Lectin Affinity Electrophoresis Research Articles

Alpha-fetoprotein microheterogeneity: a potential biochemical marker for Down’s syndrome

Our purpose was to examine the utility of analyzing alpha-fetoprotein (AFP) microheterogeneity assessed by lectin affinity in Down’s syndrome (DS) screening. Maternal sera and amniotic fluids were collected from 18 women who were carrying DS fetuses and 70 unaffected pregnancies around 16 weeks of gestation. The percentages of AFP which reacted with Lens culinaris agglutinin (AFP-L2,3) were determined by lectin affinity electrophoresis. AFP-L2,3 levels were significantly increased ( P<0.0001) in both maternal serum and amniotic fluid from DS-affected versus unaffected pregnancies. The fractional areas under the receiver operating characteristic curves were 0.835 and 0.700 ( P=0.106) for AFP-L3 and AFP MoM (multiples of the median) in maternal serum. No correlation was found between AFP-L3 and AFP MoM in maternal serum ( r=0.006). Our data suggest that the measurement of AFP-L3 in maternal serum is a potential biochemical marker for DS.

Alterations of IGF-binding proteins in patients with alcoholic liver cirrhosis

The protein synthetic activity of the liver is diminished in cirrhosis. The aim of this study was to investigate possible changes in the serum IGF–IGFBP system among patients with alcoholic liver cirrhosis (ALC). The results obtained demonstrated that serum IGF-I and IGF-II concentrations were significantly lower in patients with ALC than in healthy persons ( P=0.0008 for IGF-I and 0.0002 for IGF-II). The IGFBP profile was markedly altered and the 34 kDa IGFBP from patients had higher affinity towards 125I-IGF-II compared to the 34 kDa IGFBP of control individuals. Moreover, the 40–45 kDa IGFBP (in isolated complex with 125I-IGF-II) exhibited diminished interaction with concanavalin A, wheat germ, and breadfruit lectins. Modification of the glyco-component of the 40–45 kDa IGFBP seems to be an early event in ALC since change in reactivity towards lectins was noticed in patients with ALC classified as Child score A, whose serum IGF-I and IGF-II levels were within reference limits (the existence of carbohydrate microheterogeneity of this IGFBP was also assessed by lectin-affinity electrophoresis). It is possible that these biochemical alterations may affect the functional activity of the IGFs by changing the dynamics and distribution of these growth factors in the organism.

Clinical utility of simultaneous measurement of serum high-sensitivity des-gamma-carboxy prothrombin and Lens culinaris agglutinin A-reactive alpha-fetoprotein in patients with small hepatocellular carcinoma.

To evaluate the diagnostic efficacy of simultaneous measurements of high-sensitivity des-gamma-carboxy prothrombin (H-DCP) and Lens culinaris agglutinin A-reactive alpha-fetoprotein (AFP-L3) in small hepatocellular carcinoma (HCC). Sixty-one patients with small HCCs < or = 2 cm in diameter and 134 controls (chronic hepatitis: 59 cases; cirrhosis: 75 cases) were examined. H-DCP was measured by electrochemiluminescence immunoassay (cut-off 40 mAU/ml (milli-arbitrary units/ml) and AFP-L3% (percentage of AFP-L3/total AFP) by lectin-affinity electrophoresis coupled with the antibody-affinity blotting method (cut-off 10%). Fifty-six patients were histologically diagnosed and the remaining five patients were diagnosed clinically. Of 61 patients, 27 (44.3%) were positive for H-DCP and 14 (23.0%) were positive for AFP-L3. There was no correlation between H-DCP and AFP-L3%. Nineteen patients (31.1%) had positive H-DCP alone. Six patients (9.8%) had positive AFP-L3 alone, and in eight patients (13.1%) both markers were positive. In combination assay, 33 of 61 patients (54.1%) were positive for either marker; specificity and accuracy were 97.8% and 84.1%, respectively. There was a tendency for the AFP-L3% to be elevated in patients with moderately or poorly differentiated HCC (P= 0.0564) and multiple HCC nodules (P= 0.0316), while the H-DCP showed no elevation related to the tumour type. The detection rate of small HCC was improved by combination assay with H-DCP and AFP-L3%. Our results indicate that the markers are complementary and useful for the diagnosis and evaluation of small HCC when measured simultaneously.

