Crossed Affinity Immunoelectrophoresis Research Articles

Glycoforms of alpha 1-acid glycoprotein in sera of human immunodeficiency virus-infected persons.

In acute infections thus far studied, there is a relative increase in plasma protein glycoforms rich in biantennary complex type N-glycans (type I), while in some diseases with chronic inflammatory changes, there is increase in glycoforms with more branched N-glycans (type II). In sera of 109 human immunodeficiency virus (HIV)-infected persons, 38 rheumatoid arthritis patients, and 44 healthy subjects, the composition of alpha 1-acid glycoprotein (AGP) glycoforms was studied using crossed immunoaffinity electrophoresis with concanavalin A as a ligand. In patients in CDC classifications I, II, and III, distribution of AGP glycoforms was analogous to that in normal subjects. Type I alterations were observed in patients in group IV who had no signs of arthritis. Type II changes, analogous to those found in rheumatoid arthritis, were seen in group IV patients who developed arthritis. Most significant type I changes were associated with Pneumocystis carinii pneumonia (specificity, 100%; sensitivity, 96%).

Detection of Choristoneura fumiferana Brush Border Membrane-Binding Molecules Specific to Bacillus thuringiensis δ-Endotoxin by Crossed Affinity Immunoelectrophoresis

Brush border membrane vesicles (BBMVs) prepared from midguts of Choristoneura fumiferana larvae were pretreated separately with the three CryIA δ-endotoxins from Bacillus thuringiensis subsp. kurstaki HD-1. Crossed affinity immunoelectrophoresis of the solubilized toxin-BBMV complexes, using antibodies raised against the toxins, revealed at least two peaks for each toxin. All complexes (BBMV bound toxin) migrated in the opposite direction to that of the free toxins. It is concluded that the toxins form stable complexes with membrane acceptor molecules, possibly receptors, and that there are at least two types of acceptor molecules present in the midgut epithelial cells of C. fumiferana that bind to all three toxins. The relative size of one of the BBMV-toxin peaks correlates with the in vivo potency of the toxins against C. fumiferana.

Effects of alpha-1 acid glycoprotein on human polymorphonuclear neutrophils: influence of glycan microheterogeneity

The biological functions of alpha-1 acid glycoprotein (AGP) are poorly understood but appear to depend on glycan microheterogeneity. Variations of AGP glycan structure (in terms of concanavalin A (ConA) reactivity) have been observed during the inflammatory process. We studied these modifications in AGP from patients with chronic renal impairment and investigated the effects of AGP microheterogeneity on healthy polymorphonuclear leukocyte (PMN) chemotaxis and oxidative metabolism. AGP was extracted by a two-step procedure from sera from ten patients with various degrees of renal impairment, selected according to AGP glycan heterogeneity determined by crossed immunoaffinity electrophoresis with ConA. AGP (0.5 g/l) significantly inhibited the chemotatic response of PMN to formyl-methionyl-leucyl-phenylalanine (10 -7 mol/l) and complement fraction C5a, regardless of ConA reactivity. AGP also inhibited superoxide anion generation in response to phorbol myristate acetate (10 -7 mol/l). After stimulation by opsonized zymosan (1 g/l), the effect of AGP appeared to depend on its glycan structure ( r = 0.70, P < 0.05), decreasing with ConA non-reactivity. These data suggest that AGP can down-regulate neutrophil responsiveness, an effect that depends in part on its glycan microheterogeneity. Alterations of AGP microheterogeneity in various pathological states, particularly renal failure, may be related to the inflammatory process.

