Usefulness of IgG4 subclass antibodies for diagnosis of human clonorchiasis

The present study analyzed serum IgG subclass antibody reaction to major antigenic bands of Clonorchis sinensis to investigate improvement of its serodiagnosis. Of the four subclass antibodies, IgG1 and IgG2 antibodies were produced but not specific, IgG3 antibody was least produced, and IgG4 antibody was prominent and specific. The serum IgG antibody reaction to any of 43-50, 34-37, 26-28, and 8 kDa bands was found in 65.5% of 168 egg positive cases while IgG4 antibody reaction was found in 22.0% of them. The positive rates of IgG and IgG4 antibodies were directly correlated with the intensity of infection. All of the sera from heavily infected cases over EPG 5,000 showed positive reaction for specific IgG and IgG4 antibodies. The specific serum IgG4 antibody disappeared within 6 months after treatment. The bands of 35 kDa and 67 kDa cross-reacted with IgG antibodies but not with IgG4 antibodies in sera of other trematode infections. The present findings suggest that serum IgG4 antibody reaction to 8 kDa band is specific but not sensitive. Any method to increase its sensitivity is required for improved serodiagnosis.     

用间接免疫荧光法检测流行性出血热病人尿中特异性抗体的研究

用间接免疫荧光法检测110例不同病程、病期及病型的流行性出血热病人尿中及血清中特异性抗体。尿中IgM型抗体阳性率为62.7％。尿中IgG型抗体阳性率91.8％与血清IgG型者90.9％相似,而总阳性率(IgG或IgM有一项以上阳性者的总检出率)99.1％则高于血清IgG者。20例其它疾病及10例正常人尿抗体均为阴性。结果表明尿抗体检查法是特异且可靠的,它比血清学方法简便、灵敏、为临床诊断可早期快速得出结果,不用采血有利于病人。IgM型抗体阳性率受病程、病期、病型及尿蛋白量的影响较明显。     

Protective effect of colostrum in Mycoplasma pneumoniae infection induced in infant mice

The immunological responses and mechanism of maternal immunity in Mycoplasma pneumoniae infection of mice were investigated. ICR female mice, 4 weeks old, and infant mice, 2 to 4 days old, were infected with M. pneumoniae. Anti-M. pneumoniae antibodies in serum and colostrum were determined by enzyme-linked immunosorbent assay. The specific IgG antibody production persisted for 9 months or longer in both the young and infant mice. These infected mice were protected from rechallenge with M. pneumoniae. In addition, the infected dams conferred passive immunity on their offspring. The infant mice born to uninfected normal dams were protected from the challenge with M. pneumoniae when fed by infected foster dams. Conversely, the infant mice born to infected dams were not protected from the challenge with M. pneumoniae when the infants were fed by uninfected dams. The specific IgG antibody appeared in serum of infant mice inoculated orally with M. pneumoniae-infected mouse serum and the infants were protected from challenge with M. pneumoniae, while the infants given protein A-absorbed serum were not protected from the challenge. These results suggest that one of the factors involved in the resistance of infant mice to M. pneumoniae infection is the specific IgG antibody present in the colostrum rather than the result of transplacental transfer.     

Activation of a functional idiotype network response by monoclonal antibody specific for a virus (M-MuLV)-induced tumor antigen

BALB/c mice were injected with IgM mAb specific for Moloney murine leukemia virus (M-MuLV)-determined cell surface Ag in an attempt to inhibit Moloney sarcoma growth. The monoclonal IgM significantly inhibited sarcoma growth when given to the mice after inoculation with Moloney murine sarcoma/leukemia virus, and also potentiated the in vivo antibody response specific for M-MuLV Ag. These responses were significantly greater than the primary response to the virus alone in age- and sex-matched control mice, and were also seen in mice which were injected with the IgM antibody only and not with virus, suggesting that an Ag-independent mechanism may be involved. The M-MuLV-specific serum antibody responses induced by the monoclonal IgM, with or without prior virus inoculation, were predominantly of the IgG1 isotype, with some IgG2a; no other isotypes were found to have titers significantly higher than in the normal response to virus alone. M-MuLV-specific IgG1 was detected only in mice injected with monoclonal IgM, and not in the response to virus alone. The same sera also had high titers of anti-idiotypic antibodies, (Ab2), as well as anti-anti-idiotypic antibodies (Ab3). It appears, therefore, that passive immunization with M-MuLV-specific IgM mAb activates an idiotypic network, which results in both Ab2 and Ab3 responses; the M-MuLV-specific response may be considered a subset of Ab3.     

