Passive Cutaneous Anaphylaxis Test Research Articles

Reactivity of Human Immunoglobulins in Amebiasis

Summary Immunoglobulins (IgG, IgA, IgM) were separated from sera of two patients with symptomatic infections of Entamoeba histolytica. The techniques used were Sephadex G 200 and DEAE-cellulose column chromatography and sucrose density gradient ultracentrifugation. The relative purity of fractions was assayed by the Ouchterlony technique and immunoelectrophoresis against specific immunoglobulin antisera. Whole serum and fractions were analyzed by serodiagnostic methods and by passive cutaneous anaphylaxis (PCA) in the guinea pig and monkey. Absorption experiments were carried out to confirm the reactions. The IgG globulins were reactive in all the serodiagnostic tests for E. histolytica and in the guinea pig PCA test. The reaginic activity demonstrable by PCA in the monkey appeared to be heterogenous and was associated with an IgA fraction eluted from a Sephadex G 200 column and the IgG fraction eluted from a DEAE-cellulose column. While hypersensitivity in man associated with amebiasis has not, as yet, been demonstrated, a high proportion of amebic diagnostic sera gave positive PCA reactions in the guinea pig. The monkey appeared to be far less sensitive.

The antigenic loci of insulin.

Insulin exhibited some cross-reactivity with the isolated S-sulfo A- and B-chains in the passive cutaneous anaphylaxis test. The synthetic peptides A1–9, A10–21, and A1–21gave a passive cutaneous anaphylactic reaction with guinea pig antiserum to natural A-chain, whereas high levels of A10–21and A1–21could also inhibit a subsequent insulin challenge from reacting with antiserum to insulin. The synthetic peptides B1–8, B9–30, B24–30, and B1–30reacted with antiserum to natural B-chain but high levels of peptides (B2–8)2, B9–14, and (B17–23)2were inactive. The symmetrical double peptide (B1–8)2reacted with guinea pig antiserum to intact insulin as well as with antiserum to B-chain and certain sera from insulin-resistant diabetic patients. The results indicated that injection of ox insulin into guinea pigs caused production of anaphylactic antibodies mainly towards antigenic loci in the regions A10–21and B1–8of the insulin molecule, whereas antigenic determinants in the regions A1–9and B24–30were less important.

Further studies on the immunogenicity of d-amino acid polymers

Racemic polymers, composed of d-amino acids and decreasing amounts of l-amino acids (ranging from 42% to 3%) induced antibody formation in rabbits and delayed hypersensitivity in guinea pigs. A polypeptide composed exclusively of d-amino acids, p( d-Tyr, d-Glu, d-Lys), provoked an immune response in rabbits and in guinea pigs. In this case, the dose used for immunizing rabbits was 100 μg antigen in complete Freund's adjuvant (CFA). Antisera against either the racemic polymers or the d-amino acid polypeptide were negative in the passive cutaneous anaphylaxis (PCA) test. Antibodies against one of the racemic polymers and against the polypeptide composed exclusively of d-amino acids, were isolated by means of specific immunoadsorbents and characterized. Purified antibodies gave positive PCA reactions with homologous antigens.

Basic proteins from the acidic extract of bovine spinal cord: II. Encephalitogenic, immunologic and structural interrelationships

1. 1. Of 6 cationic proteins isolated form the acidic extract of bovine spinal cord, 3 induce experimental allergic encephalomyelitis when injected into guinea pigs simultaneously with complete Freund's adjuvant. 2. 2. The 3 encephalitogens were shown to be related immunologically, by the passive cutaneous anaphylaxis test, and structurally, by the peptide mapping technique. Quite likely, they are fragments from one molecule produced by enzymatic degradation during the isolation procedures.

Immunofluorescent antibody studies of a murine leukemia virus.

