8th International Workshop on Human Leukocyte Differentiation Antigens (HLDA8). We developed lOhybridomas secreting mAbs recognizing the native and/or denatured APRIL molecule, which facilitate the study on the functions of APRIL and the TNF subfamily as well as provides a
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