Quantitative hepatitis B core antibody and quantitative hepatitis B surface antigen:Novel viral biomarkers for chronic hepatitis B management

The management of hepatitis B virus(HBV)infection now involves regular and appropriate monitoring of viral activity,disease progression,and treatment response.Traditional HBV infection biomarkers are limited in their ability to predict clinical outcomes or therapeutic effectiveness.Quantitation of HBV core antibodies(qAnti-HBc)is a novel non-invasive biomarker that may help with a variety of diagnostic issues.It was shown to correlate strongly with infection stages,hepatic inflammation and fibrosis,chronic infection exacerbations,and the presence of occult infection.Furthermore,qAnti-HBc levels were shown to be predictive of spontaneous or treatment-induced HBeAg and HBsAg seroclearance,relapse after medication termination,re-infection following liver transplantation,and viral reactivation in the presence of immunosuppression.qAnti-HBc,on the other hand,cannot be relied on as a single diagnostic test to address all problems,and its diagnostic and prognostic potential may be greatly increased when paired with qHBsAg.Commercial qAnti-HBc diagnostic kits are currently not widely available.Because many methodologies are only semi-quantitative,comparing data from various studies and defining universal cut-off values remains difficult.This review focuses on the clinical utility of qAnti-HBc and qHBsAg in chronic hepatitis B management.

Screening and evaluation of human single-chain fragment variable antibody against hepatitis B virus surface antigen

BACKGROUND: Phage display technology has become a vital tool in studies aimed at identifying molecules binding to a specific target. It enables the rapid generation and selection of high affinity, fully human antibody product candidates to essentially any disease target appropriate for antibody therapy. In this study, we prepared the recombinant single-chain fragment variable ( ScFv) antibody to hepatitis B virus surface antigen (HBsAg) by the phage display technology for obtaining a virus-targeting mediator. METHODS: mRNA was isolated from B-lymphocytes from a healthy volunteer and converted into cDNA. The fragment variables of heavy and light chain were amplified separately and assembled into ScFv DNA with a specially constructed DNA linker by polymerase chain reaction. The ScFv DNA was ligated into the phagmid vector pCANT-AB5E and the ligated sample was transformed into competent E. coli TG1. The transformed cells were infected with M13K07 helper phage to form a human recombinant phage antibody library. The volume and recombinant rate of the library were evaluated by bacterial colony count and restriction analysis. After two rounds of panning with HBsAg. the phage clones displaying ScFv of the antibody were selected by enzyme-linked immunosorbant assay ( ELISA) from the enriched phage clones. The antigen binding affinity of the positive clone was detected by competition ELISA. HB2151 E. coli was transfected with the positive phage clone demonstrated by competition ELISA for production of a soluble form of the anti-HBsAg ScFv. ELISA assay was used to detect the antigen binding affinity of the soluble anti-HBsAg ScFv. Finally, the relative molecular mass of soluble anti-HBsAg ScFv was measured by SDS-PAGE. RESULTS: The variable heavy ( VH ) and variable light (VL) and ScFv DNAs were about 340bp, 320bp and 750bp, respectively. The volume of the library was up to 2 × 106 and 8 of 10 random clones were recombinants. Two phage clones could strongly compete with the original HBsAb for binding to HBsAg. Within 2 strong positive phage clones, the soluble anti-HBsAg ScFv from one clone was found to have the binding activity with HBsAg. SDS-PAGE showed that the relative molecular weight of soluble anti-HBsAg ScFv was 32 kDa. CONCLUSION: The anti-HBsAg ScFv successfully produced by phage antibody technology may be useful for broadening the scope of application of the antibody.

