Immunoelectrophoretic Studies Research Articles

LACK OF STIMULATION OF SUCRASE AND ISOMALTASE WITH FRUCTOSE IN CHILDREN WITH SUCROSE-ISOMALTOSE MALABSORPTION |[lpar]|S-IM|[rpar

Top of pageAbstract In normal adults fructose stimulates the activity of sucrase and maltase. In a single patient with S-IM Greene et al. (1) found an increase in the sucrase activity after fructose ingestion (~ 40-60% of the caloric intake (c.i.)) and suggested dietary fructose as a form of therapy to these patients. In order to elucidate this possibility we have by methods earlier described (2) investigated the activity and rocket immunoelectrophoretic pattern of the dissaccharidasis in small intestinal biopsies from 2 children with S-IM (age 13 and 16 yrs) before and 2 weeks after a daily intake of an otherwise isocaloric diet containing 300 g of fructose (~50% of c.i.). Before the diet both children showed a sucrase activity below detection level (1 U/g prot.) and a residual isomaltase activity of 7, 9 and 2, 9 U/g prot., respectively. After the diet there was still no sucrase activity and the changes in the in the isomaltase level were not significant. The immunoelectrophoretic studies were in complete concordance with these findings. In conclusion even a very high dietary supplement with fructose can not initiate an increase of the missing enzymes in S-IM and represents no therapeutic alternative. (1) Greene, H L et al. Biochem Med 1972; 6: 409. (2) Skovbjerg, H, Krasilnikoff, P A. Pediatr. Res 1981; 15: 214.

Modifications of the properties of human sex steroid-binding protein by nonesterified fatty acids.

The effect of unsaturated and saturated nonesterified fatty acids (NEFAs) on the electrophoretic, immunological, and steroid-binding properties of human sex hormone-binding protein (SBP) were investigated. Tests were carried out on whole serum from pregnant women and on purified SBP using polyacrylamide gel electrophoresis, crossed immunoelectrophoresis with autoradiography, and equilibrium dialysis. All three methods showed that NEFAs influence the binding of sex steroids to SBP both in whole serum and with the purified protein. Saturated NEFAs caused a 1.5-2-fold increase in binding of dehydrotestosterone, testosterone, and estradiol to SBP, while unsaturated NEFAs, such as oleic (18:1) and docosahexaenoic (22:6) acids inhibited the binding of these steroids to SBP. Thus, unsaturated NEFAs in the concentration range 1-100 microM are more inhibitory for estradiol binding than for testosterone or dehydrotestosterone binding. In addition to these binding changes, polyacrylamide gel electrophoresis and immunoelectrophoretic studies revealed a shift in SBP from the slow-moving active native form to a fast-moving inactive one. There was also a reduction in the apparent SBP concentration by Laurell immunoelectrophoresis in the presence of unsaturated NEFA (5.5 nmol of NEFA/pmol of protein). These studies indicate that unsaturated NEFAs induce conformational changes in human SBP which are reflected in its electrophoretic, immunological, and steroid-binding properties. They suggest that the fatty acid content of the SBP environment may result in lower steroid hormone binding and thus increased free hormone levels.

Effects of chloroquine on the serum complement system

Chloroquine, a well-known anti-malarial and anti-inflammatory agent, was studied with respect to its effect on the serum complement system. The drug exhibited significant in vitro anti-complementary activity only at a very high non-therapeutic dose of 48 mg/ml. Chloroquine-induced in vitro complement consumption was observed to take place even in the absence of Ca2+ and Mg2+ ions. The drug also haemolyses rabbit erythrocytes in the presence of Mg2+ -EGTA and immunoelectrophoretic studies of fresh human serum and chloroquine incubation mixture against specific anti-C3 and anti-factor B antisera have demonstrated that it cleaves both C3 and factor B. In another experiment, chloroquine failed to exert inhibitory effects on complement utilisation by immune complexes. Studies of the serum complement profile of Plasmodium falciparum-infected malaria patients receiving chloroquine therapy indicated that, in contrast to the situation in vitro, the serum C3 level is invariably decreased. Marginal reductions in the levels of C4, factor B and properdin were also found in some of these patients, while administration of chloroquine to normal human individuals failed to produce any significant change in their serum complement component profile. It is, therefore, probable that malarial parasites and not chloroquine are responsible for complement activation in patients suffering from malaria.

