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.

Immunochemical characterisation of species-specific antigens in bovine crude heparin

In order to develop immunoassays for the control of the species origin of crude heparins, polyclonal antisera were produced in rabbits against samples obtained from the last purification steps of bovine intestinal crude heparin. The reactivity of the antisera was analysed by agar gel double immunodiffusion, immunoelectrophoresis, crossed and line immunoelectrophoresis. Up to 13 antigenic components were detected in the effluents of the ion-exchange chromatographic step, and three in the final crude heparin. The major and most anodic antigen (Ag1) was recovered in bovine crude heparin purified by the two different industrial processes. This bovine specific antigen was found in high concentrations in lung, liver, small intestine, spleen and kidney. It displayed an apparent molecular weight of 45 kDa by size-exclusion chromatography. Though the identification of Ag1 is not yet fully elucidated, a single radial immunodiffusion assay has been developed for the quantification of this antigen, allowing the detection of 6 p 1000 bovine crude heparin in porcine heparin (50 mg/ml) after 2 h diffusion.

MRNA expression and protein localization of placental tissue proteins in malignant tumors

A novel combination of two conventional techniques (autoradiography, AR and line immunoelectrophoresis, LIE; ARLIE) for identification of specific proteins synthesized de novo by explants is described. The incorporation rate of [35S]-methionine was linear in proteins derived from cytosol fractions and supernatants of first trimester human trophoblast and gestational endometrium for up to 18 h. SDS-PAGE analysis of these fractions provided further evidence of the protein synthesis and secretion by the tissue explants.The ARLIE system was evaluated by investigating the synthesis and secretion of five test proteins (PP12, PP14, hPL, FA-1 and FA-2) by trophoblast and gestational endometrium. The synthesis P12 and PP14 could be demonstrated by gestational endometrium only. Similarly the synthesis of hPL could be demonstrated by the trophoblast alone. The synthesis of the fetal proteins (FA-1 and FA-2) could not be demonstrated by either tissue. The control procedure, Protein A assisted immunoprecipitation, yielded similar results for PP14 but not hPL. This novel combination (ARLIE) provides a simple technique with which to study the de novo synthesis of several proteins simultaneously which is independent of the subclass and species of origin of antibodies.

Characterisation of the aminopropeptide of procollagen (alpha 1) I in human amniotic fluid

Two fetal antigens were identified during the course of experiments to generate antibodies against endometrial proteins [1,2]. One of them, Fetal Antigen 2 (FA-2), has been shown to be identical to the aminopropeptide of procollagen (alpha 1) I [3], and biochemical characterisation has revealed two molecular mass forms, l4OkDa and 35kDa [l]. Immunohistochemical studies have localised this protein in fetal and adult skin [4], in fetal osteoblasts [5], and intracellularly in osteoblast and fibroblast cultures [4,6]. There is also evidence that it is involved in matrix and/or basement membrane changes during tumor growth and invasion [7,8]. It has been suggested that FA-2 might be useful as a marker for evaluating bone disease [5,6]. The present study examines the molecular heterogeneity of FA-2 more fully, and describes a purification protocol for isolating specific FA-2 forms. FA-2 was studied in pooled second trimester amniotic fluid using line immunoelectrophoresis, employing polyclonal antibodies, for quantification. Gel filtration confirmed two size forms, 140kDa and 34kDa, and revealed 65% was recovered in the 140kDa peak. Separation on DEAE-Sephacel also revealed two forms. 73% of the recovered FA-2 eluted as a broad peak (DEAE1) at approximately 0.2M sodium chloride, and the remainder as a sharp peak (DEAE2) at approximately 0.26M sodium chloride. Electrophoretic analysis using nondissociating (ND) -PAGE revealed three major peaks (A, B and C), and several minor peaks (fig. 1). The relation between the FA-2 forms was studied. (1) The 140kDa form has two components which migrate as peaks A and B on ND-PAGE. (2) The 34kDa form has several components which migrate as peak C and the minor peaks on ND-PAGE. (3) A and B show similar subunit composition

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.

