A biotin/avidin solid-phase sandwich enzyme immunoassay for the antibody to hepatitis B surface antigen (anti-HBs)

A biotin/avidin solid-phase enzyme immunoassay for the detection and quantitation of the antibody to hepatitis B surface antigen (anti-HBs) is described. The assay utilizes hepatitis B surface antigen (HBsAg) as a solid-phase 'capture' reagent and a mixture of biotinylated HBsAg and avidin-conjugated horseradish peroxidase as a probe 'detector' reagent. The assay was compared to a commercial radioimmune assay for anti-HBs detection. The two assays were found to measure the same molecules and to correlate well regarding anti-HBs titers.     

The utilization of biotin-antibiotin interaction for the detection of antibody to hepatitis B surface antigen (anti-HBs)

A biotin-antibiotin solid-phase enzyme-linked immunoassay for the detection and quantitation of the antibody to hepatitis B surface antigen (anti-HBs) is described. The assay utilizes hepatitis B surface antigen as a solid-phase 'capture' reagent and a mixture of biotinylated HBsAg and antibiotin-conjugated horseradish peroxidase as a detector reagent. The assay was compared to a commercial enzyme immunoassay (AUSAB EIA) which used the biotin-avidin system for anti-HBs detection. The two assays were found to measure the same molecules and to correlate well regarding anti-HBs titers.     

Comparison of alpha-fetoprotein with some other tumour markers in Malaysians with hepatocellular carcinoma

Although alpha-fetoprotein (AFP) is regarded as the reference marker for hepatocellular carcinoma (HCC), it sometimes produces false results. The objective of this study was to see if some of the readily available laboratory markers could complement AFP to improve the laboratory diagnosis of HCC. The markers tested and their sensitivities were: CA 125, 92%; ferritin, 71.3%; CA 19-9, 69.8%; beta-2-microglobulin (B2M), 53.3%; CA 72-4, 13.6%; and carcinoembryonic antigen (CEA), 10.6%. In comparison, AFP had a sensitivity of 58.8%. CA 72-4 and CEA (at the "tumour" cut-off level of 20 ng/ml) had specificities of 100%, and AFP, 97.4%. The specificities of the other markers were less impressive: CEA, 77.8% (at the cut-off level of 5 ng/ml); ferritin, 48.6%; CA 125, 48.5%; B2M, 39.6%; and CA 19-9, 37.3%. The efficiencies of the markers for HCC, which are based on the consideration of sensitivity and specificity together, were as follows: AFP, 77.6%; CA 125, 71.3%; ferritin, 60.5%; CA 19-9, 55.3; B2M, 46.9%; CEA, 40.8%; and CA 72-4, 34.5%. The receiver-operating characteristic plots confirmed AFP to be the most efficient marker for HCC. Nevertheless, it is proposed that CA 125 be combined with AFP for HCC screening because of their excellent sensitivity and specificity, respectively: a negative result for both, or even just CA 125 alone, would indicate that the disease is unlikely while a positive AFP (which would likely occur with a positive CA 125) would make its presence highly probable. A positive CA 125 and negative AFP would be equivocal for HCC. Other markers in combination with AFP are less useful.     

miRNA 表达与免疫细胞因子相关性在原发性肝癌早期诊断和预后中的作用

目的:检测miR-146a、miR-224、miR-34c、miR-200a、miR-148b、miR-375 六种miRNAs 在原发性肝癌中的表达变化及其与HBsAg、抗-HBs、HBeAg、抗-HBe、抗-HBc 和IL-12、IL-4、IL-6、IL-10、IFN-γ、TNF-α等炎症因子表达的相关性,以验证循环miRNA 是否可作为理想的血源性新型生物标志物用于原发性肝癌的早期检测。方法:收集肝炎、肝硬化患者及健康对照组静脉血,并收集原发性肝癌患者癌组织和癌旁组织。提取总RNA 后通过实时定量PCR 检测并比较各组miRNA 的相对表达水平,同时检测miRNA 表达水平变化与血清肿瘤标志物AFP、CEA、CA19-9、CA125 表达的关系;并检测miRNAs 表达水平变化与HBsAg、抗-HBs、HBeAg、抗-HBe、抗-HBc 和炎症因子IL-12、IL-4、IL-6、IL-10、IFN-γ、TNF-α表达相关性。结果:相对于健康组,miR-34c、miR-224、miR-146a 在PHC 组血清和组织中表达显著上调;miR-200a、miR-148b、miR-375 在PHC 组血清和组织中表达显著下调,差异具有统计学意义。HBsAg 与血清miR-375 和miR-146a 存在回归关系,miR-375 随HBsAg 表达水平升高而降低,而miR-146a 随HBsAg 表达升高而升高。IFN-γ与miR-146a 存在回归关系, miR-146a 随IFN-γ表达水平降低而升高,miR-375 和miR-146a 诊断能力大于CA19-9 和AFP。结论:miR-146a、miR-224、miR-34c、miR-200a、miR-148b、miR-375 在原发性肝癌血清和组织中存在表达差异,其中miR-375 和miR-146a 诊断能力优于AFP 和CA19-9,血清miR-375 和miR-146a 可能成为新的肝癌早期诊断标志。     

