Rocket-line Immunoelectrophoresis Research Articles

A protein homology detection between rattlesnakes (Viperidae: Crotalinae) from South and North America deduced from antigenically related metalloproteases

Abstract Despite the fact that the analysis of two venom proteins is a limited investigation, the occurrence of proteins antigenically related between two Crotalus species from very distant geographic locations may be of importance to a further understanding of venom immunology. In this study, shared venom antigens of crotaline snakes are determined by immunoprecipitin analysis using rocket line immunoelectrophoresis (RLIE). Metalloprotease components were detected in the venom of Uracoan Rattlesnake (Crotalus vegrandis) from Venezuela, and in the venom of the Western Diamondback Rattlesnake (Crotalus atrox) from Texas, USA. Cross-reactive components detected in both species were identified using rocket line immunoelectrophoresis (RLIE). The present study was focused on a comparison of Crotalus antigens by anode deflection. There was cross-reactivity between Uracoina-1 from C. vegrandis and C. atrox venom tested with polyvalent Bothrops/Crotalus antivenom. Moreover, a common sequence of amino acids has been found between Uracoina-1 (C. vegrandis) and a catrocollastatin precursor from C. atrox. These results confirm the existence of cross-reacting components between antigenic extracts of both snake venoms. Rattlesnakes originally spread from North to South America, and both studied species, which share a similar protein, have probably been separated since the Cretaceous, for more than 80 million years.

Immunoelectrophoretic characterization and cross‐reactivity of Rochalimaea henselae, Rochalimaea quintana and Afipia felis

The soluble antigens of Rochalimaea henselae, Rochalimaea quintana and Afipia felis were characterized by crossed immunoelectrophoresis using bacterial sonicates as antigens against pooled hyperimmune rabbit sera. A precipitin pattern was drawn for each bacterium and shown to be reproducible and stable even when normal or preimmune rabbit serum was incorporated in the intermediate gel. By this technique 56 antigens were identified from R. henselae, 49 from R. quintana, and 39 from A. felis. The serological cross-reaction between R. henselae, R. quintana and A. felis, and between these 3 bacteria and 32 pathogenic bacteria was analysed by rocket-line immunoelectrophoresis, crossed-line immunoelectrophoresis, and tandem-crossed electrophoresis. It was concluded that (i) 4-7 antigens distinguish R. henselae, R. quintana and A. felis from each other, (ii) both Gram-positive and Gram-negative bacteria cross-react with R. henselae, R. quintana and A. felis antisera, (iii) the cross-reacting antigens of Gram-negative bacteria have both precipitating and non-precipitating specificities, whereas Gram-positive bacteria have mainly non-precipitating specificities, (iv) the cross-reacting antigens are common to several species, and (v) fewer cross-reacting antigens are found in phylogenetically disparate species than in more closely related species.

Epitope mapping of region 11-70 of ovalbumin (Gal d I) using five synthetic peptides.

Five successively located peptides, in region 11-70 of the major allergen of ovalbumin (OA) Gal d I (11-19, 20-33, 34-46, 47-55, 56-70), were obtained by manual solid-phase peptide synthesis. These peptides together with the previously reported OA region 1-10 comprise a segment of 70 amino acid residues located at the N-terminal of ovalbumin. The crude peptides were purified by gel filtration and reversed-phase high-performance liquid chromatographies and their sequences were verified. Polyclonal antibodies against the peptides conjugated to carrier protein (BSA) were raised in rabbits. Rocket line immunoelectrophoresis showed that four peptides (20-33, 34-46, 47-55 and 56-70), could deflect OA-line immunoprecipitates. The peptide's affinity to rabbit polyclonal Ig was examined by quantitative precipitation inhibition and the results suggested that an epitope was encompassed in segments 34-55 and 47-55. Allergenicity was tested by inhibition of specific IgE binding of ovalbumin, using several sera and a serum pool from 16 egg-allergic patients. The results showed that the allergenicity was distributed over the whole region. These findings suggested that: (a) the region 11-70 of OA seemed not to encompass continuous epitopes; (b) the antigenicity of this region was convincing for peptides 34-46 and 47-55; (c) the allergenicity, though dependent on the patient serum used, was distributed over the whole of region 11-70; (d) peptide 11-19, although weak antigenically was capable of specific IgE inhibition; (e) human and rabbit polyclonal antibodies did not show analogous affinities to the present peptides.

