Affinity Electrophoresis Research Articles

Lectin affinity electrophoresis in a yolk sac tumour in the vagina with yolk sac tumour-type glycoform of alpha fetoprotein.

AIMS: To investigate a potential diagnostic use of alpha fetoprotein (alpha FP) isoform analysis by lectin affinity electrophoresis to distinguish between endodermal sinus tumours arising in the vagina in infants...

Immunochemical study of immunoglobulins bound to lactate dehydrogenase

Immunochemical analysis of λ type Bence Jones protein (BJP; designated as Suzuki-BJP) and IgG-λ type M-protein (designated as Miki-IgG), λ type BJP (designated as Miki-BJP) which showed non-specific binding with lactate dehydrogenase (LD, EC 1.1.1.27) was carried out in two cases. When the purified LD mixed with NADH was eluted through the CNBr-Sepharose 4B coupled to Suzuki-BJP or Miki-IgG, the affinity with these adsorbents was not demonstrated. The amino acid residue of the N-terminal in the Suzuki-BJP and λ chain of the Miki-IgG was determined to be tyrosine by primary structure analysis, on the other hand, alanine was detected in the γ chain of the Miki-IgG that did not have LD binding ability. By counter affinity electrophoresis, it was shown that LD bound to a synthetic peptide consisting of 15 amino acid residues of N-terminal which had the same β-sheet structure as the Suzuki-BJP. It seems probable that LD combines with BJP (or IgG) molecule at the NAD + binding site producing a three-dimensional structure similar to NAD +.

Sequence-dependent separation of DNA fragments by capillary electrophoresis in the presence of SYBR Green I.

BioTechniquesVol. 23, No. 1 BenchmarksOpen AccessSequence-Dependent Separation of DNA Fragments by Capillary Electrophoresis in the Presence of SYBR® Green IHiroshi Takai, Hisashi Yamakawa, Osamu Ohara & Junko Sakaguchi-InoueHiroshi TakaiBeckman Instruments, TokyoSearch for more papers by this author, Hisashi YamakawaBeckman Instruments, TokyoSearch for more papers by this author, Osamu OharaBeckman Instruments, TokyoSearch for more papers by this author & Junko Sakaguchi-Inoue*Address correspondence to Junko Sakaguchi-Inoue, 7457 Corliss Avenue North, Seattle, WA 98103, USA.Beckman Instruments, TokyoSearch for more papers by this authorPublished Online:7 Aug 2018https://doi.org/10.2144/97231bm10AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail "Sequence-Dependent Separation of DNA Fragments by Capillary Electrophoresis in the Presence of SYBR® Green I." , 23(1), pp. 58–60FiguresReferencesRelatedDetailsCited ByStudy on detection of mutation DNA fragment in gastric cancer by restriction endonuclease fingerprinting with capillary electrophoresis15 July 2011 | Biomedical Chromatography, Vol. 15Genetic mutation analysis by CE with LIF detection using inverse-flow derivatization of DNA fragmentsELECTROPHORESIS, Vol. 27, No. 19Capillary electrophoresis of double-stranded DNA fragments using a new fluorescence intercalating dye EvaGreen31 May 2006 | Journal of Separation Science, Vol. 29, No. 9Hot electron-induced electrogenerated chemiluminescence of SYBR® Green IAnalytica Chimica Acta, Vol. 541, No. 1-2Characterization of SYBR Gold Nucleic Acid Gel Stain: A Dye Optimized for Use with 300-nm Ultraviolet TransilluminatorsAnalytical Biochemistry, Vol. 268, No. 2Comparison of SYBR® Green I nucleic acid gel stain mutagenicity and ethidium bromide mutagenicity in the Salmonella/mammalian microsome reverse mutation assay (Ames test)Mutation Research/Genetic Toxicology and Environmental Mutagenesis, Vol. 439, No. 1Fluorescent Imaging of Nucleic Acids and Proteins in GelsVariability in the stability of DNA–peptide nucleic acid (PNA) single-base mismatched duplexes: Real-time hybridization during affinity electrophoresis in PNA-containing gels21 July 1998 | Proceedings of the National Academy of Sciences, Vol. 95, No. 15 Vol. 23, No. 1 Follow us on social media for the latest updates Metrics Downloaded 81 times History Published online 7 August 2018 Published in print July 1997 Information© 2018 Author(s)PDF download

Separation and quantification of corticosteroid-induced, bone and liver alkaline phosphatase isoenzymes in canine serum.