Second generation assays for the detection of antibody to HBsAg using recombinant DNA-derived HBsAg

A second generation radioimmunoassay (RIA) and enzyme-linked immunoassay (EIA) for the detection and quantitation of the antibody to hepatitis B surface antigen (anti-HBs) was developed which utilizes recombinant DNA-derived HBsAg (rHBsAg) in place of human plasma derived HBsAg. In these sandwich assays, rHBsAg immobilized on a solid phase was used to capture anti-HBs from the specimen and rHBsAg conjugated to horseradish peroxidase or radiolabeled with 125I was used as a detecting reagent. These rHBsAg-based assays were compared to a commercial radioimmunoassay for anti-HBs detection (AUSAB RIA). For a population of 1711 sera and plasma specimens, 99.2% overall agreement was demonstrated between the recombinant RIA and EIA and 98.6% agreement was observed between the recombinant assays and AUSAB-RIA. The recombinant assays demonstrated equivalent sensitivity and detectability to AUSAB RIA. Most discrepant samples were low-level reactive by AUSAB-RIA, generally less than 10 mIU/ml, and likely represent nonspecific reactivity since no other marker for hepatitis B infection was detected in these samples.     

肝病时AFP、CA19-9、CA242检测的临床价值探讨

目的：探讨肝病时对肿瘤标志物测定的影响。方法：40例正常人、柏例急性黄疸型肝炎病人、柏例急性无黄疸型肝炎病人、柏例慢性肝炎和柏例肝硬化病人，对这些人群进行肝功能、AFP、CA19—9、CA242等检测。结果：肝功能异常时AFP、CA19-9阳性率较高，高胆红素血症时CA19—9增高明显，肝硬化失代偿期AFP、CA19-9增高明显。结论：肝病时肝功能异常、高胆红素血症以及肝硬化失代偿期对肿瘤标志物测定有较大影响。     

改良荧光磁微粒酶免分析法检测三种肿瘤标志物及应用

探讨利用改良荧光磁微粒酶免分析法(A法)检测三种肿瘤标志物及临床应用价值。本文用A法检测352份血清AFP、CEA和CA19-9水平,并与化学发光微粒免疫分析法(B法)比较。A法与B法检测结果相关性好,无显著性差异(P>0.05)。A法在磁性微球基础上对制备冻干试剂、反应杯独立包装和双波长检测荧光信号进行改进;利用A法检测肿瘤标志物具有一定的临床应用价值。     

AFP联合CA19—9检查对良恶性肝病鉴别诊断的临床研究

目的探讨AFP（甲胎蛋白）联合CA19—9（糖链抗原）检查对原发性肝癌、转移性肝癌、肝硬化、病毒性肝炎的鉴别诊断价值。方法采用全自动电化学发光系统对300例各类肝病患者及50例健康对照者进行血清AFP和CA19—9检查。结果原发性肝癌患者AFP值（838．5±175．4）μg／L较其他患者明显增高,而肝硬化患者AFP中度升高,病毒性肝炎和转移性肝癌患者AFP仅轻度升高。当以AFP〉500μg／L作为原发性肝癌诊断界值时,其灵敏度为70.3％．特异性为98．8％。转移性肝癌患者CA19—9值（1265．8±113．4）U／ml较原发性肝癌、肝硬化、病毒性肝炎患者明显增高。结论AFP联合CA19—9检测在原发性肝癌和转移性肝癌,以及对其他良恶性肝病的鉴别诊断具有良好的价值。     

