Gold nanoparticle-based immunodetection of Staphylococcus aureus leukotoxin M/F′-PV in subclinical samples of bovine mastitis
| Autor: | Radhakrishnan Jayasree Padmaja, Munna Singh Thakur, Uchangi Satyaprasad Akshath, Prakash M. Halami, K. S. Abhijith |
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| Rok vydání: | 2014 |
| Předmět: |
Chromatography
biology Toxin General Chemical Engineering General Engineering medicine.disease_cause Primary and secondary antibodies Analytical Chemistry Microbiology law.invention chemistry.chemical_compound Affinity chromatography chemistry Polyclonal antibodies law Colloidal gold Staphylococcus aureus biology.protein Recombinant DNA medicine Fluorescein isothiocyanate |
| Zdroj: | Anal. Methods. 6:5214-5220 |
| ISSN: | 1759-9679 1759-9660 |
| DOI: | 10.1039/c4ay00525b |
| Popis: | In the present study, an immunosensor was designed to assess bovine mastitis at the earliest by quantifying leukotoxin M/F′-PV (LukM/F′-PV), a potent neutrophil targeting and beta-barrel pore-forming toxin secreted by bovine strains of S. aureus. Polyclonal antibodies to the recombinant LukF (rLukF) component of LukM/F′-PV was raised and purified by affinity chromatography. Further, anti-rLukF antibody was used to design a classical ELISA detection system in which we obtained 1000 ng ml−1 LOD. Considering the drawbacks of the classical detection system, antibodies were functionalized to gold nanoparticles of a large surface plasmon band providing an opportunity to design immunoassays based on nanosurface energy transfer (NSET) from dye to gold nanoparticles (GNPs). In an experimental set up, rLukF was incubated with functionalized GNPs, fluorescein isothiocyanate (FITC)-labeled secondary antibodies were added, and fluorescence quenching was monitored as a function of toxin concentration. With this method, leukotoxin was detected in the 100–0.1 ng ml−1 range, with a LOD of 0.1 ng ml−1 and R2 = 0.9908. In addition, the above proposed assay was performed to detect toxins in spiked and field samples with 89–95% recovery. Thus, the proposed method overcomes the major drawbacks of ELISA systems and can provide a window for sensitive detection of toxin before onset of mastitis. |
| Databáze: | OpenAIRE |
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