Electrophoretic analysis of Bence Jones proteinuria

Electrophoretic analysis of Bence Jonesproteinuria

The electrophoresis of Bence Jones proteinuria (BJP) by urinary protein electrophore-sis (UPE), immunoelectrophoresis (IE), immunofixation electrophoresis (IFE), sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), isoelectric focusing(IEF), two-dimensional electrophoresis (2-DE) and capillary electrophoresis (CE) is de-scribed. UPE, IE and IFE are briefly discussed as clinical laboratory methods for thedetection and typing of free light chain (LC) whilst the high resolution electrophoreticmethods (SDS-PAGE, IEF, 2-DE and CE) are considered in greater detail as researchtools for molecular characterisation of free LC and its association with nephrotoxicity.Refinements of sample processing designed to improve the standardisation of analysisof BJP by high resolution electrophoretic methods are reported.

Keywords: Bence Jones proteinuria / Urinary protein electrophoresis / Immunofixation electro-

phoresis / Sodium dodecyl sulfate-polyacrylamide gel electrophoresis / Isoelectric focusing / Two-

dimensional polyacrylamide gel electrophoresis / Capillary electrophoresis / Review EL 3326

Review

Thomas MarshallKatherine M. Williams

Analytical BiochemistryResearch Group, School ofHealth Sciences, TheUniversity of Sunderland,UK

Contents

1 Introduction . . . . . . . . . . . . . . . . . . . . . . 13072 Clinical laboratory analysis of Bence Jones

proteinuria . . . . . . . . . . . . . . . . . . . . . . . 13082.1 Protein assay of BJP . . . . . . . . . . . . . . . . 13082.2 Urinary protein electrophoresis of BJP . . . . 13092.3 Immunoelectrophoresis of BJP . . . . . . . . . 13092.4 Immunofixation electrophoresis of BJP . . . . 13103 High resolution electrophoretic analysis of

BJP . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13103.1 Sodium dodecyl sulfate-polyacrylamide gel

electrophoresis of BJP . . . . . . . . . . . . . . . 13103.1.1 Early applications . . . . . . . . . . . . . . . . . . 13103.1.2 Immunoblotting . . . . . . . . . . . . . . . . . . . . 13123.1.3 Advantages and pitfalls . . . . . . . . . . . . . . 13133.2 Isoelectric focusing of BJP . . . . . . . . . . . . 13143.2.1 Immunoblotting . . . . . . . . . . . . . . . . . . . . 13153.2.2 Nephrotoxicity . . . . . . . . . . . . . . . . . . . . . 13163.2.3 Limitations . . . . . . . . . . . . . . . . . . . . . . . 13163.3 Two-dimensional electrophoresis of BJP . . . 1317

3.3.1 Early applications . . . . . . . . . . . . . . . . . . 1317

3.3.2 Recent applications . . . . . . . . . . . . . . . . . 1317

3.4 Capillary electrophoresis of BJP . . . . . . . . 1320

4 Concluding remarks . . . . . . . . . . . . . . . . . 1321

5 References . . . . . . . . . . . . . . . . . . . . . . . 1322

1 Introduction

Electrophoretic methods have been widely used for theclinical analysis of proteinuria (protein in the urine) [1].Under normal circumstances, the renal glomeruli restrictfiltration of plasma proteins and the renal tubules reab-sorb and degrade most filtered protein such that onlytrace amounts of protein (approximately 150 mg per 24 h)appear in the urine [2]. In renal disease, the level of pro-tein in the urine tends to be persistently elevated and theprotein composition is modified in a characteristic man-ner. Renal glomerular disease (glomerulonephritis, neph-rosis, glomerulosclerosis and IgA nephropathy) results inurine containing increased amounts of albumin and highmolecular weight proteins whilst renal tubular disease(pyelonephritis, drug-induced tubulopathies and heavymetal toxicity) results in urine containing elevated levelsof low molecular weight proteins such as b2-microglobu-lin, lysozyme, retinol-binding protein, a1-microglobulin,and a1-acid glycoprotein [1, 2]. Alternatively, excess lev-els of abnormal protein in the blood can spill over into theglomerular filtrate ± this overloads the reabsorptioncapacity of the renal tubules, resulting in the excretion ofthe excess protein in the urine [2].

Correspondence: Dr. Thomas Marshall, School of Health Scien-ces, The University of Sunderland, Fleming Building, SunderlandSR1 3RG, UKE-mail: [email protected]: +4491-515-3747

Abbreviations: BJP, Bence Jones proteinuria; IE, immunoelec-trophoresis; IFE, immunofixation electrophoresis; LC, immuno-globulin light chain; UPE, urinary protein electrophoresis

Electrophoresis 1999, 20, 1307±1324 1307

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Gen

eral

Bence Jones proteinuria (BJP) is an ªoverload proteinu-riaº associated with immunoproliferative disorders [3, 4]and is characterised by the spill over of excess free mon-oclonal immunoglobulin light chain (LC) into the urine [1±5]. The initial report of BJP is of historic interest [6]. In1845 the chemical pathologist Henry Bence Jones [7]was sent a sample of urine from a patient with mollitiesossium (ªsoftening of the boneº). Bence Jones confirmedthe unusual thermal precipitation properties of the urine(as earlier noted by the physician William McIntyre) andattributed them to a ªhydrated deutoxide of albumenº(Bence Jones protein) [8, 9]. Post mortem examination ofthe patient©s bone marrow revealed a ªgelatiniform sub-stance of a blood-red colour and unctuous feelº contain-ing large nucleated cells [10] which, in retrospect, can beinterpreted as myeloma cells [6]. Subsequently, twoimmunologically distinct types of BJP (k and l) were rec-ognised and identified as free LC in association with ele-vated levels of serum protein in multiple myeloma (MM)[11±13].

Under normal circumstances, excess polyclonal free LCappears in small amounts in urine as monomers (Mr

22 000) and dimers (Mr 44 000) [14, 15] but LC fragments[16], tetramers [15, 17, 18] and glycated forms (Mr

55 000) [3, 19] have also been reported. The predominantk free LC demonstrates approximately equal amounts ofthe covalent (disulfide-linked) and the noncovalentdimeric forms whilst the l free LC is mainly covalent in itsdimeric form [14, 15, 19±21]. Renal filtration of LC mono-mer promotes dissociation of the noncovalent LC dimers(predominantly k) in the blood resulting in a higher freeLC k:l ratio in the urine than the blood [3, 15].

Monoclonal free LC is sometimes detected in urine inmonoclonal gammopathy of unknown significance [3, 4,22, 23] but is predominantly associated with malignantmonoclonal gammopathies and in particular MM, lympho-proliferative disease (e.g., Waldenströms macroglobulin-aemia, lymphoma) and amyloidosis [2±4]. BJP is mostfrequently associated with MM, a plasma cell dyscrasia ofunknown aetiology (possibly linked to genetic factors orexposure to radiation or pesticides [4]) which predomi-nates in patients over 60 years of age with an incidenceof 1:25 000 (equivalent to >10% of haematological malig-nancies and ~1% of all malignant diseases) [3, 4]. Symp-toms include bone pain and fatigue (commonly associ-ated with anaemia). Diagnosis is based upon the plasmacell content of the bone marrow (³ 10%), the detection oflytic bone lesions (~60% of patients) and the presence ofa monoclonal immunoglobulin (paraprotein) in serum orurine (~95% of patients) [2±4]. MM is commonly associ-ated with depression of normal immunoglobulin synthesis(due to inhibition of normal plasma cell clones) and neuro-

logical, vascular and renal complications [2±4]. Treatmentinvolves radiotherapy/chemotherapy (to reduce tumourmass) and the management of the complications arisingfrom hyperviscosity and renal failure [24]. Prognostic indi-cators include the plasma cell labelling index, b2-micro-globulin, thymidine kinase and C-reactive protein, but thecondition is incurable and death usually occurs withinthree years [4, 25].

