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Multivalent hybrid antibody with double specificity as a tool for locating cell surface antigens by electron microscopy
Journal of Immunological Methods, 35 (1980) 33--41
33
© Elsevier/North-Holland Biomedical Press
MULTIVALENT HYBRID ANTIBODY WITH DOUBLE SPECIFICITY AS A TOOL F O R LOCATING CELL SURFACE ANTIGENS BY ELECTRON MICROSCOPY
E. MANDACHE, ELENA MOLDOVEANU, GABRIELA MOTA, I. MORARU and V. GHETIE Babeq Institute, Spl. Independen~ei 99, R-76201 Bucharest, Rumania
(Received 31 October 1979, accepted 23 January 1980)
Multivalent hybrid antibody complexes with dual specificity were prepared by combining rabbit antibody with anti-mouse immunoglobulin (mIg) and anti-peroxidase (HRP) specificity using protein A of Staphylococcus aureus. The presence of two antibody molecules with anti-mIg and anti-HRP specificity in a single molecule of hybrid complex was demonstrated by their abilities to produce hemagglutination with both HRP-coated and mIg-coated sheep red cells, to give a reaction of complete identity with mIg and HRP and to allow mIg bearing lymphoeytes to form rosettes with HRP-coated sheep red blood ceils. Electron microscopy of mouse lymphocytes and thymocytes (previously coated with mIg anti-Thy-1 antibody) treated with hybrid antibody complex and HRP showed strong and specific staining of the cell membrane of both cell types. The hybrid antibody complex containing anti-HRP antibody is a valuable reagent for determining various antigenic markers on cell membranes by electron microscopy.
INTRODUCTION U n i v a l e n t a n t i b o d y molecules with double specificity were obtained by combining univalent Fab' fragments from antibodies of different specificity (Nisonoff ~qd Rivers, 1961). Such hybrid antibodies with different double specificities have been used for locating cell surface antigens by electron microscopy (H~immerling et al., 1968; Aoki et al., 1969; H~immerling and Rajewsky, 1971). M u l t i v a l e n t hybrid a n t i b o d y complexes with dual specificity were prepared by combining two antibodies with different specificities using protein A of S t a p h y l o c o c c u s aureus (SPA) (Ghe~ie and Mona, 1980). The soluble complex is composed of 4 molecules of IgG and 2 molecules of SpA (mol.wt. 684,000 daltons) and has the molecular formula (IgG anti-A/SpA/ IgG anti-B)2 (Mona et al., 1978; Mih~escu et al., 1979). Compared to the hybrid a n t i b o d y prepared by combination of Fab' fragments, the main advantage of the hybrid SpA-antibody complex is the simplicity of its preparation, its purity and its multivalency (Ghe~ie and Mona, 1980). Multivalent
34 hybrid SpA-antibody complexes might be useful tools for the location of antigenic markers on the cell surface. Results reported here show that multivalent hybrid antibody with antiimmunoglobulin and anti-peroxidase specificity is a sensitive reagent for detecting immunoglobulin molecules on the lymphoid cell surface by electron microscopy. MATERIALS AND METHODS
Proteins Protein A of Staphylococcus aureus (Pharmacia, Uppsala), peroxidase from horse radish (HRP) (BDH Chemicals Ltd.) and mouse immunoglobulin (mIg) obtained by preparative electrophoresis in agarose gel were used.
Antibodies Rabbit anti-HRP serum was kindly supplied by Dr. Doina Onic~ (Babe~ Institute, Bucharest). Anti-mIg serum was prepared in rabbits by repeated injections of mIg. IgG was isolated from rabbit anti-HRP and anti-mIg sera by DEAE-cellulose chromatography. Purified antibodies were obtained from these IgG preparations by adsorption on glutaraldehyde insolubilized HRP and mIg (Avrameas, 1969) and elution in 0.1 M glycine-HC1 buffer, pH 1.8.
