A standardized method for quantitating the complement-mediated immune complex solubilizing capacity of human serum

Journal of Immunological Methods, 59 ¢1983) 369-380

369

Elsevier Biomedical Press

A Standardized Method for Quantitating the Complement-Mediated Immune Complex Solubilizing Capacity of Human Serum Gunnar Baatrup, Ivan Petersen, Sven-Erik Svehag and h'an Brandslund Institute o] Medtcal Mtcrobtologv. Odense Umt'.er~itv, J, B. 14'tnsl~ws liel 19. DK- 5(900 Oden~e C. Denmark

(Receb,ed 21 June 1982. accepted 21 November 1982)

A standardized radioassay for measuring the complement-medmted ~mmune complex solubihzmg capacit,, (CMSC) and the mitml kinetics of the solubdization (IKS) reaction is described. The total complement (C)-mediated solubih~ng capacity was determined after incubation of diluted serum and ]251-BSA-anti-BSA. Percentage C-mediated sohibihzation tCMS) was measured after centrifugatlon by determining the distribution of radioactivity. The dependency of CMSC upon factors such as serum dilution and buffer system used, amount of IC added to serum, serum storage conditions and centrifugation conditions was investigated in order to optimize the assay. The CVj of the standardized assay was 0.10-0.17 depending upon the CMSC level measured. Treatment which inactivates C factors (heating), interferes with C activation (EDTA) or activates and consumes C components (zymosan) markedly reduces the CMSC. Prelinunary investigation of pathological sera showed that both IKS and CMSC were clearly reduced m SLE sera. By contrast, rheumatoid arthritis sera exhibited normal IKS and only marginal reduction in CMSC. Key words: tmmune complexes - - complement-medtated solubtlizatton -- tmmune complex solubihzatton

Introduction Miller an d Nu s s e n z w e ig (1975) d e m o n s t r a t e d a solubilizing effect o f the c o m p l e m e n t system on p r e f o r m e d i m m u n e c o m p l e x e s (IC). Since then the underlying m e c h a n i s m has been investigated and partly clarified (Rajnav61gyi et al., 1978; Schifferli et al., 1980; Fujita et al.. 1981). A c c o r d i n g to the intercalation hypothesis of Miller and Nussenzweig, the reaction is stoichiometric. A n event crucial to the solubilization reaction appears to be the assembly of factor B - d e p e n d e n t amplification C3 c o n v e r t a s e on the IC generating accelerated activation of C3. Large a m o u n t s o f nascent C 3 b then bind strongly to the IC and c o n t r i b u t e to the d i s r u p t i o n o f s o m e primary, a n t i g e n - a n t i b o d y b o n d s a n d subsequent dissociation of IC into smaller c o m p l e x e s ( C z o p and Nussenzweig, 1976). T h u s the effect is m a i n l y a t t r i b u t a b l e to p r o t e i n s of the alternative p a t h w a y (Miller, 1976; T a k a h a s h i et al., 1980), although 0022= 1759/83/0000-0000/'$03.00 '~' 1983 Elsevier Sctence Publishers

370 the reaction is facilitated by an intact classical pathway (Takahashi et al., 1978). The solubilized IC have been shown to be heterogeneous in size (Czop and Nussenzweig, 1976). It has been postulated (Takahashi et al., 1976) that this phenomenon may be of clinical relevance in certain diseases and recently 3 reports dealing ~ith the complement-mediated solubilization capacity (CMSC) of sera from patients x~ith IC-associated diseases have appeared (Aguado et al., 1981; Schifferli et al.. 1981, Sakurai et al.. 1982). In previous studies radioassay has been used to measure the CMSC. the preformed IC being BSA-anti BSA with ~z-~I-BSA as tracer. However, the assay used has not been standardized as regards centrifugation conditions, amount of IC used. IC storage, type of controls etc. The aim of the present study was to investigate the influence of several methodological variables in order to develop a reproducible and standardized assay for CMSC determinations and for studies of the kinetics of the reaction.

