IgG subclass composition of antibodies to HBsAg in circulating immune complexes from patients with hepatitis B virus infections

The IgG subclass of antibody associated with hepatitis B surface antigen (HBsAg) in circulating immune complexes (CIC) from patients with either acute or chronic hepatitis B virus (HBV) infections was measured using an isotype and antigen-specific ELISA. All patients were HBsAg positive but were negative for free anti-HBs antibody. The subclass of antibody associated with HBsAg in CIC in both groups was predominantly IgG1 and IgG4. This is in contrast to free anti-HBs in convalescent sera from patients recovering from HBV infection, which are highly restricted to IgG1 and IgG3. The finding of high levels of IgG4 antibodies in CIC suggest that CIC containing this subclass may be cleared less efficiently than CIC containing antibodies of other subclasses. Formation of these CIC may be an important factor in the progression of infection to chronicity and may also be involved in the antigen-specific immunosuppression seen in early acute and chronic HBV infections.     

乙型肝炎表面抗原-破伤风类毒素结合疫苗的制备及免疫学特性

目的 制备乙型肝炎(乙肝)表面抗原(HBsAg)与破伤风类毒素(TT)结合疫苗,并研究其免疫学特性.方法 TT经溴化氢活化后与己二酰肼形成TT-酰肼基衍生物,在碳二亚胺作用下与HBsAg共价结合.将结合物、HBsAg及乙肝疫苗免疫小鼠,并设生理盐水对照组,分别于7、14、21、28 d取血,应用化学发光法检测小鼠血清抗-HBs,于第7、28天应用酶联免疫斑点法(ELISPOT)检测脾单个核细胞(mouomlclear cells,MNC)分泌HBsAg特异性IFN-γ和IL-2能力.结果 成功制备了HBsAg-TT结合物,该结合物诱导抗-HBs阳转率及抗体滴度均较单独免疫HBsAg及乙肝疫苗组高,主要以IgG2a抗体为主,且分泌IFN-γ和IL-2的淋巴细胞数也显著增加.结论 用该方法 制备的HBsAg-TY结合疫苗,不仅可诱导较强的体液免疫,还可诱导以TH1应答为主的细胞免疫.     

Anti-HBc IgG subclasses in different populations by comparing a variety of ELISA plates

The number of IgG subclasses for hepatitis B virus (HBV) core antigen (anti-HBc), demonstrated for HBV-infected individuals, was measured by enzyme-linked immunosorbent assay (ELISA). Four commercially available hepatitis B core antigen (HBcAg) plates and one prepared plate were tested for ELISA sensitivity by the detection of 14 serum samples drawn from HBV chronic carriers, cured patients, vaccinees, and non-infected individuals. Differences in optical density (OD) values were obtained by comparing data gathered from the five plate types, suggesting that different plates may have different binding capabilities for each anti-HBc IgG subclass and, thus, contribute to the different ELISA sensitivities. Of these plates, the GB plate showed the most obvious absorbance changes for anti-HBc subclasses in different populations. These data also indicated different patterns for IgG-specific subclasses for various populations. For HBsAg+ carriers, the OD for IgG1 was greater than for IgG3. By contrast, the OD for IgG3 was higher than that for IgG1 in those subjects who were negative for HBsAg.     

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).     

IgG Subclass Deficiency in Patients with Down''s Syndrome and Aberrant Hepatitis B Vaccine Response

Seventeen adult patients with Down's syndrome (DS) and 19 adult healthy references were vaccinated with a hepatitis B vaccine in order to study the IgG subclass response. An enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies specific for IgG subclasses was employed. In spite of normal levels of total IgG1 and normal or even high levels of IgG3 in the DS patients, a significantly lower IgG1 response to the vaccine was observed in trisomic patients than in the references.     

脂质体佐剂对增强HBsAg免疫原性的作用

利用DC Chol制备粒径为 5 0～ 30 0nm的正电荷脂质体 ,作为乙肝疫苗 (HBsAg)的佐剂 ,免疫BALB/c小鼠后进行血清中特异性抗体IgG1及IgG2a、脾细胞产生细胞因子的检测。结果该脂质体佐剂所诱导的抗体亚类以IgG2a为主 ,脾细胞产生的IL 2、IL 5、IFN γ分别比铝佐剂组高 16 5倍、 10倍、 2倍。表明该脂质体佐剂可以诱导很强的细胞免疫反应 ,是一种能促进Th1和Th2均衡应答的佐剂 ,值得作进一步的研究     

Significance of maternal and infant serum antibodies to hepatitis B core antigen in hepatitis B virus infection of infancy

The significance of IgM and IgG class antibodies to hepatitis B virus (HBV) core component (anti-HBc) was investigated in a study of maternal-fetal HBV transmission. An IgM anti-HBc response was lacking in the majority (49/53) of HBV-infected infants. This antibody thus cannot be used as an indicator of transplacental infection. However, most infants who became HBsAg positive during the first 6 months of life acquire infection in the perinatal period rather than transplacentally. Passively transferred maternal IgG anti-HBc in the infant and additional IgM anti-HBc positively in the carrier mother have no modulating influence on HBV infection of infants born to HBV carrier women.     

