Mediator release assays based on human or murine immunoglobulin E in allergen standardization

BACKGROUND: The biological potency of allergens can be measured by provoking mediator release from effector cells. As established immunochemical methods in allergen standardization only determine inhibition potency or major allergen content, routine tests for biological potency may enhance standardization and batch control of allergen products. OBJECTIVE: The general performance and application potential of biological in vitro assays in batch control and standardization of allergens and as a tool for verifying activity and stability of allergen standards were analysed. METHODS: Allergen extracts of five clinically relevant allergens from three to five different manufacturers were investigated. A CAP-IgE-inhibition assay was compared with mediator release assay (MRA)s based on murine or human basophils. Rat basophilic leukaemia (RBL) cells were passively sensitized with pooled murine allergen-specific IgE-containing sera. Humanized RBL cells and human-stripped basophils were sensitized with pooled patient's sera, which were also used for the CAP-IgE-inhibition assay. Allergen specificity of the sera was determined by immunoblotting. RESULTS: A good batch-to-batch consistency was found with each assay among all manufacturers and allergens tested. Between different manufacturers, the products showed differences in activity and the various assays indicated an almost identical ranking. However, the biological assays revealed qualitative differences of biological activity or composition of allergen preparations undetectable by IgE-inhibition assay. CONCLUSIONS: MRAs provide refined information on allergen activity, either confirming the results of IgE-inhibition assay, or indicating differences requiring further investigation, and represent a highly sensitive novel tool in allergen standardization. By using permanently cultivated cell lines, repeated venepuncture to obtain human basophils is avoided. As in the RBL assay, the coefficient of variation for the release values were below 15% and for the ED50 below 25%, the assay is suitable to determine differences that are relevant for batch control purposes.     

IgE FAST inhibition: a rapid technique for the standardization of pollen allergen extract potency

An IgE FAST(TM) inhibition protocol has been developed as a rapid in vitro method for determining relative potencies of in vivo allergenic extracts. Results are obtained within one day and correlate well with those of the RAST inhibition protocol (r = .96), making the FAST assay a preferable procedure for inprocess potency determination during the manufacture of standardized allergenic extracts.     

Latex allergen IgE assays in the assessment of Veterans Affairs health care workers.

BACKGROUND: A previous multicenter study of Veterans Affairs health care workers evaluated hospital participants for latex hypersensitivity. Well-defined groups from that study allowed us to explore the diagnostic utility of newer antilatex allergen IgE immunoassays in the present study. OBJECTIVES: To determine whether an enhanced CAP (ENHCAP) assay or an enzyme-linked immunosorbent assay (ELISA) identifies latex glove symptomatic individuals with antilatex allergen IgE that had not been detected by the CAP assay used in the original study and to determine the specificity of the ENHCAP assay. METHODS: The ELISA measured IgE antibody to Malaysian nonammoniated natural rubber latex extract (MNA), Hev b1, Hev b5, and Hev b6. Four patient groups were tested: confirmed latex glove allergic, latex glove symptomatic, latex glove sensitized/asymptomatic, and latex glove nonallergic. RESULTS: The ENHCAP assay and the MNA ELISA were highly concordant with the original CAP assay. In the subgroup with latex glove symptoms that were previously negative by the CAP assay, the ENHCAP assay value was elevated in 7 (11%) of 64 samples, only 3 of which were class 2 or higher. The MNA ELISA result was positive in only 4 (6%) of these 64 samples, and 3 of these were fractionally above the cutoff value for this assay. CONCLUSIONS: The ENHCAP assay and the MNA ELISA identified a few additional positive individuals in the group that was latex glove symptomatic and originally CAP assay negative. The ENHCAP assay and the MNA ELISA produced only a modest improvement in diagnostic sensitivity over that of the original CAP assay.     

Immunoassay of specific IgE: low level assays require measurement of allergen specific assay background.

