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At least 19 recordsLinked to original sources

PubMed1977

Labeling immunoelectrophoresis: a general method for increasing the sensitivity of rocket immunoelectrophoresis with 125I-labeled anti-antibodies.

In a new procedure, rocket immunoelectrophoresis is performed at pH 8.7 with rabbit antibodies and 5% polyethyleneglycol 6000 in the agarose gel. After the pH in the gel has been changed to 5 the nonprecipitated immunoglobulins are electrophorsed out of the gel simultaneously with the electrophoresis of 125I-labeled swine antibodies against rabbit IgG into the gel. The latter antibodies tag the rabbit IgG, which is not present only in the precipitates. The radioactive precipitates are visualized by autoradiography. The method permits quantification of antigens down to an amount of approximately 0.5 ng; well-defined rockets are not formed below this limit. Compared to conventional protein staining with Coomassie brilliant blue, this represents an increase in sensitivity of up to 20 times.

Antibodies, Anti-Idiotypic↗

PubMed1979

Serologic studies of Streptococcus intermedius, Streptococcus constellatus, and Streptococcus morbillorum by crossed immunoelectrophoresis.

A reference antigen-antibody system for Streptococcus intermedius, Streptococcus constellatus, and Streptococcus morbillorum was established with crossed immunoelectrophoresis. A comparison of S. intermedius, S. constellatus, and S. morbillorum with crossed immunoelectrophoresis and crossed immunoelectrophoresis with intermediate gel indicated that S. intermedius and S. constellatus are closely related antigenically with as many as six common cytoplasmic antigens. S. morbillorum was antigenically more distinct; antiserum of one strain of S. morbillorum was monospecific, indicating that specific serogroups of S. morbillorum exist. Crossed immunoelectrophoresis and tandem crossed immunoelectrophoresis revealed that S. intermedius, S. constellatus, and S. morbillorum also share some common antigens with Streptococcus sanguis and Streptococcus mitis, but S. intermedius, S. constellatus, and S. morbillorum are antigenically distinct from Streptococcus mutans and Streptococcus bovis.

Antibodies, Bacterial↗

PubMed1979

Analysis of antigens in a commercial house-dust extract by means of quantitative immunoelectrophoresis.

Crossed immunoelectrophoresis (CIE) of a commercial house-dust extract revealed 43 immunoprecipitates. Rocket immunoelectrophoresis with 106 different allergen extracts from animal hair and dander, feathers, fungi, pollen, foodstuffs, textiles and house-dust mites showed the presence of precipitating antibodies in the rabbit anti-house-dust serum against antigens from 34 of the extracts. Crossed immunoelectrophoresis with the addition of antigen, and crossed line immunoelectrophoresis (CLIE) with these 34 allergen extracts, as well as crossed immunoelectrophoresis with intermediate gel containing rabbit antibodies against nine human serum proteins, allowed the identification of 14 antigens in the house-dust extract. These antigens were shown to originate from house-dust mites, hair and dander from various animals, fungi, feathers, human hair and dander, and human serum proteins.

Allergens↗

PubMed1975

Quantitative immunoelectrophoresis of proteins in human erythrocyte membranes. Analysis of protein bands obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

