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Implementation of the on-chip immunoassay for alpha-fetoprotein (AFP)-L3% was achieved using a fully automated microfluidic instrument platform that will prepare the chip and run the assay with a total assay time of less than 10min. Reagent/sample mixing, concentration, and reaction in microfluidic channels occur by the electrokinetic analyte transport assay (EATA) technique, enabling the integration of all assay steps on-chip. The determination of AFP-L3%, a biomarker for hepatocellular carcinoma, was achieved by the presence of Lens culinaris agglutinin in the separation channel, causing separation of the fucosylated isoform, AFP-L3, from the nonfucosylated AFP-L1 by lectin affinity electrophoresis. Laser-induced-fluorescence (LIF) detection was used to quantitate the labeled immunocomplexes. The limit of detection (LOD) was 0.1ng/ml AFP, and assay precision of less than 2% coefficient of variation (CV) was obtained for quantitation from 24 to 922ng/ml total AFP in spiked serum samples. Assay precision of less than 3% CV was obtained for AFP-L3% measurements from 8.5 to 81%. Furthermore, good correlation of test results for 68 patient serum samples with a commercially available reference method (LiBASys assay for AFP-L3%) was obtained, with r(2)=0.981 and slope=1.03.

Procalcitonin serum level has been recommended as a new marker of bacterial infectious diseases. The aim of this prospective, multicenter study was to determine the clinical usefulness of procalcitonin in differentiating patients with sepsis from those with severe sepsis. Eighty-two patients were enrolled: 20 without systemic inflammatory response syndrome (SIRS), 9 with SIRS, 34 with sepsis, and 19 with severe sepsis. The patients with severe sepsis had significantly higher procalcitonin levels (median, 36.1 ng/ml) than those with sepsis (median, 0.6 ng/ml). With a procalcitonin cutoff value of 2.0 ng/ml, sensitivity for the detection of severe sepsis and specificity for the detection of sepsis were 94.7% and 78.1%, respectively. A good correlation was found between the serum procalcitonin level and the Sepsis-Related Organ Failure Assessment (SOFA) score (r = 0.680), although no correlation was found between the C-reactive protein (CRP) level and the SOFA score. In conclusion, the procalcitonin serum level may be useful not only for aiding the diagnosis of sepsis but also for discriminating between sepsis and severe sepsis.

Background: Procalcitonin (PCT) is a biomarker for the diagnosis of sepsis and bacterial infection diseases., Methods: A new fully automated SphereLight PCT (SL-PCT) assay system for PCT concentration in human serum or plasma by using SphereLight 180 (SL180, Olympus Corp.) analyzer was developed. The SL-PCT assay is based on chemiluminescent enzyme immunoassay., Results: A linear dose response relationship was observed up to 200 ng/ml PCT concentration. The detection limit of PCT concentration was 0.06 ng/ml. Endogenous substances, anticoagulants, sodium fluoride and drugs did not interfere with assay results. There was a good correlation between the present method and the manual method in serum and plasma samples., Conclusions: These results indicate that the SL-PCT assay showed good performance in terms of the linearity, detection limit and precision. Use of this PCT measurement may improve the detection of sepsis and infectious disease.

Background: Traditionally, the follow-up of differentiated thyroid carcinoma consists of periodic withdrawal from L-T4-suppressive therapy to allow performance of a highly sensitive serum Tg measurement to detect recurrences. We investigated Lens culinaris agglutinin-reactive thyroglobulin ratios in serum to evaluate in usefulness for detection of thyroid carcinoma., Methods: The study was conducted on 93 serum sample from 23 healthy volunteers, 32 patients with benign thyroid tumor, 28 patients with thyroid carcinoma without metastasis, and 10 patients with thyroid carcinoma with lymph node metastasis., Results: The Lens culinaris Agglutinin reactive thyroglobulin ratio in patients with thyroid carcinoma was significantly lower than in patients with benign thyroid tumor with serum thyroglobulin concentration >200 ng/ml. Among cases of thyroid carcinoma with lymph node metastasis, Lens culinaris Agglutinin reactive thyroglobulin ratios were significantly lower than in patient with thyroid carcinoma without metastasis and those with benign tumor regardless of serum thyroglobulin concentration., Conclusion: Measurement of Lens culinaris Agglutinin reactive thyroglobulin ratio in serum may be useful for distinguishing between thyroid carcinoma and benign thyroid tumor.