Differences in the structure of alpha-fetoprotein and its clinical use in pediatric surgery

Techniques have been studied which distinguish two variants of human alpha-fetoprotein (AFP) on the basis of characteristics of the carbohydrate moiety of this glycoprotein. AFP in serum samples from six children with tumors of yolk sac origin showed little concanavalin-A (Con A) binding. In contrast, Con A binding of AFP was almost complete in serum samples from 14 other subjects with elevated AFP, including two with liver-cell tumors, eight with neonatal cholestasis, and four normal newborn infants. Differences were confirmed by immunoelectrophoretic studies. Thus, AFP from cells of yolk sac origin can be distinguished from AFP from liver cells or from tumors of hepatic cell origin.

Immunoglobulins and complement components in human aortic atherosclerotic intima

Concentration and preferential retention of immunoglobulins and complement components were studied in comparison with other plasma proteins in 42 human aortae with atherosclerosis. Saline and acid extracted IgG, IgA, IgM, Clq, C3c, C4, C9, C3A, C-reactive protein, α 1-antitrypsin, α 2-macroglobulin, albumin, transferrin and fibrinogen were quantitatively determined using the radial immunodiffusion. The fibrous plaques and their adjacent areas contained higher levels of each protein than intima with only fatty streaks. No significant differences were found between the fibrous plaques and their adjacent areas presenting intimal thickenings. Saline eluted IgG and IgA were significantly higher in the fibrous plaque intima than in intimal samples with fatty streaks and were the only proteins detected in the acid eluates. The complement components were present in all saline eluates, while C-reactive protein was found in 23 samples. Crossed immunoelectrophoretic studies showed the activation of saline C3 and C4. In 8 cases serum levels of the studied proteins were compared with their concentration in saline eluates obtained from intima and media. The immunoglobulins and complement components presented higher intima/serum and lower media/intima retention ratios than the other studied proteins suggesting their preferential retention in the intima. The presence of immune related proteins in the atherosclerotic intima and their preferential retention might be explained not only by an altered permeability but also in relation to their function.

An update of the isolation and characterization of culture-derived soluble antigens of Babesia bovis

In this paper recent progress concerning the identification of soluble antigens from cultures of Babesia bovis parasites is reviewed. Soluble antigens present in the supernatant of B. bovis cultures have been shown to be efficient immunogens for induction of protective immunity against bovine babesiosis. Immunochemical analysis of culture supernatants has demonstrated that at least 3 parasite antigens are released in vitro. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that at least 2 of these antigens have molecular weights within the range of 37 000–40 000 daltons. Crossed immunoelectrophoretic studies have further revealed an antigenic spectrum consisting of 1 major and 2 minor antigens with mobilities in the albumin and alpha 1 region. Within the infected erythrocyte, 2 antigens have been localized on or near the erythrocytic membrane, while the third antigen appears to be directly associated with the parasite.

Isolation, characterization, and specificity of a glycoprotein antigen from Mycobacterium kansasii.

We have isolated in highly purified form and characterized a glycoprotein antigen from culture filtrates of Mycobacterium kansasii. Immunoelectrophoretic studies demonstrated that this antigen is present only in M. kansasii among 14 species of mycobacteria studied. It is a potent tuberculin skin test antigen, but skin test reactions to it in sensitized guinea pigs display only limited specificity. Enzyme-linked immunoabsorbent antibody assays of serum samples from patients suffering from diseases caused by M. kansasii, M. intracellulare, and M. tuberculosis display no specificity with this antigen. These findings are best explained by hypothesizing a single highly antigenic determinant that is present as a moiety on many mycobacterial antigens and is widely shared among mycobacteria.

Iron-binding proteins with vitreous humour

The soluble protein composition of Macaque monkey vitreous humour was studied in order to understand its iron-binding properties. The protein content of vitreous humour was 217 μg/ml ± 4.6%, 40% of which was serum albumin and 30% an iron-binding protein of hydrodynamic properties identical to that of trasferrin or lactoferrin. Relative to serum, the vitreous humour contained a 13-fold excess of this protein(s). Isoelectric focusing, iron-binding and immunoelectrophoretic studies indicated that both vitreous humour and aqueous humour contained lactoferrin as well as serum transferrin. The iron-binding capacity of these proteins in vitreous humour was equivalent to the mass of haemoglobin iron contained in at least 570 000 monkey erythrocytes. It was concluded that the intraocular lactoferrin originated from within the eye. These iron-binding proteins may play a protective role in ocular disturbances such as viterous haemorrahge, iron foreign body toxicity and infection.

