J Clin Pathol 1984;37:255-262 J Clin Pathol: first published as 10.1136/jcp.37.3.255 on 1 March 1984. Downloaded from Detection and identification of serum monoclonal immunoglobulin by immunoisoelectric focusing. Limits of sensitivity and use during relapse of multiple myeloma D SINCLAIR, DS KUMARARATNE,* DI STOTT From the Department ofBacteriology and Immunology, Western Infirmary, Glasgow GIl 6NT SUMMARY The limits of detection of four classes of monoclonal immunoglobulin and free light chain in serum by isoelectric focusing and immunoisoelectric focusing have been determined and the sensitivity of these techniques compared with that obtained using immunoelectrophoresis and zonal electrophoresis with immunofixation. Immunoisoelectric focusing was 10-40 times more sensitive than immunoelectrophoresis and could be used to detect concentrations of monoclonal immunoglobulin that were undetectable by zonal electrophoresis with immunofixation. The rele- vance of this work in monitoring multiple myeloma during treatment and relapse is discussed. An important factor in the diagnosis of multiple immunofixation was performed as described by copyright. myeloma and other B cell malignancies is the detec- Ritchie and Smith.2 Patients' sera were referred for tion of a paraprotein in the patient' s serum. We have investigation of serum paraproteinaemia either as a shown that isoelectric focusing and immunoisoelec- clinical assessment of effectiveness of treatment or tric focusing are superior to immunoelectrophoresis for detecting monoclonal immunoglobulin after and zonal electrophoresis for the detection of mono- treatment. clonal immunoglobulin when it is present at con- In order to determine the limits of detection of centrations below 5 mg/ml or is masked by poly- monoclonal immunoglobulin, sera from patients http://jcp.bmj.com/ clonal immunoglobulin.' A potentially important with multiple myeloma or Waldenstrom' s macro- clinical application of these techniques is the detec- globulinaemia were diluted with pooled normal tion of early relapse in patients with B cell neoplasia human serum and analysed by zonal electrophoresis, who are in clinical and serological remission. In this immunoelectrophoresis, isoelectric focusing, and paper we show how isoelectric focusing and immunoisoelectric focusing. Sera containing mono- immunoisoelectric focusing may be used to monitor clonal IgA or IgM were treated with monoclonal immunoglobulin at an early stage in the 2-mercaptoethanol and focused in a narrow range relapse and we have compared the limits of sensitiv- pH gradient to facilitate focusing of these molecules. on September 25, 2021 by guest. Protected ity of isoelectric focusing and immunoisoelectric Interpretation of the immunoelectrophoretic pat- focusing for the detection of paraproteins with more tern of IgM is made easier by the use of conventional techniques. 2-mercaptoethanol.' Material and methods Results Immunoelectrophoresis, isoelectric focusing, and Fig. la shows the isoelectric focusing pattern of immunoisoelectric focusing were performed as pre- serum from a patient with multiple myeloma (IgG viously described.' Zonal electrophoresis with (K) isotype) with a total IgG concentration of 54-0 mg/ml (measured by radial immunodiffusion). This serum was serially diluted in pooled normal human *Present address: Department of Immunology, East Birmingham Hospital, Birmingham B9 5ST serum to determine the limits of detection of mono- clonal immunoglobulin. Monoclonal immunoglobu- Accepted for publication 26 Octnber 1983 lin was still clearly detectable at a dilution of 1/200 255 J Clin Pathol: first published as 10.1136/jcp.37.3.255 on 1 March 1984. Downloaded from 256 Sinclair, Kumararatne, Stott .1 .A :,::. :::- i. ",- Nec., XF :i::-: nA: .I P. .j :: N. Ali A ! *'D1i e F.!'e-ttj,.- .F :ee Fig. 1 Comparison ofthe sensitivity ofdetection ofmonoclonal IgG in serum by isoelectric focusing, copyright. immunoisoelectric focusing, and immunoelectrophoresis. Troughs containing anti-IgG and anti- light chain are indicated at the right hand side ofthe immunoelectrophoresis plate..,..Dilutions . ofthe myeloma serum in normal human serum (NHS) are indicated at each track. pH gradient for isoelectric focusing and immunoisoelectric focusing = 3-10. and very faint bands were visible at 1/1000, tion = 81 mg/ml). The monoclonal IgA could be http://jcp.bmj.com/ although these could have been missed in a routine detected by isoelectric focusing at a concentration of specimen. Immunofixation with anti-IgG 1-6 mg/ml (1/50) and by immunoisoelectric focusing (immunoisoelectric focusing) gave similar results, at the highest dilution-that is, 0-16 mg/ml; this the monoclonal IgG again being detectable down to figure, as before, represents a