THE LOCATION OF BLOOD GROUP ANTIGEN A ON CULTURED RABBIT KIDNEY CELLS AS REVEALED BY FERRITIN-LABELLED ANTIBODY

Transcription

1 J. Cell Sci. 10, (1972) 525 Printed in Great Britain THE LOCATION OF BLOOD GROUP ANTIGEN A ON CULTURED RABBIT KIDNEY CELLS AS REVEALED BY FERRITIN-LABELLED ANTIBODY ELIZABETH DIMMOCK,* D. FRANKS AND AUDREY M. GLAUERTf Strangeways Research Laboratory, Cambridge and Department of Pathology, University of Cambridge, England SUMMARY Fluorescein- and ferritin-labelled antibodies were used to locate blood group antigen A on the surfaces of cultured rabbit kidney cells (RK 13). Fluorescent anti-a revealed the antigen on some cells, but not on others. Controls indicated that specific staining could be obtained. Comparable observations were made with ferritin-labelled antibody, and the comparatively high resolution of this method allowed individual antigen molecules to be located. Measurements were made of the distances between the centres of the ferritin molecules and the cell surface, and the position of the blood group antigen relative to the visible components of the membrane is discussed in relation to this. INTRODUCTION Although much is now known about the chemical composition of biological membranes (e.g. see Cook, 1968), there is little information concerning the location of these components within membranes. The detection of the sites of individual antigens is possible with the ferritin-labelled antibody technique (Singer & McLean, 1963) and the aim of the present study was to use this method to locate a specific component, blood group A substance, of the surface membrane of a tissue cell as accurately as possible. An epithelioid line of rabbit kidney cells (RK 13) was chosen for examination since the distribution of blood group antigen A on these cells had already been studied with the mixed agglutination technique (Franks & Dawson, 1966; Dawson & Franks, 1967). MATERIALS AND METHODS Cells The characteristics and method of culruring RK 13 cells have been described in an earlier publication (Dimmock, 1970). Present address: Department of Physiology, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada, f Sir Halley Stewart Research Fellow.

2 526 E. Dimmock, D. Franks and A. M. Glauert Preparation of conjugate of fluorescein isothiocyanate and anti-a globulin A globulin fraction was separated from a high titre anti-a serum by sodium sulphate precipitation. The titre of the serum by the anti-globulin test was i: 1024 and of the redissolved globulin fraction 1:51200c The protein concentration was 96 mg/ml; 2-1 ml of the globulin solution was mixed with 3 mg of fluorescein isothiocyanate and stirred at 4 C for 17 h (Riggs et at. 1958). After conjugation, the mixture was titrated to ph 7-4, the globulin was separated from unconjugated fluorescein isothiocyanate on G 25 Sephadex and subsequently fractionated by stepwise elution from DEAE-cellulose. After concentration and dialysis against phosphatebuffered saline, the fluorescein content of each fraction was measured with a spectrophotometer by absorption at 495 run, and the molar fluorescein: protein ratio was calculated (Lachmann, 1964). The anti-a titres and staining abilities of all the fractions were measured, and 2 fractions (numbers 3 and 6) were found to give staining which was bright enough for use in a series of experiments to investigate the specificity of the staining reaction. Preparation of a conjugate of ferritin and anti-a globulin One millilitre of the globulin fraction of the anti-a serum was conjugated with 1 ml of 7 % ferritin using bis-diazotized benzidine (Williams & Gregory, 1967). After conjugation for 1 h at o C the conjugate was dialysed against buffer and the unconjugated globulin was separated from the conjugate by centrifugation (Rifkind, Hsu & Morgan, 1964) 3 times at g for 4h. Preparation of formalin-fixed rabbit liver hotnogenates Rabbit tissue preparations for absorption of fluorescein- and ferritin-conjugated anti-a preparations were made from the livers of A-positive and A-negative rabbits. The A antigen on rabbit tissue cells cross-reacts with the blood group antigen A found in humans. The rabbits were grouped by a mixed agglutination test on their buccal cells (Hawes & Coombs, 1959). Homogenates of rabbit liver were washed in saline and fixed with 1 % formalin at 4 C overnight. They were then washed in saline and stored in 20 % glycerol at 20 C. The homogenates were washed again in saline before use. Fixation of RK 13 cells It is desirable to fix cells which are to be treated with ferritin-conjugated antibody since this precludes the possibility of pinocytosis of the conjugate (Metzger & Smith, 1962), but fixation may destroy the reactivity of the antigen and consequently preliminary tests are necessary. It was found that glutaraldehyde-fixed RK 13 cells gave the expected reactions in a mixed agglutination test with several anti-a sera and no reaction with a control AB serum. It was also observed that glutaraldehyde-fixed red blood cells of blood group A were agglutinated by anti- A but not by anti-b grouping sera. Methods of staining RK 13 cells with fluorescein- and ferritin-labelled antibodies Glutaraldehyde-fixed monolayer cultures of RK 13 cells on cover-slips (Dimmock, 1970) were exposed tofluorescein-conjugatedanti-a for 10 min and subsequently washed twice. The preparations were mounted in buffered glycerol (Mrenova & Albrecht, 1966) and examined with a Reichert Zetopan microscope fitted with an HBO 200 mercury lamp. The ferritin-conjugated antibody was applied to glutaraldehyde-fixed RK 13 cells for 30 min and then the cells were washed 3 times in cold phosphate buffer, fixed in osmium tetroxide and dehydrated in ethanol. The layer of cells was removed from the coverslip during dehydration and embedded in Araldite (Dimmock, 1970). Thin sections were stained with uranyl acetate and lead citrate, or with lead citrate only, and examined in an AEI EM 6B electron microscope.

