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Serogrouping of halophilic bdellovibrios from chesapeake bay and environs by immunodiffusion and immunoelectrophoresis.

Little has been reported on the serological relationship of halophilic bdellovibrios (Bd). Immunodiffusion analysis performed with rabbit or mouse Bd antisera developed against eight halophilic Bd isolates and one terrestrial Bd isolate, when reacted with soluble antigen preparations of 45 isolates of halophilic Bd, allowed separation into seven serogroups, which were distinct from the terrestrial isolate. Soluble antigen preparations of prey bacteria, Vibrio parahaemolyticus P-5 (P-5) and Escherichia coli ML 35 (ML 35), exhibited no reactivity with the antisera by immunodiffusion. Immunoelectrophoresis revealed the presence of three distinct antigens in homologous reactions and one shared antigen in heterologous Bd reactions. Shared antigens were noted between halophilic and terrestrial Bd, in addition to between halophilic Bd strains, indicating the possible existence of an antigen(s) which may be shared among all Bd. Again, no shared antigen was noted when P-5 or ML 35 was allowed by immunoelectrophoresis to react with the antisera. Prey susceptibility testing of the seven distinct groups of halophilic Bd, using 20 test prey, produced essentially identical spectra for each group, indicating that this was not a useful technique in delineating the Bd. While immunoelectrophoresis was able to demonstrate an antigen common to all Bd tested, immunodiffusion was able to delineate strains on the basis of a "serogroup specific" antigen. This suggests that immunological tools may serve as important means to study the taxonomy of halophilic Bd, as well as in the formation of a clearer taxonomic picture of the genus Bdellovibrio.

Journal Article↗

Identification and localization of enzymes of the fumarate reductase and nitrate respiration systems of escherichia coli by crossed immunoelectrophoresis.

Crossed immunoelectrophoresis was used to analyze the components of membrane vesicles of anaerobically grown Escherichia coli. The number of precipitation lines in the crossed immunoelectrophoresis patterns of membrane vesicles isolated from E. coli grown anaerobically on glucose plus nitrate and on glycerol plus fumarate were 83 and 70, respectively. Zymogram staining techniques were used to identify immunoprecipitates corresponding to nitrate reductase, formate dehydrogenase, fumarate reductase, and glycerol-3-phosphate dehydrogenase in crossed immunoelectrophoresis reference patterns. The identification of fumarate reductase by its succinate oxidizing activity was confirmed with purified enzyme and with mutants lacking or overproducing this enzyme. In addition, precipitation lines were found for hydrogenase, cytochrome oxidase, the membrane-bound ATPase, and the dehydrogenases for succinate, malate, dihydroorotate, D-lactate, 6-phosphogluconate, and NADH. Adsorption experiments with intact and solubilized membrane vesicles showed that fumarate reductase, hydrogenase, glycerol-3-phosphate dehydrogenase, nitrate reductase, and ATPase are located at the inner surface of the cytoplasmic membrane; on the other hand, the results suggest that formate dehydrogenase is a transmembrane protein.

Adenosine Triphosphatases↗

[Immunoelectrophoresis For Anthelmintics Evaluation Against Experimental Paragonimiasis]

Paragonimiasis is one of the most important endemic diseases in Korea. However, recognition of the limitation of microscopic examination of sputum and feces for Paragonimus eggs has led to the investigation of immunoserological techniques for paragonimiasis. The other hand, bithionol preparation has been used as a drug of choice in paragonimiasis but recently niclofolan preparation has introduced as a new agent. By the time, the authors attempted the evaluation of above both agents against experimental rat paragonimiasis by immunoelectrophoresis and Ouchterlony test. The immunoeletrophoresis and Ouchterlony tests were performed according to the method Tsuji and Grabar et Williams respectively, with antigen extracted from lyophilized worm of P. westermani with 0.1 per cemt saline solution. Meanwhile, rats were infected with per os 20 metacercariae of P. westermani above two kinds of serologic tests and were undertaken at biweekly intervals. Then, bithionol was administered every other day for 5 successively for 2 weeks or 6 weeks after infection. Another groups, niclofolan was administered per os single dose same as above. 1. The sera from the rats infected with P. westermani, after treatment with bithionol or niclofolan preparations began to show the precipitin bands against P. westermani antigen 8 weeks after the infection in immunoelectrophoresis and Ouchterlony test, but from 10 or 12 weeks after infection, the number of bands were decreased or disappeared gradually. 2. In genera1, the sera from the rats treated with bithionol or niclofolan showed the precipitin bands delaying 2 weeks than control rat groups. 3. The sera from the rats administered with hydrocortisone showed precipitin bands neither in immunoelectrophoresis nor in Ouchterlony test.