Background: The early stage of pancreatic carcinoma lacks typical clinical signs and symptoms, and is difficult to diagnose. We developed an assay for active form of serum carboxypeptidase A (F-CPA) and its zymogen precursor pro CPA, and evaluated them as a marker for early-stage pancreatic carcinoma., Methods: Serum CPA from 406 patients including 169 with pancreatic carcinoma, 53 with acute pancreatitis, 23 with chronic pancreatitis, and 161 with non-pancreatic diseases were assayed by a method with N-acetyl-phenylalanyl-l-3-thiaphenylalanine as substrate and dl-benzylsuccinic acid as specific inhibitor of CPA. Activation of pro CPA was carried out using trypsin., Results: An established assay system was performed fully automatically and possesses enough performance for routine clinical assay. This system requires 31 minutes for CPA detection. No significant differences were detected between the F-CPA activity of patients with pancreatic carcinoma and that of patients with non-pancreatic diseases (p=0.168). F-CPA was mainly increased in patients with pancreatitis. Since total-CPA (T-CPA, T-CPA=pro CPA+F-CPA) was increased both in patients with pancreatic carcinoma and those with acute pancreatitis (p<0.0001, each case), the F-CPA/T-CPA ratio was low only in the patients with pancreatic carcinoma. The rate of positivity of T-CPA in the early stage of pancreatic carcinoma in which tumor was less than 2 cm was 77%, and higher than those of CA19-9 (31%), CEA (8%), and elastase 1 (46%)., Conclusion: It was suggested that assays of both T-CPA and F-CPA in serum might be useful for the surveillance of early-stage pancreatic carcinoma.

The clinical significance of serum procalcitonin (PCT) for discriminating between bacterial infectious disease and nonbacterial infectious disease (such as systemic inflammatory response syndrome (SIRS)), was compared with the significance of endotoxin, beta-D: -glucan, interleukin (IL)-6, and C-reactive protein (CRP) in a multicenter prospective study. The concentrations of PCT in patients with systemic bacterial infection and those with localized bacterial infection were significantly higher than the concentrations in patients with nonbacterial infection or noninfectious diseases. In addition, PCT, endotoxin, IL-6, and CRP concentrations were significantly higher in patients with bacterial infectious disease than in those with nonbacterial infectious disease (P<0.001, P<0.005, P<0.001, and P<0.001, respectively). The cutoff value of PCT for the discrimination of bacterial and nonbacterial infectious diseases was determined to be 0.5 ng/ml, which was associated with a sensitivity of 64.4% and specificity of 86.0%. Areas under the receiver operating characteristic curves (POCs) were 0.84 for PCT, 0.60 for endotoxin, 0.77 for IL-6, and 0.78 for CRP in the combined group of patients with bacterial infectious disease and those with nonbacterial infectious disease, and the area under the ROC for PCT was significantly higher than that for endotoxin (P<0.001). In patients diagnosed with bacteremia based on clinical findings, the positive rate of diagnosis with PCT was 70.2%, while that of blood culture was 42.6%. PCT is thus essential for discriminating bacterial infection from SIRS, and is superior in this respect to conventional serum markers and blood culture.

Background: Lens culinaris agglutinin (LCA)-reactive alpha-fetoprotein (AFP-L3) percentage of total AFP concentration [(AFP-L3/total AFP)x100] has been used as an effective marker for earlier diagnosis, for assessment of therapeutic effects and for predicting the prognosis of hepatocellular carcinoma (HCC)., Methods: A new clinical automatic analyzer, the "LiBASys", and an assay kit for the simultaneous determination of AFP-L3 percentage and concentration of AFP in human serum were developed. LiBASys performed automatic re-measurement and also met stat samples. Both AFP-L3 percentage and AFP concentration were calculated simultaneously and printed out continuously every 3.4 min per test., Results: A linear dose-response relationship was observed up to 1000 ng/ml AFP concentration. The detection limit and functional sensitivity of LCA-nonreactive AFP (AFP-L1) and AFP-L3 concentrations were 0.4 and 0.8 ng/ml, and 0.4 and 1.0 ng/ml, respectively. Interassay CVs of AFP-L3 percentage and AFP concentration ranged from 2.8% to 13.4% and 2.6% to 4.6%, respectively. Assay results exhibited good correlations with those of commercially available lectin-affinity electrophoresis (r=0.984) for AFP-L3 percentage and with the RIA method (r=0.999) for AFP concentration., Conclusions: This method simultaneously yields both qualitative and quantitative results for AFP-L3 percentage and AFP concentration.

