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PubMed1980

Mycoplasma pneumoniae antigens demonstrated by crossed immunoelectrophoresis.

Soluble Mycoplasma pneumoniae antigens were analysed by crossed immunoelectrophoresis. At least 15 precipitation arcs were developed between the soluble antigens and rabbit anti-Mycoplasma pneumoniae hyperimmune sera. Sera from Mycoplasma pneumoniae infected patients formed precipitation arcs which were identified with those formed by a chloroform-methanol extract of Mycoplasma pneumoniae. Low concentrations of human antibodies, not detectable in a complement fixation test, were found to react with these antigens. The antigens were shown to cross-react with antigens of Mycoplasma neurolyticum. Emphasis was placed on the technical development of the crossed immunoelectrophoresis for the analysis of immune-precipitable antigens of Mycoplasma pneumoniae.

Animals↗
PubMed1981

Crossed immunoelectrophoresis using immobilized thrombin in intermediate gel. A method for demonstration of thrombin-binding platelet proteins.

A method is described in which the interaction between thrombin and platelet proteins can be studied directly. Solubilized platelet proteins were examined by crossed immunoelectrophoresis against polyspecific antiplatelet antibodies, and the interaction with thrombin observed by insertion of an intermediate gel containing thrombin coupled to Sepharose 4-B. Four immunoprecipitates were absent or showed an altered position compared to that of the control containing regular Sepharose 4-B in the intermediate gel. These were No. 1 (representing platelet factor 4), No. 13 (glycocalicin-related protein, probably GP lb), No. 19 (factor XIII), and No. 2a which has not yet been identified. Immunoprecipitates No. 16, representing a complex of GP IIb and GP IIIa, and No. 6 (albumin) as well as the other immunoprecipitates seen in the control pattern were unaffected by the presence of immobilized thrombin in the intermediate gel. The interaction of glycocalicin-related protein as well as purified glycocalicin with thrombin was confirmed by use of a monospecific antiserum. It is concluded that crossed immunoelectrophoresis using immobilized thrombin in an intermediate gel represents a new and useful approach to the investigation of the interaction between thrombin and platelet proteins. This procedure may also be extended to many other interactions between agents and cell proteins.

Blood Platelets↗
PubMed1981

Direct detection of two cross-reactive antigens between porcine and rabbit zonae pellucidae by radioimmunoassay and immunoelectrophoresis.

Zonae pellucidae were isolated from oocytes obtained from rabbit and porcine ovaries. Antisera were prepared in rabbits against heat-solubilized porcine zonae and in guinea pigs against intact and heat-solubilized rabbit zonae and against purified rabbit zona proteins isolated from two-dimensional electrophoresis gels. These antisera were used in radioimmunoassay studies to show cross-reactivity between porcine and rabbit zona antigens. Immunoelectrophoresis studies also demonstrated the immunochemical cross-reactivity between two distinct antigens of rabbit and porcine zonae. These two rabbit zona antigens were demonstrated to be immunochemically identical to porcine zona antigens by crossed-line immunoelectrophoresis.

Animals↗
PubMed1975

Variants of normal human alpha2-macroglobulin. Immunoelectrophoresis and enzyme-binding effect.

Three phenotypical variants of normal human serum alpha2-macroglobulin revealed by immunoelectrophoresis and specific antibodies obtained in rabbits are presented. The variants are characterized by differences in electrophoretic mobility: one being fast, one slow, and one of an intermediate rate. To find out possible differences with respect to the effect of the trypsin and chymotrypsin on the three variants, they were treated with the enzymes before immunoelectrophoresis. Migration was accelerated in all cases, after complexing with the enzymes, but the differences in the relative positions of the variants were maintained. The trypsin- and chymotrypsin-binding capacities of these three forms seem to differ, as suggested by the results presented in this report.

Adult↗
PubMed1981

Comparative immunochemical study of ribosomal proteins from various mammalian cells by two-dimensional immunoelectrophoresis.

