Rapid preparation of peroxidase: Anti-peroxidase complexes for immunocytochemical use

Journal oflmmunological Methods, 20 (1978) 317--324 © Elsevier/North-Holland Biomedical Press

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RAPID P R E P A R A T I O N OF P E R O X I D A S E : A N T I - P E R O X I D A S E COMPLEXES F O R IMMUNOCYTOCHEMICAL USE

D.Y. MASON and RITA SAMMONS University o f Oxford, Department of Pathology, Radcliffe Infirmary, Oxford, U.K.

(Received 8 June 1977, accepted 19 October 1977)

Soluble immune complexes of horseradish peroxidase and antibody to peroxidase (PAP) have been widely used in the 'unlabelled antibody' method for the immunocytochemical detection of cellular antigens. This paper describes a simple and rapid method for preparation of these complexes by column chromatography of a mixture of the enzyme and the IgG fraction of antiperoxidase antiserum on Sephacryl S-200. The material eluting in the void column consists of stable soluble PAP complexes, with a molar peroxidase :antiperoxidase ratio of 0.8 and a molecular weight of approximately 400,000. When tested immunocytochemically this material gives identical results to those obtained with conventionally prepared PAP.

INTRODUCTION The first techniques to be developed for labelling antibodies with horseradish peroxidase (HRP) involved the use of bifunctional covalent coupling agents such as glutaraldehyde or difluoro-dinitro-diphenyl-sulphone (Avrameas and Uriel, 1966; Nakane and Pierce, 1966). These techniques suffered f r o m the disadvantage that coupling was relatively inefficient (Nakane and Kawaoi, 1974), a drawback which p r o m p t e d the devel opm ent of 'unlabeled a n t i b o d y ' i m m u n o p e r o x i d a s e m e t hods (Mason et al., 1969; Sternberger and Cuculis, 1969) in which no chemical modification o f the ant i body or e n z y m e is required. These methods, when used for the immunohistological detection o f antigens, involve the f or m a t i on o f a 'sandwich' of (1) specific antiserum (raised in species A) to the antigen being sought; (2) antiserum (raised in species B) to Ig f r o m species A; (3) antiserum (raised in species A) to HRP; and (4) purified HRP. The second antiserum (anti-Ig o f species A) acts bivalently as a bridge between antisera 1 and 3. A potential disadvantage to unlabelled ant i body m et hods is that if the anti-HRP in stage 3 is applied in the form o f whole antiserum (or the IgG fraction th er eo f ) t he binding by antiserum 2 of IgG specific for HRP is competitively reduced by the presence of excess non-specific IgG in stage 3. This may be circumvented by the use of a n t i b o d y to HRP which has been purified by absorption and elution on a solid phase immuno-absorbant of HRP (Sternberger and Cuculis, 1969). However, for reasons which are n o t clear

318 recovery of specific antibody from such immuno-absorbants is poor (Petrali et al., 1974). This problem led Sternberger et al. (1970) to develop a procedure for preparing soluble complexes of HRP and anti-HRP (peroxidase: anti-peroxidase complexes, or PAP) in which little or no non-enzymecomplexed IgG is present. The use of PAP for immunohistochemical staining not only offers greater sensitivity than conventional immunoperoxidase techniques (Petrali et al., 1974) but is simpler than the original unlabelled antibody m e t h o d in that stages 3 and 4 (see below) are reduced to a single step. The practical value of PAP is attested by the numerous reports on its use which have appeared in recent years covering a wide variety of antigens (for references see Wendelschafer-Crabb et al., 1976). The preparation of PAP is a multi-step process requiring time and a degree of immunochemical expertise. In the present report a simplified and rapid procedure for the preparation of this useful immunohistochemical reagent is described, based upon the chromatographic separation of mixtures of antiHRP and HRP. MATERIALS AND METHODS

A n tisera Rabbit anti-human lactoferrin, swine anti-rabbit IgG, rabbit anti-peroxidase and PAP were obtained from Dakopatts A/s, Copenhagen. Goat antiHRP and goat anti-rabbit IgG were obtained from Miles Laboratories. A rabbit antiserum to HRP was also prepared in this laboratory by a conventional immunisation schedule. An IgG fraction of this serum was obtained by chromatography on DEAE cellulose and PAP complexes were then prepared according to Sternberger et al. (1970).

