Mieko Kato

Cell activation by CpG ODN leads to improved electrofusion in hybridoma production

Hybridoma formation is an indispensable step in the production of monoclonal antibodies. Obtaining highly efficient fusion of an antibody-producing cell to the myeloma cell to form the hybridoma is an important step in this process. The electrofusion method is superior to chemical fusion methods such as the polyethylene glycol (PEG) method due to its high fusion efficiency. However, this method requires cell activation prior to electrofusion, a process that is time-consuming and tends to cause cell death. In this study, we achieved much higher fusion efficiency by stimulating B cells with CpG oligodeoxynucleotide (CpG ODN) over shorter periods. Splenocytes were isolated from immunized mice and cultured in the presence of a CpG ODN for 1 or 2 days. This CpG ODN stimulation evokes about one order of magnitude higher fusion efficiency than other stimulators. CpG ODN stimulation not only increases the fusion efficiency but also the number of antibody-producing cells. This leads to a substantial increase in the number of positive clones obtained. This highly efficient fusion method was used to produce a functional antibody against Gaussia luciferase. This method was found to produce greater numbers of hybridomas and to enable direct screening for antibodies with functional characteristics such as inhibition of the luminescence activity of an antigen. We were able to establish a functional antibody against Gaussia luciferase after a single fusion experiment using our electrofusion method.

A method for inducing antigen-specific IgG production by in vitro immunization.

In vitro immunization (IVI) possesses a number of advantages over conventional immunization. However, the number of positive clones derived from IVI is limited, and the affinity of the antibodies from derived clones is relatively low. Moreover, the majority of immunoglobulins produced in culture are IgMs instead of IgGs, which limits the application. Here, we report an improved protocol for IVI using mouse spleen cells. This protocol consists of multiple cycles of repeated antigen stimulation followed by cell expansion, which increases the frequency of plasma cells that produce antigen-specific IgG antibodies. The culture conditions, including the cell density, the type of stimulants, and the initial cell preparation, were found to be important for inducing the IgG response. In addition, an analysis of the genes and cytokines expressed during the IVI showed that the antigen-specific B cells were specifically activated via CD4-positive helper T cells. As evidence for this concept, our IVI protocol enabled us to establish an IgG antibody against keyhole limpet hemocyanin with a dissociation constant in the order of 10(-7)M.

Characterization of discontinuous epitope of prion protein recognized by the monoclonal antibody T2.

The anti-prion protein (PrP) monoclonal antibody T2 has previously been prepared using PrP-knockout mice immunized with mouse recombinant PrP residues 121-231, however its interaction mechanism to PrP antigen has not been cleared. Here we identified and characterized the epitope of T2 antibody. The competitive ELISA with 20-mer synthetic peptides derived from PrP121-231 showed that T2 antibody had no affinity for these peptides. The analysis with deletion mutants of PrP revealed that 10 amino acids in the N terminus and 66 amino acids in the C terminus of PrP121-231 were necessary for reactivity with T2. Two far regions are necessary for complete affinity of the T2 antibody for PrP; either region alone is not sufficient to retain the affinity. The epitope recognized by T2 antibody is discontinuous and conformational. We examined the effect of disulfide bond and salt bridges. Alkylation of cysteine residues in C terminus of PrP121-231, which breaks a disulfide bond and disrupts the structure, had diminished the reactivity. Mutations induced in the PrP121-231 to break the disulfide bond or salt bridges, markedly had reduced the reactivity with T2 antibody. It suggests that T2 antibody recognized the structure maintained by the disulfide bond and salt bridges.

Bioluminescence assay for detecting cell surface membrane protein expression.

We have developed a method to measure the amounts of cell surface-expressed membrane proteins with bioluminescence. Dinoflagellate luciferase was expressed on the surface of a mammalian cell as a chimeric fusion protein with a membrane protein of interest. Using a membrane-impermeable substrate to quantify the membrane-displayed luciferase, the expression of the membrane protein on the cell surface was determined. By inclusion of a quenching step for the luminescent activity of luciferase on the cell surface, we were able to monitor the membrane protein expression kinetics by measuring the luminescence recovery from the cell surface after quenching. The reported methods provide a convenient way to monitor the kinetics of expression and transport of membrane proteins to the cell surface. It is applicable to the high-throughput analysis of drugs or drug candidates concerning their effects on membrane protein expression.