Usefulness of Lens culinaris agglutinin A-reactive fraction of alpha-fetoprotein (AFP-L3) as a marker of distant metastasis from hepatocellular carcinoma.

The objective of this study was to clarify the relationship between the serum level of the Lens culinaris agglutinin A-reactive fraction of alpha-fetoprotein (AFP-L3) and the clinical features including sex, age, Child's classification, virus markers, tumour size, tumour stage, distant metastasis, histopathologic findings, portal thrombus and outcome of hepatocellular carcinoma (HCC). We measured the AFP-L3 levels in 170 HCC cases at the time of diagnosis using lectin-affinity electrophoresis followed by antibody-affinity blotting. The patients were divided into two groups, those who were AFP-L3 positive (n=56; AFP-L3 >/=15% relative to the total alpha-fetoprotein (AFP) concentration) and those who were AFP-L3 negative (n=114; AFP-L3 <15%). Then we examined the association between the serum AFP-L3 level and the clinical features of HCC. No significant differences were found in age, sex, and virus markers between the AFP-L3-positive and -negative groups. However, patients in the positive group had worse liver function and larger tumours compared to the negative group. They also had more advanced cancer with poor tumour histology compared to the negative group. Distant metastasis was diagnosed significantly more often in the positive group than that in the negative group. There was no significant correlation between the AFP-L3 level and portal thrombus. Although the follow-up period was brief the prognosis for the positive group clearly was poor. These results suggest that AFP-L3 is a useful indicator of distant metastasis and a poor prognosis for HCC.

A Study on the Lectin Reactivity of Alpha-Fetoprotein Produced by Hepatoid Adenocarcinomas and Yolk Sac Tumors

The carbohydrate structure of glycoproteins is considered to be tissue-specific or cell type-specific, but there have been no reports on the differences of the carbohydrate structure of alpha-fetoproteins (AFPs) produced by histologically identical tumors in different tissues. The lectin affinity electrophoresis of hepatoid adenocarcinomas and yolk sac tumors from different organs suggested that either the tumor heterogeneity or the tissue specificity is possibly involved, the lectin reactivity of the AFP sugar chain structure produced by the tumors in different tissues.

Structural studies on sugar chains of carbohydrate-deficient transferrin from patients with alcoholic liver disease using lectin affinity electrophoresis.

It is well-known that microheterogeneity of human serum transferrin observed in alcoholics manifests as sialic acid-deficient transferrin isoforms, otherwise known as carbohydrate-deficient transferrin (CDT). A recent study demonstrated that serum CDT lacked one or both of the entire carbohydrate chains but the investigation required several troublesome procedures. The aim of the present study was to confirm the sugar chain structures of serum transferrin, and of serum CDT in particular, from patients with alcoholic liver disease (ALD) using conventional lectin affinity electrophoresis which might be useful in the clinical setting. The serum CDT obtained from ALD-patients was partially purified using an anion exchanger. Serum transferrin and the partially purified serum CDT were investigated by concanavalin A (Con A)- and Datura stramonium agglutinin (DSA)-affinity electrophoresis followed by antibody-affinity blotting and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with Western blotting. By Con A-affinity electrophoresis, serum CDT was separated into weakly reactive and nonreactive transferrins which showed slower electrophoretic mobilities than those from the healthy controls. Moreover, nearly all of the serum CDT was nonreactive with DSA. On SDS-PAGE, the molecular masses of serum CDT were estimated to be approximately 75 and 72 kDa, which corresponded to those of partially and completely deglycosylated transferrin obtained from the healthy controls (78 kDa), respectively. In conclusion, these results indicated that the sugar chain structures of serum CDT from patients with ALD show not merely a loss of terminal sialic acids, but also the absence of asparagine-N-linked oligosaccharides.

