Amplification of the biotin-avidin immunofluorescence technique

Amplification of the biotin-avidin immunofluorescence technique

Journal o f Immunological Methods, 36 (1980) 335--338 335 © Elsevier/North-Holland Biomedical Press A M P LI F I C ATI O N OF THE BIOTIN-AVIDIN IMM...

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Journal o f Immunological Methods, 36 (1980) 335--338

335

© Elsevier/North-Holland Biomedical Press

A M P LI F I C ATI O N OF THE BIOTIN-AVIDIN IMMU N O FL U O RE SCE N CE TECHNIQUE

JOAN W. BERMAN and ROSS S. BASCH Department o f Pathology, New York University Medical Center, 550 First Avenue, New York, N Y 10016, U.S.A.

(Received 22 April 1980, accepted 24 April 1980)

An amplification of the immunofluorescence technique which uses biotinylated antibody and fluoresceinated avidin is described. By introducing a sandwich technique using fluorescein~conjugated goat anti-avidin, a 5-fold enhancement of staining over the conventional immunofluorescence method is achieved, and the brightness is more than twice that achieved with the simple biotin-fluoresceinated avidin reaction.

INTRODUCTION The use of f l u o r o c h r o m e s to identify cell surface markers has served as an e x t r e m e l y i m p o r t a n t tool for delineating various cell populations. The conjugation o f a n t i b o d y to these dyes, a technique first developed by Coons et al. in 1941, affords a highly specific and direct means for localization o f sites of reaction of a n t i b o d y (Coons and Kaplan, 1950). Since bot h fluorescein and rhodamine, the two most c o m m o n l y used fluors, emit high intensity fluorescence, even trace am ount s of antigen-antibody complexes can be visually d etecte d. The 'indirect' m e t h o d of Mellors et al. (1955, 1959) renders the system m or e sensitive by introducing a second immunoglobulin, directed against the determinants of the primary a n t i b o d y , which is conjugated to the f l u o r o c h r o m e . This bridging effect significantly enhances the brightness o f the staining of specific sites. Background brightness, however, is also somewhat elevated. A triple layer assay can be used to furt her intensify the brightness of the staining, but it is c u m b e r s o m e and introduces yet a n o t h e r o p p o r t u n i t y for non-specific backgrounds. More r ecen tl y, Bayer and Wilchek (1974} have made use of the high affinity binding (10 -is Kin) of the egg white protein avidin to biotin (Green, 1963) to develop a new i m m u n o f l u o r e s c e n t labelling technique. Biotin is covalently coupled to a n t i b o d y using the h y d r o x y s u c c i n i m i d e ester. The derivatized a n t i b o d y is used f or primary staining and fluoresceinated avidin is used as the second reagent. Since more f l u o r o c h r o m e can be coupled to Address all correspondence to R.S. Basch, M.D.

