An enzyme-linked immunoassay for the detection of antibodies to the mouse mammary tumor virus: Application to human breast cancer

Journal oflmmunological Methods, 32 (1980) 85--91 © Elsevier/North-Holland Biomedical Press

85

AN ENZYME-LINKED IMMUNOASSAY FOR THE DETECTION OF ANTIBODIES TO THE MOUSE MAMMARY TUMOR VIRUS: APPLICATION TO HUMAN BREAST CANCER

STEVEN S. WITKIN, NURUL H. SARKAR, ROBERT A. GOOD and NOORBIBI K. DAY Memorial Sloan-Kettering Cancer Center, New York, N Y 10021, U.S.A.

(Received 8 August 1979, accepted 13 September 1979)

An enzyme-linked immunoassay (ELISA) was developed, using the mouse mammary tumor virus (MMTV) fixed to wells of a microtiter plate, for the determination of antibodies to MMTV. The intensity of the final color change was dependent upon virus or viral antibody concentration. MMTV antibody was readily detectable in sera diluted as much as 1 : 2800. Fixed MMTV bound antibodies to an internal viral protein (p 28) as well as to viral envelope components (gp 52, gp 34), demonstrating that the virus was rendered permeable by our procedure. Applying this assay to human sera, significant differences (P < 0.005) in IgG binding to MMTV were detected between sera of breast cancer patients, benign breast disease patients and healthy individuals. 26% of breast cancer-derived sera contained MMTV binding antibody; 10% of benign sera or 8% of normal sera were also positive. The reactivity of human IgG with MMTV was blocked by prior incubation of the virus with antisera to gp 34 or, to a lesser extent, with gp 52. The results demonstrate that MMTV antibodies can be quantitated by this simple, rapid and inexpensive procedure.

INTRODUCTION T h e r e is a g r e a t n e e d f o r t h e d e v e l o p m e n t o f m e t h o d s t o i d e n t i f y w o m e n a t i n c r e a s e d r i s k f o r b r e a s t c a n c e r , t o d e t e c t e a r l y stage b r e a s t c a n c e r a n d t o m o n i t o r its c o u r s e f o l l o w i n g s u r g i c a l a n d / o r c h e m i c a l i n t e r v e n t i o n . P r e s e n t l y , o b s e r v a t i o n o f m e t a s t a s i s t o t h e l y m p h n o d e s is t h e m a j o r m e t h o d o f disc r i m i n a t i n g b e t w e e n p a t i e n t s in t e r m s o f t h e n e e d f o r p o s t - s u r g i c a l t h e r a p y . H o w e v e r , 18% o f p a t i e n t s w i t h o u t a p p a r e n t l y m p h n o d e i n v o l v e m e n t will experience tumor recurrence (Coombes, 1978). T h e i d e n t i f i c a t i o n o f a n t i g e n s o r a n t i b o d i e s in h u m a n s e r u m r e l a t e d t o t h e m o u s e m a m m a r y t u m o r virus ( M M T V ) m i g h t l e a d t o t h e d i f f e r e n t i a l diagnosis o f b r e a s t c a n c e r . B l a c k e t al. ( 1 9 7 5 ) , u s i n g a l e u k o c y t e m i g r a t i o n inhibition assay, have shown t h a t only l e u k o c y t e s from breast cancer patients w e r e r e s p o n s i v e t o M M T V . S i m i l a r l y , b y an in v i t r o l y m p h o c y t e t r a n s f o r m a t i o n a s s a y , it w a s s h o w n t h a t l y m p h o c y t e s f r o m 50% o f b r e a s t c a n c e r o r benign breast disease patients were responsive to mouse milk which cont a i n e d M M T V ( C u n n i n g h a m - R u n d l e s e t al., 1 9 7 6 ) . M e s a - T e j a d a e t al. ( 1 9 7 8 )

