Methods and reagents for determining immunoglobulin gamma (IgG) antibody isotype concentration from biological samples

Abstract

Disclosed herein are methods and reagents for determining immunoglobulin gamma (IgG) antibody isotype concentration from biological samples, and for analyzing a plurality of cell samples for IgG antibody production.

We claim: 
     
         1 . A method for analyzing a plurality of cell samples for immunoglobulin gamma (IgG) antibody production, the method comprising:
 (a) transferring a plurality of cell samples to an assay plate having a plurality of sample wells, wherein each sample well contains a cell sample that is an undiluted cell culture expressing a target IgG antibody and mixing the plurality of cell samples with analysis reagents to generate a plurality of analysis mixtures, wherein the analysis reagents comprise:
 (i) a capture bead, wherein the capture bead binds to IgG antibody; and 
 (ii) a first detection molecule comprising (A) a control IgG antibody or fragment thereof, and (B) a first detectable moiety; 
   (b) incubating the plurality of analysis mixtures for a time and under conditions to promote binding of the target IgG antibody and the first detection molecule to the capture bead; and   (c) determining the target IgG antibody concentration in each analysis mixture of the plurality of analysis mixtures by flow cytometry analysis.   
     
     
         2 . The method of  claim 1 , wherein the cell culture is propagated from a single clone. 
     
     
         3 . The method of  claim 1 , wherein the capture bead is covalently linked with Protein G or Protein A. 
     
     
         4 . The method of  claim 1 , wherein the capture bead is a magnetic bead or an agarose bead. 
     
     
         5 . The method of  claim 1 , wherein the assay plate contains lyophilized analysis reagents before the plurality of samples are transferred. 
     
     
         6 . The method of  claim 1 , wherein mixing the analysis reagents with the plurality of samples comprises adding the analysis reagents to the cell sample in the wells. 
     
     
         7 . The method of  claim 1 , wherein the assay plate is centrifuged before reading the plurality of analysis mixtures in the assay plate in a flow cytometer. 
     
     
         8 . The method of  claim 1 , wherein the mixing comprises simultaneously mixing the plurality of cell samples and all of analysis reagents, wherein the first detection molecule competes with the target IgG antibody for binding to the capture bead; and wherein the amount of the first detection molecule bound to the capture bead in each analysis mixture provides a measure of the target IgG antibody concentration in a given analysis mixture. 
     
     
         9 . The method of  claim 1 , wherein the mixing comprises stepwise addition of the capture beads and the first detection molecule, wherein the stepwise addition comprises:
 first mixing the plurality of cell samples with the capture beads for a time and under conditions to promote binding of the target IgG antibody to the capture beads to generate a plurality of first mixtures, and then adding the first detection molecules to the plurality of first mixtures for a time and under conditions to promote binding of the first detection molecules to the capture beads to generate the plurality of analysis mixtures;   wherein the amount of the first detection molecule bound to the capture bead in each analysis mixture provides a measure of the target IgG antibody concentration in a given analysis mixture.   
     
     
         10 . A method for analyzing a plurality of cell samples for immunoglobulin gamma (IgG) antibody production, the method comprising:
 (a) transferring a plurality of cell samples to an assay plate having a plurality of sample wells, wherein each sample well contains a cell sample that is an undiluted cell culture expressing a target IgG antibody and mixing the plurality of cell samples with analysis reagents to generate a plurality of analysis mixtures, wherein the analysis reagents comprise:
 (i) a capture bead, wherein the capture bead binds to IgG antibody; and 
 (ii) a first detection molecule comprising a first detectable moiety; 
   (b) incubating the plurality of analysis mixtures for a time and under conditions to promote binding of the target IgG antibody and the first detection molecule to the capture bead; and   (c) determining the target IgG antibody concentration in each analysis mixture of the plurality of analysis mixtures by flow cytometry analysis.   
     
     
         11 . The method of  claim 10 , wherein the first detection molecule lacks immunoglobulin light chains and is capable of binding to the capture bead. 
     
     
         12 . The method of  claim 11 , wherein the method further comprises contacting the plurality of analysis mixtures with a second detection molecule for a time and under conditions to promote binding of the second detection molecule to the target IgG antibody bound to the capture bead;
 wherein the second detection molecule comprises a detectably labeled anti-IgG light chain antibody that is optically distinguishable from the first detection molecule; and   wherein the amount of the second detection molecule bound to the target IgG antibody in each analysis mixture provides a measure of intact target IgG antibody concentration in a given analysis mixture.   
     
     
         13 . The method of  claim 12 , wherein the plurality of analysis mixtures are centrifuged and subjected to a wash step before the labeled anti-light chain antibody is added. 
     
     
         14 . The method of  claim 1 , wherein the analysis reagents further comprises one or more of a cell viability dye, a cell surface biomarker, or a marker of apoptosis. 
     
     
         15 . The method of  claim 1 , further comprising determining one or more of:
 (i) a number of cells in each analysis mixture;   (ii) a percentage of viable cells in each analysis mixture;   (ii) a concentration of target IgG antibody per cell in each analysis mixture; and/or   (iii) a concentration of target IgG antibody per viable cell in each analysis mixture.   
     
     
         16 . A kit comprising:
 (a) an assay plate, and   (b) analysis reagents, wherein the analysis reagents comprise:
 (i) a capture bead, wherein the capture bead binds to IgG antibody; and 
 (ii) a first detection molecule comprising a first detectable moiety. 
   
     
     
         17 . The kit of  claim 16 , wherein the first detection molecule comprises a control IgG antibody or fragment thereof. 
     
     
         18 . The kit of  claim 16 , wherein the kit further comprises a detectably labeled anti-light chain antibody that is optically distinguishable from the first detection molecule. 
     
     
         19 . The kit of  claim 16 , wherein the analysis reagents further comprises one or more of a cell viability dye, a cell surface biomarker, or a marker of apoptosis. 
     
     
         20 . The kit of  claim 16 , wherein (a) the capture bead is covalently linked with Protein G or Protein A; (b) the capture bead is a magnetic bead or an agarose bead; and/or (c) the assay plate contains lyophilized analysis reagents.