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A new fluorescent label for antibody proteins
ARCHIVES
OF
BIOCHEMISTRY
AND
A New
Fluorescent
FELIX From
87, 293-%7 (1960)
BIOPHYSICS
BOREK
the Armed
Label AND
Forces
for
ARTHUR
Institute
Antibody
Proteins
M. SILVERSTEIN
of Pathology,
Washington,
D. C.
Received October 23, 1959 A new orange-fluorescent dye, aminorosamine B, was prepared by condensation of 2 moles m-diethylaminophenol with 1 mole p-nitrobenzaldehyde, followed by catalytic reduction of the nitro group. This dye was found to be useful as a label for antibody proteins, with which it may be conjugated as an isocyanate, isothiocyanate, or diazonium derivative. The optimal dye-protein ratio was determined corresponding to a maximum intensity of staining with a minimum decrease in antibody activity. A diazorosamine B-antibody conjugate was found useful in the staining of mixed bacterial smears, in conjunction with a fluorescein-antibody conjugate. INTRODUCTION
sufficiently different from that of fluorescein so that one might be readily distinguished Following the introduction by Coons and from the other. (d) If possible, each color co-workers of an immunohistochemical should be seen independently when both technique using antibodies labeled with are present in the samearea. (e) The conjugafluorescein (l), and its varied applications in tion with the dye should leave essentially unmany fields [reviewed in Ref. (2)], the employment of a second fluorescent label of changed the original titer and immunochemical specificity of the antibody protein. contrasting color appeared desirable for two A number of Auorochromes have been reasons. First, certain tissues, like thyroid, suggestedin the last few years as candidates show a natural blue-green fluorescence, for the position of a second fluorescent which contraists poorly with the yellow-green label. For example, Clayton (6) labeled antiemission of fluorescein. Here a label of a bodies with 1-dimethylaminonaphthalene-5color farther removed from blue is needed sulfonyl chloride and nuclear fast red to (3). Secondly, the use of a label of contrastproduce yellow and red fluorescence, respecing color in conjunction with fluorescein tively. Silverstein (4) introduced orangewould be advantageous in the staining of fluorescent conjugates of rhodamine B isomixed bacterial smears, in which each kind cyanate, while Riggs et al. (7) employed the of organism might be stained with a difisothiocyanate derivative of the same dye. ferent colored label (4). The requirements that a second label Hiramoto et al. (3) used conjugates of the tetramethyl homolog of rhodamine B isoshould satisfy in order to be useful have been cyanate. Chadwick and co-workers (5) resummarized by Silverstein (4) and by Chadported application of orange-fluorescent prowick et al. (5). These are: (a) The dye used tein conjugates prepared from a sulfonyl should be ca,pableof forming stable chemical chloride derivative of lissamine rhodamine bonds with proteins such as azo, carbamido, B 200. These investigators screened eight or sulfonamido linkages. (b) The intensity of other fluorescent dyes, including a derivative fluorescence should be sufficiently high to of rhodamine B, for their applicability to permit the visualization of the antibodyantibody labeling and found none suitable, antigen complex at the relatively low con- either becauseof low intensity of fluorescence centrations often present in the area of or because of undesirable color characterinterest. (c) The fluorescent color should be istics. 293
BOREK AND SILVERSTEIN
294
We wish to report the preparation of a new fluorescent dye that, according to preliminary results, may be used to advantage as a second antibody label. The dye is an amino derivative of 2,6-tetraethyldiamino-g-phenylxanthene, and by analogy with aminorhodamine B, we have called it aminorosamine B. By conkast with aminorhodamine B, which is obtained as a mixture of two isomers (differing in the position of the amino group in the phenyl ring) (4, S), aminorosamine B cannot form isomers and is therefore purified more easily. Its conjugation to antibody protein by the diazotization and coupling method yielded a conjugate more intensely fluorescent than those prepared from the isocyanate and isothiocyanate derivatives of the same dye.
