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PubMed1981

Serotyping of Haemophilus influenzae a comparison of co-agglutination with countercurrent--immunoelectrophoresis and slide--agglutination.

30 out of 55 strains of H. influenzae could be assigned to one of the six serotypes A--F by slide agglutination, countercurrent-immunoelectrophoresis or co-agglutination. While slide-agglutination appeared less reliable due to non-specific agglutinations countercurrent-immunoelectrophoresis was of high specificity. No cross reactions were encountered in this test and results were unambiguously to evaluate. Co-agglutination is attractive because of its rapidity and simplicity without any need of special equipment. It was found to be of increased sensitivity in detecting type B capsular antigen when compared with countercurrent-immunoelectrophoresis, the problem of cross-reactions remains to be solved however. The use of co-agglutination for serotyping H. influenzae strains in combination with countercurrent-immunoelectrophoresis can be recommended.

Agglutination Tests↗

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↗

PubMedDate not supplied

Determination of LD-Ig complex with counter immunoelectrophoresis using terylene cellulose acetate membrane as supporting media.

OBJECTIVE: In order to determine abnormal LD isoenzyme bands in serum, we established a method of counter immunoelectrophoresis using terylene cellulose acetate as supporting media. SETTING: Department of Clinical Laboratory, Southwestern Hospital, Chongqing 400038 China. PRACTICE DESCRIPTION: LD isoenzymes were electrophoretically fractionated with a Paragon electrophoretic system. LD anomaly was identified by counter immunoelectrophoresis using terylene cellulose acetate as supporting media. MAIN OUTCOME MEASUREMENTS: Identification of the LD-Ig complex using a simple and practical method. RESULTS: An abnormal LD-4 band and an extra band on the cathodic side of LD-5 were observed in the serum of a post-burn patient and proved to be an LD-IgG complex. CONCLUSION: We have established a useful and efficient protocol that uses both isoenzyme electrophoresis and counter immunoelectrophoresis to identify abnormal isoenzyme bands. The method of counter immunoelectrophoresis we describe, using terylene cellulose acetate as supporting media, is simple and suitable for routine clinical use.

Antigen-Antibody Complex↗

PubMed1989

The use of PhastSystem crossed immunoelectrophoresis with immunoblotting to demonstrate a complex between glycoprotein Ib and the actin-binding protein (ABP) of human platelets.

The study shows how a technique described in an accompanying paper can be applied to solve a biological problem. The technique makes use of the observation that a monoclonal antibody that has been coprecipitated with its antigen during crossed immunoelectrophoresis can be transferred to a nitrocellulose membrane and visualized. Previous studies using crossed immunoelectrophoresis of Triton X-100 extracts of platelets have indicated that a particular immunoprecipitate (peak III) of the membrane receptor glycoprotein Ib (GP Ib) might contain a complex between the receptor and the actin-binding protein (filamin). When a monoclonal antibody (PM6/317) directed towards the actin-binding protein was added to a platelet extract prior to immunoelectrophoresis and blotting, this was visualized on the blot as a replica of the peak III immunoprecipitate. This demonstrates a colocalization of GP Ib and the actin-binding protein in the precipitate, and thus the existence of a complex between the membrane receptor and the cytoskeletal protein.

Antibodies, Monoclonal↗

PubMed1983

Rocket and crossed immunoelectrophoresis of proteins solubilized with sodium dodecyl sulfate.

A method for the immunoelectrophoretic analysis of both hydrophilic and hydrophobic proteins from whole-cell extracts solubilized with 2% (w/v) sodium dodecyl sulfate (SDS) is described. For rocket immunoelectrophoresis, Triton X-100 is added to the sample before electrophoresis to sequester non-protein-bound SDS, and polyethylene glycol (PEG) is added to the antibody gel to enhance precipitin formation. With the optimal ratio of Triton X-100 to PEG, the quantitative determination of 5 ng of protein is possible. The SDS-solubilized sample can also be analyzed by crossed immunoelectrophoresis using SDS-polyacrylamide gels in the first dimension and antibody-containing agarose gels in the second. The best results are obtained when intermediate gels without nonionic detergents are used and when ionic detergents are omitted from the cathodal gel. Precipitin peaks of high quality, reproducibility, and without artifacts are obtained using antibody concentrations 5- to 50-fold lower than with other crossed-immunoelectrophoresis procedures.

Chlamydomonas↗

PubMed1985

'Crossed immunoblotting': identification of proteins after crossed immunoelectrophoresis and electrotransfer to nitrocellulose membranes.

