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The specificity of antibodies to a peptide determinant of the tobacco mosaic virus protein induced by immunization with the peptide conjugate
BIOCHIMICAET BIOPHYSICAACTA
509
BBA R e p o r t BBA 31111 The specificity o f antibodies to a p e p t i d e d e t e r m i n a n t o f the t o b a c c o mosaic virus protein i n d u c e d b y i m m u n i z a t i o n w i t h the peptide conjugate*
FRANK J. FEARNEY*,CHERRY Y. LEUNG**,JANIS DILLAHAYOUNG*** and E. BENJAMINI** *Bio-Rad Laboratories, 32nd and Griffin A venue, Richmond, Calif. 94804 (U.S.A.), **University o f California Medical School, Department o f Medical Microbiology, Davis, Calif. 95616 (U.S.A.) and ***University o f California, Space Sciences Laboratory, Berkeley, Calif. 94 720 (U.S.A.)
(Received July 19th, 1971) SUMMARY An antigenic decapeptide having the sequence Thr-Thr-Ala-Glu-Thr-Leu-AspAla-Thr-Arg representing residues 103-112 of the tobacco mosaic virus (TMV) protein, and the C-terminal pentapeptide portion of the decapeptide were conjugated to suceinylated bovine serum albumin. Rabbit antibodies produced following immunization with these conjugates were exhibited to bind with the homologous peptides, with TMV protein and with peptides which bind with anti-TMV-protein. The results indicate that the specificity of the receptor on immunocompetent cells parallels the specificity of the antibodies produced.
In previous studies we have characterized a determinate area of tobacco mosaic virus (TMV) protein 1,2. This area consists of a decapeptide having the sequence Thr-ThrAla-Glu-Thr-Leu-Asp--Ala-Thr-Arg, and representing residues 103-112 of TMV protein. The carboxyl pentapeptide sequence (residues 108-112) was the smallest peptide which exhibited binding with anti-TMV-protein. In the present communication we present our preliminary results on obtaining antibodies to this decapeptide and pentapeptide by immunization of rabbits with the peptides conjugated to succinylated bovine serum albumin, and on the specificity of the antibodies thus produced. EXPERIMENTAL Solid phase peptide syntheses of the four pentapeptides given in Table I and of N-tyrosyl-decapeptide were performed according to the-method of Merrifield as detailed by Abbreviation: TMV, tobacco mosaicvirus. *Address for correspondence: ProfessorE. Bonjamini,Divisionof the SciencesBasicto Medicine, Department of MedicalMicrobiology,Universityof Californiaat Davis, Davis,Calif. 95616, U.S.A. •
" "
Biochim. Biophys. Acta, 243 (1971) 509-514)
510
BBA REPORT
BBAREPORT
511
Stewart and Young a . A portion of each'pentapeptide (2 ttmoles) was acetylated with a 10 molar excess of [a H] acetic anhydride (100 mC/mmole, New England Nuclear Company) as previously described 1 . The specific activities of these peptides ranged from 36 500 to 39 500 counts/min per nmole. The tyrosyl-decapeptide was iodinated with Na12SI (New England Nuclear, 100 mC/mmole) using the method of Greenwood et aL 4. The 12sI-labeled peptide had a specific radioactivity of 3.3. lOs counts/min per nmole. The conjugate of the pentapeptide or the decapeptide with succinylated bovine serum albumin was prepared using essentially the carbodiirnide coupling method employed by Stason et a / s . Succinylated bovine serum albumin was prepared by adding succinic anhydride (511 mg), over a 1.5-h period, to 412 mg bovine serum albumin in 120 ml 0.1 M sodium phosphate buffer (pH 7). The solution was magnetically stirred and kept at pH 7 with 1 M NaOH. Following succinylation, the reaction mixture was dialyzed at 3°C, using 3 changes of 2 1 of cold water. The non-dialyzable material was freeze-dried and taken up in 5 ml water. A representative conjugation of the peptide to succinylated bovine serum albumin was performed as follows: USing magnetic stirring throughout, the pentapeptide Leu-Asp-AlaThr-Arg (48/amoles) was added to 1 ml of a succinylated bovine serum albumin solution prepared as described above. The volume was brought to 10 ml with water, and 120 mg of 1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide-HClwas added. Dioxane (10 ml), and approximately 0.15 ml triethylamine were added to give a pH of 7.5 to 8.0. The mixture was stirred overnight at room temperature and then dialyzed against three changes of 2 1 of cold water at 3°. The contents of the dialysisbag were centrifuged at 11 000 rev./min for 40 min. Both the supernatant and the precipitate were analyzed for amino acid content. Based on the expected phenylalanine content of bovine serum albumin and the increase in the amino acids of the pentapeptide, 62.7 nag of bovine serum albumin as succinylated bovine serum albumin and 12 #moles of pentapeptide were present in the supernatant, with the remaining succinylated bovine serum albumin and 3.5 tamoles of pentapeptide in the precipitate. The succinylated bovine serum albumin decapeptide was soluble and analyzed as 41 mg bovine serum albumin (as succinylated bovine serum albumin) conjugated with 12 tamoles decapeptide. The supernatants only were used for immunization and contaieed 0.18 and 0.3 ttmoles peptide per mg bovine serum albumin (in the form of succinylated bovine serum albumin) for the pentapeptide and decapeptide, respectively. The peptide-conjugates containing 1.5 to 2.0 nag bovine serum albumin as succinylated bovine serum albumin in 1 ml were emulsified with 1 ml of Frcund's complete adjuvant and injected into rabbits subcutaneously or intramuscularly. Each rabbit received one injection at multiple sites at weekly intervals for three weeks. One month later, the rabbits received one additional injection at multiple sites. Three weeks after the last injection, the animals were bled from the ear vein and the serum obtained. Globulins were prepared as previously described6 . The final volume of the non-dialyzable globulin solution was diluted to anA2so nm of 17.5 units/ml. Studies on the binding of 12sl-labeled tyrosyl-decapeptide with antibodies were performed by adding a2SI-labeled tyrosyl-decapeptide (0.01 or 0.05 m o l e ) to various volumes of globulins. When inhibitions were performed, TMV protein in various amounts was added and the volume made to 0.55 nil. The reactant mixture was incubated at room temperature for 15 min at the end of which time an equal volume of saturated ammonium .Biochir~ Biophya Acta, 243 (1971) 509-514
512
BBA REPORT
sulfate was added. The resulting precipitates were centrifuged, re-dissolved in saline and reprecipitated two additional times. The final precipitates were taken up in 0.5 ml saline and an aliquot was counted for radioactivity in a Nuclear Chicago Model 186A crystal counter. Assays of the binding of [3 H] acetylated peptides with antibodies were performed as described above except that 0.1 ml of the [~H] acetylated peptides (50 nmoles/ml) were added (instead of the 12sI-labeled tyrosyl-decapeptide) and 0.5 ml of the globulins were used. The final precipitates from the repeated ammonium sulfate precipitations were taken up in 0.4 ml saline and 0.2 ml aliquots were counted for radioactivity as described by Benjamini et aL 7 using a Nuclear Chicago Mark I liquid scintillation system. RESULTS AND DISCUSSION In the past few years it has been demonstrated that immunizations with preparations consisting of antigenic peptides derived from protein antigens conjugated to protein carriers may induce antibodies capable of reacting not only with the homologous peptides but also with the proteins from which these peptides were derived9-13 . It has previously been shown that the decapeptide having the sequence Thr-ThrAla-Glu-Thr-Leu-Asp-Ala-Thr-Arg representing sequence 103-112 of TMV protein, is a major antigenic determinant of the protein