Llames L, Goyache J, Domenech A, de Avila A, Suarez G, Gomez-Lucia E
Departamento de Patología Animal I, Facultad de Veterinaria, Universidad Complutense de Madrid, Spain.
J Virol Methods. 1999 Oct;82(2):129-36. doi: 10.1016/s0166-0934(99)00092-0.
ELISA and Western blot have been used for detecting specific antibodies or antigens for routine diagnostic laboratory tests and experimental protocols, as well as for screening hybridomas secreting antibodies. Although these techniques are sensitive, some slow growing hybridomas are identified as positive only when they are grown slowly long time. We standardized the dot-ELISA, a more sensitive technique, for the detection of antibodies against BLV. The main advantages of the dot-ELISA described in this study are (a) its sensitivity, detecting hybridomas which would otherwise be considered negative and discarded from the results of indirect ELISA and/or Western blot; and (b) the possibility of economizing reagents using as little as 1 microl of the antigen and 0.5 microl of antibody and conjugate. Different BLV-antigen preparations were bound to nitrocellulose membranes (NC), including cells lysed chemically (LYS) or by sonication (SOC), semi-purified virus (PV), and supernatant from infected cultures, either without treatment (SUP) or sonicated (SOS). The antigen preparations most adequate for detecting monoclonal antibodies against BLV and polyclonal antibodies in cattle sera were undiluted cell lysates (LYS) and semi-purified BLV (PV). When testing bovine sera, the supernatant (SUP) and sonicated supernatant (SOS) antigens gave a high background due to the presence of FCS which reacted with the anti-bovine labeled antibodies. In this study, 59 BLV specific antibody secreting hybridomas were identified using the dot-ELISA, compared to only 20 detected using iELISA, and doubtful reactions due to nonspecific binding to fetal calf serum (FCS) and cellular components were measured. The results of the improved dot-ELISA described may be stored at room temperature for future reference. Results were consistently reproducible in coated nitrocellulose membranes kept at different storage temperatures (-20 degrees C, 4 degrees C, and 25-30 degrees C) 48 h, 1 week and 5 months.
酶联免疫吸附测定（ELISA）和蛋白质印迹法已用于检测特定抗体或抗原，用于常规诊断实验室检测和实验方案，以及筛选分泌抗体的杂交瘤。尽管这些技术很灵敏，但一些生长缓慢的杂交瘤只有在长时间缓慢生长时才被鉴定为阳性。我们对斑点ELISA进行了标准化，这是一种更灵敏的技术，用于检测抗牛白血病病毒（BLV）的抗体。本研究中描述的斑点ELISA的主要优点是：（a）其灵敏度，能够检测出那些在间接ELISA和/或蛋白质印迹法结果中会被视为阴性并被舍弃的杂交瘤；（b）有可能节省试剂，仅使用1微升抗原、0.5微升抗体和缀合物。不同的BLV抗原制剂被固定在硝酸纤维素膜（NC）上，包括化学裂解（LYS）或超声处理（SOC）的细胞、半纯化病毒（PV），以及未经处理（SUP）或超声处理（SOS）的感染培养物上清液。最适合检测抗BLV单克隆抗体和牛血清中多克隆抗体的抗原制剂是未稀释的细胞裂解物（LYS）和半纯化的BLV（PV）。检测牛血清时，由于胎牛血清（FCS）与抗牛标记抗体发生反应，上清液（SUP）和超声处理的上清液（SOS）抗原产生了高背景。在本研究中，使用斑点ELISA鉴定出59个分泌BLV特异性抗体的杂交瘤，相比之下，使用间接ELISA仅检测到20个，并检测到由于与胎牛血清（FCS）和细胞成分非特异性结合导致的可疑反应。所描述的改进斑点ELISA的结果可在室温下保存以供将来参考。在不同储存温度（-20℃、4℃和25 - 30℃）下保存48小时、1周和5个月的包被硝酸纤维素膜中，结果始终具有可重复性。
