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2 Journal of Neuroimmunology ELSEVIER Journal of Neuroimmunology 62 (1995) Cerebrospinal fluid free kappa light chains versus IgG findings in neurological disorders: qualitative and quantitative measurements KJB Lamers a, JGN de Jong a, PJH Jongen a, MJH Kock-Jansen b, MA Teunesen b, EMW Prudon-Rosmulder a a University Hospital Nijmegen, institute of Neurology, PO Box 9101, 6500 HB Nijmegen, The Netherlands h University Hospital Nijmegen, Central Clinical Chemical Laboratory, PO Box 9101, <5500 HB Nijmegen, The Netherlands Received 21 February 1995; revised 18 April 1995; accepted 15 May 1995 Abstract In this study free kappa light chains in cerebrospinal fluid (CSF) were determined both by an affinity mediated capillary blotting technique after isoelectric focusing (IEF) in agarose gel and by a quantitative enzyme linked immunosorbent assay (ELISA) The free kappa results were compared with the IgG findings in 4 neurological patient groups with a distinct CSF IgG pattern: (1) CSF without oligoclonal IgG bands, (2) CSF with serum derived IgG bands, (3) CSF restricted IgG bands and (4) CSF restricted and serum derived IgG bands Oligoclonal free kappa bands are nearly absent in CSF of groups 1+ 2, and present in 88% of group 3 and 84% of group 4 patients We could also establish free kappa indices from specimens in the 4 groups in analogy to IgG indices Group 1 had a median free kappa index of 11, group 2: 10 and groups 3+4: 100 The correspondence between immunoblot and index findings for free kappa is better than for IgG Free kappa index is more sensitive but somewhat less specific than IgG index for establishing intrathecal immune production Keywords: Cerebrospinal fluid; Free kappa light chain; IgG index; Free kappa index 1 Introduction been found in M S and other neuro-immunological disorders (Fagnart et a l, 1988; Rudick et al, 1986; Gallo et a l, Several diseases of the nervous system are associated with immunological abnormalities in cerebrospinal fluid (CSF) Selective increase in the C SF IgG level has been observed consistendy in M S and it has become an important laboratory test for confirmation of the diagnosis (Poser et al, 1983) A selective C SF IgG increase can be established by using the IgG Index (Link and Tibbling, 1977), formulas for intrathecal produced IgG (Reiber and Felgenhauer, 1987; Ohman et al, 1992) or by detection of C SF restricted oligoclonal IgG bands (W alker et al, 1983; Luxton et al, 1990) The IgG finding in M S is nonspecific, as also patients with central nervous system (C N S) infections or inflammatory diseases have regularly elevated C SF IgG levels Besides IgG, also selective increase of free kappa and lambda light chains (free kappa, free lambda) in C SF has 1989; Decarli et al, 1987) C SF oligoclonal free kappa bands in M S have been observed with about the same frequency as that for C SF oligoclonal IgG bands (Sindic and Laterre, 1991a) Free light chain determination in C SF also has relevance for the diagnostics of patients with early M S (Lo lli et al, 1991) and the presence of C SF free light chains correlates significantly with recent demyelinadng activity in M S (Vakaet and Thompson, 1985; Bracco et al, 1987) From these studies it is clear that intrathecally produced C SF IgG is mostly accompanied by intrathecally produced C SF free light chains and free light chain detection is a complementary, although optional, test to establish a laboratory-supported diagnosis (Sindic and Laterre, 1991b) However, in all publications the detection limit for free light chain determination is too high to measure C SF levels in normal patients Furthermore, the coincidence of free light chain levels with oligoclonal free light chains in C SF and the relation between IgG- and free light chain Corresponding author Phone +31 (80) ; Fax +31 (80) findings in C SF are not systematically investigated Therefore, we developed a sensitive E L IS A test for /95/$ Elsevier Science BV All rights reserved SSDI (95)

