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Sensitization of allergic patients normally takes place over several years and is the result of repeated exposure to low levels of allergen. Most mouse asthma models use a high dose of allergen administered over a short period. We have investigated the role of dose in the immune response to an inhaled respiratory allergen (Blomia tropicalis). We observed the effect of priming dose on the allergic response in mice intranasally immunized with low (0·5 μg) and high (50 μg) doses of B. tropicalis extract and killed 1 day after the last challenge. For both doses of allergen, T helper type 2 (Th2) cells and Th2 cytokines were evident as well as eosinophilic inflammation accompanied by mucus hyper-secretion. By contrast, IgE and IgG1 antibody responses were normally only detected at high-dose priming. To investigate the mechanism for these effects, we found group 2 innate lymphoid cells (ILC2s) were increased 48 hr after challenge in the low-dose-treated but not the high-dose-treated mice. Furthermore, we determined whether repeated low-dose exposure with different priming protocols could induce an antibody response. Repeated low-dose exposure to 0·5 μg three times weekly for 4 weeks (cumulative 6 μg) had the same effect as a shorter high-dose exposure (cumulative 80 μg) and increasing cumulative dose induced antibody responses. These data indicate that low doses of allergen are sufficient to prime Th2 cells and ILC2s, but insufficient to induce antibody responses. Cumulative exposure to small amounts of allergen induces both Th2 and antibody responses and may better reflect natural sensitization., (© 2017 John Wiley & Sons Ltd.)

ELISA is increasingly used to measure antibodies in new circumstances. Recently, it has been applied to the measurement of IgG subclass antibodies against common antigens in early childhood. These studies have raised concerns about the specificity of some of these assays. This paper details the results of experiments which have assessed the specificity of IgG1 binding to allergens of dust mite (Dermatophagoides pteronyssinus) and ryegrass (Lolium perenne) pollen by inhibition ELISA in the sera of 2-year-old children of atopic parents. Six sera which showed binding of IgG1 to D. pteronyssinus and six to L. perenne were used. All had IgG1 antibody against ovalbumin. In the children's sera, binding to D. pteronyssinus was substantially inhibited by preincubation with the homologous antigen, but not with ovalbumin, thereby confirming the specificity of the assay. However, suppression of IgG1 binding to L. perenne with the homologous antigen was comparatively small, and ovalbumin could cause an equivalent inhibition, indicating poor specificity. Furthermore, the level of IgG1 binding to L. perenne was closely correlated to the level of IgG1 binding to ovalbumin (r = 0.98; P < 0.001). When the assay was reversed, IgG1 binding to ovalbumin was only slightly inhibited by L. perenne, indicating that most antibody binding to ovalbumin was specific. Thus, binding IgG1 in both adult and child sera to D. pteronyssinus appeared to be specific, while child, but not adult, IgG1 binding to L. perenne showed poor specificity. This disparity may be due to differences in the affinities of the respective antibodies, and it illustrates the importance of determining assay specificity when making measurements in early childhood.

The quantity and functional affinities of IgG1 and IgG4 antibodies to the dietary antigens casein and ovalbumin were measured in unselected mothers and their 1-year-old infants. In these infants, the titre of IgG antibodies to both antigens was highest in the IgG1 subclass, while in their mothers the titre of IgG1 and IgG4 antibodies to these foods was similar. High affinity IgG4 responses to both casein and ovalbumin were frequently found in mothers, whilst IgG1 responses, particularly to ovalbumin, were of low functional affinity. By contrast, in the 1-year-old infants, the functional affinity of IgG1 antibody to ovalbumin was substantially higher than in their mothers (Student's paired t-test, P < 0.001), indicating that higher affinity IgG1 antibody was produced on first exposure to ovalbumin rather than following chronic exposure. The effect of antigenic load on affinity maturation was further investigated by comparing the affinity of IgG1 antibody to casein in bottle, mixed and breast fed infants. Bottle fed infants had significantly higher-affinity IgG1 antibodies to casein compared with breast or mixed fed infants (Student's unpaired t-test, P < 0.01 and 0.02), suggesting that antigen exposure via the gut was able to drive the affinity maturation process. In studying the immune response it is clear that account must be taken of the affinity as well as of the titre of the antibody produced.