Quantifying alkaline phosphatase (ALP) isoenzymes in canine serum would provide a useful index in a clinical laboratory. To achieve this goal, we tested a semi-automatic assay combining wheat germ lectin (WGL) precipitation and chemical inhibition of isoenzymes of the TNS gene with levamisole to quantify bone ALP (BALP) and corticosteroid-induced ALP (CALP), respectively. The liver ALP (LALP) isoenzyme was then calculated from the equation: TALP = BALP + LALP + CALP BALP, LALP and CALP standards from serum of puppies, bile-duct ligated dogs and dogs on 4.4 mg/kg/day prednisolone for 30 days, respectively, were used. The suitability of standard sera was tested by affinity electrophoresis. Levamisole (4.2 mM) inhibits 98% of BALP and LALP but only 42% CALP. Multiplying measured CALP by 1.8 gives the total CALP value in serum. WGL precipitated 92.3% BALP, 23.3% LALP and 26.8% heated CALP standards. These values were used to adjust precipitated ALP to obtain the exact levels of BALP. WGL was then tested on pooled serum standards in which the relative proportions of all the ALPs were known and controlled. BALP was adequately quantified except when LALP and CALP levels were extremely high. The assay was also applicable under conditions resulting in high ALP. Therefore, combining WGL and levamisole inhibition provides an adequate separation and quantification of canine ALP isoenzymes. The method has great potential for diagnostic use and should be tested further for routine implementation.

Analysis of the affinity of each haptoglobin polymer for hemoglobin by two-dimensional affinity electrophoresis

Using polyacrylamide gel electrophoresis we have often encountered a decrease in the high molecular weight polymers of haptoglobin 2-1 in the serum of patients after transfusion and with hemolytic diseases. Using two-dimensional affinity electrophoresis we have attributed this phenomenon to the high affinity of high molecular weight polymers of the haptoglobin 2–1 for hemoglobin.

Chiral separations in capillary electrophoresis using proteins as stereoselective binding agents.

Proteins are receiving increased attention in capillary electrophoresis (CE) for use as ligands in chiral separations. This review examines the types of proteins that have been employed in CE for the analysis of chiral compounds and provides a summary of applications for such an approach. Several formats for this type of analysis are discussed, including methods that use immobilized proteins (i.e., affinity capillary electrochromatography and capillary gel affinity electrophoresis) or techniques that use proteins as running buffer additives (i.e., affinity capillary electrophoresis). The role of various experimental factors in these separations are considered, and a comparison is made between the general analytical properties of CE systems that use immobilized versus solution-phase proteins.

Capillary affinophoresis of fluorescence-labeled pea lectin with laser-induced fluorescence detection.

Affinophoresis is a type of affinity electrophoresis in which an affinophore, a conjugate of an affinity ligand and a multiply charged soluble matrix, causes a mobility change in proteins that have specific affinity for the ligand. This technique enables, not only the separation of a specific molecule, but also the analysis of specific interaction between biomolecules. The affinity constant between fluorescence-labeled pea lectin and affinophores bearing mannoside as an affinity ligand, was determined by capillary electrophoresis using laser-induced-fluorescence detection. Although the labeling of the protein caused microheterogeneity as evidenced by electrophoretic mobility, the affinity constant determined for the main peak in the electropherogram of the labeled lectin agreed with those obtained for unlabeled lectin by the same type of analysis using ultraviolet-absorption detection. As a result of the use of the fluorescence detection, the consumption of the protein sample is greatly reduced to several pg per run.

Determination of the affinity constants of pea lectin for neutral sugars by capillary affinophoresis with a monoligand affinophore.

Affinophoresis is a type of affinity electrophoresis in which an affinophore, a conjugate of an affinity ligand and a multiply charged soluble matrix, causes a change in migration velocity of proteins which have a specific affinity for the ligand. A monoligand affinophore bearing a mannoside was prepared by coupling iodoacetylated p-aminophenyl alpha-D-mannoside to the free thiol group of N-succinylated glutathione, and used for the affinophoresis of pea lectin in a capillary. The electrophoretic mobility of pea lectin towards the anode increased in the presence of the affinophore as a function of its concentration in a manner that is described by the equation for affinity electrophoresis. Analysis of the suppression of the affinophoresis on the addition of neutral sugars to the system allowed the determination of the dissociation constants of the lectin for these neutral sugars. The dissociation constants obtained on affinophoresis agreed well with the values in the literature. The preparation of a monoligand affinophore for ligands bearing an amino group should facilitate the application of this type of microscale analysis (0.14 ng of protein for each run) to protein ligand interactions.