I . Moreover it was demonstrated that the C-terminal portion of the decapeptide was responsible for the antigenic activity of the latter2 . In view of the extensive characterization of antigenic specificity of the pentapeptide with respect to binding with anti-TMV-protein it was of interest to ascertain the relationship between peptide structure and immunogenicity. Since neither the pentapeptide nor the decapeptide were immunogenic14 it was necessary to approach the problem through investigations with peptide conjugates. Data presented in this communication show that immunogens consisting of succinylated bovine serum albumin to which either of the two antigenic peptides were conjugated, elicited, in rabbits, antibodies capable of reacting with the homologous peptide, with the protein TMV protein, and with peptides which bind with anti-TMV-protein. Data in Fig. 1 demonstrate that both anti-succinylated-bovine-serum-albumindecapeptide and anti-succinylated-bovine-serum-albumin-pentapeptide containedantibodies capable of binding with 12sI-labeled N-tyrosyl-decapeptide. The data also indicate that more of the test antigen was bound to anti-succinylated-bovine-serum-albumin-decapeptide than to anti-succinylated-bovine-serum-albumin pentapeptide. This phenomenon may reflect the presence of a larger antibody population directed towards the decapeptide (perhaps with higher affinities) in antiserum to succinylated-bovine-serum-albumin-decapeptide than in anti-succinylated-bovine-serum-albumin-pentapeptide, but with specificity still directed towards the C-terminal pentapeptide. On the other hand, it is possible that antisuccinylated-bovine-serum-albumin-decapeptide contains antibody populations directed not only against the C-terminal pentapeptide, but also against the N-terminal portion of the decapeptide. Data in Fig. 1 also show that the binding of 12sI-labeled tyrosyl-decapeptide with anti-TMV-protein is similar to that with anti-succinylated-bovine-serum-albumindecapeptide, suggesting that the immunogenic capacity of the decapeptide attached to succinylated bovine serum albumin is similar in character to the immunogenic capacity of Biochim. Biophy~ Acta, 243 (1971) 509-514
BBA REPORT
513
,.o 8 o o o _z_z nn 0 6000 (,.9 z 0 I--
Q 400O
Z
2000
6O 40
20
0.050.'0 o ~ o.~ o.~ o.~ ML GLOBULINS
o~ 1
~
b
~o
3b
,UG TMVP ADDED
Fig. 1. The binding of 12Sl-labeled tyrosyl-decapeptide (0.05 nmoles) to anti-TMV-protein(×); antisueeinylated-bovine-setum-albumin-decapeptide(o); anti-suecinylated-bovine-serurn-albuminpentapeptide (A) and normal rabbit globulins (o). Fig. 2. Inhibition of binding of *2Slqabeledtyrosyl-decapeptide (0.01 nmoles) with anti-sueeinylatedbovine-serum-albumin-deeapeptide(0.01 ml globulins) by TMV protein (TMVP). Identical weights of human skin gelatin or rat skin gelatin caused no inhibition. the peptide when it is situated in its native environment,/.e, in the TMV-protein molecule. Results on the inhibition of the reaction between 12sI-labeled N-tyrosyldecapeptide and anti-suecinylated-bovine-serum-albumin-decapeptide by TMV protein (Fig. 2) indicate not only that the antibodies are capable of binding with TMV protein but also that the specificity of the majority of the antibodies is against area(s) on the decapeptide which are also possessed by TMV protein. In fact the high degree of inhibition (albeit by relatively high concentrations of TMV protein) suggests that the serum contains antibodies to determinant(s) of the decapeptide situated on succinylated bovine serum albumin which are present on the decapeptide in TMV protein. The data indicate maximal inhibition of 80-85%. On the one hand this may reflect the presence of a minor population of antibodies directed against area(s) (perhaps conformation) on the decapeptide which are absent in TMV protein. On the other hand it is conceivable that still