Materials and Methods Patients and normal donor sera

Blood was obtained from 4 SEE patients (diagnosed on ARA criteria: I male, 3 females, ages 14-35). 4 patients with classical rheumatoid arthritis (AILA, criteria; 2 males; 2 females, ages 44-58) and 1 patient (female, age 17) ,xith rapidly progressing glomerulonephritis. All were inpatients at the Odense University Hospital. Blood from 50 normal donors (registered blood donors and medical studentsl was also obtained (27 males, 23 females, ages 23-65. mean 41). Blood was collected in ordinary glass tubes. The blood was allowed to clot for 75 min at 22°C, and centrifuged for 10 min at 1000 x g. Serum was immediately transferred to 3 ml PVC tubes in aliquots from 200 vl to 1000 ~1, quick-frozen at - 7 0 ° C in an ethanol bath, and stored at - 7 0 ° C . Serum samples once thawn were not reused. Negallt,e control sera

Four types of negative control sera were used. I I } Sera heated for 30 rain at 56°C. (2) EDTA-treated serum (final concentrations of EDTA 10 mM or 20 mM). (3) Zymosan-treated serum: 2 ml zymosan stock solution was centrifuged, and the supernatant discarded. The precipitate was resuspended in I ml serum and incubated for 60 min on a shaker at 37°C. The sample was centrifuged at 25,000 × g for 60 min and the supernatant used as negative control serum. (4) Zymosan treatment followed by heating (3 + I ). Antiserum to B S A

Rabbits were injected subcutaneously with 0.5 nag BSA in complete Freund's adjuvant and boosted with 0.5 mg BSA in Freund's incomplete adjuvant 3 times. Serum was collected 1 week and 2 weeks after the last booster. The antiserum, a pool of several bleedings, was absorbed twice on an HSA-Sepharose 4B column (Axelsen

371

et al., 1973). After absorption, the concentration of specific anti-BSA IgG was estimated to be 1.63 m g / m l by quantitative precipitation. Complement was inactivated by storage of the serum at 4°C for 4 months and checked by hemolytic assay. Serum was transferred to 3 ml PVC tubes coated with Tween 20 (0.5 m l / I PBS) in 400 ~1 aliquots, quick-frozen and stored at - 70°C. The antiserum was only thawed once before use.

BSA BSA (Behringwerke, Marburg) was dissolved in phosphate (0.01 M)-buffered saline (PBS), to a final concentration of 0.5 m g / m l , and distributed in 200 #1 aliquots in Tween 20 coated PVC tubes, quick-frozen and stored at - 7 0 ° C .

i_'_~l.BSA '25I-BSA was prepared by the iodo-gen method (Franker and Speck. 1978) using the BSA solution described above, and stored in aliquots of 400 p,I (6.6 x 105 cpm and 0.8 ~g '25I-BSA) at - 7 0 ° C .

Z)'mosan stock preparation 100 mg zymosan (Sigma) was boiled in 10 ml 0.9% NaCI for 30 rain, centrifuged for 10 min at 1000 × g, and the precipitate redissolved in 50 ml 0.9% NaCI containing 0.1% N a N 3 and stored at 4°C.

Immune complexes A quantitative precipitation test was performed using the rabbit anti-BSA serum ( 1 : 1 0 in PBS) and 2-fold dilutions of BSA (0.5 m g / m l ) with 3% t25I-BSA tracer. After incubation for I h at 37°C and 22 h at 4°C, maximum precipitation was determined after centrifugation of the samples (10 min at 3000 x g) and measurement of the radioactivity in the supernatants and precipitates. Maximal precipitation was found at a molar a b / a g ratio of 3.4. For routine use, an IC stock suspension was prepared at this ratio, from 200 /.tl rabbit anti-BSA serum (325 /,tg anti-BSA lgG), 80 ~1 BSA dilution (40 p,g BSA) and 400 #1 ~zSI-BSA (0.8/,tg BSA). Incubation was performed as described above. After incubation, PBS was added to a final volume of 3.0 ml. The IC were then washed twice by centrifugation at 3000 × g for l0 rain and 2.4 ml of the supernatant was replaced with PBS. To this sample was added 3.0 ml of a 20% ( w / v ) sucrose solution in PBS. The tube was whirlmixed for 30 sec, and the content dispersed by passage through a tuberculin needle 5 times. The complex-containing suspension was transferred to PVC tubes in aliquots of 100 FI to 1000 FI, quick-frozen and stored at - 70°C. The ~25I-BSA tracer amounted to 3% of the total BSA.