L
k
J
-Nt2
(1)
-NH2
w
- NHZ.HCI
(ml
- N=C=O
mu
-N=C=s -&N
Cle
(PI (SE)
EXPERIMENTAL’
PREPARATION
OF MATERIALS
4 ‘-Nitrorosamine B (I) A mixture of p-nitrobenzaldehyde (3.8 g.; 25 mmoles) and m-diethylaminophenol (8.25 g.; 50 mmoles) in 50 ml. of 50y0 sulfuric acid was heated at 130-140” for 5 hr. The reaction mixture was cooled, and 11. of 3’% sodium hydroxide was added, resulting in the precipitation of a purple-red solid. The product was filtered with suction and dried in vucuo. The yield of crude product was 10 g. (90% of theory). The material was purified by repeated precipitation from acetone with n-hexane; 1 The melting points were detemined on a Fisher-Johns apparatus and are uncorrected. Microanalyses were done in part by Dr. H. A. Bright of the National Bureau of Standards, Washington, D. C., and in part by Mr. J. F. Alicino, Metuchen, N. J. Infrared spectra were determined with a Perkin-Elmer “Infracord” spectrophotometer; visible absorption spectra, with a Beckman model DU spectrophotometer; and fluorescent emission spectra, with an Aminco-Bowman spectrophotofluorometer.
dec. 185-190”. An acid solution of I showed orange fluorescence in ultraviolet light. Anal. Calcd. for C27H3104N3: C, 70.26; H, 6.77; N, 9.10. Found: C, 70.1; H, 6.9; N, 9.0.
4’-Aminorosamine B (II) A solution of 4’-nitrorosamine B (2.11 g.; 5 mmoles) in 100 ml. of absolute ethanol was shaken with hydrogen at 20 lb./sq. in. pressure and room temperature, in the presence of Raney nickel catalyst, for 3 hr. The catalyst was removed by centrifugation, and the solution was evaporated to dryness at reduced pressure. The semisolid residue was dissolved in acetone and precipitated with n-hexane (dec. above 200”), but its microanalysis gave low values for C, H, and N. Therefore, the hydrochloride (III) of 4’.aminorosamine B was prepared by repeatedly filtering the solution of the amine (II) in N hydrochloric acid, and evaporating and drying the filtrate in vucuo over solid sodium hydroxide. The hydrochloride salt did not melt below 300”. It was found to be hygroscopic, and was dried for analysis in uucuo at 100” for 3 hr. Anal. Calcd. for C~TH~~ON&~~.>$H~O: C, 65.45; H, 6.92; N, 8.48. Found: C, 65.92; H, 6.91; N, 8.50.
RosamineB 4’-Isocyanate (IV) This derivative of 4’.aminorosamine B was prepared by t,he method used by Coons and Kaplan in the preparation of fluorescein isocyanate (l), employing a saturated solution of phosgene gas in dry acetone. An acetone solution of the product was used directly for conjugation with protein. Isolation of the solid intermediate was not attempted.
Rosamine B 4’-Isothiocyanate (V) The method used here was based on that used by Riggs in the preparation of fluorescein isothiocyanate (8). A solution of 4’-aminorosamine B (1.0 g.; 2.4 mmoles) in 8 ml. of dry acetone was added dropwise to a stirred solution of thiophosgene (3.0 g.; 26 mmoles) in 5 ml. of dry acetone, at room temperature. The reaction was continued for 1 hr., and the mixture was then allowed to stand in the cold overnight. The purple-red precipitate was filtered and washed with n-hexane. The yield was 0.90 g. (82% of theory). The product showed orange fluorescence in acid solution. The infrared spectrum (Nujol) showed an absorption band in the region of 2100 cm.+ (absent in the spectra of the nitro and amino derivatives of rosamine B), characteristic of the -N=C=S group (9). Reprecipitation from acetone with n-hexane gave a hygroscopic solid, dec. 195202”. Anal. Calcd. for C?8H3102N3S.HsO: N, 8.53. Found: N, 8.50.
FLUORESCENT
LABEL
FOR ANTIBODY
Protein Conjugates of Rosamine B 4’-Isocyanate and Isothiocyanate The isocyanate and isothiocyanate derivatives of rosamine B ‘were combined with serum proteins following the method used by Coons and Kaplan in the conjugation of fluorescein isocyanate (l), except that dioxane was omitted in preparing the solutions. The initial dye-protein ratio was 36:l instead of 14: I, on a molar basis. The resulting conjugates showed orange fluorescence of moderate intensity, when buffered at pH 7.1.
