Human Osteopontin ELISA Assay
The Human Osteopontin ELISA Assay is For Research Use Only
Size: 1×96 wells
Sensitivity: 0.510 ng/ml
Dynamic Range: 1.25 ng/ml – 40 ng/ml
Incubation Time: 130 minutes
Sample Type: Plasma, Serum
Sample Size: 100 μL
Alternative Names: OPN ELISA
Assay Principle for Human Osteopontin ELISA Assay
The Human Osteopontin ELISA Assay is a sandwich type immunoassay. The 96-well microplate is coated with a polyclonal antibody specific for human osteopontin. The standards and samples are added to the microplate wells. The microplate is then incubated at room temperature without agitation. After the first incubation is complete, the wells are washed with Wash Buffer and blotted dry. The polyclonal detection antibody conjugate is then added, and the microplate is incubated a second time at room temperature without agitation. After the second incubation is complete, the wells are washed with Wash Buffer and blotted dry. HRP conjugated streptavidin is then added, and the microplate is incubated a third time. After the third incubation is complete, the wells are washed with Wash Buffer and blotted dry. TMB Substrate is added, and the microplate is incubated a fourth time at room temperature without agitation. Once the incubation is complete, Stop Solution is added, and the optical density (OD) is measured by a spectrophotometer at 450 nm. The intensity of the color generated is directly proportional to the amount of human osteopontin in the sample.
Sample Handling and Preparation for Human Osteopontin ELISA Assay
Human plasma and urine samples are appropriate for use in this assay. Human serum samples are not recommended for use because of a proteolytic cleavage, by thrombin, during clotting processes. OD values obtained from measuring human serum samples are expected to be lower than those measured in human plasma samples. All samples should be centrifuged for 10 minutes at 13,000 rpm, and the supernatant used in the assay. Discard any remaining sample. It is recommended that all samples are assayed in duplicate. Dilute samples with 1X Diluent Buffer and gently vortex. For duplicate samples, dilute 10 µL of sample in 390 µL with 1X Diluent Buffer. If a sample has a concentration of human osteopontin greater than the highest standard, dilute the sample further in 1X Diluent Buffer and repeat the analysis.
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