ovalbumin and 3-3--5-5--tetramethylbenzidine

The 3,3',5,5'-tetramethylbenzidine colorimetric test is proposed as a method for the evaluation of shell egg freshness. A simplification of the original method was tested on albumen, and the modified method was used to study the development of albumen-tetramethylbenzidine reaction in eggs stored at three temperatures for different times. The stability of the reagent solution and the repeatability of the analytical method were also studied. The reaction mechanism showed the predominant role of iron ions on the colorimetric response, and the secondary role of copper ions added to the reaction mix in order to enhance the response. The response coefficient of variation, due to the natural variability in eggs laid by hens of two breeds and different ages, was 10.3%, which is similar to the value obtained for furosine, but lower than those previously observed for other egg freshness indices such as Haugh Unit and air-cell height.

Topics: Animals; Benzidines; Colorimetry; Egg Shell; Eggs; Food Preservation; Iron; Ovalbumin; Poultry; Quality Control; Temperature; Time Factors

3,3',5,5'-Tetramethylbenzidine (TMB) is a widely used chromogen for horseradish peroxidase-based detection systems because it yields reaction products with high absorption coefficients and lacks carcinogenicity. Unfortunately, TMB is labile and poorly soluble in aqueous buffers and such solutions must be freshly prepared before each experiment. Moreover, substrate depletion can occur under assay conditions. To overcome these problems we have developed a two-component TMB substrate system which has a lower detection limit and is more sensitive than many commercially available TMB reagents when compared in microtiter plate enzyme-linked immunosorbent assays. Both components of the substrate system are stable for at least 1 year at 4 degrees C and the usual discoloration of TMB stock solutions is prevented by the addition of a stabilizer that decomposes upon mixing of the two components.

Topics: Animals; Benzidines; Chromogenic Compounds; Drug Stability; Enzyme-Linked Immunosorbent Assay; Evaluation Studies as Topic; Female; Horseradish Peroxidase; Immunoglobulin A; Immunoglobulin G; Indicators and Reagents; Mice; Mice, Inbred BALB C; Ovalbumin; Sensitivity and Specificity; Substrate Specificity