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A biotin-labelled antigen radioimmunoassay (BILA) for antibodies to membrane antigens useful for monoclonal antibody screening
Journal of Immunological Methods, 115 (1988) 219-226 Elsevier
219
JIM 04987
A biotin-labelled antigen radioimmunoassay (BILA) for antibodies to membrane antigens useful for monoclonal antibody screening Bo Jansson, T h o m a s B r o d i n a n d H a n s O l o v SjSgren Department of Tumor Immunology, The Wallenberg Laboratory, University of Lund, Box 7031, S-220 07 Lund, Sweden (Received 11 April 1988, revised received 16 June 1988, accepted 15 July 1988)
Viable cells or protein extracts were labelled with N-hydroxysuccinimidobiotin and used as target antigens in a biotin-labelled antigen radioimmunoassay (BILA). The binding of the biotinylated antigens to capture antibodies coated on the bottoms of 96-well plastic plates were measured using 125I-labelled streptavidin as the detection step. The assay circumvents some of the problems associated with solid-phase RIA and permits screening of antibodies to undefined protein antigens present in very small amounts in complex protein solutions. Key words: Avidin; Biotin; Membrane antigen
Introduction
The most frequently used techniques for screening monoclonal antibodies against cell surface antigens make use of intact cells (Brown et al., 1979) or crude plasma membrane preparations (Morgan et al., 1980; Kelleher et al., 1983). In most assays cells or membranes are simply bound to the bottom of 96-well plastic plates. The binding of antibodies, e.g., of hybridoma supernatants, is detected by a labelled secondary antibody, staphylo-
Correspondence to: B. Jansson, Department of Tumor Immunology, The Wallenberg Laboratory, University of Lund, Box 7031, S-220 07 Lund, Sweden. Abbreviations: PBS, 10 mM NaH2PO 4, 150 mM NaCI, pH 7.3; OA, ovalbumin; BSA, bovine serum albumin; LMW, low molecular weight standard proteins; DMSO, 1,2-di-methylsulphoxide; PMSF, phenylmetliylsulphonyl fluoride; FCS, fetal calf serum; DOC, sodium deoxycholate; NHS-d-biotin, N-hydroxysuccinimidobiotin.
coccal protein A (Brown et al., 1981), or streptococcal protein G (AkerstrSm et al., 1985). There are certain limitations associated with these standard techniques. When preparations of plasma cell membranes are used as targets, the antigens have to compete with nonrelevant proteins and lipids for the very limited binding capacity of the plastic wells. If a detectable amount of antigen is to remain on the plastic surface the original crude membrane fraction has to contain a large percentage of the relevant antigen. In addition the necessity of keeping most membrane-associated antigens in detergent solution also impairs the binding of antigens to the plastic solid phase. To avoid this problem antigens and cells might need to be fixed (for example, by glutaraldehyde) in order to adhere properly to the plastic surface (Kelleher et al., 1983) and fixation always increases the risk of denaturation of antigenic determinants. When adsorbing proteins to a solid phase there is always a risk of creating
0022-1759/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
220 a n t i g e n i c n e o e p i t o p e s b e c a u s e the n a t u r a l m o l e c u lar flexibility is restricted. W e n o w d e s c r i b e an a l t e r n a t i v e screening m e t h o d m a k i n g use of b i o t i n y l a t e d antigens a n d a n o n c o v a l e n t l y a t t a c h e d c a p t u r e a n t i b o d y . This assay c i r c u m v e n t s s o m e o f the p r o b l e m s e n c o u n t e r e d when using a cell m e m b r a n e p r e p a r a t i o n as target in screening assays for m o n o c l o n a l a n t i b o d ies.
Cell lines A G r o s s v i r u s - i n d u c e d rat l y m p h o m a W / F u G1 (Shellam et al., 1974) a n d 1 , 2 - d i m e t h y l h y d r a z i n e - i n d u c e d rat c o l o n c a r c i n o m a s BN7005 a n d D M H W 4 9 ( H e d l u n d et al., 1980) were used.
Monoclonal antibodies T h e m o n o c l o n a l a n t i b o d i e s used are d e s c r i b e d in T a b l e I.
Materials and methods
Biotinylation of low molecular weight proteins ( L M W proteins)
Chemicals
L M W c a l i b r a t i o n kit p r o t e i n s f r o m P h a r m a c i a c o n t a i n i n g 64 ~tg p h o s p h o r y l a s e - b , 83 /tg b o v i n e s e r u m a l b u m i n , 147 # g o v a l b u m i n , 83 ~ g c a r b o n i c a n h y d r a s e , 8 0 / t g soya b e a n t r y p s i n i n h i b i t o r a n d 121/~g of a - l a c t a l b u m i n / v i a l w e r e d i l u t e d in 1 ml of 0.1 M N a 2 C O 3 p H 8.0 a n d l a b e l l e d with 100/~1 o f N - h y d r o x y s u c c i n i m i d o b i o t i n (1.25 m g / m l D M S O ) for I h a n d then d i a l y s e d o v e r n i g h t a g a i n s t PBS. T h e total v o l u m e was i n c r e a s e d to 2 ml b y
N-hydroxysuccinimidobiotin (NHS-d-biotin, Sigma, St. Louis, M O , H1759). S t r e p t a v i d i n (Sigma, St. Louis, M O , S-4762) was l a b e l l e d with 1251 using the c h l o r a m i n e - T m e t h o d for the labelling of I g G (Brown et al., 1981) (specific activity 21 # C i / # g ) . S t a p h y l o c o c c a l p r o t e i n A was l a b e l l e d w i t h 125I using the m e t h o d d e s c r i b e d b y B o l t o n a n d H u n t e r 1973 (specific activity 23 # C i / # g ) .
TABLE 1 MONOCLONAL ANTIBODIES USED IN THIS STUDY Monoclonal antibody
Antigen
Reference
Mouse IgG1 Mouse IgG1 Mouse IgG2b
Rat x light chain Human y heavy chain p53 antigen
Bazin et al., 1984 Kuritami et al., 1981 Gurrey et al., 1980
OX-18 OX-19 OX-1 OX-6
Mouse IgG1 Mouse IgG1 Mouse IgGl Mouse IgGl
Rat RT-1 monomorphic part Rat T cell-associated antigen Rat monocyte-associated antigen Rat MHC class 2
Fukumoto et al., 1982 Dallman et al., 1982 Sunderland et al., 1979 Fukomoto et al., 1982
Anti-OA 03B3 Q12
Rat IgG2a Rat IgG2a Rat IgM
Ovalbumin Rat RT-lu Ovalbumin
b c
1G6, 1F6, 1Cll, 10B12, 10H5, 7D8
Rat IgM
d
d
Designation
Class
MARK-1 HB60 Ab 122
a Anti-ovalbumin rat IgG2a monoclonal antibody obtained from a fusion between Y3.Ag.l.2.3 rat myeloma and spleen cells from an immunized WF rat and as described by Galfr6 et al. (1979). b 03B3 rat IgG2a monoclonal antibody binding MHC class 1 antigens of WF rats (kind gift from Magnus Lindwall the Wallenberg Laboratory, Lund). c Q12, rat IgM monoclonal Ab specific for OA. d 1G6, 1F6, 1Cll, 10B12, 10H5, 7D8 rat IgM monoclonal antibodies which recognize autoantigens and rat colon carcinoma associated antigens (unpublished observations T. Brodin, B. Jansson and H.O. Sj~Sgren).
221 the addition of 3% bovine serum albumin (BSA) in PBS.