.$I-Diazonium RosamineB (VI) and Its Protein Conjugate A solution of 4’-aminorosamine B (5.2 mg.; 1.2 X 10m2 mmole) was dissolved in 0.48 ml. of 0.1 N hydrochloric a.cid and 0.32 ml. water, and treated in the cold with a solution of sodium nitrite (1.1 mg.; 1.5 X 10-z mmole) in 0.4 ml. water. The resulting solution of the diazonium salt was allowed to stand in the cold for 10 min., and then was added dropwise to a cold, stirred solution of 1 ml. of bovine anti-PasteurelEa multocidu serum (containing approximately 7 X 1OV mmole protein) in 5 ml. of 0.15 11/1 sodium chloride and 1 ml. of 0.5 M bicarbonate-carbonate buffer at pH 9.1. The reaction mixture was stirred overnight, and then it was dialyzed for 4 days against 0.15 M saline with 0.01 M phosphate buffer at pH 7.2. The dialyzate was changed at 24-hr. intervals. After the dialysis, the solution of the conjugate was found to show only a very weak orange fluorescence in ultraviolet light. When it was acidified with phospha,te-citric acid buffer at pH 3, however, a brilliant orange fluorescence was observed.
ESTIMATION OF THE FINAL DYEPROTEIN RATIO IN THE CONJUGATES A O&ml. :aliquot of the solution of each conjugate was diluted to 2&40 ml. with phosphate buffer (pH 7.1), and its absorption spectrum was examined in the region of 500-600 mr. The maximum absorption in most cases was at 568 mp. The amount of dye in the conjugate was calculated from the optical density at the maximum and the extinction coefficient of the dye determined previously. In the case of the conjugates obtained via the isothiocyanate route, the reference extinction coefficient used was that of the product of the reaction of rosamine B 4’-isothiocyanate and histamine, containing a thiocarbamate linkage resembling those present in the conjugates. In the case of the conjugates prepared from the diazonium salt of rosamine B, the reference ext,inction
PROTEINS TABLE DYE-PROTEIN
295 I RATIOS Molar dye-protein
I Dye derivative
Isothiocyanate Diazonium
used
ratio
I cmo,
Final . fnlt”’
Before extraction
After extraction
salt
coefficient used was that of the coupling product of the diazotized 4’-aminorosamine B and phenol. i An assumption was made that these extinction coefficients were not substantially different from those of the combination of the dye with protein. In calculating the final dye-protein ratio, the protein loss was assumed to be negligible, and the average molecular weight of the serum proteins was taken as 100,000. In order to determine the amount of dye present in the conjugates in a chemically bound form, as distinguished from any free dye that may have remained in the solution after dialysis, absorption spectra of the conjugated proteins were examined after extracting the latter exhaustively with ethyl acetate. The results are shown in Table I. DETERMINATION DYE-PROTEIN DIAZO
OF THE OPTIMAL RATIO IN THE CONJUGATES
The objective of this experiment was to determine the optimal conditions of coupling of the rosamine B diazonium salt to antibody that would result in a maximum brightness of staining, with a minimum of antibody alteration. 4’-Diazonium rosamine B was coupled with a rabbit antiserum to Escherichiacoli, at six different dye-protein molar ratios increasing in twofold increments from 2:l to 64:1, using the procedure outlined above for the anti-P. multocida serum. After dialysis, aliquots of the conjugate solutions were examined for their agglutination titers, and their respective dye contents were determined spectrophotometrically as previously described. These data were correlated with the intensity of the staining reactions obtained with smears of a stock culture of E. coli. The stained smears were mounted in glycerine with 200/o phosphate-citric acid buffer at pH 3, examined in a Zeiss fluorescent microscope with OSRAM HBO light source, and the relative brightness of staining of the organisms was estimated visually. The results are shown in Table II.
