Antibody generation through B cell panning on antigen followed by in situ culture and direct RT-PCR on cells harvested en masse from antigen-positive wells

Lightwood, Daniel; Carrington, Bruce; Henry, Alistair J.; McKnight, Andrew J.; Crook, Kenneth; Cromie, Karen D.; Lawson, Alastair D. G.

doi:10.1016/j.jim.2006.08.010

Cited by 24 publications

(24 citation statements)

References 20 publications

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“…To date, several adaptations of this protocol as well as completely new technologies using advanced PCR-based methods are available for sampling and characterizing antigen specific B cells from spleen and from blood of immunized animals. However, these technologies require extensive expression cloning efforts to obtain a reasonable number of antigen specific and functional monoclonal antibodies mainly for two reasons: (i) the IgG amount in the supernatant is so low that only one or two binding assays can be performed excluding functional assays, resulting at best in a plethora of antigen binding supernatants [7]–[15], or (ii) the cultivation of a pool of different lymphocytes including polyclonal antigen specific B cells requires that each of the possible heavy (HC) and light chain (LC) pairs has to be cloned and characterized separately [16], [17].…”

Section: Introductionmentioning

confidence: 99%

A Robust High Throughput Platform to Generate Functional Recombinant Monoclonal Antibodies Using Rabbit B Cells from Peripheral Blood

Seeber¹,

Ros²,

Thorey³

et al. 2014

PLoS ONE

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We have developed a robust platform to generate and functionally characterize rabbit-derived antibodies using B cells from peripheral blood. The rapid high throughput procedure generates a diverse set of antibodies, yet requires only few animals to be immunized without the need to sacrifice them. The workflow includes (i) the identification and isolation of single B cells from rabbit blood expressing IgG antibodies, (ii) an elaborate short term B-cell cultivation to produce sufficient monoclonal antigen specific IgG for comprehensive phenotype screens, (iii) the isolation of VH and VL coding regions via PCR from B-cell clones producing antigen specific and functional antibodies followed by the sequence determination, and (iv) the recombinant expression and purification of IgG antibodies. The fully integrated and to a large degree automated platform (demonstrated in this paper using IL1RL1 immunized rabbits) yielded clonal and very diverse IL1RL1-specific and functional IL1RL1-inhibiting rabbit antibodies. These functional IgGs from individual animals were obtained at a short time range after immunization and could be identified already during primary screening, thus substantially lowering the workload for the subsequent B-cell PCR workflow. Early availability of sequence information permits one to select early-on function- and sequence-diverse antibodies for further characterization. In summary, this powerful technology platform has proven to be an efficient and robust method for the rapid generation of antigen specific and functional monoclonal rabbit antibodies without sacrificing the immunized animal.

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Section: Introductionmentioning

confidence: 99%

A Robust High Throughput Platform to Generate Functional Recombinant Monoclonal Antibodies Using Rabbit B Cells from Peripheral Blood

Seeber¹,

Ros²,

Thorey³

et al. 2014

PLoS ONE

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“…Primers were based on a previous publication (Lightwood et al, 2006) and were designed for leader sequences (5′ primer) or the C-terminus of the junction. Primers used for heavy chain amplification were 5′-GATATCAAGCTTACGCTCACCATGGAGACTGGGC-3′ and 5′-CGCGCGCTCGAGACGGTGACSAGGGTSCCYKGGCCCC-3′.…”

Section: Methodsmentioning

confidence: 99%

Evaluation of a novel multi-immunogen vaccine strategy for targeting 4E10/10E8 neutralizing epitopes on HIV-1 gp41 membrane proximal external region

et al. 2017

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The membrane proximal external region (MPER) of HIV-1 gp41 is targeted by broadly neutralizing antibodies (bnAbs) 4E10 and 10E8. In this proof-of-concept study, we evaluated a novel multi-immunogen vaccine strategy referred to as Incremental, Phased Antigenic Stimulation for Rapid Antibody Maturation (IPAS-RAM) to induce 4E10/10E8-like bnAbs. Rabbits were immunized sequentially, but in a phased manner, with three immunogens that are progressively more native (gp41-28×3, gp41-54CT, and rVV-gp160DH12). Although nAbs were not induced, epitope-mapping analyses indicated that IPAS-RAM vaccination was better able to target antibodies towards the 4E10/10E8 epitopes than homologous prime-boost immunization using gp41-28×3 alone. MPER-specific rabbit monoclonal antibodies were generated, including 9F6. Although it lacked neutralizing activity, the target epitope profile of 9F6 closely resembled those of 4E10 and 10E8 (671NWFDITNWLWYIK683). B-cell repertoire analyses suggested the importance of co-immunizations for maturation of 9F6, which warrants further evaluation of our IPAS-RAM vaccine strategy using an improved priming immunogen.

