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PubMed1982

Quantification of the C3 breakdown product C3d by rocket immunoelectrophoresis.

The levels of the C3 breakdown product C3d were measured in plasma, serum, and synovial fluid by the technique of rocket immunoelectrophoresis. Serum and plasma from 22 healthy donors, 4 patients with rheumatoid arthritis, 1 patient with systemic lupus erythematosus, and 1 patient with cryoglobulinemia were used. The synovial fluid was studied in 3 other patients with rheumatoid arthritis. Normal values for the 22 healthy donors in plasma were within a narrow range of 3.25--5.75% as compared to the standard pool that was prepared. Storage of plasma to -80 degrees C during 7--15 days did not modify the results of the test. Significant differences in C3d values were observed between serum and EDTA plasma samples. Patients with immunological diseases showed higher values of C3d in the samples (from 5 to 30% of activity as compared to the standard pool). Measurement of C3d by rocket immunoelectrophoresis showed to be an easy, satisfactory and reliable procedure able to be incorporated to routine immunological evaluations.

Arthritis, Rheumatoid↗
PubMed1982

Ultrathinfilm immunoelectrophoresis.

Immunoelectrophoresis (Laurell-rocket) in ultrathinfilm gels has been applied to the determination of human serum albumin in a model study. Agarose gels of 0.24 mm thickness were used and optimum electrophoretic conditions were determined. Precision was assessed and comparison with conventional immunoelectrophoresis and a chemical method was made. Though precision was poor (CV 4-6%) improvements in dispensing small volumes (0.2 microliter) should change this. Good correlation between methods (correlation coefficient 0.89-0.97) was obtained. The advantages of the technique are speed of analysis and low reagent costs.

Blood Protein Electrophoresis↗
PubMed1980

Quantification of proteins with a new sensitive method--zone immunoelectrophoresis assay.

Quantitative determination of proteins has been made by electrophoresis thereof in tubular canals filled with agarose gel containing antibodies. The procedure is simple and practicable. Sample volumes of a few microliter up to some hundred microliter may be added for each test. The amount of protein in a sample was found to be linearly related to the distance from the point of application (i.e. gel surface) to the front of the immunoprecipitation zone. The zone immunoelectrophoresis method shows over rocket immunoelectrophoresis the following advantages: less consumption of antibodies; much lower detection limit (0.05 microgram of protein per ml); larger concentration range with a linear calibration curve. A high correlation (0.98) between the first- and last-mentioned method was obtained for alpha 1-antitrypsin. Precision data are given for albumin and alpha-fetoprotein and typically a relative standard deviation of a few percent was obtained.

Blood Protein Electrophoresis↗
PubMed1983

Individual difference of seminal plasma detected with crossed immunoelectrophoresis.

Seminal plasma from 120 subjects was examined by crossed immunoelectrophoresis using anti-seminal plasma. The samples were divided into two groups depending on a precipitation line which migrated fast in the first dimension electrophoresis. This precipitation line showed an incidence of 48 percent and was proved to have no acid phosphatase, esterase or lactate dehydrogenase activity and was not stained with Schiff's reagent. Analysis with column chromatography, Sephadex G-200 and Sephadex G-50, revealed that molecular weight of the protein was about 10,000 dolton. The difference was not identified with the other methods; polyacrylamide gel electrophoresis and agarose immunoelectrophoresis.

Blood Proteins↗
PubMed1990

Platelet factor 4 and the platelet secreted proteoglycan: immunologic characterization by crossed immunoelectrophoresis.

Platelet factor 4 (PF4) is a hydrophobic, alpha-granule protein with potent antiheparin activity. It also binds to a chondroitin sulfate-containing proteoglycan (PG) isolated from platelets. In order to evaluate further the relationship between PF4 and the chondroitin sulfate-containing proteoglycan in resting platelets, the PF4-binding proteoglycan from human platelets has been purified using purified PF4 as an affinity ligand and used to prepare polyclonal antiserum. Two antisera have been characterized: one reacts primarily with chondroitin sulfate (CS), the other reacts with the protein core of the platelet proteoglycan after chondroitinase AC digestion. PF4 and PG core protein antigen are present in separate, dissimilar precipitin arcs when triton-solubilized platelets are analyzed by crossed immunoelectrophoresis using polyclonal antisera to purified PF4 and PG. PF4 was demonstrated in a complex with a separate chondroitin sulfate antigen by crossed immunoelectrophoresis (CIE) experiments in which either anti-PF4 or anti-CS antisera was incorporated in the intermediate gel. Both the PF4-chondroitin sulfate complex and the proteoglycan are secreted from platelets when fresh, washed human platelets are stimulated by human alpha-thrombin. This second antigen may represent the PG after posttranslational modification of a precursor form of the proteoglycan.

Blood Platelets↗
PubMed1990

Immunoelectrophoresis of mycobacterial antigens.

