Preparation of double-antibody and establishment of sandwich ELISA against milk β-casein and soybean β-conglycinin
Protein adulteration and allergens are the two major food safety issues, and can pose health threats to consumers. One of the effective precautions is extensive test, which needs simple, rapid and low -cost test method. Present methods including sodium dodecyl sulfate polyacrylamide gel electrophore...
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| Format: | Article |
| Language: | English |
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Zhejiang University Press
2013-03-01
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| Series: | 浙江大学学报. 农业与生命科学版 |
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| Online Access: | https://www.academax.com/doi/10.3785/j.issn.1008-9209.2012.11.617 |
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| author | XIAO Hailong ZHAO Kai LIN Saijun WANG Hongqing PAN Jianhong |
| author_facet | XIAO Hailong ZHAO Kai LIN Saijun WANG Hongqing PAN Jianhong |
| author_sort | XIAO Hailong |
| collection | DOAJ |
| description | Protein adulteration and allergens are the two major food safety issues, and can pose health threats to consumers. One of the effective precautions is extensive test, which needs simple, rapid and low -cost test method. Present methods including sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS)/MS are not acceptable for consumers due to expensive instruments. Immunological technique is a rapid method for screening and is suitable for consumers to use.β-casein and β-conglycinin are not only the major proteins of milk and soybean, but also important food borne allergens. In this study, the monoclonal antibodies and polyclonal antibodies against β-casein and β-conglycinin were prepared and the enzyme-linked immunosorbent assay (ELISA) kits were established for detecting adulteration and allergens. BALB/c mice were immunized four times with purified antigens adding adjuvant or not until the serum titer achieved 1∶1×10<sup>5</sup>, and the mice spleen cells and myeloma cells SP2/0 were fused as the routine cell-fusion technology. Positive cells were screened for 3-4 times with indirect ELISA by coating purified antigens, and were injected into mice peritoneal. The monoclonal antibodies were obtained after the purification of ascites. The polyclonal antibodies against β-casein and β-conglycinin were also prepared from rabbit serum immunized by antigens, respectively. The double antibody sandwich ELISA for β-casein and β-conglycinin were successfully established by optimized parameters.The results showed that the titers of purified monoclonal antibodies of β-casein and β-conglycinin were over 1∶1×10<sup>7</sup>, and the polyclonal antibody titers of both were about 1∶2×10<sup>5</sup>. The minimum detection limits of both ELISA kits were about 15 ng/mL, and no cross reaction were observed among the proteins of different species.In conclusion, the double antibody sandwich ELISA methods established for β-casein and β-conglycinin are sensitive and specific, and can afford a theoretical foundation for developing rapid, accurate and low -cost screening methods for the detection of adulteration and allergens. |
| format | Article |
| id | doaj-art-87ae9a49cd8c471db06c380b68c64527 |
| institution | Kabale University |
| issn | 1008-9209 2097-5155 |
| language | English |
| publishDate | 2013-03-01 |
| publisher | Zhejiang University Press |
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| series | 浙江大学学报. 农业与生命科学版 |
| spelling | doaj-art-87ae9a49cd8c471db06c380b68c645272025-08-20T03:34:08ZengZhejiang University Press浙江大学学报. 农业与生命科学版1008-92092097-51552013-03-013922222610.3785/j.issn.1008-9209.2012.11.61710089209Preparation of double-antibody and establishment of sandwich ELISA against milk β-casein and soybean β-conglycininXIAO HailongZHAO KaiLIN SaijunWANG HongqingPAN JianhongProtein adulteration and allergens are the two major food safety issues, and can pose health threats to consumers. One of the effective precautions is extensive test, which needs simple, rapid and low -cost test method. Present methods including sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS)/MS are not acceptable for consumers due to expensive instruments. Immunological technique is a rapid method for screening and is suitable for consumers to use.β-casein and β-conglycinin are not only the major proteins of milk and soybean, but also important food borne allergens. In this study, the monoclonal antibodies and polyclonal antibodies against β-casein and β-conglycinin were prepared and the enzyme-linked immunosorbent assay (ELISA) kits were established for detecting adulteration and allergens. BALB/c mice were immunized four times with purified antigens adding adjuvant or not until the serum titer achieved 1∶1×10<sup>5</sup>, and the mice spleen cells and myeloma cells SP2/0 were fused as the routine cell-fusion technology. Positive cells were screened for 3-4 times with indirect ELISA by coating purified antigens, and were injected into mice peritoneal. The monoclonal antibodies were obtained after the purification of ascites. The polyclonal antibodies against β-casein and β-conglycinin were also prepared from rabbit serum immunized by antigens, respectively. The double antibody sandwich ELISA for β-casein and β-conglycinin were successfully established by optimized parameters.The results showed that the titers of purified monoclonal antibodies of β-casein and β-conglycinin were over 1∶1×10<sup>7</sup>, and the polyclonal antibody titers of both were about 1∶2×10<sup>5</sup>. The minimum detection limits of both ELISA kits were about 15 ng/mL, and no cross reaction were observed among the proteins of different species.In conclusion, the double antibody sandwich ELISA methods established for β-casein and β-conglycinin are sensitive and specific, and can afford a theoretical foundation for developing rapid, accurate and low -cost screening methods for the detection of adulteration and allergens.https://www.academax.com/doi/10.3785/j.issn.1008-9209.2012.11.617β-caseinβ-conglycininmonoclonal antibodyenzyme-linked immunosorbent assay |
| spellingShingle | XIAO Hailong ZHAO Kai LIN Saijun WANG Hongqing PAN Jianhong Preparation of double-antibody and establishment of sandwich ELISA against milk β-casein and soybean β-conglycinin 浙江大学学报. 农业与生命科学版 β-casein β-conglycinin monoclonal antibody enzyme-linked immunosorbent assay |
| title | Preparation of double-antibody and establishment of sandwich ELISA against milk β-casein and soybean β-conglycinin |
| title_full | Preparation of double-antibody and establishment of sandwich ELISA against milk β-casein and soybean β-conglycinin |
| title_fullStr | Preparation of double-antibody and establishment of sandwich ELISA against milk β-casein and soybean β-conglycinin |
| title_full_unstemmed | Preparation of double-antibody and establishment of sandwich ELISA against milk β-casein and soybean β-conglycinin |
| title_short | Preparation of double-antibody and establishment of sandwich ELISA against milk β-casein and soybean β-conglycinin |
| title_sort | preparation of double antibody and establishment of sandwich elisa against milk β casein and soybean β conglycinin |
| topic | β-casein β-conglycinin monoclonal antibody enzyme-linked immunosorbent assay |
| url | https://www.academax.com/doi/10.3785/j.issn.1008-9209.2012.11.617 |
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