Detection of antibodies to cell surface antigens by a simplified cellular elisa (CELISA)

An improved method for screening human hybridoma antibodies to cell surface antigens is described. The following modifications have been developed: rapid expansion of desired screening cell types by EBV transformation; use of only 5 X 10(4) cells/well; elimination of the need for glutaraldehyde fixation; elimination of the requirement for PLL to attach cells to microplates; preparation of a large number of plates which can be stored at 4 degrees for 3 months; Protein A-peroxidase ELISA assay yielding excellent replicates, low background "noise", and high OD readings for positive wells. The techniques we have developed should greatly simplify and shorten the assay procedures for detecting human antibodies to a variety of cell surface antigens.     

Immunochemical identification of mouse IgE

Immunochemical identification of a distinct immunoglobulin class associated with mouse reaginic antibody and designated IgE has been performed. An antiserum against mouse reaginic antibody was prepared in rats by immunization with homologous peritoneal mast cells sensitized with mouse reaginic antibody. This antiserum, after adequate absorption, recognized only one component existing in mouse serum or fractions of serum containing reaginic antibody. This component was not any of the already known γ₁, γ₂, IgA or IgM immunoglobulins; its immunoglobulin nature was indicated by its antigen-binding capacity in radioimmunodiffusion analysis. Fractionation experiments (zone electrophoresis, gel filtration on Sephadex G-200) showed that there was a strict association between reaginic anaphylactic activity and IgE immunoglobulin. Molecular weight of IgE was found to be about 200,000. In biological tests, anti-IgE neutralized anaphylactic activity attributed to reaginic antibody, but not that attributed to γ₁-antibody. Anti-IgE degranulates normal and sensitized mouse peritoneal mast cells, and rat peritoneal mast cells after they have been sensitized by mouse reaginic antibody.     

Proteolytic digestion of mouse IgE

Conditions are described for the preparation of F(ab')2 and Fab fragments of mouse IgE. Papain, pepsin or trypsin each produced F(ab')2 fragments with Mr approximately equal to 130,000 which yielded Fab fragments on further digestion. The release of Fab fragments from F(ab')2 resulted from further cleavage of the H chain. Pepsin, and especially trypsin appear more suitable for the preparation of F(ab')2 because of the difficulty of separating a 93 kDa by-product from the F(ab')2 produced by papain. The best yields of purified Fab were obtained with papain. Rates of digestion were in the order, pepsin approximately equal to trypsin much greater than papain.     

Competitive inhibition of passive sensitization of mouse mast cells by IgE. A bioassay for mouse and rat IgE.

Possibility of inhibition of an efficient in vitro IgE-sensitization system was studied. The sentization of mouse peritoneal mast cells with an anti-ovalbumin IgE-rich fraction of serum, as tested by ovalbumin-induced degranulation, was inhibited by previous incubation with antisera of another or of no specificity. Fractionation and other experiments showed that the inhibiting activity correlated with IgE content. IgGl did not seem to have an effect. Sensitization was also inhibited by rat myeloma IgE, 50 ng giving a 50 per cent inhibition. Plots of the logarithms of rat and mouse IgE concentration vs their inhibitory effect on sensitization gave two parallel linear curves, indicating that mouse and rat IgE compete for the same receptor sites. It was thus possible to use this system as a sensitive bioassay for both mouse and rat IgE levels and, by comparing inhibition by mouse IgE to that by a known rat IgE standard, to obtain not only relative data but absolute mouse IgE levels. This, and also a better discrimination of IgE doses, was the major advantage of this bioassay in relation to the equally sensitive anti-IgE degranulation tests.     

Detection of mosquito saliva-specific IgE antibodies by capture ELISA

We developed an IgE-capture ELISA and measured mosquito saliva-specific IgE antibodies in 27 children sensitive to mosquito bites. Children with large 15-min bite wheals had significantly higher ( P < 0.0005) mosquito saliva-specific IgE levels than children with small wheals. In the latter group, the saliva-specific IgE level was significantly higher ( P =0.031) than the levels of six infants never exposed to mosquitoes. A positive correlation ( r =0.65; P =0.0002) was found between the size of the 15-min wheal and the mosquito saliva-specific IgE antibody levels. These results further support the role of mosquito saliva-specific IgE antibodies in the pathogenesis of mosquito-bite whealing. Compared to immunoblotting, IgE-capture ELISA provides a quantitative method to measure mosquito saliva-specific IgE antibodies.     