Structural similarities between helix pomatia galactan and the carbohydrate moiety of hemocyanin

1. 1. Immunoelectrophoretic studies and quantitative precipitin reactions reveal that antisera raised against Helix pomatia galactans crossreact with the carbohydrate moiety of hemocyanin. 2. 2. Hemocyanin is heterogeneous in respect to its galactose content and can be fractionated by affinity chromatography on the insolubilized dGal-specific lectin of Axinella polypoides. 3. 3. Galactose is a major component in the carbohydrate moiety of hemocyanin as demonstrated by gas-liquid chromatography. 4. 4. Anti- Helix pomatia hemolymph antisera show a high degree of species specificity towards various snail galactans. Thus the carbohydrate moieties of hemocyanin are immunodominant groups and they are closely related to the species specific structures in the snail galactans.

Comparative Studies on Tree-Pollen Allergens

A comparative electrophoretic and autoradiographic analysis of the different fractions of birch pollen crude extract was performed. This extract included a minimum of 17 reproducible and distinct antigens located in the gel filtration fractions BV2–BV4. Only one of these precipitates had the ability to bind IgE, as demonstrated by immunoelectrophoretic and autoradiographic techniques using several poly- and monospecific rabbit antibodies. The allergenicity of the different fractions was examined in vitro by RAST and RAST inhibition, and in vivo by passive cutaneous transfer and skin prick tests. The data suggested that <i>Betula verrucosa </i>pollen extract contains a group of isoallergens with related antigenicity but with variable molecular sizes. The findings have also confirmed the presence of identical antigenic properties of the previously isolated pI 5.18 and BV45 as deduced from their immunoelectrophoretic and autoradiographic studies.

Purification of pig serum haemopexin by haemin-sepharose affinity chromatography

Haemopexin was isolated from pig serum in pure form by affinity chromatography and ion-exchange chromatography. The affinity gel synthesized contained about 0.3 μmol/ml haemin covalently linked to AH-Sepharose 4B. The molecular weight of the protein was measured by polyacrylamide gel electrophoresis in the presence of SDS followed by sensitive silver staining. This procedure and also immunoelectrophoretic studies indicated purity. The mobility of haemopexin in polyacrylamide gels changed following reduction with dithiothreitol.

Cell surface origin of antigens shed by Leishmania donovani during growth in axenic culture.

Antisera against isolated cell surface preparations (PCSP-As) of Leishmania donovani promastigotes were used to detect extracellular antigens produced during the growth of these organisms in four different growth media. The PCSP-As precipitated two major antigenically identical but electrophoretically distinct components, in addition to several minor antigens. Immunoelectrophoretic studies employing PCSP-As, PCSP-As absorbed with intact, live promastigotes, and PCSP-As absorbed with a major extracellular antigen demonstrated the antigenic identity between the major extracellular antigens and two major components externally disposed at the surface of promastigotes. Growth curve kinetic investigations suggested that the major extracellular antigens did not appear in the growth media primarily as a result of cell lysis or damage. The carbohydrate nature of the major extracellular antigens was indicated by physicochemical characterization.

Crossed-Immunoelectrophoretic Analyses of Trypanosoma cruzi Epimastigote, Metacyclic, and Bloodstream Forms

Sonicated suspensions of epimastigote, metacyclic, or bloodstream forms of Trypanosoma cruzi were emulsified in Freund's complete adjuvant. Rabbits immunized with epimastigotes or metacyclics received five intramuscular (i.m.) injections of 1 x 10(9) sonicated trypanosomes at weekly intervals. Immunization with bloodstream forms included three i.m. injections of 5 x 10(7) and six injections of 2 x 10(8) sonicated trypanosomes. Selected antisera from these rabbits were employed in crossed immunoelectrophoretic studies against the homologous or heterologous extracts of sonicated trypanosomes. Extracts of epimastigote, metacyclic, and trypomastigotes produced 31, 29, and 11 precipitin peaks respectively against the homologous rabbit antisera. Tandem, crossed-immunoelectrophoresis of these extracts against antiepimastigote or antimetacyclic sera revealed that epimastigotes or metacyclics may each have at least four antigens that did not appear to be shared by the other, whereas each of these forms may have at least eight or nine antigens that were not detected with extracts from trypomastigotes. Cross-absorptions of antiepimastigote or antimetacyclic sera with live trypanosomes caused marked reductions in the numbers of precipitin peaks formed against the homologous extracts, but cross-absorptions with sonicated suspensions of epimastigotes or metacyclics showed that epimastigotes or metacyclics each have at least two antigens that were not detected in extracts of the other. Differentiation appeared to be accompanied by antigenic change. More antigens appear to be shared by epimastigotes and metacyclic forms than by trypomastigotes and epimastigotes or metacyclics.

