Titration method for measuring kinetic binding parameters and for discriminating specific binding from background
AssigneeMiltenyi Biotec GmbH
InventorAli KINKHABWALA
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Abstract
Method for determination of the kinetic binding parameters for a molecule binder conjugated to a fluorophore. Repeated staining of the antigen and detection of the emission radiation generates a titration series. Fitting of the emission radiation over the titration series allows for determination of kinetic binding parameters. If the emission radiation is detected with a camera, single pixel fitting using a global analysis permits discrimination of the specific binding signal from non-specific background in each pixel.
Claims7
A method for determining the on-rate constant for specific binding of a conjugate comprising a fluorescent detection moiety and an antigen binding moiety applied to a fixed biological sample expressing the corresponding antigen characterized by the steps
- a. measuring the emission radiation of the fixed biological sample as an image formed on a camera before providing the conjugate
- b. providing the conjugate to the fixed biological sample subsequently in at least two different concentrations and for a specified time interval
- c. detecting the emission radiation for each concentration as an image formed on a camera
- d. aligning the images to each other
- e. fitting a function that accounts for the amount of specific binding and background binding for each concentration to the emission radiation within each aligned pixel over the separate images
- f. obtaining from step e) the on-rate constant that specifies the specific binding function.
Dependencies are inferred from the claim text.