This study investigates the early humoral immune response following natural exposure to an inhalant antigen (Dermatophagoides pteronyssinus) in 36 babies, from birth until 1 year of age. The total IgG and subclass 1 and 4 D. pteronyssinus-specific antibody levels were assayed in sera collected at 7 days, 3 and 12 months by ELISA. After an initial fall, due to the progressive loss of maternal antibodies, an IgG specific response to D. pteronyssinus was seen between 3 and 12 months. This was restricted to the IgG1 subclass when the values at 12 months were significantly higher than those detected at the third month (P less than 0.001, paired t test). No D. pteronyssinus-specific IgG4 antibody was detected in any subject at any of the time points tested. The present study demonstrates that inhalant as well as food antigens are able to stimulate the immune system during the first year of life and that the antibodies produced are of the IgG1 subclass.

This follow-up study of 191 babies investigated the development of food allergy in an unselected population and its relationship to total and antigen-specific IgE and IgG subclass levels. Sensitization to egg, as indicated by a positive skin test or RAST, was found in 5% of 1-year-old babies, but none of the babies in this series fulfilled the clinical criteria for immediate-type milk allergy. For both bovine casein (CAS) and egg albumin, the IgG response was largely restricted to IgG1 in contrast to the predominant IgG4 response to these antigens that is found in adults. The level of IgG4, but not IgG1, antibody to CAS and ovalbumin (OV) was lower in some of the babies compared with that of their mothers (N = 166; p less than 0.05, Student's paired t test). However, there was no difference in the total serum IgG subclass levels between mothers and babies. These results demonstrate that, in the population of babies studied, (1) type I hypersensitivity to egg occurred in 5% of 1-year-old babies, (2) the predominant IgG subclass of antibodies to CAS and OV in babies is IgG1, and (3) in the 22% of babies, there was substantially (greater than 1000-fold) less IgG4 antibody to CAS and OV than in their mothers, suggesting specific exclusion of some IgG4 antibodies.

A double-blind, placebo-controlled trial of immunotherapy was conducted in patients with Dermatophagoides pteronyssinus rhinitis. Thirty patients received an extract with a high content of Der p I (Pharmalgen), 20 received a conventional mite extract (Allpyral), and 30 patients received histamine chloride (placebo). Specific IgG and subclasses were measured before and after 3 and 12 months of treatment by RIA and/or ELISA, and specific IgE by RAST. Clinical outcome was assessed by skin prick tests, nasal challenge, visual analogue, and diary-card symptom and drug scores; from these findings, a clinical index was derived. An IgG response occurred only in the Pharmalgen-treated group: D. pter IgG and IgG1 increased by 3 months (p less than 0.05) and then plateaued to 12 months (p less than 0.05). IgG4 levels increased throughout treatment (p less than 0.05 and p less than 0.01), as did the IgG/IgE ratio. A subclass switch from IgG1 to IgG4 occurred. D. pter IgE rose at 3 months (p less than 0.05). Clinical improvement occurred at 3 and 12 months in the Pharmalgen-treated group only. Pretreatment levels of IgE, IgG1, or IgG4 did not predict clinical outcome. Our findings are compatible with the hypothesis that IgG subclasses may modulate antigen-IgE interactions, although the antibody response to this potent extract need not be causally related to improvement.

The role of IgG-subclass antibodies in the spectrum of immunologic disorders has not yet been fully defined. In an attempt to understand its role in an immune complex-mediated disease, we studied patients who developed serum sickness (SSX) after treatment with an equine-derived immunoglobulin, antithymocyte globulin (ATG), for bone marrow failure. The predominant IgG subclass produced was IgG1, representing nearly 80% of all IgG anti-ATG activity present. The appearance of IgG anti-ATG antibodies and C1q-containing immune complexes was closely correlated with symptoms of SSX. Although other antibody isotypes were present and may have contributed to the patients' symptoms, it is evident that IgG1 is the predominant IgG subclass produced in human SSX caused by a heterologous protein.

The radioallergosorbent test (RAST) has been widely used in studying allergy to hymenoptera stings, but with variable results. We report here a modification of the RAST method which in a total of 157 bee keepers and family members, gave a close correlation between a positive RAST, and a history of generalized (100%) or localized (95%) allergic reactions. There was no positive RAST among forty-nine non-atopic control subjects but a striking finding was that 58% of non-allergic bee keepers had significant, but usually low, levels of IgE antibody to bee venom. IgG antibodies to bee venom have also been measured by a paper disc RAST which is quick and convenient. Levels were highest in non-allergic bee keepers. In the presence of high levels of IgG antibody, IgE antibody levels may be underestimated.