Comparison of carbohydrate structures of serum α-fetoprotein by sequential glycosidase digestion and lectin affinity electrophoresis

Serum α-fetoprotein (AFP) is a glycoprotein of which the sugar chain is considered to show structural changes with malignancies. Microheterogeneity of the serum AFP carbohydrate structure was studied in samples from 35 patients with benign and malignant diseases. Sera were digested directly, extensively, and sequentially with sialidase, ß-galactosidase and ß- N-acetylhexosaminidase. Before and after digestion, sera were examined by means of lectin affinity electrophoresis using eight lectins. Relationships between AFP carbohydrate structures and liver diseases were elucidated by the lectin-reactive profiles and the effect of glycosidase digestion. More than 94% of the AFP carbohydrate structures found in patients with benign and malignant liver diseases were biantennary complex-type oligosaccharides. Changes in the AFP carbohydrate structures at the early stage of hepatocellular carcinoma revealed the addition of α1 → 6 fucose to the reducing terminal N-acetylglucosamine and monosialylated AFPs. In both advanced hepatocellular carcinoma and AFP producing extrahepatic malignancies, AFP carbohydrate structures were characterized as the further addition of ß1 → 4 N-acetylglucosamine and heterogeneity in the galactose and N-acetylglucosamine residues. Sequential glycosidase digestion and lectin affinity electrophoresis is useful for analysing the carbohydrate structures of serum glycoprotein.

Prognostic significance of Lens culinaris agglutinin A-reactive alpha- fetoprotein in small hepatocellular carcinomas

BACKGROUND & AIMS: Lens culinaris agglutinin A-reactive fraction of alpha-fetoprotein (AFP-L3) has been reported to be a useful marker in the early diagnosis of hepatocellular carcinoma (HCC). The aim of this study was to evaluate the prognostic value of AFP-L3 for HCC. METHODS: Fifty-five patients with HCC whose AFP-L3 levels were negative before initial therapy were studied. AFP-L3 levels were measured by lectin- affinity electrophoresis coupled with antibody-affinity blotting. RESULTS: Of the 55 patients, 28, 15, and 12 underwent percutaneous ethanol injection, transcatheter arterial embolization, and hepatectomy, respectively. Thirty-two (58.2%) of the 55 patients maintained a negative AFP-L3 status during the study, and 23 patients (41.8%) became positive for AFP-L3 during posttreatment observation. Multiple recurrences of HCC and portal vein tumor thrombus were observed significantly more often in patients with positive AFP-L3 than in those with negative AFP-L3 status (P < 0.0001). In contrast, most patients with negative AFP-L3 had solitary recurrences of HCC without portal vein tumor thrombus. Overall survival was significantly lower in patients with positive AFP-L3 than in those with negative AFP-L3 status (P = 0.0006). Cox's proportional hazards model identified that AFP-L3 was an independent prognostic factor (P = 0.0005). CONCLUSIONS: AFP-L3 seems to be a significant marker of poor prognosis for HCC. (Gastroenterology 1996 Oct;111(4):996-1001)

Biochemical characterization of alpha-fetoprotein and other serum proteins produced by a uterine endometrial adenocarcinoma.

A high serum α‐fetoprotein (AFP) level was found in a patient with endometrial adenocarcinoma of the uterus, which appeared to be hepatoid on histological examination. The AFP of this unusual patient was purified hy immunoaffinity chromatography and characterized. The electrophoretic profiles on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis both before and after glycopeptidase F treatment were indistinguishable from those of a hepatoma AFP. This indicates that the patient's AFP was also composed of a single polypeptide chain of Mr 67,000 and an N‐linked sugar chain of Mr 3,000. Amino acid sequence analyses of this AFP, and of AFP from hepatoma and umbilical cord serum indicated that the N‐terminal sequences were essentially the same. The sequence, Arg‐Thr‐Leu‐His‐Arg‐Asn‐Glu‐Tyr‐Gly‐Ile, was slightly different from previous reports, but matched that deduced from the cDNA sequence. AFP isoforms due to microheterogeneity of the sugar chain were analyzed by lectin affinity electrophoresis using a series of lectins. The AFP isoform profiles were distinct from those of proteins derived from cord serum, hepatoma, yolk sac tumor and gastric cancer. The reverse‐transcription of RNA from the tumor tissue followed by a polymerase chain reaction using primers with AFP‐specific sequences gave a product of the size and nucleotide sequence expected for AFP. mRNAs possessing the requisite sequences for albumin and transferrin syntheses were also detected in the tumor. The expression of these hepatocyte‐specific proteins supported the hepatoid nature of this tumor.

Lectin affinity electrophoresis of alpha-fetoprotein detected by immunoenzymatic and chemiluminescent amplification followed by direct scanning.

