Assay for detecting free light chains by capillary zone electrophoresis

Abstract

The invention provides a method detecting free light chains (FLCs) comprising: (i) providing a sample from a subject; (ii) mixing the sample with an anti-FLC specific antibody, or fragments thereof capable of specifically binding the FLC, to form a mixture; (iii) passing the mixture through a capillary tube by capillary zone electrophoresis (CZE); and (iv) detecting the presence of the antibody or fragment thereof after passage through at least a portion of the capillary tube. Capillary tubes for use in CZE and kits comprising capilliary tubes and at least one anti-FLC antibody are also provided.

Claims

1. A method of detecting and quantifying free light chains (FLCs) comprising: (i) providing a sample from a subject; (ii) mixing the sample with (a) an anti-FLC specific antibody, or fragments thereof capable of specifically binding the FLC and (b) a predetermined amount of a labelled FLC, or a labelled fragment of said FLC, to form a mixture; (iii) passing the mixture through a capillary tube by capillary zone electrophoresis (CZE); (iv) detecting the presence of the antibody or fragment thereof after passage through at least a portion of the capillary tube; (v) measuring the peak area or the height of a FLC/anti-FLC specific antibody complex, and (vi) quantifying the peak area or height of the FLC/anti-FLC specific antibody complex, wherein the peak or height of the FLC/anti-FLC specific antibody complex is inversely proportional to the FLC concentration in the sample.

2. A method according to claim 1, wherein intact monoclonal immunoglobulins are additionally detected by CZE.

3. A method according to claim 1, wherein a label is attached to the FLC or the fragment via a charged linker.

4. A method according to claim 3, wherein the charged linker is a poly(aspartic acid) or poly(lysine).

5. A method according to claim 1, wherein the antibody is FLC or FLC specific.

6. A method according to claim 1, wherein the anti-FLC specific antibody comprises two antibodies that are mixed with the sample, wherein each of the two antibodies has a specificity for a different FLC type.

7. A method according to claim 6, comprising detecting two different FLC types, using two different antibodies each with a different fluorescent label.

8. A method according to claim 7, wherein the capillary tube comprises two detection apertures.

9. A method according to claim 5 comprising detecting the ratio of :FLC in the sample.

10. The method according to claim 1, wherein the antibody or the antibody fragments is labelled with a fluorescent label.

11. The method according to claim 1, wherein the FLC concentration in the sample is about 200 mg/L or less.

Description

(1) The invention will now be described by way of example only with reference to the following figures:

(2) FIG. 1 shows a CZE electropherogram of normal human serum.

(3) FIG. 2 shows the fluorescence staining of FLC in the position of the gamma region of the overlaid electropherogram (large peak to left).

(4) FIG. 3 shows an example of modifying the charge of the labelled antibody, showing the charge in the peak position (a) compared to the uncharged antibody with succinic anhydride (b).

(5) FIG. 4 shows the affect of acetic anhydride on peak position (a) compared to uncharged antibody on an electropherogram (b).

(6) FIG. 5 shows EDC modified F(ab).sub.2 fragments (a) 20 mm, (b) 2 mm compared to an uncharged (c) electropherogram.

(7) FIG. 6 shows the modification of the charge of IgG with succinic anhydride (a) compared to uncharged (b).

(8) FIG. 7 shows the effect on FLC peak height when mixed with a dilution series of anti-FLC antibody fragments.

(9) FIG. 8 shows an example of a discreet assay for FLC using a labelled antibody showing the differentiation in peak height between samples containing 200 (a) and 0 mg/L (b) free light chains.

(10) FIG. 9 shows a direct assay for the components of serum using CZE. The absorbance at 200 nm is shown by the line with the large peak (at right of trace). Fluorescence is shown using anti lambda FITC conjugate in comparison with pure lambda light chain at 78 mg/L and 218 mg/L.

METHODS

(11) CZE Assay

(12) There follows a summary of the separation conditions used and examples that demonstrate the proof of principle of these assays for the detection of FLC to the target sensitivity. All development assays have been performed on a Beckman PACE MDQ system with 488 nm LIF unit. The columns were purchased from Analis 30 cm length (10 cm to detector aperture), 25 um bare faced capillary. The separation voltage unless otherwise noted was 7 KV for 6.5 minutes which resulted in a current of 10 mA, column temperature was maintained at 32 C. Column preconditioning was performed between each run with each pressure rinse at 20 psi. Firstly 4.3M urea for 2 minutes followed by water 2 minutes, then 0.1M NaOH 2 minutes and finally run buffer for 2 minutes. The final run buffer was 150 mM Boric acid adjusted to pH9.9 with 1M NaOH. Sample buffer was TBS ELISA PBS wash buffer type III, working strength buffer was diluted 10 for use as the sample buffer.

