The use of monoclonal and polyspecific antibodies in the IgE sandwich ELISA.

The use of antibody to link antigen to microtitre plates in the enzyme-linked immunosorbent assay (ELISA) has been extended to include mouse monoclonal antibodies and polyspecific rabbit antibodies. Small amounts of both reagents could be used. The capacity of the microtitre plate for the antibody was found to be critical and irradiated plates generally gave better results. Both rabbit anti-IgE conjugated to horseradish peroxidase, and monoclonal mouse anti-IgE with alkaline phosphatase-conjugated rabbit anti-mouse IgG could be used as detection reagents. Comparison with the radioallergosorbent (RAST) test showed a good agreement with the sandwich ELISA. The sandwich ELISA using polyspecific rabbit antibody was substantially more sensitive than conventional ELISA and also marginally more sensitive than RAST.

Measurement of 'blocking antibodies' to pollen antigens by radiolabelled protein A from Staphylococcus aureus.

Radiolabelled protein A from Staphylococcus aureus (125I-SpA) was utilized to measure the relative concentrations of the so-called 'blocking antibodies' (BA) in sera from patients subjected to two different forms of desensitization therapy. In addition, sera from rabbits immunized actively with pollen antigens were assayed. Antigens coupled to CNBr-activated papear discs were used as solid phase, and BA bound to the solid-phase antigens were detected by 125I-SpA. The results obtained with this technique correlated excellently with those of a double-antibody technique for IgG, indicating the 125I-SpA assay to be suitable for the measurement of the antigen-specific IgG. The BA concentration in sera from patients subjected to conventional injection therapy increased significantly during the treatment period, whereas no increase could be detected in sera from patients subjected to sublingual treatment. The increases in BA concentrations of rabbit antisera to pollen antigens could be readily traced by this technique.

Immunological cross-reactions between a cat hair and skin scraping extract and a cat serum and investigation of common allergenic molecules of cat 1 with cat serum.

Investigations of cross reactions between a cat hair and skin scraping extract (CHSS) and a cat serum (CS) were done by several biochemical and immunochemical methods. Furthermore, we looked for common allergenic molecules in a cat 1 reference preparation and in a CS. In crossed (radio) immunoelectrophoresis (CIE/CRIE) of CHSS vs. anti CS-serum one to eight allergen bands could be detected depending on the human serum employed. By constructing allergograms from the CRIE patterns of 17 patients' sera one out of eight allergens could be defined as a major allergen. CRIE of the cat 1 reference vs. anti CS-serum, using the sera of two cat allergic patients, displayed four to nine allergen bands, indicating that the cat 1 reference is not a pure preparation but rather an enriched fraction.

IgE-mediated allergic reactions to potatoes.

We investigated sera of 12 patients with IgE-mediated hypersensitivity reactions to potato, using different in vitro methods. Radioallergosorbent test classes 2-4 (7.6-46.5% binding) were measured with potato allergen disks. Immunoblot detected allergen bands with an isoelectric point range of approximately 4.5-5.2 and mainly in a molecular weight range between 16.00 and 30.00 kD. But also at molecular weights of 43.00 and 65.00 kD allergen bands (IgE specificities) could be detected. Histamine release assay performed with isolated fractions of the potato extract showed a great individual variation and positive results of fractions of molecular weights between 10.00 and 80.00 kD. From our data we conclude that raw potato is a 'multiallergenic' vegetable.

Leukocyte histamine release and humoral changes during oral and subcutaneous hyposensitization of grass pollen allergic children.

Thirty-one children with seasonal hay fever, sensitive to rye grass by skin test, RAST, and leukocyte histamine release, received preseasonal hyposensitization with a rye grass pollen extract either subcutaneously (cumulative dose 40,000 PNU) or orally (cumulative dose 400,000 PNU). While there was a significant (p less than 0.005) increase in specific serum IgG-antibodies and a decrease (p less than 0.005) of leukocyte sensitivity in the subcutaneously treated group, oral treatment with an aqueous pollen extract could not be demonstrated to be immunologically effective.

