The complement hemolytic intermediate EAC1q4

Mol~ular Immurmlogy, Vol. 17, pp. 1275-1281. Pergamon Press Ltd. 1980 Prmted in Great Britain.

THE COMPLEMENT XU Biochemistry

FEN,

Research

HEMOLYTIC SHEN

Group,

and ZHU

FENG-XIN

Nanjing

INTERMEDIATE

Medical

College,

EAC&

ZENG-BIAN*

Nanjing,

People’s

Republic

of China

(First received 20 April 1979; in revised form 10 December 1979) was prepared from a small amount of pooled human sera through two steps of DEAEcellulose chromatography. Antiserum against C,, was raised in rabbits by means of multi-point intradermal injections. The existence of intermediate EAC,,, was confirmed by the following observations: (1) The human C,, on thecomplement hemolytic intermediate was demonstrated with anti-human C,, antiserum. It was shown that calcium ions were not required for the binding reaction between C,, and EA. The formation of EAC,, could be achieved in EDTA solution, and C,, was present on the intermediate EAC,, generated by incubating EAC,,,, in EDTA saline; (2) With guinea pig complement, the hemolytic reaction of the intermediate derived from EAC,,, by incubating in EDTA saline could be induced in the absence of C,,. Abstract-C,,

INTRODUCTION

The complement intermediate EAC, was discovered by Reeker in 1959. Because EAC, is easily prepared, this intermediate is widely used in complement technology. Since Lepow et al. (1963) resolved C, into three subcomponents, C,,, Cl, and Cls, EAC, was employed in C,, studies for guiding isolation and assaying in several laboratories (Yonemasu & Stroud, 1971; Reid et al., 1972; Calcott & Miiller-Eberhard, 1972; Assimeh et al., 1974). However, in the course of the C,, isolation study, we found that results obtained with EAC, were often inconsistent. It was shown that there was no need for calcium ions for the binding activity of C,,, and in EDTA solution, the intermediate EAC,,, was transformed into EAC,,, instead of EAC,. MATERlALS

AND METHODS

Materials Verona1 bujfered saline stock solution (VBS stock, u = 0.75). Solution A contained 85 g of sodium chloride and 3.75 g of sodium barbiturate, dissolved in about 1400 ml of distilled water. Solution B contained 5.75 g of barbital dissolved in about 500 ml of warm distilled water. Solutions A and B were mixed and distilled water added to a volume of 2000 ml. Complementjixation diluent (CFD). VBS stock (100 ml) was mixed with 400 ml of distilled water, to which were added 25 ml each of 1.OOM MgCl, and 0.30 M CaCl,. *Present address: Quiyang Medical took part in C,, isolation in 1977.

College.

This author

Hemolysin and sensitized sheep erythrocytes (EA). These are prepared according to the method of Kabat & Mayer (1961). Phosphate buflered saline (PBS). A 960 ml quantity of 0.15 M NaCl was mixed with 40 ml of 0.1 M phosphate buffer, pH 7.4. EDTA-Mg saline (EDTA-Mg O.OlM, pH 7.4). Na, EDTA.2H,O (1.86g) was dissolved in about 15 ml of distilled water, and adjusted to pH 7.4 with 1 N NaOH, then diluted with distilled water to 20 ml. This EDTA solution is mixed with 5 ml of 1.00 M MgCl, and 475 ml of PBS, and adjusted to pH 7.4. EDTA saline (EDTA 0.005 M, pH 7.4). EDTA saline is composed of 95 parts of PBS and 5 parts of 0.1 M EDTA (pH 7.4). Calcium-magnesium ionized water. This was prepared by adding 0.25 ml of 1.OOM MgCl,and 0.25 ml of 0.30 M CaCl, to 500 ml distilled water. 10% Glucose. Glucose (1 g) was dissolved in 10 ml of calcium-magnesium ionized water, EDTA-Mg dilutedserum (C-EDTA-Mg). The guinea pig serum was diluted with EDTA-Mg saline 1:50, prepared immediately before use. DEAE-cellulose. DEAE-cellulose (0.1 mEq/g) was treated with 0.5 N NaOH for about half an hour, washed to remove NaOH, suspended in distilled water, adjusted with 1 N HCl to neutral pH, and packed in a chromatography tube of 2 cm diameter. The packed DEAE-cellulose column was then washed with 2-3 column volumes of 0.5 M NaCl, and equilibrated with the starting buffer overnight. Starting buffer (pH 7.4 p = 0.15, containing 0.001 M EDTA). This was prepared according to 1275

