- Email: [email protected]
Comparison of peroxidase and ferritin labelling of cell surface antigens
Experimental Cell Research 71 (1972) 145-155
COMPARISON OF PEROXIDASE AND FERRITIN LABELLING OF CELL SURFACE ANTIGENS R. BRETTON’,
T. TERNYNCK
and S. AVRAMEAS
Institut de Recherches Scientqiques sur le Cancer, 94-Villejuif, France
SUMMARY In a comparative study of peroxidase and ferritin used as labels for cell surface antigens, the following observations were made: 1. Peroxidase labelled more antigenic sites than did ferritin. The cell surface label was shown to be nearly continuous with peroxidase, whereas there were large discontinuities with ferritin. 2. Highly diluted peroxidase conjugate showed discontinuous labelling of the cell surface similar to undiluted ferritin conjugate. 3. When ferritin labelled conjugate is used in concentrations much higher than is usual, it labels the cell surface continuously as much as does a peroxidase-labelled conjugate. 4. Partial removal of the cell surface coat with hyaluronidase narrowed the distance between the ferritin conjugate and the unit membrane component of the cell surface, resulting in more extensive but still discontinuous labeling. As a result of these observations, it was concluded that: (A) The heavier labelling obtained with peroxidase conjugate than with ferritin conjugate was specific and not the result of diffusion. (B) The patchy ferritin labelling and the uneven labelling with diluted peroxidase suggests a variability in the distribution of cell surface antigens, but does not support the concept that antigens reside in widely separated circumscribed segments of the cell surface. (C) Peroxidase conjugate has the advantage over ferritin conjugate in being amuchsmallermolecule.
During the past twelve years, three techniques have been developed that are applicable to the study of cell surface antigens at the ultrastructural level. In 1959, Singer [20, 211 introduced ferritin as a labelling agent. In 1966, Avrameas et al. [5, 81 and Nakane & Pierce [18, 191independently introduced the technique of using enzyme-labelled antibodies, among them peroxidase. In 1968, Hlimmerling et al. [12, 131 modified the ferritin method by preparing a hybrid antibody. Bretton & Lespinats [lo] in 1969 used 1 Permanent address: Tufts Medical School, Department of Dermatology, 185 Harrison Avenue, Boston, Mass. 02111, USA. 10-721803
peroxidase labelled antibodies for the localization of cell surface antigens. At that time, a difference was observed between peroxidase labelling and the ferritin labelling reported in the literature. It was found that the deposition of peroxidase-antibody conjugate on the cell surface was continuous. This is in contrast to the deposition of ferritin-antibody conjugate which generally has been found to be present in patches with wide, intervening unlabelled zones. Although several authors, among them Nakane & Pierce [19] have expressedthe view that peroxidase is superior to ferritin as a label for intracellular antigens because of its smaller molecular weight, ferritin is still Exptl Cell Res 71
146 R. Bretton et al.
widely used for labelling cell surface antigens. In the present study, the effectiveness of peroxidase-labelled antibodies and of ferritinlabelled antibodies as markers of cell surface antigens is compared. The cellular systems were chosen for a single purpose: visualization of differences in cell surface labelling. Therefore species-specific antibodies were used since the corresponding antigens were more evenly distributed over most of the cell surface; a well defined immunological system. where only limited parts of the cell surface would be labelled, would have made interpretation of variations extremely difficult. Furthermore, there was no endogenous peroxidase or peroxidase-like activity on the cell surface, as is seenwith red blood cells. MATERIAL
AND METHODS
Cell systems Two cell systems were used: (a) cells of a solid tumour from a BALB/c mouse plasmacytoma originally induced by Bayol F rrineral oil [14] and transolanted bv subcutaneous iniection: (b) cultured cells of an established line of green ‘monkey kidney eoithelial cell culture (CVl) used between the 50th and 100th subculture ‘[15].’ Both cell systems were dissociated with 0.135 % trypsin (Choay Laboratory, crystallized and lyophilized) in PBS, which required 36-60 min for the plasmacytoma cells and 5-6 min for the kidney cell cultures. Even though it was realized that the brief exposure to trypsin removed parts of certain cell surface constituents [l], it represents the best method available for separating cells of solid tumours and good immunological reactions were still obtained.
Preparation and labelling of antibody Pure antibodies were used that were isolated from hyperimmunized animals. The isolation of the antibodies was accomplished with an immunoadsorbent prepared by copolymerization at pH 5 of bovine serum albumin and the corresponding antigen (4 : 1) using glutaraldehyde as the insolubilizing agent and following the batchwise procedure [q. For experiments (a) with mouse plasmacytoma cells, rabbit antibody antirat IgG was used; (b) with monkey kidney cell culture sheep antibody antirabbit IgG was used; (c) on the specificity of peroxidase staining sheep antiferritin antibody was used. Isolated antibodies were labelled with peroxidase, using glutaraldehyde as the coupling agent [4]. (The advantage of glutaraldehyde as a cross linking agent is due to coupling via free amino groups which does not actively participate in enzyme antibody activity.) Ferritin was-coupled to antibodies (a) using toluene diisocyanate according to Singer [20]; (b) using glutaraldehyde as the coupling agent. With the latter, the reactants were used in the following- quantities: _ 5 mg of antibodies, 10 mg of ferritin in 2 ml of phosohate buffer 0.1 uH 6.8 in which 0.05 ml of a 1 % aqueous solution of glutaraldehyde was added. After dialysis and centrifugation, the preparation was purified by ultracentrifugations according to the technique of Singer [21]. The labelled preparations were adjusted to the same concentration of 5 mg/ml. This concentration was determined in the case of ueroxidase-labelled antibodies, by assuming that no unlabelled antibody is present in- the preparation [4] and. in the case of ferritin-labelled antibodies, by measuring the amount of unconjugated antibody present in the supernatant after centrifugation at 40 000 g for 2 h and subtracting this amount from the total amount of antibody used. The adjusted conjugates were stored at 4°C. The activity of all the conjugates, at the same antibody concentrations, was determined by the passive hemagglutination technique, using sheep erythrocytes coated with rabbit IgG [6]. Both preparations were found to agglutinate coated erythrocytes at the same dilutions. Since the procedures used for the experiments on both cell systems were identical, they will be presented together.
Antisera
Expt 1: classical sandwich technique
(a) Rat antiserum BALB/c mouse plasmacytoma serum was prepared by hyperimmunizing Wistar rats [16]; (b) rabbit antimonkey kidney cell serum, supplied by R. Wicker, was obtained by injecting a total of 100 mg of CVl cell homogenate intramuscularly and intradermally into the foot-pad of adult rabbits. The procedure was repeated twice at intervals of 2 months. Complement was inactivated in both sera by incubating them at 56°C for 30 min; (c) rabbit antiserum anti-rat IgG was prepared following an immunization procedure already described [7]. Antirabbit IgG serum and anti-ferritin serum was prepared b; immunizing two sheep. A total of 300 mg of rabbit IgG and ferritin respectively, was used over a period of 6 months.