86

d e m o n s t r a t e d th at 39% of human breast cancers, but n o t corresponding normal m a m m a r y tissue, contained an antigen immunologically related to the MMTV surface protein, gp 52. Sera f r om breast cancer patients, but n o t f r o m normals, has been shown to contain antibodies reactive with MMTV (Charney and Moore, 1971). Recently, we have dem onst rat ed that sera of breast cancer patients are more efficient in MMTV virolysis than are sera f r o m patients with benign m a m m a r y disease, colorectal cancer, or from normals (Witkin et al., 1979). All of the above procedures suffer f r om the disadvantage of being unamenable to the screening of large numbers of patients on a routine basis. In this report, we describe a simple, reliable and inexpensive enzyme-linked immunoassay for the de t e c t i on of antibodies to MMTV in human sera. M A T E R I A L S AND M E T H O D S

Sera Blood f r o m patients with breast cancer or benign m a m m a r y disease was obtained u p o n admission to Memorial Hospital and processed to obtain serum as previously described (Witkin et al., 1979). Blood from age-matched apparently healthy individuals was similarly treated. All samples were stored in aliquots a t - - 7 0 ° C until used. Antisera were prepared in rabbits to whole MMTV (Sarkar and Dion, 1975) or to purified individual MMTV proteins gp 52, gp 34 or p 28 (Marcus et al., 1978; Marcus et al., 1979). Virus MMTV, a ty pe B retrovirus, was purified by sucrose b u o y a n t density centrifugation f r o m the culture supernatant of chronically infected MuMT-73 cells (Sarkar et al., 1977).

Enzyme-linked immunoassay (ELISA ) The procedure described by Voller et al. (1976) was modified to permit t h e d e t e c t i o n and quantitation of antibodies to MMTV. Purified MMTV (at c o ncen tr atio n s indicated in the individual experiments) was diluted in 0.01 M carbonate buffer, pH 9.8 and 200 pl applied to individual wells of a m i cr o titer plate (Dynatech Laboratories, Alexandria, VA). After an overnight incubation at 4 ° C, the liquid was r e m oved and the wells were washed 3 times with 200 pl aliquots of phosphate-buffered saline-0.05% Tween 20 (PBS-Tween). Human sera diluted 1 : 8 in PBS-Tween (200 pl final vol.) or various dilutions of sera f r om immunized rabbits were then added in duplicate and the samples were incubated at r o o m t e m p e r a t u r e for 120 min. The diluted sera were removed, the wells washed 3 times with PBS-Tween and a 1 : 2 0 0 dilution in PBS-Tween of alkaline phosphatase-conjugated rabbit anti-human IgG (or goat anti-rabbit IgG) (Microbiological Associates, Walkersville, MD) was added. Following a n o t h e r 120 min incubation at r o o m

87 t e m p e r a t u r e , t h e s o l u t i o n s w e r e r e m o v e d , t h e wells again w a s h e d 3 t i m e s w i t h P B S - T w e e n a n d 2 0 0 pl o f a 1 m g / m l s o l u t i o n o f p - n i t r o p h e n y l p h o s p h a t e ( C a l b i o c h e m , L a J o l l a , CA) in 10% d i e t h a n o l a m i n e , 0.5 m M MgCl:, p H 9.8 was a d d e d . T h e r e a c t i o n s w e r e t e r m i n a t e d a f t e r 30 m i n b y t h e a d d i t i o n o f 50 pl 3 N N a O H a n d t h e i n t e n s i t y o f t h e y e l l o w c o l o r w h i c h f o r m e d was q u a n t i t a t e d b y d e t e r m i n i n g t h e a b s o r b a n c e o f t h e s o l u t i o n s at 4 0 0 n m in a spectrophotometer. Sera were a s s a y e d in parallel in wells w i t h o u t M M T V a n d t h e i r a b s o r b a n c e ( 0 . 0 2 - - 0 . 0 7 ) s u b t r a c t e d f r o m t h e e x p e r i m e n t a l values. D u p l i c a t e values differing b y m o r e t h a n 10% w e r e re-assayed. RESULTS

