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Lightning-Link® Horseradish Peroxidase in a rat model of renal interstitial fibrosis

Our Lightning-Link® Horseradish Peroxidase (HRP) conjugation kit has recently been published!

Introduction

Renal interstitial fibrosis (IF) is an important pathological feature of disease progression in chronic kidney disease (CKD). However, early detection and quantitative analysis of markers for IF due to CKD progression remains a challenge. Early detection of renal tissue remodelling prior to commencement of advanced pathological structural changes (reduced eGFR, albuminuria) would be beneficial.

Currently, the gold standard to assess renal IF in progressive CKD is invasive, requires expert evaluation and is prone to errors. Urinary markers have been identified as promising alternatives to invasive biopsies, because they arise due to the processes of extracellular matrix (ECM) production and degradation, which reflect fibrogenesis.

In this study, the authors developed a rat model to investigate early urinary markers for progressive renal IF. They used Lightning-Link® Horseradish Peroxidase (HRP) conjugation kit to label a monoclonal antibody (mAb) purified from an antibody producing hybridoma supernatant, to facilitate evaluation of early predictive non-invasive markers for renal fibrosis. Correlation analyses indicated that two urinary markers reflected and predicted tubulointerstitial fibrogenesis.

 Results

An injection of the chemotherapy drug doxorubicin (Adriamycin) successfully induced proteinuria in male Wistar rats. Subsequently, all rats including control untreated rats, underwent renal biopsy at six weeks post-injection.

Lightning-Link® HRP conjugated mAb was used to measure collagen type I (C1M) and III (C3M) degradation fragments in blood and urine using competitive ELISA at Week 6 and following sacrifice at 12 weeks.

Correlation analyses indicated that urinary C3M (uC3M) and C1M (uC1M) both reflected and were predictive for tubulointerstitial fibrogenesis.

 Summary and Conclusion

In this study, the authors developed a proteinuria-driven fibrosis rat model to investigate early urinary markers for renal IF in progressive CKD. Their strategy utilized Expedeon’s Lightning-Link® HRP conjugation kit to label a mAb purified from an antibody producing hybridoma supernatant. Once labeled, the mAb was used in ELISA to detect fibrotic changes in rat blood and urine.

A novel observation from this study is that urinary collagen type III (uC3M) can be measured prior to histological detection, which makes it an early non-invasive predictive marker for renal IF.

These data displayed urinary collagen breakdown products as sensitive early markers of renal IF that precede histological fibrotic changes. This non-invasive urine test may replace the gold standard, but invasive, renal biopsy procedure currently used to assess renal IF in progressive CKD.

 Lightning-Link® HRP conjugations kit for identification of biomarkers for early renal IF

In this study the authors relied on the Lightning-Link® HRP kit to form a stable conjugate between a purified monoclonal antibody (mAb) and HRP for use in ELISA. The Lightning-Link® HRP kit offers several advantages:

  • Direct conjugation of antibodies, proteins and peptides, or any other biomolecule with an available amine group, to Horseradish Peroxidase (HRP)
  • Quick and easy to use – Save time (30 seconds hands-on time), no special knowledge required
  • No separation steps – 100% recovery – No antibody / protein loss
  • Can be used in a wide range of applications – Flexible
  • Freeze dried – Ships at ambient temperature, long shelf-life
  • Fully scalable (10ug to 1g or more) – Easy transfer from R&D to manufacturing
  • Stringently QC tested – Consistent high quality, excellent batch-to-batch reproducibility.

All the above are key for developing a sensitive and specific point-of-care (POC) clinical assessment for early renal IF in CKD.

Reference

Hijmans, RS., Rasmussen, DG., Yazdani, S., Navis, G., van Goor, H., Karsdal, MA., Genovese, F., van den Born, J. Urinary collagen degradation products as early markers of progressive renal fibrosis. J Transl Med. 2017 Mar 20;15(1):63–73. doi: 10.1186/s12967-017-1163-2. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5358042/pdf/12967_2017_Article_1163.pdf Accessed September 2018.