Virus-specific B-lymphocytes are probably the primary targets for Aleutian disease virus

368 1- to 5-year-old mink of wild-type or black genetic background were infected with Aleutian disease virus (ADV) naturaly or using virus-containing immune complexes or purified virus. Thirty of the mink were immunized with dinitrophenol-conjugated ovalbumin (DNP-OA) before and during infection. Blood samples were taken at monthly intervals. We found that weak (and transient) monoclonal or oligoclonal immunoglobulin components were present in the plasma of serum approximately 1 month after infection, as judged by zone electrophoresis. In a few cases, we found quite stable myeloma-like hypergammaglobulinemia, which usually occurs much later in the infection. All sera with monoclonal immunoglobulin components and most of the sera with immunoglobulins of restricted heterogeneity were analysed by crossed serum line immunoelectrophoresis. In all cases, the distinct immunoglobulins were found to have antibody activity to ADV proteins. In the few sera from DNP-OA-immunized mink showing restricted immunoglobulin heterogeneity, this was also the case. The findings from the study imply that ADV-specific B lymphocytes are probably the primary targets for ADV. The resulting ADV replication introduces a “pseudo-transformation” stage, so that the infected B lymphocytes proliferate and differentiate to an extreme degree. The mechanism behind this B-cell pseudotransformation ability of ADV is a puzzle. It may, however, be important, that the p75/85 structural polypeptides of ADV contain an amino acid sequence almost identical to the GTP-binding pocket of the Ras oncogene.

Simultaneous autoradiography and line immunoelectrophoresis (ARLIE): A novel combination to identify de novo protein synthesis by pregnancy tissues

International Journal of Gynecology & ObstetricsVolume 33, Issue 3 p. 292-292 Citations from the literature clinical investigation Simultaneous autoradiography and line immunoelectrophoresis (ARLIE): A novel combination to identify de novo protein synthesis by pregnancy tissues TN Fay, TN FaySearch for more papers by this authorK Price, K PriceSearch for more papers by this authorB Teisner, B TeisnerSearch for more papers by this authorI Jacobs, I JacobsSearch for more papers by this authorJG Grudzinskas, JG GrudzinskasSearch for more papers by this author TN Fay, TN FaySearch for more papers by this authorK Price, K PriceSearch for more papers by this authorB Teisner, B TeisnerSearch for more papers by this authorI Jacobs, I JacobsSearch for more papers by this authorJG Grudzinskas, JG GrudzinskasSearch for more papers by this author First published: November 1990 https://doi.org/10.1016/0020-7292(90)90063-QAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume33, Issue3November 1990Pages 292-292 RelatedInformation

De Novo Synthesis of Placental Protein-14 (PP14) and not PP12 by Monolayer Cultures of Glandular Epithelium of Gestational Endometrium

Nine monolayer cell cultures of glandular epithelium from gestational endometrium were established from six apparently healthy women undergoing elective termination of pregnancy (7-11 weeks gestation). Radiolabel incorporation studies showed increasing incorporation of [35S]methionine into proteins present in supernatant and cytosol fractions over 48 h. The secreted proteins represented approximately 20% of the total incorporation of methionine into cytosolic proteins. De novo synthesis and secretion of placental protein-14 (PP14) and not PP12 was identified by a novel combination of line immunoelectrophoresis and autoradiography. All monolayer cultures demonstrated the presence of radiolabeled PP14, but not PP12, in the culture supernatants. These observations suggest that the glandular epithelial cells are the major site of synthesis and secretion of PP14 in human gestational endometrium.

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.

Identification of specific serum proteins synthesized de novo by monolayer cultures of glandular cells of gestational endometrium

Monolayer cell cultures (n = 3) of glandular epithelium of gestational endometrium obtained from three apparently healthy women undergoing elective termination of pregnancy (7-9 weeks gestation) were established. De novo synthesis of eight serum proteins (albumin, alpha 1-antitrypsin, ceruloplasmin, beta-lipoprotein, alpha 2-macroglobulin, fibronectin and complement factors C3 and C4) was demonstrated by the incorporation of radiolabelled substrate ([35S]methionine) employing autoradiography (AR) in combination with crossed immunoelectrophoresis (XIE), referred to as ARXIE, and line immunoelectrophoresis (LIE), referred to as ARLIE. By contrast, there was no evidence for de novo synthesis of IgA, haptoglobin and orosomucoid. Our findings suggest that the gestational endometrium may contribute to the production of several proteins considered to be synthesized and secreted mainly by the liver and reticulo-endothelial system. The simple techniques used here to identify the de novo synthesis of human serum proteins could be applied to investigate protein synthesis by a wide range of tissues and cells.

Simultaneous autoradiography and line immunoelectrophoresis (ARLIE): a novel combination to identify de novo protein synthesis by pregnancy tissues.

A novel combination of two conventional techniques (autoradiography, AR and line immunoelectrophoresis, LIE; ARLIE) for identification of specific proteins synthesized de novo by explants is described. The incorporation rate of [35S]-methionine was linear in proteins derived from cytosol fractions and supernatants of first trimester human trophoblast and gestational endometrium for up to 18 h. SDS-PAGE analysis of these fractions provided further evidence of the protein synthesis and secretion by the tissue explants. The ARLIE system was evaluated by investigating the synthesis and secretion of five test proteins (PP12, PP14, hPL, FA-1 and FA-2) by trophoblast and gestational endometrium. The synthesis P12 and PP14 could be demonstrated by gestational endometrium only. Similarly the synthesis of hPL could be demonstrated by the trophoblast alone. The synthesis of the fetal proteins (FA-1 and FA-2) could not be demonstrated by either tissue. The control procedure, Protein A assisted immunoprecipitation, yielded similar results for PP14 but not hPL. This novel combination (ARLIE) provides a simple technique with which to study the de novo synthesis of several proteins simultaneously which is independent of the subclass and species of origin of antibodies.