Transformation of hepatitis B serologic markers in babies born to hepatitis B surface antigen positive mothers

AIM:To better understand the clinical significance of hepatitis B seroiogic markers in babies born to hepatitis B surface antigen (HBsAg) positive mothers, the incidence of maternal seroiogic markers of hepatitis B via placenta and its transformation in these babies were investigated. METHODS: Mothers with positive HBsAg were selected in the third trimester of pregnancy. Their babies received immunoprophylaxis with hepatitis B immunoglobulin and hepatitis B vaccine after birth, and were consecutively followed up for hepatitis B seroiogic markers and HBV DNA at birth, mo 1, 4, 7, 12, and 24. RESULTS: Forty-two babies entered the study, including 16 born to hepatitis B e antigen (HBeAg)-positive HBsAg carrier mothers and 26 to HBeAg-negative HBsAg carrier mothers. Apart from four babies born to HBeAg-positive carrier mothers and demonstrated persistent positive HBeAg eventually became HBV carriers, all other babies developed anti-HBs before 12 mo of age. Among the other 12 babies born to HBeAg-positive carrier mothers, HBeAg was detected in 7 at birth, in 4 at mo 1, and in none of them thereafter. No antibody response to the transplacental HBeAg was detected. Among the babies born to HBeAg-negative carrier mothers, anti-HBe was detected 100% at birth and mo 1, in 88.5% at mo 4, in 46.2% at mo 7, in 4.2% at mo 12 and none in mo 24. Among all the immunoprophylaxis-protected babies born to either HBeAg-positive or HBeAg-negative carrier mothers, anti-HBc was detected in 100% at birth, mo 1 and mo 4, in 78.9% at mo 7, in 36.1% at mo 12 and in none at mo 24. CONCLUSION: HBeAg can pass through human placenta from mother to fetus and become undetectable before 4 mo of age, but no antibodies response to the transplacental HBeAg can be detected till mo 24 in the immunoprophylaxis-protected babies. The sole existence of anti-HBe before 1 year of age or anti-HBc before 2 years of age in babies born to HBsAg carrier mothers may simply represent the transplacental maternal antibodies, instead of indicators of HBV infection status.

Mutations in surface and polymerase gene of chronic hepatitis B patients with coexisting HBsAg and anti-HBs

AIM： To investigate the clinical significance and presence of mutations in the surface （S） and overlapping polymerase gene of hepatitis B patients with coexisting HBsAg and anti-HBs. METHODS： Twenty-three patients with chronic hepatitis B were studied. Of the 23 patients, i i were both positive for hepatitis B virus （HBV） surface antigen （HBsAg） and antibody to HBV surface antigen （anti-HBs）, 12 were negative for anti-HBs while positive for HBsAg. DNA was extracted from 200 μL serum of the patients. Nucleotide of the surface and overlapping polymerase gene from HBV-infected patients was amplified by PCR, and the PCR products were sequenced. RESULTS： Forty-one mutations were found within the surface gene protein of HBV in 15 patients （10 with coexisting HBsAg and anti-HBs）. Six （14.6%） out of 41 mutations were located at ＂α＂ determinant region in 5 patients （4 positive for HBsAg and anti-HBs）. Eleven mutations （26.8%） occurred in the downstream or upstream of ＂α＂ determinant region. Lamivudine （LMV）- selected mutations were found in three patients who developed anti-HBs, which occurred in amino acid positions （196, 198, 199） of the surface protein and in YMDD motif （M204I/V） of the polymerase protein simultaneously. Presence of these mutations did not relate to changes in ALT and HBV DNA levels.CONCLUSION： Besides mutations in the ＂α＂ determinant region, mutations at downstream or upstream of the ＂α＂ determinant region may contribute to the development of anti-HBs. These mutations do not block the replicating competency of HBV in the presence of high titer of anti-HBs.