We have developed a sensitive enzyme-linked immunosorbent assay (ELISA) which measures antibodies to bee venom phospholipase A2 (PLA2) and hyaluronidase (HYAL), horse IgG, bovine casein, and the bacterium Streptococcus mutans in each of the four human IgG subclasses. For this purpose, we have used mouse monoclonal antibodies (McAb) specific for each subclass and one which showed 'pan-IgG' reactivity. Binding to human IgG was similar for all the McAb and dilution of human IgG resulted in similar dilution curves for each subclass. Results were expressed as arbitrary U ml-1 by comparing the optical density obtained with each subclass-specific McAb to a reference curve for total IgG antibody constructed using the 'pan-IgG' McAb. Close agreement was found between the total amount of IgG antibody and the sum of the antibody in each of the four subclasses (PLA2 r = 0.90, horse IgG r = 0.98, bovine casein r = 0.84, S. mutans r = 0.85), confirming that these assays provide semi-quantitative measurements of the amount of subclass-specific antibody.

Previous studies have suggested that, apart from IgE-mediated reactions, some of the symptoms of food allergy may be caused by IgG antibodies to food proteins. This study was carried out to see if there were any distinctive features of the IgG sub-class antibody response to dietary antigens which occurs in food allergic patients. IgG sub-class antibodies were measured using a quantitative enzyme-linked immuno-sorbent assay (ELISA) to wheat gliadin, ovalbumin and bovine casein in twenty patients who had coeliac disease and in twenty-eight egg allergic patients. These were compared with twenty-one atopic dermatitis patients who did not have food allergy and twenty-six healthy control subjects. Coeliac disease patients tended to have raised IgG antibody levels (especially IgG1) to all three antigens but these overlapped considerably with that seen in egg allergic and atopic dermatitis patients. Coeliacs who avoided gluten had anti-gliadin antibody levels which did not differ from those seen in healthy subjects but nevertheless had raised anti-ovalbumin and casein-specific antibodies. The IgG antibody was largely restricted to IgG1 and IgG4 sub-class although the relative amount of each varied with the antigen. Although gliadin-specific antibodies were mainly IgG1, ovalbumin-specific antibodies were mainly IgG4. The increased antibody levels to all three antigens in coeliacs were caused by a raised IgG1 response, IgG4 antibodies were usually normal. Egg allergic patients also had raised IgG1 but not IgG4 antibodies to ovalbumin. These data show that the response to different dietary antigens can vary with the antigen. The fact that IgG1 and not IgG4 antibodies were raised to all three antigens in patients with coeliac disease suggests that they are a secondary consequence of the disease, perhaps reflecting increased transport of antigens across a damaged gut mucosa rather than a specific immunopathological reaction. However, the observation that antibodies to gliadin, and not ovalbumin or casein, fell following gluten avoidance shows that the response to gliadin, at least, is dependent upon continued exposure to antigen.

IgE responses are closely regulated in non-atopic humans and low IgE responder animals. After an initial period of IgE production following antigen exposure, IgE synthesis appears to be actively suppressed. Inhalation of the dust of castor beans induces persistent IgE responses in atopic and non-atopic humans alike. This phenomenon was investigated in animals. Hooded Lister rats were immunized intraperitoneally with different preparations of castor bean. These had been heated for different lengths of time, 60 and 15 mins, to inactivate the toxin ricin. Immunization with as much as 100 micrograms of the extract heated for 60 min failed to produce an IgE response, while injection of 100 micrograms of the extract heated for 15 min produced a marked IgE response to castor bean proteins. Thus the component of castor bean extract which induces the IgE response appears to be heat labile. The IgE potentiating component in castor bean was found to enhance IgE responses to other antigens such as ovalbumin and when 0.8 microgram of an unheated castor bean extract was administered together with an optimal dose of ovalbumin, there was a substantial increase in ovalbumin-specific IgE but not IgG in all animals. In addition, total serum IgE but not IgG increased up to 20-fold. The effect of castor bean was more sustainable than that of an established IgE-specific adjuvant, Bordetella pertussis, and was able to boost an IgE response that had diminished and maintain an ongoing IgE response when re-administered at weekly intervals. In addition, it was possible to reproduce the IgE potentiating effects with purified castor bean ricin at 25 ng/rat. The way that it produces this effect is not known but it is possible that ricin blocks the normal IgE suppressive mechanisms that regulate IgE responses.