Susoensions of 5 x lo6 cells/O.3 ml were incubated with their respective antiserum for 15 min at 37°C. The cells were washed in PBS-PVG (ohosnhatebuffered saline containing 1 % polyvinyl~glycol, pH 7.4). and centrifuged at 1 400 ram. The washing and’centrifugation was repeated twice. The cells then were incubated for a period of 30 min at 37°C with either peroxidase-labelled or ferritin-labelled antibody. The labelled antibodies having an initial concentration of 5 mg/ml were diluted 1: 10, 1 : 50, 1: 250 and 1 : 500. In addition, ferritin-labelled antibody at a concentration of 10 mg/ml was used. Again the cells were washed and centrifuged three times in PBS-PVG in order to remove all unbound labelled antibody. Fixed in 2.5 % glutaraldehyde in
Exptl
Cell Res 71
Peroxidase and ferritin labelling SGrensen buffer at pH 7.4 for 15 or 60 min at 4°C. Washed four times for a total of 15 min in 5 ml of Siirensen buffer pH 7.4 and centrifuged after each washing at 1 500 rpm. The peroxidase-labelled cells were incubated with 3-3’ diaminobenzidine for 5, 10, 15 or 30 min at room temperature, in order to reveal the peroxidase activity [ll], washed three times in phosphate buffer, each followed by centrifuging.
Expt 2: Double labelling with ferritin and peroxidase To determine the relative effectiveness of each conjugate to detect antigen on the same cellular preparations, the following experiments were performed: cell suspensions were incubated with their antisera as in expt 1. They ,were then divided into two groups. One group was incubated in peroxidase-labelled antibody (0.5 mg/ml) followed by ferritin-labelled antibody (10 mg/ml). With the other group, we reversed the order of incubation, i.e. ferritin followed by peroxidase. Both groups were incubated with each labelled antiserum for 30 min at 37”C, washed after each incubation, then fixed with 2.5 % glutaraldehyde for 1 h at 4°C. The DAB reaction time was kept at 10 min to insure visualization of both markers.
Expt 3: Specificity of peroxidase (a) The cell suspensions were incubated with their antiserum and then with ferritin-labelled antibody (0.5 mrriml). as described in exut 1. Subsequently. they w&e incubated with sheep aitiferritin anjibod;; labelled with oeroxidase (fig. 7). Exoosure to 3-3’ diaminobenzidine (DAB) ‘was limited to 5 min in order to avoid masking of the ferritin by excessive formation of the electron-dense peroxidase DAB reaction product. (b) CVl cell suspensions were incubated first with their antiserum and subsequently with the peroxidaselabelled antibody (0.5 mgiml), as in expt 1. The cells were then fixed, as u&al,- with 2.5 % glutaraldehyde for 15 min at 4°C. Hamster embryo fibroblasts cell suspensions also were similarly fixed with glutaraldehyde. Both cell suspensions were washed four times -after fixation, mked with one another and olaced into a test tube and treated with DAB for is min.
Controls The following control incubations were carried out on the BALB/c mouse plasmacytoma cell suspensions and the CV 1 cell cultures: (1) Normal rat serum intead of the rat antimouse plasmacytoma serum; (2) solutions of normal rabbit IgG labelled with either ueroxidase or ferritin in olace of the resoectivelv labelled rabbit antibody a&rat IgG (co;?iugatek (3) incubation only with the peroxidase or ferritinlabelled rabbit conjugate; (4) incubation only with pure peroxidase or ferritin solution; (5) incubation only with DAB.
147
Electron microscopy The cell suspensions were postfixed in phosphatebuffered 2 % osmium tetroxide for 30-40 min at room temperature, dehydrated in ethanol and embedded in Epon. Thin sections were cut on a Sorvall MT-l or LKB ultramicrotome. Sections were not stained with lead or Uranvl acetate since thev both give additional electron density to the plasma-membrane. The sections were examined with a Philips 200 Electron Microscope, 80 kV, objective aperture 50 pm.
RESULTS The results obtained with the two cell systems were identical and, therefore, will be presented together. Expt 1: Classical sandwich technique
Labelling of the cell surface antigens with peroxidase conjugate diluted 1 : 10 revealed large amounts of electron-dense material in close apposition to the cell membrane (fig. 1). The labelling was seenas a nearly continuous deposit, uneven in its distribution. Frequently, the reaction product had a beaded appearance (B). In sections perpendicular to the plasma membrane, the beads appeared to average 235 8, in diameter; but quite frequently, larger aggregateswere seenwhich were thought to have resulted from varying degreesof superimposition of closely arranged beads (fig. 1C). In areas of oblique sectioning, individual beads appeared in an uneven mosaic-like pattern (fig. 1M). Artifacts consisting of large black deposits were seen in a few areas, particularly in ‘protected’ areas where washing was inadequate, such as at the angles at the base of microvilli (fig. 1A), and in pinocytotic vesicles. As shown in table 1, on using ferritinconjugate antibody, it was found that, at any given concentration, less labelling of cell surface antigens was obtained with the ferritin conjugate than with peroxidase conjugate. Also in contrast to the peroxidase conjugate, the labelling was discontinuous Exptl Cell Res 71
148 R. Bretton et al.
Fig. 1. Labelling with peroxidase conjugate. Dilution 1: 10. The labelling is nearly continuous, consisting in most areas of bead-like structures (II). M, mosaic-like arrangement of beads; C, continuous labelling due to coalescence of beads; A, artifacts; x 60 000. Fig. 2. Labelling with ferritin conjugate. Dilution 1 : 10. The labelling is discontinuous (FE); it is absent on the villus (Ferritin-FE). x 90 000.
