筛选出一株可识别多种变异HBsAg的单克隆抗体及其应用

目的 制备筛选可识别变异表面抗原(hepatitis B surface antigen,HBsAg)的单克隆抗体(monoclonal antibody, mAb).用筛选出的单克隆抗体建立检测变异HBsAg的ELISA实验方法.方法 用血源HBsAg免疫Balb/c小鼠,通过杂交瘤细胞融合技术制备抗-HBs单克隆抗体.不同单克隆抗体包被酶标反应孔,检测真核细胞表达的野生及变异HBsAg,了解各种单克隆抗体的反应模式 .筛选出可以较好识别变异HBsAg的单克隆抗体Hb1,优化该抗体ELISA检测HBsAg的方法,与 8种HBs Ag检测试剂比较检测变异HBsAg的能力.结果 经过筛选,得到一种可以较好识别包括G145R在内大多数变异HBsAg的单克隆抗体.检测变异HBsAg的能力优于市售HBsAg 诊断试剂.结论 用本实验制备的单克隆抗体可以用于ELISA检测变异HBsAg,减少HBsAg变异株的漏检率.     

HBV G145R突变对S蛋白及“a”决定簇合成肽抗原性影响

人工合成G145R突变后HBsAg"a"决定簇多肽G145R-SP24,将G145R-SP24、重组G145R变异S蛋白及重组野生型HBsAg分组免疫BALB/c小鼠,制备相应抗血清。通过观察各免疫原与抗血清或抗G145R变异HBsAg McAb的免疫反应特征,分析并比较G145R突变后对S蛋白及"a"决定簇合成肽抗原性的影响。结果,几种免疫原均取得理想的免疫效果,ELISA证实各抗血清均有较好的特异性。G145R-SP24和G145R变异S蛋白免疫小鼠获得的抗血清效价较野生型HBsAg略低,且抗原性也发生了明显改变,G145R突变后"a"决定簇仍然具有较好的免疫原性;另外G145R-SP24与G145R变异S蛋白之间也显示出不同的抗原性,提示孤立的G145R变异HBsAg"a"决定簇与完整G145R S蛋白的"a"决定簇可能形成不同的空间构象。本研究为进一步探讨"a"决定簇突变后空间结构的变化规律与抗原性改变的特点奠定一定的基础。     

乙肝病毒表面抗原G145R变异单克隆抗体的制备及其初步鉴定

目的:研制抗乙肝病毒G145R变异表面抗原的单克隆抗体(mAb).方法:将重组乙肝病毒G145R变异表面抗原以Al(OH)3佐剂化, 常规免疫BALB/c小鼠, 采用细胞融合技术制备抗乙肝病毒G145R变异表面抗原的mAb.采用ELISA对制备物的效价与特异性进行鉴定, 将其用于部分临床标本的检测, 并与德国抗HBs mAb GL2.001进行比较.结果:获得1株稳定分泌抗G145R HBs mAb的杂交瘤细胞, 命名为4-E12.该细胞株染色体数目为90条, 所分泌mAb属IgG1亚类.其培养上清与腹水抗G145R HBs ELISA效价为(0.5～1)×103及(1～10)×106.经持续3月培养, 低血清适应以及反复冻存与复苏后, 其细胞增殖能力与上清抗G145R HBs效价与克隆化结束时大致相似.将其与德国抗G145R HBs mAb GL2.001相比, 二者对重组乙肝表面抗原G145R变异S蛋白的反应性大致相当, 灵敏度前者稍低于后者(分别为2.0 μg/L与1.0 μg/L).对野生株表面抗原的检测前者在实验浓度范围内未显示有意义的反应信号, 后者在与高浓度(1 mg/L及以上)抗原反应时S/N值达到或高于3.9.将二者用于对一组特定患者的临床检测, 其阳性率前者(17/200, 8.5%)较后者(11.5%)略低, 其差别经统计学比较无统计学意义(P>0.05).结论:成功地制备了1株抗乙肝表面抗原G145R变异体的mAb, 为乙肝G145R变异的基础、 临床与流行病学研究提供了进一步的物质基础.     

