Gorrell M D, Milliken G L, Anderson B J, Pucci A
Dev Biol Stand. 1984;56:491-4.
The success of various attempts to discriminate serologically between vaccinated cattle and those infected with field strains of Brucella abortus has been limited. A reliable assay that has this discriminating ability would greatly benefit the Australian Brucellosis Eradication Scheme. Studies of similar diagnostic problems have shown that species and strain-specific monoclonal antibodies with diagnostic potential can be produced. Therefore, the production of monoclonal antibodies specific for Br. abortus field strains was attempted. Spleen cells from Balb/C mice immunized with formalin-killed Br. abortus strain 544 were fused with P3/NSI-1 Ag4-1 cells using polyethylene glycol 4000. The resulting hybrid cells were screened by ELISA using soluble fractions of sonicated, heat-killed (60 degrees C, 1 h) bacteria as antigen. More than 200 cultures of hybrid cells contained detectable amounts of antibody to both field and vaccine strains of Brucella. However, one monoclonal antibody, Br 25, was found to bind field strains of Br. abortus biotypes 1 and 2, but not strain 19, Br. suis, Br. melitensis, or Br. ovis. The titre of Br 25 hybridoma cell culture supernatant is over 2 000, Br 25 cells injected into mice produce ascites fluid having titres of more than 100 000. A sandwich ELISA incorporating successive incubations of Br 25, soluble Brucella antigen, bovine sera and labelled anti-bovine immunoglobulin was developed. Sera from cattle that had been either vaccinated with strain 19 and infected were tested in the ELISA. In this ELISA most infected cattle were positive, whether vaccinated or not, whereas uninfected and vaccinated cattle were negative.
通过血清学方法区分接种疫苗的牛和感染牛流产布鲁氏菌野毒株的牛的各种尝试，其成功率一直有限。一种具有这种区分能力的可靠检测方法将极大地有利于澳大利亚布鲁氏菌病根除计划。对类似诊断问题的研究表明，可以生产具有诊断潜力的种属和菌株特异性单克隆抗体。因此，尝试生产针对牛流产布鲁氏菌野毒株的单克隆抗体。用福尔马林灭活的牛流产布鲁氏菌544株免疫的Balb/C小鼠的脾细胞，使用聚乙二醇4000与P3/NSI-1 Ag4-1细胞融合。用超声破碎、热灭活（60℃，1小时）细菌的可溶性部分作为抗原，通过ELISA筛选所得的杂交细胞。200多个杂交细胞培养物含有可检测到的针对布鲁氏菌野毒株和疫苗株的抗体。然而，发现一种单克隆抗体Br 25能结合牛流产布鲁氏菌生物型1和2的野毒株，但不结合19株、猪布鲁氏菌、羊布鲁氏菌或绵羊布鲁氏菌。Br 25杂交瘤细胞培养上清液的滴度超过2000，注入小鼠体内的Br 25细胞产生的腹水液滴度超过100000。开发了一种夹心ELISA，依次孵育Br 25、可溶性布鲁氏菌抗原、牛血清和标记的抗牛免疫球蛋白。用19株疫苗接种并感染的牛的血清在ELISA中进行检测。在这种ELISA中，大多数感染的牛呈阳性，无论是否接种过疫苗，而未感染且接种过疫苗的牛呈阴性。