Antitumor Effect of Intratumoral Administration of Biological Response Modifiers: Induction of Immunosuppressive Acidic Protein, a Type of α1‐Acid Glycoprotein, in Mice

The antitumor effects of biological response modifiers (BRMs) in an experimental mouse model, the “double grafted tumor system” were analyzed. Male BALB/c mice received simultaneous inoculations of Metn‐A fibrosarcoma cells on the right flank (106 cells) and left flank (2 × 105 cells) on day 0, and BRMs were injected intratumorally into the right tumor on days 3, 4 and 5. PSK (a protein‐bound polysaccharide preparation), interleuldn‐1 (IL‐1) and cepharanthin (R) cured not only the right, but also the left, non‐treated tumor in a double grafted tumor system. OK‐432 (a Streptococcus preparation) and BCG and tumor necrosis factor (TNF) cured the right tumor and inhibited the growth of the left tumor. Lentinan (a polysaccharide preparation) and IL‐6 inhibited neither the right nor the left tumor. Immunosuppressive acidic protein (IAP) in serum was increased transiently soon after intradermal injection of PSK, CR, OK‐432 and TNF in BALB/c mice. Lentinan, however, did not induce IAP. IAP in serum was gradually increased after intradermal inoculation of Meth‐A tumor in BALB/c mice. The biochemical difference between PSK‐induced IAP (early, inflammatory IAP) and Meth‐A‐induced IAP (late, tumor‐induced IAP) was investigated by crossed affinity immunoelectrophoresis with concanavalin A. IAP of murine serum was separated into 4 peaks. IAP in normal mouse was rich in high‐mannose type sugar chain (Peak 3) and contained no hybrid‐type sugar chain (Peak 4), which was present in inflammatory and tumor‐induced IAP. Inflammatory IAP was rich in biantennary sugar chain (Peak 2) and tumor‐induced IAP was rich in tri‐tetraantennary sugar chain (Peak 1).

Hereditary Increase of Plasma Histidine-Rich Glycoprotein Associated with Abnormal Heparin Binding (HRG Eindhoven)

Plasma histidine-rich glycoprotein (HRG) was found to be persistently increased in a patient with a history of recurrent arterial thromboembolic events. The mean concentration was 270% of normal pooled plasma. Increased HRG was found in eight of the 17 relatives studied, but none of them has experienced thrombo-embolism yet. Apparently, increased HRG was hereditary with autosomal dominant inheritance. A significant correlation was found between the increased plasma concentration of the protein and the age of the subjects (P < 0.02), whereas no such relation is present in a normal population. The plasma HRG of the proposita and 9 of her family members displayed abnormal binding to heparin, as assessed in a crossed affinity immuno-electrophoresis system: the usual increase in mobility after binding to heparin was absent. The binding of this variant HRG to plasminogen was normal. This case represents the first abnormal HRG variant reported and it is proposed to designate it: HRG Eindhoven.

Changes in serum glycoprotein glycosylation during experimental inflammation in mice are general, unrelated to protein type, and opposite changes in man and rat: studies on mouse serum ?1-acid glycoprotein, ?1-esterase, and ?1-protease inhibitor

Using the method of crossed affinity immunoelectrophoresis with concanavalin A in combination with digital image processing, unrelated serum glycoproteins from normal mice and from mice undergoing an experimentally induced inflammation were analyzed for their carbohydrate-derived microheterogeneity profile. This profile changed in a generalized way in mouse serum samples taken at various time intervals after the initial induction of inflammation. The changes are not related to the acute-phase behavior of the protein itself (be it positive, negative, or nonreacting), and they are opposite to the changes in microheterogeneity profiles reported previously for glycoproteins during the acute phase in human and rat sera. These findings are discussed in the context of the biosynthetic control of glycoprotein glycosylation during the acute-phase response.

Variations in the glycoforms of serum alpha 1-antichymotrypsin in liver diseases and after liver transplantation.