Reaginic antibody formation in the mouse. VI. Suppression of IgE and IgG antibody responses to ovalbumin following the administration of high dose urea-denatured antigen.

The major antigenic determinants in ovalbumin molecules (OA) were lost following denaturation in 8 M urea. The urea-denatured antigen (UD-OA) failed to combine with anti-OA antibody, but was capable of priming mouse T cells specific for OA. BDF₁ mice primed with alum-precipitated OA were given three intravenous injections of 100 μg UD-OA at 3, 5, and 7 days after the primary immunization. The treatment with UD-OA suppressed both IgE and IgG antibody responses to OA. The same treatment of OA-primed animals with intravenous injections of OA resulted in suppression of IgE antibody response but enhanced IgG antibody response. Intravenous injections of either OA or UD-OA suppressed both IgE and IgG anti-DNP antibody responses of DNP-OA-primed animals but failed to suppress anti-hapten antibody responses to DNP-keyhole limpet hemocyanin. The effect of the treatment on helper T cells and B memory cells in OA-primed animals was studied by adoptive transfer experiments. The results showed that the OA-treatment as well as UD-OA-treatment suppressed the development of both helper function of T cells and B memory cells in the spleen, but the UD-OA treatment was more effective than OA-treatment for the suppression of B cell development. Possible mechanisms for the suppression of the development of immunocompetent cells were discussed.     

In vitro evidence indicating a role for the Fc region of IgG in the mechanism for the long-term maintenance and regulation of antibody levels in vivo

The synthesis of anti-human serum albumin (HSA) antibody was induced spontaneously in cell cultures prepared from the draining lymph nodes of rabbits immunized months earlier with polymerized HSA. Serum from HSA-immunized rabbits suppressed this response. Removal of specific antibody from immune serum eliminated suppression and the addition of specific IgG restored suppression, indicating that the feedback phenomenon may be explained by an effect of specific IgG antibody. Fab and F(ab′)₂ fragments masked antigen as effectively as IgG; however, they were markedly inferior to IgG in mediating suppression. Furthermore, Fab competed with IgG and interfered with IgG mediated suppression. The addition of small amounts of antigen to antibody-suppressed cultures induced an antibody response. The level of induction was proportional to the antigen-antibody ratio. However, 80 to 100 times the antibody concentration needed to mask all antigenic determinants was needed in order to eliminate induction of antibody synthesis. High concentrations of antigen-antibody complexes at equivalence also suppressed the spontaneous response. This suppression was similar to antibody mediated suppression at the spontaneous response in that the Fc region of IgG was required.     

Ig allotype-linked regulation of class and subclass composition of natural antibodies to group A streptococcal carbohydrate

To determine the importance of genes located in or near the Ig constant regions in regulating the human antibody response, we correlated Ig allotypic markers with total Ig concentrations and natural antibody concentrations to the streptococcal group A carbohydrate (A-CHO) in 193 healthy adult blood donors. The major correlations between Ig allotypes and total Ig and specific antibody concentrations were observed with the Gm(f;n;b) haplotype. When compared with Gm(f;n;b) negative individuals, Gm(f;n;b) positives had significantly higher concentrations of total IgG2 (p less than 0.001) and IgG2 anti A-CHO (p less than 0.05), lower concentrations of total IgG1 (p less than 0.001) and IgG1 anti A-CHO (p less than 0.001), and lower concentrations of total IgM (p less than 0.001) and IgM anti A-CHO (p less than 0.05). We conclude that individuals with the Gm(f;n;b) haplotype respond preferentially with IgG2 rather than IgG1 subclass antibodies. This increased capacity to respond with IgG2 antibodies may be reflected in the magnitude of the total antibody response when the IgG2 subclass comprises a major proportion of the response, as occurs in the adult response to many polysaccharide Ag.     

