Studies on allergens of Dermatophagoides pteronyssinus by direct and indirect RAST.

Dermatophagoides pteronyssinus (DP) extract was fractionated by Sephadex G-75 and liquid isoelectric focusing (IEF) and the allergenic activity of different fractions was monitored by direct and indirect RAST. The fractionation on Sephadex G-75 showed that the allergenic activity of DP extract was related to wide molecular weight spectrum components, even though the maximum amount was recovered in effluent that contained protein with a molecular weight ranging between 25,000 and 12,500 daltons. By fractionation of the mite extract on IEF, three main peaks of allergenic activity (pI less than 3.0; pI = 4.3 +/- 0.25; pI = 6.4 +/- 0.25) were found. Cross-inhibition experiments showed a high degree of cross-reactivity between allergenic material eluted in very distant regions of molecular weight or isoelectric point. The allergenic activity of unfractionated mite extract and of its IEF fractions was destroyed by pronase - but not by neuraminidase - treatment. These results suggest that DP extract probably contains one main allergen existing in multiple molecular forms rather than several distinct allergens and that a protein moiety of the allergen is necessary for the combination with IgE.     

Immunochemical characterization of saline-extracted antigens of Mycobacterium tuberculosis H37Ra

The saline extract (SE) of Mycobacterium tuberculosis H37Ra yielded four major fractions on Sephadex G-100, with average molecular weights of 48,000 (fraction I, F I) 32,000 (fraction II, F II), 15,000 (fraction III, F III) and 5,000 (fraction IV, F IV). FI and FII gave single bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, showed complete immunological cross-reactivity with each other and were more potent in reacting with antibodies in patients' (tuberculosis) sera than sonicate, SE, purified protein derivative, F III and F IV when tested in a competition enzyme-linked immunosorbent assay.     

Studies on Alternaria allergens. I. Isolation of allergens from Alternaria tenuis and Alternaria solani.

Extracts of Alternaria tenuis and Alternaria solani were separated into dialyzable (molecular weight less than 10,000) and non-dialyzable forms. The latter was further fractionated by gel filtration through Sephadex G-100 followed by ion-exchange chromatography on DEAE-cellulose. The dialyzable material was fractionated by gel filtration through Sephadex G-50. The allergenic activities of the fractions obtained from the A. tenuis extract was measured in vitro by the radioallergosorbent test assay and the allergenic potency was measured by radioallergosorbent test inhibition assay. Allergenic activity was detected in most of the non-dialyzable fractions, the majority of the activity being in the last G-100 fraction (MW approximately 20,000) which was predominantly protein in nature. The same component may be responsible for the activity found in the dialyzate and its first G-50 fraction since the immunodiffusion studies indicated that the last G-100 fraction has antigenic components in common with those of the first G-50 fraction. In addition, cross-reactions between A. tenuis and A. solani extracts show that the two species share common antigenic determinants.     

Allergens of mammalian origin: characterization of allergen extracted from cat pelts

An aqueous extract prepared from lyophylized, defatted cat pelts elicited intense wheal-and-flare responses in the skin of patients exhibiting asthma or rhinitis on exposure to cats. On electrophoresis of the extract in agarose, protein bands with the mobility of albumin, α₂-, β-, and γ₁-protein were observed. Allergenic activity was localized in two distinct regions with albumin and α₂-mobility, respectively. Allergenic activity remained bound to DE52-cellulose columns equilibrated with phosphatebuffered (0.01M, pH 7.4) saline (0.05M), but could be eluted by increasing the salt concentration to 0.25M. Following gel filtration on Sephadex G-100, allergen was recovered in fractions containing proteins with a molecular weight range of 30,000 to 60,000 daltons. A purified fraction of cat pelt extract obtained by sequential fractionation on DE52-cellulose and Sephadex G-100 columns was subjected to polyacrylamide disc gel electrophoresis; 5 protein bands were observed. Allergenic activity was recovered in fractions containing two of these bands. Five of twelve subjects with strong skin reactivity to cat pelt extract also reacted strongly to cat serum; the major allergen in cat serum was found to have the electrophoretic mobility of α₂-protein.     