Differential reactivity of mouse monoclonal anti-HBs antibodies with recombinant mutant HBs antigens

AIM: To investigate the reactivity of a panel of 8 mouse anti-hepatitis B surface antigen (HBsAg) monoclonal antibodies (mAbs) using a collection of 9 recombinant HBsAg mutants with a variety of amino acid substitutions mostly located within the “a” region.METHODS: The entire HBs genes previously cloned into a mammalian expression vector were transiently transfected into COS7 cells. Two standard unmutated sequences of the ayw and adw subtypes served as controls. Secreted mutant proteins were collected and measured by three commercial diagnostic immunoassays to assess transfection efficiency. Reactivity of anti-HBs mAbs with mutated HBsAgs was determined by sandwich enzyme-linked immunosorbent assay (ELISA).RESULTS: Reactivity of anti-HBs mAbs with mutated HBsAgs revealed different patterns. While three mutants reacted strongly with all mAbs, two mutants reacted weakly with only two mAbs and the remaining proteins displayed variable degrees of reactivity towards different mAbs. Accordingly, four groups of mAbs with different but overlapping reactivity patterns could be envisaged. One group consisting of two mAbs (37C5-S7 and 35C6-S11) was found to recognize stable linear epitopes conserved in all mutants. Mutations outside the “a” determinant at positions 120 (P→S), 123(T→N) and 161(M→T) were found to affect reactivity of these mAbs.CONCLUSION: Our findings could have important implications for biophysical studies, vaccination strategies and immunotherapy of hepatitis B virus (HBV) mutants.

Detection of anti-preS1 antibodies for recovery of hepatitis B patients by immunoassay

AIM: To establish a convenient immunoassay method based on recombinant antigen preS1(21-119aa) to detect anti-preS1 antibodies and evaluate the clinical significance of antibodies in hepatitis B. METHODS: The expression plasmid pET-28a-preS1 was constructed, and a large quantity of preS1(21-119aa) fragment of the large HBsAg protein was obtained. The preS1 fragment purified by Ni(2+)-IDA affinity chromatography was used as coated antigen to establish the indirect ELISA based on streptavidin-biotin system for detection of the anti-preS1 antibodies in sera from HBV-infected patients. For follow-up study, serial sera were collected during the clinical course of 21 HBV-infected patients and anti-preS1 antibodies, preS1 antigen, HBV-DNA and other serological HBV markers were analyzed. RESULTS: preS1(21-119aa) fragment was highly expressed from the plasmid pET-28a-preS1 in a soluble form in E.Coli (30mg.L(-1)), and easily purified to high purity over 90% by one step of Ni(2+)-IDA-sepharose 6B affinity chromatography. The purity and antigenicity of the purified preS1(21-119aa) protein was determined by 150g.L(-1) SDS-PAGE, Western blot and a direct ELISA. Recombinant preS1(21-119aa) protein was successfully applied in the immunoassay which could sensitively detect the anti-preS1 antibodies in serum specimens of acute or chronic hepatitis B patients. Results showed that more than half of 19 acute hepatitis B patients produced anti-preS1 antibodies during recovery of the disease, however, the response was only found in a few of chronic patients. In the clinical follow-up study of 11 patients with anti-preS1 positive serological profile, HBsAg and HBV-DNA clearance occurred in 6 of 10 acute hepatitis B patients in 5-6 months, and seroconversion of HBeAg and disappearance of HBV-DNA occurred in 1 chronic patients treated with lavumidine, a antiviral agent. CONCLUSION: The high-purity preS1(21-119aa) coated antigen was successfully prepared by gene expression and affinity chromatography. Using this antigen, a conveniently detective system of anti-preS1 antibodies in sera was established. Preliminarily clinical trial the occurrence of anti-preS1 antibodies in acute hepatitis B patients suggests the clearance of HBV from serum in a short-term time, and anti-preS1 positive in chronic patients means health improvement or recovery from the disease.

