# Development of Indirect Sandwich ELLA for Detection of Insets in Food
## 3 Results
### Validation of Indirect Sandwich ELLA in This Study  
The limit of detection (LOD) and limit of quantification (LOQ) were 0.006 mg/mL (6 ppm) and 0.028 mg/mL (28 ppm), respectively. Chitosan was determined in different insect species to confirm the sensitivity of the indirect sandwich ELLA for various species (Table 1). The chitosan concentration was higher than 4.5 mg/g in the wheat products, chips, and snacks, suggesting the natural content of N-acetylglucosamine in the control samples (Table 2). Figure 1: Box plot for WGA and LELectin in dilutions of 1:10,000 and 1:1000 with concentrations of 5 mg/mL and 10 mg/mL. Figure 2: Standard curves of indirect sandwich ELLA for chitosan detection. Spiking 1 and 10 mg/g standard into the wheat snack resulted in a recovery from 80.0% to 104.0%; for the protein bar, the recovery was from 64.1% to 79.0% depending on the blank matrix concentration (Table 3). As shown in Table 4, the chitosan standards with concentrations of 0.1, 1, and 10 mg/mL, insect _Tenebrio molitor_ in concentrations of 25 and 50 mg/mL, and insect product in concentrations of 25 and 50 mg/mL were used. The chitosan CV% of intra-day precision and inter-day precision ranged from 2.45% to 15.41% and from 3.61% to 3.17%, respectively. For insect product and _Tenebrio molitor_, the CV% of the intra-day precision was calculated. It ranged from 10.29 to 30.29% and was expressed as chitosan content. Additionally, the relative error of the standard sample was low for concentration 1 and 10 mg/mL and high for 0.1 mg/mL. \begin{table}
\begin{tabular}{c c c} \hline
**Sample** & \(n\) & **Chitosanan (mg/g, Mean \(\pm\) SD)** \\ \hline _Acheta domesticus_ (AD) & 18 & 9.83 \(\pm\) 3.99 \({}^{\text{a}}\) \\ _Alphitobius diaperinus_ (ALD) & 7 & 4.49 \(\pm\) 4.52 \({}^{\text{e}}\) \\ _Blaptic dubia_ (BD) & 12 & 8.06 \(\pm\) 5.48 \({}^{\text{b}}\) \\ _Gryllus assimilis_ (GA) & 13 & 10.78 \(\pm\) 6.09 \({}^{\text{a}}\) \\ _Hermeutia Illucus_ (HI) & 6 & 9.87 \(\pm\) 3.83 \({}^{\text{a}}\) \\ _Locusta migratoria_ (LM) & 4 & 7.09 \(\pm\) 3.12 \({}^{\text{b,c}}\) \\ _Schelfordella tartara_ (ST) & 4 & 8.44 \(\pm\) 3.42 \({}^{\text{a,b}}\) \\ _Tenebrio molitor_ (TM) & 28 & 5.63 \(\pm\) 5.6 \({}^{\text{d,e}}\) \\ _Zophous morio_ (ZM) & 18 & 5.83 \(\pm\) 3.62 \({}^{\text{c,d}}\) \\ \hline \end{tabular} Different letters indicate statistical differences (\(p\) < 0.05), SD: standard deviation, \(n\): number of samples analyzed.  
\end{table} 
Table 1: Chitosan content in different insect species. \begin{table}
\begin{tabular}{c c c} \hline
**Samples** & \(n\) & **Chitosanan (mg/g, Mean \(\pm\) SD)** \\ \hline \hline \multicolumn{1}{c}{Baked amaranth chips: tomato and basil} & 1 & 4.73 \(\pm\) 0.94 \({}^{\text{a}}\) \\ \multicolumn{1}{c}{(gluten free)} & & \\ Complete high-protein pasta & 1 & 3.35 \(\pm\) 0.86 \({}^{\text{c}}\)d \\ Doritos sweet chilli flavored/hot corn & 1 & 3.23 \(\pm\) 1.07 \({}^{\text{c}}\)d \\ Fermented chocolate granola & 1 & 5.29 \(\pm\) 1.62 \({}^{\text{a}}\) \\ with coconut & 1 & 4.69 \(\pm\) 2.16 \({}^{\text{a,b,c}}\) \\ Durable graham sticks: Havlik & 1 & 2.52 \(\pm\) 0.99 \({}^{\text{d}}\) \\ Durable graham sticks: spicy & 1 & 4.52 \(\pm\) 0.71 \({}^{\text{a,b}}\) \\ Legume chips: garlic & 1 & 4.87 \(\pm\) 1.07 \({}^{\text{a}}\) \\ Legume chips: original & 1 & 3.54 \(\pm\) 0.67 \({}^{\text{b,c,d}}\) \\ Lentil wasabi: multigrain snack & 1 & 5.31 \(\pm\) 1.43 \({}^{\text{a}}\) \\ Protein bar: chocolate flavor & 1 & 4.84 \(\pm\) 1.65 \({}^{\text{a}}\) \\ \hline \end{tabular} Different letters indicate statistical differences (\(p\) < 0.05), SD: standard deviation.  
\end{table} 
Table 2: Natural content of N-acetylglucosamine in the samples.