An enzyme-linked immunosorbent assay (Elisa) for detergent solubilized Ia glycoproteins using nitrocellulose membrane discs

Journal o f I m m u n o l o g i c a l Methods, 52 (1982) 395--408

395

Elsevier Biomedical Press

AN ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) FOR D E T E R G E N T S O L U B I L I Z E D Ia G L Y C O P R O T E I N S U S I N G N I T R O C E L L U L O S E MEMBRANE DISCS

ROGER G.E. PALFREE 1 and BRUCE E. ELLIOTT2 Cancer Research Laboratories, Botterell Hall, D e p a r t m e n t o f Pathology, Queen's University, Kingston, Ontario K 7 L 3N6, Canada

(Received 11 December 1981, accepted 29 January 1982)

An enzyme-linked immunosorbent assay has been developed for the quantitative detection of detergent solubilized murine Ia. Nitrocellulose membrane discs were used to bind membrane glycoproteins applied in solutions containing detergent. The bound antigen was detected with monoclonal antibodies and horseradish-peroxidase-coupled anti-IgG. The assay produced a linear response with respect to antigen concentration, and could readily detect partially purified Ia derived from 103 to 104 mitogen stimulated spleen cells. Nitrocellulose discs efficiently bound protein in the presence of deoxycholate, taurocholate, and octylglucoside. Less binding occurred in the presence of Triton X-100 or Tween 80, but 90% binding efficiency was obtained in 0.01% solutions of these detergents. The association of protein with the discs was stable under normal conditions for antigen detection, but could be further stabilized by briefly fixing with glutaraldehyde for more rigorous procedures. The ability of this method to detect antigen in detergent solutions makes it useful in monitoring fractions during the purification of cell membrane proteins.

Key words: E L I S A - - m e m b r a n e g l y c o p r o t e i n s - - detergent solubilized - - Ia antigens -nitrocellulose

INTRODUCTION I n c o n t r a s t t o B cells, r e c o g n i t i o n o f antigen b y T cells o c c u r s in association with m a j o r h i s t o c o m p a t i b i l i t y c o m p l e x (MHC) p r o d u c t s (Zinkernagel, 1978). I n d e e d , s t r o n g c y t o t o x i c a n d h e l p e r T cell responses with exquisite specificity f o r antigenic d e t e r m i n a n t s such as t h o s e expressed b y certain H-2 m u t a n t s ( E g o r o v a n d Blandova, 1 9 7 2 ) a n d a u t o l o g o u s t u m o r cells have been demo n s t r a t e d u n d e r c o n d i t i o n s in w h i c h n o significant a n t i b o d y response is

1 Research Fellow of the Medical Research Council of Canada. 2 Research Scholar of the National Cancer Institute of Canada. 0022-1759/82/0000--0000/$02.75©1982 Elsevier Biomedical Press

396 observed (Dennis et al., 1981; Kripkie, 1981). This asymmetry in the T and B cell responses to certain antigens is most likely a result of regulatory events (Gershon et al., 1981) mediated by suppressor cells (Frost et al., 1982) and Ia restricted helper cells (Sprent et al., 1981). An understanding of the mechanisms of antigen recognition b y regulatory and effector cells demands a resolution at the molecular level of the receptor-antigen interactions involved. Although Kappler et al. (1981) have recently presented strong evidence for a single receptor molecule recognizing foreign antigen in association with MHC products, the possibility of one receptor with more than one binding site for self and foreign antigens has not been excluded. As a first step in approaching this question, many laboratories have characterized the functional specificity of a variety of long-term helper and c y t o t o x i c T cell lines with specificity for allogeneic Ia, or self Ia plus antigen X (for review, see Immunol. Rev. 54, 1981). A formal model of receptor combining sites, however, requires an analysis of receptor affinities for self and foreign antigens, and of competition of antigen binding at the receptor sites. A necessary part of such an analysis is a quantitative assay for physiologically active Ia. The wealth of literature on various enzyme-linked immunosorbent assays (ELISA) is evidence of the growing popularity of this technique (Voller et al., 1978; Engvall, 1980). As with other immunoassays it allows the specific detection and quantitation of molecules for which no functional assay is available. The primary requirement is the availability of specific antibodies to determinants on the molecule. The availability of a wide range of monoclonal antibodies against a variety of Ia determinants makes an immunoassay for Ia particularly convenient. In developing such an assay the following criteria had to be met: first, it was necessary to detect an integral membrane glycoprotein (Delovitch and McDevitt, 1975) in the presence of detergent. Since detergents can prevent the binding of antigen by monoclonal antibodies (Herrman and Mescher, 1979) and are disruptive to cells, antigen competition assays such as the microcytotoxicity assay (Elliott et al., 1979) or that of Letarte et al. (1980) using fixed cells as the solid-phase reference antigen, and assays which involve immunoprecipitation of radiolabeled antigen from detergent solutions (Emerson et al., 1980) would severely restrict the conditions under which solubilized Ia could be detected. It was desirable, therefore, to immobilize directly the Ia on a solid phase, so that subsequent incubations with antibody could be performed under mild conditions. Second, the assay had to detect Ia in preparations where it was a minor c o m p o n e n t in a varying mixture of other proteins which would c o m p e t e for binding to the solid phase. Thus, consistent quantitation demanded a high-capacity solid phase which would bind close to 100% of the applied sample irrespective of the presence of detergent. The usual plastic microwells are inefficient at binding protein (less than 8%; Palfree, unpublished result), especially in the presence of detergent. The above criteria were met by the choice of nitrocellulose membrane as solid phase.