Antigenic Relationships among Bacteriodes Species Studied by Rocket-Line Immunoelectrophoresis

Antigenic relationships among the 10 Bacteroides species, Porphyromonas gingivalis, Prevotella bivia, and Prevotella intermedia were investigated by rocket-line immunoelectrophoresis, using antigenic extracts prepared from type strains and antisera to Bacteroides fragilis, Bacteroides distasonis, Bacteroides thetaiotaomicron, Porphyromonas gingivalis, and Prevotella intermedia. Among the members of the genus Bacteroides, B. fragilis, Bacteroides caccae, Bacteroides stercoris, Bacteroides vulgatus, B. thetaiotaomicron, and Bacteroides ovatus were found to be closely related, and B. distasonis and Bacteroides merdae were the species that were most distantly related to B. fragilis; Bacteroides eggerthii and Bacteroides uniformis were in an intermediate position. Porphyromonas gingivalis, Prevotella bivia, and Prevotella intermedia shared very low percentages of common antigens with the Bacteroides species. Detection of enzymic activities in immunoprecipitates allowed us to identify some antigens to enzymes and to study the immunological similarities of the enzymes. The malate dehydrogenases of the Bacteroides species were genus specific, and the glucose-6-phosphate dehydrogenases of most species exhibited antigenic similarities. The esterases of B. distasonis and B. thetaiotaomicron appeared to be species specific, whereas the esterases and phosphatases of B. fragilis cross-reacted with the esterases and phosphatases of B. stercoris and B. vulgatus, as did the phosphatases of B. distasonis and B. merdae. The phosphatases of B. eggerthii, B. uniformis, Porphyromonas gingivalis, and Prevotella intermedia were antigenically similar.

AN IMMUNOCHEMICAL ANALYSIS OF BEER FOAM

Beer foam produced in a continuous foaming tower in volumes representative of commercial dispense, was analysed by immunoelectrophoretic and immunoblotting techniques to identify antigens involved in foam structural stability. In crossed immunoelectrophoresis (CIE), only one antigen precipitated from foam in the homologous foam antiserum. This antigen was shown to be of malt origin by rocket-line immunoelectrophoresis and was also present in 11 commercial beers (5 bitters, 4 lagers and 2 stouts). However, the foam preparation separated into more than 20 polypeptides by SDS polyacrylamide gel electrophoresis. Immunoblotting showed that at least 12 of these reacted with foam antiserum and that they originated from either malt or yeast. Similar polypeptides were also identified in the antigen precipitated in CIE, suggesting that these polypeptides were probably present in the foam as a complex. It is concluded that the stability of foam reflected molecular interactions between these polypeptides (and possibly other components such as carbohydrates) in the liquid film of the bubble structure.

Antigenic cross-reactivity among isolates of group JK corynebacteria

Using rocket and rocket-line immunoelectrophoresis and immunoblotting it was demonstrated that a group of antibiotic-resistant bacteria, including several authenticated Corynebacterium jeikeium strains, shared many cross-reacting antigens. Only weak cross-reactivity was demonstrated with representatives of three other skin corynebacteria, C. bovis, C. hofmanii and C. minutissimum or with non-coryneforms. Differences within C. jeikeium are sufficient to permit the use of immunoblotting as an epidemiological tool.

