Competitive immunoassay for vancomycin using capillary electrophoresis with laser-induced fluorescence detection

A competitive immunoassay using capillary electrophoresis with laser-induced fluorescence was developed for vancomycin. Capillary electrophoresis using a Tris-glycine running buffer provided adequate separation of the antibody-bound from the unbound fluorescent probe (tracer) in less than 4 min. Laser-induced fluorescence polarization (LIFP) provided high sensitivity detection and simultaneous monitoring of fluorescence intensity and polarization. A fluorescence polarization value of 0.30 confirmed the formation of the antibody-tracer complex. Calibration curves showed a working linear range of 2-3 orders of magnitude with a minimum detectable concentration of 0.98 ng mL(-1) (or 1.1 fg vancomycin). Clinical samples obtained from patients undergoing vancomycin treatment were analyzed for vancomycin and the results correlated well with a standard immunoassay based on latex particle detection that was routinely used by a hospital laboratory. Only 1/10 of the reagents were needed as compared with the standard immunoassay.     

Competitive immunoassay for recombinant hirudin using capillary electrophoresis with laser-induced fluorescence detection

A competitive immunoassay based on capillary electrophoresis (CE) with laser-induced fluorescence (LIF) has been developed for the determination of recombinant hirudin (r-hirudin) in biological mixtures. Hirudin, a thrombin inhibitor, is a polypeptide of 65 amino acids. To check purity levels and perform pharmacokinetic studies of (r-hirudin), specific and reproducible analysis methods are demanded. The work involved the development of separation conditions allowing for routine analysis of plasma samples. In this study, r-hirudin was labeled with fluorescein isothiocyanate (FITC), and FITC-labeled r-hirudin was purified using high-performance liquid chromatography. The purified product was then mixed with the sample followed with the addition of anti-hirudin antibody. Free, antibody-bound, and tagged r-hirudin could be separated within 5 min by CE analysis using uncoated fused-silica capillary with high reproducibility. The developed method can be used to determine r-hirudin with good precision and a detection limit lower than 20 nM. This result demonstrates the feasibility of the CE-LIF immunoassay method for the determination of r-hirudin in plasma samples.     

Direct immunoassay of estrone by capillary electrophoresis with laser-induced fluorescence detection

An immunoassay for estrone (E(1)) in women's serum, based on the competitive reaction between fluorescein-labeled complete antigen and E(1) with limited amount of anti-estrone monoclonal antibody is described. A thermally reversible hydrogel, poly-N-isopropylacrylamide (pNIPA), was added to the buffer to improve the reproducibility. With a laser-induced fluorescence (LIF) detector, the capillary electrophoretic immunoassay (CEIA) can be applied to determine E(1) at a concentration lower than 19.6 pg/mL. The E(1) levels in ten normal women's serum were measured at the range of 118.6-222.0 pg/mL.     

Competitive immunoassay for staphylococcal enterotoxin A using capillary electrophoresis with laser-induced fluorescence detection.

Staphylococcal enterotoxins are a family of toxic proteins secreted by S. aureus. Using capillary electrophoresis (CE) linked with laser-induced fluorescence, a highly sensitive and selective assay using antibody-antigen recognition was developed for the determination of Staphylococcal enterotoxin A. Staphylococcal enterotoxin A (SEA) was chemically labeled with fluorescein and the product was used as a fluorescent tracer. A competitive assay was developed to detect SEA at concentrations between 0.3 nM and 6.5 nM with standard deviations of less than 5%. The detection limit was found to be 3 amol with the potential improvement by further optimization of the assay. No cross-reactivity between staphylococcal enterotoxin B and the SEA antibody was found at the concentrations used for the CE immunoassay.     

The immunoassay of methotrexate by capillary electrophoresis with laser-induced fluorescence detection.

Immunoassay is widely employed as a highly sensitive, specific analytical method for hormones and drugs in biological samples. A technique utilizing capillary electrophoresis with laser-induced fluorescence detection was examined based on the reaction process of these immunoassays in order to develop a protocol characterized by high sensitivity and high speed. The conditions of the antigen-antibody reaction and capillary electrophoresis were variously examined using fluorescein-labeled methotrexate and the antibody of methotrexate. As a result, the immunoassay could be completed within a few minutes. Moreover, detection in the pg range could be accomplished. The sensitivity corresponded to that of radioimmunoassay. A simultaneous multi-component analysis of the immunoassay is also possible due to the high resolving power of capillary electrophoresis. In this study, the possibility of a simultaneous analysis of methotrexate and vancomycin was also investigated.     

