Scand. .1. tmmtmot. 35, 343-351. 1992
Light Chain Distribution of Anti-Borrelia burgdorferi IgG Antibodies in Lyme Arthritis M. C R U Z & A. S I D E N Department of Neurology, Karolinska Institule. Huddinge University Hospital, Huddinge, Sweden
CruzM.Siden A. Light Chain Distribution of Anli-fi('/r(7/»A»/-,i,'(/((f/('//IgG Antibodies in Lyme Arihrilis, Scand J Immunol I992:35:.M3 51 Joint lluid (Jl I and serum IgG from eighl Lyme arthritis patients was investigated by isoelectric focusing, blolting to membranes of uncoatcd nitrocellulose (NC) as well as Borretia {B.) burgdorferi •AnUgen-conted NC(immunoblot)and immunoenzymatic staining for visualization of Ihe IgG isotype and characterization ofthe kappa, lambda light chain distribution. Oligoclonal bands oflotal IgG occurred in JF as well as in serum (3 cases) or in JF only (I ease): IgG lambda components were present in all four subjects and IgG kappa in three. IgG (Fc frag) reaetive bul kappa.lambda light chain negative oligoclonal bands were present in JF oi'one further palient. Additional investigations supported t!io inierpretiition that these components were free gamma chains. Oligoclonal anti-fl. hurf'ilorfrri \^Q antibodies oecurred in JF as well as in serum (6 cases) or in JF only (I case). There were complete identities, as defined by isoclectrie points and iight ehain lypes. belween JF and serum antibodies in an individual patient while identities belween oligoclonal bands of total IgG and anti-fi. hurgdorfcri IgG were infrequent. Aniibodies of IgG kappa identity were present in all seven palients and six of ihem also had IgG lambda antibodies. The antibody response was, therefore, by definition oligo- and nol monocionai in 6 out of 7 patients. Furthermore, the anii-fi. hur^dorji-ri aniibodies of IgG kappa identity only that were present in one case did not exhibit any ei>nipletely characteristic monoclonal microheterogeneity.
Mahel Cruz. Department ol Neurology. Huddinge University Hospital. S-141H6 Huddinge. Sweden
Infection with the tick-borne spirochete Borrelia [B.) burgdorfert (lj may lead to a multisystem disease primarily affecting the skin, nervous system, heart and joints [for review, see Rel". 2]. The arthritic manifestation. Lyme arlhrilis. is characterized by Intermittent attacks of asymmetric, oligoarticular arlhrilis affecting mainly large joints, especially ihe knee [3]. Some patients may develop a chronic arthritis (4, 5]. The spiroehete has been isolaled from joinl fluid (JF) of Lyme arthritis patients [6, 7] and may persist in synovial tissue [8]. In a previous study. Cruz et at. [9] investigated ihe anU-B. hurgdorferi IgG response in Lyme arthritis JF and serum and found that 7 out of 10 patients had anti-B. burgdorferi IgG aniibodies with restricted electrophoretic heterogeneities (oligoclonal bands) in serum and/or JF. Oligoclonal bands of total IgG in either JF only or JF and serum were detected in 5 out of 10 subjeets. The
oligoelonal anti-tf. biogdorjeri IgG antibody bands were generally not identical with the oligoclonal bands of total IgG. In order to define further the anti-fi. tmrgdor/('/•/IgCi response in Lyme arthritis JF and serum, we applied agarose gel isoelectric focusing (AIF) which separates proteins according to isoelectrie point (pi) differences, SDS polyacrylamide gel eleetrophoresis (SDS PAGE) which separates proteins according to moleeular weight (mol. wt.) differences and protein blotting with immunoenzymatie staining. The proteins were blotted to uncoaled nitrocellulose (NC) membranes for visualization of total IgG patterns as well as to B. burgdorferi antigen-coated NC membranes (immunoblot) for visualization of anti-6. burgdorferi IgG antibody patterns. The immunoenzymatic staining included, besides a visualization of the IgG isotype, also a characterization of the kappa and lambda light chain distribution. 343
344
M. Cruz & A. Siden TABLE I. Basic clinieal data in eight patients with Lyme arthritis. Anti-fi. burgdorferi IgG aniibodies were measured by enzyme-linked immunosorbenl assay as previously described [10]: the upper reference OD value was 0.160 IgG eoneentration (g/l) Duration of Patient no. Age/sex arthritis (months) 1 2
3 4 5* 6 7 8
15/M 30/M 13/M 33/M 55/M 35/F 59/M 67/M
6 19 12 2 134 5 2 25
Anti-B. hurgdorferi IgG antibodies
JF
S
JF {OD)
S (OD)
14.6 13.6 18.8 11.4 7.8 7.3 14.2 19.2
18.6 16.4 17.6 II.O 10.7 12.7 19.5 lt^.4
0.350 1.180 0.250 0.750 0.010 0.270 0.550 0.299
0.320 1.250 0.360 0.115 0.010 0.270 0.600 0.291
JF. joinl fluid: S, serum: OD. optical density. * In a previous publicalion [9], the disease duration ofthis patient was erroneously given as 18 months.