and was either slight or absent on themicrovilli (fig. 2). Wherever labelling with ferritin had occurred, it was in aggregates and the ferritin cores lay at varying distances away from the cell membrane. (A comparison of glutaraldehyde with toluene diisocyanate as a conjugating agent for ferritin to antibodies was made. No significant difference was noted.) Using a dilution of peroxidaselabelled antibodies of 1: 50 instead of 1: 10 resulted in no decrease in labelling of the cell surface antigens. At a high dilution of 1: 250, and even more at a dilution of 1: 500, there was a great decrease in peroxidase labelling and the labelling was discontinuous, as seenwith ferritin labelling (fig. 3). Ferritin labelling, even when used undiluted (5 mg/ml) in place of a dilution of 1 : 10, never was Exptl Cell Res 71
continuous. At a dilution of 1: 50, there was a marked decrease in labelling, but some surface labelling was still present. At dilutions of 1: 250 and 1: 500, labelling was fundamentally absent except that it was occasionally present in ‘protected’ areas, such as at the angles at the base of microvilli and in pinocytotic vesicles(fig. 4). The highly concentrated ferritin-conjugated antibody (10 mg/ml) labelled almost the entire cell surface, resembling in distribution that of peroxidase-conjugated antibody (fig. 5). Expt 2: Double labeling with jerritin and peroxidase. The results in table 1 indicate that cells incubated first in peroxidase then in ferritinlabelled antibody revealed staining only with
Peroxidase and ferritin labelling
149
Table 1. Comparison of peroxidase and jerritin conjugates Concentrations of peroxidase-labelled antibody (POAb)and ferritin-labelled antibody (FeAb)used
Peroxidase staining
Ferritin staining
Expt I 10 mg/ml 0.5 mg/ml 0.01 mg/ml 0.002 mg/ml 0.001 mg/ml
Not done Continuous, total surface Continuous, total surface Patchy, total surface Patchy, total surface
Patchy, almost continuous Patchy, total surface Decrease in patches No labelling No labelling
Expt 2
Doublelabelling
POAb 0.5 mg/ml followed by FeAb 10 mg/ml FeAb 10 mg/ml followed by POAb 0.5 mg/ml
Continuous, total surface No labelling Staining of PO found in the same Patchy, almost continuous areas as patches of ferritin and in areas unstained by ferritin
peroxidase over the whole cell surface, indicating that there were no available sites for the ferritin conjugate. On the other hand, cells incubated first with the concentrated ferritin labelled antibody (10 mg/ml) were stained with ferritin over most of the cell surface. These areas had the peroxidase reaction product interspersed between the ferritin. The peroxidase reaction product appeared to be denser in areas whereferritin was heavily deposited. Therefore all antigenic sites were not taken up by the first conjugate ferritin. Areas on the cell membrane which were devoid of ferritin were either negative or stained with peroxidase (fig. 6). Controls with peroxidase labeled antibodies unrelated to the cell system were negative. Expt 3: Specificity of peroxidase staining (a) Cell suspensions, after incubation with
ferritin conjugate, were postincubated with an antiferritin serum labelled with peroxidase (fig. 7). If peroxidase-labelling was limited to sites where ferritin was present, it would indicate that there was no diffusion of the reaction product over the plasma membrane. In fact, the peroxidase attached itself only to ferritin-labelled antibody and did not diffuse,
leaving portions of the cell surface unlabelled (fig. 8). The short exposure to DAB (5 min) enabled us to observe both ferritin and reaction product of peroxidase. Fifteen minutes of exposure completely masked the ferritin. (b) In the experiment in which CVl cells treated with peroxidase conjugate were compared with hamster embryo cells not treated with peroxidase antibody conjugate subsequent exposure to DAB revealed that the hamster embryo cells were easily distinguished from the CVl cells, not only by their much smaller size, but also by the fact that the CV 1 cells showed the positive electron-dense reaction product distributed over nearly the entire cell surface. Hamster embryo cells, however, even those in close contact with the CVl cells (fig. 9), showed no reaction of the cell surface. It is concluded that peroxidase and/or its reaction product does not attach itself nonspecifically to areas of the plasma membrane. DISCUSSION Antibodies specific for cell surface antigens labelled with peroxidase produced a nearly continuous staining of the cell surface, inExptI Cell Res 71
150 R. Bretton et al.
Fig. 3. Labelling with peroxidase conjugate. Dilution 1: 250. The labelling is sparse and discontinuous. x 90 000. Fig. 4. Labelling with ferritin conjugate. Dilution 1 :250. One area selected to show the ferritin labelling! which
occurred rarely and was present only in some ‘protected’ areas, such as at the angles at the base of mtcrovilli and in pinocytotic vesicles. x 90 000. Fig. 5. Labelling with concentrated ferritin conjugate (10 mg/ml). Labelling is nearly continuous. Some villi are labelled. x 60 000. Exptl Cd
Res 71
Peroxidase and ferritim labelling
15 1
CELL /
7 Fig. 6. Labelling with ferritin conjugate followed by labelling with peroxidase conjugate. Ferritin labelling is seen in the same areas as peroxidase labelling. (+). Some areas are labelled only with peroxidase conjugate, which can be seen as either continuous labelling (C), or bead-like TTTuctures (B). Bare areas with neither label are also present (b ). Peroxidase was revealed only for 10 min with diaminobcnzidine to obtain an electron density less than ferritin. x 60 000. Fig. 7. Specificity of peroxidase staining. Antigen at cell surface (1); rabbit antiserum anti CVl (2); sheep antibody anti-rabbit IgG (3) labelled with ferritin (4); sheep antibody anti-ferritin (5) coupled to peroxidase (6); green monkey kidney epithelial cell culture (CV 1). Fig. 8. Labelling with ferritin conjugate followed by labelling with antiferritin-antibody conjugated with peroxidase. Peroxidase labelhng is seen only in areas of ferritin labelling (FE). Peroxidase was revealed only for 5 min with diaminobenzidine, therefore obtaining a light stain. x 90 000. Exptl Cell Res 71
152 R. Bretton et al.
Fig. 9. Specific attachment of the peroxidase stain on the cell surface. Labeled CVl cell (+), with adjacent unlabelled hamster embryo cells (-). Both cell types were exposed for 15 min to diaminobenzidine in the presence of the peroxidase conjugate. (Low power.)
