IgE-mediated cardiac hypersensitivity reactions. An experimental model.

To investigate the involvement of the heart in acute allergic reactions in a system immunologically analogous to that of humans, a model of cardiac anaphylaxis mediated by IgE antibodies was developed in the guinea pig. Hearts obtained from guinea pigs, passively sensitized with homologous antidinitrophenyl IgE antibodies, were perfused and challenged in vitro with antigen. Challenge resulted in sinus and ventricular tachycardia, atrioventricular conduction block and substantial histamine release. The results demonstrate that IgE antibodies can sensitize the heart and that the severity of cardiac dysfunction, which follows challenge with specific antigen, directly correlates with the magnitude of histamine released. Since myocardial ischemia and similar arrhythmias occur during immediate hypersensitivity reactions in humans, this experimental model will be helpful in the investigation of cardiac involvement in acute allergic reactions.     

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblotting as a serological tool in the diagnosis of syphilitic infections.

The utility of sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblotting as a serological tool in the diagnosis of human syphilitic infections was examined. In model experiments, rabbits were immunized with Treponema pallidum or T phagedenis, and the antisera were tested for cross-reactivities with both sets of antigens. A major T. pallidum antigen with a molecular weight of ca. 17,000 appeared to be the most reliable specific antigenic marker as assessed by the immunoblotting technique with peroxidase-labeled second antibodies. Antibodies to this antigen were never detected in hyperimmune rabbit anti-T. phagedenis sera or in the sera of nonsyphilitic humans. In contrast, reactive antibodies were found in all syphilitic human sera and also in liquor samples that were positive in the passive hemagglutination test. Differentiation between immunoglobulin M and immunoglobulin G antibodies was directly possible by applying the respective specific second antibodies. Immunoblotting tests were performed with sera exhibiting low passive hemagglutination test titers and equivocal fluorescent treponemal antibody and rapid plasma reagin card reactions. In more than 60% of these cases, immunoblot positivity with respect to the 17,000-molecular-weight antigen was found. The same results were obtained with partially purified 17,000-molecular-weight antigen. The immunoblot technique should be useful as an additional diagnostic tool for differentiating between true and false-positive serological reactions.     

Antibodies Against Equine ALG During Treatment of Rejection Crisis after Renal Allotransplantation

The development of antibodies to equine anti-human-lymphocyte globulin (ALG) was followed using passive hemagglutination and gel diffusion, and to Forssman antigen using direct sheep erythrocyte agglutination, in a controlled clinical investigation of ALG treatment of established rejection of renal allografts in a total of 12 patients. With passive hemagglutination a weak antibody to ALG was seen only in one case. There was no increase in titer for sheep erythrocyte antigens during treatment, which agrees well with clinical observations in which no anaphylactic reactions were seen.     

An experimental allergic conjunctivitis induced by topical and repetitive applications of Japanese cedar pollens in guinea pigs

OBJECTIVE AND DESIGN: To develop a model of experimental allergic conjunctivitis, guinea pigs were given repetitive and topical applications of Japanese cedar pollen as an antigen, and the resulting allergic reaction was characterised. SUBJECTS: Male Hartley guinea pigs. TREATMENT: Guinea pigs were sensitised by insertion of small gelatin sponge pieces (3 pieces/eye, both eyes) containing the pollen extracts + Al(OH)3 into the palpebra superior/inferior sulci of both eyes for 8 h/day for 6 days. Then the animals were challenged by dropping pollen suspension in each eye once every week. METHODS: Time-course changes of conjunctivitis intensity score (CIS), which represents oedema and redness, and scratching frequency, and gamma1 and IgE levels in sera were assessed following the respective 1st-17th challenges. Ophthalmic lavage was performed to assay for albumin leakage and migrated leukocytes at each of the odd-numbered challenges. RESULTS: At relatively early stages of the repeated challenge, only a gradual increase of CIS was observed. However, thereafter, considerably higher CIS with the maximum at 30 min after the challenge was provoked at the 12th-17th challenges. Quite interestingly, there were differences in the CIS between the right and left eyes, depending on the individuals. Scratching, and albumin leakage and neutrophil influx into the lavage fluid were also developed during the challenges. Although the anaphylactic antibodies against the antigen were detected in the sera from half of the animals, the levels were not correlated to severity of the symptoms. CONCLUSIONS: The present sensitisation/challenge procedures are unique in terms of topical application of antigen. The lack of any correlation between the levels of the anaphylactic antibodies and severity of anaphylactic symptoms, and the difference of CIS between the left and right eyes of an individual strongly suggest that the anaphylactic antibodies are formed in locally limited tissue surrounding an eye. The present method should be useful for analysing the mechanisms of allergic conjunctivitis.     