Monoclonal free LC is not simply a marker protein for themonoclonal gammopathies but is also a causative agentin the progression of renal failure and the development ofsystemic disease. Intracellular crystallisation of LC resultsin renal tubular dysfunction (Fanconi syndrome) and castnephropathy (ªmyeloma kidneyº) [26, 27]. Extracellulardeposition of acidic free LC (mean pI 4.8) and LC frag-ment (Mr 12 000±18 000), predominantly of the l type, inantiparallel b-pleated sheets in the kidney, heart, liver,nerves and gastrointestinal tract leads to amyloidosis [28,29]. Extracellular deposition of free LC (predominantly k)in a less structured manner results in light-chain deposi-tion disease [3].

Electrophoresis has played a major role in the detectionand typing of BJP for the clinical diagnosis and manage-ment of immunoproliferative diseases. The present reportconcentrates on the use of high resolution electrophoreticmethods for characterisation of BJP and its associationwith nephrotoxicity.

2 Clinical laboratory analysis of BenceJones proteinuria

Urines are screened for BJP using protein assay and uri-nary protein electrophoresis (UPE) on either celluloseacetate or agarose gels. The identity of the suspectedfree LC is then confirmed using immunoelectrophoresis(IE) or immunofixation electrophoresis (IFE). The levelsof free LC can then be quantitatively monitored using avariety of immunochemical techniques as an index ofplasma cell burden.

2.1 Protein assay of BJP

Protein assay is a valuable prerequisite to electrophoreticanalysis. It dictates (i) the need for (and degree of) sam-ple concentration for a suitable protein load and (ii) thechoice of detection method. The total protein content ofurine can be determined by (i) biuret assay [30, 31],(ii) precipitation with TCA-Ponceau S [32], (iii) turbidime-try using tannic acid [33], benzethonium chloride [34] orTCA [35], or (iv) protein dye-binding assays using Coo-massie Brilliant Blue (CBB) [36±38] or Pyrogallol red-molybdate (PRM) [39, 40]. Comparative evaluation hasfailed to identify a reliable method as the assays are

1308 T. Marshall and K. M. Williams Electrophoresis 1999, 20, 1307±1324

prone to interference and give a variable response withdifferent urinary proteins [41±44]. The differential re-sponse of the CBB and PRM protein dye-binding assaysto BJ protein can be expressed as a CBB:PRM proteinassay ratio and exploited as a simple screening test to dif-ferentiate BJP from glomerular proteinuria [45]. Furtherinvestigation has indicated that the CBB:PRM proteinassay ratio does not reliably distinguish BJP from renalfailure (Table 1). However, the existing screening testsbased upon dipsticks (which fail to adequately detect freeLC [46]) or thermal denaturation [17, 47] are likewiseunreliable [4, 48].

Immunochemical methods for quantitative determinationof free LC in BJP [3, 49] use monoclonal antibody (or anti-sera enriched in antibodies specific to free LC) andinclude radioimmunoassay [50], enzyme immunoassay[51±54], automated immunonephelometry [55±58] andimmunoturbidimetric assay [59]. An absolute measure ofthe k or l free (monoclonal) LC concentration is difficult todetermine as the antibody specificity for free monoclonalLC is variable, unpredictable and accentuated by LC poly-mers/fragments with possible cross reactivity with bound(intact immunoglobulin) or free (polyclonal) LC [3, 49, 58,60]. However, the immunochemically determined k/l ratioshows a semi-quantitative correlation with the absoluteamount of free monoclonal LC [50, 51, 58, 61, 62].

2.2 Urinary protein electrophoresis of BJP

UPE for the detection of BJP [63] involves zonal electro-phoresis using cellulose acetate strips [64] or agarosegels [65] followed by protein staining with Amido Black orCBB [3, 4, 48, 66]. The methodology is simple and well-established with commercially available electrophoresiskits, e.g., the Paragon electrophoresis system (BeckmanInstruments, Fullerton, CA, USA) and the Titan Gel sys-tem (Helena Laboratories, Beaumont, TX, USA). BJ pro-tein usually appears as a single and discrete prominentband or sometimes as multiple bands (corresponding tooligomers or rarely a biclonal gammopathy) which can beconfused with b2-microglobulin and a2-microglobulin

when renal damage produces a supplementary tubularproteinuria [3, 4, 48]. The optimal urine sample for detec-tion of BJP by UPE is still a subject of debate [46, 63, 67±69]. Comparison of random, 24 h, and early morning col-lections from 20 patients with BJP resulted in negativedetection of BJP in 6, 3 and 1 of the patients, respectively,but indicated a linear relationship between the proteincontent of the early morning urine and the 24 h samples[46]. Thus, random samples should be avoided [46, 69]and early morning urine can be used as a convenientalternative to 24 h collections, at least for the purpose ofelectrophoresis [68]. The urines should be centrifuged orfiltered to remove particulates [48] and analysed within 2h or stored in the refrigerator at 4oC [70]. In the absenceof an added preservative, urine can be stored at roomtemperature for up to one week for detection of BJP byUPE but this is not to be recommended as bacteriuria willresult in reduced BJ protein levels within 24 h [68, 71].Samples containing < 1 g/L protein are routinely concen-trated 100-fold [46], 300-fold [48] or up to 600-fold [68] byMinicon B15 ultrafiltration (Amicon Division, W. R. Grace& Co., Beverly, MA, USA) but this results in protein loss[72] and may prove particularly troublesome with BJ pro-tein because of its variable tendency to polymerise andaggregate. Ultrasensitive detection methods based upongold staining of cellulose acetate membranes [73] or nitro-cellulose blots of agarose gels [74, 75] avoid the need forsample concentration but are technically demanding andcomplicate the diagnosis as the electrophoresis patternsare dissimilar to those of traditional detection methods.UPE is a useful screening technique but fails to detectlow levels of free LC (particularly in the presence of amasking glomerular type proteinuria [3, 66]) and low mo-lecular weight proteins can be mistakenly detected as freeLC with tubular proteinuria [48]. Thus, BJP detected byUPE should be confirmed by immunoelectrophoresis orimmunofixation electrophroesis [3, 4, 48, 66].