Human serum proteins or membrane proteins from human erythrocytes were separated by crossed immunoelectrophoresis in agarose gels under non-denaturating and non-reducing conditions and precipitated by polyspecific antibodies against the respective protein mixtures. The separated proteins were subsequently transferred electrophoretically to nitrocellulose sheets and incubated with mouse monoclonal antibodies against individual protein species. Visualization of the complexes of antigen and monoclonal antibody on the nitrocellulose sheets was performed with peroxidase-conjugated second antibodies. The additional electroblotting step clearly improved the analytical resolution possibilities of crossed immunoelectrophoresis: crossed immunoblotting allows a straightforward identification of individual proteins in complex crossed immunoelectrophoresis patterns. At the same time separation and characterization of proteins is performed under non-denaturing conditions, which is not possible with blotting techniques based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Adult↗

PubMed1993

Immunoelectrophoresis and ELISA techniques for assay of plasma beta 2 glycoprotein-1 and the influence of plasma lipids.

Beta 2 glycoprotein-1 (beta 2GP1) has been identified as a cofactor for the binding of some antiphospholipid antibodies to anionic phospholipids and has been demonstrated to possess anticoagulant properties in vitro. We have investigated Laurell rocket immunoelectrophoresis (IEP) and ELISA techniques for measurement of beta 2GP1. Western blotting and crossed immunoelectrophoresis (CIE) of plasma demonstrated free beta 2GP1 and beta 2GP1 complexed with unidentified plasma constituents. The free and complexed forms were not distinguished in immunoelectrophoresis assays, allowing measurement of total beta 2GP1. Standard and detergent modified IEP and ELISA techniques were compared: significant correlation was demonstrated between unmodified and detergent modified IEP, detergent modified IEP and ELISA and unmodified IEP and ELISA. The intra-assay co-efficients of variation (CVs) of the unmodified and modified IEPs and ELISA were 7.5, 3.7 and 7.9%. Inter-assay CVs determined for the modified IEP and ELISA were 5.8 and 9.1% respectively. The purified beta 2GP1 used to standardise the assays was shown to have subfraction selectivity and different calibration values were obtained for pooled normal plasma by the unmodified IEP (242 mg/l) and modified IEP and ELISA (201 mg/l). We have also investigated the influence of some pre-test variables on beta 2GP1 levels and shown that heparin, citrate or EDTA plasma and serum samples are suitable for assay of this glycoprotein and that levels are unaffected by repeated freeze/thawing. The influence of plasma lipids on beta 2GP1 measurement was also examined and we demonstrated no significant differences between pre and postprandial samples, which suggests that fasting status is not an important consideration for assay of beta 2GP1 in healthy subjects.

Apolipoproteins↗

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↗

PubMed1982

Crossed Immunoelectrophoresis analysis of bee venom. Enzymatic identification of antigens.

Bee venom obtained by electrical stimulation has been analysed by crossed immunoelectrophoresis against high-titer rabbit antibodies. The antigenic analysis of bee venom revealed that the extract contained 17 antigens, which were detected in the crossed immunoelectrophoresis pattern after staining with Coomassie Brilliant Blue. The crossed immunoelectrophoresis pattern indicated heterogeneity in several of the antigens, as most of the precipitates (exhibiting enzyme and hemolytic activities) represented multiple forms of phospholipase A, hyaluronidase, acid phosphatase and melittin.

Acid Phosphatase↗

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↗

PubMed1985

Serum alpha-fetoprotein subfractions in pregnant women identified by the modified method of lectin affinity crossed-line immunoelectrophoresis.

Using a modified method of lectin affinity crossed-line immunoelectrophoresis, we studied the alpha-fetoprotein (AFP) subfractions in sera from 12 pregnant Japanese women, at 22 and 42 weeks of gestation. The method involves the first dimension electrophoresis in agarose gel containing concanavalin A (Con A) or lentil lectin (LCH), the second dimension immunoelectrophoresis in agarose gel containing polyclonal antibody against AFP, reaction with peroxidase-conjugated Protein A and staining with 4-methoxy-1-naphthol. We found that type a of Con A or type C of LCH was the only subfraction present in maternal circulation at the second or third trimester. These AFP subfractions were assumed to be of fetal liver-origin. The minimum concentration which yielded an immunoprecipitation peak was approximately 100 ng/ml, being twenty times more sensitive than the conventional lectin affinity crossed-line immunoelectrophoresis.

Antibodies↗

PubMed1994

Studies of serum protein complexes with nickel using crossed immunoelectrophoresis.

Crossed immunoelectrophoresis of human serum spiked with nickel in the range 0.85-24 mmol Ni/L was used to study nickel-protein complexes. These high concentrations, which are far higher than the physiological level (approximately 7.8 nmol/L), were used to saturate both high and low affinity binding sites. Addition of increasing amounts of nickel resulted in dose-dependent changes of the electrophoretic patterns of prealbumin, alpha-1-lipoprotein, alpha-1-antitrypsin and alpha-2-macroglobulin. Radioactive 63Ni was used for crossed immunoelectrophoresis autoradiography experiments for further identification of nickel-protein complexes. When a 63Ni pulse of 740 kBq/application was used, many human serum proteins were labeled. When using a 63Ni pulse of 185 kBq/application only albumin and alpha-1-antitrypsin were visualized clearly. The binding of large amounts of nickel to albumin, visualized by autoradiography, may reflect the high abundance of albumin in human serum as compared to other serum proteins. Addition of nickel to serum proteins resulted in liquid phase precipitation of serum proteins, and rocket immunoelectrophoresis was used to demonstrate that IgG in particular is precipitated. This precipitation of serum proteins may disturb the elution profile when chromatographic techniques are used to analyze nickel-protein complexes. Consequently, immunoelectrophoretic methods may also be attractive alternatives to column chromatographic techniques. The present study demonstrated that, besides the nickel-binding of albumin and alpha-2-macroglobulin, several other serum proteins have nickel-binding affinity.

