Counterimmunoelectrophoresis Method Research Articles

Seroepidemiological survey for infections by fungi causing systemic mycoses in the Xacriabá Indian Reserve, Minas Gerais State, Brazil

Xacriabá Indian Reserve is situated in the north of Minas Gerais State, Brazil, near the municipality of Manga and São Francisco River. The Indian population is miscegenated with caucasian and negroid people and have farming activities. Blood samples were collected from 180 inhabitants of the reserve (85 men and 95 women), 15 to 84 years old. Serum antibodies against antigens of Paracoccidioides brasiliensis, Histoplasma capsulatum, Cryptococcus neoformans, and Candida albicans were tested for by counterimmunoelectrophoresis method. Seropositivity was verified in 5%, 3.9%, 4.2%, and 6.7%, respectively of the 180 samples. Those seropositive for antigens of P. brasiliensis and H. capsulatum were predominantly women and had lower age and more elevated mean titers of antibodies than individuals whose sera reacted with antigens of C. neoformans and C. albicans. The results suggest the occurrence of paracoccidioidomycosis-infection and histoplasmosis-infection in the Xacriabá Indian Reserve, particularly in the first decades of life of the inhabitants.

Process standardization for optimal virus recovery and removal of substrate DNA and bovine serum proteins in Vero cell-derived rabies vaccine

Purification of a rabies vaccine by a single zonal centrifugation run was replaced by two runs with optimal standardization of the sucrose density gradient. As a result, significant reductions in the levels of substrate DNA and bovine serum protein in the Vero cell-derived human rabies vaccine were achieved. Following many trials, for the first run, loading of the 3.2- l capacity K-3 rotor with 1800 ml of 60% sucrose solution and 1400 ml of vaccine PBS buffer solution gave a satisfactory linear gradient. However, after the first run, the substrate DNA and bovine serum contents exceeded the required levels. After protamine sulphate and Tween-80 treatment of the concentrated inactivated material, a second run using the same procedure as in the first run was tried. However, these purification procedures resulted in low virus recovery. To achieve optimal virus recovery, and removal of substrate DNA and bovine serum protein, the peak fractions from the first run as indicated by the haemagglutination, sucrose concentration, and optical density values were pooled and the sucrose concentration of the pooled fractions was increased to 60%. A second (flotation) run was then carried out. Using this method, the virus recovery rate was more than 95% that of the first run, and the levels of cellular DNA and bovine serum protein were well within the acceptable limits of less than 100 pg/dose and one part per million, respectively. The substrate DNA was quantified by both radioactive labeling and non-radioactive biotin labeling methods. For the quantification of calf serum protein, a counter-immunoelectrophoresis method was developed and effectively applied. A potency assay was performed using the National Institutes of Health (NIH) and well-standardized in vitro single radial immuno diffusion (SRD) methods. Finally, an immunogenicity study was conducted with human volunteers and the results were confirmed by a rapid fluorescent focus inhibition test (RFFIT).

Comparison and variation of different methodologies for the detection of autoantibodies to nuclear antigens (ANA)

Interest in the assessment of autoantibody specificity stems from the need for an autoantibody marker capable of predicting clinical events in autoimmune disorders. However, the multiplicity of epitopes present on autoantigenic particles, the quantitative and qualitative heterogeneity of autoantibodies, as well as the nature of the tests, mean that each of the assays used in their determination have different characteristics. The aim of this study was to compare the specificities of different ANAs using four commercial assays. The routine method used for the detection of ANA is indirect immunofluorescence on Hep-2 cells. The assays used were: counterimmunoelectrophoresis (CIE), enzyme-linked immunosorbent assay (ELISA), and two immunoblotting assays. Kappa statistic was applied to evaluate the consistency between tests. Kappa index is a measure of agreement between categorical data. Kappa has a maximum of 1.00 when the agreement is perfect, a value of zero indicates no agreement better than chance, and negative values show worse than chance agreement. For SS-B antibodies, there was a good concordance between all four methods used (Kappa 0.66-0.74). For anti RNP antibodies, the results for CIE/ELISA (Kappa 0.60) were consistent as were the two immunoblot methods (Kappa 0.69). For anti Scl-70 (topoisomerase I) antibody, results from the ELISA and CIE methods were totally consistent (Kappa 1.00). In spite of the high prevalence of anti SS-A/Ro antibodies, the agreement between the methods was poor, without statistical significance. Finally, for Sm antibodies, more consistent results were obtained between CIE/ELISA (Kappa 0.51) and between one of the immunoblotting methods and ELISA (Kappa 0.54). In conclusion, CIE concurs mostly with ELISA for anti-RNP, Scl-70, Sm and SS-B antibodies, but with some disagreement for SS-A antibodies.

