Development of enzyme-free single-step immunoassays for glycocholic acid based on palladium nanoparticle-mediated signal generation.

Palladium nanoparticles (PdNPs) are composed mainly of inert noble metals, and their outstanding properties have attracted wide attention. PdNPs are not only capable of mimicking the oxidase-like characteristics of natural bio-enzymes, but they also present a clear black band in the test zone. In this work, the synthesized PdNPs promoted a transformation of colorless tetramethylbenzidine (TMB) to a blue oxidation product of TMB, providing a Km value of 0.09 mM for TMB, and revealing the good catalytic performance of the synthesized PdNPs. For both signal generation and amplification, PdNPs effectively replaced natural bio-enzymes as a new labeling tag. Thus, the PdNP-based enzyme-free single-step immunoassays were successfully developed for efficient and sensitive detection of glycocholic acid (GCA). Under optimal conditions, a noticeable linear relationship was identified by the enzyme-linked immunosorbent assay (ELISA) over a range of 8-2390 ng/mL, while the visual limit of detection (vLOD) in the constructed lateral flow immunoassay (LFA) was 10 ng/mL for GCA. The recovery rate in spiked urine samples obtained by the ELISA ranged from 84.2 to 117.9%, which was consistent with the results in LFA. The present work demonstrates the potential of PdNPs as labeling matrices in enzyme-free single-step immunoassays.

Prussian blue nanoparticles with peroxidase-mimicking properties in a dual immunoassays for glycocholic acid.

Prussian Blue nanoparticles (PBNPs) were utilized in a lateral flow immunoassay (LFA) and in an indirect competitive nanozyme-linked immunosorbent assay (icELISA), respectively, for their intense blue color and peroxidase (POx) -like activity. The PBNPs with good POx-like activity was linked to the antibody. Under the optimal parameters, both the PBNP-icELISA and PBNP-LFA perform very well. The icELISA has an IC50 value of 190 ng/mL, the working range extends from 29 to 1200 ng/mL, and the limit of detection is 22 ng/mL. The visual cut-off limit is 10 ng/mL. The dual immunoassay was used to quantify glycocholic acid in spiked human urine. Excellent recoveries and correlation between the two methods were observed.

Development of a simple, rapid and high-throughput fluorescence polarization immunoassay for glycocholic acid in human urine.

In this paper, a simple, rapid and high-throughput fluorescence polarization immunoassay (FPIA) based on polyclonal antibodies (PAb) is described for the determination of glycocholic acid (GCA) in human urine. Three fluorescein-labeled GCA (tracers) with different structures and spacer bridges were synthesized and purified by thin-layer chromatography (TLC). The structure effect of tracers on the assay was investigated and the sensitivity of best tracer in the optimized FPIA demonstrated an IC50 value of 306 ng/mL. The working range of FPIA was 36 ∼ 2 600 ng/mL and the limit of detection (LOD) was 9 ng/mL. The developed FPIA was time-saving that could be completed within 10 min. Human urine samples spiked with GCA were analyzed by this method, followed by confirmation with commercial enzyme immunoassay analysis (EIA). Excellent recoveries and correlation between these two methods were observed (R2 = 0.996), suggesting the developed FPIA could be applied to screening of GCA in human urine samples without complicated cleanup.

Biotinylated single-chain variable fragment-based enzyme-linked immunosorbent assay for glycocholic acid.

