Rapid identification of monospecific monoclonal antibodies using a human proteome microarray

Abstract

To broaden the range of tools available for proteomic research, we generated a library of 16,368 unique full-length human ORFs that are expressible as N-terminal GST-His(6) fusion proteins. Following expression in yeast, these proteins were then individually purified and used to construct a human proteome microarray. To demonstrate the usefulness of this reagent, we developed a streamlined strategy for the production of monospecific monoclonal antibodies that used immunization with live human cells and microarray-based analysis of antibody specificity as its central components. We showed that microarray-based analysis of antibody specificity can be performed efficiently using a two-dimensional pooling strategy. We also demonstrated that our immunization and selection strategies result in a large fraction of monospecific monoclonal antibodies that are both immunoblot and immunoprecipitation grade. Our data indicate that the pipeline provides a robust platform for the generation of monoclonal antibodies of exceptional specificity.

Fig. 1.

Construction of a human proteome microarray. A human ORF collection was mobilized into a yeast galactose-inducible GST fusion vector (pEGH-A) using Gateway-mediated site-specific recombination. Individual clones were verified to have correctly sized inserts by BsrGI digestion. Clones with confirmed identities were transformed into yeast, and large scale protein expression and purification were performed. Protein size and purity were tested by anti-GST immunoblotting. Protein samples were printed on a glass slide in duplicate, and printed spots were visualized by anti-GST antibody.

Fig. 2.

Evaluation of human proteome microarray quality. a, a representative image of a protein microarray. A protein microarray was incubated with anti-GST antibody, and printed spots were identified by probing with an anti-rabbit Alexa Fluor 555 conjugate. b, a histogram of foreground and background signal intensities. The x axis represents log₁₀ scale. c, endogenous distribution of target proteins. d, protein expression levels with different subcellular localization. e, protein expression level by different protein families. f, protein expression level by protein length. The small boxes in d and e represents mean values.

Fig. 3.

Strategy for identification of highly specific mAbs. Various different live human cell lines were used for immunization of BALB/c mice. The resulting hybridomas were tested for secretion of IgG and were used for ICC for the cell lines used. ICC-positive supernatants were combined in 12 × 12 two-dimensional pools, which were then used to probe the human proteome microarrays. Data from pooled samples were deconvoluted, and the candidate monospecific mAbs were then probed to the human proteome microarrays individually. Examples of antigens recognized by monospecific mAbs are shown.

Fig. 4.

Analysis of highly specific mAbs in different research applications. To validate the specificities of individual mAbs, a series of experiments was performed. a, representative ICC data are shown for endogenous proteins in HeLa (XRCC5, RAB8A), HL-60 (DLAT), and HCT116 cells (ANXA2). b, shRNA knockdown of target antigens. Plasmids driving expression of target proteins tagged on the N terminus with the V5 epitope were then transfected either with corresponding shRNA or without shRNA expression constructs. The resulting cell lysates were analyzed by immunoblot with each mAb in question and anti-V5 antibody for validation of antigen specificity. In each sample, the first lane shows expression construct, the second lane shows co-transfection of expression and shRNA expression construct, and the third lane shows no transfection showing endogenous protein detection. c, IP assays. Immunoprecipitation was performed to test whether mAbs recognize native antigens. V5 fusion constructs were transfected in HeLa cells. Along with input cell lysate, IP was performed with or without mAbs (negative control). As a positive control, anti-V5 antibody was used to pull down target V5 fusion antigen proteins. First lane, input; second lane, mAb IP; third lane, no antibody (negative control); fourth lane, anti-V5 IP (positive control). d, assays for which highly specific mAbs were proven effective. The diagram summarizes the assays for which individual purified highly specific mAbs were confirmed to be specifics. IB, immunoblot.