Characteristics o f the MMTV-ELISA assay V a r y i n g c o n c e n t r a t i o n s o f M M T V ( 0 . 2 5 - - 1 0 pg) w e r e f i x e d t o wells o f a m i c r o t i t e r p l a t e a n d i n c u b a t e d f o r 1 2 0 m i n w i t h a 1 : 500 d i l u t i o n o f s e r u m e i t h e r f r o m an u n i m m u n i z e d r a b b i t or f r o m a r a b b i t i m m u n i z e d w i t h w h o l e M M T V . R a b b i t I g G b i n d i n g t o M M T V was q u a n t i t a t e d b y o u r E L I S A assay. T h e results (Fig. 1) d e m o n s t r a t e d t h a t n o r m a l r a b b i t I g G did n o t b i n d t o M M T V u n d e r o u r e x p e r i m e n t a l c o n d i t i o n s . In c o n t r a s t , I g G f r o m t h e M M T V - i m m u n i z e d r a b b i t was b o u n d to M M T V . O v e r an 0 . 1 - - 1 0 p g r a n g e o f M M T V c o n c e n t r a t i o n , I g G b i n d i n g was p r o p o r t i o n a l to t h e a m o u n t o f f i x e d M M T V p r e s e n t . This result d e m o n s t r a t e d t h e s p e c i f i c i t y o f o u r assay f o r a n t i b o d y t o M M T V a n d , in a d d i t i o n , illustrated t h a t t h e a m o u n t o f M M T V t h a t b o u n d to t h e wells was a f u n c t i o n o f t h e initial M M T V i n p u t .

2.0

c 0 0

t.6

t.2 c

~ 0.8

0.4

J

0.0¶

0.1

1.0

10

(zg) Fig. 1. Rabbit anti-MMTV IgG or unimmunized rabbit IgG binding to MMTV fixed to wells of a mierotiter plate. Serum from an unimmunized rabbit (o) or from a rabbit immunized with MMTV (e), both diluted 1 : 500, were incubated for 120 rain in wells containing various concentrations of fixed MMTV. The binding of rabbit IgG to the MMTV was quantitated by the ELISA assay using alkaline phosphatase-eonjugated goat antirabbit IgG. MMTV

88

The kinetics of rabbit anti-MMTV IgG binding to fixed MMTV is illustrated in Fig. 2. For this experiment, the time of incubation of a 1 : 500 dilution of antisera to whole MMTV with fixed MMTV (0.5 /~g/well) was varied. All other reaction steps were as described in Methods. The rate of IgG binding to MMTV was approximately linear over the incubation interval of 15--300 min. Incubation for as little as 15 min, however, resulted in easily detectable binding levels. The sensitivity of our assay was assessed by measuring IgG binding to fixed MMTV (0.5 pg/well) using various dilutions of the rabbit anti-MMTV sera. Serum dilutions as low as 1 : 2800 were easily accessible for MMTV IgG (Fig. 3). These results also demonstrated that the final absorbance obtained varies directly as a function of MMTV antibody concentration.

Components o f bound MMTV accessible to antibody It was of interest to determine the effect of our MMTV binding and ELISA assay procedures on the availability of MMTV components for antibody binding. Accordingly, the binding of IgG from rabbits immunized with the purified MMTV protein gp 52, gp 34 or p 28 was measured, gp 52 is the major external envelope protein, gp 34 is located within the envelope while