Standard procedure for measuring CMSC and initial kinetics of solubilizanon (IKS) The term 'initial kinetics' is here defined as the slope of the solubilization curve between the 10 rain and 20 min values, expressed as % increase in CMSC per min. The CMSC is defined as the % CMS measured after 120 min of incubation of serum and preformed IC.

372 (I) The IC suspension was thawed at 22°C and kept at 37°C for 1 h before testing. Serum samples were thawed at 22°C just before testing. (2) Half a milliliter test serum or negative control serum w.as diluted 1:2 in PBS 2÷ (EDTA-treated serum is diluted in PBS) and tested in duplicate. (3) The diluted sera were transferred to a 37°C waterbath at 20 sec intervals, and preincubated for 10 min. The IC suspension was whirlmixed for 30 sec and dispersed by passage through a tuberculin needle (0.4 mm) 5 times. 14) At time zero, 40 p,l IC 12.2 #g anti-BSA lgGI suspension was added to the tubes in the same order and at the same time intervals. The tubes were immediately capped and mixed by rotation. (51 In IKS studies, 200 #1 samples were withdrawn after 10, 20. 60 and 120 min incubation. Before samples were collected, the tubes were mixed b~ rotation. In the CMSC studies, onl~ one sample was withdrax~n after 120 rain. The samples ~ere immediately transferred to PVC tubes containing 2.8 ml ice-cold PBS and 20 mM EDTA. (6) The tubes were placed in precooled rotor head holders and centrifuged at 3000 × g for 10 min at 4°C. (7) The first 1.0 ml supernatant was discarded and the next 1.0 ml 11/3 supernatant) transferred to a 3 ml PVC tube. The pipette followed the surface during the pipetting procedure. The remaining 1.0 ml was designated I,/3 supernatant + precipitate. (8~ All the tubes were counted in a gamma-counter and the % CMS calculated from the equation: (3 × I / 3 sup.)

x

100

(2 x I / 3 sup.) + ( 1 / 3 s u p . + precip.)

= % CMS

Reagents and solutions EDTA: tetrasodium and NaN 3 from Sigma (St. Louis, MOI. HSA: purified, Behringwerke AG (Marburg). t-Tyrosine, FAS central drug store (Denmark). NaI" extra pure, and Tween 20, Merck (Darmstadt). D( + )-Saccharose: Fluka AG, Chemische Fabrik (Buchs). Iodo-gen: Pierce Chemical Co. (Rockford, I L). PBS: ~,aterfree KH_,PO4 12.1 g, Na_,HPO 4 49.4 g, NaCI 64.8 g and distilled H_,O to a final volume of 10 I, pH adjusted to 7.4. PBS 2~ (Dulbecco's phosphate-buffered saline): 0.2205 g CaCI ,, 1.015 g MgCI_,, 10.00 g NaN 3. PBS to a final volume of 10 I, pH adjusted to 7.4. (Ca-'+= 15 × 10 -5 M, Mg -'+= 5 × 10 -4 M). EDTA-PBS: PBS containing 20 mM EDTA. Tween-PBS: 10 I PBS with addition of 5 ml Tween 20 and 10 g NaN 3. pH 7.4.

Results

The influence of serum dilution on IKS and C M S C Dilution of serum in PBS 2 ÷ affects both IKS and CMSC. This was demonstrated

373

in 2-fold d i l u t i o n series, where the ratio between volume of u n d i l u t e d serum and a m o u n t of IC (40 #1 IC s u s p e n s i o n / 0 . 5 ml u n d i l u t e d serum) was kept constant. T h e C M S C was found to increase with increasing serum dilution up to 1 : 4. after which a s t e a d y decline in C M S was o b s e r v e d (Fig. 1). However. a serum dilution of i : 8 in PBS-" +, still gave a higher C M S C than u n d i l u t e d serum. W h e n the i n c u b a t i o n was p r o l o n g e d to 3 h the i : 8 serum dilution reached the same C M S value as the 1 : 4 dilution. At a I : 8 serum dilution, the C M S curve tended to have a sigmoid shape. T h e I K S was m a r k e d l y r e t a r d e d at serum dilutions exceeding 1 : 2.

