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Bovine reaginic antibody. II. Preparation of an anti-reaginic immunoglobulin.

A method of preparing an antiserum to bovine reaginic immunoglobulins is described. This procedure which relies on the ability of bovine reaginic immunoglobulin to attach to rat mast cells, results in an antiserum which upon absorption with rat and bovine materials did not react with rat serum or with bovine IgG1, IgG2, IgM and IgA. The antiserum did, however, react in a gel diffusion assay with PCA positive bovine globulin and a reaginic immunoglobulin-rich fraction of bovine serum. Further, anti-reaginic immunoglobulin activity was shown by binding to and degranulation of rat mast cells previously incubated with PCA positive bovine globulin. The antiserum prevented sensitization of calf skin in a PCA reaction while anti-human IgE did not.

Animals

Reagin synthesis in inbred rats. II. Genetic control of reaginic antibody synthesis.

Specific reaginic (IgE) and hemagglutinating (IgG) antibodies were quantified after immunization with ovalbumin aluminum hydroxide gel in BN and ACI inbred rats, as well as their F1, F1 X BN backcross, F2, and F3, and F3 progeny. The dissimilarity of these immune responses indicated that reaginic (IgE) antibody synthesis was influenced by polygenic factors, but not sex, and was controlled by loci different from that governing hemagglutinating (IgG) antibody synthesis. In addition, tissue typing of the BN, ACI, F1, and F3 hybrids suggested that reaginic antibody synthesis was not linked to the major rat histocompatibility locus.

Animals

Comparison between the automated reagin test and reagin screen test methods of VDRL screening tests for syphilis in use in a routine laboratory.

A comparison is made between the automated reagin test (ART) using Technicon AutoAnalyzer equipment and the reagin screen test (RST) introduced by Lederle using a new antigen formulation. Treponemal haemagglutination tests (TPHA) were done simultaneously as second screen tests. The absorbed fluorescent treponemal antibody test (FTA-ABS) was used in all seropositive cases. Altogether 1154 sera were tested; 1028 were negative with all tests, 66 were positive with all tests, 42 were positive with TPHA and negative with ART and RST, and 16 were positive with TPHA and RST and negative with ART. It was concluded that the use of the RST and TPHA together would be the more sensitive screen test.

Antibodies, Bacterial

Reaginic antibody production to Ascaris suum allergen, ASC-1. I. The function of glutaraldehyde-polymerized antigen in the induction of reaginic (IgE) antibodies in the rat.

Rats immunized with a purified Ascaris suum allergen (Asc-1) or with its dinitrophenylated derivate (DNP-Asc-1) produced high levels of reaginic (IgE) antibodies. A second injection of antigen given 30 days later did not result in an anamnestic IgE antibody response. Immunization of adult-thymectomized, lethally-irradiated and bone-marrow reconstituted (ATxB) rats with soluble Asc-1 or DNP-Asc-1 failed to stimulate reaginic antibody production. The administration of glutaraldehyde-polymerized antigen induced in some but not all ATxB rats, low but detectable levels of IgE antibodies. These levels increased following a second injection of nonpolymerized antigen in A1 (OH)3 gels. Priming of animals with polymerized carrier and Bordetella pertussis did not stimulate a primary anticarrier IgE response but led to an enhanced antihapten IgE response following the administration of soluble DNP-Asc-1 in A1(OH)3. The results are consistent with the notion that a sharply reduced but clearly functional T-derived helper cell population could be triggered by the polymerized but not by the soluble form of the immunogen.

Allergens

Bovine reaginic antibody III. Cross-reaction of antihuman IgE and antibovine reaginic immunoglobulin antisera with sera from several species of mammals.

Using antisera specific for the heavy chain of human IgE and bovine reaginic immunoglobulin, the degree of cross-reaction amongst sera from pig, rat, rabbit, guinea pig, goat, cow, horse, dog, cat and human was tested. Antihuman IgE antiserum gave strong reactions with pig, rabbit, cow, goat and human sera (100% to 15.1%) and weak reactions with rat, guinea pig, horse, dog and cat sera (10.1% to 3.22%). Antibovine reagin antiserum produced a considerable amount of cross-reaction with sera from pig, rat, rabbit, goat, horse and human (43.6% to 20.1%) with limited reactions with guinea pig, dog and cat sera (13.9% to 9.3%).

Animals

Suppression of reaginic antibodies with modified allergens. II. Abrogation of reaginic antibodies with allergens conjugated to polyethylene glycol.

