IgG subclass distribution of anti-ADAMTS13 antibodies in patients with acquired thrombotic thrombocytopenic purpura

Summary.  Background : ADAMTS13-neutralizing IgG autoantibodies are the major cause of acquired thrombotic thrombocytopenic purpura (TTP). Objective : To analyze the IgG subclass distribution of anti-ADAMTS13 antibodies and a potential relationship between subclass distribution and disease prognosis. Methodology : An enzyme-linked immunosorbent assay-based method was used to quantify the relative amounts of IgG subclasses of anti-ADAMTS13 antibodies in acquired TTP plasma. Results : IgG₄ (52/58, 90%) was the most prevalent IgG subclass in patients with acquired TTP, followed by IgG₁ (52%), IgG₂ (50%), and IgG₃ (33%). IgG₄ was found either alone (17/52) or with other IgG subclasses (35/52). IgG₄ was not detected in 10% of the patients. There was an inverse correlation between the frequency and abundance of IgG₄ and IgG₁ antibodies ( P  < 0.01). Patients with high IgG₄ levels and undetectable IgG₁ are more prone to relapse than patients with low IgG₄ levels and detectable IgG₁. Conclusions : All IgG subclasses of anti-ADAMTS13 antibodies were detected in patients with acquired TTP, with IgG₄, followed by IgG₁, antibodies dominating the anti-ADAMTS13 immune response. Levels of IgG₄ could be useful for the identification of patients at risk of disease recurrence.     

IgG4 subclass antibodies impair antitumor immunity in melanoma

Host-induced antibodies and their contributions to cancer inflammation are largely unexplored. IgG4 subclass antibodies are present in IL-10–driven Th2 immune responses in some inflammatory conditions. Since Th2-biased inflammation is a hallmark of tumor microenvironments, we investigated the presence and functional implications of IgG4 in malignant melanoma. Consistent with Th2 inflammation, CD22⁺ B cells and IgG4⁺-infiltrating cells accumulated in tumors, and IL-10, IL-4, and tumor-reactive IgG4 were expressed in situ. When compared with B cells from patient lymph nodes and blood, tumor-associated B cells were polarized to produce IgG4. Secreted B cells increased VEGF and IgG4, and tumor cells enhanced IL-10 secretion in cocultures. Unlike IgG1, an engineered tumor antigen-specific IgG4 was ineffective in triggering effector cell–mediated tumor killing in vitro. Antigen-specific and nonspecific IgG4 inhibited IgG1-mediated tumoricidal functions. IgG4 blockade was mediated through reduction of FcγRI activation. Additionally, IgG4 significantly impaired the potency of tumoricidal IgG1 in a human melanoma xenograft mouse model. Furthermore, serum IgG4 was inversely correlated with patient survival. These findings suggest that IgG4 promoted by tumor-induced Th2-biased inflammation may restrict effector cell functions against tumors, providing a previously unexplored aspect of tumor-induced immune escape and a basis for biomarker development and patient-specific therapeutic approaches.     

Specific IgG subclass antibodies, IgE and IgG S-TS antibodies to wheat gluten fraction B in patients with coeliac disease

Antibodies were measured in the sera of fifteen patients with untreated coeliac disease and twenty-eight patients with inflammatory bowel disease. Increased levels of specific IgG, IgG1, IgG2, and IgG4 antibody to wheat gluten fraction B, measured by an enzyme-linked immunosorbent assay, were shown in the coeliac disease group, but not in the inflammatory bowel disease group. No specific IgE antibody to fraction B was detected but 33% of the patients with coeliac disease had specific short-term sensitizing (anaphylactic) IgG antibody activity (IgG S-TS) to fraction B. There was no correlation between the IgG2 or IgG4 specific antibody and the presence of IgG S-TS activity.     

An autoanalyzer test to determine immunoglobulin class and IgG subclass of blood group antibodies

An automated antiglobulin test was used to characterize the immunoglobulin class and IgG subclass of red blood cell-bound antibodies. Immunoglobulin classes and IgG subclasses of some clinically significant antibodies (anti-D and anti-CD, anti-Fy, anti- Jka) as well as of clinically insignificant anti-Chido antibodies were determined. Eighteen anti-Duffy antibodies were structurally homogeneous, and were mostly composed only of IgG1. Of the 16 anti-Fya antibodies studied only three had IgG2, and four had IgM molecules. Ten anti-Jka antibodies were heterogeneous, but all samples were either IgG1 or IgG3, or both. The most prominent immunoglobulin of 12 hyperimmune anti-Rh (anti-D and anti-CD) antibodies was IgG1. Under the conditions of our test, we also detected IgG3 and very low concentrations of IgG2 and IgG4 molecules in the anti-Rh antibodies.     