Comparison and variation of different methodologies for the detection of autoantibodies to nuclear antigens (ANA)

Interest in the assessment of autoantibody specificity stems from the need for an autoantibody marker capable of predicting clinical events in autoimmune disorders. However, the multiplicity of epitopes present on autoantigenic particles, the quantitative and qualitative heterogeneity of autoantibodies, as well as the nature of the tests, mean that each of the assays used in their determination have different characteristics. The aim of this study was to compare the specificities of different ANAs using four commercial assays. The routine method used for the detection of ANA is indirect immunofluorescence on Hep-2 cells. The assays used were: counterimmunoelectrophoresis (CIE), enzyme-linked immunosorbent assay (ELISA), and two immunoblotting assays. Kappa statistic was applied to evaluate the consistency between tests. Kappa index is a measure of agreement between categorical data. Kappa has a maximum of 1.00 when the agreement is perfect, a value of zero indicates no agreement better than chance, and negative values show worse than chance agreement. For SS-B antibodies, there was a good concordance between all four methods used (Kappa 0.66–0.74). For anti RNP antibodies, the results for CIE/ELISA (Kappa 0.60) were consistent as were the two immunoblot methods (Kappa 0.69). For anti Scl-70 (topoisomerase I) antibody, results from the ELISA and CIE methods were totally consistent (Kappa 1.00). In spite of the high prevalence of anti SS-A/Ro antibodies, the agreement between the methods was poor, without statistical significance. Finally, for Sm antibodies, more consistent results were obtained between CIE/ELISA (Kappa 0.51) and between one of the immunoblotting methods and ELISA (Kappa 0.54). In conclusion, CIE concurs mostly with ELISA for anti- RNP, Scl-70, Sm and SS-B antibodies, but with some disagreement for SS-A antibodies. J. Clin. Lab. Anal. 11:388–392, 1997. © 1997 Wiley-Liss, Inc.

Avaliação do teste de látex comparativamente à cultura e à imunoeletroforese cruzada no diagnóstico de meningites bacterianas

About 326 cerebrospina1 fluid samp1es of patients with clinical signs of meningitis were studied through a commercial latex reagent for bacteria1 antigen detection. The puru1ent cerebrospina1 fluid samp1es were analysed for Neisseria menigitidis groups B and C, Haemophilus influenzae b, and Streptococcus pneumoniae antigens. The results showed a better performance of the latex agglutination test in comparison to culture for Neisseria menigitidis group B and group C, and for Streptococcus pneumoniae. Except frr Streptococcus pneumoniae which was not tested through counterimmunoe1ectrophoresis, the 1atex reagent had better performance than counterimmunoe1ectrophoresis on1y for Neisseria meningitidis group B. Sensitivity of latex test, as compared with culture, showed highest levels for Neisseria meningitidis group B, and for Streptococcus pneumoniae. When latex test results were compared with counterimmunoelectrophoresis ones, highest 1eve1sof sensitivity were observed for Neisseria meningitidis group B and Haemophilus influenzac b. Concerning specificity, fair values were observed for all bacterial agents relating to culture, and for all but Streptococcus pneumoniae which was not tested for counterirnrnunoelectrophoresis; fair values were a1so observed when the results of this method were considered. The degree of kappa agreement (k), when compared with culture, exhibited better K indices for Haemophilus influenzae b, and better K indices for this bacterial agent and for Neisseria meningitidis group C as well, when the resu1ts of latex agglutination test were compared with counterimmunoelectrophoresis method.

Investigation of antibodies to extractable nuclear antigens in dogs

Summary Determination of antibodies to specific nuclear antigens, termed extractable nuclear antigen (ena), was investigated in healthy dogs and in dogs with autoimmune, inflammatory, and neoplastic diseases. Using a counter-immunoelectrophoresis method, the dogs' sera were tested for antibodies against the nuclear antigens single-stranded dna, Sm, Ro, La, ribonucleoprotein, Scl, and proliferating cell nuclear antigen. Antibodies to the Ro antigen were found in 1 dog with discoid lupus erythematosus, in 1 dog with pemphigus erythematosus, and in 1 dog with facial pyoderma and chronic superficial keratitis. In 15 dogs, antibodies were detected to ena, but the precipitin lines were too weak to identify the specific ena. These antibodies were found in some dogs with systemic lupus erythematosus, discoid lupus erythematosus, pemphigus erythematosus, dermatomyositis, vitiligo, lymphoma; in the dog with facial pyoderma and chronic superficial keratitis; and in 1 healthy dog. The highest percentage of dogs with antibodies to ena in a large series (> 8) of this study was in dogs with systemic lupus erythematosus (4 of 13; 31%).