eluding the microvilli. The surface area stained with this peroxidase conjugate was much thicker in some areas than in others, suggesting an uneven distribution of the cell surface antigen. On labelling the same antibody with ferritin, on the other hand, there was discontinuous staining of the cell surface, with little or no reaction on the microvilli. These results demonstrate that peroxidase antibody conjugate labels more antigenic sites at the cell surface than does ferritin antibody conjugate. The heavier labelling obtained with peroxidase conjugate might be explained by the fact that peroxidase has a much smaller molecular weight than ferritin, namely 40 000 Exptl Cell Res 71
vs 650 000. Becauseof the large size of ferritin molecules, the cell coat may have hindered the penetration of the ferritin conjugate, but not that of the much smaller peroxidaseconjugate macromolecule, so that the latter could become bound to more of the cell surface antigens. This is supported by the results of an experiment which showed that, after partial removal of the cell coat with hyaluronidase, more ferritin penetrated down towards the level of the cell membrane and, therefore, more was bound since it was exposed to a greater number of antigenic groups, even if the assumption is made that there was a loss of antigenic sites from the cell after hyaluronidase treatment. Furthermore, be-
Peroxidase and ferritin labelling
153
continuous staining with ferritin conjugates and have therefore searched for reasons to explain the discontinuous staining. Sternberger [22], in reviewing the problem, foundno satisfactory reason for the relatively sparse localization of ferritin conjugate, even on the surface of cells where accessibility poses no problem. He cites various explanations given by different authors. Thus, Borek & Silverstein [9] and Vogt & Kopp [23] have explained the discontinuous and rather sparse localization of ferritin conjugate as being due to the relatively poor yield or conjugated immunologically active antibody remaining after separation from unconjugated immunoglobulin. Lee & Feldman [17] have explained the discontinuous staining as the result of using dilute solutions of labelled antibody; or as the result of blocking of conjugated by unconjugated antigen; or as artifacticious. It is interesting that in addition to all the above cited reasons, so far a possible steric hindrance exerted by the cell surface coat on the large ferritin molecules has not been considered. As shown in expt 1, the labelling with the peroxidase conjugate is also discontinuous if only small amounts of conjugate are used, as predicted by Lee & Feldman [17]. We suggest that the reason for the discontinuous labelling is that eventhough cell surface antigens are present on nearly the entire cell surface, they are present in variable amounts in different areas.Thus, if only small amounts of conjugate are available, only areas with the heaviest concentration of cell surface Ferritin staining antigen react, resulting in discontinuous labelThe discontinuous staining of the cell surface ling. with ferritin conjugates has been interpreted When very concentrated solutions of conby some authors, such as Hammerling et al. jugated ferritin antibodies were used, as in [12] and Aoki et al. [2, 31, as an indication expt 1, a nearly continuous but uneven labelthat antigen resides in widely separated, cir- ling of the cell surface was obtained, similar cumscribed areas of the cell surface. Other au- to that seen with peroxidase. The disadvantthors, however, have assumedthat the surface age resulting from a high molecular weight of cells was sufficiently rich in antigen to show probably applies to all ferritin conjugates, cause of the high molecular weight of ferritin, the ferritin conjugate, after having attached itself to cell surface antigens, may have been more easily washed off than the peroxidase conjugate. However, thorough washings are absolutely necessary in order to remove all conjugate not bound to antigen. In expt 2, in which the ferritin conjugate was first incubated, followed by the peroxidase conjugate, we observed some areas with neither label. The lack of labelling by the peroxidase conjugate in those areas meant that there was no antigen present to which it could attach. One interpretation would be that the ferritin labelled antibody and the antigen to which it became bound were washed away as a whole unit. In areas where peroxidase staining was present and ferritin absent, one could conclude either that the ferritin labelled antibody became detached from its cell surface antigen, or that it was unable to reach these antigenic sites. On the other hand, the presence of peroxidase staining wherever ferritin was present enabled us further to conclude that ferritin labelled antibody did not occupy all antigenic sites available, possibly due to the large molecular size of ferritin conjugate, and hence steric hindrance to attachment, and the secondarily incubated peroxidase labelled antibodies were attached to these free antigenic sites (fig. 6). Non-specific trapping was ruled out by a negative control with a peroxidase labelled normal sheep IgG.
Exptl Cell Res 71
154 R. Bretton et al.
including the hybrid antibody introduced by Hammerling et al. [12, 131. Peroxidase staining
The peroxidase conjugate, in comparison with the ferritin conjugate, has the advantage of a lower molecular weight which would reduce steric hindrance. In addition, since the enzyme is not consumed in the histochemical reaction, many molecules of electron-dense reaction product can be deposited at the antigenic sites and the intensity of staining or the size of the reaction product may be controlled by varying the duration of incubation [19] (figs 6, 8, 9). It is obviously important to show that the peroxidase conjugate, like the ferritin conjugate, attached itself specifically to the cell surface antigens without non-specific diffusion or attachment over the cell membrane. As shown in expt 3a, with peroxidase conjugated with antibody anti-ferritin, the peroxidase reaction product remained specifically localized to the ferritin, and in expt 3b, the possibility of non-specific attachment on the cell surface of peroxidase oxidized 3-3’ diaminobenzidine was excluded by very careful washing. In addition, control experiments, in which cell suspensions were directly exposed to peroxidase, eliminated the possibility that peroxidase attached itself nonspecifically to the cell surface. Furthermore, the possibility of false labelling, as may occur in cell systemshaving endogenous peroxidase or peroxidase-like reactions on their cell surface, was excluded. The presence of such endogenous reactions can be easily detected by a direct reaction with 3-3’ diaminobenzidine, and this was negative in our control experiments. In conclusion, our data indicate that all immunological methods utilizing labelled antibodies have a threshold of sensitivity Exptl Cell Res 71
below which the antigen cannot be detected. This sensitivity depends in part on the local density of antigen and negative results cannot necessarily be interpreted to be a complete absence of antigen. Methods of different sensitivity reveal different distributions of antigen, with the less sensitive methods failing to show the areas with the smallest quantities of antigen. This paper is dedicated in memory of Nicole Granboulan. The authors wish to exuress their gratitude to Dr W. Bernhard for his interest and critical advice. We are arateful to Mrs Marie-Jeanne Burglen for her technical assistance and to Jacqueline Ney-for her kindness in tvuina the manuscrint. - k. -Bretton is the-recipient of a National Institute of Health Special Research Fellowship (5 FOfCA41-801-02) from the National Cancer Institute.
REFERENCES 1. Allen, A & Snow, C, Biochem j 117 (1970) 32P. 2. Aoki, T, H&nrnerling, U, de Harven, E, Boyse, E A & Old, L J, J exptl med 130 (1969) 979. 3. Aoki, T, Stuck, B, Old, L J, Hammerling, U & de Harven, E, Cancer res 20 (1970) 244. 4. Avrameas, S, Immunochemistry 6 (1969) 43. 5. Avrameas, S & Lespinats, G, Compt rend acad sci 265 (1967) 1149. 6. Avrameas. S, Taudou. B & Chuilon, S. Immunochemistry 6 (1969) 67.. 7. Avrameas, S & Temynck, T, Immunochemistry 6 (1969) 53. 8. Avrameas, S, & Uriel, J, Compt rend acad sci 262 (1966) 2543. 9. Borek, F & Silverstein, A M, J immunol 87 (1961) 555. 10. Bretton, R & Lespinats, G, Compt rend acad sci 268 (1969) 3223. 11. Graham, R C Jr & Karnovsky, M J, J histochem cytochem 14 (1966) 291. 12. Hiimmerling, U, Aoki, T, de Harven, E, Boyse, E A & Old, L J, J exptl med 128 (1968) 1461. 13. Hammerling, U, Aoki, T, Wood, H A, Old, L J, Boyse, E A & de Harven, E, Nature 223 (1969) 1158. 14. Hermann, G, Lespinats, G & Bartholomew ,R W, Path01 biol 14 (1966) 914. 15. Jensen, F C, Girardi, A I, Gilden, R V & Koprowski, 3, Proc natl acad sci US 52 (1964) 53. 16. Lespinats, G, J natl cancer inst 45 (1970) 845. 17. Lee, R E & Feldman, J D, J cell biol 23 (1964) 396. 18. Nakane, P K & Pierce, G B, Enzyme-labeled antibodies for the ultrastructural localization of antigens. Proc 6th intern tong for electron
Peroxidase and ferritin labeling microscopy, vol. 2, p. 51. Maruzen Co., Tokyo (1966). 19. Nakane, P K & Pierce, G B, J cell biol 33 (1967) 307. 20. Singer, S J, Nature 183 (1959) 1523. 21. Singer, S J & McLean, J D, Lab invest 12 (1963) 1002.