1. Using crossed immunoaffinity electrophoresis with free concanavalin A in the first dimension, we studied the microheterogeneity of alpha 1-antichymotrypsin due to various glycoforms in sera from patients with various liver diseases and after liver transplantation. 2. Studies by isoelectric focusing and immunoblotting and by crossed immunoelectrophoresis without concanavalin A in the first dimension allowed us to show that there is no dramatic variation in electrophoretic heterogeneity of alpha 1-antichymotrypsin in the serum of patients with liver diseases or after liver transplantation when compared with that of normal subjects. Therefore the heterogeneity observed in crossed immunoaffinity electrophoresis is due to various interactions with concanavalin A. 3. The results were expressed as the ratio of concanavalin A non-reactive glycoforms plus concanavalin A weakly reactive glycoforms to concanavalin A reactive glycoforms, called R alpha 1-ACT. R alpha 1-ACT was significantly higher in patients with cirrhosis (n = 53) when compared with normal subjects (n = 30). The median R alpha 1-ACT was 1 (range 0.72-1.25) in normal subjects. It was 1.6 (range 1.18-3.02), 1.45 (range 0.65-4.12) and 2.24 (range 1.03-19) in cirrhosis of Child's grade A, B and C, respectively. There was a dramatic decrease in glycoforms with mostly biantennary glycans in some patients with Child's grade C cirrhosis. Serum levels of alpha 1-antichymotrypsin were lower than normal only in some patients with Child's grade C cirrhosis. 4. Among the patients with acute viral hepatitis studied (n = 17), five were studied longitudinally.(ABSTRACT TRUNCATED AT 250 WORDS)

Counterflow affinity isotachophoresis on cellulose acetate membranes

Counterflow isotachophoresis on cellulose acetate membranes of human alpha-fetoprotein (AFP) was performed with concanavalin A, lentil lectin, and castor bean lectin driven by electroendosmotic counterflow. This counterflow caused a uniform stream of lectin to migrate towards the cathode against AFP with carrier ampholytes in steady-state position. Retardation of microheterogeneity forms bound to lectins was observed, giving results comparable to standard crossed affinity immunoelectrophoresis. Smaller amounts of lectins and more diluted samples of AFP could be used in the described method.

Microheterogeneity of α1-antichymotrypsin in the management of giant-cell arteritis and polymyalgia rheumatica

1. Using crossed immunoaffinity electrophoresis with free concanavalin A in the first dimension, we studied the glycan microheterogeneity of alpha 1-antichymotrypsin in sera from patients with giant-cell arteritis and/or polymyalgia rheumatica, and its variation in the serum of several of these patients during induction of disease remission by prednisone therapy and possible further recurrence of giant-cell arteritis and/or polymyalgia rheumatica. 2. In the serum of patients with active disease we observed increased proportions of concanavalin A nonreactive and concanavalin A weakly reactive fractions. The results were expressed as the ratio of concanavalin A non-reactive fraction plus concanavalin A weakly reactive fraction to concanavalin A reactive fraction, called R alpha 1-ACT. An R alpha 1-ACT higher than 1.8 (upper normal value) was found in 30/31 sera from patients with active disease (sensitivity 97%) and in 2/22 sera from patients with inactive disease (specificity 91%). 3. The erythrocyte sedimentation rate and the serum C-reactive protein level, common markers of biological inflammation, are usually elevated in active giant-cell arteritis and/or polymyalgia rheumatica. The two parameters are commonly used to guide the therapy during the course of the disease, but they have no specificity. An erythrocyte sedimentation rate of greater than 30 mm/h was found in 30/31 sera from patients with active disease (sensitivity 97%) and in 5/22 sera from patients with inactive disease (specificity 77%).(ABSTRACT TRUNCATED AT 250 WORDS)

Immunochemical identification and preliminary characterization of a nonfimbrial hemagglutinating adhesin of Bacteroides gingivalis