Glycocholic acid (GCA) has been identified as a novel selective and sensitive biomarker for hepatocellular carcinoma (HCC). In this work, a recombinant antibody, scFv-G11, which was shown previously to have selective reactivity for GCA, was labeled with biotin using a chemical and an enzymatic method, respectively. The enzymatic method proved superior giving sensitive scFv-biotin preparations. Based on biotinylated scFv against GCA and a biotin-streptavidin system for signal amplification, an indirect competitive biotin-streptavidin-amplified enzyme-linked immunosorbent assay (BA-ELISA) has been established for the sensitive and rapid detection of GCA. Several physiochemical factors that influenced assay performance, such as organic cosolvent, ionic strength, and pH, were studied. Under the optimized conditions, the indirect competitive BA-ELISA based on the obtained biotinylated scFv antibodies indicated that the average concentration required for 50% inhibition of binding (IC50) and the limit of detection (LOD) for GCA were 0.42 µg mL-1 and 0.07 µg mL-1, respectively, and the linear response range extended from 0.14 to 1.24 µg mL-1. Cross-reactivity of biotinylated scFv antibodies with various bile acid analogues was below 1.89%, except for taurocholic acid. The recoveries of GCA from urine samples via this indirect competitive BA-ELISA ranged from 108.3% to 131.5%, and correlated well with liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS), which indicated the accuracy and reliability of biotinylated scFv-based ELISA in the detection of GCA in urine samples. This study also demonstrates the broad utility of scFv for the development of highly sensitive immunoassays.

Development of an Indirect Competitive Enzyme-Linked Immunosorbent Assay for Glycocholic Acid Based on Chicken Single-Chain Variable Fragment Antibodies.

Glycocholic acid (GCA) is an important metabolite of bile acids, whose urine levels are expected to be a specific diagnostic biomarker for hepatocellular carcinoma (HCC). A high-throughput immunoassay for determination of GCA would be of significant advantage and useful for primary diagnosis, surveillance, and early detection of HCC. Single-chain variable fragment (scFv) antibodies have several desirable characteristics and are an attractive alternative to traditional antibodies for the immunoassay. Because chicken antibodies possess single heavy and light variable functional domains, they are an ideal framework for simplified generation of recombinant antibodies for GCA detection. However, chicken scFvs have rarely been used to detect GCA. In this study, a scFv library was generated from chickens immunized with a GCA hapten coupled to bovine serum albumin (BSA), and anti-GCA scFvs were isolated by a phage-displayed method. Compared to the homologous coating antigen, use of a heterologous coating antigen resulted in about an 85-fold improvement in sensitivity of the immunoassay. This assay, under optimized conditions, had a linear range of 0.02-0.18 µg/mL, with an IC50 of 0.06 µg/mL. The assay showed negligible cross-reactivity with various related bile acids, except for taurocholic acid. The detection of GCA from spiked human urine samples ranged from 86.7% to 123.3%. These results, combined with the advantages of scFv antibodies, indicated that a chicken scFv-based enzyme-linked immunosorbent assay is a suitable method for high-throughput screening of GCA in human urine.

Macromolecular Crowding Agents-Assisted Imprinted Polymers For Analysis Of Glycocholic Acid In Human Plasma And Urine.

Glycocholic acid (GCA) is a newly identified biomarker for hepatocellular carcinoma (HCC) patients. In this study, a method based on macromolecular crowding strategy has been applied for preparation of a molecularly imprinted polymer (MIP), which possesses high adsorption capacity for GCA. Polymethyl methacrylate was used as a macromolecular crowding agent, N-(3-aminopropyl)-methacrylamide hydrochloride as a functional monomer and ethylene dimethacrylate as a cross-linker. The morphology and binding characteristics of MIP were assessed by scanning electron microscopy and absorption experiments. The MIP was used as an adsorbent material to separate GCA, and the molecularly imprinted solid-phase extraction (MISPE) was carefully optimized. The MISPE combined with high-performance liquid chromatographic analysis was successfully used to determine the GCA in plasma and urine samples. When spiked levels ranged from 0.2 to 20 µmol L-1 , the recoveries were between 94.3 and 100.5%. As a proof of principle, this proposed method has been validated on a small subset of HCC patients (n = 10) and healthy volunteers (n = 10). The average GCA concentrations of HCC patients in plasma and urine were about 25 and 2.8 times than that of healthy volunteers. Copyright © 2016 John Wiley & Sons, Ltd.