155
22. Stemberger, L A, J histochem cytochem 15 (1967) 139. 23. Vogt, A & Kopp, R, Z Bakt 198 (1965) 270. Received July 5, 1971 Revised version received September 30, I97 I
Exptl Cell Res 71
COMPARISON OF PEROXIDASE AND FERRITIN LABELLING OF CELL SURFACE ANTIGENS R. BRETTON’,
T. TERNYNCK
and S. AVRAMEAS
Institut de Recherches Scientqiques sur le Cancer, 94-Villejuif, France
SUMMARY In a comparative study of peroxidase and ferritin used as labels for cell surface antigens, the following observations were made: 1. Peroxidase labelled more antigenic sites than did ferritin. The cell surface label was shown to be nearly continuous with peroxidase, whereas there were large discontinuities with ferritin. 2. Highly diluted peroxidase conjugate showed discontinuous labelling of the cell surface similar to undiluted ferritin conjugate. 3. When ferritin labelled conjugate is used in concentrations much higher than is usual, it labels the cell surface continuously as much as does a peroxidase-labelled conjugate. 4. Partial removal of the cell surface coat with hyaluronidase narrowed the distance between the ferritin conjugate and the unit membrane component of the cell surface, resulting in more extensive but still discontinuous labeling. As a result of these observations, it was concluded that: (A) The heavier labelling obtained with peroxidase conjugate than with ferritin conjugate was specific and not the result of diffusion. (B) The patchy ferritin labelling and the uneven labelling with diluted peroxidase suggests a variability in the distribution of cell surface antigens, but does not support the concept that antigens reside in widely separated circumscribed segments of the cell surface. (C) Peroxidase conjugate has the advantage over ferritin conjugate in being amuchsmallermolecule.
During the past twelve years, three techniques have been developed that are applicable to the study of cell surface antigens at the ultrastructural level. In 1959, Singer [20, 211 introduced ferritin as a labelling agent. In 1966, Avrameas et al. [5, 81 and Nakane & Pierce [18, 191independently introduced the technique of using enzyme-labelled antibodies, among them peroxidase. In 1968, Hlimmerling et al. [12, 131 modified the ferritin method by preparing a hybrid antibody. Bretton & Lespinats [lo] in 1969 used 1 Permanent address: Tufts Medical School, Department of Dermatology, 185 Harrison Avenue, Boston, Mass. 02111, USA. 10-721803
peroxidase labelled antibodies for the localization of cell surface antigens. At that time, a difference was observed between peroxidase labelling and the ferritin labelling reported in the literature. It was found that the deposition of peroxidase-antibody conjugate on the cell surface was continuous. This is in contrast to the deposition of ferritin-antibody conjugate which generally has been found to be present in patches with wide, intervening unlabelled zones. Although several authors, among them Nakane & Pierce [19] have expressedthe view that peroxidase is superior to ferritin as a label for intracellular antigens because of its smaller molecular weight, ferritin is still Exptl Cell Res 71
146 R. Bretton et al.
widely used for labelling cell surface antigens. In the present study, the effectiveness of peroxidase-labelled antibodies and of ferritinlabelled antibodies as markers of cell surface antigens is compared. The cellular systems were chosen for a single purpose: visualization of differences in cell surface labelling. Therefore species-specific antibodies were used since the corresponding antigens were more evenly distributed over most of the cell surface; a well defined immunological system. where only limited parts of the cell surface would be labelled, would have made interpretation of variations extremely difficult. Furthermore, there was no endogenous peroxidase or peroxidase-like activity on the cell surface, as is seenwith red blood cells. MATERIAL
AND METHODS
Cell systems Two cell systems were used: (a) cells of a solid tumour from a BALB/c mouse plasmacytoma originally induced by Bayol F rrineral oil [14] and transolanted bv subcutaneous iniection: (b) cultured cells of an established line of green ‘monkey kidney eoithelial cell culture (CVl) used between the 50th and 100th subculture ‘[15].’ Both cell systems were dissociated with 0.135 % trypsin (Choay Laboratory, crystallized and lyophilized) in PBS, which required 36-60 min for the plasmacytoma cells and 5-6 min for the kidney cell cultures. Even though it was realized that the brief exposure to trypsin removed parts of certain cell surface constituents [l], it represents the best method available for separating cells of solid tumours and good immunological reactions were still obtained.
Preparation and labelling of antibody Pure antibodies were used that were isolated from hyperimmunized animals. The isolation of the antibodies was accomplished with an immunoadsorbent prepared by copolymerization at pH 5 of bovine serum albumin and the corresponding antigen (4 : 1) using glutaraldehyde as the insolubilizing agent and following the batchwise procedure [q. For experiments (a) with mouse plasmacytoma cells, rabbit antibody antirat IgG was used; (b) with monkey kidney cell culture sheep antibody antirabbit IgG was used; (c) on the specificity of peroxidase staining sheep antiferritin antibody was used. Isolated antibodies were labelled with peroxidase, using glutaraldehyde as the coupling agent [4]. (The advantage of glutaraldehyde as a cross linking agent is due to coupling via free amino groups which does not actively participate in enzyme antibody activity.) Ferritin was-coupled to antibodies (a) using toluene diisocyanate according to Singer [20]; (b) using glutaraldehyde as the coupling agent. With the latter, the reactants were used in the following- quantities: _ 5 mg of antibodies, 10 mg of ferritin in 2 ml of phosohate buffer 0.1 uH 6.8 in which 0.05 ml of a 1 % aqueous solution of glutaraldehyde was added. After dialysis and centrifugation, the preparation was purified by ultracentrifugations according to the technique of Singer [21]. The labelled preparations were adjusted to the same concentration of 5 mg/ml. This concentration was determined in the case of ueroxidase-labelled antibodies, by assuming that no unlabelled antibody is present in- the preparation [4] and. in the case of ferritin-labelled antibodies, by measuring the amount of unconjugated antibody present in the supernatant after centrifugation at 40 000 g for 2 h and subtracting this amount from the total amount of antibody used. The adjusted conjugates were stored at 4°C. The activity of all the conjugates, at the same antibody concentrations, was determined by the passive hemagglutination technique, using sheep erythrocytes coated with rabbit IgG [6]. Both preparations were found to agglutinate coated erythrocytes at the same dilutions. Since the procedures used for the experiments on both cell systems were identical, they will be presented together.
Antisera
Expt 1: classical sandwich technique
(a) Rat antiserum BALB/c mouse plasmacytoma serum was prepared by hyperimmunizing Wistar rats [16]; (b) rabbit antimonkey kidney cell serum, supplied by R. Wicker, was obtained by injecting a total of 100 mg of CVl cell homogenate intramuscularly and intradermally into the foot-pad of adult rabbits. The procedure was repeated twice at intervals of 2 months. Complement was inactivated in both sera by incubating them at 56°C for 30 min; (c) rabbit antiserum anti-rat IgG was prepared following an immunization procedure already described [7]. Antirabbit IgG serum and anti-ferritin serum was prepared b; immunizing two sheep. A total of 300 mg of rabbit IgG and ferritin respectively, was used over a period of 6 months.