Olive (Olea europea) pollen allergens--II. Isolation and characterization of two major antigens

A dialyzed extract of olive (Olea europea) pollen was fractionated by anion exchange chromatography on DEAE-Sepharose CL-6B using a discontinuous gradient of ammonium bicarbonate. The most important protein allergen was obtained from the 0.3 M fraction after gel filtration on Sephadex G-100 and separation by lentil-lectin Sepharose-4B. The major allergen of olive pollen was contained in the effluent and was designated Olea Antigen I. This material inhibited the RAST activity of 15 patients' sera that were tested. Analytical IEF demonstrated a major band at pH 5.3 and two minor ones at pH 5.6 and 5.0. When these were run into SDS-polyacrylamide gel electrophoresis in a second dimension, all were separated into two bands of mol. wt 17 and 19 K. A second protein, which is the next most important allergen, Olea Antigen II, was obtained from the 0.5 M fraction by chromatofocusing in a 4-7 pH range followed by filtration on Bio-gel P-30. Olea Antigen II had a mol. wt of 8 K as assessed by SDS-PAGE. IEF analysis displayed one main band at pH 3.6 and two minor bands at pH 3.8 and 4.0, respectively. OL-1, an anti-Olea europea monoclonal antibody (MAb) previously reported by us Lauzurica et al. (1988) reacted with the 17 and 19 K antigens from the crude extract and with Olea Antigen I but not with Olea Antigen II.     

Low molecular weight allergens of the pollen of Parietaria officinalis

The allergenic composition of a low mol. wt fraction of the pollen extract of Parietaria officinalis (PO) was investigated. Fraction C, that was eluted after oxytocin (mol. wt 1040) when the pollen extract was gel filtered on Sephadex or on Biogel, was cross-reactive in the RAST with the major allergen P015 and was capable of eliciting histamine release from leukocytes of sensitive donors. RAST inhibition (RAST I) analysis of the eluate of gel filtration on Sephadex G-10 revealed several peaks of IgE binding activity. Analysis of fine specificity of response of individual patients carried out by skin-prick tests and by RAST I, revealed individual patterns of reactivity, indicating that allergens contained in fraction C were minor allergens.     

Fractionation and analysis of allergenicity of allergens from Prosopis juliflora pollen

Prosopis juliflora pollen allergen extract was prepared, and its crude allergen extract was fractionated by Sephadex G-100 gel filtration. Six different fractions were obtained which was confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Protein and carbohydrate content of each fraction were estimated. Fraction E (MW 20,000) showed a 25% carbohydrate concentration. The amino acid analysis indicated that this fraction was rich in glutamic acid and alanine. Antigenicity or allergenicity of fractionated allergens were checked by gel diffusion test, rocket immunoelectrophoresis, skin prick test, and radioallergosorbent test. All these test indicate that fraction E consisted mainly of allergenic molecules (MW 20,000) of P. juliflora pollen.     

Purification and characterization of Par o I, major allergen of Parietaria officinalis pollen.

Par o I, a major allergen of Parietaria officinalis, was purified from the pollen extract. The purified allergen was obtained by ultrafiltration, Sephadex gel filtration and DE-52 ion exchange chromatography: the purified preparation yields a single band in polyacrylamide gel isoelectric focusing (PAG-IEF), sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting, a single immunoprecipitation arc in crossed immunoelectrophoresis (CIE) and crossed radioimmunoelectrophoresis (CRIE) and a single peak in size exclusion high-performance liquid chromatography (HPLC). Par o I is a glycoprotein with a protein to carbohydrate ratio of 100:21. The molecular weight, determined by SDS-PAGE, Sephadex G-50 gel filtration and size exclusion HPLC, varied between 13.5 and 14.5 kDa according to the method employed. The isoelectric point was 4.6. The amino acid composition and the sequence of the first twelve N-terminal residues were determined. The allergenicity was assayed in vivo and in vitro. 29/29 Parietaria-allergic patients were skin positive to Par o I and possessed high level of specific serum IgE antibody as it determined by radioallergosorbent test (RAST). Par o I contained dominant epitopes for human IgE as inhibited to 85% the pollen extract RAST performed with a pool of sera of allergic patients. The RAST inhibitory activity was not abolished by deglycosylation.     

Widespread IgE-mediated hypersensitivity in Northern Sudan to the chironomid Cladotanytarsus lewisi (''green nimitti'').

Hypersensitivity to Chironomidae (non-biting midges) has been a problem in Northern Sudan since about 1927 and is probably due to the working of dams which have produced lake-like conditions on parts of the Blue and main Niles where breeding has evidently increased. Studies were undertaken to determine whether this hypersensitivity is mediated by IgE. Sixteen Sudanese, with bronchial asthma associated with exposure to the chironomid, Cladotanytarsus lewisi ('green nimitti'), were investigated. All patients gave a positive immediate-type skin reaction to an extract of the midge and the majority had markedly elevated concentrations of circulating IgE. Serum from all patients passively sensitized human lung fragments in vitro for the release of histamine and slow-reacting substance of anaphylaxis by the 'nimitti' antigen. This tissue-sensitizing activity could be removed by immunoabsorption with an anti-IgE. These results indicate that this widespread and important hypersensitivity in the Sudan is IgE-mediated and thus may potentially be treated by desensitization.     

