Prediction of antigen-responding VHH antibodies by tracking the evolution of antibody along the time course of immunization.

Antibody maturation is the central function of the adaptive immune response. This process is driven by the repetitive selection of mutations that increase the affinity toward antigens. We hypothesized that a precise observation of this process by high-throughput sequencing along the time course of immunization will enable us to predict the antibodies reacting to the immunized antigen without any additional in vitro screening. An alpaca was immunized with IgG fragments using multiple antigen injections, and the antibody repertoire development was traced via high-throughput sequencing periodically for months. The sequences were processed into clusters, and the antibodies in the 16 most abundant clusters were generated to determine whether the clusters included antigen-binding antibodies. The sequences of most antigen-responsive clusters resembled those of germline cells in the early stages. These sequences were observed to accumulate significant mutations and also showed a continuous sequence turnover throughout the experimental period. The foregoing characteristics gave us >80% successful prediction of clusters composed of antigen-responding VHHs against IgG fragment. Furthermore, when the prediction method was applied to the data from other alpaca immunized with epidermal growth factor receptor, the success rate exceeded 80% as well, confirming the general applicability of the prediction method. Superior to previous studies, we identified the immune-responsive but very rare clusters or sequences from the immunized alpaca without any empirical screening data.

Cytosine detection by a fluorescein-labeled probe containing base-discriminating fluorescent nucleobase.

We report on a new method for the detection of a base at a specific site in a DNA sequence by monitoring the fluorescence emission of fluorescein. To achieve this goal, we developed a new base-discriminating fluorescent (BDF) nucleobase, naphthodeazaadenine ((ND)A). The fluorescence spectrum of the duplex possessing a cytosine base as a complementary base of (ND)A showed a fluorescence peak at 383 nm when using an excitation wavelength of 350 nm. When the complementary base of (ND)A was one of the other bases, the fluorescence intensity was very low. The fluorescence emission spectrum of (ND)A overlapped with the fluorescence excitation spectrum of fluorescein in the wavelength range of 400-500 nm. Thus, we designed FRET-BDF probes containing (ND)A as the FRET donor and fluorescein as the acceptor. The interaction of these two fluorophores, which are separated by defined base pairs, allowed an efficient energy transfer that resulted in a dominant fluorescence emission of fluorescein at 520 nm when using an excitation wavelength of 350 nm. Fluorescence emission from FRET-BDF probes was observed only when the complementary base of (ND)A is C, thus achieving a clear distinction of a C base on the complementary DNA strand. However, the general utility of our method is limited due to the quenching of the (ND)A fluorescence by a G/C base pair flanking (ND)A.

Design of base-discriminating fluorescent nucleoside and its application to t/c SNP typing.

We report a novel method for base detection using a base-discriminating fluorescent (BDF) nucleoside. We developed BDF probes containing methoxybenzodeazaadenine MDA and methoxybenzodeazainosine MDI, which give strong fluorescence only when the base on the complementary strand is cytosine and thymine, respectively. Thus, the MDA- and MDI-containing ODNs can be used as a very effective BDF probe for the detection of single base alterations, such as SNPs and point mutations. The present method using BDF probes is a very powerful tool for SNP typing that does not require any enzymes and time-consuming steps, and can avoid hybridization errors. In addition, a combination of MDA- and MDI-containing BDF probes facilitates the T/C SNP typing of a heterozygous sample.