Purification of equine IgG3 by lectin affinity and an interaction analysis via microscale thermophoresis.

The availability of purified antibodies is a prerequisite for many applications and the appropriate choice(s) for antibody-purification is crucial. Numerous methods have been developed for the purification of antibodies from different sources with affinity chromatography-based methods being the most extensively utilized. These methods are based on high specificity, easy reversibility and biological interactions between two molecules (e.g., between receptor and ligand or antibody and antigen). However, no simple techniques have yet been described to characterize and purify subclasses of immunoglobulins (Ig) from some animals of biotechnology importance such as equines, which are frequently used to produce biotherapeutic antibodies. The sera of these animals present a large number of Ig classes that have a greater complexity than other animals. The implementation of an effective protocol to purify the desired antibody class/subclasses requires meticulous planning to achieve yields at a high purity. The IgG3 subclass of equine-Ig has recently been used as antigen in a new diagnostic test for allergic responses to horse sera-based therapies. Here, we defined a simple method using Jacalin lectin immobilized on Sepharose beads to prepare highly pure equine IgG3 antibodies with a determination of the affinity constants for Jacalin lectin and horse IgG3.

Bactericidal Activity of a Cationic Peptide on Neisseria meningitidis.

BACKGROUND: The increasing prevalence of antibiotic resistant bacteria has raised an urgent need for substitute remedies. Antimicrobial peptides (AMPs) are considered promising candidates to address infections by multidrug-resistant bacteria through new mechanisms of action that require a careful evaluation of their performance. OBJECTIVE: Identification of effective AMPs against Neisseria meningitidis, which represents a pathogen of great public health importance worldwide that is intrinsically resistant to some AMPs, such as polymyxin B. METHODS: A cationic 11-residue peptide (KLKLLLLLKLK), referred to as poly-Leu, was synthesized and its antimeningococcal activity was compared to cecropin A and poly-P (KLKPPPPPKLK) through a variety of assays. Flow cytometry was used to measure propidium iodide uptake by N. meningitidis serotype B as an indicator of the effectiveness of each peptide when added to cultures at different concentrations. RESULTS: The addition of the poly-Leu peptide led to a 90.3% uptake of the dye with an EC50 value of 7.9 µg mL-1. In contrast, uptake was <10% in cells grown in the absence of peptides or with an identical concentration of cecropin and poly-Pro peptides. Electron micrographs indicated that the integrity of the cellular wall and internal membrane was impacted in relation to peptide concentrations, which was confirmed by the detection of released alkaline phosphatase from the periplasmic space due to disruption of the external membrane. CONCLUSION: Poly-Leu peptide demonstrated definitive antimicrobial activity against N. meningitidis.