0.6

0.9

0,5 c 0 0

0.8 c

0.4

°o

0.71

I

2

0.6

0.3

3

0.2

0.5

0.1

o

6'0

2. o Time (rain.}

3 0'0

1:100

, 1:300 Rabbit

f : l O, 0 0

Anti-MMTV

1:3000

Serum

Fig. 2. K i n e t i c s o f anti-MMTV IgG b i n d i n g t o MMTV fixed to wells o f a m i c r o t i t e r plate. A 1 : 500 d i l u t i o n o f r a b b i t a n t i s e r u m t o MMTV was i n c u b a t e d for various p e r i o d s o f t i m e w i t h 0.5 pg o f MMTV fixed t o wells o f a m i c r o t i t e r plate. The a n t i s e r u m was t h e n r e m o v e d , the wells w a s h e d 3 t i m e s w i t h PBS-Tween and the a m o u n t o f r a b b i t IgG b o u n d to the M M T V was q u a n t i t a t e d b y t h e E L I S A assay. Fig. 3. E f f e c t o f a n t i b o d y d i l u t i o n o n t h e b i n d i n g o f anti-MMTV IgG to MMTV. Various d i l u t i o n s o f s e r u m f r o m a r a b b i t i m m u n i z e d against MMTV were i n c u b a t e d for 120 m i n w i t h 0.5 pg o f MMTV fixed t o wells o f a m i c r o t i t e r plate. M e a s u r e m e n t o f r a b b i t IgG b o u n d t o MMTV was as d e s c r i b e d in Fig. 1.

89 p 28 is a c o m p o n e n t o f t h e i n t e r n a l viral c o r e . A n t i b o d i e s f r o m all 3 sera b o u n d to M M T V (Table 1), while a n t i b o d y f r o m an i m m u n i z e d r a b b i t was u n r e a c t i v e . T h u s , o u r p r o c e d u r e , p r o b a b l y the inclusion o f T w e e n 20, r e n d e r e d t h e virus p e r m e a b l e to a n t i b o d i e s a n d p e r m i t t e d t h e d e t e c t i o n o f a n t i b o d i e s t o i n t e r n a l as well as t o e x t e r n a l virion p r o t e i n s .

A n t i b o d y to M M T V in human sera T h e E L I S A assay was n e x t a p p l i e d t o t h e d e t e c t i o n o f a n t i b o d y to M M T V in h u m a n s e r u m . Sera w e r e o b t a i n e d f r o m 54 p a t i e n t s w i t h b r e a s t cancer, 58 p a t i e n t s w i t h benign m a m m a r y disease a n d 63 h e a l t h y individuals a n d t e s t e d w i t h o u r assay using 0.5 p g M M T V / w e l l (Table 2). T h e d i s t r i b u t i o n o f h u m a n I g G b i n d i n g to M M T V was significantly d i f f e r e n t f o r t h e 3 p a t i e n t classes (X 2 test, P < 0 . 0 0 5 ) . Using the m e a n plus 2 S.D. o f t h e n o r m a l s e r u m values (0.8 a b s o r b a n c e ) to distinguish b e t w e e n specific a n d b a c k g r o u n d IgG b i n d i n g to M M T V , 14 o f 54 b r e a s t c a n c e r p a t i e n t sera (26%), 6 o f 58 benign b r e a s t disease p a t i e n t sera (10%) and 5 o f 63 n o r m a l sera (8%) were positive. All 5 positive n o r m a l sera plus 4 o f t h e 6 benign sera h a d a b s o r b a n c e values b e l o w 1.4. E i g h t o f t h e 14 p o s i t i v e b r e a s t c a n c e r sera a n d 2 benign sera y i e l d e d an a b s o r b a n c e a b o v e 1.4, r a n g i n g u p t o 2.5 a b s o r b a n c e . Specificity o f human IgG binding to M M T V In o r d e r t o d e t e r m i n e if h u m a n I g G was in f a c t r e a c t i n g w i t h M M T V , t h e effect on human IgG reactivity of the prior incubation of bound MMTV with a n t i s e r a t o M M T V p r o t e i n s was e x a m i n e d . M M T V (0.5 p g / w e l l ) was incub a t e d f o r 90 m i n at r o o m t e m p e r a t u r e w i t h sera f r o m an u n i m m u n i z e d rabbit or f r o m r a b b i t s i m m u n i z e d w i t h M M T V gp 52, gp 34 or p 28. T h e a n t i s e r a h a d all p r e v i o u s l y b e e n t i t r a t e d f o r M M T V b i n d i n g (see T a b l e 1), and a d j u s t e d so as to c o n t a i n e q u i v a l e n t c o n c e n t r a t i o n s o f M M T V b i n d i n g anti-

TABLE 1 BINDING OF ANTIBODIES SPECIFIC FOR MMTV PROTEINS TO MMTV FIXED TO WELLS OF A MICROTITER PLATE Serum

Unimmunized rabbit Rabbit anti-gp 52 Rabbit anti-gp 34 Rabbit anti p 28

Absorbance (400 nm) a I

II

0.017 0.931 1.545 1.892

0.008 0.958 1.506 1.884

a MMTV was fixed to wells at a concentration of 0.5 pg/200 pl. Rabbit IgG binding to MMTV was quantitated using alkaline phosphatase-conjugated goat anti-rabbit IgG. The absorbance of the yellow color formed after addition of p-nitrophenyl phosphate was determined at 400 nm.