Urinary metabolic profiling identifies a key role for glycocholic acid in human liver cancer by ultra-performance liquid-chromatography coupled with high-definition mass spectrometry.

BACKGROUND: Metabolomics has been proposed to be a hallmark of cancer, yet a systematic characterization of a metabolite and metabolic pathways in human hepatocarcinoma (HCC) remains a challenge. METHODS: Using ultra-performance liquid-chromatography/quadrupole-time-of-flight coupled with high-definition mass spectrometry (UPLC-Q-TOF-HDMS) in conjunction with multivariate data analysis methods, we identified and measured the metabolite profile of glycocholic acid from urine samples obtained from patients with HCC diseases. Bioinformatic tools were used to construct the metabolite network that can identify a key role for glycocholic acid in HCC. RESULTS: Biochemical analyses revealed that glycocholic acid expression was up-regulated in urine samples associated with HCC. Its pathway analysis suggested the modulation of multiple vital physiological pathways, including primary bile acid biosynthesis, secondary bile acid biosynthesis, metabolic pathways, and bile secretion. The network generation clearly enhances the interpretation and understanding of mechanisms for glycocholic acid. CONCLUSIONS: Metabolomics can contribute to evaluating the potential of metabolites in HCC patients and may provide new insight into pathophysiologic mechanisms.

A monoclonal antibody-based enzyme-linked immunosorbent assay of glycolithocholic acid sulfate in human urine for liver function test.

Urinary levels of sulfated metabolites of lithocholic acid (LCA) are expected to be a useful index of liver function. Thus, a sensitive, specific, and feasible enzyme-linked immunosorbent assay (ELISA) of these sulfated LCA metabolites (LCA-Suls) should be established. A newly generated monoclonal antibody specific to glycolithocholic acid sulfate (glycine-amidated LCA-Sul (GLCA-Sul)) was immobilized on microtiter plates via a second antibody. A urine specimen and an alkaline phosphatase-labeled antigen were added to the plate, which was then incubated at room temperature for 3h. After this competitive reaction, bound enzyme activity was measured colorimetrically using p-nitrophenyl phosphate as a substrate. The detection limit for GLCA-Sul was 0.4 pg/assay. Nonamidated LCA-Sul and taurine-conjugated LCA-Sul showed 40 and 11% cross-reactivities, respectively, while 3-sulfates of cholic acid (CA; 0.02%), chenodeoxycholic acid (CDCA; 0.63%), and deoxycholic acid (DCA; 2.2%) exhibited very low cross-reactivities. Applicability of the ELISA system to clinical samples was well validated by parallelism, recovery test, and intra/inter-assay variance. Enzymatic deconjugation with bile acids sulfatase resulted in dramatically decreased urinary levels, supporting the specificity of the ELISA toward GLCA-Sul. The mean GLCA-Sul levels in early morning urine from healthy volunteers were 314 ng/mg Ucre (males: n=16) and 507 ng/mg Ucre (females: n=9). Patients with liver diseases, including chronic hepatitis (CH) and liver cirrhosis (LC) exhibited significantly higher values (mean 5222 ng/mg Ucre: n=21). The present 'monoclonal ELISA' is predicted to be useful as a novel noninvasive diagnostic tool for liver function and hepatobiliary diseases.

N-acetylglucosaminides. A new type of bile acid conjugate in man.