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“…Purification of CD138 + cells from rat bone marrow CD138 + cells were enriched from bone marrow preparations using a rabbit anti-rat CD138 reagent that we had previously produced at UCB. We generated a monoclonal antibody specific for the rat CD138 molecule through immunization of rabbits with rat CD138 extracellular domain protein followed by screening of activated B cell culture supernatants and subsequent variable region cloning from single antigen-specific B cells using a method similar to that described by Lightwood et al 23 The purified rabbit anti-CD138 antibody was then labeled with phycoerythrin (PE) using a Lightning-link kit (Innova Biosciences). CD138 + cells were then enriched from bone marrow samples via magneticactivated cell sorting (MACS) (Miltenyi Biotec) with anti-PE microbeads following the manufacturer's protocol.…”

Section: Number Of Htnfspecific Recombinant Antibodies (Via Tap)mentioning

confidence: 99%

“…[17][18][19][20] B cell panning has also been used to select for antigen-specific memory B cells before recovery of variable region genes by reverse transcription (RT)-PCR. [21][22][23] Alternatively, memory B cell culturing and screening followed by micromanipulation of single antigen-specific B cells 24 or single-cell memory B cell cultures 25 have also been successfully employed as methods of monoclonal antibody generation.…”

Section: Introductionmentioning

confidence: 99%

The rapid generation of recombinant functional monoclonal antibodies from individual, antigen-specific bone marrow-derived plasma cells isolated using a novel fluorescence-based method

Clargo

Hudson

Ndlovu

et al. 2013

mAbs

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Single B cell technologies, which avoid traditional hybridoma fusion and combinatorial display, provide a means to interrogate the naturally-selected antibody repertoire of immunized animals. Many methods enable the sampling of memory B cell subsets, but few allow for the direct interrogation of the plasma cell repertoire, i.e., the subset of B cells responsible for producing immunoglobulin in serum. Here, we describe the use of a robust and simple fluorescence-based technique, called the fluorescent foci method, for the identification and isolation of antigen-specific IgG-secreting cells, such as plasma cells, from heterogeneous bone marrow preparations. Following micromanipulation of single cells, cognate pairs of heavy and light chain variable region genes were recovered by reverse transcription (RT)-polymerase chain reaction (PCR). During the PCR, variable regions were combined with a promoter fragment and a relevant constant region fragment to produce two separate transcriptionally-active PCR (TAP) fragments that were directly co-transfected into a HEK-293F cell line for recombinant antibody expression. The technique was successfully applied to the generation of a diverse panel of high-affinity, functional recombinant antibodies to human tumor necrosis factor (TNF) receptor 2 and TNF derived from the bone marrow of immunized rabbits and rats, respectively. Progression from a bone marrow sample to a panel of functional recombinant antibodies was possible within a 2-week timeframe.

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Antibody generation through B cell panning on antigen followed by in situ culture and direct RT-PCR on cells harvested en masse from antigen-positive wells

Cited by 24 publications

References 20 publications

A Robust High Throughput Platform to Generate Functional Recombinant Monoclonal Antibodies Using Rabbit B Cells from Peripheral Blood

A Robust High Throughput Platform to Generate Functional Recombinant Monoclonal Antibodies Using Rabbit B Cells from Peripheral Blood

Evaluation of a novel multi-immunogen vaccine strategy for targeting 4E10/10E8 neutralizing epitopes on HIV-1 gp41 membrane proximal external region

The rapid generation of recombinant functional monoclonal antibodies from individual, antigen-specific bone marrow-derived plasma cells isolated using a novel fluorescence-based method

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