Comparison of rocket and crossed immuno-electrophoresis assays for determination of the level of actin complexing of Gc globulin.

OBJECTIVE: Gc globulin (vitamin D-binding protein) is a component of the extracellular actin scavenger system. The level of Gc globulin is reduced in patients with fulminant hepatic failure, septic shock and trauma. Furthermore, low levels of Gc globulin in patients with fulminant hepatic failure and multiple trauma have been found to correlate with the morbidity and mortality of patients. Owing to a large increase in the turnover of Gc globulin upon complex formation with actin, it may be important to determine both the total Gc globulin concentration and the degree of complexing with actin for estimating the clinical prognosis of a patient. For this reason, we have compared a crossed immuno-electrophoresis method (CIE), suitable for visualizing the degree of complexing with actin, with a rocket immuno-electrophoresis method (RIE), previously used for determination of the complex degree. MATERIAL AND METHODS: Sera from healthy donors and from patients with acetaminophen-induced liver disease or trauma were investigated using CIE, RIE and enzyme-linked immunosorbent assay (ELISA). RESULTS: Using the CIE, no Gc globulin-actin complexes were detected among healthy donors. Complexes were present in 21 of 39 patients with liver disease and 3 of 37 trauma patients. High complex ratios (> 20 %) were found in 6 of 7 patients with hepatic encephalopathy. Using the RIE, complexes were detected in most samples. CONCLUSION: The results show that the CIE method may be used for determining the degree of actin complexing in conjunction with ELISA or RIE in determining the levels of total Gc globulin.

Molecular analysis of plasma alpha 1,3-fucosyltransferase deficiency and development of the methods for its genotyping.

Four patients with mental illness were found to be deficient in plasma alpha1,3-fucosyltransferase for the first time in Japan [Exp Clin Immunogenet 1999;16:125-130]. Complete sequencing of FUT6 genes in these individuals revealed the presence of two point mutations, i.e., G739 to A (Glu-->247 to Lys) and C945 to A (Tyr-->315 to stop). In addition to two reported alleles having G739 to A (pf1) and G739 to A and C945 to A (pf3), a new mutated allele having C945 to A (pf2) was found to be present and all the individuals who lack alpha1,3-fucosyltransferase activity in plasma were found to possess pf genes homozygously (pf/pf). In order to detect such lethal mutations in FUT6 genes easily, PCR-RFLP methods have also been developed and applied for the screening of FUT6 deficiency in a large number of samples which resulted in the demonstration of three additional FUT6-deficient individuals. The absence of alpha1,3-fucosylated molecules on alpha(1)-acid glycoprotein in plasma from all the 7 individuals was confirmed to result from the plasma alpha1,3-fucosyltransferase deficiency.

Application of image analysis techniques to two-dimensional crossed immunoelectrophoresis study.

Two-dimensional crossed immunoelectrophoresis (2D-CIEP) is a technique widely used for studying composition of protein mixtures in biological and clinical studies. A low cost image analysis system with the use of an optical flatbed scanner and a IBM-compatible PC was set up in this work for capturing 2D-CIEP patterns. A computer package CIEPEASY was developed for modification and analysis of the image acquired to determine various peak parameters such as the migration distance, peak height and peak area of the constituting components for both qualitative and quantitative studies. In this approach, more composition information of the standard and sample gel patterns could be extracted from the proposed image analysis system. The time required for data collection and interpretation of 2D-CIEP images was shortened significantly and the results obtained have a higher accuracy than those obtained by using conventional methods. Moreover, a linear relationship between the peak area and the amount of antigen present in a sample was confirmed accurately and reported for the first time in the literature.

Electrophoretic methods for studying protein-protein interactions.

Protein-protein interactions are involved in many biological processes ranging from DNA replication, to signal transduction, to metabolism control, to viral assembly. The understanding of those interactions would allow the effective design of new drugs and further manipulation of those interactions. Several useful analytical methods are available for the study of protein-protein binding, and among them, electrophoresis is commonly used. We describe two types of electrophoresis: gel electrophoresis and capillary electrophoresis. Gel electrophoresis is a well-established method used to study protein-protein interactions and includes overlay gel electrophoresis, charge shift method, band shift assay, countermigration electrophoresis, affinophoresis, affinity electrophoresis, rocket immunoelectrophoresis, and crossed immunoelectrophoresis. These techniques are briefly described along with their advantages and limitations. Capillary electrophoresis, on the other hand, is a relatively new method and affinity capillary electrophoresis has demonstrated its value in the measurement of binding constants, the estimation of kinetic rate constants, and the determination of stoichiometry of biomolecular interactions. It offers short analysis time, requires minute amounts of protein samples, usually involves no radiolabeled compounds, and, most importantly, is carried out in solution. We summarize the principles of affinity capillary electrophoresis for studying protein-protein interactions along with current limitations and describe in depth its application to the determination of stoichiometries of tight and weak binding protein-protein interactions. The protocol presented in the experimental section details the use of affinity capillary electrophoresis for the determination of stoichiometry of protein complexes.

