Quantitation of the membrane attack complex of complement in an air-driven ultracentrifuge

Journal oflmmunological Methods, 31 (1979) 351--360 © Elsevier/North-Holland Biomedical Press

351

QUANTITATION OF THE MEMBRANE ATTACK COMPLEX OF C O M P L E M E N T IN A N A I R - D R I V E N U L T R A C E N T R I F U G E *

RI C HAR D M. BARTHOLOMEW **, ECKHARD R. PODACK *** and A L F R E D F. ESSER ***,§

Department o f Molecular Immunology, Research Institute o f Scripps Clinic, La Jolla, CA 92037, [LS.A. (Received ] June 1979, accepted 3 August 1979)

A sensitive assay of complement (C) activation via either the classical or alternative pathway was developed by evaluating assembly of the terminal complexes (C5b-9)2 or SC5b-9. Activation of serum containing [125I]C7 resulted in the formation of a stable, radiolabeled complex which was separable from its precursors by sedimentation in an airdriven ultracentrifuge. The radioactivity in the sediment was directly proportional to the amount of complex formed and assembly of the complex could be detected after C activation by aggregated IgG in concentrations as low as 10 pg/ml. Mild detergents such as Triton X-100 could be included in the reaction mixture, because they affected neither the assembly nor the integrity of the complexes. The assay, which detects both assembly of the membrane attack complex (MAC or (C5b-9)2) on target membranes and formation of SC5b-9 in fluid phase, measures the potential of certain substances to trigger the cytolytic phase of C regardless of whether the classical or alternative pathway was activated. However, by using serum depleted of either factor B or C l q , activation of either pathway can be assessed individually.

INTRODUCTION Because measurement of complement (C §§) l e v e l s is t h e basis f o r so m a n y tests o f clinical a n d e x p e r i m e n t a l i m p o r t a n c e , a great m a n y assays have b e e n d e v e l o p e d f o r t h i s p u r p o s e . M e t h o d s t o e s t a b l i s h t h a t C h as b e e n c o n s u m e d in t e s t s e r u m i n c l u d e t h e c l a s s i c a l C f i x a t i o n t e s t ( M a y e r , 1 9 6 1 } , * This is publication number 1728 from the Research Institute of Scripps Clinic. This work was supported by NIH Grants AI 14099 and AI 07007 and in part by contract No. 1 CP 71018 within the Viral Oncology Program of the National Cancer Institute. ** Recipient of Public Health Service Fellowship No. 1 F32 CA 05916. *** Established Investigator of the American Heart Association. § To whom to address correspondence. §§ Abbreviations: Complement components are abbreviated according to WHO recommendations (1968); MAC, membrane attack complex of complement or (C5b-9)2 ; EDTA, ethylenediamine t e t r a a c e t a ~ ; EAC1-7, antibody sensitized sheep erythrocytes with the first 7 components of the classical complement sequence bound; GVB, gelatin-veronal buffer (143 mM NaCl, 3.5 mM barbital, 0.5 mM Mg 2+, 0.15 mM Ca 2., 0.1% gelatin, pH 7.3); SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

352 C1 binding (Augener et al., 1971) or activation (Cooper and Ziccardi, 1977), C3 consumption or cleavage (Cooper et al., 1970) and activation of factor B (GStze and Mfiller-Eberhard, 1971). In addition, one can test for formation of the terminal C complexes -- the membrane attack complex (MAC), which is composed of the C components (C5b-9)2, or its fluid phase equivalent SC5b-9 -- by using a hemolytic assay, or by measuring consumption of the terminal components, or by demonstration of these components on sucrose density gradients (Kolb and Miiller-Eberhard, 1973). Unfortunately, the results of these tests are sometimes ambiguous or the techniques are often n o t suitable. Therefore, we have developed an assay that is sensitive and simple to perform. The essence of this technique is to deplete the serum of one of the precursor components of MAC, restore this c o m p o n e n t in a radiolabeled form, then after activation separate the heavy weight MAC from the lighter weight precursors in the serum by high speed centrifugation. As a result, one can evaluate the degree of C activation, the participation of each pathway and the extent of MAC formation on cells. MATERIALS AND METHODS