Proteins isolated from ribosomal subunits of various mammalian cels were analysed comparatively by two different methods: a two-dimensional polyacrylamide gel electrophoresis system and a recently described two-dimensional immunoelectrophoresis technique. For this purpose, antisera were raised in rabbits against the total mixture of ribosomal proteins from murine cells. These sera were characterized by ring-test, double immunodiffusion and two-dimensional immunoelectrophoresis. They were shown to contain antibodies to a large number of ribosomal proteins. Immunoelectrophoretic analysis of 60S and 40S subunit proteins from rabbit, lamb, canine and human cells using anti-murine sera revealed a striking conservation of their antigenic properties. These results corroborated those obtained by two-dimensional polyacrylamide gel electrophoresis.

Animals↗
PubMed1991

Purification of beta-glucuronidase and structural assessment of the carbohydrate chains by lectin affinity immunoelectrophoresis.

The purification of rat liver beta-glucuronidase from a lysosomal fraction by methods including affinity chromatography, chromatofocusing and preparative PAGE steps is described. Molecular weights of 300,000 and 150,000 were estimated by two dimensional gradient PAGE/immunoelectrophoresis of the lysosomal extract. Isoelectrofocusing in agarose gel followed by immunoelectrophoresis in the second dimension revealed the presence of at least five maxima in the range pH 4.3-7.4. The structural assessment of the carbohydrate chains of lysosomal and microsomal beta-glucuronidase was performed by lectin affinity immunoelectrophoresis. Reaction with Concanavalin A indicated the presence of bi-antennary complex, oligomannosidic and hybrid type structures, whereas the absence of tri- and tetra-antennary complex type structures was deduced from the lack of interaction with phytohemagglutinin-L. The reaction with Lens culinaris agglutinin, Pisum sativum agglutinin and Lotus tetragonolobus lectin revealed that part of the glycans contained a fucose alpha(1-6)-linked to the N-acetylglucosamine attached to asparagine. The presence of terminal beta(1-4)-galactose residues was detected with Ricinus communis agglutinin I.

Animals↗
PubMed1985

Antigenic analysis of Trypanosoma cruzi strains by crossed immunoelectrophoresis: demonstration and isolation of antigens particular to some strains.

Antigenic differences among soluble extracts of Y, CL, SF, and Colombian strain epimastigotes of Trypanosoma cruzi have been demonstrated by crossed immunoelectrophoresis with an intermediate gel containing the heterologous antiserum. Using crossed immunoelectrophoresis with the homologous antiserum, over 30 precipitin lines could be demonstrated for each strain and, even though marked differences were observed in experimental infections, the strains shared a significant number of antigens. In addition, some strain-particular antigens were isolated using affinity chromatography. These antigens could be valuable in the study of biological, immunological, and pathological characteristics of experimental and natural T. cruzi infections.

Animals↗
PubMed1979

Immunoelectrophoresis in the diagnosis of neuroectodermal and mesodermal intracranial tumours, especially those of the posterior fossa.

Immunoelectrophoresis of extracts of 200 intracranial tumours against rabbit anti-glioblastoma serum gave positive results (= precipitation) in all cases of tumours of neuroectodermal origin such as glioblastoma, astrocytoma, oligodendroglioma, ependymoma, neurinoma, and spongioblastoma. No immunoelectrophoretic precipitation was seen for any of the tumours of mesenchymal origin, for instance meningioma and metastases of cancer. On the basis of these findings, immunoelectrophoresis is considered to be a reliable method for differentiation between tumour tissue of neuroectodermal and non-neuroectodermal origin. Among the 41 posterior fossa tumours some unusual observations were made. Cerebellar angioblastoma (Lindau tumour) showed an atypically located precipitation line, which for the present is interpreted as an immunological reaction to vascular wall tissue. Furthermore, among the group of so-called medulloblastomas, two subgroups were distinguished on the basis of three parameters. The first of these subgroups comprises tumours whose immunoelectrophoretic pattern resembles that of gliomas, which are histologically characterized by neuroectodermal structures and which occur in younger children (5--10 years). The tumours of the second subgroup, which do not show this neuroectodermal immunoelectrophoretic pattern, have a sarcomatous character histologically, and occur in patients aged between 10 and 50 years. The view that medulloblastoma comprises a number of different types of tumour seems to be confirmed by this finding.