Glycosylation of alpha1-acid glycoprotein in inflammatory disease: analysis by high-pH anion-exchange chromatography and concanavalin A crossed affinity immunoelectrophoresis.

High-pH anion-exchange chromatography with pulsed amperometric detection is a highly sensitive technique that can be used for detecting changes in sialylation and fucosylation, as well as different branching patterns of N-linked oligosaccharides in glycoproteins. We examined the N-glycans of alpha1-acid glycoprotein obtained from twelve patients with various inflammatory conditions with this technique, as well as traditional concanavalin A crossed affinity immunoelectrophoresis. We found the chromatographic profiles of N-glycans in all patients with rheumatoid arthritis to be very similar, but significantly different from normal controls. N-glycans from patients with ulcerative colitis also showed specific alterations in their chromatographic profiles. However, some heterogeneity was found between these patients, perhaps reflecting changes in glycosylation secondary to certain states of the disease, or to medical treatment. We conclude that this technique is useful for detailed mapping of glycosylation changes in alpha1-acid glycoprotein in clinical samples, and that it may be used to further increase our knowledge about glycosylation changes in response to inflammatory disease.

[The isolation and purification of the protective antigen and the edema factor from the culture filtrate of Bacillus anthracis STI-1]. / Vydelenie i ochistka protektivnogo antigena i otechnogo faktora iz kul'tural'nogo fil'trata Bacillus anthracis STI-1.

A rapid method for producing a protective antigen (PA) and edema factor (EF), components of anthrax toxin, is described. The specific features of the method are as follows: addition of a mixture of protease inhibitors to the culture fluid; simultaneous concentration on a fiber filter and adsorption on hydroxylapatite, followed by non-linear gradient a phosphate concentration; purification of elution of a phosphate concentration; purification of eluates from salts via electrodialysis. Resultant from protein desorption, PA and EF agents are electrophoretically homogeneous by no less than 80%, 10 liters of filtrate yielded 8 mg of PA and 5 mg of EF. The resultant agents were tested for their biological properties and protective activity.

Electrophoretic and chromatographic differentiation of two forms of albumin in equilibrium at neutral pH: new screening techniques for determination of ligand binding to albumin.

Analysis of normal human serum by crossed hydrophobic interaction immunoelectrophoresis with Phenyl-Sepharose revealed a biphasic appearance of the albumin peak. The molecular mechanism behind this apparent albumin heterogeneity was investigated. Analysis of defatted purified albumin showed that a major fraction bound to the Phenyl-Sepharose and that addition of ligands (e.g. long-chain fatty acids, bilirubin, sulfonamides and warfarin) before electrophoresis blocked this binding to different degrees. A quantitative relation between ligand binding and the amount of nonbinding albumin was found. Thus the technique might be suitable for screening of ligand binding to albumin. Analysis of serum samples from newborns with hyperbilirubinemia revealed a positive correlation between the fraction of the nonretarded albumin and the bilirubin concentration. By chromatography on Phenyl-Sepharose, defatted albumin was separated into a binding and a nonbinding form and this technique was subsequently used to determine the kinetics of the intramolecular conversion. After rechromatography, each of the fractions could again be separated into two fractions, indicating the presence of an equilibrium. By varying the passage time for albumin on the column or varying the period between the first and the second separation it was possible to calculate the conversion rates. The half-life for the conversion was found to be as long as 1 1/4 h. It is the first time that a conformational change for albumin with such a long conversion time has been described experimentally.

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.

Bronchial provocation with cat allergen: long-term outcome of the late allergic reaction and the individual IgE CRIE pattern.

In bronchial provocation tests with allergen, about 50% of the patients experienced a late allergic reaction (LAR), which has been associated with a specific IgE pattern as determined by crossed radioimmunoelectrophoresis (CRIE). Long-term outcome of this LAR is still unknown. Six patients allergic to cat, with documented LAR and specific IgE CRIE patterns were rechallenged in the same conditions after a mean interval of 2.5 years. Forced expiratory volume in one second (FEV1) and resistance results of the bronchial provocation tests (BPT) were compared, as well as specific IgE level and IgE CRIE patterns. All six patients were still suffering from asthma when exposed to a cat, although they were not being treated for asthma. Three patients (50%) had lost their LAR without specific treatment. They were older, with a longer history of asthma, but presented a very similar early allergic reaction to similar allergen doses. The other 3 were more reactive to the allergen but presented similar LAR after a slightly worse early allergic reaction (EAR). Specific IgE levels had decreased and the IgE CRIE pattern showed a diminished intensity of staining in the three patients who had lost their LAR, as opposed to the other three. These data suggest that asthmatic patients can lose their LAR over time without treatment. The occurrence of a LAR seems to be associated with a specific IgE CRIE pattern and IgE level. Further analysis of the individual antigen fractions might help to understand the mechanism of allergic reactions.