Horseradish peroxidase (HRP) This enzyme was obtained from Boehringer (Grade I).

Immunoprecipitation Equivalence ratios for anti-peroxidase antisera were determined by incubating 25 pl aliquots of antiserum with a range of dilutions of HRP in 0.15 M pH 7.2 phosphate buffered saline (PBS). After one hour at room temperature the precipitates which formed were washed in cold PBS, dissolved in 0.2 M NaOH and their optical densities read at 280 mt~. The L-plate m e t h o d of Allison and Humphrey (1960) was used to determine the diffusion constant of PAP (see Sternberger et al., 1970).

Assay of peroxidase The substrate, which was freshly prepared before each assay, consisted of 120 ml of PBS (pH 5.0), containing 1.0 ml of 1% o-dianisidine in absolute

319 m e t h a n o l a n d 1.2 ml o f 0.3% HzO2. A l i q u o t s o f s a m p l e s t o be a s s a y e d were a d d e d to 2.9 ml o f s u b s t r a t e a n d t h e r e a c t i o n f o l l o w e d at 30°C and 460 m y in a U n i c a m S P 8 0 0 0 r e c o r d i n g s p e c t r o p h o t o m e t e r . O n e u n i t o f a c t i v i t y corr e s p o n d s t o an O.D. increase o f 0.1 p e r m i n u t e .

Chromatographic preparation of PAP complexes In o r d e r t o p r e p a r e PAP c o m p l e x e s an a l i q u o t o f r a b b i t a n t i - H R P was i n c u b a t e d f o r 1 h at r o o m t e m p e r a t u r e w i t h an excess o f H R P (three to t e n fold). T h e m i x t u r e was t h e n c e n t r i f u g e d f o r 3 m i n in an E p p e n d o r f 5412 b e n c h t o p c e n t r i f u g e , and t h e s u p e r n a t a n t applied t o an e q u i l i b r a t e d c o l u m n o f S e p h a c r y l S-200 S u p e r f i n e ( P h a r m a c i a ) at 4°C w i t h p H 7.2 PBS as eluting b u f f e r . T h e o p t i c a l d e n s i t y o f f r a c t i o n s was m e a s u r e d at 2 8 0 m~ ( f o r p r o t e i n ) and 4 0 0 m g ( f o r H R P ) . F r a c t i o n s c o n t a i n i n g PAP c o m p l e x e s were p o o l e d , c o n c e n t r a t e d b y v a c u u m dialysis or in A m i c o n d i s p o s a b l e c o n c e n t r a t o r s and s t o r e d at 4°C. In s e p a r a t e e x p e r i m e n t s H R P a l o n e and a n t i - H R P alone was passed d o w n t h e s a m e c o l u m n and f r a c t i o n s assayed as o u t l i n e d above. B o t h c o m m e r c i a l a n t i - H R P ( D a k o ) a n d a n t i - H R P p r e p a r e d in this l a b o r a t o r y gave identical results.

Immunocytochemical staining T h e PAP p r e p a r a t i o n s o b t a i n e d b y c o l u m n c h r o m a t o g r a p h y w e r e t e s t e d in t h e s a n d w i c h t e c h n i q u e f o r the d e t e c t i o n o f l e u c o c y t e l a c t o f e r r i n in s m e a r s o f h u m a n p e r i p h e r a l b l o o d (Mason et al., 1 9 7 5 ) and o f i m m u n o g l o b u l i n in p a r a f f i n sections o f a H o d g k i n ' s disease l y m p h n o d e w h i c h c o n t a i n e d n u m e r o u s Ig p o s i t i v e p l a s m a cells and r e t i c u l u m cells ( T a y l o r , 1976). In o r d e r to q u a n t i t a t i v e l y c o m p a r e c h r o m a t o g r a p h i c a l l y p r e p a r e d PAP