MATERIALS AND METHODS Patients and samples. Paired JF and serum samples were obtained from eight patients with Lyme arthritis. These patients were selected for ihe present investigation beeause their sera and/or JF exhibited, according to results from a previous study [9], oligoclonal bands of total IgG (1 ease), oligoclonal anli-fi. burgdorferi IgG antibody bands (3 eases) or both these types of IgG eomponents (4 cases). Table I gives certain basic clinical data. A clinically definite erythema chronicum migrans prior to the arthritic manifestations was present in four cases. The joint symptoms were recurrent or ehronic and had mono-/oligoarticular or polyartieular distributions with predominance for Ihe knee joints. Reagents. Rabbit antisera against human IgG (Fc frag, code A089; gamma chain, code A424). IgM (my chain, code A091), IgA (alpha chain, code A262), bound kappa (code A192) and bound lambda (code A194) light chains, free kappa (code AlOO) and free lambda (code AlOi) light chains were obtained from Dako (Copenhagen. Denmark). Vectastain ABC kil (biotinylated goat anii-rabbit IgG and avidin-biotin peroxidase eomplex) were purchased from Veclor Laboralories (Burlinghame, Ala.. USA). NC membranes 0.45 ;(m were obtained from Schleicher & Schuell (Dassel. Germany) and bovine serum albumin (BSA) from Sigma (St Louts, Mo., USA). Isogcl agarose (FMCBioProducts. Rockiand. NY. USA) and Ampholines pH 3.5 10 (Pharmacia LKB Biotechnology, Uppsala, Sweden) were used for AIF. Sonicated whole B. burgdorferi antigen was obtained from Dr K. Hansen (Stalens Seruminstitute. Copenhagen, Denmark) and prepared as previously described [10]. Detection of light chain di.strihution of total IgG and anti-B. hurgdorferi IgG antihodies. Casting of gels for AIF and Ihe focusing procedure were performed as previously described [II]. Prior lo application, the JF and serum samples were each diluted to two different IgG concentrations (30 mg/l and 50 mg/1) with phosphale buffered saline (PBS) pH 7,2. One 200 mm x IOO
mm X I mm AIF gel was used for the paired JF/serum samples from one patient: 15 jA of the dilutions containing 30 mg/l IgG were applied in triplicate on one half of the gel (for deteeiion of light chain distribution of total IgG) and 15 ^\ of the dilutions containing 50 mg.l IgG were applied in triplicate on the other half of the gel (for detection of light ehain distribution of antiB. hurgdorferi IgG antibodies). The coating of NC membranes with B. burgdorferi antigen and the protein transfer procedures from the gel were performed as previously described [9), Briefly, the transfer by capillary forces of total protein to uncoated NC and by affinity-driven transfer (immunoblot) of anti-B. hurgdorferi aniibodies to antigen-coated NC were perlbrmed in parallel. This was achieved by applying the PBS welted sheets of uncoated NC and anligen-coated NC on the respective halves of the gel. After 1 h transfer at room temperature (20 C). the NC sheets were cut in three parts (each containing proteins from one of the paired JF.serimi triplicates) before being taken away from the gel surface. Empty protein binding spaces were then blocked with 3"'" (wt/vol) BSA in PBS for 30 min followed by a short rinse in PBS. The three NC strips from each half of the gel were immunoenzymatically stained for detection of toial IgG as well as its light chain distribution (NC strips from the gel hairwhere30mg/l IgG concentraiions wereapplied) and for detection of anti-ff. hurgdorferi IgG antibodies as well as their light chain distribution (NC strips from the gel half where 50 mg/l IgG concentrations were applied). These stainings were performed by incubating the NC membranes at room temperature in the following solutions prepared in PBS; (I) 2 h in the first antisera—rabbit anti-buman IgG (Fc frag) l/IOOO. bound kappa light chains I/IOOO and bound lambda light chains 1/6(X): (2) 2 h in the second antiserum— hiotinylaled goat anli-rabbit IgG 1,1000: (3) I h in avidin-biotin peroxidase complex I /200. and (4) peroxidase staining as previously described [12], Three washing cycles of 10 min eaeh with PBS were performed belween every step. In one case, where IgG (Fc frag)