MI.M.M. 17/10-F

I216

XU FEN.SHEN FENG-XIN andZHU

Lepow et al. (1963) as following. Anhydrous* NaH,PO, (3.6g) and anhydrous Na,HPO, (19.875g) were dissolved in distilled water, and mixed with 20 ml of 0.15 M EDTA (pH 7.4) prepared from purified anhydrous Na,EDTA, then diluted with distilled water to 3000 ml. Sodium acetate huj@ (O.O2M, pH 5.6). Purified anhydrous sodium acetate (2.968g) was mixed with 19.20 ml of 0.2 N acetic acid, and diluted with distilled water to 2000 ml. Yeast. Yeast was prepared from fresh baker’s yeast (Health brand, Shanghai) according to the method presented by Lachmann et al. (1973). EDTA saline dilutedserum (C-EDTA). Guinea pig serum was diluted 1:50 with EDTA saline immediately prior to use. Complement C,. Functionally pure C, adapted for C, fixation and transfer test is prepared from guinea pig serum by DEAEcellulose chromatography according to a modification of a procedure recommended by Borsos et al. (1961). It was kindly provided by Lecturer Fei Ru-Zheni-. Anti-Zg antiserum. IgG was isolated from human serum according to Vaerman et al. (1963). IgA was isolated from human colostrum as described by Mach & Pahud (1971). The antisera were raised in rabbits kindly presented by Lecturer Fei Ru-Zhent. C,,.,. Cl,-, was prepared from guinea pig serum according to the procedure described in Results (cf. Fig. 2). The C,,, used in human C,, isolation is obtained from the same human serum (cf. Fig. 1). Methods HcJmolytic assay ,for C,,. The sample to be assayed (0.05 ml),O.lO ml of 2”,, EA. 0.05 ml of Clrs mixture and 0.10 ml of lOO,<,glucose were mixed in order. and incubated for 15 min at 37°C. Then 0.45 ml of C-EDTA-Mg was added to the reaction mixture and it was incubated at 37°C. The results were read after 90 min. c,, agglutination test. 2% EA (0.5 ml)was mixed with 0.2 ml of test sample, incubated for 15 min at 37°C and then put aside at O-4 C for over 3 hr with intermittent shaking. The mixture was centrifuged and the red cells washed once with 0.5 ml of CFD. They were diluted to a 0.50(; suspension by adding 2 ml of CFD. Two drops of diluted C,, antiserum and 2 drops of EAC,,,

*Refined by H,S treatmcnt f Nanjing

Chinese

Medicine

and recrystallisation. College.

I,ENG-BIAN

prepared as mentioned. were added to the wells 01 the micro-reaction plate. The plate was shaken for 1 min. and put aside for subsequent observation. lt~~nzzmogk/hzr/i~~rr~~:g/lr/illr!iro/l /(‘.\I. A 0.5 ml quantity of 2”,, red cells uas mixed with 0.2 ml of‘ the sample to be assayed and processed test. except identically as in the C, I, agglutination that Ig antiserum was used instead of (I,, antiserum. Hcrwol?~tic intcrnwrlitrtc E.4 C‘,. EAC, was prepared according to the method of Lachmann (11ai. (1973). REX

