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HEMATOPATHOLOGY Original Article Production and Immunodiagnostic Applications of Antihuman Light Chain Monoclonal Antibodies MASAHIRO ABE, M D , * f TETSUYA GOTO, M D , * f STEPHEN J. KENNEL, P H D , * DENNIS WOLFENBARGER, MT,* SALLIE D. MACY, MT,* DEBORAH T. WEISS, BS,* AND ALAN SOLOMON, MD*,§ cell and surface Ig of B-lymphocyte populations, respectively. These antibodies facilitated the immunohistochemical detection and characterization of light-chain-associatcd amyloid (AL amyloid) and other types of light-chain-related tissue deposits. Furthermore, the unti-C, -specific MoAbs were used to measure serum and urinary Igx and IgX concentrations. Quantification of Bence Jones protein excretion, even in the presence of other urinary proteins, was possible using the highly sensitive anti-C„ and anti-Cx MoAbs reactive only with free light chains. The ability to identify and characterize, through the use of these antihuman light chain MoAbs, light-chain-related epitopes at the protein, cellular, and tissue level has clinical importance in the diagnosis and treatment of patients with monoclonal plasma cell and related B-cell immunoproliferative diseases.(Key words: Monoclonal antibodies; Light chains; Bence Jones proteins; Multiple myeloma; AL amyloidosis; Plasma cells; B cells) Am J Clin Pathol 1993; 100:67-74. Hybridomas producing antihuman light chain monoclonal antibodies (MoAbs) were derived from fusion of SP2/0 mouse myeloma cells with splenic lymphocytes from mice repeatedly immunized with purified Kand X-type Bence Jones proteins representative of the major V, (V,„ V.M, V„m, V„IV) and Vx (VM, Vxll/V, Vxll„ Vxlv, Vxv,) subgroups or gene families. Monoclonal antibodies were obtained that had specificity for constant-region (C,) determinants common to all K or X light chains (C, and Cx, respectively) as well as for variable-region (V,) epitopes unique to each of the V, or Vx subgroups. The capability of these reagents to recognize C, and V, determinants on monoclonal immunoglobulin (Ig) molecules was demonstrated in fluid-phase antigen-capturing enzymelinked immunosorbent assay (ELISA), solid-phase ELISA, and immunoblotting. In addition, these antilight chain MoAbs were used to establish immunocytochemically the K or X type and V, -subgroup nature of light chains expressed by the cytoplasmic Ig of monoclonal plasma resentative of the major V,. and Vx gene families.15 The results of these serologic analyses have been confirmed from those obtained from amino acid and nucleotide sequence data, and reveal that the classification of light chains has considerable biologic and clinical importance. Our studies with these polyclonal antisera and those of other investigators who have prepared antilight chain monoclonal antibodies (MoAbs) have demonstrated that certain disease processes are associated with particular VL subgroups or epitopes.6"9 The usefulness of these reagents for immunodiagnosis, however, has been precluded by their limited availability. To address this limitation and as part of a longstanding effort to elucidate the immunochemical properties of human light chains, we have prepared a large panel of MoAbs that recognize structurally distinct CL- and VL-associated light chain epitopes expressed by secreted forms of monoclonal Ig molecules and by monoclonal plasma cells and B lymphocytes. These reagents From the * Human Immunology & Cancer Program. Department of have been used in serologic, immunocytochemical, and immuMedicine. University of Tennessee Medical Center/Graduate School of nohistochemical analyses to identify and characterize monocloMedicine. Knoxville. Tennessee, the tFirst Department of Medicine. nal Igs, cell populations, and light chain-related tissue deposits. University of Tokushima. Tokushima. Japan, and the ^Biology Division, Oak Ridge National Laboratory, Oak Ridge. Tennessee. MATERIALS A N D METHODS Received April 7, 1992; accepted for publication May 22, 1992. Proteins Supported by Public Health Research Grant CA 10056 from the National Cancer Institute and an Ina M. Barger Memorial grant for Monoclonal urinary light chains and serum Igs, ie, K and X cancer research (IM-430) from the American Cancer Society. Bence Jones proteins, IgG and IgA myeloma proteins, and IgM § American Cancer Society (Clinical) Research Professor. macroglobulins were obtained from patients with multiple myAddress reprint requests to Dr. Solomon: University of Tennessee