Anti-B. burgdorferi IgG Antibodies positive but kappa/lambda light chain negative oligoclonal bands were observed in JF. additional detection procedures were aiso fjerformcd with the following primary antisera: rabbit anti-human IgG (gamma ehain) I/IOOO. IgM (my chain) 1,500. IgA (alpha chain) 1/500. free kappa and free lambda light chains 1/250, The remaining steps ofthe immunoenzymatie staining were identical to those already described, SDS PACE, electrohlo! and immunoenzymalic slaining. JF and serum samples from Ihe patient, with IgG (Fc frag) positive but kappa/lambda light chain negative oligoclonal bands, were separated by SDS PAGE (lO'l^i polyaerylamide) as previously described [13] in native as well as reduced (with beta-mercaptoethanol) form. Two slab gels were used for the same run as follows: 0,5 //g of native JF IgG were applied in each of six well.sin onehal! of age! and 0,5/ig of reduced JF IgG in six wells in the other half of the gel. the same procedure was followed regarding native and reduced serum IgG applied on the seeond slab gel. After electrophoresis, Ihe separated prolein were electroblotled to NC membranes [I4j which were then blocked with yVu {wt/vol,) BSA in PBS for 1 h al room temperature. Eaeh membrane with the cloctroblolted proteins was cut in to 12 strips (6 strips carrying native IgG and 6 strips carrying reduced IgG), This was followed by immunoenzymatie staining by incubating the NC strips in the following solutions prepared in PBS: (I) 2 h in the first antisera -rabbit anli-human IgG (Fc frag) I/IOOO, IgG (gamma chain) l/iOOO, bound kappa lights chains 1/1000. bound lambda light chains l.'6OO. and free kappa and free lambda light chains 1/ 250; (2) 2 h in Ihe second antiserum—biotinylated goat anti-rabbit IgG I/IOOO; (3) I h in avidin-biotin peroxidase complex 1 /200, and (4) peroxidase staining. Three washing cycles of tO min eaeh with PBS were performed between every step.
RESULTS Oligoclonal bands of total IgG Oligoclonal IgG (Fc frag) reactive bands were delected in serum and/or JF from five patients
345
(Table II, patients nos 1-5), Three of them (nos 13) had such bands in JF as well as in serum, IgG lambda bands were present in all these three cases and two of them, in addition,, showed IgG kappa bands having more acidic pi values than those of lambda identity; there was a complete identity, as defined by pi values and light chain types, between the J F and serum IgG components in the individual subject. The remaining two patients (nos 4 and 5) had oligoclonal IgG (Fc frag) reactive bands in JF only, IgG kappa as well as IgG lambda bands were detected in one of these
IgG
4.5 6
9.5
s Jf
s jf
s Jf
FtG. I, Kappa (K ) and lambda (/,) light chain distribution of total IgG in serum (s) and joint fluid (jf) IVom one Lyme arthritis patient having an intrasynovial production of oligoclonal IgG, Total protein transfer to uncoated NC after AIF (the anode was al the top); the approximate shape ofthe pH gradient is shown to the left, Oligoelonal components of total IgG with kappa or lambda light chains are indicated by arrow-heads.
TAHLF: II, Light chain distribution of otigoclonal bands of total IgG and of oligocional anti-B, burgdorferi IgG aniibodies in eight patients wilh Lyme arthritis Oligoclonal bands of total IgG (light chain identities)
1 2 3 4 5 6 7 8
S
JF
+ (L) + (K/L) + (K/L)
+ + + +
JF
Patient no. + + + + +
(L) (K/L) (K/L) (K/L) (no K/no L)
Oligoclonal anti-ff. hurgdorferi \gG bands (light cham identities) S (K/L) (K/L) (K) (K/L)
+ (K/L) + (K/L) + (K)
+ (K/L) + (K/L) + (K/L)
+ (K/L) -1- (K/L) + (K/L)
-
JF. joint fluid; S. serum; K. kappa lighl chain; L. lambda light chain.
