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Technical Briefs |
1
Graduate School of Allied Health Sciences, Tokyo Medical and Dental University, Yushima 1-5-45, Bunkyo-ku, Tokyo 113-8519, Japan
2
Department of Nephrology, Sendai Shakaihoken Hospital, Tsutsumimachi, 3-16-1, Aoba-ku, Sendai 981-8501, Japan
3
Department of Laboratory Medicine, Asahikawa Medical College, Midorigaoka-higashi 2-1-1-1, Asahikawa, Hokkaido 078-8510, Japan
a Author for correspondence: fax 81-3-5803-0166, e-mail
k.shiba.mtec{at}tmd.ac.jp
The monoclonal free light chain of immunoglobulin, Bence Jones protein (BJP), is associated with malignant monoclonal gammopathies, in particular with multiple myeloma, lymphoproliferative diseases such as Waldenström macroglobulinemia and malignant lymphoma, amyloidosis associated with light chain, and light chain deposition disease (1)(2). The detection of urinary BJP is useful for diagnosing and evaluating the prognosis for monoclonal gammopathies (3)(4)(5)(6)(7). BJP can be detected as a sharp band by urinary protein electrophoresis (UPE). UPE on cellulose acetate, cellulose nitrate, and agarose has been reported. More recently, sodium dodecyl sulfate-agarose gel electrophoresis and capillary electrophoresis of urinary proteins have been reported (8)(9)(10)(11)(12)(13).
Electrophoresis on cellulose acetate membranes is carried out for serum protein fractions in many clinical laboratories, and it is a simple and easily reproducible technique. After UPE on cellulose acetate, membranes are stained with solutions containing Acid-violet 17 and Coomassie Brilliant Blue; however, extensive preconcentration of urine before electrophoresis generally is recommended because of the low concentrations of urinary proteins. This procedure for concentrating urine is time-consuming and has problems such as protein loss, aggregation, and degradation. Although highly sensitive staining methods using colloidal gold solution without preconcentration of urine have been reported (14)(15)(16), these methods require 2–3 h for protein staining.
We previously reported a rapid and highly sensitive colloidal silver
staining solution suitable for cellulose acetate membranes
(17). In this study, we further modified the staining method
and developed a more rapid and sensitive colloidal silver staining
method to detect small amounts of BJP that does not require
preconcentration of urine. We used urine samples obtained from
inpatients with multiple myeloma (n = 9), benign monoclonal
gammopathy (n = 1), primary macroglobulinemia (n = 1), light
chain deposition disease (n = 1), and primary amyloidosis (n
= 5). Informed consent was obtained from all patients. The urinary
total protein concentration was determined by the Pyrogallol red dye
method (Wako Pure Industrial Chemical) on a Hitachi 7070
automated analyzer (Hitachi). Colloidal silver solution was prepared
according to the following method. To a solution containing 12 mL of
2.5 mL/L Tween 20 and 15 mL of 28 mmol/L iron(II) sulfate
heptahydrate, 5 mL of 280 mmol/L trisodium citrate dihydrate was added
and mixed. One milliliter of 350 mmol/L silver nitrate was then added
and vigorously shaken by hand to prevent premature flocculation of
silver particles. Finally, 2 mL of 150 mL/L acetate acid was added and
mixed. This solution was prepared fresh before use. After
electrophoresis at a constant current of 0.7 mA/cm per membrane for 25
min in veronal buffer (60 mmol/L, pH 8.6; ionic strength,
0.06), the proteins were fixed in 0.4 mol/L trichloroacetic acid–0.03
mol/L sulfosalicylic acid for 5 min, immersed in 10 mL/L acetic acid
for 5 min, and stained with freshly prepared colloidal silver solution
for 20 min with continuous shaking. Finally, the membranes were washed
in distilled water for 10 min. For immunofixation (IFE), each track was
overlayed with cellulose acetate strips impregnated in antisera
specific against IgG, IgA, IgM, and free and bound 
and 
(Dako).
Immunofixed strips were washed in 9 g/L NaCl and stained with colloidal
silver solution. Routine IFE was performed according to the
manufacturer’s procedure (Helena Laboratories) with urine samples
concentrated 
100-fold in a Minicon BS15 (Millipore) before
electrophoresis.