Brown, Eric R. (Northwestern University Medical School, Chicago, Ill.), Peter Buinauskas, and Steven O. Schwartz. Immunofluorescent antibody studies of a murine leukemia virus. J. Bacteriol. 92:978-982. 1966.-Correlation was close between in vitro complement fixation, immunodiffusion, and passive cutaneous anaphylaxis tests with the S-63 and GC murine leukemia viruses and immunofluorescence reactions with these viruses. When fluorescein-isothiocyanate-conjugated convalescent sera obtained from mice initially infected with S-63 leukemia virus were used, the reactive site was within the cytoplasm of the infected cell. By electron microscopic examination, virus particles were demonstrated in the same areas within the cells that exhibited specific fluorescence with the conjugates. Spleen and mesenteric nodes contained the most virus, whereas kidney tissues contained the least amount. Fluorescence was not observed within the nuclei of infected cells.

Isolation and Characterization of Active Fragments Obtained by Cleavage of Immunoglobulin G with Cyanogen Bromide

Abstract Treatment of rabbit immunoglobulin G with cyanogen bromide in 0.3 M HCl under specified conditions resulted in the cleavage of about half of its methionyl peptide bonds. A 5 S fragment was isolated from the reaction mixture by gel filtration in a quantitative yield. A comparison of the molecular weight and the amino acid composition of this fragment with the pepsin-produced 5 S fragment indicates that the latter is somewhat bigger than the CNBr 5 S fragment. The CNBr 5 S fragment reacted fully with goat antiserum against papain digest Fragment I of rabbit immunoglobulin G, but, in contrast to native immunoglobulin G, did not react with goat antiserum against papain digest Fragment III. The CNBr 5 S fragment of rabbit antibody to bovine serum albumin retained the full capacity to precipitate the antigen, but did not respond in the passive cutaneous anaphylaxis test; the ability to fix complement was very markedly reduced. Reduction of the CNBr 5 S fragment of the antibody with cysteine or 2-mercaptoethylamine led to the formation of a 3.3 S fragment, capable of reacting with bovine serum albumin but not of precipitating it. Reoxidation of the CNBr 3.3 S fragment resulted in a partial reformation of the 5 S fragment.

The effect of alum-precipitated pyridine ragweed extract on guinea pigs: II. Comparison of passive cutaneous anaphylaxis with Arthus reactivity after single low dose

To investigate further the enhanced antigenicity of alum-precipitated pyridine (APPR) as compared to aqueous ragweed extract (ARE), passive cutaneous anaphylaxis (PCA) experiments were performed using sera obtained from the guinea pigs prior to the studies for Arthus reactivity previously reported. 1 The APPR immunized animals produced a greater total number of Arthus reactions, whether moderate or maximum, and the sera from these same animals gave a larger total percentage of positive PCA tests, 39 of 120 (32.5) per cent, than did the ARE immunized animals, 5 of 36 (13.8 per cent). Chi square for this difference was 4.737 and is significant at the 5 per cent level. The APPR immunized animals appeared to develop two types of antibodies more consistently than the ARE immunized. Their sera gave positive PCA reactions earlier after immunization and with lower single doses of APPR than did ARE immunized animals. With immunizing doses of 10 μg of protein N or below, antibodies persisted longer in APPR immunized animals. On day 136, 10 of 10 animals immunized with this dose of APPR gave positive Arthus reactions and their sera produced 7 of 10 positive PCA tests. On the other hand, none of the ARE immunized animals developed Arthus reactions nor did their sera produce positive PCA tests.

Benzylpenicilloyl-Specific Serum Antibodies to Penicillin in Man

Summary The molecular classes of benzylpenicilloylspecific (BPO) antibodies detected in human sera by the presently described passive hemagglutination method (HA) were studied by mercaptoethanol (ME) degradation, ultracentrifugal fractionation in sucrose, and neutralization with monospecific rabbit anti-immunoglobulin sera. Sera whose HA titers were reduced to zero by ME treatment were shown to contain virtually only IgM antibodies. Sera whose HA titers were not reduced by ME were shown to contain mainly IgG antibodies, but small amounts of IgM and antibodies of other molecular classes were also present. The HA method was capable of detecting as little as 0.0003 µg/ml anti-benzylpenicilloyl (BPO) antibody protein in rabbit antisera and it may be more sensitive for human sera. By contrast, the passive cutaneous anaphylaxis (PCA) test in guinea pigs was 200-fold less sensitive for rabbit sera and 1500-fold for human sera. By means of this assay method, BPO-specific antibodies were detected in the sera of 97% of patients selected at random; most sera contained only IgM antibodies and 14% contained IgG antibodies in addition to IgM. BPO specificity of these antibodies was proved by hapten inhibition. Because of its specificity, sensitivity and simplicity, this method may be applicable to diverse immunologic studies.