Bile acids were extracted from human urine and were separated into groups of nonamidated and glycine- and taurine-conjugated compounds. Each group was subfractionated in a reversed-phase high performance liquid chromatography system, and the fractions were analyzed by negative ion fast atom bombardment mass spectrometry and also by gas chromatography-mass spectrometry after enzymatic removal of glycine and taurine moieties. The major glycosides of the non-amidated bile acids were more polar than reference bile acid glucosides and gave quasimolecular ions at m/z 592, 594, and 610 consistent with N-acetylglucosaminides of unsaturated dihydroxy and saturated di- and trihydroxy bile acids. Gas chromatography-mass spectrometry analyses of methyl ester trimethylsilyl ether derivatives showed fragments typical for N-acetylglucosaminides (m/z 173 and 186) in addition to those also given by glucosides (m/z 204 and 217). The N-acetylglucosaminides were inert toward alpha- and beta-glucosidase but were cleaved completely with N-acetylglucosaminidase. The released sugar moiety was identified as N-acetylglucosamine. One of the liberated bile acids was identified as ursodeoxycholic acid. The other acids were not identical to any known primary or secondary bile acid in humans. Fast atom bombardment mass spectrometry analyses of the glycine-and taurine-conjugated bile acid glycosides only showed ions consistent with the presence of glucosides (m/z 626 and 676). These compounds were sensitive only toward beta-glucosidase which liberated a trihydroxy bile acid as the major compound. Based on the recover of 13C- and 14C-labeled chenodeoxycholic acid glucoside added as internal standard, the daily excretion of nonamidated bile acid glycosides was estimated to be about 137 micrograms or 0.29 mumol, N-acetylglucosaminides constituting about 90%. The daily excretion of the glucosides of amidated bile acids was about 150 micrograms or 0.25 mumol, glycine conjugates constituting about 90%.

Radioimmunoassay of urinary 3 beta-hydroxy-5-cholenoyl glycine in hepatobiliary disease.

A radioimmunoassay for 3 beta-hydroxy-5-cholenoyl glycine in human urine has been developed. The antiserum was elicited with the antigen in which the steroid hapten is linked to a bovine serum albumin through the C-19 position. The [125I]-tyrosine derivative of the hapten was used as radioligand. The standard curves were linear ranging from 10 to 320 ng/mL. The cross-reactivities with other bile acids were not detectable and below 0.3% with cholesterol. Sample preparation includes extraction of 3 beta-hydroxy-5-cholenoyl glycine from urine and solvolysis of the sulfates--main form present in urine. Urinary excretion of 3 beta-hydroxy-5-cholenoyl glycine was 0.373 +/- 0.133 mumol/day in healthy adults. Urinary excretion of 3 beta-hydroxy-5-cholenoyl glycine increased in chronic liver dysfunction, hepatoma and obstructive jaundice in this order.

Sulfated and nonsulfated bile acids in urine of patients with biliary atresia: analysis of bile acids by high-performance liquid chromatography.

To elucidate urinary bile acid patterns in patients with biliary atresia (BA), 15 sulfated and nonsulfated bile acids in urine were separately measured by high-performance liquid chromatography. This relatively simple technique for fluorescence detection utilizes the enzyme 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) to reveal urinary bile acid patterns. By this method, recovery rates of sulfated and nonsulfated bile acids in urine were satisfactory, and this analysis was shown to be applicable to clinical situations. In 10 patients with BA, the mean level of total bile acids in urine (23.35 +/- 18.51 mumol/day) was seven times higher than the mean level in eight normal infants (3.05 +/- 2.05 mumol/day). In the infants with BA, the mean level of total sulfated bile acids was about half of the total bile acid level. The main components of urinary nonsulfated bile acids in BA were glycocholic acid (6.21 +/- 5.55 mumol/day) and taurocholic acid (2.28 +/- 1.33 mumol/day), whereas the main components of the urinary sulfated bile acids were glycochenodeoxycholic acid (4.58 +/- 6.97 mumol/day) and taurochenodeoxycholic acid (3.67 +/- 3.54 mumol/day). Chenodeoxycholic acid, which is relatively toxic to the liver, may more easily be conjugated with sulfate and, hence, excreted into urine at a faster rate than cholic acid. Marked individual variations in urinary bile acid patterns were observed not only in BA patients but also in normal controls.

