Identification of autoantigens in body fluids by combining pull-downs and organic precipitations of intact immune complexes with quantitative label-free mass spectrometry.

The research article presents a method for identifying autoantigens, proteins that cause an immune response in autoimmune diseases, by isolating intact autoantibody-antigen complexes from body fluids. Through this method, they were able to identify three new autoantigens in the autoimmune disease uveitis.

Research Objective

• The objective of the study was to design a method for autoantigen identification which could lead to a better understanding of autoimmune diseases.
• Autoimmune diseases result from an immune response to an organism’s own proteins, known as autoantigens. However, the nature and composition of these autoantigens, both initially and during disease progression, remain largely unknown.
• The researchers therefore developed a strategy for autoantigen identification based on the isolation of intact autoantibody-antigen complexes from body fluids.

Methodology

• The researchers began with an organic precipitation of high molecular weight proteins and free immunoglobulins. These processes helped to filter out irrelevant components and concentrate on the desired immune complexes.
• After these precipitation steps, the released autoantigens were identified by means of quantitative label-free liquid chromatography mass spectrometry. This allowed for fine-tuned identification of the autoantigens.
• The researchers tested this process by spiking a highly complex body fluid with a predefined antibody-antigen complex, confirming the method’s ability to detect these complexes even at low levels of abundance.

Study Findings

• As a proof of concept, the researchers applied their technique to study autoimmune uveitis, an inflammatory disease caused by autoreactive T-cells attacking the inner eye and accompanied by autoantibodies.
• With their approach, the researchers identified three novel autoantigens in the equine recurrent uveitis model (secreted acidic phosphoprotein osteopontin, extracellular matrix protein 1, and metalloproteinase inhibitor 2).
• The presence of autoantibodies to these newly identified autoantigens was confirmed in 15-25% of patient samples using an enzyme-linked immunosorbent assay.

Implications of Research

• The researchers argue that this workflow can lead to the identification of novel autoantigens in autoimmune diseases, thereby increasing understanding of these diseases.
• By identifying the autoantigens targeted by the immune system in autoimmune diseases, potential treatments can be developed to stop or mitigate the harmful immunological reactions.
• The method thus provides a versatile and useful tool for autoimmune research and may open up new avenues for diagnosis and treatment of autoimmune diseases in the future.