Blood Proteins↗

PubMed1995

Detection of biotinylated proteins in crossed immunoelectrophoresis gels: studies on platelet membrane receptors and microparticles.

Biotinylation can be used as an alternative for surface labeling of cell membrane proteins. The use of the water soluble N-hydroxysulfosuccinimide (NHSS)-biotin or the more lipophilic N-hydroxysuccinimide (NHS)-biotin reagent has been investigated in the present study labeling two central receptor complexes on the platelet surface, i.e. the glycoprotein (GP) Ib-IX and the GP IIb-IIIa complexes involved in platelet adhesion and aggregation. Lack of labeling of the intracellularly located albumin was used as a negative control. The labeling has been studied using crossed immunoelectrophoresis in the PhastSystem format after extraction of the labeled cells in Triton X-100, and it is shown that, using enzyme-conjugated avidin and chromogenic substrates, the biotinylated proteins can be visualized directly in the dried electrophoresis gel without the need for a transfer to a blotting membrane as is used after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Suitable conditions for biotinylation and for visualization in the crossed immunoelectrophoresis gels are described. Further, surface-biotinylation of platelets was used to observe shedding of microparticles as a consequence of formation of the complement membrane attack complex. For this purpose the formation and composition of the biotinylated microparticles were observed by flow cytometry and crossed immunoelectrophoresis.

Antibodies, Monoclonal↗

PubMed1986

Human inter-alpha trypsin inhibitor and immunologically related inhibitors investigated by quantitative immunoelectrophoresis. I. Method and reference material.

The concentration in serum of inter-alpha trypsin inhibitor (I alpha I) and its immunologically related inhibitor, the prealbumin-like proteinase inhibitor (pA-PI), was investigated by quantitative crossed immunoelectrophoresis (CIE) in 33 healthy persons. The corresponding urinary excretion over a 4 h period of the immunologically related urinary proteinase inhibitor (UPI) was measured by rocket immunoelectrophoresis. The concentration of pA-PI increased significantly (p less than 0.001) with age whereas that of I alpha I and the urinary excretion of UPI were independent of age. There was no significantly difference in the concentration of the inhibitors in serum between the sexes, but females were found to have a lower urinary excretion of UPI than males (p less than 0.05). Furthermore we found no significant variation in the concentration of the inhibitors during the course of the day or from day to day over a 5 day period. Results obtained by rocket immunoelectrophoresis correlated well with enzymatically measured trypsin inhibitory activity.

Adult↗

PubMed1990

Radial immunodiffusion and immunoelectrophoresis compared for identifying autoantibodies to lactate dehydrogenase in human serum.

Variant electrophoretic patterns of lactate dehydrogenase isoenzymes were studied. By radial immunodiffusion and immunoelectrophoresis, immunoglobulin and light chain class of autoantibodies to lactate dehydrogenase were identified in nine sera: seven of these sera demonstrated IgG (5 lambda, 2 kappa) autoantibodies to lactate dehydrogenase, the other two demonstrated IgA (both kappa) autoantibodies to lactate dehydrogenase, the other two demonstrated IgA (both kappa) autoantibodies to lactate dehydrogenase. We conclude that radial immunodiffusion and immunoelectrophoresis are equally effective for identifying auto-antibodies to lactate dehydrogenase in serum. Radial immunodiffusion, however, is easier to perform than immunoelectrophoresis.

Autoantibodies↗

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↗

PubMed1983

Platelet factor XIII is an active enzyme after solubilization and crossed immunoelectrophoresis.

Crossed immunoelectrophoresis of platelets against antiplatelet antibodies has proved to be a valuable tool in the study of platelet proteins (1-8). The advantage of this separation system is that the proteins are separated under nondenaturating conditions and thus to some extent would be expected to maintain their functional properties. Previously, the binding of several proteins to immobilized thrombin (5) and immobilized heparin (9) during crossed immunoelectrophoresis of platelet proteins solubilized in a Triton X-loo-containing buffer has been described. Furthermore, it has been demonstrated that fibrinogen is able to bind to immunoprecipitates containing the glycoprotein IIb-IIIa-complex (7). These studies indicate that the proteins contained in the immunoprecipitates represent biologically active entities. In the present study we provide direct evidence for this by demonstrating enzymatic activity associated with the immunoprecipitate containing Factor XIII in immunoplates obtained after crossed immunoelectrophoresis of solubilized platelets against anti-platelet antibodies.

Blood Platelets↗

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↗