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.

The subunit composition of factor XIII proteins in normal and factor XIII deficient plasma and serum analysed by line immunoelectrophoresis

Line immunoelectrophoresis was used for the characterization of human coagulation factor XIII in normal and factor XIII-deficient plasma and serum. In normal plasma, two immunoreactive factor XIII proteins termed XIII-p ( M r 330 000 Da) and XIII-s ( M r 170 000 Da) were identified within the beta-globulin fraction. Their reactions with monospecific antisera against known factor XIII subunits, suggests that the factor XIII-p and -s proteins recognized represent the factor XIII a 2b 2 tetramer and -b 2 dimer, respectively. The concentration of the XIII-p protein was, in normal plasma, approximately 6 mg/l, in plasma from a case of congenital factor XIII, approximately 1 mg/l and in normal serum approximately 3 mg/l. The concentration of the XIII-s protein (approximately 18 mg/l) was unaffected by the coagulation process and normal in plasma and serum from the case of congenital factor XIII deficiency. Thus, the present investigation indicates the presence of an excess of the anticatalytic, DNA-binding factor XIII carrier protein in normal as well as in factor XIII-deficient plasma and serum.

Two fetal antigens (FA-1 and FA-2) and endometrial proteins (PP12 and PP14) isolated from amniotic fluid; preliminary observations in fetal and maternal tissues

Rabbit antihuman antibodies were derived by the injection of fractions of second trimester amniotic fluid known to contain proteins of endometrial/decidual origin. Using standard separation and absorption procedures, two antibody preparations were generated which demonstrated specificities against two and three proteins, respectively, in line immunoelectrophoresis and crossed immunoelectrophoresis. Analysis against proteins of fetal, maternal, endometrial and placental origin revealed that the bispecific antiserum reacted only with placental protein 14 (PP14; also known as progestagen-dependent endometrial protein, PEP) and one other hitherto undescribed antigen referred to as Fetal Antigen 1 (FA-1) molecular mass 60 kDa; electrophoretic mobility: slow; α 1– α 2; fast, albumin. The trispecific antiserum demonstrated specificities against placental protein 12 (PP12), alphafetoprotein (AFP) and another previously undescribed antigen referred to as Fetal Antigen 2 (FA-2) molecular mass 35 and 140 kDa; electrophoretic mobility: albumin. Following purification, monospecific antisera against each of these proteins (with the exception of AFP) were derived in new rabbits. Maternal and fetal blood, amniotic fluid and aqueous extracts from endometrial/decidual and placental tissues were analysed in rocket immunoelectrophoresis using these antisera to examine the distribution in these tissues. The analyses demonstrated a pattern of distribution typical for proteins of endometrial/decidual origin in these compartments in the case of PP12 and PP14, but suggested that the primary source of origin of FA-1 and FA-2 may be the fetus.

Production of monospecific antisera by immunization with precipitin line leads to misinterpretation of immunoblot.

Immunoblotting displays an artifact in the preparation of monospecific antibodies from immunocomplexes cut out of line immunoelectrophoresis. Agarose induces the formation of antibodies towards a 68 KDa protein which is contained in commercial agarose.

Biochemical and immunochemical evidence for the origin of the spermatophore material inGlossina morsitans morsitans Westwood

Protein patterns in secretions from fully differentiated male accessory reproductive glands (ARG), spermatophore (Sp) and testes (Te) of the tsetse,Glossina morsitans morsitans Westwood, were determined by isoelectrofocusing. Isoelectrofocusing patterns of total ARG proteins and those of Sp were remarkably similar. At least 27 bands were detected in ARG and Sp. Out of these, 13 were major protein bands and isoelectrofocused in the pl range of 4 and 6.55. About 10 of these 13 were found to be acidic. Ouchterlony immunodiffusion and straight line immunoelectrophoresis showed that male accessory reproductive gland secretory proteins and spermatophore share common immunological characteristics which are different from those of the testes.