Susoensions of 5 x lo6 cells/O.3 ml were incubated with their respective antiserum for 15 min at 37°C. The cells were washed in PBS-PVG (ohosnhatebuffered saline containing 1 % polyvinyl~glycol, pH 7.4). and centrifuged at 1 400 ram. The washing and’centrifugation was repeated twice. The cells then were incubated for a period of 30 min at 37°C with either peroxidase-labelled or ferritin-labelled antibody. The labelled antibodies having an initial concentration of 5 mg/ml were diluted 1: 10, 1 : 50, 1: 250 and 1 : 500. In addition, ferritin-labelled antibody at a concentration of 10 mg/ml was used. Again the cells were washed and centrifuged three times in PBS-PVG in order to remove all unbound labelled antibody. Fixed in 2.5 % glutaraldehyde in
Exptl
Cell Res 71
Peroxidase and ferritin labelling SGrensen buffer at pH 7.4 for 15 or 60 min at 4°C. Washed four times for a total of 15 min in 5 ml of Siirensen buffer pH 7.4 and centrifuged after each washing at 1 500 rpm. The peroxidase-labelled cells were incubated with 3-3’ diaminobenzidine for 5, 10, 15 or 30 min at room temperature, in order to reveal the peroxidase activity [ll], washed three times in phosphate buffer, each followed by centrifuging.
Expt 2: Double labelling with ferritin and peroxidase To determine the relative effectiveness of each conjugate to detect antigen on the same cellular preparations, the following experiments were performed: cell suspensions were incubated with their antisera as in expt 1. They ,were then divided into two groups. One group was incubated in peroxidase-labelled antibody (0.5 mg/ml) followed by ferritin-labelled antibody (10 mg/ml). With the other group, we reversed the order of incubation, i.e. ferritin followed by peroxidase. Both groups were incubated with each labelled antiserum for 30 min at 37”C, washed after each incubation, then fixed with 2.5 % glutaraldehyde for 1 h at 4°C. The DAB reaction time was kept at 10 min to insure visualization of both markers.
Expt 3: Specificity of peroxidase (a) The cell suspensions were incubated with their antiserum and then with ferritin-labelled antibody (0.5 mrriml). as described in exut 1. Subsequently. they w&e incubated with sheep aitiferritin anjibod;; labelled with oeroxidase (fig. 7). Exoosure to 3-3’ diaminobenzidine (DAB) ‘was limited to 5 min in order to avoid masking of the ferritin by excessive formation of the electron-dense peroxidase DAB reaction product. (b) CVl cell suspensions were incubated first with their antiserum and subsequently with the peroxidaselabelled antibody (0.5 mgiml), as in expt 1. The cells were then fixed, as u&al,- with 2.5 % glutaraldehyde for 15 min at 4°C. Hamster embryo fibroblasts cell suspensions also were similarly fixed with glutaraldehyde. Both cell suspensions were washed four times -after fixation, mked with one another and olaced into a test tube and treated with DAB for is min.
Controls The following control incubations were carried out on the BALB/c mouse plasmacytoma cell suspensions and the CV 1 cell cultures: (1) Normal rat serum intead of the rat antimouse plasmacytoma serum; (2) solutions of normal rabbit IgG labelled with either ueroxidase or ferritin in olace of the resoectivelv labelled rabbit antibody a&rat IgG (co;?iugatek (3) incubation only with the peroxidase or ferritinlabelled rabbit conjugate; (4) incubation only with pure peroxidase or ferritin solution; (5) incubation only with DAB.
147
Electron microscopy The cell suspensions were postfixed in phosphatebuffered 2 % osmium tetroxide for 30-40 min at room temperature, dehydrated in ethanol and embedded in Epon. Thin sections were cut on a Sorvall MT-l or LKB ultramicrotome. Sections were not stained with lead or Uranvl acetate since thev both give additional electron density to the plasma-membrane. The sections were examined with a Philips 200 Electron Microscope, 80 kV, objective aperture 50 pm.
RESULTS The results obtained with the two cell systems were identical and, therefore, will be presented together. Expt 1: Classical sandwich technique
Labelling of the cell surface antigens with peroxidase conjugate diluted 1 : 10 revealed large amounts of electron-dense material in close apposition to the cell membrane (fig. 1). The labelling was seenas a nearly continuous deposit, uneven in its distribution. Frequently, the reaction product had a beaded appearance (B). In sections perpendicular to the plasma membrane, the beads appeared to average 235 8, in diameter; but quite frequently, larger aggregateswere seenwhich were thought to have resulted from varying degreesof superimposition of closely arranged beads (fig. 1C). In areas of oblique sectioning, individual beads appeared in an uneven mosaic-like pattern (fig. 1M). Artifacts consisting of large black deposits were seen in a few areas, particularly in ‘protected’ areas where washing was inadequate, such as at the angles at the base of microvilli (fig. 1A), and in pinocytotic vesicles. As shown in table 1, on using ferritinconjugate antibody, it was found that, at any given concentration, less labelling of cell surface antigens was obtained with the ferritin conjugate than with peroxidase conjugate. Also in contrast to the peroxidase conjugate, the labelling was discontinuous Exptl Cell Res 71
148 R. Bretton et al.
Fig. 1. Labelling with peroxidase conjugate. Dilution 1: 10. The labelling is nearly continuous, consisting in most areas of bead-like structures (II). M, mosaic-like arrangement of beads; C, continuous labelling due to coalescence of beads; A, artifacts; x 60 000. Fig. 2. Labelling with ferritin conjugate. Dilution 1 : 10. The labelling is discontinuous (FE); it is absent on the villus (Ferritin-FE). x 90 000.