The C M S C is dependent upon the amount oJ IC used in the assay The C M S C values o b t a i n e d when n o r m a l d o n o r sera were tested showed a m a r k e d dependenc~ u p o n the a m o u n t of IC used in the assay. The 2 d o n o r sera shown in Fig. 2 differed c o n s i d e r a b l y in C M S C , but both were ~ i t h i n the n o r m a l range. W h e n m i n u t e a m o u n t s of IC ( c o n t a i n i n g less than I.I p,g a n t i - B S A / m l 1 : 2 d i l u t e d serum) were used, the differences between the C M S C registered for different d o n o r sera t e n d e d to be reduced. lnlTuence of centri]ugation conditions on the measured C M S It has been d e m o n s t r a t e d that solubilized IC are heterogeneous in size (Czop and Nussenzweig. 1976). The measured C M S should c o n s e q u e n t l y be expected to be d e p e n d e n t upon the c e n t r i f u g a t i o n c o n d i t i o n s used. T h e effect of c e n t r i f u g a t i o n at 1200 x g for 10 rain ( C z o p a n d Nussenzweig, 1976) and 3000 x g for 10 min, respectively on the C M S measured at s t a n d a r d times of i n c u b a t i o n was investigated. T h e C M S C for both fresh d o n o r serum a n d h e a t - i n a c t i v a t e d serum reached higher values after c e n t r i f u g a t i o n at 1200 x g (Fig. 3). However, the difference between

100 -

50

100 -

80

z,.O

80

60

30

o~ 60

•- 4o

20

u

20

10

20

o

o

11

12

14

Serum

18 dttutton

116

12

~

40

z.O pl

120

200

IC suspenstonlmt

dduted

280

360

s.erum

12

Fig. 1. Influence of serum d i l u t i o n in PBS 2. on IKS and CMSC. The r a u o between a m o u n t of IC and v o l u m e of undJluted serum ,,+,as kept constant. C M S C (o) and IKS ( O ) of untreated serum. C M S C (A) a n d IKS (e,) for heat-treated serum d i l u t e d I : 2 in PBS 2 ÷. All points are based on d u p l i c a t e d e t e r m i n a tions (vertical bars). Fig. 2. C M S C d e p e n d e n c e upon a m o u n t of IC used in the s t a n d a r d assay (see Methods). One d o n o r (~) had a C M S C in the u p p e r n o r m a l range, the other ( O ) in the Iov+er n o r m a l range. Samples (200 p.I) were taken after 120 nun i n c u b a t i o n at 3 7 ° C and tested m d u p l i c a t e s (,.ertical bars).

374

donor serum and negative control serum was more pronounced when centrifugtation was performed at 3000 × g. In addition, the mean difference between the highest and lowest values (16 triple determinations at each g rate) was 6.8% when centrifugation was performed at 1200 × g and 2.3% at 3000 × g. Two different methods of harvesting supernatant and precipitate were compared. In one (Takahashi et al., 1980), 2.7 ml supernatant is withdrawn from a total volume of 3.0 ml, and the radioactivity in the supernatant and precipitate determined. This procedure was compared with that described in Materials and Methods. With the former procedure the CMS percentage was calculated according to the formula:

supernatant × 100 supernatant + precipitate

=

c~

CMS

and in the latter by the formula given in Methods. The ,% of CMS registered by the 2 different procedures was the same. The mean difference between the highest and lowest CMSC values of triplicates was 3.5% in the first procedure, and 2.3c~ in the second (16 triple determinations in each procedure).

706050. L0.

80 60

20

/

u~ l.O-

L--I

10 0

20

i

,

.

10 20 60 In(ubQtion fame Im~nJ ot

120

]'/°C

,0 2'0 Incubahon time (ram) at

37°C

Fig. 3. Influence of c e n t n f u g a u o n c o n d i t i o n s on C M S C measured in one normal d o n o r serum using the s t a n d a r d p r o c e d u r e (3000 x g, IO min) (A) and a modified s t a n d a r d p r o c e d u r e ( 1200 × g. IO nun) (v). The same serum, heat-inactivated, was also tested using the same centrifugation c o n d i t i o n s (3000 x g, IO min~ I © ) and 1200x g for I0 min (E3). Fig. 4. Solubilization p e r f o r m e d with a freshly prepared IC suspension stored u n d e r different conditions. O n e aliquot was diluted I : 2 in PBS and stored for I h at 4 " C (A). A n o t h e r aliquot was diluted I : 2 in PBS a n d frozen at - 7 0 ° C for I h before testing ((3). A third aliquot was diluted I :2 m PBS c o n t a i n i n g 20~ ( w / ' v ) sucrose, frozen at - 7 0 ° C for I h before testing (z~ and a fourth aliquot was diluted 1 : 2 in PBS c o n t a i n i n g 0.2 M $1ycine and frozen for I h at - 70°C (o). To heat-inactivated control serum (ra) was a d d e d [C which had not been frozen, s u s p e n d e d in PBS. The test v.as performed on the same normal s e r u m a c c o r d i n g to the s t a n d a r d procedure. Vertical bars represent d u p l i c a t e determinations.