Allergens of mammalian origin. II. Characterization of allergens extracted from rat, mouse, guinea pig, and rabbit pelts.

Aqueous extracts prepared from lyophilized, defatted rat, mouse, guinea pig, and rabbit pelts elicited intense wheal-and-flare responses in the skin of a high proportion of patients who were clinically sensitive to these animals. The major allergens in each extract were nondialyzable. Skin test reactions to rat, mouse, and guinea pig serum were common in patients allergic to these animals. The fractions of rat, mouse, and rabbit pelt extract showing maximum allergenic activity contained proteins with the electrophoretic mobility of serum albumin. Fractions of guinea pig pelt extract with maximum allergenic activity were of prealbumin mobility and contained little stainable protein. On Sephadex G-100 gel filtration, most allergen from rat, mouse, and guinea pig pelt extracts was recovered in fractions containing proteins with a molecular weight range of 10,000 to 25,000 daltons. Allergen in rabbit pelt extract had a slightly higher molecular weight range of 18,000 to 38,000 daltons.     

Isolation of hCG and its characterization by radioimmunoassay, enzyme-immunoassay, and radio-receptor assay

The nature of human chorionic gonadotropin (hCG) molecules present during early pregnancy of Indian women is poorly understood. Therefore, a study has been undertaken to isolate hCG and characterize different forms of hCG from urine. The hCG molecules from urine of pregnant women (45-75 days post LMP) were adsorbed onto kaolin, eluted with ammonium hydroxide, and precipitated using acetone and then lyophilized. The lyophilized extract was subjected to Sephadex G-100 chromatography followed by ion-exchange fractionation. Three major fractions of protein (i.e., Peaks I, II, and III) associated with carbohydrate activity were obtained. Peaks II and III eventually resolved into a single peak I following repeated ion exchange chromatography, which suggested the presence of aggregates of molecules. Further purification on an affinity column resolved all three peak fractions into one unadsorbed and two adsorbed (A and B) fractions. These adsorbed fractions were characterized by radioreceptor assay (RRA), radioimmunoassay (RIA), and enzyme linked immunosorbent assay (ELISA). The activity was standardized against WHO reference preparation 75/589. Peaks I (A and B) were found to have maximum at about 75% of immunologically potent hCG, followed by peaks II (40%) and III (5%). The molecular sizes of peaks I, II, and III on a Sephadex G-200 column corresponded to 27,500D, 66,000D, and 84,000D, respectively. Relative mobilities of all adsorbed fractions in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) showed the presence of hCG-alpha (mol. wt. 19,539D) and hCG-beta (28,870D) subunits. The presence of both subunits of hCG were also revealed by Western blot analysis. For the first time, we report the low molecular weight hCG molecule, of 27,500D by size exclusion chromatography, which has immunological and biological activity as measured by RIA, ELISA, and RRA.     

Fractionation and immunological characterization of allergens and allergoids of Prosopis juliflora pollen.

Allergoids of Prosopis juliflora pollen were prepared by formalinization of crude allergen and glycoprotein. Fractionation of crude allergen and allergoids on Sephadex G-100 resulted in separation of proteins of varying molecular size and a glycoprotein of 81 to 13 KD. Allergoids prepared from the glycoprotein fractionated into two proteins of approximately 200 KD and more than 200 KD. Crossed immunoelectrophoresis indicated 12 and gel diffusion test 3 precipitating antigens incrude allergen extract; by these tests allergoids depicted 8 and 3 precipitin bands, respectively. The precipitin analysis showed heterogeneity of allergenic determinants and also variation in cross-immunogenicity of the formalinized derivatives. The skin prick and radioallergosorbent tests depicted greater activity of fractionated crude allergens than the allergoids. The above tests suggest altered and concealed antigenic determinants as result of formalinization of P. juliflora pollen which, however, showed reduced allergenic activity relative to the native allergen.     

