A Three-Layer Immunoradiometric Assay for Determination of IgG Subclass Antibodies in Human Sera ("IgG Subclass RAST")

We report the development of a three-layer immunoradiometric assay (TIRA) for measurement of IgG antibodies of all four subclasses in human sera. The first layer consists of diluted human serum, the second layer is monoclonal mouse antibodies to human IgG subclasses., and the third layer is ¹²⁵I-labelled rabbit anti-mouse IgG. Monoclonal anti-IgG1. anti-IgC3 and anti-IgG4 reacted only with their complementary IgG subclass, whereas the anti-lgG2 showed slight cross-reactivity to immunoglobins of other subclasses and classes and to light chain proteins. The observed cross-reactivity was found to be without importance, when the TIRA was applied to measurement of IgG subclass antibodies. Equipotency was established by use of appropriate dilutions of the monoclonal antibodies, and the assay was calibrated by use of human reference serum. The TIRA therefore permits reliable inter-individual and intra-individual comparisons of the IgG antibody response in all four subclasses. Von-sped fie binding obtained with pooled normal human serum was below 0.33'#. Inter-assay coefficient of variation was between 18 and 27%, The TIRA was applied to measurement of IgG subclass antibodies to timothy grass pollen in sera from grass pollen allergies undergoing immunotherapy.     

The Subclass Nature and Clinical Significance of the IgG Antibody Response in Patients Undergoing Allergen-Specific Immunotherapy

The purpose of this paper is to discuss the methodological difficulties in quantitation of human IgG subclass antibodies to allergens, to describe the subclass nature of the IgG antibody response in patients undergoing allergen-specific immunotherapy, and to discuss the possible immunological functions and clinical significance of allergen-specific IgG antibodies of different subclasses. Based on results obtained by use of assays with documented specificity it is concluded that the IgG antibody response during allergen-specific immunotherapy is IgG1 and IgG4 restricted, although low levels of IgG2 and IgG3 antibodies to some allergens may occur. In most patients the early IgG antibody response is IgG1 dominated and the late IgG4 dominated. A too early or too pronounced IgG4 dominated antibody response seems to indicate a poor clinical outcome of immunotherapy with inhalant allergens, whereas a pronounced early IgG1 antibody production has been found to be associated with a decrease in synthesis of IgE antibodies to an insect venom. It is therefore proposed that an early IgG1 dominated response is necessary to induce suppression of the ongoing IgE antibody production, which in its turn may be a prerequisite for long-lasting clinical effect. The possibility of induction of an early IgG1 dominated response in every patient by use of alternative immunotherapy procedures is discussed.     

Characterization of IgG-Subclass Antibodies to Individual Allergens by Crossed Radioimmunoelectrophoresis

A crossed radioimmunoelectrophoretic (CRIE) method for detection of human IgG subclass specificities against individual antigens is described. After crossed immunoelectrophoresis (CIE) of the antigens the immunoplates are incubated with serum, washed and incubated with mouse monoclonal anti-human IgG-subclass antibodies. After washing, the plates are finally incubated with the 125I-labelled detector protein, rabbit anti-mouse IgG, and washed. The plates are then placed on an X-ray film for autoradiography. The specificity of the method was tested by inhibition with antigens in the first layer and by inhibition with myeloma sera containing only one IgG subclass protein in the second layer. The specificity of the third layer was assured by affinity purification of the rabbit anti-mouse IgG antibodies. The method was shown to be sensitive and specific. The test systems were timothy (Phleum pratense) pollen antigens and house dust mite (Dermatophagoides pteronyssinus) antigens.     

Determination of Specific IgG Antibody by Crossed Radioimmunoelectrophoresis

A crossed radioimmunoelectrophoretic method was developed for detection of honey bee venom specific IgG antibodies in patient sera. At the serum concentration 1/200 the contrast between specific binding and background was the most favourable. The detection limit was fairly low, approximately 30 kU/1 (IgG RAST units). A reference system based on the reference kits in Phadebas IgG-RAST® was elaborated.     