Separation

LTS

qf’ C, q und C,,,

The crude C,, was isolated according to the method of Lepow cut ul. (1963), somewhat modified. About 20 ml of fresh human serum was cautiously adjusted with 0.15 M HCI toPH 5.6-6.0, and immediately dialysed against icecold 0.02 M sodium acetate buffer, pH 5.6 (2000 ml), for about 5 hr. The dialysed serum was centrifuged at 0°C and the euglobulin precipitate was washed three times with the cold dialysis buffer by decantations, and twice more washed with this buffer by resuspension and centrifugation. The washed precipitate was dissolved in 2 ml of 0.3 M VBS and mixed with 0.2 ml of 0.15 M EDTA, pH 7.4, and then dialysed against 200 ml of starting buffer in the cold for about 3 hr. The equilibrated euglobulin solution was applied to a DEAE-cellulose column and the column was eluted with the starting buffer at O-4 C. The ratio of the volume of the serum to that of the cellulose column was about 1:5. The eluting rate was 15-20 ml per hr. After about three column volumes of effluent had been collected, the column was thoroughly washed with starting buffer and was then developed with 0.3 M NaCl in starting buffer (Al = 0.45) for the elution of C,,.,. In order to neutralize EDTA in the starting buffer (0.001 M), each fraction was mixed with one-tenth volume of 0.022 M CaCl,. For the second protein peak, in addition to CaCl,, two volumes of distilled water were added, lowering the ionic strength to 0.15. Then aliquots from each fraction of the first protein peak were pooled, serving as C,, and used to define the distribution of Clrr activity in the second protein peak. The verified C,r\ fractions were in turn employed for assaying C,, in the first protein

1277

EG,,

4 w”

/ St buffer plus 0.3M NoCl

St. buffer IO-

c5

\

11

\ .\

1 ,._.....-.b,

.\,.

.’

-+**+++c+-

Cl,

‘.*L.-._._

,-.. IO

20

IO ___

Cl

,tL+C+C+II

Ig

I: 2

1.4 C WI

Fractions Fig. 1. Elution pattern of human C,, and C,, (18 ml) in DEAE-cellulosechromatography. In the first peak the C,, was assayed with the hemolytic method, and the immunoglobulins were detected by double diffusion in agar. In the second peak the C, and C,, activities were assayed by the hemolytic method. The column volume was 90 ml.

peak. The result is shown in Fig. 1. The C,, obtained was found to be contaminated with much immunoglobulin. The Ct,, from human serum was often contaminated with C, activity and was merely used in diluted form. The elution pattern of C,, and Ct,, of guinea pig serum is somewhat different from that of human serum, as shown in Fig. 2. The Clrs obtained was free of C, activity. In the early experiments on C,, isolation, we noticed that the results of assaying C, activity in the Clrs peak with EA and EAC, were very different, as shown in Table 1. Therefore we selected EDTA-Mg diluted serum for assaying C, and C,,. The assay

Purijication

\ St. buffer plus 0.3M N&l \

St. buffer \,

\

D

W"

0.5

*\

\ \

I/

19

.‘.\. \..,_

20

IO C

\

1 ‘\_._ t -++++-----

Cl c (,‘,

/

1

IO

20 _++*

procedure is described in Materials and Methods. This reagent has been used by Lepow et al. (1963) for the C, assay. The reliability of this reagent in assaying C,, is shown in Table 2. EDTA-Mg chelates calcium ions in the serum and blocks C, activity. Thus C-EDTA-Mg is a simple way to provide C,-C,. But, using this reagent, the hemolytic reaction appeared to be progressive and often resulted in high blank values, so that this was mainly used as a qualitative method. However, if the reaction time was controlled and the result was read with respect to the control test, this method appeared to be reliable.

______

-+++-++++*

Fractions Fig. 2. Elution pattern of guinea pig C,, and C,,, (IO ml) in DEAE-cellulose chromatography (column volume 60 ml). The C,,, C, and C,, activities were assayed by the hemolytic method.

of c I q

Fractions with C,, activity as shown in Fig. 1 were pooled. One volume of pooled crude C,, solution was dialysed overnight against 10 vol. of 0.02 M phosphate buffer, pH 7.4, plus 7.5 vol. distilled water. After adjusting the conductance to that of 0.02 Iw phosphate buffer, the crude C,, solution was applied onto a DEAE~ceIlulo~ column (column volume 60 ml), previously equilibrated with 0.02 A4 phosphate buffer and eiuted with the same buffer solution at 0-4”C. The eluting rate was about 4 ml per 20 min. After 2-3 column volumes of eluate were collected, the eluting solution was changed to 0.15 M phosphate buffer, pH 7.4. The result is shown in Fig. 3. Each fraction was assayed for C,, and immunoglobulin by means of double diffusion in agar, agglutination test and hemolytic assay. The

1278

XU FEN. SHEN

_--. .-.. .____.._

_.