筛选血清HBV-DNA阴性且HBsAg与HBsAb阳性CHB患者的血清危险因素及评分与预后的相关性研究

目的筛选乙肝病毒(hepatitis B virus,HBV)DNA阴性的乙肝表面抗原(hepatitis B surface antigen,HBsAg)和乙肝表面抗体(hepatitis B surface antibody,HBsAb)阳性的慢性乙型肝炎(chronic hepatitis B,CHB)患者的血清危险因素,探讨危险因素评分与预后的相关性。方法筛选2020年1月~2022年6月江苏大学附属金坛第一人民医院收治的HBV-DNA阴性的CHB患者193例,其中HBsAg和HBsAb阳性的119例患者作为观察组,HBsAg阳性且HBsAb阴性的74例患者作为对照组。收集所有患者的临床资料并计算外周血炎性指标[嗜中性粒细胞/淋巴细胞比值(neutrophil to lymphocyte ratio,NLR),血小板-淋巴细胞比值(platelet-to-lymphoccyte ratio,PLR),全身免疫炎症指数(systemic immune inflammatory index,SII)]和肝纤维化指标[谷氨酰转肽酶-血小板比值(gamma-glutamyl transpeptidase to platelet ratio,GPR),门冬氨酸转移酶纤维化指数(aspartate aminotransferase to platelet ratio index,APRI),肝纤维化4因子指数(fibrosis 4 score,FIB-4)],采用t检验和U检验比较两组间各指标的差异。采用ROC曲线评价差异指标的预测价值,多因素回归分析影响HBsAg和HBsAb阳性模式的危险因素。随访观察组患者二年内HBsAg和HBsAb变化,将20例HBsAg水平明显降低的患者作为预后良好组,24例HBsAg水平没有明显降低甚至HBsAb消失的患者作为预后不良组。结果与对照组相比,观察组的PLT水平[(181.07±63.31)×10^(9)/L vs(158.27±61.55)×10^(9)/L]和TP水平[70.20(65.73,74.90)g/L vs 67.00(64.45,71.25)g/L]明显升高,差异有统计学意义(t=2.459,U=3254.00,均P<0.05);而观察组的HBsAg水平[1.60(0.37,13.15)IU/ml vs 138.78(8.66,161.94)IU/ml]和APRI比值[0.31(0.22,0.47)vs 0.46(0.32,0.71)]明显降低,差异有统计学意义(U=1685.50,2972.00,均P<0.05)。ROC曲线确定PLT,TP,APRI和HBsAg在两组间的差异的截断值分别为157.50×10^(9)/L,69.45 g/L,0.29和22.56 IU/ml。单因素回归分析结果显示,PLT≥157.50×10^(9)/L,TP≥69.45 g/L,APRI≤0.29,HBsAg≤22.56IU/ml与CHB患者的HBsAg和HBsAb阳性模式相关(χ^(2)=8.231~50.862,均P<0.05);多因素回归分析显示,HBsAg≤22.56 IU/ml[OR(95%CI):9.853(4.722~20.560)]和TP≥69.45 g/L[OR(95%CI):2.358(1.132~4.912)]是HBsAg和HBsAb阳性模式的独立危险因素(均P<0.05)。基于上述危险因素建立的评分模型预测HBsAg和HBsAb阳性模式发生的ROC曲线下面积(area under curve,AUC)为0.703,灵敏度和特异度分别为66.7%和73.9%。随访结果显示,预后良好组主要分布在评分模型的高分段(2分),而预后不良组主要分布在评分模型的低分段(0分和1分),差异有统计学意义(χ^(2)=13.838,P=0.001)。结论HBsAg≤22.56 IU/ml和TP≥69.45 g/L是影响HBV-DNA阴性且HBsAg与HBsAb阳性的CHB患者的独立危险因素,据此建立的评分模型对上述患者的预后有一定的预测价值。