Biotinylation of ovalbumin admixed to fetal calf serum Ovalbumin was added to fetal calf serum diluted in 0.1 M N a 2 C O 3 p H 8.0 buffer. The total protein concentration was adjusted to 1 m g / m l and 1 ml was labelled for 1 h with 100/~1 of 1.25 m g / m l NHS-d-biotin and dialysed against PBS.
Biotinylation of viable cells Cell surface labelling with biotin was performed after careful washing of the cells (5 × 10 6) with PBS either in suspension ( W / F u G1 l y m p h o m a and spleen cells of W F rats) or as adherent cells ( D M H W49 colon carcinoma of W F rat) in a cell culture bottle. The red blood cells were removed from rat spleen cell suspensions by hypotonic shock. Nonadherent cells were suspended in 0.5 ml PBS and supplied with 50/zl (10 m g / m l NHS-d-biotin in DMSO), agitated for 20 min at room temperature and washed in cold R P M I 5% ( v / v ) FCS. The cells were then lysed by incubation for 10 min at room temperature in 2 ml 1% Triton TX-100, 2 m M PMSF and 0.1% sodium azide in PBS. Adherent colon adenocarcinoma cells, D M H W49, were labelled in the same way as the W / F u G1 cells making use of a cell culture flask with a dense monolayer (approximately 1 x 10 6 cells). After extensive washing 2 ml of PBS containing 200 /xl of 10 m g / m l NHS-d-biotin were added and the mixture slowly agitated for 20 min at room temperature. The cells were washed and lysed in the same way as the W / F u G1 cells. Debris and nuclei were pelleted at 1000 x g for 10 min. The D M S O and excess NHS-d-biotin were removed by desalting on a short G 25-M column (PD-10, Pharmacia, Sweden). The supernatant was cleared in a Beckman airfuge (30 PSI, A95 rotor for 10 min). Labelled antigen was used immediately or stored as frozen aliquots at - 80 ° C. Frozen-thawn biotin-labelled antigen preparations were always recentrifuged before use to avoid an increase in background binding that was otherwise noted.
Biotin-labelled antigen assay (BILA) Rabbit anti-mouse Ig antiserum (primary capture antibody, 10 mg ammoniumsulphate-precipitated antibodies/ml Dakopatts, Denmark) was diluted 1 / 1 0 0 in 5 m M Hepes, p H 8.1 and adsorbed to a 96-well polyvinyl plate (Costar cat. no. 2595). The antibody in a volume of 50 #1 was left to adhere overnight at 4 o C. Next day the plates were washed with 2 × 200 /~1 PBS with 0.05% Tween 20/well and residual protein binding sites were blocked with 50% ( v / v ) FCS in PBS for 15 min. Following the blocking procedure 50/~1 of hybridoma culture supernatant or ascites fluid (secondary capture antibody) diluted in R P M I 1640 supplied with 5% ( v / v ) FCS were added. The monoclonal antibody was left for 1 h in room temperature to bind to the anti-mouse antibody. After another washing step, 50 #1 biotinylated antigens in detergent solution or in 3% ( w / v ) BSA 0.05% ( v / v ) Tween 20 in PBS (LMW, O A / F C S ) were added. The antigen was incubated for at least 1 h before final washing and detection with 125Istreptavidin (21 ~ C i / ~ g ) diluted in 3% ( w / v ) BSA 0.05% ( v / v ) Tween 20 in PBS. U n b o u n d streptavidin was washed off and the radioactivity in each well was analysed in a gamma-counter. Standard RIA assay L M W proteins were diluted in 5 m M Hepes p H 8.1 and were left overnight to adhere to the plastic plate (Costar cat. no. 2595) at 4 ° C . Residual protein binding sites were blocked by incubation for 30 min with 50% fetal calf serum in PBS. The plates were sequentially incubated with primary antibody, secondary antibody ( D A K O rabbit anti-mouse diluted 1/500) in R P M I 1640 supplied with 10% FCS and staphylococcal protein A labelled with 125I (23/~Ci/~g). After each incubation the plates were washed with 2 x 200 ~1 PBS containing 0.05% Tween 20. All incubations were performed in a reaction volume of 50 #1.
Results
The sensitivities of the BILA and the standard R I A were evaluated using biotin-labelled ovalbumin in a mixture of different proteins and antiovalbumin antibody (Figs. 1, 2 and 3). M a x i m u m
222
BILA 30000 ~" Q.
[] LMW • LMW
v'~3
~
B
S
1/10, 370 1/100, 3 7
ng ng
A
20000 •
.E .E .~I t-
10000 '
e,,~
~
0
~
.
• 1
10
Log
2
3
dilutions
4
5
6
of
anti
OA
Fig. 1. BILA assays. Plates coated with D A K O rabbit antimouse immunoglobulin antibodies were incubated with mouse anti-OA monoclonal capture antibody and a standard mixture of proteins (LMW) including OA at a concentration of 73.5 fig O A / 2 8 9 ~,g (25% w / w ) total protein/ml solution. Antigen binding is presented as cpm bound 125I-streptavidin. Serial dilutions of anti-OA hybridoma supernatant were tested for binding of biotinylated LMW standard proteins at two concentrations. Biotinylated BSA (100 ~ g / m l ) was used as a negative control (mean CPM ± SEM is indicated, n = 4).
binding in the BILA assay was found when antiovalbumin supernatants were diluted 1 / 1 0 and the antigen incubation time exceeded 1 h (Fig. 1).
OA constituted 25% ( w / w ) of the total protein content. Using a 1 / 1 0 0 dilution of the anti-OA it was possible to detect as little as 0.37 ng of OA in the mixture of biotinylated antigens (Fig. 2a). In the standard RIA assay the protein mixture was adsorbed to the 96-well plate and at a 1 / 1 0 0 dilution of anti-OA, 3.7 ng but not 0.37 ng could be detected (Fig. 2b). Experiments were then performed to investigate whether a protein antigen could be assayed when present in an even smaller portion of a complex protein mixture. Fetal calf serum and OA were mixed and diluted in 0.1 M sodium carbonate before labelling with biotin in the same way as previously described for LMW. Using the BILA it was possible to detect as little as 0.5 ng OA in a total protein mixture of 50 /~g (0.001%, w / w ) (Fig. 3a). When the same protein mixtures were used as target antigens in a solidphase RIA (Fig. 3b) undiluted anti-OA supernatant exhibited a detection limit of 0.5 ttg in a protein mixture containing 50 ~g of protein (1%). The effect of detergent on the binding of biotin-labelled antigen was tested (Figs. 4a and 4b). A mixture of DOC, Triton TX-100 and SDS gave the lowest background and was used as a standard
b a
BILA []
Q. O
•
Anti-OA Anti-OA
IX.
1/10 1/100
ov t,-
20000
t.m
"0 10000
0
RIA
2000
30000-
1000
'10 0 ..Q
E
~, 370
Amount
,
37
.
37
,
,
-
0.37 0.04 0004
of biotinylated OA (ng/well)
l
t
J
i
370
37
37
037
Amount
i
J
0.04 0.004
of O A ( r i g / w e l l )
Fig. 2. a: BILA assays. Plates coated with D A K O rabbit anti-mouse immunoglobulin antibodies were incubated with mouse anti-OA monoclonal capture antibody and a standard mixture of proteins (LMW) including OA at a concentration of 73.5 ~g O A / 2 8 9 /*g (25% w / w ) total protein/ml solution. Serial dilutions of biotinylated LMW proteins were tested for binding to anti-OA as capture antibody and 03B3 as control antibody. Antigen binding is presented as cpm bound 125I-streptavidin. b: RIA assay. Results of a regular solid-phase RIA for ovalbumin in a dilution series of a standard protein mixture (LMW protein solution contained 73.5 #g O A / 2 8 9 txg (25% w / w ) total protein/ml solution). 50 #1 of serially diluted LMW proteins were bound to 96-well plates and detected using anti-OA supernatant. Background cpm values were estimated with control antibody 03B3 diluted 1/10. The test plates were sequentially incubated with rabbit anti-mouse sera 1/500 and staphylococcal protein A labelled with 1251 (mean c p m ± S E M is indicated, n = 2).