1. We have defined conditions that permit quantitative immunoelectrophoresis in agarose gels of dodecyl sulfate-solubilized erythrocyte membrane proteins. 2. Using human serum albumin, transferrin, MN-glycoprotein (glycophorin) and crude spectrin as test proteins, we found that accurate analyses are possible if samples and gels are 1% in non-ionic detergent (Berol EMU-043) or Triton X-100) and if no more than 100 nmol free dodecyl sulfate is applied per sample. 3. Dodecyl sulfate treated membranes analyzed by crossed immunoelectrophoresis using rabbit antibodies against membrane material yielded optimal precipitation patterns in gels containing 1% of non-ionic detergent. 4. Crossed immunoelectrophoresis in the presence of 1% of Berol revealed precipitates when 10 protein bands defined and isolated by preparative dodecyl sulfate-polyacrylamide gel electrophoresis were run against anti-membrane antibodies. Seven of these bands showed more than one precipitation arc, indicating the presence of more than one antigenic component. 5. Crossed-line immunoelectrophoresis showed that dodecyl sulfate-polyacrylamide gel electrophoresis bands 1, 2 and 2.1 shared common antigenic components. The MN-glycoprotein was present in bands 3, 4A, 4B and 5, where antigenic components of the major intrinsic erythrocyte membrane protein, band 3, were also found. 6. After absorption of the anti-membrane antibody with intact erythrocytes, immunoelectrophoresis showed the disappearance of the MN-glycoprotein precipitates. An increase in the area below the precipitate corresponding to the major intrinsic protein (band 3) was also observed, indicating exposure of some antigens of this protein on the outer surface of intact cells. 7. After absorption of the antibody preparation with washed erythrocyte membranes, immunoprecipitates were not seen in any experiments, indicating that all antigenic determinants observed are exposed at one or both surfaces of the membrane. 8. Our analyses indicate that the peptide moieties of serum lipoproteins do not constitute a significant component of erythrocyte membranes.

Antibody Specificity↗

PubMed1979

Characterization of human platelet proteins solubilized with Triton X-100 and examined by crossed immunoelectrophoresis. Reference patterns of extracts from whole platelets and isolated membranes.

Whole human platelets and platelet membranes have been solubilized in 1% Triton X-100, and the solubilized proteins examined by crossed immunoelectrophoresis using rabbit antibodies raised against either whole platelets or isolated membranes. 90% of the platelet proteins were solubilized by this extraction. About twenty immunoprecipitates were observed using the extracts obtained from whole platelets, whereas normally eight immunoprecipitates were seen with extracts from isolated membranes. Albumin, factor VIII and fibrinogen were identified with monospecific antibodies. Correlation of the patterns obtained for platelets or membranes was obtained by addition experiments, by crossed-line immunoelectrophoresis and by crossed immunoelectrophoresis of a mixture of extracts from unlabeled whole platelets and membranes isolated from platelets labeled by lactoperoxidase-catalyzed 125I iodination. Four sialoglycoproteins were identified by their reduced electrophoretic migration after neuraminidase treatment, and six proteins interacted with various lectins, indicating them to be glycosylated. Seven amphiphilic proteins were identified by charge-shift crossed immunoelectrophoresis, and nine by crossed hydrophobic interaction immunoelectrophoresis with phenyl-Sepharose. The topographical arrangement of the membrane proteins was examined with lactoperoxidase-catalyzed 125I-labeled platelets as antigens, and by antibodies absorbed with a suspension of whole platelets. Four and six radioactively labeled precipitates could be identified using the platelet and membrane extracts, respectively, indicating them to be exposed at the outer platelet surface. This was confirmed by the use of antibodies absorbed with intact platelets.

Blood Platelets↗

PubMed1978

Demonstration of K88ac and K88ab antigens of Escherichia coli by means of immunoelectrophoresis and immunodiffusion.

Five strains of Escherichia coli were tested for the presence of the K88ac or K88ab antigens by immunoelectrophoresis and immunodiffusion. The K88ac antigen of 0A2 and Sojka Abbotstown gave an anodic line in the immunoelectrophoresis test and a line in immunodiffusion with homologous K88ac antisera. The K88ab antigens of 0G7, 0E68, and Moon 263 also gave anodic lines in immunoelectrophoresis, and were detectable by immunodiffusions. The 0 groups of these strains were also demonstrated by immunoelectrophoresis and immunodiffusion with homologous 0 antisera. Lack of complete inactivation at 100 degrees C of both the K88ac and K88ab antigens was noted in this study.