Analysis of alpha-fetoprotein by a procedure combining crossed immunoaffinoelectrophoresis and immunoblotting.

A newly developed procedure combining crossed immunoaffinoelectrophoresis and an immunoblotting assay (CIAE-IBA) has been used to discriminate between and precisely quantitate lectin-bound and lectin-free forms of alpha-fetoprotein (AFP) derived from human serum at levels of AFP as low as 5.6 ng/ml (adult reference range, 0 to 15). Twenty-two serum specimens with elevated levels of AFP were examined by a reference CIAE method, using either of two lectins, concanavalin A (Con A) or Lens culinaris agglutinin (LCA); aliquots of the specimens were then diluted in AFP-free serum to normal or near-normal levels of AFP and examined by CIAE-IBA, which included a final protein transfer to nitrocellulose. The quantitative results produced by the two methods were essentially the same. Overall, the sensitivity of the CIAE-IBA procedure surpasses that of other CIAE methods of AFP fractionation. The CIAE-IBA procedure may be used for study of the heterogeneity of AFP and may be applicable as well to the study of other proteins.

Comparison of two immunoassays for the complement protein C4b-binding protein in health and disease.

An enzyme-linked immunoassay for human C4b-binding protein (concentration range 25-400 ng/ml) was developed using two monoclonal antibodies; the intra- and interassay coefficients of variation were less than 7.2%. In 50 normal subjects, 20 patients with liver cirrhosis and 34 full-term newborns, the plasma levels of C4b-binding protein were very similar to those measured by Laurell electroimmunoassay; in 24 patients with elevated erythrocyte sedimentation rates, levels measured by enzyme-linked immunoassay were higher then those measured by the Laurell method (270 +/- 70% vs. 223 +/- 67%). In these patients crossed immunoelectrophoresis showed a pattern different from that of normal individuals, which may explain the lower values found with the Laurell method.

Crossed immunoelectrophoresis used for bacteriological diagnosis in patients with endocarditis.

Sera from 151 patients suspected of having endocarditis were obtained during a period of 3 1/2 years at Rigshospitalet, Copenhagen. The sera were examined by crossed immunoelectrophoresis for antibodies to bacteria causing endocarditis. The patients were divided into four groups: 1. Patients with definite endocarditis, 2. Patients with culture-negative endocarditis, 3. Patients with uncertain endocarditis, and 4. Patients without endocarditis. In sera from patients suffering from endocarditis caused by viridans streptococci, precipitating antibodies were demonstrated by crossed immunoelectrophoresis (diagnostic specificity = 86%; diagnostic sensitivity = 100%) while other bacterial etiologies of endocarditis were less reliably demonstrated by this method.

Glycan microheterogeneity of alpha 1-antitrypsin in serum and meconium from normal and cystic fibrosis patients by crossed immunoaffinoelectrophoresis with different lectins (Con A, LCA, WGA).

In order to test whether abnormalities of glycosylation occur in cystic fibrosis (CF), the glycan microheterogeneity of alpha 1-antitrypsin (alpha 1-AT) was studied in serum and meconium from normal individuals and patients with cystic fibrosis, by crossed immunoaffinoelectrophoresis (CIAE) using free Concanavalin A (Con A), Lens culinaris lectin (LCA) and wheat germ agglutinin (WGA). Three main results emerged from this study: (1) modification of glycosylation in serum alpha 1-AT from patients with cystic fibrosis were only significant with free Con A and WGA; this probably results from a reduced synthesis of the bi-antennary side-chains or by their increased catabolism. (2) Differences in isoforms found in alpha 1-AT from normal individuals and patients with CF using free Con A, LCA, were more pronounced in the meconium than in the serum; this may provide a useful test in diagnosis of cystic fibrosis. (3) There was parallelism between the behaviour of alpha 1-AT in serum and meconium from patients with CF using LCA, Con A; this may be explained by different types or levels of disfunction affecting a glycosylation mechanism.

Concanavalin A crossed affinoimmunoelectrophoretic analysis of the major pig serum proteins during fetal development.

