Detection of ricin by colorimetric and chemiluminescence ELISA.
A highly sensitive and specific ELISA was developed to detect ricin in biological fluids. The assay utilizes an affinity-purified goat polyclonal antibody to adsorb ricin from solution. The same antibody (biotinylated) is then used to form a sandwich, and avidin-linked alkaline phosphatase allows color development and measurement of optical density at 405 nm. Our routine assay uses a standard curve over the range of 0-10 ng/ml ricin, with accurate quantitation below 1 ng/ml (100 pg/well) in assay buffer as well as in a 1:10 dilution of human urine or 1:50 dilution of human serum spiked with ricin. Ricin measured in spiked samples demonstrated accuracy typically within 5% of the expected value in all matrices. The coefficient of variation ranged from 3-10% at 10 ng/ml to 8-25% at 2.5 ng/ml. Two variations on the routine assay were also investigated. First, lengthened incubation times and additional time for color development allowed accurate quantitation in serum dilutions as low as 1:2. Second, increased concentrations of biotinylated antibody and avidin-linked enzyme from 1:250 to 1:70 enhanced the sensitivity of the assay 10-fold, achieving a detection limit of at least 100 pg/ml (10 pg/well). The assay was also configured to a format based upon chemiluminescence, which allowed quantitation in the 0.1-1 ng/ml range, but was subject to slightly greater variability than the colorimetric assay.
Poli
M A
MA
Toxinology Division, U.S. Army Medical Research Institute of Infectious Diseases, Frederick, MD 21702-5011.
Rivera
V R
VR
Hewetson
J F
JF
Merrill
G A
GA
D000818:Animals; D000906:Antibodies / Q000737:chemistry; D002846:Chromatography, Affinity; D003124:Colorimetry; D004797:Enzyme-Linked Immunosorbent Assay; D006041:Goats; D006801:Humans; D008163:Luminescent Measurements; D012015:Reference Standards; D015203:Reproducibility of Results; D012276:Ricin / Q000032:analysis* / Q000097:blood / Q000276:immunology / Q000652:urine
D000906:Antibodies; D012276:Ricin