Quantification of Limonin and Limonoate A-ring Monolactone During Growth and Development of Citrus Fruit and Vegetative Tissues by Radioimmunoassay

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RIAs were developed using a polyclonal antibody raised against limonin-7-(O-carboxymethyl)oxime conjugated to BSA (1, 2). Using an iodinated tracer, sensitivity was 150 fmol limonin (less than 1 ppb) and only deoxylimonin showed significant cross-reactivity. The assay developed using 3H-limonol as tracer had a detection limit of 0.22 ng (2.2 ppb). In both systems, there was no cross-reactivity with limonoate A-ring monolactone, therefore this compound could also be quantified. Since the RIAs required no prior purification of samples, up to 400 samples could be analyzed per day. Therefore, they were used to analyze natural variation of limonin levels in fruit and vegetative tissues, changes during growth and development, and variation in processed juice. RIA techniques can provide several advantages as compared to many other quantitative or qualitative methods. They are sensitive down to the fmol range, allow processing of hundreds of samples per day, and can be highly specific, depending on characteristics of antibodies used. The specificity of RIA frequently permits substances to be measured or detected in crude samples.