397 In this report we give the general procedure for the assay, compare it with the microcytotoxicity assay, and explore some of the conditions under which protein is bound and retained by the nitrocellulose discs. MATERIALS AND METHODS

Reagents Horseradish-peroxidase (HRP) was Sigma Type II further purified to an RZ of approx. 3 on Con A-Sepharose and Sephacryl S-200. Normal mouse IgG and the IgG fraction of goat anti-mouse IgG (Fc fragment; heavy chain specific) were from Cappel Laboratories. HRP was coupled to goat antimouse IgG with the bifunctional agent SPDP according to the method of Nilsson et al. (1981). Monoclonal anti-Ia.8 was from Ralph Steinman (clone B21-2, 1.2 mg/ml). Anti-Ia.19 (clone 116/32R5, 1.8 mg/ml) and anti-Ia.15 (clone 7/227.R7) were purified from culture supernates of hybridoma lines (Lemke et al., 1979) on protein A-Sepharose (Eye, 1978). Glutaraldehyde, 50%, was from Eastman Kodak, bovine albumin (fraction V; BSA), lipopolysaccharide (LPS), phenylmethylsulfonyl-fluoride (PMSF), a-methyl-Dmannoside, and the detergents Triton X-100, Tween 80, sodium lauryl sulfate (SDS), sodium deoxycholate, sodium taurocholate, and 1-0-n-octyl#-D-glucopyranoside (octylglucoside) were supplied by Sigma Chemical Co. Lentil (Lens culinaris) lectin was purified by the method of Howard et al. (1971) as modified by O'Brien et al. (1979), and coupled to CNBr activated-Sepharose (approx. 4 mg lectin/ml Sepharose). [3SS]methionine (1230 Ci/mmol) was from Amersham Corporation. Other chemicals were obtained from Fisher Scientific. Protein A-Sepharose, Con A-Sepharose, CNBr activated-Sepharose, Sephacryl S-200 and N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) were products of Pharmacia Fine Chemicals. Nitrocellulose membrane, Schleicher and Schuell BA 85, was obtained through Mandel Scientific Co. Genescreen was a gift from New England Nuclear. Flow Laboratories supplied multiwell Lin bro plates. Cells The preparation and culture of spleenic lymphocytes was as previously described (Nagy and Elliott, 1979). To label with [3SS]methionine, 3 day LPS blasts were incubated at 10S/ml in met-- medium containing [35S]met (approx. 0.1 mCi/ml) for 2 h, washed, and incubated a further 6 h at 6 X 106 / ml in complete medium. Membrane vesicles These were prepared by nitrogen cavitation and differential centrifugation as described in detail by Nagy and Elliott (1979).

398

Cell glycopro tein fraction Vesicles (approx. 1 mg/ml) or whole cells (107--108/ml) were solubilized 30 min at 4°C in 50 mM Tris-HC1, 0.15 M NaC1, pH 8.2, containing 0.5% deoxycholate and 10 - 3 M PMSF. Insoluble material was removed by centrifugation at 100,000 X g for 30 min, and the supernatant was applied to a lentil lectin-Sepharose column. After washing with 10 column volumes of buffer containing the appropriate detergent, glycoproteins were eluted with 5% a - m e t h y l mannoside. ELISA assay procedure Discs (approx. diam. 4 mm) were punched from nitrocellulose membrane and floated on the surface of distilled water. Moist discs were placed on absorbent filter paper and 10 or 20 pl samples were applied and allowed to soak through evenly. Gentle pressure from the pipet tip was occasionally necessary in order to make good contact between disc and filter paper, b u t discs must n o t be punctured. Loaded discs were placed in wells of a Linbro tray (96 V - b o t t o m wells) containing 100 pl 3% BSA, pH 7.5, and incubated 1 h at 37°C to block excess binding sites. The BSA was replaced with 100 pl of first arLtibody diluted in Tris-NaC1, pH 7.5 (or phosphate-buffered saline, PBS) containing 0.05% Tween 80 and 0.3% BSA, and incubated 8 h or overnight at 4°C. The appropriate dilution was determined for each antibody preparation, since linearity of the assay requires antibody in excess. Discs were washed briefly 3 times in the small wells with buffer containing 0.05% Tween 80, and transferred to larger Linbro wells (24 f l a t - b o t t o m wells) for a further 3 washes, each in 1 ml for 10 min at room temperature with agitation. The transfer was facilitated by use of a long-bore Pasteur pipet tipped with a short length of Tygon tubing. With fine curved forceps, the discs were returned to 96-well trays for incubation with HRP-anti Ig, again 100 t~l for 8 h or overnight at 4°C. Under certain conditions, incubation times may be shorter (see Results). Discs were washed as before and transferred to clean test tubes for enzyme assay. HRP activity was assayed using o-phenylenediamine as substrate at 0.4 mg/ml in citrate buffer, pH 5, containing 0.001% H202 (Wolters et al., 1976; Engvall, 1980}. Reaction volumes were 0.5 ml, and reactions were stopped by addition of 0.25 ml 4N H2SO4. The specific ELISA signal was calculated as {Es ( s p ) - E

b (sp) / - - { E s ( c t l ) - - E b (ctl)/

where E represents the extinction at 492 nm; s denotes sample; b, blank (no sample); sp, specific antibody; ctl, control (non-specific) antibody.

Microcy totoxicity assay A microcytotoxicity test (Frelinger et al., 1974) was adopted to quantify antibody absorbed with different antigen preparations as described previously (Elliott et al., 1979).

399 Standardization o f the ELISA In the absence of purified Ia for use as standard, we have adopted a simple procedure for relating concentrations of crude Ia preparations to cell surface Ia through the microcytotoxicity assay. Membrane vesicle preparations are frozen in aliquots until required. The Ia concentration of each preparation is compared with normal spleen cells or LPS blasts of the appropriate H-2 haplotype in a microcytotoxicity assay. The vesicle dilution and the cell concentration necessary to absorb enough antibody to reduce target lysis by 50% are equated to express the vesicle Ia concentration in terms of functionally equivalent cell concentration. The vesicle preparations can then be used as reference standards in the ELISA. An equal volume of 1% taurocholate in PBS is added to a vesicle aliquot to solubilize the Ia; insoluble material is removed by ultracentrifugation; and a dilution series is applied to nitrocellulose discs for construction of an ELISA standard curve. Ia concentrations in test solutions may then be related to vesicle Ia concentrations and hence to functionally equivalent cell concentrations. Glu taraldehyde fixation After sample application, discs were incubated 10 min in 3% BSA, rinsed briefly, and reacted with 100 pl 0.25% glutaraldehyde in PBS for 15 min at r o o m temperature. Discs were rinsed 3 times and residual active groups and binding sites blocked by addition of 100 ul 0.1 M glycine (pH 7.5) and 100 t~l 3% BSA to each well, and incubation for 1--11/~ h at 37°C. RESULTS Specificity and linearity o f the ELISA for Ia The dependence of the final ELISA signal on the concentration of antigen applied to the disc is shown in Fig. 1. An excellent linear correlation was obtained. Deviations from linearity could generally be traced to limiting antib o d y conditions; in the case of Fig. l b , the second antibody (HRP-anti-IgG) was slightly limiting at the highest antigen concentration. In Fig. l a the antigen was derived from a B10.BR (H-2 k) plasma membrane vesicle preparation b y solubih~zation in deoxycholate and further purification on lentil-lectin-coupled Sepharose. That used in Fig. l b was obtained by direct solubilization of B10 (H-2 b) LPS blasts and partial purification on lentil lectin-Sepharose. A monoclonal antibody with Ia.19 specificity, detecting products of I-A k b u t n o t I-A b, produced a signal with material from B10.BR {Fig. la) b u t n o t B10 (Fig. lb). Conversely, the anti-Ia.8 antibody detected Ia in material from B10 (I-A b) but n o t B10.BR (I-Ak). Normal mouse IgG did n o t detect either. The assay p r o d u c e d an unacceptably high background when crude material solubilized from LPS blasts was used. This was n o t due to endogenous peroxidase activity and was obtained in the absence of a first antibody incubation. When unpurified material from solubilized vesicles was used, the

400

0.6 o.

°.8 0.7f b o6 05 0.4 0.5

0.5 04

, ~ / ,~/

0.2

/

0.2 0.1

- &~

I 2

OI

4

8

I 2

4

8

UNIT ANTIGEN CONCENTRATION

Fig. 1. E L I S A o f partially purified Ia k and Iab: antigen c o n c e n t r a t i o n dependence. Ia-enriched g l y c o p r o t e i n fractions were prepared f r o m B10.BR (H-2 k) vesicles (a), and B10 (H-2 b) LPS blast cells (b). Ten m i c r o l i t e r samples of various dilutions were applied to nitrocellulose discs, and assayed for Ia using, as first a n t i b o d y : A, anti-Ia.19; 4 , antiIa.8; ©, normal m o u s e IgG. Unit c o n c e n t r a t i o n of antigen corresponds to an a m o u n t of material applied to the disc which was derived from a p p r o x i m a t e l y 2.5 × 104 cells (a), or 105 cells (b). Each p o i n t is the m e a n of triplicate d e t e r m i n a t i o n s + S.E.M.

control signal was somewhat higher than those shown in Fig. 1, but still allowed consistent estimations of Ia. Shed Ia in LPS blast culture supernatants (prepared as described previously, Nagy et al., 1982) was readily detectable. Its detection was slightly more efficient when taurocholate was added to the supernatant to a 0.1% concentration before applying samples to the nitrocellulose discs. This was probably a result of disruption of the lipid micelles in which Ia is shed (Emerson and Cone, 1981).