Microdetermination of alpha-fetoprotein (AFP) glycoforms by lectin affinity electrophoresis followed by chemiluminescence reaction using horseradish peroxidase (POD) or alkaline phosphatase (ALP) in antibody-affinity blotting was developed. The intensity of chemiluminescence obtained by ALP was greater than that by POD; however, the coefficient of variation with POD was less than that with ALP. The optimized sensitivity of the chemiluminescence method with POD was two times that of the most sensitive colorimetric method currently available in terms of the chemiluminescence intensity per unit AFP concentration. The lower detection limit by the chemiluminescence method with POD (0.5 ng/ml) was much lower than that by the colorimetric method (3 ng/ml). Both methods gave identical percentages of lentil lectin- and erythroagglutinating phytohemagglutinin-reactive minor bands using a serum with 52 ng/ml AFP. This result indicates that microdetermination of AFP glycoforms by chemiluminescence after lectin-affinity electrophoresis was more sensitive than currently available methods and that it is potentially useful for clinical application.

Developments in lectin affinity immunoelectrophoresis of serum glycoproteins

Two lectin affinity immunoelectrophoretic techniques, i.e., crossed affinity immuno-electrophoresis and lectin affinity electrophoresis combined with antibody-affinity blotting, are reviewed. These procedures are suitable in structural studies of serum glycoprotein glycans. Applications of these techniques in fields of biochemical and clinical interest are discussed.

Synthetic Glycogels for Affinity Electrophoresis: A Facile and Efficient Method for Investigating Sugar-Lectin Interaction

A novel and efficient method for analyzing sugar-lectin interaction using affinity electrophoresis (AEP) is described. Polyacrylamide gels covalently conjugated with 2-acetamido-2-deoxy-D-glucopyranose (GlcNAc) residues were successfully prepared by radical copolymerization of highly reactive 3-(N-acryloylamino)propyl 2-acetamido-2-deoxy-beta-D-glucopyranoside with acrylamide in the presence of N,N'-methylenebisacrylamide (BIS). When the glycogels carrying various densities of GlcNAc branches were employed for polyacrylamide gel electrophoresis (PAGE) of lectins, the mobilities of wheat germ agglutinin (WGA) were specifically reduced by increasing the concentrations of the GlcNAc residues in gels, although concanavalin A (Con A) showed no significant change in the mobility. It was also demonstrated that the association constant of WGA with immobilized GlcNAc residue can be determined by combined use of this stable glycogel and an automated gel-scanning system associated with fluorometric spectroscopy. The association constant of WGA with the GlcNAc moiety was estimated to be 1.24 x 10(4) M-1.

Interaction of lectins with their ligand carbohydrate of alpha-fetoprotein: analysis by mixed-lectin affinity electrophoresis.

Serum or ascites alpha-fetoprotein (AFP) from patients with hepatocellular carcinoma and from a cord blood were analyzed by affinity electrophoresis with two lectins mixed in agarose gel in a combination of concanavalin A (Con A) and Lens culinaris agglutinin A (LCA-A) or of erythroagglutinating phytohemagglutinin (E-PHA) and Allomyrina dichotoma lectin (allo A). Con A- and LCA-A-reactive AFP-C2-L3 was not further retarded by mixing with either of the other lectin. It showed a mobility identical with that of AFP-C2 or AFP-L3. E-PHA- and allo A-reactive AFP-P4-A3 showed similar results. It migrated with intermediate mobilities of AFP-P4 and AFP-A3 depending on the concentrations of the two lectins mixed in the gel. The results indicate that the two mixed lectins compete with each other for the topologically different lectin-binding sites on the oligosaccharide of AFP molecule.

Immune response to different doses of a hapten of fluorescein isothiocyanate analyzed by two-dimensional affinity electrophoresis.

