Target-selective homologous recombination cloning for high-throughput generation of monoclonal antibodies from single plasma cells

<p>Abstract</p> <p>Background</p> <p>Molecular cloning of functional immunoglobulin genes from single plasma cells is one of the most promising technologies for the rapid development of monoclonal antibody drugs. However, the proper insertion of PCR-amplified immunoglobulin genes into expression vectors remains an obstacle to the high-throughput production of recombinant monoclonal antibodies.</p> <p>Results</p> <p>We developed a single-step cloning method, target-selective homologous recombination (TS-HR), in which PCR-amplified immunoglobulin variable genes were selectively inserted into vectors, even in the presence of nonspecifically amplified DNA. TS-HR utilizes Red/ET-mediated homologous recombination with a target-selective vector (TS-vector) with unique homology arms on its termini. Using TS-HR, immunoglobulin variable genes were cloned directly into expression vectors by co-transforming unpurified PCR products and the TS-vector into <it>E. coli</it>. Furthermore, the high cloning specificity of TS-HR allowed plasmids to be extracted from pools of transformed bacteria without screening single colonies for correct clones. We present a one-week protocol for the production of recombinant mouse monoclonal antibodies from large numbers of single plasma cells.</p> <p>Conclusion</p> <p>The time requirements and limitations of traditional cloning procedures for the production of recombinant immunoglobulins have been significantly reduced with the development of the TS-HR cloning technique.</p

Prophage-triggered membrane vesicle formation through peptidoglycan damage in Bacillus subtilis

Bacteria release membrane vesicles (MVs) that play important roles in various biological processes. However, the mechanisms of MV formation in Gram-positive bacteria are unclear, as these cells possess a single cytoplasmic membrane that is surrounded by a thick cell wall. Here we use live cell imaging and electron cryo-tomography to describe a mechanism for MV formation in Bacillus subtilis. We show that the expression of a prophage-encoded endolysin in a sub-population of cells generates holes in the peptidoglycan cell wall. Through these openings, cytoplasmic membrane material protrudes into the extracellular space and is released as MVs. Due to the loss of membrane integrity, the induced cells eventually die. The vesicle-producing cells induce MV formation in neighboring cells by the enzymatic action of the released endolysin. Our results support the idea that endolysins may be important for MV formation in bacteria, and this mechanism may potentially be useful for the production of MVs for applications in biomedicine and nanotechnology

Guinea pig immunoglobulin VH and VL naïve repertoire analysis.

The guinea pig has been used as a model to study various human infectious diseases because of its similarity to humans regarding symptoms and immune response, but little is known about the humoral immune response. To better understand the mechanism underlying the generation of the antibody repertoire in guinea pigs, we performed deep sequencing of full-length immunoglobulin variable chains from naïve B and plasma cells. We gathered and analyzed nearly 16,000 full-length VH, Vκ and Vλ genes and analyzed V and J gene segment usage profiles and mutation statuses by annotating recently reported genome data of guinea pig immunoglobulin genes. We found that approximately 70% of heavy, 73% of kappa and 81% of lambda functional germline V gene segments are integrated into the actual V(D)J recombination events. We also found preferential use of a particular V gene segment and accumulated mutation in CDRs 1 and 2 in antigen-specific plasma cells. Our study represents the first attempt to characterize sequence diversity in the expressed guinea pig antibody repertoire and provides significant insight into antibody repertoire generation and Ig-based immunity of guinea pigs

ELEVATION OF HETEROPHILIC ANTIBODIES TO RABBIT ERYTHROCYTES IN HUMAN PATHOLOGIC SERA : QUANTITATIVE STUDIES BY ELISA USING A GLYCOSPHINGOLIPID ANTIGEN

Human heterophilic hemagglutinin to rabbit erythrocytes was quantitatively determined by the hemagglutination test and enzyme linked immunosorbent assay (ELISA) using rabbit erythrocyte pentaglycosyl ceramide (CPH), Gal (α 1-3) Gal (β 1-4) GlcNAc (β 1-3) Gal (β 1-4) Glc-Cer as the antigen. The antibody levels in sera from 74 Hanganutziu-Deicher (H-D) antibody-positive patients were significantly higher than those in sera from 55 healthy donors, and the correlation between the antibody levels detected by ELISA and hemagglutinin titers was significant. The antibody levels detected by ELISA correlated to the IgG antibody levels, while the hemagglutinin titers correlated to the IgM antibody levels. These results suggested that IgG levels as well as IgM levels against rabbit erythrocytes were elevated in H-D antibody-positive patients with various tumors and infectious diseases. In 4 out of 67 cancer patients collected randomly, abnormally high levels of anti-CPH IgG antibody were detected

Rapid production of antigen-specific monoclonal antibodies from a variety of animals

<p>Abstract</p> <p>Background</p> <p>Although a variety of animals have been used to produce polyclonal antibodies against antigens, the production of antigen-specific monoclonal antibodies from animals remains challenging.</p> <p>Results</p> <p>We propose a simple and rapid strategy to produce monoclonal antibodies from a variety of animals. By staining lymph node cells with an antibody against immunoglobulin and a fluorescent dye specific for the endoplasmic reticulum, plasma/plasmablast cells were identified without using a series of antibodies against lineage markers. By using a fluorescently labeled antigen as a tag for a complementary cell surface immunoglobulin, antigen-specific plasma/plasmablast cells were sorted from the rest of the cell population by fluorescence-activated cell sorting. Amplification of cognate pairs of immunoglobulin heavy and light chain genes followed by DNA transfection into 293FT cells resulted in the highly efficient production of antigen-specific monoclonal antibodies from a variety of immunized animals.</p> <p>Conclusions</p> <p>Our technology eliminates the need for both cell propagation and screening processes, offering a significant advantage over hybridoma and display strategies.</p