Qinggang Wang

Capillary electrophoresis-based immunoassay

Capillary electrophoresis-based immunoassay (CEIA) is a developing analytical technique with a number of advantages over conventional immunoassay, such as reduced sample consumption, simpler procedure, easy simultaneous determination of multiple analytes, and short analysis time. However, there are still a number of technical issues that researchers on CEIA have to solve before the assay can be more widely used. These issues include method to improve the concentration sensitivity of the assay, requirement for robust separation strategy for different analytes, and method to increase the throughput of the assay. The approaches to solve these issues are reviewed. Several studies have been devoted to develop general separation strategies for CEIA, and to enhance the sensitivity of detection. The recent development of microchip-based CEIA is encouraging and is likely to address more drawbacks of CEIA, particularly on the throughput issue.

SIMULTANEOUS FORMATION OF PEPTIDES AND NUCLEOTIDES FROM N-PHOSPHOTHREONINE

An intramolecular mutual activation between a phosphoryl group and carboxyl group results in the simultaneous formation of nucleotides and peptides by the reaction of nucleosides with N-(O,O-diisopropyl)phosphothreonine in anhydrous pyridine. These results suggest pathways for the simultaneous prebiotic synthesis of peptides and oligonucleotides.

Use of capillary electrophoresis-based competitive immunoassay for a large molecule.

A systematic study on the optimization of capillary electrophoresis-based immunoassay (CEIA) was performed using bovine serum albumin (BSA) and monoclonal anti-BSA. The immunocomplex could not be resolved from free BSA or anti-BSA with UV detection. When fluorescein isothiocyanate-labeled BSA (FITC-BSA) was used as tracer, the free and bound FITC-BSA were well separated giving definite peaks with laser induced fluorescence detection. The factors affecting the separation of the free and bound FITC-BSA, including voltage, pH and ionic strength of the running buffer, were systematically analyzed. Competitive CEIAs were demonstrated in uncoated and coated capillaries with whole or Fab fragment of the antibody. The coefficient of variation for the quantification of BSA in coated capillary was less than that in uncoated capillary. This study demonstrated that competitive CEIA could be applied to quantify high-molecular-mass protein in biological fluids.

Noncompetitive immunoassays using protein G affinity capillary chromatography and capillary electrophoresis with laser-induced fluorescence detection

A new and simple approach to perform immunoassay using protein G affinity capillary chromatography and laser-induced fluorescence detection was described. A noncompetitive assay for monoclonal anti-bovine serum albumin (BSA) was used to test the performance of the system. Fluorescein isothiocyanate labeled BSA was used as a tracer to determine anti-BSA in pM level. Capillaries with inner diameter of 150 microns were packed with recombinant protein G-bound perfusive support. The packed capillary was used to capture the immunocomplexes, which were subsequently desorbed by 100 mM glycine (pH 9.0). Open tube capillary electrophoresis-based immunoassay (CEIA) for anti-BSA was also performed. Using standard samples, calibration curves for anti-BSA was established in both assays. Compared with CEIA, this system improved the concentration sensitivity for about 100-fold due to the pre-concentration of immunocomplex in the protein G column, while the mass sensitivity was similar in the two methods.

Capillary Electrophoresis Based Immunoassay for Monoclonal Antibody with Diode Laser Induced Fluorescence Detection

ABSTRACT A capillary electrophoresis based immunoassay (CEIA) for monoclonal antibody using diode laser induced fluorescence (LIF) detection was described. A direct assay for monoclonal anti-BSA in mouse serum was used as a model. BSA was labeled with Cy5 and used as the immunoreagent. The 635 nm line of a diode laser was used as the excitation source for LIF detection. The calibration curve for anti-BSA in mouse serum had a linear dynamic range of 4-40 nM. The concentration limit of detection (LOD) was 1.2 nM. Incubation time and CE conditions such as buffer concentration, pH and separation voltage were optimized, and the performances of different lasers as excitation sources were also compared.