Detection of specific IgE to quinolones

BACKGROUND: In the last years, immediate reactions to quinolone antibiotics have been observed with increasing frequency, mainly urticaria, angioedema, and shock. No test was available because of the high incidence of false-positive results on skin tests. Thus the pathogenesis, value of diagnostic procedures, and cross-reactivity have not been evaluated in a systematic way. OBJECTIVE: We sought to assess whether these reactions are IgE mediated and whether an in vitro test for quinolone-specific IgE is useful in the diagnosis and understanding of cross-reactivity. METHODS: We assayed specific serum IgE to quinolones using epoxy-activated sepharose 6B as the solid phase in 55 patients with immediate adverse reactions; specificity of IgE binding was demonstrated by inhibition tests. RESULTS: The test yielded positive results in 30 (54.5%) patients who were tested 1 to 48 months after the reaction had occurred. The quinolone-specific IgE seems to disappear more slowly in atopic patients. The cross-reactivity between various quinolones allowed us to identify a common structural motif within quinolones that might be responsible for clinical and serologic cross-reactivity. CONCLUSION: A substantial portion of immediate reactions to quinolones appear to be IgE mediated. Cross-reactivity of IgE among different quinolones is frequent and suggests that a common avoidance of quinolones should be attempted in all patients with respective symptoms.     

Detection of Allergen-Specific IgE Antibody Responses

Allergen-specific IgE production is the central event in the pathogenesis of atopic disorders and increases in specific IgE serum antibodies are an indicator of immediate hypersensitivity responses in humans and in animal models of allergy. Consequently, accurate and user-friendly methods are needed to measure serum levels of allergen-specific IgE. This review examines historical and recent developments in in vivo and in vitro methods for the detection of allergen-specific IgE in humans and in animal models. Routinely, in vitro methods such as enzyme-linked immunosorbant assays or radioallergosorbant tests and in vivo methods such as the skin prick test (SPT) for humans and the passive cutaneous anaphylaxis assay (PCA) used in animals are utilized to detect allergen-specific IgE. While in vivo assays are usually more accurate than in vitro assays since they provide a functional readout of IgE activity, they are relatively costly and require considerable expertise. On the other hand in vitro assays are limited by the fact that the amount of allergen-specific serum IgG exceeds IgE antibody by several orders of magnitude, resulting in competition for allergen binding. Consequently, methods that use allergen as a direct capture step are limited by the availability of free allergen binding sites for IgE. In order to circumvent this problem, in vitro methods usually require prior depletion of IgG or use high amounts of allergen in order to facilitate availability of free binding sites for IgE detection. Clearly, these approaches are limited for small sample volumes and allergens that are in short supply. New methods such as protein microarray could potentially overcome this problem by providing high allergen concentrations in a relatively small reaction volume. Currently, in vitro methods are rarely used in isolation for prognosis but are used primarily to complement the information obtained from in vivo assays. With the emergence of new technologies it is conceivable that in vitro assays may in the future replace in vivo assays, however until then in vivo assays remain the gold standard of allergen-specific IgE detection.     

尘螨变应原的免疫印渍法分析

尽管粉尘螨浸液中的许多蛋白具有变应原性,但进一步用免疫印渍法分析,它们又分成若干能和哮喘患者的IgE结合的较小组分,这些组分是异质的并显示出不同的图谱。与皮肤挑刺试验、ELISA比较:免疫印渍法中IgE抗F_1和皮肤挑刺试验阳性、阳性符合率达91%,免疫印渍法和ELISA法测IgE结果在统计上显著相关(r=0.7029 p<0.01)。因此本文提出:具有小分子量的变应原组分可形成众多的分子量较大的变应原,其中F_1(MW1800 OD)是主要变应原组分。     

Detection of human IgE to sulfamethoxazole by skin testing with sulfamethoxazoyl-poly-L-tyrosine

