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Validation of ELISA for the detection of gums in foodstuffs

Project Code: A01015

22/04/2009

Department of Biological Sciences, University College Chester
Elyse Ireland, H ;
Leatherhead Food International
Haines, J;
Department of Biological Sciences, University College Chester
Bonwick, G;
Leatherhead Food International
Patel, P;
Department of Biological Sciences, University College Chester
Williams, J

The development of novel and efficient methods for accurate identification of polysaccharide and plant gums within complex matrices is needed to ensure consumer protection and quality control of food manufacturing processes. Sensitive assays based upon the specificity of antigen-antibody interactions, such as the enzyme-linked immunosorbent assay (ELISA) have the potential to meet these demands. ELISAs have been previously developed by University College Chester for Acacia senegal gum, Acacia seyal gum and Combretum erythrophyllum gum and Leatherhead Food International for alginates and carrageenan.

The primary aim of this project was to validate immunoassays previously developed for the detection of A. senegal gum, A. seyal gum, C. erythrophyllum gum, alginates and carrageenan in foodstuffs. A secondary aim was to establish the market potential for the ELISAs developed during the project.

In support of these aims the following objectives were identified:

Production of a validated method for the quantification of gum arabic in foodstuffs.
Production of a validated method for the quantification of carrageenan in foodstuffs.
Production of a validated method for the quantification of alginates in foodstuffs.
Survey on the interest of gum ELISAs to industry.
Production of reports and publication.

The performance of the assays was investigated against foodstuffs to which known concentrations of the gums had been added. The intra-laboratory studies were used as a basis for development of standard operating procedures (SOPs) for the assays. The assays were subjected to independent inter-laboratory trial operated under conditions specified by AOAC International.

ELISAs were successfully developed for the measurement of the A. senegal, A. seyal and C. erythrophyllum gums in confectionery. There was no cross-interference from the non-related gum species examined which confirmed the specificity of the assays. In the originator’s laboratory, the assays showed reasonable performance in terms of precision (intra-assay < =15 %CV, inter-assay < =10 %CV) and quantitative recovery (65-100% ± 15%) in foods. The ELISAs for A. senegal, A. seyal and C. erythrophyllum gums met the specifications for stage 1 of the AOAC International Peer Verified Method (PVM) programme and were therefore suitable for inter-laboratory trial.

The performance of A. senegal, A. seyal and C. erythrophyllum gum ELISAs with standards during the inter-laboratory trial was consistent with that observed in the originator’s laboratory. Repeatability of the assays was excellent for the standard curve (%CV values <10%). The repeatability of spiked samples was much more variable (%CV ranging from 0.6 - 173 %). Recovery of the gums from the confectionery was lower than observed during intra-laboratory study, was consistently significantly lower than 100% and ranged from 36 – 69%. All of the assays failed to meet the criteria required for acceptance as a Peer-Verified Method of AOAC International.

ELISAs were successfully developed for the detection of alginate and carrageenan in foodstuffs. The specificity of the assays was confirmed using a range of commercial food-grade alginate and carrageenan samples. There was no cross-interference from the non-related gum species examined. In the originator’s laboratory, the assays showed good performance in terms of precision (intra-assay <=10 %CV, inter-assay <=15 %CV) and quantitative recovery (typically 100% ± 20%) in foods. Both the alginate and carrageenan ELISAs met the specifications for stage 1 of the AOAC International PVM programme and were suitable for inter-laboratory trial.

Unfortunately the performance of all ELISAs failed to totally match that observed in the originator’s laboratory. The data indicated that either transport to, or storage at, the participant laboratories had been detrimental to the assay components. Overall, the inter-laboratory trial demonstrated that further investigation of factors affecting the robustness of the ELISAs was required. Following an improvement in assay robustness, a repetition of the inter-laboratory trial would be necessary in order for these assays to meet the specifications of stage 2 of the AOAC PVM programme and thus achieve PVM status. Each of the ELISAs could be used as a qualitative assay for the detection of the analyte. The results of the market survey conducted during this study indicated that there is some interest in the assays being available as test kits, particularly for alginate and carrageenan.

In summary it is considered that all the main objectives of this study were successfully met and as a result the aims were achieved. Unfortunately it is clear that further work is necessary with each of the ELISAs before they are likely to achieve PVM status.

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