Fusion of Zif268 to the C-Terminus of Scfvs Promotes Expression of the Active Form in the Cytoplasm of Escherichia coli

The expression of functional scFvs in Escherichia coli cytoplasm at high yields remains a challenge, because the reducing environment of the cytoplasm inhibits disulfide bond formation, which is essential for efficient and appropriate folding of scFvs. Thus, to address this challenge, we aimed to develop a method for the efficient functional expression of scFvs in E. coli cytoplasm. The scFv against rabbit IgG (scFv(A10B)) fused with Zif268 at its C-terminus was expressed at high levels in the cytoplasm of E. coli in a soluble and active form. The reactivity Zif268-fused scFv against the antigen was identical to that of un-fused scFv(A10B). In contrast, un-fused scFv(A10B) can be produced in a functional form only when expressed in the oxidizing environment of the periplasmic space, which limits expression quantities. Fusion with maltose-binding protein (MBP) did not improve expression. We compared the productivity and functionality of un-fused and Zif268-fused proteins by using several scFvs derived from hybridomas and a chicken scFv phage display library. We found that Zif268-fused scFvs are expressed at a high level in the cytoplasm of E. coli as soluble and active proteins.

Salt bridges in prion proteins are necessary for high-affinity binding to the monoclonal antibody T2

We studied the role of the 2 salt bridges (Asp143-Arg147 and Asp146-Arg150) in helix 1 of mouse prion protein (PrP) on the formation of the complex between PrP and the monoclonal antibody T2. We introduced 6 charge-changing mutations to the amino acid residues associated with the salt bridges. Analysis of the circular dichroism spectra of the mutant PrPs showed that the salt bridge mutations did not change the secondary structures. We analyzed the kinetics of the association and dissociation of the PrPs with the T2 antibody. The results showed that the association kinetics were not significantly different among the variants except Arg150Lys, while the dissociation rate of the neutralized-charge variants was 2 orders of magnitude higher than that of the wild type. These results indicate that salt bridges make the interaction of PrP with T2 tighter by slowing down dissociation.

Enzymatic Assembly for scFv Library Construction

Recombinant monoclonal antibodies can be established by displaying single-chain variable fragment (scFv) antibody libraries on phages and then biopanning against the target. For constructing superior scFv libraries, antibody light-chain variable region (VL) and heavy-chain variable region (VH) fragments must be assembled into scFvs without loss of diversity. A high-quality scFv library is a prerequisite for obtaining strong binders from the scFv library. However, the technical challenges associated with the construction of a diverse library have been the bottleneck in the establishment of recombinant antibodies through biopanning. Here, we describe a simple and efficient method for assembling VL and VH fragments through the concerted action of λ-exonuclease and Bst DNA polymerase. We successfully used this method to construct a diverse chicken scFv library.

Single-step colony assay for screening antibody libraries

We describe a method, single-step colony assay, for simple and rapid screening of single-chain Fv fragment (scFv) libraries. Colonies of Escherichia coli expressing the scFv library are formed on a hydrophilic filter that is positioned in contact with a membrane coated with an antigen. scFv expression is triggered upon treatment of colonies with an induction reagent, following which scFvs are secreted from the cells and diffused to the antigen-coated membrane. scFvs that exhibit binding affinity for the antigen are captured by the membrane-immobilized antigen. Lastly, detection of scFv binding of the antigen on the membrane allows identification of the clones on the filter that express antigen-specific scFvs. We tested this methodology by using an anti-rabbit IgG scFv, scFv(A10B), and a rat immune scFv library. Experiments conducted using scFv(A10B) revealed that this method improves scFv expression during the colony assay. By using our method to screen an immune library of 3×103 scFv clones, we established several clones exhibiting affinity for the antigen. Moreover, we tested 7 other antigens, including peptides, and successfully identified positive clones. We believe that this simple procedure and controlled scFv expression of the single-step colony assay could make the antibody screening both rapid and reliable and lead to successful isolation of positive clones from antibody libraries.

Potentiation of antigen-specific antibody production by peptides derived from Ag85B of Mycobacterium tuberculosis.

To generate high-titer monoclonal antibodies, strong immuno-stimulation must be used for eliciting an intense cellular immune response. Here, we report that antigen-specific antibody production was potentiated by Peptide-25 derived from Ag85B of Mycobacterium tuberculosis, and that the production of antigen-specific IgG1 in particular was markedly potentiated; specifically, this occurred because the use of Peptide-25 resulted in an increase in the number of antigen-specific antibody-producing cells. We studied the activation of T cells by the peptide by examining gene expression. The observed expression pattern of GATA-3 and T-bet suggests that the peptide modulates the Th1/Th2 balance during immunization. This potentiation, which was remarkably high in BALB/c mice, could be applied in the immunization performed for monoclonal antibody production in vivo and in vitro.