A Comparison of Some Commonly Used Methods for Detecting Antibodies to Bovine Albumin in Human Serum

Summary Fifteen human and two rabbit sera that contained anti-bovine serum albumin (BSA) as measured by the ammonium sulfate test were studied with a variety of other methods for detecting antibody. These included radioimmunoelectrophoresis, P-80 precipitin test, microimmunodiffusion, hemagglutination, passive cutaneous anaphylaxis and the Prausnitz-Küstner passive transfer for reaginic activity. The rabbit anti-BSA sera bound amounts of I131-BSA comparable to the human antisera and gave strongly positive reactions when tested with each procedure. Radioimmunoelectrophoresis studies confirmed that each of the human sera selected on the basis of the ammonium sulfate test contained antibodies capable of binding I131-BSA, but among all the other techniques there was considerable variation with regard to their capacity to detect and measure anti-BSA activity in the human sera. The P-80 test revealed that the human sera had reduced precipitating efficiency as compared to similar rabbit anti-BSA sera. Microimmuno-diffusion was relatively insensitive in that it detected antibody in only one human serum. The hemagglutination test was positive in 10 of the 15 sera, but the titers were relatively low and did not reflect the major differences observed with the ammonium sulfate test. Reaginic activity was present in four human sera and only three of these contained γA-globulin which bound I131-BSA when tested with radioimmunoelectrophoresis. Conversely, one human anti-BSA serum contained γA-globulin which bound I131-BSA but did not have reaginic activity. Thus, reaginic activity was not necessarily related to the presence or absence of γA-globulins with the capacity to bind I131-BSA. The passive cutaneous anaphylaxis tests were uniformly negative employing the human anti-BSA sera. Of the techniques used in this study, only radioimmunoelectrophoresis and the ammonium sulfate method detect the primary interaction between antigen and antibody. In contrast, the other in vitro and in vivo tests depend upon measuring secondary events which in varying degree may occur sometime after the primary antigenantibody reaction.

Antigenic Behavior of the S-63 Mouse Leukemia Virus

Abstract The present studies are an extension of earlier reports on the antibody response of mice to a leukemogenic virus (S-63). These investigations were designed to show whether a correlation exists between in vivo and in vitro determinations, and whether protective antibodies against the virus could be produced in immune-tolerant animals. Viral neutralization and protection studies in mice were compared with passive cutaneous anaphylaxis (PCA), immunodiffusion, microprecipitin, and immunofluorescent tests using similar antigens and antibodies. As shown in tables 1, 2 and 3, antibody could be demonstrated by both in vivo and in vitro methods, and these studies showed (table 2) that the antibody was protective. The S-63 leukemia virus was isolated in 1963 from a mouse ascites cell leukemia line. That cell line had been carried in the laboratory for over 3 years. The virus has been carried in ICR mice since its isolation. Immunologic studies were carried out to determine the relation between the virus and the originating ascites cell. Studies of antibodies produced in normal rabbits against the S-63 virus were unsatisfactory for purposes of these experiments because of cross reaction with normal mouse tissue. Immune-tolerant animals have yielded valuable information: Antibodies produced in immune-tolerant rabbits injected with the S-63 virus do not cross react against intact ascites cells from which the virus was originally isolated. Conversely, antibodies similarly produced against the ascites cells do not react against the virus antigen. Antibodies produced in immune-tolerant rabbits against ascites cells protect mice against ascites cell-induced leukemia, but not against the disease induced by S-63 virus. Mice challenged with ascites cells and treated with antiascites cell gamma globulin are protected against the disease. The best therapeutic advantage is obtained when treatment is started either at the time of cell inoculation or in the early stages of the disease. Antibodies which protect newborn animals from infection develop in adult ICR mice recovering from S-63 virus leukemia. These antibodies produced by convalescent mice are similar, from the standpoint of protection, to those produced in immune-tolerant rabbits.