2.3 Immunoelectrophoresis of BJPIE is the traditional method for confirming and typing a BJprotein detected by UPE. Replicate aliquots of the same

Electrophoresis 1999, 20, 1307±1324 Electrophoresis of BJP 1309

Table 1. The CBB:PRM protein assay ratio in BJP and other proteinurias

Patient group CBB values (g/L) CBB:PRM ratio (mean � SD)

BJP (n = 43) 0.07± 6.60 0.24±1.15 (0.59 � 0.17)Pregnancy-induced hypertension (n = 26) 0.03± 4.75 0.33±1.87 (0.86 � 0.33)Renal failure (n = 24) 0.30±10.50 0.46±0.89 (0.67 � 0.11)Diabetes (n = 14) 0.04± 0.55 0.53±1.17 (0.87 � 0.20)Glomerulonephritis (n = 6) 0.60± 7.75 0.60±0.95 (0.82 � 0.12)

The CBB:PRM ratio was calculated from the protein concentration values determined withthe CBB [43] and the PRM [39] protein dye-binding assays. The BJP CBB:PRM ratio wassignificantly different (P < 0.02) from that of glomerulonephritis, diabetes and pregnancy-induced hypertension but not renal failure.

concentrated urine and an appropriate control are electro-phoresed in agarose gels containing preformed troughsto which monospecific antiserum (IgG, IgA, IgM, k or l) issubsequently added prior to a 24±48 h incubation period.Diffusion and equivalence of antigens and antisera resultin precipitin arcs which are detected by protein staining[76]. Precast agarose gels and electrophoresis kits arecommercially available (e.g., the Helena Titan Gel sys-tem) but the resulting IE patterns can be highly complex,requiring considerable skill for interpretation [3, 66] andthe method is being increasingly superseded by immuno-fixation electrophoresis [48].

2.4 Immunofixation electrophoresis of BJP

IFE is the current method of choice for confirming and typ-ing a BJ protein detected by UPE [48]. Replicate aliquotsof the same concentrated urine are electrophoresed inadjacent tracks on cellulose acetate strips [77] or agarosegels [78]. Following electrophoresis, a protein fixative sol-ution is applied to one track (to give a total protein electro-phoresis profile) and a different monospecific antiserum(k, l, IgG, IgA or IgM) is applied to each of the fiveremaining tracks. Interaction of the LC with the appropri-ate antiserum results in precipitation of an LC-antibodycomplex which thereby fixes the LC (upon subsequentwashing to remove nonprecipitated protein) for detectionby protein staining (Fig. 1). IFE is more sensitive thanUPE for detection of BJP as the binding of the antibody tothe free LC amplifies the protein staining [66]. It ispreferred to IE because of its speed, sensitivity and theease of interpretation of the results [3, 48, 66, 78]. How-ever, IFE exploits free and bound LC antisera (free anti-sera is expensive and of low titre) and will not distinguish

free LC comigrating with monoclonal immunoglobulin orfree LC in the presence of high concentrations of poly-clonal LC [3, 79]. The high sensitivity of IFE can result ina ªladderº or ªtiger stripeº pattern associated with protei-nuric polyclonal free LC [80, 81] and can reveal microhe-terogeneity of monoclonal free LC [82]. These problemsare accentuated by excessive concentration of urine [66]and can sometimes be compensated by sample dilution[83]. More recently immunoblotting has been advocatedas a more sensitive method for detection of BJP than IFE(or IE) following UPE [84, 85].

3 High resolution electrophoretic analysisof BJP

High resolution electrophoretic methods including sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), isoelectric focusing (IEF), two-dimensional elec-trophoresis (2-DE) and capillary electrophoresis (CE)have been used for molecular characterisation of BJ pro-tein and the link between free LC and nephrotoxicity(Table 2). These research methods are generally consid-ered to be too technically demanding for routine clinicallaboratory use [1] although CE in combination with immu-nosubtraction is being developed as a rapid automatedalternative to IFE.

3.1 Sodium dodecyl sulfate-polyacrylamide gelelectrophoresis of BJP

SDS-PAGE separates proteins according to relative mo-lecular mass (Mr) by the process of molecular sieving.The proteins should be completely dissociated into theirconstituent polypeptides by treatment with SDS and areducing agent but the latter is usually omitted (ªnonre-ducing conditionsº) for urinalysis in order to retain the sizedistribution profile important for differentiating glomerularfrom tubular proteinuria [1]. BJP has been widely investi-gated by SDS-PAGE (Table 2). The SDS-PAGE patternsare characterised by the detection of prominent bands inthe LC dimer (D) and/or LC monomer (M) positions undernonreducing conditions and LC M and/or LC fragment (F)under reducing conditions (Fig. 2). The Mr positions of theLC components vary from sample to sample and arestrongly influenced by the choice of reducing or nonreduc-ing conditions (Fig. 2, Table 3). However the interpreta-tion of the patterns is complicated by the presence ofother urinary proteins including polyclonal free LCFig. 2).

3.1.1 Early applications

Early applications of SDS-PAGE to BJP validated themethod relative to gel filtration [86] and UPE/IE [87].


Figure 1. IFE of BJP using the Beckman Paragon sys-tem. The diagrammatic representation corresponds to kfree LC in the presence of albumin. Track 1 (UPE) indi-cates the total protein profile whilst tracks 2±6 correspondto immunofixation with the respective antibody. Theanode (+) is to the top of the electrophoresis gel.

SDS-PAGE was recommended in preference to gel filtra-tion (using Sephadex G-75 or G-200) for determination ofthe size distribution profile of urinary proteins as it allowedhigh sample throughput over a wide separation range(Mr 300 000±10 000) using a single gel system [86]. SDS-PAGE, under reducing or nonreducing conditions, of onlya limited number of BJP (n = 4) was sufficient to demon-strate both LC M and LC D and to reveal variations in theirRf values from sample to sample [86]. In a more compre-hensive study of BJP (n = 37), SDS-PAGE revealed thatthe k subtype was predominantly monomeric and the lpredominantly dimeric but there was no clear correlationbetween the LC subtype and associated proteinuria, thelatter tending to be of a nonspecific glomerular type indi-cative of glomerular lesions in addition to the expectedtubular damage [87]. In two patients (both k), the propor-

tion of LC D to LC M increased over a four-month periodof medication (cyclophosphamide) possibly suggestingªescapeº of the neoplastic clone from treatment [87].SDS-PAGE (first dimension) in combination with agarosegel crossed immunoelectrophoresis (second dimension)was subsequently used to investigate BJP (n = 10) toidentify the free LC and determine its molecular weight(Mr 20 000±26 500) and size distribution (seven patientsshowed LC M only; 2, LC M + LC D; and 1, LC D only)[88]. In a methodological development, SDS-PAGE ofBJP (n = 12) was demonstrated on horizontal ultrathin4±25% polyacrylamide gradient gels using the Multiphorelectrophoresis system (Amersham Pharmacia BiotechUK, Little Chalfont, Bucks, UK) ± the free LC and LC frag-ment (Mr 15 000) were detected by silver staining follow-ing electrophoresis of unconcentrated urine and were dis-


Table 2. High-resolution electrophoretic analysis of BJP

Method Year Authors [Reference] System Detection Purpose/Analysis

SDS-PAGE 1972 Pesce et al. [86] Beckmann CBB Validation of method1974 Virella et al. [87] Gel rods Amido Black LC type and form1980 Bradhorst and Wetter [88] Gel rods CBB LC form and Mr

1986 Schiwara et al. [89] Multiphor CBB, Aga) Ag stain and ultrathin gels1987 Walker et al. [91] Gel slabs CBB, Ag, Bb) Glycosylation of LC1988 Jackson et al. [90] PhastSystem Ag Automated analysis1990 Bellotti et al. [29] Gel slabs B LC amyloidogenicity1990 Boege et al. [56] Multiphor Ag Use as screening method1994 Diemert et al. [92] PhastSystem B Predicting nephrotoxicity