375

Freezing the IC preparation under different conditions affects CMS Fig. 4 shows C M S curves for an untreated d o n o r serum tested by use of IC stored at different conditions. Virtually identical c u ~ ' e s are seen for freshly prepared IC and IC frozen in either PBS c o n t a i n i n g 0.1 M glycine or 10% (w./v) sucrose. Solubilization of IC frozen and stored at - 7 0 ° C in PBS only was significantl,~ reduced. N o difference in C M S was o b s e ~ ' e d when aliquots from the same serum were reacted with IC suspended in PBS, or PBS plus sucrose or glycine, p r o v i d e d the IC had not been ex p o s e d to freezing. Solubilization of IC suspensions stored at 4 ° C was already reduced after 2 - 3 days of storage. In contrast, storage of IC suspensions in PBS plus 10% sucrose at - 7 0 ° C did not affect the C M S C of a given d o n o r serum when tested over a period of o n e month.

Negatiee control sera In all experiments, the 4 types of negative control sera (see Materials and M e t h o d s ) were prepared from the same bleeding, the untreated serum being used as positive control. Us i n g freshly p r e p a r e d IC, all 4 types of negative control sera gave C M S values between 8%. and 15% at all i n c u b a t i o n times, p r o v i d i n g the IC a m o u n t per ml I : 2 diluted serum c o r r e s p o n d e d to an a n t i - B S A co n t en t of 4.4 ~g. With IC c o n t a i n i n g 2.2 # g anti-BSA, the C M S was 15-20%. after 10 rain. falling to 10-15%. after 120 rain incubation. Freezing the IC in PBS resulted in a C M S level in the negative controls ~,hich was 5-10,~ lower than when n o n - f r o z e n IC were tested. IC frozen in PBS plus 0.1 M glycine or 10% ( ~ / v ) sucrose gave a ' b a c k g r o u n d solubilization" ~ i t h negative c ont r o l sera of 30-35% after 10 min incubation, falling to ab o u t 15%. after 120 rain inc u b at i o n . Results with a 10 m M E D T A treated negative control serum are shown in Tab l e I. When IC d eep -f r o z e n in PBS c o n t a i n i n g glycine or sucrose were thawed and kept

TABLE I SOLUBILIZATION VALUES OBTAINED WITH EDTA TREATED CONTROL SERUM INCUBATED WITH IC STORED UNDER DIFFERENT CONDITIONS Serum

EDTA treated NHS EDTA treated NHS EDTA treated NHS EDTA treated NHS EDTA treated NHS EDTA treated NHS Untreated. NHS

IC suspension m:

PBS. frozen IOt~. sucrose, frozen 0.1 M glycine, frozen PBS. not frozen 10% sucrose, not frozen 0.1 M glycme, not frozen 10qo sucrose, frozen

a Mean of duplicate deterrmnations and range.