3 Location of antigen A on cells in culture 527 RESULTS The application offluoresceinisothiocyanate-labelled anti-a to RK 13 cells The application of fraction 3 or fraction 6 of the fluorescein isothiocyanate-labelled anti-a to RK 13 cells resulted in the fluorescent staining of some but not all cells (Fig. 1). Somewhat less than half the cells were stained and the unstained cells were scattered irregularly throughout the preparations. Table 1. Summary of the results of experiments with fractions 3 and 6 of thefluoresceinisothiocyanate-anti-a conjugate Type of test Fraction Blocking agent Reagent Absorbing agent Results Control 3 None None Blocking test 3 Anti-A None N.T. ( + ) False blocking test 3 AB serum None N.T. + + Absorption test 3 None A ve + + ( ) Absorption test 3 None A +ve ( + )/ ( ) Blocking test on absorbed fraction 3 Anti-A A ve Blocking test on absorbed fraction 3 Anti-A A +ve Blocking test on absorbed fraction 3 AB serum A ve (+) Blocking test on absorbed fraction AB serum A +ve + +/ Control 3 6 None None Blocking test Blocking test 6 6 Anti-A AB serum None None (+)/ Absorption test Absorption test 6 6 None None A +ve A ve + + Fluorescence: very bright; bright; + + faint; + very faint; ( + ) very, very faint; ( ) almost certainly none; definitely none; N.' r. not tested. A ' F ' ^ Undiluted diluted fraction i in 2 The application of unconjugated anti-a serum to the cells for 30 min before the application of fraction 3 of the conjugate resulted in the almost total inhibition of staining (Table 1). By contrast serum from an AB person had only a very slight effect on the brightness of the staining. Fraction 3 of the conjugated antibody was absorbed with rabbit liver homogenates by incubation 3 successive times for 15, 30 and 60 min respectively. Absorption with blood group A-negative rabbit liver homogenate slightly reduced the brightness of the staining, while absorption with the A-positive liver homogenate abolished the staining completely or almost completely. The application of unconjugated anti-a serum before application of the absorbed conjugate fraction resulted in the total inhibition of staining by the conjugate fraction absorbed with the A-negative liver preparation; and as expected there was no staining by the conjugate fraction absorbed with the A-positive liver preparation after application of the unconjugated serum. The substitution of serum from an AB person had no significant dimming effect on the staining given by the conjugate fraction absorbed with the A-negative liver homogenate (Table 1).

