Abstract
BackgroundCurrent diagnostic methods for tuberculosis (TB), a major global health challenge that kills nearly two million people annually, are time-consuming and inadequate. During infection a number of bacterial molecules that play a role in the infective process are released and have been proposed as biomarkers for early TB diagnosis. Antigen 85 (Ag85) is the most abundant secreted TB protein, and a potential target for this diagnostic approach. One of the bottlenecks in the direct detection of such bacterial targets is the availability of robust, sensitive, specific antibodies.MethodsUsing Ag85 as a model, we describe a method to select antibodies against any potential target using a novel combination of phage and yeast display that exploits the advantage of each approach.ResultsThe efficiency of this approach was attested to by the 111 specific antibodies identified in initial screens. These were assessed for binding to the different Ag85 subunits, affinity, and activity in sandwich assays.ConclusionsThe novelty of this approach lies in the possibility of screening the entire output of a phage antibody selection in a single experiment by yeast display. This can be considered analogous to carrying out a million ELISAs. The monoclonal antibodies (mAbs) identified in this way show high binding affinity and selectivity for the antigens and offer an advantage over traditional mAbs produced by relatively expensive and time consuming techniques. This approach has wide applicability, and the affinity of selected antibodies can be significantly improved, if required.
Highlights
Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a global health problem with 2–3 millions deaths and 8 million active infections annually [1]
Phage Display Selection of single chain Fvs (scFvs) Antibodies Our previously described naıve phage antibody library [22] was used to select Antigen 85 (Ag85) antibodies using an automated Kingfisher magnetic bead system (Thermo Lab Systems), allowing selection to be carried out in solution as previously described [24]. 0.5 mg of biotinylated Ag85 were used in the first round and 0.05 mg in the second
Phage display is used to preselect the vast diversity of our naıve phage antibody library to a diversity compatible with subsequent analysis and selection by yeast display
Summary
Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a global health problem with 2–3 millions deaths and 8 million active infections annually [1]. The standard screening method is the Tuberculin Skin Test, which lacks sensitivity and specificity, making it less useful for people at low risk [4]. The gold diagnostic standard remains clinical examination with sputum examination and cultures for acid-fast bacilli. These require expertise, and are not sensitive enough to detect more than 65–70% of cases. Current diagnostic methods for tuberculosis (TB), a major global health challenge that kills nearly two million people annually, are time-consuming and inadequate. One of the bottlenecks in the direct detection of such bacterial targets is the availability of robust, sensitive, specific antibodies
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