296
BOREK TABLE STAINING Molar
OF CONJUGATES ratio
Final
NO. Initial
Before extraction
2:l 4:l 8:l 16:l 32: 1 64:l
1.6:1 3.2:1 6.2:1 13:l 24:l 48:l
_____
1 2 3 4 5 6
II
EFFICIENCY dye-protein
AND
After extraction
I
I Aggl$in$tiz: (control: ?46)
Staining brigbtIleSS (estimate)
~__
DOUBLE
1.5:1 2.7:1 5.2:1 10.5:1 18:l 31.4:1
STAINING
OF
M
+-
4i6
++
?Q % % 3s
+-I-+ ++++ ++-i-+ +++
SMEARS
Preliminary examination of the usefulness of conjugates of rosamine B in conjunction with fluorescein was made, employing mixed smears of bacteria. Bovine anti-P. multocidaserum conjugated with diazorosamine B and rabbit anti-E. coli labeled with fluorescein isothiocyanate were used in the double staining of smears containing bothP. multocida and E. coli. Examination of the stains under different conditions revealed some interesting facts. At pH 7, the fluorescein-st,ained coli organisms were brilliantly fluorescent, while at this pH (and even more so at pH 9) the rosamine-stained multocida organisms fluoresced only weakly. Upon lowering the pH of the mounting medium, the yellow-green emission of fluorescein was now found to be almost completely quenched, while those organisms stained with rosamine B were easily visible due to their bright orange fluorescence. It was possible, by taking advantage of this pH variation and of the availability of suitable secondary glass filters,* first to visualize one label while quenching the other, and then to effect the reverse situation. COMPARISON FLUORESCENT
WITH
OTHER LABELS
The fluorescent emission spectra of the solutions of 4’-aminorosamine B and its protein conjugates buffered at pH 3 were examined in the range 200-800 rnp, using a monochromatic activat2 Zeiss exciter filter UG5, upper barrier filter OG4, and lower barrier filter GG4 were used to visualize the fluorescein-stained organisms. Exciter filter BG12, upper barrier filter OG4, and lower barrier filter OG5 were used for the rosaminestained organisms.
SILVERSTEIN ing light at 300 rnp. All of them contained a single emission peak at 62@-630 mp. On the other hand, rhodamine B and lissamine RB 200 conjugates, when activated at 300 rnp or 350 rnp, gave rise to two emission peaks: one weaker (between 590 and 610 mp), and another stronger (between 650 and 700 mp). According to Emmart (lo), fluorescein conjugates when activated at 495 mp emit at 550 rn+ The intensity of fluorescence of the rosamine B-protein conjugates was found to be comparable to, and in some instances higher than, that of rhodamine B and tetramethylrhodamine B conjugates. The lissamine RB 200 conjugates were found to be more intensely fluorescent, but at the same time they also showed more tendency for nonspecific staining of tissue sections. Like all other available orange-fluorescent dyes, rosamine B shows fluorescence of a lower order of intensity than fluorescein. However, while fluorescein stains are known to fade completely after a few minutes of exposure to ultraviolet light, the diazorosamine B stains merely change their color from orange to yellow after approximately 5 min. of exposure, but retain most of their intensity even after being exposed for 30 min. to strong ultraviolet light. DISCUSSION
The data in Table II indicate that the optimal range of the initial dye-protein ratios for the diazorosamine B conjugate, when it is used to stain bacterial smea.rs, lies between 16: 1 and 32: 1. The use of diazotization for the coupling of the dye to protein has a considerable advantage.3 The procedure is simple and time tested, and does not involve the use of toxic reagents. Moreover, the addition of diazonium salts to proteins is known to be a mild reaction proceeding without appreciable protein denaturation, even upon introduction of 30 or more groups per molecule of protein (ll), and to a large extent without a significant change in the immunologic properties of the y-globulins (12). In contrast, the coupling of isocyanate derivatives has been found by Coons to result in a considerable denaturation of protein, and to be limited in the number of substituent groups that may be introduced into the protein molecule without causing its denaturation 3 As found by Coons, diazotization fluorescein and subsequent coupling results in a nonfluorescent conjugate
of aminoto protein (2).
