*2.3. Sequence Analysis and Phylogenic Construction*

The PCR product was purified and sequenced and a 750 bp fragment was obtained. The sequence was aligned using the NCBI analysis tool and showed 97% similarity with other *AfIR* genes listed in GeneBank. The sequence was compared with 50 sequences and a phylogenetic tree was constructed. An Egyptian *AfIR* gene was closely similar to *AfIR* gene MH752587 obtained from *Aspergillus* sp. PS-2018c isolate BN038G *AFlR*, Arizona, USA (Figure 2).

#### *2.4. Antigenicity Test*

A 256 residue amino acid sequence was deduced from the gene sequence (Table 2). Eight peptides showed antigenic activity by Kolaskar & Tongaonkar Antigenicity [16] Figure 3. Peptide lengths ranged from 8 to 14 amino acids. Their positions started from amino acid numbers 26, 66, 107, 136, 170, 186, 205, and 236 (Table 2). Epitope prediction using Kolaskar and Tongaonkar Antigenicity Prediction identifies the protein epitopes that are useful for diagnostic purposes and also in the development of peptide vaccines [17].

**Figure 2.** Phylogenetic tree for the amplified aflatoxin B1 based on the DNA nucleotide sequence and compared with the other 50 *AFB1*genes listed on gene bank. The phylogeny was constructed using Mega 6 program.


**Table 2.** Predicted peptides with antigenic activity, their length, and positions.

**Figure 3.** The predicted antigenic activity of the recombinant protein (afIR).

#### *2.5. IgG Polyclonal Antibody Labeling and Purification*

Serum obtained from immunized rabbits was fractionated using affinity chromatography protein G-Sepharose column and one band of conventional IgG with a molecular weight of 130 kDa was obtained. Moreover, two bands of a 42 kDa heavy chain and a 19 kDa light chain were separated under reducing conditions. Glutaraldehyde was used to prepare conjugates using a ratio of 4:1 of IgG and enzyme alkaline phosphatase (AP). IgG-AP conjugates were purified by gel filtration on a Sephacryl S200 column. AP (EC 3.1.3.1) is a stable enzyme its activity can be measured by many different substrates. The most common method of labeling immunoglobulin G (IgG) antibody with this enzyme uses the homobifunctional reagent glutaraldehyde [18].