Differences between intra- and extracellular interleukin-1

The intracellular (IC) and extracellular (EC) pools of interleukin 1 (IL-1) of human monocyte cultures were found to differ in their molecular size and charge characteristics. EC activity was found by Sephadex G-75 chromatography to consist mainly of a single peak in the 15,000–17,000 mol. wt range. In contrast, IC activity was distributed in four peaks (mol. wts of approx. 15,000, 26,000, 45,000 and> 70,000). Treatment of a pool of the IC 26,000, 45,000 and> 70,000 mol. wt species with CHAPS, a zwitterionic detergent, yielded a large amount of the 15,000 mol. wt species, thus suggesting that a portion of the larger species consists of aggregates of the 15,000 mol. wt molecule. Both IL-1 pools were found by isoelectrofocusing to be composed of three molecular species with pIs of 5.5, 6.1 and 6.7. However, the proportions of these species differed markedly between the EC and IC pools. The large majority of IC activity (˜90%) was found at pI 5.5, while 55–60% of EC activity had a pI of 6.7 and 35–40% had a pI of 5.5. The differences in their biophysical properties support the notion that the IC and EC pools of IL-1 also differ in their functions.     

Characterization of a T-lymphocyte inhibitor in the serum of tumour-bearing mice.

Sera from mice with large tumours from a variety of tissue types and sources have been shown to contain substances capable of suppressing the proliferative response of normal mouse lymphocytes to concanavalin A (Con A), bacterial endotoxin (LPS) and allogeneic cells. The present paper deals with studies on the nature of these inhibitory materials using mainly a methylcholanthrene-induced rhabdomyosarcoma in DBA/2J mice. It was found that a material responsible for inhibition of the Con A response eluted with immunoglobulins on Sephadex G-150 and eluted with monomeric immunoglobulin on Sephadex G-200. The component of tumour-bearer serum responsible for the suppression of the LPS response of normal lymphocytes eluted from Sephadex G-150 with the alpha and beta globulins and albumin (molecular weight less than 150,000). The immunoglobulin-containing serum fraction from tumour-bearing animals inhibited the mixed lymphocyte response, Con A response, and specific immune response to purified protein derivative (PPD) in allogeneic cell systems. It also inhibited the in vitro primary response of mouse cells to sheep red blood cells, and, to a lesser extent, the response to a T cell-independent antigen (DNP-dextran). The inhibitory activity continued to elute with monomeric IgG on Sephadex G-200 when columns were run in 1640 medium and adjusted to pH 2-5, indicating that an acid dissociable complex was not responsible for inhibitory activity. Inhibitor activity could be removed by absorption on immuno-adsorbents containing goat anti-mouse immuno-globulin, and could be recovered by acid elution from the absorbent. Inhibitor activity was not removed by immunoadsorption on columns prepared with antisera to chicken immunoglobulin.     

Properties of the Enterotoxic Component in Escherichia coli Enteropathogenic for Swine

The enterotoxic component in sterile syncase broth filtrates of Escherichia coli strains 340 (O9:K.:NM) and P307 (O8:K87,88a,b:H19) was studied. The enterotoxic activity in both strains was retained by an ultrafiltration membrane with a molecular weight retention of 100,000 (XM-100A) and eluted from a Sephadex G-200 column in the void volume. The enterotoxic activity in strain 340 was resistant to heating at 75 C for 30 min, but the activity in strain P307 was destroyed by heating at 60 C for 30 min. The P307 Sephadex G-200 column eluate possessing the enterotoxic activity, when desalted, contained 45.8% carbohydrate and 9.3% protein, and had an ED(50) of 2.2 mg/rabbit ileal loop. Immunodiffusion studies showed that this material contained both endotoxin and acid-polysaccharide capsular material. The enterotoxic activity was acidlabile and was destroyed by Pronase, but was resistant to trypsin and eluted as a single peak in the void volume of a 4% agarose column. The enterotoxic component could not be separated from the endotoxin; in fact, the data indicated that the two components are closely associated and that the enterotoxic activity resides in material of a protein nature.     

Correlation between structural characteristics and immunological properties of the terpolymer L-glutamic acid60-L-alanine30-L-tyrosine10.

The structure of the terpolymer L-glutamic acid⁶⁰-L-alanine³⁰-L-tyrosine¹⁰ (GAT) has been investigated. GAT appears very heterogeneous in size as determined by gel filtration on a Sephadex G-1 00 column. A dramatic downward shift in the average molecular weight (m.w.) is observed after gel filtration under denaturing conditions (Sepharose 6B, 6 M guanidine hydrochloride or polyacrylamide gel electrophoresis containing 0.1 % sodium dodecyl sulfate). We conclude that under nondenaturing conditions, GAT is a multimeric structure; the dissociation of the structure is reversible. GAT was fractionated based on the size of the polypeptide chains under denaturing conditions. After removal of guanidine hydrochloride three fractions were obtained with ～ 100 000 (GAT fraction I), 45 000 (GAT fraction II) and < 10 000 (GAT fraction III) m.w., respectively. The immunological properties of these three fractions have been compared with those of unfractionated GAT. Fraction II resembles unfractionated GAT, showing similar immunogenicity in responder mice and suppressive properties in nonre-sponder animals. Fraction I is less immunogenic, otherwise it resembles unfractionated GAT. Fraction III is the most dissimilar of the three fractions investigated. It retains the suppressive activity of GAT but is unable to stimulate a specific antibody response in responder animals. GAT fraction III is not a general tolerogen and is specifically suppressive for nonresponder mice.     

