Summary of SMN ELISA Characteristics.

The hSMN Standard Curve: Dynamic range was generated using N = 6 curves from N = 3 experiments. Reproducibility ranged from 4.3–10.6% CVs. The sensitivity limit was derived from the extrapolated value of SMN signal taken at 2 standard deviations above the background level of the assay. Freeze Thaw: The freeze thaw experiment was conducted using a single normal human PBMC sample with a 1∶4 lysate dilution. No reduction in SMN signal was seen with up to two freeze thaw cycles. Recovery was calculated as the mean percentage of SMN signal from a series of up to three sequential freeze thaw cycles compared to the signal from the sample analyzed at the time of cell lysis. PBMC Dilutional Linearity: PBMCs were lysed and diluted from 1∶4 to 1∶32 to assess linearity of sample signal upon dilution. Dilution values were ±5–12% for all analyses with means ranging from 98 to 105%. PBMC SMN Spike Recovery: Using a 1∶4 lysis dilution PBMC samples were spiked with 0, 267, 667 or 1667 pg/mL of the recombinant human SMN used as a standard in the ELISA. The SMN spike signal recovery was calculated based on the expected value from adding the additional SMN to the value produced by the non-spiked samples. Values ranged by +5–37% over the non-spiked signal with mean values ranging from 99–105%, suggesting that accurate analysis of SMN in samples with increased SMN levels is possible, and may benefit from greater sample dilutions. The SMN spike was added into the PBMC mixture at the time of lysis. Minimum Sample Dilutions: Given the differences in sample masses and protein concentrations, different tissue dilutions of µL lysis buffer/mg tissue were used for the SMN protein analysis ranging from 1∶2 to 1∶20.