Détection des anticorps anti-adénovirus dans le sérum de chevaux Polo au Nigeria

Serum samples obtained from 107 Polo horses showing clinical signs of viral respiratory disease were tested for precipitating antibodies to adenovirus by agar gel precipitation test and counter-immunoelectrophoresis method. The results obtained demonstrate serological evidence of adenovirus infection in Polo horses in Nigeria. The counter-immunoelectrophoresis method was observed to be about 3 times more sensitive than the agar gel precipitation test with 19.3 vs 64.5%. It could thus be used to screen a large number of serum samples within a short period.

Routine testing for antinuclear antibodies: A comparison of immunofluorescence, counterimmunoelectrophoresis and immunoblotting

We compared the classical immunofluorescence test (IFT) and counter-immunoelectrophoresis method (CIE) with the new immunoblotting technique (IBT) for the detection of antinuclear antibodies (ANA). Sera from 200 patients were tested in all three assays. Patients were classified as having either rheumatic disease including rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), mixed connective tissue disease (MCTD), primary Raynaud's phenomenon or nonrheumatic disease. Within these broad categories, we observed that IFT and IBT showed a more or less comparable sensitivity and specificity (IFT: 0.54 and 0.82, respectively; IBT: 0.39 and 0.79, respectively). The CIE method combines a high specificity (0.99) with an extremely low sensitivity (0.08). By combining positive results obtained by IFT and IBT, a higher specificity (0.97) but a diminished sensitivity (0.24) is obtained. As IBT allows simultaneous discrimination between ANA of different specificities, we also tested for a correlation between the presence of anti-Sm, anti-RNP and anti-SS-B and the disease category. Only anti-SS-B discriminated significantly between rheumatic- and nonrheumatic disease. Anti-RNP was found in 50% of the SLE patients and in 50% of the MCTD patients; anti-Sm in 17% of the SLE patients and 25% of the MCTD patients. Anti-SS-B was found in 33% of the SLE patients. However, predictive rates of these ANA were low: 0.37 (anti-RNP), 0.67 (anti-Sm) and 0.43 (anti-SS-B). We conclude that from a practical point of view IFT is the preferable assay to screen for the presence of ANA. To characterize ANA specificities, however, the IBT is far superior to the CIE technique.

Exoantigens of Trypanosoma cruzi. I. Conditions for their detection and immunogenic properties in experimental infections.

Exoantigens of Trypanosoma cruzi were produced in experimentally infected BALB/c mice. The exoantigens were detected by the counterimmunoelectrophoresis method (CIE), with antisera raised in rabbits by immunization with total homogenates of culture forms of T. cruzi, in plasma from infected animals obtained by centrifugation and filtration. Control experiments indicated that exoantigens are not somatic components of T. cruzi leaked during the preparative procedure. Exoantigens were detected in male and female mice, 11-90 days old, between 6 and 60 days of infection, and in all mice with patent parasitemia. After 13 days of infection, mice developed antibodies to exoantigens; by CIE up to three populations of antibodies were revealed in different groups of animals. In mice between 13 and 60 days of infection, the coexistence of exoantigens and homologous antibodies was also observed. The exoantigens are not strain specific since a cross reactivity between antigens from three strains of T. cruzi (Tulahuén, Higueras, and Alejandro) was seen. Finally, the presence of antibodies to exoantigens in humans with chronic Chagas' disease was demonstrated.

Immunochemical Measurement of Serum Prostatic Acid Phosphatase (PAP). Clinical Evaluation of Radioimmunoassay and Counter Immunoelectrophoresis

Gupta, Manjula K., Rajaraman, Srinivasan, Gupta, Satyendra K., Hewitt, Clarence B., and Deodhar, Sharad D.: Immunochemical measurement of serum prostatic acid phosphatase (PAP). Clinical evaluation of radioimmunoassay and counter Immunoelectrophoresis. Am J Clin Pathol 76: 430-436, 1981. Two radioimmunoassay procedures (RIA-1 and RIA-2) were evaluated for the quantitation of prostatic acid phosphatase in serum and compared with the enzymatic method and counter immunoelectrophoresis method for their specificity and sensitivity. Sera from 168 patients were analyzed and these included: normals, 27; untreated prostatic cancer patients Stage A, 2; Stage C, 3; Stage D, 17; cancer of prostate treated with different modalities, 42; sarcoma of prostate, 1; prostatitis, 3; nonprostatic carcinoma, 17; and benign prostatic hyperplasia (BPH), 56. RIA-1 procedure appeared more sensitive (82% sensitivity) and specific (94.5% specificity) than the RIA-2 procedure (68% sensitivity and 91.8% specificity), but the differences were not statistically significant. The enzymatic method was found to be least sensitive (63.6% sensitivity) but also the most specific (100% specificity). Only 69 of the specimens were analyzed by counter immunoelectrophoresis, which showed sensitivity of 87% and specificity of 51.4%. False positives were observed more often in patients with nonprostatic cancer and BPH. The variations in diagnostic specificity of immunologic assays suggest the need of characterization of each antibody specificity. (