The two immunogenic preparations, ovalbumin (OA) and the nondialysable constituents of the aqueous extract of ragweed pollen (RAG), were conjugated with polyethylene glycols of molecular weights of 6,000 and 20,000 (PEG6 and PEG20) with the aid of cyanuric chloride. The i.v. administration of OA-PEG6 or OA-PEG20 into normal mice or into mice sensitized to a state of immediate hypersensitivity to dinitrophenylated OA (DNP3-OA) resulted, respectively, in the suppression of the primary or secondary IgE responses to DNP and OA. Similarly, the administration of RAG-PEG6 abrogated the primary as well as the ongoing anti-RAG reaginic responses in mice sensitized to RAG. The unresponsiveness of spleen cells from animals which had received a tolerogenic dose of OA-PEG6 was also maintained after transfer into X-irradiated (550 rad) mice. The suppressive effects of the tolerogenic PEG conjugates of OA and RAG were shown to be immunologically specific. It is, therefore, suggested that PEG-modified allergens may prove useful for the abrogation of the IgE response to a variety of allergens responsible for conditions of common hypersensitivity in man.

Allergens

Reaginic antibody to contact sensitizing agents. Occurrence of cells which supress IgG and not reagin responses.

Mice painted with the contact sensitizing agent picryl chloride produce IgG and reaginic or IgE-type antibody. They concomitanly produce suppressor cells which can inhibit IgG responses of normal mice to picryl chloride. These cells were produced between 3 and 7 days after primary sensitization (skin painting) and were T cells, as evidenced by their sensitivity to anti-theta serum and by their passage through nylon wool columns. Although they inhibited IgG responses they did not significantly change IgE titres. This correlates with a previous observation that repeated painting with picryl chloride increased IgE and not IgG responses.

Animals

Studies on immune responses to parasite antigens in mice. II. Aspects of the T cell dependence of circulating reagin production to Ascaris suum antigens.

Heat-labile, rat skin-fixing antibodies were detected readily in the sera of young female mice dosed intranasally with the body fluid of Ascaris suum (ABF) and the adjuvant, Bordetella pertussis vaccine (BPV). In addition, washed cell suspensions prepared from spleen and the lymph nodes regional to the lungs were positive in an adoptive cutaneous anaphylaxis assay, an assay which may detect activities of reagins associated with mast cells rather than reaginic antibody-secreting cells. The intraperitoneal route was a poor means of inducing circulating anti-ABF reagins and an intraperitoneal injection of ABF + BPV delayed the appearance of circulating reagins in mice dosed at the same time with ABF + BPV intranasally. Hypothymic female BALB/c. nu/nu ('nude') mice failed to produce circulating reagins to ABF but an injection of normal thymocytes or cortisone-resistant thymocytes from syngeneic female mice led to higher titers of circulating reagins than found in normal female BALB/c. nu/+ littermates. Using cells from young male or female syngeneic donors and male and female BALB/c. nu/mu recpiients, evidence was obtained for a defect in the thymus of young male mice and conceivably this defect may extend to the peripheral T cell population in such mice. Cyclophosphamide pretreatment or adrenalectomy increased circulating reagin titers in normal mice dosed intranasally with ABF + BPV, and pretreatment with lipopolysaccharide intranasally markedly reduced titers of circulating anti-ABF reagins. In the discussion, emphasis is given to the hypothesis that potent allergens are T cell-stimulating, relatively persistent antigens which, when located in submucosal lymphoid sites and under conditions of limited antibody production as a result of limited recruitment of 'helper' T cells systemically, lead to the induction and sustained production of IgE by resident Bxi cells and their progeny.

Adrenalectomy

Suppression of reaginic antibody formation. III. Relationship between immunogenecity and tolerogenicity of hapten-carrier conjugates.

From the study of the effect of epitope density on the immunogenicity of haptenated ovalbumin (DNP-OA) it was concluded that the lightly haptenated conjugate, DNP0-5-OA, induced, on the one hand, only low titers of anti-DNP hemagglutinating antibody and no reaginic antibodies to the hapten and, on the other, high reaginic and high hemagglutinating antibody responses to the carrier. The conjugate with a slightly higher degree of haptenation, i.e., DNP2.3-OA, induced both reaginic and hemagglutinating antibodies to both the hapten and the carrier. By contrast, the heavily haptenated conjugate, DNP20-OA, elicited reaginic and hemagglutinating antibodies only against the hapten but not against the carrier. Specific suppression of anti-hapten reaginic antibody formation had been achieved by treatment of mice with a tolerogen consisting of the hapten (DNP) conjugated covalently to isologous gamma globulins (MgammaG). The epitope density of the DNPx-MgammaG conjugates was shown to play a dominant role in determining whether or not the conjugate was tolerogenic. Thus, lightly haptenated conjugates (DNP0.5-MgammaG, DNP1.3-MgammaG or DNP1.9-MgammaG) were not tolerogenic, moderately haptenated conjugates (DNP4.2-MgammaG, DNP8-MgammaG, and DNP 14-MgammaG) were tolerogenic, and heavily haptenated conjugates (DNP32-MgammaG and DNP53-MgammaG) were immunogenic, being capable of priming the recipients for the DNP hapten. Further evidence for the nonimmunogenicity of DNP 8-MgammaG conjugate was inferred from its rate of clearance in tolerized and normal mice. Thus, the half-life of 125I-labeled DNP8-MgammaG in circulation was not significantly different for normal and tolerized mice; it was 3.7 and 3.5 days, respectively, which is within the range of data reported for clearance of normal MgammaG. These results suggest that DNP8-MgammaG was catabolized at a rate similar to that of nonconjugated, isologous MgammaG. Moreover, there was no significant difference in the localization of DNP8-MgammaG in identical difference in the localization of DNP8-MgammaG in identical organs (spleen, thymus, kidney, and liver) of normal and tolerized mice. All the multivalent DNPx-MgammaG conjugates were shown to be able to elicit passive cutaneous anaphylaxis (PCA) reaction on i.v. challenge of rats which had been pre-sensitized i.d. with anti-DNP reaginic antibodies.