223 a
O u.
o9 O kl.
BILA
b
==
RIA [] [] • •
m
0 0.005 o O "o
'
[]
Anti-OA
undiluted
50
o
[]
O3B3
undiluted
0.05
O
0.5
~
5.0
...........
I
,
"6 ""
.
.
.
.
.
.
/ well / well well / well
'
500
T=
50 50 0
O3B3, 50 ug protein Anti-OA, 0.5 ug protein Anti-OA, 5 ug protein / Anti-OA, 50 ug protein
5000
.
0~
5000
i
0
10000
20000
o~ o c
0
200
400
600
c
Antigen
Z
binding
(CPM)
Antibody
binding
(CPM)
Fig. 3. Estimation of the amount of ovalbumin (OA) present in a complex protein mixture that was detectable by the BILA or standard RIA assay, a: Samples of FCS admixed with small amounts of OA were labelled with NHS-d-biotin (materials and methods section) and assayed. Undiluted culture supernatant from the anti-OA hybridoma was used as the capture antibody and 50 /~g of biotin-labelled proteins were added to each well. Bound antigen was detected using streptavidin labelled with 1251. As a background control the monoclonal 03B3 of the same isotype was used. b: Results of a standard RIA assay of small amounts of OA admixed to FCS. 50 ~1 containing 50, 5, or 0.5 #g of total protein including various amounts of OA admixed to FCS were bound to 96-well plates and detected usinganti-OA supernatant. Background cpm values were estimated with the control antibody 03B3. The test plates were sequentially incubated with rabbit anti mouse sera and staphylococcal protein A labelled with 125I (mean cpm + SEM is indicated, n = 2).
detergent solution when performing assays on cell membrane antigens. Selective binding of biotinylated molecules present in extracts of normal and neoplastic cells was observed with monoclonal antibodies of the appropriate specificity (Fig. 5). Using spleen cell extracts binding was observed with antibodies showing specificity for immunoglobulin, M H C and the CD5 T cell marker. Weak binding to the
leucocyte marker-specific antibody OX-1 was also observed. In colon tumour extracts molecules could be detected which reacted with antibodies to M H C class 1, p53 and some of the monoclonals which by immunohistological techniques had been shown to bind to colon tumour sections. When using rat tissue preparations containing rat immunoglobulin care was taken to ensure that all the rabbit anti-rat and mouse Ig binding sites
a LMW proteins
b Lymphoma
DOC+TX+SDS
Doc+TX+SDS
BSA DOC Tween Triton
20
AntI-OA
[]
O3B3
10000
Antigen
binding (CPM)
20000
I
[ ] HB 60 •
DOC Tween
Triton
X-100
I
r
BSA
•
cell extract
OX-18
20 X-100
10000
20000
Antigen binding (CPM)
Fig. 4. Influence of detergents on the antigen binding and the background cpm values in the BILA. Both the biotin-labelled LMW protein preparation and the biotin-labelled cell ( W / F u G1) extract were diluted in 10 mM Tris-HC1, pH 8.1 containing: 0.5% v / v Triton X-100 (Triton X-100), 0.5% v / v Tween 20 (Tween 20), 0.5% w / v sodium deoxycholate (DOC), 1% w / v bovine serum albumin (BSA) or 0.5% ( v / v ) Triton X-100 0.5% w / v sodium deoxycholate 0.05% SDS (DOC-TX-SDS). a: Monoclonal anti-OA 1/100 and background control antibody 03B3 1/100 were used as capture antibodies with 1/10 dilutions of LMW protein as biotin-labelled antigen, b: Monoclonal antibodies OX-18 (1/100) against rat MHC class 1 and control antibody HB60 1/1130 were used as capture antibodies with 1 / 1 0 dilutions of biotinylated W / F u G1 cell membrane extracts (mean cpm _.+SEM is indicated, n = 2).
224 A
Rat s p l e e n
cell
extract
MARK-1 ' HB
B Rat DMH W49 C a r c i n o m a e x t r a c t o1
H
.~_
ox-6
Medium
"1o O .O
60
)
ox-19
~
p53
-~
Ox-18
ox-1
c o
ox-18
o t-
H
O3B3
O 2000
4000
Antigen
6000
8000
binding
10000
(CPM) C
._~ "o O .Q
i 1000
12000
Antigen Rat BN 7005 c a r c i n o m a
i 2000
binding
i 3000
4000
(CPM)
extract
Q12 7D8
m ~
"E 10HS 10B12 tO
1Cll
O
1F6
O
1G6
H i
•
2000
i
4000
Antigen
,
i
•
6000
i
8000
binding
•
i
10000
,
12000
(CPM)
Fig. 5. BILA assays with biotinylated membrane extracts of rat cells using rabbit anti-mouse Ig antibodies as a primary capture antibody and a n u m b e r of different monoclonal antibodies. Before the addition of biotinylated antigen residual binding sites for rat Ig on the primary capture antibody were blocked using 50 /~1 normal rat serum diluted 1 / 2 0 in PBS and incubated for 15 min. a: Mouse monoclonal antibodies (ascites diluted 1/100) as secondary capture antibodies tested against an extract of biotinylated spleen cell membranes, b: Mouse (ascites diluted 1/100) and rat (undiluted hybridoma culture supernatant) monoclonal antibodies as secondary capture antibodies tested against an extract of biotinylated W49 colon tumor cell membranes, c: Rat monoclonal lgM anti-tumour antibodies as secondary capture antibodies exemplifying screening tests against biotinylated m e m b r a n e proteins from BN7005 colon carcinoma tumour. Fresh hybridoma culture medium was used undiluted (mean cpm_+ SEM is indicated, n = 2).
were blocked by incubating the wells for 15 min with normal rat serum diluted 1 / 2 0 in RPMI. Both non-biotin-labelled antigen preparations and free antibody were used to inhibit the observed binding and confirm the specificity of each test system (Fig. 6).
Discussion
When producing monoclonal antibodies to cell membrane-associated proteins there is a need for a technique that can detect monoclonal antibodies binding to proteins present in complex mixtures. Very often the protein of interest is not yet characterized and is merely a minor component of the plasma cell membrane. A detergent-solubilized
preparation of plasma membranes from the cells will not only contain nonrelevant plasma membrane molecules but frequently proteins from the cytoplasm and nucleus also. Typical R I A or ELISA assays are based on adherence of a protein solution to a solid phase. The adsorbing area and the protein binding capacity of the solid phase or the crosslinking reagents imposes restrictions on the efficiency of these techniques. When making use of labelled antigens instead of labelled antibodies and particularly when cell surface labelling is being used, the above problems should theoretically be minimized. The total amount of target protein is no longer a limiting step and it should be possible to raise the total protein content until reasonable signal to background cpms are registered. Indeed our experi-
225
Inhibition assay HB60 Ox-18
a 1/10
I
Spleen BSA
1
anti-OA
1/10
anti-OA
1/2
I
b I ]
CA O3B3 BSA
1/2 i
20
Per
i
40
cent
i
i
60
80
100
inhibition
Fig. 6. Inhibition assay. Binding inhibition assays were performed with rabbit anti-mouse as primary capture antibody, a: As secondary capture antibodies OX-18 ascites diluted 1/100 were used. Biotinylated extracts of spleen cell membranes corresponding to 12 500 cells/well were incubated with medium containing BSA (BSA), a spleen cell preparation of 1.5 x 106 viable spleen cells (spleen) or monoclonal antibody OX-18 ascites diluted 1 / 1 0 (OX-18 1/10) or monoclonal antibody HB60 ascites diluted 1 / 1 0 (HB60). Before the addition of biotinylated antigen residual binding sites for rat Ig on the capture antibody were blocked using 50 #1 normal rat serum diluted 1 / 2 0 in PBS and incubated for 15 min. b: As a secondary capture antibody anti-ovalbumin diluted 1 / 1 0 was used. Biotinylated L M W proteins (37 ng OA/well) were incubated with ovalbumin 50 ~ g / w e l l (OA), bovine serum alb u m i n 50 /tg/well (BSA) anti-OA supernatant diluted 1 / 2 or 1 / 1 0 (anti-OA 1/2, 1 / 1 0 ) and 03B3 supernatant diluted 1 / 2 (03B3 1 / 2 ) (mean cpm + SEM is indicated, n = 2).
