| Soybean antigen protein can cause allergic reaction, which not only threatens human health, but also causes huge economic losses of livestock and poultry. In the present study, allergenicity of soybean antigen protein on intestines of piglets was studied through causing allergy by supplementing high purity soybean protein antigen to piglets’diets. Meanwhile, an indirect enzyme-linked immunosorbent assay (ELISA) was established for detecting the serum antibody levels of piglets sensitized by soybean antigen protein. This may provide a clinical diagnosis method for allergenicity caused by soybean protein antigen.Allergenicity of soybean antigen protein on intestines of piglets.30 healthy piglets, weaned at day 21, were randomly divided into three groups (10 weaned piglets in each group):control group, glycinin (11S) allergen group and β-conglycinin (7S) allergen group. The first and second sensitization experiments were carried out at the age of 21 to 27 days and 32 to 34 days in piglets, respectively. The ratioes of 11S (purity:80%) basic diet and 7S (purity:85%) basic diet was 4% in both 11S allergen group and 7S allergen group. Blood samples were collected from the anterior vena cave on days 21,28,35 with ethylene diamine tetraacetic acid (EDTA) anticoagulant tube for measuring levels of plasma D-lactic acid (D-LA), diamine oxidase (DAO) and 5-hydroxytryptamine (5-HT). The skin sensitivity test was carried out at 35 d of age and five piglets were randomly selected in each group at 36 d of age. Duodenum, proximal jejunum, middle jejunum, distal jejunum and distal ileum was chosen for the 6 cm of length. The distribution and amount of secretory IgA positive expression were determined using immunohistochemistry method.Establishment of an indirect ELISA method for measuring serum antibody of soybean antigen protein in sensitized piglets. The groups and treatments were same to above experiment. Blood samples were collected from experimental piglets between 7:00～8:00 at the age of 21,28 and 35 days. After being placed for 2 h at room temperature, serum was collected by centrifuging at 3000 rpm for 10 min and The skin sensitivity test of piglets was conducted at day 35. The optimal coating quality concentrations of antigen and dilution times of sera were determined by board titration, in order to develop an indirect ELISA method for measuring 11S and 7S. Moreover, the standard of the serum test, and the repeatability, accuracy and sensitivity of the ELISA method were determined.The results were as follows:1. After skin sensitivity test, we found that skin reactions where injected by 11S and 7S were more apparent, the evaluation results were above "+". The reaction of piglets in 7S allergen group was particularly evident, the evaluation results were above "++". These results suggested that soybean antigen protein had allergic reactions at the age of 35 day of piglets.2. Compared with control group, the plasma D-LA, DAO and 5-HT levels in the sensitization group were significantly higher (P<0.01) on days 28,35. The present results showed that the levels of plasma D-LA and the activity of DAO had significantly correlations with intestinal injury. Also we found that the concentration of plasma 5-HT showed the tread which was low first, high again, and low last. The physiological function of intestines had certain relevance with the changes of plasma 5-HT contents.3. The distribution and positive expression intensity of secretory IgA in duodenum, proximal jejunum, middle jejunum, distal jejunum and distal ileum of piglets were determined by immunohistochemistry method. The results showed optical density (OD) and positive expression intensity of secretory IgA in middle jejunum were significantly higher than that in other intestinal segments. Positive expression intensity in 7S allergens group was more apparent, followed by 11S allergens group. The OD of positive expression of secretory IgA in five intestinal segments were middle jejunum> duodenum> proximal jejunum, distal ileum> distal jejunum from strong to weak.4. The indirect ELISA method for measuring the serum antibodies of the piglets’ soybean protein antigen was established. The optimal conditions for measuring the serum antibodies of 11S were as follows:the quality concentration of coating was 2.0 μg·mL-1, the action time of the coating was 2 h at 37 ℃; confining liquid was 10 g·L-1 BSA blocked for 1 h at 37 ℃; the test serum were diluted into 1:1600, reacted for 1 h at 37 ℃; the enzyme labeled antibody was diluted into 1:1500, reacted for 1 h at 37 ℃; the optimal reaction time of substrate was 15 min. The optimal conditions for measuring the serum antibodies of 7S were as follows:the quality concentration of coating was 2.5 μg·mL-1, the action time of the coating was 2 h at 37 ℃; confining liquid was 10 g·L-1 BSA, blocked for 1 h at 37 ℃; the test serum were diluted into 1:1600, reacted for 1 h at 37 ℃; the enzyme labelled antibody was diluted into 1:1500, reacted for 1 h at 37 ℃; the optimal reaction time of substrate was 25 min. Meanwhile, the coefficient of variation in both intra-assay and inter-assay were less than 10% with well reproducibility and high sensitivity.5. The sensitivity and accuracy of established indirect ELISA were detected in clinical. The indirect ELISA sensitivity of 11S antibody was 87.5%(7/8), the accuracy was 81.3% (13/16).The indirect ELISA sensitivity of 7S antibody was 87.5%(7/8), the accuracy was 87.5%(14/16).These results indicated that soybean antigen protein had allergic reaction on piglets, which changed the intestinal physiological function of piglets. When allergy reaction occurs, a large amount of secretory IgA were released from intestinal mucosa. The indirect ELISA method with good repeatability and high sensitivity was established for measuring the serum antibodies of the piglets’soybean protein antigen. And this method could be used to determine allergy induced by soybean protein antigen in clinical diagnosis. |
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