Early effect of ultrarush venom immunotherapy on the IgG antibody response

BACKGROUND: We have previously shown in several allergy models that allergic and tolerance status with respect to allergens is associated with a somewhat different dominant specificity of IgG antibodies. The objective was to test this hypothesis in the compelling model of ultrarush venom immunotherapy (VIT), which induces clinical tolerance after only a few hours of treatment. METHODS: Antibody titers and specificity were evaluated through solid-phase ELISA using streptavidin-biotin technology in 12 patients allergic to wasp venom before and during the ultrarush procedure (at 12 h, 24 h, and 15 days). The results were compared with those from another group of 20 patients treated with venom injections for at least 2 years. RESULTS: No significant change was observed in IgG titers during the early phase of VIT. The capacity of individual sera to prevent the antigen binding of pooled IgG from allergic patients changed rapidly, with mean percentage inhibitions falling from 80+/-15%, before starting VIT, to 26+/-14%, 35+/-15%, and 34+/-5% after 12 h, 24 h, and 15 days of treatment, respectively (P<0.001 by one-way ANOVA). The capacity of individual sera to prevent the antigen binding of pooled IgG from patients receiving prolonged VIT changed, with mean percent inhibitions increasing from 47+/-8%, before starting VIT, to 76+/-7%, 83+/-6%, and 87+/-6% after 12 h, 24 h, and 15 days of treatment, respectively (P<0.001 by one-way ANOVA). CONCLUSIONS: During the initial phase of ultrarush VIT, a change in IgG specificity, i.e., a change in the set of epitopes dominantly recognized by IgG on wasp-venom antigens, occurred concomitantly with early clinical tolerance and was already detectable a few hours after the onset of treatment. Although it may be an epiphenomenon, this change represents the earliest humoral modification described so far during this procedure. The mechanism is unknown, but it appears to be a selective depletion of the highest avidity antibody fraction by the venom injected in large doses at this stage of therapy. Finally, our data now show the previously documented association between a particular IgG specificity and the clinical status (allergy vs tolerance) to be true also with ultrarush VIT, a model in which the clinical ability to display allergic symptoms is rapidly reversed.     

Asymmetric IgG Antibodies Induced by Different Immunotherapies in a Murine Model of Allergy

Specific immunotherapy (SIT) is the only potentially curative treatment for those allergic processes mediated by IgE. We compared the effects of different SITs in mice sensitised with ovalbumin (OVA) Al (OH)₃ : 1) OVA entrapped in particles of poly (D,L-lactic-co-glycolic acid) (PLGA-OVA), 2) Soluble OVA (OVA-sol) and 3) Polymerised OVA (OVA-pol). Serum levels of specific IgE, IgG1, IgG2a and asymmetric IgG, the cutaneous anaphylaxis test (PCA), and the IL-10, IFNγ and IL-4 levels in culture supernatants of splenocytes challenged with OVA were assessed. Mice treated with PLGA-OVA had higher levels of asymmetric antibodies than non-desensitised mice; a low IgG1 and high IgG2a level was observed together with inhibitory effect in the PCA reaction that reversed in the absence of asymmetric IgG. IL-10 and IFNγ levels were higher in supernatants from mice treated with PLGA-OVA and OVA-sol than those obtained from non-desensitised controls. Our results suggest that among the different SITs evaluated, PLGA-OVA is the one that best showed an increase in the asymmetric IgG molecules and an effective deviation of the immune response. Furthermore, the increase in the proportion of asymmetric antibodies would be of importance when designing new vaccination strategies for allergy.     