9O TABLE 2 BINDING OF ANTIBODIES FROM HUMAN SERUM TO MMTV FIXED TO WELLS OF A MICROTITER PLATE Absorbance (400 nm)

<0.5 <0.8 <1.0 <1.5

% total sera a Breast cancer

Benign breast disease

Normal

37 26 20 13

28 10 9 3

27 8 5 0

a The analysis involved 54 breast cancer sera, 58 benign breast disease sera and 63 normal sera. An absorbance of 0.8 was taken as the upper range of normal.

body. At the end of the incubation, the rabbit sera were removed, the wells w a s h e d 3 t i m e s w i t h P B S - T w e e n a n d 2 0 0 p l o f a 1 : 10 d i l u t i o n o f h u m a n s e r u m in P B S - T w e e n w a s a d d e d . F o l l o w i n g a n a d d i t i o n a l 9 0 m i n i n c u b a t i o n , t h e a m o u n t o f h u m a n IgG b o u n d to M M T V was q u a n t i t a t e d by our usual p r o c e d u r e . T h e r e s u l t s ( T a b l e 3) d e m o n s t r a t e d t h a t a n t i s e r a t o M M T V p r o t e i n s , e s p e c i a l l y gp 3 4 a n t i s e r a , d i m i n i s h e d h u m a n I g G b i n d i n g t o M M T V . T h e g r e a t e r d e c r e a s e in h u m a n I g G b i n d i n g f o r all s e r a t e s t e d f o l l o w i n g p r e i n c u b a t i o n w i t h gp 3 4 o r gp 5 2 , as o p p o s e d t o n o r m a l r a b b i t s e r u m o r p 28 a n t i s e r u m , p r o v i d e s strong e v i d e n c e t h a t h u m a n IgG r e a c t e d p r e d o m inantly with the MMTV envelope.

TABLE 3 E F F E C T OF PREINCUBATION WITH ANTIBODIES TO MOUSE MAMMARY TUMOR VIRUS ON THE REACTIVITY OF HUMAN SERA TO MOUSE MAMMARY TUMOR VIRUS Patient

13 288 1 818 13 090 147 13 158 $2 $11

Diagnosis

Breast cancer Breast cancer Breast cancer Breast cancer Benign breast disease Normal Normal

