SBIR-STTR Award

The poor reproducibility of published research is a major focus of the scientific community inrecent years. Western analysis Abs are often validated against antigen (Ag) materials such asmammalian cell lysates which have undergone treatment (e.g., chemical exposure, UV radiationetc.) to induce (or reduce) modification of residues on a protein of interest. Several stages duringthe generation of this material can pose challenges. First, it can be difficult to identify theappropriate treatment and cell line for the residue of interest. Second, treated cell lysates can betechnically challenging to generate reproducibly. Even with careful adherence to a protocol, theselysates may have varying degrees of modification present in "treated" and "untreated" samples.This can lead to incorrect specificity determination for the Ab. A reliable method for validatingPTM-specific Abs would be one where each Ag sample contains the target sequence residue witheither 100% modification or 100% non-modification. We have developed a system that providesthat certainty.

Public Health Relevance Statement:
NARRATIVE Antibodies (Abs) are the most frequently used tools in science research and in clinical assays. But there is still an absence of universally accepted guidelines or standardized methods for validating these reagents. We have invented a surrogate validation method to validate antibodies against post-translationally modified protein sites. To determine quantitatively just how bad the problem may or may not be, we propose to test and validate the 10 most cited antibodies from several different manufacturers.

Project Terms:

Antibodies (Abs) are commonly employed in biomedical research to identify and quantify targetproteins. However, the specificity, sensitivity and reliability of these Abs can be variable, which is asignificant concern in the scientific community. The use of an incorrect Ab can result in misleadingor inaccurate results, impeding progress in various areas of biomedical research. In Phase I of thisproject, we developed tools to validate the specificity of commercial Abs used to detectphosphorylated sites in proteins in a method we developed and published that we namedMILKSHAKE. We will present some of these results from this Phase I effort, which demonstrates thecritical need to address this concern. This proposed SBIR Phase II project aims to both extend andcommercialize the Phase I effort. We will continue the Ab validation work initiated in Phase I byextending the analysis to other types of post-translational modifications (PTMs) of proteins inWestern analysis and flow cytometry. This Phase II project also seeks to improve and scale thispipeline further, and to develop new methods for validating Abs against glycosylated proteins, whichare often dynamic and challenging to detect using traditional techniques. We will also employ aninnovative tool for measuring Ab promiscuity using a technology we term "˜Sundae' to betterinvestigate the specificity of the Ab paratope. Sundae will be demonstrated for two therapeutic Abs.The Phase II project has several key objectives, including the development of new Ab-specificassays and the establishment of a centralized database for sharing validated Ab data. The projectteam comprises experts in Ab validation, proteomics, and molecular biology, and will collaborateclosely with our current and future industry partners and academic researchers to ensure oursuccess.

Public Health Relevance Statement:
NARRATIVE While there are already some established methods for antibody validation, there are still issues with the specificity, cross-reactivity, reproducibility, sensitivity, and accuracy of them. This is especially true for antibodies directed against post-translationally modified proteins. The proposed Phase II effort aims to address these issues by investigating the effects of post-translational modifications, comparing our MILKSHAKE method with other established methods for quantifying specificity and sensitivity, using our Sundae method for evaluating the importance of paratope specificity, and our Epivolve method for the discovery and validation of glycosylation-specific antibodies. The proposal also includes a means for the dissemination and commercialization of this Phase II project, which will increase the impact of this effort. Overall, the proposed research has the potential to contribute to the development of more reliable and accurate antibody validation methods and improve the reproducibility of biomedical research.

Project Terms: