Sensitive detection of C-reactive protein in serum by immunoprecipitation-microchip capillary gel electrophoresis.

Sepsis represents a significant cause of mortality in intensive care units. Early diagnosis of sepsis is essential to increase the survival rate of patients. Among others, C-reactive protein (CRP) is commonly used as a sepsis marker. In this work we introduce immune precipitation combined with microchip capillary gel electrophoresis (IP-MCGE) for the detection and quantification of CRP in serum samples. First high-abundance proteins (HSA, IgG) are removed from serum samples using affinity spin cartridges, and then the remaining proteins are labeled with a fluorescence dye and incubated with an anti-CRP antibody, and the antigen/antibody complex is precipitated with protein G-coated magnetic beads. After precipitation the complex is eluted from the beads and loaded onto the MCGE system. CRP could be reliably detected and quantified, with a detection limit of 25 ng/µl in serum samples and 126 pg/µl in matrix-free samples. The overall sensitivity (LOQ = 75 ng/µl, R(2) = 0.9668) of the method is lower than that of some specially developed methods (e.g., immune radiometric assay) but is comparable to those of clinically accepted ELISA methods. The straightforward sample preparation (not prone to mistakes), reduced sample and reagent volumes (including the antibodies), and high throughput (10 samples/3 h) are advantages and therefore IP-MCGE bears potential for point-of-care diagnosis.

Biomimetic delivery strategies at the urothelium: targeted cytoinvasion in bladder cancer cells via lectin bioconjugates.

PURPOSE: Urothelial cells, including bladder cancer (BCa) cells, represent a highly valuable but challenging target for localized antineoplastic therapy. This study describes a novel, biomimetic approach to improve intravesical drug delivery, based on glycan-specific targeting. In direct analogy to the invasion mechanism used by uropathogenic bacteria, we evaluate the potential of lectin bioconjugates to facilitate binding and uptake of large payload molecules at this penetration-hostile barrier. METHODS: Wheat germ agglutinin (WGA) served as a targeting ligand and was covalently coupled to fluorescein-labeled bovine serum albumin (fBSA), yielding multivalent protein bioconjugates. Cytoadhesion, uptake and intracellular processing were characterized on a panel of urothelial cell lines of non-malignant and malignant origin. RESULTS: Conjugation to WGA rendered the fBSA payload protein strongly cytoadhesive, with a clear preference in binding to cancerous cells. The highly specific, lectin-mediated recognition process was followed by rapid internalization, and extensive but non-exclusive accumulation in acid and LAMP-2-positive compartments. Stage of malignancy and mechano-structural cell configuration were important determinants for the sorting between different processing pathways. CONCLUSION: Lectin-bioconjugates allow for triggering endogenous uptake routes and influencing the intracellular distribution in BCa cells. They hold considerable promise for enhancing the delivery of small molecule drugs and complex biomolecules in intravesical therapy.

A fluorescent derivatization method of proteins for the detection of low-level impurities by microchip capillary gel electrophoresis.

A novel pre-chip fluorescent derivatization method is presented for protein sizing and quantification by microchip CGE. The derivatization reaction employed a water-soluble and stable fluorescent dye and was performed under conditions that favored the formation of homogeneous reaction products. The method delivered in terms of protein sizing similar results as microchip CGE with on-chip staining but showed an extended linear dynamic range for protein quantification encompassing four orders of magnitude. The sensitivity of the method was similar to standard silver-stained planar gels. The characterization of derivatization reaction products by MS and preparative isoelectric focusing indicated that a constant degree of dye molecule tagging was obtained over a broad range of protein/dye ratios. The method allowed detecting and quantifying an impurity spiked into an antibody preparation down to a level of 0.05%. Advantages of this method compared with CGE approaches with pre-column derivatization include a shorter analysis time and an increased robustness and ease of use.

Immunoprecipitation combined with microchip capillary gel electrophoresis: Detection and quantification of ß-galactosidase from crude E. coli cell lysate.

A sensitive and selective analytical method for the determination and quantification of endogenous ß-galactosidase in crude E. coli cell lysates by immunoprecipitation combined with automated microchip capillary gel electrophoresis (IP-MCGE) with laser-induced fluorescence (LIF) detection was developed. Total cell lysates were derivatized minimally with a fluorescence dye, incubated with anti-ß-galactosidase antibodies, and the antigen/antibody complex was precipitated with protein G-coated magnetic beads. After capturing the complex, it was eluted from the beads under denaturing conditions and loaded directly onto a multisample microchip for analysis. The effects of antibody selection and the importance of preclearing steps were studied in detail. For quantification, an external calibration through spiking pure ß-galactosidase into E. coli lysate was performed. Recovery rates of immunoprecipitation after spiking experiments and the amount of unknown endogenous ß-galactosidase in E. coli lysates were determined. As proof of principle, E. coli cultures grown on nutrition media with several glucose/lactose ratios were analyzed. Differences in the expression level of ß-galactosidase could be detected and measured with the developed method. Detected amounts of ß-galactosidase in different culture media correlated with the ß-galactosidase activities in these cultures.