Generation of a large complex antibody library from multiple donors

Little, Melvyn; Welschof, Martin; Braunagel, Michael; Hermes, Ingrid; Christ, Christiane; Keller, Alice; Rohrbach, Petra; Kürschner, Timo; Schmidt, Stefanie; Kleist, Christian; Terness, Peter

doi:10.1016/s0022-1759(99)00164-7

Cited by 64 publications

(29 citation statements)

References 11 publications

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“…Studies are in progress to determine whether the highly specific germline IgG clones really secrete pathogenic Abs that retain a germline sequence or whether they represent anergized autoreactive B cells also found in nonimmunized subjects that possess the genetic potential to produce anti-␣ IIb ␤ 3 Abs (61). However, despite several attempts, we have been unable to select anti-␣ IIb ␤ 3 phage Abs from a complex naive library made from mRNA isolated from pooled blood of 50 healthy donors (33). Autoreactive B cells, even if present in naive blood samples, are certainly not proliferating, and, as a result, the RNA may not be in sufficient amounts to be amplified.…”

Section: Discussionmentioning

confidence: 99%

“…PBL from patient EB and spleen tissue from patient TE were obtained as a source of mRNA for the generation of IgG libraries constructed largely according to published protocols (32,33). Briefly, mRNA was extracted using Dynabeads oligo(dT) 25 (Dynal Biotech, Compiegne, France) from total RNA purified with Trizol LS Reagent (Life Technologies, Cergy Pontoise, France) from ϳ5 ϫ 10 7 cells.…”

Section: Rna Isolation and Creation Of Combinatorial Scfv Librariesmentioning

confidence: 99%

“…To generate first-strand cDNA, three C-region primers, kindly provided by Dr. D. Nugent (Orange County, CA), specific for anti-sense sequences of the C␥1, C , and C genes were used to amplify Ig-specific mRNA (7). The V domains of the ␥-, -, and -chains were amplified by RT-PCR using primers previously described by us (33) and Klentaq polymerase (Sigma-Aldrich, St. Quentin, France). Reactions were conducted using a Gene-Amp 9600 PCR system (PerkinElmer, Foster City, CA) and a denaturing cycle of 3 min at 95°C, 30 cycles of 30 s at 95°C, and 3 min at 68°C, completed with a final cycle of 5 min at 68°C.…”

Section: Rna Isolation and Creation Of Combinatorial Scfv Librariesmentioning

confidence: 99%

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Human IgG Monoclonal Anti-αIIbβ3-Binding Fragments Derived from Immunized Donors Using Phage Display

Jacobin

Laroche‐Traineau

Little

et al. 2002

The Journal of Immunology

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Previous studies of the immune response in polytransfused Glanzmann thrombasthenia (GT) patients and in autoimmune thrombocytopenic purpura (AITP) have relied on serum analysis and have shown the frequent development of Abs directed against the αIIbβ3 integrin. However, little is known about the molecular diversity of the humoral immune response to αIIbβ3 due to the paucity of mAbs issuing from these pathologies. We have isolated human IgG anti-αIIbβ3 binding fragments using combinatorial libraries of single-chain IgG created from the B cells of a GT and an AITP patient, both with serum Abs. Ab screening was performed using activated platelets or activated αIIbβ3-expressing Chinese hamster ovary cells. Sequencing of selected phage Abs showed that a broad selection of genes from virtually all V gene families had been used, indicating the diversity of the immune response. About one-half of the VH and VL segments of our IgG anti-αIIbβ3 fragments displayed extensive hypermutations in the complementarity-determining region, supporting the idea that an Ag-driven immune response was occurring in both patients. The H chain complementarity-determining region 3 analysis of phage Abs revealed motifs other than the well-known RGD and KQAGDV integrin-binding sequences. To our knowledge, our study is the first to illustrate multiple human IgG anti-αIIbβ3 reactivities and structural variations linked to the anti-platelet human immune response. Human αIIbβ3 Abs preferentially directed against the activated form of the integrin were further characterized because platelet αIIbβ3 inhibitors are potential therapeutic reagents for treating acute coronary syndromes. Currently available αIIbβ3 antagonists do not specifically recognize the activated form of the integrin.

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Section: Discussionmentioning

confidence: 99%

Section: Rna Isolation and Creation Of Combinatorial Scfv Librariesmentioning

confidence: 99%

Section: Rna Isolation and Creation Of Combinatorial Scfv Librariesmentioning

confidence: 99%

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Human IgG Monoclonal Anti-αIIbβ3-Binding Fragments Derived from Immunized Donors Using Phage Display

Jacobin

Laroche‐Traineau

Little

et al. 2002

The Journal of Immunology

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“…For the amplification of VH of IgG (c) and IgM (l), V j , and V k gene, a total of 71 reactions (28, 16, and 27 reactions for VH, V j , and V k gene, respectively) were carried out using each gene specific primer set. The primers used were from Little et al [14] with the following slight modifications to generate a linker sequence between VH and VL gene, and to allow overlapping sequences for cloning into the yeast surface display vector, pCTCON. For the insertion of a (G 4 S) 3 linker between VH and VL gene, the following sequence was added to the 5 0 end of the VH reverse primer (5 0 -CGA GCC CCC GCC ACC CGA ACC GCC CCC ACC TCT-3 0 ) and the 5 0 end of the V j and V k forward primer (5 0 -GGT TCG GGT GGC GGG GGC TCG GGC GGG GGT GGC TCA GAT CT-3 0 ).…”

Section: Methodsmentioning

confidence: 99%

Construction and characterization of a pseudo-immune human antibody library using yeast surface display

Lee

Park

et al. 2006

Biochemical and Biophysical Research Communications

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“…The determined affinities (K a ) of both anti-estradiol and anti-progesterone scFv were approximately 10 8 M -1 . Little et al (1999) generated a large complex library of single-chain antibodies based on four individual libraries from each of 50 non-immunised donors, the final combined library comprised 4 × 10 9 of members. The DNA encoding the heavy and light-chain variable domains of the IgM and IgG repertoires was amplified by PCR using two different sets of primers for each individual library.…”

Section: Single-pot Librariesmentioning

confidence: 99%

Generation of hapten-specific recombinant antibodies: antibody phage display technology: a review

Brichta¹,

Hnilova²,

Viskovic³

2005

Vet. Med.

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Production of antibodies has been revolutionized by the development of modern molecular biology methods for the expression of recombinant DNA. Phage display technology represents one of the most powerful tools for production and selection of recombinant antibodies and has been recognized as a valuable alternative way for the preparation of antibodies of a desired specificity. In comparison to poly-and monoclonal antibodies, recombinant antibodies using the phage display technology can be prepared faster, in more automatic process and with reduced consumption of laboratory animals. This review summarizes current trends of phage display technology with focus on the generation of hapten-specific recombinant antibodies and gives the examples of successful applications of phage display in the environmental analysis of low molecular weight compound.

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Generation of a large complex antibody library from multiple donors

Cited by 64 publications

References 11 publications

Human IgG Monoclonal Anti-αIIbβ3-Binding Fragments Derived from Immunized Donors Using Phage Display

Human IgG Monoclonal Anti-αIIbβ3-Binding Fragments Derived from Immunized Donors Using Phage Display

Construction and characterization of a pseudo-immune human antibody library using yeast surface display

Generation of hapten-specific recombinant antibodies: antibody phage display technology: a review

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