ments suggested that this was so in our model system and small amounts of ovalbumin could be detected in the presence of a vast excess of other proteins. A positive reaction was seen in protein mixtures containing ovalbumin in as small a proportion as 0.5 n g / 5 0 #g total protein compared to 0.5 fig/50 /tg sample when using an ordinary solid-phase assay (Figs. 3a and 3b). The necessary amount of biotin-labelled antigen used when screening antibodies with the BILA assay is very difficult to predict and therefore it is advisable to use as much antigen as possible. However, as little as a few n g / m l of the relevant antigen present in a large excess of other proteins could be detected with the anti-OA monoclonal antibody (Fig. 1). The upper limit of total antigen protein that could be used was indicated by an experiment in which it was shown that the biotin-labelled antigen concentration could be increased from 1 0 0 / ~ g / m l (as
used in the experiment described in Fig. 5c) to 1 m g / m l without increasing the specific binding of the antibodies tested (data not shown). The binding and precipitation of adhesive, proteins is always a problem when working with membrane proteins. However, the inclusion of detergents and a centrifugation step immediately before the assay of frozen and thawed preparations, minimizes the background problems. The BILA assay is probably not suitable for testing serum antibodies since the proportion of specific antibodies among serum immunoglobulins is comparatively low. The anti-OA monoclonal antibody (9.4 ffg/ml) diluted 1 / 1 0 0 showed only a small decrease in the binding of labelled antigen (Fig. 2a). Thus the amount of antibody bound to each well could be estimated to within a few nanograms. This amount showed some variation dependent on the immunoglobulin subclass of the monoclonal antibody and variations in the capacity of the capture antibodies to bind the subclass in question. The NHS-d-biotin labelled most proteins satisfactorily but other biotinylating reagents can be used if preferred (Billingsley et al., 1987). The biotin reagents solubilized in D M S O are particularly useful, since they can selectively label cell surface molecules (Hurley et al., 1985). Labelled cells ( W / F u G1) show high viability (95-100% as measured by trypan blue exclusion) and their viability does not seem to be affected by exposure for 1 h to 10% D M S O and 3 mg N H S - d - b i o t i n / 5 x 106cells in 0.5 ml PBS. However, 20 rain exposure at room temperature, 0.5 mg N H S - d - b i o t i n / 5 x 1 0 6 cells and 0.5 ml PBS were used as standard conditions. The NHS-d-biotin ester is a non-toxic reagent that is easy to handle and can, if necessary any, be stored in frozen aliquots for at least 6 months. When labelling antigens there is always a risk of denaturation but the same risk must be faced when antigens are bound to a solid surface. We have no data to exclude the possibility that the streptavidin and monoclonal antibody binding sites may sometimes be located so close to each other that steric hindrance or displacement of binding could take place. However, since all of the tested monoclonal antibodies to known biotinlabelled antigens have been used successfully in
226 t h e B I L A a s s a y s u c h i n t e r f e r e n c e d o e s n o t s e e m to occur frequently. T h e use o f s o l u b i l i z e d cell m e m b r a n e s d e m a n d s the use of d e t e r g e n t s in t h e a n t i b o d y - a n t i g e n b i n d i n g step. T h i s m a y c a u s e p r o b l e m s since d e t e r g e n t may interfere with the antigen-antibody binding. N o s u c h c o m p l i c a t i o n w a s o b s e r v e d in o u r e x p e r i ments. In RIA/ELISA procedures commonly used for s c r e e n i n g m o n o c l o n a l a n t i b o d i e s , t h e r e is also a n e e d for the p r e s e n c e of s o m e d e t e r g e n t to e l i m i n a t e n o n - s p e c i f i c b i n d i n g o f the a n t i b o d y to t h e p l a s t i c surface, p a r t i c u l a r l y w h e n t h e a n t i b o d y is o f the I g M i s o t y p e ( E m m e r i c h , 1984). T h e b i o t i n - l a b e l l e d a n t i g e n test is a useful a n d o f t e n a m o r e s e n s i t i v e a l t e r n a t i v e to o r d i n a r y s c r e e n i n g p r o c e d u r e s w h i c h m a k e use o f a n t i g e n a d s o r b e d o n a solid p h a s e . It gives a d d i t i o n a l i n f o r m a t i o n a b o u t the v a l u e of t h e a n t i b o d y a n d selects t h o s e a n t i b o d i e s t h a t are likely to b e u s e d for a f f i n i t y c h r o m a t o g r a p h y . T h e b i o t i n l a b e l l i n g o f a n t i g e n s is also a p o t e n t i a l l y v a l u a b l e t e c h n i q u e for m o n i t o r i n g t h e p u r i f i c a t i o n o f m e m b r a n e antigens without labelling more than one reagent with isotope.
Acknowledgements W e t h a n k L o t t a R o l a n d e r a n d Pia KarlstriSm for t h e i r e x p e r t t e c h n i c a l assistance. T h i s w o r k was s u p p o r t e d b y t h e S w e d i s h M e d i cal R e s e a r c h C o u n c i l , J o h n a n d A u g u s t a P e r s s o n s Foundation, and the Medical Faculty, University of Lund.
References AkerstriSm, B., Brodin, T,, Reis, K. and Bjrrck, L. (1985) Protein G: A powerful tool for binding and detection of monoclonal and polyclonal antibodies. J. Immunol. 135, 2589. Bazin, H., Xhurdbise, L.-M., Burtonboy, G., Lebacq, A.-M., Declerq, L. and Cormont, F. (1984) Rat monoclonal antibodies. 1. Rapid purification from in vitro culture supernatants. J. Immunol. Methods 66, 261.