Serum and C reagents Human serum originated from the C o m m u n i t y Blood and Plasma Service (San Diego, CA) and was depleted of either C7 or C8 by affinity chromatography in the presence of 10 mM EDTA (Podack et al., 1977a). This procedure also depleted the serum of C l q , which was replaced to its level in normal serum. Factor B and factor B-depleted serum were donated by Dr. R.D. Schreiber, Scripps Clinic and Research Foundation (La Jolla, CA). All sera were stored at --70°C until used. Purified SC5b-9 (Kolb and MiillerEberhard, 1975a), C7 (Podack et al., 1976), C8 (Kolb and Mtiller-Eberhard, 1976) and C l q (Kolb et al., 1979) were obtained as described previously. C activators Zymosan (Nutritional Biochemical Corporation, Cleveland, OH) was refluxed in saline for 60 min at a concentration of 100 mg/ml and stored at 4°C suspended in saline. Inulin (DIFCO Laboratories, Detroit, MI) was used as a saline suspension at a concentration of 200 mg/ml. IgG was fractionated from human serum by a m m o n i u m sulfate precipitation and ion exchange chromatography (Miiller-Eberhard et al., 1956), then aggregated (agg-IgG) at a concentration of 10 mg/ml by heating at 63°C for 12 min. Moloney murine leukemia virus was prepared by Electro Nucleonics Inc., Bethesda, MD, and supplied by the Office of Program Resources and Logistics, Viral Oncology, National Cancer Institute. The viral envelope protein P15E was isolated and purified as previously described (Bartholomew et al., 1978). Preparation o f [12si] C 7 and [12si] C8 reagen t Purified C7 or C8 (70 pg) was radiolabeled with 500 /aCi [12SI]Na by

353 using the solid-state lactoperoxidase m e t h o d of David and Reisfeld (1974). [~25I]C7 (or C8) (106 cpm, 2.5 pg) was added to C7- (or C8-) depleted serum prepared as described above. Normal C l q and bivalent metal ion concentrations were restored by adding 70 pg C l q / m l and Ca 2÷ and Mg 2÷ to final concentrations of 4.5 mM and 15 mM respectively.

Preparation o f EA C1--7 EA (1.5 × 10 s cells/ml) were suspended in 10 ml GVB and mixed with 0.5 ml C8-depleted serum which had been reconstituted in C l q , Ca 2÷ and Mg 2÷ to normal levels. After a 30 min incubation at 37°C the EAC1--7 were washed with GVB and adjusted to 1.5 × 108 cells/ml.

Measurement o f C1 activation Methods for preparing radiolabeled C1 in serum and details of its use to measure specific cleavage of [12sI]Cls to [12sI]Cl~ have been published (Bartholomew and Esser, 1977).

Assay for (C5b-9)2 or SC5b-9 assembly To 100 pl of [12sI]C7 (or C8) reagent, the test sample in 50 pl buffer was added and incubated for 30 min at 37 ° C. In the case of particulate activators such as zymosan or inulin, the mixture was first cleared by low speed centrifugation. One hundred pl of the resulting mixture was overlaid on 50 pl cushions of 15% sucrose in 175 pl capacity centrifuge tubes and centrifuged at 160,000 ×gmax for 90 min in an air-driven ultracentrifuge (Airfuge, Spinco Division of Beckman Instruments, Inc., Palo Alto, CA). The supernates were absorbed into tissue paper and discarded. Radioactivity in the sediments was determined by cutting off and analyzing the b o t t o m s of each tube. Some assays were performed with 6 mM deoxycholate or 1% Triton X-100 included in all buffers.

Hemagglutination-inhibition assay The distribution of C5b-9 in the ultracentrifuged mixture was measured by the hemagglutination inhibition assay (Kolb and Mfiller-Eberhard, 1975b). Briefly, 25 pl of serially diluted samples (final dilution 1 : 2048) were mixed with 25 pl of 1 : 200 diluted antiserum (antineo) to detect complex-specific neoantigens. After a 30 min preincubation at 25°C, EAC1--7 (25 pl, 1.5 X l 0 s cells/ml) were added to each well, followed by further incubation at 37°C for 30 min and at 4°C overnight.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PA GE) C5b-9 assay samples to be analyzed by SDS-PAGE were all prepared in the presence of 6 mM deoxycholate (see above) to prevent aggregation of the complex (Biesecker et al., 1979) and to facilitate removal of contaminating proteins not part of SC5b-9. All samples were washed thrice with 6 mM deoxycholate by centrifugation in the Airfuge and the pellets were then