In an attempt to understand the role of the different IgG subclasses in allergic disease, we have studied the subclass of IgG antibody to dust mite (HDM) and grass pollen (GP) produced as a result of natural exposure. Studies were carried out on 40 atopic children and 100 atopic adults who had never received immunotherapy. Thirty-two non-atopic adult controls were also studied. The specificity of the assay for IgG antibodies to dust mite was confirmed by inhibition with the homologous extract but not mite culture medium or fetal calf serum. IgG1 antibodies to HDM could be detected in most atopics (94%) and non-atopics (97%), and similar results were obtained for GP (81% and 100%, respectively). IgG4 antibodies to HDM were detected in more atopics (66%) than non-atopics (53%) and the difference was more marked for GP (72% vs. 19%). While the levels of IgG1 antibodies were not significantly different in the two groups, the levels of IgG4 antibodies were much lower in the non-atopics (P less than 0.001, Mann-Whitney U-test). These data show that all subjects were capable of recognizing and mounting an IgG1 antibody response to these inhaled antigens. Atopic individuals differed from normal subjects in the frequency with which they made IgG4 antibodies in response to natural exposure to both dust mites and pollen.

Antibodies to individual bee venom antigens were studied in detail in nine bee sting-allergic patients who received venom immunotherapy without side effects, in two patients who failed to reach maintenance, and in two whose sensitivity returned. The study was confined to patients who had IgE antibodies to at least one of four purified bee venom antigens at the start of treatment. IgE and IgG antibodies to phospholipase A2 (PLA2), hyaluronidase (HYAL), and acid phosphatase (ACID P) and IgE antibodies to melittin (MEL) were measured, and changes in the antibody levels were followed during bee venom immunotherapy. Two contrasting patterns of antibody response were seen in the nine successfully treated patients. In five patients there was a rise in serum IgG antibodies to the same antigens as the IgE antibodies. In two patients' serum IgE antibody to HYAL or ACID P fell without a marked IgG antibody response to these antigens, although high levels of IgG antibody to PLA2 were present in both. Although the first pattern is consistent with a "blocking" role for IgG antibody, clearly the second is not. Not all patients can be conveniently divided into these two categories, and two patients did not show any significant change in either IgG or IgE antibody but were nevertheless able to tolerate the maintenance dose of 100 micrograms of venom. Two patients who failed to reach the maintenance dose of 100 micrograms because of their allergic reactions to the injections of venom were distinguished by (1) very high serum IgE antibody and (2) a low ratio of IgG/IgE antibody. Passive immunization with IgG antibody from a hyperimmune beekeeper was, however, protective in these patients, although it did not raise their overall serum IgG antibody level very much. We are unable to explain either the failure of conventional therapy or the beneficial effect of passive immunization in these two patients. Two bee sting--allergic beekeepers lost their sensitivity to stings, but later, when their sera contained IgE antibody to another bee venom antigen, they reacted to stings and inhalation of beehive dander. These data suggest that either falling IgE antibody or IgG- "blocking" antibody could be responsible for providing clinical protection to bee venom--allergic subjects. Renewed clinical sensitivity was observed when the IgE response was modulated, with patients making IgE antibody first to one antigen and then to another.

We have measured in allergic and non-allergic beekeepers IgE and IgG antibodies to the bee venom allergens, phospholipase A2 (PLA2) and hyaluronidase (HYAL), by the radioallergosorbent test (RAST) and a 125I-radiolabelled antigen-binding assay. The absolute amount of IgG antibody in a reference serum was determined by saturation analysis using 125I-radiolabelled PLA2 and HYAL. Using monoclonal anti-IgE coated microtitre plates, the absolute amount of IgE antibody to the same antigen was also determined by saturation analysis. Regardless of the IgE response to the different allergens, IgG antibody concentrations to PLA2 were invariably higher than those to HYAL. In addition, the ratio of IgG to IgE antibody was higher for PLA2 (220:1) than for HYAL (10:1). Higher levels of IgG antibody to both allergens (especially HYAL) were found in those who had had prolonged exposure to bee stings. These data suggest that the level of IgG antibody produced is related to the dose administered, while the amount of IgE antibody may be regulated by other factors.