Clinical utility of Lens culinaris agglutinin-reactive alpha-fetoprotein in small hepatocellular carcinoma: special reference to imaging diagnosis

Background/Aims: Blood concentration levels of alpha-fetoprotein like the Lens culinaris agglutinin-reactive fraction (AFP-L3) are a useful marker for predicting the long-term prognosis of hepatocellular carcinoma. This study investigated the relationship between serum AFP-L3 and various imaging modalities. Methods: Sixty-three patients with small hepatocellular carcinomas ≤2 cm in diameter were studied. Serum AFP-L3 concentrations were measured by lectin-affinity electrophoresis coupled with antibody-affinity blotting and expressed as % AFP-L3 (the percent of AFP-L3 as total AFP). A clinical “cutoff level” of 10% was used in this study to indicate the presence of hepatocellular carcinoma. Selective hepatic intraarterial digital subtraction angiography (DSA), ultrasonographic angiography with carbon dioxide microbubbles (USAG), and computed tomography during arterial portography (CTAP) were performed to evaluate the hemodynamics of hepatic nodules. Results: Fourteen (22.2%) of the 63 patients were positive for % AFP-L3. The % AFP-L3 levels ( n=45, 4.4%) of patients with hypervascular tumors were significantly higher than those ( n=15, 0.0%) of patients with isovascular or hypovascular tumors as determined by USAG ( p=0.0061). The % AFP-L3 levels ( n=53, 4.4%) of patients with a negative portal blood supply were significantly higher than the % AFP-L3 levels ( n=7, 0.0%) of patients with a positive portal blood supply as determined by CTAP ( p=0.0140). The % AFP-L3 levels of patients with tumors with a long doubling time (DT) were significantly lower than for patients with tumors with a short DT ( p=0.0176). Conclusion: AFP-L3 is a positive indicator which may be more specific for small advanced hepatocellular carcinoma.

Characterization of sugar chain structures of human alpha-fetoprotein by lectin affinity electrophoresis. ibarahp@oka.urban.ne.jp.

Alpha-Fetoprotein (AFP) glycoforms, defined as AFP with different chemical structures of carbohydrate, were analyzed by affinity electrophoresis with several lectins of known specificities against complex-type oligosaccharides. Serum AFP samples from cord blood on full-term delivery and from patients with hepatocellular carcinoma and extrahepatic malignancies including gastrointestinal tumors and yolk sac tumors were used. Two-dimensional lectin affinity electrophoresis and also lectin affinity chromatography coupled with lectin affinity electrophoresis were employed. More than ten AFP glycoforms were identified or characterized using the above-mentioned AFP samples. Known specificities of the lectins against complex-type oligosaccharides were refined or their additional specificities were found in this study. Lectin appeared to have specificity against carbohydrates by recognizing not only specific residues but also the whole carbohydrate molecule containing the residues, resulting in differential affinities for the lectin.

Multimodal application of lectin affinity electrophoresis of alpha-fetoprotein.

Multimodal application of lectin affinity electrophoresis of alpha-fetoprotein (AFP) glycoforms is reviewed. Crossed affinity immunoelectrophoresis developed by Bøg-Hansen and others was extended to tandem-lectin affinity electrophoresis by tandem lining of two different lectin gels and to mixed-lectin affinity electrophoresis. By introducing an antibody-affinity blotting technique for detection of separated glycoforms of AFP, several two-dimensional combinations of lectin affinity electrophoresis became possible: two different lectins for the first and second dimension electrophoresis, lectin-gradient affinity electrophoresis, and electrophoretic separation of lectin isoforms in the first-dimension electrophoresis, followed by affinity electrophoresis against the separated lectin isoforms. Usefulness of the different modalities of lectin affinity electrophoresis for several analytical purposes has been described.

Lectin-gradient gel affinity electrophoresis of glycoproteins.

