Clinical biochemistry · 2025
A novel fluorescent-labeled serum protein electrophoresis method for detection of monoclonal free light chains in serum.
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Pang Gaoyang, Kang Xiaojun, Li Yan, Zhang Xiaojuan, Ren Huijun
Аннотация
BACKGROUND: Current guidelines recommend a combination of serum protein electrophoresis (SPE), immunofixation electrophoresis (IFE), and serum free light chain (FLC) assays for monoclonal immunoglobulin (M-protein) screening. However, the high cost of this comprehensive panel limits its widespread use. In routine clinical practice in China, SPE alone is commonly employed for general population screening, but it suffers from low sensitivity for detecting monoclonal free light chains (LC-M-proteins), leading to a high rate of missed diagnoses. There is a clear clinical need for a novel method that enhances LC-M-protein detection while preserving the practicality of SPE.
METHODS: We established a fluorescent-labeled serum protein electrophoresis (FSPE) technique. The method utilizes 2-(diphenylphosphino)ethylamine (DPEA) to selectively reduce the C-terminal thiols of LC-M-proteins (4 °C, 60 min, dark), followed by specific fluorescent labeling with sulfo-Cyanine5 maleimide (4 °C, 30 min, dark). After agarose gel electrophoresis separation, synchronous identification and quantification of LC-M-proteins and intact M-proteins are achieved via dual-channel (fluorescence and protein staining) imaging.
RESULTS: Method validation demonstrated that DPEA enables efficient and selective reduction. The detection limit of FSPE for LC-M-proteins reached 400 mg/L. Quantitative analysis showed excellent performance (linearity: R2 = 0.9887; total precision: relative standard deviation, RSD = 6.61%). FSPE results were highly correlated with the reference Freelite™ FLC assay (Spearman's rs = 0.895, p < 0.01), although Bland-Altman analysis revealed quantitative differences between individual samples.
CONCLUSION: FSPE represents a significant upgrade to conventional SPE, effectively addressing its critical flaw of missing LC-M-proteins while achieving sensitivity comparable to IFE. It retains the key advantages of SPE, namely operational simplicity and low cost. Therefore, FSPE emerges as a potential standalone method for efficient M-protein screening.
METHODS: We established a fluorescent-labeled serum protein electrophoresis (FSPE) technique. The method utilizes 2-(diphenylphosphino)ethylamine (DPEA) to selectively reduce the C-terminal thiols of LC-M-proteins (4 °C, 60 min, dark), followed by specific fluorescent labeling with sulfo-Cyanine5 maleimide (4 °C, 30 min, dark). After agarose gel electrophoresis separation, synchronous identification and quantification of LC-M-proteins and intact M-proteins are achieved via dual-channel (fluorescence and protein staining) imaging.
RESULTS: Method validation demonstrated that DPEA enables efficient and selective reduction. The detection limit of FSPE for LC-M-proteins reached 400 mg/L. Quantitative analysis showed excellent performance (linearity: R2 = 0.9887; total precision: relative standard deviation, RSD = 6.61%). FSPE results were highly correlated with the reference Freelite™ FLC assay (Spearman's rs = 0.895, p < 0.01), although Bland-Altman analysis revealed quantitative differences between individual samples.
CONCLUSION: FSPE represents a significant upgrade to conventional SPE, effectively addressing its critical flaw of missing LC-M-proteins while achieving sensitivity comparable to IFE. It retains the key advantages of SPE, namely operational simplicity and low cost. Therefore, FSPE emerges as a potential standalone method for efficient M-protein screening.