Percentage solubd~zatton at different times of incubauon 10 mm

20 rain

60 mm

120 mm

15+_0.5 J 33_+0.0 27_+0.5 20 +_0.5 2 6 + _ 0 .0 19-+2.0 23 _+1.5

14+ 1.5 27+2.0 28+0.5 19 + 1.5 27+0.5 20_+0.0 44_+2.0

I0+0.0 16+_ 1.5 14+0.5 I1 -+ 1.0 14+_ 1.5 12-+0.5 66+_0.5

13+ 1.0 18+3.0 12+0.0 12 _+2.0 13£ 1.0 12-+0.5 64:t:4.0

3'76

for 60 min at 37°C before testing, the CMS level seen with negative controls was reduced by 5-10% after 10 and 20 min incubation. Preincubation of IC for 120 rain at 37°C before the test run did not further reduce this 'background" CMS. Internal standards and reproducibility of the assay A standard curve was obtained by diluting the serum from a healthy donor in a zymosan and heat-treated sample from the same bleeding. On decreasing the volume ratio between untreated and inactivated serum, the CMS approached the negative control level (Fig. 5). Donor serum diluted similarly in PBS containing 60 mg of H S A / m l gave an identical CMS curve. Based on the standard curve, and the use of a serum from a donor with high CMSC (87%.), standards with CMSC values of 45%.. 60%.. 70% and 80q~. were prepared. Each standard was distributed in 32 aliquots (0.6 ml each), and quick-frozen at - 7 0 ° C . The standard preparations were diluted 1:2 in PBS -'+ and tested 16 times in duplicates according to the standard assay procedure (see Methods). The order in which the standards were tested was different each time. and the 2 series (duplicate determinations) were arranged in U-form. The calculated mean. S.D.. CVt and CV,, are shown in Table 11. At reaction times 10 and 20 rain, the highest CV L, CV,, and S.D. values were seen when the standard giving 80% CMS was tested. The mean, S.D. and CV~ for the measurements of IKS are given in Table III. The CV~ is increased slightly, as compared to in the CMSC studies, especially for the standards. which have a very fast solubilization reaction (70% and 80%. CMSC standards). Measurements of I K S and C M S C in patients' sera by the standardized assay Sera from 4 SLE patients and 4 rheumatoid arthritis patients, who were IC positive as determined by the PEG - CC assay (Brandslund et al., 1981) and from I glomerulonephritis patient were examined to test the ability of the standardized assa5 to discriminate between the CMSC of normal donor sera and pathological sera. All patients' sera were tested in parallel with normal donor sera and heat-in-

80

_ 60 ~

~

20 0 ,Y 08 /.~, 5'3 62 71 8'0 Ratio of donor" serum f0 dduent Fig. 5. Standard cu~'es for CMSC. One cu~'e (,',) was obtained b.~ dilution of normal donor serum in the same serum after treatment by zymosan and heatin 8 and the other ( O ) by diluting the same normal donor serum in an HSA solution (60 mg H S A / m l PBS). Solubilization was performed according to the standard procedure. Vertical bars represent duplicate determinations.

377 T A B L E IT STATISTICAL PAR_,-%METERS FOR T H E CMS BASED ON 16 D U P L I C A T E D E T E R M I N A T I O N S U S I N G F O U R S T A N D A R D SERA A N D T H E S T A N D A R D I Z E D ASSAY Incubation ume

Standard sera (no.)

Mean CMS

S.D.

CV~

CV t

10 rain

I~ 2 3 4 I 2 3 4 I 2 3 4

27% 28% 26~ 31% 30q~. 34~46% 65~ 48~ 61q~ 72~ 79~

3.4 4.2 4.0 5.0 2.9 25 4.7 6.0 3.3 4.4 4.0 4.2

0.10 0.12 0.13 0.15 0.13 0.15 0.18 0.21 0.05 0.11 0.07 0.07

0.11 I).17 0.15 (9.23 0.08 0.06 0.20 0.34 0.10 0.17 0.15 0.17

20 m m

120 min

Standard sera nos. I, 2. 3 and 4 v.lth CMSC 1120 rain) of 45q, 60q~, 70% and 80% respectl~el) calculated from a standard cur',e.

activated control sera at different dilutions with different amounts

of IC and with IC

frozen

The difference

in P B S p l u s

10% ( w / v )

sucrose

and

in PBS respectively.

in

CMSC between patients' sera and normal donor sera was most pronounced with the standardized assay procedure (see Materials and Methods). The IKS and CMSC of the SLE sera were markedly the rheumatoid CMSC.

reduced compared

arthritis sera showed

The glomerulonephritis

serum

with normal

nearly normal showed

donor

sera. By contrast,

IKS and only slight reduction

clearly reduced

in

IKS and CMSC.