Benzylpenicillin preparations can evoke a systemic anaphylactic reaction in guinea pigs

All of the five commercially available benzylpenicillin preparations obtained from different sources and a PcG preparation prepared by filtration of a commercial PcG on Sephadex G10 elicited the systemic anaphylactic reactions in guinea pigs which had been immunized with benzylpenicilloyl (BPO)-Ascaris extract conjugate (BPO-As) mixed with aluminum hydroxide gel. These preparations could evoke no such reactions in guinea pigs immunized with BPO-bovine gamma globulin conjugate (BPO-BGG) emulsified with complete Freund's adjuvant. The severity of the systemic anaphylactic reactions correlated significantly with the titers of either 8-day passive cutaneous anaphylactic (8-day PCA) reactions or 4-hr PCA reactions evoked with the same benzylpenicillin preparations. In vitro benzylpenicillin preparation contracted the tracheas of the guinea pigs immunized with BPO-As. These results indicated that the commercially available benzylpenicillin preparations have enough antigenicity to evoke systemic anaphylactic reactions in guinea pigs immunized with BPO-As mixed with aluminum hydroxide gel. Such guinea pigs represent an animal model for investigation of penicillin allergy.     

Studies on monovalent anaphylactogens with carbohydrate auxiliary groups: exclusion of an artefact

N1-DNCP-N6-lactobionoyl-1,6-hexanediamine is an elicitor of anaphylactic reactions in the guinea pig passively sensitized by antisera raised with a DNCP-bovine gammaglobulin conjugate. If the antisera contained anticarbohydrate antibodies against the carbohydrate residues of the globulin, significantly cross-reacting with the lactobionoyl moiety, anaphylactogenic cell triggering would be by classical hetero-specific bridging which involved the DNCP- and the lactobionoyl residues as ligands. This possibility was excluded by showing that a bis-lactobionoyl conjugate is unable to elicit anaphylactic reactions.     

Technical aspects of the Staphylococcus aureus teichoic acid antibody assay: gel diffusion and counterimmunoelecrophoretic assays, antigen preparation, antigen selection, concentration effects, and cross-reactions with other organisms.

Because variable results are being reported from laboratories performing the teichoic acid antibody assay in patients with serious infections due to Staphylococcus aureus, we have thoroughly reviewed all technical aspects of the test. This paper reports on the importance of agar and antigen preparation in standardizing results of the assay and reducing the prevalence of false-negative and -positive tests. Once standardized, the counterimmunoelectrophoretic method is as accurate as the gel diffusion method for both initial screening and generating titers; practically, however, unless numerous tests are to be performed, the gel diffusion technique will suffice for most purposes. The cell wall of the Lafferty strain of S. aureus was used as the standard antigen in assays for serum antibodies. We studied whether it is an appropriate antigen and found that antibody titers obtained with the Lafferty strain antigen were, in three patients with endocarditis, the same as those obtained with antigens from the individual blood stream isolates. We have also confirmed that pooled human gamma globulin can be used, by back titration against newly prepared lots of antigen, to select optimal antigen concentration and is as good as more specific, higher titer serum specimens for that purpose. Finally, cell wall antigens from Staphylococcus epidermidis and a variety of streptococci may react with normal human sera, but such antigens are distinct by immunoprecipitation from those from S. aureus.     

Serodiagnostic application of immunohistoperoxidase reactions on antigen-coupled agarose beads.

Agarose beads to which antigens were covalently bound were subjected to indirect immunohistoperoxidase procedures. For detection and titration of serum antibodies against bovine gamma globulin (BGG) this method appeared to be specific and sensitive. One advantage is that no special instruments are needed. As an example of diagnostic applicability the system was successfully used for demonstration of antibodies in human serum containing antibodies against the trematode Schistosoma mansoni. At the microscopical level the technique is suitable for study of basic problems. Microspectrophotometric absorbance scanning of the beads revealed that the method can provide quantitative information, and is probably capable of quantifying stoichiometric relations in immunological reactions.     

