# Development of Indirect Sandwich ELLA for Detection of Insets in Food
## 4 Discussion
In contrast, the ELLA method is suitable as a screening tool, providing preliminary results before specific insect identification methods are applied. Our developed ELLA method was validated based on ELISA. The sensitivity, specificity, recovery, LOD, and LOQ were used. The results show good performance in terms of these criteria. In food applications, these data are not known for insect determination; thus, a comparison with allergen determination was made. The LOD and LOQ in our method were higher than those for allergen determination and ranged from 0.02 to 2.5 ppm [37]. For example, for soy determination, it was 0.08 to 0.25 ppm [38]; for crustacea, it was 0.001 ppm to 0.003 ppm [39]; and for peanuts, it was 0.06 to 0.19 ppm [40]. To compare, proteomic methods for _Acbeta domesticus_ determination using peptide markers achieved LODs ranging from 8000 to 25,000 ppm. [17]. For multiplex real-time PCR, 1 ppm LOD was confirmed [11]. Likewise, the recovery was high for the snack product in 10 mg/g concentration of chitosan. The concentration in 1 mg/g of chitosan and spike matrix based on the protein bar was lower (Table 3). However, the values obtained correspond to AOAC requirements, which should be within the range of 50-150% [41]. These results show that there are possible interactions in the food matrix between chitin, chitosan, and proteins. The interaction between chitin and proteins was studied in insects [42], but in food, this interaction is not known. The developed ELLA method is based on the determination of terminal carbohydrates of N-acetylglucosamine, which is contained in chitin as well as in the deacetylated chitin derivative chitosan [18]. Chitosan was used as a standard in this study. Different insects have variable contents of chitin due to the fact that the insect is collected and processed during the larval stage, before developing the exoskeleton [14]. Based on electrophoretic methods, it was determined that the chitin content in edible insects' ranges between 43 and 84 mg/g in HI, ALD, TM, GA, and AD [43]. Using extraction methods, chitin was determined in LM, ranging from 86.4 to 123.3 mg/g [44]. Different chitosan contents were also confirmed in our study, where nine insect species were evaluated. The content of chitosan was in the range of 4.49 to 10.78 mg/g. The lowest concentration was in ALD, and the highest in GA. The results are in conformity with the chitin content determined by electrophoretic methods [43]. The absolute amount of chitosan is lower than chitin. This finding was also confirmed for LM, where the chitosan content was 58.95 to 69.75% of the total chitin [44]. The chitosan content also varies in different insect bodies: in _Schistocerca gregaria_, it was determined within the range of 8 to 10% [45]. The lower content of chitosan determined by the indirect sandwich ELLA is caused by the determination of terminal N-acetylglucosamine. Another study evaluated the total amount of chitin after long-term hydrolysis [28; 46]. Intra-day CV and inter-day CV were also comparable with the ELISA method. For soy protein, the intra-day CV in a concentration of 1.0 mg/mL was 3.96%, while the inter-day CV was 12.1. For crustacea, the intra-day and inter-day CVs were 5.69 to 14.32 and 9.0 to 12.59, respectively [39]. For peanuts, the intra-day and inter-day CVs reached 0.84 to 5.21 and 0.87 to 6.12, respectively [28]. Comparing our results (Table 3), the indirect sandwich ELLA intra-day CV was higher, and the inter-day CV varied. In spite of that, the recorded values matched the repeatability \(\leq\)20% and reproducibility \(\leq\)30% values required by the AOAC [41], which implied that our indirect sandwich ELLA exhibited sufficient precision. Numerous products based on processed insects potentially exist. In this case, it is important to validate the ELLA methods for residue or cross-reactivity analysis of commercial products [38]. Eleven commercial samples from the Czech retail market were evaluated and compared with samples without insects (Table 2).