Cells Spleen lymphocytes and th.ymocytes were isolated from CBA mice. The lymphocytes were subsequently purified by the Ficoll-sodium metrizoate technique (BCyum, 1968). Cells were washed and suspended in IC-65 medium (equivalent to TC-199 medium) (Cantacuzino Institute, Bucharest). Sheep red blood cells (E) were coated with HRP (EHRP), mIg (EmIg) or SpA (ES) (Ghe~ie et al., 1975) by the chromium chloride method (Gold and Fudenberg, 1967).
Preparation o f hybrid antibody with anti-HRP and anti-mIg specificities The preparation of hybrid anti-HRP/SpA/anti-mIg complexes starting either from an IgG fraction of antiserum or from purified antibody preparations was performed as described by Ghe}ie and Mo}a (1980). Briefly, antibody with specificity for HRP was mixed with SpA in 0.05 M NaC1 solution to final concentrations of 1 mg IgG/ml and 2 mg SpA/ml (molar ratio IgG/SpA = 1/7). After incubation for 1 h at 37°C the mixture was brought to pH 5.5 in 0.02 M acetate buffer by repeated ultrafiltration and further chromatographed on CM-Sephadex C-50 equilibrated with the same buffer.
35 The adsorbed IgG/SpA complex was eluted with phosphate buffered saline (PBS), pH 7.2, and the solution was concentrated to 4 mg IgG/ml. An equal volume of a solution containing the same concentration of rabbit antibody with specificity for mIg was then added and the mixture was incubated for 1 h at 37°C. The mixture was gel filtered on Sepharose 6B equilibrated with 0.1 M Tris-HC1 buffer pH 8.0, 0.2 M NaC1. The peak containing the hybrid a n t i b o d y complex (mol.wt. 669,000 daltons) was concentrated and further used in electron microscope studies.
Cell treatment Mouse spleen l y m p h o c y t e s (3 X 107) were suspended in 0.9 ml IC-65 medium and 0.1 ml hybrid antibody solution (10--200 pg) was added. After 30 min incubation at 4°C the cells were washed thrice with IC-65 medium and resuspended to 2 X 107 cells/0.9 ml. To this suspension 0.1 ml o f H R P (10--100/ag) in PBS was added and the mixture was incubated for 30 min at 4°C. After repeated washings the cells were pelleted by centrifugation. Mouse t h y m o c y t e s (107 cells/ml) were treated with 10 gl of mouse antiThy-1 serum (kindly supplied by Dr. A. Sulica, Babe~ Institute, Bucharest). After 30 min incubation at 4°C the cells were repeatedly washed with IC-65 medium, finally resuspended at 3 X 107 cells/0.9 ml, and were then processed in the same way as the mouse spleen lymphocytes. Control experiments with cell samples n o t treated either with hybrid antibody complex (for spleen lymphocytes) or with anti-Thy-1 serum (for t h y m o c y t e s ) were also performed.
Ultrastructural peroxidase processing The cell pellet was fixed for 5 min with 1.5% glutaraldehyde in 0.067 M sodium cacodylate buffer, pH 7.4. For uniform fixation the cells were resuspended in the fixing medium and centrifuged. The cells were then washed 3 times in 0.05 M Tris-HCl buffer, pH 7.4 and incubated for 15 min at r o o m temperature in a medium containing 0.05% 3,3'-diaminobenzidine and 0.02% H202 in 0.05 M Tris-HC1 buffer, pH 7.4, for peroxidase activation according to Graham and Karnovsky (1966). The cells were then washed and postfixed for 30 min in 1% OsO4 in sodium cacodylate buffer, pH 7.4, then washed and pelleted again. The pellet was incorporated in 1% Difco agar at 65°C and divided into 1 mm 3 samples. This procedure was followed b y dehydration and embedding in Epon 812 and processing for electron microscopy.
Labelled cells morphometry A planimetric determination of the labelled areas fraction (AAi) of the cellular membrane was performed using a Weibel grid with a hundred points.
36
Rosette technique Aliquots of cells treated with hybrid antibody complex or with hybrid antibody complex and HRP were rosetted with EHRP (106 cells/5 X 107 EHRP/0.5 ml). The mixture was centrifuged at 4°C for 10 min at 100 X g and further incubated at 4°C for 1 h. The pellet was then resuspended and about 300 cells were counted. Cells binding more than 3 erythrocytes were counted as rosettes. Aliquots of t h y m o c y t e s treated with anti-Thy-1 serum were rosetted with ES (106 cells/5 X 107 ES/0.5 ml) and further processed as described above.
