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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↗

PubMed1980

Candida albicans group A-specific soluble antigens demonstrated by quantitative immunoelectrophoresis.

Soluble cytoplasmic extracts of Candida albicans groups A and B were prepared and compared by quantitative immunoelectrophoresis experiments performed with a commercial anti-C. albicans group A immune serum. Although crossed immunoelectrophoresis, tandem crossed immunoelectrophoresis, and line immunoelectrophoresis revealed many cross-reactions between the two groups, some components seemed to be specific to group A. However, the complexity of the extracts studied did not allow us to demonstrate specific constituents with these methods. Crossed-line immunoelectrophoresis with and without absorption of antibodies in situ was then used, and four specific antigens unique to group A cytoplasmic extract were demonstrated, one of which appeared to be quantitatively important. The value of various quantitative immunoelectrophoretic methods applied to complex antigenic preparations is discussed.

Absorption↗

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↗

PubMed1981

Partial purification and characterization of the major species-specific protein antigens of Rickettsia typhi and Rickettsia prowazekii identified by rocket immunoelectrophoresis.

Species-specific antigens from Rickettsia typhi and Rickettsia prowazekii were readily solubilized by French pressure cell extraction or sonication of Renografin density gradient-purified rickettsiae and were identified by rocket immunoelectrophoresis. As measured by quantitative rocket immunoelectrophoresis, the species-specific typhus rocket antigens (STRAs) appeared to be proteins; they were denatured by heating at 56 degrees C for 30 min but not by 50 degrees C treatment, and they were sensitive to pronase and trypsin but were not affected by periodate oxidation, glycosidases of various specificities, phospholipase A, or lipase. STRAs from both R. typhi and R. prowazekii were separated from common antigens by DE52 column chromatography of 100,000-X-g supernatant fractions of rickettsial extracts. The purified STRAs were characterized by crossed immunoelectrophoresis, by polyacrylamide gel electrophoresis on Davis and sodium dodecyl sulfate gels, and by an enzyme-linked immunosorbent assay. The two purified STRAs were proteins with similar native electrophoretic mobilities in agarose and polyacrylamide gels, and these proteins had similar polypeptide patterns on sodium dodecyl sulfate gels. Most of the STRA activity migrated as a single protein band on sodium dodecyl sulfate-polyacrylamide and Davis polyacrylamide gels, although minor protein bands with STRA activity were also detected. The major STRA proteins constituted 10 to 15% of the total cellular protein of R. typhi and R. prowazekii. According to sensitive enzyme-linked immunosorbent assay titrations, the STRA of R. prowazekii had substantial cross-reactivity with rabbit antiserum prepared against R. typhi, as shown also by rocket immunoelectrophoresis, whereas the STRA of R. typhi reacted only very weakly with antiserum prepared against R. prowazekii according to the enzyme-linked immunosorbent assay and not at all according to rocket immunoelectrophoresis.

Antigens, Bacterial↗

PubMed1986

Catalases, peroxidases, and superoxide dismutases in Mycobacterium leprae and other mycobacteria studied by crossed immunoelectrophoresis and polyacrylamide gel electrophoresis.