354 incubated overnight at 37°C in 1% SDS and 4 M deionized urea. Electrophoresis in 12 cm 7.5% polyacrylamide gels was p e r f o r m e d as described by Weber and Osborn (1969). Protein bands were visualized by staining the gels with 0.02% Coomassie brilliant blue R-250. RESULTS The assay for f or m at i on of the terminal reaction complexes of complement, SC5b-9 or MAC respectively, is based on the large size difference between [12sI]C7 in its isolated f or m and [12"~I]C7 that is assembled into one of the stable complexes. The f o r m a t i o n of the m e m b r a n e - b o u n d MAC and the fluid phase SC5b-9 can be written in the following way: 2C5 + 2C6 + 2C7 + 2C8 + 6C9

C5 convertase m e m b r a n e * 2C5a + (C5b, C6, C7, C8, C93)2

C5+C6+C7+C8+3C9+3S--

C5 convertase , C 5 a + S 3 C 5 b , C6, C7, C8, C93 fluid phase

(1)

(2) Upon activation of the [125I]C7 reagent to form either the 1.7 × 106 dalton MAC (Eq. 1) or the i × 106 dalton SC5b-9 (Eq. 2) we found routinely that a p p r o x i m a t e l y 35--40% of the total [12sI]C7 i m put was recovered in the sedimented material whereas in the presence of EDTA to block C activation only 5--8% could be pelleted. Th at the sedimented [J2sI]C7 after c o m p l e m e n t activation was part of a C5b-9 c o m p l e x was d e m o n s t r a t e d directly by SDS-PAGE. For this experiment, normal human serum was used in place of the C7-depleted serum so that, u p o n activation, C5b-9 complexes could form in am ount s detectable on SDS-PAGE. Fig. 1 shows the SDS-PAGE pattern that resulted when the sediment from zymosan-activated human serum was analyzed. For comparison, the distribution of protein bands of purified SC5b-9 and MAC is also shown. The sediment f r om activated serum contained each of the terminal c o m p l e m e n t protein bands typical of the SC5b-9 complex. The S-protein is diminished due to dissociation by d e o x y c h o l a t e (Podack and Miiller-Eberhard, 1978), which was necessary in this e x p e r i m e n t to prevent complex aggregation and to facilitate removal of contaminating proteins that were not part of SC5b-9. Also present towards the top of the gel are large molecular weight proteins, mainly IgM and az-macroglobulin, both of which have S-rates similar to that of SC5b-9, and an unidentified band migrating between C5b and C6 which is also present in MAC. When 10 mM EDTA was added to the serum to prevent complex f o r m a t i o n , protein bands corresponding to the terminal c o m p o n e n t s could no longer be detected by SDSPAGE.

355

.IC

C5b.,,,. C5C-"

C6... S-" C9-..

co, -

Fig. 1. S D S - P A G E of t h e s e d i m e n t a b l e m a t e r i a l f r o m z y m o s a n - a c t i v a t e d serum. A 1 ml a l i q u o t of z y m o s a n - a c t i v a t e d s e r u m was m a d e 6 mM in s o d i u m d e o x y c h o l a t e a n d centrifuged in 150 pl a l i q u o t s as d e s c r i b e d in t h e text. T h e r e s u l t i n g pellets were w a s h e d 3 t i m e s w i t h a 70 m M Tris-acetate (pH 8.1) b u f f e r c o n t a i n i n g 90 m M NaC1, 2 mM E D T A a n d 6 mM d e o x y c h o l a t e , t h e n c o m b i n e d , dissolved in 50 ~l 8 M urea, 2% SDS a n d analyzed b y SDS-PAGE. Purified SC5b-9 ( 1 0 0 p g ) a n d p u r i f i e d M A C ( 1 0 0 p g ) are i n c l u d e d for c o m p a r i s o n a n d were p r e p a r e d for e l e c t r o p h o r e s i s as d e s c r i b e d in Materials a n d M e t h ods.