higher concentrations of TMV protein would result in increased inhibition. It is interesting to speculate at this point that the high TMV protein concentrations required for high degrees of inhibition reflect a situation where the affinities of the anti-succinylated-bovine-serum-albumin specific to the decapeptide are higher to the 12sl-labeled N-tyrosyl-decapeptide than to TMV protein. Data presented in Table I demonstrate the relative binding of several peptides with anti-succinylated-bovine-serum-albumin-pentapeptide. The data demonstrate that in a general way, those peptides which bind with anti-succinylated-bovine-serum-albumindecapeptide also bind with anti-TMV-protein. This phenomenon indicates that the specificity of the antibodies induced by immunization with TMV protein parallels the specificity of those antibodies produced by immunization with the antigenic pentapeptide conjugated to succinylated bovine serum albumin. This parallel specificity would be expected to occur if the receptor on the immunocompetent cell possesses the same specificity as the resulting antibodies. Biochim. Biophys. Acta, 243 (1971) 509-514
514
BBA REPORT
ACKNOWLEDGMENTS We wish to thank Mrs. Margareta Harris for sensitization of the rabbits and Mrs. Seguidina San Juan for performing the immunological assays. This work was supported in part by U.S. Public Health Service, National Institutes of Health grant No. AI-06040, and grant No. AM-15174. REFERENCES 1 J.M. Stewart, J.D. Young, E. Benjamini, M. Shimizu and C.Y. Letmg, Biochemistry, 5 (1966) 3396. 2 J.D. Young, E. Benjamini, J.M. Stewart and C.Y. Leung, Biochemistry, 6 (1967) 1455. 3 J.M. Stewart and J.D. Young, Solid Phase Pept~de Synthesis, W.H. Freeman and Co., San Francisco, 1969. 4 F.C. Greenwood, W.M. Hunter and J.S. Glover, Biochem. J., 89 (1963) 114. 5 W.B. Stason, M. VoUoton and E. Haber, Biochim. Biophys. Acta, 133 (1967) 582. 6 E. Benjamini, J.D. Young, W.J. Peterson, C.Y. Leung and M. Shimizu, Biochemistry, 4 (1965) 2081. 7 E. Benjamini, M. Shimizu, J.D. Young and C.Y. Leung, Biochemistry, 8 (1969) 2242. 8 J.D. Young, E. Benjamini and C.Y. Leung, Biochemistry, 7 (1968) 3133. 9 R. Arnon and M. Sela, Proc. Nat. Acacl ScL U.S., 62(1) (1969) 163, 10 E. Maron, C. Shiozawa, R. Arnon and M. Sela, Biochemistry, 10 (1971) 763. 11 F.A. Anderer, Biochim. Biophys. Acta, 71 (1963) 246. 12 F.A. Anderer and H.D. Schlumberger, Biochim. Biophys. Acta, 97 (1965) 503. 13 F.A. Anderer and H.D. Schlumberger, Biochira. Biophy~ Acta, 115 (1966) 222. 14 L. SpRier, E. Benjamini, J.D. Young, H. Kaplan and H.H. Fudenberg, J. Exptl Meg, 131 (1970) 133.
Biochim. Biophys. Acta, 243 (1971) 509-514
509
BBA R e p o r t BBA 31111 The specificity o f antibodies to a p e p t i d e d e t e r m i n a n t o f the t o b a c c o mosaic virus protein i n d u c e d b y i m m u n i z a t i o n w i t h the peptide conjugate*
FRANK J. FEARNEY*,CHERRY Y. LEUNG**,JANIS DILLAHAYOUNG*** and E. BENJAMINI** *Bio-Rad Laboratories, 32nd and Griffin A venue, Richmond, Calif. 94804 (U.S.A.), **University o f California Medical School, Department o f Medical Microbiology, Davis, Calif. 95616 (U.S.A.) and ***University o f California, Space Sciences Laboratory, Berkeley, Calif. 94 720 (U.S.A.)
(Received July 19th, 1971) SUMMARY An antigenic decapeptide having the sequence Thr-Thr-Ala-Glu-Thr-Leu-AspAla-Thr-Arg representing residues 103-112 of the tobacco mosaic virus (TMV) protein, and the C-terminal pentapeptide portion of the decapeptide were conjugated to suceinylated bovine serum albumin. Rabbit antibodies produced following immunization with these conjugates were exhibited to bind with the homologous peptides, with TMV protein and with peptides which bind with anti-TMV-protein. The results indicate that the specificity of the receptor on immunocompetent cells parallels the specificity of the antibodies produced.