Antigens, Bacterial↗

PubMed1977

Membrane asymmetry and expression of cell surface antigens of Micrococcus lysodeikticus established by crossed immunoelectrophoresis.

Crossed immunoelectrophoresis of Triton X-100-solubilized plasma membranes of Micrococcus lysodeikticus established the presence of 27 discrete antigens. Individual antigens were identified as membrane components possessing enzyme activity by zymogram staining procedures and by reactivity of certain antigens with a selection of four lectins in the crossed-immunoelectrophoresis (immunoaffinoelectrophoresis) system. Absorption experiments with intact, stable protoplasts and isolated membranes established the asymmetric nature of the M. lysodeikticus plasma membranes. Of the 14 antigens with determinants accessible solely on the cytoplasmic face of the membrane, four possessed individual dehydrogenase activities, and a fifth was identifiable as a component possessing adenosine triphosphatase (EC 3.6.1.3) activity. Evidence from absorption studies with isolated membranes suggested that antigens such as the adenosine triphosphatase complex were more readily accessible to reaction with antibodies than was succinate dehydrogenase (EC 1.3.99.1), for example. Twelve antigens were located on the protoplast surface as determined by antibody absorption, and the succinylated lipomannan was identified as a major antigen. At least five other antigens possessed sugar residues that interacted with concanavalin A. With the antisera generated to isolated membranes, there was no evidence suggesting that any of these antigens was not detectable on either surface of the plasma membrane. From absorption experiments with washed, whole cells of M. lysodeikticus, it was concluded that the immunogens on the protoplast surface were also detectable on the surface of the intact cell. However, some of the components such as the succinylated lipomannan appeared to be exposed to a greater extent than others. The cytoplasmic fraction from M. lysodeikticus was used as an antigen source to generate antibodies, and 97 immunoprecipitates were resolvable by crossed immunoelectrophoresis. In the cytoplasm-anticytoplasm reference immunoelectrophoresis pattern of precipitates, three of the immunoprecipitates unique to the cytoplasmic fraction were identifiable by zymogram staining procedures as catalase (EC 1.11.1.6), isocitrate dehydrogenase (EC 1.1.1.42), and polynucleotide phosphorylase (EC 2.3.7.8). The identification of membrane and cytoplasmic antigens (including the above-mentioned enzymes) provides a sensitive analytical system for monitoring cross-contamination and antigen distribution in cellular fractions.

Adenosine Triphosphatases↗

PubMed1979

Methods for serotyping nasopharyngeal isolates of Haemophilus influenzae: slide agglutination, Quellung reaction, countercurrent immunoelectrophoresis, latex agglutination, and antiserum agar.

Nasopharyngeal isolates of H. influenzae were typed by the slide agglutination test, the Quelling reaction, the latex agglutination test, countercurrent immunoelectrophoresis, and the antiserum agar test. These tests gave essentially comparable results, with countercurrent immunoelectrophoresis and latex agglutination being slightly more sensitive. Cross-reactive problems encountered with latex agglutination and the expense of performing countercurrent immunoelectrophoresis or the antiserum agar test made these tests less practical than the slide agglutination test to identify single strains that were already isolated. The Quellung reaction and slide agglutination were the most rapid tests used to type an organism. For mass screening of multiple samples, countercurrent immunoelectrophoresis was the simplest technique. The antiserum agar test was slow but was the best technique to screen nasopharyngeal swab cultures to identify the presence of any encapsulated strains in the mixed flora. Whether any of the above techniques were as sensitive as the immunofluorescence test was not evaluated in this study.

Agar↗

PubMed1978

False spurs in quantitative immunoelectrophoresis.

Antigens which show reactions of identity in double-diffusion tests in gel may cause spur formation in crossed immunoelectrophoretic techniques. To differentiate these misleading spurs from spurs of double-diffusion tests which show the true immunochemical relationship between antigens, we have denoted the former type "false spurs". False spurs are often formed in tandemcrossed immunoelectrophoresis and fused rocket immunoelectrophoresis when antigens with different electrophoretic mobility are compared. On the basis of model experiments, criteria are set up for accepting a spur as a true spur, and procedures are described for avoiding formation of false spurs in quantitative immunoelectrophoresis.