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4 KJB Lamers et ai /Journal of Neuroimmunology 62 ( 995) Table 2 Median values and ranges of immunological parameters in CSF and serum in the 3 patient groups Parameter Unit Group I: IgG normal (n = 37) Group 2: IgG mirror (n = 31) Group : IgG restricted (plus) (n = 38) Median (Range) Median (Range) Median (Range) CSF IgG m g/l 21 (9-51) 42 (10-150) 35 (12-130) CSF free kappa Mg/1 25 (58-85) 50 (88-410) 330 ( ) Serum IgG g/1 11 (60-21) 11 (41-20) 10 (49-23) Serum free kappa m g/l 42 (27-95) 61 (16-23) ) Ratio albumin X (25-10) 77 (30-49) 52 (24-19) Ratio IgG X 1(T 3 17 (11-44) 36 (13-35) 39 (14-11) Ratio free kappa X 10~3 50 ( 12-11) 96 (33-55) 68 (56-330) Index IgG ( ) 046 ( ) 067 (044-29) Index free kappa 11 (05-29) 10 (05-44) 10 (16-89) 227 Free kappa immunoblot For oligoclonal free kappa the same method as for oligoclonal IgG was used with minor modifications A l ways 10 / i \ unconcentrated (or diluted) C SF and 75 j j l I (1:100) diluted serum were applied upon the agarose gel Instead of passive protein transfer affinity-mediated capillary blotting was performed The NC membrane was incubated overnight with rabbit antihuman free kappa, diluted (1:500) with P B S (code A10G Dakopatts, Copenhagen) The N C membrane was washed in P B S and blocked during 1 h in 3% bovine serum albumin/pbs and again washed in PB S The focused proteins in agarose were blotted onto the NC membrane for 30 min After blocking of the N C membrane for 60 min in 2% milk powder in 015 M NaCl the membrane was incubated with (1:500) diluted rabbit antihuman total kappa, H R P labelled, in 02% milk powder/nacl for 2 h (code P129 Dakopatts, Copenhagen), followed by color development Q-free kappa L CSF normal A CSF mirror Q-albumin - 3 Fig 2 Relation between Q albumin ( X 10 3) and Q free kappa ( X 10 3) in patients of group (3 + 4) (top) and in patients of groups 1 and 2 (bottom)

5 22 KJB, Latners et ai / Journal of Neuroimmunology 62 (1995) Free kappa ELISA Free kappa light chains were measured by a solid phase sandwich E L IS A Microtiter plates were coated overnight with 200 fa (1:1000) diluted rabbit antihuman free kappa (Dakopatts, code A 100, Copenhagen) Reagents are the same as in an earlier described method (Haraldson et al, 1991) After washing, blocking with 2% bovine albumin and washing 100 px native C SF and (1:10 and 1:100) diluted C SF and corresponding serum (1:2000) diluted 100 Index -free kappa were incubated 90 min at 37 C After washing, 100 /j l \ H RP conjugated rabbit antihuman total kappa (1:1000) diluted (Dakopatts, code P I 29, Copenhagen) was incubated 90 min at 37 C Color development was performed with OPD-re agent Standard curves were performed with dilutions of an oligoclonal free kappa pool CSF The pooled C SF was left 2 days at 4 C, centrifuged and filtered through a 02 pan filter and divided in aliquots of 05 ml and stored at 70 C, The C SF pool was calibrated on a commercial purified free kappa standard (27 mg/ml, Tago, Burlingame, CA, U SA ) C SF pool is 100% W e used always 9 standard dilutions (from 01% to 2% of the C SF pool) 1% C SF pool solution corresponds with 146 g/i Tago free kappa light chain concentration With help of the C SF pool standards the reproducibility of the assay was much better than with Tago standards The lower limit of sensitivity is 2 /xg/1 To evaluate intra-assay precision, we studied two diluted C SF samples containing 3 and 7 /xg/1 free kappa, repeating the assays 20 times on the same day The coefficient of variation (C V ) was respectively 61% and 29% The inter assay precision was determined by daily assay of 1 C SF sample, containing 3 jag/1 free kappa for 9 days The CV was 81% Concentrations remained stable during storage at -~70 C and 20 C for 3 months To test the specificity of the assay we used C SF specimens with a high free kappa level in our E L IS A test In a modified E L IS A test we used in stead of H RP conjugated rabbit antihuman total kappa as second anti- Groupnr Fig 4 Distribution of index free kappa values measured in