IEF 1979 Clyne et al. [106] Columns (Sucrose) A 280 nm Nephrotoxicity in rat1984 Coward et al. [108] Multiphor (P)c) CBB, IFd) LC and renal function1984 Melcion et al. [109] Thin layer (P) CBB, B LC pI and nephrotoxicity1985 Norden et al. [110] Ultrophor (A)e) B IEF versus IFE1986 Cheong et al. [112] PhastSystem (P) CBB Automated analysis1986 Johns et al. [113] Thin layer (P) CBB LC pI and nephrotoxicity1986 Palant et al. [114] Slabs (Sephadex) CBB Glomerulosclerosis1987 Norden et al. [18] Ultrophor (A) B Identification of LC1988 Jackson et al. [90] PhastSystem (P) Ag, B Automated analysis1989 Norden et al. [111] Ultrophor (A) B Renal impairment1990 Bellotti et al. [29] Thin layer (P) B LC amyloidogenicity1990 VØgh et al. [115] Thin layer (A) IF Lymphoproliferation1994 Boege et al. [116] PhastSystem (P) Ag, IF Predicting nephrotoxicity1994 Diemert et al. [92] PhastSystem (P) B Predicting nephrotoxicity

2-DE 1980 Latner et al. [120] Simplified CBB Methodological1980 Tollaksen and Anderson [121] ISO-DALT CBB Methodological1982 Edwards et al. [122] ISO-DALT CBB Methodological1992 Harrison [82] ISO-DALT Ag LC microheterogeneity1993 Harrison et al. [126] ISO-DALT Ag Clonality analysis1995 Tichy et al. [127] PROTEAN II Ag Time course study1995 Stulik et al. [128] PROTEAN II Ag, B IgD myeloma1998 Williams et al. [129] Multiphor CBB, Ag, B LC heterogeneity1998 Marshall and Williams [130] Multiphor CBB, Ag Methodological

CE 1994 Jenkins et al. [131] Applied Biosystems A 200 nm Methodological1997 Jenkins [132] Applied Biosystems A 200 nm Unconcentrated urine1997 Friedberg and Shihabi [133] Beckmann A 214 nm Interference

a) Ag, silver; b) B, blotting; c) P, polyacrylamide; d) IF, immunofixation; e) A, agarose.

tinguished from other microproteins by their yellow-brownstaining properties [89].

3.1.2 Immunoblotting

Early studies demonstrated the clinical potential of SDS-PAGE to detect free LC in unconcentrated urine with high

sample throughput. However, the problems of free LCidentification and technical complexity were only ade-quately addressed by the introduction of immunoblottingand the PhastSystem (Amersham Pharmacia Biotech),an automated electrophoresis system combining SDS-PAGE on precast 8±25% polyacrylamide mini gels (43 ´50 mm) with LC detection (by silver staining) and typing


Figure 2. SDS-PAGE of BJP. The CBB-stained patterns correspond to unconcentrated urine(7.5 mL) from eleven patients (1±11). The samples were prepared for electrophoresis (A) in theabsence of 2-mercaptoethanol (nonreducing conditions) or (B) in its presence (reducing conditions).The protein concentrations as determined by CBB protein assay were as follows: (1) 0.43 g/L,(2) 0.44 g/L, (3) 0.52 g/L, (4) 0.58 g/L, (5) 1.24 g/L, (6) 1.35 g/L, (7) 1.60 g/L, (8) 1.76 g/L, (9) 2.25g/L, (10) 2.68 g/L, and (11) 4.50 g/L. Mr indicates relative molecular mass ´ 10±3. The LC type (asindicated by IFE and immunoblotting) was l (patients 1, 2, 4 and 6) or k (patients 3, 5, 7±11). Abbrevi-ations: alb, albumin; D, LC dimer; M, LC monomer; and F, LC fragment. Note: (i) Both D and M aredetected under nonreducing conditions but their Mr positions vary from sample to sample (A); (ii) theelectrophoretic mobility of the free LC (M) is less under reducing than nonreducing conditions; (iii) LCfragment is only detected in some samples, often only under reducing conditions (B, patient 6);(iv) intact paraprotein (A, patient 7) results in the detection of monoclonal heavy chain under reducingconditions (B, arrowhead patient 7) but bands of similar Mr may be present in its absence (B, arrow-heads patients 6 and 10); and (v) high molecular weight polymers are sometimes detected at highprotein load under nonreducing conditions (A, patient 10).

Table 3. The Mr values of Bence Jones proteins as determined by SDS-PAGE

LC type Nonreducing conditions Reducing conditionsMonomer Dimer Monomer

k 25 800 � 600 51 300 � 2 000 27 000 � 900(n = 37)l 26 700 � 1 400 53 600 � 2 900 29 000 � 2 100(n = 33)

Seventy BJP were analysed by SDS-PAGE, under reducing or nonreducing conditions,using the Multiphor electrophoresis system and homogeneous 12.5% gels. The Mr valuesof the LC monomer were significantly higher (P < 0.001) under reducing conditions ascompared to nonreducing conditions reflecting cleavage of intrachain disulphide bonds.The Mr values of the k LC monomer and dimer were significantly lower (P < 0.002) than therespective l values.

(by immunoblotting) to give results within 2 h [90]. Thismethod is limited by a maximum sample volume of 1 mLwhich necessitates a BJ protein content of 1 g/L for cleardetection of free LC [90]. The versatility of immunoblottinghas been exploited by concanavalin A binding to investi-gate the carbohydrate content of IgG BJ proteins (n = 20)[91]. The BJ proteins were all glycosylated through O-gly-cosidic links to serine or threonine residues [91]. Immuno-blotting, in combination with SDS-PAGE (under reducingconditions), has also been used to correlate the amyloido-genicity of free LC with its size distribution by comparisonof patients (n = 35) with myeloma-associated amyloidosis(A+) with patients (n = 51) without amyloidosis (A±) [29].Amyloidosis was associated with an increased frequencyof the l LC (77% in the A+, 45% in the A±) and the pres-ence of LC fragment Mr 12 000±18 000 (86% in the A+,29% in the A±) [29]. SDS-PAGE has been evaluated, rel-ative to immunonephelometric determination of individualproteins (including the k:l ratio), as a screening techniquefor BJP (n = 40) ± the detection of LC M (Mr 23 000) andLC D (Mr 47 000) proved 100% specific but only 53%sensitive [56]. Problems with SDS-PAGE included (i) diffi-culty in identifying BJP in the presence of an obscuringrenal proteinuria, (ii) an inability to distinguish monoclonaland polyclonal forms of free LC, and (iii) lack of quantita-tion, but an immunospecific detection method was notexploited [56]. More recently, immunoblotting and SDS-PAGE have been combined using the PhastSystem in aninvestigation of BJP (n = 58) to correlate the proportion ofLC M (Mr 22 500) and LC D (Mr 45 000) with nephrotoxic-ity [92]. The free LC was predominantly dimeric in 62% ofthe BJP, and LC fragment (Mr 14 000) detected in 43%,but the LC size distribution profile was not predictive ofthe onset of renal impairment [92]. Thus, the size distribu-tion of free LC, as indicated by SDS-PAGE, appears tocorrelate with amyloidosis [29] but not nephrotoxicity [92].

