Abstract
The application provides a method of identifying or monitoring a plasma cell associated disease, comprising purifying immunoglobulin free light chains (FLCs) from a sample from a subject with anti-FLC specific antibodies or fragments thereof and subjecting the purified sample to a mass spectrometry technique to identify the presence of one or more peaks corresponding to one or more monoclonal FLCs in the sample.
Claims
1.-11. (canceled)
12. A kit comprising anti-kappa free light chain antibodies, fragments thereof; anti-lambda free light chain antibodies thereof; and a mass spectrometry target.
13. A kit according to claim 12, wherein the antibodies are immobilised on the mass spectrometry target.
14. (canceled)
Description
[0140] The invention will now be described by way of example only, with reference to the following figures:
[0141] FIG. 1 shows a mass spectrometry run in positive ion mode covering singly charged ion range 22.5 to 23.5 kDa of normal samples without the presence of a plasma cell associated disease, following purification with anti-free lambda antibodies.
[0142] FIG. 2 shows a mass spectrometry run in positive ion mode covering the singly charged ion range 22.5 to 23.5 kDa of normal samples following purification with anti-free kappa antibodies.
[0143] FIG. 3 shows an overlay of the printouts for FIGS. 1 and 2.
[0144] FIG. 4 shows the effect of co-purification with anti-free lambda and anti-free kappa antibodies on a normal sample.
[0145] FIG. 5 shows a mass spectrometry run with an abnormal sample following purification with anti-free lambda. This shows an abnormal peak showing the presence of the abnormal monoclonal protein.
[0146] FIG. 6 shows a mass spectrometry run of an abnormal sample where the abnormal clonal production of free lambda is present, following purification with anti-free kappa.
[0147] FIG. 7 shows the overlay of the printouts shown in FIGS. 5 and 6.
[0148] FIG. 8 shows a mass spectrometry run of an abnormal sample comprising abnormal clonal production of free lambda, following co-purification with anti-free lambda and anti-free kappa.
[0149] FIG. 9 Crosslinking of sheep anti-human IgG antibodies by BS(PEG)5, as shown by reducing SDS-PAGE analysis. L.sub.1=free immunoglobulin light chain, H.sub.1=free immunoglobulin heavy chain, H.sub.1L.sub.1 and H.sub.2L.sub.2=crosslinked heavy and light chain moieties.
[0150] FIG. 10 Antibodies crosslinked with BS(PEG)5 retain biological activity. Sheep anti-human IgG antibodies were crosslinked with increasing concentrations of BS(PEG)5 and analysed for their IgG binding activity by ELISA.
[0151] FIG. 11A shows a mass spectrometry run for a sample from a subject with IgG kappa MGUS in positive ion mode for double charged ions.
[0152] FIG. 11B shows a mass spectrometry run for a sample from a subject with IgG kappa MGUS in positive ion mode for single charged ions.
[0153] FIG. 12A shows a mass spectrometry run for a sample from a subject with IgA lambda MGUS in positive ion mode for double charged ions.
[0154] FIG. 12B shows a mass spectrometry run for a sample from a subject with IgA lambda MGUS in positive ion mode for single charged ions.
[0155] FIG. 13A shows a mass spectrometry run for a sample from a subject with IgA lambda MGUS in positive ion mode for a double charged ion.
[0156] FIG. 13B shows a mass spectrometry run for a sample from a subject with IgA lambda MGUS in positive ion mode for a single charged ion.
[0157] FIG. 14A shows a mass spectrometry run from a normal subject in positive ion mode for double charged ions.
[0158] FIG. 14B shows a mass spectrometry run of the normal sample in FIG. 14A in positive ion mode for single charged ions.
[0159] FIG. 15A shows a mass spectrometry run of a normal sample in positive ion mode for double charged ions.
[0160] FIG. 15B shows a mass spectrometry run of the normal sample in FIG. 15A in positive ion mode for single charged ions.