Objective: The purpose of this study was to evaluate the use of the Lens culinaris agglutinin-reactive alpha-fetoprotein ratio for the detection of fetal Down syndrome in combination with traditional serum markers., Study Design: We obtained maternal serum from 530 women with unaffected pregnancies and 31 women who were pregnant with a fetus with Down syndrome at 14 to 20 weeks of gestation. Various combinations of Lens culinaris agglutinin-reactive alpha-fetoprotein ratio, alpha-fetoprotein, human chorionic gonadotropin, and unconjugated estriol were evaluated by score, without regard for the maternal age-related fetal Down syndrome risk., Results: The best combination of serum markers, according to our scoring method, was a combination of all 4 markers. This combination showed a sensitivity of 83.9%, with a 5.1% false-positive rate for Down syndrome. Moreover, in older women, high sensitivity was obtained without increasing the false-positive rate., Conclusion: The screen using all 4 markers showed high sensitivity in all age groups without increasing the false-positive rate.

Background: Alpha-fetoprotein (AFP) from hepatocellular carcinoma (HCC) displays differential affinity to lectin Lens culinaris agglutinin (LCA) compared to that from chronic hepatitis/liver cirrhosis. According to their binding capability to LCA, total AFP can be separated into three different glycoforms, AFP-L1, AFP-L2, and AFP-L3. AFP-L1 is the non-LCA-bound fraction, which constitutes the major glycoform of AFP in serum of chronic hepatitis and liver cirrhosis. AFP-L3 is the LCA-bound fraction of AFP. It has been reported that malignant liver cells produce AFP-L3, even when HCC is at its early stages, and especially when the tumor mass is supplied by the hepatic artery. Clinical research has determined that AFP-L3 is a highly specific marker for HCC. The AFP-L3 can be detected in the serum of approximately 35% of the patients with small HCC (<2 cm). The AFP-L3-positive HCC has potential for rapid growth and early metastasis. Compared to imaging techniques, it has been shown to have 9-12 months of lead-time in early HCC recognition. Combined sensitivity of AFP-L3 for HCC is 56%, with a specificity of >95%., Methods: Automated assay for measuring AFP-L3 has been developed and introduced in clinical use. The new automated method for measurement of ALP-L3 is based on liquid phase binding of the AFP-L3 glycoform with LCA and two specific monoclonal antibodies labeled with peroxidase and polysulfated tyrosine peptide, respectively., Conclusion: AFP-L3 is a new generation of tumor marker for HCC and yields useful information on HCC for clinical decision making.

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.

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 < or = 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.

A new immunoassay for alpha-fetoprotein based upon liquid-phase binding reactions is described. In this procedure, a sample that contains alpha-fetoprotein is mixed with a solution of two anti-alpha-fetoprotein monoclonal antibodies complexed with peroxidase and sulfated peptide, respectively. After incubation, immune complex is separated from other components by anion exchange column chromatography. Immune complex is quantified using fluorometric detection by peroxidase enzymatic activity. Peroxidase activity correlated with a alpha-fetoprotein with a 1:1 relationship. The stoichiometric immunoreaction allowed a large analytical range (0.4-7500 ng/ml) with a linear dose-response relationship, high sensitivity and good precision. Endogenous substances did not interfere with assay performance. Assay results showed good correlation with other established methods. These results indicate that the method is useful for clinical alpha-fetoprotein determinations.

Serum alpha-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. beta-galactosidase and beta-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 alpha 1-->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 beta 1-->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.

Diagnostic kits for determination of alpha-fetoprotein (AFP) carbohydrate chain microheterogeneity were developed using lectin affinity electrophoresis with Lens culinaris agglutinin-A (LCA-A) and erythro-agglutinating phytohemagglutinin-E4 (PHA-E4). Separated AFP bands by electrophoresis were detected with high sensitivity by antibody-affinity blotting and immunoenzymatic amplification. Densitometry was used to apportion lectin reactive AFPs. The within-run S.D. for proportions of AFP bands was below 3%. Band intensity was linearly related to AFP concentration between 2 and 200 ng/ml. Profiles of lectin reactive AFPs were compared in serum samples from 55 patients having liver diseases. The average values of lectin reactive AFPs for chronic hepatitis and liver cirrhosis patients were both below 13%, but those of hepatocellular carcinoma patients were above 25%. Correlation of data with disease states suggests that the methods can greatly facilitate the discrimination between benign and malignant liver diseases.

