Highly enhanced ELISA sensitivity using acetylated chitosan surfaces
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METHODOLOGY ARTICLE
Open Access
Highly enhanced ELISA sensitivity using acetylated chitosan surfaces Tania García-Maceira1*, Fé I. García-Maceira1, José A. González-Reyes2 and Elier Paz-Rojas1
Abstract Background: 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. 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. Results: Chitin 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). Conclusions: As 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. Keywords: Chitosan surface, Chitin binding domain, Antibody orientation, ELISA
Background 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]. Disease biomarkers detection on clinical samples have great importance for diagnosis as well as for the * Correspondence: [email protected] 1 Canvax Biotech; Parque Científico y Tecnológico Rabanales 21, c/Astrónoma Cecilia Payne s/n, Edificio Canvax, 14014 Córdoba, Spain Full list of author information is available at the end of the article
monitoring of disorders. However, the effectiveness of the detection is frequently limited by the sensitivity and quantification capacity of the assay. Due to its high specificity and sensitivity, ELISA technique is probably the most used technique for these purposes, although for many biomarkers this technique has shortcomings based on criteria like kinetic properties and/or antibody availability [3, 4]. To cope with this issue, several methods have been devel
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