eloma, Waldenstrom's macroglobulinemia, chronic lymphoMedical Center, 1924 Alcoa Highway, Knoxville, Tennessee 37920. Monoclonal immunoglobulin (Ig) light chains represent a unique biomarker of malignant plasma cell and other lymphocyte-related immunoproliferative disorders, including lightchain-associated amyloidosis (AL amyloidosis) and have long been implicated in many of the pathophysiologic manifestations of these diseases.1,2 The detection and characterization of these components therefore have important diagnostic, therapeutic, and prognostic implications. We have employed serologic methods to classify light chains at the protein and cellular levels using polyclonal antihuman light chain antisera prepared by immunizing rabbits with selected human monoclonal light chains, ie, Bence Jones proteins. 34 Through the use of these reagents, it has been possible to identify readily not only the two constant-region (CL) light chain isotypes, K and X, but also the variable-region (VL) subgroups and subsubgroups rep- 67 68 HEMATOPATHOLOGY Original Article cytic leukemia (CLL), or AL amyloidosis. The proteins were isolated by zone (block) electrophoresis and purified by gel filtration as previously described.3 The purity of the proteins was determined by electrophoresis in agarose and polyacrylamide gels; the light chain type and heavy chain class were established by immunofixation electrophoresis (Paragon® Electrophoresis System, Beckman, Brea, CA) or by immunoelectrophoresis. The light chain VL-subgroup nature of K and X monoclonal Igs was determined serologically using rabbit anti-V, and anti-Vx subgroup-specific antisera, respectively.3 Normal (polyclonal) human light chains were prepared from pooled serum IgG3 (Cohn FRII, Sigma Chemical, St. Louis, MO), normal (polyclonal) IgG, IgA, and IgM proteins were obtained from Sigma Chemical, and pooled normal human serum was obtained from Chemicon (Temecula, CA). Antibody Preparation Monoclonal antibodies were prepared using heat-precipitated Bence Jones proteins representative of the four V, (V,,, V«„, V,M1> V,1V) and five Vx (Vx„ Vx]l/V, Vxl„, V xlv , V xvl ) subgroups.8 Five mice were immunized with each protein: 6- to 8-week-old pathogen-free, female Balb/c mice were injected intraperitoneally (IP) every 2 to 4 weeks with 100 n% of the immunogen emulsified in 0.2 mL of either complete Freund's adjuvant or a mixture containing 50 fig of monophosphoryl lipid A and 50 /ig of trehalose dicorynomycolate (RIBI ImmunoChem Research, Hamilton, MT). Ten days after injection, serum antilight chain antibody titers were determined in a fluid-phase antigen-capturing enzyme-linked immunosorbent assay (ELISA) on specimens of blood obtained by retroorbital bleeding. Three days before fusion, mice were injected intrasplenically with 100 j*g of protein suspended in 0.1 mL of phosphate-buffered saline. Hybridomas were generated from the fusion of 1.6 X 108 immune spleen cells with 2.4 X 10s SP2/0 mouse myeloma cells in the presence of polyethylene glycol according to the method of de St. Groth and co-workers.10 Hybridoma culture supernatants were screened for MoAb production in fluid-phase antigen-capturing ELISA using biotinylated Bence Jones proteins. Based on these results, colonies were selected, expanded, and recloned at least twice by limited dilution. Hybridomas were grown in pristane-primed Balb/c mice and the MoAbs isolated from ascitic fluid by ammonium sulfate precipitation and ion-exchange chromatography." The MoAb heavy chain isotype was identified by ELISA with subclass-specific rabbit-antimouse Igs (Mono AB-ID/EIA kit, Zymed Laboratories, San Francisco, CA). The association constants of the MoAbs were determined from Scatchard plots using nonlinear regression analyses of data obtained by immunoprecipitation of radioiodinated Bence Jones proteins.12 Enzyme-Linked Immunosorbent Assay Procedures To determine the mouse polyclonal antihuman light chain antibody response and the antilight chain reactivity and specificity of the MoAbs, a fluid-phase antigen-capturing ELISA was employed in which 96-well microtiter plates (Corning Glass, Corning, NY) were coated with a polyclonal goat antimouse IgG antiserum (Sigma Chemical). After blocking and washing, appropriately diluted samples of mouse serum, culture fluid supernatant, ascitic fluid, or purified MoAbs were added to each well. Subsequent steps included the addition of a biotinylated13 Bence Jones protein or intact Ig, an avidin-biotin complex (Vectastain, Vector