FLUORESCENT
LABEL
FOR
(1). The use of a sulfonyl chloride derivative was found by Chadwick et al. to lead to a marked decrease in the antibody titer (5). The results of the experiments in bacterial staining indicate that although rosamine B is not suitable as a substitute for fluorescein, it can be used as a second label in conjunction with the latter. The fact that fluorescein is an anionic dye, while rosamine B is of the cationic type, may account for the observation that the fluorescent intensities of their respective conjugates change in opposite direction with varying pH. ACKNOWLEDGMENT We wish to thank Colonel W. C. Eveland, MSC, U.S.A., of the Bacteriology Branch of the Armed Forces Institute of Pathology for supplying us with antisera and bacterial smears.
A. H., AND KAPLAN, 91, 1 (1950).
297
PROTEINS
2. COONS, A. H., Intern. Rev. Cytol. 6, 1 (1956). 3. HIRAMOTO, R., ENGEL, K., AND PRESSMAN, D., Proe. Sot. Ezptl. Biot. Med. 97, 611 (1958). 4. SILVERSTEIN, A. M., J. Histochem. and Cytothem. 6, 94 (1957). 5. CHADWICK, C. S., MCENTEGART, M. G., NAIRN, R. C., Immunology 1,315 (1958). 6. CLAYTON,
R. M.,
Nature
7. RIGGS, J. L., SEIWALD, J. H., DOWNS, C. M., Am. J. Pathol. 34,108l 8. RIGGS, J. L., MASTER’S 1957.
M.
H.,
J. Exptl.
AND
174, 1059 (1954). R. J., BURCKHALTER, AND METCALF, T. G., (1958). THESIS,
Univ.
Kansas,
9. MILLER, F. A., in “Organic Chemistry” (Gilman, H., ed.), Vol. III, p. 145. John Wiley & Son, Inc., New York, 1953; cf. Ref. (7) or (8). 10. EMMART, E. W., 1 (1958).
Arch.
Biochem.
11. BOYD, W. C., AND HOOKER, Chem. 104, 329 (1934).
REFERENCES 1. COONS, Med.
ANTIBODY
Biophys. S. B.,
12. EAGLE, H., SMITH, D. E., AND J. Exptl. Med. 63, 617 (1936).
J.
VICKERS,
73, Biol. P.,
OF
BIOCHEMISTRY
AND
A New
Fluorescent
FELIX From
87, 293-%7 (1960)
BIOPHYSICS
BOREK
the Armed
Label AND
Forces
for
ARTHUR
Institute
Antibody
Proteins
M. SILVERSTEIN
of Pathology,
Washington,
D. C.
Received October 23, 1959 A new orange-fluorescent dye, aminorosamine B, was prepared by condensation of 2 moles m-diethylaminophenol with 1 mole p-nitrobenzaldehyde, followed by catalytic reduction of the nitro group. This dye was found to be useful as a label for antibody proteins, with which it may be conjugated as an isocyanate, isothiocyanate, or diazonium derivative. The optimal dye-protein ratio was determined corresponding to a maximum intensity of staining with a minimum decrease in antibody activity. A diazorosamine B-antibody conjugate was found useful in the staining of mixed bacterial smears, in conjunction with a fluorescein-antibody conjugate. INTRODUCTION
sufficiently different from that of fluorescein so that one might be readily distinguished Following the introduction by Coons and from the other. (d) If possible, each color co-workers of an immunohistochemical should be seen independently when both technique using antibodies labeled with are present in the samearea. (e) The conjugafluorescein (l), and its varied applications in tion with the dye should leave essentially unmany fields [reviewed in Ref. (2)], the employment of a second fluorescent label of changed the original titer and immunochemical specificity of the antibody protein. contrasting color appeared desirable for two A number of Auorochromes have been reasons. First, certain tissues, like thyroid, suggestedin the last few years as candidates show a natural blue-green fluorescence, for the position of a second fluorescent which contraists poorly with the yellow-green label. For example, Clayton (6) labeled antiemission of fluorescein. Here a label of a bodies with 1-dimethylaminonaphthalene-5color farther removed from blue is needed sulfonyl chloride and nuclear fast red to (3). Secondly, the use of a label of contrastproduce yellow and red fluorescence, respecing color in conjunction with fluorescein tively. Silverstein (4) introduced orangewould be advantageous in the staining of fluorescent conjugates of rhodamine B isomixed bacterial smears, in which each kind cyanate, while Riggs et al. (7) employed the of organism might be stained with a difisothiocyanate