基因重组干扰素治疗急性乙型肝炎疗效观察

目的　以基因重组干扰素治疗急性乙型肝炎病人,观察其减少急性乙肝转慢率的效果。方法　用基因重组α干扰素（３００ 万Ｕ,肌内注射,隔日１ 次,１２ 周为一个疗程） 治疗１９ 名急性乙肝病人,对治疗后未产生抗ＨＢｓ 者,加用乙肝疫苗（３０ μｇ,肌内注射,每周注射１ 次,连用３ 周）;对照组为病情相似的１５ 例病人,服用一般保肝药物。结果　治疗组１８ 例（９５－０％ ,１８１９）ＨＢｓＡｇ 阴转,但均未产生抗ＨＢｓ,再用乙肝疫苗后,１７ 例（９４－０％ ,１７１９） 产生抗ＨＢｓ。２４～２４０ 周随访期间,１８ 例ＨＢｓＡｇ 阴转者无复发;１ 例ＨＢｓＡｇ 未阴转者,至随访结束时仍为阳性。对照组８ 例（５３－０ ％ ,８１５）ＨＢｓＡｇ 阴转,同时,８７－５ ％（７８）的病例产生抗ＨＢｓ,另７ 例ＨＢｓＡｇ 未阴转者,在２４ ～２４０ 周的随访期间,仅１ 例ＨＢｓＡｇ阴转,余６ 例ＨＢｓＡｇ 持续阳性。结论　对于急性乙型肝炎发病８ 周后ＨＢｓＡｇ 仍未阴转者,采用干扰素合用乙肝疫苗,对防止转慢及ＨＢｓＡｇ 阴转后抗ＨＢｓ 的产生可能有一定作用     

Primary hepatocellular carcinoma and hepatitis B virus infection in korea

Serological evidence of hepatitis B virus (HBV) infection and serum alphafetoprotein (AFP) were assayed in sera from 112 Korean patients with primary hepatocellular carcinoma (PHC) and from 63 age- and sex-matched controls. Serological evidence of HBV infection was found in 100% of PHC patients and in 97% of controls. The majority of PHC patients (87%) were positive for hepatitis B surface antigen (HBsAg). In contrast, only 14% of control individuals were positive for HBsAg, but 82% were positive for antibody to HBsAg (anti-HBs). Hepatitis B e antigen (HBeAg) was detected in a high percentage (38%) of HBsAg-positive PHC patients, but in none of the nine HBsAg-positive control individuals. Serum AFP was detectable in 83% of PHC patients but in only one of 63 controls (1.5%). These results document that HBV infection may be the mjor factor in the development of PHC in this country.     

14株抗乙型肝炎表面抗原单克隆抗体与变异表面抗原的反应模式及应用

目的制备抗乙型肝炎表面抗原(hepatitis B surface antigen,HBsAg)的单克隆抗体,检测单克隆抗体与15种变异HBsAg的反应模式。用筛选出的单抗建立快速检测变异HBsAg的ELISA实验方法,并做初步评价。方法用中国乙型肝炎病毒感染者血清中分离的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。检测不同单克隆抗体与野生及变异HBsAg的反应性。筛选出两种可以较好识别变异HBsAg的单克隆抗体McAb2和McAb3,建立两种抗体ELISA检测HBsAg的方法。结果制备了14株抗-HBs单抗。经过初筛,有4种可以较好识别包括G145R在内的大多数变异HBsAg。优化了McAb2和McAb3检测HBsAg的条件,检测HBsAg的灵敏度较好,检测变异HBsAg的能力优于2种现行国产HBsAg检测试剂盒。结论用本实验制备的单抗可以很好地识别包括G145R在内的大多数变异HBsAg。     

Highly Sensitive Detection of Hepatitis B Virus Surface Antigen by Use of a Semiautomated Immune Complex Transfer Chemiluminescence Enzyme Immunoassay

The performance of hepatitis B surface antigen (HBsAg) screening assays is continuously improved to reduce the risk of transfusion-associated hepatitis B. In this study, a semiautomated immune complex transfer chemiluminescence enzyme immunoassay (ICT-CLEIA) for the detection of HBsAg, which is as sensitive as hepatitis B virus (HBV) DNA PCR, was developed; the ICT-CLEIA assay performance was compared with the performance of the Architect HBsAg QT assay and HBV DNA PCR. The specificities in the initial assay and after retesting were 99.50% (1,988/1,998 samples) and 99.95% (1,997/1,998 samples), respectively. The analytical detection limit was determined to be 0.2 mIU/ml using the 2nd International WHO HBsAg standard, and the cutoff value (0.5 mIU/ml) of the ICT-CLEIA assay was 8.0 standard deviations (SD) above the mean of the HBsAg-negative specimens. The ICT-CLEIA assay could detect HBsAg even in the presence of anti-HBs antibodies and demonstrated a 23.6-day-shorter window period using commercially available HBsAg seroconversion panels than the Architect HBsAg QT assay. Furthermore, the monitoring of the viral kinetics by the ICT-CLEIA assay and the HBV DNA PCR produced very similarly shaped curves during both the HBsAg seroconversion and reverse seroconversion periods. Therefore, the ICT-CLEIA assay may be useful not only for an earlier detection of HBV reactivation but also for the monitoring of hepatitis B patients.     