The five mycobacteria Mycobacterium lepraemurium, M. leprae, M. bovis BCG, M. smegmatis, and M. intracellulare were studied. Catalase and peroxidase activities were demonstrated in polyacrylamide and crossed immunoelectrophoresis gels for M. lepraemurium, M. intracellulare, and BCG, but not for M. leprae. Peroxidase and catalase activities were associated with the same precipitate line in crossed immunoelectrophoresis for M. lepraemurium, M. intracellulare, and BCG, showing that in these mycobacteria the two enzyme activities resided in the same molecule. M. smegmatis peroxidase and catalase activities were closely associated on polyacrylamide gel electrophoresis, but on the crossed immunoelectrophoresis catalase and peroxidase activities were associated with two different precipitate lines. Catalases without peroxidase activity were demonstrated in crossed immunoelectrophoresis and polyacrylamide gel electrophoresis in M. intracellulare and M. smegmatis. The catalase without peroxidase activity in M. intracellulare was heat resistant and therefore classified as an m-catalase. In M. smegmatis the catalase without peroxidase activity was only partially heat resistant. All of the catalases with peroxidase activity were heat-sensitive t-catalases. Superoxide dismutase activity in the crossed immunoelectrophoresis was associated with the M. leprae antigen no. 4 and with cross-reacting antigens in the other mycobacteria studied. Several superoxide dismutases were demonstrated in Mycobacterium duvalii. They were antigenically different from the other superoxide dismutases in this study, as shown by lack of reactivity with a monospecific antibody to M. lepraemurium superoxide dismutase. Molecular weights were estimated for all the enzymes in this study by sodium dodecyl sulfate-polyacrylamide gels.

Catalase↗

PubMed1983

Comparison of immunodiffusion and crossed-immunoelectrophoresis in the diagnosis of invasive candidiasis.

A retrospective single blind study was conducted to compare the efficacy of crossed-immunoelectrophoresis using concanavalin A intermediate gel with double immunodiffusion tests in the diagnosis of invasive candidiasis. On the basis of cytoplasmic antibody detection, crossed-immunoelectrophoresis with concanavalin A differentiated reliably between Candida albicans fungemia and invasive candida infection, whereas double immunodiffusion did not. With regard to sensitivity, specificity, and predictive value crossed-immunoelectrophoresis with concanavalin A was superior to double immunodiffusion. Analysis of double immunodiffusion precipitin bands revealed fuzzy and sharp precipitin bands which corresponded to mannan (fuzzy) and cytoplasmic (sharp) antibodies in the crossed-immunoelectrophoresis with concanavalin A assay. Therefore, the finding of sharp bands in the double immunodiffusion procedure supports the diagnosis of invasive candidiasis.

Adolescent↗

PubMed1980

Rapid detection of rinderpest virus antigens by counter-immunoelectrophoresis.

Counter-immunoelectrophoresis was compared with immunodiffusion for its ability to detect rinderpest virus antigens. Counter-immunoelectrophoresis detected antigens in lymph node biopsies from all of 9 infected cattle whereas immunodiffusion detected them in 8 only. Counter-immunoelectrophoresis was between 4 and 16 times more sensitive than immunodiffusion for detecting rinderpest virus antigens in the tissues of diseased animals and could detect positive reactions within 40 min. Counter-immunoelectrophoresis could offer an opportunity for rapid laboratory confirmation of rinderpest.

Animals↗

PubMed1983

Quantification of human serum lipoprotein Lp(a): zone immunoelectrophoresis assay, a new sensitive method as compared to electroimmuno assay.

This study aimed at improving the immunological procedures for the quantification of human serum lipoprotein Lp(a). Lipoprotein Lp(a) was estimated by two immunoelectrophoretic methods: electroimmunoassay and zone immunoelectrophoresis assay. Whereas the electroimmunoassay gave curvilinear calibration lines (in peak height versus concentration plots) the zone immunoelectrophoresis assay revealed linearity in the same concentration range. The results obtained are in good agreement with each other (r = 0.975). The inter-assay coefficient of variation for the zone immunoelectrophoresis assay was 12% as determined by the use of a lyophilised reference serum. Zone immunoelectrophoresis assay is recommended for the routine quantification of lipoprotein Lp(a). Although the levels for total cholesterol (p less than 0.0025), HDL-cholesterol (p less than 0.001) and triglycerides (p less than 0.025) differed significantly between male (n = 24) and female (n = 22) individuals, no difference could be detected comparing the frequency distributions of Lp(a)-levels found for the male and the female group.

Adolescent↗

PubMed1996

A retrospective study of the relative utility of electrophoresis, immunoelectrophoresis, immunofixation, and nephelometry in the investigation of serum proteins.

