Development of low affinity techniques for applications in clinical chemistry

An increase in the plasma level of creatine kinase MB (CKMB) has long been used to indicate acute myocardial infarction (AMI). For optimal treatment an early diagnosis is important. An increase in the ratio between the two isoforms of CKMB is seen much earlier than an increase in the total level of CKMB. One possible method for separating these isoforms could be weak affinity chromatography (WAC). This thesis is about WAC and about utilising WAC for clinical chemistry purposes. Both lectins and antibodies have been used in WAC. Further, a screening method for obtaining antibodies with low affinity has been developed based on WAC. The performance of WAC has been studied by solving the mathematical equations in chromatographic theory. Finally, antibodies with specificity for one of the CKMB isoforms, CKMB2, have been developed and characterised

Affinity screening for weak monoclonal antibodies

When selecting for monoclonal antibodies of a desired affinity, affinity chromatography can be a feasible alternative. This is of particular interest when low affinity monoclonal antibodies (dissociation constant (Kd) > 10(-4) M) are screened, as they are not easily recognised by traditional immunoassay procedures. In this study we have evaluated this approach by monitoring low affinity monoclonal antibodies on high performance liquid affinity chromatography columns with oligosaccharides, dinitrophenol and digoxin as immobilised antigen. Crude monoclonal antibodies in ascites or cell culture supematants, directed against these antigens, were retarded or adsorbed according to affinity or avidity on the antigen columns. Based on antibody retention, we were able to select hybridomas with the desired low affinity characteristics

Transport study of interleukin-1 inhibitors using a human in vitro model of the blood-brain barrier

International audienceThe proinflammatory cytokine Interleukin-1 (IL-1), with its two isoforms α and β, has important roles in multiple pathogenic processes in the central nervous system. The present study aimed to evaluate and compare the blood-to-brain distribution of anakinra (IL-1 receptor antagonist), bermekimab (IL-1α antagonist) and canakinumab (IL-1β antagonist). A human in vitro model of the blood-brain barrier derived from human umbilical cord blood stem cells was used, where isolated CD34+ cells co-cultured with bovine pericytes were matured into polarized brain-like endothelial cells. Transport rates of the three test items were evaluated after 180 ​min incubation at concentrations 50, 250 and 1250 ​nM in a transwell system. We report herein that anakinra passes the human brain-like endothelial monolayer at a 4-7-fold higher rate than the monoclonal antibodies tested. Both antibodies had similar transport rates at all concentrations. No dose-dependent effects in transport rates were observed, nor any saturation effects at supraphysiological concentrations. The larger propensity of anakinra to pass this model of the human blood-brain barrier supports existing data and confirms that anakinra can reach the brain compartment at clinically relevant concentrations. As anakinra inhibits the actions of both IL-1α and IL-1β, it blocks all effects of IL-1 downstream signaling. The results herein further add to the growing body of evidence of the potential utility of anakinra to treat neuroinflammatory disorders