Purpose: HSP47 is a collagen-specific chaperone protein that is necessary for the collagen deposition and collagen fibril formation. Hepatic stellate cells (HSCs) are the primary cellular mediators of liver fibrosis, upon exposure to inflammatory stimuli, HSCs are activated and transformed into myofibroblasts which synthesize and secrete procollagen. Cleaved procollagen accumulates as insoluble collagen, causing fibrosis. The local expression, and in some instances the plasma level, of HSP47 is shown to be elevated in fibrotic diseases in rodent models, including liver, lung and kidney fibrosis. In the rodent models, knockdown of HSP47 decreased collagen accumulation and prevented the progression of fibrosis. Due to the mechanism and involvement of HSP47 in the development of fibrosis, there is value in the validation of an immunoassay for measuring circulating HSP47 as a potential fibrosis biomarker. Commercial kits are readily available for plasma HSP47 measurement. Here we describe the screening of commercial kits along with validation of the lead kit for use in clinical sample analysis. Proof of utility was evaluated using plasma samples from normal healthy human subjects and stage 2-4 diabetic kidney disease (DKD) patients in which kidney fibrosis is a common comorbidity.
Methods: Five commercial HSP47 kits were evaluated employing the calibrator proteins from each kit across all kits. The most sensitive kits were selected for further screening. The kits were further evaluated for dilution linearity and matrix interference using plasma samples. The HSP47 kit assay best suited to the clinical needs was optimized and taken into validation for accuracy precision, recovery, stability and other validation parameters in preparation for exploratory clinical use.
Results: Amongst the five kits screened, the Abcam HSP47 kit emerged as meeting the need in terms of sensitivity, performance and ease of use. The minimum required dilution was found to be 16-fold allowing for dilution of matrix interference and a broad analytical range when dilution was factored in. The Abcam HSP47 kit was taken into validation and the kit met the validation parameters, with accuracy and precision CV’s of <20%, which were acceptable as defined by the validation SOP. Storage and Freeze-thaw stability were also within the SOP acceptance limits. Analysis of samples from normal healthy and DKD subjects demonstrated that there is a difference in the mean measured level of HSP47 in normal healthy 20.4 ng/mL (range 12.8 to 36.2 ng/mL) and DKD 88.8 ng/mL (range 14.5 to 350 ng/mL).
Conclusion: The kit screening was successful. The Abcam HPS47 kit was identified as suitable and taken into validation. The assay had good performance during validation and successfully passed the validation criteria for exploratory clinical use. The HSP47 assay was used for analysis of normal and DKD plasma samples. The results show overlap between the normal range and disease ranges, but also demonstrate that HSP47 levels are higher in the disease samples, suggesting that the assay may have clinical utility.
Diane Shevell– Biomarker Scientist, Bristol-Myers Squibb Company
Sheng Gao– Senior Research Investigator II, Bristol-Myers Squibb Company
Jianing Zeng– Associate Director, Bristol-Myers Squibb, Princeton