Journal Article↗

[Immunoelectrophoresis for Fasciola hepatica]

In an attempt to investigate the specific antigenic substance of Fasciola hepatica, Ouchterlony tests and immunoelectrophoretic analyses were carried out. Crude Fasciola antigen was prepared and fractionated by Sephadex G-200 column to Antigen I, II and III according to protein content. Crude antigens of Paragonimus westermani, Clonorchis sinensis and Paramphistomum sp. were also prepared for control and absorption study. Antiserum was prepared by injecting 0.5 ml of crude Fasciola antigen with same amount of complete Freund's adjuvant in rabbits, 10 times at an interval of l week. The results obtained in this study were as follows: 1. Crude Fasciola antigen reacted with antiserum with 9 precipitin bands by Ouchterlony test and with 11 bands by immunoelectrophoresis. 2. Cross reaction was observed between Paragonimus, Clonorchis and Paramphistomum antigens and anti-Fasciola rabbit serum respectively. By Ouchterlony test, 3-4 cross reacting bands were found. 3. Anti-Fasciola sera which were absorbed with respective Paragonimus, Clonorchis and Paramphistomum antigens, reacted with Fasciola crude antigen. Ouchterlony test gave 5-6 precipitin bands. Further reaction between Fasciola antigen and antiserum absorbed with the above 3 antigens concomitantly gave 5 precipitin bands by Ouchterlony test and 7 bands by immunoelectrophoretic analyses. 4. Fractionated Fasciola antigens (Antigens I, II & III) reacted with anti-Fasciola rabbit serum in immunoelectrophoresis. Antigen I, II and III gave 2, 3 and 5 precipitin bands respectively. Anti-Fasciola rabbit serum which was absorbed with 3 trematodes antigens gave, by immunoelectrophoresis, 1 band with Antigen I, 2 bands with Antigen II and III of Fasciola hepatica. From the above results, it is concluded that Fasciola hepatica possessed the specific antigenic substance not cross-reacted with other trematodes.

Journal Article↗

Diagnosis of cystic echinococcosis: ultrasound imaging or countercurrent immunoelectrophoresis?

Cystic echinococcosis is a major zoonotic diseases in the Islamic Republic of Iran. This study was carried out in 3 general hospitals in Shiraz. We examined the records of all 1227 surgical patients with a surgically-proven diagnosis of cystic echinococcosis for the 20-year period 1978-98. The results of countercurrent immunoelectrophoresis were compared with pathology and ultrasound reports to determine whether serological tests could be helpful for diagnosis. Countercurrent immunoelectrophoresis could detect only 62.0% of cases, whereas the pathology and ultrasound results were positive for 96.3% of cases. This study confirms the usefulness of ultrasound and suggests that only in doubtful cases would countercurrent immunoelectrophoresis be useful for diagnosing cystic echinococcosis.

Biopsy↗

[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↗

Crossed affino-immunoelectrophoresis or affino-blotting with lectins: advantages and limitations for glycoprotein studies.

In contrast to the conventional combination of physical, chemical and enzymatic methods used for a structural analysis of glycans in glycoproteins, alternative methods involve affinity electrophoresis as a tool for the detection, characterization, and quantitation of glycoproteins and their carbohydrate moiety, owing to interactions with lectins. Two major approaches involve (i) crossed affino-immunoelectrophoresis and variations thereof, whereby lectin/glycoprotein interactions occur during the electrophoretic runs, or (ii) affino-blotting, where the glycoproteins are electrophoretically separated and then immobilized onto a solid support prior to their interaction with lectins. A critical comparison of these two series of techniques is the scope of the present paper. These techniques are of high interest by virtue of their ability at differentiating a classical glycan structure from unusual oligosaccharide side chains. The former structures will usually be qualitatively and quantitatively described with the easy and fast procedures as well as the simple equipment required for crossed affino-immunoelectrophoresis or affino-blotting, whereas the latter will be good candidates for further structural analyses.

Animals↗

Identification of lipoglycan antigens from the Acholeplasma laidlawii cell membrane in crossed immunoelectrophoresis.