3.1.3 Advantages and pitfalls

A major advantage of SDS-PAGE is that it exploits proteindenaturation which minimises (nonreducing conditions) oreliminates (reducing conditions) the tendency for BJ pro-tein to polymerise and form protein complexes that resultin multiple anomalous bands upon electrophoresis undernondenaturing conditions. However, standardisation ofsample preparation is of particular importance and it isnecessary to be aware of possible pitfalls: (i) Freezingand thawing of urine differentially precipitates some BJproteins, particularly those containing LC fragment (Fig.3), in a manner analogous to the precipitation of Tamm-Horsfall mucoprotein and a1-acid glycoprotein [93]. Urineshould be centrifuged (to remove particulates) prior tofreezing and thawing (for storage purposes) but not after-

wards [1]. Whilst this is a major potential source of proteinloss with nondenaturing electrophoretic methods (UPE,IE, IFE, IEF), the problem can be avoided with SDS-PAGE as the protein suspension disperses upon samplepreparation in the SDS buffer. (ii) The choice of samplepreparation method [86, 90, 94±99] influences the SDS-PAGE pattern of urine [1] and this includes BJP (Fig. 4).Failure to adequately saturate the proteins with SDSresults in polymers and aggregates (under nonreducingconditions) whilst treatment with iodoacetamide [96, 97]can induce the appearance of anomalous protein bands(Fig. 4). (iii) Sample concentration by lyophilisation orultrafiltration undoubtedly leads to protein loss [1]. Urinaryprotein concentration by dye precipitation [100±102] givesexcellent protein recovery without modification of the uri-nary protein profile when applied to BJP (Fig. 5). (iv) Sil-ver staining gives highly complex patterns when applied


Figure 3. The precipitation of BJ proteins upon freezingand thawing as demonstrated by SDS-PAGE underreducing conditions and CBB staining. One mL aliquots ofurine from four patients (P1±P4) were frozen (±70oC) andsubsequently thawed at room temperature. The urine waswhirlimixed and a sample of the suspension (track 1)compared, at equal sample volume, with the respectivesupernate (track 2) and pellet redissolved in 1 mL of SDSsample denaturing buffer (track 3) following centrifugationat 10 000 ´ g for 2 min. Mr indicates relative molecularmass ´ 10±3. Abbreviations: alb, albumin; M, LC mono-mer; and F, LC fragment. The protein concentrationsdetermined by CBB protein assay were: (P1) 4.5 g/L, (P2)2.25 g/L, (P3) 0.62 g/L, and (P4) 0.24 g/L. Note: (i) Tracks(2) and (3) reflect the proportion of soluble to insoluble BJprotein as compared to the combined amount in the urinesuspension (track 1); (ii) freezing and thawing can resultin the precipitation of BJ protein (in a relatively pure form);and (iii) precipitation is predominantly associated withsamples containing LC fragment.

to BJP (Fig. 5) but eliminates the need for sample con-centration (when the protein concentation is < 0.5 g/L).(v) The technical demands are minimised by commercialelectrophoresis systems which combine simplicity andreproducibility by use of precast polyacrylamide gels, gelbuffer strips and dedicated staining methods (Fig. 6).

3.2 Isoelectric focusing of BJP

IEF separates proteins according to isoelectric point (pI)and is usually performed in agarose or polyacrylamidegels. The proteins may be separated in their native state(nondenaturing conditions) or pretreated with reducingagents and urea (in the absence of SDS) for separation ofpolypeptide constituents (denaturing conditions). In thelatter case, the IEF gels usually contain 8 M urea. IEF isnot ideally suited for complex protein mixtures as each

individual protein can give multiple bands due to chargemicroheterogeneity and this results in highly complexbanding patterns that are difficult to interpret. IEF has


Figure 4. The effect of sample preparation upon SDS-PAGE of BJP. Two BJ proteinuric urines (1, 2) were pre-pared for electrophoresis as follows: (A) ªuntreatedº (w/oSDS, w/o heat) [90]; (B) plus SDS (f.c. 1%), w/o heat(room temperature, 15 min) [86]; (C) plus SDS, plus heat(37oC, 1 h) [94]; (D) plus SDS, plus heat (95oC, 5 min)[95]; (E) plus SDS, plus iodoacetamide (f.c. 0.1 mol/L),plus heat (95oC, 5 min) [96, 97]; or (F) plus SDS, plusurea (f.c. 8 M), plus heat (95oC, 5 min) [98, 99]. The dena-tured urines were compared at equal sample volume. Mr

indicates relative molecular mass ´ 10±3. Abbreviations:alb, albumin; D, LC dimer; and M, LC monomer. Note:(i) Preparation (D) is our standard recommended method;(ii) in comparison, preparations (C) and (F) give similarresults but (C) is time-consuming and (F) involves theaddition of solid urea which dilutes the sample, therebyreducing the protein load; and (iii) in contrast, prepara-tions (A) and (B) fail to fully dissociate protein complexesand preparation (E) induces additional protein bands(arrowheads, E track 1).

Figure 5. The concentration of BJ proteins by dye pre-cipitation with CBB. BJ proteinuric urine from six patients(1±6) was analysed by SDS-PAGE (nonreducing condi-tions) without concentration (±C) or after fivefold concen-tration (+C) [100±102]. The SDS-PAGE patterns (lowerhalf magnified to enhance detail) were detected by (A)CBB staining or (B) silver staining. Abbreviations: alb,albumin; D, LC dimer; and M, LC monomer. The proteinconcentrations as determined by CBB protein assay wereas follows: (1) 0.16 g/L, (2) 0.22 g/L, (3) 0.22 g/L, (4) 0.22g/L, (5) 0.33 g/L, and (6) 0.33 g/L. Note: (i) comparison of(+C) and (±C) at equal sample volumes (10 mL) with CBBstaining (A) demonstrates concentration of the BJ protein;(ii) redilution of (+C) (1 in 5) prior to comparison with anequal volume of ±C (: 0.25 mg protein) and silver stain-ing (B) indicates excellent protein recovery following dyeprecipitation; and (iii) sample concentration is unneces-sary with silver staining but the resulting patterns arecomplex (B) and more difficult to interpret than the re-spective CBB-stained patterns (A).

been widely applied to BJP (Table 2) because nephrotox-icity has been associated with free LC [103±105] and inparticular its pI [106±108].

Early application of IEF to BJP involved pI determinationof dimeric BJ protein (n = 12, pI 5.2±6.6) isolated by gelfiltration and tested for nephrotoxicity by intraperitonealinjection into aciduric, female Sprague Dawley rats [106].BJ proteins of higher pI, predominantly l and associatedwith patients with impaired renal function, showed greaterevidence of nephrotoxicity ± it was concluded that BJ pro-teins with pI values greater than the pH of the urine wereacutely nephrotoxic [106], perhaps due to an electrostaticinteraction with Tamm-Horsfall mucoprotein [107]. Thisclaim was supported by a clinical study which demon-strated a significant negative correlation between creati-nine excretion and the isoelectric point (pI 4.5±8.2) of BJ

proteins (n = 23) isolated from agarose UPE gels [108].However, there was no significant difference between theapparent nephrotoxicity of k and l subtypes [108].

