Multi-wavelength assessment of hemolysis-related absorbance patterns in ELISA and ratio-based index

Enzyme-linked immunosorbent assay (ELISA) is widely used for quantitative serum analysis, but hemolysis can alter optical absorbance and is considered a potential preanalytical interference factor. However, wavelength-dependent effects of hemolysis on ELISA chromogenic readouts remain insufficiently characterized. This study quantitatively evaluated ELISA readout patterns across varying degrees of hemolysis using a multi-wavelength approach without assuming a specific interference mechanism. Ninety porcine serum samples with different hemolysis levels were analyzed using a commercial classical swine fever (CSF) antibody indirect ELISA. Serum absorbance was measured at hemoglobin-associated wavelengths (414, 541, and 576 nm) and at 620 nm, while the final ELISA reaction mixture was measured at 405, 414, 541, 576, and 620 nm. Baseline-corrected absorbance values were calculated, and a spectral separation index (RSoret=EA405/EA414) was used to describe wavelength structure near the Soret region. Strong linear relationships were observed among ELISA wavelengths across all samples, indicating stable inter-wavelength proportionality despite wide variation in hemoglobin-associated serum absorbance. A small subset of samples (7. 8%) with higher hemolysis showed a relative reduction in the 405-nm signal compared with hemoglobin-associated bands. These results provide a quantitative description of multi-wavelength absorbance behavior in hemolyzed sera and support ratio-based indicators as complementary tools for identifying atypical 405-nm ELISA readout patterns.