Improving selection of alphaIIbbeta3-binding phage antibodies with increased reactivity derived from immunized donors. 2003

M J Jacobin, and R Robert, and O Pouns, and J Laroche-Traineau, and A Nurden, and K Peter, and M Little, and G Clofent-Sanchez
CNRS UMR 5533, Hôpital Cardiologique, Avenue de Magellan, 33604 Pessac, France.

Although many studies of the immune response in polytransfused Glanzmann thrombasthenia (GT) patients and in autoimmune thrombocytopenic purpura (AITP) have demonstrated the frequent development of Abs directed against the alphaIIbbeta3 integrin, little is known about the induced anti-alphaIIbbeta3 autoantibodies at the molecular level. Phage display is a powerful technology for selecting and engineering mAbs expressed on the surface of filamentous bacteriophage. Combinatorial libraries of single-chain IgG were constructed from splenocytes from two patients with AITP and one patient with GT. In a previous study, activated platelets or alphaIIbbeta3-expressing CHO cells selection was performed to isolate human IgG anti-alphaIIbbeta3 binding fragments using combinatorial libraries created from the B cells of a GT and an AITP patient. However, we have experienced practical problems such as enrichment of truncated antibodies during selection. We decided to test prolonged treatments with elution agents after screening on the purified form of the alphaIIbbeta3 integrin activated with the RGD peptide. We obtained a higher percentage of clones with full-size antibody fragments as well as an enrichment of more specific alphaIIbbeta3-binding phage-Abs. Some of them, recognizing the activated form of the integrin, would be interesting to further study as potential diagnostic or therapeutic agents in acute coronary syndromes. Sequencing of selected phage-Abs revealed that they used different VH and VL genes with, for the majority of them, a high level of extensive hypermutations in the complementarity determining regions, indicating the diversity of the antigen-driven immune response that occurred in GT and AITP patients.

UI MeSH Term Description Entries
D007114 Immunization Deliberate stimulation of the host's immune response. ACTIVE IMMUNIZATION involves administration of ANTIGENS or IMMUNOLOGIC ADJUVANTS. PASSIVE IMMUNIZATION involves administration of IMMUNE SERA or LYMPHOCYTES or their extracts (e.g., transfer factor, immune RNA) or transplantation of immunocompetent cell producing tissue (thymus or bone marrow). Immunologic Stimulation,Immunostimulation,Sensitization, Immunologic,Variolation,Immunologic Sensitization,Immunological Stimulation,Sensitization, Immunological,Stimulation, Immunologic,Immunizations,Immunological Sensitization,Immunological Sensitizations,Immunological Stimulations,Sensitizations, Immunological,Stimulation, Immunological,Stimulations, Immunological,Variolations
D007143 Immunoglobulin Heavy Chains The largest of polypeptide chains comprising immunoglobulins. They contain 450 to 600 amino acid residues per chain, and have molecular weights of 51-72 kDa. Immunoglobulins, Heavy-Chain,Heavy-Chain Immunoglobulins,Ig Heavy Chains,Immunoglobulin Heavy Chain,Immunoglobulin Heavy Chain Subgroup VH-I,Immunoglobulin Heavy Chain Subgroup VH-III,Heavy Chain Immunoglobulins,Heavy Chain, Immunoglobulin,Heavy Chains, Ig,Heavy Chains, Immunoglobulin,Immunoglobulin Heavy Chain Subgroup VH I,Immunoglobulin Heavy Chain Subgroup VH III,Immunoglobulins, Heavy Chain
D007147 Immunoglobulin Light Chains Polypeptide chains, consisting of 211 to 217 amino acid residues and having a molecular weight of approximately 22 kDa. There are two major types of light chains, kappa and lambda. Two Ig light chains and two Ig heavy chains (IMMUNOGLOBULIN HEAVY CHAINS) make one immunoglobulin molecule. Ig Light Chains,Immunoglobulins, Light-Chain,Immunoglobulin Light Chain,Immunoglobulin Light-Chain,Light-Chain Immunoglobulins,Chains, Ig Light,Chains, Immunoglobulin Light,Immunoglobulins, Light Chain,Light Chain Immunoglobulins,Light Chain, Immunoglobulin,Light Chains, Ig,Light Chains, Immunoglobulin,Light-Chain, Immunoglobulin
D008297 Male Males
D008875 Middle Aged An adult aged 45 - 64 years. Middle Age
D009154 Mutation Any detectable and heritable change in the genetic material that causes a change in the GENOTYPE and which is transmitted to daughter cells and to succeeding generations. Mutations
D009842 Oligopeptides Peptides composed of between two and twelve amino acids. Oligopeptide
D002999 Clone Cells A group of genetically identical cells all descended from a single common ancestral cell by mitosis in eukaryotes or by binary fission in prokaryotes. Clone cells also include populations of recombinant DNA molecules all carrying the same inserted sequence. (From King & Stansfield, Dictionary of Genetics, 4th ed) Clones,Cell, Clone,Cells, Clone,Clone,Clone Cell
D005260 Female Females
D006224 Cricetinae A subfamily in the family MURIDAE, comprising the hamsters. Four of the more common genera are Cricetus, CRICETULUS; MESOCRICETUS; and PHODOPUS. Cricetus,Hamsters,Hamster

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