Disposable immunosensor for the determination of domoic acid in shellfish

The construction of an electrochemical immunosensor coupled to differential pulse voltammetry (DPV) for the detection of domoic acid (DA), a neurotoxic aminoacid responsible for the human syndrome known as "Amnesic Shellfish Poisoning" (ASP), is proposed here. The method involves the use of disposable screen-printed electrodes (SPEs) for the immunosensor development based on a "competitive indirect test". Domoic acid conjugated to bovine serum albumin (BSA-DA) was coated onto the working electrode of the SPE, followed by incubation with sample (or standard toxin) and anti-DA antibody. An anti-goat IgG-alkaline phosphatase (AP) conjugate was used for signal generation. A spectrophotometric enzyme-linked immunosorbent assay (ELISA) was used in a preliminary phase of development, prior to transferring the assay to the SPEs. Results showed a detection limit equal to 5 ng/ml of toxin. The electrochemical system is simple and cost-effective due to the disposable nature of the SPEs, and the analysis time is 150 min, shorter than that for the spectrophotometric method. The suitability of the assay for DA quantification in mussels was also evaluated. Samples were spiked with DA before and after the sample treatment to study the extraction efficiency and the matrix effect, respectively. After treatment, samples were analysed using a 1:250 v/v dilution in PBS-M (phosphate saline buffer pH 7.4 + CH3OH 10%) to minimise the matrix effect and allow for the detection of 20 microg/g of DA in mussel tissue. This represents the maximum acceptable limit defined by the Food and Drug Administration [Compliance Programme 7303.842. Guidance Levels, Table 3, p. 248, http://www.fda.org]. The optimised ELISA systems were then used, in parallel with a conventional HPLC method, to detect and confirm DA in shellfish extract in order to verify the performance of the electrochemical system. Very good recoveries were obtained, demonstrating the suitability of the proposed assay for accurate determination of the DA concentration in mussel samples.     

Polyclonal antibodies to domoic acid, and their use in immunoassays for domoic acid in sea water and shellfish

Ovine antibodies raised against conjugates linked through the secondary amino group of domoic acid (1) were used, together with activated-ester-derived conjugates of domoic acid (DA) as the plate coater, to develop a robust indirect competitive enzyme-linked immunosorbent assay (cELISA) for DA in shellfish and seawater. The ELISA was used to analyze shellfish samples for DA, and was compatible with several extraction procedures. The ELISA had a detection limit below 0.01 ng ml(-1), a limit of quantitation (LOQ) of 0.15 ng ml(-1) and a working range of 0.15-15 ng ml(-1) DA. The LOQ is equivalent to 38 ng g(-1) DA in shellfish flesh, assuming a 250-fold dilution during extraction. This is more than 500 times lower than the maximum permitted level (20 microg g(-1) flesh). The ELISA is designed for use alongside regulatory analyses, and, following formal validation, should be available for pre-screening of regulatory shellfish flesh samples. The ELISA was also shown to be appropriate for analysis of DA in algal cultures and in samples of seawater, and thus has the potential to provide early warning of developing algal blooms.     

The amnesic shellfish poison domoic acid enhances neurotoxicity by excitatory amino acids in cultured neurons

Summary A recent episode of human intoxication by cultured mussels containing a rare excitatory amino acid named domoic acid, received particular attention for its neurological implications. The intoxication produced neurological problems, such as headache, confusion, and loss of memory, particularly severe at times. Neuronal damage was found in the hippocampus and amygdala of four patients. We now report that in neuronal cultures the neurotoxicity of a domoic acid-containing mussel extract is the result of domoic acid potentiation of the excitotoxic effect of glutamic acid and aspartic acid present in high amounts in mussel tissue. Moreover, we show that subtoxic concentrations of domoic acid are sufficient to potentiate glutamic acid and aspartic acid neurotoxicity. We present evidence suggesting that the neurotoxic synergism may be due to a reduction of Mg^{+ +} block at the NMDA receptor-associated channel, following activation of NON-NMDA receptors by domoic acid.     