Measurement of renin activity in human fluids using a fluorogenic substrate and high performance liquid chromatography (HPLC) is described. A nine amino acid peptide containing the fluorogenic residue, N-(2-pyridyl) glycine (Pg) is used as a substrate. The peptide sequence is homologous with the cleavage site of human angiotensinogen. This substrate is hydrolyzed by renin to generate fluorogenic and non-fluorogenic products. The amount of fluorogenic product is directly measured by reversed phase HPLC. Optimization of assay conditions and measurement of human serum renin levels are described. Assay results correlated well with those from radioimmunoassay. The method is simple, convenient, highly sensitive and can be used for routine clinical renin assays.

p-Nitrophenyl O-(6-O-benzyl)-alpha-D-glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl- (1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1-- --4)-alpha-D-glucopyranoside (BG5P) is hydrolyzed by both human salivary alpha-amylase (HSA) and human pancreatic alpha-amylase (HPA) to O-(6-O-benzyl)-alpha-D-glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl- (1----4)-alpha-D-glucopyranose (BG3) and p-nitrophenyl alpha-maltoside (G2P). Glucoamylase and alpha-glucosidase cannot hydrolyze BG5P because of the modification of the OH group of the 6-position of the non-reducing-end glucose residue with the benzyl group. Taking advantage of these characteristics of the substrate, BG5P, we developed a method to assay the total alpha-amylase activity in human fluids using glucoamylase and alpha-glucosidase as the coupled enzymes. This method is simple and can be used as the standard method for routine clinical assays of alpha-amylase activity.

p-Nitrophenyl alpha-maltopentaoside, having a benzyl group on O-6 of the terminal (nonreducing) D-glucosyl group was prepared by use of a reductive ring-opening reaction. Highly regioselective reduction of p-nitrophenyl O-(2,3-di-O-benzoyl-4,6-O-benzylidene-alpha-D-glucopyranosyl)-(1----4)- tris[O-(2,3,6-tri-O-benzoyl-alpha-D-glucopyranosyl)-(1----4)]-2,3,6-tri- O- benzoyl-alpha-D-glucopyranoside by dimethylamine-borane and p-toluenesulfonic acid, followed by debenzoylation, gave p-nitrophenyl O-(6-O-benzyl-alpha-D-glucopyranosyl)-(1----4)-tris[O-alpha-D-glucopyran osyl- (1----4)]-alpha-D-glucopyranoside. An experiment was done on the mode of action of human pancreatic and salivary alpha amylases on this derivative. The compound is suitable as a substrate for the assay of alpha amylase when used with glucoamylase and alpha-D-glucosidase as coupling enzymes.

The enzymatic measurement of alpha-amylase in human serum with chemically modified amylose as substrate was studied. Several substitutions of carboxymethylamylose were prepared from amylose, suitable amounts of monochloroacetic acid, and sodium hydroxide. The relationship between the degree of substitution of the carboxymethylamylose and the kinetic parameters of human pancreatic, salivary and porcine pancreatic alpha-amylases was determined. The action of alpha-amylases on such modified amyloses in the presence of glucoamylase was measured by a specific enzymatic assay having glucose as the product. The kinetic parameters of human pancreatic alpha-amylase were similar to those of human salivary alpha-amylase. It was possible to determine suitable sensitivities for the alpha-amylase assay using this substrate. These results suggested a subsite model.

A mixture of p-nitrophenyl O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D- glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D- glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D- glucopyranoside (FG5P) and p-nitrophenyl alpha-D-glucoside (GP) was incubated with cyclomaltodextrin glucanotransferase (CGTase) [EC 2.4.1.19]. Analysis of the digest by HPLC showed that the products were p-nitrophenyl O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D- glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D- glucopyranosyl-(1----4)-alpha-D-glucopyranoside (FG4P) and p-nitrophenyl alpha-D-maltoside (G2P), and no other product could be detected. Based on the reaction, a sensitive method to assay for CGTase was developed.