Analysis of C5b-9 formation A hemagglutination inhibition assay was used to analyze the C5b-9 distribution in ultracentrifuged samples (Fig. 2). The a m o u n t of C5b-9 complexes in an unfractionated sample was compared to the a m o u n t of C5b-9 complexes in the corresponding supernate and sedimented material after centrifugation. C5b-9 complexes were quantitatively sedimented during the centrifugation, with only negligible amounts remaining in the supernate. Each

356

A B C D E Serial Dilutions of Antigen ( Fig. 2. C5b-9 distribution in ultracentrifuged samples. A 200 pl sample of serum was incubated with the desired activator at 37°C for 45 rain (data shown are for 100 pg Moloney leukemia virus). One-half of the sample (100 pl) was ultracentrifuged, the supernate was removed and the resulting sediment resuspended in GVB to its original volume. Fifty pl aliquots of the unfractionated sample (A), the resuspended pellet (B) and the supernate (C) were serially diluted with GVB and their C5b-9 content directly compared by using the C5b-9 complex-specific hemagglutination-inhibition assay. An unfractionated sample of serum incubated with virus and 10 mM EDTA (D) and a sample containing 4/lg purified C5b-9 (E) served as controls. Sample wells in which the cells settle as buttons, rather than agglutinate, signify the presence of C5b-9 complexes. a c t i v a t o r , w h e n a d d e d in e x c e s s , i n d u c e d f o r m a t i o n o f e q u i v a l e n t a m o u n t s o f c o m p l e x e s ( d a t a n o t s h o w n ) , a n d y i e l d e d b a c k g r o u n d levels o f c o m p l e x e s in s a m p l e s c o n t a i n i n g 10 mM E D T A .

The limiting c o m p l e m e n t c o m p o n e n t is C7 To i n c r e a s e t h e s e n s i t i v i t y o f t h e assay, [12sI]C7 was a d d e d a t a c o n c e n t r a t i o n o f 7 p g / m l or less. T h e c o n c e n t r a t i o n o f C7 in s e r u m is a p p r o x i m a t e l y 70 p g / m l ( P o d a c k et al., 1 9 7 7 a ) . D o s e - r e s p o n s e e x p e r i m e n t s s h o w e d t h a t , in the presence of excess activator, c o m p l e x f o r m a t i o n increased linearly with i n c r e a s i n g c o n c e n t r a t i o n s of [12sI]C7 u n t i l C7 r e a c h e d e q u i m o l a r c o n c e n t r a t i o n s to t h e o t h e r t e r m i n a l C c o m p o n e n t s . T h e s e d i m e n t e d r a d i o a c t i v i t y a m o u n t e d t o 35% o f t h e t o t a l C7 i n p u t a n d d r o p p e d to l o w e r levels w h e n C7 was in e x c e s s o v e r t h e o t h e r t e r m i n a l c o m p o n e n t s .

Responses to different activators In c o m p a r i n g S C 5 b - 9 or M A C f o r m a t i o n i n d u c e d b y z y m o s a n , i n u l i n , agg-IgG a n d M - M u L V (Fig. 3), we f o u n d l i t t l e v a r i a t i o n in t h e a m o u n t s o f

357

i

~ 2o

,~

~.~.lO

1

°1 1

Ii

,,o

Agg-lgG (/~g)

1,5

Fig. 3. Analysis o f C5b-9 f o r m e d by several d i f f e r e n t C activators. Activators in 50 pl samples were i n c u b a t e d with 100 pl C7-reagent at 37°C for 45 min and t h e n assayed for C5b-9 f o r m a t i o n as described. (A) 50 pg agg-IgG; (B) 2 mg z y m o s a n ; (C) 50 pg M o l o n e y l e u k e m i a virus; (D) 2 mg inulin; (E) 2 mg z y m o s a n , 0.5% T r i t o n X-100. ~ = i n c u b a t e d in the p r e s e n c e o f Ca2+/Mg2+; • = i n c u b a t e d in the p r e s e n c e o f 10 mM EDTA. Fig. 4. D o s e - r e s p o n s e curve for C5b-9 f o r m a t i o n by agg-IgG. Increasing a m o u n t s o f agg-IgG c o n t a i n e d in 50 pl were i n c u b a t e d 45 rain at 37°C with 100 pl C7-reagent containing 0.25 pg [12sI]C7 (10 s c p m ) . C5b-9 f o r m a t i o n was t h e n m e a s u r e d as described.

radiolabeled C7 that sedimented. The presence of Triton X-100 or deoxycholate during activation and subsequent centrifugation steps did n o t interfere with the assay.