Laboratories, Burlingame, CA), and a 2.2'-azino-bis[3-ethylbenzthiazoline-6 sulfonic acid] substrate solution (Kirkegaard and Perry Laboratories, Gaithersburg, MD). Color development was terminated by the addition of 2% oxalic acid. To select hybridoma clones for further propagation and to test the reactivity of the MoAbs against a large panel of Bence Jones proteins, a solid-phase ELISA was employed. Wells were coated with a solution of monoclonal light chains, and after blocking and washing, hybridoma culture supernatant (or appropriate dilutions of ascitic fluid or purified MoAb) was added to each well. Detection of bound MoAb was accomplished using a peroxidase-labeled goat antimouse IgG antiserum (BioRad, Richmond, CA) and the 2.2'-azino-bis[3-ethylbenzthiazoline-6 sulfonic acid] substrate. A quantitative sandwich ELISA was used to measure the concentration of bound or free light chains. Microtiter plates were coated with purified mouse antihuman light chain MoAbs, and, after blocking and washing, the wells were filled with appropriately diluted serum or urine specimens or with 1.0- to 1000-ng/mL solutions of a reference standard consisting of either (1) a monoclonal IgG* or IgGX protein or (2) an equimolar mixture of 20 different K or X Bence Jones proteins representative of each of the VL subgroups. After incubation and washing, the wells were filled with peroxidase-labeled goat F(ab')2 antihuman K or X light chain-specific antiserum (Tago, Burlingame, CA). 2.2'-azino-bis[3-ethylbenzthiazoline-6 sulfonic acid] was used as a substrate. For all three methods, color development was measured after 15 minutes at 415 nm using an ELISA plate reader (BioTek). The reactivity was graded as: 0: no difference from background or +, ++, +++: >0.5, >0.9, and >1.3 optical density units above background, respectively. In the quantitative ELISA, the data were analyzed using the Kineti-Calc® software program (BioTek Instruments, Winoski, VT). Immunoblotting Sodium dodecyl sulfite/polyacrylamide gel electrophoresis (SDS/PAGE) was performed on Bence Jones proteins and monoclonal IgGs using the Phast system (LKB-Pharmacia, Piscataway, NJ). The proteins were transferred by electroblotting onto 0.45-/*m Immobilon membranes (Millipore, Bedford, MA) and, after blocking, were exposed first to culture fluidcontaining supernatant or dilutions of purified mouse antihuman light chain MoAbs and then to an alkaline-phosphataselabeled horse antimouse IgG antiserum (ABC kit, Vector). Bound protein was detected using the Alkaline Phosphatase Substrate II kit (Vector). Immunocytochemistry and Immunohistochemistry Monoclonal B-cell populations were isolated using FicollHypaque gradient centrifugation from peripheral blood, bone marrow, or lymph nodes. They were then either cytocentrifuged onto poly-L-lysine-coated slides or prepared for flow cytometry. Additional material for study included 4-fim sections of fresh-frozen lymph nodes or of deparaffinized, formalinfixed tissue or bone marrow. Immunoperoxidase or alkaline phosphatase techniques used to examine the light chain nature of cytoplasmic or surface Igs (clg or slg, respectively) of cells contained in the cytocentrifuge or tissue specimens were similar to those described previously.4 For flow cytometry, cells were incubated with appropriately diluted MoAbs, after which they were exposed first to a fluores- A.J.C.P.-July 1993 69 ABE ET AL. MoAbs Antihuman L cein isothiocynate- (FITC) conjugated F(ab')2 goat antimouse IgG antiserum absorbed with human Ig (Tago) and then to a PE-conjugated F(ab')2 goat antihuman K or X chain antiserum (Tago). After washing, the stained cells were examined in an Epics Profile flow cytometer (Coulter Laboratories, Hialeah, FL). Backgroundfluorescencewas determined by staining cells with (1) an unrelated mouse MoAb, (2) the FITC-conjugated antiserum, and (3) PE-conjugated F(ab')2 prepared from nonimmune goat IgG. RESULTS Generation and Characterization of Antihuman Light Chain Monoclonal Antibodies Initial attempts to generate antihuman light chain MoAbs using Freund's adjuvant met with limited success in that the resultant antibodies were of low affinity (Ka, <106 M"') and recognized primarily V-region antigenic determinants unique to the protein used for immunization, ie, antiidiotype specificity. In contrast, the use of the RIBI adjuvant resulted in MoAbs of broader specificity and higher affinity (Ka, >106 M_1) for light-chain-associated determinants. The maximum