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

Abstract

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.

Brief introduction of current technologies in isolation of broadly neutralizing HIV-1 antibodies

“…B cell hybridomas can be an important source for screening of monoclonal antibodies. Highthroughput screening is used to characterize mouse IgG antibodies; including sub-isotypes, binding activity, and neutralizing activities (Liu et al, 2015;Szafran et al, 2016). Fluorescent antigen can be used to sort antigen binding hybridoma cells from a mixture as opposed to the traditional way of screening using multi-micro well plate screening and limiting dilution.…”

Flexible and powerful strategy for qualitative and quantitative analysis of valepotriates in Valeriana jatamansi Jones using high‐performance liquid chromatography with linear ion trap Orbitrap mass spectrometry

“…Moreover, the obtained data were processed using SIEVE 2.0, including denoising, baseline correction, overlapping‐peak resolution, and peak alignment, and then the m/z , the retention time, and the peak intensity of samples from 15 regions were obtained. Hierarchical clustering analysis (HCA) was used to compare the compositions and contents of 92 chemical components in the samples, while principal component analysis (PCA) was used to analyze the diversity of the samples by region. This quantitative information was propitious for quality evaluation by distinguishing the diversity of the composition in Valeriana jatamansi Jones from 15 regions.…”

High-throughput Imaging as a versatile and unbiased discovery tool

“…While many HTI applications use RNAi or small molecule screens, alternative approaches are similarly feasible. As examples, Hasse et al report on a versatile system to systematically express fluorescently tagged proteins at large scale in cultured cells to analyze the their function and location using HTI [9] and Szafran et al, have developed an HTI pipeline for large scale screening of complex mixtures of antibodies by using a novel supervised machine learning algorithm that can classify each antibody based on immunuofluorescence staining features extracted by automated image analysis [10].…”