IgG subclass distribution of anti-HBs antibodies following vaccination with cDNA HBsAg.

Serum samples were obtained during follow-up of nine young adults vaccinated over 1 year with cDNA hepatitis B antigen. The absolute amounts of anti-HBs IgG subclass antibodies present in the sera were determined by comparing the optical densities (OD) obtained using an antigen-specific ELISA with those obtained by serial dilutions of known amounts of human IgG1-4. The calibration curves for each IgG subclass were corrected for the corresponding coating efficiency. Our data suggest that HBs antibody responses of vaccinated subjects occur in all IgG subclasses but IgG1 and IgG2 are the major subclasses involved.     

Immunoglobulin G subclass restriction of antibodies against hepatitis B surface antigen.

Immunoglobulin G (IgG) antibodies against hepatitis B surface antigen were found to be restricted to subclasses IgG1 and IgG3 in serum samples of 17 individuals. Quantitative determinations showed that in 10 samples the minor serum subclass IgG3 contained more antibodies than the predominant subclass IgG1. Ranges of concentrations were between 0.72 and 16.50 micrograms/ml for IgG3 antibodies and between 0.55 and 6.19 micrograms/ml for antibodies of subclass IgG1.     

IgG and IgA subclass distribution of antitoxin antibody responses after oral cholera vaccination or cholera disease

The immunoglobulin subclass distribution of cholera antitoxin antibody responses in serum was studied in Swedish volunteers after different routes of immunization with a cholera B subunit-whole cell vaccine (B + WCV) and in Bangladeshi patients convalescing from cholera disease. Both oral and parenteral immunization induced antitoxin antibodies of all the different IgG subclasses (IgG1, IgG2, IgG3 and IgG4) whilst the IgA antibodies were restricted to the IgA1 subclass. A single oral dose of B + WCV induced proportionally higher levels of IgG4 antitoxin in previously cholera-immunized volunteers than in a matched group who had not been cholera-vaccinated before, suggesting that repeated immunization preferentially stimulate formation of IgG4 antibodies. The IgG and IgA subclass distribution of antitoxin antibodies in orally vaccinated Swedes closely resembled that in Bangladeshi cholera patients.     

Utilizing self-prepared ELISA plates for a cross-population study of different anti-HBe IgG subclass profiles

Fourteen serum samples obtained from hepatitis B virus (HBV) chronic carriers and patients recovered from hepatitis B infection were used with four sodium dodecyl sulfate-treated enzyme-linked immunosorbent assay (ELISA) plates available commercially, and one self-prepared HBcAg analog for evaluation of anti-HBe subclass pattern absorbance. The self-prepared plates had the best performance and were thus used for samples obtained from 104 (60 male and 44 female) HBV chronic carriers and 439 (247 male and 192 female) recovered individuals. Tests for aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were also carried out in 21 of the subjects (>25 IU/ml). Statistical comparison of these patients with elevated ALT/AST levels with other ALT/AST-normal chronic carriers revealed no significant differences in the anti-HBe OD, although the mean optical density (OD) of patients with elevated ALT/AST levels was higher. The results suggest that the anti-HBe IgG subclass profiles in the chronic carriers did not change with inflammation of the liver, and were independent of sex and age. In contrast to previous anti-HBc findings, the distribution pattern of anti-HBe subclasses in HBV chronic carriers was IgG1 > IgG4 > IgG3 while in the recovered individuals it was IgG1 > IgG3 > IgG4, for both males and females. Subclasses IgG1 and IgG2 were the most and least prevalent isotypes, respectively, in both study groups. The results of the study suggest that induction of IgG1 and/or IgG3 antibodies is important for effective virus neutralization, while IgG2 antibodies are of limited importance. Significantly higher OD values for anti-HBe IgG4 were observed when comparing samples from the chronic carriers and recovered individuals, which may reflect the effects of persistence. Further, in contrast to previous anti-HBs results, the concentrations of total IgG and IgG1 were higher in the samples from chronic carriers relative to those from recovered individuals.     