A sensitive and specific enzyme-linked immunosorbent assay (ELISA) for allergen-specific IgG antibodies is described. Various solid-phase supports (microtiter plates), coating procedures, binding kinetics, and presentation of allergen in the assay were investigated. Using optimal conditions the indirect ELISA, in which the allergen is coated onto the well, was capable of detecting 2.4 ng/ml specific IgG antibodies to bee venom phospholipase A2(PLA2). The sandwich ELISA, in which the allergen was immobilized via specific antibody precoated onto the well, detected 0.24 ng/ml IgG antibodies to PLA2.

IgG and IgE antibody levels have been followed for a period of 2 years in patients receiving immunotherapy with bee and wasp venom. 106 adult patients who had had anaphylactic reactions to wasp stings had initially low IgG antibody levels to wasp venom which rose with therapy (p less than 0.001). IgE antibody levels also showed an initial rise but subsequently fell (p less than 0.001). The pattern was similar to that previously reported in children who had had anaphylactic reactions to bee stings, but who, after a course of immunotherapy, were able to tolerate stings with impunity. 60 adults who had had anaphylactic reactions to bee stings showed a different pattern, with initially high IgG antibody levels which did not rise further. Since two thirds of this group were beekeepers or members of beekeeper's families, the high initial IgG antibody levels could have been a response to the frequent stings to which such individuals are prone. The fact that high levels did not protect against anaphylaxis shows, however, that the classical concept of 'blocking antibody' is in need of revision.

The IgG sub-class antibody response to bee venom in the four sub-classes was investigated in ten patients during and after venom immunotherapy. All patients tolerated a bee sting challenge 1, 2 and 3 years after the start of treatment as well as 1 and 2 years after treatment was stopped. Anti-phospholipase A2 (PLA2) antibodies were of IgG1 and IgG4 sub-class and rose early in treatment, IgG1 anti-PLA2 fell to pre-treatment levels after 3 years in contrast to IgG4 anti-PLA2 levels, which remained high during maintenance therapy and declined relatively little in the 2 years after the termination of treatment. This data shows that IgG4 antibodies are maintained in the absence of monthly maintenance injections and suggests that they may provide long lasting clinical protection from insect stings.

High functional affinity and high titer IgG4 antibodies to phospholipase A2 were produced by allergic patients in response to bee-venom immunotherapy. In contrast, the affinity of IgG1 antibodies decreased after immunotherapy, and both the titer and affinity of IgG1 antiphospholipase A2 remained significantly lower compared to IgG4 1 to 2 years after treatment. Analysis of affinity heterogeneity suggested a loss of IgG1 high-affinity antibody-producing clones during immunotherapy and a preferential expansion of IgG4 clones. High-affinity IgE antibodies were found in untreated allergic patients, and preliminary results suggest that immunotherapy may result in an early marked decrease in the affinity of IgE antibodies.

We have measured IgG1, 2, 3 and 4 subclass antibodies to gliadin and casein in normal controls, treated and untreated coeliac patients. The amount of antibody in each subclass was determined separately as a titre and as a concentration, by reference to a standard curve. Both methods of quantitation, carried out in separate assays, yielded comparable results (IgG antibody r = 0.86, IgG1 antibody r = 0.89). In all the coeliac patients, the majority of antibody was IgG1, and little IgG2, 3, 4 antibody was detected. Untreated coeliac patients had higher titres of IgG and IgG1 antibodies to gliadin and casein compared with normal controls. In the treated coeliac patients compared with the normal controls, there was no significant difference between the titres of IgG and IgG1 antibodies to gliadin but there were higher titres to casein.

The immunological response to individual bee-venom allergens was studied in blood samples collected at frequent intervals from four bee-venom allergic patients who had suffered systemic allergic reactions to injections of bee venom during immunotherapy. All had high IgE antibody levels, at the upper end of the range found in bee-sting allergic patients, and all had antibodies to the minor allergens at the time of the reactions. These did not, however, provide a simple explanation for the reactions that occurred. We were able to observe two interesting phenomena--in one patient IgE antibodies to the individual venom antigens appeared to be 'switched off' sequentially. In another, IgE antibodies to hyaluronidase rose substantially after 4 years of therapy. We believe that these results provide evidence to support the view that the regulation of IgE antibodies is controlled by mechanisms that are both isotype- and antigen-specific.

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.