patients of groups 1, 2 and (3 +4), The line represents the upper limit ( + 2 SD) of the control group body, H R P conjugated goat antihuman IgG (Fc), gamma chain specific The free kappa positive specimens all gave a negative reaction with this second antibody This negative reaction excludes a cross reaction of intact IgG molecules with rabbit antihuman free kappa 3 Results 3 J Free kappa investigation with immunoblot A ll C SF and serum samples were tested for the presence of oligoclonal free kappa bands Fig 1 shows examples of immunoblot findings in patients out of the 4 groups and also in one patient with an IgG kappa paraproteinemia There is no cross reaction of antibodies to oligoclonal free kappa with oligoclonal IgG molecules Table 1 summarizes the results of the free kappa immunoblots in the CSF-free kappa (ug/l) i Intrathecal CSF-IgG (mg/l) Fig 3 Relation between intrathecal CSF IgG (mg/1) and CSF free kappa ( lg/1) in patients of group (3 + 4, CSF restricted plus),

6 KJB Lamers et al / Journal of Neuroimmunology 62 (1995) groups Generally, oligoclonal free kappa bands are very scarce in serum: the 6 specimens from group 2 with positive serum free kappa had an identical free kappa pattern in serum and C SF In patients with local IgG synthesis (groups 3 + 4), C SF oligoclonal free kappa is frequent (88% in group 3 and 84% in group 4), The free kappa oligoclonal pattern was always different from the IgG oligoclonal pattern in all positive specimens Table 4 IgG index and free kappa index results in the three patient groups Group Oligoclonal IgG pattern Number IgG index increased 1 Normal Mirror Restricted (plus) Free kappa index increased 52 Free kappa investigation with ELISA In a number of patients in each group we measured in CSF and serum also the quantitative levels of free kappa, IgG and albumin, and calculated ratios and indices W e selected 37 patients from group 1, 31 patients from group 2 and 38 patients from groups The results of the free kappa immunoblots in groups were not different Therefore we handled groups as one group In Table 2 we present the median values and the ranges of the immunological parameters for the 3 groups The median serum levels of free kappa in the 3 groups are comparable but the median C SF levels are different: 25, 50 and 330 /xg/i for groups 1, 2 and (3 + 4 ) respectively, Furthermore, the median level of index free kappa in group (3 + 4) (local IgG synthesis) is 10-times higher than the median level of index free kappa of groups 1 and 2 (no local IgG synthesis) A ll 37 patients in group 1 had normal white cell number in CSF, an albumin ratio ^ 10 X 10-3, no oligoclonal IgG bands and normal IgG index The mean + SD of the free kappa index for this IgG-normar group is 13 ±05 Therefore, we considered patients of group 1 as a nonimmunological control group for free kappa index with 2 SD limits: In Fig 2, the relation between ratio serum/csf (Q ) albumin and Q free kappa is presented for the 3 groups The correlation coefficient between Q albumin and Q free kappa is 064 for group l t 049 for group 2 and 0064 for group (3 + 4) The correlation coefficient between intrathecal C SF IgG and C SF free kappa is 091 (Fig 3) In Fig 4 the free kappa indices for the 3 patient groups are presented 33 Correspondence between immunoblot and index findings 4 In Table 3 we present the results of immunoblots and indices of free kappa and IgG in the 3 patient groups In the total group of 106 patients the index and immunoblot correspond in 102 patients for free kappa (96%) and in 92 patients for IgG (87%) In Table 4 we present the results of IgG- and free kappa indices in the 3 patient groups Based on the acceptance that Ig G isoelectric focusing is the gold standard for indication of an intrathecal IgG production, it can be concluded that for free kappa index 4 false negatives and 5 false positives were found and for IgG index 12 false negatives and 2 false positives 4 Discussion A selective increase in C SF of free kappa has been found in patients with M S and in patients with