Absorbance (400 nm) following preincubation with normal rabbit serum

1.53 0.97 1.30 1.73 1.49 1.64 0.97

Percent decrease in absorbance following preincubatlon with : Anti gp 52

Anti gp 34

Anti p28

25 13 46 35 46 11 9

54 51 72 40 54 12 33

16 0 40 0 38 9 19

91 DISCUSSION An E L I S A assay f o r t h e d e t e r m i n a t i o n o f a n t i b o d i e s reactive to M M T V was d e v e l o p e d and c h a r a c t e r i z e d . T h e p r o c e d u r e is u n c o m p l i c a t e d , rapid, d o e s n o t r e q u i r e r a d i o c h e m i c a l s or e x p e n s i v e e q u i p m e n t a n d can be performed on many samples simultaneously. In studies utilizing h u m a n sera it was d e t e r m i n e d t h a t a n t i b o d i e s w h i c h b i n d to M M T V w e r e p r e s e n t in a small m i n o r i t y o f sera o b t a i n e d f r o m norm a l individuals or f r o m w o m e n w i t h benign b r e a s t disease. T h e p e r c e n t a g e o f positives increased 3-fold w h e n sera f r o m b r e a s t c a n c e r p a t i e n t s w e r e e x a m ined. T h e s e results, in c o n c o r d a n c e w i t h t h e studies o f o t h e r investigators, suggest t h a t an MMTV-like c o m p o n e n t is p r e s e n t in h u m a n breast cancer. T h e d e t e c t i o n o f a n t i b o d y r e a c t i v e w i t h M M T V in a f e w n o r m a l sera a n d in sera f r o m benign b r e a s t disease p a t i e n t s , w h o m a y be at increased risk o f d e v e l o p i n g b r e a s t cancer, raises t h e intriguing possibility t h a t this E L I S A assay m a y i d e n t i f y t h o s e individuals w h o are m o r e s u s c e p t i b l e t o t h e develo p m e n t o f m a m m a r y t u m o r s . In a d d i t i o n , t h e assay m a y be valuable in t h e early d e t e c t i o n o f b r e a s t c a n c e r or in m o n i t o r i n g its r e c u r r e n c e f o l l o w i n g m a s t e c t o m y . T h e i n c i d e n c e o f b r e a s t c a n c e r in h u m a n s is m a n y t i m e s less t h a n t h e p e r c e n t a g e o f n o r m a l or benign b r e a s t diseased w o m e n positive f o r M M T V a n t i b o d i e s . This suggests t h a t f u t u r e c o n s e q u e n c e s o f M M T V - l i k e a n t i g e n e x p r e s s i o n , if a n y , m a y be d e p e n d e n t u p o n e n v i r o n m e n t a l a n d / o r genetic f a c t o r s . L o n g - t e r m m o n i t o r i n g o f M M T V a n t i b o d y - p o s i t i v e individuals is n o w in progress. ACKNOWLEDGEMENTS T h e e x c e l l e n t t e c h n i c a l assistance o f Ms. N a n c y L o k y s is g r a t e f u l l y a c k n o w l e d g e d . We t h a n k Ms. P a t s e y Y e o f o r sera a n d d a t a c o o r d i n a t i o n a n d Dr. Y. Wang f o r statistical analyses. T h e s e studies w e r e s u p p o r t e d b y grants f r o m t h e U S P H S , N I H , CA 0 8 7 4 8 , CA 1 8 4 8 8 , NS 1 1 4 5 7 , T h e A m e r i c a n C a n c e r S o c i e t y , No. 1M-185, a n d t h e Special Projects C o m m i t t e e o f t h e M e m o r i a l S l o a n - K e t t e r i n g C a n c e r Center. REFERENCES Black, M.M., R.E. Zachran, B. Shore, D.H. Moore and H.P. Leis, Jr., 1975, Cancer 35, 121. Charney, J. and D.H. Moore, 1971, Nature 2291 627. Coombes, R.C., 1978, Invest. Cell Pathol. 1,347. Cunningham-Rundles, S., W.F. Feller, C. Cunningham-Rundles, B. Dupont, H. Wanebo, R. O'Reilly and R.A. Good, 1976, Cell. Immunol. 25,322. Marcus, S.L., S.W. Smith, J. Racevskis and N.H. Sarkar, 1978, Virology 86,398. Marcus, S.L., R. Kopelman and N.H. Sarkar, 1979, J. Virol. 31,341. Mesa-Tejada, R., I. Keydar, M. Ramanarayan, T. Ohno, C. Fenoglio and S. Spiegelman, 1978, Proc. Natl. Acad. Sci. U.S.A. 75, 1529. Sarkar, N.H. and A.S. Dion, 1975, Virology 64,471. Sarkar, N.H., A.A. Pomenti and A.S. Dion, 1977, Virology 77, 12. Voller, A., D. Bidwell and A. Bartlett, 1976, in: Manual of Clinical Immunology, eds. N. Rose and W. Friedman (American Society for Microbiology, Washington, DC) p. 506. Witkin, S.S., R.A. Egeli, N.H. Sarkar, R.A. Good and N.K. Day, 1979, Proc. Natl. Acad. Sci. U.S.A. 76. 2984.