Abstract
BackgroundThe enzyme-linked immunosorbent assay (ELISA), is the most widely used and reliable clinical routine method for the detection of important protein markers in healthcare. Improving ELISAs is crucial for detecting biomolecules relates to health disorders and facilitating diagnosis at the early diseases stages. Several methods have been developed to improve the ELISA sensitivity through immobilization of antibodies on the microtiter plates. We have developed a highly sensitive ELISA strategy based on the preparation of acetylated chitosan surfaces in order to improve the antibodies orientation.ResultsChitin surfaces were obtained by mixing small quantities of chitosan and acetic anhydride in each well of a microtiter plate. Anti-c-myc 9E10 low affinity antibody fused to ChBD was cloned and expressed in CHO cells obtaining the anti-c-myc-ChBD antibody. We found that anti c-myc-ChBD binds specifically to the chitin surfaces in comparison with anti-c-myc 9E10, which did not. Chitin surface was used to develop a sandwich ELISA to detect the chimeric human protein c-myc-GST-IL8 cloned and expressed in Escherichia coli. The ELISA assays developed on chitin surfaces were 6-fold more sensitive than those performed on standard surface with significant differences (p<0,0001).ConclusionsAs shown here, acetylated chitosan surfaces improve the antibody orientation on the substrate and constitute a suitable method to replace the standard surfaces given the stability over time and the low cost of its preparation.
Highlights
The enzyme-linked immunosorbent assay (ELISA), is the most widely used and reliable clinical routine method for the detection of important protein markers in healthcare
The results show an improved detection limit with the consequent improvement of the ELISA sensitivity, possibly due to an increase immobilization and better orientation of antibodies fused to the chitin binding domain
The recombinant protein His6-c-myc-glutation-S-transferase protein (GST)-IL8h was detected in the sandwich ELISA using anti-human IL-8, as well as with an anti-c-myc-chitin binding domain (ChBD) as capture antibody
Summary
The enzyme-linked immunosorbent assay (ELISA), is the most widely used and reliable clinical routine method for the detection of important protein markers in healthcare. We have developed a highly sensitive ELISA strategy based on the preparation of acetylated chitosan surfaces in order to improve the antibodies orientation. The ELISA is a powerful and widely used technique which has been used for decades to detect different molecules, especially protein analytes, in diagnostic and research context. This highly versatile technique allows the detection of biomolecules with high specificity and sensitivity, associating the readout with a subsequent enzymatic reaction producing colorimetric, fluorescence or luminescence signals [1, 2]. The use of silver nanoparticles, streptavidin-coated microparticles, or the signal amplification with the tyramide system, can be mentioned [8,9,10]
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