Comparison of three variations of the radioallergosorbent test-inhibition assay for measuring allergenic activity of grass pollen extracts.

Three variations of the radioallergosorbent (RAST)-inhibition assay (RI) have been compared for measuring the allergenic activity of grass pollen extracts. The main difference between the three assays consisted in the solid supports to which the allergens were coupled. These supports were paper disks, microcrystalline cellulose, and Matrex. It could be demonstrated that all three variations of RI yielded almost identical results as expressed by their F value (F0). Correlations between the three methods were highly significant (P less than 0.001). Three independent valid assays showed an excellent reproducibility of each test system (P less than 0.01). Three different preparations of each of the supports yielded highly reproducible F0 (0.76 +/- 3% SD). Evaluation of the 50% inhibition values achieved with the different assays based on protein showed differences between the three supports. Provided the same allergen extract is coupled to the different solid supports and the results are related to a reference preparation, different laboratories using different forms of RI will be able to compare their results.

Comparison of histamine release assay and RAST inhibition as tools for allergen extract standardization.

We compared allergen-induced histamine release (HRA) from actively sensitized human leukocytes and RAST inhibition (RI) for their capacity to measure the activity of allergen extracts. Four different grass pollen preparations were investigated for their allergenic activities by employing leukocytes and sera from 13 patients sensitive to grass pollen allergens. We could demonstrate that both test systems detect differences in allergenic activities of the preparations with similar accuracy and reproducibility. HRA is more sensitive than RI but shows greater variability of the results. Pooling of sera from individual patients diminishes individual sensitivities in RI. By this means the precision of RI is further increased. As reagents for RI can be standardized and stored under stable conditions, RI can be performed at different times and different laboratories under identical conditions. For these reasons, RI seems to be more suitable for routine standardization of allergen extracts than HRA.

Studies on allergen and allergoid preparations from purified timothy (Phleum pratense) pollen extracts. III. Comparative investigations by skin prick tests and nasal provocation tests.

The allergenic activities of allergen (GEN) and allergoid (GOID) preparations from partially purified timothy pollen extract were investigated in quantitative skin prick tests and nasal provocation tests. In skin prick test GOID yielded significantly less activity than GEN. Approximately 90 times more protein of GOID than of GEN was necessary to elicit wheals of the same mean size as 1 mg/ml histamine control solution (1 HEP). In nasal provocation the patients seemed to react differently with respect to their nasal sensitivity to GOID. One group of patients showed equal sensitivity to GOID and GEN, whereas the second group showed higher thresholds to GOID. These differences in nasal sensitivity to GOID could be of practical value to predict the optimal dosage schedule for the treatment of patients with GOID.

Molecular weight determination of allergen extracts and isolation of allergenic molecules by high-performance liquid chromatography.

The usefulness of size-exclusion high-performance liquid chromatography (HPLC) for the molecular weight determination of allergen extracts as a method of process control was investigated by measuring seven different batches of timothy pollen extracts. Preparative HPLC was used for the isolation of the major allergen Cat 1, from a crude cat hair and skin scraping (CHSS) extract. The chromatograms of the seven batches of the timothy pollen extracts looked very similar, suggesting the same molecular weight distribution in each extract. The major allergen, Cat 1, could be isolated from the crude CHSS extract by preparative HPLC in acceptable purity, determined by crossed radioimmunoelectrophoresis.

Comparison of classical Lowry, modified Lowry and a dye-binding assay for the estimation of protein in allergen extracts and influence of different parameters on the modified Lowry assay.