Immunological analysis Double diffusion was performed in agarose gels. Microhemagglutination assay of hybrid antibody complex with EHRP or EmIg was performed in microtiter plates as described (Mih~escu et al., 1979). The titers were expressed as pg hybrid a n t i b o d y / m l of the final dilution giving a positive reaction. RESULTS The ability of anti-HRP/SpA/anti-mIg hybrid complex to react with both HRP and mIg was demonstrated by hemagglutination of E coated with HRP (EHRP) or mIg (EmIg) (Table 1). Double diffusion with IgG anti-HRP/SpA/IgG anti-mIg hybrid complex showed a reaction of comple.te identity between the precipitin lines with HRP and mIg in contrast to a mixture of IgG anti-HRP and IgG anti-mIg where a reaction of non-identity was observed. Mouse spleen lymphocytes or t h y m o c y t e s coated with mouse anti-Thy-1
TABLE 1 Hemagglutinating activity of anti-HRP/SpA/anti-mIg hybrid complex Ligands
Hybrid prepared from IgG fraction of antisera Hybrid prepared from purified antibody IgG anti-HRP IgG anti-mIg a NA = no agglutination.
Titer (pg ligand/ml) with EHRP
EmIg
2.0
7.8
0.3 2.9 NA a
0.9 NA a 8.2
37 TABLE 2 Ability of mouse lymphoid cells treated with anti-HRP/SpA]anti-mIg hybrid complex to form rosettes with sheep red blood cells coated with HRP Ligands
IgG anti-HRP/SpA/anti-mIg IgG anti-HRP (control) IgG anti-mIg (control)
Per cent of EHRP a formed by Spleen lymphocytes
Thymocytes
Thymocytes treated with anti-Thy-1
39.0 b ~1 <:1
(1 <1 <1
54.1 c <1 <1
a If E were used instead of EHRP no rosettes were formed. b If the cells were treated with HRP (5 pg/107 cells/ml) after the attachment of hybrid antibody to the cell surface, complete inhibition of the rosette formation was observed. c Thymocytes treated with mouse anti-Thy-1 sera showed 85% rosette formation with ES indicating that the hybrid antibody was not bound to the whole population of thymocytes bearing antibody to Thy-1 antigen.
Fig. 1. Spleen lymphocyte treated with hybrid antibody showing peroxidase positive labelled areas (arrows). × 25,000.
38
Fig. 2. T h y m o c y t e s c o a t e d with anti-Thy-1 a n t i b o d y and then treated with hybrid antib o d y . The arrows indicate the few labelled areas, x 20,000.
Fig. 3. Spleen l y m p h o c y t e s showing patch-like labelled areas. The large l y m p h o c y t e has the labelled areas c o n c e n t r a t e d in a z o n e with microviUous projections (arrows). X 22,000.
39
antibody and treated with IgG anti-HRP/SpA/IgG anti-mIg hybrid complex and with EHRP were able to form rosettes with the indicator cells (Table 2). Electron microscopy of mouse lymphocytes and thymocytes (previously treated with mouse anti-Thy-1 antibody) which had been reacted with the hybrid antibody complex and HRP (optimum concentration 100 pg/107 cells/ml) showed specific staining of the cell membrane of both cell types. The spleen lymphocytes showed strong positive labelling when treated with a high concentration of hybrid (200 pg/3 X 107 cells/ml) (Fig. 1). The thymocytes initially coated with anti-Thy-1 antibody and treated with the hybrid antibody (200 ttg/3 X l 0 T cells/ml) and HRP showed ultrastructural labelling of their cell membranes similar to that of lymphocytes but less extensive (Fig. 2). Control preparations showed no membrane labelling. The labelling pattern on ultrathin sections consisted of electron-dense lines of different lengths on the outer aspect of the plasma membrane, suggesting a patch-like distribution. Some cells showed patch-like labelled areas spread at random all over the cell surface while other spleen lymphocytes showed polarization of these areas to irregular regions of the cellular
Fig. 4. Granulocyte unaffected by the labelling process beside a labelled lymphocyte. x 20,000.