Analysis of vasopressin using capillary electrophoresis with laser-induced fluorescence detector based on competitive immunoassay

A competitive immunoassay based on CE-LIF has been developed for the determination of vasopressin in biological mixtures. Vasopressin participates in the hormonal control of water metabolism and the constriction of arterioles in humans. Thus, detection of vasopressin is important in diagnosing pathological conditions and physiological water metabolism. The peptides were fluorescently tagged with FITC and purified by HPLC. The purified product was then mixed with the cerebrospinal fluid sample followed with the addition of anti-vasopressin antibody. It was possible to separate antibody-bound and free FITC-tagged vasopressin within 10 min by CE-LIF analysis using uncoated fused-silica capillary with high reproducibility.     

Sensitive and efficient determination of gabapentin in human serum based on capillary electrophoresis with laser-induced fluorescence detection

A sensitive and efficient analytical method for gabapentin (GBP) in human serum based on capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection has been established. 6-Oxy-(N-succinimidyl acetate)-9-(2′-methoxycarbonyl) fluorescein (SAMF), a new synthesized fluorescent reagent, was used for precolumn derivatization of the non-fluorescent drug in serum. γ-Aminobutyric acid (GABA) was used as an internal standard (I.S.). The best derivative condition was obtained in phosphate buffer (pH 8) at room temperature for 10 min. Optimal separation and detection were obtained with a background electrolyte (BGE) of 3.5 × 10^?2 M phosphate buffer (pH 5.5) and laser-induced fluorescence detection excited at 473 nm. The method developed for GBP was linear over the concentration range of 4.0 × 10^?9 to 4.0 × 10-7 M. The concentration limit of detection was 2.0 × 10^?10 M (signal-to-noise ratio = 3). The sensitive method was used for the determination of GBP in serum samples.     

Sensitive and rapid determination of nitric oxide in human serum using microchip capillary electrophoresis with laser-induced fluorescence detection

Microfluidic chip capillary electrophoresis with laser-induced fluorescence detection is employed for direct determination of trace nitric oxide in human blood using diaminorhodamines as the fluorescence probe. Factors influencing the separation and detection processes were systematically studied. Complete and fast separation of the highly fluorescent triazole formed was achieved within 45 s, and the relative standard deviations values of migration time and peak area were less than 3%. The detection limit of NO was 3.0 nmol.L^-1 (at a signal-to-noise ratio of 3) and the liner range was from 1.0?×?10^-8?mol.L^-1 to 3.0?×?10^-6?mol.L^-1. The method has been applied to the determination of NO in serum of healthy persons and patients suffering from diseases, with recoveries varying from 92.65 to 98.43%.     

Capillary electrophoretic competitive immunoassay with laser-induced fluorescence detection for methionine-enkephalin

Immunoassays are commonly used in bioresearch for the detection and quantification of small proteins and macromolecules in biological fluids and other complex matrices. In this report, a competitive immunoassay using capillary electrophoresis (CE) with laser-induced fluorescence was developed for methionine-enkephalin (ME). The method is based on the competitive reaction between the ME and fluorescein conjugated ME (ME-F) with anti-ME antibody, capillary electrophoresis separation of the ME-antibody bound and free ME-F, followed by the laser-induced fluorescence detection of the fluorescent species. With the optimized separation conditions, it was possible to separate the antibody bound and free fluorescien conjugated ME by a capillary electrophoresis-laser-induced fluorescence (CE-LIF) analysis using an uncoated fused-silica capillaries. The results concluded that the assay specificity, selectivity and accuracy were excellent.     

Direct immunoassay of estriol in pregnancy serum by capillary electrophoresis with laser-induced fluorescence detector

A simple and sensitive capillary electrophoretic immunoassay (CEIA) was described for the determination of estriol (E₃) in pregnant women's serum. The method was based on the competitive reaction of fluorescein-labeled E₃ antigen and E₃ with limited amounts of monoclonal antibody. The addition of the thermally reversible hydrogel, poly-N-iso propylacrylamide (pNIPA) in the buffer serving as a replaceable packing material, improved the reproducibility of the method. With laser-induced fluorescence detector (LIF), this method can be applied to determine E₃ at concentrations lower to 31.6 pg ml⁻¹. Recoveries from human steroid-free serum matrix were greater than 94% with relative standard deviation (R.S.D.) values less than 3.5%. Serum E₃ levels of ten normal pregnant women were measured at the range of 10.2-15.6 ng ml⁻¹.     

癌胚抗原毛细管电泳-化学发光均相免疫分析

设计了一种测定人血清中癌胚抗原的毛细管电泳-化学发光检测均相免疫分析新方法。采用四苯硼酸钠增强luminol-H2O2-HRP体系化学发光的原理,化学发光检测经毛细管电泳分离的,用辣根过氧化物酶(HRP)标记的癌胚抗体及免疫复合物。测定癌胚抗原的线性范围2.0~80.0μg/L(R=0.9921),检出限为0.1μg/L(绝对检出限为0.75 fg)。     

癌胚抗原毛细管电泳-化学发光均相免疫分析

建立了一种测定人血清中癌胚抗原的毛细管电泳-化学发光检测的均相免疫分析新方法.采用四苯硼钠增强luminol-H2 O2-HRP体系化学发光的原理,化学发光检测经毛细管电泳分离的,用辣根过氧化物酶(HRP)标记的癌胚抗体及免疫复合物.测定癌胚抗原的线性范围2.0～80.0 μg/L(R=0.9921),检出限为0.1 μg/L(绝对检出限为0.75 fg).     