and was either slight or absent on themicrovilli (fig. 2). Wherever labelling with ferritin had occurred, it was in aggregates and the ferritin cores lay at varying distances away from the cell membrane. (A comparison of glutaraldehyde with toluene diisocyanate as a conjugating agent for ferritin to antibodies was made. No significant difference was noted.) Using a dilution of peroxidaselabelled antibodies of 1: 50 instead of 1: 10 resulted in no decrease in labelling of the cell surface antigens. At a high dilution of 1: 250, and even more at a dilution of 1: 500, there was a great decrease in peroxidase labelling and the labelling was discontinuous, as seenwith ferritin labelling (fig. 3). Ferritin labelling, even when used undiluted (5 mg/ml) in place of a dilution of 1 : 10, never was Exptl Cell Res 71
continuous. At a dilution of 1: 50, there was a marked decrease in labelling, but some surface labelling was still present. At dilutions of 1: 250 and 1: 500, labelling was fundamentally absent except that it was occasionally present in ‘protected’ areas, such as at the angles at the base of microvilli and in pinocytotic vesicles(fig. 4). The highly concentrated ferritin-conjugated antibody (10 mg/ml) labelled almost the entire cell surface, resembling in distribution that of peroxidase-conjugated antibody (fig. 5). Expt 2: Double labeling with jerritin and peroxidase. The results in table 1 indicate that cells incubated first in peroxidase then in ferritinlabelled antibody revealed staining only with
Peroxidase and ferritin labelling
149
Table 1. Comparison of peroxidase and jerritin conjugates Concentrations of peroxidase-labelled antibody (POAb)and ferritin-labelled antibody (FeAb)used
Peroxidase staining
Ferritin staining
Expt I 10 mg/ml 0.5 mg/ml 0.01 mg/ml 0.002 mg/ml 0.001 mg/ml
Not done Continuous, total surface Continuous, total surface Patchy, total surface Patchy, total surface
Patchy, almost continuous Patchy, total surface Decrease in patches No labelling No labelling
Expt 2
Doublelabelling
POAb 0.5 mg/ml followed by FeAb 10 mg/ml FeAb 10 mg/ml followed by POAb 0.5 mg/ml
Continuous, total surface No labelling Staining of PO found in the same Patchy, almost continuous areas as patches of ferritin and in areas unstained by ferritin
peroxidase over the whole cell surface, indicating that there were no available sites for the ferritin conjugate. On the other hand, cells incubated first with the concentrated ferritin labelled antibody (10 mg/ml) were stained with ferritin over most of the cell surface. These areas had the peroxidase reaction product interspersed between the ferritin. The peroxidase reaction product appeared to be denser in areas whereferritin was heavily deposited. Therefore all antigenic sites were not taken up by the first conjugate ferritin. Areas on the cell membrane which were devoid of ferritin were either negative or stained with peroxidase (fig. 6). Controls with peroxidase labeled antibodies unrelated to the cell system were negative. Expt 3: Specificity of peroxidase staining (a) Cell suspensions, after incubation with
ferritin conjugate, were postincubated with an antiferritin serum labelled with peroxidase (fig. 7). If peroxidase-labelling was limited to sites where ferritin was present, it would indicate that there was no diffusion of the reaction product over the plasma membrane. In fact, the peroxidase attached itself only to ferritin-labelled antibody and did not diffuse,
leaving portions of the cell surface unlabelled (fig. 8). The short exposure to DAB (5 min) enabled us to observe both ferritin and reaction product of peroxidase. Fifteen minutes of exposure completely masked the ferritin. (b) In the experiment in which CVl cells treated with peroxidase conjugate were compared with hamster embryo cells not treated with peroxidase antibody conjugate subsequent exposure to DAB revealed that the hamster embryo cells were easily distinguished from the CVl cells, not only by their much smaller size, but also by the fact that the CV 1 cells showed the positive electron-dense reaction product distributed over nearly the entire cell surface. Hamster embryo cells, however, even those in close contact with the CVl cells (fig. 9), showed no reaction of the cell surface. It is concluded that peroxidase and/or its reaction product does not attach itself nonspecifically to areas of the plasma membrane. DISCUSSION Antibodies specific for cell surface antigens labelled with peroxidase produced a nearly continuous staining of the cell surface, inExptI Cell Res 71
150 R. Bretton et al.
Fig. 3. Labelling with peroxidase conjugate. Dilution 1: 250. The labelling is sparse and discontinuous. x 90 000. Fig. 4. Labelling with ferritin conjugate. Dilution 1 :250. One area selected to show the ferritin labelling! which
occurred rarely and was present only in some ‘protected’ areas, such as at the angles at the base of mtcrovilli and in pinocytotic vesicles. x 90 000. Fig. 5. Labelling with concentrated ferritin conjugate (10 mg/ml). Labelling is nearly continuous. Some villi are labelled. x 60 000. Exptl Cd
Res 71
Peroxidase and ferritim labelling
15 1
CELL /
7 Fig. 6. Labelling with ferritin conjugate followed by labelling with peroxidase conjugate. Ferritin labelling is seen in the same areas as peroxidase labelling. (+). Some areas are labelled only with peroxidase conjugate, which can be seen as either continuous labelling (C), or bead-like TTTuctures (B). Bare areas with neither label are also present (b ). Peroxidase was revealed only for 10 min with diaminobcnzidine to obtain an electron density less than ferritin. x 60 000. Fig. 7. Specificity of peroxidase staining. Antigen at cell surface (1); rabbit antiserum anti CVl (2); sheep antibody anti-rabbit IgG (3) labelled with ferritin (4); sheep antibody anti-ferritin (5) coupled to peroxidase (6); green monkey kidney epithelial cell culture (CV 1). Fig. 8. Labelling with ferritin conjugate followed by labelling with antiferritin-antibody conjugated with peroxidase. Peroxidase labelhng is seen only in areas of ferritin labelling (FE). Peroxidase was revealed only for 5 min with diaminobenzidine, therefore obtaining a light stain. x 90 000. Exptl Cell Res 71
152 R. Bretton et al.
Fig. 9. Specific attachment of the peroxidase stain on the cell surface. Labeled CVl cell (+), with adjacent unlabelled hamster embryo cells (-). Both cell types were exposed for 15 min to diaminobenzidine in the presence of the peroxidase conjugate. (Low power.)
