Immunoglobulin G in Platelet-Derived Wound Healing Factors

We intended to reformulate an existing platelet-derived wound healing formula to target each phase of the healing wound with the appropriate phase-specific molecules. A decreased perfusion of the skin, often associated with conditions such as thalassemia, sickle cell disease, diabetes mellitus, and chronic vascular disease, is the most common etiology of cutaneous ulcers and chronic wounds. We had previously shown that a PDWHF topically applied to a chronic nonhealing ulcer of a β-thalassemia homozygote stimulated and accelerated closure of the wound. The PDWHF was prepared from a pooled platelet concentrate of a matching blood group, consisting of a combination of platelet α-granule-derived factors. Processing of the apheresis-pooled platelets yielded various amounts of proteins ( 3.36 g / mL ± 0.25 (SD) ( N = 10 )) by the better lysis buffer method. Immunoglobulin G was found to be the most abundant α-granule-secreted protein. Equally broad quantities of the IgG ( 10.76 ± 12.66 % (SD) ( N = 10 )) and IgG/albumin ratios ( 0.6 ± 0.4 (SD) ( N = 10 )) were quantified. We have developed a method using a reformulated lysis buffer followed by size exclusion chromatography and affinity chromatography to extract, identify, quantify, and purify IgG from activated platelets. IgG purification was confirmed by Western blot and flow cytometry. It was thought unlikely that the platelet IgG could be accounted for by adsorption of plasma protein, though the variable quantities could account for diversity in wound healing rates. The IgG could protect the wound even from subclinical infections and functionally advance healing. It may be useful in the management of skin ulcers in the early phase of wound healing.

Engineered pH-Sensitive Protein G / IgG Interaction

ABSTRACTWhile natural protein-protein interactions have evolved to be induced by complex stimuli, rational design of interactions that can be switched-on-demand still remain challenging in the protein design world. Here, we demonstrate a computationally redesigned natural interface for improved binding affinity could further be mutated to adopt a pH switchable interaction. The redesigned interface of Protein G-IgG Fc domain, when incorporated with histidine and glutamic acid on Protein G (PrG-EHHE), showed a switch in binding affinity by 50-fold when pH was altered from mild acidic to mild basic. The wild type (WT) interface only showed negligible switch. The overall binding affinity at mild acidic pH for PrG-EHHE outperformed the WT PrG interaction. The new reagent PrG-EHHE will be revolutionary in IgG purification since the traditional method of using an extreme acidic pH for elution can be circumvented.Abstract Figure

Quantitative LC-MS/MS method for nivolumab in human serum using IgG purification and immobilized tryptic digestion

A simple and rapid LC-MS/MS method for quantification of nivolumab in human serum using immobilized trypsin coupled to IgG purification.

Endoglycosidase S enables a highly simplified clinical chemistry assay procedure for direct assessment of serum IgG undergalactosylation in chronic inflammatory disease

Over the past 30 years, it has been firmly established that a wide spectrum of (autoimmune) diseases such as rheumatoid arthritis, Crohn’s and lupus, but also other pathologies like alcoholic and non-alcoholic steatohepatitis (ASH and NASH) are driven by chronic inflammation and are hallmarked by a reduced level of serum IgG galactosylation. IgG (under)galactosylation is a promising biomarker to assess disease severity, and monitor and adjust therapy. However, this biomarker has not been implemented in routine clinical chemistry due to a complex analytical procedure that necessitates IgG purification, which is difficult to perform and validate at high throughput.We addressed this issue by using endo-β-N-acetylglucosaminidase from Streptococcus pyogenes (endoS) to specifically release Fc N-glycans in whole serum. The entire assay can be completed in a few hours and only entails adding endoS and labeling the glycans with APTS. Glycans are then readily analyzed through capillary electrophoresis. We demonstrate in two independent patient cohorts that IgG undergalactosylation levels obtained with this assay correlate very well with scores calculated from PNGaseF-released glycans of purified antibodies. Our new assay allows to directly and specifically measure the degree of IgG galactosylation in serum through a fast and completely liquid phase protocol, without the requirement for antibody purification. This should help advancing this biomarker towards clinical implementation.

Simple Protocol for immunoglobulin G Purification from Camel “Camelus dromedarius” Serum

The present study aimed to describe and standardize a simple and efficient protocol for purification of camel IgG from serum, which can be applied for Camilidae antibody production in research laboratories, the preindustrial stage. Camel serum IgG was separated with caprylic acid and ammonium sulfate, then the effect of four variables studied: caprylic acid concentration, pH, stirring time, and stirring intensity. Camel IgG prepared by standardized caprylic acid fractionation method for camel serum was compared with commercial anti-sera products. Camel IgG purification from undiluted sera using caprylic acid at concentration of 8% v/v gave the best results. Purification at different pH values using caprylic acid at 8% v/v revealed that pH 5.5 was optimal. Investigating purification at different stirring time intervals using 8% v/v caprylic acid at pH 5.5 demonstrated that stirring for 90 min gave the optimum results. Finally, studying purification at different stirring intensities using 8% v/v caprylic acid at pH 5.5 for 90 min, the best stirring intensity was at 450 rpm. Overall, the results suggest that caprylic acid purification of camel serum IgG is more effective and safe than ammonium sulfate method in simplicity, purity, and lower non-IgG proteins in the final preparation with lower protein aggregates.