A cell-bound hemagglutinating adhesin (HA-Ag2) of Bacteroides gingivalis was identified by crossed immunoaffinity electrophoresis as one of the common antigens of the species. A polyclonal antiserum with a restricted specificity for HA-Ag2 was produced by immunizing with the relevant immunoprecipitate excised from crossed-immunoelectrophoresis gels. The immunoglobulin G fraction of this monospecific antiserum inhibited hemagglutination. The antiserum was used against a cell surface extract of B. gingivalis in immunoblotting experiments, and we detected two antigens with apparent molecular masses of 33 and 38 kilodaltons in B. gingivalis ATCC 33277 and W83. Monoclonal antibody, C1.17, produced in another laboratory against B. gingivalis 381 and characterized as showing reactivity with a hemagglutinin of this strain (Y. Naito, K. Okuda, T. Kato, and I. Takazoe, Infect. Immun. 50:231-235, 1985), was also used to produce immunoblots of extracts of strains ATCC 33277 and W83. The apparent molecular masses of the major polypeptides recognized by monoclonal C1.17 in the immunoblots were the same as those detected by the polyclonal monospecific antiserum, i.e., 33 and 38 kilodaltons. Significantly, none of the polypeptides identified in this study corresponded to the polypeptide appearing in the 41- to 43-kilodalton region and identified by Yoshimura and co-workers (F. Yoshimura, K. Takahashi, N. Yoshinobu, and T. Suzuki, J. Bacteriol. 160:949-957, 1984) as the fimbrial protein characteristic of the species. Enzyme-linked immunosorbent assay inhibition experiments with the monospecific antiserum indicated that the cell surface extracts from strains ATCC 33277 and W83 were strong inhibitors, whereas the fimbria-enriched preparations from both strains failed to inhibit binding of antibodies to the cell surface antigens. As a whole, our study indicates that a nonfimbrial surface protein complex demonstrating erythrocyte-binding capacity, HA-Ag2, is common to three strains of B. gingivalis and is composed of at least two associated polypeptides with apparent molecular masses of 33 and 38 kilodaltons which share at least one antigenic determinant.

Concanavalin A crossed affinity immunoelectrophoresis and image analysis for semiquantitative evaluation of microheterogeneity profiles of human serum transferrin from alcoholics and normal individuals.

The microheterogeneity profile of human serum transferrin from normal and alcoholic subjects was investigated qualitatively and quantitatively by means of Concanavalin A crossed affinity immunoelectrophoresis and an image analysis program. Differences in amounts of nonreacting transferrin molecules were found, suggesting an increase in triantennary glycosylation of transferrin from alcoholics compared with normal individuals. The increased amount of a highly retarded fraction in crude sera from alcoholics was demonstrated to be artefactual, probably due to entrapment or coprecipitation as the fraction disappeared after repeating the analysis with immunosorbent-purified transferrin. In conclusion, affinity electrophoresis represents a simple approach for demonstration of variations in the neutral monosaccharides of glycans and can discriminate between transferrin from alcoholics and normal individuals.

Con A non‐reactive fractions of human amniotic fluid alpha‐fetoprotein in prenatal diagnosis of fetal neural tube defects and fetal abdominal wall defects

During a 6-year period 19,647 consecutively received amniotic fluid samples were analyzed for the alpha-fetoprotein (AFP) concentration. In samples with an AFP concentration above the 95% upper normal limit the fraction of AFP non-reactive with concanavalin A (con A) was determined by crossed affinity immunoelectrophoresis. Of 436 samples from normal pregnancies with a moderate elevation of AFP (between the 95% limit and 2.5 times the median) the con A fraction was normal in 92 to 98% of the cases, depending on the gestational age. At significantly elevated AFP concentrations (above 2.5 times the median), the fractions were normal in 78% of the 50 cases with a normal outcome. In 80 of 89 cases of fetal malformations, abnormally low fractions were found, and in all 19 cases of abdominal wall defects and congenital nephrosis the fractions were low. No significant difference was found in the sensitivity for detection of fetal malformations by con A analysis compared to quantitative analysis for acetylcholinesterase (AChE). However, significantly more false positive results were found in normal pregnancies for the con A analysis compared to the AChE analysis. Both analyses performed significantly better than ultrasound scanning in terms of sensitivity. It is concluded that AChE is the test of choice in prenatal diagnosis of fetal neural tube defects. However, a sensitivity of 1.00 and a predictive value of a negative test of 1.00 were found for the con A analysis for fetal abdominal wall defects. The AchE analysis performed less well in this context.