3.2.1 Immunoblotting

The introduction of immunoblotting (with immunoenzy-matic detection) allowed BJ protein to be specificallydetected in whole urine [109]. Immunoblotting revealed aspectrum of charge microheterogeneity with individual BJproteins (n = 17) ± there was no simple correlation be-tween isoelectric point (pI 5.2±8.0) and nephrotoxicity butsubclassification of the patients indicated a high pI inassociation with chronic irreversible renal failure [109].IEF, in combination with immunoblotting, has beenfavourably evaluated relative to IFE for detection of BJP(n = 164) as it eliminates protein loss (due to adsorption


Figure 6. SDS-PAGE of BJP with the Multiphor electrophoresis system. The unconcentrated urinefrom ten patients (1±10) was prepared for electrophoresis in the absence of 2-mercaptoethanol (A, C;nonreducing conditions) or in its presence (B, D; reducing conditions). The samples were comparedat equal protein loads and detected by CBB staining (A, B; 5 mg protein) or silver staining (C, D; 0.25mg protein). Mr indicates relative molecular mass ´ 10±3. Abbreviations: alb, albumin; D, LC dimer;and M, LC monomer. Note: (i) Commercial precast gels (and gel buffer strips) give highly reproduci-ble results; and (ii) the dedicated PlusOne silver stain enhances detection sensitivity at least 20-fold(A, C) but the patterns can be obscured by vertical streaks following SDS-PAGE under reducing con-ditions (D).

of free LC on ultrafiltration cell membranes) during sam-ple concentration but the LC patterns are complex andrequire cautious interpretation [110, 18]. Thus, in compre-hensive studies of BJP, combining IEF and immunoblot-ting, the free LCs gave multiple prominent bands (attrib-uted to polymerisation and in vivo fragmentation) withbimodal distribution (pI 6±6.05 or 7±7.95) [110] whichwere superimposed upon (i) a normal/tubular proteinuriatype LC pattern, characterised by three broad bands pI7.1±7.3 (k & l), pI 7.8±8.0 (k) and pI 8.3±8.5 (k), (ii) threeor more evenly spaced bands of intact monoclonal immu-noglobulin, and (iii) a diffuse zone (pI 6.0±8.5) of intactpolyclonal immunoglobulin [18]. Correlation of the IEFpatterns with creatinine clearance and urinary excretionof a1-microglobulin and b2-microglobulin indicated thatthe pI of the free LC was a poor index of renal impairment[111]. Application of the PhastSystem demonstrated auto-mated IEF of BJP on pH 3±9 micro-slab PhastGels incombination with CBB staining [112] or silver staining andimmunoblotting [90] but sample volume (1 mL) remained alimiting factor.

3.2.2 Nephrotoxicity

The correlation between pI and nephrotoxicity was furtherinvestigated in an IEF study of 43 LC isolates [113].Approximately 50% of the BJ proteins showed microhet-erogeneity of pI (2±4 bands covering 0.5±1.5 pH units)but there was no obvious association between the iso-electric points (pI 3.5±9.5; 53% > pI 7.0), degree of sialy-lation (present in 62%) or the polymerisation state of theBJ protein and renal failure [113]. Furthermore, there wasno correlation between the pI of the BJ protein and thereversibility of renal failure although proximal tubular dys-function was more commonly associated with basic thanacidic free LC [113]. In contrast, advanced nodular glo-merulosclerosis in MM was associated with a BJ proteinof unusually high isoelectric point (pI 8.4) suggesting thatthe interaction between basic BJ protein and glomerularpolyanion results in irreversible renal damage [114].

The amyloidogenicity of the free LC has been correlatedwith pI, using immunoblotting and IEF under denaturingconditions, by comparison of patients with or without mye-loma-associated amyloidosis (A+, n = 35; A±, n = 51; re-spectively) [29]. Amyloidosis was associated with an in-creased frequency of the l LC (77% in the A+, 45% in theA±), the presence of LC fragment Mr 12 000±18 000(86% in the A+, 29% in the A±) and a significantly lower(P = < 0.001) isoelectric point of the free LC (pI 4.8 � 1.1in the A+, pI 6.2 1.6 in the A±); the latter may contributeto binding to the calcium-P-component characteristic ofamyloid deposits [29]. The combination of IEF and immu-nofixation has been clinically evaluated for detection of

BJP in a study of 637 patients with true or suspected lym-phoproliferative diseases [115]. The method confirmedmicroheterogeneity of the free LC and gave a higher rateof detection of BJP than conventional electrophoreticmethods with a tenfold increase in sensitivity (0.02 g/Lfree LC). More recently, the correlation between free LCand nephrotoxicity has been further investigated by IEFand titration curve analysis using the PhastSystem [92,116]. Multiple LC bands (distributed over more than onepH unit) were detected in the majority of patients with BJP(n = 64) and similar results were observed under proteindenaturing conditions to eliminate polymerisation [116].BJ proteins were characterised by a bimodal distributionof pI (pH 4.5±6.5 in 55% of patients, n = 51; pH 6.5±8.0 in45% patients, n = 41) but there was no predictive correla-tion between pI and renal impairment [92]. Titration curveanalysis revealed three types of pH-dependent LC mobil-ity, of which Type I (strongly ionic) carried the greatestrisk of renal failure and was characterised by a steeplytransgressed pI at pH < 5 [116].

Thus, early indications of a possible correlation betweenthe pI of free LC [106, 108] and its nephrotoxicity have notbeen substantiated [92, 111, 113]. Animal experimentsinvolving the infusion of isolated human BJ protein [117,118] or the transplantation of immunoglobulin-secretingtumours [119] into rats indicated an effect of BJ proteinupon glomerular filtration rate [117], inulin clearance[118], and renal histology [119] but did not support theconcept that cationic BJ proteins are particularly nephro-toxic [117, 119]. It has been suggested that pI in itself isnot the important factor but rather the net surface chargeprevailing under physiological conditions in the microen-vironment associated with the site of toxicity [92, 111,116].

3.2.3 Limitations

Whilst IEF has been widely applied to BJP in studies ofnephrotoxicity the limitations of the method have not beenadequately acknowledged. Highly basic BJ protein maynot be detected upon IEF in conventional carrier ampho-lytes and this problem can only be adequately addressedby recourse to immobilised pH gradients (IPGs). IEF ofthe native BJ protein under nondenaturing conditions maydetect LC oligomers (and complexes with other proteinssuch as albumin) rather than revealing a true spectrum ofthe pI isoforms (Fig. 7). Surprisingly few IEF studies ofBJP have avoided this problem by the use of proteindenaturing conditions. In addition, the efficacy of detec-tion of basic LC by immunoblotting has not been ade-quately evaluated ± there may be charge-related prob-lems associated with transfer, binding and visualisation ofbasic LC.


3.3 Two-dimensional electrophoresis of BJP

Early 2-DE methods combined IEF (first dimension) withgradipore electrophoresis (second dimension) for two-dimensional separation of urinary proteins under nonde-naturing conditions. High resolution 2-DE combines IEF(in 8 M urea) with SDS-PAGE after sample pretreatmentwith protein denaturing reagents. Alternative 2-DE tech-niques combine other 1-DE methods under a range ofprotein denaturing conditions but high resolution 2-DEhas been most widely applied to urine and BJP (Table 2).The advantage of 2-DE over 1-DE (UPE, SDS-PAGE,IEF) is that it exploits two independent electrophoreticparameters (pI, Mr) to resolve the free LC into polypeptidespots which can be identified by immunoblotting (Figs. 8and 9).