Immunologic studies of factor IX (christmas factor) I. Counterimmunoelectrophoresis method for factor IX antigen

A precipitating monospecific antiserum for factor IX antigen was produced by immunization of rabbits with a partly purified factor IX and solid-phase immunoadsorption. The monospecific antiserum was used to determine the presence of factor IX antigen in hemophilia B and normal plasmas by a counterimmunoelectrophoresis method. Factor IX antigen appears to be markedly reduced or absent in most hemophilia B patients when studied by this method.

Evaluation of Methods to Detect Antibodies against Aspergillus fumigatus

Seventy-five sera, including sera from 23 patients with aspergillosis, 17 with tuberculosis, asthma or carcinoma, and 35 normal controls, were studied for antibody activity against Aspergillus fumigatus by agar gel double diffusion (DD), counterimmunoelectrophoresis (CIE), indirect hemagglutination (IHA) and indirect immunofluorescence (IF). Metabolic antigens produced from a selected strain of A. fumigatus were used for DD, CIE and IHA, while slide cultures of the same organism were used for IF. The results indicated that sera from the patients with aspergillosis had high IHA and IF titers, while only 91 and 87% were positive for CIE and DD methods, respectively. IHA was more sensitive than DD and CIE, and IF was equally sensitive but had false-positive reactions. CIE was simple and fast, but had false-negative and occasionally false-positive results.

Immunodiffusion method for identification of group A streptococci.

The development of a method suitable for identification of group A streptococci by microprecipitation in gels is described. The method is based on preparation of specific sera containing high antibody levels agains the antigenic determinant characteristic of group A streptococcal polysaccharide. In a comparative study with a counterimmunoelectrophoresis method, the proposed test proved to be specific, easily read, and less complicated. Results were obtained in 2 h.

Application of Counterimmunoelectrophoresis in Rapid Detection of Cytomegalovirus Antibodies

Determination of cytomegalovirus antibodies by counterimmune-electrophoresis correlated well with results of complement-fixation antibody studies. The sensitivity and specificity of the counterimmunoelectrophoresis method (vs. complement fixation) was 88.9%. Fourfold antibody increases detected by complement fixation were also detected by counterimmunoelectrophoresis. In addition, no cross-reaction was observed in the counterimmunoelectrophoresis assay between other DNA viral antisera and cytomegalovirus antigen (Ad-169).

Studies in chronic allergic bronchopulmonary aspergillosis. 3. Immunological findings.

Precipitin tests by two different methods, double-diffusion (DD) and counterimmunoelectrophoresis (CIE), and measurements of total and specific IgE against Aspergillus fumigatus were made in 50 patients with chronic allergic bronchopulmonary aspergillosis and in three control groups--atopics with a positive immediate prick test to A. fumigatus but no evidence of allergic aspergillosis, atopics with a negative prick test to A. fumigatus, and non-atopics. Precipitins were found in 84% and 78% of the patients with aspergillosis by the DD and CIE methods respectively. Precipitins were also found in 6 out of 27 (22%) patients with a positive prick test to A. fumigatus but no evidence of aspergillosis and in 1 of 24 patients with a negative prick test to A. fumigatus. The means of specific and total IgE values were significantly higher in the group of patients with aspergillosis than in the three other groups of patients. The increase in specific but not total IgE showed a statistically significant correlation with positive precipitin tests in the patients with aspergillosis. Total IgE but not specific IgE values were significantly higher (0-02 less than P less than 0-05) in patients who had had a transient radiographic shadow in the previous three months. Positive precipitin tests were also significantly correlated with the number of transient shadows in the past and with the interval of time since the last transient shadow.

Detection of Aspergillus fumigatus precipitins: a comparison of counter immunoelectrophoresis and double diffusion.

A counter immunoelectrophoresis method was compared with a double diffusion test for detecting Aspergillus fumigatus antibodies. Of 70 patients, 23 gave positive results in both tests and the remainder gave negative results. Fifteen patients with proven aspergillosis gave positive results in both tests.