(13) TABLE-US-00003 PBS buffer diluted 10X for use as sample buffer Chemical Formula Weight g/L Sodium Chloride (Analar) NaCl 7.2 Disodium hydrogen Na.sub.2HPO.sub.4 0.161 orthophosphate Sodium di-hydrogen NaH.sub.2PO.sub.42H.sub.2O 0.01 orthophosphate Tween-20 0.5 mL Kathon 0.01 mL Water (18.2 M) H.sub.20 to 1 L Sample injection time was 10 seconds at 0.5 psi pressure, this equates to a sample volume of approximately 15 nL. Test samples were stored onboard at 10 degrees. Wavelengths monitored were 200 nm UV or 488 nm fluorescent.

(14) Fluorescent labels used were FITC and DyLight 488.

(15) Anti-FLC antibody fragments were used to exemplify the invention.

(16) Charged Modification of Antibodies or Antibody Fragments

(17) Negative charge modification can be obtained by conversion of positive amino groups (e.g. on Lysine residues) by acetylation with Acetic anhydride or succinylation with Succinic anhydride. The former modifies a positive to a neutral group (a change of one positive charge, +1 to 0) and the latter converts a positive to a negative group (+1 to 1, a change of two positive charges). The coupling reaction was as follows. The protein to be conjugated at a concentration of 1 mg ml-1 (antibody or FLC) was buffer-exchanged into 0.1 M sodium bicarbonate buffer pH 8.5 by dialysis. To this was added a 5- or 30-fold molar excess (theoretical lysine content) of either Acetic or Succinic anhydride. The mixture was then reacted for 10 min at RT (room temperature) and unreacted anhydride was quenched by the addition of Tris (0.1 M). Finally the sample was desalted and buffer exchanged by dialysis into a suitable buffer for further modification or immunoassay. The molar ratios of reagent were determined experimentally to produce high or low negative charge modification.

(18) Positive charge modification can be accomplished by the amidation of carboxyl groups on Aspartic and Glutamine acid residues. Proteins (1 mg ml-1) dissolved in distilled water were reacted with 2-100 mM 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) in the presence of 200 mM Ethylenediamine dihydrochloride for 2 h at RT. The reaction was quenched with Acetic acid and buffer exchanged by dialysis into a suitable buffer. The level of positive charge modification was controlled by the EDC concentration.

(19) Native agarose gel electrophoresis and analytical Anion-exchange chromatography was used for the analysis of the charge-modified proteins.

(20) Immunoassay

(21) Typical conditions used for the assays were: Run buffer0.15M Borate pH9.9 Sample buffer1:10 Type III (PBS) Column25 m i.d. 30 cm Analis bare faced silica Column temperature 32 degrees Onboard sample storage temperature 10 degrees C. Sample load 10 sec at 20 psi Separation voltage 7 kV for 6.5 minutes Ramp time 0.5 minute Base line zero 0.5 mins Monitor LIF 488 nm

(22) Both competition and indirect assays were studied.

(23) Results

(24) Fab and F(ab).sub.2 labelled with FITC or DyLight 488 nm were found to selectively label the gamma-globulin peak of the electropherogram.

(25) FIG. 2 shows an example of an anti FLC Fab labelled with FITC overlaid in a normal reference serum electropherogram. Separation was run at 25 KV, 50 mM Borate, pH 10.5. Fluorescence was monitored at 488 nm. The area of the peak was found to be a function of the concentration of the FLC in the sample.

(26) The position of the labelled antibody was found to be adjustable using, for example, succinic anhydride, Fab 100:1 succinic anhydride. This is shown in FIG. 3. Increasingly the charge with succinic anhydride (for example from 30:1 to 1:1), was shown to increase the migration rate of the labelled peak.

(27) Acetic anhydride (1:1 Fab:acetic anhydride) reduced the speed of the peak through the capillary tube column (FIG. 4).

(28) EDC was found to increase the rate of movement of F(ab).sub.2 fragments (2 mM EDC), as shown in FIG. 5. This was carried out at 7 KV, 150 mM Borate pH 9.9. Fab fragments were shown to run at the same or slower speed than the gamma region.

(29) Adding, for example, 5:1 succinic anhydride to IgG, was also shown to increase the rate of migration of the IgG compared to non-modified antibody (FIG. 6).

(30) Immunoassays

(31) F(ab).sub.2 (modified with 4 mM EDC) anti FLC antibody and labelled FLC, was compared to different dilutions of antibody. Two peaks were observed. A complex peak and an unlabelled antibody peak.

(32) The effect of labelled FLC peak height when mixed with a dilution series of anti-FLC, is shown in FIG. 7. This showed that a competition assay, using labelled FLC could be produced

(33) A direct assay is shown in FIG. 9.

(34) In the direct assay uncharged whole IgG gave the best result, there was clear differentiation between the free labelled antibody and antibody complexed to the light chain. The use of labelled antibody fragments was less successful as the added charge had an overriding effect on their mobility when complexed with light chain.

CONCLUSIONS

(35) The results show that FLC and other immunoglobulins can be detected using CZE. The immunoassays can be run at the same time as CZE electropherogram to increase the diagnostic information available from such assays.