用多克隆抗体构建的夹心免疫传感器检测心肌肌钙蛋白I

用表面等离子体子共振生物传感器构建对心肌肌钙蛋白I特异性的免疫传感器检测心肌肌钙蛋白I,并建立两种检测方法:直接法的最低检测限为2.5μg/L,基于传感膜上的夹心免疫法的灵敏度为0.5μg/L,检测范围为0.5～20μg/L,批内及批间精密度分别为3.5%～4.9%,6.1%～7.4%;用夹心法及国外试剂盒对40名健康献血者和20例急性心肌梗死患者血清心肌肌钙蛋白I水平进行检测,两者符合率为95%.     

Synthetic Strategies for Artificial Lipidation of Functional Proteins

Biosynthesis of natural lipidated proteins is linked to important signal pathways, and therefore analyzing protein lipidation is crucial for understanding cellular functions. Artificial lipidation of proteins has attracted attention in recent decades as it allows modulation of the amphiphilic nature of the protein of interest, and is used in the design of drug-delivery systems containing antibodies anchored on a lipid bilayer carrier. However, the intrinsic hydrophobicity of lipids makes the synthesis of lipid–protein conjugates challenging with respect to the yield and selectivity of the lipidation. In this Minireview, the development of chemical and enzymatic synthetic strategies for the preparation of a range of lipid–protein conjugates that do not compromise the functions of the proteins are discussed as well as applications of the conjugates.     

Preparation of Well‐Defined Antibody–Drug Conjugates through Glycan Remodeling and Strain‐Promoted Azide–Alkyne Cycloadditions

Antibody–drug conjugates hold considerable promise as anticancer agents, however, producing them remains a challenge and there is a need for mild, broadly applicable, site‐specific conjugation methods that yield homogenous products. It was envisaged that enzymatic remodeling of the oligosaccharides of an antibody would enable the introduction of reactive groups that can be exploited for the site‐specific attachment of cytotoxic drugs. This is based on the observation that glycosyltransferases often tolerate chemical modifications in their sugar nucleotide substrates, thus allowing the installation of reactive functionalities. An azide was incorporated because this functional group is virtually absent in biological systems and can be reacted by strain‐promoted alkyne–azide cycloaddition. This method, which does not require genetic engineering, was used to produce an anti‐CD22 antibody modified with doxorubicin to selectively target and kill lymphoma cells.     

Anti‐CRLF2 Antibody‐Armored Biodegradable Nanoparticles for Childhood B‐ALL

B‐precursor acute lymphoblastic leukemia (B‐ALL) lymphoblast (blast) internalization of anti‐cytokine receptor‐like factor 2 (CRLF2) antibody‐armored biodegradable nanoparticles (AbBNPs) are investigated. First, AbBNPsaere synthesized by adsorbing anti‐CRLF2 antibodies to poly(D,L‐lactide‐co‐glycolide) (PLGA) nanoparticles of various sizes and antibody surface density (Ab/BNP) ratios. Second, AbBNPs are incubated with CRLF2‐overexpressing (CRLF2+) or control blasts. Third, internalization of AbBNPs by blasts is evaluated by multicolor flow cytometry as a function of receptor expression, AbBNP size, and Ab/BNP ratio. Results from these experiments are confirmed by electron microscopy, fluorescence microscopy, and Western blotting. The optimal size and Ab/BNP for internalization of AbBNPs by CRLF2+ blasts is 50 nm with 10 Ab/BNP and 100 nm with 25 Ab/BNP. These studies show that internalization of AbBNPs in childhood B‐ALL blasts is AbBNP size‐ and Ab/BNP ratio‐dependent. All AbBNP combinations are non‐cytotoxic. It is also shown that CD47 is very slightly up‐regulated by blasts exposed to AbBNPs. CD47 is “the marker of self” overexpressed by blasts to escape phagocytosis, or “cellular devouring”, by beneficial macrophages. The results indicate that precise engineering of AbBNPs by size and Ab/BNP ratio may improve the internalization and selectivity of future biodegradable nanoparticles for the treatment of leukemia patients, including drug‐resistant minority children and Down's syndrome patients with CRLF2+B‐ALL.     

Isolation and Enrichment of Pathogens with a Surface‐Modified Aluminium Chip for Raman Spectroscopic Applications

We developed a Raman‐compatible chip for isolating microorganisms from complex media. The isolation of bacteria is achieved by using antibodies as capture molecules. Due to the very specific interaction with the targets, this approach is promising for isolation of bacteria even from complex matrices such as body fluids. Our choice of capture molecules also enabled the investigation of samples containing yet unidentified bacteria, as the antibodies can capture a large variety of bacteria based on their analogue cell wall surface structures. The capability of our system is demonstrated for a broad range of different Gram‐positive and Gram‐negative germs. Subsequent identification is done by recording Raman spectra of the bacteria. Further, it is shown that classification with chemometric methods is possible.     

Detection and quantification of plasma amyloid-β by selected reaction monitoring mass spectrometry

Amyloid-β (Aβ) in human plasma was detected and quantified by an antibody-free method, selected reaction monitoring mass spectrometry (SRM-MS) in the current study. Due to its low abundance, SRM-based quantification in 10 μL plasma was a challenge. Prior to SRM analysis, human plasma proteins as a whole were digested by trypsin and high pH reversed-phase liquid chromatography (RPLC) was used to fractionate the tryptic digests and to collect peptides, Aβ_1–5, Aβ_6–16, Aβ_17–28 and Aβ_29–40(42) of either Aβ_1–40 or Aβ_1–42. Among those peptides, Aβ_17–28 was selected as a surrogate to measure the total Aβ level. Human plasma samples obtained from triplicate sample preparations were analyzed, obtaining 4.20 ng mL⁻¹ with a CV of 25.3%. Triplicate measurements for each sample preparation showed CV of <5%. Limit of quantification was obtained as 132 pM, which corresponded to 570 pg mL⁻¹ of Aβ_1–40. Until now, most quantitative measurements of Aβ in plasma or cerebrospinal fluid have required antibody-based immunoassays. Since quantification of Aβ by immunoassays is highly dependent on the extent of epitope exposure due to aggregation or plasma protein binding, it is difficult to accurately measure the actual concentration of Aβ in plasma. Our diagnostic method based on SRM using a surrogate peptide of Aβ is promising in that actual amounts of total Aβ can be measured regardless of the conformational status of the biomarker.