Comparison of antigens from agents of actinomycetoma by immunodiffusion and electrophoresis procedures

Antigenic extracts were prepared from culture filtrates of the principal agents known to cause actinomycetoma, namely Actinomadura madurae, Actinomadura pelletieri, Nocardia asteroides, Nocardia brasiliensis, Nocardia otitidis-caviarum, and Streptomyces somaliensis. These antigenic preparations were compared by immunodiffusion (ID), counterimmunoelectrophoresis (CIE), line immunoelectrophoresis (LIE) and rocket line immunoelectrophoresis (RLIE), with rabbit antisera prepared against each of the extracts. Cross-reactivity between antigenic extracts from the different actinomycetes, measured by determining the number of precipitin lines in homologous and heterologous systems, was common. Reactions were always stronger and more precipitin lines were present when antigenic extracts were tested against homologous antisera. Similarities between A. madurae and A. pelletieri antigens were of a low order and cross-reactivity did not exceed 33%. A. pelletieri resembled N. asteroides more closely than A. madurae, with 44% of detectable antigenic components cross-reacting. The three species of Nocardia had common antigenic epitopes, but the overall degree of similarity was of a low order (between 12 and 27% by LIE and RLIE). Antigenic extracts of S. somaliensis had few components in common with the other species tested and only one of the 34 lines present in the RLIE system for N. asteroides showed any reaction of identity with an antigenic component in the S. somaliensis extract. Single cross-reacting lines were also present in the CIE and LIE systems.

Guanine content and Dermatophagoides pteronyssinus allergens in house dust samples

The study of the relationship between the guanine content and Dermatophagoides pteronyssinus ( Dp) allergens in house dust samples is reported. Mattress and carpet dust of bedrooms from 22 different homes constituted the house dust samples. The guanine content was determined by quantitative measurements and the mite allergenicity by two immunochemical assays with a partially purified extract of Dp as internal reference: RAST inhibition and crossed and rocket line immunoelectrophoresis. A large scale range of guanine content was obtained among the 22 house dust samples studied (0.01 to 1.78 mg/0.1 gm of dust). Data of RAST inhibition, analyzed according to parallel line bioassay, demonstrated no significant difference between the slopes of the reference and the house dust sample lines, but a 100-fold variation in the relative Dp potencies was observed. By crossed and rocket line immunoelectrophoresis technique, the presence and the amounts of major Dp allergens ( Der p I and Dp 4) were established in most house dust extracts. A significant correlation was found between the guanine content of the house dust samples and their relative Dp potencies ( r = 0.86) on the one hand, and with their relative content of Der p I and Dp 4, two major Dp allergens ( r = 0.75 and r = 0.74, respectively) on the other hand; in each case, a quantitative relationship was established. These results suggest that the guanine determination could assess mite allergens in house dust and may be a useful tool in large-scale investigations of house dust.

Dirofilaria immitis: Identification and partial characterization of parasite antigens in the serum of infected dogs

We have recently developed a sensitive and specific immunodiagnostic test for canine Dirofilaria immitis infection based on detection of soluble parasite antigens in dog sera by monoclonal antibody-based enzyme immunoassay. In addition to their importance as markers of infection, these antigens may contribute to the pathogenesis of heartworm disease in dogs. In the present study, a variety of methods were used to identify and characterize circulating D. immitis antigens. Two antigens were identified in infected dog sera that formed lines of identity in rocket-line immunoelectrophoresis with soluble antigens extracted from adult D. immitis. Circulating D. immitis antigens were also demonstrated in infected dog sera by immunoblot analysis with polyclonal and monoclonal antibodies. These antigens had apparent molecular weights that ranged from 50 to 250 kDa. Most of the circulating D. immitis antigens contained the epitope defined by monoclonal antibody 1418BF2.1 which is used in our enzyme immunoassay for circulating D. immitis antigen. Studies of parasite antigens released during in vitro culture indicated that the circulating D. immitis antigens in dog sera that are detected by our enzyme immunoassay are primarily derived from adult female worms.

α-Amylase Production by Pre-mature Wheat (Triticum aestivumL.) Embryos

Embryo/scutellar tissue of pre-mature wheat grains usually contains little α-amylase but readily produces the enzyme upon removal from the caryopsis. Enzyme production is accompanied by cytological changes in the scutellar epithelium cells characteristic of germinating mature embryos, although α-amylase production by pre-mature tissue is not always associated with germinative growth of the embryonic axis. Production of α-amylase is influenced by embryo age, stimulated by GA3 and overall is inhibited by ABA. Examination by isoelectric focussing and rocket-line immuno-electrophoresis reveals the presence of both α-AMYl and α-AMY2 isoenzymes, the latter being the major constituent. In the presence of ABA certain α-AMY2 isoenzymes not detected previously are observed.