Lectin-gradient agarose gel affinity electrophoresis was developed for identification of glycoforms of glycoproteins in lectin affinity electrophoresis. Gradation of lectin was done by stacking agarose gel blocks with increasing concentrations of lectin (discontinuous system) and by keeping the plate in a moist chamber at 4 degrees C overnight (continuous system) before electrophoresis. On the visualization of separated glycoform lines, the antibody-affinity blotting was superior for low concentrations of alpha-fetoprotein. Fluoresceine isothiocyanate (FITC) labeling of whole serum proteins, enzyme activity staining for alkaline phosphatase, and prestaining for lipoproteins were also applicable for visualization of proteins at higher concentrations. The conventional Western blotting can not be recommended because of the competition between lectin and glycoproteins in binding to nitrocellulose membrane. Lectin-gradient affinity electrophoresis also had a wide application for optimization of the condition of lectin affinity electrophoresis.

Serum alpha-fetoprotein levels in healthy Japanese adults.

With advances in lectin affinity electrophoresis of alpha-fetoprotein (AFP), the detection of significant changes in serum AFP at low levels in cirrhotics has become important for early detection of hepatocellular carcinoma. Serum AFP levels of 616 healthy individuals without abnormal liver function tests or virus markers of hepatitis B and C were determined by enzyme immunoassay with IMx-AFP Dainapack using automated IMx apparatus set at twice the ordinary sensitivity and compared with those of 241 individuals with abnormal liver function tests and/or positive hepatitis virus markers. The coefficient of variation in this assay was less than 10% at AFP levels as low as 0.2 ng/ml with a lower detection limit of 0.1 ng/ml. The AFP level of healthy population showed a Gaussian distribution curve after logarithmic transformation with a median and 2.5-97.5 percentile reference range of 2.2 (0.6-5.6) ng/ml. There was no significant difference in the AFP level between males and females. Individuals with abnormal liver function tests alone showed no significant increase in serum AFP unless they were associated with positive hepatitis virus markers.

Developmental alterations of alpha-fetoprotein sugar chain in amniotic fluids analyzed by lectin affinity electrophoresis.

Affinity electrophoresis of human alpha-fetoprotein (AFP) in amniotic fluid from pregnant women between 6 to 42 weeks of gestation and in the serum of a yolk sac tumor was performed with concanavalin A (Con A), lentil lectin (LCA), erythroagglutinating phytohemagglutinin (E-PHA) and Allomyrina dichotoma lectin (allo A). Separated AFP bands were detected by sensitive antibody-affinity blotting. In the first trimester, amniotic fluid AFP showed elevated percentages of Con A-nonreacting AFP (AFP-C1) and LCA weakly-reacting AFP (AFP-L2) as previously reported. Additionally, high percentages of E-PHA strongly-reacting AFP (AFP-P5) and E-PHA-reacting AFP (AFP-P4) were observed. E-PHA-nonreacting AFP (AFP-P1), E-PHA weakly-reacting AFP (AFP-P3f), allo A-nonreacting AFP (AFP-A1) and asialo-AFP, AFP-A1 s, were present only in amniotic fluids from 6 to 17 weeks of gestation. With advancing gestation, percentages of AFP-C1, AFP-P4 and AFP-P5 decreased and AFP-L2, AFP-P3f, AFP-A1, and AFP-A1 s disappeared by the end of 18 weeks. The glycoforms of serum AFP of the yolk sac tumor resembled those of amniotic fluid AFP in the early gestational stages.