Animals↗

PubMed1979

Serology of Neisseria gonorrhoeae. Demonstration of strain-specific antigens by immunoelectrophoresis, immunofluorescence and co-agglutination techniques.

Among a group of four selected strains of Neisseria gonorrhaeae, common and strain-specific antigens were demonstrated by immunoelectrophoresis, indirect immunofluorescence (IFL) and co-agglutination (COA). In preparations of ultrasonically-disrupted organisms, the strain-specific antigens appeared in crossed-line immunoelectrophoresis (C-LIE) with antigen containing intermediate agar gel as a two-peak precipitin line, one peak close to the antigen well, the other towards the anode. A similar pattern was found for each of the four strains studied. These strain-specific precipitin lines were also identified in rocket-line immunoelectrophoresis (R-LIE) tests, which were found useful for comparative studies. Using whole cells, strain-specific antigens were also demonstrated by COA and IFL tests with the use of cross-absorbed antibodies. The results corresponded to those obtained with C-LIE and R-LIE. Immunosorption experiments indicated identity between the strain-specific antigens shown by COA tests and those demonstrated by C-LIE and R-LIE tests.

Agglutination Tests↗

PubMed1979

Detection of M-components by an easy immunofixation procedure: comparison with agarose gel electrophoresis and classical immunoelectrophoresis.

Commercial anti-gamma-alpha-mu and anti-kappa-lambda (mixture of commercial anti-kappa and anti-lambda) were used for immunofixation after agarose gel electrophoresis of 100 serum samples diluted 1 : 5. This simple screening method detected M-components in 39 sera. M-components were detected in 33 sera by agarose gel electrophoresis, and in 30 sera by classical immunoelectrophoresis. More than one band was detected in 15 sera by the screening method, in 3 sera by agarose gel electrophoresis, and in 2 sera by immunoelectrophoresis. The screening method was superior to the combined use of agarose gel electrophoresis and classical immunoelectrophoresis for detection of M-components, and easier to perform and interpret.

Animals↗

PubMed1978

Countercurrent immunoelectrophoresis in the diagnosis of viral infections of the central nervous system.

Countercurrent immunoelectrophoresis was utilized in the study of 621 specimens of cerebrospinal fluid to determine the correlation of detection of viral antigens with the clinical diagnosis of aseptic meningitis and related viral infections. A panel of viral antisera was immunoelectrophoresed against 119 specimens from patients with suspected viral infections of the central nervous system (group I), 32 from patients with bacterial meningitis (group 2), and 470 from patients with no suspected infection of the nervous system (group 3). One or more precipitin bands were detected in 79% of specimens from group 1, 19% from group 2, and 4% from group 3. Paired acute- and convalescent-phase sera from 32 (78%) of 41 patients with precipitin bands detected by countercurrent immunoelectrophoresis demonstrated a fourfold or greater change in complement-fixing antibodies to the detected antigen. With refinements in antisera, countercurrent immunoelectrophoresis may become useful in the rapid laboratory diagnosis of viral infection of the central nervous system.

Acute Disease↗

PubMed1979

Outer membrane antigens of Neisseria meningitidis group B serotype 2 studied by crossed immunoelectrophoresis.