乙型肝炎病毒表面抗原基因点突变对HBsAg抗原性的影响

目的 研究乙型肝炎病毒表面抗原(HBsAg)常见基因点突变对HBsAg抗原性的影响,了解我国目前常用的HBsAg检测试剂对HBV S基因突变株的检测灵敏性,减少漏检,有效控制乙肝病毒(HBV)感染的传播.方法 构建HBsAg重组野毒株和重组变异株表达质粒,分别将重组野毒株HBsAg表达质粒pSS1adw2及pSS1adr和重组变异株HBsAg表达质粒pSS1adw2-145Arg、pSS1adr-126 Ser和pSS1adr-126 Asn转染COS-7细胞,进行瞬时转染.采用市售HBsAg ELISA检测试剂盒对细胞上清进行抗原性检测.结果 野毒株HBsAg和两种126位变异株HBsAg具有较好的抗原性;145位点突变后、导致HBsAg的抗原性下降.结论 推测是由于145位点变异影响了"a"抗原决定簇的空间结构,从而降低了其与抗-HBs的结合能力.     

G145R变异乙型肝炎表面抗原诱导的抗体性质研究

乙型肝炎表面抗原（HBsAg）变异可导致抗原性改变。HBsAga决定簇最常见的变异位点是G145R。G145R变异可能改变原来的抗原表位，并形成新的抗原表位。本研究探讨G145RHBsAg能否刺激机体产生抗体及免疫应答的情况。     

DNA免疫法研制抗猪繁殖与呼吸综合征病毒M蛋白中和性单克隆抗体

目的：研制抗PRRSVM蛋白的单克隆抗体（McAb），以期获得中和性单克隆抗体。方法：将PRRSVM蛋白的基因克隆入真核表达载体pcDNA3．1，利用构建成的重组真核质粒免疫BALB／c小鼠，采用杂交瘤技术制备抗PRRSVM蛋白的单抗，建立间接ELISA方法筛选阳性克隆。利用试剂盒检测Ig亚类。通过免疫印迹（Western blot）、间接免疫荧光试验（IFA）鉴定McAb的特异性。间接ELISA和病毒中和试验检测杂交瘤细胞上清McAb效价和腹水McAb效价。结果：获得3株可分泌特异性抗PRRSVM蛋白抗体的杂交瘤细胞株，分别命名为1A7、4G3、51：3。1A7和4G3的Ig亚类为IgM。ELISA检测杂交瘤细胞培养上清效价为1：54～1：1024，腹水效价为1：3200～1：10240。同时用Western blot、IFA检测，结果均是阳性。4G3和1AT相对亲和力不同，证明其识别不同抗原位点。1AT和4G3具有病毒中和活性，中和效价达到1：96。结论：获得2株特异性抗PRRSVM蛋白的中和性单抗，为PRRSV诊断和免疫预防研究奠定了重要基础。     

抗人甘露聚糖结合凝集素(MBL)单克隆抗体的制备、鉴定与检测方法的建立

目的 制备抗甘露聚糖结合凝集素(MBL)的单克隆抗体(McAb),建立免疫学检测方法.方法 以纯化MBL为抗原,免疫Ualb/c小鼠,制备单克隆抗体,鉴定其特性,建立并验证夹心ELISA定量检测法.结果 筛选出2株稳定分泌抗MBL的单抗杂交瘤细胞株,免疫球蛋白亚类均为IgG1.两单抗间抑制率＜50%,结合位点不同两单抗特异识别MBL.选择包被抗体B10浓度为8μg/mL,酶标抗体B3稀释度为1:500,建立夹心EIJSA定量检测法.该法检出范围为1～100 μg/mL,与其他抗原无交叉反应.重复性试验的批内批间变异均小于10%,回收率在101%～103%之间,cv小于7%.检测结果与国外试剂盒比较无显著差异.结论 制备的抗MBL单克隆抗体可用于MBL的免疫学检测.     

