Establishment Of Evaluating Methods For Type I Anaphylaxis In Guinea Pig For Tuberculosis Vaccine Evaluation

Adverse effects associated with immunotoxicity caused by vaccine inoculation are the main issue in the safety evaluation of vaccine, and the type I anaphylaxis is the most common and dangerous among those adverse effects. Methods of evaluating the type Ianaphylaxis at present are mainly based on rat or mouse model, which are subjective and difficult to quantization and standardization and lack of reasonable evaluating models. In this study, we optimized the model of type I anaphylaxis based on guine pig which is the most sensitive animal to type I anaphylaxis, and establish a guinea pig model to evaluate type I anaphylaxis caused by vaccine and a series of methods to detect indicators quantitively and objectively. Our research involved several aspects as followed:1. Determination of anaphylactic antibodies in sera:Guinea pig model of type I anaphylaxis was established through standardizing the way of immunization, IgE and IgG in sera were preliminarily isolated through saturated ammonium sulphate precipitate and affinity chromatograph of rProtein A, and reference, standard was established preliminarily. A continuous passive cutaneous anaphylaxis test (PCA) was performed and results showed that both IgE and IgG play roles in typeâ… anaphylaxis of guinea pig, and the hypersensitive reaction induced by IgG is fast and short than that induced by IgE. The ELISA to determine the level of IgGl in serum of vaccine-immunized guinea pigs was established and Results showed that the level of specific IgGl in vaccine group was significantly higher than that of control group(P< 0.05), but the total IgGl increased obveriously only in OVA group(P<0.05).2. Determination of anaphylactic mediators:We collected the BALF and determined the level of histamine and leukotriene C4(LTC4), collected peritoneal mast cells and basophile granulocytes in peripheral blood and stimulated in vitro to determined the level of histamine and LTC4 in supernatant. Results showed that concentrations of histamine and LTC4 in positive model group were significantly higher than that of control group(P<0.05).3. Determination by cytologic and histologic methods:Ba and Eo of OVA group were significantly higher than that of control group through Wright-Giemsa staining, Ba of OVA group possessed a higher proportion in PB than control group through neutral red stanining. Concentration of IgE in tissue homogenate determined by ELISA was the highest in liver, lung and kidney. Results of immunohistochemistry showed that IgE was secreted mainly in pulmonary vascular wall, bronchial epithelial cells, and red substance of spleen cells, no IgE specific secretion was found in heart, liver and kidney.In conclusion, we established guinea pig model of type I anaphylaxis and indicators and evaluated hepatitis B vaccine and smegmatis vaccine, results showed that the type I anaphylaxis could be evaluated using that model and those indicators. This study revealed the type and functional characteristic of anaphylactic antibodies in type I anaphylaxis, established the guinea pig model, methods and quantitive indicators to evaluate anaphylaxis caused by vaccine firstly in China, established reference standard for quality evaluation, established the foundation for evaluation of vaccine immunotoxicity especially type I anaphylaxis.