The allergen-specific backgrounds of a paper disk radioimmunoassay (RIA) system and a cellulose sponge fluorescent enzyme immunoassay (FEIA) system were evaluated using three inhalants, timothy, short ragweed, and cat, with reagents obtained from the same manufacturer. Radioimmunoassay was performed with Phadebas RAST reagents by the modified RAST method, and FEIA by the Pharmacia CAP System. Horse serum, 5% HSA, assay diluent, and serum pools from nonallergic and allergic patients, were assayed. The lower limit of detection (LLD) was defined using both the Z distribution (as is conventional) and the t distribution. The solid phases, analytes, and assays differed (p less than .001) in background results. For RIA, background was lowest for timothy and highest for cat; for FEIA, background was lowest for cat and highest for short ragweed. For RIA, background assessed with the allergic serum pool was higher than the other analytes; for FEIA, responses of the five analytes did not differ. For timothy and short ragweed, background of RIA was lower than FEIA. For FEIA, the highest LLD calculated using the Z distribution was 11 SD lower than the manufacturer's recommended quantitative cutoff; for RIA, the highest LLD calculated was 2.5 SD higher than the recommended analytic cutoff. The analytic false positive rate for RIA may differ between allergic and nonallergic patient populations. Laboratories reporting results near either assay's background should set LLD based on assay of allergen-specific negative controls in each assay run.     

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.     

Effect of reagins and allergen extracts on radioallergosorbent assays for mite allergen

The reproducibility of the radicallororbent (RAST) inhibition and direct binding assays with mite allergen were investigated in the presence of heterogeneous extracts and non-mite-sensitive atopic sera. Both contain components similar to potential contaminants which would occur in the assay of mite allergen and dust allergen extracts. The standardized inhibition and direct binding assays employed had a day to day (n= 4) coefficient of variation [(s.d. ± 100)/mean] of 15% and 24% respectively. The inhibition assay for mite allergen was reproducible in the presence of protein concentrations of added plant, fungal, arthropod and animal extracts in excess of the protein concentrations that occur under the operational mite assay conditions. The mite inhibition assay was also reproducible in the presence of non-mite allergen extracts, with and without additional sera containing IgE specific for the non-mite allergens. The binding of a constant quantity of mite allergen to the activated solid phase in the direct binding assay was reproducible in the presence of added bovine serum albumin, and of a fungal or arthropod extract, representing the heterogeneous components of an allergen extract at the concentrations of total protein known to occur in the direct binding assay of mite extracts.     

Identification of a German cockroach-specific allergen by human IgE and rabbit IgG

To further study the immunology of insect hypersensitivity, we identified and partially characterized the principal allergens in whole body German cockroach (WBGCR) (Blattella germanica) and compared this extract to whole body antigens prepared from other insects. WBGCR extract was fractionated over a calibrated Sephadex G-200 column; peak allergenic activity was contained in fraction 3 (GCR3), containing components with apparent molecular weights ranging from 12,500 to 75,000 daltons. The antigenicity, allergenicity, and specificity of GCR3 components were tested by using rabbit antisera raised to GCR3 or true armyworm (Pseudaletia unipuncta). Radioimmunoassay, cross-inhibition and immunoblot studies revealed, particularly in the IgE system, that GCR3 contained an allergen with a pI of 6.7 and MW of 36,000 daltons that was unique to WBGCR extracts and not present in other insect species, including true armyworm, caddis fly, lakefly, yellow jacket, or honeybee. This GCR3 component may represent a specific marker for the diagnosis of cockroach hypersensitivity in an insect-sensitive population of individuals.     

Suppression of murine IgE responses with amino acid polymer/allergen conjugates. II. Suppressive activities in adjuvant-induced IgE responses

Treatment with conjugates of polysarcosine and grass pollen allergen extracts efficiently suppressed the induction of IgE responses in mice. The suppressive activity was shown to be allergen-specific and required covalent linking of the polysarcosine. Inhibitory effects could be overcome by booster injections of native allergen when these were given 3-4 weeks after treatment with conjugates. Administration of conjugates had only marginal effects on established IgE responses. The variance of these results with those of other studies on IgE suppression and the suitability of murine models for investigating reaginic antibody suppression are discussed.     

Experimental studies on red cell-based assays for total IgE and allergen-specific IgE

The potential of red cell-based assays for IgE and allergen-specific IgE has been examined using mouse monoclonal anti-human IgE antibodies and chimaeric human IgE anti-4-hydroxy-3-iodo-5-nitrophenacetyl. Experiments concerned with developing a red cell IgE antibody-capture assay for allergen-specific IgE have pointed to the advantages of presenting the allergen on a second agglutinable red cell.     

