Methods to generate site-specific conjugates of antibody and protein.

Antibody-protein conjugates have been useful tools for studying biological systems and also played important roles in developing therapeutics and diagnostics. In particular, because of the increased interest in therapeutics of complexity higher than monoclonal antibodies, various methods have been reported for generating bispecific antibodies, immunotoxins, and antibody-enzyme conjugates in which antibodies are site-specifically conjugated with other proteins. Compared with conjugating antibodies with synthetic molecules, controlling the modification sites is difficult in the antibodies conjugated with protein cargos due to the presence of several reactive groups in both molecules. Enzymatic reactions are often used to generate antibody-protein conjugates owing to their high specificity for both reactants and products. Chemical modifications involving genetic introduction of natural or unnatural amino acid residues have also been used for site-specific conjugation of antibodies. Recent studies have developed methods to modify native antibodies using peptides having affinity for antibodies, and these methods do not need antibody engineering for conjugation reactions. In this review, we have summarized enzymatic and chemical approaches to generate site-specific antibody-protein conjugates.

Coupling hCG-based protease sensors with a commercial pregnancy test strip for simple analyses of protease activities.

Proteases play an essential role in many cellular processes, and consequently, abnormalities in their activities are related to various diseases. Methods have been developed to measure the activity of these enzymes, but most involve sophisticated instruments or complicated procedures, which hampers the development of a point-of-care test (POCT). Here, we propose a strategy for developing simple and sensitive methods to analyze protease activity using commercial pregnancy test strips that detect human chorionic gonadotropin (hCG). hCG was engineered to have site-specific conjugated biotin and a peptide sequence, which can be cleaved by a target protease, between hCG and biotin. hCG protein was immobilized on streptavidin-coated beads, resulting in a protease sensor. The hCG-immobilized beads were too large to flow through the membrane of the hCG test strip and yielded only one band in the control line. When the peptide linker was hydrolyzed by the target protease, hCG was released from the beads, and the signal appeared in both the control and test lines. Three protease sensors for matrix metalloproteinase-2, caspase-3, and thrombin were constructed by replacing the protease-cleavable peptide linker. The combination of the protease sensors and a commercial pregnancy strip enabled the specific detection of each protease in the picomolar range, with a 30-min incubation of the hCG-immobilized beads and samples. The modular design of the protease sensor and simple assay procedure will facilitate the development of POCTs for various protease disease markers.

Advantages of Adult Mouse Dorsal Root Ganglia Explant Culture in Investigating Myelination in an Inherited Neuropathic Mice Model.

A co-culture of neurons and Schwann cells has frequently been used to investigate myelin sheath formation. However, this approach is restricted to myelin-related diseases of the peripheral nervous system. This study introduces and compares an ex vivo model of adult-mouse-derived dorsal root ganglia (DRG) explant, with an in vitro co-culture of dissociated neurons from mouse embryo DRG and Schwann cells from a mouse sciatic nerve. The 2D co-culture has disadvantages of different mouse isolation for neurons and Schwann cells, animal number, culture duration, and the identification of disease model. However, 3D DRG explant neurons and myelination cells in Matrigel-coated culture are obtained from the same mouse, the culture period is shorter than that of 2D co-culture, and fewer animals are needed. In addition, it has simpler and shorter experimental steps than 2D co-culture. This culture system may prove advantageous in studies of biological functions and pathophysiological mechanisms of disease models, since it can reflect disease characteristics as traditional co-culture does. Therefore, it is suggested that a DRG explant culture is a scientifically, ethically, and economically more practical option than a co-culture system for studying myelin dynamics, myelin sheath formation, and demyelinating disease.

One-pot colorimetric detection of molecules based on proximity proteolysis reaction.

Various types of molecules serve as biomarkers of diseases, and numerous methods have been reported to detect and quantify them. Recently, research efforts have been made to develop point-of-care (POC) tests, which contribute to early diagnoses of diseases, particularly in resource-limited settings. An assay performed in a homogeneous phase is an obvious route to develop these methods. Here, simple homogeneous methods based on proximity proteolysis reactions (PPR) are reported to detect biological molecules. A typical PPR system has been designed such that the proteolysis reaction between protease and zymogen is enhanced in the presence of a target analyte. The activated zymogen generates a color signal by hydrolyzing a chromophore. A protease and zymogen are linked to target binders using specific hybridization between complementary single-stranded DNAs, and several molecules, including proteins, antibodies, aptamers, and small molecules, are used as target binders. The developed assay methods successfully detected several kinds of analytes at subnanomolar concentrations with the one-step procedure and color signal. The modular design of the PPR-based assay will enable the development of simple POC diagnostics for various biomarkers.

Chromosome 11q13 deletion syndrome.

Chromosome 11q13 deletion syndrome has been previously reported as either otodental syndrome or oculo-oto-dental syndrome. The otodental syndrome is characterized by dental abnormalities and high-frequency sensorineural hearing loss, and by ocular coloboma in some cases. The underlying genetic defect causing otodental syndrome is a hemizygous microdeletion involving the FGF3 gene on chromosome 11q13.3. Recently, a new form of severe deafness, microtia (small ear) and small teeth, without the appearance of eye abnormalities, was also reported. In this report, we describe a 1-year-old girl presenting with ptosis of the left upper eyelid, right auricular deformity, high-arched palate, delayed dentition, simian line on the right hand, microcephaly, and developmental delay. In this patient, we identified a deletion in the chromosome 11q13.2-q13.3 (2.75 Mb) region by using an array-comparative genomic hybridization analysis. The deletion in chromosome 11q13 results in a syndrome characterized by variable clinical manifestations. Some of these manifestations involve craniofacial dysmorphology and require a functional workup for hearing, ophthalmic examinations, and long-term dental care.

Aspects of bioanalytical method validation for the quantitative determination of trace elements.

Bioanalytical methods are used to quantitatively determine the concentration of drugs, biotransformation products or other specified substances in biological matrices and are often used to provide critical data to pharmacokinetic or bioequivalence studies in support of regulatory submissions. In order to ensure that bioanalytical methods are capable of generating reliable, reproducible data that meet or exceed current regulatory guidance, they are subjected to a rigorous method validation process. At present, regulatory guidance does not necessarily account for nuances specific to trace element determinations. This paper is intended to provide the reader with guidance related to trace element bioanalytical method validation from the authors' perspective for two prevalent and powerful instrumental techniques: inductively coupled plasma-optical emission spectrometry and inductively coupled plasma-MS.