Comparison of ELISA and microcytotoxicity assay In the m i c r o c y t o t o x i c i t y assay antibody is allowed to bind to lymphocyte surface antigens, and the cells are subsequently lysed by incubation with complement. Preincubation of the antibody with another source of antigen, such as whole cells, membrane vesicles, or solubilized antigen, reduces the free antibody concentration and the degree of cell lysis (Elliott et al., 1979). Fig. 2a shows the inhibition of anti-Ia mediated lysis by preincubation of antibody with membrane vesicles or soluble Ia. The soluble Ia was the lentil lectin-selected glycoprotein fraction from taurocholate solubilized LPS blasts. It was eluted from the lectin column after the detergent had been changed to 0.01% Tween 80, since this concentration is tolerated by cells and does n o t interfere with the assay. A comparison of the dilutions of the Ia preparations which produced the same degree of inhibition (50% inhibition of cytotoxicity) provides an estimate of the relative Ia concentrations. The same Ia preparations were used in the ELISA (Fig. 2b). The data are

401 121

b

,oo[

I0

~j 8o

08

O6

\

g N

\

2O

02 I

I

I

I

2

4

8

16

I ~~..o.....~-~ :52 6 4

1D

128 2 5 6 .'512 1024

ANTIGEN DILUTION

I I

I 2

L 4

I 8

J 16

/ $2 6 4

I I I I 128 256 512 1024

ANTIGEN DILUTION

Fig. 2. Microcytotoxicity assay and ELISA of the same vesicle and soluble Ia preparations. (a) Monoclonal anti-Ia.15 (20 t~l) was absorbed with an equal volume of dilutions of B10.BR membrane vesicles (0) or a lentil lectin°selected glycoprotein fraction from CBA LPS blasts (©) for 15 min at room temperature and 1V2 h at 0°C. The soluble glycoprotein fraction was in 0.01% Tween 80. The absorbed antibody was tested in a 2-stage microcytotoxicity assay using CBA spleen targets. (b) Samples (10 #l) of dilutions of taurocholate solubilized B10.BR membrane vesicles (o) or the CBA blast glycoprotein fraction (©) were applied to nitrocellulose discs for ELISA using monoclonal anti-Ia.19 as specific antibody. The protein concentrations in the undiluted preparations were 1.2 mg/ml (vesicles) and approx. 30 ttg/ml (soluble glycoprotein fraction). The material in the glycoprotein fraction was derived from approx. 5 × 107 CBA LPS blast cells/ml soluble Ia.

s h o w n on a semi-log p l o t f o r c o m p a r i s o n with Fig. 2a, b u t t h e relative Ia c o n c e n t r a t i o n s are m o r e a c c u r a t e l y o b t a i n e d f r o m linear p l o t s o f t h e kind s h o w n in Fig. 1. F r o m t h e d a t a in Fig. 2 a n d similar d a t a f o r Ia p r e p a r a t i o n s f r o m H-2 b strains, t h e Ia c o n c e n t r a t i o n s in d e t e r g e n t solubilized g l y c o p r o t e i n f r a c t i o n s h a v e b e e n c o m p a r e d with Ia c o n c e n t r a t i o n s in m e m b r a n e vesicle p r e p a r a t i o n s b y b o t h E L I S A a n d m i c r o c y t o t o x i c i t y assay ( T a b l e 1). T h e results s h o w t h e 2 assays t o be q u i t e c o n s i s t e n t w i t h e a c h o t h e r . T h e assays a p p e a r to have similar sensitivities, a l t h o u g h t h e E L I S A is m o r e precise. Fig. 2b s h o w s t h a t a 1 2 8 - f o l d d i l u t i o n o f t h e solubilized g l y c o p r o t e i n fract i o n was clearly d e t e c t a b l e in the E L I S A . A t this dilution, t h e a m o u n t o f m a t e r i a l a p p l i e d to each disc was derived f r o m b e t w e e n 103 a n d 104 blast cells.

Effect o f detergent on binding and retention o f protein by nitrocellulose discs E f f i c i e n t b i n d i n g o f p r o t e i n was n o t a c h i e v e d b y m e r e l y i m m e r s i n g the discs in dilute p r o t e i n solutions. I n s t e a d , t h e m o i s t discs w e r e p l a c e d o n abs o r b e n t filter p a p e r , a n d 10 or 20 pl s a m p l e s w e r e a p p l i e d so t h a t t h e s o l u t i o n s o a k e d t h r o u g h evenly. O v e r f l o w i n g at t h e edges o f discs was n o t a p r o b l e m . In o r d e r t o s t u d y t h e e f f i c i e n c y w i t h w h i c h p r o t e i n b o u n d , a n d t o d e t e r m i n e t h e e f f e c t s o f v a r i o u s d e t e r g e n t s o n s a m p l e binding, r a d i o l a b e l e d p r o t e i n was used.