IGF-Ⅰ、Anti-HBs及IL-4对乙型肝炎的诊断价值

目的探讨胰岛素样生长因子Ⅰ(IGF-Ⅰ)、乙型肝炎病毒表面抗原抗体(Anti-HBs)以及微环境因子白细胞介素-4(IL-4)对乙型肝炎的诊断价值,以期为临床治疗提供参考。方法本研究选择大连市第五人民医院2019年2月—2022年2月收治的71例乙型肝炎患者作为研究组研究对象,另选同期于该院进行体检的50例健康个体为对照组研究对象。比较2组研究对象IGF-Ⅰ、Anti-HBs及IL-4水平;采用绘制ROC曲线的方式分别计算IGF-Ⅰ、Anti-HBs及IL-4水平对乙型肝炎的诊断效能;通过Pearson相关分析探究乙型肝炎患者IGF-Ⅰ、Anti-HBs及IL-4水平的相关性。结果研究组患者IGF-Ⅰ及IL-4水平均高于对照组,而Anti-HBs水平低于对照组,差异有统计学意义(P<0.05)。分别绘制IGF-Ⅰ、AntiHBs及IL-4水平诊断乙型肝炎的ROC曲线,结果显示IGF-Ⅰ、Anti-HBs及IL-4水平诊断乙型肝炎的AUC分别为0.9133(P<0.001)、0.9500(P<0.001)、0.7067(P=0.006)。Pearson相关分析结果显示,乙型肝炎患者IGF-Ⅰ、AntiHBs及IL-4呈明显相关性,其中IGF-Ⅰ与Anti-HBs呈负相关关系(r=-0.5363,P<0.001),与IL-4呈正相关关系(r=0.2826,P<0.001),Anti-HBs与IL-4呈负相关关系(r=-0.2408,P<0.001)。结论IGF-Ⅰ、Anti-HBs以及IL-4水平在乙型肝炎患者中呈现异常表达状态,且相互之间存在明显的相关性,可以考虑将它们应用于乙型肝炎的鉴别诊断中。

昆明地区HBsAg与抗-HBs双阳性慢性HBV感染患者流行病学调查及PreS/S基因突变特征

目的探讨昆明地区HBsAg与抗-HBs(Anti-HBs)双阳性乙型肝炎病毒(HBV)感染患者流行病学及PreS/S基因突变特征。方法选择2018年5月—2022年4月于云南大学附属医院住院及门诊定量检测乙肝两对半的438例慢性HBV感染患者为观察对象,将202例HBsAg(+)/抗-HBs(+)患者作为观察组,将236例HBsAg(+)/抗-HBs(-)患者作为对照组,对比两组患者临床特征。使用巢式聚合酶链反应扩增出PreS/S基因全长序列,确定基因型并分析PreS/S基因突变特征。结果观察组患者在<10岁及高于80岁人群中的流行率较高,而对照组在20~49岁人群中的流行率较高。与对照组比较,观察组患者年龄、HBsAg<250 IU/mL比例、C型HBV感染及乙肝携带者比例均显著增加(P<0.05)。成功扩增PreS/S并测序成功的观察组样本共132例,其中B型基因81例,C型基因51例;对照组样本中B型基因110例,C型基因65例。B型基因患者中,与对照组比较,观察组N末端区域突变率明显增加(P<0.05)。C型基因患者中,与对照组比较,观察组MHR、a决定簇及N末端区域突变率明显增加(P<0.05)。在观察组中,81例B型基因患者中PreS基因缺失突变14例(17.28%),51例C型基因患者中PreS基因缺失突变19例(37.25%),而在对照组中未发现PreS基因缺失突变。结论HBsAg/抗-HBs双阳性HBV感染者在<10岁及高于80岁HBsAg阳性人群多见,HBsAg/抗-HBs双阳性可能与HBsAg突变及PreS基因突变有关。

HBsAg(+)供体肾移植HBsAg(-)受体的相关研究进展

肾移植已成为肾病终末期患者的首选治疗方法,其进一步的推广与发展仍面临部分问题,供体不足仍是限制肾移植手术开展的重要因素之一。乙肝感染者的肾脏是重要的供体来源,其作为血液传播性疾病,应用乙肝表面抗原阳性供体肾脏可能造成乙肝病毒在供受体之间传播,在部分实验中观察到受体乙肝表面抗原转阳等情况,导致受体术后生存率的减少与生活质量的降低。因此需要在不同供受体情况下选择应用相应个体化的预防与治疗手段,减少病毒传播的风险,以及术后随访的过程中通过对相应指标的监测与随访及时发现潜在的病毒传播情况并采取相应对策,期望将不同供受体的移植情况进行系统性总结,为肾移植围手术期治疗方案提供理论与应用参考。

