Generation of recombinant guinea pig antibody fragments to the human GABA(C) receptor

Department of Biological Sciences, University of Illinois at Chicago, 60607, USA.
Journal of immunological methods (Impact Factor: 1.82). 02/2011; 368(1-2):36-44. DOI: 10.1016/j.jim.2011.02.010
Source: PubMed


To generate monoclonal antibodies to the human ρ1 GABA(C) receptor, a ligand-gated chloride ion channel that is activated by the neurotransmitter γ-aminobutyric acid (GABA), we recovered the immunoglobulin variable heavy chain (V(H)) and light chain (V(L)) regions of a guinea pig immunized with a 14-mer peptide segment of the N-terminal extracellular domain of the ρ1 subunit. Oligonucleotide primers were designed and used to amplify the V(H) and V(L) regions of guinea pig RNA by the reverse transcriptase polymerase chain reaction. The amplified and cloned V(H) and V(L) regions were transferred together into a phagemid vector, yielding a library of 5×10(6) members, which displayed chimeric fragments of antigen binding (Fabs) with guinea pig variable and human constant regions fused to protein III of M13 bacteriophage. Through affinity selection of this phage-display library with the biotinylated 14-mer peptide segment of GABA(C), we isolated four different antibody fragments that bound specifically to the immunogenic peptide. Phage particles displaying two of these antibodies, but not negative controls, bound selectively to the surface of neuroblastoma cells expressing the ρ1 GABA(C) receptor. Such antibody fragments will be useful in future studies involving targeting of specific neural tissues that express the GABA(C) receptor.

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    • "However, antibodies selected from synthetic repertoires often resulted in low affinity and/or non-specificity compared to antibodies derived from immunized animals. To overcome these limitations, phage antibody libraries were made from immunized animals and monoclonal antibodies with high binding affinity have been obtained [20-24]. Unlike antibody display technology, our method enables the preservation of original heavy-light chain pairings, which is useful in analyzing protective antibodies in infectious disease or identifying autoimmune antibodies in humans. "
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    ABSTRACT: Background 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. Results 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. Conclusions Our technology eliminates the need for both cell propagation and screening processes, offering a significant advantage over hybridoma and display strategies.
    BMC Biology 09/2012; 10(1):80. DOI:10.1186/1741-7007-10-80 · 7.98 Impact Factor

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