Polyvalent antiserum to culture filtrate of H37 Ra M. tuberculosis was raised in rabbits. Monospecific antiserum was raised against M. tuberculosis antigen-5, prepared from the culture filtrates by immunoabsorbent affinity chromatography. On immunoelectrophoresis, antigen-5 demonstrated single precipitin arc against polyvalent and monospecific antisera. The culture filtrate antigen demonstrated multiple precipitin arcs against polyvalent antiserum and single precipitin are against monospecific antiserum. Antigen-5 could be isolated and characterized from the culture filtrate of H37 Ra M. tuberculosis. Immunoelectrophoresis could be one of the method to characterize the mycobacterial antigens prepared in the laboratory.

Animals↗
PubMed1979

Identification of alpha1-lipoproteins in crossed immunoelectrophoresis.

Evans Blue dye binds selectively, but with different avidities, to five major antigens in human serum. The anodic mobility of the antigen-dye complexes is greater than that of the antigens alone in crossed immunoelectrophoresis, which is of practical value for identification. We used this characteristic to show that in some human sera there is a population of alpha1-lipoprotein molecules that migrates electrophoretically in the beta-lipoprotein region, where in conventional zone electrophoresis it could be mistaken for beta-lipoprotein. We also demonstrate that horses, unlike rabbits, rarely make precipitins to human serum alpha1-lipoprotein. Equine antiserum to human serum therefore generally is useless for detecting this important serum antigen in immunoelectrophoresis.

Evans Blue↗
PubMed1979

Pseudorabies: adaptation of the countercurrent immunoelectrophoresis for the detection of antibodies in porcine serum.

A countercurrent immunoelectrophoresis test was developed for the detection of precipitating antibodies to pseudorabies virus in pig serum. The precipitation reaction occurred only between the pseudorables antigen and the homologous porcine antiserum. The sensitivity of the method was compared to that of the serum neutralization test. On the basis of its sensitivity, its specificity and the rapidity with which the results were obtained, the countercurrent immunoelectrophoresis may become a potentially valuable screening method to test large numbers of porcine serum.

Animals↗
PubMed1986

Comparative studies of Leishmania soluble antigens by crossed-immunoelectrophoresis: demonstration of specific antigens to L. mexicana and L. braziliensis.

The antigenic relationship between two species of Leishmania (L. mexicana amazonensis and L. braziliensis braziliensis) was analysed using crossed-immunoelectrophoresis with an intermediate gel. Rabbit hyperimmune sera were prepared by subcutaneous inoculations of soluble antigens obtained from each species, emulsified in Freund's adjuvant. The antisera were then tested by immunodiffusion and electrophoresis; soluble antigens obtained from promastigote forms were submitted to ultrasonication and ultracentrifugation. Immunoelectrophoresis with homologous and heterologous antisera demonstrated the existence of some antigens which were species specific and some which were common to both species. The importance of these antigens is discussed.

Animals↗
PubMed1986

An evaluation of the applicability of crossed immunoelectrophoresis in seroepidemiological studies of hydatid disease in livestock.

Hydatidosis is both an economic and public health problem all over the world. Although various tests have been used on serodiagnosis of the disease in laboratory infected sheep, there is a problem in detecting antibody to the parasite in sheep. The antibody response of people infected with E. granulosus differs from the antibody response of livestock. Crossed immunoelectrophoresis was used to search for antibodies in 44 goats known to have had the infection. Crossed immunoelectrophoresis was able to detect two antibodies produced by a goat naturally infected with hydatid disease. Three of 44 known cases were detected by the test method. None of the naturally infected animals' sera exhibited a pattern of arcs similar to the reference pattern. The test's sensitivity was poor.

Animals↗
PubMed1985

Precipitating antibodies to non-treated dengue type 2 viral antigens in sera of Thai hemorrhagic fever patients by crossed immunoelectrophoresis.

Crossed immunoelectrophoresis of dengue type 2 virus revealed at least two precipitating antigens which shared some antigenic determinants. Glycoprotein components of both antigens were detected by binding to concanavalin A. Sera from dengue hemorrhagic fever patients showed precipitating antibodies to both antigens which could be quantitated according to the precipitate patterns formed in the intermediate gel of crossed immunoelectrophoresis. All secondary dengue hemorrhagic fever patients demonstrated an increase in precipitin titers in convalescence sera. Most patients with mild illness contained precipitating antibodies in acute phase sera whereas severe cases did not. Convalescent sera from severe cases showed only low titers. These precipitating antibodies may be associated with protection since they were produced early only in those with mild form of illness.

Adolescent↗
PubMed1979

Immunoelectrophoresis of ligandin.

Procedures are described that facilitate the immunoelectrophoretic analysis of the carcinogen-binding protein ligandin. Rocket immunoelectrophoresis is performed at pH 4.9 in agarose gel containing carbamylated antiserum thereby promoting more rapid migration of ligandin, pI 9.0, into the gel. This method is used as a basis for quantitative crossed immunoelectrophoresis using either electrophoresis or isoelectrofocusing of ligandin in the first dimension.