Animals↗
PubMed1983

Demonstration of 125I-labelled thrombin binding platelet proteins by use of crossed immunoelectrophoresis and autoradiography.

A possible receptor for thrombin on the platelet membrane has been identified. Whole platelets were treated with 125I-labelled thrombin followed by washing of the platelets, solubilization in Triton X-100, crossed immunoelectrophoresis and autoradiography. A heavily labelled antigen which migrated slightly more slowly than albumin was observed. No corresponding arc was seen on the same immunoplate when stained with Coomassie brilliant blue, indicating that the antigen possessed weak antigenic properties and/or was present in very small amounts. When 125I-labelled thrombin that had been inactivated by phenylmethylsulphonyl fluoride was used, no such labelled arc was seen. The radiolabelled immunoprecipitate does not represent any of the antigens identified hitherto in the immunoelectrophoretic patterns obtained with platelets or platelet material. The electrophoretic mobility of the antigen was influenced neither by neuraminidase treatment of the platelets prior to the 125I-labelled thrombin exposure nor by inclusion of concanavalin A, wheat-germ lectin or lentil lectin in the gel during the first-dimension electrophoresis. This suggests that the antigen does not represent a glycoprotein. Upon subcellular fractionation the radioactively labelled arc was observed in the cytosol fraction following crossed immunoelectrophoresis and autoradiography. Analysis of the secreted proteins after induction of the release reaction with 125I-labelled thrombin revealed labelling of immunoprecipitates representing thrombospondin, albumin and the 'line' form of platelet factor 4. This confirms that stable complexes of 125I-labelled thrombin and platelet proteins can exist in the presence of Triton X-100 and during electrophoresis.

Animals↗
PubMed1975

Application of pepsin-digested horse antibodies for quantitative immunoelectrophoresis.

The majority of the precipitating antibodies in hyperimmune horse serum belong to the beta globulins, as demonstrated by reversed immunoelectrophoresis. As these antibodies migrate in agarose gel during electrophoresis in conventional pH = 8.6-8.9 buffers, horse antiserum cannot be used satisfactorily for quantitative immunoelectrophoresis. On pepsin digestion of horse antiserum F(ab)' 2 fragments are generated which migrate with the gamma globulins. This cleaved product works well in quantitative immunoelectrophoretic techniques.

Animals↗
PubMed1976

Detection of circulating carcinoembryonic antigen (CEA) by counter-immunoelectrophoresis.

A sensitive method is described for detecting circulating carcinoembryonic antigen (CEA) by combined use of counter-immunoelectrophoresis and intensifiers. Perchloric acid extraction of serum was followed by counter-immunoelectrophoresis against monospecific antiserum to CEA. A precipitin line formed was intensified further by electrophoretic binding of anti-gammaG gamma-globulin and Amido Black staining. Less than 10 ng CEA/ml was detectable. Using this method, a total of 84 sera from patients with or without malignancy was examined. The results obtained were in general agreement with those reported by radioimmunoassay. The method appears to be useful as a diagnostic aid, since it requires neither labeled preparations of purified CEA nor special equipment for assay.

Antigens, Neoplasm↗
PubMed1980

Comparison of immunofixation and immunoelectrophoresis methods in the identification of monoclonal immunoglobulins in serum.

1.A comparison has been made of the relative ability of immunofixation and immunoelectrophoresis to identify monoclonal IgG, IgA and IgM in three sera with high concentration and in three sera with low concentration of these proteins. 2. Immunofixation identified the proteins unambiguously in all six sera examined, whereas immunoelectrophoresis gave ambiguous results in one of the sera with high protein concentration and in three of the sera with low protein concentration. The superiority of immunofixation resulted from its greater resolution. It was condluded that immunofixation is the preferred technique for the identification of monoclonal immunoglobulins. 3. The sensitivity of immunofixation was greater at 40 degrees C than at 10 degrees C or 25 degrees C.

Electrophoresis↗
PubMed1980

Direct analysis of the lectin reactivity of alpha-fetoprotein in maternal serum by crossed affinity radio-immunoelectrophoresis.

