Abstract
The chemical, technological and allergy properties of goat’s milk are significantly affected by the level of αs1-casein. Detection and quantification of αs1-casein requires high-specificity methods to overcome high-sequence similarity between this protein and others in the casein family. Unavailability of antibodies with high affinity and specificity towards goat αs1-casein hinders the development of immuno-based analytical methods such as enzyme-linked immunosorbent assay (ELISA) and biosensors. Here, we report the generation of polyclonal antibodies (or immunoglobulins, IgGs) raised towards goat αs1-casein N- (Nter) and C-terminal (Cter) peptide sequences. The Nter and Cter peptides of goat αs1-casein were immunized in rabbits for the generation of antisera, which were purified using protein G affinity chromatography. The binding affinity of the antisera and purified IgGs were tested and compared using indirect ELISA, where peptide-BSA conjugates and goat αs1-casein were used as the coating antigens. The Nter antiserum displayed higher titer than Cter antiserum, at 1/64,000 and 1/32,000 dilutions, respectively. The purification step further yielded 0.5 mg/mL of purified IgGs from 3 mL of antisera. The purified Nter IgG showed a significantly (p < 0.05) higher binding affinity towards peptide-BSA and goat αs1-casein, with lower Kd value at 5.063 × 10−3 μM compared to 9.046 × 10−3 μM for the Cter IgG. A cross-reactivity test showed that there was no binding in neither Nter nor Cter IgGs towards protein extracts from the milk of cow, buffalo, horse and camel. High-quality antibodies generated will allow further development of immuno-based analytical methods and future in vitro studies to be conducted on goat αs1-casein.
Highlights
Goat αs1 -casein is a phosphorylated protein and has been identified as a major allergen in goat’s milk
Immuno-based analytical methods such as enzyme-linked immunosorbent assay (ELISA) and biosensors are ideal platforms for this purpose, but they demand high quality antibodies as their biorecognition molecules
Antisera titer is defined as antisera dilution, resulting in an uninhibited assay signal three times that of the background signal; in this case, preimmune sera is investigated under the given assay conditions. It was determined through an end-point titration ELISA
Summary
Goat αs1 -casein is a phosphorylated protein and has been identified as a major allergen in goat’s milk. The αs1 -casein production levels in different goats may vary greatly due to the high polymorphism of the CSN1N1 gene that encodes the αs1 -casein. Various levels of αs1 -casein synthesis in goat’s milk have been described, ranging between 0 to 3.5 g/L for null alleles (N, O) and strong alleles (A, B, C, H, L, M), respectively [1]. Determining the level of goat αs1 -casein in milk samples is important for the safety and quality assessment of the goat’s milk or goat’s. Immuno-based analytical methods such as enzyme-linked immunosorbent assay (ELISA) and biosensors are ideal platforms for this purpose, but they demand high quality antibodies as their biorecognition molecules
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