Adverse reactions to sulfamethoxazole (SMX) occur in 4% to 6% of normal individuals. Many of these reactions resemble immunopathologic reactions, but skin test or in vitro evidence of a role for IgE is limited. Earlier RAST studies in our laboratory provided evidence that the N4-SMX hapten was a major determinant in immediate hypersensitivity reactions to SMX. We tested the hypothesis that IgE to this hapten is present on the mast cells of patients who have experienced immediate hypersensitivity reactions temporally related to exposure to SMX. A multivalent skin test reagent, SMX168-poly-L-tyrosine, and a univalent hapten, SMX-tyrosine, were synthesized. Forty-four patients with histories of allergic reactions to SMX and six subjects who had been exposed to the drug, but who had not reacted, were skin tested. Twenty-seven percent of the history-positive patients were skin test positive. None of the control individuals was positive. The immunologic responses to SMX in three patients who had experienced allergic reactions during SMX/trimethoprim therapy were analyzed in serial skin test and RAST assessments. One to three years after the clinical reactions, IgE to SMX could be demonstrated by skin testing in all three patients with a SMX-poly-L-tyrosine skin test reagent. Skin test reactions were inhibited by the monovalent reagent, SMX-tyrosine, in a dose-dependent manner. SMX-specific IgE antibodies could also be detected by RAST in serum obtained within days of the reactions from two of the three individuals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of Epstein-Barr virus specific IgE antibody

To confirm the existence and investigate the biological significance of IgE virus-specific antibodies, we studied Epstein-Barr virus (EBV)-specific IgE antibody by enzyme-linked immunosorbent assay with an anti human IgE monoclonal antibody. We detected EBV-specific IgE antibody in sera not only from patients with the EBV associated diseases of infectious mononucleosis and nasopharyngeal carcinoma, but also from patients with bronchial asthma, collagen disease and healthy volunteers. However, there was no significant difference in the titers of IgE antibody specific for EBV among these groups. No significant relationship between the titers of EBV-specific IgG and IgE antibody, or between the titers of EBV-specific IgE and the total IgE levels in the sera was observed.     

Detection of Chlorella-specific IgE in mould-sensitized children

The content of IgE, specific to the unicellular green alga Chlorella sp., was analysed in sera from 46 atopic children sensitized to moulds, using radioallergosorbent test (RAST), immunoblotting and crossed immunoelectrophoresis/crossed radioimmunoelectrophoresis (CIE/CRIE). Chlorella-specific IgE was found in 23/46 sera by RAST, in 28/41 sera by immunoblotting and in 6/30 sera by CIE/CRIE. The Chlorella components most frequently binding IgE as analysed by gradient gel electrophoresis and immunoblotting were of molecular weights of approximately 13, 17, 19, 26 and 49 kD. Twenty-nine precipitating antigens, including seven IgE-binding precipitates were detected by CIE/CRIE. The study shows that low concentrations of specific IgE are formed to the green alga Chlorella in sera from atopic individuals sensitized to moulds.     

Measurement of antigen-specific mouse IgE by a fluorometric reverse (IgE-capture) ELISA

A reverse, or IgE-capture, enzyme-linked immunosorbent assay (ELISA) for measuring ovalbumin-specific IgE antibody in the serum of immunized mice has been developed. Microplate wells were first coated with a commercial anti-mouse IgE rat monoclonal antibody, and then incubated with two-fold serial dilutions of test sera with 10% normal mouse serum as diluent for the capturing of only IgE class molecules. Biotinylated ovalbumin and then beta-D-galactosidase-conjugated streptavidin were added and, finally, 4-methylumbelliferyl-beta-D-galactoside was used as the enzyme substrate. The fluorescence intensity of the reaction product (4-methylumbelliferone) was determined on a microplate fluorescence reader. The sensitivity of this assay was equal to that of passive cutaneous anaphylaxis (PCA). In contrast to indirect ELISAs this IgE-capture assay is free from competition by non-IgE antibodies. Furthermore, it requires much less antigen than the PCA assay.     

Production of monoclonal mouse IgE antibodies with DNP specificity by hybrid cell lines

IgE anti-DNP antibody-secreting hybridomas were obtained by polyethylene glycol fusion of murine myeloma cells with spleen cells from mice, primed with DNP-KLH and challenged with DNP-Nippostrongylus brasiliensis extract. The in vivo and in vitro continuously growing hybridomas are producing high amounts of monoclonal IgE anti-DNP antibodies, which are heat-labile, hapten-specific and have rat mast cell sensitizing capacity.     

Isolation of IgE from normal mouse serum.