maximum value. With a dilution of at least 1/200. immunoelectrophoresis (Fig. 2b) monoclonal IgA If we assume that most of the, IgG is monoclonal was detectable with certainty at concentrations immunoglobulin, this represents a detection limit down to only 4 05 mg/ml (1/20), although the dilu- on September 25, 2021 by guest. Protected below 0-27 mg/ml. This figure is a maximum value tion containing 1-62 mg/ml (1/50) was abnormal but since polyclonal IgG will also be present in the equivocal. This represents an improvement of be- serum of the myeloma patient, albeit in low concen- tween 10 and 25 fold for immunoisoelectric focusing tration because of the suppression of normal compared with immunoelectrophoresis. immunoglobulin production often associated with Monoclonal IgM (original concentration 17-9 multiple myeloma. mg/ml) could be detected at concentrations down to Fig. lb shows the immunoelectrophoresis of the 0 45 mg/ml (1/40) by isoelectric focusing and less same IgG dilutions. Monoclonal IgG could be than 0-11 mg/ml (1/160) by immunoisoelectric identified with certainty, at best, at concentrations focusing (Fig. 3a), while immunoelectrophoresis down to 2-7 mg/ml. Thus isoelectric focusing was at (Fig. 3b) could confirm monoclonal IgM, at best, at least 10 times more sensitive than immunoelec- concentrations down to 3-6 mg/ml (1/5). This rep- trophoresis in detecting monoclonal IgG in this case. resents at least a 32 fold increase in sensitivity by Fig. 2a shows the isoelectric focusing and immunoisoelectric focusing compared with immunoisoelectric focusing patterns of a serum con- immunoelectrophoresis. taining monoclonal IgA (A) (original IgA concentra- Monoclonal IgD (original concentration 54 J Clin Pathol: first published as 10.1136/jcp.37.3.255 on 1 March 1984. Downloaded from Detection of identification ofserum monoclonal immunoglobulin by immunoisoelectric focusing 257 Anti - serum Nect 1/10 1120 *r | t ! ~~~~~~~~~1/150( 0 8t&& l!8~~~~NH I k.w.CZ) Q) 53 detection (a (b) Isoelectric focusing Immuno-isoelectrnc focusing IrmmunoeLectrophor-esis (11t;) (IIEF) (IEP) original LIgA]=Tlmg/m L mt of detecton IEP 4 05mg/ml ;1/20) EF = 62mg/ml (!/50) mIIEF= Ot6mg/ml 0/500) Fig. 2 Comparison of the sensitivity ofdetection of monoclonal IgA in serum by the three methods described in Fig. 1. copyright. pH gradient for isoelectric focusing and immunoisoelectric focusing = 5-8. mg/ml), a less common paraprotein, could be Immunoelectrophoresis showed only a slight detected at a concentration of 1 mg/ml (1/50) by abnormality at the anodic end of the kappa precipi- isoelectric focusing and less than 0-05 mg/ml (1/ tin arc. Isoelectric focusing of the serum showed a 1000) by immunoisoelectric focusing (Fig. 4a) com- single abnormal band which was identified as kappa http://jcp.bmj.com/ pared with a detection limit of 2 mg/ml (1/25) by light chain by immunoisoelectric focusing. There immunoelectrophoresis (Fig. 4b). This represents a was no detectable monoclonal IgG, a paraprotein 40 fold increase in sensitivity by immunoisoelectric which had been suspected owing to the rising IgG focusing. concentration; nor was monoclonal immunoglobulin Fig. 5 compares immunoelectrophoresis, isoelec- of any other class found. Isoelectric focusing of con- tric focusing, and immunoisoelectric focusing in the centrated urine also showed a single band at the and this detection of light chains in serum. Free light chain same isoelectric point as the "serum band,," on September 25, 2021 by guest. Protected is visible at dilutions down to 1/20 by immunoelec- was identified as free kappa light chain by trophoresis, but down to 1/320 by immunoisoelec- immunoisoelectric focusing. tric focusing, representing a 16 fold improvement in Table 1 shows the results of using the four techni- sensitivity. ques on sera from four patients with paraproteins of To show the value of isoelectric focusing and different classes. In each of these cases, except immunoisoelectric focusing in an individual case, patient 4, only immunoisoelectric focusing gave Fig. 6 shows a comparison of zonal electrophoresis, positive identification of a monoclonal immuno- immunoelectrophoresis, and immunoisoelectric globulin. Such examples of sera containing low con- focusing for the analysis of serum from a patient centrations of monoclonal immunoglobulin detect- with a solitary plasmacytoma and rising IgG con- able by immunoisoelectric focusing but not by zonal centrations but with no paraproteins as shown by or immunoelectrophoresis have often been encoun- conventional techniques. No paraprotein was found tered in our laboratory. by zonal electrophoresis of the serum, but Table 2 shows the results from 27 patients who immunofixation of the strip with antikappa serum had undergone