Serologic and IgG subclass characterization of Cartwright (Yt) and Gerbich (Ge) antibodies

Examples of anti-Yta and anti-Ge were tested for reactivity with red cells treated with proteolytic enzymes and with antisera of known IgG specificity. Eight of 14 examples of anti-Yta did not react as well with cells treated with either papain or ficin; treatment with trypsin did not reduce reactivity. Four examples of the anti-Yta were IgG4; the others tested did not react with IgG subclass antisera. Of 13 examples of anti-Ge, seven failed to react with red cells treated with trypsin, ficin, or papain. Nine examples were IgG1, one was IgG1 plus IgG3, and three did not react with IgG subclass antisera.     

Quantitation of IgG subclass antibodies to pneumococcal capsular polysaccharides by ELISA, using Pneumovax-specific antibodies as a reference

A quantitative enzyme-linked immunosorbent assay (ELISA) method has been developed to assay the levels of IgG subclasses to pneumococcal capsular polysaccharides (PCP) by using a reference standard. This standard solution containing specific antibodies to a polyvalent pneumococcal vaccine (Pneumovax) was purified from the serum of an immunized healthy adult by affinity chromatography. In order to determine the predominant response to Pneumovax in the four IgG subclasses, specific IgG subclasses in preimmune and postimmune sera from six healthy adults were assessed quantitatively by the ELISA. With regard to peak concentrations after immunization, there was a marked increase in the IgG2 subclass, compared with those of IgG1 and IgG3. Such a quantitative assay of Pneumovax-specific IgG subclass antibodies is useful for the direct evaluation of immune responses to immunization with a polyvalent pneumococcal vaccine, and at the same time, for estimating the IgG2 response to PCP antigens in individuals.     

桥本甲状腺炎声像图与甲状腺功能指标的相关分析

目的:探讨桥本甲状腺炎超声图像特征与甲状腺功能指标的相关性.方法:回顾性分析68例临床确诊为桥本甲状腺炎患者的甲状腺二维及彩色多普勒(CDFI)超声图像特征,并与同期血甲状腺功能指标(包括FT3、FT4、TSH)结果相对照.结果:68例桥本甲状腺炎患者根据甲状腺功能指标检验结果分正常组、亢进组、减低组;各组间二维图像分型及血流分级均有统计学意义(P＜0.01):局灶性回声减低型主要见于正常组(7/8,87.5％),弥漫性回声增强型主要见于减低组(4/5,80％);CDFI方面,0级主要见于正常及减低组(21/22,95.45％),Ⅰ～Ⅱ级主要见于正常和亢进组(37/42,88.1％),Ⅲ级仅见于亢进组(4/4,100％).其中弥漫性回声减低型甲状腺实质内的血流分级与甲状腺功能有统计学意义(P＜0.01).结论:桥本甲状腺炎超声图像与甲状腺功能指标有一定的相关性,使用二维及CDFI能间接反应桥本甲状腺炎的甲状腺功能状态,协助临床进行诊断治疗.     

Nephelometry of human IgG subclass concentration in serum

An immunoassay method for determination of human IgG subclass concentration by rate nephelometry was developed by using the Beckman Immunochemistry Analyzer, subclass-specific antisera, and a human serum standard. Twelve sera derived from normal as well as gammapathological patients were analyzed. The total IgG concentration, as determined by using a readily available kit, correlated with the sum of the concentration of the individual subclasses. Most of the gammapathological cases were shown to be the IgG1 subclass, which was confirmed by radial immunodiffusion.     