Indoor allergens: relevance of major allergen measurements and standardization

Major allergen measurements have relevance for the standardization of allergen extracts for immunotherapy and for epidemiologic studies into the cause of allergic diseases. Standardization is still centered around overall IgE-binding potencies (biological standardization). Major allergen levels show significant correlation with IgE-binding potencies, but ratios of the two can differ 5- to 10-fold between individual extracts. Major allergen quantities needed for effective and safe subcutaneous immunotherapy are proposed to be between 5 and 20 microg per maintenance shot. Although this figure is not really based on dose-finding studies, it has reached the status of a guiding principle. It is necessary to add major allergen measurements to standardization requirements to design adequate dosage schemes and elucidate the dose-response relation between major allergen dose and therapeutic effect. This will also help clarify to what extent sublingual immunotherapy requires higher doses of major allergen. Fine specificity of different assays toward isoforms and other variants of single allergens often results in diverging allergen measurements. Standardization should be based on certified major allergen references and accompanying assays that are cross-reactive enough to recognize all variants to facilitate comparability. This will also ensure that primary and secondary prevention strategies aiming at regulating allergen exposure will stay on solid ground.     

Mediator release from human lung mast cell subtypes in chronic bronchitis and emphysema

Human lung mast cells were obtained from pulmonary tissue of normal individuals and patients with chronic bronchitis or emphysema by enzymatic dispersion. Based on their density two mast cell subtypes, a formalin-sensitive (FS) and a formalin-insensitive (FI) cell type, could be separated.Although differences in anti-IgE-induced histamine release could be demonstrated for the mast cell subtypes of normal individuals, these experiments could not be performed for both mast cell subtypes from both patient groups. LTC₄ and PGD₂ release could be demonstrated for the FS- and FI-mast cell respectively. The release of PGD₂ from FI-mast cells of patients with chronic bronchitis was enhanced as compared with normal subjects.     

Suppression of murine IgE responses with amino acid polymer/allergen conjugates. IV. Suppressive activities in established IgE model systems

Conjugates of poly-N-methylglycine (polysarcosine) and grass pollen extracts, previously found to be capable of suppressing immature IgE antibody responses in mice, were shown to be highly effective at inhibiting the capacity of immune splenocytes to produce a secondary response in sub-lethally irradiated recipient animals. Anamnestic IgE responses in mice primed without adjuvant were also suppressed, but the effects were modest and of short duration. The predictive value of murine models for selecting clinically appropriate specific IgE suppressive agents and treatment schedules that might be successfully employed for clinical use are discussed.     

Influence of bronchial allergen challenge on histamine release by human basophils

BACKGROUND: Basophils can be primed by cytokines such as interleukin (IL) -3, IL-5 or granulocyte macrophage-colony stimulating factor (GM-CSF). It has been described that the concentrations of these cytokines are enhanced at sites of allergic inflammation as well as systemic in allergic asthma. OBJECTIVE: To investigate the priming status of basophils as detected by thapsigargin-induced histamine release during bronchial allergen challenge. METHODS: Ten subjects allergic to house dust mite were challenged via an aerosol delivery system. Spontaneous leucocyte histamine release as well as histamine release induced by various stimuli was measured in vitro at several time points. In addition, lung function parameters, serum IL-5 and blood eosinophil counts were evaluated. RESULTS: We found no effect of bronchial allergen challenge upon spontaneous leucocyte histamine release, nor upon histamine release induced by anti-immunoglobulin (Ig) E, house dust mite extract, C5a, fMLP, IL-3, PMA+ thapsigargin or IL-3+ thapsigargin. However, the priming status of basophils as measured by thapsigargin-induced histamine release was enhanced at 24 h after bronchial allergen challenge. Analysis of the individual data showed a heterogeneous initial response (30 min, 6 h) followed by a predominant increase at 24 h after allergen challenge. This increase in the thapsigargin-induced histamine release correlated with the increase in serum IL-5 levels at 24 h after allergen challenge. CONCLUSION: The priming status of human basophils as measured by thapsigargin-induced histamine release is enhanced 24 h after allergen challenge.     