Competitive immunoassay by capillary electrophoresis with laser-induced fluorescence for the trace detection of chloramphenicol in animal-derived foods

A competitive immunoassay using CE with an LIF detector was developed for the detection of chloramphenicol (CAP). The method was based on the competitive reactions between fluorescently labeled CAP hapten and free CAP, with a limited amount of anti-CAP antibody. The poly(N-isopropylacrylamide) (pNIPA) hydrogel was added in the separation buffer as a dynamic modifier to reduce adsorption and enhance reproducibility. The linear range and LOD for CAP were 0.008-5 mug/L and 0.0016 mug/L, respectively. An ELISA using the same immuno-reagents was also developed for the analysis of CAP, with an LOD of 0.03 mug/L. The sensitivity of this CE immunoassay (CEIA)-LIF was almost 20 times greater than that of the ELISA. Using CEIA-LIF, equilibrium was reached in 15 min and the analytical results were obtained within 5 min by CE separation. Sample preparation for CEIA-LIF was not time-consuming and the matrix effect was easy to remove. An LOD of 0.1 mug/kg CAP in food matrices was easily achieved. This method is thus proposed as a fast and sensitive means of detecting trace amounts of CAP residues in animal-derived foods.     

Detection of prion protein using a capillary electrophoresis-based competitive immunoassay with laser-induced fluorescence detection and cyclodextrin-aided separation

The development of capillary electrophoresis (CE)-based competitive immunoassay for prion protein (PrP) using carboxymethyl beta-cyclodextrin (CM-beta-CD) as a buffer additive is described here. The assay was based on the competitive binding of PrP and a fluorescein-labeled peptide from the prion protein with a limiting amount of specific antibody. The amount of both free and fluorescein-labeled peptide bound to antibody (immunocomplex) were determined by CE with laser-induced fluorescence detection. In the presence of PrP, the peak height ratio of the immunocomplex and the free peptide was altered compared to the control. These changes were directly proportional to the amount of PrP present. The fluorescently labeled peptide spanning amino acid positions 140-158 of the PrP and its corresponding monoclonal antibody is reported here. The reaction times of the antibody with either the peptide or the recombinant PrP was less than 1 min and is a large improvement over the 16-18 h required to achieve equilibrium for polyclonal antibodies. CM-beta-CD was explored as a buffer additive to suppress analyte adsorption and enhance separation selectivity in the CE analysis. A fast (1.1 min), selective (resolution 4.7), and reproducible (relative standard deviations of migration time for free and bound fluorescein isothiocyanate (FITC)-peptide 0.56% and 0.64%, respectively) separation was obtained with 0.6% CM-beta-CD in 25 mM N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS) at pH 8.8. The concentration detection limit of the assay for recombinant PrP was determined to be 80 ng/mL (or mass detection limit 1 pg). When blood samples from scrapie-infected sheep and from normal sheep were tested, the results of the blood assay were consistent with scrapie status of the sheep as determined post mortem by Western blot analysis. Development of this assay will lead to a potentially robust, rapid, and specific preclinical diagnosis for transmissible spongiform encephalopathies (TSEs) in animals and humans.     

Separation and determination of carnosine-related peptides using capillary electrophoresis with laser-induced fluorescence detection

A capillary electrophoresis (CE) method with laser-induced fluorescence (LIF) detection was developed for the separation and detection of carnosine-related peptides (carnosine, anserine, and homocarnosine). A sensitive and fluorogenic regent, 3-(4-carboxybenzoyl) quinoline-2-carboxaldehyde (CBQCA) was selected as a precapillary labeling reagent for imidazole dipeptides to form isoindole derivatives. The optimized molar ratio between CBQCA and peptide was found to be 75:1, and 50 mmol/L borate buffer (pH 9.2) was used for the derivatization in order to achieve good efficiency. Three imidazole dipeptides were baseline-separated within 20 min by using 112 mmol/L sodium borate (pH 10.4-10.8) as running buffer. Concentration detection limits (signal-to-noise ratios) for carnosine, anserine, and homocarnosine were 4.73, 4.37, and 3.94 nmol/L, respectively. This method has been applied to the analysis of human cerebrospinal fluid (CSF) and meat dry powder of pig and sheep. Recoveries were in the range of 82.9-104.8% for homocarnosine in CSF. For carnosine and anserine, the recoveries are 98.3% and 80.2% in meat dry powder of pig and 111.2% and 112.8% in meat dry powder of sheep, respectively.     