Comparison of Direct and Indirect Solid-Phase Microradioimmunoassays for the Detection of Viral Antigens and Antiviral Antibody

Viral antigens were fixed to the surface of microtiter wells, and serial dilutions of antiviral antibody were added. The amount of antiviral antibody bound to viral antigens was determined by measuring the extent to which the antiviral antibody either inhibited the specific binding of (125)I-labeled antiviral immunoglobulin G (IgG) (direct technique) or enhanced the specific binding of (125)I-labeled anti-IgG (indirect technique). Immune complexes composed of viral antigens and antiviral antibody (human) could be detected by the binding of (125)I-labeled rheumatoid factor. Specific binding was influenced by the concentration of protein in the diluents used during the different steps of the procedure. A high concentration of protein in the diluent used with the viral antigens decreased specific binding, whereas a high concentration of protein in the diluent used with (125)I-labeled anti-IgG increased specific binding by decreasing nonspecific attachment of the labeled anti-IgG. Under the conditions employed, the titer of a given antiviral serum was several hundredfold greater by the indirect than by the direct technique.     

Biological characterization of Campylobacter fetus lipopolysaccharides

Abstract Ten patients with chronic liver disease, seven healthy seropositive individuals with a remote history of rubella, and three patients with acute rubella were examined for serum levels of IgG subclasses and subclass antibodies against rubella virus structural proteins. One patient with AICAH had no detectable total or rubella specific IgG3 or IgG4. The liver disease patients were hypergammaglobulinemic and had greatly raised IgG1 levels. Patients with acute rubella lacked antibodies to the rubella virus E2 protein and showed no IgG4 antibody response. The liver disease patients showed a somewhat weaker IgG4 antibody response against the core (C) protein than healthy controls. However, differences are suggested within the subclasses in antibody reactivity against the individual rubella virus antigens. It is concluded that test systems that discriminate reactivities against individual antigens have to be used for characterization of viral antibody subclass profiles.     

Role of antigen-specific T cell help in the generation of in vivo antibody responses. II. Sustained antigen-specific T cell help is required to induce a specific antibody response.

The injection of mice with a goat or rabbit antibody to mouse IgD stimulates a large polyclonal IgG response, approximately 10% of which is specific for antigenic determinants on the anti-IgD antibody molecule. The large goat IgG (GIgG)-specific antibody response in mice injected with goat antibody to mouse IgD requires that GIgG-specific B cells undergo much greater clonal expansion than B cells specific for other Ag. One possible explanation for the greater clonal expansion of GIgG-specific B cells is that B cells that lack GIgG specificity can only be stimulated with GIgG-specific T help during the relatively short time that anti-IgD binds to, and is processed and presented by, these B cells before they cease to express membrane mIgD. In contrast, GIgG-specific B cells can continue to bind, process, and present GIgG through mIgM after they lose mIgD. To test the hypothesis that extended stimulation with Ag-specific T help is required to generate a specific antibody response, we determined time requirements for Ag-specific T cell help for the development of such a response. Mice were injected with rabbit antibody to mouse IgD plus one or more daily injections of FITC conjugated to a F(ab')2 fragment of rabbit IgG (FITC-(Fab')2), which has a short in vivo half-life, and IgG1 anti-FITC antibody production was analyzed. In this system, each additional injection of FITC-F(ab')2 extends the period during which FITC-specific B cells can process this Ag and present it to rabbit IgG-specific T cells. Each additional injection of FITC-F(ab')2 stimulated a several-fold increase in IgG1 anti-FITC antibody levels, and injections on 5 consecutive days were required to induce a maximal anti-FITC response. These observations provide evidence that sustained Ag-specific T cell help is required to stimulate the degree of B cell clonal expansion that characterizes a specific antibody response.     