As shown in Fig. 1A
, we could accurately detect and identify BJP from
unconcentrated urine that had been obtained from a patient with benign
monoclonal gammopathy (total urinary protein, 57 mg/L; BJP, 5 mg/L). On
the other hand, routine IFE detected a small amount of BJP that was not
apparent after UPE. Patient profiles and data are summarized in
Table 1
. We could detect and identify BJP for all 17 samples by UPE on
cellulose acetate membrane followed by staining with the colloidal
silver solution. Furthermore, when we used IFE together with colloidal
silver staining after UPE, we could identify the type of BJP.
Using routine IFE with concentrated urine samples, we could identify
BJP for 16 samples.
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Because clear electrophoretic images could be obtained without
preconcentration of urine samples, we generated a calibration curve
using the areas for densitometric images of albumin calibrators, and
the BJP concentration was calculated from the curve for
semiquantification. The electrophoretic results for the albumin
calibrators (2.5–200.0 mg/L) and a urine sample are shown in the left
panel of Fig. 1B
. A typical calibration curve based on our albumin
calibrators is shown in the right panel of Fig. 1B
. The amounts of free
and light chains in urine were determined by immunonephelometry
using antisera against the free form (Dako) and N Protein Standard SL
(Behringwerke AG) on a Behring Nephelometer Analyzer II (Behringwerke).
We performed the assay according to the manufacturer’s
recommendations. We examined the correlation between the BJP
concentrations obtained using the present method (y) and
those obtained using the immunonephelometric assay (x). The
linear regression equation for BJP was: y =
0.777x + 57.661 (r = 0.999). We then
examined the correlation for urine samples having BJP concentrations
<1000 mg/L. The linear regression equation for BJP was:
y = 0.989x + 21.758 (r =
0.991).
With our highly sensitive silver staining method, even 5 mg/L
BJP could be detected by staining for only 20 min. In addition, the
colloidal silver staining solution we developed can also be used for
IFE to determine the type of BJP and to identify other proteins. Even
when IFE is performed, the entire procedure is completed in only
1.5 h. The sensitivity of our method has been shown to be higher
than that of routine IFE with 100-fold concentrated urine. In
preliminary studies to ascertain background noise, urine samples from
74 patients without BJP and 32 healthy volunteers were analyzed by UPE
combined with IFE using our colloidal silver staining solution. We did
not detect BJP and had no false-positive results (data not shown).
Given the occurrence of false positives in cases such as hepatitis
where oligoclonal banding is common, interpretation of the result
together with other clinical data is important.
Increased BJP indicates a poor prognosis and aggravation of monoclonal
gammopathies. The quantification of urinary BJP is important
for observing the course and evaluating treatment effects. In patients,
the urinary total protein concentration varies. Because low urinary
total protein concentrations cannot be determined precisely, the BJP
concentration cannot be calculated by multiplying the amount of total
protein by the percentage of the BJP band. When we used or
solutions as calibrators for semiquantification of BJP, their
electrophoretic patterns were smeared. In this study, therefore, we
used albumin as a calibrator, for which the electrophoretic pattern was
a single sharp band. The calibration curve based on albumin calibrators
has been shown to be acceptably linear. More accurate quantification is
possible in overflow-type urine samples, which show a high correlation
between the values obtained by our semiquantitative method and that
obtained by immunonephelometry. However, for electrophoretic images of
urine samples containing high amounts of protein, a decrease in the
quantification accuracy must also be taken into consideration.
In the recent study by Levinson (18), BJP concentrations
were expressed as semiquantitative values based on data obtained by UPE
and IFE. If the results obtained by our method are also considered to
be semiquantitative rather than quantitative, our method may be useful
for observing and evaluating the clinical course of monoclonal
gammopathies.
Acknowledgments
This study was supported in part by a research grant from the Kurozumi Medical Foundation. We thank Yoji Hirabayashi of SRL Inc. for performing the immunonephelometry assay and Naoko Yusa (Department of Nephrology, Sendai Shakaihoken Hospital) for collection of urine samples and clinical information.
References
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