Membranes from the wall-less prokaryote Acholeplasma laidlawii contain a component termed lipoglycan or lipopolysaccharide (LPS). The lipoglycan has extraction properties, which are similar to those of LPS of gram-negative bacteria, but it is chemically distinct from bacterial LPS. The membrane-bound lipoglycan of A. laidlawii did not seem to be particularly immunogenic and antibodies against it could not always be detected by rocket immunoelectrophoresis (RIE) or crossed immunoelectrophoresis (CIE) in hyperimmune sera raised against membranes. The immunoprecipitate corresponding to the lipoglycan, obtained by CIE of Tween 20-solubilized A. laidlawii membranes, has been identified and shown to be both a cathodically and anodically migrating component at pH 8.6. The shape of the immunoprecipitate in both RIE and CIE showed that the lipoglycan antigen is composed of at least two components, which are immunologically related.

Acholeplasma laidlawii↗

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↗

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↗

Crossed immunoelectrophoresis of mouse serum.

Because crossed immunoelectrophoresis (X-IEP) is quantitative and offers more information than immunoelectrophoresis (IEP), we selected X-IEP to study various physiologic and pathologic effects on the antigen composition of mouse serum. We therefore needed and X-IEP map of normal mouse serum. This paper presents one that we have developed, along with brief descriptions of how the mouse serum antigens were identified. Cross-reactivity between several mouse and human serum antigens was especially helpful. Some data from using ratios of the areas of precipitation of detected antigens to that of an internal standard antigen, alpha-macroglobulin, are presented. They show that X-IEP readily detects quantitative abnormalities among any of the several serum antigens detected by this technique, and that 'normal' values for these antigens in one population of mice can be established in the same way of using ratios with just a few analyses.

Animals↗

Double-decker rocket immunoelectrophoresis for direct quantitation of complement C3 split products with C3d specificities in plasma.

A double-decker rocket immunoelectrophoresis (DD-RIE) method for direct quantitation of complement split products with C3d determinants in human plasma is described. The usefulness of the DD-RIE method for monitoring C3 activation has been assessed and compared with conventional crossed immunoelectrophoresis (CIE) for C3c determination in a patient with iatrogenic septic shock and patients with rheumatoid arthritis. In contrast with CIE the DD-RIE method is quantitative by reference to a standard curve based on an internal reference C3d preparation and its sensitivity and assay capacity are superior to CIE. All reagents and antibody preparation are commercially available and the production of standards is easy. No overlapping was observed between C3d values in plasma from healthy persons and patients with active classical rheumatoid arthritis. The DD-RIE is highly suitable for routine use in laboratories of clinical immunology.

Complement C3↗

A single step immunoelectrophoresis method for the quantitation of complement C3c in biological fluids.

An intermediate gel rocket immunoelectrophoresis (IRI) suitable for direct quantitation of complement split products with C3c specificity is described. The technique represents a simple, quick, and reproducible method for the assessment of complement activity in biological fluids in diseases. The advantages of the quantitative approach of the IRI method as compared to the conventional crossed immunoelectrophoresis (CIE) are the increased sensitivity, specificity, and precision of the method; also, the assay capacity is superior to CIE. The IRI-C3c assay is therefore highly suitable as a scientific tool, and may also be useful for routine laboratory investigations.

Complement Activation↗

Electroimmunoassay-immunoblotting (EIA-IB) for the utilization of monoclonal antibodies in quantitative immunoelectrophoresis: the method and its applications.

Immunoprecipitates formed in conventional electroimmunoassays were solubilized by brief immersion of the agarose gels in sodium dodecylsulfate. The proteins were then transferred to nitrocellulose sheets by electroblotting. The blotted proteins were readily amenable to analyses by non-precipitating monoclonal antibodies and the immunoblots were developed with second antibody/biotin-streptavidin-peroxidase staining. The electroimmunoassay-immunoblot (EIA-IB) method is of value in (1) specificity assays of monoclonal antibodies in crossed immunoelectrophoresis; (2) analysis of specific molecular interactions between proteins; (3) rapid screening and simple identification of monoclonal antibodies by line immunoelectrophoresis-immunoblotting.

Animals↗

Integration of monoclonal antibodies in quantitative immunoelectrophoresis by indirect immunoprecipitation.