Antigenic and Allergenic Determinants of Ovalbumin

The effects of enzymic cleavage and perturbing the conformation of the allergenic and antigenic determinants of hens egg white albumin (OA) were examined. Hens egg white extract of a total protein concentration 8.43 g/l was prepared. Isoelectric focusing in sodium dodecyl sulfate and polyacrylamide gel peptide maps for the crude egg white extract showed 26 spots visualized by staining with Coomassie blue. The OA was purified using a TSK-2000 gel filtration chromatography column. The specific allergenic reactivity of the purified OA as measured by RAST inhibition and direct RAST was relatively high: 3 micrograms gave an inhibition of approximately 10%. The cleavage of OA with cyanogen bromide resulted in 4 fractions, all capable of binding specific IgE with the first peak showing the highest inhibition. Thermal denaturation of OA had no direct effect on the antigenic reactivity. RAST inhibition values for the denatured protein were similar to those of the native protein. Carboxymethylation of OA gave a product with only 20% of the inhibition reactivity. Further treatment with trypsin did not abolish the allergenic and antigenic reactivities as shown by RAST inhibition and by deflection of OA line in rocket line immunoelectrophoresis. On the other hand, limited pepsin hydrolysis destroyed the antigenic structure of the molecule. The reactivity of OA is thus relatively stable and could easily be retained making it possible to identify the allergenic determinants of enzymic hydrolysates used for elucidating the antigenic structure of the molecule.

Cloning of Mycobacterium bovis BCG DNA and expression of antigens in Escherichia coli.

A gene bank of Mycobacterium bovis BCG DNA in Escherichia coli was constructed by cloning Sau3A-cleaved mycobacterium DNA fragments into the lambda vector EMBL3. The expression of mycobacterial antigens was analyzed by Western blotting with hyperimmune rabbit sera. Among 770 clones tested, several were found that produced various mycobacterial antigens in low amounts, with concentrations generally close to the detection limit. One particular clone was chosen for further investigation. This clone produced a 64-kilodalton (kDa) antigen. By placing the lambda promoter PL in front of the structural gene of this antigen, an overproducing E. coli strain was obtained. Rocket-line immunoelectrophoresis experiments showed that antigens cross-reacting with the 64-kDa protein are present in a wide variety of mycobacteria and also in so-called purified protein derivatives which are routinely used for skin tests. Preliminary experiments indicate the presence of antibodies against the 64-kDa antigen in sera from tuberculosis patients.

Identification and Antigenic Comparison of Enzymes in the Genus Candida by Means of Quantitative Imunoelectrophoretic Methods: Taxonomic Significance

Summary Zymogram staining techniques were used to identify Candida tropicalis immunoprecipitates corresponding to esterases, phosphomonoesterase, chymotrypsin and leucine aminopeptidase in crossed immunoelectrophoresis reference pattern. In the same way, immunoprecipitates were identified corresponding to chymotrypsin and leucine aminopeptidase in the Candida albicans crossed immunoelectrophoresis reference pattern. In addition, precipitation peaks which were not stainable with Coomassie blue were found for phosphomonoesterase, α esterase, β esterase and glucosidase. Cross-reactions of phosphomonoesterase, esterases and leucine aminopeptidases from Candida tropicalis and from 19 strains of 10 other species were studied by means of crossed-line immunoelectrophoresis and rocket-line immunoelectrophoresis. Rocket-line immunoelectrophoresis appeared very suitable for the rapid comparison of various antigenic enzymes from several strains. One esterase and one leucine aminopeptidase did not show any cross-reaction and seemed to be species-specific. Another leucine aminopeptidase and the phosphomonoesterase cross-reacted only with strains of species related to Candida tropicalis . The other esterase, which showed also a chymotrypsin-like activity, cross-reacted with all the strains tested. Phosphomonoesterase, esterase and leucine aminopeptidases from Candida albicans and 22 other Candida strains were compared by rocket-line immunoelectrophoresis. The esterases and one leucine aminopeptidase were species-specific. Phosphomonoesterase and the second leucine aminopeptidase from Candida albicans cross-reacted only with Candida tropicalis strains. Thus, the antigenic specificities of these enzymes are a useful taxonomic tool.