レクチン親和電気泳動を用いた肝癌細胞株培養液上清中の糖蛋白糖鎖変異に関する研究

レクチン親和電気泳動を用いた肝癌細胞株培養液上清中の糖蛋白糖鎖変異に関する研究

Time course of histological changes in patients with a sustained biochemical and virological response to interferon-α therapy for chronic hepatitis C virus infection : Tsubota, A., Kumada, H., Chayama, K., Arase, Y., Saitoh, S., Koida, I., Suzuki, Y., Kobayashi, M., Murashima, N., Ikeda, K. Department of Gastroenterology, Toranomon Hospital, Minato-ku, Tokyo, Japan Journal Of Hepatology 1997; 27 (1): 49–55

Background/Aims: Blood concentration levels of alpha-fetoprotein like the Lens culinaris agglutinin-reactive fraction (AFP-L3) are a useful marker for predicting the long-term prognosis of hepatocellular carcinoma. This study investigated the relationship between serum AFP-L3 and various imaging modalities.Methods: Sixty-three patients with small hepatocellular carcinomas ≤2 cm in diameter were studied. Serum AFP-L3 concentrations were measured by lectin-affinity electrophoresis coupled with antibody-affinity blotting and expressed as % AFP-L3 (the percent of AFP-L3 as total AFP). A clinical “cutoff level” of 10% was used in this study to indicate the presence of hepatocellular carcinoma. Selective hepatic intraarterial digital subtraction angiography (DSA), ultrasonographic angiography with carbon dioxide microbubbles (USAG), and computed tomography during arterial portography (CTAP) were performed to evaluate the hemodynamics of hepatic nodules.Results: Fourteen (22.2%) of the 63 patients were positive for % AFP-L3. The % AFP-L3 levels (n=45, 4.4%) of patients with hypervascular tumors were significantly higher than those (n=15, 0.0%) of patients with isovascular or hypovascular tumors as determined by USAG (p=0.0061). The % AFP-L3 levels (n=53, 4.4%) of patients with a negative portal blood supply were significantly higher than the % AFP-L3 levels (n=7, 0.0%) of patients with a positive portal blood supply as determined by CTAP (p=0.0140). The % AFP-L3 levels of patients with tumors with a long doubling time (DT) were significantly lower than for patients with tumors with a short DT (p=0.0176).Conclusion: AFP-L3 is a positive indicator which may be more specific for small advanced hepatocellular carcinoma.

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...

Microheterogeneity with Concanavalin A Affinity of Serum Transferrin in Patients with Alcoholic Liver Disease

Microheterogeneity of transferrin (Tf) with concanavalin A (ConA) affinity was investigated by sensitive lectin-affinity electrophoresis and antibody-affinity blotting technique of sera obtained from patients with alcoholic liver disease (ALD) and normal subjects. Serum Tf was separated by ConA into three bands-a strongly ConA-reactive major band (C1), a weakly reactive minor band (C2), and a non-reactive trace band (C3). The C3 fraction was significantly increased in patients with ALD before alcohol abstinence, compared with normal subjects and patients with ALD after 4 weeks of abstinence. Furthermore, a significant correlation was found between the C3 fraction and serum carbohydrate-deficient transferrin or gamma-glutamyl-transpeptidase. These results indicate that the microheterogeneity of serum Tf in patients with ALD may be a more complex abnormality of elongation and processing on the glycans than merely a loss of terminal sialic acids. Determination of the C3 fraction is a useful marker for ALD.

Microheterogeneity with Concanavalin A Affinity of Serum Transferrin in Patients with Alcoholic Liver Disease

Microheterogeneity of transferrin (Tf) with concanavalin A (ConA) affinity was investigated by sensitive lectin-affinity electrophoresis and antibody-affinity blotting technique of sera obtained from patients with alcoholic liver disease (ALD) and normal subjects. Serum Tf was separated by ConA into three bands–a strongly ConA-reac-tive major band (C1), a weakly reactive minor band (C2), and a non-reactive trace band (C3). The C3 fraction was significantly increased in patients with ALD before alcohol abstinence, compared with normal subjects and patients with ALD after 4 weeks of abstinence. Furthermore, a significant correlation was found between the C3 fraction and serum carbohydrate-deficient transferrin or γ-glutamyl-transpeptidase. These results indicate that the microheterogeneity of serum Tf in patients with ALD may be a more complex abnormality of elongation and processing on the glycans than merely a loss of terminal sialic acids. Determination of the C3 fraction is a useful marker for ALD.

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.

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.

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.