This study shows that the capsular polysaccharide, protein, and lipopolysaccharide antigens from the outer membrane of Neisseria meningitidis group B serotype 2 may be identified by crossed immunoelectrophoresis. By using this technique, seven precipitates were resolved when outer membrane preparations were reacted against goat anti-whole cell group B type 2 antiserum. Most of these precipitates were identified by comparison with purified reference preparations. Different outer membrane preparations, reflecting different growth conditions, varied in their compositions of lipopolysaccharide, protein, and polysaccharide. Detergent treatment altered the protein and lipopolysaccharide precipitation patterns. In the presence of detergent, the lipopolysaccharide did not precipitate, and the electrophoretic migration of the protein antigens decreased. Crossed immunoelectrophoresis is a useful qualitative method for analysis of the antigenic components of the meningococcal outer membrane. The crossed immunoelectrophoresis with intermediate gel technique is presently being used to measure the human immune response to the different cell surface components.

Antigens, Bacterial↗

PubMed1978

[Immunodiagnostic results of amoebiasis. The value of counter immunoelectrophoresis in comparison to three other immunotests (author's transl)].

63 cases of extraintestinal amoebiasis, 17 cases of intestinal amoebiasis, and 25 selected control persons were examined for serum antibodies to antigen from E. histolytica using the counter-immunoelectrophoresis, the complement fixation test, the indirect haemagglutination test and the latex agglutination test. In addition counter-immunoelectrophoresis was applied to 41 sera from patients with extraintestinal amoebiasis whose other serological results were included in a previous study. Even with the employment of our immunological methods, the test results can be used for diagnostic purposes only when they are in accordance with the clinical picture and the exposure. Complete failures of the immuno-diagnostic tests did not occur in our study. On the other hand, there are persons with no symptoms of amoebiasis who have antibodies in two tests, among them sometimes the counter-immunoelectrophoresis, with levels corresponding to those of intestinal or extra-intestinal amoebiasis.

Amebiasis↗

PubMed1977

Analysis of murine C-type virus structural proteins by rocket and crossed immunoelectrophoresis.

We have examined the structural proteins of Rauscher murine leukemia virus (R-MuLV) by means of rocket immunoelectrophoresis and crossed immunoelectrophoresis, using polyspecific antisera to Tween/ether-disrupted purified R-MuLV. Fifteen different precipitation lines were recognized in virus lysates. Using five reference antisera prepared to purified R-MuLV-structural proteins, the precipitation lines of p 10, p 15, p 30 and gp 69/70 were identified. These techniques, although less sensitive than radioimmunoassay, have several advantages, such as simplicity, direct control of precipitation reactions and possibility of using crude antigen preparations.

Animals↗

PubMed1978

Laurell crossed immunoelectrophoresis and affinity chromatography for the purification of a parasite antigen.

Laurell crossed immunoelectrophoresis (two-dimensional electroimmunodiffusion) was used to prepare minute amounts of purified parasite antigens complexed with their precipitating antibodies obtained from rabbits. These complexes, emuslified in Freund's complete adjuvant, were then used to prime rabbits for selective production of precipitins to the complexed antigens when the animals were later boosted with whole parasite extract. The IgG antibody from the monospecific antiserum recovered was then utilized in affinity chromatography to isolate from the crude parasite antigen large amounts of specific antigen in one step. Thus the combination of preparatory crossed immunoelectrophoresis for immunization using complexed antigen and affinity chromatography with monospecific antibody offers a powerful procedure for the rapid isolation of specific antigens.

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↗

PubMed1975

Subtilopeptidase A isoenzyme system. Interaction with serum components and its importance for quantitative immunoelectrophoresis.

A method was developed which involved electroimmunoassay and crossed immunoelectrophoresis of subtilopeptidase A (EC 3.4.21.14). Initial trials with unfractionated antiserum were not successful and interaction of the enzyme with non-immunoglobulin serum components were shown to be the cause of the failures. Quantitative immunoelectrophoresis was possible when purified immunoglobulins were used. A pH of 6.5 (lower than the usual pH 8.6) was necessary to obtain a proper baseline definition. Subtilopeptidase A was confirmed as a multiple isoenzyme system. Qualitative inter-batch variations were detected. Di-isopropyl phosphorofluoridate inhibition altered the electrophoretic pattern, but no loss of antigenic determinants was observed.

Animals↗