More than meets the Kappa for Antibody Superantigen Protein L (PpL)

Immunoglobulin superantigens play an important role in the affinity purification of antibodies and underlie the microbiota-immune axis at mucosal areas Focussing on the Staphylococcal Protein A (SpA), Streptococcal Protein G (SpG), and the Finegoldia Protein L (PpL) that were previously thought to bind to only specific regions of human antibodies, a systematic and holistic analysis of the antibody regions using 63 antibody permutations involving six Vκ and seven VH region IgG1 revealed showed novel PpL-antibody interactions. While SpA and SpG showed relatively consistent interactions with the antibodies, our findings showed PpL binding to certain VH-Vκ2, 5 and 6 interactions had contribution by other antibody regions. The findings of this have implications on PpL-based affinity antibody purifications and antibody design as well as provides novel insights to PpL-based microbiota-immune axis effects.

IMMOBILIZATION OF CELLULASES ON CHITOSAN: APPLICATION FOR SUGARCANE BAGASSE HYDROLYSIS

In this work, a commercial cellulolytic cocktail was immobilized on glutaraldehyde activated chitosan gel. The chitosan concentration in the gel preparation, pH, immobilization time and enzymatic loading were evaluated. Immobilized cellulases showed better hydrolysis performance when an enzyme loading of 134 mg protein/g carrier was used for immobilization at pH 9.0 for 30 minutes. Hydrolysates with a glucose content of 13.43 and 10.35 g/L were obtained when Avicel and pretreated sugarcane bagasse were used as substrate, respectively. Immobilized cellulase lost 60% of its hydrolysis performance after 8 cycles using Avicel, and 75% after 6 cycles for sugarcane bagasse. The hydrolysis performance associated with the reuse of the immobilized cellulases indicates that an improvement in the immobilization of cellulases, coupled with an improvement in the pretreatment of lignocellulosic biomass, will allow the development of a continuous hydrolysis system with the enzyme retained in the reactor.

A plug-and-play platform of ratiometric bioluminescent sensors for homogeneous immunoassays

Heterogeneous immunoassays such as ELISA have become indispensable in modern bioanalysis, yet translation into point-of-care assays is hindered by their dependence on external calibration and multiple washing and incubation steps. Here, we introduce RAPPID (Ratiometric Plug-and-Play Immunodiagnostics), a mix-and-measure homogeneous immunoassay platform that combines highly specific antibody-based detection with a ratiometric bioluminescent readout. The concept entails analyte-induced complementation of split NanoLuc luciferase fragments, photoconjugated to an antibody sandwich pair via protein G adapters. Introduction of a calibrator luciferase provides a robust ratiometric signal that allows direct in-sample calibration and quantitative measurements in complex media such as blood plasma. We developed RAPPID sensors that allow low-picomolar detection of several protein biomarkers, anti-drug antibodies, therapeutic antibodies, and both SARS-CoV-2 spike protein and anti-SARS-CoV-2 antibodies. With its easy-to-implement standardized workflow, RAPPID provides an attractive, fast, and low-cost alternative to traditional immunoassays, in an academic setting, in clinical laboratories, and for point-of-care applications.

Enzymatic Hydrolysis and Fermentation of Banana Pseudostem Hydrolysate to Produce Bioethanol

Banana pseudostem (BPS) is an agricultural waste with a high holocellulose content, which, upon hydrolysis, releases fermentable sugars that can be used for bioethanol production. Different pretreatment methods, namely, 3% (w/v) NaOH, 5% (v/v) H2SO4, and liquid hot water, applied on the BPS resulted in the availability of 52%, 48%, and 25% cellulose after treatment, respectively. Saccharification of the pretreated BPS with 10 FPU/g dry solids (29.3 mg protein/g d.s) crude enzyme from Trichoderma harzianum LMLBP07 13-5 at 50°C and a substrate loading of 10 to 15% released 3.8 to 21.8 g/L and from T. longibrachiatum LMLSAUL 14-1 released 5.4 to 43.5 g/L glucose to the biomass. Ethanol was produced through separate hydrolysis and fermentation (SHF) of alkaline pretreated BPS hydrolysate using Saccharomyces cerevisiae UL01 at 30°C and 100 rpm. Highest ethanol produced was 17.6 g/L. Banana pseudostem was shown as a potentially cheap substrate for bioethanol production.

Purification of polyclonal Immunoglobulin G from human serum using LigaGuardTM and LigaTrapTM adsorbents

This study presents the chromatographic purification of immunoglobulin G (IgG) from human plasma using a two-column process integrating the peptide-based adsorbents LigaGuardTM, which captures non-Ig plasma proteins in flow-through mode, and LigaTrapTM, which isolates IgG in bind-and-elute. Buffer composition and column loading were optimized for both adsorbents. Two process configurations were evaluated. In the first design, plasma was fed to a LigaGuardTM column to capture plasma proteins, the effluent was loaded on the LigaTrapTM column, and the bound IgG was eluted with 63.8% global recovery and 99.7% purity; in comparison, Protein G agarose afforded ~67% recovery and 97.2% purity. In the alternative design, the LigaGuardTM column was utilized to polish the LigaTrapTM elution stream, affording 82.3% global recovery and 98.8% purity. Collectively, these results demonstrate the potential of a fully chromatographic process for purifying polyclonal IgG from plasma feedstocks.