The interaction between concanavalin A (Con A) and alpha-fetoprotein (AFP), transferrin (TF), fetuin, alpha 1-antitrypsin (AT) and alpha 1-acid glycoprotein (AGP) has been analyzed by crossed affinoimmunoelectrophoresis (CAIE) in fetal extracts or sera, from 26-day-old porcine fetuses to birth, and in adult pigs. Most of the TF and AFP (100 and 85-90%, respectively) reacted with Con A during the entire developmental period. AGP showed both two reactive and one nonreactive Con A isoforms, whose proportions change greatly during development. In younger fetuses 100% of the protein was Con A-nonreactive. This variant represented 64% in 50-day-old fetuses, 80% in newborn pigs and 20% in adult sera. Fetuin and AT showed a maximum of three Con A-reactive microforms and one Con A-nonreactive microform, which was always a minor form. These microforms were detected mainly in young fetuses. Although the proportion of Con A-reactive variants of fetuin and AT changes during fetal development, the predominant microform was always that with intermediate affinity against Con A. The same microform was also predominant in adult AT, whereas the more reactive microform in respect to Con A predominates in adult fetuin.

Crossed immunoelectrophoresis of fungal antigens in tissues as a means of diagnosing systemic aspergillosis and zygomycosis in cattle.

A novel method for diagnosing bovine aspergillosis and zygomycosis is described. Rabbit hyperimmune antisera raised against somatic antigens of Aspergillus fumigatus and Absidia corymbifera were used in crossed immunoelectrophoresis with supernatants from disintegrated tissues from acute necrohaemorrhagic mycotic lesions from cattle. The method specifically identified 4 of 5 lesions with aspergillosis and 2 of 5 lesions with zygomycosis. One lesion dually infected with aspergillosis and zygomycosis was negative. The method worked with unabsorbed sera, was specific, and required only standard electrophoretic equipment. It can therefore supplement chemical detection of fungi in tissues in the diagnosis of bovine aspergillosis and zygomycosis.

Factors influencing the reaction of alpha 1-fetoprotein with concanavalin A and Lens culinaris agglutinin in crossed affinoimmunoelectrophoresis.

Concanavalin A (Con A) and lentil lectin (LCA) analysis of alpha-fetoprotein (AFP) glycosylation heterogeneity is used in a variety of clinical situations. We studied the influence of analytical conditions on the separation of AFP glycoforms by using lectin-crossed affinoimmunoelectrophoresis, regardless of the AFP concentration, which can vary over a wide range in biological fluids. We defined the optimal concentration of Con A (2 g/L) and LCA (0.35 g/L) in the first-dimension gel, together with the optimum antigen (AFP)/antibody ratio in the second-dimension gel. The presence of protein in the diluent used for AFP samples was found to change the shape of crossed affinoimmunoelectrophoresis patterns without changing the percentage composition of AFP fractions. The within-run CV was less than 4% for both lectins, and the between-run CV was less than 6.3%. The minimal quantity of AFP that provided a visible pattern with both lectins was 4 ng, corresponding to 50 microL of an 80 micrograms/L AFP sample. These technical conditions allow the cellular origin of AFP to be determined, regardless of the concentration in the sample. Typical AFP lectin patterns of secreting tumors are compared with fetal and cord serum AFP.

[Free A-I apolipoprotein and bound A-I apolipoprotein determined by "multiple" crossed immunoelectrophoresis in patients with acute hepatitis and obstructive jaundice].

Crossed immunoelectrophoresis (CIEP) using anti-A-I apolipoprotein antiserum gave a large immunoprecipitin peak of bound A-I apolipoprotein (bound A-I) with alpha mobility and a small immunoprecipitin peak of free A-I with pre-beta mobility. The concentrations of free A-I and bound A-I were determined in 7 patients with acute hepatitis (AH) and in 3 patients with obstructive jaundice (OJ) by a "multiple" CIEP designed so that four samples could be simultaneously determined on one agarose-gel plate. The concentrations of bound A-I and free A-I in 40 normolipidemic healthy individuals were 126.5 +/- 16.3 mg/dl and 7.2 +/- 2.2 mg/dl (means +/- SD), respectively. The characteristic findings in CIEP for apo A-I obtained on patients with AH and OJ were the pronounced decrease in bound A-I and the appearance of double peaks with pre-beta mobility, the one on the cathodic side of which was proved to be bound A-I. Hypo-A-I apolipoproteinemia recognized in patients with AH and OJ was due to the decrease of bound A-I. In parallel with the recovery of the hepatic function after treatment, the reduced concentration of bound A-I was gradually normalized, accompanied by restoration of the level of total apo A-I. However, the concentration of free A-I increased in AH and OJ complicated with hypertriglyceridemias, and was not affected by the change in liver function. These findings suggest that free A-I originates from organs other than the liver whereas bound A-I certainly originates from the liver. The determination of bound A-I is clinically useful in AH and OJ.