336 avidin (without loss of its ability to bind to biotin) than to immunoglobulin, this system results in very bright, specific staining yet has little of the background problems inherent in the other methods. We now report a further amplification of the biotin-avidin system. Using a sandwich technique of biotinylated antibody and uncoupled avidin followed by a fluoresceinated or rhodaminated goat anti-avidin immunoglobulin, we can achieve a 3-fold enhancement in brightness over the conventional indirect immunofluorescence. If a fluorochrome-conjugated avidin intermediate is used, we attain a 5-fold enrichment. Using this amplified system, we can now identify labelled antigenic sites that were previously too dim to perceive, as well as intensify any already apparent brightness. MATERIALS AND METHODS Rabbit anti-mouse brain antiserum (RaMB) was prepared according to the m e t h o d of Golub (1972). The IgG portion was obtained by fractionating the 40% a m m o n i u m sulfate precipate on a Sephacryl S-200 column (2.5 cm × 100 cm, Pharmacia) equilibrated with PBS-0.1% NAN3. The IgG fraction of normal rabbit serum (NRS) was similarly obtained, and both antisera were subjected to limited digestion with pepsin to yield F(ab)'2 fragments (Nisonoff et al., 1960). The hydroxysuccinimide ester of biotin (Sigma) was prepared according to the m e t h o d of Becker and Wilchek (1972). Two mg of the ester in 100 pl DMSO were added to 1 ml of each a n t i b o d y (1 mg/ml) in 0 . 1 M Na borate, pH 8.5, and stirred at 4°C for 10 min. The biotinylated antibody was then separated from the solvent by chromatography on a Sephadex G-50 Fine column (1 cm × 14 cm, Pharmacia) equilibrated in PBS-0.1% NAN3. For the immunofluorescence studies, a Thy-1 negative, stem-cell antigen (SC-1) positive, cloned t u m o r line of RL(fl-4, 4.2.12, was used (Basch et al., 1980). Two X 106 cells were incubated for 15 min at 4°C with 60 pl of either biotinylated or unbiotinylated RaMB or NRS (21 ~g immunoglobulin}. The samples were then washed three times with PBS-I% BSA-0.1% NAN3. Those that had been stained with unbiotinylated antisera were then incubated with 60 t~l of an F(ab)'2 goat anti-rabbit immunoglobulin (garIg; Cappel Laboratories) at a dilution of 1 : 50, while the cells that were treated with biotinylated reagents received 60 pl of either avidin or fluoresceinated avidin (fl-avidin; Vector Laboratories) at a final concentration of 1 : 100 (600 ng). After 15 min at 4°C, the cells were again washed 3 times, and the biotinylated samples were fixed with 4% formaldehyde. Some samples which had received biotinylated a n t i b o d y and fluoresceinated avidin were also fixed, while all others were treated with a third reagent, fluoresceinated goat anti-avidin (fl-gaav) at a I : 50 dilution (Cappel Laboratories). After an additional 15 min in the cold, the cells were washed and fixed. All samples were analyzed in a FACS-II flow microfluorimeter (Becton and Dickinson, Fullerton, CA) using the 488 nm emission of the 2 W argon laser operating at 580 V and 200 mW.

337 RESULTS T h e d a t a are p r e s e n t e d in Fig. 1 a n d in T a b l e 1. T h e abscissa in t h e figure is a linear scale o f f l u o r e s c e n c e i n t e n s i t y (in a r b i t r a r y units). T h e n e t increase in i n t e n s i t y (specific a n t i b o d y m i n u s n o r m a l r a b b i t s e r u m ) is s h o w n in t h e first c o l u m n o f t h e t a b l e . Full a m p l i f i c a t i o n increased t h e e f f e c t i v e yield 5-fold w h e n c o m p a r e d to c o n v e n t i o n a l i n d i r e c t i m m u n o f l u o r e s c e n c e a n d m o r e t h a n d o u b l e d it if c o m p a r e d t o t h e simple b i o t i n - f l u o r e s c e i n a t e d avidin r e a c t i o n . T h e s e results are n o t a l t e r e d b y s u b s t i t u t i n g t h e m e a n f l u o r e s c e n c e i n t e n s i t y o f e a c h s a m p l e f o r t h e m o d a l value used in T a b l e 1. T h e results can also be c a l c u l a t e d in t e r m s o f t h e r a t i o o f specific to n o n - s p e c i f i c fluorescence. T h e s e are p r e s e n t e d in t h e s e c o n d c o l u m n o f t h e Table. A l t h o u g h fl-gaav increases t h e b r i g h t n e s s o f t h e specific staining, it also increases t h e b r i g h t n e s s o f t h e n o n - s p e c i f i c c o n t r o l , so t h a t w h e n this r e a g e n t is used w i t h u n c o n j u g a t e d avidin t h e r a t i o o f specific to n o n - s p e c i f i c f l u o r e s c e n c e is o n l y

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Fig. 1. Histogram of the fluorescent staining of SC-1 positive cells by RaMB. For each panel, the continuous line represents specific staining, while the dotted line represents staining with normal rabbit serum. The specific antibody used for each panel is as follows. A , R a M B + fl-garIg; B, Biotinylated R0~MB + fl-avidin; C, Biotinylated RaMB + avidin + fl-gaav; D, Biotinylated RaMB + fl-avidin + fl-gaav. Full scale of the ordinate is 1000 cells for the specifically stained sample but 4000 cells for the control.

