*2.2. Enzyme-Linked Immunosorbent Assay of Prepared Standards for Determination of Aflatoxin Content in Chicken Feed Samples*

Aflatoxin B1 cELISA, using AFB1-ELISA low matrix kit (Helica Biosystems Inc.®, Santa Ana, CA, USA) is the last segment of the improved aflatoxin test procedure (Section 4.1) and its details are in Section 4.1.5. Curve-fitting characteristics of this immunoassay are shown in Figure 1. The four-parameter logistic curve (4PLC) of spiked aflatoxin B1 (AFB1) concentrations was characterized by two plateau regions and an inflection point (Figure 1). This curve was used to study linearity of measurements of AFB1 spiked in the modified extract to aqueous buffer mixture, 80% acetonitrile extraction solvent: PBS-T mixture (100:650, *v*/*v*).

Response (inhibition), y = a − d/[1 + (x/c)b] + d (1)

where x = AFB1 concentration and a–d are described in Table 1.

associated coefficients (r and r<sup>2</sup>

Slope at inflection point (b)

).

Minimum signal intensity (d)

Coefficients of correlation (r)

Coefficients of determination (r<sup>2</sup>

)

**Figure 1.** Four-parameter logistic standard curve of duplicate analysis of aflatoxin B1 standards in modified acetonitrile extract solvent: phosphate buffered saline-20 mixture (100:650, *v*/*v*). Extraction of aflatoxin B1 from organic into aqueous phase using the modified extraction solvent mixture did not affect the assay performance since the percent inhibition values were within the range specified by the manufacturer. **Figure 1.** Four-parameter logistic standard curve of duplicate analysis of aflatoxin B1 standards in modified acetonitrile extract solvent: phosphate buffered saline-20 mixture (100:650, *v*/*v*). Extraction of aflatoxin B1 from organic into aqueous phase using the modified extraction solvent mixture did not affect the assay performance since the percent inhibition values were within the range specified by the manufacturer.

Numerous methods have been developed for analysis of aflatoxins in food and feed with high performance liquid chromatography (HPLC) as the gold standard. However, enzyme-linked immunosorbent assay (ELISA) has also been widely used given its many **Table 1.** Curve fitting characteristics for the AFB1-ELISA generated by AFB1 standards in modified acetonitrile extract solvent: phosphate buffered saline-20 mixture (100:650, *v*/*v*) showing both values of the parameters (a–d) and linearityassociated coefficients (r and r<sup>2</sup> ).

**Table 1.** Curve fitting characteristics for the AFB1-ELISA generated by AFB1 standards in modified acetonitrile extract solvent: phosphate buffered saline-20 mixture (100:650, *v*/*v*) showing both values of the parameters (a–d) and linearity-

**Parameters Values of 4-Parameter Logistic Curve**

98.9 1.58 0.072 ng/mL 5.6 0.998 0.997

Concentration at inflection point ((50% B/B0), IC<sup>50</sup> (c)


and specialized equipment, skilled personnel for quantification by ELISA together with improved upstream sample handling procedures described herein, combined with high throughput (42 samples per run), guarantee rapid and reliable estimation of aflatoxin in complex and amorphous matrices such as animal feed. In addition, incorporation of a water slurring step in the extraction procedure reduces the number of test replications, considerably reducing sample analysis turnaround time. Numerous methods have been developed for analysis of aflatoxins in food and feed with high performance liquid chromatography (HPLC) as the gold standard. However, enzyme-linked immunosorbent assay (ELISA) has also been widely used given its many advantages over HPLC and other chromatographic techniques. ELISA is cost-effective because it does not need clean-up columns and expensive instrumentation, user-friendly, high-throughput, accurate and reproducible. One disadvantage of ELISAs is susceptibility to matrix effects, causing the reported analyte level to be falsely elevated or depressed. We used an ELISA method designed to be resistant to matrix interferences and which was previously validated to test disparate sample types, such as pet food [44], sorghum [45], maize [46], nuts, spices and many others [47]. Lack of requirement for sample clean-up and specialized equipment, skilled personnel for quantification by ELISA together with improved upstream sample handling procedures described herein, combined with high throughput (42 samples per run), guarantee rapid and reliable estimation of aflatoxin in complex and amorphous matrices such as animal feed. In addition, incorporation of a water slurring step in the extraction procedure reduces the number of test replications, considerably reducing sample analysis turnaround time.