Carrier and hapten functions in immune deviation. III. Possible antigenic competition between hapten and carrier determinants at the macrophage level

The effects of the intravenous injection of DNP-Ficoll on delayed hypersensitivity reactions to bovine gamma globulins (BGG) and anaphylactic reactions to the hapten induced by immunization with DNP-BGG in Freund's adjuvant were studied in guinea pigs. Animals treated with DNP-Ficoll exhibited stronger delayed hypersensitivity to BGG and lower anaphylactic reactions to the hapten than control animals treated with saline, Ficoll alone or with an unrelated protein antigen. These results could be explained by antigen competition between hapten and carrier determinants occurring at the macrophage level.     

Comparison of the neutralizing and ELISA antibody titres to measles virus in human sera and in gamma globulin preparations

Measles virus infection as well as measles vaccination induces a long-lasting immune protection. Specific antibodies have been proven to be associated with this immune protection, since measles immunity can be transferred by immune globulin application (passive immunisation). The neutralisation test (NT) is regarded as the gold standard method for measles immunity because it measures functional neutralising antibody, while with the ELISA, which is often based on cell culture grown native virus antigens, predominantly antibodies to the nucleoprotein antigen were detected. To compare the results of NT and ELISA 199 individual sera and 364 gamma globulin samples, which were made from plasma pools, were tested. Qualitative results showed that the sensitivity of the ELISA was 141/144 (97.9%) and specificity was 48/55 (87.3%) when compared to the NT and focused to the patient samples. For the gamma globulin samples the sensitivity and specificity was 100%. As expected no measles NT negative plasma pool samples were found. The present study showed that with increasing NT-titre, the ELISA-values also rise. False negative ELISA results were obtained in 1.5% of patient sera, mainly containing low levels of neutralising antibody. In both antibody tests seropositive specimens revealed a quite good to moderate correlation. Taken together, the measles IgG ELISA is adequately for immunity testing and identifying of seronegative individuals for vaccination.     

Demonstration by immunoelectro-osmophoresis of precipitating antibodies to a purified rubella virus antigen.

The nonionic detergent Triton X-100 was used to extract antigens of rubella virus from infected tissue culture cells. Three virus-specific antigens were demonstrated by crossed immunoelectrophoresis by using a pool of human gamma globulin as antiserum. The most dominant of these antigens were purified by ion-exchange chromatography on diethylaminoethyl-cellulose. This antigen was of glucoprotein nature and had slow electrophoretic motility and low binding capacity to diethylaminoethyl-cellulose. Thus, it seems likely that the antigen is identical with the precipitating antigen of rubella virus designated b-antigen or tro-osmophoresis with precipiting antibody in sera obtained from patients recovering from acute postnatal rubella. The precipitin reaction that could be correlated to the hemaglutination-inhibition titers of the same sera appeared 12 days after onset of the disease and remained positive for several years.     

A rapid and reliable in vivo method for anti-GPLA (class I and class II antigens) antibody titration and GPLA typing

A reverse Arthus reaction (RAR) may be successfully used in guinea pigs to detect histocompatibility alloantigens of the GPLA type. Epidermal cells carry class I GPLA antigens, and Langerhans cells also bear class II alloantigens. It is therefore possible to elicit an RAR by intradermal injection of relevant alloimmune sera in the skin of guinea pigs of known GPLA haplotype. RAR is detected by increased vascular permeability due to IgG1 antibody and hemorrhage due to IgG2 antibody. Compared with an in vitro protein A-rosetting method RAR is easier and quicker. It proved more sensitive for class II antigens in which Langerhans cells are the target for anti-class II antibody and rather less sensitive for class I antigens. RAR is a convenient method for following the course of GPLA alloimmunization, allowing titration of antibodies against both classes of antigen. It may also be used to type guinea pigs of unknown GPLA haplotype.     

Appearance and Persistence of Antibodies Against Different Virus Components After Regular Measles Infections

Different measles virus-specific antibody activities in acute, early (11 to 40 days after rash) and late (4 to 20 years postinfection) convalescent sera and gamma globulin were determined. Early immunoglobulin G antibodies gave a poor neutralization, which was increased 10- to 60-fold by addition of anti-gamma globulin.There was a high degree of correlation between titers of hemolysis-inhibiting (HLI) and hemagglutinating-inhibiting (HI) antibodies. However, in one out of fifteen late convalescent sera an HLI antibody titer of 640 in the presence of titer of only 20 in HI tests with Tween 80-either-treated antigen was found. Similar findings were made with sera from two patients with multiple sclerosis included in a parallel study. A somewhat higher titer of HI antibodies was demonstrable in these three sera when untreated material was used as antigen. These findings are interpreted in the following way. Antibodies against the hemagglutinin can block not only virus-specific agglutination but also lysis of red cells. In contrast, antibodies against the hemolysin, besides blocking the biological activity of this component, carry only a slight HI activity. This HI activity can be detected only by use of antigen preparations containing hemagglutinin-associated hemolysin.Complement-fixation (CF) and immunodiffusion tests (the latter were carried out with antigen preparations treated with 0.25% sodium dodecyl sulfate) demonstrated that, in almost all cases, antibodies against nucleocapsid structures dominated quantitatively among antibodies appearing in connection with and persisting after regular measles infections. Generally, only low titers of antibodies reacting with purified small particle hemagglutinin (HA; 10 to 14S) or additional structural or nonstructural components were identified in CF and immunodiffusion tests.     