I‘uhe number EAh Method

Table

-~

FENG-XIN

and ZHU

1. C, activity in C,Fb peak” ---.._____l_.-...-~

I

2

3

4

5

6

__

_

_

-

_

_

-II__

7 _

_____~-_.~_...-~_.__-_

EAC,”

ZENG-BIAN

~-_~-____.-.“_

8

9

i

+

-+

-i-

IO

I1

12

I.3

_

__-___

i

+

+

-~

+

+

+

&

-

-

-

“The sign + denotes hemolysis. - denotes no hemolysis “The EAC, formed is assayed with C-EDTA-Mg ‘The EAC,, formed is assayed with C, and C-EDTA.

Table 2. Hemolytic Reaction _~.______

activity __.

of EDTA-Mg

Hemolytic

components” -_I...__.__.~

EA EA i C,, EA f C,,\ EA + C,, i- C,,,

diluted

VBS stock solution and the binding activity of C,, was assayed with the agglutination test. The control was processed identically, but the ionic strength remained at 0. I.5 unchanged. The result is shown in Table 3, In the isolation procedure shown in Fig. 4, the more gradual salt concentration gradient was used for elution. In order to make the adjustment of calcium ion concentration more convenient, EDTA solution (pH 7.4) was added into 0.02 M phosphate buffer to a final concentration of 0.001 M. In the resulting C,, solution, immunoglobulin was not detectable. This preparation was used in the agglutination study. Its hemolytic activity is presented in Table 4.

serum

reaction _.__I.

i-

.-_-

“These components were first incubated at 37 C for 15 min, then allowed to react with C-EDTA-Mg.

unabsorbed nonspecific anti-IgG or anti-IgA antiserum was used to detect immunog~obu~in impurities. Under neutral pH and low ionic strength conditions, the hemolytic activity of C, is very easily inactivated. In the above-mentioned purification procedure, in order to separate C,, from IgG, the condition of 0.02 M phosphate buffer was selected. This is the condition which easily induces the loss of C, activity. However, the binding activity of C,, was shown to be stable under such conditions. This is tested as following. The crude C,, solution was mixed with 6.5 vol. distilled water to lower the ionic strength to 0.02, and kept at 4°C for 3 hr. Then the ionic strength was raised to 0.15 by adding

0.02M

qj”ahserum

againsf C ,y

C,, antisera were raised in three rabbits. The fractions of C,, shown in Fig. 3 were free of immunoglobulin after fraction No. 41. Fractions 42-48 were pooled, and used to immunize one rabbit. Fractions 49-55 were pooled, and used to immunize two rabbits. Each rabbit was immunized with 3 ml of C,, solution mixed with 0.5 ml of complete Freund’s adjuvant and 2.5 ml of incomplete Freund’s adjuvant by means of

phosphate buffer pH 74

0.15M buffer

0.15

s L!J

Pwpara~ion

phosphate pti 24

0.10

0.05

Ig

-

Cl,

+

+

++

*

-

_

_

_

_

_-

-

-

_--+___.______-___

+ t

+

,+c++,

Fractions

Fig. 3. DEAE-celluiose chromatography of crude C,,. using a column of 40 ml. and 12 ml human serum. Elution pattern of C,, and immunoglobulins. The C,, was finally separated from immunoglobulins.

1279

30

20

IO

40

50 + +

C ,q (hemolytic assay) ~agglutination test) Ig (ogglurinati~n test1 Fractions

Fig. 4. DEAE-cellulose chromatography of crude C,,. (column volume 60 ml) Elution pattern of C,, and immunoglobulins. The majority of immunoglobulin was first washed away together with some C,, activity (not shown) and the C,, free of immunoglobulin was finally eluted. gait gradient = 0.3 M NaCl-0.02 M phosphate buffer 0.02 M phosphate buffer (300 ml) ’ Table 3. Effect of low ionic strength on binding activity of C,, __I._~._.___l 1:20 I:80 1:40 1:160 Titer of C,, antiserum I:320 EAC,, from C,, in low g EAC,, from C,, control

+++ t++

+++

++

i

+t+

++

f

1640 -

-

i

Table 4. Hemolytic titer of C,, preparation”

EAb

I:8 -___ i-

EAC,”

+

C,, dilution

I:16

I:32

1:64

+

_

-

+

+

+

I:128

I:4096

... _

-

+

+

No C,,

Method +

f

“The sign + denotes hemolysis, - denotes no hemolysis. ‘The EAC, formed is assayed with C-EDTA-Mg. “The EAC,, formed is assayed with C, and C-EDTA. Table 5. Fixation of C,, by EA (a~lutination