Isotype specific immune responses in murine experimental toxocariasis

In this work, a murine experimental model of toxocariasis has been developed in BALB/c, C57BL/10 and C3H murine strains orally inoculated with 4,000 Toxocara canis embryonated eggs, in order to investigate the isotype-specific immune responses against excretory-secretory antigens from larvae. T. canis specific IgG+M, IgM, IgG, IgA, IgG1, IgG2a and IgG3 were tested by ELISA. The dynamics of the specific immunoglobulins (IgG+IgM) production showed a contrasting profile regarding the murine strain. Conversely to the results obtained with the IgM isotype, the IgG antibody class showed similar patterns to those obtained with IgG+IgM antibodies, only in the case of the BALB/c strain, being different and much higher than the obtained with IgG+IgM antibodies, when the C3H murine strain was used. The antibodies IgG+IgM tested in BALB/c and C57BL/10 were both of the IgM and IgG isotypes. Conversely, in the C3H strain only IgG specific antibody levels were detected. The IgG1 subclass responses showed a similar profile in the three murine strains studied, with high values in BALB/c, as in the case of the IgG responses.     

树鼩IgG纯化鉴定及其多克隆抗体制备和检测

目的:比较两种抗体纯化方法在分离纯化树鼩IgG抗体的应用,制备抗IgG的多克隆抗体及检测。方法:采用两种商品化IgG抗体纯化试剂盒分离树鼩血清IgG抗体,采用SDS-PAGE和蛋白定量测定提纯IgG。以树鼩IgG作为抗原,与等量弗氏完全佐剂(第一次)、弗氏不完全佐剂(第二次)混合皮下注射免疫兔,对分离血清进行多克隆抗体纯化及Western Blot检测及定量分析。结果:两种方法均能有效分离纯化树鼩IgG,在经过Montage PROSEP-A试剂纯化后的IgG在纯度和含量方面均优于Protein A/G Matrix试剂。通过纯化后的树鼩IgG免疫兔制备的抗IgG抗体能有效识别树鼩IgG。结论:纯化的树鼩IgG具有良好免疫原性,由此制备的抗体具有高度特异性。研究结果为利用树鼩作为实验动物提供了必要的实验基础。     

Transition in the Character of Immunological Memory in Mice after Immunization

The immunological memory in antibody response of mice to bovine serum albumin was investigated at the level of IgM and IgG antibody-forming cells. The antigen at a dose much lower than required for eliciting a detectable level of the primary antibody response could latently activate the immune machinery to an extent adequate for specific recall, whereas higher doses of antigen were effective in evoking strong anamnestic response. The potentiality to develop the anamnestic response was found even in the latent phase of the primary antibody response and was maintained for more than 2 months. The immunological memory acquired in an early phase after the primary immunization mainly involved IgM antibody response and late memory concerned IgG response.     

Airway IgG counteracts specific and bystander allergen-triggered pulmonary inflammation by a mechanism dependent on Fc gamma R and IFN-gamma

Besides IgE, the Ab isotype that gives rise to sensitization and allergic asthma, the immune response to common inhalant allergens also includes IgG. Increased serum titers of allergen-specific IgG, induced spontaneously or by allergen vaccination, have been implicated in protection against asthma. To verify the interference of topical IgG with the allergen-triggered eosinophilic airway inflammation that underlies asthma, sensitized mice were treated by intranasal instillation of specific IgG, followed by allergen challenge. This treatment strongly reduced eosinophilic inflammation and goblet cell metaplasia, and increased Th1 reactivity and IFN-gamma levels in bronchoalveolar lavage fluid. In contrast, inflammatory responses were unaffected in IFN-gamma-deficient mice or when applying F(ab')(2). Although dependent on specific allergen-IgG interaction, inflammation triggered by bystander allergens was similarly repressed. Perseverance of inflammation repression, apparent after secondary allergen challenge, and increased allergen capture by alveolar macrophages further characterized the consequences of topical IgG application. These results assign a novel protective function to anti-allergen IgG namely at the local level interference with the inflammatory cascade, resulting in repression of allergic inflammation through an FcgammaR- and IFN-gamma-dependent mechanism. Furthermore, these results provide a basis for topical immunotherapy of asthma by direct delivery of anti-allergen IgG to the airways.     