Use of alpha-fetoprotein in prenatal diagnosis.

Alpha fetoprotein (AFP) measurement is possible by rocket immunoelectrophoresis and counterimmunoelectrophoresis methods which can measure concentrations as low as .1-1 mg/ml. A several 100-fold difference in AFP between fetal serum and amniotic fluid (AF) exists; hence cross-contamination can cause false positive results. 90% of proteins in AF however come from the maternal serum (MS) compartment and since AFP is small enough to cross into the MS compartment its levels can be used to detect prenatally fetal defects by measuring MS AFP levels. In second trimester AFP falls rapidly because normal AF volume is expanding and fetal production rates remain relatively constant. Polyhydramnios may change AFP in either direction (e.g. depressed with maternal diabetes and elevated with fetal swallowing defect). AF AFP is elevated in some cases of twin pregnancy; similarly MS AFP is more constantly elevated than AF. For proper laboratory results accurate pregnancy date estimation is essential because of rapidly changing second trimester AFP values. If careful attention is given to possible fetal blood contamination pregnancy date and twin pregnancy AF AFP measures are 90% accurate or better in mothers at high risk for neural tube defects (NTD). MS AFP is measured in units 1000-fold lower than AF (mcg/ml) and 1 million-fold lower than fetal serum (mg/ml). MS AFP does not rise above nonpregnancy until Week 10 of gestation. Elevated MS AFP in first trimester is probably due to direct fetomaternal transfer (placental injury or separation). AFP in MS reaches maximum 30 weeks postconception or 30-32 weeks of pregnancy. 3 problems in comparing and applying AFP screening results are: 1) differing definitions of upper limits of normal pregnancy AFP values; 2) differing incidence of NTD in populations studied; and 3) possibility of genetic variations in normal pregnancy values. Low for pregnancy date values indicate decidua without remaining fetal parts.

Detection of complement activation by counterimmunoelectrophoresis (CIE)

Counterimmunoelectrophoresis (CIE) was used as a method of detecting activation of the third component of the complement system (C3). Highly purified C3, normal human serum (NHS), EDTA-treated plasma and serum activated with aggregated human immunoglobulin (agg-IgG) or inulin were used as sources of C3 and/or C3 split products. Activation of the alternative pathway of complement was assayed in the presence of EGTA (10 mM) and MgCl 2 (0.3 mM), conditions which block activation of the classical pathway. When purified native C3, fresh NHS and fresh EDTA-plasma were tested in CIE against either antisera to whole C3 or to C3 split products, only one precipitin line was found, which was identified as native C3. However, when serum activated with agg-IgG or inulin were tested against the same reagents, two precipitin lines were seen. The first, with more cathodal mobility was identical to that of native C3. The second line had a more anodal mobility, was distinctly separated from the first and contained C3c and C3d as shown immunochemically with specific antisera. Native C3 and split products of C3 were identified by this CIE method in patients showing evidence of activated complement by having subnormal total complement (CH50) levels. When C3 split products were identified, the C3c-C3d precipitin line could always be distinguished from native C3 by its different electrophoretic mobility, even when C3 concentrations in serum varied from 0.25 mg/ml to 1.5 mg/ml. The sensitivity of CIE was compared to that of CH50 by assaying at different time intervals after agg-IgG was added to fresh NHS. C3c-C3d split products were detected by CIE before any fall in CH50 and at all times when a significant decrease in CH50 was present. This study shows that the CIE technique is a highly sensitive, specific and rapid method for detecting activation of the complement system via classical or alternative pathways in human disease.

Determination of DNA antibodies in normal and pathological sera by a new counterimmunoelectrophoresis method

A quantitative counter-immunoelectrophoresis technique has been applied to the evaluation of antibodies against native and single-stranded DNA. Anti-DNA antibodies have been found at high dilutions in patients with systemic lupus erythematosus, without correlation with the existence of renal lesions or with the degree of DNA binding assessed by Farr assay. Significant precipitates were also observed at significantly lower dilutions in other pathological situations and in normal subjects, posing the problem of the nature of the precipitates in these cases.

Hepatitis-associated (Australia) antigen in the Republic of Ireland: a preliminary report.

FIVE hundred and forty patients with hepatitis, or their contacts, and 10,145 voluntary blood donors were tested for hepatitis-associated (Australia) antigen using the semi-micro immunodiffusion and discontinuous counterimmunoelectrophoresis methods. The results indicate that the incidence of the antigen in voluntary blood donors in the Republic of Ireland is lower than in other European countries at the present time. Hepatitis-associated antigen was not detected in seven faecal samples tested from patients with ‘infectious’ hepatitis.