Enhancement of the urinary excretion of non-sulphated and sulphated radioactive bile acids by sodium acetate in the bile duct obstructed rat.

The renal clearances of [14C]glycocholate, [14C]taurocholate and [3H]glycochenodeoxycholate-3-sulphate were determined in bile duct obstructed rats. Comparisons of the bile acid clearances with glomerular filtration rates (GFR) indicate that most of the filtered bile acids are reabsorbed. Inhibition studies with p-aminohippurate (PAH) and probenecid suggest that a proportion of the bile acids in urine is secreted. Attempts were made to increase the renal clearance of the bile acids by the administration of pharmacological agents. An infusion of sodium acetate (0.3 mol/l) increased the clearance of the radioactive bile acids and augmented the urinary excretion of endogenous 3 alpha-hydroxy bile acids and reduced their concentration in plasma.

Unconjugated, glycine-conjugated, taurine-conjugated bile acid nonsulfates and sulfates in urine of young infants with cholestasis.

A direct assay system for conjugated bile acids using an enzymatic procedure and high-performance liquid chromatography was used for the analysis of urinary bile acid profiles in young infants with intrahepatic cholestasis (idiopathic neonatal hepatitis syndrome) or extra-hepatic biliary atresia. The major urinary bile acids were cholate and chenodeoxycholate conjugates, but a small amount of deoxycholate and 3 beta-hydroxy-5-cholenate conjugates were detected. Although there was no significant difference in total bile acid excretion between patients with intrahepatic cholestasis and extrahepatic biliary atresia, mean ratios of cholate to chenodeoxycholate and sulfated to total urinary bile acids were different between the two groups examined (5.63 +/- 2.83 vs. 2.50 +/- 1.25, p less than 0.05, 15.8 +/- 9.9 vs. 34.5 +/- 9.9%, p less than 0.005). The proportion of taurine-conjugated chenodeoxycholate in the sulfate fraction to the total bile acid was lower in intrahepatic cholestasis, compared with that in biliary atresia (7.7 +/- 7.5 vs 22.7% +/- 7.8%, p less than 0.005). The greater ratio of cholate to chenodeoxycholate and the reduced excretion of sulfated urinary bile acids in intrahepatic cholestasis was due to decreased taurine-conjugated chenodeoxycholate sulfate excretion.

Renal bile acid excretion as a cause of neoplastic lesions in the urinary tract after total portacaval shunt in the normal rat?

Rats with a total portacaval anastomosis (PCA, PC-SS) develop preneoplastic and neoplastic lesions in the urinary tract. In contrast to this, animals with a modified shunt (mPCA) do not develop these lesions. To evaluate the possible role of bile acids excreted with the urine for tumor development, total plasma bile acid concentration and 24 hours urinary bile acid excretion were measured radioimmunologically in rats with total and modified shunts. Additionally the renal 14C-glycocholic acid excretion into the urine was studied after oral administration. Total plasma BA increased from 4.89 +/- 1.0 mumol/l in sham-operated controls to 77.7 +/- 39 mumol/h in PCA and 52.9 +/- 36.7 mumol/l in mPCA rats (p less than 0.001 vs controls, PCA vs mPCA = n.s.). Urinary bile acid excretion rose from 0.2 +/- 0.29 mumol/24 hours in controls to 4.47 +/- 4.49 in PCA and 2.55 +/- 2.22 mumol/24 hours in mPCA rats (p less than 0.001 vs control; PCA vs mPCA = n.s.) 14C-glycocholic acid was excreted within 24 hours into the urine in 13.6 +/- 11.5% in PCA and 26.3 +/- 23.5% of the administered dose in mPCA-rats (controls; 2.98 +/- 0.67%, p less than 0.001; PCA vs mPCA = n.s.). Since renal BA-excretion is similar in both shunted groups, urinary BA does not seem to be of primary significance for cancer development in the urinary tract of totally shunted rats.