Individual Specificity of Blocking Antibodies in Molar and Normal Term Placenta-Bound IgG

ABSTRACT: In view of the protective and enhancing effect of blocking antibodies (BA) on the survival of trophoblastic cells, the presence or absence of individually specific BA against paternal HLA antigens in trophoblast-bound immunoglobulin G (IgG) of molar as well as normal term placentas were investigated using a mixed lymphocyte culture reaction (MLR) blocking assay and complement-dependent lymphocytotoxicity test. It was found in this study that IgG could be eluted from molar trophoblasts. Further, it was strongly suggested that each molar and normal placental eluate-IgG had individual, specific cytotoxic and blocking effects against paternal lymphocytes and on MLR between spouses, respectively. In conclusion, in molar as well as in normal term placenta-bound IgG, BA that are heterogeneous and partly contain IgG specific to the paternal HLA antigens, including HLA-D/DR antigens, are likely to exist. BA may also have a protective and enhancing effect on the survival of trophoblastic cells by binding with these cells.     

A critical study of the use of staphylococci containing protein A for separation of IgG and IgM antibodies

This study was to determine the best conditions for using staphylococci bearing protein A to separate IgG from IgM. The validity of the technique was evaluated for detection of IgM with antimicrobial activity and for typing monoclonal IgM. The results indicate that separation of IgG and IgM is not entirely satisfactory in normal sera and worse in hyperglobulinemic sera. The detection and titration of IgM antimicrobial antibodies (rubella and hepatitis B core (HBc) specific IgM) was unreliable because IgG was only partially absorbed by staphylococcal cells, while a significant portion of IgM was bound. The use of higher concentrations of staphylococci did not improve the results because the more IgG was absorbed, the more IgM was also bound. It is shown that with anti-HBc specific IgM the risk of misinterpretation is very high with a sensitive radioimmunoassay technique allowing detection of trace amounts of nonabsorbed IgG. In contrast staphylococcal protein A proved useful in typing monoclonal IgM.     

Crossed Radioimmunoelectrophoretic Studies of Bee Venom Allergens

Crossed immutioelectrophoresis (CIE) glass slides of boney bee venom showing 18 immunoprecipitates were used in crossed radioimmunoelectrophoresis (CRIE) experiments with sera from 25 patients allergic to honey bee venoms. Phospholipase A, acid phosphatase, hyaluronidase and melittin were demonstrated in the immunoprecipitates during zymograpbic techniques and direct hemolysis of sheep erythrocytes. The CRIE experiments verified the allergenicity of these proteins. In addition, another antigen (Ag-1) with allergenic activity was identified. This allergen is probably identical to the previously described allergen C in honey bee venom. The presence and complexity of multiple forms of the known allergenic proteins in honey bee venom, was well illustrated in the CIE/CRIF system employed.     

An Automated Particle Counting Immunoassay (PACIA) for Determination of Blocking Antibodies against Timothy Grass Pollen in Sera from Desensitized Allergies

An automated particle counting immunoassay (PACIA) for measurement of blocking antibodies (antigen neutralizing capacity) against timothy grass pollen extract in sera from desensitized allergics is described. Latex particles coated with F(ab')2-anti-timothy are agglutinated by timothy. Serum containing anti-timothy antibodies inhibits the agglutination. Non-agglutinated particles are counted in a modified AutoCounter. Nineteen of 20 sera from timothy allergics who had undergone immunotherapy with purified timothy extract for 30 weeks, showed significant agglutination-inhibition. None of 42 normal human sera gave significant inhibition. The inhibiting antibody could be removed by absorption with protein A and was thus of non-IgE nature, i.e. blocking antibody. The results obtained correlated statistically significantly with those found with a double-antibody method (rS = 0.62, n = 20, t = 3.35, P less than 0.01) and with the cumulated dosage of timothy allergen extract administered to the individual patient (rS = 0.56, n = 20, t = 2.87, P less than 0.02). Between-assay coefficient of variation was from 6.4% to 18.3%. The capacity is 40 samples per hour. The method has also been applied to measurement of blocking antibodies to honey bee and wasp venom.     

Immunobiochemical characteristics of IgG antibodies in myasthenia

The abzyme activity of mAb35 and pQ1-209 was compared with that of normal rabbit IgG and serum IgG from patients with various forms of myasthenia. It was found that mAb35 and pQ1-209 and IgG from patients with myasthenia possess catalytic activity. IgG from myasthenia patients with thymomas possess creatine phosphokinase activity, which 2- fold surpassed the control.     