antibody response (as determined from measurement of the mouse immune response to Bence Jones proteins of the same VL subgroup as the injected human protein) was usually achieved after a 4- to 8-month period of immunization. Splenic lymphocytes were harvested only from animals that exhibited the highest serum titer of polyclonal antihuman light chain antibody. Five mice were injected with each protein; a significant immune response developed in at least two. Typically, each fusion resulted in ~200 antibody-producing hybridomas, and, based on results obtained by ELISA, selected clones were recloned and expanded for in vivo passage. The specificity of the antihuman light chain MoAbs was tested influid-and solid-phase ELISA against a reference panel of >70 K- and X-type Bence Jones proteins, and >20 monoclonal IgG, IgA, and IgM proteins as well as polyclonal light chains and intact Igs. Two groups of MoAbs were identified: the first recognized a CL-related epitope associated with K or X light chains (C, or Q, respectively) and reacted with free light chains as well as those associated with intact Ig proteins, ie, bound light chains. Other anti-CL antibodies were obtained that reacted only with free monoclonal or polyclonal light chains but not with intact monoclonal or polyclonal Ig molecules (Table 1). These reagents could detect in quantitative ELISA light chain concentrations as low as 1.0 ng/ml. The second group of MoAbs recognized antigenic determinants present on the V region of K or X light chains (V, or Vx). This group included those with antiidiotype specificity as well as antibodies that detected V, or Vx antigenic determinants unique to each of the major VL subgroups of K or X chains, respectively. Based on the results of ELISA, we selected from these reagents two panels of antibodies having optimum reactivity with each of the VL subgroups. The first consisted of anti-V, MoAbs that were specific for either KI, KII, KIII, or KIV light chains; the second included anti-Vx MoAbs that reacted exclusively with XI, XII/V, XIV, or XVI proteins. In the case of t n e vxm subgroup, no single anti-VXII1 MoAb could detect all XIII proteins. However, antibodies specific for each of the three VMII subsubgroups14—VXIIIa, VMIIb, and VMllc—were identified and mixed to create an anti-VMU-specific reagent (Table 2). The anti-VL subgroup-specific MoAbs were used to determine by ELISA the V,- or Vx-subgroup nature of Bence Jones proteins and light chains associated with monoclonal IgG, IgA, and IgM proteins. The anti-Vx subgroup-specific MoAbs reacted well in both fluid- and solid-phase ELISA, as did the anti-V„ reagents, with the exception of anti-V,,, which had optimum reactivity in solid-phase ELISA. All of the mouse antihuman light chain MoAbs were of the IgG class. Their heavy chain isotypes and association constants, which ranged from 0.3 to 80 X 107 M"\ are given in Table 3. Application of Antihuman Light Chain MoAbs The capability of the antihuman light chain MoAbs to recognize CL and VL determinants associated with serum or urinary Ig molecules, clgs or slgs of plasma cell and B-cell populations, and light-chain-associated tissue deposits was tested using various immunologic methods. TABLE 1. REACTIVITY OF ANTIHUMAN LIGHT CHAIN MONOCLONAL ANTIBODIES Immunoglobulin Antitotal K (14-6E4)* Antifree K (LKC8) +++t +++ Antitotal X (21-3F4) Antifree X (18-9G11) Monoclonal BJPK BJPX Ig&c IgGX IgA/c IgAX IgM/c IgMX Polyclonal Light chains x/X Ig&c/X IgAx/X Iglvk/X 0 0 0 +++ 0 +++ 0 +++ 0 0 0 0 0 0 0 0 +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ 0 0 0 * Clone designation. t Reactivity in fluid-phase ELISA. Vol. 100-No. I 0 +++ 0 +++ 0 +++ 0 0 0 0 0 0 +++ 0 0 0 70 HEMATOPATHOLOGY Original Article TABLE 2. SPECIFICITY OF MOUSE ANTIHUMAN V«- AND Vx-SUBGROUP-SPECIFIC MONOCLONAL ANTIBODIES Anti-Vu (5-8C)* Subgroup Anti-VK„ (20-15H2) Anti-VK„, (27-4A6) Anti-VKlv (11-1F4) 0 ++ 0 0 0 0 ++ 0 0 0 0 +++ +t V.,(10)t V.„ (8) V.„, (9) V>1V (6) 0 0 0 Anti-Vv (28-20E12) Anti-Vwv (23-7F7) Anti-Vm, (22- I5D11, 29-3D12, 24-8Dl2)§ Anti-V)jV (21-18F9) Anti-VM, (19-6D6) +++ 0 0 0 0 0 +++ 0 0 0 0 0 +++ 0 0 0 0 0 +++ 0 0 0 0 0 +++ VM(10) Vx,„v (6) Vx... 0 4 ) 1 Vx,v (2) Vxv, (6) * Clone designations. t Number of monoclonal lighl chains teslcd. $ Reactivity influid-phaseEL1SA. § Mixture of anti-Xllla. -lllb. and -lllc monoclonal antibodies. || Includes 5. 