derivative of the same dye. ferent colored label (4). The requirements that a second label Hiramoto et al. (3) used conjugates of the tetramethyl homolog of rhodamine B isoshould satisfy in order to be useful have been cyanate. Chadwick and co-workers (5) resummarized by Silverstein (4) and by Chadported application of orange-fluorescent prowick et al. (5). These are: (a) The dye used tein conjugates prepared from a sulfonyl should be ca,pableof forming stable chemical chloride derivative of lissamine rhodamine bonds with proteins such as azo, carbamido, B 200. These investigators screened eight or sulfonamido linkages. (b) The intensity of other fluorescent dyes, including a derivative fluorescence should be sufficiently high to of rhodamine B, for their applicability to permit the visualization of the antibodyantibody labeling and found none suitable, antigen complex at the relatively low con- either becauseof low intensity of fluorescence centrations often present in the area of or because of undesirable color characterinterest. (c) The fluorescent color should be istics. 293
BOREK AND SILVERSTEIN
294
We wish to report the preparation of a new fluorescent dye that, according to preliminary results, may be used to advantage as a second antibody label. The dye is an amino derivative of 2,6-tetraethyldiamino-g-phenylxanthene, and by analogy with aminorhodamine B, we have called it aminorosamine B. By conkast with aminorhodamine B, which is obtained as a mixture of two isomers (differing in the position of the amino group in the phenyl ring) (4, S), aminorosamine B cannot form isomers and is therefore purified more easily. Its conjugation to antibody protein by the diazotization and coupling method yielded a conjugate more intensely fluorescent than those prepared from the isocyanate and isothiocyanate derivatives of the same dye.
L
k
J
-Nt2
(1)
-NH2
w
- NHZ.HCI
(ml
- N=C=O
mu
-N=C=s -&N
Cle
(PI (SE)
EXPERIMENTAL’
PREPARATION
OF MATERIALS
4 ‘-Nitrorosamine B (I) A mixture of p-nitrobenzaldehyde (3.8 g.; 25 mmoles) and m-diethylaminophenol (8.25 g.; 50 mmoles) in 50 ml. of 50y0 sulfuric acid was heated at 130-140” for 5 hr. The reaction mixture was cooled, and 11. of 3’% sodium hydroxide was added, resulting in the precipitation of a purple-red solid. The product was filtered with suction and dried in vucuo. The yield of crude product was 10 g. (90% of theory). The material was purified by repeated precipitation from acetone with n-hexane; 1 The melting points were detemined on a Fisher-Johns apparatus and are uncorrected. Microanalyses were done in part by Dr. H. A. Bright of the National Bureau of Standards, Washington, D. C., and in part by Mr. J. F. Alicino, Metuchen, N. J. Infrared spectra were determined with a Perkin-Elmer “Infracord” spectrophotometer; visible absorption spectra, with a Beckman model DU spectrophotometer; and fluorescent emission spectra, with an Aminco-Bowman spectrophotofluorometer.
dec. 185-190”. An acid solution of I showed orange fluorescence in ultraviolet light. Anal. Calcd. for C27H3104N3: C, 70.26; H, 6.77; N, 9.10. Found: C, 70.1; H, 6.9; N, 9.0.
4’-Aminorosamine B (II) A solution of 4’-nitrorosamine B (2.11 g.; 5 mmoles) in 100 ml. of absolute ethanol was shaken with hydrogen at 20 lb./sq. in. pressure and room temperature, in the presence of Raney nickel catalyst, for 3 hr. The catalyst was removed by centrifugation, and the solution was evaporated to dryness at reduced pressure. The semisolid residue was dissolved in acetone and precipitated with n-hexane (dec. above 200”), but its microanalysis gave low values for C, H, and N. Therefore, the hydrochloride (III) of 4’.aminorosamine B was prepared by repeatedly filtering the solution of the amine (II) in N hydrochloric acid, and evaporating and drying the filtrate in vucuo over solid sodium hydroxide. The hydrochloride salt did not melt below 300”. It was found to be hygroscopic, and was dried for analysis in uucuo at 100” for 3 hr. Anal. Calcd. for C~TH~~ON&~~.>$H~O: C, 65.45; H, 6.92; N, 8.48. Found: C, 65.92; H, 6.91; N, 8.50.