402 TABLE 1 Comparison of microcytotoxicity assay and ELISA. Source of soluble Iaa

Reference Iab

Relative Ia concentration c Microcytotoxicity ELISA

CBA LPS blasts B10 LPS blasts

B10.BR vesicles B6 vesicles

0.11 0.30

0.13 0.27

a Soluble Ia was the lentil lectin-selected glycoprotein fraction. The undiluted preparations contained between 20 and 40 ug protein/ml. b The undiluted membrane vesicle preparations contained protein at 1.2 mg/ml (B10.BR) and 3.4 mg/ml (B6). For ELISA the vesicles were solubilized in 0.5% taurocholate. c The relative Ia concentration is the ratio of soluble Ia concentration to the vesicle Ia concentration. A g l y c o p r o t e i n f r a c t i o n f r o m LPS blasts labeled with [35 S ] m e t h i o n i n e was selected b y passage o f t a u r o c h o l a t e solubilized p r o t e i n s t h r o u g h a lentil l e c t i n - S e p h a r o s e c o l u m n . T h e a d s o r b e d g l y c o p r o t e i n was eluted in 0.01% T w e e n 80 with 5% a - m e t h y l m a n n o s i d e , and diluted 1:5 i n t o solutions o f various d e t e r g e n t s over a range o f c o n c e n t r a t i o n s . Samples were applied t o discs of nitrocellulose or G e n e s c r e e n (a n e w p r o d u c t f r o m N e w England Nuclear). A f t e r b l o c k i n g the discs with BSA and rinsing t h e m twice in 1 ml PBS, b o u n d r a d i o a c t i v i t y was d e t e r m i n e d (Fig. 3). In a l m o s t all cases G e n e s c r e e n was n o better, and o f t e n worse, t h a n nitrocellulose in its p r o t e i n binding properties. T h e m o s t striking d i f f e r e n c e o c c u r r e d w h e n s o l u t i o n s c o n t a i n e d d e o x y c h o l a t e (Fig. 3e). B e t w e e n 95 and 100% o f applied r a d i o a c t i v i t y was b o u n d b y n i t r o c e l l u l o s e discs at all d e o x y c h o l a t e c o n c e n t r a t i o n s tested, b u t the higher c o n c e n t r a t i o n s severely affected binding t o G e n e s c r e e n , so t h a t less t h a n 20% b o u n d in 0.5% d e o x y cholate. T h e e f f e c t o f t a u r o c h o l a t e was essentially the same as t h a t o f d e o x y c h o l a t e ( d a t a n o t shown). T h e r a d i o a c t i v i t y was b o u n d e f f i c i e n t l y b y nitrocellulose f r o m s o l u t i o n s c o n t a i n i n g octylglucoside, with a t e n d e n c y t o w a r d m o r e e f f i c i e n t binding at the higher d e t e r g e n t c o n c e n t r a t i o n s (Fig. 3d). This was p r o b a b l y d u e t o a r e d u c t i o n in surface t e n s i o n allowing a m o r e even p e n e t r a t i o n into the p o r e s o f t h e m e m b r a n e . Binding was p o o r w h e n samples c o n t a i n e d T r i t o n X - 1 0 0 (Fig. 3a) o r T w e e n 80 (Fig. 3b) at 0.05% or m o r e , b u t was over 90% in 0.01% d e t e r g e n t . T h e s e binding characteristics were r e f l e c t e d in the E L I S A assay f o r Ia. T h e signal was u n a f f e c t e d by d e o x y c h o l a t e or o c t y l g l u c o s i d e over the same c o n c e n t r a t i o n range, b u t was r e d u c e d b y a p p r o x i m a t e l y 50% w h e n the sample was applied in 0.05% Triton. Fig. 4 shows t h e r e t e n t i o n o f b o u n d r a d i o a c t i v i t y by nitrocellulose discs u p o n s u b s e q u e n t washing in solutions c o n t a i n i n g d e t e r g e n t . B o u n d radioactivity was n o t significantly r e m o v e d b y washing in d e o x y c h o l a t e , tauroc h o l a t e or octylglucoside, b u t washing in T r i t o n greatly r e d u c e d the a m o u n t

403 ioo

~okk~~ ,,

=

60

c - - o Genescreen

: BA85

40

~o~ ,~, °

_ 0

,

0-.01

.05 I

5

LL OI

I 05

I I

i .05

i I

J 5

80 60

\

~o \

C

20 0

I

ol

II

05

d

N I

5

L 01

i 5

01

L

i

i

05

I

5

D E TERGENT CONCENTRATION ( % w/v )

Fig. 3. Effect of detergents on glycoprotein adsorption by nitrocellulose and Oenescreen. A glycoprotein fraction from LPS blasts labeled with [35S]methionine (approx. 20 ug protein/ml, 17,000 cpm/#g) in 0.01% Tween 80 was diluted 1:5 into Tris-NaC1 containing detergent. Twenty microliter samples were applied to discs, and discs were blocked with BSA and washed twice with 1 ml Tris-NaC1, each 10 rain at 22°C. Each disc was placed in 5 ml Scintiverse for liquid scintillation counting of bound radioactivity (maximum count rates were obtained after 12 h). The discs were nitrocellulose BA 85 (solid lines) and Genescreen (dashed lines). Detergents were: a, Triton X-100; b, Tween 80; c, SDS; d, octylglucoside; e, deoxycholate. Points are triplicate means -+S.E.M.