抗HPT单克隆抗体的制备与生物学特性鉴定

目的：制备抗潮霉素B磷酸转移酶(HPT)的单克隆抗体(McAb)，建立一种快速检测转基因作物中该选择标记基因HPT编码蛋白的方法。方法：用基因重组潮霉素B磷酸转移酶(HPT)抗原免疫BALB/C小鼠，采用杂交瘤技术制备McAb。选择不同的抗原决定簇与兔抗HPT多抗配对，建立双抗夹心ELISA检测HPT抗原。结果：筛选出四株稳定分泌抗HPT单抗的杂交瘤细胞株，IgG亚类鉴定均为IgG1，ELISA检测证实单抗可特异性识别细胞培养上清、重组子菌体裂解产物中及纯化出的HPT蛋白。结合位点测定实验表明4株单抗是针对不同的抗原决定簇，与多克隆抗体组成双抗夹心ELISA法，均可较好地检测到HPT抗原。检测的灵敏度为30ng／ml，且与别的无关抗原无交叉反应性。结论：4株杂交瘤细胞株特异性好，亲和力强，组成双抗夹心ELISA法可用于快速、灵敏检测HPT抗原。     

乙型肝炎病毒S基因129Leu和145Arg变异株的抗原表达与DNA免疫研究

目的 研究我国发现的乙肝病毒S基因突变株(129Leu和145Arg)表达HBsAg及DNA免疫的特性. 方法 用自乙肝疫苗免疫无保护婴儿血清中克隆的2株变异S基因(其中"a"决定簇突变,分别为nt540A→T,aa129Gln→Leu和nt587G→A,aa145Gly→Arg)片段,置换已知核苷酸序列并可表达及复制的HBV1.2个拷贝全基因野毒株重组质粒p3.8Ⅱ的S基因.将重组质粒转染HepG2细胞,用Abbott试剂和24种单抗检测转染细胞培养上清中HBsAg的结合力.用重组质粒DNA肌肉免疫小鼠. 结果 129Leu变异株转染细胞上清对HBsAg酶联免疫反应检测的吸光度(A)值高于p3.8Ⅱ野毒株,而145Arg变异株的A值低于p3.8Ⅱ;用24种单抗测定的总趋势为129Leu表达的HBsAg 的S/C值高于p3.8Ⅱ,而145Arg的结果则低于p3.8Ⅱ.三者表达的HBeAg水平相似.用DNA免疫经129Leu诱生的抗-HBs水平低于野毒株p3.8Ⅱ,但略高于145Arg. 结论 129Leu变异株表达的HBsAg与HBs多抗及单抗的结合力较野毒株明显增高,但129Leu 变异株DNA免疫诱生的抗-HBs水平却低于野毒株p3.8Ⅱ.免疫原性低下可能有利于129Leu致持续性感染.     

恶性疟原虫乳酸脱氢酶单克隆抗体的制备及其特性鉴定

为制备抗恶性疟原虫乳酸脱氢酶（LDHp)单克隆抗体（McAb),并对其特异性进行鉴定。用纯化的LDHp重组抗原免疫BALB/c小鼠,采用杂交瘤技术制备McAb,筛选出分泌高滴度McAb的杂交瘤细胞株,测定其免疫球蛋白亚类及其效价,ELISA,Westen blotting试验分析其特异性。结果,制备出2A5和1H10两株能稳定分泌抗LDHpMcAb的杂交瘤细胞株,两株单抗均为IgG2b,2A5和1H10培养上清的ELISA效价分别为1：512和1：256,腹水效价分别为1：25600和1：12800,两株单抗与间日疟,红细胞,弓形虫,日本血吸虫等抗原均不发生交叉反应,能识别恶性疟原虫的33Kda虫源蛋白。证明制备的抗LDHp杂交瘤细胞株能分泌高滴度和高特异性的单抗。     