TABLE 1 Comparison of immunocytochemical potency of conventionally and chromatographically prepared PAP. The two PAP preparations were tested on peripheral blood smears in conjunction with antilactoferrin antiserum (see Methods). The intensity of neutrophil cytoplasmic staining was scored on a -- to +++ scale. Dilution of PAP

PAP 1 PAP 2

1/400

1/1600

1/6400

1/25600

+++ +++

++/+++ ++

+ -+

---

PAP 1 = chromatographic PAP. Enzymatic activity 115,800 units/ml. PAP 2 = conventional PAP (Dako). Enzymatic activity 92,400 units/ml.

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with conventional PAP, different dilutions of the two samples were used in staining a batch of blood smears for lactoferrin. The concentration of the other antisera and the time of development of the peroxidase reaction were kept constant for all slides. The enzymatic content of the two PAP samples before dilution was assayed and shown to be of comparable strength (table 1). RESULTS

Mixtures of HRP and anti-HRP when chromatographed on Sephacryl S-200 (fig. 1) gave a peak of protein and HRP eluting in the void volume (Vo), followed by a second protein peak (at 1.25 X Vo) representing uncomplexed IgG, and a third peak of HRP and protein (at 1.5 × Vo) corresponding to free enzyme. The same yield of material in the first peak was obtained when different ratios of HRP to anti-HRP (from three to ten fold antigen excess) were used, although the magnitude of the peak of free enzyme increased in proportion to the degree of antigen excess. The first peak, when concentrated and tested by Ouchterlony immunodiffusion, gave precipitin lines (fig. 2) against both anti-rabbit IgG and antiHRP. These lines showed a reaction of identity, although in some prepara-

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Fig. 1. C h r o m a t o g r a p h i c s e p a r a t i o n o f a m i x t u r e of HRP and a n t i - H R P o n a Sephacryl $ 2 0 0 S u p e r f i n e c o l u m n ( 9 0 x 2.2 cm). T h e void v o l u m e ( V o ) and the e l u t i o n p o s i t i o n s o f IgG a n d o f HRP (determined during c a l i b r a t i o n o f t h e c o l u m n ) are indicated. N o t e t h a t the a n t i - H R P u s e d in all s e p a r a t i o n s was a n IgG f r a c t i o n , a n d t h u s c o n t a i n e d n o m a t e r i a l eluting in a d v a n c e o f t h e IgG peak.

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Anti-IgG PAP 19 Anti-Px

-q$ Anti-IgG PAP 29 Anti-Px Fig. 2. Immunoprecipitation of PAP complexes prepared chromatographically (PAP 1) and by conventional methods (PAP 2) against goat anti-rabbit IgG and goat anti-HRP (anti-Px). PAP 2 was obtained from Dakopatts A/s. tions a faint spur towards the anti-HRP well indicated the presence of a small a m o u n t of non-enzyme-complexed IgG in addition to the major c o m p o n e n t of PAP complexes. This pattern may be compared with that obtained when PAP prepared by the conventional technique is tested against the same antisera (fig. 2). A line of partial identity is also formed, but a prominent spur towards the anti-IgG well indicates the presence of substantial amounts of free HRP. The molar ratio of HRP to anti-HRP in the PAP complexes was 0.8 (tak~.1~ of 19.6 and a RZ value of 3.0 for HRP, and an -2s0 ~1~ of 14.3 for ing an -400 anti-HRP). The enzyme activity was 43.5% of that expected on the basis of its absorption at 400 mp, indicating a degree of enzyme inhibition by antibody. An estimate of the molecular weight was obtained by measuring the diffusion constant (2.57). This is slightly higher than the value of 2.48 reported by Sternberger et al. (1970). Since the latter authors estimated the molecular weight of their PAP preparation at 440,000, it suggests a slightly lower molecular weight for the chromatographically prepared PAP, i.e. of the order of 400,000. PAP complexes isolated by gel chromatography were tested immunocytochemically and gave intense cytoplasmic staining when used in conjunction with anti-lactoferrin and anti-Ig antisera. When the two types of PAP were