OBJECTIVES: To assess the relative value of visual inspection of protein electrophoresis patterns, immunoelectrophoresis, immunofixation, and specific protein quantitation in the investigation of serum proteins. DESIGN AND METHODS: A retrospective study of protein results on 372 sera analyzed by electrophoresis and immunoelectrophoresis. A subsection of specimens (276) also had immunoglobulin quantitation by immunonephelometry and 30 were studied by immunofixation. RESULTS: Total gamma globulin levels were correctly assessed visually in 84.4% of cases. Discrete bands seen visually were mainly monoclonal in 74 of 80 (92.5%). Six monoclonal proteins were not detected visually. Twenty-five monoclonal proteins required immunofixation for typing, and 60 were typed by immunoelectrophoresis. The visual assessment of the level of normal immunoglobulins in the presence of a monoclonal gammopathy was correct in 58% of cases. CONCLUSIONS: The majority of monoclonal gammopathies could be evaluated by a combination of protein electrophoresis and immunoelectrophoresis. Except for quantitating monoclonal immunoglobins, protein electrophoresis was unreliable as a quantitative procedure and nephelometry was preferred.

Antibodies, Monoclonal↗

PubMed1992

Purification of lysosomal arylsulfatase B from rat liver and its reactivity with lectins in affinity immunoelectrophoresis.

1. Arylsulfatase B (ASB) from lysosomal fraction of rat liver were isolated and purified 260-fold with a recovery of about 5%. 2. The enzyme in gradient PAGE 4-30% followed by immunoelectrophoresis migrated as a single peak of M(r) 84,000. The pI, measured by isoelectrofocusing in agarose followed by immunoelectrophoresis, was equal to 6.7. 3. ASB reacted with Con A, LCA, PSA, LTL, WGA, RCAI and did not react with PHA, SBA, HPA, CAA and PAL in crossed affino-immunoelectrophoresis or rocket immunoelectrophoresis. These results permit of preliminary elucidation of ASB glycan structure.

Animals↗

PubMed2005

Cross-reactivity among edible nuts: double immunodiffusion, crossed immunoelectrophoresis, and human specific igE serologic surveys.

BACKGROUND: As many as one third of all food allergen anaphylactic events are related to tree nut ingestion. Although concurrent allergen sensitivity to tree nuts is common, cross-reactivity among nut antigens is less well defined. OBJECTIVE: To survey serologic cross-reactivities among 7 tree nuts (walnut, pecan, hazelnut, cashew, Brazil nut, pistachio, and almond) and peanut. METHODS: Human specific IgE enzyme-linked immunosorbent assay inhibition was used to identify nut allergen cross-reactivities. Single-nut rabbit antisera were used in double immunodiffusion, crossed-line immunoelectrophoresis, and crossed immunoelectrophoresis with intermediate gel studies of nut antigen cross-reactivity. RESULTS: Nut specific IgE enzyme-linked immunosorbent assay inhibition demonstrated no cross-reactivities between peanut and tree nuts. Among tree nuts, 2 groups with allergen cross-reactivity were defined: (1) walnut, pecan, and hazelnut and (2) hazelnut, cashew, Brazil nut, pistachio, and almond. Double immunodiffusion, crossed-line immunoelectrophoresis, and crossed immunoelectrophoresis with intermediate gel results supported the same groupings of cross-reactive tree nuts and identified several less prominent nut-nut antigen cross-reactivities between groups and with peanut. CONCLUSION: With few exceptions (notably limited peanut cross-reactivity with pistachio and walnut), peanut antigens did not serologically cross-react with tree nuts. Walnut, pecan, and hazelnut form a group of strongly cross-reactive tree nuts. Hazelnut, cashew, Brazil nut, pistachio, and almond form a group of moderately cross-reactive tree nuts. Cross-reactivities between these groups are less pronounced (notably limited cross-reactivity of walnut and pecan with Brazil nut). The strongest cross-reactivities among tree nuts follow botanical family associations: (1) walnut and pecan in the family Juglandaceae and (2) cashew and pistachio in the family Anacardiaceae.