HBsAg与抗-HBs同时阳性的慢性乙型肝炎患者临床特点分析

目的分析HBsAg与抗-HBs同时阳性的现象及其临床特点,并探讨其产生的原因。方法收集2011年2月-2014年2月东南大学附属第二医院体检者2260例,其中被诊断为慢性乙型肝炎的患者830例。采用化学发光微粒子免疫分析法筛选HBsAg与抗-HBs同时阳性的患者188例,分为HBeAg阳性组(n=101)和HBeAg阴性组(n=87)。同时选取200例HBsAg阳性、抗-HBs阴性者作为对照,其中HBeAg阳性组80例,HBeAg阴性组120例。检测HBV血清学标志物、肝功能、病毒载量并结合临床进行分析。计数资料组间比较采用χ2检验。结果 HBV血清学标志物在HBsAg与抗-HBs双阳性情况下共有5种模式,其中以HBsAg、抗-HBs、HBeAg及抗-HBc阳性,且抗-HBe阴性多见,占47.9%(90/188),肝功能指标总异常率为69.1%(130/188),HBV DNA总阳性率为56.9%(107/188)。HBeAg阳性的2组HBV DNA均存在高水平复制,其中HBsAg与抗-HBs双阳性组HBV DNA阳性率与对照组比较,差异无统计学意义(χ2=2.632,P>0.05);HBeAg阴性组中,HBsAg与抗-HBs双阳性组HBV DNA定量>1×105IU/ml的比例与对照组比较,差异有统计学意义(χ2=10.740,P<0.05)。对HBV S区进行测序分析发现,测序的80例HBsAg与抗-HBs双阳性患者中有27例患者的HBV S区发生变异,突变率33.7%,且S区变异位点主要有P29L、S61L、P62L、I126T/S、Q129N、M133K、F134L、G145R/K、L175S和L186H等。结论 HBsAg与抗-HBs同时阳性者在乙型肝炎患者中有一定比例,其主要原因可能是病毒株变异所致。这种情况并不代表疾病好转,且抗-HBs出现并不一定能完全有效清除HBsAg,病毒DNA往往存在持续复制,需引起重视。

乙肝患者血清HBsAg与HBsAb双阳性的临床分析

目的通过分析乙型病毒性肝炎(以下简称"乙肝")患者乙型肝炎病毒(HBV)血清标志物中乙型肝炎表面抗原(HBsAg)与乙型肝炎表面抗体(HBsAb)同时阳性罕见模式的病例,探讨其存在原因及临床价值。方法应用微粒子酶免疫化学发光分析法(MEIA)检测乙肝患者血清标志物(HBV-M);采用用荧光定量PCR方法检测HBV-DNA,采用PCR-高分辨率熔解曲线(PCR-HRM)分析技术检测HBV基因基本核心启动子(BCP)双变异nt1762A-T/nt1764G-A;比色法检测谷丙转氨酶和谷草转氨酶,并结合患者的临床特点进行综合分析。结果在15 600例乙肝患者中,HBsAg与HBsAb双阳性的检出率为2.3%,其阳性率在不同性别组及不同年龄组中差异无统计学意义(P> 0.05);在所有HBsAg与HBsAb双阳模式中,HBsAg、HBsAb、HBeAb、HB-cAb阳性模式比例最高,占57.9%;HBsAg与HBsAb双阳模式中HBeAg阳性组乙肝患者的HBV DNA阳性率高于HBeAg阴性组;而HBeAg阴性组BCP双变异nt1762A-T/nt1764G-A发生率较HBeAg阳性组高;两组差异具有统计学意义(P <0.05);慢性乙肝患者HBsAg与HBsAb双阳检出率均高于无症状携带者、乙肝后肝硬化、重型乙肝及乙肝肝癌(P <0.05)。结论 HBsAg与HBsAb双阳性出现,并不预示乙肝病毒传染性减弱,而提示病毒基因有可能出现变异,临床有必要通过检测血清HBV DNA来确定患者体内病毒是否处于复制状态,为此部分HBsAg与HBsAb双阳性患者的个性化治疗提供一定临床帮助。