T A B L E III STATISTICAL P A R A M E T E R S FOR THE IKS W H E N SOLUBILIZATION WAS P E R F O R M E D A C C O R D I N G TO THE S T A N D A R D P R O C E D U R E U S I N G IC SUSPENSION WITH 10ft W,,V SUCROSE ADDED. Standards

Meant SD CV r

No. I t

No. 2

No. 3

No. 4

0.29 -" 0.40 0.15

0.63 0.44 0.18

1.96 0.46 0.39

3.40 0.70 0.46

Standard sera no. I, 2, 3 and 4 with CMSC of 45%, 60%, 7 0 f and 8 0 f respecti,,eb, calculated from a standard curve. ~" The values represent increase in percentage CMS per rain betv, een I0 and 20 min of incubation. All values are based on 16 duphcate determinations.

378

Discussion

We have standardized a method for measuring CMSC and IKS in sera to test the integrity of the alternative complement pathwa3. The influence of several variables in the different steps of the test procedure on the registered CMSC was investigated in order to optimize the assa3. The solubilization reaction shows marked Ca-'+ and Mg 2÷ dependency as previously reported I Miller and Nussenzweig. 19751. ,Addition of these ions in adequate concentrations to PBS resulted in about 25~ increase in CMSC when serum was diluted in this buffer (PBS= +1. in contrast, dilution of serum in PBS, PBS containing 60 mg H S A / m l or in complement inactivated sert, m caused a decrease in CMSC, even at I : 2 dilution. The reason for choosing a serum dilution of no more than 1:2 in PBS -'÷ for the standard procedure is that the difference in CMSC between sera from normal donors and patients ma3 be masked when serum is further diluted in PBS-"*. The anaount of IC incorporated in I ml serum greatly influences the measured CMSC. This suggests that we are dealing ~ith a stoichiometric reaction as suggested b3 Czop and Nussenzweig (1976). Schifferli et al. (1981) recently investigated the CMSC of sera from different patient groups using IC containing 20 ~g anti-BSA per ml I : 3 diluted serum. The BSA/'anti-BSA ratio was not given, but it can be assumed that relativel3 large amounts of IC were used as indicated by the wide normal range of CMSC in this study. In the experiments of Aguado et al. (1981) 12 ~g IC protein per ml 1:2 diluted serum was used. which probably (depending upon the BSA/anti BSA ratiol exceeds the amount used here. These authors also report a very wide normal range (71-143%) of CMSC. Our studies indicate that it is preferable to use small amounts of IC in investigations of pathological sera as this gives a narrow normal reference range, and a high mean value of CMSC. This increases the likelihood of discriminating between normal sera and pathological sera with reduced CMSC. If on the other hand, it is desired to investigate sera expected to haxe elevated CMSC. or interindividual differences among normal donor sera. larger amounts of IC should be used as this will retard the solubilization reaction and gi~e a less steep slope of the initial CMS curve and a lower CMSC. The same effect can be achieved by freezing the IC in PBS for I h at - 7 0 ° C before testing. This may cause rearrangement of the initial complex making it more resistant to CMS. The centrifugation is the most critical step in the procedure with respect to reproducibility of the assay. Only by controlling the centrifugation conditions carefull_~, is it possible to reduce the CV, to approximately the same level as the C\.~. Correction for variations in the g rate ma) be achieved by use of CMS-positive standard preparations included in each run. The method chosen for separation of supernatant and precipitate may also influence the reproducibility and accuracy of the assay and the CMSC measured. Czop and Nussenzweig (19761 introduced erythrocytes as a marker to improve reproducibility. We have preferred another approach which has the ad,,antage of being eas.~ and rapidly performed. Onl~ the second 1.0 ml of the supernatant is