40 membrane (Fig. 3). It is worth mentioning that the few non-lymphocyte cells present in both spleen l y m p h o c y t e and t h y m o c y t e samples were completely unaffected by treatment with antibody hybrid complex (Fig. 4). By counting 60 spleen l y m p h o c y t e s (as judged by their ultrastructure) we noted t h a t 53.3% of the cells were labelled. Morphometry of spleen lymphocytes showed that the average labelled fraction of the cell surface was 48.9 + 2.7 {mean of 14 measurements). DISCUSSION The presence of two antibody molecules with different specificities linked by SpA to form a single hybrid complex molecule was clearly demonstrated by their abilities to produce hemagglutination of both EHRP and EmIg, to give on double diffusion a reaction of complete identity between two antigenically unrelated proteins (HRP and mIg), and to allow Ig-bearing mouse spleen B l y m p h o c y t e s or Thy-1 bearing t h y m o c y t e s charged with mIg anti-Thy-1 to form EHRP rosettes (see also Ghe~ie and Mona,
1980). The anti-peroxidase specific antibody seems to be the best choice as a marker c o m p o n e n t of such hybrids since purified HRP is highly active and after reaction with specific antibody remains stable w i t h o u t loss of activity. The labelled areas obtained with this anti-HRP/SpA/anti-mIg hybrid are clear electron
41 H~mmerling, U., T. Aoki, E. De Harven, E.A. Boyse and L.J. Old, 1968, J. Exp. Med. 128, 1461. Mih~escu, S., A. Sulica, J. SjSquist and V. Ghe~ie, 1979, Rev. Roum. Biochim. 16, 57. Mo]~a, G., V. GheJ~ie and J. SjSquist, 1978, Immunochemistry 15,625. Nisonoff, A. and M.M. Rivers, 1961, Arch. Biochem. Biophys. 93,460. Parish, C.R., 1975, Transplant. Rev. 25, 98.
33
© Elsevier/North-Holland Biomedical Press
MULTIVALENT HYBRID ANTIBODY WITH DOUBLE SPECIFICITY AS A TOOL F O R LOCATING CELL SURFACE ANTIGENS BY ELECTRON MICROSCOPY
E. MANDACHE, ELENA MOLDOVEANU, GABRIELA MOTA, I. MORARU and V. GHETIE Babeq Institute, Spl. Independen~ei 99, R-76201 Bucharest, Rumania
(Received 31 October 1979, accepted 23 January 1980)
Multivalent hybrid antibody complexes with dual specificity were prepared by combining rabbit antibody with anti-mouse immunoglobulin (mIg) and anti-peroxidase (HRP) specificity using protein A of Staphylococcus aureus. The presence of two antibody molecules with anti-mIg and anti-HRP specificity in a single molecule of hybrid complex was demonstrated by their abilities to produce hemagglutination with both HRP-coated and mIg-coated sheep red cells, to give a reaction of complete identity with mIg and HRP and to allow mIg bearing lymphoeytes to form rosettes with HRP-coated sheep red blood ceils. Electron microscopy of mouse lymphocytes and thymocytes (previously coated with mIg anti-Thy-1 antibody) treated with hybrid antibody complex and HRP showed strong and specific staining of the cell membrane of both cell types. The hybrid antibody complex containing anti-HRP antibody is a valuable reagent for determining various antigenic markers on cell membranes by electron microscopy.