Isotopic and enzymatic IgE assays in non-allergic subjects

In order to establish normal values in an adult population for serum IgE concentration, sera were obtained from 446 ambulatory Canadian caucasian subjects with negative allergy histories. A standard isotopic procedure, the Phadebas paper radioimmunosorbent test (PRIST), was compared with the new enzymatic counterpart, the Phadezyme PRIST. By the isotopic method, serum IgE concentrations in women and men were comparable from one age group to another with no age-related trend in the seven age groups examined (15-20, 21-30, 31-40, 41-50, 51-60, 61-70, above 70). The median and 95th percentile units (U)/ml respectively were 17.5 and 145 for 224 women and 25.5 and 275 for 222 men. Mean values +/- 1 SD for women were 43 +/- 102 and for men, 58 +/- 137. Levels were significantly higher in men as a group. Sera with IgE concentrations above 100 U and a sampling of additional sera were tested for specific IgE antibodies to 13 common allergens by the radioallergosorbent test (RAST). After exclusion of RAST-positive sera, the mean U/ml values +/- 1 SD were 22 +/- 29 for 204 women and 37 +/- 54 for 196 men. Geometric mean U/ml values for these sera were 14.6 for women and 22.3 for men and the median and 95th percentile U/ml respectively for the women were 15 and 66, for the men, 24 and 135. These 95th percentile values are considered the upper limits of normal in this population. The RAST identification of antibodies to allergens to which sensitization was demonstrated provided a potential explanation for serum IgE concentrations above 100 U/ml in less than 30% of the sera in this population with negative allergy histories. The isotopic method and the counterpart enzymatic method (Phadezyme PRIST) were highly comparable; the correlation coefficient (r) for all the sera was +0.93 (P less than 0.001). IgE levels were significantly higher in male smokers than non-smokers by both methods but these differences were not significant in women.     

Cord blood house dust mite allergen in newborns: relationship to maternal blood levels of allergen and allergen specific IgG and IgE.

BACKGROUND: House dust mite allergen has previously been detected in the cord blood of some newborns but not others. The factors that affect the presence and levels in cord blood of this and possibly other allergens are unknown. OBJECTIVE: To test the hypothesis that the levels of maternal allergen and allergen specific IgG affect the presence and levels of newborn allergen. METHODS: Enzyme-linked immunosorbent assays were used to measure the levels of house dust mite allergen Der p 1 and Der p 1 specific IgG and IgE in paired blood samples from 98 mothers and full-term newborns. RESULTS: Der p 1 was detected in 27 mothers and 12 newborns. None of the 71 mothers who lacked Der p 1 had Der p 1-positive newborns. When present, cord blood Der p 1 levels correlated with and were approximately one third of Der p 1 levels in maternal blood. Cord blood Der p 1 levels also tended to correlate with maternal blood levels of Der p 1 specific IgG but showed little, if any, correlation with maternal blood levels of Der p 1 specific IgE. CONCLUSIONS: When detectable, the levels of an allergen in the blood of newborns correlate with the levels of that allergen in the blood of their mothers and tend to be related to maternal levels of allergen specific IgG.     

Haplotype-based banking of human pluripotent stem cells for transplantation: potential and limitations

High expectations surround the area of stem cells therapeutics. However, the cells' source-adult or embryonic-and the cells' origin-patient-derived autologous or healthy donor genetically unrelated-remain subjects of debate. Autologous origins have the advantage of a theoretical absence of immune rejection by the recipient. However, this approach has several limitations with regard to the disease of the recipient and to potential problems with the generation, expansion, and manipulation of autologous induced pluripotent stem cells (iPS cells) preparation. An alternative to using autologous cells is the establishment of a bank of well-characterized adult cells that would be used to generate iPS cells and their derivatives. In the context of transplantation, such cells would come from genetically unrelated donors and the immune system of the recipient would reject the graft without immunosuppressive therapy. To minimize the risk of rejection, human leukocyte antigen (HLA) compatibility is certainly the best option, and the establishment of an HLA-organized bank would mean having a limited number of stem cells that would be sufficient for a large number of recipients. The concept of haplobanking with HLA homozygous cell lines would also limit the number of HLA mismatches, but such an approach will not necessarily be less immunogenic in terms of selection criteria, because of the limited number of HLA-compatible loci and the level of HLA typing resolution.     

Human immune system mice: current potential and limitations for translational research on human antibody responses

It has recently become possible to generate chimeric mice durably engrafted with many components of the human immune system (HIS mice). We have characterized the maturation and function of the B cell compartment of HIS mice. The antibody response of HIS mice to T cell-dependent B cell antigens is limited, and contributing factors may be the general immaturity of the B cell compartment, infrequent helper T cells selected on human MHC class II antigens, and incomplete reconstitution of secondary lymphoid organs and their microenvironments. In contrast, HIS mice generate protective antibody responses to the bacterium Borrelia hermsii, which acts as a T cell-independent antigen in mice, but do not respond to purified polysaccharide antigens (PPS). We speculate that the anti-B. hermsii response of HIS mice is derived from an abundant B cell subset that may be analogous to B1 B cells in mice. We suggest that failure of HIS mice to respond to PPS is due to the lack of a B cell subset that may originate from adult bone marrow and is highly dependent on human interleukin-7 for development.