Localization of hepatitis B surface antigen epitopes present on variants and specifically recognised by anti-hepatitis B surface antigen monoclonal antibodies

Small hepatitis B surface antigen (HBsAg) is considered to be the best marker for the diagnosis of Hepatitis B virus infection. However, HBsAg variants with mutations within the "a" determinant may be poorly or not detected by diagnostic assays. Three anti-HBsAg monoclonal antibodies (6H6B6, 27E7F10, and 2G2G10), directed against conformational epitopes, were tested for their ability to detect the wild-type HBsAg as well as variant forms and their respective epitopes were localised on the HBsAg sequence by using the phage-displayed peptide library technology. Whereas 6H6B6 did not detect mutations T123N, S143L, D144A and G145R, 27E7F10 binding was affected by mutations P120T and G145R. In contrast, 2G2G10 reacted strongly with all tested variants including variant with the G145R mutation. Part of the 6H6B6 epitope was located in the major hydrophilic region (MHR) at residues 101-105, the 27E7F10 epitope (residues 214-219) was located near the C-terminal end of the antigen and the 2G2G10 epitope at residues 199-208, within the theoretical fourth transmembrane helix. The 2G2G10 epitope localisation brings information about the HBsAg structure and the validity of established topological models. Finally, 2G2G10 is a valuable tool for HBsAg variant detection that is used as capture phase in a new bioMérieux diagnostic assay, which is currently in development.     

Mutation of the "a" determinant of HBsAg with discordant HBsAg diagnostic kits

Sera from 1003 in- and out-patients were investigated for hepatitis B surface antigen (HBsAg) mutation at Siriraj Hospital, Bangkok, Thailand. Individual samples were screened using two commercial HBsAg kits on automatic machine-based assays set up in parallel. The first kit was a sandwich ELISA kit that used monoclonal capture/monoclonal conjugate and color detection whereas the second was a sandwich MEIA, using monoclonal capture/polyclonal indicator and fluorochrome determination. Six specimens were found discordant by negative EIA and positive MEIA; two specimens of which were detectable of HBV DNA. Three out of four discordant results were confirmed by an anti-HBs neutralization assay. Based on direct sequencing, one HBsAg/anti-HBs sample with multiple mutations in the S gene was found. The mutation included the common glycine to arginine escape mutation at amino acid position 145 of the "a" determinant. The observation should alert the blood bank to the necessity of using screening kits capable of detecting HBV mutant carriers as well as providing verification for the phenomenon of vaccine-escape mutation in Thailand.     

European collaborative evaluation of the Enzygnost HBsAg 6.0 assay: performance on hepatitis B virus surface antigen variants

Amino acid changes within the major antigenic determinant of the hepatitis B virus (HBV) surface antigen (HBsAg) may modify eventually the antigenic properties of the protein and may have impact on the sensitivity of diagnostic assays. Modifications in the design of an assay can, however, improve significantly its ability to detect HBV mutants. One hundred forty‐seven clinical samples containing HBsAg variants, and 54 supernatants of cells expressing recombinant HBsAg mutants were tested by two generations of a commercial HBsAg test (Enzygnost® HBsAg 5.0 and 6.0, Siemens Healthcare Diagnostics Products, Marburg, Germany), and the results were compared. A significant improvement was demonstrated for the second test by comparing the mean and individual sample/cut‐off values, as well as by the detection of several samples displaying amino acid changes in residues 120 and 145 of the HBsAg which were recorded as negative by the former test. The results showed that modifications in design of the assay improved considerably the ability of the test to detect HBsAg mutants, and that difficulties in detecting such HBV variants should not be expected with the routine use of the test in diagnostic laboratories and in blood transfusion centers. J. Med. Virol. 83:95–100, 2011. © 2010 Wiley‐Liss, Inc.     

A novel hepatitis B virus variant S 129 (Gln-->Leu): lack of correlation between antigenicity and immunogenicity.