Sensitive determination of ephedrine and pseudoephedrine by capillary electrophoresis with laser-induced fluorescence detection

A CE-LIF method was developed for the separation and sensitive detection of ephedrine and pseudoephedrine after derivatization by 4-chloro-7-nitrobenzo-2-oxa-1,3-diazol (NBD-C1). The derivatization and separation conditions were investigated in detail and the optimum conditions were obtained. Under the optimum experiment conditions, good linearity relationships (correlation coefficients: 0.9942 for ephedrine and 0.9970 for pseudoephedrine) between the peak heights and concentrations of the analytes were obtained (0.7-140 microM). The detection limits were 0.16 microM for ephedrine and 0.17 microM for pseudoephedrine, which indicated that the sensitivities were at least ten times improved over those reported in the literature obtained by UV detection. The method was applied to the analysis of ephedrine and pseudoephedrine in ephedra herb plants and preparations with good results.     

Determination of aluminum in serum by capillary zone electrophoresis with laser-induced fluorescence detection

Summary A novel method of separating and detecting trace aluminum by capillary zone electrophoresis is described. Aluminum is reacted with lumogallion [4-chloro-3-(2,4-dihydroxyphenylazo)-2-hydroxybenzen-1-sulphonic acid] so that the complex can be selectively and sensitively detected by a laser-induced fluorescence detector after capillary electrophoretic separation. Using the proposed method, limits of detection in the sub parts per billion range are achieved. The technique is applied to the determination of aluminum in human serum.     

A new quantitative method for circulating DNA level in human serum by capillary zone electrophoresis with laser-induced fluorescence detection

We present a method for the quantification of circulating DNA in serum by capillary zone electrophoresis (CZE) with laser-induced fluorescence detection (LIF). The serum was digested by proteinase to release free DNA. SYBR Gold was utilized as DNA intercalating dye, fluorescein as internal standard (ISTD). CZE-LIF was applied for the separation and quantification of total circulating DNA. Good linearity (R = 0.9992) in the low range of DNA concentrations (0.5-40 ng/mL) and a detection limit of 0.5 ng/mL for DNA (S/N = 3) were obtained. Our data demonstrated that CZE-LIF system has a good linearity with excellent sensitivity and satisfactory reproducibility in the quantification of circulating DNA in serum. This method was successfully used for the quantification of circulating DNA levels in serum. We observed that the circulating DNA levels in certain cancer patients were significantly higher than that in healthy individuals. Compared to current methods, our protocol does not need the extraction of DNA from serum. Our preliminary results have illustrated that CZE-LIF system is simple, rapid, and sensitive, and it is well suitable for large-scale quantification of circulating DNA levels in clinical diagnosis.     

Multiplexed microRNA detection by capillary electrophoresis with laser-induced fluorescence

In this study, we developed a novel assay that simultaneously detects multiple miRNAs (microRNAs) within a single capillary by combining a tandem adenosine-tailed DNA bridge-assisted splinted ligation with denaturing capillary gel electrophoresis with laser-induced fluorescence. This proposed method not only represents a significant improvement in resolution but also allows for the detection of multiple miRNAs within a single capillary based on the length differences of specified target bridge DNA. The assay's linear range covers three orders of magnitude (1.0 nM to 1.0 pM) with a limit of detection (S/N=3) as low as 190 fM (2.5 zmol). Five miRNAs of Epstein-Barr virus (EBV) were also detected in EBV-infected nasopharyngeal carcinoma cells, while they did not appear in non-virus infected cells. Moreover, the electropherogram indicated that the screening of isomiRs (isomer of miRNA) of BART2 by CE-LIF is feasible by our proposed method. The developed electrophoresis-based method for miRNA detection is fast, amplification-free, multiplexed and cost-effective, making it potentially applicable to large-scale screening of isomiRs.     

Amino acids determination using capillary electrophoresis with on-capillary derivatization and laser-induced fluorescence detection

Free amino acids have been derivatized on-capillary with 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ) and analyzed using a laboratory-made capillary electrophoresis apparatus with laser-induced fluorescence detection. Several parameters that control on-capillary derivatization of amino acids, including pH, mixing time, reaction time, concentration of the derivatization reagents (potassium cyanide and FQ) and solvent of FQ, as well as the temperature of mixing and reaction were optimized. Repeatabilities better than 1.8% for migration time and 7.8% for peak height were obtained. Assay detection limits for the different amino acids ranged from 23 nM for glycine to 50 nM for lysine and glutamic acid. The methods developed were applied to the analysis of several amino acids in pharmaceutical preparations and plasma samples. Results showed a good agreement with those obtained using an amino acid autoanalyzer for the same samples.