eluding the microvilli. The surface area stained with this peroxidase conjugate was much thicker in some areas than in others, suggesting an uneven distribution of the cell surface antigen. On labelling the same antibody with ferritin, on the other hand, there was discontinuous staining of the cell surface, with little or no reaction on the microvilli. These results demonstrate that peroxidase antibody conjugate labels more antigenic sites at the cell surface than does ferritin antibody conjugate. The heavier labelling obtained with peroxidase conjugate might be explained by the fact that peroxidase has a much smaller molecular weight than ferritin, namely 40 000 Exptl Cell Res 71
vs 650 000. Becauseof the large size of ferritin molecules, the cell coat may have hindered the penetration of the ferritin conjugate, but not that of the much smaller peroxidaseconjugate macromolecule, so that the latter could become bound to more of the cell surface antigens. This is supported by the results of an experiment which showed that, after partial removal of the cell coat with hyaluronidase, more ferritin penetrated down towards the level of the cell membrane and, therefore, more was bound since it was exposed to a greater number of antigenic groups, even if the assumption is made that there was a loss of antigenic sites from the cell after hyaluronidase treatment. Furthermore, be-
Peroxidase and ferritin labelling
153
continuous staining with ferritin conjugates and have therefore searched for reasons to explain the discontinuous staining. Sternberger [22], in reviewing the problem, foundno satisfactory reason for the relatively sparse localization of ferritin conjugate, even on the surface of cells where accessibility poses no problem. He cites various explanations given by different authors. Thus, Borek & Silverstein [9] and Vogt & Kopp [23] have explained the discontinuous and rather sparse localization of ferritin conjugate as being due to the relatively poor yield or conjugated immunologically active antibody remaining after separation from unconjugated immunoglobulin. Lee & Feldman [17] have explained the discontinuous staining as the result of using dilute solutions of labelled antibody; or as the result of blocking of conjugated by unconjugated antigen; or as artifacticious. It is interesting that in addition to all the above cited reasons, so far a possible steric hindrance exerted by the cell surface coat on the large ferritin molecules has not been considered. As shown in expt 1, the labelling with the peroxidase conjugate is also discontinuous if only small amounts of conjugate are used, as predicted by Lee & Feldman [17]. We suggest that the reason for the discontinuous labelling is that eventhough cell surface antigens are present on nearly the entire cell surface, they are present in variable amounts in different areas.Thus, if only small amounts of conjugate are available, only areas with the heaviest concentration of cell surface Ferritin staining antigen react, resulting in discontinuous labelThe discontinuous staining of the cell surface ling. with ferritin conjugates has been interpreted When very concentrated solutions of conby some authors, such as Hammerling et al. jugated ferritin antibodies were used, as in [12] and Aoki et al. [2, 31, as an indication expt 1, a nearly continuous but uneven labelthat antigen resides in widely separated, cir- ling of the cell surface was obtained, similar cumscribed areas of the cell surface. Other au- to that seen with peroxidase. The disadvantthors, however, have assumedthat the surface age resulting from a high molecular weight of cells was sufficiently rich in antigen to show probably applies to all ferritin conjugates, cause of the high molecular weight of ferritin, the ferritin conjugate, after having attached itself to cell surface antigens, may have been more easily washed off than the peroxidase conjugate. However, thorough washings are absolutely necessary in order to remove all conjugate not bound to antigen. In expt 2, in which the ferritin conjugate was first incubated, followed by the peroxidase conjugate, we observed some areas with neither label. The lack of labelling by the peroxidase conjugate in those areas meant that there was no antigen present to which it could attach. One interpretation would be that the ferritin labelled antibody and the antigen to which it became bound were washed away as a whole unit. In areas where peroxidase staining was present and ferritin absent, one could conclude either that the ferritin labelled antibody became detached from its cell surface antigen, or that it was unable to reach these antigenic sites. On the other hand, the presence of peroxidase staining wherever ferritin was present enabled us further to conclude that ferritin labelled antibody did not occupy all antigenic sites available, possibly due to the large molecular size of ferritin conjugate, and hence steric hindrance to attachment, and the secondarily incubated peroxidase labelled antibodies were attached to these free antigenic sites (fig. 6). Non-specific trapping was ruled out by a negative control with a peroxidase labelled normal sheep IgG.
Exptl Cell Res 71
154 R. Bretton et al.
including the hybrid antibody introduced by Hammerling et al. [12, 131. Peroxidase staining
The peroxidase conjugate, in comparison with the ferritin conjugate, has the advantage of a lower molecular weight which would reduce steric hindrance. In addition, since the enzyme is not consumed in the histochemical reaction, many molecules of electron-dense reaction product can be deposited at the antigenic sites and the intensity of staining or the size of the reaction product may be controlled by varying the duration of incubation [19] (figs 6, 8, 9). It is obviously important to show that the peroxidase conjugate, like the ferritin conjugate, attached itself specifically to the cell surface antigens without non-specific diffusion or attachment over the cell membrane. As shown in expt 3a, with peroxidase conjugated with antibody anti-ferritin, the peroxidase reaction product remained specifically localized to the ferritin, and in expt 3b, the possibility of non-specific attachment on the cell surface of peroxidase oxidized 3-3’ diaminobenzidine was excluded by very careful washing. In addition, control experiments, in which cell suspensions were directly exposed to peroxidase, eliminated the possibility that peroxidase attached itself nonspecifically to the cell surface. Furthermore, the possibility of false labelling, as may occur in cell systemshaving endogenous peroxidase or peroxidase-like reactions on their cell surface, was excluded. The presence of such endogenous reactions can be easily detected by a direct reaction with 3-3’ diaminobenzidine, and this was negative in our control experiments. In conclusion, our data indicate that all immunological methods utilizing labelled antibodies have a threshold of sensitivity Exptl Cell Res 71
below which the antigen cannot be detected. This sensitivity depends in part on the local density of antigen and negative results cannot necessarily be interpreted to be a complete absence of antigen. Methods of different sensitivity reveal different distributions of antigen, with the less sensitive methods failing to show the areas with the smallest quantities of antigen. This paper is dedicated in memory of Nicole Granboulan. The authors wish to exuress their gratitude to Dr W. Bernhard for his interest and critical advice. We are arateful to Mrs Marie-Jeanne Burglen for her technical assistance and to Jacqueline Ney-for her kindness in tvuina the manuscrint. - k. -Bretton is the-recipient of a National Institute of Health Special Research Fellowship (5 FOfCA41-801-02) from the National Cancer Institute.
REFERENCES 1. Allen, A & Snow, C, Biochem j 117 (1970) 32P. 2. Aoki, T, H&nrnerling, U, de Harven, E, Boyse, E A & Old, L J, J exptl med 130 (1969) 979. 3. Aoki, T, Stuck, B, Old, L J, Hammerling, U & de Harven, E, Cancer res 20 (1970) 244. 4. Avrameas, S, Immunochemistry 6 (1969) 43. 5. Avrameas, S & Lespinats, G, Compt rend acad sci 265 (1967) 1149. 6. Avrameas. S, Taudou. B & Chuilon, S. Immunochemistry 6 (1969) 67.. 7. Avrameas, S & Temynck, T, Immunochemistry 6 (1969) 53. 8. Avrameas, S, & Uriel, J, Compt rend acad sci 262 (1966) 2543. 9. Borek, F & Silverstein, A M, J immunol 87 (1961) 555. 10. Bretton, R & Lespinats, G, Compt rend acad sci 268 (1969) 3223. 11. Graham, R C Jr & Karnovsky, M J, J histochem cytochem 14 (1966) 291. 12. Hiimmerling, U, Aoki, T, de Harven, E, Boyse, E A & Old, L J, J exptl med 128 (1968) 1461. 13. Hammerling, U, Aoki, T, Wood, H A, Old, L J, Boyse, E A & de Harven, E, Nature 223 (1969) 1158. 14. Hermann, G, Lespinats, G & Bartholomew ,R W, Path01 biol 14 (1966) 914. 15. Jensen, F C, Girardi, A I, Gilden, R V & Koprowski, 3, Proc natl acad sci US 52 (1964) 53. 16. Lespinats, G, J natl cancer inst 45 (1970) 845. 17. Lee, R E & Feldman, J D, J cell biol 23 (1964) 396. 18. Nakane, P K & Pierce, G B, Enzyme-labeled antibodies for the ultrastructural localization of antigens. Proc 6th intern tong for electron
Peroxidase and ferritin labeling microscopy, vol. 2, p. 51. Maruzen Co., Tokyo (1966). 19. Nakane, P K & Pierce, G B, J cell biol 33 (1967) 307. 20. Singer, S J, Nature 183 (1959) 1523. 21. Singer, S J & McLean, J D, Lab invest 12 (1963) 1002.
155
22. Stemberger, L A, J histochem cytochem 15 (1967) 139. 23. Vogt, A & Kopp, R, Z Bakt 198 (1965) 270. Received July 5, 1971 Revised version received September 30, I97 I
Exptl Cell Res 71