Billingsley, M.L., Penneypacker, R., Hoover, C.G. and Kincaid, R.L. (1987) Biotinylated proteins as probes of protein structure and protein-protein interactions. Biotechniques 5, 22. Bolton, A.E. and Hunter, W.M. (1973) The labelling of proteins to high specific radioactivities by conjugation to a a251 containing acylating agent. Biochem. J. 133, 529. Brown, J.P., Tamerius, J.D. and HellsstriSm, I. (1979) Indirect 1251-labelled protein A assay for monoclonal antibodies to cell surface antigens. J. Immunol. Methods 31,201. Brown, J.P., Woodbury, R.C., Hart, C.E., Hellstr~Sm, I. and Hellstr/3m, K.E. (1981) Quantitative analysis of melanomaassociated antigen p97 in normal and neoplastic tissues. Proc. Natl. Acad. Sci. U.S.A. 78, 539. Dallman, M.J., Mason, D.W. and Webb, M. (1982) The roles of host and donor cells in the rejection of skin allografts by T cell-deprived rats injected with syngeneic T cells. Eur. J. Immunol. 12, 511. Emmrich, F. (1984) Reduction of non-specific human lgM binding in solid phase assays. J. Immunol. Methods 72, 501. Fukumoto, T., McMaster, W.R. and Williams, A.F. (1982) Mouse monoclonal antibodies against rat major histocompatibility antigens. Two Ia antigens and expression of la and class 1 antigens in rat thymus. Eur. J. Immunol. 12, 237. Galfrr, G., Milstein, C. and Wright, B. (1979) Rat x rat hybrid myelomas and a monoclonal anti-Fd portion of mouse IgG. Nature 277, 131. Hedlund, G. and SjiSgren, H.O. (1980) Induction of transplantation immunity to rat colon carcinoma isografts by implantation of intact fetal colon tissue. Int. J. Cancer 26, 71. Hurley, L.H., Finkelstein, E. and Hoist, B.D. (1985) Identification of surface proteins on bovine leucocytes by a biotin avidin protein blotting technique. 85, 195. Kelleher, J.P., Mathews, H.L., Woods,L.K., Farr, R.S. and Minden, P. (1983) A solid-phase radioimmunoassay to detect antibodies produced by hybridomas to antigens derived from human melanoma cells. Cancer Immunol. lmmunother. 14, 185. Morgan, Jr., A.C., Galloway, D.R., Wilson, B.S. and Reisfield, R.A. (1980) Human melanoma associated antigens: A solid-phase assay for detection of specific antibody. J. Immunol. Methods 39, 233. Shellam, G.R. (1974) Studies on a Gross-virus induced lymphoma in the rat. I. The cell mediated response. Int. J. Cancer 14, 65. Sunderland, C.A., McMaster, W.R. and Williams, A.F. (1979) Purification with monoclonal antibody of a predominant leukocyte-common antigen and glycoprotein from rat thymocytes. Eur J. Immunol. 9, 155.
219
JIM 04987
A biotin-labelled antigen radioimmunoassay (BILA) for antibodies to membrane antigens useful for monoclonal antibody screening Bo Jansson, T h o m a s B r o d i n a n d H a n s O l o v SjSgren Department of Tumor Immunology, The Wallenberg Laboratory, University of Lund, Box 7031, S-220 07 Lund, Sweden (Received 11 April 1988, revised received 16 June 1988, accepted 15 July 1988)
Viable cells or protein extracts were labelled with N-hydroxysuccinimidobiotin and used as target antigens in a biotin-labelled antigen radioimmunoassay (BILA). The binding of the biotinylated antigens to capture antibodies coated on the bottoms of 96-well plastic plates were measured using 125I-labelled streptavidin as the detection step. The assay circumvents some of the problems associated with solid-phase RIA and permits screening of antibodies to undefined protein antigens present in very small amounts in complex protein solutions. Key words: Avidin; Biotin; Membrane antigen
Introduction
The most frequently used techniques for screening monoclonal antibodies against cell surface antigens make use of intact cells (Brown et al., 1979) or crude plasma membrane preparations (Morgan et al., 1980; Kelleher et al., 1983). In most assays cells or membranes are simply bound to the bottom of 96-well plastic plates. The binding of antibodies, e.g., of hybridoma supernatants, is detected by a labelled secondary antibody, staphylo-
Correspondence to: B. Jansson, Department of Tumor Immunology, The Wallenberg Laboratory, University of Lund, Box 7031, S-220 07 Lund, Sweden. Abbreviations: PBS, 10 mM NaH2PO 4, 150 mM NaCI, pH 7.3; OA, ovalbumin; BSA, bovine serum albumin; LMW, low molecular weight standard proteins; DMSO, 1,2-di-methylsulphoxide; PMSF, phenylmetliylsulphonyl fluoride; FCS, fetal calf serum; DOC, sodium deoxycholate; NHS-d-biotin, N-hydroxysuccinimidobiotin.
coccal protein A (Brown et al., 1981), or streptococcal protein G (AkerstrSm et al., 1985). There are certain limitations associated with these standard techniques. When preparations of plasma cell membranes are used as targets, the antigens have to compete with nonrelevant proteins and lipids for the very limited binding capacity of the plastic wells. If a detectable amount of antigen is to remain on the plastic surface the original crude membrane fraction has to contain a large percentage of the relevant antigen. In addition the necessity of keeping most membrane-associated antigens in detergent solution also impairs the binding of antigens to the plastic solid phase. To avoid this problem antigens and cells might need to be fixed (for example, by glutaraldehyde) in order to adhere properly to the plastic surface (Kelleher et al., 1983) and fixation always increases the risk of denaturation of antigenic determinants. When adsorbing proteins to a solid phase there is always a risk of creating
0022-1759/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
220 a n t i g e n i c n e o e p i t o p e s b e c a u s e the n a t u r a l m o l e c u lar flexibility is restricted. W e n o w d e s c r i b e an a l t e r n a t i v e screening m e t h o d m a k i n g use of b i o t i n y l a t e d antigens a n d a n o n c o v a l e n t l y a t t a c h e d c a p t u r e a n t i b o d y . This assay c i r c u m v e n t s s o m e o f the p r o b l e m s e n c o u n t e r e d when using a cell m e m b r a n e p r e p a r a t i o n as target in screening assays for m o n o c l o n a l a n t i b o d ies.
Cell lines A G r o s s v i r u s - i n d u c e d rat l y m p h o m a W / F u G1 (Shellam et al., 1974) a n d 1 , 2 - d i m e t h y l h y d r a z i n e - i n d u c e d rat c o l o n c a r c i n o m a s BN7005 a n d D M H W 4 9 ( H e d l u n d et al., 1980) were used.
Monoclonal antibodies T h e m o n o c l o n a l a n t i b o d i e s used are d e s c r i b e d in T a b l e I.
Materials and methods
Biotinylation of low molecular weight proteins ( L M W proteins)
Chemicals
L M W c a l i b r a t i o n kit p r o t e i n s f r o m P h a r m a c i a c o n t a i n i n g 64 ~tg p h o s p h o r y l a s e - b , 83 /tg b o v i n e s e r u m a l b u m i n , 147 # g o v a l b u m i n , 83 ~ g c a r b o n i c a n h y d r a s e , 8 0 / t g soya b e a n t r y p s i n i n h i b i t o r a n d 121/~g of a - l a c t a l b u m i n / v i a l w e r e d i l u t e d in 1 ml of 0.1 M N a 2 C O 3 p H 8.0 a n d l a b e l l e d with 100/~1 o f N - h y d r o x y s u c c i n i m i d o b i o t i n (1.25 m g / m l D M S O ) for I h a n d then d i a l y s e d o v e r n i g h t a g a i n s t PBS. T h e total v o l u m e was i n c r e a s e d to 2 ml b y
N-hydroxysuccinimidobiotin (NHS-d-biotin, Sigma, St. Louis, M O , H1759). S t r e p t a v i d i n (Sigma, St. Louis, M O , S-4762) was l a b e l l e d with 1251 using the c h l o r a m i n e - T m e t h o d for the labelling of I g G (Brown et al., 1981) (specific activity 21 # C i / # g ) . S t a p h y l o c o c c a l p r o t e i n A was l a b e l l e d w i t h 125I using the m e t h o d d e s c r i b e d b y B o l t o n a n d H u n t e r 1973 (specific activity 23 # C i / # g ) .