[0161] Kappa FLC and lambda FLC can be purified either separately or co-purified, using anti-kappa FLC antibodies and anti-lambda FLC antibodies, or mixtures thereof.
[0162] The purified FLCs are spotted onto a mass spectrometry plate and analysed by MALDI-TOF.
[0163] FIGS. 1 to 8 show how the presence of a monoclonal free light chain in the serum of a patient, may be easily identified by the presence of a peak, above the background, normal, production of free light chains. This peak may be identified even in areas of where there is overlap between kappa and lambda peaks.
[0164] Anti-human IgG antibodies can be crosslinked by the homobifunctional crosslinker BS(PEG)5.
[0165] The antibodies were incubated with increasing concentrations of BS(PEG)5 (0-40 molar excess) and analysed by reducing SDS-PAGE analysis. As shown in FIG. 9, in the absence of BS(PEG)5, the reducing agent (50 mM DTT) leads to the disassociation of the antibody into its heavy and light chain parts. In contrast, incubation of the antibody with increasing concentrations (0-40 Molar excess) of BS(PEG)5 produces a concomitant increase in crosslinking of the heavy and light chains to form reduction-resistant heavy-light chain pairs.
[0166] Antibodies crosslinked with BS(PEG)5 retain biological activity.
[0167] Purified human IgG Lambda was coated onto microtitre plates at 3-2000 ng/mL. Following crosslinking with 0-40 Molar excess of BS(PEG)5, sheep Anti-human IgG antibodies were applied. The amount of bound antibody was determined using donkey anti-sheep antibodies conjugated to horse radish peroxidase reporter enzyme and 3,3′,5,5′-Tetramethylbenzidine chromogenic substrate. As shown in FIG. 10, at concentrations of BS(PEG)5 up to 40× molar excess, no significant effect on human IgG binding was observed, as compared to the uncrosslinked antibody.
[0168] Testing of Samples from Subjects with MGUS and Normal Samples
[0169] Human serum samples (3 IPE positive MGUS FIGS. 11 to 13 (A-C) and 2 healthy controls) FIGS. 14 to 15 were diluted with PBS-T buffer (25 mM Sodium phosphate, 150 mM NaCl, 0.1% tween 20, pH 7.0) and incubated with antibody coated magnetic beads resuspended and washed sequentially 3×in PBS-T and twice with deionised water. The beads were eluted with an acidic buffer for 15 mins at RT. One of the elution was mixed with a matrix (α-cyano-4-hydroxycinnamic acid, 10 mg/gl) and then spotted onto a polished steel MALDI target plate using the Mosquito HTS spotter (TTP), and analysed on the Bruker Biotyper MALDI-TOF mass spectrometer (Microflex LT/SH Smart). Mass spectra were acquired in positive ion mode covering the m/z range of 10,000 to 30,000 which includes the singly charged (+1, m/z 22-25 kDa) and doubly charged (=2, m/z 10-14 kDa) ions. Data was analysed with Bruker Flex analysis software.
TABLE-US-00002 Kappa Lambda FLC Figure Sample Description IFE (mg/L) (mg/L) Ratio 11 MGUS with IgG kappa 57.73 6.95 8.31 abnormal FLC ratio 13 MGUS with IgA lambda 12.82 153.20 0.08 abnormal FLC ratio 12 MGUS with IgA lambda 5.36 19.43 0.28 normal FLC ratio
[0170] This shows that using mass spectrometry it is possible to detect monoclonal kappa and lambda free light chains even at the free light chain levels normally seen in normal samples.
[0171] The preliminary results also show that using the positive mode for double charged ions allows the abnormal monoclonal FLCs to be detected better than the single positive mode.
[0172] The ability to rapidly identify the presence of low levels of FLCs in samples, even in subject with MGUS which are often difficult to identify, is surprising and opens up a new approach to being able to identify subjects with MGUS and other monoclonal gammopathies.