IgG subclass concentrations in sera from 200 normal adults and IgG subclass determination of 106 myeloma proteins: an interlaboratory study

The IgG subclass protein concentrations in sera from 200 normal subjects were determined independently in two laboratories, using the same technique (radial immunodiffusion), the same subclass-specific antibodies and the same calibrator. The coefficients of correlation (rs) between IgG subclass concentrations determined in the two laboratories were 0.487, 0.883, 0.928 and 0.926 for IgG1, IgG2, IgG3 and IgG4, respectively (p less than 0.0005 in all four cases). By the chi-square test for goodness of fit, the frequency distributions observed in the two laboratories were found to differ significantly for all four subclasses (p less than 0.01). In one laboratory, the distributions of IgG1 and IgG2 were not significantly different from normal distributions, whereas the distributions of IgG3 and IgG4 deviated significantly. In the other laboratory, all four subclass distributions were significantly different from normal distributions. In the first laboratory, the IgG2 concentrations were log-normal distributed, whereas in the second, IgG1, IgG2 and IgG4 concentrations were log-normal distributed. We conclude that use of identical reagents does not ensure identical frequency distributions. This finding emphasizes the need for standardization of the measurement technique too. Furthermore, we argue that at present, intralaboratory reference intervals for IgG subclass protein concentrations are necessary. The reference intervals should be based on non-parametric statistics. The subclass of 106 monoclonal IgG proteins, which were demonstrated by agarose gel electrophoresis, was identified by RID for 89 samples. The subclass of the remaining 16 M-components was readily determined by qualitative immunoelectrophoretic analysis using the same subclass-specific antibodies.     

Sequential changes in the relative affinity of antibodies synthesized during the immune response

The anti-2,4-dinitrophenyl (DNP) antibodies synthesized by suspensions of lymph node cells obtained at various intervals from rabbits that had been immunized with DNP-bovine γ-globulin increased progressively in their affinity for the dinitrophenyl determinant. This change accompanied and was apparently responsible for a similar change in the binding properties of anti-DNP antibodies isolated from the serum. The rate of change in affinity was related to the dose of immunogen: increasing the dose delayed the change. The antibodies formed during a brief (5 hr) incubation in vitro were heterogeneous in their binding properties. Therefore, the mixing in the circulation of molecules synthesized at different times may contribute to, but is not alone responsible for, the heterogeneity in the serum antibodies. Variability in binding did not appear to be related to heterogeneity in immunoglobulin class. Indeed, the variations in relative affinity occurred entirely within the γG-immunoglobulins.     

桥本甲状腺炎患者白介素-2及受体与甲状腺过氧化物酶抗体的关系

目的：探讨桥本甲状腺炎（HT）患者外周血中白介素-2（IL-2）及白介素-2受体（IL-2R）与甲状腺过氧化物酶抗体（TPoAb）的关系。方法：将86例HT患者分为初诊、复诊2组,并另选20名健康体检者作为正常对照组。采用酶联免疫吸附（ELISA）夹心法检测受检者外周血中的IL-2及IL-2R,并采用微粒子酶免化学发光分析法检测TPoAb浓度水平,观察IL-2及IL-2R与TPoAb的关系。结果：IL-2在初诊HT组、复诊HT组与正常对照组中的含量分别为（25.01±3.31）ng/L、（23.17±5.89）ng/L和（24.05±3.92）ng/L,3组间两两比较,差异无统计学意义（P〉0.05）;IL-2R在初诊HT组、复诊HT组和正常组水平分别为（1125.94±743.87）ng/L、（722.93±640.06）ng/L和（293.48±113.35）ng/L,3组间两两比较,差异均有统计学意义（P均〈0.05）。复诊HT组的TPoAb含量为（169.75±44.36）IU/L,高于正常组的（8.30±1.70）IU/L,低于初诊HT组的（460.96±299.05）IU/L,3组间两两比较,差异均有统计学意义（P均〈0.05）。HT患者外周血中的IL-2R含量与TPoAb水平呈正相关（r=0.696,P〈0.01）。结论：HT患者外周血IL-2R含量升高,且与TPoAb水平呈正相关,检测IL-2R含量有助于观测患者的免疫功能状态。     

IgE and IgG4 subclass in atopic families.

Raised levels of IgG4 were present in twelve (35%) of thirty-four asthmatic children and raised levels of IgE in twenty-three (68%). Eighteen of 104 first degree relatives also had raised levels of IgG4, thirteen had no history of atopic disease and nine failed to give positive skin reactions to Dermatophagoides pteronyssinus or to mixed grass pollens. Fifteen relatives had raised IgE, five without symptoms. No relationship was noted between either raised levels of IgE or IgG4 and infant feeding. Although these immunoglobulin patterns were not consistently associated with symptoms they did tend to be associated in atopic families and it is suggested that the same polygenic factors may govern the IgE and IgG4 levels.     

Characterization of the thyroid microsomal antigen, and its relationship to thyroid peroxidase, using monoclonal antibodies.