Enriched populations of T or B cells, prepared by nylon wool filtration of mesenteric node lymphocytes from mice infected with Trichinella spiralis, were capable of transferring immunity to normal and irradiated syngeneic mice. In cell recipients there was an early loss of fecundity by the worms and an accelerated expulsion from the intestine. Treatment with anti-Thy 1.2 serum, to deplete contaminating T cells, severely reduced or abolished the protective activity of enriched B-cell fractions. Replacement of contaminating T cells by normal T cells restored the capacity of B-cell fractions to reduce worm fecundity but did not result in worm expulsion. As it was shown that comparatively small numbers of T cells (3 x 10(6)) alone were effective in transferring immunity to irradiated mice it is suggested that the T cells act not as helper cells, but are involved in the generation of changes in the intestinal environment that are detrimental to worm survival.     

Fc-receptors for IgE on human lymphocytes. Detection with a rosetting assay using a recombinant human/mouse IgE antibody and characterization with monoclonal antibodies

Two monoclonal antibodies, M-L25 and M-L47, were produced against the human lymphoid Fc receptor for IgE (Fc epsilon R). These antibodies were identified by their ability to selectively inhibit the binding of IgE to Fc epsilon R+ lymphoid cells as demonstrated by a newly developed IgE rosetting assay. In this method, NIP coated ox erythrocytes were complexed with a NP-specific recombinant chimeric human/mouse IgE antibody and employed as indicator cells for the detection of Fc epsilon R+ cells. The anti-Fc epsilon R antibodies stained 4.6 +/- 2.3% of normal peripheral blood mononuclear cells, 0.4 +/- 0.3% of T cells, 22.2 +/- 11.7% of the non-T cell fraction, and 34.9 +/- 2.9% of tonsil cells. Less than 0.1% of monocytes, basophilic and eosinophilic granulocytes, platelets, and thymus cells were labelled. This indicates an antigenic heterogeneity of the low affinity Fc epsilon R on lymphocytes and the Fc epsilon R found on monocytes, platelets, and eosinophilic granulocytes. The lymphoid Fc epsilon R was immunoprecipitated by M-L25 from the lysate of surface iodinated lymphoid cells. Three polypeptide chains were identified having an apparent MW of 40, 82, and 100 kd under non-reducing, and of 42, 115, and 145 kd under reducing conditions, suggesting a multichain structure of the human lymphoid Fc epsilon R.     

Rapid detection of antigen-specific mouse IgE by enzyme-linked immunosorbent assay (ELISA)

A rapid, sensitive, antigen-specific mouse IgE capture ELISA is described. A monoclonal rat anti-mouse IgE was used as the capture antibody, and a DNP-coupled BSA-biotinylated conjugate along with a peroxidase-avidin-biotin complex was utilized as the detection system. The lower detection limit of this assay is 8.5 ng/ml of antigen-specific IgE. With some modifications, this assay can be employed to screen for antigen specific antibodies of other isotypes and subtypes.     

The effect of rat mast cell sensitization with mouse IgE antibody on anaphylactic histamine release by anti-mouse IgE and Con A

The peritoneal mast cells of rat (Wistar X August) F1 were incubated with purified mouse IgE antibodies and then challenged (in the presence of 50% D2O) with antigen, anti-mouse IgE and concanavalin A. It was found that incubation of mast cells with IgE antibodies led to different level of cell sensitization (in mast cells from different rats), expressed in antigen-induced histamine release (0-52%). Moreover, a) significant antigen-induced histamine release was usually accompanied by high Con A- and anti-IgE-induced histamine release from these cells; the magnitude of release was comparable to Con A- and anti-IgE-induced release from control, nonsensitized cells of the same rat; b) low antigen-induced histamine release was accompanied by the decrease of Con A- and anti-IgE-induced release, as compared to the release from control cells. This fall of reactivity to Con A and anti-IgE was statistically significant and was irreversible during 120 min.     

Detection of human IgE antibody by a modified rat mast cell degranulation technique

A rat mast cell degranulation (R.M.C.D.) technique is described in detail. This method is shown to detect specific antibodies to rye grass pollen and purified factors thereof (rye Group I antigen). The antibody detected belongs to the IgE immunoglobulin class as evidenced by specific degranulation when the rat mast cells are passively sensitized with the sensitive subject''s serum and challenged with a monospecific rabbit antimyeloma IgE antiserum. No significant mast cell degranulation is observed with serum alone, antigen alone, or when anti-IgG, IgA or IgM are used in this system in an appropriate set of controlled experiments.