Antigenicity of hapten-polysaccharide conjugates

The immunogenicity of penicilloyl polysaccharides with molecular weights from 5 000 to 400 000 was studied in two guinea pig strains. The conjugates quite regularly induced penicilloyl specific and carrier specific dermal delayed-type hypersensitivity. On the other hand, circulating antibodies were found only occasionally. In a few animals anaphylactic antibody could be demonstrated but precipitating or hemagglutinating antibodies were found in no instance. Since penicilloyl polysaccharides are relatively instable, more stable benzyldextran and nitrobenzyldextran conjugates were studied as well. These antigens also induce dermal delayed-type hypersensitivity. The capacity of the nitrobenzyldextran to induce hapten specific and carrier specific reactions was quite similar to the capacity of penicilloylated polysaccharides. Penicilloyl polysaccharides are able to elicit penicilloyl specific cutaneous anaphylactic reactions in passively immunized guinea pigs. A relatively highly penicilloylated dextran conjugate exhibited an eliciting potency quite comparable to a highly penicilloylated poly-L-lysine. Haptenic and carrier specificities in sensitisation and the possible tolerogenicity of these antigens are discussed; the question to what extent the immunogenicity of hapten-polysaccharide conjugates may be regarded as intrinsic is treated in particular.     

Identification and characterisation of the IgE-binding proteins 2S albumin and conglutin gamma in almond (Prunus dulcis) seeds

BACKGROUND: Almond proteins can cause severe anaphylactic reactions in susceptible individuals. The aim of this study was the identification of IgE-binding proteins in almonds and the characterisation of these proteins by N-terminal sequencing. METHODS: Five sera were selected from individuals with a positive reaction to food challenge. Sodium dodecylsulphate-polyacrylamide gel electrophoresis and immunoblotting were performed on almond seed proteins. Purified IgE-binding proteins were tested for immunoblot inhibition with sera pre-incubated with extracts of hazelnut and walnut. RESULTS: N-terminal sequences of the 12-, 30- and 45-kD proteins were obtained. The 45- and 30-kD proteins shared the same N terminus, with 60% homology to the conglutin gamma heavy chain from lupine seed (Lupinus albus) and to basic 7S globulin from soybean (Glycine max). The sequences of the N-terminal 12-kD protein and of an internal peptide obtained by endoproteinase digestion showed good homology to 2S albumin from English walnut (Jug r 1). Immunoblot inhibition experiments were performed and IgE binding to almond 2S albumin and conglutin gamma was detected in the presence of cross-reacting walnut or hazelnut antigens. CONCLUSIONS: Two IgE-binding almond proteins were N-terminally sequenced and identified as almond 2S albumin and conglutin gamma. Localisation and conservation of IgE binding in a 6-kD peptide obtained by endoproteinase digestion of 2S albumin was shown.     

The study of antigenic properties of certain corpuscular and soluble fractions isolated from tumors of inbred mice

Summary The presence of specific antigens was studied in certain fractions of mice tumors, which were induced and transplanted on purebred animals and, thus, had no isogenic variations.It was established by anaphylactic reaction with desensitization in guinea pigs that there is a specific antigen present in the fractions of mitochondria, microsome, globulin and methanol precipitate of the hepatoma tissue and breast cancer in mice C₃HA. This antigen differed from the antigens which were present in the same fractions obtained from the normal liver or a mixture of organs of healthy mice.The presence of specific tumor antigens in the cellular organoids, as well as in the proteins of the hyaloplasm shows the widespread distribution of this antigen in various parts of the tumor cell.Presented by Active Member AMN SSSR, N. N. Zhukov-Verezhnikov     

The mechanism of anaphylaxis: observations on the failure of antibodies from certain species to sensitize guinea pigs in direct and reversed passive anaphylaxis

Using the criteria of generalized anaphylaxis, the Schultz-Dale test, and mast cell degranulation occurring on contact with antigen in vitro, a study has been made of the ability of antisera, prepared in various species, to sensitize guinea pig tissues passively. Whereas small amounts of guinea pig and rabbit antibodies and rather larger amounts of monkey, dog and human antibodies were effective, antibodies from goat, horse, rat, fowl and a human auto-immune thyroid antiserum were ineffective. These results are in general agreement with those in the literature.