TABLE 1 MONOCLONAL ANTIBODIES USED IN THIS STUDY Monoclonal antibody
Antigen
Reference
Mouse IgG1 Mouse IgG1 Mouse IgG2b
Rat x light chain Human y heavy chain p53 antigen
Bazin et al., 1984 Kuritami et al., 1981 Gurrey et al., 1980
OX-18 OX-19 OX-1 OX-6
Mouse IgG1 Mouse IgG1 Mouse IgGl Mouse IgGl
Rat RT-1 monomorphic part Rat T cell-associated antigen Rat monocyte-associated antigen Rat MHC class 2
Fukumoto et al., 1982 Dallman et al., 1982 Sunderland et al., 1979 Fukomoto et al., 1982
Anti-OA 03B3 Q12
Rat IgG2a Rat IgG2a Rat IgM
Ovalbumin Rat RT-lu Ovalbumin
b c
1G6, 1F6, 1Cll, 10B12, 10H5, 7D8
Rat IgM
d
d
Designation
Class
MARK-1 HB60 Ab 122
a Anti-ovalbumin rat IgG2a monoclonal antibody obtained from a fusion between Y3.Ag.l.2.3 rat myeloma and spleen cells from an immunized WF rat and as described by Galfr6 et al. (1979). b 03B3 rat IgG2a monoclonal antibody binding MHC class 1 antigens of WF rats (kind gift from Magnus Lindwall the Wallenberg Laboratory, Lund). c Q12, rat IgM monoclonal Ab specific for OA. d 1G6, 1F6, 1Cll, 10B12, 10H5, 7D8 rat IgM monoclonal antibodies which recognize autoantigens and rat colon carcinoma associated antigens (unpublished observations T. Brodin, B. Jansson and H.O. Sj~Sgren).
221 the addition of 3% bovine serum albumin (BSA) in PBS.
Biotinylation of ovalbumin admixed to fetal calf serum Ovalbumin was added to fetal calf serum diluted in 0.1 M N a 2 C O 3 p H 8.0 buffer. The total protein concentration was adjusted to 1 m g / m l and 1 ml was labelled for 1 h with 100/~1 of 1.25 m g / m l NHS-d-biotin and dialysed against PBS.
Biotinylation of viable cells Cell surface labelling with biotin was performed after careful washing of the cells (5 × 10 6) with PBS either in suspension ( W / F u G1 l y m p h o m a and spleen cells of W F rats) or as adherent cells ( D M H W49 colon carcinoma of W F rat) in a cell culture bottle. The red blood cells were removed from rat spleen cell suspensions by hypotonic shock. Nonadherent cells were suspended in 0.5 ml PBS and supplied with 50/zl (10 m g / m l NHS-d-biotin in DMSO), agitated for 20 min at room temperature and washed in cold R P M I 5% ( v / v ) FCS. The cells were then lysed by incubation for 10 min at room temperature in 2 ml 1% Triton TX-100, 2 m M PMSF and 0.1% sodium azide in PBS. Adherent colon adenocarcinoma cells, D M H W49, were labelled in the same way as the W / F u G1 cells making use of a cell culture flask with a dense monolayer (approximately 1 x 10 6 cells). After extensive washing 2 ml of PBS containing 200 /xl of 10 m g / m l NHS-d-biotin were added and the mixture slowly agitated for 20 min at room temperature. The cells were washed and lysed in the same way as the W / F u G1 cells. Debris and nuclei were pelleted at 1000 x g for 10 min. The D M S O and excess NHS-d-biotin were removed by desalting on a short G 25-M column (PD-10, Pharmacia, Sweden). The supernatant was cleared in a Beckman airfuge (30 PSI, A95 rotor for 10 min). Labelled antigen was used immediately or stored as frozen aliquots at - 80 ° C. Frozen-thawn biotin-labelled antigen preparations were always recentrifuged before use to avoid an increase in background binding that was otherwise noted.
Biotin-labelled antigen assay (BILA) Rabbit anti-mouse Ig antiserum (primary capture antibody, 10 mg ammoniumsulphate-precipitated antibodies/ml Dakopatts, Denmark) was diluted 1 / 1 0 0 in 5 m M Hepes, p H 8.1 and adsorbed to a 96-well polyvinyl plate (Costar cat. no. 2595). The antibody in a volume of 50 #1 was left to adhere overnight at 4 o C. Next day the plates were washed with 2 × 200 /~1 PBS with 0.05% Tween 20/well and residual protein binding sites were blocked with 50% ( v / v ) FCS in PBS for 15 min. Following the blocking procedure 50/~1 of hybridoma culture supernatant or ascites fluid (secondary capture antibody) diluted in R P M I 1640 supplied with 5% ( v / v ) FCS were added. The monoclonal antibody was left for 1 h in room temperature to bind to the anti-mouse antibody. After another washing step, 50 #1 biotinylated antigens in detergent solution or in 3% ( w / v ) BSA 0.05% ( v / v ) Tween 20 in PBS (LMW, O A / F C S ) were added. The antigen was incubated for at least 1 h before final washing and detection with 125Istreptavidin (21 ~ C i / ~ g ) diluted in 3% ( w / v ) BSA 0.05% ( v / v ) Tween 20 in PBS. U n b o u n d streptavidin was washed off and the radioactivity in each well was analysed in a gamma-counter. Standard RIA assay L M W proteins were diluted in 5 m M Hepes p H 8.1 and were left overnight to adhere to the plastic plate (Costar cat. no. 2595) at 4 ° C . Residual protein binding sites were blocked by incubation for 30 min with 50% fetal calf serum in PBS. The plates were sequentially incubated with primary antibody, secondary antibody ( D A K O rabbit anti-mouse diluted 1/500) in R P M I 1640 supplied with 10% FCS and staphylococcal protein A labelled with 125I (23/~Ci/~g). After each incubation the plates were washed with 2 x 200 ~1 PBS containing 0.05% Tween 20. All incubations were performed in a reaction volume of 50 #1.
Results
The sensitivities of the BILA and the standard R I A were evaluated using biotin-labelled ovalbumin in a mixture of different proteins and antiovalbumin antibody (Figs. 1, 2 and 3). M a x i m u m
222
BILA 30000 ~" Q.
[] LMW • LMW
v'~3
~
B
S
1/10, 370 1/100, 3 7
ng ng
A
20000 •
.E .E .~I t-
10000 '
e,,~
~
0
~
.
• 1
10
Log
2
3
dilutions
4
5
6
of
anti
OA
Fig. 1. BILA assays. Plates coated with D A K O rabbit antimouse immunoglobulin antibodies were incubated with mouse anti-OA monoclonal capture antibody and a standard mixture of proteins (LMW) including OA at a concentration of 73.5 fig O A / 2 8 9 ~,g (25% w / w ) total protein/ml solution. Antigen binding is presented as cpm bound 125I-streptavidin. Serial dilutions of anti-OA hybridoma supernatant were tested for binding of biotinylated LMW standard proteins at two concentrations. Biotinylated BSA (100 ~ g / m l ) was used as a negative control (mean CPM ± SEM is indicated, n = 4).
binding in the BILA assay was found when antiovalbumin supernatants were diluted 1 / 1 0 and the antigen incubation time exceeded 1 h (Fig. 1).
OA constituted 25% ( w / w ) of the total protein content. Using a 1 / 1 0 0 dilution of the anti-OA it was possible to detect as little as 0.37 ng of OA in the mixture of biotinylated antigens (Fig. 2a). In the standard RIA assay the protein mixture was adsorbed to the 96-well plate and at a 1 / 1 0 0 dilution of anti-OA, 3.7 ng but not 0.37 ng could be detected (Fig. 2b). Experiments were then performed to investigate whether a protein antigen could be assayed when present in an even smaller portion of a complex protein mixture. Fetal calf serum and OA were mixed and diluted in 0.1 M sodium carbonate before labelling with biotin in the same way as previously described for LMW. Using the BILA it was possible to detect as little as 0.5 ng OA in a total protein mixture of 50 /~g (0.001%, w / w ) (Fig. 3a). When the same protein mixtures were used as target antigens in a solidphase RIA (Fig. 3b) undiluted anti-OA supernatant exhibited a detection limit of 0.5 ttg in a protein mixture containing 50 ~g of protein (1%). The effect of detergent on the binding of biotin-labelled antigen was tested (Figs. 4a and 4b). A mixture of DOC, Triton TX-100 and SDS gave the lowest background and was used as a standard
b a
BILA []
Q. O
•
Anti-OA Anti-OA
IX.