infections of the CNS The incidence of C SF oligoclonal free kappa in neuroimmunological disorders is not systematically studied W e investigated the coincidence of free kappa and IgG oligoclonal bands in C S F and serum in 4 neurological patient groups Generally, free kappa bands are very scarce in serum In 88% of patients with C SF IgG restricted bands (group 3) and in 84% patients with C SF IgG restricted and additional bands (group 4), C SF free kappa bands are present These findings can confirm observations from other studies that C S F oligoclonal free kappa bands can be demonstrated in a high percentage in patients with local IgG synthesis (Sindic and Laterre, 1991b; Gallo et Table 3 Correspondence of immunoblot and index results of free kappa and IgG in the three patient groups Group Oligoclonal IgG pattern Number Free kappa IgG Immunoblota: Index b: Normal Mirror 31 3 I Restricted (plus) Total a +, positive;, negative h +, increased;, normal

7 K J H I inters el a I / Journal of Neuroimmunology 62 (1995) ul 1989; Lolli et a! 1991) In patients with identical IgG bands in CSF and serum (group 2), CSF iree kappa bands are only present in 8% From these data it is obvious that in contrast to intraiheeally produced oligotional IgG, systcmically produced oligoclonal IgG is seldom accompanied with oligoclonal free kappa This phenomenon can probably he explained by the rapid removal of these small free kappa proteins from blood to urine as is found for Bence Jones proteinuria in patients with myeloma In case a laboratory receives only CSF and no serum of a patient it is more conclusive to perform both oligoclonal IgG and oligoclonal free kappa isoelectric focusing When both investigations arc positive, this is a strong indication for intrathecal IgG production When oligoclonal IgG is positive and oligoclonal free kappa is negative, this is a strong indication for systemic IgG production With respect to the quantitative levels, in patients without immunological CNS disturbances, the levels in CSF of tree kappa are mostly below the detection limit of the assay in literature (Fagnart et al, 1988; Lolli et al, 1991; Rudick et al, 1986) We have developed a sensitive method in order to determine normal CSF free kappa levels Our median free kappa serum level (42 mg/1) is nearly 3-times higher than the value of 158 mg/1 reported by Fagnart et al (1988), comparable with the value of 336 mg/1 reported by Rudick et al (1986), and 4-times lower than the value reported by Sollink (1975) Absolute levels of free light chains in body fluids are difficult to compare owing to both the differences in specific anti sera and the absence of a valid free kappa light chain standard The use of an index mitigates the problem of an appropriate standard As free light chains are smaller molecules than albumin, theoretically the index must be higher than 1,0 since the filtration process of the blood CSF barrier is size dependent and proportional for free light chains The median index of 11 for free kappa and the relationship between Q albumin and Q free kappa for the control group can confirm the validity of the use of a free kappa index for establishing intrathecal free kappa production Based on the acceptance that IgG isoelectric focusing is the gold standard for indication of intrathecal IgG production, we can establish for free kappa index a sensitivity of 90% and a specificity of 93%, and for IgG index a sensitivity of 6K% and a specificity of 97% The pathophysiological significance of intrathecal production of free light chains remains obscure Their presence is not due to local degradation of immunoglobulins but excess of free light chains is likely a marker of the ongoing intrathecal humoral immune response In conclusion: CSF oligoclonal free kappa is observed with about the same frequency as that for CSF restricted oligoclonal IgG (groups 3 and 4 in Table 1) In case a laboratory receives only CSF and no serum of a patient oligoclonal free kappa investigation is more conclusive than oligoclonal IgG The correspondence between