Demonstration of monoclonal lymphoplasmacytic proliferations by immunofluorescence on routine formalin-fixed, paraffin-embedded tissue.

This study explores the use of immunofluorescence on routine formalin-fixed paraffin-embedded tissue to distinguish monoclonal from polyclonal lymphoplasmacytic proliferations. Sixteen tissues containing plasma cell or lymphocyte and plasma cell proliferations were studied. Five-micron sections were deparaffinized in xylene, rehydrated, and treated with 0.1% trypsin for two hours. After washing with phosphate-buffered saline, separate sections were stained with fluorescein-conjugated antibody to IgG, IgA, IgM, IgD, and IgE, and a single section was stained with both fluorescein-conjugated anti-kappa and rhodamine-conjugated antilambda. The latter section was useful to distinguish nonspecific adsorption of the fluorochromes. Where possible, results were correlated with immunoelectrophoretic studies of serum and urine. Eleven specimens with monoclonal and four specimens with polyclonal lymphoplasmacytic proliferations were readily identified, including a case of giant lymph node hyperplasia with a monoclonal IgDK plasma cell component (confirmed by specific absorption studies). Identification of monoclonality by immunofluorescence preceded immunoelectrophoretic identification in one case. One other case gave equivocal results by fluorescence. Further, the method worked on formalin-fixed decalcified tissues, although a somewhat heavier background staining was noted. This method offers a simple, reliable technique to establish or confirm the diagnosis of monoclonal lymphoplasmacytic lesions.

Aging of the erythrocyte. X. Immunoelectrophoretic studies on the denaturation of superoxide dismutase

Superoxide dismutase (SOD) content estimated by rocket immunoelectrophoresis did not differ in various density (age) fractions of bovine erythrocytes though its specific activity decreased with increasing cell density. No changes were found, either, in the crossed immunoelectrophoretic pattern of SOD with increasing red cell age. Effects of different denaturing agents on the immunoelectrophoretic pattern and activity of the enzyme were compared, including H 2O 2 and free radicals generated by ionizing radiation. The yield for inactivation was higher than that for loss of antigenic properties in all cases studied which could correspond to the in vivo situation. However, changes in electro-phoretic mobility were revealed for all the denaturing agents, which is contrary to a main causative role of these agents in the in vivo inactivation of SOD. The results suggest spontaneous “subtle denaturation” as a chief cause for the SOD inactivation during erythrocyte aging.

Detection of circulating antigens in onchocerciasis.

This report describes the presence of circulating Onchocerca volvulus antigens (COA) in sera of patients with onchocerciasis. By using a double diffusion immunoelectrophoresis method, COA could be detected in 24 of 77 sera analyzed (31%). In contrast, when more sensitive assays such as the radioimmunoprecipitation-PEG assay or sandwich radioimmunoassay were used to detect COA, about 75% of the sera from O. volvulus-infected patients were found positive; moreover, a highly significant correlation between the two assays was observed. The parasite specificity of the COA was demonstrated directly by identity reaction with a component of O. volvulus somatic antigens. COA was never found when hyperimmune antisera against other parasite antigenic extracts were used instead of anti-O. volvulus hyperimmune serum. However, when anti-O, volvulus hyperimmune serum was used against sera obtained from patients infected with various other helminths we found a cross-reactivity between COA and the circulating antigens of other human filarids (Wuchereria bancrofti, Loa loa, Brugia malayi), but not with other nematode or trematode parasites (Ascaris lumbricoides, Schistosoma mansoni, Fasciola hepatica). Further immunoelectrophoretic studies demonstrated one precipitin are localized in the cathodic region which seemed specific for COA, which raises the possibility of preparing a monospecific hyperimmune serum to circumvent cross-reactivities.

Antibodies against platelet membrane glycoproteins. I. Crossed immunoelectrophoresis studies with antibodies that inhibit ristocetin-induced platelet aggregation.

