Mass Spectrometry Calibrator

Abstract

The invention provides a method of quantifying the amount of kappa or lambda immunoglobulin light chain in a sample from a subject comprising: i. providing a sample from a subject; ii. mixing the sample with a predetermined amount of lambda light chain calibrator or kappa light chain calibrator to form a mixture; iii. performing mass spectrometry on the mixture; and iv quantifying one or both of a) the amount of lambda light chain in the sample by comparing the relative amount of lambda light chain in the mixture as determined by the mass spectrometry to the relative amount of calibrator kappa light chain in the mixture as determined by mass spectrometry; and/or b) the amount of kappa light chain in the sample by comparing the relative amount of kappa light chain in the mixture as determined by mass spectrometry to the relative amount of calibrator lambda light chain in the mixture as determined by mass spectrometry, most typically MALDI-TOF spectrometry or liquid chromatography-mass spectrometry.

Claims

1. A method of quantifying the amount of kappa or lambda immunoglobulin light chain in a sample from a subject comprising: i. providing a sample from a subject; ii. mixing the sample with a predetermined amount of lambda light chain calibrator or kappa light chain calibrator to form a mixture; iii. performing mass spectrometry on the mixture; and iv. quantifying one or both of a) the amount of lambda light chain in the sample by comparing the relative amount of lambda light chain in the mixture as determined by the mass spectrometry to the relative amount of calibrator kappa light chain in the mixture as determined by mass spectrometry; and/or b) the amount of kappa light chain in the sample by comparing the relative amount of kappa light chain in the mixture as determined by mass spectrometry to the relative amount of calibrator lambda light chain in the mixture as determined by mass spectrometry, MALDI-TOF spectrometry, or liquid chromatography-mass spectrometry.

2. A method according to claim 1 wherein the calibrator is polyclonal lambda light chains or polyclonal kappa light chains.

3. A method according to claim 1 wherein the calibrator is monoclonal kappa light chains or monoclonal lambda light chains.

4. A method according to claim 1, wherein the kappa or lambda light chain immunoglobulin in the sample is bound to one or more immunoglobulin heavy chains.

5. A method according to claim 1, wherein the lambda light chains or the kappa light chains of the calibrator prior to mixing are bound to one or more immunoglobulin heavy chains.

6. A method according to claim 1, wherein the kappa or lambda light chains in the sample are free kappa or lambda light chains.

7. A method according to claim 1, wherein the lambda light chains or kappa light chains in the calibrator are free light chains.

8. A method according to claim 1, wherein the lambda light chains in the calibrator or kappa light chains on the calibrator are mass modified.

9. A method according to claim 8 wherein the calibrator, has one or more additional amino acids compared to lambda light chains or kappa light chain in the sample, or is attached to polyethylene glycol.

10. A method according to claim 1, wherein the mixture is purified in at least one purification step prior to performing mass spectrometry.

11. A method according to claim 1, wherein the sample or mixture is immunopurified.

12. A method according to claim 11, wherein the sample or mixture is immunopurified with an anti-heavy chain class specific antibody, an anti-total light chain type specific antibody, an anti-free light chain type specific antibody or an anti-heavy chain class-light chain type specific antibody or fragment thereof.

13. A method according to claim 12, wherein the antibody is anti-IgG specific, anti-IgA specific, anti-IgD specific, anti-IgM specific or anti-IgE specific.

14. A method according to claim 12 wherein the antibody is, anti-total lambda specific, anti-total kappa specific, anti-free lambda specific or anti-free lambda specific.

15. A method according to claim 11, wherein the sample or mixture is immunopurified with anti-human specific antibodies.

16. A method according to claim 1, wherein the sample is selected from blood, serum, plasma, cerebrospinal fluid, and urine.

17. A method according to claim 1, comprising treating the sample or mixture with a reducing agent prior to performing mass spectrometry.