INTRODUCTION U n i v a l e n t a n t i b o d y molecules with double specificity were obtained by combining univalent Fab' fragments from antibodies of different specificity (Nisonoff ~qd Rivers, 1961). Such hybrid antibodies with different double specificities have been used for locating cell surface antigens by electron microscopy (H~immerling et al., 1968; Aoki et al., 1969; H~immerling and Rajewsky, 1971). M u l t i v a l e n t hybrid a n t i b o d y complexes with dual specificity were prepared by combining two antibodies with different specificities using protein A of S t a p h y l o c o c c u s aureus (SPA) (Ghe~ie and Mona, 1980). The soluble complex is composed of 4 molecules of IgG and 2 molecules of SpA (mol.wt. 684,000 daltons) and has the molecular formula (IgG anti-A/SpA/ IgG anti-B)2 (Mona et al., 1978; Mih~escu et al., 1979). Compared to the hybrid a n t i b o d y prepared by combination of Fab' fragments, the main advantage of the hybrid SpA-antibody complex is the simplicity of its preparation, its purity and its multivalency (Ghe~ie and Mona, 1980). Multivalent
34 hybrid SpA-antibody complexes might be useful tools for the location of antigenic markers on the cell surface. Results reported here show that multivalent hybrid antibody with antiimmunoglobulin and anti-peroxidase specificity is a sensitive reagent for detecting immunoglobulin molecules on the lymphoid cell surface by electron microscopy. MATERIALS AND METHODS
Proteins Protein A of Staphylococcus aureus (Pharmacia, Uppsala), peroxidase from horse radish (HRP) (BDH Chemicals Ltd.) and mouse immunoglobulin (mIg) obtained by preparative electrophoresis in agarose gel were used.
Antibodies Rabbit anti-HRP serum was kindly supplied by Dr. Doina Onic~ (Babe~ Institute, Bucharest). Anti-mIg serum was prepared in rabbits by repeated injections of mIg. IgG was isolated from rabbit anti-HRP and anti-mIg sera by DEAE-cellulose chromatography. Purified antibodies were obtained from these IgG preparations by adsorption on glutaraldehyde insolubilized HRP and mIg (Avrameas, 1969) and elution in 0.1 M glycine-HC1 buffer, pH 1.8.
Cells Spleen lymphocytes and th.ymocytes were isolated from CBA mice. The lymphocytes were subsequently purified by the Ficoll-sodium metrizoate technique (BCyum, 1968). Cells were washed and suspended in IC-65 medium (equivalent to TC-199 medium) (Cantacuzino Institute, Bucharest). Sheep red blood cells (E) were coated with HRP (EHRP), mIg (EmIg) or SpA (ES) (Ghe~ie et al., 1975) by the chromium chloride method (Gold and Fudenberg, 1967).
Preparation o f hybrid antibody with anti-HRP and anti-mIg specificities The preparation of hybrid anti-HRP/SpA/anti-mIg complexes starting either from an IgG fraction of antiserum or from purified antibody preparations was performed as described by Ghe}ie and Mo}a (1980). Briefly, antibody with specificity for HRP was mixed with SpA in 0.05 M NaC1 solution to final concentrations of 1 mg IgG/ml and 2 mg SpA/ml (molar ratio IgG/SpA = 1/7). After incubation for 1 h at 37°C the mixture was brought to pH 5.5 in 0.02 M acetate buffer by repeated ultrafiltration and further chromatographed on CM-Sephadex C-50 equilibrated with the same buffer.
35 The adsorbed IgG/SpA complex was eluted with phosphate buffered saline (PBS), pH 7.2, and the solution was concentrated to 4 mg IgG/ml. An equal volume of a solution containing the same concentration of rabbit antibody with specificity for mIg was then added and the mixture was incubated for 1 h at 37°C. The mixture was gel filtered on Sepharose 6B equilibrated with 0.1 M Tris-HC1 buffer pH 8.0, 0.2 M NaC1. The peak containing the hybrid a n t i b o d y complex (mol.wt. 669,000 daltons) was concentrated and further used in electron microscope studies.
