Generation of High Affinity Anti-Peptide Polyclonal Antibodies Recognizing Goat α<sub>s1</sub>-Casein
The chemical, technological and allergy properties of goat’s milk are significantly affected by the level of α<sub>s1</sub>-casein. Detection and quantification of α<sub>s1</sub>-casein requires high-specificity methods to overcome high-sequence similarity between this protei...
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| Format: | Article |
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MDPI AG
2020-06-01
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| Series: | Molecules |
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| Online Access: | https://www.mdpi.com/1420-3049/25/11/2622 |
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| author | Aliah Zannierah Mohsin Rashidah Sukor Jinap Selamat Anis Shobirin Meor Hussin Intan Hakimah Ismail Nuzul Noorahya Jambari Farina Mustaffa-Kamal |
| author_facet | Aliah Zannierah Mohsin Rashidah Sukor Jinap Selamat Anis Shobirin Meor Hussin Intan Hakimah Ismail Nuzul Noorahya Jambari Farina Mustaffa-Kamal |
| author_sort | Aliah Zannierah Mohsin |
| collection | DOAJ |
| description | The chemical, technological and allergy properties of goat’s milk are significantly affected by the level of α<sub>s1</sub>-casein. Detection and quantification of α<sub>s1</sub>-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 α<sub>s1</sub>-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 α<sub>s1</sub>-casein N- (Nter) and C-terminal (Cter) peptide sequences. The Nter and Cter peptides of goat α<sub>s1</sub>-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 α<sub>s1</sub>-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 (<i>p</i> < 0.05) higher binding affinity towards peptide-BSA and goat α<sub>s1</sub>-casein, with lower K<sub>d</sub> value at 5.063 × 10<sup>−3</sup> μM compared to 9.046 × 10<sup>−3</sup> 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 α<sub>s1</sub>-casein. |
| first_indexed | 2024-03-10T19:22:10Z |
| format | Article |
| id | doaj.art-d0156fe02ca04b22bc2bc88c817bd06a |
| institution | Directory Open Access Journal |
| issn | 1420-3049 |
| language | English |
| last_indexed | 2024-03-10T19:22:10Z |
| publishDate | 2020-06-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Molecules |
| spelling | doaj.art-d0156fe02ca04b22bc2bc88c817bd06a2023-11-20T02:55:40ZengMDPI AGMolecules1420-30492020-06-012511262210.3390/molecules25112622Generation of High Affinity Anti-Peptide Polyclonal Antibodies Recognizing Goat α<sub>s1</sub>-CaseinAliah Zannierah Mohsin0Rashidah Sukor1Jinap Selamat2Anis Shobirin Meor Hussin3Intan Hakimah Ismail4Nuzul Noorahya Jambari5Farina Mustaffa-Kamal6Institute of Tropical Agriculture and Food Security, Universiti Putra Malaysia, Serdang 43400, MalaysiaInstitute of Tropical Agriculture and Food Security, Universiti Putra Malaysia, Serdang 43400, MalaysiaInstitute of Tropical Agriculture and Food Security, Universiti Putra Malaysia, Serdang 43400, MalaysiaFaculty of Food Science and Technology, Universiti Putra Malaysia, Serdang 43400, MalaysiaFaculty of Medicine, Universiti Putra Malaysia, Serdang 43400, MalaysiaInstitute of Tropical Agriculture and Food Security, Universiti Putra Malaysia, Serdang 43400, MalaysiaFaculty of Veterinary Medicine, Universiti Putra Malaysia, Serdang 43400, MalaysiaThe chemical, technological and allergy properties of goat’s milk are significantly affected by the level of α<sub>s1</sub>-casein. Detection and quantification of α<sub>s1</sub>-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 α<sub>s1</sub>-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 α<sub>s1</sub>-casein N- (Nter) and C-terminal (Cter) peptide sequences. The Nter and Cter peptides of goat α<sub>s1</sub>-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 α<sub>s1</sub>-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 (<i>p</i> < 0.05) higher binding affinity towards peptide-BSA and goat α<sub>s1</sub>-casein, with lower K<sub>d</sub> value at 5.063 × 10<sup>−3</sup> μM compared to 9.046 × 10<sup>−3</sup> 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 α<sub>s1</sub>-casein.https://www.mdpi.com/1420-3049/25/11/2622IgG purificationgoat’s milk allergyα<sub>s1</sub>-caseinanti-peptide polyclonal antibodyimmunogenic peptides |
| spellingShingle | Aliah Zannierah Mohsin Rashidah Sukor Jinap Selamat Anis Shobirin Meor Hussin Intan Hakimah Ismail Nuzul Noorahya Jambari Farina Mustaffa-Kamal Generation of High Affinity Anti-Peptide Polyclonal Antibodies Recognizing Goat α<sub>s1</sub>-Casein Molecules IgG purification goat’s milk allergy α<sub>s1</sub>-casein anti-peptide polyclonal antibody immunogenic peptides |
| title | Generation of High Affinity Anti-Peptide Polyclonal Antibodies Recognizing Goat α<sub>s1</sub>-Casein |
| title_full | Generation of High Affinity Anti-Peptide Polyclonal Antibodies Recognizing Goat α<sub>s1</sub>-Casein |
| title_fullStr | Generation of High Affinity Anti-Peptide Polyclonal Antibodies Recognizing Goat α<sub>s1</sub>-Casein |
| title_full_unstemmed | Generation of High Affinity Anti-Peptide Polyclonal Antibodies Recognizing Goat α<sub>s1</sub>-Casein |
| title_short | Generation of High Affinity Anti-Peptide Polyclonal Antibodies Recognizing Goat α<sub>s1</sub>-Casein |
| title_sort | generation of high affinity anti peptide polyclonal antibodies recognizing goat α sub s1 sub casein |
| topic | IgG purification goat’s milk allergy α<sub>s1</sub>-casein anti-peptide polyclonal antibody immunogenic peptides |
| url | https://www.mdpi.com/1420-3049/25/11/2622 |
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