Affinity ranking of antibodies using flow cytometry: Application in antibody phage display-based target discovery

Geuijen, Cecilia A.W.; Horst, Marieke Clijsters-van der; Cox, Freek; Rood, P. M. L.; Throsby, Mark; Jongeneelen, Mandy; Backus, H. H. J.; Deventer, Els van; Kruisbeek, Ada M.; Goudsmit, Jaap; Kruif, John de

doi:10.1016/j.jim.2005.04.016

Cited by 29 publications

(19 citation statements)

References 18 publications

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“…Serial dilutions of CD3 mAb further confirmed the findings above (Figure 2D), since the reduced binding to γ +/− T cells persisted in saturation conditions, but it was gradually lost near the endpoint, supporting the existence of less CD3 binding sites [9]. …”

Section: Resultssupporting

confidence: 74%

Human CD3γ, but not CD3δ, haploinsufficiency differentially impairs γδ versus αβ surface TCR expression

et al. 2013

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BackgroundThe T cell antigen receptors (TCR) of αβ and γδ T lymphocytes are believed to assemble in a similar fashion in humans. Firstly, αβ or γδ TCR chains incorporate a CD3δε dimer, then a CD3γε dimer and finally a ζζ homodimer, resulting in TCR complexes with the same CD3 dimer stoichiometry. Partial reduction in the expression of the highly homologous CD3γ and CD3δ proteins would thus be expected to have a similar impact in the assembly and surface expression of both TCR isotypes. To test this hypothesis, we compared the surface TCR expression of primary αβ and γδ T cells from healthy donors carrying a single null or leaky mutation in CD3G (γ+/−) or CD3D (δ+/−, δ+/leaky) with that of normal controls.ResultsAlthough the partial reduction in the intracellular availability of CD3γ or CD3δ proteins was comparable as a consequence of the mutations, surface TCR expression measured with anti-CD3ε antibodies was significantly more decreased in γδ than in αβ T lymphocytes in CD3γ+/− individuals, whereas CD3δ+/− and CD3δ+/leaky donors showed a similar decrease of surface TCR in both T cell lineages. Therefore, surface γδ TCR expression was more dependent on available CD3γ than surface αβ TCR expression.ConclusionsThe results support the existence of differential structural constraints in the two human TCR isotypes regarding the incorporation of CD3γε and CD3δε dimers, as revealed by their discordant surface expression behaviour when confronted with reduced amounts of CD3γ, but not of the homologous CD3δ chain. A modified version of the prevailing TCR assembly model is proposed to accommodate these new data.

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Section: Resultssupporting

confidence: 74%

Human CD3γ, but not CD3δ, haploinsufficiency differentially impairs γδ versus αβ surface TCR expression

et al. 2013

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“…Surface plasmon resonance (SPR) technology normally used for antibody affinity measurement requires purified antibody and is not available in many laboratories [26]. FCM methods have been used to measure the affinity of cell-membrane-expressed antigens [26][27][28], and it shows that ranking of the antibodies based on FCM data correlates well with ranking based on K D values as measured by SPR and can therefore be used to discriminate between high-and low-affinity antibodies.…”

Section: Discussionmentioning

confidence: 99%

“…FCM methods have been used to measure the affinity of cell-membrane-expressed antigens [26][27][28], and it shows that ranking of the antibodies based on FCM data correlates well with ranking based on K D values as measured by SPR and can therefore be used to discriminate between high-and low-affinity antibodies. The NlpA-based bacteria display technology combined with FCM have been utilized to recombinant antibodies screening and epitopes mapping in our previous studies [23,29].…”

Section: Discussionmentioning

confidence: 99%

Bi-specific antibodies with high antigen-binding affinity identified by flow cytometry

Xu¹,

Wang

Zhao³

et al. 2015

International Immunopharmacology

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“…13 However, either high affinity of the antibody or high expression level of surface-bound antigen or both was required presumably due to the low sensitivity of the analysis. 10,25 Moreover, both solution affinity and reverse rate could only be determined from two independent sets of measurements, 10,25 while they were able to be determined simultaneously from a single set of adhesion frequency measurements in the current study. On the other hand, BIAcore analysis was widely used in the determination of antibody affinity when the surface-bound antigen was purified out and subsequently immobilized onto the chip surface, 10,25 which is not available in the case that the antigen of interest is constitutively expressed on cell membrane (e.g., CD20 antigens onto Daudi cell surface).…”

mentioning

confidence: 82%

Molecular Modeling and Affinity Determination of scFv Antibody: Proper Linker Peptide Enhances Its Activity

Gu¹,

Jia

Feng

et al. 2009

Ann Biomed Eng

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Abstract-One of existing strategies to engineer active antibody is to link V H and V L domains via a linker peptide. How the composition, length, and conformation of the linker affect antibody activity, however, remains poorly understood. In this study, a dual approach that coordinates molecule modeling, biological measurements, and affinity evaluation was developed to quantify the binding activity of a novel stable miniaturized anti-CD20 antibody or singlechain fragment variable (scFv) with a linker peptide. Upon computer-guided homology modeling, distance geometry analysis, and molecular superimposition and optimization, three new linker peptides PT1, PT2, and PT3 with respective 7, 10, and 15 residues were proposed and three engineered antibodies were then constructed by linking the cloned V H and V L domains and fusing to a derivative of human IgG1. The binding stability and activity of scFv-Fc chimera to CD20 antigen was quantified using a micropipette adhesion frequency assay and a Scatchard analysis. Our data indicated that the binding affinity was similar for the chimera with PT2 or PT3 and~24-fold higher than that for the chimera with PT1, supporting theoretical predictions in molecular modeling. These results further the understanding in the impact of linker peptide on antibody structure and activity.

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Affinity ranking of antibodies using flow cytometry: Application in antibody phage display-based target discovery

Cited by 29 publications

References 18 publications

Human CD3γ, but not CD3δ, haploinsufficiency differentially impairs γδ versus αβ surface TCR expression

Human CD3γ, but not CD3δ, haploinsufficiency differentially impairs γδ versus αβ surface TCR expression

Bi-specific antibodies with high antigen-binding affinity identified by flow cytometry

Molecular Modeling and Affinity Determination of scFv Antibody: Proper Linker Peptide Enhances Its Activity

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