Cell treatment Mouse spleen l y m p h o c y t e s (3 X 107) were suspended in 0.9 ml IC-65 medium and 0.1 ml hybrid antibody solution (10--200 pg) was added. After 30 min incubation at 4°C the cells were washed thrice with IC-65 medium and resuspended to 2 X 107 cells/0.9 ml. To this suspension 0.1 ml o f H R P (10--100/ag) in PBS was added and the mixture was incubated for 30 min at 4°C. After repeated washings the cells were pelleted by centrifugation. Mouse t h y m o c y t e s (107 cells/ml) were treated with 10 gl of mouse antiThy-1 serum (kindly supplied by Dr. A. Sulica, Babe~ Institute, Bucharest). After 30 min incubation at 4°C the cells were repeatedly washed with IC-65 medium, finally resuspended at 3 X 107 cells/0.9 ml, and were then processed in the same way as the mouse spleen lymphocytes. Control experiments with cell samples n o t treated either with hybrid antibody complex (for spleen lymphocytes) or with anti-Thy-1 serum (for t h y m o c y t e s ) were also performed.
Ultrastructural peroxidase processing The cell pellet was fixed for 5 min with 1.5% glutaraldehyde in 0.067 M sodium cacodylate buffer, pH 7.4. For uniform fixation the cells were resuspended in the fixing medium and centrifuged. The cells were then washed 3 times in 0.05 M Tris-HCl buffer, pH 7.4 and incubated for 15 min at r o o m temperature in a medium containing 0.05% 3,3'-diaminobenzidine and 0.02% H202 in 0.05 M Tris-HC1 buffer, pH 7.4, for peroxidase activation according to Graham and Karnovsky (1966). The cells were then washed and postfixed for 30 min in 1% OsO4 in sodium cacodylate buffer, pH 7.4, then washed and pelleted again. The pellet was incorporated in 1% Difco agar at 65°C and divided into 1 mm 3 samples. This procedure was followed b y dehydration and embedding in Epon 812 and processing for electron microscopy.
Labelled cells morphometry A planimetric determination of the labelled areas fraction (AAi) of the cellular membrane was performed using a Weibel grid with a hundred points.
36
Rosette technique Aliquots of cells treated with hybrid antibody complex or with hybrid antibody complex and HRP were rosetted with EHRP (106 cells/5 X 107 EHRP/0.5 ml). The mixture was centrifuged at 4°C for 10 min at 100 X g and further incubated at 4°C for 1 h. The pellet was then resuspended and about 300 cells were counted. Cells binding more than 3 erythrocytes were counted as rosettes. Aliquots of t h y m o c y t e s treated with anti-Thy-1 serum were rosetted with ES (106 cells/5 X 107 ES/0.5 ml) and further processed as described above.
Immunological analysis Double diffusion was performed in agarose gels. Microhemagglutination assay of hybrid antibody complex with EHRP or EmIg was performed in microtiter plates as described (Mih~escu et al., 1979). The titers were expressed as pg hybrid a n t i b o d y / m l of the final dilution giving a positive reaction. RESULTS The ability of anti-HRP/SpA/anti-mIg hybrid complex to react with both HRP and mIg was demonstrated by hemagglutination of E coated with HRP (EHRP) or mIg (EmIg) (Table 1). Double diffusion with IgG anti-HRP/SpA/IgG anti-mIg hybrid complex showed a reaction of comple.te identity between the precipitin lines with HRP and mIg in contrast to a mixture of IgG anti-HRP and IgG anti-mIg where a reaction of non-identity was observed. Mouse spleen lymphocytes or t h y m o c y t e s coated with mouse anti-Thy-1
TABLE 1 Hemagglutinating activity of anti-HRP/SpA/anti-mIg hybrid complex Ligands
Hybrid prepared from IgG fraction of antisera Hybrid prepared from purified antibody IgG anti-HRP IgG anti-mIg a NA = no agglutination.
Titer (pg ligand/ml) with EHRP
EmIg
2.0
7.8
0.3 2.9 NA a
0.9 NA a 8.2
37 TABLE 2 Ability of mouse lymphoid cells treated with anti-HRP/SpA]anti-mIg hybrid complex to form rosettes with sheep red blood cells coated with HRP Ligands
IgG anti-HRP/SpA/anti-mIg IgG anti-HRP (control) IgG anti-mIg (control)
Per cent of EHRP a formed by Spleen lymphocytes
Thymocytes
Thymocytes treated with anti-Thy-1
39.0 b ~1 <:1
(1 <1 <1
54.1 c <1 <1
a If E were used instead of EHRP no rosettes were formed. b If the cells were treated with HRP (5 pg/107 cells/ml) after the attachment of hybrid antibody to the cell surface, complete inhibition of the rosette formation was observed. c Thymocytes treated with mouse anti-Thy-1 sera showed 85% rosette formation with ES indicating that the hybrid antibody was not bound to the whole population of thymocytes bearing antibody to Thy-1 antigen.