DETERMINATION OF LEVONORGESTREL IN HUMAN SERUM BY LIQUID CHROMATOGRAPHIC-ELECTROSPRAY TANDEM MASS SPECTROMETRY

A rapid, sensitive, and specific high-performance liquid chromatography electrospray tandem mass spectrometric method has been developed for the determination of levonorgestrel in human serum using norethindrone as an internal standard. Levonorgestrel was detected using multiple reaction monitoring mode after extracted from human serum by an ether extraction procedure. The calibration curve was linear over the range of 5–500 ng/mL (r 2 ≥ 0.999) with the limitation of detection of 1 ng/mL. The intra-day precision and accuracy, expressed as CV and RE, ranged from 0.5% to 10.1% and −0.2% to 2.2%. The inter-day precision and accuracy ranged from 2.1% to 13.6% and −2.3% to 3.3%. The mean recovery was 89.5% for levonorgestrel, and 90.2% for the internal standard.

Analysis of the reaction products of N-(O,O-diisopropyl)phosphorylthreonine with uridine by capillary zone electrophoresis with diode array detection and capillary electrophoresis-mass spectrometry

Abstract The reaction productions of N-(O,O-diisopropyl)phosphorylthreonine with uridine were analyzed by capillary zone electrophoresis with diode array detection using 50 m M boric acid buffer at pH 8.5. Peaks in the electropherograms were identified by the on-line UV spectra as well as some authentic standards. The influence of buffer composition and capillary coating on separations were studied. The products were also analyzed by capillary electrophoresis-mass spectrometry with a sheath-flow electrospray ionization interface. Identification was achieved by reconstructed ion electropherograms. The existence of nucleotides, homooligonucleotides and their derivatives in the products was confirmed by both methods, which supports a hypothesis for the origin of life.

FEASIBILITY STUDY OF ENZYME-AMPLIFIED SANDWICH IMMUNOASSAY USING PROTEIN G CAPILLARY AFFINITY CHROMATOGRAPHY AND LASER INDUCED FLUORESCENCE DETECTION

The feasibility of performing the enzyme-amplified sandwich immunoassay with protein G capillary affinity chromatography and laser induced fluorescence (LIF) detection was investigated using the determination of human immunoglobulin G (hIgG) as a model system. The incubated mixture of samples containing hIgG and alkaline phosphatase (ALP) conjugated goat anti-human IgG F(ab′)2 fragment was loaded onto the capillary column packed with recombinant protein G bound perfusive support in neutral pH. After nonretained compounds were eluted, the fluorogenic ALP substrate, fluorescein diphosphate (FDP), was loaded onto the capillary column followed by stop-flow incubation. Finally, the product of ALP-mediated hydrolysis of FDP, fluorescein, was swept out of the capillary column and detected with a LIF detector using the 488 nm line of an argon ion laser as the excitation source. Chromatographic conditions were optimized. The calibration curve for hIgG was linear over the range of 0.5–50 pmol/L (r2=0.999) with the limit of detection of 0.2 pmol/L.

SANDWICH IMMUNOASSAY FOR MONOCLONAL ANTIBODY USING PROTEIN G IMMUNOAFFINITY CAPILLARY CHROMATOGRAPHY AND DIODE LASER INDUCED FLUORESCENCE DETECTION

A sandwich immunoassay for monoclonal antibody using protein G immunoaffinity capillary chromatography and LIF detection was described. Monoclonal anti-bovine serum albumin (BSA) was used as a model to test the performance of the system. Cy5 was used as the fluorescent tag and the 635 nm line of a diode laser was used as the excited source. Capillaries with inner diameter of 150 μm were packed with recombinant protein G bound perfusive support and used to capture the immunocomplexes, which were subsequently desorbed by an acidic buffer and then detected using LIF. The results demonstrated the feasibility to perform sandwich immunoassay using this system.

Determination of gestrinone in human serum by liquid chromatography–electrospray tandem mass spectrometry

A rapid, sensitive and specific high-performance liquid chromatography-electrospray tandem mass spectrometric method has been developed for the determination of gestrinone (R 2323) in human serum using mifepristone (RU 486) as an internal standard. R 2323 was extracted from human serum by an ether extraction procedure. Multiple reaction monitoring was used to detect R 2323 and RU 486. The calibration curve was linear over the range of 3.5-177 ng/ml (r2 > or =0.99) with the limitation of detection of 0.8 ng/ml. The intra-day precision and accuracy, expressed as C.V. and RE, ranged from 2.3-13.7 to -4.8-3.0%. The inter-day precision and accuracy ranged from 5.5-14.8 to -6.7-3.1%. The mean recovery was 91.0% for R 2323, and 90.6% for the internal standard. The method was successfully applied to the pharmacokinetic study of R 2323.