抗Spodoptera f.病毒单克隆抗体的初步研制

DEVELOPMENT OF MONOCLONAL ANTIBODIES TO SPODOPTERA FRUGIPERDA VIRUS

本文介绍了 Spodoptera frugiperda 病毒多角体及病毒粒子的提纯方法。粗提材料先通过40%蔗糖,在10000r/min 下离心15分钟,随后悬浮于0.01M Tris 缓冲液,25～65%蔗糖的连续梯度中,于40000 r/min 下离心30分钟。取出多角体的条带,洗除游离残存的蔗糖,透析过夜。将多角体在4℃,0.01MNa_2CO_3,0.01MEDTA,0.17M NaCl,pH=1下,作用1小时,随后调节 pH 到8.5～8.9,2000g,离心10分钟。该制剂在4℃下,25～60%蔗糖梯度中,24000r/min,离心2小时。随后取出病毒粒子,重新悬浮于蒸馏水中,于—70℃下贮藏备用。每个 BALB/C 小白鼠免疫4～5次,最后一次注射是在融合前4～7天。将2×10~8脾细胞与2×10~7P_3×63或 SP-2骨髓瘤细胞混合,并用50%PEG(Sigma)融合,用间接酶联免疫法进行筛选阳性杂交瘤细胞,强阳性者进行克隆化。以多角体及病毒粒子为抗原者,均建立了10株能产生特异性单克隆抗体的杂交瘤细胞株,该抗体的效价较高,并进行了血清学的分类。

Polyhedra of Spodoptera frugiperda is purified by banding on 40% sucrose gradients,10,000 r/min for 15 min,then suspension is layered on 25-65%conti- nuous sucrose gradients in 0.01M Tris buffer and centrifuged at 40,000 r/min for 30min.The polyhedra band is removed and then by washing free of residual suc- rose,dialysised overnight. Virus particles ars removed from polyhedra by incubating the polyhedra for 1 hr.at 4℃ in 0.01M Na_2CO_3,0.01M EDTA,0.17M NaCl,pH=11(Summers and Smith,1975),then by adjusting pH to 8.5-8.9,centifuged 2,000g 10 min.The preparations are layered on 25-60% sucrose gradients and centrifuged for 2 hr.at 24,000 r/min at 4℃.The virus particles are resuspended in water and stored at-70℃. Each BALB/c mouse is immunized four or five times.The last booster is given 4-7 days prior to cell fusion.Pooled spleen cells(2X10^8)mixed with P_3X 63 or SP-2 mycloma cells(2X10^7)and fused with 50% PEG(Sigma).The indirect enzyme linked immunosorbent assay(ELISA)method is used to screen for positive hybridomas. The positive hybridoma are cloned. Ten cell lines,which produce monoclonal antibodies,specific to the antigen (Polyhedra or Virus),are established.The antibodies titles and serological classi- fication are determined.