immunoblot and index findings for free kappa is better than for IgG (Table 3) Free kappa index is more sensitive but somewhat less specific than IgG index for establishing intrathecal immune production (Table 4) References Andersson, M, Alvarez-Cermeno, J, Bemardi, G, Cogato, I, Fredman, P, Frederiksen, J, Frederikson, S, Gallo, P, Grimaldi, LM, Gr0nning, M, Keir, Gt Lamers, K, Link, H, Magalhaes, A, Massaro, AR, Ohman, S, Reiber, H, Ronnb&ck, L, Schluep, M, Schuller, E, Sindic, CJM, Thompson, EJ, Trojano, M and Wurster, U (1994) The role of cerebrospinal fluid analysis in the diagnosis of multiple sclerosis: a consensus report J Neurol Neurosurg Psych 57, Bracco, F, Gallo, P, Menna, R, Battistin, L and Tavolato, B (1987) Free light chains in the CSF in multiple sclerosis J Neurol 234, Decarli, C, Menegus, MA and Rudick, RA (1987) Free light chains in multiple sclerosis and infections of the CNS Neurology 37, Fagnart, OC, Sindic, CJM and Laterre, C (1988) Free kappa and lambda light chain levels in the cerebrospinal fluid of patients with multiple sclerosis and other neurological diseases J Neuroimmunol 19, Gallo, P, Tavolato, Bt Bergenbrant, S and Siden, A, (1989) A study with special reference to the occurrence of free light chains in cerebrospinal fluid with and without oligoclonal immunoglobulin G J Neurol Sci, 94, Haraldson, A, Kock-Jansen, Jaminon, M, van Eck-Arts, PBJM, de Boo, T, Weemaes, CMR and Bakkeren, JAJM (1991) Determination of kappa and lambda light chains in serum immunoglobulins G, A and M Ann Clin Biochem 28, Keir, G Luxton, RW and Thompson, EJ (1990) Isoelectric focusing of cerebrospinal fluid immunoglobulin G: an annotated update Ann Clin Biochem 27, Link, H and Tibbling, G (1977) Principles of albumin and IgG disorders Evaluation of IgG synthesis within the central nervous system in multiple sclerosis Scand J Clin Lab Invest 37, Lolli, F, Siracusa, G, Amato, MP, Fratiglioni, L, Dal Pozzo, G, Galli, E and Amaducci, L (1991) Intrathecal synthesis of free immunoglobulin light chains and IgM in initial multiple sclerosis Acta Neurol Scand 83, Luxton, RW, McLean, BN and Thompson, EJ (1990) Isoelectric focusing versus quantitative measurements in the detection of intrathecal local synthesis of IgG Clin Chim Acta 187, I Ohman, S, Ernerudh, H, Forsberg, P, Henriksson, A, von Schenk, H and Vrethem, M (1992) Comparison of seven formulae and isoelectrofocusing for determination of intrathecally produced IgG in neurological disease Ann Clin Biochem 29, Poser, CM, Paty, DW, Scheinberg, L, McDonald, WI, Davis, FA, Ebers, GC, Johnson, KP, Sibley, WA, Silberberg, DH and Tourtellotte, WW (1983) New diagnostic criteria for multiple sclerosis: Guidelines for research protocols Ann Neurol 13, Reiber, H and Felgenhauer, K (1987) Protein transfer at the blood cerebrospinal fluid barrier and the quantitation of the humoral immune response within the central nervous system Clin Chim Acta 163, Rudick, RA, Pallant, A, Bidlack, JM and Herndon RM (1986) Free kappa light chains in multiple sclerosis spinal fluid Ann Neurol 20, Sindic, CJM and Laterre, C (1991a) Free light chains in CSF from MS

8 KJB Lamers et al / Journal of Neuroimmunology 62 (1995) patients: an affinity-mediated blot study In Wictholter et al (Eds), Current Concepts in MS, Elsevier Science Publishers» pp Sindic, CJM and Laterre, C (1991b) Oligoclona! free kappa and lambda bands in the cerebrospinal fluid of patients with MS and CNS infections An immu no affinity-mediated capillary blot study J Neuroimmunol 33, Sollink, K (1975) Free light chains of immunoglobulins in normal serum and urine determined by radioimmunoassay Scand J Clin Lab Invest 35, Vakaet, A and Thompson, EJ (1985) Free light chains in the cerebrospinal fluid: an indicator of recent immunological stimulation J Neurol Neurosurg Psych 48, Walker, RWH, Keir, G, Johnson, MH and Thompson, EJ (1983) A rapid method for detecting oligoclonal IgG in unconcentrated CSF, by Agarose isoelectric focusing, transfer to cellulose nitrate and immunoperoxidase staining J Neuroimmunol 4,