Microtitre plate assay for paralytic shellfish toxins using saxiphilin: gauging the effects of shellfish extract matrices, salts and pH upon assay performance.
Saxiphilin is a hydrophilic protein with a high affinity and specificity for paralytic shellfish toxins (PSTs) found in the circulatory fluid of many invertebrates and ectothermic vertebrates. Saxiphilin has been found to be closely related to the iron binding transferrins, a group of proteins that range in molecular weight between 70 and 90 kDa. One saxiphilin isoform, that from the centipede Ethmostigmus rubripes, has been used to develop a microtitre plate assay for PSTs which relies upon detection of bound tritiated saxitoxin (STX). In this study, this assay was challenged with differing conditions of salt concentration and identity, pH and addition of non-toxic extracts of commercial shellfish prepared following the Association of Official Analytical Chemists endorsed procedure, elements the assay would encounter if used for PST monitoring and may compromise assay performance. The assay tolerated up to 15% of the total reaction, volume being shellfish extract before assay signal started to diminish. The presence of these extract matrices had little effect upon assay accuracy and precision when measuring STX, as a typical PST. Also, the detection of STX in the presence of shellfish matrices could be confidently reproduced on different days by different experimenters. The elements present in the shellfish extracts were measured by inductively coupled plasma spectroscopy, with the most common cationic elements being Na followed by K, Mg and Ca. Only trace amounts of other cationic elements were also present. From these results, the effects upon the assay by the four most common salts of these elements, NaCl, KCl, CaCl(2) and MgCl(2) were measured. Both NaCl and KCl did not impair assay performance at concentrations as high as 550 mM. It should be noted, however, that greater than 80 mM of either of these salts must be present in the assay for it to achieve the maximal signal. Adding CaCl(2) and MgCl(2) to the assay had dramatic effects upon performance. In the case of CaCl(2), the NaCl that was present in standard assay conditions enhanced its negative impact upon the assay. With MgCl(2), NaCl counteracted its inhibitory effect to some extent. After taking into account sample dilutions of shellfish extracts however, the potential for an interfering effect by Ca or Mg is minimal. A pH of 5.4 or less is necessary for there to be any significant impact upon the assay, with the assay signal being stable up to an alkaline pH as high as 9. Using the conditions herein, this assay can be used to reliably detect 1.3 microg STXeq/100 g shellfish tissue if it were to be used for monitoring for PST contaminated shellfish. These results demonstrate that this assay is a highly robust diagnostic tool for the measurement of PSTs in shellfish extracts.