379 ha~,ested a n d taken as a representative s a m p l e of the s u p e r n a t a n t as described. This was considered j u s t i f i e d as we o b s e ~ , e d a linear g r a d i e n t of r a d i o a c t i v i t y t h r o u g h o u t the s u p e r n a t a n t . All m e t h o d s used here to inactivate serum c o m p l e m e n t a p p e a r e d to be equally efficient in reducing the C M S . The level of C M S C registered in the negative control sera d e p e n d e d upon the serum dilution used, the a m o u n t of IC i n t r o d u c e d in the assay and the c o n d i t i o n s of IC storage. it is necessaD' to be able to use IC stored in a frozen state in a s t a n d a r d i z e d assay for routine use. However, freezing and thawing of IC in PBS was found to reduce the measured C M S C of sera. It is therefore a d v i s a b l e to a d d 10~, ( v , / v ) sucrose or 0.1 M glycine to the IC suspension before freezing. W h e n IC frozen in PBS c o n t a i n i n g sucrose were i n t r o d u c e d into the assay the ' b a c k g r o u n d " solubilization b)' negative control sera was slightly increased, especially for the l0 and 20 rain samples. Newly thawed IC may exist in a state of s u b o p t i m a l aggregation, as suggested the o b s e ~ ' a t i o n that. when the thawed IC were allowed to i n c u b a t e for I h at 37°C p r i o r to testing, the p e r c e n t a g e C M S was slightly reduced in the early s a m p l e s a n d the I K S values were m o r e reproducible. The r e p r o d u c i b i l i t y of the m e t h o d d e s c r i b e d is a c c e p t a b l e when the C M S C is m e a s u r e d after 60 or 120 rain reaction at 37°C. The reproducibilit.~ of I K S m e a s u r e m e n t s can be i m p r o v e d by m i n o r m o d i f i c a t i o n of the assay which retards the initial rate of the C M S reaction as previously described. O u r p r e l i m i n a r y investigations of S L E and r h e u m a t o i d arthritis sera indicate that S L E sera m a y show a m a r k e d reduction of both C M S C , as previously reported by Schifferli et al. (1981). A g u a d o et al. (1981) a n d Sakurai et al. (1982), and IKS. N o such clearly reduced C M S c a p a c i t y was o b s e r v e d in o u r pilot stud)' of r h e u m a t o i d arthritis sera. F u r t h e r m o r e , IC which were not c o m p l e m e n t - s o l u b i l i z e d were found in SLE sera, while no such c o m p l e x e s were d e m o n s t r a b l e in sera from r h e u m a t o i d arthritis patients ( u n p u b l i s h e d data).

Acknowledgements W e are i n d e b t e d to Dr. J.C. Jensenius for s u p p l y i n g some of the reagents, and Mrs. EIsebeth O r t h m a n n for efficient secretarial assistance. T h e project was s u p p o r t e d by The D a n i s h League against R h e u m a t i s m , The D a n i s h Medical Research Council a n d The Regional Research Council of the C o u n t y of F u n e n .

References Aguado. M.T.. L.H. Perrin, P.A. Miescher and P.H. Lambert. 1981. Arthritis Rheum. 24, 1225. Axelsen, N.H., J. Kroll and B. Weeke, 1973, Scand. J. lmmunol. 2 (Suppl. I), 98. Brandslund, I., H.C. Sierstext, J.C. Jensenius, and S.-E. Svehag, 1981. Methods in Enzymolog~. Vol. "/4. part C (Academic Press, Nev, York) p. 551. Czop, J, and V. Nussenzweig, 1976. J. Exp. Med. 143. 615.

380 Franker. P.J. and J.C. Speck. Jr.. 1978, Bitxhem. Biophys. Res. C o m m u n . 80. 849. Fujita. T., Y. Kata(a and N. Tamura, 1981. J. Exp. Med. 1.54. 1743. Miller. G.W.. 1976. J. lmmunol. 117. 1374. Miller. G.W. and V. Nussenzv, elg, 1975. Proc. Natl. Acad. Sci. U.S.A. 72,418 Rajnavi31gyi. E., G. Ftist, J. Ember. G.A. Medgyesi and J. Gergel~, 1978, J. Immunochem. 15. 335. Sakurai. T., T. Fugita, I. Kono, T. Kabashima, and K. Yamane. 1982. Clin. Exp. Immunol. 48. 37. Schifferli. J.A.. S.R. BarlolottJ and D.K. Peters, 1980, Clin. Exp. Immunol. 42, 387. Schifferli, J.A.. S.M. Morris. A. Dash and D.K. Peters. 1981. Clin. Exp. Irnmunol. 46, 557. Takahashi, M.. J. Czop, A. Ferreira and V. Nussenzweig, 1976, Transplant. Rev. 32, 121. Takahash], M., B.F. Tack and V. Nussenz,~eig, 1977, J. Exp. Med. 145, 86. Takahashi, M., S. Takahashi. V. Brade and V. Nussenzv, eig, 1978, Clin. Inxest. 62, 349. Takahashi, M.. S. Takahashl and S. Hirose. 1980. Prog. Allergy 27, 134..