Methods and reagents for determining immunoglobulin gamma (IgG) antibody isotype concentration from biological samples
Inventors
Liu, Zhaoping • Duensing, Thomas
Assignees
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Publication Number
US-11733237-B2
Publication Date
2023-08-22
Expiration Date
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.
Core Innovation
The invention provides a method for determining immunoglobulin gamma (IgG) antibody isotype concentration from an undiluted biological sample. The method incubates in a microtiter plate well an undiluted biological sample comprising B cells or cell hybridoma supernatant with or without cells, together with IgG antibodies, and a detection reagent comprising a defined concentration ratio of three or more different target IgG protein isotypes, or fragments thereof, wherein the target isotypes are detectably labeled.
The method further includes incubating in the same reaction mixture three or more different bead-bound IgG isotype-specific populations of capture reagent, where each bead-bound IgG isotype-specific population is separately distinguishable and comprises binding molecules that selectively bind to specific IgG antibody isotypes. In a single reaction mixture, the incubation forms bead-bound IgG isotype-specific binding complexes, and without wash steps the method detects in a flow cytometer signal from the bead-bound IgG isotype-specific binding complexes to determine a concentration for any of the IgG antibodies present.
Quantitation is based on an inverse relationship between the amount of an IgG antibody isotype protein present in the biological sample and each detected signal from the relevant bead-bound IgG isotype-specific binding complex. The disclosed variants include standard-curve generation using control samples and competitive binding, and optionally measuring cell viability and/or cell number.
Claims Coverage
The independent claim is directed to a multiplexed, no-wash flow cytometry method for determining IgG antibody isotype concentration from an undiluted biological sample using detectably labeled target IgG isotypes (or fragments), separately distinguishable bead-bound isotype-specific capture reagent populations, and an inverse relationship between detected signal and antibody isotype concentration. Dependent claims further add control/standard-curve generation via competitive binding, increasing the number of labeled target isotypes, adding a detectable cell viability marker with viability and/or cell number measurement, and specifying particular mammalian cell-source/isotype sets.
No-wash multiplexed IgG isotype quantitation using inverse flow cytometer signal
A method for determining IgG antibody isotype concentration from an undiluted biological sample by incubating in a microtiter plate well a defined, detectably labeled detection reagent containing three or more target IgG protein isotypes (or fragments) and three or more separately distinguishable bead-bound IgG isotype-specific capture reagent populations that selectively bind specific IgG antibody isotypes, followed by detecting flow cytometer signals from the bead-bound IgG isotype-specific binding complexes without wash steps, wherein IgG isotype protein amount is inversely proportional to each detected signal.
Standard curves via competitive binding with serial dilution control mixtures
A method that incubates serial dilutions of a control sample containing three or more unlabeled IgG protein isotypes to generate control mixtures, incubates the control mixtures with three or more bead-bound IgG isotype-specific capture reagents to produce control IgG isotype-specific binding complexes via competitive binding, and generates a standard curve for each IgG protein isotype to determine the IgG antibody concentration in the biological sample by reference to the curves.
Defined concentration ratio of four or more labeled target IgG isotypes
The method where the detection reagent comprises a defined concentration ratio of four or more different target IgG protein isotypes, or fragments thereof, wherein the target IgG protein isotypes are detectably labeled and the concentration of four or more IgG antibody isotypes is determined in each biological sample.
Cell viability or cell number measurement with a detectable viability marker
A method where the undiluted biological sample comprises cells, the detection reagent further comprises a detectable cell viability marker, and the method further measures cell viability and/or cell number in the flow cytometer.
Mammalian cell-source specific IgG isotype sets
A method where the biological sample comprises specific mammalian cell types (rabbit/sheep; goat/pig/bovine; horse; or monkey) with corresponding selected IgG isotypes chosen from specified groups for those species.
Rat IgG isotype selection for quantitation
A method where the biological sample comprises rat cells and the different IgG isotypes are selected from rat IgG1, IgG2a, IgG2b, and IgG2c.
Overall, the claims cover a no-wash, multiplex bead-capture and flow cytometry approach that determines IgG isotype concentration using separately distinguishable bead-bound isotype-specific capture reagents with detectably labeled target IgG isotypes and an inverse signal-to-concentration relationship. Dependent claims further refine quantitation with competitive-binding standard curves, expand the number of labeled isotypes, add optional viability/cell number measurement, and restrict or specify selected isotype sets for particular mammalian cell-source samples.
Stated Advantages
Determines IgG antibody isotype concentration from an undiluted biological sample.
Enables multiplexed determination of concentration for any of the IgG antibodies present using bead-bound IgG isotype-specific capture reagent populations that are separately distinguishable.
Operates without wash steps.
Uses an inverse proportional relationship between IgG antibody isotype protein amount and detected flow cytometer signal to determine concentration.
Allows quantitation using standard curves generated with control samples using competitive binding.
Optionally measures cell viability and/or cell number using a detectable cell viability marker.
Documented Applications
Analysis of biological samples comprising B cells or cell hybridoma supernatant (with or without cells) for IgG antibody isotype concentration.
Quantitation from biological samples comprising specific mammalian cell types (rabbit, sheep, goat, pig, bovine, horse, or monkey) using corresponding selected IgG isotypes.
Quantitation from biological samples comprising rat cells using rat IgG1, IgG2a, IgG2b, and IgG2c.
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