Solid phase radioimmunoassay for quantitation of antigen-specific IgG in human sera with 125I-protein A from Staphylococcus aureus.

Radiolabeled protein A from Staphylococcus aureus (Staph A) has been used to develop a solid phase, noncompetitive radioimmunoassay for quantitation of specific IgG antibody. The assay involves two incubations: First, agarose-insolubilized antigen is mixed with serum samples for 1 to 4 hr during which specific antibody is bound; second, after a washing procedure, the solid phase immune complexes are incubated for 4 to 18 hr with 125I-Staph A, during which the radiolabeled detection protein binds to the insolubilized specific IgG antibody. In a comparative study of the IgG antiphospholipase A antibody content of 23 human sera drawn from honeybee venom-sensitive patients, resulted of the Staph A assay correlated highly (r = 0.981, p less than 0.001, N = 23) with those obtained from a liquid phase, competitive radioimmunoprecipitation (double antibody) assay. The two assays demonstrated comparable precision, sensitivity, and reproducibility. In contrast, the use of 125I-Staph A in the solid phase radioimmunoassay was superior to 125I rabbit anti-human IgG because of lower negative serum (blank) values, shorter time required to reach equilibrium binding, and greater precision and reproducibility. In principle, the 125I Staph A assay may be applied ot IgG quantitation for crude allergen extracts as well as purified antigens. Furthermore, the sera of a number of mammalian species may be studied without further modification.     

内蒙地区发热患者血清中冠状病毒抗体的检测

目的分析内蒙地区发热患者中冠状病毒的感染情况。方法以SARS冠状病毒感染Vero细胞涂片为冠状病毒抗原片,用间接免疫荧光法分别检测55例发热患者和68例正常人血清中冠状病毒的IgG、IgM抗体。结果发热患者血清中冠状病毒IgG抗体和IgM抗体阳性率分别为29.1%(16/55)和10.9%(6/55),而正常人血清中只检测到2.9%(2/68)的IgG抗体,且未检测到IgM抗体,2组患者的IgG和IgM抗体阳性率比较差异均有显著性;随机选取7例患者的IgG阳性血清进行SRAS冠状病毒的特异性抗体封闭实验,结果有6例血清仍为阳性,有1例血清转为阴性,说明冠状病毒IgG抗体阳性血清中85.7%为普通冠状病毒特异性,14.3%为SARS冠状病毒特异性。结论普通冠状病毒是内蒙地区发热患者的主要病原体之一,部分患者还存在SARS冠状病毒的既往感染。     

IgE and IgG are synergistic in antigen-mediated release of thromboxane from human lung macrophages.

The mechanism of IgE-mediated release of thromboxane A2 from human lung macrophages has been studied using a monoclonal chimeric human/mouse IgE antibody and its specific antigen. The cells could be sensitized at 37 degrees C but not at 4 degrees C by incubation with IgE, and released a significant amount of thromboxane A2 (TXA2), measured as the stable hydrolysis product TXB2, in response to an anti-chimeric IgE antibody. In contrast, stimulation of IgE-sensitized macrophages with the specific antigen produced less than 10% of this response. A similar time course for the release of TXB2 and the formation of inositol monophosphate in the presence of LiCl was observed. Cleavage of the Fc domain of the anti-chimeric IgE antibody substantially eliminated its capacity to stimulate IgE-sensitized cells. However, the weak or undetectable response to chimeric IgE plus specific antigen was substantially potentiated by an antigen-specific chimeric IgG antibody. IgG-sensitized macrophages did not respond to antigen challenge by the release of TXB2. Preincubation of the cells with a monoclonal antibody against the low affinity receptor for IgE (Fc epsilon RII/CD23) did not prevent IgE sensitization. We conclude that cell-bound IgE antibody cannot induce the release of TXB2 but has fixed antigen which then must interact with specific IgG antibody and IgG receptors to induce mediator release.     