ELISA detection of human IgG subclass antibodies to Streptococcus mutans

A sensitive enzyme-linked immunosorbent assay (ELISA) has been developed to measure IgG subclass antibodies against whole cells of Streptococcus mutans and to a purified streptococcal antigen (SA I/II). Bacterial cells were bound to the solid phase using methyl glyoxal and mouse monoclonal antisera against IgG and each IgG subclass were used to detect antibodies. Natural antibodies to S. mutans were predominantly of the IgG1 and IgG2 subclasses, though IgG3 and IgG4 antibodies were detectable in most subjects, and were the majority response in a few subjects. Antibodies to SA I/II were predominantly of the IgG1 subclass with virtually no activity detectable in the IgG3 and IgG4 subclasses. Inhibition studies suggested some restriction of IgG subclass responses to bacterial antigens since SA I/II and c polysaccharide could inhibit binding of all subclasses to whole cells of S. mutans equally, whereas glucosyltransferase, lipoteichoic acid and dextran showed greatest inhibition of the IgG3 and IgG4 subclasses.     

Cell-mediated immunity to hepatitis B surface antigen in man.

An aqueous preparation of hepatitis B virus (HBV) vaccine was used as an intradermal skin test antigen to assess delayed hypersensitivity to hepatitis B surface antigen (HBsAg). Thirty-five persons were tested including 10 individuals seronegative for all HBV markers, 10 positive for HBsAg (chronic carriers) and 15 positive for antibody to HBsAg (anti-HBs), five of whom had received the HBV vaccine. All patients were also studied for lymphocyte blastogenic responses to phytohaemagglutinin, concanavalin A, pokeweed mitogen and purified HBsAg. Only one individual had a positive delayed skin test reaction to HBsAg. This person had received the HBV vaccine and had high titres of anti-HBs in serum. However, neither this individual nor any other subject exhibited a positive lymphocyte blastogenic response to HBsAg in vitro. Thus, delayed hypersensitivity skin test reactivity to HBsAg was not detected after natural infection with HBV and was rarely present in hyperimmunized individuals. In vitro assays of immune responsiveness failed to demonstrate cellular immunity to HBsAg even in hyperimmunized persons. These studies provide no evidence that cell-mediated immunity to HBsAg plays a role in the immunopathogenesis of acute or chronic type B hepatitis.     

IgG subclasses in circulating immune complexes with hepatitis B e antigen in chronic hepatitis B

IgG subclasses of antibodies to hepatitis B e antigen (anti-HBe) complexed to HBeAg were determined in 126 HBsAg-positive sera. In the assay HBeAg complexes were bound to microtitre plates by monoclonal anti-HBe and indicated by biotinylated monoclonals to each of the four human IgG subclasses. To evaluate the specificity of the complexed IgG, serum dilutions were also tested for HBeAg and for subclasses of anti-HBe IgG. Two groups of sera were investigated: (i) 64 sera from 64 HBsAg carriers; and (ii) 62 sera from 13 HBeAg-positive patients, of whom five seroconverted to anti-HBe. At least four sera were available from each of these patients. Complexed anti-HBe IgG was detected in 22 of 30 HBeAg-positive, and in three of HBeAg-negative carrier sera. There was no significant association between presence of complexed anti-HBe and levels of HBeAg in these sera. Complexes with multiple subclass composition were found in 13 of the 25 sera with complexed anti-HBe. The most common IgG subclasses found complexed to HBeAg were IgG1 (75%) and IgG4 (67%). A significant association (P less than 0.05) was found between the presence of free and complexed anti-HBe IgG1 in the carrier sera, indicating that the IgG1 antibodies, complexed to HBeAg, were specific for HBeAg. In the five patients who seroconverted to anti-HBe, anti-HBe IgG1 was detected in the HBeAg-positive phase before seroconversion. In the eight patients with persistent HBeAg antigenemia, free anti-HBe IgG1 was detected in only two sera from two different patients. In one patient, complexed anti-HBe IgG1/IgG4 was detected in all serum samples drawn during a period of 111 months. In conclusion, complexed anti-HBe might be detected several years before apparent seroconversion to anti-HBe in conventional anti-HBe assays. In contrast 'free' anti-HBe IgG1, when detected in HBeAg-positive sera with our anti-HBe subclass assay, seemed to signal ensuing apparent seroconversion to anti-HBe.     