IgG Antibodies to Human Cytomegalovirus Late Protein UL94 in Patients with Systemic Sclerosis

Human cytomegalovirus (HCMV) has been proposed as an amplifying agent for at least some of the spectrum of systemic sclerosis (SSc; scleroderma). In support of this hypothesis, antibodies to the HCMV late protein UL94 have been detected in the majority of SSc patients in a study involving Caucasian subjects from Italy. The aim of this investigation was to determine whether elevated levels of anti-UL94 antibodies are present in African American and Caucasian SSc patients from the U.S. We further wished to determine whether there was a significant difference in the levels of anti-UL94 antibodies between the diffuse and the limited forms of the disease. IgG antibodies to a UL94 peptide were measured in 254 Caucasian and 90 African American subjects by an enzyme-linked immunosorbent assay (ELISA). In both Caucasian and African American subjects, the mean antibody level in the diffuse form of SSc was significantly higher than that in the respective control subjects (714 vs. 466?ng/ml, p=0.005; 1226 vs. 512?ng/ml, p<0.0001). Also, among Caucasian SSc patients, the mean antibody level in the diffuse form of SSc was significantly higher than that in the limited form of the disease (714 vs. 465?ng/ml, p=0.02). These results show that increased levels of antibodies to the HCMV late protein UL94 are associated with SSc and they may be a marker for the severity of the disease.     

IgG Subclass Antibody Response in Grass Pollen-Allergic Patients Undergoing Specific Immunotherapy

All four subclasses of IgG antibodies to timothy grass pollen extract were measured by a three-layer immunoradiometric assay in sera from 20 grass pollen-allergic patients who underwent specific immunotherapy in a 3-year prospective study. Both IgG1 and IgG4 antibody levels rose significantly during the first 8 weeks of immunotherapy. IgG1 antibody level passed its peak (median 5.4 U/ml) after 12 weeks. At this time, the ratio between the medians of IgG1 and IgG4 antibodies was 2.25. IgG4 antibody level reached its peak (median 11.6 U/ml) just before termination of immunotherapy. At this time IgG1/IgG4 ratio was 0.43. Two years after the end of immunotherapy, IgG1 and IgG4 antibody levels were 0.0 and 1.8 U/ml in median, respectively. The amounts of IgG2 and IgG3 antibodies detected in the sera were less than 1.6 U/ml and were considered insignificant. Preseasonal serum IgG1 and IgG4 antibody levels did not correlate significantly with symptom scores in the subsequent season. Serum IgG4 level obtained after 12 weeks of immunotherapy was significantly correlated to symptom score in the third season, i.e. the season just after termination of therapy (rs = 0.529, t = 2.567, P = 0.02). In this work, a serum IgG4 antibody level higher than 8.0 U/ml after 12 weeks of therapy predicted poor clinical result at the end of immunotherapy with 100% sensitivity and 87% specificity. An IgG4/IgG1 ratio greater than 1.0 after 12 weeks' therapy had the same predictive value.     

Modulation of Cancer Patients' Immune Responses by Anti-idiotypic Antibodies

The immunomodulatory role of anti-idiotypic antibodies (Ab2) in patients with gastrointestinal cancer has been demonstrated in two types of clinical trials. In the first, cancer patients were treated with a monoclonal antibody (MAb) defining a tumor-associated antigen (Ag). MAb administration initiated an idiotypic network as demonstrated by the induction of both Ab2 and anti-anti-idiotypic antibodies (Ab3) in the treated patients. The Ab3 bound to tumor cells and isolated tumor Ag with the same specificity as the Mab (Ab1) at the beginning of the idiotypic cascade. A beneficial role of Ab3 is postulated for patients showing delayed clinical responses to MAb therapy.