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tested in parallel over a range of dilutions (table 1) similar titres were obtained. Chromatographically prepared PAP complexes were retested by Ouchterlony immunodiffusion and in the immunocytochemical system after a period of three m o n t h s at 4°C in order to assess their stability on storage. There was no evidence in either test of deterioration during this time. DISCUSSION

The technique described above yields soluble complexes of horseradish peroxidase and anti-horseradish peroxidase suitable for use in unlabelled antibody methods for antigen localisation. This m e t h o d of PAP preparation has several advantages over the conventional m e t h o d described by Sternberger et al. {1970). It is considerably simpler and faster, an aspect emphasized by the comparative summary of the two techniques in table 2. Not only are the individual steps simpler than those of the original technique (in which care is needed both when acidifying and neutralising the complexes to avoid excessive swings in pH) but the total

TABLE 2 C o m p a r i s o n of t w o t e c h n i q u e s for t h e p r e p a r a t i o n of PAP. S t e r n b e r g e r et al. ( 1 9 7 0 )

Chromatographic technique

1 I n c u b a t i o n of H R P a n d a n t i - H R P at equivalence proportions

1 I n c u b a t i o n of H R P a n d a n t i - H R P in a n t i g e n excess

2 C e n t r i f u g a t i o n to s e p a r a t e i n s o l u b l e PAP c o m p l e x e s

2 C e n t r i f u g a t i o n to r e m o v e insoluble PAP

3 Washing of c o m p l e x e s

3 A p p l i c a t i o n of s u p e r n a t a n t to Sephacryl column

4 S u s p e n s i o n in H R P s o l u t i o n

4 I d e n t i f i c a t i o n of first p r o t e i n peak

5 S o l u t i o n b y a d j u s t m e n t of pH t o pH 2.3

5 C o n c e n t r a t i o n of peak

6 N e u t r a l i s a t i o n to pH 7.4 7 P r e c i p i t a t i o n of P A P w i t h s a t u r a t e d ammonium sulphate 8 Washing w i t h 50% s a t u r a t e d a m m o n i u m sulphate 9 S o l u t i o n of insoluble c o m p l e x e s in distilled w a t e r 10 Dialysis against a c e t a t e b u f f e r 11 C e n t r i f u g a t i o n to r e m o v e insoluble material Time r e q u i r e d :