Using antibodies labelled with ¹³¹I, no gross differences were found between those which sensitized and those which did not, either in respect of uptake on guinea pig mesentery in vitro, or retention in the tissue after uptake in vivo. By means of antigen labelled with ¹³¹I it was also shown that the antibody adsorbed on guinea pig mesentery could combine equally well with antigen, whether the antibody came from rabbit (sensitizing) or rat (non-sensitizing). These experiments also indicated that the amount of adsorbed antibody necessary for sensitization is very small.

Reversed passive anaphylaxis was studied with a variety of different antigen-antibody systems, using the criteria listed above. It occurred when, but only when, the antigen was a γ-globulin, and when both antigen and antibody globulins came from species whose antibodies were able to sensitize guinea pigs for direct passive anaphylaxis.

It is concluded that the explanation of the differences between antibodies or antigens which sensitize guinea pigs passively, and those which do not, is to be found in the nature of the antigen-antibody combination as well as in the manner of `fixation' on the tissues. The possible importance of ability to activate guinea pig complement is discussed.

     

The occurrence and treatment of false positive reactions in enzyme-linked immunosorbent assays (ELISA) for the presence of fungal antigens in clinical samples.

Non-specific positive reactions have been revealed in enzyme-linked immunosorbent assays (ELISA) of sera for the presence of fungal antigen. These false positives were recognized by their occurrence in tests for both Candida albicans and Aspergillus fumigatus antigens and by their response to dithiothreitol, combined with their reaction with non-immune rabbit globulin. A scheme is proposed which differentiates between true and false positive reactions. Use of fractionated anti-fungal globulin in conjugates reduced the incidence of false positive results in sera from hospitalized patients and eliminated them from sera of healthy subjects. The test scheme was applied to two panels of sera containing samples from patients with (a) invasive candidosis and (b) invasive aspergillosis. The relevance of ELISA tests for the detection of fungal antigen in human serum is discussed.     

Radioimmunoassay procedure for quantitating bacterial antibody in human sera.

A RIA system was developed to detect antibodies in human sera against bacteria. Sonicates of Escherichia coli and Fusobacterium polymorphum were used as antigens to sensitize plastic-coated beads; antibodies to these antigens were detected with 125I-labeled antihuman globulin. Serum antibody titers against E. coli were determined by the serial dilution method; from the results the standard curve principle was applied in determining the relative amounts of antibodies against E. coli in serum samples tested at a single dilution. The coefficient of variation of the RIA procedure was less than 10%. Serum titers obtained by the RIA and indirect immunofluorescence test were compared; RIA was more sensitive, quantitative and objective. Absorption studies, using E. coli and F. polymorphum absorbentes against E. coli and F. polymorphum. This RIA procedure offers a combination of desirable advantages; it is sensitive, specific, objective, quantitative, and easy to perform.     

Detection of soluble antigens of Toxoplasma gondii by a four-layer modification of an enzyme immunoassay.

A sensitive four-layer modification of an enzyme immunoassay for the detection of soluble antigens of Toxoplasma gondii is described. Microtiter plates were sensitized with rabbit anti-toxoplasma immunoglobulins (6 micrograms/ml) used as the primary antibodies; guinea pig anti-toxoplasma immunoglobulins (6 micrograms/ml) were used as the secondary trapping antibodies. Horseradish peroxidase-conjugated anti-guinea pig immunoglobulins were used as the indicator antibodies. The specificity of the antigen assay was confirmed by using guinea pig immunoglobulins from preimmunization sera. The sensitivity of the antigen assay was found to be at least 10 ng of antigen protein per ml. The suitability of the method for detecting antigens of T. gondii in different specimens was studied by experimental toxoplasma infection in mice. Antigenic components of T. gondii could be detected in different tissue specimens from infected animals from the first day after infection onwards. Toxoplasma antigen in serum and urine samples from infected mice reached detectable levels on day 2 after infection followed by a linear increase in antigen concentration in succeeding samples. This method might offer a valuable aid for a rapid etiological diagnosis also in human cases of acute toxoplasmosis.