346
M. Cru= & A. Siden
free K
IgG
y
free K
8 |f
8 jf
pH
4.5 6
9.5 S jf
S jf
S jf
S jf
Fi(i, 2, Serum (s) and joint liuid (jf) Irom palicnl no, 5 examined under ihe same experimental conditions as in Fig, I. Four prominent IgG (Fc frag) as well asganinui cfiain reactive bands arc indicated by arrow-heads. These bands were not stained by antiscra against bound or free light chains: nor were there any indications of free liglit chain components.
cases, the kappa components having more alkaline pi values (Fig. I), The second patient (no. 5) exhibited IgG (Fc frag) reactive bands which were not stained by the light chain antisera, however (Fig, 2), The JF and serum samples from Ihis latlerpatienl were also examined for IgG (gamma chain), IgM (my chain). IgA (alpha chain), free kappa and free lambda light chain reactivities. Only the gamma chain specific antiserum stained the IgG (Fc frag) reactive bands and the light chain anti.sera did not indicate the presence of free light chains (Fig. 2), Oligoclonal IgG (Fc frag I gamma chain) reactive hut kappajlambda light chain negative components To confirm further the AIF results regarding palicnt no, 5. the JF and serum proteins were separated according to mol, wt, by SDS-PAGE under native as well as reducing conditions with the following main results (Fig. 3). The antisera against gamma chains and kappa/lambda lighl chains stained components at an approximate mol. wt. of 150 kDa in native JF and serum. These bands were nol present in the reduced samples which instead showed broad and prominent light
chain reactive zones in the 25 30 kDa mol. wt. range. The native JF exhibited one gamma chain reactive band at a mol. wt, of 55-60 kDa and the reduced JF one sueh component at about 30 kDa. There were no prominent kappa/lambda light chain reactive bands within Ihe 25-30 kDa mol. wt. range in native JF and .serum, Oligoclonal unli-B. burgdorferi IgG antibodies Six of the seven anti-B, burgdorferi IgG antibody positive patients had oligoclonal antibodies in JF as well as in serum (Table II, patients nos 1 3 and 6-8) and the seventh patient (no, 4) had such components in JF only. Finally, patient no. 5, whose JF exhibited IgG (Fc frag/gamma chain) reactive but kappa/lambda light chain negative bands, had no oligoclonal anti-B, hurgdorferi IgG anlibodies, Imtnunoblot for identification of the antibody light chain types (Table II, patients nos 1-4 and 6-8) revealed oligoclonal IgG kappa as well as IgG lambda anti-B. burgdorferi components in six patients (nos 1, 2, 4 and 6- 8) and IgG kappa bands only in one subject (no. 3). Among the six patients wilh anlibodies of both light chain types, five had such anti-B. burgdorferi bands in JF as
Anti-B. burgdorferi IgG Antibodies
Reduced MW
T
K
A
7
K
A
94
67
30
20 14 jf s jf
s jf s
jf s jf s jf s
Fi(i. 3. SDS PAGForniilivc and reduced joint lluid (jD and serum (s) samples from palionl No. .S. The anode was al the hotlom and Ihc approximalc mol. wl. values are shown to the left. After eleclroblotlin}!. NC strips were stained for deteetion of gamma {•;). kappa (K)and lambda (/.) chain reactivities. Symbols; > = 150 kDa gamma chain as well as kappa lambda light chain reactive components; > =25 30 kDa kappa' lambda light chain reactive components: • = 55 60 kDa gamma ehain reactive components.
well as in seriini and one patient exhibited oligoclonal anti-6. burgdorferi antibodies in JF only. The sLtbjecl (no. }>) with only IgO kappa anli-B. burgdorferi aniibodies had such components in JF as well as in serum. Complete identities, as defined by pi values and light chuin types, between the antibody palterns in JF and serum existed in every individual patient with anti-/J. burgdotferi IgG responses in bolh comparttiienis. Fig. 4A and B gives the patlerns of total IgG and anli-B. hurgdorferi IgG antibodies, respectively, in the patient wiih the most prominent oligoclonal antibody response: IgG kappa and IgG lambda aniibody band spectra (Fig. 4B) covering the pi intervals o\' pH 6.5 7.5 and 6.5 H.5, respectively. The oligoclonal IgG kappa antiB. burgdorferi antibodies in the remaining six cases were focused within the pi intervals of pH
7-9 or 8 y. In one of these patients (no. 3). the components focused as two separated band spectra but in ihe remaining cases they exhibited the typical microhelerogeneous character exempliHed in Fig. 4B. The oligoclonal IgG lambda antiB. burgdorferi aniihodiQ^ in five out of six subjects showed a relatively uniform pattern as excrnplilied in Fig. AC: a restricted number (3-5) of bands focused within the pi interval of pH 8-8.5. In one o\~ these eases (Fig. 4C) there appeared, in addition, a second group of IgG lambda antibody bands in the pi interval of pH 6.5-7. Among the seven patients being positive for oligocional anti-fi. burgdorferi IgG antibodies, three exhibited no detectable oligoclonal bands of total IgG w hile four patients had both these types ofcomponents in JF and serum or JF only. In one subject, the oligoclonal bands of total IgG were identical, as defined by p! values and light chain
348
M. Cruz & A. Siden
W
o
rt H
(0
B-=.