Animals↗
PubMed1984

Serotypes of red clover necrotic mosaic virus. III. Immunoelectrophoresis under different conditions.

The effects of 4 buffers at different pH and of different molarities was tested on the immunoelectrophoresis of representatives of three serotypes and their natural mixtures of red clover necrotic mosaic virus (RCNMV). No substantial differences were observed with buffers at pH 7.2, 8.0 and 8.6. The representatives of the three RCNMV serotypes showed different mobilities in 0.1 and 0.01 mol/l ionic buffers in the agarose gel from cathode to anode. In nonionic 0.01-0.1 mol/l N-2-hydroxyethylpiperazine-N'-ethanesulphonic acid (HEPES) buffer, the RCNMV isolates moved from the anode to the cathode. Isolate TpM48 (serotype B) moved the most rapidly, while isolates No. 6 (serotype C) and TpM34 (serotype A) showed lower mobilities distinct from one another. This made possible a reliable differentiation of serotype B from serotypes A and C as well as of serotype A from serotype C. Agarose gels in 0.001 mol/l buffers proved to be unsuitable for the immunoelectrophoresis of RCNMV.

Hydrogen-Ion Concentration↗
PubMed1983

beta-2-microglobulin. Quantitation by rocket immunoelectrophoresis and evaluation of serum levels in renal transplant patients.

An assay using Laurell rocket immunoelectrophoresis has been developed for quantitation of beta-2-microglobulin (beta 2m) and serial levels of beta 2m have been measured in sera obtained daily from 52 patients following renal transplantation. The RIEP assay was performed using 1% low-endosmosis agarose gels containing 4% polyethylene glycol 6000 and the IgG fraction of rabbit antihuman beta 2m. Elevated beta 2m levels pretransplant fell markedly in the first 48 hr posttransplant in all patients with primary functioning grafts. Base-line control values for serum beta 2m were obtained in patients when stable graft function was established. Serum beta 2m levels increased during renal graft rejection, and significant elevations of serum beta 2m were detected at the onset of all graft rejection episodes as compared with control values. It is suggested that serum beta 2m quantitation by rocket immunoelectrophoresis is a simple and valuable assay to assess posttransplant renal graft function.

Beta-Globulins↗
PubMed1983

[Importance of immunoelectrophoresis in agar and disc electrophoresis in polyacrylamide gel for characterizing different strains of Bordetella pertussis].

The antigenic composition of typical and atypical B. pertussis strains obtained in the foci of pertussis infection, as well as experimentally obtained antibiotic-resistant B. pertussis strains, has been studied by the methods of immunoelectrophoresis in agar and electrophoresis in polyacrylamide gel (PAAG). Immunoelectrophoresis in agar has been found capable of differentiating B. pertussis culture from a group of unidentified morphologically similar Gram-negative bacilli by their antigenic composition and thus suitable for use as an additional criterion in the identification of atypical B. pertussis strains. PAAG electrophoresis has permitted finding differences in the set of protein antigens in the control strain and in its clones obtained by multiple subculturing in media with antibiotics added.

Agar↗
PubMed1983

[Laboratory procedures with adenoviruses. IX. Hexon antibody determination by countercurrent immunoelectrophoresis].

101 paired sera from patients with proved adenovirus infections (mostly conjunctivitis) were tested quantitatively by counter immunoelectrophoresis, with hexon of adenovirus 5 as a genus-specific antigen, in comparison with complement-fixation. Both methods were equally sensitive to demonstrate antibodies; also the titers showed a marked correlation. The number of titer rises, however, was higher in counter immunoelectrophoresis; the titer rises mostly occurred around the 10th day after onset of disease. No differences between patients infected with different adenovirus types were found.

Adenoviridae Infections↗
PubMed1980

An assay for antibodies in leprosy sera reacting with ribonucleoprotein (RNP), a mycobacterial ribosomal antigen, using crossed immunoelectrophoresis with intermediate gel.

The technique of crossed immunoelectrophoresis with intermediate gel has been adapted to provide a quantitative assay for antibodies to a mycobacterial ribosomal antigen termed ribonucleoprotein (RNP) antigen. This antigen is no. 1 in the M. smegmatis reference system and corresponds to antigen no. 5 of M. leprae. The assay method measures changes in the rate of migration of the reference precipitin peak caused by the addition of serum in the intermediate gel and utilizes a lepromatous serum pool (LSP) both in the reference gel and as a standard in the intermediate gel. The anti-RNP activity in 24 leprosy sera differentiated the pauci-bacillary tuberculoid group (11 of 12 < 35% LSP) from the multi-bacillary lepromatous group (11 of 12 > 35% LSP). These findings confirm the work of others which indicates that assay of anti-RNP activity may have applications in the classification of leprosy patients and in the serodiagnosis of lepromatous leprosy infections. This method should also be applicable in other systems in which an antigen of high electrophoretic mobility forms a major precipitin in crossed immunoelectrophoresis.

Antibodies, Bacterial↗