Fusarium solani: Evidence for common antigenic/allergenic determinants with other Fungi Imperfecti

Aqueous extracts of Fusarium solani and other members of the Fungi Imperfecti were evaluated for the presence of common antigenic/allergenic determinants using skin-prick testing, radio-allergo-sorbent test (RAST) inhibition, and immunoelectrophoretic methods. Prevalence of skin reactivity in forty-four atopic individuals, tested with commercially available fungal extracts, ranged from 27.3% for Alternaria tenuis to 6.8% for Penicillium notatum. No specific patterns of reactivity emerged from statistical analyses of skin test data. In contrast, RAST inhibition demonstrated common allergenic determinants. P. notatum and Aspergillus glaucus inhibited F. solani RAST by 79% and 84%, respectively. This was supported by crossed line immunoelectrophoresis; both P. notatum and A. glaucus had antigenic determinants in common with F. solani. Collectively, these studies suggest that F. solani, P. notatum, and A. glaucus have several common antigenic/allergenic determinants.

The development of monospecific antibodies against human thrombo-plastin apoprotein (apoprotein III) and their application in the immunocytochemical detection of the antigen in blood cells

Human thromboplastin apoprotein (apoprotein III) purified by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was purified a further 2–4 fold by PAGE in the presence of digitonin. Subsequent line immunoelectrophoresis of the protein revealed several lines, only one of which contained inhibitory antibodies. New inhibitory antibodies which were raised by using this particular line to immunize rabbits produced only a single line in immunoelectrophoresis of apoprotein III, with precipitated inhibitory antibodies being present only in the line. When these antibodies were used in electroblot immunobinding studies of crude thromboplastin after SDS-PAGE staining was found mainly in a single band of MW about 50 000, but also to some extent in immunologically related higher MW material. Prior deglycosylation of the thromboplastin using trinitrobenzenesulfonic acid resulted in a shift of the bulk of the main band representing an apparent MW reduction of 16%, and a corresponding shift in the position of protein with the capacity to bind inhibitory antibodies. Besides being a good criterion of specificity of the antibodies this also suggests that non-carbohydrate parts of apoprotein III may be involved in the interaction with Factor VII. Immunoperoxidase staining of unstimulated or endotoxin stimulated blood cells using the antibodies revealed the presence of significant amounts of apoprotein III only in stimulated monocytes, apparently available on the surface of the cells since it was detectable also by preembedding staining of fixed cells in suspension. The result is strong evidence that apoprotein III is synthesized de novo in monocytes upon endotoxin stimulation.

Combination of line immunoelectrophoresis and sodium dodecyl sulfate‐polyacrylamide gel electrophoresis for molecular weight evaluation of isozymes: Application to iso‐α‐amylases identified in barley and rice seeds

AbstractA procedure for enzyme purification by means of line immunoelectrophoresis compatible with subsequent sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of the purified enzyme and Coomassie Blue staining is described. Electrophoresis was carried out in an agarose gel backed to GelBond film. The enzyme activity was detected after immunoelectrophoresis on the wet gel and the immunoprecipitate by protein staining on the dried gel. The strip bearing the maximum amount of precipitate was excised and the dried agarose gel scratched off the GelBond support. The dried gel was extracted with the Laemmli sample buffer, heated at 100 °C for 10 min and submitted to sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. Proteins were stained with Coomassie Blue. Difficulties due to the presence of the immunoglobulin G subunits were encountered. The validity of the procedure was checked with an α‐amylase from barley purified by immunoaffinity chromatography. The procedure was used to compare the molecular weight of α‐amylase isozymes identified in barley and in rice seeds. In barley, α‐amylase I, the minor enzyme group appearing during germination, had a slightly higher molecular weight than α‐amylase II, the main enzyme group. In germinating rice seeds, the minor enzyme group, antigen D, was found to have a molecular weight slightly lower than the one of the other rice α‐amylases. A new α‐amylase antigen identified in rice as the constituent with the lowest pI was called pre‐A. Its molecular weight was found to be the same as the one of the main enzyme group, antigen (A + B).

Restricted heterogeneity of the early antibody response to Aleutian disease virus in mink kits.

Mink kits born of Aleutian disease (AD) negative dams were infected neonatally with different isolates of Aleutian disease virus (ADV). They were then sacrificed at different days after infection (10-45 days) and their sera were analysed by different techniques for concentrations of immunoglobulins and quality of the produced antibodies to ADV. The infected mink kits produced significantly higher concentrations of IgM and IgG immunoglobulins than non-infected-age matched control mink (p less than 0.001). Eight percent of the sera from the infected mink kits exhibited a restricted heterogeneous gammaglobulin profile as shown by serum electrophoresis. These findings were further investigated by other techniques such as SDS-PAGE and crossed serum line immunoelectrophoresis. It is concluded that mink kits when infected neonatally with ADV start to develop hypergammaglobulinemia soon after infection, and that a small percentage of the mink react with a gammaglobulin response of restricted heterogeneity.