I00

80

=~ g

-

°

~

O

60

40 (3 -2 o

I I ~ n

.l

20

_1 .05

I

I

.I DETERGENT

.5 CONCENTRATION

(% W/V)

Fig. 4. Effect of detergent washes on retention of glycoprotein by nitrocellulose discs. Discs were loaded with equal amounts of [3sS]glycoprotein, blocked with BSA, and washed twice with 1 ml Tris-NaCl containing detergent (each was 10 min at 22°C). Radioactivity remaining with discs was determined as described in Fig. 3. The detergents were: ©, deoxycholate; o, taurocholate; A, octyglucoside; A, SDS; •, Tween 80; m, Triton X-100.

404 retained. Tween 80 was n o t so harsh; more than 80% of applied radioactivity remained with the disc after washing with 0.05% T w een 80. Thus, 0.05% Tween 80 could be used in a n t i b o d y solutions and washes t h r o u g h o u t the ELISA in order to reduce non-specific binding. As expected, the use of T r ito n instead of T w e e n resulted in a reduced ELISA signal, but this could be overcome with a brief glutaraldehyde fixation step after sample application (see Materials and Methods). Fixation, however, was n o t part of the routine procedure.

Dependence of antigen binding efficiency on sample protein concentration Samples o f radioactive glycoprotein fraction in 0.1% t aurochol at e were mixed 1:1 with solutions of serum albumin before application to nitrocellulose discs (Table 2). Over 9(F/o of the applied radioactivity was bound when samples contained up to 2.5 mg albumin/ml. When assayed for Ia in the ELISA, however, the specific signal began to decrease with a sample albumin c o n c e n t r a t i o n ~>0.5 mg/ml. T he ELISA signal was unaffect ed by an albumin c o n c e n t r a t i o n of 0.2 mg/ml ( n o t shown in Table 2). Thus the assay is capable of consistent q u a n t i t a t i o n of Ia in the presence of a wide range o f c o n c e n t r a t i o n s of irrelevant protein.

Optimization of the assay Th e a n t i b o d y incubation periods and the washing steps were critical for m a x i m u m sensitivity. T o find the o p t i m u m ant i body incubation times, discs containing equal amounts of antigen were incubated with first (Iaspecific or control) a n t i b o d y for various times keeping the second (HRPanti-mouse IgG) a n t i b o d y incubation constant, or vice versa (Table 3). In either case an incubation of at least 8 h was necessary to achieve a m a x i m u m signal, although a b o u t 65% of the m a x i m u m could be e x p e c t e d with two 4 h a n t i b o d y incubations. A similar time d ependence has been r e p o r t e d for a n t i b o d y binding to antigen coated p o l y s t y r e n e tubes (Kucich et al., 1980). Particular a t t e n t i o n was paid to the washing schedule. T hree or four brief washes in the microwells was sufficient f or a qualitative det ect i on of antigen

TABLE 2 Effect of sample protein concentration on the proportion of sample bound to nitrocellulose discs, and on the subsequent ELISA signal. BSA in samplea (mg/ml) 0.00 0.05 0.5 % cpm bound ELISA signal (A492)

95 0.32

96 0.34

97 0.28

2.5 91 0.20

5 78 0.18

10

15

50 0.15

32 0.09

a Samples contained an LPS blast glycoprotein fraction (approx. 10 ug protein/ml) in 0.05% taurocholate. A volume of 20 al was appled to each disc.

405 TABLE 3 Time dependence of antibody binding at 4°C as indicated by ELISA signal (A4n).

First antibodya Second antibodyb

Incubation time (h) 2 4

6

8

0.52 0.54

0.74 0.74

0.82 0.77

0.65 0.70

10 0.84 0.82

14 0.84 0.81

a A mixture of monoclonal antibodies against I-Ak product in a CBA glycoprotein fraction. The incubation time for the second antibody was constant at 14 h. b HRP-goat anti-mouse IgG (Fc). The first antibody incubation time was 10 h.

at high c o n c e n t r a t i o n , but a f u r t h e r three 1 ml washes in larger Linbro wells p r o d u c e d a 3-fold r e d u c t i o n in ELISA c o n t r o l values, which was essential for quantitative work. When o-dianisidine was used as substrate in the peroxidase assay (Avrameas and Guilbert, 1972) the reaction p r o d u c t precipitated on the discs and seemed to inhibit the reaction. This was n o t a problem with o-phenylenediamine, which was used routinely. With this substrate, color developm e n t in the reaction was linear (data n o t shown). Typical reaction times were b etween 10 and 20 min. S h o r t e r times, resulting from high antigen concentrations, were inconvenient when carefully timed reactions were to be p e r f o r m e d on large numbers o f discs, whereas longer times, necessary for color d e v e l o p m e n t with very low antigen concentrations, resulted in higher c o ntr o l values. DISCUSSION We have described an ELISA f o r soluble Ia, and have shown its application to the d e t e c t i o n of Ia amongst detergent solubilized proteins from m e m b r a n e vesicles and in glycoprotein fractions from vesicles or whole cells. In preliminary work we have also used the m e t h o d t o d e t e c t Ia shed into the m e d i u m in cultures of LPS blasts. ~ h e assay was f o u n d to be at least as sensitive as the m i c r o c y t o t o x i c i t y assay (Fig. 2), and could d e t e c t solubilized Ia f r o m b etween 103 and 104 LPS blast cells. T h e m e t h o d was developed f or quantitative d e t e c t i o n o f soluble Ia with physiologic activity with respect to its binding to T cell receptors. Although d e t e c t i o n in th e ELISA does n o t guarantee such activity, the use o f m onoclonal antibodies selected for their ability to d e t e c t Ia on normal cell surfaces strongly suggests t hat the det ect ed Ia retains m uch of its native conformation. A major r e q u i r e m e n t f or the assay was its ability to quant i fy detergent solubilized cell surface antigen. T h e use of nitrocellulose discs has achieved this. Their high capacity allows efficient binding of small a m o u n t s of antigen

