R. Djurup

The Ige and IgG subclass antibody response in patients allergic to yellow jacket venom undergoing different regiments of venom immunotherapy

We developed a three-layer immunoradiometric assay for quantitation of IgG antibodies of all four subclasses to YJV. We studied the IgE and IgG subclass antibody response to YJV in 31 patients allergic to YJV who were undergoing three different kinds of venom immunotherapy. Group A received weekly single injections with alum-adsorbed venom, group B received weekly clustered injections with aqueous venom, and group C received fortnightly clustered injections with aqueous venom during the increasing dose phase of our study. All patients received alum-adsorbed venom during maintenance therapy. Results from the first 6 months of observation are reported. After 6 months of therapy the IgE antibody level rose significantly in group A, was unchanged in group B, and tended to fall in group C. The fall in IgE antibody level in group C correlated significantly to the pretreatment IgE antibody level. The IgG subclass antibody assays measured IgG antibodies of different subclasses in comparable units. No IgG2 or IgG3 antibodies to YJV were found. Before the start of immunotherapy, 23 patients had significant concentrations of IgG1 antibodies to YJV, and 14 had significant concentrations of IgG4 antibodies. In group A the IgG1 antibody level rose significantly after 6 months, and the IgG4 antibody level rose significantly after 3 months. In group B the IgG1 antibody level rose after 2 weeks and the IgG4 antibody level rose after 3 weeks. In group C the IgG1 antibody level rose after 2 weeks and the IgG4 antibody concentration rose after 8 weeks. When the maintenance dose was reached, the IgG1 antibody level in group C was significantly higher than that in group A. The possibility that IgG1 antibodies formed during venom immunotherapy take part in a feedback inhibition of the IgE antibody production is discussed.

Clustered immunotherapy with Yellow Jacket venom. Evaluation of the influence of time interval on in vivo and in vitro parameters.

To evaluate difference in clinical efficacy, side effects, in vivo and in vitro parameters, 25 patients allergic to Yellow Jacket were treated with clustered immunotherapy using either 7 or 14 days interval between clusters. Twenty‐one patients completed the 6 months’ treatment period and four were withdrawn due to adverse reactions (2 cases of anaphylactic shock). Sixteen patients were challenged by in‐hospital sting and the clinical efficacy was complete. Local side effects were observed in the majority of patients, but only rarely limited the course of immunotherapy. Skin sensitivity estimated as the venom concentration eliciting a wheal equal to histamine HCl 0.1 mg/ml using intradermal test was significantly reduced after 6 months of treatment. Specific IgE showed an initial increase, thereafter declining to pretreatment levels. IgG subclasses were determined by a triple antibody assay. Only subclasses 1 and 4 showed response. Subclass 4 showed a steady increase contrary to subclass 1 which decreased after reaching maintenance dose. No unambiguous relation between either the absolute value or the change of IgG1 and IgG4 at the time of challenge was observed in the patients who tolerated a sting. Furthermore, the IgG response was not correlated to the cumulative dose of venom administered. No simple regulatory function of IgG subclasses in the skin and IgE response was found, and the occurrence of local side effects did not seem to be determined by IgG antibodies. We conclude that clustered immunotherapy with Yellow Jacket venom is highly effective and that the frequency of side effects is acceptable. Neither the IgE nor the IgG response is significantly different with a 7‐ and 14‐day interval between clusters, indicating that a protective dose of venom might be reached within 1 month.

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.

Hyposensitization in asthmatics with mPEG-modified and unmodified house dust mite extract. III. Effect on mite-specific immunological parameters and correlation to changes in mite-sensitivity and symptoms.