An evaluation of passive cutaneous anaphylactic reactions with Trichinella and Toxocara antibody-antigen systems.

SummaryThe test for passive cutaneous anaphylaxis (PCA) proved relatively simple and easy to perform. Within limits, the minimal amount of challenge Trichinella antigen was dependent on the amount of antibody used for sensitization. The optimal concentrations of Trichinella and Toxocara antigens to use in PCA tests with these experimental infections were determined. Studies with rabbit and guinea pig antisera indicate that antibodies involved in PCA reactions may differ from those involved in bentonite flocculation or hemagglutination tests. PCA reactions with heterologous antigens are common. The simplicity of the procedure is considered of distinct value and advantageous, but the amount of antigen required for a reaction and the possibility of cross-reactions are drawbacks.

HISTO-IMMUNOLOGICAL LOCALIZATION OF HYALURONIDASE IN THE BULL TESTIS.

Summary. A highly purified and potent hyaluronidase preparation extracted from bull testis was employed to induce heterologous antibody by sensitizing adult rabbits. Specific anti-enzyme antibodies were demonstrated by Ouchterlony's method, and by passive cutaneous anaphylaxis (pca), and haemagglutination and complement fixation tests. In order to localize the cellular site of the accumulation of the enzyme, direct and indirect immunofluorescent techniques were applied to tissue sections and to isolated germ cells of adult bull testis. Specific fluorescence adequately checked by control reactions appeared in the perinuclear area of spermatids, in the acrosomes of spermatozoa and with less certainty in the cytoplasm of other cells tentatively identified as spermatocytes. Non-specific fluorescence could be seen in the basal cell line of the seminiferous tubules.

COMPARATIVE STUDIES OF FRANCISELLA TULARENSIS AND FRANCISELLA NOVICIDA.

Owen, C. R. (U.S. Public Health Service, Rocky Mountain Laboratory, Hamilton, Mont.), E. O. Buker, W. L. Jellison, D. B. Lackman, and J. F. Bell. Comparative studies of Francisella tularensis and Francisella novicida. J. Bacteriol. 87:676-683. 1964.-Comparative studies of various properties of Francisella tularensis (= Pasteurella tularensis) and F. novicida were performed. The two organisms are very similar morphologically. Growth of both was markedly enhanced by addition of cystine to media, but F. novicida is less fastidious than F. tularensis. The virulence of F. novicida for mice and cavies is lower than that of fresh isolates of F. tularensis. In complement-fixation tests, some cross-reaction occurred when rabbit antisera were used; complement-fixation tests with cavy antisera were specific. Agglutination tests with sera from both rabbits and cavies were specific. Nonliving vaccines of the two organisms (extracts, whole dead cells) conferred no cross-protection to mice; living attenuated vaccines conferred cross-protection which was more transitory than was specific protection. Passive cutaneous anaphylaxis (PCA) tests were highly specific. Absorption of antisera with homologous organisms removed all PCA reactivity, while absorption with heterologous organisms left it almost intact. Hemagglutination and hemagglutination-inhibition tests were specific. It was concluded that the two organisms are sufficiently similar to belong in the same genus but sufficiently different to be retained in separate species.

MILK ALLERGY

The examination of sera from milk allergic children, children allergic to substances other than milk, and normal children, revealed no significant differences relative to coated tanned cell hemagglutinin titer or the presence of precipitating antibody as measured by gel diffusion for milk components. The passive cutaneous anaphylaxis test revealed a high percentage of positive responses with sera from milk allergic individuals only. The presence of milk hemagglutinins could not be demonstrated in the cord sera from 30 newborns. The results of this study suggest that the passive cutaneous anaphylaxis technique deserves further evaluation as a laboratory method for the confirmation of milk allergy.

