Novel monoclonal antibody against alphaX subunit from horse CD11c/CD18 integrin.

The researchers successfully generated a novel monoclonal antibody which specifically targets the alphaX subunit of the horse CD11c/CD18 integrin, a protein found on horse dendritic cells. This development presents opportunities for advancing horse dendritic cell biology studies and developing dendritic cell targeted vaccines.

Expression and Immunogen Generation

• The researchers initially undertook the successful expression of the alphaX I-domain of the horse integrin CD11c in Escherichia coli, a common bacterium widely used for protein production.
• The expressed alphaX I-domain was then isolated and purified.
• This purified protein was biochemically characterized to reveal its properties and structure.
• The protein was finally used as an immunogen – an agent capable of inducing an immune response – to generate specific monoclonal antibodies.

Monoclonal Antibody Generation and Assessment

• A specific monoclonal antibody named mAb-1C4 was created against the alphaX I-domain.
• mAb-1C4 is an IgG2a type antibody known to bind and interact with the recombinant I-domain. An ELISA assay, a test that measures the interaction between antibodies and antigens, indicated that the half-maximal effective concentration (EC50) of this interaction was 2.4 nanograms.
• When the investigators performed a Western Blot – a method used to detect specific proteins in a sample – on lysates from horse peripheral blood mononuclear cells (PBMCs), mAb-1C4 recognized a 150 kilodalton protein, which corresponds to the size of the CD11c molecule.

Immunohistochemistry and Identification of Dendritic Cells

• The scientists went a step further and performed immunohistochemistry using the mAb-1C4 on horse lymph node tissue sections. This allowed them to check whether the antibody marked cells in situ, i.e., in their original position in the tissue.
• Some of the labelled cells exhibited dendritic morphology, suggesting that these might be dendritic cells, critical components of the immune system.
• Given the labelling intensity and the location of the marked cells in the paracortical and interfollicular areas of the lymph nodes, the researchers identified these as putative horse dendritic cells.

Implications of the Study

• Development of a monoclonal antibody to horse CD11c broadens the scope of study for horse dendritic cell biology, an underexplored area due to the lack of commercial antibodies.
• It also opens new avenues for biotechnological applications like dendritic cell targeting-based vaccines.