CO
o
CO
(0
CQ
Light Chain Distribution of Anti-Borrelia burgdorferi IgG Antibodies in Lyme Arthritis M. C R U Z & A. S I D E N Department of Neurology, Karolinska Institule. Huddinge University Hospital, Huddinge, Sweden
CruzM.Siden A. Light Chain Distribution of Anli-fi('/r(7/»A»/-,i,'(/((f/('//IgG Antibodies in Lyme Arihrilis, Scand J Immunol I992:35:.M3 51 Joint lluid (Jl I and serum IgG from eighl Lyme arthritis patients was investigated by isoelectric focusing, blolting to membranes of uncoatcd nitrocellulose (NC) as well as Borretia {B.) burgdorferi •AnUgen-conted NC(immunoblot)and immunoenzymatic staining for visualization of Ihe IgG isotype and characterization ofthe kappa, lambda light chain distribution. Oligoclonal bands oflotal IgG occurred in JF as well as in serum (3 cases) or in JF only (I ease): IgG lambda components were present in all four subjects and IgG kappa in three. IgG (Fc frag) reaetive bul kappa.lambda light chain negative oligoclonal bands were present in JF oi'one further palient. Additional investigations supported t!io inierpretiition that these components were free gamma chains. Oligoclonal anti-fl. hurf'ilorfrri \^Q antibodies oecurred in JF as well as in serum (6 cases) or in JF only (I case). There were complete identities, as defined by isoclectrie points and iight ehain lypes. belween JF and serum antibodies in an individual patient while identities belween oligoclonal bands of total IgG and anti-fi. hurgdorfcri IgG were infrequent. Aniibodies of IgG kappa identity were present in all seven palients and six of ihem also had IgG lambda antibodies. The antibody response was, therefore, by definition oligo- and nol monocionai in 6 out of 7 patients. Furthermore, the anii-fi. hur^dorji-ri aniibodies of IgG kappa identity only that were present in one case did not exhibit any ei>nipletely characteristic monoclonal microheterogeneity.
Mahel Cruz. Department ol Neurology. Huddinge University Hospital. S-141H6 Huddinge. Sweden
Infection with the tick-borne spirochete Borrelia [B.) burgdorfert (lj may lead to a multisystem disease primarily affecting the skin, nervous system, heart and joints [for review, see Rel". 2]. The arthritic manifestation. Lyme arlhrilis. is characterized by Intermittent attacks of asymmetric, oligoarticular arlhrilis affecting mainly large joints, especially ihe knee [3]. Some patients may develop a chronic arthritis (4, 5]. The spiroehete has been isolaled from joinl fluid (JF) of Lyme arthritis patients [6, 7] and may persist in synovial tissue [8]. In a previous study. Cruz et at. [9] investigated ihe anU-B. hurgdorferi IgG response in Lyme arthritis JF and serum and found that 7 out of 10 patients had anti-B. burgdorferi IgG aniibodies with restricted electrophoretic heterogeneities (oligoclonal bands) in serum and/or JF. Oligoclonal bands of total IgG in either JF only or JF and serum were detected in 5 out of 10 subjeets. The
oligoelonal anti-tf. biogdorjeri IgG antibody bands were generally not identical with the oligoclonal bands of total IgG. In order to define further the anti-fi. tmrgdor/('/•/IgCi response in Lyme arthritis JF and serum, we applied agarose gel isoelectric focusing (AIF) which separates proteins according to isoelectrie point (pi) differences, SDS polyacrylamide gel eleetrophoresis (SDS PAGE) which separates proteins according to moleeular weight (mol. wt.) differences and protein blotting with immunoenzymatie staining. The proteins were blotted to uncoaled nitrocellulose (NC) membranes for visualization of total IgG patterns as well as to B. burgdorferi antigen-coated NC membranes (immunoblot) for visualization of anti-6. burgdorferi IgG antibody patterns. The immunoenzymatic staining included, besides a visualization of the IgG isotype, also a characterization of the kappa and lambda light chain distribution. 343
344
M. Cruz & A. Siden TABLE I. Basic clinieal data in eight patients with Lyme arthritis. Anti-fi. burgdorferi IgG aniibodies were measured by enzyme-linked immunosorbenl assay as previously described [10]: the upper reference OD value was 0.160 IgG eoneentration (g/l) Duration of Patient no. Age/sex arthritis (months) 1 2
3 4 5* 6 7 8
15/M 30/M 13/M 33/M 55/M 35/F 59/M 67/M
6 19 12 2 134 5 2 25
Anti-B. hurgdorferi IgG antibodies
JF
S
JF {OD)
S (OD)
14.6 13.6 18.8 11.4 7.8 7.3 14.2 19.2
18.6 16.4 17.6 II.O 10.7 12.7 19.5 lt^.4
0.350 1.180 0.250 0.750 0.010 0.270 0.550 0.299
0.320 1.250 0.360 0.115 0.010 0.270 0.600 0.291
JF. joinl fluid: S, serum: OD. optical density. * In a previous publicalion [9], the disease duration ofthis patient was erroneously given as 18 months.