RosamineB 4’-Isocyanate (IV) This derivative of 4’.aminorosamine B was prepared by t,he method used by Coons and Kaplan in the preparation of fluorescein isocyanate (l), employing a saturated solution of phosgene gas in dry acetone. An acetone solution of the product was used directly for conjugation with protein. Isolation of the solid intermediate was not attempted.
Rosamine B 4’-Isothiocyanate (V) The method used here was based on that used by Riggs in the preparation of fluorescein isothiocyanate (8). A solution of 4’-aminorosamine B (1.0 g.; 2.4 mmoles) in 8 ml. of dry acetone was added dropwise to a stirred solution of thiophosgene (3.0 g.; 26 mmoles) in 5 ml. of dry acetone, at room temperature. The reaction was continued for 1 hr., and the mixture was then allowed to stand in the cold overnight. The purple-red precipitate was filtered and washed with n-hexane. The yield was 0.90 g. (82% of theory). The product showed orange fluorescence in acid solution. The infrared spectrum (Nujol) showed an absorption band in the region of 2100 cm.+ (absent in the spectra of the nitro and amino derivatives of rosamine B), characteristic of the -N=C=S group (9). Reprecipitation from acetone with n-hexane gave a hygroscopic solid, dec. 195202”. Anal. Calcd. for C?8H3102N3S.HsO: N, 8.53. Found: N, 8.50.
FLUORESCENT
LABEL
FOR ANTIBODY
Protein Conjugates of Rosamine B 4’-Isocyanate and Isothiocyanate The isocyanate and isothiocyanate derivatives of rosamine B ‘were combined with serum proteins following the method used by Coons and Kaplan in the conjugation of fluorescein isocyanate (l), except that dioxane was omitted in preparing the solutions. The initial dye-protein ratio was 36:l instead of 14: I, on a molar basis. The resulting conjugates showed orange fluorescence of moderate intensity, when buffered at pH 7.1.
.$I-Diazonium RosamineB (VI) and Its Protein Conjugate A solution of 4’-aminorosamine B (5.2 mg.; 1.2 X 10m2 mmole) was dissolved in 0.48 ml. of 0.1 N hydrochloric a.cid and 0.32 ml. water, and treated in the cold with a solution of sodium nitrite (1.1 mg.; 1.5 X 10-z mmole) in 0.4 ml. water. The resulting solution of the diazonium salt was allowed to stand in the cold for 10 min., and then was added dropwise to a cold, stirred solution of 1 ml. of bovine anti-PasteurelEa multocidu serum (containing approximately 7 X 1OV mmole protein) in 5 ml. of 0.15 11/1 sodium chloride and 1 ml. of 0.5 M bicarbonate-carbonate buffer at pH 9.1. The reaction mixture was stirred overnight, and then it was dialyzed for 4 days against 0.15 M saline with 0.01 M phosphate buffer at pH 7.2. The dialyzate was changed at 24-hr. intervals. After the dialysis, the solution of the conjugate was found to show only a very weak orange fluorescence in ultraviolet light. When it was acidified with phospha,te-citric acid buffer at pH 3, however, a brilliant orange fluorescence was observed.