4 528 E. Dimmock, D. Franks and A. M. Glauert The application of ferritin-labelled anti-a to RK 13 cells The fine structure of RK 13 cells has been described by Dimmock (1970) and is not affected by the application of ferritin-conjugated anti-a. Ferritin was never identified in preparations of cells to which it had not been applied. When unabsorbed ferritin-labelled anti-a globulin was applied to RK 13 cells, ferritin was found scattered along the surfaces of some but not all cells (Fig. 2). It was seen along both upper and lower surfaces and between adjacent cells. RK 13 cells were treated with a mixture of ferritin and unconjugated antibody to investigate the possibility that ferritin may stick to the cells even when antibody cannot be responsible for the binding (Cohen, Zuelzer & Evans, i960). Very little ferritin was seen in such preparations; an occasional ferritin molecule was seen on the upper surface of a cell and sometimes on the lower surface. More ferritin was seen in the regions between cells, probably because of non-specific trapping. After absorption of ferritin-labelled anti-a with an A-negative liver preparation the conjugate stained some but not all cells (Fig. 3), and the ferritin was more sparsely distributed than in preparations treated with the unabsorbed conjugate (compare Fig. 2). In contrast, there was almost no ferritin present on cells stained with the conjugate absorbed with the A-positive liver homogenate, where the result resembled that obtained with a mixture of ferritin and antibody. When anti-a globulin was applied to the RK 13 cells before the application of the ferritin-conjugated antibody a total inhibition of ferritin staining was not obtained, but an almost total abolition of staining was obtained by applying the unconjugated antibody before the conjugate absorbed with the A-negative liver homogenate. These results are consistent with the results obtained with the third fraction of the fluorescent antibody conjugate of the same serum. It appears, therefore, that the staining with the ferritin-antibody conjugate after absorption with an A-negative liver preparation is specific for blood group A substance, and that ferritin present on the upper surfaces of the cells in such preparations is conjugated to antibody which is specifically attached to A substance. This ferritin is irregularly distributed along the upper surfaces of the cells; it is found on the level parts of the cell surface and on the microvilli but not within the coated vesicles. The ferritin molecules appear to be distributed singly or in pairs; occasionally they are in chains (Fig. 3, arrow) or clumps. Measurements of the perpendicular distance of the centres of ferritin molecules to the nearest point on a clearly defined membrane surface gave values ranging from 6 to 30 nm, with a majority between 8 and 15 nm. DISCUSSION The staining of some but not all RK 13 cells with fluorescein-labelled anti-a indicates the immunological nature of the staining (Franks & Dawson, 1966) and the control experiments show that the reaction between fraction 3 of the fluorescent conjugate, absorbed with A-negative liver homogenate, and RK 13 cells is a specific

