ABSTRACT
An immuno-separated assay for ochratoxin A detection coupled with a nano-affinity cleaning up for LC-confirmation was developed. Firstly, ochratoxin A was modified to quantum dot beads for immuno-fluorescent reporters. Secondly, Fe3O4 magnetic nanoparticles were conjugated with protein G for immuno-magnetic adsorbents. The immuno-separation of fluorescent reporters by magnetic adsorbents could be completed by ochratoxin A, so the fluorescent reporters released from the immune complex indicate a linear correlation with the concentration of ochratoxin A. Furthermore, the immuno-separated ochratoxin A can be eluted from magnetic adsorbent for LC-conformation. The optimized assay showed results as follows: the quantitative range of the immuno-separated assay was 0.03-100 ng mL-1 of ochratoxin A. The recoveries for spiked samples ranged from 78.2% to 91.4%, with the relative standard deviation (RSD) being 11.9%~15.3%. Statistical analysis indicated no significant difference between the HPLC-FLD results based on commercial affinity column and by nano-affinity cleaning up.
ABSTRACT
Vardenafil is a phosphodiesterase-5 (PDE-5) inhibitor for the treatment of erectile dysfunction (ED). Undeclared vardenafil and related analogues adulterated in herbal products are a threat to public health. To screen vardenafil and its analogues in herbal matrix rapidly, an immunoassay based on a group specific monoclonal antibody (McAb) was developed. Glutaraldehyde was used to link vardenafil to immunogen and coating-antigen, respectively. Through the assessment of the structural specificity of eight anti-vardenafil McAbs, the McAb of 4B9 was characterized as being specific to the common structure of vardenafil and its analogues. An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was established based on this McAb, the limit of detection of vardenafil was 5.0 ng mL(-1), the calibration curve was linear from 5.0 to 40 ng mL(-1) (R(2)=0.952) with an IC(50) value of 18.2 ng mL(-1). In the extracts of 20 Chinese traditional drugs, the detection capability (CCbeta) of vardenafil was 0.08 mg g(-1), the recoveries were 76-116% and the coefficients of variation (CV%) were 9.7%-16.2%. The ic-ELISA was in good agreement with LC-UV when detected herbal products containing vardenafil and its analogue. The method is a suitable tool for screening vardenafil and its analogues as illegal additives in herbal products.
Subject(s)
Antibodies, Monoclonal/immunology , Enzyme-Linked Immunosorbent Assay/methods , Imidazoles/analysis , Phosphodiesterase Inhibitors/analysis , Piperazines/analysis , Glutaral/chemistry , Herbal Medicine , Imidazoles/chemistry , Imidazoles/immunology , Limit of Detection , Phosphodiesterase Inhibitors/chemistry , Phosphodiesterase Inhibitors/immunology , Piperazines/chemistry , Piperazines/immunology , Plant Preparations/chemistry , Sulfones/analysis , Sulfones/chemistry , Sulfones/immunology , Triazines/analysis , Triazines/chemistry , Triazines/immunology , Vardenafil DihydrochlorideABSTRACT
OBJECTIVE: To develop a more reliable and stable method for determining monoclonal antibody (mAb) affinity constant based on competitive antibody/antigen binding. METHODS: The Kd value was calculated based on the relationship between the binding proportion of the antigen and the original concentration of the mAb that competed for the binding site of the antigen. RESULTS: The Kd values measured with this improved method under two different conditions were 2.61x10(-12) mol/L and 2.39x10(-12) mol/L, and those with Friguent method in these two conditions were 5.57x10(-10) mol/L and 1.41x10(-10) mol/L, respectively. CONCLUSION: Compared with Friguent method, the Kd values measured with this improved method are closer to the actual value, and the measurement results under different experiment conditions are more stable.