3.3.1 Early applications

Early applications of 2-DE to urinary proteins were of ageneral methodological nature but included analysis of alimited number of BJP to demonstrate (i) coelectrophore-sis of serum and urinary free LC [120], (ii) charge hetero-geneity of the free LC [120±122], and (iii) a higher Mr of l-relative to k-free LC [122]. The latter was initially consid-ered sufficient to distinguish and thereby type the free LC[122] but subsequent studies indicated that this could notbe done with an acceptable level of reliability [123]. Thedetection of multiple LC spots upon 2-DE of BJP was ini-tially thought to question the concept of monoclonality[121] but the phenomenon appears to reflect chargemicroheterogeneity of paraproteins [120, 123, 124]. 2-DEis ideal for investigations of microheterogeneity as thepolypeptide isoforms are detected as individual spots.However, with respect to free LC, it is important to distin-guish apparent microheterogeneity (oligomeric forms,protein complexes) from true microheterogeneity (chargevariants of an individual polypeptide). Thus, the sensitivityof IEF reveals ªpseudo-oligoclonalº bands (the ªladderº orªtiger stripeº pattern) characterised by multiple (3±7bands of normal polyclonal-free LC (predominantly k LC)[125]. The phenomenon has been investigated by 2-DEwhich ªreveals a regularly spaced, isomassic, restrictedzonal distribution in the light chain regionº [80] or ªdistinctspots, each representing a group of molecules of similarisoelectric pointº [120] and has been attributed to comi-gration of charge-related superfamilies of normal poly-clonal LC produced by the limitation in amino acid substi-tutions imposed by recombination constraints on the LCgenes [80]. By our definition, this is not a true microheter-ogeneity. Likewise, multiple bands of BJ protein detectedupon UPE may not reflect true microheterogeneity asdouble LC bands generate a single anodal band afterDTT treatment (suggesting aggregation) and the exciseddouble bands each show the same pattern of chargemicroheterogeneity upon 2-DE [82, 126]. The molecularbasis of the latter remains unclear but it appears to be acommon feature of BJ proteins when analysed by 2-DEunder reducing [82, 120±122, 126±130] or nonreducingconditions [130]. Carbamylation studies suggest a singlecharge difference between adjacent isoforms [82, 126].Size microheterogeneity of BJ protein equivalent to 700±1300 Da has also been reported following 2-DE [82] andappears to be a distinct phenomenon unrelated to free LCfragments.

3.3.2 Recent applications

Recent applications of 2-DE to BJP have incorporatedIPGs and immunoblotting in small-scale studies of multi-ple myeloma (n = 2) [127] and IgD myeloma (n = 1) [128]


Figure 7. IEF of BJP with the Multiphor electrophoresissystem. The unconcentrated urine from five patients (1±5), containing essentially only BJ protein and a trace ofalbumin, was incubated at 37oC for 2 h in the absence ofdithiothreitol (±DTT) or in its presence (+DTT) [82]. Thesamples were loaded (10 mg protein) at either the cathode(LHS) or the anode (RHS) of an Ampholine PAGplate, pH3.5±9.5, and focused for 1.5 h at 1500 V, 50 mA and30 W prior to protein detection by CBB staining. The pHgradient was calibrated (pI) using IEF-Mix 3.5±9.3 (M,Sigma) and the albumin position determined using puri-fied human serum albumin (alb; Sigma). Note: (i) The BJproteins give pI spectra which vary from sample to sam-ple and are characterised by two or more bands of acidic(1, 3), neutral (2) or basic (4, 5) pI which may be closelystacked (1, 3), laddered (4, 5) or more widely spread (2);(ii) incubation with DTT (to promote protein dissociation)may have little effect (2) or reduce the number of promi-nent bands (1, 3, 5) or induce a cathodal shift (4) some-times with the appearance of additional bands (3); and(iii) the pattern of the IEF calibration proteins (M) is unaf-fected by the loading position (anode or cathode) butacidic BJ proteins are better loaded at the cathode (toavoid a ªbowingº effect) whilst basic BJ proteins are betterresolved when loaded at the anode.


Figure 8. 2-DE and immunoblotting of BJP at high protein load. The urinary protein (100 mg) fromtwo patients was analysed using the simplified method of 2-DE [129] and the resulting patternsdetected by CBB staining (A, B). Briefly, IEF (first dimension) in 5% w/v polyacrylamide gel rods con-taining 9 M urea, 0.5% Nonidet P-40 and 2% carrier ampholytes (Pharmalyte pH 2.5±5.0, Ampholine


pH 5.0±7.0, Pharmalyte 2-D pH 3±10; 2:3:6 v/v/v) was followed by SDS-PAGE (second dimension) in 6±20% w/v polyacryl-amide linear gradient gels. Duplicate gels were replicate blotted onto Immobilon-P polyvinylidinedifluoride (PVDF) mem-branes (Millipore) for immunodetection (C, D) using peroxidase and diaminobenzide as previously described [129]. Theresidual nonblotted protein was subsequently detected by CBB staining of the 2-DE gels (E, F). Mr indicates relative molec-ular mass ´ 10±3. Abbreviations; alb, albumin; L, light chain; and F, light chain fragment. Note: (i) The presence of multipleabnormal spots (A, B) corresponds to the pI isoforms of the free light chain (L) and its fragments (F); (ii) immunoblottingconfirms the identity of the free light chain spots (C, D; respectively); (iii) replicate blotting allows typing of the free lightchain (C = l; D = k) but leaves sufficient protein for determination of the urinary protein profile by CBB staining (E, F; re-spectively); and (iv) there was little cross-reactivity between the two primary antibodies, i.e. anti-k (C) or anti-l (D) revealedonly trace amounts of polyclonal free light chain (results not shown).

Figure 9. 2-DE and immunoblotting of BJP at low protein load. The urinary protein (2.5 mg) from twopatients (A, C and B, D) was analysed using the simplified method fo 2-DE [129] and the residualnonblotted protein was detected by silver staining (A, B) after replicate blotting for immunodetection(C, D). Mr indicates relative molecular mass ´ 10±3. Abbreviations: alb, albumin; L, light chain; and F,light chain fragment. Note: (i) 2-DE in combination with (A, B) silver staining and (C, D) immunoblot-ting allows detection and typing of free light chain at urinary protein levels within the normal range(0.025 g/L); (ii) replicate blotting, even at these low protein levels, leaves sufficient protein for determi-nation of the urinary protein profile by silver staining (A, B); and (iii) immunoblotting is equally effectivewith acidic (A, C) and basic (B, D) free light chain.

with detection of multiple free LC fragments [128] andadditional acidic isoforms (pI < 4.95) of potential prognos-tic value [127]. We have combined 2-DE and immunoblot-ting to demonstrate complex patterns of LC charge andsize heterogeneity in BJP (n = 30) and compared conven-tional carrier ampholytes with IPGs, the latter beingpreferred to nonequilibrium pH gradient electrophoresis(NEPHGE) for detection of basic free LC (Fig. 10) [129,130]. In this sample population, 15 (50%) showed chargemicroheterogeneity alone and a further 10 (33%) showedboth charge microheterogeneity and the presence of frag-ments [129]. There was no correlation between LC typeand the degree of charge microheterogeneity but sevenof the 10 BJP with LC fragments were of the l LC type[129].

Whilst 2-DE is a technically demanding method clearlyunsuited for routine clinical laboratory use, it provides anexcellent research tool for characterisation of the micro-heterogeneity of BJ proteins. The indication that doubleBJ bands detected by UPE reflect aggregation rather thangenuine LC charge variants [82, 126] suggests that 2-DEshould likewise be used to characterise the multiplebands detected upon IEF of BJP under nondenaturingconditions (Fig. 7), i.e. the observed pI spectra may notreflect the true pI range of the BJ protein. The detection ofBJ protein aggregates (Mr 50 000±900 000) upon poly-acrylamide gel electrophoresis under nondenaturing con-ditions [92] and the effect of DTT upon the pI spectrum ofBJ proteins (Fig. 7) are consistent with this possibility.