5 Location of antigen A on cells in culture 529 reaction between anti-a and blood group antigen A. The results of the experiments with ferritin-labelled anti-a are in agreement with those with fluorescein-labelled anti-a, suggesting that ferritin staining with the absorbed conjugate is also specific for blood group antigen A, and that the ferritin molecules indicate the sites of the antigen on the membranes of RK 13 cells. The density of staining with ferritin-conjugated anti-a is not very great, indicating that blood group A substance is sparsely distributed on the surfaces of RK 13 cells. Some other authors have reported similar densities; for example, in the distribution of Rh 0 (D) antigen on human red blood cells (Lee & Feldman, 1964). Various factors such as the type of antibody (Haberman, Blanton & Martin, 1967; Lee & Feldman, 1964) and the thickness of the sections, which may vary considerably (Williams & Meek, 1966), will affect the apparent density of the ferritin staining. There are reports, however, of variations in the quantity of ferritin present which it seems entirely reasonable to accept as a genuine indication of antigen distribution. Lee & Feldman (1964) found a considerable difference between the amount of ferritin-anti-a and ferritin-anti-rh 0 (D) bound to human red blood cells. Their findings are in agreement with evidence provided by other methods. Aoki, Hammerling, De Harven, Boyse & Old (1969) found differing densities of ferritin present both in different areas of the same cell surface and on different cell types in an extensive survey of the distribution of H-2, 6, and thymus leukemia antigens on various mouse cells. It is possible that the distribution of A antigen and therefore of ferritin along the surfaces of RK 13 cells might show some variation and association with certain surface features. Evidence of this was looked for but it appears that the antigen is randomly distributed and shows no particular association with any identifiable feature such as the microvilli of the RK 13 cell surface. This is in accordance with the observation that blood group antigen A is uniformly distributed on the human erythrocyte surface (Lee & Feldman, 1964). Ferritin-labelled antibodies enable the sites of individual antigen molecules to be located, the resolution depending on the size of the ferritin-antibody conjugate which can be estimated approximately. Ferritin is a spherical molecule with a diameter of 11 nm and it has a central, iron-containing core with a diameter of about 5-5 nm; this core is visible in electron micrographs of thin sections and thus the centre of the molecule can be located. Feinstein & Rowe (1965) estimated that IgG antibody is in a folded configuration with an overall length of 10-5 nm when combined with 1 antigen molecule, and is extended, with a length of 20 nm, when combined with 2 antigen molecules, while Green (1969) suggested that the length of the IgG molecule from the antigen-combining site to the tips of the Fc fragment is 12 nm. The sizes of IgM molecules have been measured by Hoglund & Levin (1965), who give values of 20 x 30 nm. More recently Feinstein & Munn (1969) concluded that IgM molecules are approximately 30 nm long in the extended configuration. Thus the maximum distance of the antigen from the centre of the ferritin molecule is = 16 nm, = 17-5 nm or = 25-5 nm with an IgG molecule, and = 35-5 nm for an IgM molecule. These are maximum figures, since it is possible that the ferritin molecule is attached at some point along the antibody molecule, rather

6 530 E. Dimmock, D. Franks and A. M. Glauert than at the farthest point from the combining site with antigen. Furthermore the ferritin-antibody conjugate may be inclined at an angle to the beam within the section, with a consequent reduction in the apparent distance of the ferritin from the antigen. It can therefore be concluded that the antigen lies at a distance of 35-5 nm or less from the centre of the ferritin molecule. If it is known that only IgG is present, then this distance is reduced to 25-5, 17-5 or 16 nm, depending upon the configuration of the antibody. In the studies of the location of blood group antigen A on RK 13 cells the serum used contained IgG antibody in high titre, but no attempt was made to remove the IgM antibody, so that the antigen may be 35-5 nm from the centre of the ferritin. Measurements from the micrographs indicate that there is a peak in the distribution of ferritin molecules at a distance of 8-15 nm from the membrane. It seems likely that IgG molecules predominate in this serum, and assuming that the antibody is in the unextended configuration, it can be concluded that the blood group antigen A is located either within the visible structure of the membrane, which in these micrographs appears as a single dark line about 10 nm thick, or in the unstained surface layer of the membrane (Dimmock, 1970). Studies with fixation methods that reveal more of the membrane structure and on micrographs at higher magnification will be necessary before it will be possible to localize the antigen more precisely. We are grateful to Dame Honor Fell, F.R.S., for her continued interest in this work and to Mr R. A. Parker for skilled technical assistance. One of us (E.D.) is grateful to the Medical Research Council for a Scholarship for Training in Research Methods, and also to Dr L. B. Jaques and his secretarial staff for help with the typescript. REFERENCES AOKI, T., HAMMERLING, U., DE HARVEN, E., BOYSE, E. A. & OLD, L. J. (1969). Antigenic structure of cell surfaces. An immunoferritin study of the occurrence and topography of H-2, 6 and TL alloantigens on mouse cells. J. exp. Med. 130, COHEN, F., ZUELZER, W. W. & EVANS, M. M. (i960). Identification of blood group antigens and minor cell populations by the fluorescent antibody method. Blood 15, COOK, G. M. W. (1968). Chemistry of membranes. Br. med. Bull. 24, DAWSON, A. & FRANKS, D. (1967). Factors affecting the expression of blood group antigen A in cultured cells. Expl Cell Res. 47, DIMMOCK, E. (1970). The surface structure of cultured rabbit kidney cells as revealed by electron microscopy. J. Cell Sci. 7, FEINSTEIN, A. & MUNN, E. A. (1969). Conformation of the free and antigen-bound IgM antibody molecules. Nature, Lond. 224, FEINSTEIN, A. & ROWE, A. J. (1965). Molecular mechanism of formation of an antigen-antibody complex. Nature, Lond. 205, FRANKS, D. & DAWSON, A. (1966). Variation in the expression of blood group antigen A in clonal cultures of rabbit cells. Expl Cell Res. 42, GREEN, N. M. (1969). Electron microscopy of the immunoglobulins. Adv. Immxin. n, HABERMAN, S., BLANTON, P. & MARTIN, J. (1967). Some observations on the ABO antigen sites of the erythrocyte membranes of adults and newborn infants. J. Immun. 98, HAWES, M. D. & COOMBS, R. R. A. (1959). Demonstration of the A antigen on buccal epithelial cells of A-like rabbits. Int. Archs Allergy appl. Immun. 15, HOGLUND, S. & LEVIN, 0. (1965). Electron microscopic studies of some proteins from normal human serum. J. molec. Biol. 12,

