Development of an antigen microarray for high throughput monoclonal antibody selection

Staudt, Nicole; Müller-Sienerth, Nicole; Wright, Gavin J.

doi:10.1016/j.bbrc.2013.12.033

Cited by 20 publications

(23 citation statements)

References 25 publications

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“…HCA allows for the miniaturization of the process using multiwell assay plates and the high sensitivity of the image based approach allows for earlier hybridoma screening which reduces time of production and reagent use. The ability to multiplex monoclonal antibody generation from a single animal and cell fusion is well suited to be combined with reported methods that allow for the rapid recombinant cloning of the antibody variable regions from hybridoma cultures and microprinting of protein arrays that allow for the rapid identification of antibody binding targets [4,32]. The ability of HCA to define distinct hybridomas in primary and secondary characterization would only further increase the efficiency of these methods.…”

Section: Discussionmentioning

confidence: 99%

Use of HCA in subproteome-immunization and screening of hybridoma supernatants to define distinct antibody binding patterns

Szafran

Mancini

Nickerson

et al. 2016

Methods

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Understanding the properties and functions of complex biological systems depends upon knowing the proteins present and the interactions between them. Recent advances in mass spectrometry have given us greater insights into the participating proteomes, however, monoclonal antibodies remain key to understanding the structures, functions, locations and macromolecular interactions of the involved proteins. The traditional single immunogen method to produce monoclonal antibodies using hybridoma technology are time, resource and cost intensive, limiting the number of reagents that are available. Using a high content analysis screening approach, we have developed a method in which a complex mixture of proteins (e.g., subproteome) is used to generate a panel of monoclonal antibodies specific to a subproteome located in a defined subcellular compartment such as the nucleus. The immunofluorescent images in the primary hybridoma screen are analyzed using an automated processing approach and classified using a recursive partitioning forest classification model derived from images obtained from the Human Protein Atlas. Using an ammonium sulfate purified nuclear matrix fraction as an example of reverse proteomics, we identified 866 hybridoma supernatants with a positive immunofluorescent signal. Of those, 402 produced a nuclear signal from which patterns similar to known nuclear matrix associated proteins were identified. Detailed here is our method, the analysis techniques, and a discussion of the application to further in vivo antibody production.

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Section: Discussionmentioning

confidence: 99%

Use of HCA in subproteome-immunization and screening of hybridoma supernatants to define distinct antibody binding patterns

Szafran

Mancini

Nickerson

et al. 2016

Methods

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“…All proteins were expressed in mammalian cells as recombinant proteins as described [11]. The extracellular regions of zebrafish cell surface and secreted proteins which were previously used in protein interaction screens [13,14,15] were subcloned into a plasmid with C-terminal rat Cd4 domains 3+4, an enzymatically biotinylatable peptide and 6-His tags [16].…”

Section: Methodsmentioning

confidence: 99%

“…Monoclonal antibodies were raised and screened by microarray printing as described [11]. Amplification of both the rearranged antibody light and heavy chain was performed using total RNA extracted from ~10 6 hybridoma cells and both the amplified rearranged light and heavy antibody variable regions were recombined with an overlapping “linker” fragment by PCR and cloned into a single plasmid [10].…”

Section: Methodsmentioning

confidence: 99%

“…Amplification of both the rearranged antibody light and heavy chain was performed using total RNA extracted from ~10 6 hybridoma cells and both the amplified rearranged light and heavy antibody variable regions were recombined with an overlapping “linker” fragment by PCR and cloned into a single plasmid [10]. Plasmids encoding functional antibodies were identified by colony PCR and expression selection [10,11]. The plasmids encoding all recombinant antibodies can be obtained from Addgene [19].…”

Section: Methodsmentioning

confidence: 99%

“…Recently, we reported systematic and scalable methods for the selection and cloning of recombinant monoclonal antibodies [10,11,12]. Using a pooled immunisation approach, we demonstrated that up to five monoclonal antibodies could be selected in parallel and cloned into a single, convenient expression plasmid for distribution and storage.…”

Section: Introductionmentioning

confidence: 99%

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A panel of recombinant monoclonal antibodies against zebrafish neural receptors and secreted proteins suitable for wholemount immunostaining

Staudt

Müller-Sienerth

Fane-Dremucheva

et al. 2015

Biochemical and Biophysical Research Communications

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Cell surface receptors and secreted proteins play important roles in neural recognition processes, but because their site of action can be a long distance from neuron cell bodies, antibodies that label these proteins are valuable to understand their function. The zebrafish embryo is a popular vertebrate model for neurobiology, but suffers from a paucity of validated antibody reagents. Here, we use the entire ectodomain of neural zebrafish cell surface or secreted proteins expressed in mammalian cells to select monoclonal antibodies to ten different antigens. The antibodies were characterised by Western blotting and the sensitivity of their epitopes to formalin fixation was determined. The rearranged antigen binding regions of the antibodies were amplified and cloned which enabled expression in a recombinant form from a single plasmid. All ten antibodies gave specific staining patterns within formalin-treated embryonic zebrafish brains, demonstrating that this generalised approach is particularly efficient to elicit antibodies that stain native antigen in fixed wholemount tissue. Finally, we show that additional tags can be easily added to the recombinant antibodies for convenient multiplex staining. The antibodies and the approaches described here will help to address the lack of well-defined antibody reagents in zebrafish research.

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Application of microfluidic technology in antibody screening

Wang

2022

Biotechnology Journal

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Specific antibodies are widely used in the biomedical field. Current screening methods for specific antibodies mainly involve hybridoma technology and antibody engineering techniques. However, these technologies suffer from tedious screening processes, long preparation periods, high costs, low efficiency, and a degree of automation, which have become a bottleneck for the screening of specific antibodies. To overcome these difficulties, microfluidics has been developed as a promising technology for high‐throughput screening and high purity of antibody. In this review, we provide an overview of the recent advances in microfluidic applications for specific antibody screening. In particular, hybridoma technology and four antibody engineering techniques (including phage display, single B cell antibody screening, antibody expression, and cell‐free protein synthesis) based on microfluidics have been introduced, challenges, and the future outlook of these technologies are also discussed.

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Development of an antigen microarray for high throughput monoclonal antibody selection

Abstract: HighlightsDevelopment of an antigen array for high throughput monoclonal antibody selection.Identification of cross-reactive antibodies at an early stage of screening.Convenient screening method to identify functional recombinant antibodies.

Cited by 20 publications

References 25 publications

Use of HCA in subproteome-immunization and screening of hybridoma supernatants to define distinct antibody binding patterns

Use of HCA in subproteome-immunization and screening of hybridoma supernatants to define distinct antibody binding patterns

A panel of recombinant monoclonal antibodies against zebrafish neural receptors and secreted proteins suitable for wholemount immunostaining

Application of microfluidic technology in antibody screening

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