In previous studies we have characterized a determinate area of tobacco mosaic virus (TMV) protein 1,2. This area consists of a decapeptide having the sequence Thr-ThrAla-Glu-Thr-Leu-Asp--Ala-Thr-Arg, and representing residues 103-112 of TMV protein. The carboxyl pentapeptide sequence (residues 108-112) was the smallest peptide which exhibited binding with anti-TMV-protein. In the present communication we present our preliminary results on obtaining antibodies to this decapeptide and pentapeptide by immunization of rabbits with the peptides conjugated to succinylated bovine serum albumin, and on the specificity of the antibodies thus produced. EXPERIMENTAL Solid phase peptide syntheses of the four pentapeptides given in Table I and of N-tyrosyl-decapeptide were performed according to the-method of Merrifield as detailed by Abbreviation: TMV, tobacco mosaicvirus. *Address for correspondence: ProfessorE. Bonjamini,Divisionof the SciencesBasicto Medicine, Department of MedicalMicrobiology,Universityof Californiaat Davis, Davis,Calif. 95616, U.S.A. •
" "
Biochim. Biophys. Acta, 243 (1971) 509-514)
510
BBA REPORT
BBAREPORT
511
Stewart and Young a . A portion of each'pentapeptide (2 ttmoles) was acetylated with a 10 molar excess of [a H] acetic anhydride (100 mC/mmole, New England Nuclear Company) as previously described 1 . The specific activities of these peptides ranged from 36 500 to 39 500 counts/min per nmole. The tyrosyl-decapeptide was iodinated with Na12SI (New England Nuclear, 100 mC/mmole) using the method of Greenwood et aL 4. The 12sI-labeled peptide had a specific radioactivity of 3.3. lOs counts/min per nmole. The conjugate of the pentapeptide or the decapeptide with succinylated bovine serum albumin was prepared using essentially the carbodiirnide coupling method employed by Stason et a / s . Succinylated bovine serum albumin was prepared by adding succinic anhydride (511 mg), over a 1.5-h period, to 412 mg bovine serum albumin in 120 ml 0.1 M sodium phosphate buffer (pH 7). The solution was magnetically stirred and kept at pH 7 with 1 M NaOH. Following succinylation, the reaction mixture was dialyzed at 3°C, using 3 changes of 2 1 of cold water. The non-dialyzable material was freeze-dried and taken up in 5 ml water. A representative conjugation of the peptide to succinylated bovine serum albumin was performed as follows: USing magnetic stirring throughout, the pentapeptide Leu-Asp-AlaThr-Arg (48/amoles) was added to 1 ml of a succinylated bovine serum albumin solution prepared as described above. The volume was brought to 10 ml with water, and 120 mg of 1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide-HClwas added. Dioxane (10 ml), and approximately 0.15 ml triethylamine were added to give a pH of 7.5 to 8.0. The mixture was stirred overnight at room temperature and then dialyzed against three changes of 2 1 of cold water at 3°. The contents of the dialysisbag were centrifuged at 11 000 rev./min for 40 min. Both the supernatant and the precipitate were analyzed for amino acid content. Based on the expected phenylalanine content of bovine serum albumin and the increase in the amino acids of the pentapeptide, 62.7 nag of bovine serum albumin as succinylated bovine serum albumin and 12 #moles of pentapeptide were present in the supernatant, with the remaining succinylated bovine serum albumin and 3.5 tamoles of pentapeptide in the precipitate. The succinylated bovine serum albumin decapeptide was soluble and analyzed as 41 mg bovine serum albumin (as succinylated bovine serum albumin) conjugated with 12 tamoles decapeptide. The supernatants only were used for immunization and contaieed 0.18 and 0.3 ttmoles peptide per mg bovine serum albumin (in the form of succinylated bovine serum albumin) for the pentapeptide and decapeptide, respectively. The peptide-conjugates containing 1.5 to 2.0 nag bovine serum albumin as succinylated bovine serum albumin in 1 ml were emulsified with 1 ml of Frcund's complete adjuvant and injected into rabbits subcutaneously or intramuscularly. Each rabbit received one injection at multiple sites at weekly intervals for three weeks. One month later, the rabbits received one additional injection at multiple sites. Three weeks after the last injection, the animals