7 Location of antigen A on cells in culture 531 LACHMANN, P. J. (1964). The reaction of sodium azide with fluorochromes. Immunology 7, LEE, R. E. & FELDMAN, J. D. (1964). Visualization of antigenic sites of human erythrocytes with ferritin-antibody conjugates.^. Cell Biol. 23, METZGER, J. F. & SMITH, C. W. (1962). The application of immune electron microscopy to the demonstration of antigenic sites in biologic systems. Lab. Invest. 11, MRENOVA, N. & ALBRECHT, P. (1966). Stabilization offluorescencein preparates treated by the fluorescent antibody technique. Nature, Land. 212, RIFKIND, R. A., Hsu, K. C. & MORGAN, C. (1964). Immunochemical staining for electron microscopy.,7. Histochem. Cytochem. 12, RIGGS, J. L., SEIWALD, R. J., BURCKHALTER, J. H., DOWNS, C. M. & METCALF, T. G. (1958). Isothiocyanate compounds as fluorescent labeling agents for immune serum. Am. J. Path. 34, SINGER, S. J. & MCLEAN, J. D. (1963). Ferritin-antibody conjugates as stains for electron microscopy. Lab. Invest. 12, WILLIAMS, M. A. & GREGORY, D. W. (1967). The use of bis-diazotized benzidine for preparing ferritin-conjugated antibodies for electron microscopy. Jl R. microsc. Soc. 86, WILLIAMS, M. A. & MEEK, G. A. (1966). Studies on thickness variation in ultrathin sections for electron microscopy. Jl R. microsc. Soc. 85, (Received 16 April Revised 3 September 1971)

8 532 E. Dimmock, D. Franks and A. M. Glauert Fig. i. Black and white print of a fluorescence colour photograph of RK 13 cells stained with fluorescein isothiocyanate-conjugated anti-a globulin at a dilution of 1 in 2. Stained cells appear green (light with prominent nuclei, arrow, in black and white). Unstained cells appear purplish (grey in black and white prints). Fixation with phosphate-buffered glutaraldehyde. x 420. Fig. 2. Electron micrograph of a thin section through the upper surface of an RK 13 cell stained with ferritin-conjugated anti-a globulin. Ferritin (small black dots) is present along the surface. Some ferritin is present in clumps (arrow). Fixation with phosphate-buffered glutaraldehyde and phosphate-buffered osmium tetroxide. Section stained with uranyl acetate and lead citrate, x Fig. 3. The upper surface of an RK 13 cell treated with ferritin-conjugated anti-a antibody which had been absorbed with A-negative formalinized rabbit liver homogenate. Ferritin is sparsely scattered along the cell surface. A linear array of ferritin molecules (arrow) is present. Fixation and staining as for Fig. 2. x

9 Location of antigen A on celb in culture 533

10