A point mutation has been detected in the "a" determinant of hepatitis B surface antigen (HBsAg) in an infant immunised with hepatitis B vaccine after exposure to hepatitis B virus (HBV). This A-to-T point mutation at nucleotide 540 resulted in a glutamine-to-leucine substitution at amino acid residue 129 (129L). The S gene fragment (nucleotide 58-1072) of this isolate was cloned and used to substitute the wild-type S gene in a plasmid (p3.8II), containing 1.2 copy of full-length HBV genome with expression and replication efficiency. This plasmid p3.8II-129L was used to transfect HepG2 cells. HBsAg expressed by p3.8II-129L showed higher binding efficiency compared with the original plasmid containing the wild-type clone. A panel of 24 anti-HBs monoclonal antibodies (MAbs) was used to characterise the binding efficiency of HBsAg expressed by p3.8II-129L. Eighteen showed higher binding to the antigen, whereas HBsAg expressed by p3.8II-145R gave a consistently lower absorbance or were negative. Surprisingly, when the immunogenicity of plasmid constructs was used for DNA immunisation in Balb/c mice, the anti-HBs response induced by p3.8II-129L was significantly lower than that of the wild-type p3.8II. The lack of correlation between the antigenicity profile (binding of expressed HBsAg to anti-HBs in vitro), and the immunogenicity (induction of anti-HBs by plasmid DNA in vivo) of HBsAg with leucine substitution at position 129 indicates that biological characteristics other than the binding efficiency of HBsAg to anti-HBs could occur in HBsAg variants. These different aspects of the biological characteristics of S mutants merit further investigation.     

乙型肝炎病毒表面抗原国家定量标准品的研制

目的 研制乙型肝炎病毒表面抗原(HBsAg)国家定量标准品及定量线性参考品.方法 收集各地乙型肝炎患者和健康献血员血样,用不同厂家乙型肝炎、丙型肝炎病毒血清学试剂盒筛选.6个协作单位用7种试剂以世界卫生组织(WHO)HBsAg定量标准品标定1份HBsAg国家定量标准品,稀释后得到HBsAg国家定量线性参考品.结果经7种试剂共21次协作标定,得到HBsAg国家定量标准品的浓度为1226 IU/ml,各次检测结果的变异系数均小于15%.将国家定量标准品系列稀释后得到由8个不同浓度血清组成的HBsAg国家线性参考品,经检测后表明其浓度与理论浓度的差值均<15%,通过加速试验检测该参考品的稳定性效期暂定为5年.将其作为HBsAg国家线性参考品并初步限定检测结果的允许值范围.结论 标定1份HBsAg国家定量标准品并建立了由8份血清组成的HBsAg国家定量线性参考品.     

常见乙型肝炎病毒表面抗原突变体的抗原性分析

目的：研究乙型肝炎病毒（HBV）表面抗原突变株和野毒株对表面抗体结合能力的差异,探讨免疫漏检基因变异株的机制及对策。方法：应用基因重组技术构建HBsAg常见基因突变株和野毒株表达质粒,分别在COS－7细胞中瞬时表达,定量后用常规HBsAg免疫诊断试剂盒检测,观察重组抗原与不同抗－HBs的结合能力。结果：T126 S变异与单克隆抗体的结合力增高,G145R,K141 E和2株三位点同时变异株则明显下降,对M133T变异与单克隆抗体的结合力影响不大。变异株与多克隆抗体的结合力也有变化,但仍能检测到大部分变异株抗原。结论:“a”抗原决定簇氨基酸的置换影响了HBsAg与抗－HBs的结合能力,并且氨基酸的置换位置和特性对抗原性的影响各有不同。因此,建议应用HBsAg多克隆抗体或开发研制“免疫逃逸突变株”的特异性抗体,以完善HBsAg免疫诊断试剂的抗体组成,减少免疫诊断对常见基因变异株的漏检。     

Comparing the immunogenicity of hepatitis B virus S gene variants by DNA immunization.

