IFN-γ-ELISpot方法用于评价猪伪狂犬病疫苗免疫效果

doi:10.11843/j.issn.0366-6964.2017.10.017

Abstract

Abstract:

Due to the deficiency of antibody detection methods in evaluation of immunity against swine pseudorabies virus, to fully evaluate the cell-mediated immunity of PRV vaccine in combination with humoral immunity, we try to develop an Enzyme Linked Immuno-spot Assay (ELISpot) method for the detection of PRV specific IFN-γ level. In this study, according to the basic protocol of ELISpot, we tried to optimize the best conditions of stimulant concentration, peripheral blood mononuclear cells (PBMC) density and incubation time. After that, an animal experiment was performed, twenty PRV gB antibody-free piglets were divided into four groups which were administered with attenuated live vaccine, inactivated vaccine, live vaccine plus inactivated vaccine and normal saline (as control group). The immune response was evaluated by our in-house IFN-γ ELISpot,gB-ELISA and neutralization test, together with the results of challenge test. After optimization, the PRV IFN-γ ELISpot protocol was determined. The optimal stimulant concentration, cells density/well and incubation time were 2 μg·well^-1, 10⁶ cell·well^-1, and 36 h. Results obtained from the animal experiment revealed that live plus inactivated vaccine group showed the high level of both humoral and cellular immunity, the serum neutralization (SN) antibody titer was 1∶14.48 and the ELISpot result was 151.8±26.61, while those of SN antibody and ELISpot in live vaccine group and inactivated vaccine group were 1∶32.36, 40.4±9.07 and 1∶1.36, 189.6±27.36, respectively. The challenge test showed that the protective effect of live vaccine plus inactivatel vaccine was best as body temperature, viral shedding and clinical signs are the slighter in this group than those in other groups. The inhouse IFN-γ ELISpot results had a good relation with challenge test, providing an effective method for the evaluation of both cell immune response and optimal immune procedures for porcine pseudorabies vaccination.

CLC Number:

S858.285.3

TAN Xin, KU Xu-gang, YU Xue-xiang, GUO Heng-ke, HE Dong-xian, FAN Sheng-xian, HE Qi-gai. The IFN-γ-ELISpot for Evaluating the Immune Effect of Swine Pseudorabies Vaccine[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(10): 1939-1948.

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The IFN-γ-ELISpot for Evaluating the Immune Effect of Swine Pseudorabies Vaccine

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