Cross Reactions↗

PubMedDate not supplied

Specific antibody detection in human aspergillosis: a GEMO* multicentre evaluation of a rapid immunoelectrophoresis method (Paragon). Group d'Etude des Mycoses Opportunistes.

A new immunoelectrophoresis system, the Paragon system, was evaluated in three different hospital centres with the aim of improving standardization of the serodiagnosis of human aspergillosis. To select the most efficient antigen, various commercial and home-made antigens were first tested on 19 sera from 19 patients with highly probable aspergillosis. The value measured using the Paragon anti-Aspergillus antibody detection system was then compared with the results obtained by conventional serological diagnostic methods (conventional immunoelectrophoresis, enzyme linked-immunosorbent assay, indirect immunofluorescence): this step was performed using the first 19 sera as well as 16 other sera from 13 patients with suspected aspergillosis. Concordant results were obtained in 28 cases. The discrepancies observed with seven sera were probably related to differences in the nature of the antigens. Paragon immunoelectrophoresis proved to be a practicable technique requiring only a small amount of serum and giving results within a shorter time than competitive methods (24-48 h). Its major drawbacks compared with conventional immunoelectrophoresis are some difficulties in reading, fewer precipitin lines and the relatively high cost of routine analysis.

Antibodies, Fungal↗

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↗

PubMed1985

Immunochemical characterization of antigens of Parietaria judaica pollen. Identification of allergens by means of crossed radio immunoelectrophoresis.

A crude extract of Parietaria judaica pollen was obtained by means of extraction, centrifugation and dialysis, and studied by means of quantitative immunoelectrophoresis. Crossed immunoelectrophoresis, using a high-titer purified rabbit antibody fraction, showed that the pollen extract contained at least 26 antigens of which 18 moved towards the anode, 6 moved towards the cathode and 1 moved both towards the anode and the cathode. The allergens in the extract were identified by means of crossed radio immunoelectrophoresis. Nine of the 26 antigens were able to bind specific human IgE to their corresponding immunoprecipitates, and 4 of these antigens can be classified as major allergens. The apparent molecular weights of 17 antigens were determined by a combination of size exclusion chromatography and immunochemical analysis. Ten antigens had Kav values corresponding to molecular weights in the range of 10-40 kilodaltons, 1 antigen possessed an apparent molecular weight of less than 10 kilodaltons, and six antigens had molecular weights above 40 kilodaltons. Most of the antigens, as analysed by column isoelectric focusing, had pI values between 4 and 7. Crossed line immunoelectrophoresis using an extract of Parietaria officinalis shows that the antigens of this extract exhibit a high degree of identity with those of P. judaica.

Adult↗

PubMed1983

Demonstration and partial characterization of chronic lymphocytic leukemia lymphocyte-associated antigens by crossed immunoelectrophoresis of Triton X-100 extracts.

Crossed immunoelectrophoresis was used to study the antigens of chronic lymphocytic leukemia lymphocytes. A reference pattern was obtained and 20 samples from 18 patients were compared with this pattern. Extensive variation in the expression of individual antigens was observed. The antigens in the reference pattern were further characterized by modifications of crossed immunoelectrophoresis, viz. labelling of intact lymphocytes by lactoperoxidase-catalyzed iodination, affinity crossed immunoelectrophoresis with phenyl-Sepharose and Lentil lectin-Sepharose, charge-shift crossed immunoelectrophoresis and postelectrophoretic incubation in (125I) Lentil lectin. Six antigens were identified as surface membrane glycoproteins, 5 as cytoplasmic proteins while 5 could not be classified. Two antigens were finally identified as HLA-ABC and HLA-DR by application of small amounts of monospecific rabbit antiserum and monoclonal antibody, respectively.

Aged↗