Forty‐six adult asthmatics allergic to D. pteronyssinus (Dp) participated in a 2‐year study. Thirty‐one underwent hyposensitization (HS‐group). Fifteen were treated with Dp‐extract (Dp‐group), and 16 with a similar extract modified by monomethoxypolyethylene glycol with reduced allergenicity (mPEG‐Dp‐group). Fifteen patients served as controls. Dp‐specific antibodies and histamine release from blood basophils were determined and compared with Dp‐sensitivity in lungs and skin. In addition, IgG and IgE against the major allergen Der p I were followed in a subgroup. Dp‐specific IgG, IgG., and IgG4 increased significantly in both HS‐treated groups after 1 and 2 years (median; 2.5‐ to 11.6‐fold). IgG4 was not induced if maintenance dose during the first year was less than 20,000 BU. Median skin sensitivity decreased 4.4‐ to 8.2‐fold after 1 year and 7.4‐ to 21.4‐fold after 2 years. Der p I specific IgG response was unrelated to the occurrence or change in IgE with the same specificity. The mPEG‐Dp‐extract tended to have less effect on skin sensitivity and immunological parameters, differences reaching statistical significance for skin sensitivity only. In the HS‐group, the decrease in bronchial sensitivity was significantly correlated to a decrease in IgE (r = 0.36), IgG1/IgG4 (r = 0.49), Dp‐specific histamine release (r = 0.58), and to an increase in Dp‐specific IgG, (r =−0.36) and IgG4/IgE (r =−0.48). In patients improving clinically. Dp‐specific IgG4/IgE increased, and median Dp‐specific IgE was reduced to 80 % compared with an increase to 150–160% seen in the unchanged or deteriorated group (P < 0.05). Findings indicate an improvement of effect, if the allergen dose is sufficient to reduce specific IgE and/or induce an IgG and especially IgG4 response.

Adrenoceptors: Molecular Nature and Role in Atopic Diseases

Since Szentivanyi proposed the idea that asthma and other atopic diseases are due to a p adrenergic defect there has been much interest in the role of the adrenergic receptors in allergy. The radioactive ligand binding techniques developed within the last few years have greatly increased our knowledge concerning the molecular nature of the adrenoceptors and the events following receptor stimulation. The adrenoceptors have shown to be very dynamic structures. Their number and affinity may be altered due to various physiological and pharmacological stimuli. Their role in the pathogenesis of atopic diseases has not been definitely settled, but there seem to be a true beta adrenergic hyporesponsiveness and alpha hyperresponsiveness in asthma. This article briefly describes the radioligand binding technique and summarizes our present knowledge of the nature of the alpha and beta adrenoceptors and their possible role in atopic diseases.

An Automated Particle Counting Immunoassay (pacia) for Determination of Blocking Antibodies Against Timothy Grass-pollen in Sera From Desensitized Allergics

AN automated particle counting immunoassay (PACIA) for measurement of blocking antibodies (antigen neutralizing capacity) against timothy grass pollen extract in sera from desensitized allergies is described.

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 IgE-containing immune complexes in human sera. Evaluation of polyethylene glycol precipitation of monomeric and complex IgE and of the detectability of IgE in the complexes.

We studied the polyethylene (PEC) precipitability of monomeric human IgE, and of human IgE artificially complexed with rabbit anti‐human IgE. At conditions where precipitation of monomeric IgE did not occur, from 0.2 to 20% of the complexed IgE was precipitated. The PEG precipitability of the complexes was inversely related to the IgE/anti‐IgE ratio used for preparation of the complexes. From 1.5 to 19.2% of the IgE in the redissolved precipitates could be detected by use of a two‐site IgE immunoradiometric assay, the percentage being highest for complexes formed at equivalence. We conclude that exact quantitation of circulating IgE immune complexes (IC) probably is impossible by any PEC precipitation assay. However, the optimized assay was found to be useful for identification of IgE IC in sera with total IgE concentrations below 5,000 U/ml. IgE IC were found in 5/20 sera from patients with Feltys syndrome, in 5/39 sera from patients with extrinsic allergy and high levels of specific IgE, and in 1/17 sera from immunized wasp allergies. No IgE IC were found in 20 normal human sera.

Clinical and Immunological Aspects of a Case of Monoclonal Hyper-ige - Isolation of Ige-protein, Estimation of Basophil Cell Bound Ige and Histamine-release

A case of monoclonal IgE type lambda with IgE levels about 1 mg/ml has been followed for 6 years. Except for a slight asthma no signs of malignancies, parasitic infestations or other known diseases compatible with hyper‐IgE have been found. By the combination of fractional ammonium sulphate precipitation, gel filtration, chromatofocusing, and subtraction affinity chromatography the IgE protein was isolated in an immunochemically pure and homogeneous form. Immunofluorescence of bone marrow cells showed about 1 % IgE plasma cells. The amount of basophil bound IgE was 42 ng/106 cells, and histamine release from basophils challenged with anti‐IgE was not different from that in atopic control persons, indicating a within‐allergic‐patients normal amount of IgE receptors. The protein‐A reactivity was 0.4% equivalent to well‐known IgE myeloma proteins. No antigen specificity of the IgE protein was found. Only a few cases of asymptomatic hyper‐IgE are known, and it cannot be ruled out that this represents a premyeloma condition, since a similar case terminated in a malignant lymphoma.