3. and 6 proteins of the Xllla. All lb. and Mile subsubgroups. respectively. For protein analysis, the K or X type and the VL subgroup of Bence Jones proteins and light chains of monoclonal Igs subjected to SDS/PAGE could be established in immunoblotting analyses using the antilight chain MoAbs. The results of one such study are illustrated in Figure 1, in which the KIV or XII/V nature of the K or X Bence Jones protein, respectively, was evidenced. The anti-C L reagents were used to quantitate by sandwich EL1SA serum or urinary concentrations of bound or free light chains, respectively. Analyses using the antitotal-K or -X MoAbs revealed that in pooled normal human serum the level of Ig* was 10.0 mg/mL, and that of IgX was 3.9 mg/mL. The concentration of polyclonal free-K and -X chains in random urine specimens (obtained from six normal individuals) was measured using the antifree-K or -X MoAbs, and averaged 1.7 Mg/mL and TABLE 3. CHARACTERIZATION OF MOUSE ANTIHUMAN LIGHT CHAIN MONOCLONAL ANTIBODIES Specificity Total K Free K Total X Free X v., v.„ v.„, V.,v Vx, Vxu/v V v XlUn VxiIIb Vxltlc Vxtv Vxv Clone Designation 14-6E4 LKC8 21-3F4 18-9011 5-8C 20-15H2 27-4A6 1I-1F4 28-20E12 23-7F7 22-15D11 29-3D12 24-8D12 21-18F9 19-6D6 ND = not determined. Heavy Chain Isotype Association Constant (XIO1 mol/L) IgGl IgGl IgG2a IgGl lgG3 IgGl IgGl IgGl IgGl IgG2a IgG2b IgG2a IgGl IgG2a IgGl 2.2 3.1 33.0 0.6 1.0 ND ND ND ND 80.0 7.0 ND 1.1 10.0 0.3 0.7 jug/mL, respectively. The antifree light chain reagents also were used to quantitate Bence Jones protein excretion, and were particularly helpful in cases where the presence of this component was obscured by other urinary proteins, eg, in patients with AL amyloidosis and nephrotic syndrome. The results of analyses quantitating urinary K or X Bence Jones proteins before and after chemotherapy in two such patients are illustrated in Figure 2. At the cellular level, the antitotal-K and -X chain antibodies were tested against cell suspensions prepared from the bone marrow of patients with multiple myeloma or AL amyloidosis and from the peripheral blood, bone marrow, or lymph nodes of patients with CLL and related B-cell neoplasms. Among more than 100 cases studied, the monoclonal Ig immunophenotype of the plasma cell or lymphocyte population was appar- >*^£ .' I * * *> # v* -^ anti-x anti-X anti-«IV anti-A.ll/V FIG. 1. Immunoblotting (Western blotting) analyses, (left) The lanes containing K\, KII, KIII, and KIV Bence Jones proteins are as indicated. The upper and lower membranes were immunostained with an anti-C, MoAb and an anti-V„IV subgroup-specific MoAb, respectively, (right) The lanes containing XI, XII/V, XIII, XIV, and XVI Bence Jones proteins are as indicated. The upper and lower membranes were immunostained with an anti-Cx MoAb and an anti-VXI|/v subgroup-specific MoAb, respectively. A.J.C.P.-July 1993 ABE ET AL. Antihuman lations was more evident; eg, the cells from patient RW reacted with the anti-X and anti-V MI/v MoAbs but not with the other antibodies in the panel (Fig. 3). For more than 30 patients whose serum or urine contained a monoclonal Ig protein, there was complete concordance between the light chain classification determined immunocytochemically and that found through our serologic and sequence analyses of the isolated protein. In addition to studies of fresh-frozen cell suspensions or tissue, these antibodies could be used to immunostain formalin-fixed, paraffin-embedded tissue (Fig. 4). Analyses by flow cytometry involved usage of both the antic s and anti-V L subgroup-specific MoAbs as primary reagents for examination of peripheral blood cells or isolated nodal lymphocytes derived from patients with CLL and other forms of B-cell neoplasia. The immunocytochemical demonstration of the slg/cl+ or slg/clll+ nature of cells from two such patients is shown in Figure 5. The antilight chain MoAbs also were used to demonstrate immunohistochemically the deposition of light chains as renal tubular casts, basement membrane precipitates, or amyloid fibrils. Due to the variation among patients with AL amyloidosis in the composition of the light chains found in the amyloid deposits (ie, the predominant occurrence of VL fragments versus the presence of components containing the VL plus a portion of the C L domain 15 ), the immunohistochemical detection of AL amyloid often required the use of the anti-V L subgroupspecific antibodies (Fig. 6). That the AL amyloid deposits in patient VT consisted only of a VMIIa fragment while those in patient IR contained both VMIIa plus Cx structure was confirmed