C,, antiserum I:10 C,, antiserum 1:20

assay)

EA(CFD)

E(CFD)

EA(EDTA)

E(EDTA)

+ -

-

+

_ -

Table 6. Agglutination activity of crude C,, Titer of antiserum Antiserum 1:20

I:40

I:80

1:160

I:320 +

EA and crude C,,

Anti C,, Anti Ig

-i-f+ f-t-+

+++ +++

+i-+ ++-I-

EA and inactivated crude C,,

Anti C,, Anti Ig

++ +++

+++

+-t+

E and crude C,,

Anti C,, Anti Ig

+++

+++

-I-++

-I-+ -t-

+++

+++

+++

+

Anti C,, Anti Ig

++ +++

+++

“t-l-+

+

-

E and inactivated crude C,,

+ +++ _ +

-

+

1540

12x0

XU FEN. SHEN Table 7. Hemolytic

-

Intermediate EAC,q, EAC,, EAC,,,

Reaction

reactions

FENG-XIN

of‘ EAClq4

components

+ +

multi-point intradermal injections, as recommended by Vaitukaitis ez al. (1971). These antisera all exhibit a single precipitin line with whole serum, crude C,, and purified C,, preparations. In the agglutination assay with crude C 1q, the titer of anti C,, serum was 1: 160. After inactivating C ,4 at 56C for 30 min, the titer of anti C,, serum was reduced to I:20 (Table 6). l”he formution

of EAC,,

Calcium ions were shown not to be required in the process of C,, fixation by EA. The chelating agent EDTA showed no marked influence on the combination between EA and C,,. This was demonstrated as following. EA and E were suspended in CFD or EDTA saline, respectively, as 2”;, suspensions. Aliquots (0.5 ml) of each suspension were mixed with 0.1 ml of a purified C,, solution. These mixtures were incubated for 15 min at 37°C and over 6 hr at 4°C and then centrifuged. Each sample of red cells was suspended in 2 ml of CFD, and assayed with C , I, antiserum. The result is presented in Table 5. The titer of the C,, antiserum was 1:lO. However, in assaying some crude C,, preparations by the agglutination test, the titer of C ,4 antiserum might reach 1: 160. The higher titer Table 8. JIemolytic

-.__.

._

.-

EAC,,, EA -“C-EDTA-Mg

1:X

0.062

0.555 0.053

activity

of EACi,,

The presence of C,, on EAC, was demonstratedwith~~~,C~,andC-EDTA.EAC~and C,,, were first incubated at 37°C for 15 min, then incubated with C, (30 units) for 5 min and finally with C-EDTA. The results are summarized in Table 7. In the following experiment the hemolytic activity of a C,,, preparation was compared in EAC,,, and EA. It can be seen that the hemolytic activity of Crrs on EAC iq4 was not dependent on C,,, as shown in Table 8. The e~~i~~e~l~e?fC,, Hemolytic and EAC,,

activity

nn ‘EAC,,’

intermediates EAC 1423, EACi,, from EA and human serum were

of C,,, on EAC‘,,,

-

I:128 0 .~~____.__ - 0.219 0.260 0.078

was employed as C?,, source.

Table 9. C,,

reaction

Anti C,, Intermediate I:20 ~_EAC,**, EAC,,, EAC,, EA

The hemolytic

Hemolytic fraction Titer of C,_ I:64 1:32 1:16 _. --. 0.343 0.377 0.449 0.056 0.053 0.064

Intermediate” 1:4

ZENG-BIAN

seemed to be caused by immunoglobulins in the crude C,, solution. These relations are shown in Table 6. The low titer of C,, antiserum in the EAC,, reaction agglutination with purified C,, preparation is explained by the limiting amount of hemolysin on EA. For the preparation of EA the dose of hemolysin is only used at a subagglutinating Ievel, so the C,, which can attach to EA is very limited and the agglutination activity exhibited is feeble. The non-specihcallyfixed immunoglobulin on the surface of red cells can markedly increase the C,, combining sites. This phenomenon was used in a later study as a means of recognizing C,, in chromatographic eluates instead of the hemolytic assay. The latter is time-consuming and inconvenient.

