Abraxis: 515575: Caffeine by Immunoassay, Microtiter Plate
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Official Method Name
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Abraxis Caffeine ELISA Plate Kit PN 515575 |
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Current Revision
| 2008 |
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Media
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WATER |
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Instrumentation
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Immunoassay |
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Method Subcategory
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Organic |
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Method Source
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Citation
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Brief Method Summary
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Caffeine is detected using a colorimetric immunoassay (ELISA) procedure. A sample (0.05 mL), enzyme conjugate (Caffeine-HRP), and an antibody solution against Caffeine are added to plate wells pre-coated with monoclonal antibodies (goat anti-mouse). Both the Caffeine (if present) in the sample and Caffeine-HRP conjugate compete to bind to the antibody solution in proportion to their concentrations. The anti-Caffeine antibodies are then bound to the immobilized monoclonal antibodies on the plate. After an incubation, the unbound molecules are washed and decanted. A substrate is then added which is catalyzed by the enzyme and converted from a colorless to a blue solution. The reaction is terminated with the addition of a dilute acid. The concentration of Caffeine in the sample is determined by measuring its absorbance at a specific wavelength (450nm) using a photometer and comparing its absorbance to the absorbance of the calibrators. |
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Scope and Application
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This method determines Caffeine in water (groundwater, surface water, well water), which is an indicator of human source pollution. |
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Applicable Concentration Range
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0.15-5.0 ng/mL |
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Interferences
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Cross-Reactivity: 1,7-Dimethylxanthine, and high concentrations Theophylline and Theoxanthine may produce false positive responses for Caffeine. |
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Quality Control Requirements
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(A) Calibration with 5 standards and 1 blank, all analyzed in duplicate. (B) Precision: 3 matrix samples with different levels in the range for quantitative analysis analyzed daily for 3 days with 5 replicates in each of 5 assays. (C) Accuracy: 5 matrix samples spiked with the target analyte at 3 different levels in the range for quantitative analysis. (D) Validation: Analysis of 4 positive and 4 negative samples by an independent method, for confirmation. |
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Sample Handling
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Samples are collected in glass containers with Teflon-lined caps; for compliance monitoring, samples should be held no longer than 14 days. If samples are held frozen, they can be held for longer periods of time. Immunoassay reagents are stored refrigerated until use. Samples containing gross particulate matter should be filtered using a 1 micron filter or centrifuged before use. Drinking water samples are typically dechlorinated with 0.008% sodium thiosulfate. |
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Maximum Holding Time
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14 days at 4 degrees C, longer if frozen |
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Relative Cost
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Less than $50 |
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Sample Preparation Methods
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