1/10 1/100
ov t,-
20000
t.m
"0 10000
0
RIA
2000
30000-
1000
'10 0 ..Q
E
~, 370
Amount
,
37
.
37
,
,
-
0.37 0.04 0004
of biotinylated OA (ng/well)
l
t
J
i
370
37
37
037
Amount
i
J
0.04 0.004
of O A ( r i g / w e l l )
Fig. 2. a: BILA assays. Plates coated with D A K O rabbit anti-mouse immunoglobulin antibodies were incubated with mouse anti-OA monoclonal capture antibody and a standard mixture of proteins (LMW) including OA at a concentration of 73.5 ~g O A / 2 8 9 /*g (25% w / w ) total protein/ml solution. Serial dilutions of biotinylated LMW proteins were tested for binding to anti-OA as capture antibody and 03B3 as control antibody. Antigen binding is presented as cpm bound 125I-streptavidin. b: RIA assay. Results of a regular solid-phase RIA for ovalbumin in a dilution series of a standard protein mixture (LMW protein solution contained 73.5 #g O A / 2 8 9 txg (25% w / w ) total protein/ml solution). 50 #1 of serially diluted LMW proteins were bound to 96-well plates and detected using anti-OA supernatant. Background cpm values were estimated with control antibody 03B3 diluted 1/10. The test plates were sequentially incubated with rabbit anti-mouse sera 1/500 and staphylococcal protein A labelled with 1251 (mean c p m ± S E M is indicated, n = 2).
223 a
O u.
o9 O kl.
BILA
b
==
RIA [] [] • •
m
0 0.005 o O "o
'
[]
Anti-OA
undiluted
50
o
[]
O3B3
undiluted
0.05
O
0.5
~
5.0
...........
I
,
"6 ""
.
.
.
.
.
.
/ well / well well / well
'
500
T=
50 50 0
O3B3, 50 ug protein Anti-OA, 0.5 ug protein Anti-OA, 5 ug protein / Anti-OA, 50 ug protein
5000
.
0~
5000
i
0
10000
20000
o~ o c
0
200
400
600
c
Antigen
Z
binding
(CPM)
Antibody
binding
(CPM)
Fig. 3. Estimation of the amount of ovalbumin (OA) present in a complex protein mixture that was detectable by the BILA or standard RIA assay, a: Samples of FCS admixed with small amounts of OA were labelled with NHS-d-biotin (materials and methods section) and assayed. Undiluted culture supernatant from the anti-OA hybridoma was used as the capture antibody and 50 /~g of biotin-labelled proteins were added to each well. Bound antigen was detected using streptavidin labelled with 1251. As a background control the monoclonal 03B3 of the same isotype was used. b: Results of a standard RIA assay of small amounts of OA admixed to FCS. 50 ~1 containing 50, 5, or 0.5 #g of total protein including various amounts of OA admixed to FCS were bound to 96-well plates and detected usinganti-OA supernatant. Background cpm values were estimated with the control antibody 03B3. The test plates were sequentially incubated with rabbit anti mouse sera and staphylococcal protein A labelled with 125I (mean cpm + SEM is indicated, n = 2).
detergent solution when performing assays on cell membrane antigens. Selective binding of biotinylated molecules present in extracts of normal and neoplastic cells was observed with monoclonal antibodies of the appropriate specificity (Fig. 5). Using spleen cell extracts binding was observed with antibodies showing specificity for immunoglobulin, M H C and the CD5 T cell marker. Weak binding to the
leucocyte marker-specific antibody OX-1 was also observed. In colon tumour extracts molecules could be detected which reacted with antibodies to M H C class 1, p53 and some of the monoclonals which by immunohistological techniques had been shown to bind to colon tumour sections. When using rat tissue preparations containing rat immunoglobulin care was taken to ensure that all the rabbit anti-rat and mouse Ig binding sites
a LMW proteins
b Lymphoma
DOC+TX+SDS
Doc+TX+SDS
BSA DOC Tween Triton
20
AntI-OA
[]
O3B3
10000
Antigen
binding (CPM)
20000
I
[ ] HB 60 •
DOC Tween
Triton
X-100
I
r
BSA
•
cell extract
OX-18
20 X-100
10000
20000
Antigen binding (CPM)
Fig. 4. Influence of detergents on the antigen binding and the background cpm values in the BILA. Both the biotin-labelled LMW protein preparation and the biotin-labelled cell ( W / F u G1) extract were diluted in 10 mM Tris-HC1, pH 8.1 containing: 0.5% v / v Triton X-100 (Triton X-100), 0.5% v / v Tween 20 (Tween 20), 0.5% w / v sodium deoxycholate (DOC), 1% w / v bovine serum albumin (BSA) or 0.5% ( v / v ) Triton X-100 0.5% w / v sodium deoxycholate 0.05% SDS (DOC-TX-SDS). a: Monoclonal anti-OA 1/100 and background control antibody 03B3 1/100 were used as capture antibodies with 1/10 dilutions of LMW protein as biotin-labelled antigen, b: Monoclonal antibodies OX-18 (1/100) against rat MHC class 1 and control antibody HB60 1/1130 were used as capture antibodies with 1 / 1 0 dilutions of biotinylated W / F u G1 cell membrane extracts (mean cpm _.+SEM is indicated, n = 2).
224 A
Rat s p l e e n
cell
extract
MARK-1 ' HB
B Rat DMH W49 C a r c i n o m a e x t r a c t o1
H
.~_
ox-6
Medium
"1o O .O
60
)
ox-19
~
p53
-~
Ox-18
ox-1
c o
ox-18
o t-
H
O3B3
O 2000
4000
Antigen
6000
8000
binding
10000
(CPM) C
._~ "o O .Q
i 1000
12000
Antigen Rat BN 7005 c a r c i n o m a
i 2000
binding
i 3000
4000
(CPM)
extract
Q12 7D8
m ~
"E 10HS 10B12 tO
1Cll
O
1F6
O
1G6
H i
•
2000
i
4000
Antigen
,
i
•
6000
i
8000
binding
•
i
10000
,
12000
(CPM)
Fig. 5. BILA assays with biotinylated membrane extracts of rat cells using rabbit anti-mouse Ig antibodies as a primary capture antibody and a n u m b e r of different monoclonal antibodies. Before the addition of biotinylated antigen residual binding sites for rat Ig on the primary capture antibody were blocked using 50 /~1 normal rat serum diluted 1 / 2 0 in PBS and incubated for 15 min. a: Mouse monoclonal antibodies (ascites diluted 1/100) as secondary capture antibodies tested against an extract of biotinylated spleen cell membranes, b: Mouse (ascites diluted 1/100) and rat (undiluted hybridoma culture supernatant) monoclonal antibodies as secondary capture antibodies tested against an extract of biotinylated W49 colon tumor cell membranes, c: Rat monoclonal lgM anti-tumour antibodies as secondary capture antibodies exemplifying screening tests against biotinylated m e m b r a n e proteins from BN7005 colon carcinoma tumour. Fresh hybridoma culture medium was used undiluted (mean cpm_+ SEM is indicated, n = 2).
were blocked by incubating the wells for 15 min with normal rat serum diluted 1 / 2 0 in RPMI. Both non-biotin-labelled antigen preparations and free antibody were used to inhibit the observed binding and confirm the specificity of each test system (Fig. 6).
