More comprehensive standards for monitoring glycosylation.

Biologics manufacturers must continually monitor the attachment of carbohydrates, called glycans, to their products, because any variability can impact safety and efficacy. To help the industry meet this challenge, the United States Pharmacopeial Convention (USP) offers glycan reference standards and validated methods for glycoprofiling using high-performance liquid chromatography (HPLC). The industry has recently adopted more advanced technologies for glycan analysis, including ultra-high performance liquid chromatography (UHPLC) and mass spectrometry. In this study, we confirm that USP's glycan reference standards are compatible with UHPLC by demonstrating comparable peak separation and glycan identification to HPLC methods. The improved resolving power and shorter run-times of UHPLC also allowed us to identify many of the minor glycan components present in USP's glycan reference standards. These more comprehensively characterized glycan reference standards will enable manufacturers to assess the micro-heterogeneity that can negatively impact the safety and efficacy of biological products.

Discordant sex between fetal screening and postnatal phenotype requires evaluation.

OBJECTIVE: Non-invasive prenatal screening (NIPS) utilizes circulating cell-free DNA (cfDNA) to screen for fetal genetic abnormalities. NIPS is the first widely-available prenatal screen to assess genotypic sex. Most pediatricians have limited familiarity with NIPS technology and potential etiologies of discordant results. Increased familiarity may provide diagnostic insight and improve clinical care. STUDY DESIGN: We reviewed all patients with discordant genotypic fetal sex assessed by cfDNA and neonatal phenotypic sex referred to our medical center. RESULT: Four infants with discordant cfDNA result and phenotypic sex were identified. Etiologies include vanishing twin syndrome, difference of sexual development, sex chromosome aneuploidy and maternal chimerism. CONCLUSIONS: We present four cases illustrating potential etiologies of discordant cfDNA result and postnatal phenotypic sex. Unanticipated cfDNA results offer the perinatologist a unique opportunity for early diagnosis and targeted treatment of various conditions, many of which may not have otherwise been detected in the perinatal period.

Glutathione S-transferase pull-down assays using dehydrated immobilized glutathione resin.

We have developed an affinity-precipitation technique to facilitate conducting glutathione S-transferase (GST) pull-down assays. The dehydrated immobilized glutathione resin format, when combined with microcentrifuge spin columns, is a powerful tool that enables the simultaneous performance of resin hydration, the binding of the GST fusion protein, and the pull-down step with the appropriate protein partner in a semihigh-throughput fashion (multiple samples processed at the same time). The entire assay process is shortened and recovery is enhanced when coupled with a spin-column format, providing a convenient way to study protein-protein interactions. We successfully tested the resin format/technique in three common pull-down applications utilizing radiolabeled, overexpressed, and activated endogenous interacting protein partners.

Improved immunomatrix methods to detect protein:protein interactions.

Immunoprecipitation (IP) and coimmunoprecipitation (co-IP) are key techniques for studying protein-protein interactions. These methods utilize immobilized Protein A or Protein G to isolate antibody-bound target antigens. The main disadvantage of traditional IP and co-IP is that the conditions used to elute the precipitated antigen also release the antibody thus contaminating the antigen and destroying the antibody support. To overcome these problems, we describe two methods to generate a reusable antibody support by cross-linking the antibody to immobilized Protein A or Protein G, or by coupling it directly to the resin (see Scheme 1). Antibody cross-linking can be done in 1 h while antibody coupling requires 4 h. IP or co-IP is accomplished by incubating the antibody resin with the protein sample. Washes and elutions are carried out in a spin column to reduce resin loss and decrease assay time. Target proteins are eluted with 0.1 M glycine (pH 2.8) and the resin-bound antibody is re-equilibrated in phosphate-buffered saline (PBS) for reuse. Our studies have demonstrated that the immobilization efficiency for the antibody coupling method was similar for several species of antibody. Furthermore, we illustrate that using both methods of antibody immobilization yield IP and co-IP results similar to traditional protocols but eliminate the antibody heavy and light chain contamination.