A triple antibody assay for the quantitation of plasma IgG subclass antibodies to acetylcholine receptors in patients with myasthenia gravis

Monoclonal anti-human IgG subclass antibodies have been used in an immunoprecipitation assay for the determination of anti-acetylcholine receptor IgG subclasses in plasma from patients with myasthenia gravis. Solubilized acetylcholine receptors labelled with 125I-alpha-bungarotoxin were incubated with patient plasma. Monoclonal mouse antibodies to human IgG subclasses 1-4 were added to the incubation and finally precipitated with anti-mouse IgG antibody. A maximal IgG subclass precipitation of 62-76% was determined with 125I-labelled myeloma IgG subclasses 1-4 added to normal human plasma. The anti-IgG subclass antibodies were added in excess which ensured that the precipitation of IgG2, IgG3 or IgG4 were unchanged, and that of IgG1 was only reduced by 17%, when the plasma IgG concentration was increased by a factor of two. The anti-IgG subclass antibodies were highly specific for their complementary subclasses. Determination of the IgG subclass of the anti-acetylcholine receptor antibodies from 8 patients with myasthenia gravis showed that IgG1 and IgG3 antibodies are predominant. This may support the hypothesis that complement mediated lysis of the neuromuscular end-plate plays a pathogenetic role in myasthenia gravis.     

Immunoglobulin G (IgG) and IgA subclass pattern of human antibodies to Shigella flexneri and Salmonella serogroup B and D lipopolysaccharide O antigens

The subclass distribution of human serum antibodies to the O-antigenic lipopolysaccharides of Salmonella serogroups B and D and to Shigella flexneri serotypes 1b, 2a, and 4a lipopolysaccharide antigens were analyzed in an enzyme-linked immunosorbent assay with monoclonal antibodies to the immunoglobulin subclasses. The patients had culture-verified Salmonella (17 Swedes) or Shigella flexneri (23 Vietnamese; 11 children and 12 adults) infections. Consecutive samples drawn during 1 year postinfection were investigated. Antibodies to the Salmonella antigens were mainly of the immunoglobulin G1 (IgG1), IgA1, and IgA2 subclasses. For the Salmonella serogroup B O polysaccharide, the IgA1 and IgA2 subclasses had peak values earlier than (6/9) or coinciding with the IgG1 (3/9) peak value. Furthermore, the IgA2 response to Salmonella serogroup B was positively correlated to the duration of the carrier state (P less than 0.001); the corresponding IgA1 response was less well correlated but was still significant (P less than 0.02). In the case of the Shigella flexneri O polysaccharide, specific antibodies appeared mainly in the IgG1 and IgA1 subclasses. Some IgG2 was also found, surprisingly even in very young patients. No subclass shift with time within the immunoglobulin classes was noted in any of the groups.     

不同剂量乙肝疫苗与HBIG联合免疫阻断HBV母婴传播的效果对比

目的 探讨10 μg和20 μg乙肝疫苗与HBIG联合免疫阻断HBV母婴传播的效果.方法 124例HBsAg阳性孕妇所生的婴儿随机分为两组,即10 μg乙肝疫苗组和20 μg乙肝疫苗组.婴儿于出生6h内及30 d分别注射200 IU HBIG,同时分别于出生24 h内、1个月及6个月注射3次10 μg或20 μg重组酵母乙肝疫苗.检测婴儿出生时以及1岁时血清HBV标志物.结果 两组新生儿血清HBsAg、HBeAg及抗-HBe阳性率与滴度之间差别均无统计学意义（P＞0.05）.所有新生儿血清HBV DNA水平均小于检测下限（500 U/ml）.出生12个月时,所有124例婴儿血清HBsAg和HBeAg检测结果均为阴性;血清HBV DNA水平均在检测下限以下;10 μg和20 μg乙肝疫苗组血清抗-HBs阳性率分别为90.3％和96.8％,差异无统计学意义（P＞0.05）;抗-HBs水平分别为325.5±342.2 mIU/ml和463.7±353.3 mIU/ml,后者显著高于前者（P=0.01）.而且,20 μg乙肝疫苗组产生高应答抗-HBs（＞ 100 mIU/ml）的比例显著高于10μg乙肝疫苗组（P =0.035）.结论 20 μg乙肝疫苗联合HBIG方案阻断HBV母婴传播的效果优于10 μg乙肝疫苗联合HBIG方案.     

