*2.6. Validation of the Modified Recombinant AflR Gene Antiserum ELISA Technique with HPLC and VICAM*

In definition, validation is establishing the performance specifications of a new diagnostic tool such as a new test, laboratory developed test or modified method. But verification is defined as one-time process to determine performance characteristics of a test before use in patient testing [19].

ELISA was unable to distinguish among antigens due to the presence of common epitopes on protein surfaces [20–22]. Sampling/sub-sampling variation significantly affects the accuracy of aflatoxin analysis [23]. Extracts of 36 samples were used for validation to minimize the impacts of such variation.

Recombinant antiserum detected *AfIR* recombinant protein within a concentration range 0–1000 pg/mL with a linear correlation between *AfIR* antigenic protein and absorbance at 405 nm (y = 0.0014x − 0.0148; R<sup>2</sup> = 0.9946) (Figure 4). Non-significant differences among three samples of the same product, peanut, flour, or milk powder, were observed after HPLC (*p* > 0.05) (Table 3). The VICAM method showed similar results. However, the modified ELISA showed significant differences among toxin detections in these product samples. The serum-based analysis confirmed specific PCR results. No false negatives were observed in I-ELISA results and false positives were either nil or negligible.

**Figure 4.** I-ELISA standard curve for *AflR* recombinant protein using purified serum IgG.


**Table 3.** Comparative results obtained by HPLC, VICAM, specific PCR, and I-ELISA (ng/g).

The mean values indicated in the same column within variable with different superscripts (a, b, and c) were significantly different (*p* < 0.05); +: present of fungal infection.

Although the correlation between the data in Figure 5A,B (comparing HPLC against VICAM and ELISA) reflected that the correlation of HPLC against VICAM (Figure 5A) was better than ELISA. On the other hand, a good correlation was observed between ELISA and VICAM (Figure 5C). However, the represented modified ELISA technique is easier to use, economic as it does not need sophisticated chemicals or highly trained technicians, have a good sensitivity to detect low infection levels determining aflatoxin B1 in foods and can represent a successful alternative in case other approaches are hard to be reached in less developed communities. Previous observations were reported for validation of a competitive direct SUNQuik ELISA for aflatoxin in peanuts using a reference HPLC method and other methods, including a minicolumn and the VICAM Afla test system [24]. The comparison between HPLC, VICAM, and validated method I-ELISA with respect to limit of detection, precision and accuracy, time of analysis, cost of analysis, and use of organic solvents is summarized in Table 4.

**Figure 5.** Correlations between HPLC and VICAM (**A**), HPLC and ELISA (**B**), ELISA and VICAM (**C**).


**Table 4.** Comparison between HPLC, VICAM, and validated method I-ELISA.

#### *2.7. Limitations of the Modified Recombinant AflR Gene Antiserum ELISA Technique*

Our new established method has many limitations that must be clarified to determine and specify the application field of this method. First, our new ELISA technique does not measure the aflatoxins itself, hence, this type of test cannot be used for official control. However, it could be useful for auto control and rapid results and decision-making within a farm/company. Second, although this method is quantitative test, all positive results need to be confirmed with a confirmatory method such as HPLC or LC-MS as it is based on the measurement of a recombinant protein controlled by the gene responsible for aflatoxin biosynthesis, but not on the toxin itself.

#### **3. Conclusions and Future Perspective**

Aflatoxin B1 detection is an increasingly important health and economic issue. Accurate detection is essential to assess health problems in both humans and animals. Conventional detection methods are time-consuming and require expensive chemicals and apparatus (HPLC and VICAM). An accurate and rapid detection method that requires fewer chemicals is needed. We developed a specific quantitative detection technique (I-ELISA) using recombinant *AflR* protein. *AflR* is involved in aflatoxin biosynthesis. Comparison of results achieved from the new modified ELISA with other standardized methods HPLC and VICAM, revealed that the new ELISA technique can be used at many applications as an economic alternative to detect low levels of aflatoxin contamination. This modified technique may address problems associated with the reliable and rapid detection of aflatoxin B1 contamination in food products. The technique could be used to develop highly sensitive (0–1000 pg/mL) testing capabilities. In future, hybridoma cell culture antibody production technique can be used for production of antibodies against AfIR protein for large-scale manufacturing of rapid I-ELISA kit. This method will yield a production scale ranging from milligram to gram level with competitive pricing.

#### **4. Materials and Methods**
