Identification and monitoring of monoclonal immunoglobulins by molecular mass

Patent metadata

Field	Value
Publication number	US-2016041184-A1
Application number	US-201414777236-A
Country	US
Kind code	A1
Filing date	Mar 10, 2014
Priority date	Mar 15, 2013
Publication date	Feb 11, 2016
Grant date	—

Abstract

Disclosure herein are methods for determining whether or not an immunoglobulin is present above the polyclonal background level in a biological sample, and methods for determining whether an immunoglobulin contains a kappa or lambda light chain. These methods are useful for screening biological samples for the presence or absence of monoclonal gammopathy, and for diagnosing and monitoring monoclonal gammopathy in a subject.

First claim

What is claimed is:

1 . A method of determining whether or not an immunoglobulin is present above the polyclonal background in a sample, the method comprising:
a. providing a sample comprising variable region-containing immunoglobulins; b. subjecting the sample to a mass spectrometry technique to obtain a mass spectrum of the sample; and c. determining whether or not an immunoglobulin is present above the polyclonal background level by detecting the presence or absence of an M-protein peak in the mass spectrum.

2 . The method of claim 1 , wherein variable region-containing immunoglobulins are selected from immunoglobulin light chains, immunoglobulin heavy chains, and antigen binding fragments (Fabs) of immunoglobulins, and mixtures thereof.

3 . The method of claim 1 , further comprising determining the presence or absence of monoclonal gammopathy in the sample based on the presence or absence of an M-protein peak in the mass spectrum.

4 . A method of diagnosing monoclonal gammopathy in a subject, the method comprising:
a. subjecting a subject sample comprising variable region-containing immunoglobulins to a mass spectrometry technique to obtain a mass spectrum; b. determining whether or not an immunoglobulin of the subject is present above the polyclonal background level by detecting the presence or absence of an M-protein peak in the mass spectrum; and c. diagnosing the presence or absence of monoclonal gammopathy in the subject based on whether or not an immunoglobulin is present above the polyclonal background.

5 . A method of monitoring a treatment of monoclonal gammopathy in a subject, the method comprising:
a. providing a first sample comprising variable region-containing immunoglobulins before the treatment; b. providing a second sample comprising variable region-containing immunoglobulins after the treatment; c. subjecting the first and the second samples to a mass spectrometry technique to obtain a first mass spectrum of the first sample and a second mass spectrum of the second samples; d. determine a first level of an immunoglobulin based on the first mass spectrum, and a second level of the immunoglobulin based on the second mass spectrum; and e. comparing the first level and the second level.

6 . The method of any one of claims 1 to 5 , wherein the mass spectrometry technique comprises a liquid chromatography-mass spectrometry (LC-MS).

7 . The method of any one of claims 1 to 5 , wherein the mass spectrometry technique comprises a matrix assisted laser desorption ionization-mass spectrometry (MALDI-MS).

8 . The method of any one of claims 1 to 5 , wherein the sample is a whole blood, serum, plasma, or urine sample, or a man-made reagent.

9 . The method of any one of claims 1 to 5 , further comprising isolating variable region-containing immunoglobulins from the sample by chemical-based fractionation or by affinity purification.

10 . The method of any one of claims 1 to 5 , further comprising screening the sample by electrophoresis.

11 . A method of determining whether or not a light chain immunoglobulin is a kappa light chain, the method comprising:
a. fragmenting a preselected immunoglobulin light chain precursor ion using a tandem mass spectrometry technique to generate a distribution spectrum of fragment ions; and b. comparing the m/z's of one or more of the fragment ions to m/z's of one or more fragment ions that are expected to result from the constant region of the kappa light chain.

12 . The method of claim 11 , further comprising selecting an immunoglobulin light chain precursor ion using a mass spectrometry technique.

13 . A method of determining whether or not a light chain immunoglobulin is a lambda light chain, the method comprising:
a. fragmenting a preselected immunoglobulin light chain precursor ion using a tandem mass spectrometry technique to generate a distribution spectrum of fragment ions; and b. comparing the m/z's of one or more of the fragment ions to m/z's of one or more fragment ions that are expected to result from the constant region of the lambda light chain.

14 . The method of claim 13 , further comprising selecting an immunoglobulin light chain precursor ion using a mass spectrometry technique.

15 . A method of determining whether or not a light chain immunoglobulin is present above the polyclonal background level in a sample, the method comprising:
a. isolating total immunoglobulins from the sample; b. decoupling light chain immunoglobulins from heavy chain immunoglobulins in the total immunoglobulins to generate a decoupled immunoglobulin sample; and c. subjecting the decoupled immunoglobulin sample to a mass spectrometry technique to determine the presence or absence of an M-protein peak.

16 . The method of claim 15 , further comprising determining the presence or absence of monoclonal gammopathy in the sample based on the presence or absence of an M-protein peak in the mass spectrum.

17 . A method of diagnosing monoclonal gammopathy in a subject, the method comprising:
a. providing a sample of the subject; b. isolating total immunoglobulins from the sample; c. decoupling light chain immunoglobulins from heavy chain immunoglobulins in the total immunoglobulins to generate a decoupled immunoglobulin sample; d. subjecting the decoupled immunoglobulin sample to a mass spectrometry technique to determine the presence or absence of an M-protein peak; and e. determining whether or not the subject has monoclonal gammopathy based on the presence or absence of an M-protein peak.

18 . A method of monitoring a treatment of monoclonal gammopathy in a subject, the method comprising:
a. providing a first sample of the subject before the treatment; b. providing a second sample of the subject during or after the treatment; c. isolating total immunoglobulins from the first and second samples; d. decoupling light chain immunoglobulins from heavy chain immunoglobulins in the total immunoglobulins to generate a first and a second decoupled immunoglobulin samples; e. subjecting the first and the second decoupled immunoglobulin samples to a mass spectrometry technique to determine a first level of an immunoglobulin in the first decoupled immunoglobulin sample, and a second level of the immunoglobulin in the second decoupled immunoglobulin sample; and f. comparing the first level and the second level.

19 . A method of determining whether or not a light chain immunoglobulin is present above the polyclonal background level in a sample, the method comprising:
a. isolating total immunoglobulins from the sample; b. treating total immunoglobulins with proteases to generate Fabs of immunoglobulins; and c. subjecting the Fabs of immunoglobulin to a mass spectrometry technique to determine the presence or absence of an M-protein peak.

20 . The method of claim 19 , further comprising determining the presence or absence of monoclonal gammopathy in the sample based on the presence or absence of an M-protein peak in the mass spectrum.

21 . A method of diagnosing monoclonal gammopathy in a subject, the method comprising:
a. providing a sample of the subject; b. isolating total immunoglobulins from the sample; c. treating total immunoglobulins with proteases to generate Fabs of immunoglobulins; d. subjecting the Fabs of immunoglobulins to a mass spectrometry technique to determine the presence or absence of an M-protein peak; and e. determining whether or not the subject has monoclonal gammopa

Assignees

• Barnidge David R
• Murray David L
• Mayo Foundation

Inventors

• Barnidge David R
• Murray David L

Classifications

• G01N33/6848 Primary

  Methods of protein analysis involving mass spectrometry · CPC title

• H01J49/164

  Laser desorption/ionisation, e.g. matrix-assisted laser desorption/ionisation [MALDI] (sample holders H01J49/0418) · CPC title

• H01J49/0027

  Methods for using particle spectrometers · CPC title

• G01N2800/52

  Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis · CPC title

• G01N33/6851

  Methods of protein analysis involving laser desorption ionisation mass spectrometry · CPC title