4 days

Less t h a n 24 h o u r s

323 p r o c e d u r e takes less t h a n 24 h, in c o n t r a s t t o the f o u r days r e q u i r e d for S t e r n b e r g e r et al.'s m e t h o d . Because o f the g o o d flow characteristics o f S e p h a c r y l S-200 it is possible t o c o m p l e t e the entire process within a single working day. A c o m p a r i s o n o f the characteristics o f the t w o t y p e s o f PAP (table 3) indicates t h a t the c h r o m a t o g r a p h i c a l l y p r e p a r e d material has a p p r o x i m a t e l y half t h e H R P c o n t e n t o f the c o n v e n t i o n a l p r e p a r a t i o n , and a s o m e w h a t lower m o l e c u l a r weight (as e s t i m a t e d f r o m the relative diffusion constants). However t i t r a t i o n o f t h e t w o t y p e s o f PAP in parallel (table 1) shows no differe n c e in i m m u n o c y t o c h e m i c a l p o t e n c y . In a d d i t i o n to the practical c o n s i d e r a t i o n s n o t e d above the c h r o m a t o graphic m e t h o d o f f e r s t w o f u r t h e r advantages. Since an acidification step is n o t required the risk o f e n z y m e or a n t i b o d y d e n a t u r a t i o n is minimised. Seco n d l y the c o m p l e x e s o b t a i n e d b y c o l u m n c h r o m a t o g r a p h y are likely to be particularly stable since t h e y will c o n t a i n o n l y a n t i b o d y which is sufficiently avid to survive d i l u t i o n d u r i n g c h r o m a t o g r a p h y . S t e r n b e r g e r ( 1 9 7 4 ) suggests t h a t PAP c o m p l e x e s t e n d to dissociate o n dilution. This m a y a c c o u n t for the fact t h a t agar i m m u n o d i f f u s i o n testing o f c o n v e n t i o n a l l y p r e p a r e d PAP c o m p l e x e s (fig. 2) reveals the presence o f free e n z y m e , a finding c o n f i r m e d b y thin layer or c o l u m n gel c h r o m a t o g r a p h y (personal observations), whereas no free p e r o x i d a s e was p r e s e n t in t h e c h r o m a t o g r a p h i c a l l y p r e p a r e d PAP. T h e fact t h a t p r o l o n g e d storage at 4°C did n o t lead to d e t e r i o r a t i o n provides f u r t h e r evidence f o r the stability o f c h r o m a t o g r a p h i c a l l y p r e p a r e d PAP complexes.

TABLE 3 Comparison of immunochemical characteristics of the two types of PAP. The values given for the enzymatic activity of conventionally prepared PAP are from the original paper of Sternberger et al. (1970). Assay of commercial PAP in this laboratory gave a figure of 41% for enzymatic activity.

HRP : IgG molar ratio Enzymatic * activity Ouchterlony analysis Diffusion constant Molecular weight * Calculated from

Sternberger et al. (1970)

Chromatographic technique

1.5-- 1.7 38--98% IgG complexed to HRP, plus free HRP 2.48 440,000

0.8 43.5% IgG complexed to HRP 2.57 ? approx. 400,000

enzymatic activity per mole of HRP in PAP × 100 enzymatic activity per mole of HRP used for preparation of PAP

324 ACKNOWLEDGMENTS We are grateful t o Dr. T. Parry and to t h e D e p a r t m e n t o f Medical Illustration for assistance in t h e p r e p a r a t i o n o f figures, and t o Mrs. J o a n B r a i d w o o d for t y p i n g this m a n u s c r i p t . This w o r k is s u p p o r t e d b y t h e L e u k a e m i a Research F u n d . REFERENCES Allison, A.C. and J.H. Humphrey, 1960, Immunology 3, 95. Avrameas, S. and J. Uriel, 1966, C.R. Acad. Sci. (Paris) 262, 2543. Mason, D.Y., C. Farrell and C.R. Taylor, 1975, Br. J. Haematol. 31,361. Mason, T.E., R.F. Phifer, S.S. Spicer, R.A. Swallow and R.B. Dreskin, 1969, J. Histochem. Cytochem. 17,563. Nakane, P.K. and A. Kawaoi, 1974, J. Histochem. Cytochem. 22, 1084. Nakane, P.K. and G.B. Pierce, 1966, J. Histochem. Cytochem. 14,929. Petrali, J.P., D.M. Hinton, G.C. Moriaty and L.A. Sternberger, 1974, J. Histochem. Cytochem. 22,782. 8ternberger, L.A., 1974, Immunocytochemistry (Prentice-Hall, Englewood Cliffs, NJ) p. 140. Sternberger, L.A. and J.J. Cuculis, 1969, J. Histochem. Cytochem. 17,190. Sternberger, L.A., P.H. Hardy, Jr., J.J. Cuculis and H.G. Meyer, 1970, J. Histochem. Cytochem. 18, 315. Taylor, C.R., 1976, Eur. J. Cancer 12, 61. Wendelschafer-Crabb, G., S.L. Erlandsen and D.H. Walker, Jr., 1976, J. Histochem. Cytochem. 24,517.