Dog alpha-1-acid glycoprotein: Purification and biochemical characterization

Dog alpha-1-acid glycoprotein was purified to homogeneity from dog serum in a three-step procedure involving precipitation with sulphosalicylic acid, isoelectric focusing and size exclusion chromatography. The molecular heterogeneity in the peptide part and in the carbohydrate part of the molecule was investigated with analytical isoelectric focusing in a narrow pH range and crossed immunoaffinity electrophoresis with concanavalin A (con A) in the first-dimension gel. Up to seven molecular forms with different isoelectric points were found, whereas only a single con A-dependent molecular form was detected.

Application of monoclonal antibodies for affinity electrophoresis

Monoclonal antibodies (MoAbs) to human alpha-fetoprotein (AFP) produced by 13 different murine hybridoma clones were used in affinity electrophoresis. The binding of MoAbs to AFP was studied in different experimental systems: crossed affinity immunoelectrophoresis, rocket affinity immunoelectrophoresis and zone affinity electrophoresis in agarose. Experimental data indicated that MoAb-AFP and AFP-MoAb-AFP complexes were formed during electrophoresis. Affinity electrophoresis seems to be a convenient and sensitive method for studies on the reaction of monoclonal antibodies with their antigens. In addition, the technique offers the possibility of calculating the dissociation constants of MoAb-AFP and of AFP-MoAb-AFP complexes and evaluating the reaction of MoAbs with different antigenic determinants.

Crossed affinity immunoelectrophoresis of the escherichia coli pyruvate dehydrogenase complex

The crossed immunoelectrophoretic pattern obtained with the intact pyruvate dehydrogenase complex of Escherichia coli can be modified when this technique is combined with affinity gel electrophoresis using reactive dyes coupled to agarose as ligands. The patterns that arise have been interpreted with respect to localization of the three component enzymes. This was realized by using antibodies with different specificity, active enzyme staining and E 2–E 3 subcomplex behaviour. Dissociation of E 1 subunits occurs more easily than that of E 3 but remains incomplete in this system. The free reactive Procion Blue-MX dyes tested inactivate the complex even at neutral pH. The dyes react with all three components but E 3 (80%) and E 2 (15–20%) retain part of their catalytic activity. Modification leads to an enhanced dissociation of E 1.

Amniotic fluid analysis in prenatal diagnosis of neural tube defects: a comparison between six biochemical tests supplementary to the measurement of amniotic fluid alpha-fetoprotein.

Concanavalin A (con A) and lens culinaris agglutinin (LCA) microheterogeneity pattern of AFP (crossed affinity immunoelectrophoresis), alpha-2-macroglobulin and synaptic membrane protein D-2 (rocket immunoelectrophoresis) and qualitative (polyacrylamide gel electrophoresis) and quantitative (enzyme kinetic reaction rate) acetylcholinesterase were analysed in 87 consequtive samples from normal pregnancies and 37 abnormal samples (fetal neural tube defect or abdominal wall defect). Very few false positive results were obtained in normal pregnancies with any of the tests. In all cases of neural tube defects the correct result was obtained with qualitative acetylcholinesterase analysis, whereas only 2/3 of the abdominal wall defects were correctly predicted. Testing with con A or LCA was less optimal in neural tube defects, whereas all abdominal wall defects could be predicted correctly. Acetylcholinesterase in the quantitative test and protein D-2 did not decrease the rate of false results. Determination of the alpha-2-macroglobulin concentration performed well in the present study, but is not recommended because of the very high susceptibility to contamination of amniotic fluid with fetal or maternal blood.

Heparin-binding platelet proteins demonstrated by crossed affinity immunoelectrophoresis.