Rocket line immunoelectrophoresis: An improved assay for simultaneous quantification of a mink parvovirus (aleutian disease virus) antigen and antibody

A rocket line immunoelectrophoretic assay (RLIE) was developed for the simultaneous quantification of viral antigens and antiviral antibodies of the important mink parvovirus, Aleutian disease virus (ADV). The sensitivity of the RLIE assay was found to be 5 log 2 higher than that of the counter current immunoelectrophoresis which is the assay routinely used for diagnostic purposes.

Changes in Levels of α-Amylase Components in Barley Tissues during Germination and Early Seedling Growth

Kernels of Klages barley (Hordeum vulgare L.) were germinated for 1 to 4 days on moist sand at 18 degrees C. Representative kernels from each time period were dissected to give the following fractions: scutellum, subscutellar endosperm, aleurone-scutellum interface, remaining aleurone, subaleurone endosperm, and core endosperm. These tissues were analyzed for alpha-amylase components by isoelectric focusing and rocket-line immunoelectrophoresis. Although aleurone and scutellar tissues appeared to synthesize the same alpha-amylase components, enzyme was detected first in the scutellum. A larger proportion of scutellar alpha-amylase was excreted into the endosperm compared to aleurone synthesized alpha-amylase. Aleurone cells appeared to synthesize appreciably more alpha-amylase than did scutellar tissue.

Immunochemical analysis of cod fish allergen M: locations of the immunoglobulin binding sites as demonstrated by the native and synthetic peptides.

The major allergen of codfish (Allergen M) is a muscle protein belonging to the family of calcium binding parvalbumins. The primary structure of the molecule was established and the molecular weight was estimated from the sequence data to be 12,328. Allergen M consists of 113 amino acid residues and one residue of glucose. A molecular arrangement of three domains (AB, CD and EF--the latter two bind one Ca2+ ion each) was described for Allergen M, analogous to carp parvalbumin pI 4.25. The suggested structure was based on the extensive intramolecular amino acid homologies and the immunochemical cross-reactivities of the intact molecule and the two major isolated fragments. The immunological structure of Allergen M was studied by: 1. Modification of certain amino acids residues and study of the reactivity of the modified derivatives. 2. Examination of the immunochemical reactivity of a large number of overlapping peptides obtained by limited and selective tryptic hydrolyses. 3. Solid phase peptide synthesis (SPPS) of segments selected in regard to the reactivity of pre-examined native peptides. The immunological reactivity of the derivatives of Allergen M was assigned by: 1. Rocket line immunoelectrophoresis and quantitative precipitation using rabbit anti-Allergen M in precipitating antibody-mediated reactions and, 2. RAST/RAST-inhibition and PK test/PK-test inhibition using sera from individuals allergic to codfish in IgE-mediated reactions. The modification of Tyr-30 and Arg-75 in isolated and purified peptides indicated that the former was part of a reactive site whereas the latter did not contribute to the activity. Masking of Arg-residue or unchelating the two calcium ions from the native Allergen M, with the resulting perturbation of the tertiary structure, decreased the allergenicity by approximately 25%. Two major fragments of Allergen M were produced and purified: TM1 (residues 1-75) comprising domains AB and CD, and TM2 (residues 76-113) covering domain EF. Both were immunologically reactive; TM1 showing intermediate reactivity between Allergen M and TM2. A high degree of immunological cross reactivity was evident between TM1 and TM2. The finding was in concert with the high intramolecular amino acid homologies of Allergen M, and suggested that the reactive sites were repetitively distributed along the polypeptide chain. The immunological reactivity of several long-sequence overlapping peptides obtained by limited and selective trypsin hydrolysis of Allergen M was studied. The immunologically reactive sites were accordingly assigned to the following regions of the chain: 1. Residues 33-44 on the junction between the AB and CD domains.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunological identity between bovine preparations of thiol: Protein-disulphide oxidoreductase, glutathione-insulin transhydrogenase and protein-disulphide isomerase

Five preparations of bovine thiol:protein-disulphide oxidoreductase/glutathione-insulin transhydrogenase (EC 1.8.4.2) and one preparation of bovine liver protein-disulphide isomerase (EC 5.3.4.1) from four different laboratories showed immunological identity in double immunodiffusion and rocket-line immunoelectrophoresis. Consequently, thiol:protein-disulphide oxidoreductase/glutathione-insulin transhydrogenase and protein-disulphide isomerase, formerly classified as two separate enzymes, should be considered as alternative activities of the same enzyme.