3.4 Capillary electrophoresis of BJP

CE is essentially electrophoresis in HPLC mode usingnarrow-bore fused silica capillaries (25±50 mm) to mini-mise convection and enhance heat dissipation upon rapidhigh voltage separation and detection (A 220±214 nm) ofnanolitre volumes of injected sample. Capillary zone elec-trophoresis (CZE) occurs in free solution and exploits theelectroosmotic flow (EOF) induced by the negativecharge of the fused silica. Solvent flows towards the cath-ode but anions (negatively charged) are electricallyattracted to the anode (positively charged) and progres-sively retarded in accordance with their charge:massratio, achieving a high resolution separation analogous toUPE on cellulose acetate or agarose gel. Capillary gelelectrophoresis (CGE) is analogous to PAGE or SDS-PAGE upon inclusion of SDS. The inner surface of thecapillary is coated with a polymer which prevents EOFand provides a network for separation according to mo-lecular size, i.e. in the absence of EOF, anions are sub-ject to molecular sieving as they migrate toward theanode. Capillary isoelectric focusing (CIEF) also uses pol-ymer-coated capillaries (to eliminate EOF) to achieverapid separation (< 10 min) according to pI.

To our knowledge, the application of CE to the investiga-tion of BJP is limited to the use of CZE (Table 2). Themethod is rapid and can be automated with high samplethroughput. Consequently, it has the potential to replaceUPE in the routine clinical laboratory [131]. The CE migra-


Figure 10. 2-DE of BJP with immobilised pH gradients. The urinary protein (30 mg) was analysedusing the Multiphor/Immobiline 2-DE electrophoresis system. Briefly, the rehydrated Immobiline pH3±10 (A) or pH 4±7 (B) drystrips (first dimension) were transferred to an ExcelGel XL SDS 8±18 forhorizontal SDS-PAGE (second dimension) with SDS gel buffer strips and the resulting protein pat-terns detected by CBB staining. Mr indicates relative molecular mass ´ 10±3. Abbreviations: alb, albu-min; L, light chain; and F, light chain fragment. Note: (i) The combination of immobilised pH gradientsand precast gels and buffer strips gives highly reproducible results; and (ii) the use of narrow pHranges greatly enhances resolution to confirm charge heterogeneity of the free light chain and itsfragments.

tion times of urinary proteins (albumin, prealbumin, a1-acid glycoprotein, transferrin, b2-microglobulin, Tamm-Horsfall mucoprotein and free LC) have been determinedand a good correlation reported between CE and UPE fordetermination of albumin (r = 0.93) and BJ protein (r =0.95) [131]. The migration time of BJ protein was variable(8.2±9.2 min) [131]. CZE has been recommended for thedetection of BJ protein (down to 0.004 g/L), as a single ordouble peak (in the absence or presence of intact IgG), inunconcentrated urine containing 0.04±9.70 g/L total pro-tein [132]. However, sample pretreatment by ethanol pre-cipitation or Minicon ultrafiltration (with double recycling)is essential to eliminate interference when analysing BJP(n = 8) by CZE with UV detection methods [133].

Thus, the potential advantages of CZE (rapid and auto-mated separation) may be outweighed by the disadvan-tage of sample processing. Furthermore, CZE posesproblems with the identification and typing of the free LCas detection is nonspecifically based upon UV absorption.The recent application of immunosubtraction capillaryelectrophoresis (IS-CE) to serum paraproteins [134, 135]demonstrates a means of overcoming this problem. IS-CE involves analysis of the sample with or without prein-cubation with antibody-coated Sepharose beads specificto either IgG, IgA, IgM, l LC, or k LC in order to type thefree LC by deletion of the corresponding paraprotein peak[134, 135]. Thus, in summary, CE has enormous clinicalpotential but it is currently less sensitive and flexible thantraditional electrophoretic methods incorporating IFE[136].

4 Concluding remarksThe electrophoretic analysis of BJP by UPE, IFE, IEF andCE has been carried out predominantly under nondena-turing ªnative proteinº conditions. This is potentially prob-lematic as the free LC tends to aggregate, particularly fol-lowing sample concentration, storage and processing.High resolution electrophoretic methods using proteindenaturing conditions would eliminate this problem andthe improved sensitivity would avoid the need for sampleconcentration. SDS-PAGE is such a method but is gener-ally considered to be too technically demanding for rou-tine clinical laboratory use. Automated electrophoreticsystems (e.g. the PhastSystem) simplify SDS-PAGE buttend to be expensive. To minimise cost, we have sug-gested that existing UPE/IFE systems could be adaptedfor SDS-PAGE [1]; note an analogous method, SDS-agarose gel electrophoresis (SDS-AGE), has recentlybeen described for analysis of BJP based upon theHydrasis Hydragel (Sebia) automated UPE system [137].In a similar adaptation of existing methodology, UPE oncellulose acetate has been combined with SDS-PAGE inthe PhastSystem for 2-DE of BJP [138].

IEF is unsuitable for routine clinical laboratory analysis ofBJP as the banding patterns are highly complex and diffi-cult to interpret. However, the nephrotoxic effect of pro-tein [139, 140] and the involvement of free LC in the de-velopment of renal failure [141, 142] warrant furtherapplication of IEF but using IPGs and protein denaturingconditions to detect highly basic LC and overcome theproblem of aggregates. SDS-PAGE may also prove val-uable in investigations of nephrotoxicity as it detectsminor variations in the Mr of BJ proteins and these mayindicate differential cleavage of intra-chain disulphidebonds reflecting subtle conformational changes in proteinstructure. Such changes could predispose the BJ proteinto limited proteolysis, resulting in aggregation and LCdeposition in amyloidosis and cast nephropathy.

2-DE is undoubtedly the optimal research tool for physico-chemical characterisation of BJ protein as it clearlyresolves the free LC and its fragments into discrete spotsunder protein denaturing conditions. We are currentlyusing 2-DE to investigate the molecular basis of thecharge microheterogeneity of BJ protein and the genera-tion of free LC fragments by limited proteolysis. Suchchanges may occur spontaneously in an autocatalyticmanner as BJ protein has enzymic properties demon-strating (i) peptide-hydrolysing activity with synthetic sub-strates [143, 144], vasoactive intestinal peptide [144] andvasopressin [145], and (ii) DNA-hydrolysing activity [146].It may be possible to investigate these activities by 2-DEusing established methods for the detection of enzymesfollowing gel electrophoresis [147, 148]. 2-DE and otherhigh resolution electrophoretic methods are also beingused increasingly to detect and characterise the carbohy-drate constituents of glycoproteins by the use of lectinprobes following immunoblotting. This may be of particu-lar interest in studies of nephrotoxicity as BJ proteins areglycosylated [91, 149±152] and differential glycosylationis of pathophysiological significance [153] in dictating tis-sue uptake of free LC [154] and fibrillogenesis [155].

Ultimately, 2-DE could be combined with blotting andmass spectrometry (microsequencing mass profiling) in aproteomic type of analysis [156] to generate computer-ised databases which would be accessible on the Internetand available for prognostic purposes, i.e. to compare thephysicochemical characteristics of a BJ protein with thoseof existing BJ proteins catalogued in conjunction with clin-ical data for use as an index of nephrotoxicity.

The authors wish to thank N. A. Abbott for assistance withimmunoblotting.

Received November 17, 1998


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