through our sequence analyses of the purified amyloid extracts. free K* 0.03 0.08 0.23 0.17 0.18 0.14 0.11 4-14-91 BJP IgG FB e © ££ hssJL 9-4-90 I «-Sp 10.3 0.01 11-5-90 9 '•^Wp 12.5 0.11 12.5 0.18 18.0 0.05 12.8 0.03 23.0 0.03 15.1 0.02 MM 1 iH ! it 1-3-91 3-5-91 6-4-91 till 1 7-18-91 8-18-91 71 Lightchain MoAbs DISCUSSION BJP * nig/ml FIG. 2. Immunoquantification of Bence Jones proteinuria in patients with nephrotic syndrome. Agarose gel electropherograms of lyophilized, reconstituted (50 mg/mL) serial urine specimens from AL amyloidosis patients RM and FB, whose urine contained a K Bence Jones protein plus a monoclonal IgG* protein and a \ Bence Jones protein, respectively. In both cases, the first specimen was obtained before administration of chemotherapy. Protein concentrations were measured by a sulfosalicylic acid (SA) turbidity method and by quantitative ELISA employing MoAbs specific for free K or X light chains as indicated. The location of the IgG and Bence Jones proteins (BJP) were determined by immunofixation electrophoresis. ent by the predominant K or X reactivity of the clg or slg of these cells. When the cells were immunostained with anti-V^ or antiVx subgroup-specific MoAbs, the monoclonality of such popu- A variety of polyclonal antihuman light chain antibodies with specificities for CL- and VL-associated epitopes have been generated for clinical and research purposes; however, the relative scarcity of these reagents, especially those directed to VL antigenic determinants, as well as the broader specificity and technical problems associated with polyclonal antisera, have restricted their use. The ability to produce antilight chain MoAbs of the desired specificity in virtually unlimited amounts would provide an invaluable source of material for diagnostic, prognostic, and possible therapeutic purposes. Previously, few MoAbs with light-chain specificity have been obtained.16"20 As part of a systematic effort to generate a battery of these reagents, we developed an immunization protocol in which a series of mice were injected with well-characterized Kand X-type Bence Jones proteins representative of the major VL subgroups, V„„ V,„, V, m , V,IV and VM, VM1/V, VMU, VXIV, VXVI. The availability of large numbers of these serologically and chemically classified light chains, and of monoclonal IgG, IgA, and IgM proteins made it possible to determine by ELISA the appropriate clones for selection and propagation and to establish the specificity of the resultant antibodies for light-chain-associated C L and VL epitopes. The antihuman light chain MoAbs generated included those reacting with C L determinants—anti-C, or anti-Q—found on free or bound light chains, and others that recognized Q or Cx determinants on free light chains exclusively. Additionally, a second group of antilight chain MoAbs were produced that reacted with VL-associated idiotype and subgroup-associated epitopes. Through extensive screening and clonal selection, Vol. 100-No. 1 72 HEMATOPATHOLOGY Original Article ' ^U Iff" IW #* 'hMm+fmm OE V RW HB BR GR m antl-i antl J.I anti-xii/v antlxiv antkw MYELOMA CH LYMPHOMA JR MW anti-t anti-A A.J.C.P.-July 1993 AS 73 ABE ET AL. Antihuman Lightchain MoAbs FIG. 3. Immunocytochemical characterization of the Vx subgroups expressed by the light chains of clg and slg in monoclonal plasma cell and B-lymphocyte populations, respectively. Plasma cells derived from the bone marrow of patients with multiple myeloma (RW, HB) and AL amyloidosis (GR) and peripheral blood lymphocytes from patients with CLL (DE, BR). Cells were examined using the avidin-biotin complex alkaline phosphatase technique and the antihuman light chain MoAbs as indicated. Based on the patterns of reactivity, the light chains of cells from patients DE, RW, HB, BR, and GR were classified as XI, XI1/V, Mil, XIV, and XVI, respectively (hematoxylin counterstain; X 1,000). 