MATERIALS AND METHODS Patients and samples. Paired JF and serum samples were obtained from eight patients with Lyme arthritis. These patients were selected for ihe present investigation beeause their sera and/or JF exhibited, according to results from a previous study [9], oligoclonal bands of total IgG (1 ease), oligoclonal anli-fi. burgdorferi IgG antibody bands (3 eases) or both these types of IgG eomponents (4 cases). Table I gives certain basic clinical data. A clinically definite erythema chronicum migrans prior to the arthritic manifestations was present in four cases. The joint symptoms were recurrent or ehronic and had mono-/oligoarticular or polyartieular distributions with predominance for Ihe knee joints. Reagents. Rabbit antisera against human IgG (Fc frag, code A089; gamma chain, code A424). IgM (my chain, code A091), IgA (alpha chain, code A262), bound kappa (code A192) and bound lambda (code A194) light chains, free kappa (code AlOO) and free lambda (code AlOi) light chains were obtained from Dako (Copenhagen. Denmark). Vectastain ABC kil (biotinylated goat anii-rabbit IgG and avidin-biotin peroxidase eomplex) were purchased from Veclor Laboralories (Burlinghame, Ala.. USA). NC membranes 0.45 ;(m were obtained from Schleicher & Schuell (Dassel. Germany) and bovine serum albumin (BSA) from Sigma (St Louts, Mo., USA). Isogcl agarose (FMCBioProducts. Rockiand. NY. USA) and Ampholines pH 3.5 10 (Pharmacia LKB Biotechnology, Uppsala, Sweden) were used for AIF. Sonicated whole B. burgdorferi antigen was obtained from Dr K. Hansen (Stalens Seruminstitute. Copenhagen, Denmark) and prepared as previously described [10]. Detection of light chain di.strihution of total IgG and anti-B. hurgdorferi IgG antihodies. Casting of gels for AIF and Ihe focusing procedure were performed as previously described [II]. Prior lo application, the JF and serum samples were each diluted to two different IgG concentrations (30 mg/l and 50 mg/1) with phosphale buffered saline (PBS) pH 7,2. One 200 mm x IOO
mm X I mm AIF gel was used for the paired JF/serum samples from one patient: 15 jA of the dilutions containing 30 mg/l IgG were applied in triplicate on one half of the gel (for deteeiion of light chain distribution of total IgG) and 15 ^\ of the dilutions containing 50 mg.l IgG were applied in triplicate on the other half of the gel (for detection of light ehain distribution of antiB. hurgdorferi IgG antibodies). The coating of NC membranes with B. burgdorferi antigen and the protein transfer procedures from the gel were performed as previously described [9), Briefly, the transfer by capillary forces of total protein to uncoated NC and by affinity-driven transfer (immunoblot) of anti-B. hurgdorferi aniibodies to antigen-coated NC were perlbrmed in parallel. This was achieved by applying the PBS welted sheets of uncoated NC and anligen-coated NC on the respective halves of the gel. After 1 h transfer at room temperature (20 C). the NC sheets were cut in three parts (each containing proteins from one of the paired JF.serimi triplicates) before being taken away from the gel surface. Empty protein binding spaces were then blocked with 3"'" (wt/vol) BSA in PBS for 30 min followed by a short rinse in PBS. The three NC strips from each half of the gel were immunoenzymatically stained for detection of toial IgG as well as its light chain distribution (NC strips from the gel hairwhere30mg/l IgG concentraiions wereapplied) and for detection of anti-ff. hurgdorferi IgG antibodies as well as their light chain distribution (NC strips from the gel half where 50 mg/l IgG concentrations were applied). These stainings were performed by incubating the NC membranes at room temperature in the following solutions prepared in PBS; (I) 2 h in the first antisera—rabbit anti-buman IgG (Fc frag) l/IOOO. bound kappa light chains I/IOOO and bound lambda light chains 1/6(X): (2) 2 h in the second antiserum— hiotinylaled goat anli-rabbit IgG 1,1000: (3) I h in avidin-biotin peroxidase complex I /200. and (4) peroxidase staining as previously described [12], Three washing cycles of 10 min eaeh with PBS were performed belween every step. In one case, where IgG (Fc frag)
