(T1130-06-38) Development and Validation of a Novel ECL Method for the Quantitative Detection of Complement C3 in Human Serum

Purpose: The complement system plays critical roles in both innate and adaptive immune responses. The third complement factor (C3) is situated at the junction of all three complement pathways and is essential for activating the complement system (Law S.K.A. and Reid K.B.M., 1995). However, to accurately measure C3 level in human body fluids can be a challenge due to the abundance of C3 and its sensitivity to heat, which can trigger the proteolytic reaction to convert C3 into downstream substrates (Seya T, Nagasawa S., 1988). To overcome the difficulties, a novel electrochemiluminescence (ECL) immunoassay method was developed and validated to quantitatively detect C3 in human serum.

Methods: Comparing with traditional ELISA method, ECL immunoassay platform provides higher sensitivity and broader dynamic range and was chosen for the method. Due to the high endogenous level of C3 in human serum, assay buffer was used as surrogate matrix. A normal base pool (NBP) made with gender pooled normal human serum samples was included in the method as dilution quality control (DQC) to monitor the assay performance when analyzing human serum samples. A native C3 protein purified from normal human serum was chosen as reference standard. The standard stock was spiked into surrogate matrix to prepare for standard curve and high, middle, and low-quality controls (HQC, MQC and LQC). A pair of anti-human C3 antibodies, including a biotinylated capture antibody that binds to streptavidin coated assay plate and a SULFO-TAG conjugated detection antibody, were used to develop a sandwich immunoassay (Figure 1A) and applied for fit-for-purpose validation.

Results: A novel ECL method was successfully developed and validated to measure C3 level in human serum. A representative reference standard curve was shown in Figure 1B. The validated quantitation range of the method is 655 pg/mL (Lower Limit of Quantification, LLOQ) to 400,000 pg/mL (Upper Limit of Quantification, ULOQ). The quality controls, including buffer QCs and DQC, were used to assess the accuracy and precision of the method and the stability of the samples. All assessed samples show less than 20% inter-assay precision (%CV) and inter-assay accuracy (%RE) within ± 20% variation (Figure 2A). The samples are stable for up to 16 hours when stored at 4°C or up to 4 hours if kept at room temperature. In addition, up to 5 times freeze-thaws of samples can be tolerated (Figure 2B). Two potential interference reagents, complement C3a and C3a desArg, were assessed at a native ratio of C3 vs C3a/C3a desArg at 2000:1 according to previous report (Hubens WHG, et al. 2021). Both reagents show no significant impact on assay performance at MQC level (Figure 3A). To further investigate possible interference substance present in serum matrix, normal human serum samples were used to assess selectivity and parallelism. Reference standard was spiked into 10 normal individual samples, the NBP/DQC, and a C3 removed serum sample (C3 dpl) at LLOQ level pre-sample dilution. All tested samples show ± 20% of %RE from un-spiked nominal controls, with only one exception at 22% (Figure 3B). Finally, all six tested samples show parallelism within 1:5000 to 1:1000000 dilution range (Figure 3C), which embraces the sample dilution factor at 1:50000, when %RE is limited to ± 30%.

Conclusion: A Novel ECL method with high sensitivity and wide dynamic range was successfully developed and validated for detecting C3 in human serum. The method shows high consistent performance and accuracy. It can be reliably utilized for complement studies. The method also has the potential to be modified for detecting C3 in other types of human body fluids.

References: 1. Law S.K.A. and Reid K.B.M. Complement 2nd Edition. 1995. (ISBN 0199633568).
2. Seya T, Nagasawa S. Heat-induced thiol-disulfide interchange reaction on the third component of human complement, C3. J Biochem. 1988 May;103(5):792-6. doi: 10.1093/oxfordjournals.jbchem.a122348.
3. Hubens WHG, Beckers HJM, Gorgels TGMF, Webers CAB. Increased ratios of complement factors C3a to C3 in aqueous humor and serum mark glaucoma progression. Exp Eye Res. 2021 Mar; 204:108460.

Figure 1: (A) Schematic of the sandwich ECL immunoassay. (B) A representative reference standard curve was fitted with a 4-parameter logistic regression model with a weighting factor of 1/Y2.

Figure 2: (A) Summary table of fit-for-purpose validation accuracy and precision assessment results. (B) Short-term stability and Freeze-thaw stability results summary.

Figure 3: Fit-for-purpose validation results of (A) Specificity Assessment; (B) Normal Human Serum Selectivity; (C) Normal Human Serum Parallelism.