Protein values of dialysed allergen extracts determined by Lowry, modified Lowry (trichloroacetic acid precipitation of the proteins) and dye-binding assay were compared. The influence of different parameters on the modified Lowry was examined. The reproducibility of the modified Lowry was checked with three independent measurements. For the examination of recovery a constant amount of 6-grass pollen allergen proteins was added to the samples of the standardized human serum albumin prepared for the calibration curve. The samples were measured by modified Lowry. The mean of the ratio between the protein values of the dialysed allergen extracts obtained by modified Lowry and those obtained by classical Lowry was 3.59 (coefficient of variation Cv = 45%). The mean of the ratio between the protein values of the allergen extracts obtained by modified Lowry and dye-binding assay was 1:0.71 (Cv = 31%). Phenol interfered with the modified Lowry. Phenolic allergen extracts showed higher "protein values" than non-phenolic allergen extracts. This influence could be reduced by a second precipitation of the dissolved precipitate. The precipitation of non-phenolic dialysed aqueous allergen extracts was complete after the first trichloroacetic acid precipitation. By incubating samples with the Folin-Ciocalteu's reagent at 55 degrees C in a waterbath, the time necessary for developing the colour could be reduced from 45 min to 5 min. Protein measurements by modified Lowry of a 6-grass pollen allergen extract in three different laboratories showed good reproducibility. For these extract 785 micrograms protein/ml (Cv = 4%) could be measured.(ABSTRACT TRUNCATED AT 250 WORDS)

Escherichia coli as a biologically active ingredient of suppositories. Solid phase extraction and quantification in a double antibody ELISA.

Techniques for isolation and quantification of an active ingredient of biological origin from a pharmaceutical product (Posterisan suppositories) were developed. By means of ELISA (enzyme-linked immunosorbent assay) in connection with a computerized evaluation, the antigenic material, a bacterial culture suspension (BCS) of Escherichia coli as a raw material was shown to be specifically and reproducibly detectable and quantifiable. As a limit of detection, a bacteria concentration of 6 x 10(4) cells/ml was determined, corresponding to 0.02% of the concentration in the product. After treating of the suppositories with organic solvents, the E. coli antigens were extracted with silica columns. The complete validations of both methods, the ELISA itself and the extraction procedure of the antigens from the matrix, in accordance with pharmaceutically accepted principles are presented. The eventual application of the new technique to the analysis of other pharmaceuticals with similar ingredients as well as the possibility of substituting conventional methods like total cell counting is discussed.

Influence of a co-stimulation of human leucocytes with an Escherichia coli preparation and fixed immunoglobulins on cytokine release in the presence of hydrocortisone.

Pharmaceuticals of biological origin consisting of bacterial culture suspensions (BCS) as active ingredients have long been used for the treatment of hemorrhoidal diseases and chronic anal pruritogenic eczemas. However, some of these pharmaceuticals often contain glucocorticoids such as hydrocortisone as an anti-inflammatory supplement. Therefore, the question arises whether the claimed immunostimulatory capacity of the bacterial culture suspension might be altered by the steroid. Up to now, numerous reports support the evidence that the stimulation of the different Fc-receptor subtypes on leucocytes result in profound immunoregulatory activities influencing phagocytosis and antigen processing, antibody-dependent cytotoxicity or secretory functions thereby enhancing the overall activities of the immune system towards foreign antigens/pathogens. With these findings in mind it was investigated whether the immunomodulatory capacity(s) of the BCS in the presence of hydrocortisone will be modified by solid-phase bound immunoglobulins (Igs). For this purpose freshly prepared human peripheral blood leucocytes (PBLs) were incubated with different concentrations of the BCS (0.1, 1, 10 micrograms/ml), either with or without fixed human immunoglobulins in the presence of increasing concentrations of hydrocortisone. As a parameter of PBL activation the secretion of different cytokines was measured, e.g. tumor necrosis factor alpha (TNF-alpha), interleukin-10 (IL-10) and granulocyte-macrophage colony stimulating factor (GM-CSF). Cytokines were determined with specific sandwich ELISAs. With this modified cell culture system it was demonstrated that the immunosuppressive activities, normally caused by hydrocortisone, were partially antagonized by the combination of BCS plus fixed Igs. TNF-alpha and GM-CSF were significantly more produced, even in the presence of hydrocortisone, whereas the synthesis of IL-10 was diminished by fixed Igs. However, this effect could be reversed with increasing concentrations of hydrocortisone. These results raise the possibility that in the natural environment, e.g. the rectal mucosa, antigens derived from the BCS are bound by specific Igs, thereby modifying secretory and effector functions of locally present leucocytes in another way as free antigens. The biological relevance of these in vitro data with respect to the therapeutic benefit of the BCS preparations with hydrocortisone will be discussed considering recent findings in the literature.