Anti-B. burgdorferi IgG Antibodies positive but kappa/lambda light chain negative oligoclonal bands were observed in JF. additional detection procedures were aiso fjerformcd with the following primary antisera: rabbit anti-human IgG (gamma ehain) I/IOOO. IgM (my chain) 1,500. IgA (alpha chain) 1/500. free kappa and free lambda light chains 1/250, The remaining steps ofthe immunoenzymatie staining were identical to those already described, SDS PACE, electrohlo! and immunoenzymalic slaining. JF and serum samples from Ihe patient, with IgG (Fc frag) positive but kappa/lambda light chain negative oligoclonal bands, were separated by SDS PAGE (lO'l^i polyaerylamide) as previously described [13] in native as well as reduced (with beta-mercaptoethanol) form. Two slab gels were used for the same run as follows: 0,5 //g of native JF IgG were applied in each of six well.sin onehal! of age! and 0,5/ig of reduced JF IgG in six wells in the other half of the gel. the same procedure was followed regarding native and reduced serum IgG applied on the seeond slab gel. After electrophoresis, Ihe separated prolein were electroblotled to NC membranes [I4j which were then blocked with yVu {wt/vol,) BSA in PBS for 1 h al room temperature. Eaeh membrane with the cloctroblolted proteins was cut in to 12 strips (6 strips carrying native IgG and 6 strips carrying reduced IgG), This was followed by immunoenzymatie staining by incubating the NC strips in the following solutions prepared in PBS: (I) 2 h in the first antisera -rabbit anli-human IgG (Fc frag) I/IOOO, IgG (gamma chain) l/iOOO, bound kappa lights chains 1/1000. bound lambda light chains l.'6OO. and free kappa and free lambda light chains 1/ 250; (2) 2 h in Ihe second antiserum—biotinylated goat anti-rabbit IgG I/IOOO; (3) I h in avidin-biotin peroxidase complex 1 /200, and (4) peroxidase staining. Three washing cycles of tO min eaeh with PBS were performed between every step.
RESULTS Oligoclonal bands of total IgG Oligoclonal IgG (Fc frag) reactive bands were delected in serum and/or JF from five patients
345
(Table II, patients nos 1-5), Three of them (nos 13) had such bands in JF as well as in serum, IgG lambda bands were present in all these three cases and two of them, in addition,, showed IgG kappa bands having more acidic pi values than those of lambda identity; there was a complete identity, as defined by pi values and light chain types, between the J F and serum IgG components in the individual subject. The remaining two patients (nos 4 and 5) had oligoclonal IgG (Fc frag) reactive bands in JF only, IgG kappa as well as IgG lambda bands were detected in one of these
IgG
4.5 6
9.5
s Jf
s jf
s Jf
FtG. I, Kappa (K ) and lambda (/,) light chain distribution of total IgG in serum (s) and joint fluid (jf) IVom one Lyme arthritis patient having an intrasynovial production of oligoclonal IgG, Total protein transfer to uncoated NC after AIF (the anode was al the top); the approximate shape ofthe pH gradient is shown to the left, Oligoelonal components of total IgG with kappa or lambda light chains are indicated by arrow-heads.
TAHLF: II, Light chain distribution of otigoclonal bands of total IgG and of oligocional anti-B, burgdorferi IgG aniibodies in eight patients wilh Lyme arthritis Oligoclonal bands of total IgG (light chain identities)
1 2 3 4 5 6 7 8
S
JF
+ (L) + (K/L) + (K/L)
+ + + +
JF
Patient no. + + + + +
(L) (K/L) (K/L) (K/L) (no K/no L)
Oligoclonal anti-ff. hurgdorferi \gG bands (light cham identities) S (K/L) (K/L) (K) (K/L)
+ (K/L) + (K/L) + (K)
+ (K/L) + (K/L) + (K/L)
+ (K/L) -1- (K/L) + (K/L)
-
JF. joint fluid; S. serum; K. kappa lighl chain; L. lambda light chain.
