A method for inducing antigen-specific IgG production by in vitro immunization

Kato, Macky; Hu, Yan; Tsuji, Noriko M.; Chiba, Tomoki; Hanyu, Yoshiro

doi:10.1016/j.jim.2012.08.019

Cited by 11 publications

(10 citation statements)

References 31 publications

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“…Antigen-induced in vitro production of antibodies was attempted previously, however human PBMCs were used in their experiments and minimum of 8 days of lymphocyte culture was needed to obtain antibodies [22, 26]. Hitherto, there is no report that presents in vitro immunization of PBMCs from camelid animals.…”

Section: Discussionmentioning

confidence: 99%

“…In the present study B cells were exposed to H-OspA only long enough to recognize the antigen and induce synthesis of mRNA copies that encode antigen-specific sdAbs. Shortening of the lymphocyte culturing to 4 day (as used in the present work) does not give sufficient time to differentiate B lymphocytes into plasma cells, which are primarily associated with production of IgM isotypes antibodies [22]. …”

Section: Discussionmentioning

confidence: 99%

“…Antigen-induced in vitro production of antibodies was suggested as an alternative method to generate conventional antibodies [20–22]. However, this alternative has never been used in the sdAb production pipeline.…”

Section: Introductionmentioning

confidence: 99%

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Joining the in vitro immunization of alpaca lymphocytes and phage display: rapid and cost effective pipeline for sdAb synthesis

et al. 2017

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BackgroundCamelids possess unique functional heavy chain antibodies, which can be produced and modified in vitro as a single domain antibody (sdAb or nanobody) with full antigen binding ability. Production of sdAb in conventional manner requires active immunization of Camelidae animal, which is laborious, time consuming, costly and in many cases not feasible (e.g. in case of highly toxic or infectious antigens).ResultsIn this study, we describe an alternative pipeline that includes in vitro stimulation of naïve alpaca B-lymphocytes by antigen of interest (in this case endothelial cell binding domain of OspA of Borrelia) in the presence of recombinant alpaca interleukins 2 and 4, construction of sdAb phage library, selection of antigen specific sdAb expressed on phages (biopanning) and confirmation of binding ability of sdAb to the antigen. By joining the in vitro immunization and the phage display ten unique phage clones carrying sdAb were selected. Out of ten, seven sdAb showed strong antigen binding ability in phage ELISA. Furthermore, two soluble forms of sdAb were produced and their differential antigen binding affinity was measured with bio-layer interferometry.ConclusionA proposed pipeline has potential to reduce the cost substantially required for maintenance of camelid herd for active immunization. Furthermore, in vitro immunization can be achieved within a week to enrich mRNA copies encoding antigen-specific sdAbs in B cell. This rapid and cost effective pipeline can help researchers to develop efficiently sdAb for diagnostic and therapeutic purposes.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Joining the in vitro immunization of alpaca lymphocytes and phage display: rapid and cost effective pipeline for sdAb synthesis

et al. 2017

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“…His6-tag was introduced into these two vectors at the 5ʹ -end of the gene sequences. Site-specific incorporation of pNO 2 Phe into hTNF was carried out by mutating the codon for Tyr 87 or Lys 11 , to a TAG amber codon by using the Fast Mutagenesis Kit (Vazyme, C215-01/02) (cite previous publication on UAA incorporation methodology). 14 These mutant proteins were designated as pNO 2 Phe 87 hTNF and pNO 2 Phe 11 hTNF, respectively.…”

Section: Plasmid Constructionmentioning

confidence: 99%

“…In vitro immunization with antigens and cytokines triggers specific human naïve B-cell response in short periods, [7][8][9][10] but the approach has not been used widely, primarily because the procedures are complicated and can be repeated only in a few cases. 11 Recently, we developed a method for the rapid generation of antigen-specific B cells to improve in vitro immunization and make protocols simpler. 12 Our system has proved to be an effective method for the de novo acquisition of antigen-specific B cells of interest.…”

Section: Introductionmentioning

confidence: 99%

De novo generation of specific human IgGs by in vitro immunization using autologous proteins containing immunogenic p-nitrophenylalanine

Tong

Tian

et al. 2018

mAbs

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In vitro immunization can to used to produce monoclonal antibodies(mAbs), but this technology is limited by poor reproducibility and the requirement of pre-immunized lymphocytes. To improve this approach, we recently developed a method for rapid generation of antigen-specific B cells. Here, we report the application of this system to the production of human IgGs against tumor necrosis factor (TNF). We expressed mutant proteins with site-specific incorporated p-nitrophenylalanine (pNO 2 Phe), which stimulated an in vitro immune response in human immune cells. After constructing an antigenspecific antibody library from in vitro immunized B cells identified by fluorescence-activated cell sorting, we demonstrated that many point mutation events of the variable region occurred in our step-by-step co-cultivation system for affinity maturation in vitro. To mimic the class switching, we panned for high-affinity antigen-binding fragments by the phage display method, assembled them and identified hTNF-neutralizing human IgGs. This approach may provide a general method for raising high-affinity monoclonal antibodies against self-proteins. Furthermore, it supports mechanistic understanding in breaking human self-tolerance with pNO 2 Phe.

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Potentiation of antigen-specific antibody production by peptides derived from Ag85B of Mycobacterium tuberculosis

Kato¹,

Hanyu

2015

Journal of Immunological Methods

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A method for inducing antigen-specific IgG production by in vitro immunization

Cited by 11 publications

References 31 publications

Joining the in vitro immunization of alpaca lymphocytes and phage display: rapid and cost effective pipeline for sdAb synthesis

Joining the in vitro immunization of alpaca lymphocytes and phage display: rapid and cost effective pipeline for sdAb synthesis

De novo generation of specific human IgGs by in vitro immunization using autologous proteins containing immunogenic p-nitrophenylalanine

Potentiation of antigen-specific antibody production by peptides derived from Ag85B of Mycobacterium tuberculosis

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