Method of calibrating a flow cytometer or fluorescence microscope for quantitating binding antibodies on a selected sample, and microbead calibration kit therefor

Claim 1

A microbead calibration kit for calibrating a flow cytometer or microscope in terms of the number of binding antibodies per microbead as a function of fluorescence intensity value measured on the flow cytometer of fluorescence microscope, for subsequent measurement of a sample to which the same antibodies are bindable, said calibration kit comprising: (a) a set of populations of microbeads having a coefficient of variation of diameter of about 2 percent or less, with said diameter of said microbeads being substantially equivalent to the diameter of the sample to be measured; (b) at least two populations of said set of populations being capable of binding, directly or via a linking group, a fluorescently labeled antibody, wherein the number of fluorescently labeled antibodies binding to each microbead in said at least two populations is the same within a said population thereof, but different from population to population among the respective said populations; (c) another population of microbeads in said set, having the same fluorescence spectra and fluorescence intensity as said sample prior to binding of fluorescently labeled antibodies thereto, said microbead population being selected from the group consisting of blank microbeads and auto-fluorescent microbeads; and (d) container means enclosing each of said populations of microbeads.

Claim 8

A method for calibrating a flow cytometer or fluorescence microscope in terms of the number of binding antibodies per microbead as a function of fluorescence intensity value measured on the flow cytometer of fluorescence microscope, and subsequent measuring of a sample to which said antibodies are bindable, comprising the steps of: (i) providing a microbead calibration kit comprising a set of populations of microbeads having a coefficient of variation of diameter of about 2 percent or less, with said diameter of said microbeadebeing substantially equivalent to the diameter of the sample to be measured, said populations being capable of binding, directly or via a linking group, a fluorescently labeled antibody, wherein the number of fluorescently labeled antibodies, binding to each microbead in said populations is the same within a said population, but different from population to population among the respective said populations in said set; (ii) oontacting each of said populations of microbeads with a fluorescently labeled antibody to effect direct or indirect binding of said antibody to said microbeads for saturation of the binding sites associates with said microbeads, to produce antibody-bearing microbeads having directly or indirectly bound thereto antibodies of a known or determinable number per microbead; (iii) determining the fluorescence intensity of each of said populations of antibody-bearing microbeads on said flow cytometer or fluorescence microscope; (iv) constructing a calibration plot of the number of fluorescently labeled antibodies, as a function of fluorescence intensity measured on said cytometer of fluorescence microscope for said antibody-bearing microbeads; (v) contacting said sample with a second portion of said fluorescently labeled antibody capable of binding to said sample, to yield a fluorescently labeled antibody-bearing sample; (vi) determining the fluorescence intensity of said fluorescently labeled antibody-bearnng sample on said flow cytometer or fluorescence microscope; and (vii) determining from said calibration plot the number of fluorescently labeled antibodies binding to the sample, based on the fluorescence intensity of the sample measured on said flow cytometer of fluorescence microscope.

Claim 9

A method for calibrating a flow cytometer or fluorescence microscope in terms of the number of binding antibodies per microbead as a function of fluorescence intensity value measured on the flow cytometer of fluorescence microscope, and subsequent measuring of a sample to which said antibodies are bindable, comprising the steps of: (i) providing a microbead calibration kit comprising a set of populations of microbeads having a coefficient of variation of diameter of about 2 percent or less, with said diameter of said microbeadebeing substantially equivalent to the diameter of the sample to be measured, said populations being capable of binding, directly or via a linking group, a fluorescently labeled antibody, wherein the number of fluorescently labeled antibodies, binding to each microbead in said populations is the same within a said population, but different from population to population among the respective said populations in said set; (ii) oontacting each of said populations of microbeads with a fluorescently labeled antibody to effect direct or indirect binding of said antibody to said microbeads for saturation of the binding sites associates with said microbeads, to produce antibody-bearing microbeads having directly or indirectly bound thereto antibodies of a known or determinable number per microbead; (iii) determining the fluorescence intensity of each of said populations of antibody-bearing microbeads on said flow cytometer or fluorescence microscope; (iv) constructing a calibration plot of the number of fluorescently labeled antibodies, as a function of fluorescence intensity measured on said cytometer of fluorescence microscope for said antibody-bearing microbeads; (v) contacting said sample with a second portion of said fluorescently labeled antibody capable of binding to said sample, to yield a fluorescently labeled antibody-bearing sample; (vi) determining the fluorescence intensity of said fluorescently labeled antibody-bearnng sample on said flow cytometer or fluorescence microscope; and (vii) determining from said calibration plot the number of fluorescently labeled antibodies binding to the sample, based on the fluorescence intensity of the sample measured on said flow cytometer of fluorescence microscope.