Splenocytes of Mice with Induced Immunological Tolerance to Plant Antigens for Construction of Hybridomas Secreting Tomato Spotted Wilt Virus-Specific Antibodies

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Abstract
Immune tolerance or suppression of immune response to plant antigens was successfully induced by injecting neonatal BALB/c mice four times with healthy plants extracts on days 1, 3, 5, and 7 after birth. Subsequently, two mice each were injected once with partially purified tomato spotted wilt virus (TSWV) at age 5, 7, or 9 wk, respectively. Splenectomy and fusion of spleen cells with P3/NS1/1-Ag-4-1 myeloma cells were performed 4 days after TSWV immunization for each group. Fusion products were evaluated by ELISA on plates coated with TSWV and on plates on which TSWV was trapped by adsorbed virus-specific rabbit polyclonal antiserum. Percentages of TSWV-specific hybridomas were 83, 50, and 40% for groups of mice immunized at 5, 7, and 9 wk of age, respectively. For control mice that did not receive neonatal injections of normal host antigens, the TSWV-specific hybridomas were 0, 7, and 7%, when immunized at 5, 7, and 9 wk, respectively. Conventional immunization of adult mice over a period of time with multiple injections of immunogens resulted in about 3-12% of hybridomas being TSWV-specific. The selection of virus-specific hybridomas may best be achieved when mice, which have been rendered immunologically tolerant to normal host antigens, are immunized at the age of 5 wk. This method is useful in the generation of hybridomas for antigens that are difficult to purify or concentrate and in the preparation of hybridomas to minor distinct epitopes that are difficult to separate.