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free K
IgG
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free K
8 |f
8 jf
pH
4.5 6
9.5 S jf
S jf
S jf
S jf
Fi(i, 2, Serum (s) and joint liuid (jf) Irom palicnl no, 5 examined under ihe same experimental conditions as in Fig, I. Four prominent IgG (Fc frag) as well asganinui cfiain reactive bands arc indicated by arrow-heads. These bands were not stained by antiscra against bound or free light chains: nor were there any indications of free liglit chain components.
cases, the kappa components having more alkaline pi values (Fig. I), The second patient (no. 5) exhibited IgG (Fc frag) reactive bands which were not stained by the light chain antisera, however (Fig, 2), The JF and serum samples from Ihis latlerpatienl were also examined for IgG (gamma chain), IgM (my chain). IgA (alpha chain), free kappa and free lambda light chain reactivities. Only the gamma chain specific antiserum stained the IgG (Fc frag) reactive bands and the light chain anti.sera did not indicate the presence of free light chains (Fig. 2), Oligoclonal IgG (Fc frag I gamma chain) reactive hut kappajlambda light chain negative components To confirm further the AIF results regarding palicnt no, 5. the JF and serum proteins were separated according to mol, wt, by SDS-PAGE under native as well as reducing conditions with the following main results (Fig. 3). The antisera against gamma chains and kappa/lambda lighl chains stained components at an approximate mol. wt. of 150 kDa in native JF and serum. These bands were nol present in the reduced samples which instead showed broad and prominent light
chain reactive zones in the 25 30 kDa mol. wt. range. The native JF exhibited one gamma chain reactive band at a mol. wt, of 55-60 kDa and the reduced JF one sueh component at about 30 kDa. There were no prominent kappa/lambda light chain reactive bands within Ihe 25-30 kDa mol. wt. range in native JF and .serum, Oligoclonal unli-B. burgdorferi IgG antibodies Six of the seven anti-B, burgdorferi IgG antibody positive patients had oligoclonal antibodies in JF as well as in serum (Table II, patients nos 1 3 and 6-8) and the seventh patient (no, 4) had such components in JF only. Finally, patient no. 5, whose JF exhibited IgG (Fc frag/gamma chain) reactive but kappa/lambda light chain negative bands, had no oligoclonal anti-B, hurgdorferi IgG anlibodies, Imtnunoblot for identification of the antibody light chain types (Table II, patients nos 1-4 and 6-8) revealed oligoclonal IgG kappa as well as IgG lambda anti-B. burgdorferi components in six patients (nos 1, 2, 4 and 6- 8) and IgG kappa bands only in one subject (no. 3). Among the six patients wilh anlibodies of both light chain types, five had such anti-B. burgdorferi bands in JF as
Anti-B. burgdorferi IgG Antibodies
Reduced MW
T
K
A
7
K
A
94
67
30
20 14 jf s jf
s jf s
jf s jf s jf s
Fi(i. 3. SDS PAGForniilivc and reduced joint lluid (jD and serum (s) samples from palionl No. .S. The anode was al the hotlom and Ihc approximalc mol. wl. values are shown to the left. After eleclroblotlin}!. NC strips were stained for deteetion of gamma {•;). kappa (K)and lambda (/.) chain reactivities. Symbols; > = 150 kDa gamma chain as well as kappa lambda light chain reactive components; > =25 30 kDa kappa' lambda light chain reactive components: • = 55 60 kDa gamma ehain reactive components.
well as in seriini and one patient exhibited oligoclonal anti-6. burgdorferi antibodies in JF only. The sLtbjecl (no. }>) with only IgO kappa anli-B. burgdorferi aniibodies had such components in JF as well as in serum. Complete identities, as defined by pi values and light chuin types, between the antibody palterns in JF and serum existed in every individual patient with anti-/J. burgdotferi IgG responses in bolh comparttiienis. Fig. 4A and B gives the patlerns of total IgG and anli-B. hurgdorferi IgG antibodies, respectively, in the patient wiih the most prominent oligoclonal antibody response: IgG kappa and IgG lambda aniibody band spectra (Fig. 4B) covering the pi intervals o\' pH 6.5 7.5 and 6.5 H.5, respectively. The oligoclonal IgG kappa antiB. burgdorferi antibodies in the remaining six cases were focused within the pi intervals of pH
7-9 or 8 y. In one of these patients (no. 3). the components focused as two separated band spectra but in ihe remaining cases they exhibited the typical microhelerogeneous character exempliHed in Fig. 4B. The oligoclonal IgG lambda antiB. burgdorferi aniihodiQ^ in five out of six subjects showed a relatively uniform pattern as excrnplilied in Fig. AC: a restricted number (3-5) of bands focused within the pi interval of pH 8-8.5. In one o\~ these eases (Fig. 4C) there appeared, in addition, a second group of IgG lambda antibody bands in the pi interval of pH 6.5-7. Among the seven patients being positive for oligocional anti-fi. burgdorferi IgG antibodies, three exhibited no detectable oligoclonal bands of total IgG w hile four patients had both these types ofcomponents in JF and serum or JF only. In one subject, the oligoclonal bands of total IgG were identical, as defined by p! values and light chain
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