Depletion of human serum albumin in embryo culture media for in vitro fertilization using monolithic columns with immobilized antibodies.

Affinity depletion of abundant proteins such as HSA is an important stage in routine sample preparation prior to MS/MS analysis of biological samples with high range of concentrations. Due to the charge competition effects in electrospray ion source that results in discrimination of the low-abundance species, as well as limited dynamic range of MS/MS, restricted typically by three orders of magnitude, the identification of low-abundance proteins becomes a challenge unless the sample is depleted from high-concentration compounds. This dictates a need for developing efficient separation technologies allowing fast and automated protein depletion. In this study, we performed evaluation of a novel immunoaffinity-based Convective Interaction Media analytical columns (CIMac) depletion column with specificity to HSA (CIMac-αHSA). Because of the convective flow-through channels, the polymethacrylate CIMac monoliths afford flow rate independent binding capacity and resolution that results in relatively short analysis time compared with traditional chromatographic supports. Seppro IgY14 depletion kit was used as a benchmark to control the results of depletion. Bottom-up proteomic approach followed by label-free quantitation using normalized spectral indexes were employed for protein quantification in G1/G2 and cleavage/blastocyst in vitro fertilization culture media widely utilized in clinics for embryo growth in vitro. The results revealed approximately equal HSA level of 100 ± 25% in albumin-enriched fractions relative to the nondepleted samples for both CIMac-αHSA column and Seppro kit. In the albumin-free fractions concentrated 5.5-fold by volume, serum albumin was identified at the levels of 5-30% and 20-30% for the CIMac-αHSA and Seppro IgY14 spin columns, respectively.

A comparative study of capillary electrophoresis and isothermal titration calorimetry for the determination of binding constant of human serum albumin to monoclonal antibody.

This paper focuses on the investigation of the interactions between the anti-HSA-mAb and its protein antigen using CZE, ACE, and isothermal titration calorimetry. The CZE revealed the formation of the anti-HSA-mAb·HSA and anti-HSA-mAb·(HSA)2 complexes and the binding constants determined by plotting the amount of the bound anti-HSA-mAb as a function of the concentration of HSA. The ACE provided information on the binding strength from the change in effective electrophoretic mobility of the anti-HSA-mAb. These two separation techniques estimated the presence of two binding sites. The equilibrium dissociation constant values obtained by CZE and ACE were found to be 2.26 × 10(-6) M for anti-HSA-mAb·HSA, 1.22 × 10(-6) M for anti-HSA-mAb·(HSA)2 and 4.45 × 10(-8) M for anti-HSA-mAb·HSA, 1.08 × 10(-7) M for anti-HSA-mAb·(HSA)2 , respectively. The dissociation constant data obtained by ACE were in congruence with the values obtained by isothermal titration calorimetry (2.74 × 10(-8) M, 1.04 × 10(-7) M).

Investigation on chemotactic drug targeting (chemotaxis and adhesion) inducer effect of GnRH-III derivatives in Tetrahymena and human leukemia cell line.

GnRH-III has been shown to exert a cytotoxic effect on the GnRH-R positive tumor cells. The chemotactic drug targeting (CDT) represents a new way for drug delivery approach based on selective chemoattractant guided targeting. The major goal of the present work was to develop and investigate various GnRH-III derivatives as potential targeting moieties for CDT. The cell physiological effects (chemotaxis, adhesion, and signaling) induced by three native GnRHs (hGnRH-I, cGnRH-II, and lGnRH-III) and nine GnRH-III derivatives were evaluated in two model cells (Tetrahymena pyriformis and Mono Mac 6 human monocytes). According to our results, the native GnRH-III elicited the highest chemoattractant and adhesion inducer activities of all synthesized peptides in micromolar concentrations in monocytes. With respect to chemoattraction, dimeric derivatives linked by a disulfide bridge ([GnRH-III(C)](2) ) proved to be efficient in both model cells; furthermore, acetylation of the linker region ([GnRH-III(Ac-C)](2) ) could slightly improve the chemotactic and adhesion effects in monocytes. The length of the peptide and the type of N-terminal amino acid could also determine the chemotactic and adhesion modulation potency of each fragment. The application of the chemoattractant GnRH-III derivatives was accompanied by a significant activation of phosphatidylinositol 3-kinase in both model cells. In summary, our work on low-level differentiated model cells of tumors has proved that GnRH-III and some of its synthetic derivatives are promising candidates to be applied in CDT: these compounds might act both as carrier, delivery unit, and antitumor agents.