Studies on the physico-chemical characteristics of polymorph migration stimulator.

Polymorph migration stimulator is a supernatant factor produced by the interaction between glucocorticosteroids and human blood monocytes in culture. Studies on the physical characteristics of this factor show that it is soluble and stable at high and low temperatures. Its activity is reduced by acid and alkali treatment and destroyed by the proteolytic enzyme protease. Experiments involving dialysis, ultrafiltration and Sephadex G-100 gell filtration indicate that the molecular weight of the polymorph migration stimulator is between 12,000 and 15,000. It is suggested that this factor may mediate the anti-inflammatory effects of glucocorticosteroids on phagocytic cells.     

Purification and physicochemical characterization of Schistosoma mansoni egg allergen recognized by mouse sera obtained at an acute stage of infection

A major allergenic component recognized by mouse sera obtained at an acute stage of infection was purified from soluble egg antigen preparation (SEA) of Schistosoma mansoni by anion-exchange chromatography on DE52 and gel chromatography on Sephadex G-150. The purified allergen showed homogeneity by immunoelectrophoresis and by polyacrylamide gel electrophoresis. Its apparent molecular weight was 210,000 by gel chromatography on Sephadex G-200. This purified allergen could bind to Con A-Sepharose 4B, indicating its glycoprotein nature. After amino acid analysis, aspartic acid, glutamic acid, serine and theonine were found as the major amino acids. The allergenic activity was destroyed by heating at 100 degrees C for 60 min and by pronase or periodate treatment. By double diffusion in agar gel, this purified allergen gave a strong single band against acute (8 w) stage serum, which fused to the major band formed by crude SEA. On the other hand, it showed a very faint band against chronic (22 w) stage serum, which is apparently different from the main band formed between crude SEA and the chronic stage serum. When specific IgE or IgG antibody titers in the serum of human schistosomiasis mansoni cases were measured by ELISA using this purified allergen, the results showed good correlation with those obtained by using crude SEA. Thus, this purified allergen is not only a major allergen in the acute stage of murine schistosomiasis but also an allergen in human schistosomiasis mansoni.     

Reaginic antibodies and immunity to Nippostrongylus brasiliensis in the rat: II. Some properties of the antibodies and antigens

1. Passive transfer of immunity to Nippostrongylus brasiliensis with pooled antiserum from immune rats was neutralized in vivo by intravenous injection of small amounts of a saline extract of adult worms. This inhibition of protection was associated with systemic anaphylaxis and appeared to result from the neutralization of protective antibodies.

2. Serum from infected rats was fractionated by G-200 Sephadex gel-filtration. Reaginic antibodies were shown to be intermediate in molecular size between 7S and 19S globulins in sera from both singly and multiply infected animals. In immunoelectrophoresis they migrated with fast immunoglobulins but could not be related to either IgG or IgA rat immunoglobulins. The same serum fractions gave both homologous passive cutaneous anaphylaxis (PCA) and systemic anaphylaxis.

3. Blocking antibodies were found both in the 7S and 19S fractions after separation on G-200 Sephadex. These antibodies were found in sera from rats immunized with worm extracts as well as in sera from singly and multiply infected animals.

4. The saline extract of adult worms was fractionated on G-200 Sephadex. The isolated antigenic material (allergen) for both homologous PCA and systemic anaphylaxis seemed to be a protein with a molecular weight of approximately 12,000–17,000.

     

An improved method for the isolation of the major protein of the gonococcal outer membrane in an antigenically reactive form

The major outer membrane protein (protein I) has been isolated from Neisseria gonorrhoeae strain P9 in an immunologically reactive form. Membranes were sequentially extracted with the detergents sodium cholate and Empigen BB. Protein I was enriched in the Empigen-soluble fraction and was separated from other proteins and lipopolysaccharide by gel filtration chromatography on Sephadex G-200. The purified protein retained its antigenic activity with antiserum raised against the unfractionated outer membrane complex.