Sensitivity o f the method In C7-depleted serum r econs t i t ut e d with 2.5 pg/ml [12sI]C7, 1 pg of soluble agg-IgG was sufficient to induce measurable C5b-9 f o r m a t i o n (Fig. 4). A linear dose-response curve was obtained with up to 12 pg agg-IgG, at which c o n c e n t r a t i o n C5b-9 f o r m a t i o n became maximal. The sensitivity of the assay depends on the specific activity of the radiolabeled C7 and the a m o u n t o f C7 used for the r e c o n s t i t u t i o n o f C7-depleted serum. C5b-9 formation by the classical or alternative pathway Assembly o f the terminal C complexes occurs after activation of either the classical or alternative c o m p l e m e n t pathway. Complex f o r m a t i o n in serum thus represents the com bi ne d response of both activation mechanisms. However, each p a t hw ay can be measured individually by employing different depleted sera. Fig. 5 shows SC5b-9 f o r m a t i o n in normal, C l q and factor B-depleted sera after incubation with buffer, agg-IgG, an activator of the classical p a t h w a y , and inulin, an activator of the alternative pathway. Each activator induced f o r m a t i o n of SC5b-9 in normal serum. When C l q - d e p l e t e d serum was tested, agg-IgG was ineffective, while the response to inulin remained. Likewise, f a c t or B-depleted serum did n o t form SC5b-9 in response to inulin, but was maximally activated by agg-IgG. These results

358

Serum

~

CIq Depleted Serum

30 20

oli A

B

C

A

B

Factor B DepletedSerum

C

JD N A

8

C

Fig. 5. SC5b-9 f o r m a t i o n by the classical or alternative p a t h w a y . 100 ;ll aliquots o f h u m a n s e r u m , C l q - d e p l e t e d , and f a c t o r B - d e p l e t e d s e r u m were i n c u b a t e d at 37°C for 45 min with: (A) GVB; (B) 50 pg agg-IgG (an activator o f the classical p a t h w a y ) ; and (C) 2 m g inulin (an activator o f the alternative p a t h w a y ) . SC5b-9 f o r m a t i o n was m e a s u r e d as described.

d o c u m e n t that each pathway can be abrogated in serum without measurably affecting the other. Hence, the m e t h o d can be used to not only measure the potential of complement activators to initiate assembly of C5b-9, but also to study which activation mechanism was involved in the triggering. M A C formation on detergent micelles Assembly of C5b-9 complexes requires the proteolytic cleavage of C5 by the C5 convertase of either the classical or alternative pathway. These enzymes are extremely inefficient in the fluid phase. Most activators such as agg-IgG or zymosan are so large that they supply the necessary binding support for the convertases. However, we were surprised to find that purified P15E, a 15,000 dalton envelope protein from murine leukemia viruses,

TABLE 1 D E M O N S T R A T I O N T H A T MAC F O R M A T I O N BY P 1 5 E R E Q U I R E S T R I T O N X-100 Sample a

C1 activation (% C l g )

1 pg P 1 5 E + 0.05% T r i t o n X-100 1 pg P 1 5 E + < 0 . 0 0 1 % T r i t o n X-100 0.05% T r i t o n X-100 5 0 / l g agg-IgG + 0.05% T r i t o n X-100

63 69 9 83

b

MAC f o r m a t i o n c (% C7 s e d i m e n t e d ) 36 12 9 38

a A m o u n t s o f P 1 5 E are e s t i m a t e d f r o m activity m e a s u r e m e n t s . b C1 activation m e a s u r e d by analyzing c o n v e r s i o n o f [ 12 s I ] C l s to [ 12 s I ]CI~ as d e s c r i b e d by B a r t h o l o m e w and Esser (1977). c MAC f o r m a t i o n m e a s u r e d as d e s c r i b e d in the t e x t .