Hemolysis .._-______.

C,. C-EDTA C,,, C,, C-EDTA C,,, C,,,, C,. C-EDTA _-.__

and ZHU

+++ +++ +++

1:40 ___._-~... +t+ +++ +++ _

of EAC,,

serum --

I:160 1:80 _--_ .__-- ..-_ + * ++ i-f _

I:320 _ _

Immune adherence -t -t + +++ -i--t+

UImmune adherence was carried out by mixing two drops of CFD, one drop of 2:; suspension of selected 0 type human erythrocytes and one drop of a 0.5% suspension of EAC on a microreaction plate. and observing the agglutination pattern. A positive result implies the presence of C,, on the intermediate.

1281

EAC,,, prepared according to La~hmann et ~1. (1973). The behaviour of these human complement hemolytic intermediates in C,, agglutination test is shown in Table 9. DISCUSSION

Because the C,,, C,, and Cr, are linked together through calcium ions in the C, molecule, it is assumed that the activity of C, subcomponents is also calcium ion-dependent, and the intermediate generated by treating the intermediate EAC,, with the chelating agent EDTA is assumed to be EAC,. Since EAC, is easily prepared, this intermediate is widely used for assaying C, and its subcomponents (Lepow et al., 1963; Yonemasu & Stroud, 1971; Reid et al., 1972; Calcott & Miiller-Eberhard, 1972; Lachmann et al., 1973; Assimeh et al., 1974, 1978). Our results demonstrate that there is no need . . of calcmm ions m the C,, fixation reaction, as shown in Table 5. In Tables 4,7 and 8, it may be seen that C,, is not required in EAC, hemolysis. Moreover, the results presented in Table 9 show that the C,, is also retained on EAC,,. Therefore it is concluded that EACi4, instead of EAC, is generated from EAC,,, by treating with EDTA saline. For confirming the presence of C,, on EA by the agglutination reaction, it may be seen from Tables 5 and 6 that C,, purification is critical. Traces of heavy metal impurities in reagents may exert makedly unfavourabIe influences upon active protein in the course of purification.

In our previous work in connection with isolation of highly active cathepsin C, those reagents which are consumed in large amounts, such as ammonium sulfate, were refined in such a way that they were dissolved in water, treated with hydrogen sulfide, filtered, recrystallized and dried. Such treatment resulted in a surprising effect. (Xu Fen, Song Quan-yu, Qui Zhi-chang & Jin, Shao-hua, unpublished results). Therefore solid reagents used in the isolation of C,,, except sodium hydroxide, were subjected to such a refinement procedure. The purified C,, was h~molytically active.

REFERENCES Assimeh S, N., Bing D. H. & Painter R. H. (1974) J. Zmmun. 113, 225-234. Assimeh S. N., Chapuis R. M. & lsliker H. (1978) ~rn~zunoc~lernjstr~ 15, 13-17. Becker E. L. (1959) J. Immun. 82, 43-51. Borsos T., Rapp H, I. & Cook C. T. (1961) J; Immw. 87, 330-336. Caicott M. A. & Miiller-Eberhard H. J. (1972) Bfochemisfry 11,3443-3450.

Kabat

E. A. & Mayer M. M. (19611 ~.y~erirnent#~ 2nd Edn. Thomas, Springfield. Lachmann P. J.. Hobar J. & Aston W. P. (1973)1n Handbook ofE.xywrimenta/ l#trnz~n~~#~~ (Edited by Weir D M.) Vol. 1, Chap. 5. Blackwell, Oxford. Lepow I. H., Naff G. El.,Todd E. W., Pensky J. & Hinz E. F. (1963) J. exp. Med. 117, 983-1008. Mach J. P. & Pahud J. P. (1971) J. Immun. 106, 552-563. Reid K. B. M., Lowe D. M. & Porter R. R. (1972) Biochenz. J. 130,749-763. Vaerman J. P., Heremans .I. F. & Vaerman C. (1963) lmm~noehem~str~.

J. Immun.

91, 7-10.

Vaitukaitis J., Robbins J. B., Nieschlag E. & Ross G. H. (1971) J. clin. Endocrin. 33, 988-991. Yonemasu K. & Stroud K. M. (1971) J. ~rn~z~~. 106, 304-313.