用于固定抗体的新型温敏性核—壳结构聚合物微球的合成及其初步应用研究

利用无皂乳液聚合和种子聚合的方法合成了一种以聚苯乙烯为核,聚(N-异丙基丙烯酰胺-co-N-丙烯酸琥珀酰亚胺酯)为壳的单分散的核-壳结构的聚合物微球.用扫描电镜和透射电镜观察了球的形貌特征,发现微球具有清晰的核-壳结构和较好的单分散性,红外光谱显示了在1738cm-1处有酯羰基的特征吸收峰.动态光散射测定发现该聚合物微球具有温敏性,当温度高于聚N-异丙基丙烯酰胺的最低临界溶液温度(LCST)时,球的流体力学直径变小.利用微球壳层所含有的琥珀酰亚胺酯基与伯氨基的高反应活性,将抗体Rabbit IgG化学固定在球的壳层上.由于壳层的聚N-异丙基丙烯酰胺具有温敏性,反应温度不同结合的抗体的量也不同,在0℃和36.5℃,微球对抗体的结合率分别为61.6%和38.6%.     

Nanomagnet Bioconjugates with anti-CRP Polyclonal Antibodies as Nanobioprobes for Enhanced Impedimetric Detection of CRP

Selective purification of biological materials for their detection in complex sample matrix is a general challenge for many researchers working in the field of diagnostics. Magnetic nanoparticles functionalized with biological molecules that impart biomolecular selectivity are therefore of major interest as capture probes thus allowing for magnetic separations. Nanoparticles can also be used for the enhanced detection of biomarkers. In this work, an ultrasensitive sandwich-based impedimetric immunosensor was fabricated for the detection of C-reactive protein antigen (CRPAg). Stable and oriented immobilization of anti-CRP monoclonal antibody was achieved onto electrografted phenylethylamine derivatized with succinic anhydride and phenylboronic acid via carbodiimide chemistry. The detection of CRPAg was achieved using Electrochemical Impedance Spectroscopy (EIS). The enhancement of the impedance charge-transfer resistance (R_CT) signal was achieved using the sandwich approach. The anti-CRP polyclonal antibody was immobilized in an oriented manner onto magnetic nanoparticles functionalized with phenylboronic acid. The increase in the change in charge-transfer resistance (ΔR_CT) values was linearly proportional to the concentration of CRPAg in the range 10 to 200 ng mL⁻¹ covering the clinical range for CRPAg detection and with a detection limit of 0.34 ng mL⁻¹.     

Synthesis and optical characteristics of 4-styrylpyridinium dyes and their conjugates with antibody

Two new styryl-type dyes modified with the succinimide ester group were prepared for conjugation with antibodies. The optical characteristics of the obtained compounds in different solvents were studied, and the solvatochromic properties of these dyes were revealed. The applicability of the dyes as protein labels was demonstrated in cyto-fluorometry and fluorescence microscopy.     

Production of Monoclonal antibodies to membrane components of human colorectal cancer HCT-116 cell line for diagnostic purposes

Colorectal cancer (CRC) is the second leading cause of cancer-related death worldwide. The survival rate of people diagnosed in the early stages of disease is 92 % whereas; less than 11 % of patients survive if reached stage IV. Moreover, only 24 % of cases are diagnosed during stage I. Thus, the early diagnosis of the CRC will save many lives, reduce suffering of patients. Biomarkers that are in use for the diagnosis of CRC such as Carcinoembryonic Antigen (CEA), Carbohydrate Antigen (CA 19–9) suffer from low sensitivity, expressed in other neoplasms, and not expressed in early stages of cancer. This study aims to find a highly sensitive and specific biomarker for early detection of CRC. Hybridoma clones were established by fusing spleen cells from mice hyperimmunized with human cell membrane homogenate of HCT116 with murine myeloma cells X63Ag8. One clone was selected after fusion and designated as D2C5. Isotyping of secretory antibody revealed an IgG1 kappa light chain and was reactive to a protein with molecular weight about 55 kDa using immunoblot. In situ reactivity of the D2C5 showed homogenous and cytoplasmic pattern staining in stage IV and III, discrete and heterogeneous pattern was seen in stage II samples. Moreover, nuclear staining pattern was seen in one slide of stage II samples. In addition, mucinous type was detected. Low peripheral staining was observed in normal colon tissue. These observations suggest the localization of the target protein in the cytoplasm of epithelial cells with high expression in more advanced cases, suggesting a cytokeratin pattern of staining.     

Switching positions: Assessing the dynamics of conjugational heterogeneity in antibody–drug conjugates using CE-SDS

Antibody–drug conjugates (ADCs) are a prospective class of new oncology therapeutics with the ability to deliver a cytotoxic drug to a targeted location. The concept appears simple, but ADCs are highly complex due to their intrinsic heterogeneity. Randomly conjugated ADCs, for instance, are composed of conjugated species carrying between 0 and 8 linker-drug molecules, with several positional isomers that vary in drug distribution across the antibody. The drug load, expressed as drug-to-antibody ratio (DAR), is a critical quality attribute and should be well controlled, together with the distribution of drug molecules. Here, the impact of the duration of disulfide bond reduction on the DAR was investigated by quantitating the (isomeric) DAR species in ADCs produced with varying reduction times. Although hydrophobic interaction chromatography showed a constant DAR value as a function of reduction time, data obtained by non-reducing CE-SDS revealed an unexpected dynamic in the positional conjugated isomers. The insights obtained have improved our understanding of the correlation between the disulfide bond reduction, an important step in the manufacturing of a cysteine-conjugated ADC, and the conjugational heterogeneity.