DNA immunization was used to compare the immunogenicity of hepatitis B virus S gene variants. Four recombinant plasmid DNAs containing the full-length virus genome with different S gene inserts were used to immunize BALB/c and C57/BL/6 mice. These inserts were cloned from 129L (residue 129, glutamine to leucine), 129H (residue 129, glutamine to histidine) 145R (residue 145, glycine to arginine) variants and the wild-type virus. The titer of hepatitis B virus core antibodies (anti-HBc) in immunized mice was used as the control for the efficiency of DNA immunization. Serum hepatitis B surface antibody (anti-HBs) titer and cytokines induced in splenocytes stimulated with hepatitis B surface antigen (HBsAg) were monitored as specific immune responses induced by different plasmid DNAs. 129L DNA induced significantly lower anti-HBs antibodies (IgG, IgG1, and IgG2a) and less interferon-gamma, compared to those in mice immunized with the 129H variant and the wild-type HBV DNA (p < 0.05). Computer modeling showed that a change from glutamine to leucine at 129 residue led to higher hydrophobicity and could result in decreased immunogenicity. Results indicate that DNA immunization can be used to compare the humoral and cellular immunogenicity among different HBV S variants.     

Anti-hepatitis B surface antigen IgG1 subclass is predominant in individuals who have recovered from hepatitis B virus infection, chronic carriers and vaccinees

Patterns of each IgG-specific subclass for hepatitis B virus (HBV) core antigen (anti-HBc) are remarkably different among individuals with different infection status, i.e., completely recovered or chronic carrier. Each of the IgG-specific subclasses of HBV surface antigen (anti-HBs) was tested for ELISA sensitivity using four commercially available hepatitis B surface antigen (HBsAg) kits and one self-prepared plate. The specificity in 18 serum samples obtained from chronic HBV carriers, recovered individuals, vaccinees and non-infected individuals was investigated. Differences in absorbance values were obtained by comparing results from these different plates. Data on the absorbance values of anti-HBs IgG subclasses obtained indicated that one to four subjects had a false-negative or false-positive result using the four commercial plates. Only the self-prepared plate demonstrated 100% specificity and sensitivity for anti-HBs subclasses. Moreover, the results indicate that anti-HBs subclass IgG1 was predominant in cured patients, chronic carriers and vaccinees. The samples from both chronic carriers and vaccinees exhibited a significantly higher concentration of total IgG and IgG1 than samples in recovered individuals (P<0.05).     

单纯乙型肝炎疫苗免疫后携带者表面抗原氨基酸置换的频率和特征

目的 掌握中国单纯乙型肝炎疫苗免疫后携带者和未免疫携带者表面抗原基因变异的流行状况及特点。方法 以直接测序和特异基因序列固相聚合酶链反应（SS-SPPCR）方法，分别检测97例单纯乙型肝炎疫苗免疫后携带者、88例未免疫育龄妇女和95例未免疫儿童携带者，乙型肝炎病毒α抗原决定簇氨基酸置换的流行率。结果 直接测序检测的乙型肝炎疫苗免疫后携带者氨基酸置换流行率显著高于未免疫育龄妇女和儿童携带者对照，流行率分别为30.9%(30/97),10.2%(9/88)和5.3%(5/95)。145、126和133是最常见的氨基酸酸置换位点，应用更为敏感的SS-SPPCR法分别检测145和126位氨基酸点突变的流行率，各组间差异不明显，其中145位氨基酸置换的流行率分别为39.2%,33.0%和32.6%，经直接测序法检测，免疫后携带者基因变异流行率高于未免疫携带者5.41倍，按基因型和血清亚型分层分析，基因型B和adw2血清亚型携带者受免疫选择后出现基因变异的风险显著较高，分别为34.55和33.39；而基因型C和adr血清亚型携带者基因变异风险较低，分别为2.73和2.45。结论 单纯乙型肝炎疫苗具有免疫选择表面抗原基因变异株的作用；基因变异株在未免疫携带者中主要是弱势准种；基因变异风险与病毒基因型和血清亚型有关。