Antibody Validation Assay with PATH® Protein Microarray Substrate

IN Discussion Topics, PATH non-porous nitrocellulose

The analysis of antibody interactions to 19,500 recombinant full-length human proteins featured on HuProt™ Human Proteome Microarray provides a robust and rapid method to evaluate antibodies’ quality.


Antibodies are the most commonly used research tools. Applied in basic research as well as in clinical and diagnostic medicine, their quality, consistency, and availability are fundamental factors in determining their usability for scientific investigations.

Numerous publications have highlighted the need to develop and adopt standardized validation criteria for the classification of research-grade antibodies. In particular, specificity, affinity, cross-reactivity, and batch to batch variability need to be assessed to ensure that reliable, reproducible data can be derived.

In an effort to develop a standardized validation method, a group of researchers generated more than 1400 monoclonal antibodies, targeting 737 unique human transcription factors and transcription factors-associated proteins. A validation pipeline was established to determine the ability of each antibody to detect endogenous proteins. 
HuProt™ Human Proteome Microarray, (CDI Laboratories, Inc.) featuring 19,500 recombinant full-length human proteins spotted on PATH® Protein Microarray Slides, (Grace Bio-Labs, Inc.) was utilized to measure antibody specificity and cross-reactivity.

Validation pipeline

ELISA-positive hybridomas were initially screened with a minichip comprising the antigens used for immunization. Specificity and cross-reactivity of selected monoclonal antibodies were analyzed with the HuProt™ Human Proteome Microarray. It was also possible to distinguish antibodies targeting native proteins from antibodies directed against linear epitopes, by comparing data obtained from HuProt™ microarray analysis of native versus denaturated proteins. The last distinction could be used to classify monoclonal antibodies for the different uses they are intended for. Antibodies interacting exclusively with folded proteins can be, in fact, used in Immunoprecipitation studies, while antibodies directed at linear epitopes are suited for immunoblotting experiments. Secondary validation steps through immunoblotting and immunoprecipitation were also performed on inducible transiently transfected cells. Finally, binding kinetics were also determined for highly specific antibodies.

Proteins printed on PATH® retain their native conformation

Proteins in their native conformation are more likely to be recognized by antibodies raised against full-length proteins but may not interact with antibodies that specifically recognize linear epitopes. Thus, it was important to establish if proteins printed on the HuProt array are indeed folded and if a denaturation step with urea would enable binding of linear epitopes. The experimental data indicated that the majority of the HuProt proteins are indeed in their native conformation and only after a denaturation step antibodies that specifically bind linear epitopes were able to recognize their specific targets.


This enormous amount of work led to the selection of 373 monoclonal antibodies out of an initial 230,225 hybridomas, corroborating the idea that the production of high-quality antibodies requires a stringent evaluation process. Protein array based specificity and cross-reactivity analysis should be incorporated into the validation pipeline. Proteins in the HuProt human proteome microarray are predominantly in native conformation, this feature can be used to categorize antibodies based on their suitability for different applications. Furthermore, high-density protein microarrays are the only platforms able to provide this type of high-throughput analysis.

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