Generation of high affinity anti-peptide polyclonal antibodies recognizing goat αs1-casein
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. Unavailabil...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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Multidisciplinary Digital Publishing Institute
2020
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| Online Access: | http://psasir.upm.edu.my/id/eprint/89101/ http://psasir.upm.edu.my/id/eprint/89101/1/IGC.pdf |
| _version_ | 1848860773021908992 |
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| author | Mohsin, Aliah Zannierah Sukor, Rashidah Selamat, Jinap Meor Hussin, Anis Shobirin Ismail, Intan Hakimah Jambari, Nuzul Noorahya Mustaffa Kamal, Farina |
| author_facet | Mohsin, Aliah Zannierah Sukor, Rashidah Selamat, Jinap Meor Hussin, Anis Shobirin Ismail, Intan Hakimah Jambari, Nuzul Noorahya Mustaffa Kamal, Farina |
| author_sort | Mohsin, Aliah Zannierah |
| building | UPM Institutional Repository |
| collection | Online Access |
| description | 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. |
| first_indexed | 2025-11-15T12:50:33Z |
| format | Article |
| id | upm-89101 |
| institution | Universiti Putra Malaysia |
| institution_category | Local University |
| language | English |
| last_indexed | 2025-11-15T12:50:33Z |
| publishDate | 2020 |
| publisher | Multidisciplinary Digital Publishing Institute |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | upm-891012021-09-21T01:08:54Z http://psasir.upm.edu.my/id/eprint/89101/ Generation of high affinity anti-peptide polyclonal antibodies recognizing goat αs1-casein Mohsin, Aliah Zannierah Sukor, Rashidah Selamat, Jinap Meor Hussin, Anis Shobirin Ismail, Intan Hakimah Jambari, Nuzul Noorahya Mustaffa Kamal, Farina 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. Multidisciplinary Digital Publishing Institute 2020 Article PeerReviewed text en http://psasir.upm.edu.my/id/eprint/89101/1/IGC.pdf Mohsin, Aliah Zannierah and Sukor, Rashidah and Selamat, Jinap and Meor Hussin, Anis Shobirin and Ismail, Intan Hakimah and Jambari, Nuzul Noorahya and Mustaffa Kamal, Farina (2020) Generation of high affinity anti-peptide polyclonal antibodies recognizing goat αs1-casein. Molecules, 25 (11). art. no. 2622. pp. 1-13. ISSN 1420-3049 https://www.mdpi.com/1420-3049/25/11/2622 10.3390/molecules25112622 |
| spellingShingle | Mohsin, Aliah Zannierah Sukor, Rashidah Selamat, Jinap Meor Hussin, Anis Shobirin Ismail, Intan Hakimah Jambari, Nuzul Noorahya Mustaffa Kamal, Farina Generation of high affinity anti-peptide polyclonal antibodies recognizing goat αs1-casein |
| title | Generation of high affinity anti-peptide polyclonal antibodies recognizing goat αs1-casein |
| title_full | Generation of high affinity anti-peptide polyclonal antibodies recognizing goat αs1-casein |
| title_fullStr | Generation of high affinity anti-peptide polyclonal antibodies recognizing goat αs1-casein |
| title_full_unstemmed | Generation of high affinity anti-peptide polyclonal antibodies recognizing goat αs1-casein |
| title_short | Generation of high affinity anti-peptide polyclonal antibodies recognizing goat αs1-casein |
| title_sort | generation of high affinity anti-peptide polyclonal antibodies recognizing goat αs1-casein |
| url | http://psasir.upm.edu.my/id/eprint/89101/ http://psasir.upm.edu.my/id/eprint/89101/ http://psasir.upm.edu.my/id/eprint/89101/ http://psasir.upm.edu.my/id/eprint/89101/1/IGC.pdf |