IgG heterophile antibody causes false positivity for CA19-9, which is overcome with bovine immunoglobulin

Immunoassay using antibodies is widely applied to measure various clinical parameters such as tumor markers. However, many mechanisms of interference for this method have been reported. We studied serum sample showing false positive CA19-9 values in the range of 24 to approximately 286U/ml depending on the reagent lots used. PEG-pretreated serum did not show false positivity. Using high performance liquid chromatography (HPLC), the CA19-9 false positive peak was obtained around 160kD, which is the same as IgG. Antibody-coated microbeads, labeled antibody and buffer were used in different combinations between different lots, and the buffer was found to be the cause of false positive findings. The amount of bovine IgG contaminated in bovine serum albumin(BSA) in the reagent buffer markedly differed between lots of BSA, and reagent with a low amount of bovine IgG showed false positive results. Bovine immunoglobulin was superior to mouse IgG in the attempt to avoid a false positive reaction. We concluded that the cause of false positive CA19-9 findings in this serum sample was IgG heterophile antibody, which reacts with both of mouse and bovine IgG. The heterophile antibody had greater affinity to bovine IgG than to mouse IgG. The difference between CA19-9 values obtained by different reagent lots was due to different amounts of bovine IgG contaminated in BSA used in the reagent. Bovine immunoglobulin was superior to mouse IgG in the attempt to absorb the heterophile antibody and avoid false positive reaction in this case.     

A method for coupling the hepatitis B surface antigen to aldehyde-fixed erythrocytes for use in passive hemagglutination

A method for coupling the hepatitis B surface antigen (HBsAg) to aldehyde-fixed erythrocytes for use in passive hemagglutination assays for anti-HBs is described. This provides a stable and inexpensive reagent having a sensitivity for anti-HBs detection which is only slightly less than solid phase radioimmunoassay.     

Evaluation of assay methods for hepatitis B surface (HBsAg) antigen and its antibody (anti-HBS) in viral hepatitis B (VHB)-HBsAg-positive

The sensitivity of methods for the detection of HBsAg and its anti-HBs was compared in serial 1200 sera samples from 30 patients with VHB-HBsAg-positive. HBsAg was tested by gel-diffusion (GD), counter-immunoelectrophoresis (CIE), reversed haemogglutination (rHA), radioimmunoassay (RIA), and enzyme immunoassay (EIA). RIA and EIA methods are statistically significantly more sensitive compared with the other methods (P<0.0005). By these methods the minimal concentrations of HBsAg in sera can be proved. Although there is no statistically significant difference in the sensitivity between RIA and EIA, the latter is more sensitive if the subtype ay-HBsAg is considered (12 sera samples). In 24 patients the subtype was ay, in two ad, and in four it could not be differentiated.In 70% of patients anti-HBs was proved by RIA and in 10% by CIE, i.e., in 73% and 9% of sera samples, respectively. In 117 sera samples of these patients the sensitivity of RIA and EIA was compared for determination of anti-HBs. No statistically significant difference between the methods for determination of anti-HBs was found (50.42% 40.17%). No immune response to HBsAg has been observed in 9 cases, but 6 of them have remained permanent carriers of this antigen.     