In a recent trial, patients with advanced colorectal cancer responded to immunization with Ab2 that functionally mimicked in vitro and in vivo (animals) a gastrointestinal tumor-associated Ag by developing highly specific Ab3 with anti-tumor binding reactivities. Thus, Ab2 are promising agents in immunotherapy approaches to cancer. These studies suggest an immunoregulatory role for Ab2 in cancer patients. Modulation of cellular immune responses by Ab2 in cancer patients will be an important consideration in future studies.     

Measurement and interpretation of pneumococcal IgG levels for clinical management

The detection of pneumococcal IgG antibodies is helpful for the evaluation of response to pneumococcal vaccination and need for revaccination. Results generated by the clinical assay which is currently used, in which the 23 valent polysaccharide vaccine is the antigen, were compared to those obtained by a capsular polysaccharide serotype-specific assay that measures IgG antibodies to 9 common serotypes causing invasive disease. Discrepancies in 21/47 (45%) of the results were observed in a direct comparison between the two assays. In each case a positive titre was obtained on the clinical assay but IgG levels on the serotype-specific assay were below the putative protective level of 0.2 micro g/ml for at least one of the 9 serotypes assayed. The generation of false positives by the current clinical assay is due to its lack of specificity. Antibodies to C-polysaccharide and all of the 23 serotypes included in the pneumococcal polysaccharide vaccine are incorporated into the final titre whereas the serotype-specific assay adsorbs out noncapsular polysaccharide antibodies. The discrepancies between the two assays highlight the importance of standardized assays that measure putative correlates of protection and demonstrate the need to re-evaluate the current clinical assay. A tool that allows the interpretation of the results of the serotype-specific assay is provided and its potential for assessing individual susceptibility levels to vaccine preventable pneumococcal infection is discussed.     

Occurrence,properties, and function of asymmetric IgG molecules isolated from non-immune sera

We have previously demonstrated that 10-20% of the IgG isolated from non-immune sera is asymmetrically glycosylated, in such a way that it fails to trigger immune effector mechanisms. As a result, a major portion of the non-immune asymmetric IgG molecules of the host could be self-specific, acting as auto-protective antibodies. In order to test this hypothesis, we investigated whether asymmetric IgG molecules are capable of recognizing self-antigens. About 40% of F(ab')₂ fragment from normal rat IgG was able to react specifically with autologous rat cells. Moreover, upon being purified from normal rat sera, 78% of the asymmetric IgG sub-population showed self-reactivity. We demonstrated that about 14% of rat asymmetric IgG-F(ab')₂ fragments was able to react with bacteria isolated from the intestine of uninfected rats. Lastly, in order to test whether there is a correlation between the decline of immune responses during ageing and asymmetric antibody production, we assayed IgG isolated from sera of young and old rats. There was an increase in the asymmetric:symmetric IgG ratio with ageing. We therefore suggest that asymmetric antibodies may exert a beneficial action by protecting self-antigens as well as normal intestinal flora from a deleterious immune response.     

IgG subclass distribution of thyroglobulin antibodies in patients with thyroid disease

The IgG subclass distribution of thyroglobulin antibodies (TgAb) has been studied in Hashimoto and Graves’ patients by several investigators with conflicting results, in part explainable by methodological problems. We have recently developed a quantitative ELISA to measure in absolute terms the serum concentration of TgAb subclasses. The aim of the present study was to apply this method in a large series of patients with autoimmune as well as, for the first time, non-autoimmune thyroid diseases. We examined 28 patients with Hashimoto's thyroiditis, 30 with Graves’ disease, 21 with thyroid carcinoma and 18 with non-toxic goitre, all selected for the presence of TgAbs. The results indicated that TgAbs in thyroid diseases were not restricted to any particular isotype, but comprised all four IgG subclasses. IgG1 was represented similarly in the four groups. The same was true for IgG3, even though its contribution to the total antibody content was very small. IgG4 was the dominant subclass in patients with Graves’ disease, thyroid carcinoma and non-toxic goitre, probably reflecting a prolonged antigenic challenge. In Hashimoto's thyroiditis IgG2 was dominant, possibly because T helper lymphocytes infiltrating the thyroid are typically Th1 type.