359 seemed to induce MAC formation {Bartholomew et al., 1978). Additional experiments indicated that in addition to P15E, Triton X-100 micelles were required for MAC formation, but not for C1 activation. As shown in Table 1, P15E activated C1 in the presence or absence of Triton X-100, but required the detergent to induce MAC assembly. Dose-response experiments showed that Triton X-100 had to be present in the form of micelles (>0.01%) to allow complex formation. Therefore, it seems that in this system Triton micelles served as the support for the C5 convertase. DISCUSSION We have described an assay for the quantitation of MAC and SC5b-9 assembly in serum depleted of C7 and then reconstituted with [12sI]C7 of high specific activity. The radiolabeled complexes formed upon activating this serum are easily separated from the free C7 by differential centrifugation in an air-driven ultracentrifuge, and the radioactivity of the sedimented material corresponds to the extent of complement activation. Thus, the assay directly measures the initiation of the membrane attack pathway, which in turn depends on the formation of a C5 convertase and its activation of C5 by proteolytic cleavage. Since this assay measures the membrane attack unit, rather than its cytolytic function, it can be utilized to quantitate MAC formation on complement-resistant cells such as t u m o r cells (Ohanian et al., 1973} or bacteria (Inoue, 1972}. The assay is n o t adversely affected by detergents such as Triton X-100 and, therefore, is useful under conditions that would lyse erythrocytes and thus preclude subsequent hemolytic assays of complement activity. In fact, this assay was successful in monitoring a complement activator on retroviruses during its isolation from detergent-disrupted virus (Bartholomew et al., 1978}. This stability of C5b-9 complexes to Triton X-100 ensures that the assay is useful for measuring MAC formed on cells, artificial bilayers and similar systems that may require solubilization of the complex from a membrane fragment prior to analysis. Because radiolabeled C8 in conjunction with C8-depleted serum was used in a similar assay, C5, C6, or C9 and their respective depleted sera should also function adequately and extend the method's versatility to reagents which might be more accessible to other investigators. Finally, two major limitations of the assay system should be mentioned. Obviously this assay is not applicable to systems that prohibit the differential sedimentation of the C5b-9 complex. In such cases, prior extraction of the formed complexes with deoxycholate may be feasible. The other drawback of the system is the relatively small volume of testable material dictated by the 175 gl capacity of the airfuge tubes. We have found that as little as 10 /~l of depleted serum can be used in the assay, thus permitting analysis of a larger sample volume. However, one must consider that diluting serum more that 10-fold abrogates the alternative pathway of complement (Leon, 1956).

360 ACKNOWLEDGEMENT We w i s h t o t h a n k V i c k i W a p n o w s k i f o r s k i l l e d s e c r e t a r i a l h e l p . REFERENCES Augener, W., H.M. Grey, N.R. Cooper and H.J. Mfiller-Eberhard, 1971, Immunochemistry 8, 1011. Bartholomew, R.M. and A.F. Esser, 1977, J. Immunol. 119, 1916. Bartholomew, R.M., A.F. Esser and H.J. Mfiller-Eberhard, 1978, J. Exp. Med. 147, 884. Biesecker, G., E.R. Podack, C.A. Halverson and H.J. Mfiller-Eberhard, 1979, J. Exp. Med. 149,448. Cooper, N.R. and R.J. Ziccardi, 1977, J. Immunol. 119, 1664. Cooper, N.R., M.J. Polley and H.J. Mfiller-Eberhard, 1970, Immunochemistry 7, 341. David, G.S. and R.A. Reisfeld, 1974, Biochemistry 13, 1014. GStze, O. and H.J. Mfiller-Eberhard, 1971, J. Exp. Med. 134,905. Inoue, K., 1972, Res. Immunochem. Immunobiol. 1, 177. Kolb, W.P. and H.J. Mfiller-Eberhard, 1973, J. Exp. Med. 138,438. Kolb, W.P. and H.J. Miiller-Eberhard, 1975a, J. Exp. Med. 141, 724. Kolb, W.P. and H.J. Mfiller-Eberhard, 1975b, Proc. Natl. Acad. Sci. U.S.A. 72, 1687. Kolb, W.P. and H.J. Mfiller-Eberhard, 1976, J. Exp. Med. 143, 1131. Kolb, W.P., L.M. Kolb and E.R. Podack, 1979, J. Immunol. 122, 2103. Leon, M.A., 1956, J. Exp. Med. 103,285. Mayer, M.M., 1961, in: Experimental Immunochemistry, eds. E.A. Kabat and M.M. Mayer (Thomas, Springfield, IL) p. 133. Mfiller-Eberhard, H.J., H.G. Kunkel and E.C. Franklin, 1956, Proc. Soc. Exp. Biol. Med. 93, 146. Ohanian, S.H., T. Borsos and H.J. Rapp, 1973, J. Natl. Cancer Inst. 50, 1313. Podack, E.R. and H.J. Miiller-Eberhard, 1978, J. Immunol. 121, 1025. Podack, E.R., W.P. Kolb and H.J. Miiller-Eberhard, 1976, J. Immunol. 116, 263. Podack, E.R., W.P. Kolb and H.J. Mfiller-Eberhard, 1977a, J. Immunol. 119, 2024. Podack, E.R., C. Halverson, A.F. Esser, W.P. Kolb and H.J. Mfiller-Eberhard, 1977b, J. Immunol. 120, 1792. Weber, K. and M. Osborn, 1969, J. Biol. Chem. 244, 4406.