1 CR HF L F "? L L F 1 * 3 V 40P 4 -D>Nfc£bia LFLZ Anti-n FIG. 4. Immunophenotyping analyses of formalin-fixed, paraffin-embedded tissue. (Left) Bone marrow specimens from patients CH and JR with clgx+ and dgX+ multiple myeloma, respectively, and (right) lymph node specimens from patients MW and AS with slg*+ and sIgX+ low-grade lymphoma, respectively (immunoperoxidase method, primary antibodies—anti-x and anti-X MoAbs, as indicated; X 1,000). panels of antibodies were obtained that had specificity for the four V, and five Vx subgroups. We have demonstrated, in several types of immunologic analyses (ELISA, immunoblotting, immunocytochemistry, and immunohistochemistry), the capability of the antilight chain MoAbs to recognize the K or X type and the VL-subgroup nature of monoclonal Ig proteins, plasma cells, and B lymphocytes and have demonstrated the clinical applicability of these reagents. The anti-C, and anti-Q Moabs were used not only to establish protein and cell monoclonality but also to quantitate serum and urine Ig concentrations. Due to the obscuring presence of non-Ig proteins in nephrotic urine, it has been virtually impossible to measure the amount of Bence Jones protein in 2 HF BG BG A# LFL2 LFL2 Anti-x Anli>« FIG. 5. Flow cytometric analyses. Mononuclear cells isolated from the peripheral blood of two patients (HF and BG) with slgic* CLL were first stained with anti-V„, or anti-V,,,, subgroup-specific MoAbs and then with the FITC- or PE-conjugated anti-lg antibodies. Data are expressed. as bivariate dot plots with logfluorescenceintensity of FITC (surface V„) on the Y axis, and logfluorescenceintensity of PE (surface *•) on the X axis. The vertical and horizontal lines define the boundaries of positive and negative cells based on control staining with FITC and PE. antl-x anti-Mlla FIG. 6. Immunohistochemical characterization of AL amyloid deposits. Use of anti-CL (anti-X) and -V, (anti-Xllla) MoAbs to characterize the light chain nature of the green birefringent Congo red (CR)-stained amyloid deposits in the spleen and heart of patients VT and IR, respectively. Based on the patterns of reactivity, the amyloid in patient VT consisted of a Vx fragment, while that of IR contained Q- as well as Vx-related structure (immunoperoxidase technique, primary antisera as indicated; patient VT. X400: patient IR, X250). Vol. 100-No. I 74 HEMATOPATHOLOGY Original Article such specimens using electrophoretic or other serologic techniques. The availability of anti-CL antibodies with specificity forfree K or X light chains has made such analyses feasible; these data are especially important in documentation of response to treatment or relapse of patients with multiple myeloma or AL amyloidosis.' Because these reagents can also be used to quantitate free polyclonal (as well as monoclonal) urinary K and X light chains, it is possible to assess polyclonal B-cell activation in autoimmune and other diseases, such as multiple sclerosis,21"23 and to monitor kidney function in patients with other disorders associated with renal tubular dysfunction and polyclonal light chain proteinuria, eg, systemic lupus erythematosus and diabetes mellitus.24"27 The anti-VL subgroup-specific MoAbs also have clinical relevance. The ability to determine immunocytochemically the V, or Vx subgroup is of diagnostic and prognostic importance. Not only can monoclonality be proven even in cases in which cell numbers are minimal, but the ability to identify residual monoclonal plasma cell or B-cell populations provides an objective means to evaluate response to treatment or to predict disease relapse. In addition, these reagents (as well as those with CL specificity) can be used to identify and characterize light chainrelated tissue deposits, such as those found in myeloma (cast) nephropathy, light chain deposition disease, and especially AL amyloidosis.2 Finally, because certain VL subgroups are associated with particular disease processes, including autoimmune disorders, AL amyloidosis, and B-cell neoplasia,6"9 it is important to establish the VL nature of monoclonal proteins or cells. In addition to their diagnostic and prognostic usefulness, the antihuman light chain MoAbs may also have therapeutic potential.28,29 These antibodies could serve as immunoadsorbants to remove extracorporeally nephrotoxic or amyloidogenic light chains, or to purge neoplastic B cells from bone marrow specimens used for autologous transplantation.30 Such applications would be of obvious clinical benefit, and are currently under investigation. Acknowledgments. The authors thank Mary deBram Hart for assistance in the technical aspects of these studies and Julie Ottinger for assistance in manuscript preparation. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. REFERENCES 1. Solomon A. Monoclonal immunoglobulins as biomarkers of cancer. In: Sell S, ed. Cancer Markers. Clifton, NJ: Humana Press, 1980, pp 57-87. 2. Solomon A. Clinical implications of monoclonal light chains. Semin Oncol 1986;13:341-349. 3. Solomon A. Light chains of human immunoglobulins. Methods Enzymol 1985;116:101-121. 