Immunochemical detection of a common antigen among Streptococcus uberis isolates

Fifty-three isolates of Streptococcus uberis from various sources were examined for the presence of a common antigen. Initially, a serum was produced in rabbits which, by using rocket line immunoelectrophoresis, proved to react with identity to all of the S. uberis crude extracts as well as group B and E streptococcal extracts. The antigen(s) responsible for this cross-reactivity was partially purified by Sephacryl S-200 gel chromatography and analyzed by fused rocket immunoelectrophoresis. Further analysis by immunodiffusion showed that probably two antigens in the gel chromatography-consolidated fractions were common to the S. uberis and group B and E isolates, but that one of the antigens present was unique to S. uberis. Trypsin destroyed the immunoreactivity of this antigen. Isolation of this common antigen could possibly alleviate some of the tedium associated with the identification of this organism.

Taxonomy of the Azotobacteraceae Determined by Using Immunoelectrophoresis

The similarities of various strains of Azotobacter spp. and Azomonas spp. to reference strains of Azotobacter paspali, Azotobacter vinelandii, Azotobacter chroococcum, Azomonas agilis, Azomonas insignis, and Azomonas monocytogenes were determined by rocket line immunoelectrophoresis. The strains of Azotobacter paspali and Azotobacter vinelandii used were immunologically more homogeneous than the strains of Azotobacter chroococcum studied, possibly due to the more diverse geographical origins of the Azotobacter chroococcum strains. Low values were obtained for the mean immunological distances (1 – proportion of immunoprecipitation bands shared between strains) between Azotobacter paspali and Azotobacter vinelandii strains, suggesting that these two species are immunologically closely related. Immunological distances from the Azotobacter chroococcum reference strain were similar for Azotobacter paspali and for other undisputed members of the genus Azotobacter, which makes it reasonable to retain Azotobacter paspali in this genus. When the three Azotobacter antisera were used, all Azotobacter species had mean immunological distances of less than 0.5, whereas the Azomonas species were immunologically more distant, showing that the six species of Azotobacter form an immunologically related group which is distinct from the Azomonas species. Our results with the three Azomonas antisera show that each species of Azomonas is immunologically distant from the other species, as well as from the Azotobacter species. We compare our immunoelectrophoretic results with the molecular biological results of De Smedt et al. (Int. J. Syst. Bacteriol. 30:106–122, 1980) and the numerical taxonomic analysis of Thompson and Skerman (Azotobacteraceae: the Taxonomy and Ecology of the Aerobic Nitrogen-Fixing Bacteria, Academic Press, Inc., London, 1979).

Enzymic Mechanism of Starch Breakdown in Germinating Rice Seeds

The formation of multiple forms of amylases in germinating rice (Oryza sativa L. cv Kimmaze) grains was examined by means of isoelectric focusing, cross-immunoelectrophoresis, and rocket-line immunoelectrophoresis followed by a reaction of enzymic characterization by using beta-limit dextrin or starch as substrate. The constituents detected by isoelectric focusing were identified as three electrophoretically heterogeneous antigens. The major alpha-amylase bands A and B corresponded to a same antigen, the main portion of which was produced within 2 days' germination. The bulk of alpha-amylase D appeared between 2 and 4 days' germination. Component E, a debranching enzyme according to its action on the beta-limit dextrin, already exists in the ungerminated seeds; its amount decreases within the first 2 days of germination and increases again thereafter.Evidence showing that beta-amylase (band C) is produced by the scutellum at an early stage of germination was provided. The enzyme appeared in a suspension of the scutellum after a prolonged incubation.