More Reliable Tools Needed to Research CLN3 Protein in Batten Disease, Study Shows

Research into the CLN3 protein, whose nonfunctional form underlies juvenile Batten disease, may be compromised by the use of unreliable antibodies that lack specificity, researchers suggest.

The study, “Lack of specificity of antibodies raised against CLN3, the lysosomal/endosomal transmembrane protein mutated in juvenile Batten disease,” was published in the journal Bioscience Reports.

Neuronal ceroid lipofuscinoses (NCLs), also known as Batten disease, are a group of inherited lysosomal storage disorders with progressive neurodegeneration mostly affecting children. The most common form, juvenile NCL3 (juvenile Batten disease) is caused by mutations in the CLN3 gene.

The CLN3 gene provides instructions for the production of the CLN3 protein, also called battenin. This protein is primarily located in the membranes surrounding lysosomes and endosomes — cell compartments that digest and recycle cellular materials. However, the role of CLN3 is still unclear.

A tool often used by researchers to study a protein are antibodies that bind to and recognize that specific protein. Antibodies can help researchers determine protein levels, where it it expressed in tissues and cells, and its exact location inside the cell.

Antibodies against CLN3 have been used to uncover the functions of this protein and understand the processes that lead to juvenile Batten disease. But according to the authors, the exact specificity of these antibodies to detect CLN3 was never fully addressed.

Researchers tested both commercially available and researcher-generated anti-CLN3 antibodies against proteins taken from healthy mice and CLN3-deficient mice, that to an extent can mimic the human form of the disease.

The results revealed that all forms of anti-CLN3 antibodies available lack specificity and can detect the same proteins in control samples and samples deficient for CLN3. This means that these antibodies detect not only CLN3, but also other unrelated proteins.

This observation was consistent for different mouse tissues, including different regions of the brain (cerebellum and cortex), kidney, and fibroblast cells.

Importantly, these antibodies did not specifically bind to and detect the human form of the CLN3 protein that was expressed at high levels in mouse cells grown in the laboratory.

Problems with antibody specificity can arise when other proteins, which have a sequence similar to the target protein, are present in the same samples. These nontargets can cross-react with the antibody and generate a nonspecific signal that might be misinterpreted as the correct protein.

A misinterpretation of this kind can compromise the conclusions and validity of an entire study.

Researchers advise that the specificity of an antibody should always be checked using tissue samples from animals or cells that completely lack the protein of interest.