医院就诊人群HBsAg与抗HBs双阳性HBV感染者的流行病学与分子病毒学特征

目的探讨乙型肝炎病毒表面抗原(HBsAg)和抗HBsAg抗体(抗HBs)同时阳性的HBV感染者的流行病学及分子病毒学特征。方法分析52 070例医院就诊人群HBV血清学标志物检测结果,以HBsAg和抗HBs双阳性患者为实验组,HBsAg阳性、抗HBs阴性患者为对照组,比较2组患者的流行病学特征。半巢氏PCR扩增2组患者HBV S蛋白编码区并测序,比较2组间在不同基因区、基因型及临床诊断时的统计学差异。结果医院检测人群HBsAg阳性率为20.40%(10 621/52 070),其中HBsAg、抗HBs双阳性率为2.48%(263/10 621)。HBsAg、抗HBs双阳性在0~9岁和≥80岁人群流行率较高,而HBsAg阳性、抗HBs阴性则相反。实验组S蛋白氨基酸突变率显著高于对照组(1.52%vs 0.81%,P<0.01),差异主要存在于主要亲水区(MHR)(1.68%vs 0.57%,P<0.01)。实验组中除肝癌(HCC)患者外(1.97%vs 2.21%,P>0.05),乙肝携带者、慢性乙型肝炎(CHB)患者和肝硬化(LC)患者的S蛋白突变率均显著高于对照组同疾病患者(分别为1.47%vs 0.65%,1.28%vs0.84%,2.21%vs 0.44%,P均<0.05)。结论 HBsAg、抗HBs双阳性以0~9岁和≥80岁HBsAg阳性人群更多见。HBV感染者血清HBsAg和抗HBs共存可能与S蛋白(主要为MHR)的突变造成的免疫逃逸有关。HBsAg、抗HBs双阳性和HBsAg阳性、抗HBs阴性患者之间S蛋白的突变率差异与患者肝脏疾病所处的阶段有关。

HBV感染者血清HBsAg、抗-HBs共存与S基因变异

目的 :探讨无锡地区HBV感染者HBsAg与抗 -HBs的共存模式与S基因变异、病毒复制及免疫治疗的关系。方法 :采用Abbott及ELISA测定HBVM ;套式PCR检测HBVDNA并作序列分析 ;荧光定量法检测HBVDNA含量。结果 :30例adr亚型 ,1例adw亚型 ;2 1份血清出现S区多部位变异 ,造成HBsAg肽 36、47、6 3、77、89、90、115、12 6、12 9、139、15 4位氨基酸替换 ;该变异不影响病毒复制 ;变异组免疫药物的使用率 6 6 .6 % ;抗 -HBs阳性血清加入HBsAg阳性血清后 ,再测抗 -HBs阴转。结论 :S基因变异导致HBsAg抗原性的改变和与抗 -HBs结合力的降低 ,以及检测灵敏度的提高、亚型的转变都是造成HBsAg与抗 -HBs共存的原因 ;

非竞争性ELISA法测定人源抗HBsAg Fab功能性亲和常数

目的 测定完全人源化基因工程抗体HBsAgFab的亲和常数。方法 采用非竞争性ELISA固相法 ,经确定最佳抗原包板浓度、最佳抗原包板时间及最佳抗原与抗体结合反应时间后 ,得到了HBsAg与抗体片段抗HBsAgFab及完整抗体抗HBsAgIgG的抗原抗体结合反应曲线 ,计算出抗HBsAgFab及抗HBsAgIgG的亲和常数。结果 人源基因工程抗体抗HBsAgFab的功能性亲和常数在 10 7～10 8M 1水平 ,比完整抗HBsAgIgG仅仅小约 1个数量级 (10 8～ 10 9M 1)。结论 该基因工程抗体与抗原结合能力较强 ,为今后开发应用Fab进行生物导向治疗提供了理论基础。