18. A method according to claim 1, wherein the relative amount of lambda light chain in the sample to the relative amount of the calibrator kappa light chain, or the relative amount of kappa light chain in the sample to the relative amount of calibrator lambda light chain, is determined using the ratio of the area of the peak from the sample light chain to the area of the peak of the calibrator light chain or comparing the ratio of the area of the peak of lambda or kappa from the sample to the combined area of the lambda or kappa from the sample plus the area from the calibrator kappa or lambda peaks.

19. A method according to claim 1, wherein the calibrator kappa light chain or calibrator lambda light chain is distinguishable from the same light chain type in the sample by mass spectrometry, and the amount of calibrator light chain identified by mass spectrometry is used to quantify the amount of the same type of light chain in the sample.

20. A method according to claim 1, wherein the ratio of kappa to lambda light chains in the sample is measured.

21. A method according to claim 1, wherein the subject has a B cell proliferative disease.

22. A computer, comprising machine readable medium adapted to receive a first signal indicating an amount of kappa light chain in a sample, or an amount of lambda light chain in a sample, and a second signal indicating an amount of lambda light chain calibrator or kappa light chain calibrator obtained by a method according to claim 1 and compare the two signals to a predetermined calibration factor, to indicate the amount of kappa light chain or lambda light chain in the sample.

23. A kit for use in a method according to claim 1, comprising an antibody selected from: (a) an anti-IgG specific antibody and a predetermined amount of IgG kappa or IgG lambda immunoglobulin; (b) an anti-IgA specific antibody and a predetermined amount of IgA kappa or IgA lambda immunoglobulin; (c) an anti-IgM specific antibody and a predetermined amount of IgM kappa or IgM lambda immunoglobulin; (d) an anti-IgD specific antibody and a predetermined amount of IgD kappa or IgD lambda immunoglobulin; or (e) an anti-IgE specific antibody and a predetermined amount of IgE kappa or IgE lambda immunoglobulin.

24. The method according to claim 21, wherein the B cell proliferative disease is a monoclonal gammopathy disease.

Description

[0081] The invention will now be described by way of example only with reference to the following figures.

[0082] FIG. 1A shows SiLuLite IgG1 lambda added as an internal reference (calibrator) on the same MALDI mass spectrum with a sample from a patient with IgG kappa multiple myeloma. FIG. 1B shows the relationship between the MALDI peak intensity ratio of the calibrator to sample versus the IgG1 kappa concentration.

[0083] FIG. 2A shows SiLuLite IgG4 kappa added as an internal reference (calibrator) on the same MALDI mass spectrum with a sample from a patient with IgG lambda multiple myeloma. FIG. 2B shows the relationship between the MALDI peak intensity ratio of the calibrator to sample versus the IgG1 lambda concentration.

[0084] FIG. 3 shows the peak area ratios for equal amounts of purified polyclonal IgG kappa and IgG lambda mixed together prior to immunoprecipitation and MALDI-TOF analysis.

[0085] FIG. 4A shows purified monoclonal IgA lambda added as an internal reference (calibrator) on the same MALDI mass spectrum with a sample from a patient with IgA1 kappa multiple myeloma. FIG. 4B shows the relationship between the MALDI peak intensity ratio of the calibrator to sample versus the IgA1 kappa concentration.

[0086] FIG. 5 shows the relationship between the MALDI-TOF peak area ratio of polyclonal IgG kappa (calibrator) mixed with polyclonal IgG lambda as the analyte.

[0087] FIG. 6 shows the use of polyclonal IgG kappa as the calibrator with monoclonal SiLuLite IgG lambda as the analyte in the range 0-2 g/L.

[0088] In each of these examples an internal reference (or calibrator) protein is added at a fixed concentration to different concentrations of the analyte. Each sample was subjected to substrate-specific immunoprecipitation with paramagnetic beads coated with polyclonal anti-IgG, or anti IgA. After separation of the light from the heavy chain, light chain mass spectra were acquired using a MALDI-TOF mass spectrometer. Either the peak signal intensity or peak area was determined for each combination and this was then plotted against the concentration of the analyte (sample).