TLC对复方制剂中马兜铃酸限量检查方法学研究

目的:建立简便、快捷、准确的复方制剂中马兜铃酸限量薄层色谱(TLC)检查法。方法:采用精确定量点样薄层色谱法(TLC)对复方止咳散中毒性成分马兜铃酸进行限量检查。结果:本方法测得马兜铃酸含量在0.0808μg/g以下,符合《中国药典》2010年版中细辛药材检查项下的马兜铃酸限量标准要求。结论:本方法简便、快速、准确,适用于复方制剂中马兜铃酸限量的检查。     

A kainic acid receptor from frog brain purified using domoic acid affinity chromatography

A kainic acid receptor was purified from Triton X-100/digitonin-solubilized frog brain membranes. The purification was carried out in two steps: ion exchange chromatography using DEAE-Sepharose CL-6B and affinity chromatography with domoic acid immobilized on Sepharose 4B. The specific binding activity of the affinity-purified receptor is 481-fold higher than that of the crude solubilized preparation and 1617-fold higher than that of the whole membrane fraction. Scatchard analyses of the affinity-purified receptor showed a curvilinear plot which fit a two-site model with dissociation constants of 5.5 and 34 nM and Bmax values of 1700 pmol/mg protein and 4400 pmol/mg protein for the high and low affinity components, respectively. The dissociation constants of the purified receptor are similar to those of the crude soluble preparation (4.8 and 39 nM). Inhibition constants for several kainic acid analogs were also similar for the purified and crude preparations. The active purified receptor migrated with a Mr = 570,000 on gel filtration analysis using Sepharose 6B. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the affinity-purified receptor showed a single broad band with silver stain, migrating with a Mr = 48,000.     

Analysis of natural nucleosides and their derivatives by thin-layer chromatography

Recently published data on the separation and quantification of natural nucleosides and some of their derivatives by thin-layer chromatography on silica gel have been summarized. The use of more than 20 mobile systems for the separation of more than 52 nucleosides and derivatives was discussed; in a few cases, the conditions for their densitometry quantitative analysis after TLC separation were considered. The works performed at GosNIIgenetika on the determination of inosine, thymidine, and acadesine with domestic Sorbfil plates were reviewed in detail.     

Identification of amino acid thiohydantoins directly by thin-layer chromatography and indirectly by gas-liquid chromatography after hydrolysis.

A method is described for the identification of amino acid thiohydantoins by two-dimensional t.l.c. An indirect method for the determination of amino acid thiohydantoins is described which, after hydrolysis, the corresponding amino acids are determined by g.l.c.     

Bile acid kinetics in man studied by radio thin-layer chromatography and densitometry coupling

A method based on coupling of the techniques of radioscanning a TLC plate and densitometry has been developed for the determination of pool sizes and fractional turnover rate of bile acids in man after intraduodenal administration of ¹⁴C-labelled acid. The validity of the method has been checked by comparison of the results obtained with those of an enzymatic spectrophotometric analysis, and a measurement of the radioactivity by liquid scintillation counting, after elution of the separated bile acid from a TLC plate. Advantages of the proposed method over the previous one include a reduced number of manipulations, the possibility of automation, a better reproducibility, and the possibility of elaborating the radiometric data obtained for the primary bile acid for better characterising its metabolism inside the enterohepatic circulation.     

Determination of content and fatty acid composition of unlabeled phosphoinositide species by thin-layer chromatography and gas chromatography

Recent advances in research on the physiological roles of phosphoinositides in eukaryotic organisms indicate a need to distinguish molecular phosphoinositide species on the basis of their characteristic head groups as well as their glycerolipid moieties. Accurate identification of phosphoinositide species in biological samples poses an analytical challenge, because structurally similar inositol phosphate head groups must be resolved, as must lipid-associated fatty acids. Although intact phosphoinositide species have been successfully analyzed, such analyses employ state-of-the-art liquid chromatography/mass spectrometry and require expensive equipment not accessible to many researchers. Described here is a cost-efficient and reliable alternative developed by adaptation of a combination of classic methods for lipid analysis, thin-layer chromatography and gas chromatography.     

Separation of sterols by vapor-programmed thin-layer chromatography

By vapor-programmed thin-layer chromatography on silica gel, it was possible to separate cholestanol from cholesterol and stigmastanol from beta-sitosterol. The method was applied for the analysis of beta-sitosterol-3-(14)C.     

Revisited mycolic acid pattern of Mycobacterium confluentis using thin-layer chromatography

The profile of mycolic acids from Mycobacterium confluentis has not been adequately published. However, the definition of the composition of mycolic acids is a critical element for describing new mycobacterial species. Thus, an erroneously published profile can lead to confusing citations. The aim of this article is to make the protocols clear, by using thin layer chromatography as a tool, for defining the discrete pattern of mycolic acids of any newly reported mycobacterial species. By using this method, and corroborated using nuclear magnetic resonance analysis, we demonstrated that M. confluentis contains α-mycolates (type I) and epoxymycolates (type V mycolic acids).