MAb directed to the thyroid microsomal antigen have been developed. All bound to 101- and 107-kD bands in Western blot analysis using thyroid microsomal fraction as antigen. The MAb also bound to microsomal proteins immunoprecipitated by serum from patients having a high titer of anti-microsomal antibody but no antibodies to thyroglobulin or thyrotropin-stimulating hormone receptor. The pattern of binding was related to the amount of reducing agent. The 101- and 107-kD bands were increased by addition of dithiothreitol whereas, in its absence, numerous bands of higher molecular weight were present, suggesting a multimeric protein structure. Despite the inability to immunoprecipitate thyroid peroxidase (TPO) enzymatic activity, the MAb bound intensively in Western blot to denatured purified hog TPO and to denatured immunopurified human TPO. Trypsin digestion of the 101-107-kD antigen produced a doublet of 84-88 kD that was still immunoreactive with MAb. One of five polyclonal sera tested (with a microsomal antibody titer greater than 1/20,480 measured by the tanned red cell hemagglutination technique) also recognized the 84-88 kD trypsin fragments. Addition of V8 protease led to a disappearance of the 107-kD protein, but not the 101-kD protein, proving that this antigen is formed by two different polypeptides. The MAb bound strongly to thyroid epithelium, whereas binding to papillary carcinoma was absent or low and moderate for follicular and Hurthle cell carcinoma. This study indicates that the thyroid microsomal antigen and TPO are identical and are constituted of two different polypeptides. On SDS-PAGE the antigen appears as two contiguous bands which share common epitopes but are not identical, as proven by their size and difference in susceptibility to proteolytic digestion. The immunoreactivity of the molecule is highly dependent on a trypsin-sensitive site, which appears important in the recognition of the antigen by polyclonal sera and may have biological importance. The expression of microsomal antigenicity is variable among various thyroid carcinomas.     

Evaluation by enzyme-linked immunosorbent assay of IgG anti-D and IgG subclass concentrations in immunoglobulin preparations

BACKGROUND: Anti-D immunoglobulin preparations are injected to prevent hemolytic disease of the newborn. The concentration of IgG anti-D in these preparations is usually determined by an automated hemagglutination technique using as a reference a calibrated preparation of anti-D, but the method requires special equipment and cannot be routinely applied to measure the IgG subclasses of anti-D in these preparations. STUDY DESIGN AND METHODS:Taking advantage of a recently described enzyme-linked immunosorbent assay (ELISA) for the determination of the anti-D concentration in sera of alloimmunized pregnant women, IgG anti-D and IgG subclass concentrations were measured in the international reference preparation (IRP) coded 68/419, 10 anti-D immunoglobulin preparations, and sera of 15 D-immunized volunteers. RESULTS: An IgG anti-D concentration of 61.5 +/- 4.8 microg per ampoule (mean +/- SD) was found by ELISA in IRP 68/419.This result was in agreement with previous determinations obtained by radioimmunoassay (60 microg/ampoule). The IgG subclass concentration of anti-D in this preparation was 48.4 microg of IgG1 (78.6%), 3.0 microg of IgG2 (4.8%), 9.7 microg of IgG3 (15.8%), and 0.4 microg of IgG4 (0.7%). The mean proportion of IgG subclasses of anti-D in 10 immunoglobulin preparations was similar (81.7% for IgG1, 5.0% for IgG2, 12.7% for IgG3, and 0.6% for IgG4). In the sera of 15 immunized volunteers, the IgG anti-D concentration varied from 3.1 to 68.4 microg per mL. The mean IgG subclass composition of anti-D was 79.3 percent for IgG1, 2.2 percent for IgG2, 18.1 percent for IgG3, and 0.4 percent for IgG4. The proportions of IgG3 anti-D in these sera were found to range between 1 percent and 87 percent, as in the sera of D-alloimmunized pregnant women. CONCLUSION: ELISA provides an alternative to the radioimmunoassay and the automated hemagglutination technique. In addition, it allows the evaluation of the absolute concentration of each IgG subclass of anti-D in immunoglobulin preparations and necessitates only the conventional equipment required for an immunoenzymatic assay.     