were bled from the ear vein and the serum obtained. Globulins were prepared as previously described6 . The final volume of the non-dialyzable globulin solution was diluted to anA2so nm of 17.5 units/ml. Studies on the binding of 12sl-labeled tyrosyl-decapeptide with antibodies were performed by adding a2SI-labeled tyrosyl-decapeptide (0.01 or 0.05 m o l e ) to various volumes of globulins. When inhibitions were performed, TMV protein in various amounts was added and the volume made to 0.55 nil. The reactant mixture was incubated at room temperature for 15 min at the end of which time an equal volume of saturated ammonium .Biochir~ Biophya Acta, 243 (1971) 509-514
512
BBA REPORT
sulfate was added. The resulting precipitates were centrifuged, re-dissolved in saline and reprecipitated two additional times. The final precipitates were taken up in 0.5 ml saline and an aliquot was counted for radioactivity in a Nuclear Chicago Model 186A crystal counter. Assays of the binding of [3 H] acetylated peptides with antibodies were performed as described above except that 0.1 ml of the [~H] acetylated peptides (50 nmoles/ml) were added (instead of the 12sI-labeled tyrosyl-decapeptide) and 0.5 ml of the globulins were used. The final precipitates from the repeated ammonium sulfate precipitations were taken up in 0.4 ml saline and 0.2 ml aliquots were counted for radioactivity as described by Benjamini et aL 7 using a Nuclear Chicago Mark I liquid scintillation system. RESULTS AND DISCUSSION In the past few years it has been demonstrated that immunizations with preparations consisting of antigenic peptides derived from protein antigens conjugated to protein carriers may induce antibodies capable of reacting not only with the homologous peptides but also with the proteins from which these peptides were derived9-13 . It has previously been shown that the decapeptide having the sequence Thr-ThrAla-Glu-Thr-Leu-Asp-Ala-Thr-Arg representing sequence 103-112 of TMV protein, is a major antigenic determinant of the protein I . Moreover it was demonstrated that the C-terminal portion of the decapeptide was responsible for the antigenic activity of the latter2 . In view of the extensive characterization of antigenic specificity of the pentapeptide with respect to binding with anti-TMV-protein it was of interest to ascertain the relationship between peptide structure and immunogenicity. Since neither the pentapeptide nor the decapeptide were immunogenic14 it was necessary to approach the problem through investigations with peptide conjugates. Data presented in this communication show that immunogens consisting of succinylated bovine serum albumin to which either of the two antigenic peptides were conjugated, elicited, in rabbits, antibodies capable of reacting with the homologous peptide, with the protein TMV protein, and with peptides which bind with anti-TMV-protein. Data in Fig. 1 demonstrate that both anti-succinylated-bovine-serum-albumindecapeptide and anti-succinylated-bovine-serum-albumin-pentapeptide containedantibodies capable of binding with 12sI-labeled N-tyrosyl-decapeptide. The data also indicate that more of the test antigen was bound to anti-succinylated-bovine-serum-albumin-decapeptide than to anti-succinylated-bovine-serum-albumin pentapeptide. This phenomenon may reflect the presence of a larger antibody population directed towards the decapeptide (perhaps with higher affinities) in antiserum to succinylated-bovine-serum-albumin-decapeptide than in anti-succinylated-bovine-serum-albumin-pentapeptide, but with specificity still directed towards the C-terminal pentapeptide. On the other hand, it is possible that antisuccinylated-bovine-serum-albumin-decapeptide contains antibody populations directed not only against the C-terminal pentapeptide, but also against the N-terminal portion of the decapeptide. Data in Fig. 1 also show that the binding of 12sI-labeled tyrosyl-decapeptide with anti-TMV-protein is similar to that with anti-succinylated-bovine-serum-albumindecapeptide, suggesting that the immunogenic capacity of the decapeptide attached to succinylated bovine serum albumin is similar in character to the immunogenic capacity of Biochim. Biophy~ Acta, 243 (1971) 509-514
BBA REPORT
513
,.o 8 o o o _z_z nn 0 6000 (,.9 z 0 I--