Respiratory allergies (bronchial asthma and rhinitis) due to sensitization of type I allergy to red spider mite (Panonychus ulmi KOCH).

The inhabitants of a fruit growing area often report spontaneously of sensitization to the red spider mite (RSM) (Panonychus ulmi KOCH). These are for the most part sensitizations with low clinical symptoms (rhinitis, conjunctivitis and erythema). Severe clinical developments with bronchial asthma have been observed. We investigated six patients working in a fruit growing area sensitized by RSM. The sensitizations corresponded to a Type I allergy. Skin tests and provocation tests (nasal as well as bronchial) with RSM showed immediate reactions and RAST positive results were obtained using RSM allergen disks. RAST measurements of sera from nine house-dust mite Dermatophagoides pteronyssinus allergic patients using RSM allergen disks showed RAST-class > 1 for eight patients. RAST inhibition and immunoprint suggest a possible cross-reaction between RSM and D. pteronyssinus.

Studies of allergen and allergoid preparations from purified timothy (Phleum pratense) pollen extracts. I. Physicochemical characteristics and binding to allergen-specific human IgE.

Physicochemical and in vitro allergenic properties of two different preparations of timothy (Phleum pratense) pollen were compared. The refined allergen extract, timothy B, contained proteins of molecular weights from 10,000 to 45,000 daltons. The allergoid preparation, which was prepared by mild treatment of the partially purified extract with formaldehyde, possessed only a trace of activity in the RAST inhibition assay. The formaldehyde treatment also resulted in a shift of the net charge of proteins to the more acidic site, as demonstrated by isoelectric focusing. Furthermore, it was shown that the activities of naturally occurring enzymes of native allergen extracts were reduced considerably. Only traces of acid phosphatase activity could be demonstrated in timothy B allergoid.

Studies on allergen and allergoid preparations from purified timothy (Phleum pratense) pollen extracts. II. Anaphylaxis studies in rats and histamine release from human leukocytes.

In vivo and in vitro allergenic activities of allergen and allergoid preparations from partially purified timothy pollen extract were measured in three different systems. In heterologous PCA titration, the allergen preparation was found to be 32 times more allergenic than the allergoid preparation. By intravenous chaled that treatment of allergen preparations with formaldehyde led to a preparation with the properties of an allergoid. In addition, it was found that the carbohydrate fraction of the refined allergen preparation of timothy a reduction in blood pressure. Thus, the allergen preparation was at least 50 times more potent in inducing systemic anaphylaxis in sensitized rats than the allergoid preparation. In the histamine release assay of grass-sensitive human leukocytes, approximately 1,000 times more allergoid protein than allergen protein was required to achieve 30% histamine release. Although to different degrees, in all three test systems in could be demonstrated that treatment of allergen preparations with formaldehyde led to a preparation with the properties of an allergoid. In addition, it was found that the carbohydrate fraction of the refined allergen preparation of timothy pollen decreased the blood pressure even in nonsensitized animals. The effect of this carbohydrate fraction on blood pressure was immediate but short-lived and immunologically nonspecific.

High-performance liquid chromatographic molecular weight determination of allergen extracts. Examination of the influence of the column material on allergenic activity and allergen patterns.

