Introduction
Chondrex, Inc. has developed peanut allergen ELISA kits for detecting peanut allergens Ara h 1, Ara h 2, Ara h 3, and Ara h 6. Recovery assays were performed using standards and peanut extract samples, with results falling within the 90-110% range. However, when using biological fluids such as anti-allergen antibodies and/or serum proteins, they may bind allergen either specifically or non-specifically. This binding can mask antibody epitopes, leading to reduced reactivity in the assay. In this study, we performed recovery assays using pooled mouse serum to evaluate the performance of our kits.
Procedures
Pooled mouse serum was diluted with Solution B from the kits. We mixed 50 ?l of each diluted serum (100%, 50%, 25%, and 12.5%) with 50 ?l of the highest standard solution or Solution B, and added these mixtures to each well, resulting in final serum concentrations of 50%, 25%, 12.5%, and 6.3%. The assay was performed afterward. Mouse serum can lead to non-specific binding, Therefore, the serum-Solution B values were subtracted from the sample values to obtain corrected allergen levels. These corrected results were then compared to the added standard values to calculate the recovery data.
Results and Discussion
The recovery results varied depending on the type of kit and serum concentration. The Ara h 1 ELISA showed a recovery range of 22-60%, correlating with serum concentration. When using a 10% serum with a 1:10 dilution, the recovery rate can be around 50%. The Ara h 2 ELISA showed a recovery range of 70-90%. The Ara h 3 ELISA showed a recovery range of 58-81%. The Ara h 6 ELISA had a recovery range of 70-80%. These levels are acceptable for 50% serum samples, with a recovery around 70%. The Ara h 6 ELISA also exhibited non-specific reactions in serum plus Solution B samples within the 20-50 pg/ml range, with OD values between 0.01-0.02. While this should be considered, these very low OD values (and the calculated Ara h 6 values) can generally be considered negligible.
Overall, when we use 10% serum samples with a 1:10 dilution in Solution B, the assay results are acceptable: 70-90% for the Ara h 2, 3, and 6 ELISAs, and 50% for the Ara h 1 ELISA.
We are confident that plasma samples can be used with these kits. However, for plasma, the use of a gel separator may affect the assay results, as gel components could be present in the samples. EDTA or heparin is preferred, though serum is highly recommended.
We suggest using the same sample dilution (serum concentration) and adding one sample spiked with a standard to observe how recovery is affected by sample type. The assay results can then be corrected based on the recovery data. For example, if the sample result is 50 pg/ml and the recovery rate is 50%, the actual allergen level can be calculated as 100 pg/ml (50 pg/ml x 100% / 50%) if absolute values are required. If comparative data is sufficient, negative and positive controls, along with test samples, can be compared.