The immune response to different doses of a hapten of fluorescein isothiocyanate (FITC) in BALB/c mice was analyzed by two-dimensional affinity electrophoresis (2-D AEP). The mice were immunized with different doses (0.5 microgram, 5 micrograms, 50 micrograms, 500 micrograms and 2.5 mg) of FITC-conjugated bovine serum albumin (BSA). The antibodies to FITC bovine serum albumin (BSA) were separated into a large number of IgG spots due to differences in their isoelectric points (pI) and binding affinity to FITC ligand immobilized in the gel. The IgG spots, showing identical affinity to the ligand but different pI, have been considered as an IgG family. The affinity and quantity of IgG families changed with the increase in FITC-BSA dosage. With a low dose (5 micrograms) most of the families showed high affinity (Kd < 1 microM < Kd < 50 microM) and low affinity (Kd > 50 microM) antibodies were produced. The increase of FITC-BSA up to 500 micrograms markedly increased the quantity of IgG spots showing a variety of affinity to FITC. However, 2.5 mg FITC-BSA did not increase the quantity and heterogeneity of IgG spots significantly. The changes in the heterogeneity and quantity of anti-FITC antibodies and the subclass switch were observed over the course of immunization. The heterogeneity and the quantity of IgG1, IgG2b and IgG3 antibodies increased markedly during the first and the second immunization, whereas an increase in the heterogeneity and the quantity of IgG2a antibody was observed in the third immunization. This suggests that the subclass switch to IgG1, IgG2b and IgG3 and the somatic mutation of IgG1, IgG2b and IgG3 occur during the first and the second immunization, but the subclass switch to IgG2a and the somatic mutation of IgG2a seem to occur later than that of the other IgG subclasses.

Evaluation of a reference material for glycated haemoglobin.

The use of lyophilized blood as a reference material for glycated haemoglobin was investigated with respect to IFCC criteria for calibrators and control materials. Ninety-two laboratories, using 11 methods, detected no changes in glycated haemoglobin content when the lyophilizate was stored for one year at 4 degrees C. Affinity chromatography, HPLC, electrophoresis and immunoassay detected no changes following 18 months storage at -84 and -20 degrees C. Samples for HPLC are stable at 4 degrees C for one year, and 5 years at -20 degrees C. For the other three methods, samples are stable for 5 years at 4 degrees C. At 4 degrees C, reconstituted samples are stable for 2 days (HPLC) and 7 days (other three). Lyophilization does not cause matrix effects and inhomogeneity, since mean glycated haemoglobin and reproducibility for lyophilized samples and whole blood were similar. The coefficient of variation for vial filling precision was 0.59%. We conclude that lyophilized blood samples can be used as calibrators and control materials. Their use as calibrators, following assignment of the HbA(1c) value by HPLC, may contribute, in the interim, to the standardized interpretation of long term diabetic control.

Affinity electrophoretic determination of oligosaccharide specificity of Butea monosperma agglutinin.

The oligosaccharide specificity of newly isolated Butea monosperma agglutinin (BMA) was determined by two-dimensional lectin affinity electrophoresis of alpha-fetoprotein (AFP) with concanavalin A, lentil lectin, erythroagglutinating phytohemagglutinin and Allomyrina dichotoma lectin, of which the specificities to known AFP oligosaccharides had been established. Effects of neuraminidase treatment on the reactivities of AFP to the lectins were also studied. The results indicated that BMA had the highest affinity for the exposed Gal residues of nonreducing termini of biantennary complex-type oligosaccharides, and that the affinity was reduced to zero in the following order by the presence of monosialyl residue of the Man alpha1->3 arm, monosialyl residue of the Man alpha1->6 arm, monosialyl residue of the Man alpha 1->3 arm and bisecting G1cNAc, and disialyl residues. BMA did not recognize Neu5Ac alpha2->6 and Neu5Ac alpha2->3 substitutions of Gal. These characteristics of BMA were shown to be useful in identifying malignancy-associated alteration of AFP sugar chains.

Estimation of the affinity between haptoglobin and hemoglobin by two-dimensional affinity electrophoresis.

Estimation of the affinity between haptoglobin and hemoglobin by two-dimensional affinity electrophoresis.

Enzymo-rocket electrophoretic assay and crossed affinity enzymoimmunoelectrophoresis and its ap-lication in diagnosis of plimary liver cancer

A sensitive and accurate quantitative method of alphafetoprotein (AFP), enzymo-rocket dectrophoretie assay (EREA), was developed by introducing horseradish peroxidase-labeled anti-btmmn AFP antibody into rocket electrophoresis. The lower limit of quantitation by this method is about 10 ng/ml of AFP. The dose-nesponse curve covers a broader range of concentrations of AFP (10--4000 ng/ml) than RIA (0---400 ng/ml). 3fine accm-acy and precision is comparable to that of RIA (r=0.986). Sermn AFP measured in 100 patients with primary liver cancer by this method, 88% had levels over 25 ng/ml. The crossed affinity enzymoimmtmoelectrophoresis is a combination of lentil lectin (LCA) affinity electrophoresis and enzymo-rocket electrophoresis, it has been possible to separate the AFP into two variants, LCA-reactive (LCA-R) and LCA-nonreactive (LCA-N) fractions. The advantages of this method are high sensitivity, rapid (6--7 h), and can be effectively used to differentiate the primary liver cancer and benign liver disease.