Fig. 1. Spleen lymphocyte treated with hybrid antibody showing peroxidase positive labelled areas (arrows). × 25,000.
38
Fig. 2. T h y m o c y t e s c o a t e d with anti-Thy-1 a n t i b o d y and then treated with hybrid antib o d y . The arrows indicate the few labelled areas, x 20,000.
Fig. 3. Spleen l y m p h o c y t e s showing patch-like labelled areas. The large l y m p h o c y t e has the labelled areas c o n c e n t r a t e d in a z o n e with microviUous projections (arrows). X 22,000.
39
antibody and treated with IgG anti-HRP/SpA/IgG anti-mIg hybrid complex and with EHRP were able to form rosettes with the indicator cells (Table 2). Electron microscopy of mouse lymphocytes and thymocytes (previously treated with mouse anti-Thy-1 antibody) which had been reacted with the hybrid antibody complex and HRP (optimum concentration 100 pg/107 cells/ml) showed specific staining of the cell membrane of both cell types. The spleen lymphocytes showed strong positive labelling when treated with a high concentration of hybrid (200 pg/3 X 107 cells/ml) (Fig. 1). The thymocytes initially coated with anti-Thy-1 antibody and treated with the hybrid antibody (200 ttg/3 X l 0 T cells/ml) and HRP showed ultrastructural labelling of their cell membranes similar to that of lymphocytes but less extensive (Fig. 2). Control preparations showed no membrane labelling. The labelling pattern on ultrathin sections consisted of electron-dense lines of different lengths on the outer aspect of the plasma membrane, suggesting a patch-like distribution. Some cells showed patch-like labelled areas spread at random all over the cell surface while other spleen lymphocytes showed polarization of these areas to irregular regions of the cellular
Fig. 4. Granulocyte unaffected by the labelling process beside a labelled lymphocyte. x 20,000.
40 membrane (Fig. 3). It is worth mentioning that the few non-lymphocyte cells present in both spleen l y m p h o c y t e and t h y m o c y t e samples were completely unaffected by treatment with antibody hybrid complex (Fig. 4). By counting 60 spleen l y m p h o c y t e s (as judged by their ultrastructure) we noted t h a t 53.3% of the cells were labelled. Morphometry of spleen lymphocytes showed that the average labelled fraction of the cell surface was 48.9 + 2.7 {mean of 14 measurements). DISCUSSION The presence of two antibody molecules with different specificities linked by SpA to form a single hybrid complex molecule was clearly demonstrated by their abilities to produce hemagglutination of both EHRP and EmIg, to give on double diffusion a reaction of complete identity between two antigenically unrelated proteins (HRP and mIg), and to allow Ig-bearing mouse spleen B l y m p h o c y t e s or Thy-1 bearing t h y m o c y t e s charged with mIg anti-Thy-1 to form EHRP rosettes (see also Ghe~ie and Mona,
1980). The anti-peroxidase specific antibody seems to be the best choice as a marker c o m p o n e n t of such hybrids since purified HRP is highly active and after reaction with specific antibody remains stable w i t h o u t loss of activity. The labelled areas obtained with this anti-HRP/SpA/anti-mIg hybrid are clear electron
41 H~mmerling, U., T. Aoki, E. De Harven, E.A. Boyse and L.J. Old, 1968, J. Exp. Med. 128, 1461. Mih~escu, S., A. Sulica, J. SjSquist and V. Ghe~ie, 1979, Rev. Roum. Biochim. 16, 57. Mo]~a, G., V. GheJ~ie and J. SjSquist, 1978, Immunochemistry 15,625. Nisonoff, A. and M.M. Rivers, 1961, Arch. Biochem. Biophys. 93,460. Parish, C.R., 1975, Transplant. Rev. 25, 98.