To investigate whether the column material employed in size-exclusion high-performance liquid chromatography (HPLC) influences the allergenic activity and antigen/allergen patterns of allergen extracts, the molecular weights of a mite and a birch pollen allergen extract were determined using a Bio-Sil TSK 250 column with a guard column. The allergenic activities were measured by RAST inhibition and the antigen/allergen patterns were determined by crossed (radio)immunoelectrophoresis. The original extracts and the corresponding eluates after HPLC runs showed the same allergenic activity and the same number of antigen/allergen precipitation lines. Only slight differences in the peak heights of some precipitates were observed.

Kinetics of allergen release from house dust mite Dermatophagoides pteronyssinus.

Purified bodies of Dermatophagoides pteronyssinus were extracted over different extraction times. The release kinetics of proteins, carbohydrates, and allergens were assessed by different methods. Additional attempts have been made to elucidate the extraction process also on a molecular basis. For this purpose the extracts were analyzed by isoelectric focusing, SDS gel electrophoresis, crossed radioimmunoelectrophoresis, and an immunoprint technique. Carbohydrates, proteins, and allergens were released immediately from the mite bodies but demonstrated different release kinetics. The release of carbohydrates and proteins was linear up to 48 hours but demonstrated different slopes. The major allergen P1 demonstrated steady release up to 48 hours, whereas the overall allergenic activity as determined by RAST inhibition demonstrated typical saturation kinetics reaching a plateau after 4 to 5 hours of extraction. It was demonstrated by parallel-line assay of the RAST-inhibition curves that the allergenic determinants remained unchanged up to 48 hours. Different electrophoretic methods demonstrated that all relevant proteins and allergens were released immediately from the mite bodies. The electrophoretic patterns changed only slightly up to 4 hours. Prolonged extraction times up to 48 hours did not affect the patterns. This makes a loss of proteins or allergens by degradation unlikely.

Application of the first international standard of Dermatophagoides pteronyssinus (house dust mite) in the evaluation of allergen extracts produced from two different source materials.

The first international standard (IS) of the house dust mite Dermatophagoides pteronyssinus was used to compare side-by-side two allergen extracts prepared from two different house dust mite sources, namely whole mite cultures (WMC) and purified mite bodies (PMB), by employing several biochemical and immunochemical methods. In most methods employed, IS and WMC resembled more to each other than to PMB. These similarities comprehend protein contents, protein patterns, antigen and allergen patterns as well as total in vitro activity obtained in RAST inhibition (RI). The differences between WMC and PMB could be reproduced in different batches. Standardization in relation to IS of D. pteronyssinus yields activity ratios ranging from 2.4 to 70.1 between WMC and PMB depending on the variation of RI employed, indicating quantitative differences between the two preparations. When coupled to BrCN activated paper disks and employed in direct RAST, both preparations yielded almost identical binding values of specific IgE. Also in skin prick test, WMC and PMB produced very similar results in 12 mite-allergic patients. It was not possible to differentiate between the two preparations when used in concentrations which are usually employed for diagnostic purposes. These findings demonstrate the diversity of allergens in mite extracts derived from different sources and reveal the problems which are involved with the use of the WHO D. pteronyssinus IS.

Contact urticaria to rubber gloves is IgE-mediated.

Serological studies were performed in 3 patients with typical contact urticaria to latex surgical gloves. Specific IgE antibodies to natural latex as well as to vulcanized glove material have been detected by radioimmunoassay. Contact urticaria to latex is considered to be of the immunologic type.

[Analysis of individual antibody reactivity of patients allergic to grass pollen using isoelectric focusing and immunoblotting]. / Analyse der individuellen Antikörperreaktivität von Graspollenallergikern mittels Isoelektrofokussierung und Immunblotting.

Isoelectrofocussing and immunoblotting provide means to determine individually the specific antibody response of grasspollen allergics, especially of the IgE-class. Furthermore, these methods permit the assessment of the allergenic potency of single components of antigen extracts.