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目的:为了了解所制备的单纯疱疹病毒荧光标记单克隆抗体的临床应用价值。方法:本研究利用杂交瘤技术制备了多株抗单纯疱疹病毒型共有及型特异性单克隆抗体(６Ｂ３－２－５、Ｄａ５,５Ｂ４－６),并通过ＢＡＬＢ/Ｃ鼠体内诱生制备了较大量的单抗,经去脂澄清处理,利用辛酸－硫酸铵二步法纯化了单抗,经ＳＤＳ－ＰＡＧＥ及薄层扫描分析显示其纯度;在ｐＨ９．０－９．５条件下,采用低温袋内透析法标记了单抗５Ｂ４－６及６Ｂ３－２－５,然后经透析及ＳｅｐｈｅｄｅｘＧ－５０柱去除游离荧光素;标记单抗采用直接免疫荧光法(ＤＦＡ)检测各类临床标本１１０份及正常标本２６份,并与间接免疫荧光法(ＩＦＡ)及组织培养分离法作了初步比较。结果:１．纯化后的各单抗在解链状态下为二条链,一条为轻链,分子量各为 ２０．６ ＫＤ(５Ｂ４－６)、２４．６ ＫＤ(６Ｄ３－２－５)及 ２６．１ＫＤ(Ｄａ５),一条为重链,分子量各为５７．８ ＫＤ(５Ｂ４－６)、５４．８ ＫＤ(６Ｂ３－２－５)及５２．７ＫＤ(Ｄａ５),由此推算出其单抗分子量各为１５７ ＫＤ、１５９ ＫＤ、１５８ ＫＤ。纯化回收率各为４３．７７%、４７．２１%及 ３８．８%。２．标记单抗 Ｆ/Ｐ值各为ｌ．４ 及１．４６,利用(ＤＦＡ)法测定其效价各为 １:３２?
Objective: In order to understand the clinical application value of the prepared monoclonal antibody of herpes simplex virus fluorescently labeled. Methods: In this study, a number of common and type-specific monoclonal antibodies against herpes simplex virus (6B3-2-5, Da5 and 5B4-6) were prepared by hybridoma technique and were prepared in vivo by BALB / c mice A larger amount of mAb was purified by defatting and purified with octanoic acid-ammonium sulfate two-step method. The purity of McAb was determined by SDS-PAGE and TLC scanning. Under the conditions of pH 9.0-9.5, Low temperature bag dialysis method labeled monoclonal antibodies 5B4-6 and 6B3-2-5, and then by dialysis and SephedexG-50 column to remove free fluorescein; labeled monoclonal antibody using direct immunofluorescence (DFA) detection of various clinical specimens 110 And 26 normal samples were compared with indirect immunofluorescence (IFA) and tissue culture separation were made a preliminary comparison. Results: 1. After purification, each of the mAbs had two chains, one of which was light chain, the molecular weights of which were 20.6 KD (5B4-6), 24.6 KD (6D3-2-5) and 26.1 KD (Da5 ), A heavy chain, the molecular weight of 57.8 KD (5B4-6), 54.8 KD (6B3-2-5) and 52.7KD (Da5), which deduced that the molecular weight of its monoclonal antibody 157 KD, 159 KD, 158 KD. Purification recoveries were 43.77%, 47.21% and 38.8%, respectively. 2. Marker mAb F / P value of each l. 4 and 1.46, using the (DFA) method to determine the potency of each 1:32?