Novel anti-clusterin monoclonals as tools for characterising clusterin expression in biofluids and tissues

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The clusterin gene ( CLU ) is a top GWAS risk locus for Alzheimer’s disease (AD) while biomarker studies consistently report elevated plasma clusterin levels in AD, suggesting an important role in the disease. Clusterin is a multifunctional protein, a negative regulator of the complement terminal pathway but also involved in lipid and cholesterol transport. Although a handful of commercial ELISA kits are available to quantify clusterin in human biofluids, there is a lack of validated reagents for isolation, detection, and characterisation of clusterin in tissues and fluids, required for exploring roles in AD. To address this, we generated a panel of anti-clusterin monoclonal antibodies (mAbs) and applied them to relevant biofluids and tissues. Clusterin was isolated from human plasma and used to immunise mice. Clusterin-positive hybridomas were taken to monoclonality; eight mAbs (clones: 1A4, 1D5, 2D5, 3C9, 4C7, 5D11, 6C12, 6D1) were selected, purified by protein G affinity chromatography and validated in ELISAs, western blot (WB), immunostaining, surface plasmon resonance (SPR), and functional (classical haemolytic and reactive lysis) assays. All selected mAbs bound strongly to native clusterin in ELISA; mAbs 2D5 and 4C7 were specific for clusterin in plasma WB and were used to develop a sensitive (detection limit 8 ng/ml) and specific sandwich ELISA to measure clusterin in plasma, serum, cerebrospinal fluid (CSF), and cell lysates, as already described (PMIDs: 36149090, 37480051, 38359836). Assay reproducibility was excellent (intra-assay CV = 3.93%; inter-assay CV = 8.5%). Immunoaffinity purification of clusterin from serum using immobilised mAb 2D5 yielded pure, functional clusterin in a single step. None of the anti-clusterin mAbs prevented complement inhibition by clusterin in haemolysis assays. The mAbs were used to detect and quantify clusterin in control and AD brain tissue, astrocytoma lines, and induced pluripotent stem cell (iPSC)-derived astrocytes. One mAb (4C7) specifically detected the known intracellular isoforms of clusterin. We have developed novel mAbs against human clusterin that enable accurate quantification of clusterin in human biofluids and tissues, and visualisation of clusterin in AD and control brain tissue and iPSC-derived astrocytes. These mAbs provide essential tools for elucidating the role of clusterin in AD.

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