Relationships between relative binding affinity and electrophoretic behavior of rabbit antibodies to streptococcal carbohydrates

After repeated intravenous injections with Group C streptococcal vaccine, most rabbit antisera were shown to contain one or more IgG antibody components, as revealed by microzone electrophoresis. A procedure for the fractionation of multiple IgG antibody components from such streptococcal antisera is described. Separation is achieved on the basis of differences in relative binding affinities of the antibody components to immunoabsorbent columns. The evidence suggests that the electrophoretic mobility, and thus the net charge of an antibody, bears a reciprocal relationship to its binding affinity for the streptococcal Group C antigens. Furthermore, the relative binding affinity affords another means to assess the functional homogeneity of streptococcal antibodies. A possible relationship between light chain variable-region subclasses and binding affinities of streptococcal antibodies is discussed.     

Immunoblot analysis of IgM and IgG subclass responses in murine toxoplasmosis

The diagnosis of toxoplasmosis still relies heavily on serological methods, particularly the dye test. In order to further elucidate the role of humoral immunity and to distinguish parasite antigens which might be used in new, more specific diagnostic tests, the antibody response in murine toxoplasmosis was examined. Specific antibody was measured by the dye test in sequential serum samples collected at intervals from 3 to 98 days post inoculation. The peptides involved in the immune response were characterised by probing blots with IgM and the four murine IgG isotypes. Antibody was detectable by day 10 and rose to a titre of ≤ 16,000 by day 28. Immunoblotting revealed multiple bands on both IgM and IgG blots as early as day 3. Six intense bands with approximate molecular weights of 80, 67, 43, 35, 30 and 28 kDa developed early and persisted throught the course of infection. Several of these were thought to be surface antigens of the parasite. The initial and strongest IgG response by immunoblotting was seen with the IgG2b isotype, which is believed to play an important role in antibody-dependent cell-mediated cytotoxicity.     

Impact of intrinsic affinity on functional binding and biological activity of EGFR antibodies

Aberrant expression and activation of EGF receptor (EGFR) has been implicated in the development and progression of many human cancers. As such, targeted therapeutic inhibition of EGFR, for example by antibodies, is a promising anticancer strategy. The overall efficacy of antibody therapies results from the complex interplay between affinity, valence, tumor penetration and retention, and signaling inhibition. To gain better insight into this relationship, we studied a panel of EGFR single-chain Fv (scFv) antibodies that recognize an identical epitope on EGFR but bind with intrinsic monovalent affinities varying by 280-fold. The scFv were converted to Fab and IgG formats, and investigated for their ability to bind EGFR, compete with EGF binding, and inhibit EGF-mediated downstream signaling and proliferation. We observed that the apparent EGFR-binding affinity for bivalent IgG plateaus at intermediate values of intrinsic affinity of the cognate Fab, leading to a biphasic curve describing the ratio of IgG to Fab affinity. Mathematical modeling of antibody-receptor binding indicated that the biphasic effect results from nonequilibrium assay limitations. This was confirmed by further observation that the potency of EGF competition for antibody binding to EGFR improved with both intrinsic affinity and antibody valence. Similarly, both higher intrinsic affinity and bivalent binding improved the potency of antibodies in blocking cellular signaling and proliferation. Overall, our work indicates that higher intrinsic affinity combined with bivalent binding can achieve avidity that leads to greater in vitro potency of antibodies, which may translate into greater therapeutic efficacy.     

上呼吸道感染患儿肺炎支原体IgM类与低亲和力IgG类抗体的测定

目的探讨上呼吸道感染患儿肺炎支原体（MP）IgM类（MP—IgM）与低亲和力IgG类（MP—IgG）抗体水平及在MP感染早期诊断中的价值。方法分别用被动颗粒凝集试验（PPA）、酶联免疫吸附试验（ELISA）和间接免疫荧光法（IIF）对小儿上呼吸道感染者血清MP—IgM水平进行测定,同时用IIF法研究其低亲和力MP—IgG水平,对所测结果进行对比研究。结果3种方法所测MP—IgM以ELISA法阳性率最高（60．0％,51／88）,PPA法次之（50．0％,44／88）,而IIF法最低（40．9％,36／88）。各方法间仅PPA法与ELISA法所测结果具有显著相关（P〈0、001）和中等程度的一致性（0．7〉Kappa〉0．4）,而PPA法与IIF法、ELISA法与IIF法检测结果缺乏一致性。88例患儿中,有21例（23．9％）存在低亲和力MP—IgG抗体,其中17例同时有MP—IgM阳性,两者结果亦具有显著的相关（P〈0．001）和一定程度的一致性（0．7〉Kappa〉0．4）。结论低亲和力MP-IgG类抗体有类似于MP—IgM类抗体的早期诊断价值,与MP—IgM联合检测有助于区分MP近期感染与感染后复发或再次感染。