Platelet proteins that interact with heparin were studied using crossed affinity immunoelectrophoresis. Platelet proteins solubilized in Triton X-100 were applied to crossed immunoelectrophoresis against anti-platelet antibodies, and an intermediate gel containing heparin covalently linked to Sepharose 4B was inserted. Six immunoprecipitates were absent or showed an altered position compared to control immunoplates, indicating that the corresponding antigens were bound to the immobilized heparin. These precipitates represented platelet factor 4, thrombospondin, glycoprotein Ib, and three antigens termed G4, 17 and 25. The subcellular location of the heparin-binding proteins was either in the surface membrane (glycoprotein Ib and the antigens 17 and 25), or in the alpha-granules (platelet factor 4, thrombospondin and G4). both forms of platelet factor 4 appearing after crossed immunoelectrophoresis, i.e. a line-form and a peak-form, bound strongly to the heparin. Glycoprotein Ib showed a weak binding whereas its proteolytic split product glycocalicin did not significantly bind to the heparin in the present system. It is concluded that the platelets contain at least six heparin-binding proteins which are present on the cellular surface or are able to be exposed to the extracellular medium after the release-reaction has occurred.

Differences in the carbohydrate moieties of plasma and amniotic fluid fibronectins as revealed by crossed immunoaffinity electrophoresis with concanavalin A.

Human fibronectins isolated from pooled human plasma and amniotic fluid were studied as to differences in their carbohydrate moieties. The chemical analyses showed that amniotic fluid fibronectin is different from adult plasma fibronectin in carbohydrate content and composition while there seems to be no significant differences in amino acid composition. Crossed immunoaffinity electrophoresis with free concanavalin A, as well as rocket immunoelectrophoresis with immobilized concanavalin A intermediate gel, indicated that amniotic fluid fibronectin has little or no reactivity with this lectin while adult plasma fibronectin is strongly reactive. Fetal cord plasma fibronectin apparently interacted with concanavalin A, but its reactivity was weaker than that of adult plasma fibronectin. Fibronectin isolated from ascitic fluid of an ovarian cancer patient which was examined in additional experiments showed much weaker Con A-reactivity than fetal cord plasma fibronectin. These results suggest that fibronectins from various body fluids differ in their carbohydrate structures.

Comparison of amniotic fluid alpha-fetoprotein reactivity to Lens culinaris agglutinin and concanavalin A in crossed-affinity immunoelectrophoresis: ancillary tests in the prenatal diagnosis of severe fetal malformations.

Crossed-affinity immunoelectrophoresis of alpha-fetoprotein in amniotic fluid from 135 normal and 39 abnormal pregnancies (mainly neural-tube defects) was performed with a lectin, agglutinin from Lens culinaris. Results were compared with findings reported for another lectin, concanavalin A. Three fractions of alpha-fetoprotein were obtained by reaction with Lens culinaris agglutinin. The most weakly reactive fraction correlated strongly with the concanavalin A nonreactive fraction. With both lectins, significantly lower concentrations of these fractions were found in all samples from abnormal pregnancies than in those from normal pregnancies. In eight cases with a fetal abnormality the total alpha-fetoprotein concentrations were below the lower limit for abnormal samples. All eight samples revealed fractions weakly reactive with Lens culinaris agglutinin below the lower 0.1% limit for normal samples; seven of these samples had fractions nonreactive with concanavalin A below this limit. Although a decrease in the two described fractions in normal pregnancies was found with increasing gestational age, we find either method to be valuable as an ancillary test in the prenatal diagnosis of fetal neural-tube defects and other malformations.

Systematic use of affinity difference between immobilized lectin gels for demonstration of glycoprotein molecular variants : The example of radish β-fructosidase

The glycoprotein β-fructosidase from radish seedlings was used as an example. The extended charge heterogeneity of this enzyme does not allow analytical investigations on the microheterogeneity of the carbohydrate moiety by lectin crossed affinity immunoelectrophoresis. This microheterogeneity was investigated by a new approach of affinity chromatography on to columns of immobilized concanavalin A and Lens culinaris agglutinin displaying different affinities. This series of gels allowed a new approach for the detection and control of the microhetergeneity of the glycoprotein sugar moiety. The general chromatographic procedure requires (1) flat gradient elution to observe the heterogeneous forms, (2) re-chromatography and/or crossed chromatography for monitoring the biological significance of the heterogeneous elutions observed and (3) systematic “crossed chromatography” for the detection of further heterogeneous forms.