Discussion
When producing monoclonal antibodies to cell membrane-associated proteins there is a need for a technique that can detect monoclonal antibodies binding to proteins present in complex mixtures. Very often the protein of interest is not yet characterized and is merely a minor component of the plasma cell membrane. A detergent-solubilized
preparation of plasma membranes from the cells will not only contain nonrelevant plasma membrane molecules but frequently proteins from the cytoplasm and nucleus also. Typical R I A or ELISA assays are based on adherence of a protein solution to a solid phase. The adsorbing area and the protein binding capacity of the solid phase or the crosslinking reagents imposes restrictions on the efficiency of these techniques. When making use of labelled antigens instead of labelled antibodies and particularly when cell surface labelling is being used, the above problems should theoretically be minimized. The total amount of target protein is no longer a limiting step and it should be possible to raise the total protein content until reasonable signal to background cpms are registered. Indeed our experi-
225
Inhibition assay HB60 Ox-18
a 1/10
I
Spleen BSA
1
anti-OA
1/10
anti-OA
1/2
I
b I ]
CA O3B3 BSA
1/2 i
20
Per
i
40
cent
i
i
60
80
100
inhibition
Fig. 6. Inhibition assay. Binding inhibition assays were performed with rabbit anti-mouse as primary capture antibody, a: As secondary capture antibodies OX-18 ascites diluted 1/100 were used. Biotinylated extracts of spleen cell membranes corresponding to 12 500 cells/well were incubated with medium containing BSA (BSA), a spleen cell preparation of 1.5 x 106 viable spleen cells (spleen) or monoclonal antibody OX-18 ascites diluted 1 / 1 0 (OX-18 1/10) or monoclonal antibody HB60 ascites diluted 1 / 1 0 (HB60). Before the addition of biotinylated antigen residual binding sites for rat Ig on the capture antibody were blocked using 50 #1 normal rat serum diluted 1 / 2 0 in PBS and incubated for 15 min. b: As a secondary capture antibody anti-ovalbumin diluted 1 / 1 0 was used. Biotinylated L M W proteins (37 ng OA/well) were incubated with ovalbumin 50 ~ g / w e l l (OA), bovine serum alb u m i n 50 /tg/well (BSA) anti-OA supernatant diluted 1 / 2 or 1 / 1 0 (anti-OA 1/2, 1 / 1 0 ) and 03B3 supernatant diluted 1 / 2 (03B3 1 / 2 ) (mean cpm + SEM is indicated, n = 2).
ments suggested that this was so in our model system and small amounts of ovalbumin could be detected in the presence of a vast excess of other proteins. A positive reaction was seen in protein mixtures containing ovalbumin in as small a proportion as 0.5 n g / 5 0 #g total protein compared to 0.5 fig/50 /tg sample when using an ordinary solid-phase assay (Figs. 3a and 3b). The necessary amount of biotin-labelled antigen used when screening antibodies with the BILA assay is very difficult to predict and therefore it is advisable to use as much antigen as possible. However, as little as a few n g / m l of the relevant antigen present in a large excess of other proteins could be detected with the anti-OA monoclonal antibody (Fig. 1). The upper limit of total antigen protein that could be used was indicated by an experiment in which it was shown that the biotin-labelled antigen concentration could be increased from 1 0 0 / ~ g / m l (as
used in the experiment described in Fig. 5c) to 1 m g / m l without increasing the specific binding of the antibodies tested (data not shown). The binding and precipitation of adhesive, proteins is always a problem when working with membrane proteins. However, the inclusion of detergents and a centrifugation step immediately before the assay of frozen and thawed preparations, minimizes the background problems. The BILA assay is probably not suitable for testing serum antibodies since the proportion of specific antibodies among serum immunoglobulins is comparatively low. The anti-OA monoclonal antibody (9.4 ffg/ml) diluted 1 / 1 0 0 showed only a small decrease in the binding of labelled antigen (Fig. 2a). Thus the amount of antibody bound to each well could be estimated to within a few nanograms. This amount showed some variation dependent on the immunoglobulin subclass of the monoclonal antibody and variations in the capacity of the capture antibodies to bind the subclass in question. The NHS-d-biotin labelled most proteins satisfactorily but other biotinylating reagents can be used if preferred (Billingsley et al., 1987). The biotin reagents solubilized in D M S O are particularly useful, since they can selectively label cell surface molecules (Hurley et al., 1985). Labelled cells ( W / F u G1) show high viability (95-100% as measured by trypan blue exclusion) and their viability does not seem to be affected by exposure for 1 h to 10% D M S O and 3 mg N H S - d - b i o t i n / 5 x 106cells in 0.5 ml PBS. However, 20 rain exposure at room temperature, 0.5 mg N H S - d - b i o t i n / 5 x 1 0 6 cells and 0.5 ml PBS were used as standard conditions. The NHS-d-biotin ester is a non-toxic reagent that is easy to handle and can, if necessary any, be stored in frozen aliquots for at least 6 months. When labelling antigens there is always a risk of denaturation but the same risk must be faced when antigens are bound to a solid surface. We have no data to exclude the possibility that the streptavidin and monoclonal antibody binding sites may sometimes be located so close to each other that steric hindrance or displacement of binding could take place. However, since all of the tested monoclonal antibodies to known biotinlabelled antigens have been used successfully in
226 t h e B I L A a s s a y s u c h i n t e r f e r e n c e d o e s n o t s e e m to occur frequently. T h e use o f s o l u b i l i z e d cell m e m b r a n e s d e m a n d s the use of d e t e r g e n t s in t h e a n t i b o d y - a n t i g e n b i n d i n g step. T h i s m a y c a u s e p r o b l e m s since d e t e r g e n t may interfere with the antigen-antibody binding. N o s u c h c o m p l i c a t i o n w a s o b s e r v e d in o u r e x p e r i ments. In RIA/ELISA procedures commonly used for s c r e e n i n g m o n o c l o n a l a n t i b o d i e s , t h e r e is also a n e e d for the p r e s e n c e of s o m e d e t e r g e n t to e l i m i n a t e n o n - s p e c i f i c b i n d i n g o f the a n t i b o d y to t h e p l a s t i c surface, p a r t i c u l a r l y w h e n t h e a n t i b o d y is o f the I g M i s o t y p e ( E m m e r i c h , 1984). T h e b i o t i n - l a b e l l e d a n t i g e n test is a useful a n d o f t e n a m o r e s e n s i t i v e a l t e r n a t i v e to o r d i n a r y s c r e e n i n g p r o c e d u r e s w h i c h m a k e use o f a n t i g e n a d s o r b e d o n a solid p h a s e . It gives a d d i t i o n a l i n f o r m a t i o n a b o u t the v a l u e of t h e a n t i b o d y a n d selects t h o s e a n t i b o d i e s t h a t are likely to b e u s e d for a f f i n i t y c h r o m a t o g r a p h y . T h e b i o t i n l a b e l l i n g o f a n t i g e n s is also a p o t e n t i a l l y v a l u a b l e t e c h n i q u e for m o n i t o r i n g t h e p u r i f i c a t i o n o f m e m b r a n e antigens without labelling more than one reagent with isotope.
Acknowledgements W e t h a n k L o t t a R o l a n d e r a n d Pia KarlstriSm for t h e i r e x p e r t t e c h n i c a l assistance. T h i s w o r k was s u p p o r t e d b y t h e S w e d i s h M e d i cal R e s e a r c h C o u n c i l , J o h n a n d A u g u s t a P e r s s o n s Foundation, and the Medical Faculty, University of Lund.
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