重庆市2008～2012年无偿献血者HBsAg,ALT及抗HIV、抗HCV、抗TP抗体检测结果的分析

目的分析重庆市2008~2012年无偿献血者乙型肝炎病毒表面抗原（HBsAg）,丙氨酸氨基转移酶（ALT）及抗丙型肝炎病毒（HCV）、抗人类免疫缺陷病毒（HIV）、抗梅毒螺旋体（TP）抗体检测结果,为招募低危无偿献血者,减少血液报废提供依据。方法选择2008~2012年于重庆市血液中心接受酶联免疫吸附测定（ELISA）筛查的无偿献血者的血液标本551 133例,检测其HBsAg,ALT及抗HIV、抗HCV、抗TP抗体。采用实验室信息管理系统ITSWELL对检测结果进行读取、保存、汇总,并判断标本是否合格。结果共计检出不合格标本37 534例（6.81%）,其中,HBsAg,ALT及抗HCV、HIV、TP抗体不合格率分别为1.10%、3.79%、0.51%、0.33%、1.08%。结论加强血液检测试剂筛选及无偿献血者队伍的建设将有助于临床的输血安全。

乙型肝炎表面抗原(HBsAg)胶体金试剂的研制

目的 研制乙型肝炎表面抗原 (HBsAg)胶体金试剂。方法 用未加氢氧化铝佐剂的乙型肝炎血源疫苗原材料免疫BALB/c小鼠制备抗 -HBs单克隆抗体 ;应用胶体金标记抗 -HBs单克隆抗体 ;测定HBsAg胶体金试剂的稳定性 ;对该试剂进行临床考核及检定等。结果 经中国药品生物制品检定所检定 ,其灵敏度达到 1ng/ml,特异度为 10 0 % (2 0 / 2 0 ) ,精密度为 10 0 % (10 / 10 ) ,符合国家对HBsAg试剂盒的要求 ,临床考核合格 ,已通过国家药品监督管理局的新药评审 ,获国家新药证书。结论 HBsAg胶体金试剂可用于献血员HBsAg筛查、健康人群体检、乙型肝炎病毒 (HBV)感染的流行病学调查和临床诊断。

HBsAg Mab胶体金探针制备与鉴定的实验研究

目的:研制合格的乙肝表面抗原单克隆抗体(HBsAg Mab)标记的胶体金探针。方法:采用柠檬酸三钠还原法制备15nm的胶体金;所制备的胶体金通过透射电镜和紫外分光计度计鉴定其大小和均匀度。通过CVAI曲线,确定胶体金标记HBsAg Mab蛋白用量;采用斑点免疫吸附试验对探针进行鉴定。结果:制备的15nm胶体金颗粒均匀;紫外分光光度计400-700nm扫描结果最大吸收波长为518nm,峰宽较窄;纯化的HBsAg Mab浓度为65mg/mL;在PH为8．2时,每毫升胶体金的蛋白最适保护量为32．5μg;采用斑点免疫吸附试验鉴定探针质量合格,可保存3个月。结论:制备的HBsAg Mab胶体金探针质量合格,为进一步研究提供手段。

重组G145R HBsAg亲和纯化方法的建立及其应用

目的建立重组乙型肝炎病毒G145R变异HBsAg抗体亲和纯化方法。方法用抗-HBs单克隆抗体(D12- McAb)制备亲和层析胶,对2A8细胞(分泌G145R变异HBsAg)培养上清盐析物进行亲和纯化。采用SDS-PAGE、Western blot及ELISA,对纯化产物的纯度、特异性、含量和回收率进行鉴定,并与同法纯化的HBsAg阳性血清及r-wHBsAg提取物进行比较。结果D12-McAb对重组真核表达G145R变异HBsAg、HBsAg阳性血清及r-wHBsAS三者具有相似的亲和性,产物纯度分别为90.3%、95.2%和93.1%,回收率分别为43.3%、72.0%和66.4%。结论已成功地建立了重组G145R变异HBsAg抗体亲和纯化方法,为G145R变异以及其他HBV免疫逃逸变异感染的深入研究奠定了重要的技术基础。