Determination Of Four Kinds Of Major Allergens In Foods By Enzyme-linked Immunosorbent Assay

Food allergy has now become an emerging public health problem. In the developed countries, more than 8% of children and 2% of adults are affected by food allergy. Since the avoidance is currently the most effective means of prevention and treatment for allergy sufferers, a high flux, rapid and sensitive allergen detection method appear particularly important. There are eight categories of common food allergens, and this paper aims to establish ELISA method for the detection of shrimp, peanuts, eggs and milk allergens in foods.Ovalbumin (OVA) is one of the major egg allergens. The ovalbumin was applied to immunize BALB/c mice. The mouse with the highest titer was selected, and then splenocytes were isolated before fused with SP2/0 murine myeloma cells. Seventeen hybridoma cell lines specific to OVA were obtained and named as OVA-NO.1-17. Monoclonal antibodies, named as anti-OVA-NO.1-17, were then prepared through the ascites of mice injected intraperitoneally with the screened hybridoma cells. The monoclonal antibodies specific to OVA were labeled with HRP by the sodium periodate method. After paired screening, the best method was chosen with anti-OVA-NO.17 and HRP-anti-OVA-NO.15 as the capture antibody and detection antibody, respectively. After optimization, the ELISA method was established with the detection limit of 0.51 ng/mL and the linear range of 1.95-500 ng/mL. Recovery results and real sample analysis show the stability and reliability of the established ELISA method, which can be applied to the real sample detection.Casein, as the highest protein content of milk, is one of the major allergens in milk. Following similar method above, fourteen hybridoma cell lines specific to casein were obtained and named as casein-NO.1-14. Monoclonal antibodies were then prepared from the ascites and labeled with HRP. By paired screening, anti-casein-NO.8 was chosen as the capture antibody and HRP-anti-casein-NO.11 was as the detection antibody, the limit of detection the ELISA method was 0.55 ng/mL, and the linear range was from 0.78 to 100 ng/mL.Peanut agglutinin (PNA) is one of the allergens in peanut. Using glycosylated PNA as the immunogen, anti-PNA polyclonal antibodies from rabbit and mouse were successfully prepared. A novel sandwich-ELISA was developed for the determination PNA, by using mouse anti-PNA polyclonal antibody and rabbit anti-PNA polyclonal antibody as the capture antibody and detection antibody, respectively. The limit of detection was low (0.49 ng/mL), and the linear range was from 0.78 to 100 ng/mL. And recovery results and real sample analysis verified that this method has good precision and repeatability, which can be as a reliable tool for the detection of PNA in foods.Tropomyosin is the most major allergen in shrimp. After degreasing, extraction, salting out and gel filtration chromatography, tropomyosin with a purity of more than 90% was prepared from shrimp. With the purified tropomyosin as the immunogen, anti-tropomyosin polyclonal antibodies from rabbit and mouse were successfully prepared. An sandwich-ELISA was established, using mouse anti-tropomyosin polyclonal antibody and rabbit anti-tropomyosin polyclonal antibody as the capture antibody and detection antibody, respectively, with the detection limit of 6.25 ng/mL and the linear range of 6.25-1600 ng/mL.