406 in the presence of other protein at concentrations up to approximately 200 pg/ml, and the binding occurs efficiently in the presence of commonly used detergents. This feature of the assay should make it applicable to the detection of many other membrane proteins which must be handled in detergent solutions. Although deoxycholate, taurocholate and octylglucoside did not affect the binding of antigen to discs, Tween 80 or Triton X-100, a detergent used frequently in membrane protein solubilization, had an adverse effect on sample binding. It would be expected that the closely related NP-40 would have a similar effect. At a concentration of 0.01%, however, Tween or Triton caused little reduction in sample binding. Thus it is possible to work with the non-ionic detergents provided a suitable sample dilution can be made before its application to discs. N o t only did Triton X-100 inhibit sample binding to nitrocellulose discs, it was also the detergent most effective at removing sample from discs during a series of washes. This could be prevented by briefly fixing the b o u n d sample with glutaraldehyde. With this step another application of the ELISA is possible: a simple m e t h o d to study mild conditions for dissociation of antigen and antibody. One would simply look for a reduction in the ELISA signal resulting from the use of various test solutions to wash discs between first and second antibody incubations. Information from such an experiment might be particularly useful when antigen is to be purified on an affinity column of immobilized monoclonal antibody. Nitrocellulose membrane has been in use for some time for binding nucleic acid, and particularly for blotting nucleic acids from gels after their electrophoretic separation (Southern, 1975). Similarily, separated proteins can be blotted or electrophoretically transferred from gels to nitrocellulose for subsequent staining, autoradiography, or immunological detection (Towbin et al., 1979). It has been noted that the adsorptive properties of nitrocellulose membranes vary according to their source or conditions of storage, b u t can be significantly improved by treatment with aliphatic alcohols (Schneider, 1980). Genescreen is a new product from New England Nuclear which is considered to be more easily handled than nitrocellulose membranes in the blotting of nucleic acids and proteins from gels. For binding proteins in the presence of detergent, however, it would appear that Genescreen is less suited than nitrocellulose. We have also noticed that solution penetration of Genescreen during sample application is slower. As applied to the determination of Ia, the method is limited to the assay of partially purified preparations. The reason for the high background obtained with crude cell extracts is n o t known. It was n o t due to endogenous peroxidase activity and was independent of the first antibody, so it was caused by non-specific binding of the second. The monoclonal antibodies used to detect Ia were of the IgG class, so the second antibody was chosen to have specificity for the Fc portion of mouse IgG. Although this would minimize reactivity with endogenous immunoglobulin, predominantly non-

407 IgG in the cell preparations used, total prevention of specific binding could n o t be expected. It is also possible that Fc-receptor material was present with some affinity for the goat antibody, b u t inclusion of non-specific antib o d y did n o t appear to affect the high background. Preliminary results indicate that crude T cell extracts do n o t produce such high backgrounds in the assay. In the absence of purified Ia, its concentration in test solutions can only be related to some arbitrary standard. It would be reasonable to a t t e m p t to relate directly Ia concentrations to the a m o u n t of Ia expressed by lymphocytes, b u t the high background prevented the use of crude cell lysates in the ELISA. We have chosen instead to use material solubilized from membrane vesicles to calibrate the assay. The vesicles may be prepared in bulk and stored frozen (--70°C) over long periods of time with no apparent loss of serologically detectable Ia. Aliquots may be thawed and solubilized as required to produce a reference dilution series by which to relate Ia concentrations in test solutions to vesicle Ia concentrations. Furthermore, the Ia concentration in each vesicle preparation may be related to the amount of surface Ia expressed on whole cells through the microcytotoxicity assay as described in the Materials and Methods section. This provides an independent standard by which to assess storage losses and batch to batch variation in vesicle Ia content. There are a n u m b e r of points to consider when using this standardization procedure. The microcytotoxicity assay does not permit an accurate quantitative comparison of vesicle Ia with cell surface Ia, because they behave differently during the assay. After absorption on cells, only free antibody is transferred to the targets. Vesicles, however, behave like soluble Ia since they are so small as to remain in suspension during the assay, and vesicleb o u n d antibody is transferred along with free antibody after the absorption. Although the use of high affinity antibody would minimize the redistribution of antibody from vesicles to target cells, the system is still complicated by the divalent nature of the antibody. It is therefore necessary, when determining the functionally equivalent cell concentration of a vesicle preparation, to control the antibody and incubation times as well as the source of reference cells. Furthermore, it should be noted that cell equivalents determined in this way refer only to cell surface Ia and not total cell Ia. Thus the cell equivalent of a detergent solubilized Ia preparation frequently exceeds the n u m b e r o f cells from which it was derived. Because of the rather arbitrary Ia standardization, it is difficult to compare concentrations of different Ia alloantigens. Comparisons may be made with the ELISA using cross-reactive antibody, but only if its affinity is the same for both alloantigens. Standardization through the microcytotoxicity assay does n o t overcome this problem. Even if the antibody dependence of the assay could be minimized, the reference cells may not express the same amounts of surface Ia, and an appropriate cross-reactive antibody would again be required to test this. A knowledge of the various antigen-antibody