Characterization of the specificity of monoclonal antibody in crossed immunoelectrophoresis has been achieved by mixing the monoclonal antibody with mycobacterial antigen in the circular antigen well. After electrophoresis in the first dimension, the separated antigens and antigen complexed with monoclonal antibody were run into an intermediate gel containing rabbit anti-mouse immunoglobulin and a top gel with polyvalent rabbit anti-BCG immunoglobulin. Monoclonal antibody with bound antigen precipitated in the intermediate gel while the other antigens precipitated in their reference positions. The method was simple, efficient and sensitive. Selected monoclonal antibodies were used to demonstrate the characteristic features of the method. In rocket immunoelectrophoresis the rocket height of a monoclonal antibody may be significantly altered by adding the relevant antigen. This principle can be exploited when the polyvalent antibodies do not precipitate the antigen, and it may be used for efficient screening of monoclonal hybridoma culture supernatants. This approach may permit the quantitation of an antigen when only monoclonal antibody is available.

Animals↗

Preparation of Aspergillus fumigatus antigens and their analysis by two-dimensional immunoelectrophoresis.

The water-soluble components of Aspergillus fumigatus mycelium were partially separated by fractional precipitation with ammonium sulphate. The total protein and neutral-sugar content were determined for each of the four fractions prepared and their immunological activity was examined by double diffusion. Partial chemical characterisation of these isolates by polyacrylamide-gel electrophoresis was linked to their precipitability by allowing the separated components to diffuse from the gel into an agarose medium containing an appropriate antiserum. The distribution and reactivity of antigens was monitored by two-dimensional immunoelectrophoresis with rabbit sera raised against either mycelial or culture-filtrate antigens and human sera, obtained from patients with aspergilloma and from patients with allergic bronchopulmonary aspergillosis. This technique was also used to establish that considerable variation exists in the precipitation profile seen among patient specimens. Several antigens were found to possess sugar residues that interacted with concanavalin A, when this lectin was used in an intermediate gel in two-dimensional immunoelectrophoresis.

Animals↗

Concanavalin A reactivity pattern of human amniotic fluid AFP examined by crossed affino-immunoelectrophoresis. A definite test for neural tube defect?

The percentage of alpha-fetoprotein (AFP) not reacting with concanavalin A (con A) was determined by crossed line affino-immunoelectrophoresis in amniotic fluid from 25 pregnancies with neural tube defects (NTD) and other fetal abnormalities giving rise to elevated AFP levels, and from 128 pregnancies with normal outcome. The percentage of con A non-reactive AFP is significantly lower in the presence of fetal abnormalities (mean 3.4%, range: 0.0-6.3, n = 25), compared to the percentage found in normal pregnancies (mean 17.2%, range: 6.6-35.8%, n = 128). In amniotic fluid samples from normal pregnancies, the percentage of con A non-reactive AFP in 84 cases with total AFP levels lying within the 95% reference interval was not significantly different from the percentage found in 44 cases with levels above the 95% reference interval. The percentage of non-reactive AFP in fetal serum and cerebrospinal fluid was of the same magnitude as found in amniotic fluids of pregnancies with fetal abnormalities. It is concluded that analysis of the percentage of con A non-reactive AFP by crossed line affino-immunoelectrophoresis is a simple, reliable and apparently diagnostic test for NTD and other abnormalities with leakage of fetal serum or cerebrospinal fluid into the amniotic fluid. The test should therefore be used in all cases with only marginally elevated AFP levels, so false positives may be avoided.

Amniotic Fluid↗

Crossed immunoelectrophoresis and electroimmunoassay of human IgG subclasses.

Human IgGl, IgG2, IgG3 and IgG4 in WHO pool 67/97, a normal serum pool, Cohn Fraction II and individual sera were examined by crossed immunoelectrophoresis and electroimmunoassay in agarose with IgG subclass specific rabbit antisera. In these methods the fact that IgG subclasses differ in the electrophoretic field is utilized: IgG4 is located anodically, IgG3 cathodically, and IgG2 and IgG1 both anodically and cathodically. The mean, S.D. and range of serum IgG1, IgG2, IgG3 and IgG4 in 20 normal adults found by the electroimmunoassay were given and related to the amount in the WHO pool 67/97. The IgG subclasses values obtained by electroimmunoassay agreed with the values obtained by single radial diffusion. The reproducibility of double determinations (interplate variations) was 1.5--5.5 per cent. Repeated freezing, thawing and storage of the serum at room temperature did not influence quantitation of IgG subclasses. Cohn Fraction II was found to contain smaller amounts of IgG1, IgG2, and IgG4 than those found in the normal serum pools. Crossed immunoelectrophoresis and electroimmunoassay also easily reveal failing quality of IgG subclass antisera. To obtain good antisera in rabbits against IgG subclasses immunization should be done with several myeloma proteins with different electrophoretic mobility within the same subclass.

Humans↗