Affinity experiments with the lentil (Lens culinaris) lectin have revealed the existence of two distinct molecular populations of alpha-fetoprotein: lectin reactive and lectin non-reactive. Using a combination of crossed lectin immunoelectrophoresis and radio-immunoelectrophoresis, it has been possible to obtain directly the lentil lectin affinity patterns of alpha-fetoprotein present in maternal sera. The lentil lectin reactivity of maternal alpha-fetoprotein decreases almost linearly with the gestational age from week 15 to 35.

Female↗
PubMed1983

Determination of pepsin (EC 3.4.23.1) and gastricsin (EC 3.4.23.3) in gastric juice by rocket immunoelectrophoresis.

Antisera were raised in rabbits against chromatographically purified preparations of pepsin and gastricsin. With these antisera the contents of pepsin and gastricsin in gastric juice were determined by rocket immunoelectrophoresis. The potential content of pepsin and gastricsin of a secondary standard of gastric mucosal extract was calibrated against the chromatographically purified enzymes. This secondary standard was used for routine analyses. The intra-assay and between-assay precision was 3-4% and 6-9%, respectively. Ten healthy volunteers underwent a standard pentagastrin test. The amounts of pepsin and gastricsin determined by rocket immunoelectrophoresis corresponded to the amounts observed by ion exchange chromatography of gastric juice. After stimulation with pentagastrin the secretion of both pepsin and gastricsin was increased about 10 times.

Adult↗
PubMed1985

Accurate quantitation of native Gc in serum and estimation of endogenous Gc: G-actin complexes by rocket immunoelectrophoresis.

Complex formation between purified Gc and G-actin caused increased rocket height on immunoelectrophoresis with monospecific Gc antiserum, and artifactually high calculated Gc levels. The increase in rocket height varied in log: linear fashion with the amount of G-actin present, up to a plateau attained at equimolarity. The raw Gc values could therefore be corrected to within +/- 10% of known levels by addition of excess G-actin and use of standard plots obtained with Gc after saturation with G-actin. This also allowed quantitation of the percentage of Gc complexed with G-actin. In subsequent studies of whole human sera, comparison of normal controls with pregnant subjects and patients with liver disease showed evidence of differences both in absolute quantities of Gc and the relative proportion circulating as complex with G-actin. This appeared to be due to increased release of cellular actin into the extracellular space. These results show that rocket immunoelectrophoresis can be modified to provide accurate Gc levels, and also information concerning different molecular forms of this protein.

Actins↗
PubMed1981

Determination of relative antigen-antibody affinities by quantitative immunoelectrophoresis.

The relative affinities of an antibody population of different antigens or of different antibody populations for one antigen can be determined by quantitative immunoelectrophoresis. Antigen-antibody interactions of greater average affinity result in a greater increase in rocket area as a function of the amount of antigen applied to the wells. This is measured as the slope of the line in plots of rocket area versus antigen amount. Quantitative immunoelectrophoresis of different antigens, which nevertheless have the same affinity for antibody, produces plots with the same slopes. The relative magnitudes of the slopes of these lines reflect the relative average affinities of different antibody populations for an antigen.

Animals↗
PubMed1981

Radial counter-immunoelectrophoresis for rapid sero-diagnosis of typhoid fever.

Serum samples were collected from 24 confirmed cases of typhoid fever and 23 normal healthy controls. Convalescent sera from the patients were obtained, wherever possible, one week after the first sample. In all, 13 paired sera, 11 acute phase only and 23 normal serum samples were tested for ability to elicit precipitins to Salmonella typhi by radial counter-immunoelectrophoresis using cellulose acetate membranes. In addition, conventional counter-immunoelectrophoresis (CIE) was performed using agar-gel layer for comparison. One of 24 acute phase sera gave positive results whereas all 13 convalescent sera were positive by both methods. The radial CIE test may be useful for rapid sero-diagnosis of typhoid fever as it takes only 4 min and can be used to screen large numbers of serum samples from patients suspected of typhoid fever.

Antigens, Bacterial↗