Verification of a monoclonal antibody-based ELISA for monitoring lipid oxidation in chicken myofibrils

Lipid oxidation is a major quality deterioration problem in all muscle foods, affecting both the organoleptic value and toxicological status of the food. Detection of lipid oxidation needs to be accomplished in a very specific, sensitive and rapid manner. The standard method for measuring lipid oxidation is the 2-thiobarbituric acid reactive substances (TBARS) assay. While this assay is simple, criticisms arise from its lack of specificity. Hexanal, a secondary product of lipid oxidation, has often been used as an indicator of lipid oxidation. Headspace-gas chromatography has typically been used to measure hexanal; however, this method is expensive and tedious. As a sensitive, rapid and reproducible alternative, an enzyme-linked immunosorbent assay (ELISA) has been previously described for the detection of hexanal in meat products. The objective of this study was to verify the use of the monoclonal ELISA for monitoring lipid oxidation in chicken protein through the quantification of hexanal.; A solid-phase microextraction (SPME) method combined with gas chromatography/mass spectrometry (GC/MS) was developed and evaluated for its suitability in the detection of hexanal in chicken myofibrils. Results indicated that recovery of hexanal from spiked myofibrils was 95% using a 5 min sampling time, with a total analysis time of about 12 min/sample. Using SPME, the limit of detection for hexanal in myofibrils was 10 ppb with a coefficient of variation ranging from 1 to 13%, and a working linear response from 10 ppb to 10 ppm (r 2 = 0.995). The ability of the hexanal-specific monoclonal CI-ELISA to monitor lipid oxidation in freeze-dried chicken protein was compared to the thiobarbituric acid reactive substances assay (TBARS) and the solid-phase microextraction - gas chromatography/mass spectrometry (GC/MS-SPME) method for hexanal. Protein extraction and solubilization procedures were optimized to maximize the formation of hexanal-protein adducts and percent extractable protein. Freeze-dried myofibrils (MF) with added methyl linoleate (0.6 mmol/g protein) were stored for 5 days at 50°C at a_w of 0.30 and 0.75. Hexanal was measured by GC/MS-SPME and CI-ELISA, and malonaldehyde was measured by TBARS. Lipid oxidation reached a maximum after 4 days of storage at both water activities, then decreased. At low water activity, 34.7 and 39.7 ppm hexanal were detected by GC/MS-SPME and CI-ELISA respectively, after 4 days of storage. The CI-ELISA was well correlated with the GC/MS-SPME (r = 0.78) and TBARS (r = 0.87) methods. The correlation of the hexanal-specific CI-ELISA to both GC/MS-SPME and TBARS verified the ability of the CI-ELISA to be used as an index of lipid oxidation, offering the convenience for use in a kit to be utilized within a food processing facility.