338 TABLE 1 QUANTIFICATION OF THE EFFECTS OF AMPLIFICATION ON THE STAINING OF RLC;1-4.2.12 CELLS The A fluorescence is calculated as the difference between the modal fluorescence intensity obtained with specific antiserum and that with the normal rabbit serum. The data are also presented as the ratio of the modal fluorescence intensities

A . R ~ M B + gl-g~rIg B. Biotinylated RaMB + fl-avidin C. Biotinylated RaMB + avidin + fl-g~av D. Biotinylated R(~MB + fl-avidin + gl-g(~av

A fluorescence intensity

Ratio of specific to non-specific staining

11 23.5 34 57

2.0 3.5 2.8 4.4

2.8. This increase in b a c k g r o u n d staining is m o r e t h a n c o m p e n s a t e d for in the fully amplified s y s t e m (fl-avidin + fl-gaav) w h e r e the ratio greatly e x c e e d s t h a t in a n y o t h e r sample. The data p r e s e n t e d here have been c h o s e n to illustrate q u a n t i t a t i v e l y the e f f e c t o f the a m p l i f i c a t i o n s y s t e m . O b v i o u s l y , n o a m p l i f i c a t i o n is necessary to d e m o n s t r a t e t h e r e a c t i v i t y o f this s e r u m with the cells used. We have used the a m p l i f i c a t i o n to s t u d y several antigens w h o s e r e p r e s e n t a t i o n o n the cell surface is far m o r e sparse t h a n SC-1 o n R L ~ I - 4 . In these experim e n t s , a m p l i f i c a t i o n m a k e s the d i f f e r e n c e b e t w e e n success and failure in t h e a t t e m p t to i d e n t i f y antigen positive cells. ACKNOWLEDGEMENTS This w o r k was s u p p o r t e d b y G r a n t C A 2 4 4 7 2 o f the N I H , USPHS. JWB was partially s u p p o r t e d b y Training G r a n t 5 T 3 2 CA 0 9 1 6 1 . The F A C S - I I was m a d e available to us t h r o u g h the g e n e r o s i t y of Drs. M a t t h e w S c h a r f f and D o n a l d Marcus o f the A l b e r t Einstein College o f Medicine. We a p p r e c i a t e Dr. William R o t h m a n ' s assistance a n d R a n d a Klein's help with the preparat i o n o f this m a n u s c r i p t . REFERENCES Baseh, R.S., T. Panagiotatos, J.W. Berman and J. Buxbaum, 1980, J. Cell Physiol., in press. Bayer, E. and M. Wilchek, 1974, Meth. Enzymol. 34,365. Becker, J.M. and M. Wilchek, 1972, Biochim. Biophys. Acta 264,165. Coons, A.H. and M.H. Kaplan, 1950, J. Exp. Med. 91, 1. Coons, A.H., H.J. Creech and R.N. Jones, 1941, Proc. Soc. Exp. Biol. 47, 200. Golub, E.S., 1972, d. Exp. Med. 136, 373. Green, N.M., 1963, Biochem. J. 89,585. Mellors, R.C., J. Aria-Stella and D. Pressman, 1955, Am. J. Pathol. 31,687. Mellors, R.C., R. Heimer, J. Corcosand and L. Korngold, 1959, J. Exp. Med. 110,875. Nisonoff, A., F.C. Wissler, L.N. Lipman and P.C. Woernley, 1960, Arch. Biochem. Biophys. 89,230.