408 i n t e r a c t i o n c o n s t a n t s m a y p e r m i t a m o r e a b s o l u t e Ia q u a n t i t a t i o n t h a n the relative s t a n d a r d i z a t i o n c u r r e n t l y used. C u r r e n t l y we are using the E L I S A t o m o n i t o r c o l u m n fractions in the p r o d u c t i o n a n d analysis o f Ia p r e p a r a t i o n s . We are also e x p l o r i n g the feasibility o f using t h e E L I S A t o q u a n t i f y Ia u p t a k e by T cells in o r d e r t o analyse T cell r e c e p t o r affinities f o r Ia. In o t h e r w o r k we i n t e n d t o use the assay t o s t u d y d i f f e r e n c e s in the e x p r e s s i o n a n d s h e d d i n g o f surface antigen by t u m o r lines with altered m e t a s t a t i c capabilities. ACKNOWLEDGEMENTS This w o r k was s u p p o r t e d by grants f r o m the N a t i o n a l C a n c e r I n s t i t u t e of C a n a d a and the Medical R e s e a r c h Council o f Canada. REFERENCES Avrameas, S. and B. Guilbert, 1972, Biochimie 54,837. Delovitch, T.L. and H.O. McDevitt, 1975, Immunogenetics 2, 39. Dennis, J.W., T.P. Donaghue, D.A. Carlow and R.S. Kerbel, 1981, Cancer Res. 41, 4010. Egorov, T.K. and Z.K. Blandova, 1972, Genet. Res. 19, 133. Elliott, B.E., B. Tak~cs and Z. Nagy, 1979, Eur. J. Immunol. 9,646. Emerson, S.G. and R.E. Cone, 1981, J. Immunol. 127,482. Emerson, S.G., D.B. Murphy and R.E. Cone, 1980, J. Immunol. 125, 406. Engvall, E., 1980, in: Methods in Enzymology 70, part A, eds. H.V. Vunakis and J.J. Langone (Academic Press, New York) p. 419. Eye, P.L., S.J. Prowse and C.R. Jenkin, 1978, Immunoehemistry 15,429. Frelinger, J.A., S.E. Niederhuber, C.S. David and D.C. Shreffler, 1974, J. Exp. Med. 140. 1273. Frost, P., P. Prete and R.S. Kerbel, 1982, Int. J. Cancer, in press. Gershon, R.K., D.D. Eardley, S. Durum, D.R. Green, F.-W. Shen, K. Yamauchi, H. Cantor and D.B. Murphy, 1981, J. Exp. Med. 153, 1533. Herrman, S.H. and M.F. Mescher, 1979, J. Biol. Chem. 254, 8713. Howard, I.K., H.J. Sage, M.D. Stein, N.M. Young, M.A. Leon and D.F. Dyckes, 1971, J. Biol. Chem. 246, 1590. Kappler, J.W., B. Skidmore, J. White and P. Marrack, 1981, J. Exp. Med. 153, 1198. Kripkie, M.L., 1981, Adv. Cancer Res. 34, 69. Kucich, U., W.R. Abrams and H.L. James, 1980, Anal. Biochem. 109,403. Lemke, H., G.J. Hammerling and U. Hammerling, 1979, Immunol. Rev. 47, 175. Letarte, M., H.-S. Teh and G. Meghji, 1980, J. Immunol. 125,370. Nagy, Z.A. and B.E. Elliott, 1979, J. Exp. Med. 150, 1520. Nagy, Z.A., B.E. Elliott, D. Carlow and B. Rubin, 1982, Eur. J. Immunol., in press. Nilsson, P., N.R. Bergquist and M.S. Grundy, 1981, J. Immunol. Methods 41, 81. O'Brien, K.J., R.M. Condie, C.A. Prody and R.D. Edstrom, 1979, J. Biol. Chem. 254, 168. Schneider, Z., 1980, Anal. Biochem. 108, 96. Southern, E.M., 1975, J. Mol. Biol. 98, 503. Sprent, J.E., A. Lerner, J. Bruce and F.W. Symington, 1981, J. Exp. Med. 154,188. Towbin, H., T. Staehelin and J. Gordon, 1979, Proc. Natl. Acad. Sci. U.S.A. 76, 4350. Voller, A., A. Bartlett and D.E. Bidwell, 1978, J. Clin. Pathol. 31,507. Wolters, G., L.J. Kuijpers, J. Kacaki and A. Schuurs, 1976, J. Clin. Pathol. 29,873. Zinkernagel, R., 1978, Immunol. Rev. 42, 15.