Immunochemical Studies with Gangliosides

Summary The injection of purified gangliosides caused the production of specific antibodies in rabbits when administered with a carrier substance such as rabbit or sheep erythrocytes, Freund's adjuvant, whole human serum or bovine serum albumin. A separate antibody was also produced by the injection of erythrocytes coated with asialogangliosides. Anti-ganglioside and anti-asialoganglioside antibodies were demonstrated by hemagglutination, complement fixation and passive cutaneous anaphylaxis tests. Gangliosides exhibited blood group M and Rho(D) activity in hemagglutination inhibition tests and some, although weaker, blood group substance A and N activity. The intact ganglioside molecule was required for blood group activity. Asialogangliosides and crystalline N-acetylneuraminic acid were inactive in this regard.

The Passive Cutaneous Anaphylaxis (PCA) Test in Adiaspiromycosis

Adiaspiromycosis is the name proposed by Emmons and Jellison (1960) to replace haplomycosis (Emmons, 1948) for infections with the fungal agents Emmonsia parva (Emmons & Ashburn, 1942) and Emmonsia crescens Emmons & Jellison (1960). Natural infections with these fungi are restricted to the lungs of a great variety of mammals (FIG. 1). Emmonsia is one of the few mycotic agents not reported in man but known to be present in a great variety of wild animals. Diagnosis in man would probably depend on post mortem microscopic examination, as the spherules, measuring 400-500 t, are too small for X-ray resolution, and, since they remain fixed in lung tissue, they are not present in sputum. However, small concretions, carbon, oil globules and other material are common in the human lung and a few spherules of this fungus could well escape notice. Because of the difficulties in etiologic diagnosis of infection, a diagnostic immunologic test seemed highly desirable. The passive cutaneous anaphylaxis (PCA) test in cavies has been described by Ovary (1958) and his colleagues (Ovary and Bier, 1953; Ovary and Briot, 1951) and this test has been adapted with some success to the study of adiaspiromycosis. The results of these studies and the discovery of 2 possible human infections are reported here.

The Passive Cutaneous Anaphylaxis (PCA) Test in Adiaspiromycosis

The Passive Cutaneous Anaphylaxis (PCA) Test in Adiaspiromycosis

Antigenicity of milk proteins of prepared formulas measured by precipitin ring test and passive cutaneous anaphylaxis in the guinea pig

The antigenicity of the major whey proteins and of casein in a variety of infant milk preparations has been measured by the precipitin ring and by the passive cutaneous anaphylaxis tests. All milk formulas showed casein, alphalactalbumin, and beta-lactoglobulin activity. Some products also showed bovine serum albumin activity. It is suggested that the term “heat denatured milks” be abandoned as a designation implying nonantigenicity or nonallergenicity of the proteins in processed milk preparations. The data suggest that the use of heat modified milks in the diet of patients allergic to casein, alpha-lactalbumin, or beta-lactoglobulin is contraindicated.

Antigenic relationship of Geotrichum candidum strains.

Twelve cultures ofGeotrichum candidum have been examined by agglutination, agglutinin adsorption, passive cutaneous anaphylaxis and dermal sensitivity tests. Because of widespread cross reactivity between the strains in these tests, it was concluded that surface and internal sharing of antigens exists.

Molecular Bases of Passive Sensitization

Summary The effect of chemical procedures designed to break the disulfide linkages of γ-globulins on passively sensitize guinea pig skin was studied with a human γ-globulin-rabbit anti-HGG system. Human (antigen) and rabbit (antibody) γ-globulins lost sensitizing activity after treatment with mercaptoethanol followed by sulfhydryl blocking agents such as iodoacetate or iodoacetamide, as determined by passive cutaneous anaphylaxis tests in guinea pigs. Treatment with either mercaptoethanol or iodoacetate alone did not affect their sensitizing activity. Mercaptoethanol-iodoacetate treated proteins, either as antigen or antibody elicited cutaneous anaphylactic reactions in guinea pigs previously sensitized with unmodified γ-globulins as antigen or antibody and also showed precipitin reactions with the complementary reagents comparable to reactions using unmodified proteins. The complement-fixing properties of the HGG-anti-HGG systems were unaffected by the treatment of either antigen or antibody by mercaptoethanol and iodoacetate. The results suggested that irreversible cleavage of disulfide linkages in human and rabbit γ-globulin molecules was correlated with a decrease in their affinity for guinea pig tissues.