Cyanogenic Glycosides in Cassava

Cassava is a well-known cyanogenic plant, which contains the cyanogenic glycosides linamarin and lotaustralin, the former being the principal cyanogenic glycoside. The ability to detect and quantify cyanogenic glycosides, capable of generating cyanide, could contribute to prevention of acute and subacute cyanide poisoning from the consumption of improperly processed plants. Enzyme-linked immunosorbent assay (ELISA) can be used to detect and measure the amount of linamarin directly within the fresh cassava or cassava-related products with minimal sample preparation using antibodies as the key detector. However, there has been no antibody and ELISA developed for linamarin detection in foods, hence the need. The aim of the project was to generate polyclonal antibodies (pAbs) obtained by immunizing two New Zealand white rabbits with linamarin (hapten) conjugated to a carrier protein, bovine serum albumin (BSA) using cyanuric chloride as chemical linker to be used with the ELISA. Monoclonal antibodies (mAbs) development was attempted by immunizing four BALB/c mice with linamarin-CC-KLH and 3 BALB/c mice with linamarin-CC-BSA but failed to produce the desired antibodies. PAbs generated by both immunised rabbits were highly immunoreactive with antiserum titre of 1:100,000. The pAbs were able to establish positive inhibition assay towards free linamarin with high specificity and sensitivity (limit of detection of 0.0015 µg/ml and IC50 of 2.1 µg/ml). The optimised ELISA was able to determine the amount of linamarin in fresh cassava and processed products available in the UK market, ranging from 0.003 mg/kg to 43.08 mg/kg fresh and dry weight depending on the products. Levels of linamarin of some fresh cassava products are way beyond the safe limit of 10 mg/kg cyanide allowed in food, a potential health risk to consumers. In contrast, highly processed cassava products contained less than 1.0 mg/kg of linamarin, indicating the effectiveness of proper food processing and preparation in reducing the linamarin content in foods.