Animals

Enhancement of reaginic and hemagglutinating antibody production by an extract of Bordetella pertussis containing histamine sensitizing factor.

The effect of an extract containing the histamine-sensitizing factor (HSF) of Bordetella pertussis on the immune response of mice to ovalbumin was investigated with respect to dose of antigen and adjuvant. Of particular interest was the enhancement of reaginic antibody production. In comparison to the Al(OH)3 induced production of reaginic antibody where low doses of antigen and adjuvant yield high titers of reagin, the HSF extract demonstrated optimal adjuvant activity at high doses of both adjuvant and antigen. The reaginic antibody response was maximal usually by 2 to 3 weeks post-immunization and persisted for long periods of time. The hemagglutinating antibody response was maximal at 8 to 10 weeks post-immunization. The initial treatment of mice with HSF extract plus antigen resulted in the production of memory cells since a subsequent immunization with ovalbumin alone evoked a secondary reaginic response. These observations may have implications in clinical allergy since substances similar to the pertussis factor might be produced by other microbial organisms and these substances could modulate the immunologic response of individuals to common allergens.

Adjuvants, Immunologic

Reaginic sensitization of mice and rats.

The reagin response to hemocyanin (KLH) and egg albumin (EA) was studied in inbred strains (Balb/c, 129, C3H/A, C57BL/6J) and Swiss-Copacabana mice. Both antigens induced apparent and in certain experimental conditions, persistent reagin production, KLH being more effective. Al(OH)3 gel as an adjuvant was more suitable for IgE antibody production, while the Freund's complete adjuvant appeared more appropriate for IgG1 antibody response. The reaginic response to EA and Al(OH)3 in rats Wistar, Sprague-Dawley and F1 hybrids (Wistar x August) was weak and transient. The best sensitization, though relatively low as compared with mouse sensitization, was obtained in Sprague-Dawley and F1 hybrids. The results suggest, that a mouse model of sensitization is quite suitable for studying the persistent reagin response, while a rat model may be useful for investigating the early suppression of reagin response.

Adjuvants, Immunologic

Induction and yield of reaginic antibodies in mouse serum and ascitic fluid.

Reaginic antibody in ascitic fluid was isolated from mice immunized with ovalbumin and TAl(OH)3 or with avalbumin and pertussis adjuvant. Partial purification of this reagin by ammonium sulphate precipitation, gel filtration, and ion-exchange column chromatography yielded a gammaglobulin fraction highly active in the passive cutaneous anaphylaxis reaction. This was identical in purity and specific activity to a fraction of partially purified reagin isolated from mouse antiserum. The recovery of reaginic activity after purification from ascitic fluid was 19 per cent compared to the 7-8 per cent recovery from anti-serum. These studies demonstrate that ascitic fluid in mice immunized with methods known to produce reaginic antibody provides an excellent source of mouse IgE.

Animals

Production of circulating reaginic (IgE) antibodies by oral administration of ovalbumin to rats.

Reaginic antibody synthesis following parenteral and/or oral administration of ovalbumin and Bordetella pertussis organisms as adjuvant has been evaluated in LOU/M/Wsl inbred rats. These rats are able to produce high reaginic antibody serum levels after intraperitoneal injection of this antigen. Primary oral administration of ovalbumin doses between 10 and 100 mg with Bordetella pertussis organisms given as adjuvant by the intraperitoneal or the oral route led to characteristic reagnic responses. Secondary reaginic responses were obtained by oral administration of ovalbumin without any adjuvant in animals sensitized by the oral or the intraperitoneal route. A hundred micrograms of ovalbumin was enough to induce reaginic responses but more constant and higher reaginic levels were obtained with a 50 mg dose in the experimental model employed.

