Preparation Of The Antibodies Of Peanuts And Shrimp Allergen And Simultaneous Detemination By Piezoelectric Immunosensor

Peanuts and shrimp are listed in the list of eight common allergic foods. Compared to the other allergic foods, peanuts and shrimp have the higher incidence of allergy and the more severe clinical symptom, and have been concerned much in the field of public health and food safety. The existing methods of allergen testing, which are cumbersome to operate, are unable to meet the rapid, convenient, on-line testing requirements. Therefore, it is important to study and establish a convenient, safe, rapid detection method.The study include purificating peanuts and shrimp allergens, preparing peanut and shrimp polyclonal antibody, exploring the possibility of colloidal gold used as a new type of immune adjuvant, testing immune affinity constant of peanut antibody and shrimp antibody by using the self-organization quartz crystal microbalance immunosensor, coating the two antibody, separately screening the allergens.The allergen proteins of the peanut and freshwater shrimp were purified mainly by ammonium sulfate, isoelectric point precipitate and then AKTA protein purification system. Colloidal gold was prepared through reducing HAuCl4·3H2O by sodium citrate. The immune adjuvants were divided into two groups to prepare PcAb, respectively. Colloidal gold could significantly improve the level of the antibody to peanut and Freshwater shrimp allergens. The titre of peanuts with collolidal gold was doubled compared to that of Freund's adjuvant and the IC50 was 34.28 ng/mL. For the case of freshwater shrimp with collolidal gold, the titre was raised a quarter and the IC50 was 43.95 ng/mL.Self-assembly piezoimmunosensor determinated peanut antibodies affinity constant is 2.86×10~7 L /mol and the affinity constant of mixed antibodies to peanut is 1.37×10~7L/mol. Shrimp antibodies affinity constant is 5.16×10~7 L /mol and the affinity constant of mixed antibodies to shrimp is 2.57×10~7L/mol. Piezoimmunosensor coated both peanut and shrimp antibodies added a series concentration of peanut allergens and shrimp allergens respectively. Their respective standard curve and sensitivity could be determined. It had a high recovery rate and a good reproducibility.The self--assembly piezoimmunosensor could determine peanut and shrimp allergens simultaneously. It has fast, convenient and good practice advantage.