Development of an antigen microarray for high throughput monoclonal antibody selection

We have developed an approach that enables early screening of hybridoma supernatants generated from an animal immunised with up to five different antigens followed by cloning of the antibody into a single expression plasmid

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

2014

Scholarcy highlights

  • The high binding affinity and specificity of monoclonal antibodies for their targets have made them invaluable tools for biomedical research and an increasingly important class of drugs that have been exploited to treat a range of diseases
  • To select new monoclonal antibodies to a defined antigen, host animals are immunized and the resulting antibody-secreting B-lymphocytes are fused to a myeloma cell line to create a hybridoma
  • To increase the number of monoclonal antibodies that could be selected in parallel from a single mouse immunized with multiple antigens, we aimed to print small protein microarrays at the base of 96-well microtitre plates
  • Despite the importance of monoclonal antibodies in basic biomedical research, selecting new antibodies can be a very time- and labour-intensive process with uncertain chances of success
  • To streamline this process and reduce the number of animals used, we have developed a pooled immunisation strategy followed by recombinant cloning of the antibodies into a single expression plasmid
  • We report the development of an antigen microarray that significantly reduces the volume of supernatant required for functional screening
  • By developing an antigen array, we have reduced the volume of hybridoma supernatant required for screening so that the number of antigens per mouse can be increased from a maximum of five to over one hundred, an increase in scale that could not be achieved by miniaturising the screening ELISAs in either 384 or 1536-well plates
  • By combining pooled animal immunisation, hybridoma generation, antigen microarrays and a convenient method to identify plasmids encoding functional recombinant antibodies, we have addressed two major bottlenecks that prevented increasing the scale of a method for the parallel selection of high-quality monoclonal antibodies

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