Platelet membrane glycoproteins have been isolated by lectin-affinity chromatography and antibodies prepared against them. Platelets that have lost glycocalicin no longer respond to ristocetin-human VIIIR:WF, bovine VIIIR:WF, or to glycocalicin or glycoproteins Ia and Ib antibodies but are still agglutinated by glycoproteins IIb and IIIa antibodies. Glycoproteins Ia and Ib and glycocalicin antibodies, IgG and Fab' fragments, inhibited ristocetin-human VIIIR:WF-induced aggregation of fixed, washed platelets and of platelets in plasma while glycoproteins IIb and IIIa antibodies were without effect. Cross immunoelectrophoretic studies showed that glycocalicin was present on whole platelets in only trace amounts. Glycocalicin antibodies, however, recognized a slower migrating component. Platelets incubated in an EDTA-free medium no longer respond to ristocetin-human VIIIR:WF. Membranes isolated from such platelets contained glycocalicin which cross-reacted with a remnant of the slower migrating component. Glycoproteins Ia and Ib antibodies gave more complex patterns but it was possible to identify the slower moving component recognized by the glycocalicin antibodies. These results show that glycocalicin is not normally found as such on whole platelets but is present as a precursor which is most likely glycoprotein Ib. On degradation of this precursor, glycocalicin is released from the membrane and VIIIR:WF-receptor activity is lost.

Immunoelectrophoretic studies on human small intestinal brush border proteins—the longitudinal distribution of peptidases and disaccharidases

The longitudinal distribution of different brush border enzymes along the human small intestine was studied by crossed immunoelectrophoresis. The results are based on biopsies taken every 50 cm in three intestines obtained at autopsy and on peroral or peroperative biopsies from the ligament of Treitz, proximal jejunum and distal ileum from 11 patients undergoing jejunoileal bypass operation for obesity. Lactasephlorizin hydrolase (EC 3.2.1.23-62) and sucrase-isomaltase (EC 3.2.1.48-10) had their highest level in jejunum with decreasing activity towards the proximal and distal ends of the intestine, while maltase (EC 3.2.1.20) increased along the intestine and reached its highest activity in the distal ileum. A carboxypeptidase (EC 3.4.12.X) is demonstrated as an enzymatic entity of the human intestine. This enzyme had a rather flat distribution curve while microvillus aminopeptidase (EC 3.4.11.2), dipeptidyl peptidase IV (EC 3.4.14.X) and aspartate aminopeptidase (EC 3.4.11.7) all increased along the length axis and reached maximum values in distal ileum.

Immunoelectrophoretic studies on human small-intestinal brush-border proteins

The amounts of lactase (beta-D-galactosidase, EC 3.2.1.23), sucrase (sucrose alpha-D-glucohydrolase, EC 3.2.1.48), maltase (alpha-D-glucosidase, EC 3.2.1.20) microvillus aminopeptidase (EC 3.4.11.2) and dipeptidyl peptidase IV (EC 3.4.14.-) in tangentially sectioned biopsies from jejunum were studied by quantitative immunoelectrophoresis and enzymic assays. All enzymes had their maximum activities near the mid-region of the villi and their lowest activities at the bases of the crypts. The ratio between enzyme activity and immunoreactive protein was constant along the villus-crypt axis. This result is consistent with a continuous brush-border-enzyme synthesis as the enterocytes migrate up the villi.

Electrophoretic and immunoelectrophoretic studies of proteins in the aqueous phase of human dental plaque

The presence and amount of some anionic proteins in human plaque fluid were investigated by polyacrylamide gel electrophoresis and quantitative immunoelectrophoresis. Both methods showed albumin to be the major protein component. Of the 8–17 protein bands detected by gel electrophoresis, albumin, amylase, IgG, lactoferrin and IgA were consistently present. Quantitative immunoelectrophoretic studies detected IgA at a concentration similar to that in unstimulated submandibular saliva; IgG, albumin and lactoferrin were present in higher concentrations. IgA was detected at higher concentration by antiserum with specificity for secretory component than by antiserum with specificity for heavy chain only. The mean ratios of some plaque fluid constituents were: IgA:albumin (1:25); IgG:IgA (3.8:1); IgG:albumin (1:7); lactoferrin:IgA (3.6:1); lactoferrin:albumin (1:7). The concentrations of the plaque fluid proteins studied suggest that crevicular fluid greatly influences the protein composition of plaque fluid. The biological activity of IgA or IgG cannot be inferred because of the proteolytic activity in plaque fluid.