Adjuvants, Immunologic

The adjuvant activity of saponin and aluminium hydroxide for promoting reagins.

Saponin and aluminium hydroxide were compared as adjuvants for promoting serum reagins in cattle using BHK cell lysate as allergen and the passive cutaneous anaphylaxis test to demonstrate the antibodies. Saponin was the better adjuvant and provoked reagins in all the injected cattle while aluminium hydroxide did so in only 2 or 5 animals. The titres were also higher in the former case. A combination of both adjuvants stimulated reagin titres marginally higher than saponin alone while BHK cell lysate without adjuvant failed to provoke demonstrable reagins.

Adjuvants, Immunologic

Reaginic antibody production to protein antigens of Escherichia coli and Pseudomonas aeruginosa by mice.

Water-soluble antigens isolated from acetone-dried, gram-negative bacteria elicited reaginic antibody formation in mice. Antibodies specific for Escherichia coli antigens reacted with antigens isolated from several enterobacterial species tested, but not with antigens isolated from Pseudomonas aeruginosa. Reaginic antibodies induced by antigens isolated from a P. aeruginosa strain reacted with antigens isolated from several P.aeruginosa serotypes as well as with a purified protein component of the envelope of P. aeruginosa. The anti-Pseudomonas reagins did not cross-react with enterobacterial antigens. Antigenicity of the bacterial extracts was destroyed by trypsin treatment and reduced by heating, which suggested that the antigens were protein in nature. Whole bacterial cells adsorbed out reaginic antibodies, indicating that the antigens are located at or near the surface of the bacteria.

Animals

Enhancement of reaginic antibody formation in the mouse by concanavalin A.

SWR mice injected with Concanavalin A (Con A) and Ovalbumin (Ov) one immediately after the other or with mixtures of Con A and Ov produced reaginic antibodies to Ov (titer 1:80) after primary immunization and reaginic antibody levels of 1:640 after secondary immunization. Mixtures of Con A and Ov were shown to contain soluble complexes which could be precipitated by 20% polyethylene glycol (PEG). Mixtures of Con A and human serum albumin or Con A and bovine serum albumin did not form soluble complexes and did not induce detectable reagin formation. The possible role of soluble of Con A-Ov complexes in stimulating reaginic antibody is discussed.

Adjuvants, Immunologic

A study of serum immunoglobulins including IgE and reaginic antibodies in patients with bronchial asthma in relation to the spectrum of the disease.

Immunoglobulins G, A, M, D, and E were studied in the sera obtained from sxity-nine bronchial asthma patients, who were graded objectively according to atopic score. Among other associated atopic diseases, they often had allergic rhinitis. Ninety-one per cent of these patients had elevated serum IgE levels and the mean level of serum IgE was more than 3-5 times higher than that observed in the normal subjects. Moreover, as their atopic score increased, the serum IgE levels were also elevated, and every patient with high atopic score, had elevated serum IgE levels. This indicates the association of atopic state with serum IgE level. Furthermore, reaginic antibodies were studied in some of these patients by intradermal tests, Prausnitz-Küstner (PK) reaction, conjunctival and bronchial provocation tests. Intradermal and PK tests were comparable and proved to be the most sensitive method of detecting reagins. The intensity of these reactions correlated significantly with serum IgE LEvel, although this relationship is not invariably present. The reaginic activities in the sera of the patients with atopic bronchial asthma also compared well with the positive bronchial test (induced asthma) by inhalation of specific aerosol although this association is also not always present. Eleven patients with high atopic score had isolated serum IgA deficiency. There was a significant rise of mean serum IgM in comparison to the controls.

Adolescent

Qualitative evaluation of the reagin screen test.

After a preliminary study of 557 sera used as a procedural training exercise, the Reagin screen test (RST) for the macroscopic detection of reagin (as an aid to detecting syphilis) was qualitatively compared to the rapid plasma Reagin (RPR) (circle) card test and Veneral Disease Research Laboratory (VDRL) slide test on 435 random portions of sera using fluorescent treponemal antibody adsorption (FTA-ABS) results as a comparative base. A comparison of total agreement (positive and negative) with the FTA-ABS results led to the following order. RPRCT (I5.5%), VDRL (79.8%), and RST (74.5%). Of the total samples shown to be reactive by the FTA-ABS procedure, the percentage of these interpreted as nonreactive (i.e., "false negative") by the procedures compared was considerably higher with the RST procedure (29.3%) than with either the RPRCT (10.9%) or the VDRL (7.1%) procedures. Minor problems encountered with procedural techniques are also mentioned.

Antibodies