Hepatitis B virus markers, alpha-fetoprotein and survival in fulminant viral hepatitis

The serological markers of hepatitis B virus and serum alpha-fetoprotein (AFP) levels have been studied in 28 consecutive cases of fulminant hepatitis, correlating the data with survival. On admission, 20 patients were found to be positive for HBsAg and eight for anti-HBs. All anti-HBs-positive cases showed high titers of anti-HBc, and six patients were positive for specific anti-HBc-IgM. DNA polymerase activity was detected in serum of 11 HBsAg-positive (55%) and four anti-HBs-positive (50%) patients. HBeAg was detected in six (21.4%) subjects (five HBsAg-positive and one anti-HBs-positive), whereas anti-HBe was present in nine (32.1%) subjects (six HBsAg-positive and three anti-HBs-positive). AFP levels greater than 60 ng/ml were found in sera of 14 patients (50%). No significant difference was evidenced in the survival rate between HBsAg-positive and anti-HBs-positive and between HBeAg-positive and HBe Ag-negative patients. However, a statistically significant difference (P less than 0.05) in the survival rate was found in patients positive and negative for DNA polymerase activity and in those with AFP levels higher and lower than 60 ng/ml (P less than 0.005). Pathogenetic and prognostic significance of these findings are discussed.     

Comparison of two testing methods to determine hepatitis B surface antibody response to hepatitis B vaccine among health-care workers

Because of variations in reported seroconversion rates and to compare enzyme-linked immunoassay (EIA) and radioimmunoassay (RIA) methods to assess hepatitis B vaccine response, hepatitis B surface antibody (anti-HBs) was tested in 116 of 174 high-risk hospital employees enrolled in a hepatitis B vaccine program. All individuals were vaccinated with three injections of Heptavax-B, 1.0 ml containing 20 micrograms of HBsAg intramuscularly in the deltoid. The same lot of appropriately stored vaccine was used. Of the 41 individuals tested within zero to six months postvaccination, 35 (85%) and 38 (93%) were positive by EIA and RIA, respectively. Of the 75 individuals tested 7-24 months postvaccination, 61 (81%) and 71 (95%) were positive by EIA and RIA, respectively. Results of EIA tests performed at two laboratories were similar. Of 109 individuals positive by RIA, 13 did not have protective antibody levels. In contrast, of 96 individuals positive by EIA, only one did not have a protective antibody level. RIA may be a more sensitive test for anti-HBs, but a positive EIA result correlates better with protective antibody levels.     

癌胚抗原的时间分辨免疫荧光分析及其诊断试剂的研制

目的利用时间分辨免疫荧光分析(TRFIA)技术,建立一种人血清癌胚抗原(CEA)的快速全自动检测方法并研制其诊断试剂。方法采用双抗体夹心法(用1株抗CEA的mAb M86160M用于包被微孔板,另1株抗CEA的mAbM86110M用于标记铕)建立CEA-TRFIA,增强液采用β-二酮体为主的发光增强系统。结果CEA-TRFIA的线性测量范围为(1~560)μg/L,分析的灵敏度为0.28μg/L,批内和批间的变异系数(CV)分别为7.2%~8.6%和8.9%~13.2%。与AFP、CA12-5、CA19-9和白蛋白均无交叉反应,与CA15-3的交叉反应值为1.62μg/L。将1000份血清标本用本法与国外罗氏化学发光试剂盒同时检测,其相关系数(r)为0.946。结论CEA-TRFIA的各项指标均达到临床检测的要求,可替代国外同类检测试剂盒,用于临床血清CEA水平的测定。     

AFP,CA19—9联检在原发性肝癌和肝硬化鉴别诊断中的临床意义

为提高原发性肝癌的诊断率和与肝硬化，其他慢性肝病的鉴别诊断，本文采用放射免疫分析法和免疫放射分析法。对５０例健康体检者、４８例原发性肝癌、３９例肝硬化，３６例其他一肝病患者，分别检验其血清中甲胎蛋白，糖类抗原１９－９肿瘤标志物的含量，结果进行比较。     

乙型肝炎卡介苗联合疫苗与单价乙型肝炎疫苗免疫效果的比较

目的：比较乙型肝炎卡介苗联合疫苗与单价乙型肝炎疫苗的免疫效果。方法：实验动物采用豚鼠，按0、1、2月三针免疫程序接种，并于每针免疫后1个月采血，ELISA方法检测血清抗体滴度。实验分三部分进行。实验一：三种不同规格的联合疫苗与单价乙型肝炎疫苗的比较；实验二：同一规格连续三批联合疫苗与单价乙型肝炎疫苗的比较；实验三：联合疫苗与两种单价疫苗同时免疫的比较。结果：在三个实验中，联合疫苗组第一针血清抗体滴度均低于对照组，但无统计学差异：联合疫苗组第二、三针血清抗体滴度均高于对照组，也无统计学差异，实验组各组之间无明显差异。结论：联合疫苗组三针免疫程序的HBsAg的效力与单价乙型肝炎疫苗组相似。