[0089] Commercially available and mass defined SiLu™ Lite IgG1 Lambda was used as an internal reference and a G1K13 M-spike (IgG1 kappa) as the sample. The two light chain m/z peaks are clearly separated on the mass spectrum from each other (FIG. 1A) and a strong correlation observed between the peak intensity ratio and the concentration of the monoclonal protein sample (FIG. 1B). Where the peak intensity ratio is the ratio between the peak intensity of the lambda light chains of the reference calibrator material and the peak intensity of the kappa light chains from the IgG kappa monoclonal protein in the sample. Similarly a commercially available SiLu™ Lite IgG4 Kappa from Sigma was used as the Internal Reference calibrator and the analyte sample was a G1L08 M-spike (IgG1 lambda), with very similar results (FIGS. 2A and B). These results have shown that a monoclonal IgG of either kappa or lambda light chain can be used as a calibrator against another monoclonal IgG of opposite light chain by MALDI-TOF MS.

[0090] To test whether the intensity peak areas are constant between kappa light chains and lambda lights chains, equal amounts of purified polyclonal IgG kappa and polyclonal IgG lambda were mixed together and diluted. The diluted samples were then immunoprecipitated using anti-IgG antibodies immobilized on beads and reduced to separate the kappa and lambda light chains from the IgG heavy chains, prior to spotting on a MALDI target plate. The plate was then subject to mass spectrometry and the polyclonal light chain peak areas for the kappa and lambda light chains determined and plotted as a function of concentration (Table below and FIG. 3). The results show that the peak area (kappa to lambda) ratios are constant throughout the 10 fold concentration range (7.44 to 0.74 g/L), and are not overtly affected by ionisation or differential binding to the anti-IgG antibodies on the beads.

TABLE-US-00001 Conc. Kappa/Lambda (g/L) Average ST DEV % CV 7.44 1.81 0.05 3.0 5.76 1.81 0.05 2.6 4.09 1.80 0.08 4.3 2.42 1.63 0.05 3.2 1.41 1.59 0.06 4.0 1.08 1.60 0.12 7.2 0.91 1.48 0.12 8.4 0.74 1.41 0.15 10.7

[0091] In order to demonstrate that the effect is not heavy chain class specific a monoclonal IgA1kappa antibody was used as an internal reference and an IgA lambda as the sample. The two light chain m/z peaks are again clearly separated on the mass spectrum from each other (FIG. 4A) and a strong correlation observed between the peak intensity ratio and the concentration of the monoclonal protein sample (FIG. 4B). This shows that the ability to use lambda or kappa as a calibrator is not heavy chain class specific.

[0092] Use of polyclonal light chains as an internal reference for the opposite polyclonal light chain is shown in FIG. 5. This example used polyclonal IgG kappa as the calibrator or internal reference with poly IgG lambda as the analyte. The peak area ratios obtained for the calibrator and sample in the sample concentration range of 9.9 to 0.2 g/L are shown in the table below and plotted in FIG. 5.

TABLE-US-00002 Poly GL conc. g/L Peak area ratio 9.9 9.8 7.9 9.7 6.0 9.0 4.1 7.42 3.1 6.02 2.1 4.66 1.2 2.41 0.2 0.44

[0093] FIG. 6 confirms the ability to use polyclonal light chains as a calibrator for monoclonal light chains as the analyte. Purified polyclonal IgG kappa was used as an internal reference at 0.5 g/L and mixed with SiLuLite IgG lambda monoclonal protein in the range 2.0 to 0.1 g/L. The peak area ratios obtained for the calibrator and sample are shown in the table below and plotted against sample concentration in FIG. 6.

[0094] These examples have shown that different combinations of immunoglobulin kappa or lambda containing molecules can be used as internal reference or calibrator molecules against other immunoglobulins of opposite light chain in MALD-TOF MS. This utility is valid for both monoclonal and polyclonal proteins.

TABLE-US-00003 SiLuLite IgG g/L Peak Area Ratio 2.0 1.045 1.6 0.851 1.2 0.626 0.8 0.326 0.3 0.055 0.2 0.022 0.1 0.007