IL-4 is required to generate and sustain in vivo IgE responses

Antibodies of the IgE isotype play a predominant role in immediate hypersensitivity reactions. IL-4, a T cell-derived lymphokine that stimulates increased Ia expression by resting B cells and increased IgG1 secretion by LPS-activated B cells in vitro, has also been shown to regulate in vitro and in vivo polyclonal IgE responses. We report that large quantities of a purified anti-IL-4 mAb inhibit primary in vivo polyclonal IgE responses by 99% in mice infected with Nippostrongylus brasiliensis or injected with anti-IgD antibodies, and totally inhibit secondary Ag-specific IgE responses to TNP-keyhole limpet hemocyanin without effect on either IgG1 or IgG2a responses to these stimuli. The lack of effect of anti-IL-4 antibody on IgG1 secretion cannot be explained simply by inadequate neutralization of IL-4, inasmuch as the doses of anti-IL-4 antibody used blocked an N. brasiliensis-induced increase in B cell Ia expression by more than 85%, whereas in vitro studies indicate that enhancement of B cell Ia expression requires less IL-4 than induction of IgG1 secretion. In addition to demonstrating that IL-4 plays a necessary role in the generation of an in vivo IgE response, we show that IL-4 has an important role in sustaining established IgE responses, because anti-IL-4 antibody, when given at the peak of an N. brasiliensis- or TNP-keyhole limpet hemocyanin-induced IgE response, accelerates the declines in total serum IgE and in IgE anti-TNP antibody levels, respectively. These observations suggest that the effects of IL-4 on in vivo immune responses may be more specific than might have been predicted from in vitro observations, and that regulation of IL-4 production or action may be useful for the prevention or therapy of immediate hypersensitivity disorders.     

Control of nonspecific staining in the fluorescent antibody technique for the detection of salmonellae in foods.

A fluorescent antibody conjugate, prepared from the IgG (immunoglobulin G) fraction of Salmonella polyvalent flagellar antiserum, gave better specific staining intensities and significantly lower nonspecific staining than did conjugates prepared from globulin fractions of ammonium sulfate-fractionated Salmonella polyvalent antisera. IgG was purified by affinity chromatography against protein A, a normal cell wall component of Staphylococcus aureus. Affinity chromatography yielded high-purity IgG in a one-step purification procedure. The conjugate prepared from affinity-purified IgG was compared with commercially available fluorescent antibody conjugates for the detection of salmoneallae in retail samplings of meats and poultry and gave better correlations with the cultural method than did the commercial conjugates.     

High-dose,unlabeled, nonspecific antibody pretreatment: influence on specific antibody localization to human melanoma xenografts

Summary Nonspecific uptake of radiolabeled monoclonal antibodies in normal tissues is a significant problem for tumor imaging. A potential means of decreasing nonspecific antibody binding is to blockade nonspecific antibody binding sites by predosing with cold, nonspecific isotypematched antibody, before injecting specific antibody. Nontumor-specific murine monoclonal antibody LK2H10 (IgG1) or Ab-1 (IgG2a) was given i.v. at doses of 0 to 3.5 mg to nude mice with xenografts of human melanoma. These mice were then given i.v. 4 g of ¹³¹I anti-high molecular weight antigen of melanoma (HMWMAA) monoclonal antibody 763.24T (IgG1) or 225.28S (IgG2a), respectively. These mice were also given a tracer dose of ¹²⁵I LK2H10 or Ab-1, respectively. Specific tumor uptake of anti-HMWMAA antibodies was see in all cases. No drop in tumor or nontumor uptake was demonstrated for either of the tumor-specific or nonspecific monoclonal antibodies due to nonspecific monoclonal antibody pretreatment. These data suggest that high doses of isotype-matched unlabeled nonspecific monoclonal antibody given before ¹³¹I tumor-specific monoclonal antibody, will not enhance tumor imaging. Present address: Hybritech, San Diego, CA, USA