Q 400O
Z
2000
6O 40
20
0.050.'0 o ~ o.~ o.~ o.~ ML GLOBULINS
o~ 1
~
b
~o
3b
,UG TMVP ADDED
Fig. 1. The binding of 12Sl-labeled tyrosyl-decapeptide (0.05 nmoles) to anti-TMV-protein(×); antisueeinylated-bovine-setum-albumin-decapeptide(o); anti-suecinylated-bovine-serurn-albuminpentapeptide (A) and normal rabbit globulins (o). Fig. 2. Inhibition of binding of *2Slqabeledtyrosyl-decapeptide (0.01 nmoles) with anti-sueeinylatedbovine-serum-albumin-deeapeptide(0.01 ml globulins) by TMV protein (TMVP). Identical weights of human skin gelatin or rat skin gelatin caused no inhibition. the peptide when it is situated in its native environment,/.e, in the TMV-protein molecule. Results on the inhibition of the reaction between 12sI-labeled N-tyrosyldecapeptide and anti-suecinylated-bovine-serum-albumin-decapeptide by TMV protein (Fig. 2) indicate not only that the antibodies are capable of binding with TMV protein but also that the specificity of the majority of the antibodies is against area(s) on the decapeptide which are also possessed by TMV protein. In fact the high degree of inhibition (albeit by relatively high concentrations of TMV protein) suggests that the serum contains antibodies to determinant(s) of the decapeptide situated on succinylated bovine serum albumin which are present on the decapeptide in TMV protein. The data indicate maximal inhibition of 80-85%. On the one hand this may reflect the presence of a minor population of antibodies directed against area(s) (perhaps conformation) on the decapeptide which are absent in TMV protein. On the other hand it is conceivable that still higher concentrations of TMV protein would result in increased inhibition. It is interesting to speculate at this point that the high TMV protein concentrations required for high degrees of inhibition reflect a situation where the affinities of the anti-succinylated-bovine-serum-albumin specific to the decapeptide are higher to the 12sl-labeled N-tyrosyl-decapeptide than to TMV protein. Data presented in Table I demonstrate the relative binding of several peptides with anti-succinylated-bovine-serum-albumin-pentapeptide. The data demonstrate that in a general way, those peptides which bind with anti-succinylated-bovine-serum-albumindecapeptide also bind with anti-TMV-protein. This phenomenon indicates that the specificity of the antibodies induced by immunization with TMV protein parallels the specificity of those antibodies produced by immunization with the antigenic pentapeptide conjugated to succinylated bovine serum albumin. This parallel specificity would be expected to occur if the receptor on the immunocompetent cell possesses the same specificity as the resulting antibodies. Biochim. Biophys. Acta, 243 (1971) 509-514
514
BBA REPORT
ACKNOWLEDGMENTS We wish to thank Mrs. Margareta Harris for sensitization of the rabbits and Mrs. Seguidina San Juan for performing the immunological assays. This work was supported in part by U.S. Public Health Service, National Institutes of Health grant No. AI-06040, and grant No. AM-15174. REFERENCES 1 J.M. Stewart, J.D. Young, E. Benjamini, M. Shimizu and C.Y. Letmg, Biochemistry, 5 (1966) 3396. 2 J.D. Young, E. Benjamini, J.M. Stewart and C.Y. Leung, Biochemistry, 6 (1967) 1455. 3 J.M. Stewart and J.D. Young, Solid Phase Pept~de Synthesis, W.H. Freeman and Co., San Francisco, 1969. 4 F.C. Greenwood, W.M. Hunter and J.S. Glover, Biochem. J., 89 (1963) 114. 5 W.B. Stason, M. VoUoton and E. Haber, Biochim. Biophys. Acta, 133 (1967) 582. 6 E. Benjamini, J.D. Young, W.J. Peterson, C.Y. Leung and M. Shimizu, Biochemistry, 4 (1965) 2081. 7 E. Benjamini, M. Shimizu, J.D. Young and C.Y. Leung, Biochemistry, 8 (1969) 2242. 8 J.D. Young, E. Benjamini and C.Y. Leung, Biochemistry, 7 (1968) 3133. 9 R. Arnon and M. Sela, Proc. Nat. Acacl ScL U.S., 62(1) (1969) 163, 10 E. Maron, C. Shiozawa, R. Arnon and M. Sela, Biochemistry, 10 (1971) 763. 11 F.A. Anderer, Biochim. Biophys. Acta, 71 (1963) 246. 12 F.A. Anderer and H.D. Schlumberger, Biochim. Biophys. Acta, 97 (1965) 503. 13 F.A. Anderer and H.D. Schlumberger, Biochira. Biophy~ Acta, 115 (1966) 222. 14 L. SpRier, E. Benjamini, J.D. Young, H. Kaplan and H.H. Fudenberg, J. Exptl Meg, 131 (1970) 133.
Biochim. Biophys. Acta, 243 (1971) 509-514