T cell regulation of thyroglobulin autoantibody IgG subclasses in Hashimoto's thyroiditis

Microsomal and thyroglobulin (Tg) antibodies in patients with autoimmune thyroid disease are usually predominantly of subclasses IgG1 and/or IgG4 and the distribution pattern is characteristic for the serum of an individual. We have studied the role of T cells in synthesis of total IgG and Tg antibody IgG subclasses (measured by ELISA) in cultures of peripheral blood lymphocytes (PBL) from Hashimoto patients. Unfractionated PBL incubated with the T dependent activator pokeweed mitogen (PWM) synthesized IgG of all four IgG subclasses in the proportions 69% IgG1, 20% IgG2, 8% IgG3 and 3% IgG4; these values are similar to the proportions of the subclasses in serum. In contrast, the IgG subclass of Tg antibody was predominantly IgG1 in one patient, approximately equal proportions of IgG1 and IgG4 in four patients, and almost completely restricted to IgG4 in one patient; these patterns were similar to the subclass distribution of the autoantibodies in the individual patients' serum. B cells incubated alone secreted little Tg antibody but the response could be restored to the original levels and proportions of IgG1 and/or IgG4 Tg antibody by the addition of T cells either from the same individual or from another donor. Further, removal of suppressor T cells had little effect on the proportions of IgG1 and IgG4 Tg antibody although the total amounts of Tg antibody of both subclasses were sometimes increased. Our studies indicate that T cells are required in this in vitro system to elicit Tg antibody synthesis and to control the magnitude of the antibody response. However, the characteristic IgG subclass distribution of Tg antibody in an individual is determined at the level of the B cell.     

IgG subclasses in human chronic schistosomiasis: over-production of schistosome-specific and non-specific IgG4

IgG subclasses were determined quantitatively in sera from 63 Egyptian men who were infected with Schistosoma mansoni. Total and antigen-specific IgG was measured pre- and post-treatment. Total IgG subclass antibodies were determined by immunoradiometric assay (IRMA) using monoclonal antibodies (MoAbs). The anti-worm and anti-egg specific S. mansoni IgG subclass antibodies were quantitatively measured by ELISA using specific MoAbs and standards obtained by affinity chromatography. Our data show that total IgG of the patients was elevated in the range of two to three times above normal. The magnitude of increase differed markedly among the four subclasses of IgG. The IgG1, IgG2 and IgG3 concentrations were approximately two to four times higher than normal, whereas the IgG4 concentrations was 20 times normal (9000 mg/l). IgG1 and IgG4 tended to dominate the IgG subclass distribution of anti-soluble worm antigen preparation (SWAP) antibodies followed by IgG2 and IgG3. On the other hand, IgG1 and IgG2 dominated the IgG subclass distribution of anti-soluble egg antigen (SEA) antibodies. As with IgG1, IgG2 and IgG3, most IgG4 was non-specific. The role of IgG subclasses in the pathogenesis of schistosomiasis is not clear. However, the high concentration of IgG4 might act as IgE blocking antibody, possibly as anti-idiotypes that may play a role in down-regulation of the immune system when it is challenged with an excess of antigen.     

乙肝疫苗联合乙肝免疫球蛋白对阻断母婴垂直传播乙型肝炎病毒的临床分析

目的探讨孕妇产前用乙肝免疫球蛋白（HBIG）与乙型肝炎疫苗联合免疫阻断母婴传播的效果。方法将504例HBsAg（＋）孕妇分为A（预防组）,B（对照组）两组。A组：246名HBsAg阳性孕妇孕晚期每月分别注射基因重组型乙肝疫苗10μg、HBIG200IU（200IU/ml）,新生儿出生后采股静脉血,同时在出生后24h内注射HBIG200IU,然后在0、1、6月龄接种基因重组型乙肝疫苗,每次10μg。B组：258例产前未注射HBIG和基因重组型乙肝疫苗的HBsAg阳性孕妇,其所生新生儿在0、1、6（30μg、30μg、30μg）月龄只用基因重组型乙肝疫苗免疫。A、B两组婴儿都分别在0、3、6、9、12、24月龄静脉采血,用酶联免疫吸附试验（ELISA）检测HBV标志物,同时随访。结果A组的宫内感染率为3.25%,B组为4.16%,差异无统计学意义（χ^2=1.43,P〉0.05）。A组没有发生慢性HBV感染的婴儿,而B组中有7例婴儿发生慢性HBV感染,B组婴儿发生慢性HBV的感染率显著高于A组（χ^2=4.41,P〈0.05）。结论产前用HBIG和新生儿HBIG联合免疫可降低慢性HBV感染率,阻断宫内感染的慢性化,提高产程感染的阻断效果。