4. Solomon A, Weiss DT, Macy SD, Antonucci RA. Immunocytochemical detection of kappa and lambda light chain V region subgroups in human B-cell malignancies. Am J Pathol 1990;137:855-862. 5. Solomon A, Weiss DT. Serologically defined V region subgroups of human Might chains. J Immunol 1987;139:824-830. 6. Solomon A, Frangione B, Franklin EC. Bence Jones proteins and light chains of immunoglobulins. Preferential association of the V xv , subgroup of human light chains with amyloidosis AL(A). J Clin Invest 1982;70:453-460. 7. Ledford DK, Gofii F, Pizzolato M, et al. Preferential association of /clllb light chains with monoclonal human IgMx autoantibodies. J Immunol 1983;131:1322-1325. 8. Solomon A. Light chains of immunoglobulins: Structural-genetic correlates. Blood 1986;68:603-610. 9. KippsTJ. The CD5 B cell. Adv Immunol 1989;47:117-185. 10. Fazekas de St Groth S. Scheidegger D. Production of monoclonal 24. 25. 26. 27. 28. 29. 30. antibodies: Strategy and tactics. J Immunol Methods 1980;35:1-21. Kennel SJ, Foote U , Lankford PK. Analysis of surface proteins of mouse lung carcinomas using monoclonal antibodies. Cancer Res 1981;41:3465-3470. Kennel SJ, Chen JP, Lankford RK, Foote LJ. Monoclonal antibodies from rats immunized with fragment D of human fibrinogen. Thrombosis Res 1981;22:309-320. Klein-Schneegans A-S, Kuntz L, Fonteneau P, Loor F. An indirect asymmetrical sandwich ELISA using anti-allotype antibodies for the specific and quantitative measurement of mouse IgG2a of Igh-l b allotype. J Immunol Methods 1989;125:207-213. Eulitz M, Murphy C, Weiss DT, Solomon A. Serological and chemical differentiation of human XIII light chain variable regions. J Immunol 1991;146:3091-3096. Glenner GG. Amyloid deposits and amyloidosis: The ^-fibrilloses. N Engl J Med 1980;302:1283-1292, 1333-1343. Lowe J, Hardie D, Jefferis R, et al. Properties of monoclonal antibodies to human immunoglobulin kappa and lambda chains. Immunology 1981;42:649-659. Ling NR, Lowe J, Hardie D, et al. Detection of free K chains in human serum and urine using pairs of monoclonal antibodies reacting with C. epitopes not available on whole immunoglobulins. Clin Exp Immunol 1983;52:234-240. Greenstein JL, Solomon A, Abraham GN. Monoclonal antibodies reactive with idiotypic and variable-region specific determinants on human immunoglobulins. Immunology 1984;51:17— 25. Walker MR, Solomon A, Ling NR, et al. Immunogenic and antigenic epitopes of immunoglobulins. XVIII. Subpopulations of human lambda chains defined with a panel of monoclonal antibodies. Immunology 1986;59:467-471. Walker MR, Solomon A, Weiss DT, et al. Immunogenic and antigenic epitopes of IgE: XXV. Monoclonal antibodies that differentiate the Mcg+/Mcg" and Oz+/Oz" C region isotypes of human XL chains. J Immunol 1988;140:1600-1604. Hopper JE, O'Brien J, Papagiannes E. Restriction of blood and marrow CLL-B cells to free L-chain Ig secretion: Implication for normal B-cell function and control. Am J Hematol 1988;29:125-133. Hopper JE, Sequeira W, Martellotto J, et al. Clinical relapse in systemic lupus erythematosus: Correlation with antecedent elevation of urinary free light-chain immunoglobulin. J Clin Immunol 1989;9:338-350. Mehta PD, Cook SD, Troiano RA, Coyle PK. Increased free light chains in the urine from patients with multiple sclerosis. Neurology 1991;41:540-544. Spriggs B, Epstein WV. Clinical and laboratory correlates of L chain proteinuria in systemic lupus erythematosus. J Rheumatol 1974;1:287-292. Soiling K. Polymeric forms of free light chains in serum from normal individuals and from patients with renal diseases. Scand J Clin Lab Invest 1976;36:447-452. Soiling K, Soiling J, RomerFK. Free light chains of immunoglobulins in serum from patients with rheumatoid arthritis, sarcoidosis, chronic infections and pulmonary cancer. Acta Med Scand 1981;209:473-477. Groop L, Makipernaa A, Stenman S, et al. Urinary excretion of kappa light chains in patients with diabetes mellitus. Kidney Int 1990;37:1120-1125. Tutt AL, Stevenson FK, Smith JL, Stevenson GT. Antibodies against urinary light chain idiotypes as agents for detection and destruction of human neoplastic B lymphocytes. J Immunol 1983;131:3058-3063. Waldmann TA. Monoclonal antibodies in diagnosis and therapy. Science 1991;252:1657-1662. Gribben JG, Freedman AS, Neuberg D, et al. Immunologic purging of marrow assessed by PCR before autologous bone marrow transplantation for B-cell lymphoma. N Engl J Med 1991;325:1525-1533. AJ.C.P.-July 1993