A Novel Strategy for Generation of Monoclonal Antibodies from Single B Cells Using RT-PCR Technique and in Vitro Expression
ABSTRACT Monoclonal antibodies (Mabs) are important biomolecules in immunology and have widespread applications in prognosis, diagnosis, and therapeutics. Here, we describe a novel approach called single-cell RT-PCR-linked in vitro expression (SICREX), which enables the high-throughput generation and screening of Mabs. This approach entails the isolation of B cells from immunized mouse spleen or human peripheral blood using magnetic microbeads conjugated with a B-cell-selective marker, anti-CD19. The light chain (Lc) and Fd portion of heavy-chain (Hc) genes of each cell are separately amplified by RT-PCR and then combined with the sequences of a T7 promoter, a ribosome binding site (rbs), and a T7 terminator by an overlapping PCR technique. The paired full-length DNA fragments of Lc and Hc genes from single B cells are simultaneously expressed by an Escherichia coli in vitro transcription and translation system followed by an enzyme-linked immunosorbent assay to find positive fragments possessing the affinity for the antigen. From spleen cells of an immunized mouse with calcium binding protein 40, a Fab fragment with K(d) of 1.6 (+/- 0.3) x 10(-8) against the antigen was obtained. From human peripheral blood, Fab fragments against a blood group B-BSA were obtained in a similar manner. The SICREX approach is simple, rapid and versatile, allowing the high-throughput generation of naturally paired Lc and Hc with antigen-binding activity from various animal sources.
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ABSTRACT: The bandwidth of an E-shaped microstrip antenna is optimized using the genetic algorithm (GA) based on fuzzy decision making. The method of moments is employed for the analysis of the microstrip antenna at the frequency band of 1.8 GHz to 2.6 GHz by the optimization parameters of supply locations and slot dimensions. The fuzzy inference system is used for the control of the parameters of the genetic algorithm. In the implemented fuzzy system, inputs are parameters like population, and outputs are parameters such as crossover and mutation rates. Simulation results show the genetic algorithm to optimize the bandwidth of the E-shaped microstrip patch by 33.3%. The numerical results of the optimized antenna are compatible with measurements. VSWR is also measured and compared with the numerical data.Antennas and Propagation Society International Symposium, 2004. IEEE; 07/2004
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ABSTRACT: Antibodies that bind their respective targets with high affinity and specificity have proven to be essential reagents for biological research. Antibody phage display has become the leading tool for the rapid isolation of single-chain variable fragment (scFv) antibodies in vitro for research applications, but there is usually a gap between scFv isolation and its application in an array format suitable for high-throughput proteomics. In this chapter, we present our antibody phage display system where antibody isolation and scFv immobilization are facilitated by the design of the phagemid vector used as platform. In our system, the scFvs are fused at their C-termini to a cellulose-binding domain (CBD) and can be immobilized onto cellulose-based filters. This made it possible to develop a unique filter lift screen that allowed the efficient screen for multiple binding specificities, and to directly apply library-derived scFvs in an antibody spotted microarray.12/2008: pages 223-248;
- Journal of Bioscience and Bioengineering 11/2009; 108. DOI:10.1016/j.jbiosc.2009.08.102 · 1.79 Impact Factor