ESTIMATION OF THE FINAL DYEPROTEIN RATIO IN THE CONJUGATES A O&ml. :aliquot of the solution of each conjugate was diluted to 2&40 ml. with phosphate buffer (pH 7.1), and its absorption spectrum was examined in the region of 500-600 mr. The maximum absorption in most cases was at 568 mp. The amount of dye in the conjugate was calculated from the optical density at the maximum and the extinction coefficient of the dye determined previously. In the case of the conjugates obtained via the isothiocyanate route, the reference extinction coefficient used was that of the product of the reaction of rosamine B 4’-isothiocyanate and histamine, containing a thiocarbamate linkage resembling those present in the conjugates. In the case of the conjugates prepared from the diazonium salt of rosamine B, the reference ext,inction
PROTEINS TABLE DYE-PROTEIN
295 I RATIOS Molar dye-protein
I Dye derivative
Isothiocyanate Diazonium
used
ratio
I cmo,
Final . fnlt”’
Before extraction
After extraction
salt
coefficient used was that of the coupling product of the diazotized 4’-aminorosamine B and phenol. i An assumption was made that these extinction coefficients were not substantially different from those of the combination of the dye with protein. In calculating the final dye-protein ratio, the protein loss was assumed to be negligible, and the average molecular weight of the serum proteins was taken as 100,000. In order to determine the amount of dye present in the conjugates in a chemically bound form, as distinguished from any free dye that may have remained in the solution after dialysis, absorption spectra of the conjugated proteins were examined after extracting the latter exhaustively with ethyl acetate. The results are shown in Table I. DETERMINATION DYE-PROTEIN DIAZO
OF THE OPTIMAL RATIO IN THE CONJUGATES
The objective of this experiment was to determine the optimal conditions of coupling of the rosamine B diazonium salt to antibody that would result in a maximum brightness of staining, with a minimum of antibody alteration. 4’-Diazonium rosamine B was coupled with a rabbit antiserum to Escherichiacoli, at six different dye-protein molar ratios increasing in twofold increments from 2:l to 64:1, using the procedure outlined above for the anti-P. multocida serum. After dialysis, aliquots of the conjugate solutions were examined for their agglutination titers, and their respective dye contents were determined spectrophotometrically as previously described. These data were correlated with the intensity of the staining reactions obtained with smears of a stock culture of E. coli. The stained smears were mounted in glycerine with 200/o phosphate-citric acid buffer at pH 3, examined in a Zeiss fluorescent microscope with OSRAM HBO light source, and the relative brightness of staining of the organisms was estimated visually. The results are shown in Table II.
296
BOREK TABLE STAINING Molar
OF CONJUGATES ratio
Final
NO. Initial
Before extraction
2:l 4:l 8:l 16:l 32: 1 64:l
1.6:1 3.2:1 6.2:1 13:l 24:l 48:l
_____
1 2 3 4 5 6
II
EFFICIENCY dye-protein
AND
After extraction
I
I Aggl$in$tiz: (control: ?46)
Staining brigbtIleSS (estimate)
~__
DOUBLE
1.5:1 2.7:1 5.2:1 10.5:1 18:l 31.4:1
STAINING
OF
M
+-
4i6
++
?Q % % 3s
+-I-+ ++++ ++-i-+ +++
SMEARS
Preliminary examination of the usefulness of conjugates of rosamine B in conjunction with fluorescein was made, employing mixed smears of bacteria. Bovine anti-P. multocidaserum conjugated with diazorosamine B and rabbit anti-E. coli labeled with fluorescein isothiocyanate were used in the double staining of smears containing bothP. multocida and E. coli. Examination of the stains under different conditions revealed some interesting facts. At pH 7, the fluorescein-st,ained coli organisms were brilliantly fluorescent, while at this pH (and even more so at pH 9) the rosamine-stained multocida organisms fluoresced only weakly. Upon lowering the pH of the mounting medium, the yellow-green emission of fluorescein was now found to be almost completely quenched, while those organisms stained with rosamine B were easily visible due to their bright orange fluorescence. It was possible, by taking advantage of this pH variation and of the availability of suitable secondary glass filters,* first to visualize one label while quenching the other, and then to effect the reverse situation. COMPARISON FLUORESCENT
WITH
OTHER LABELS
The fluorescent emission spectra of the solutions of 4’-aminorosamine B and its protein conjugates buffered at pH 3 were examined in the range 200-800 rnp, using a monochromatic activat2 Zeiss exciter filter UG5, upper barrier filter OG4, and lower barrier filter GG4 were used to visualize the fluorescein-stained organisms. Exciter filter BG12, upper barrier filter OG4, and lower barrier filter OG5 were used for the rosaminestained organisms.
SILVERSTEIN ing light at 300 rnp. All of them contained a single emission peak at 62@-630 mp. On the other hand, rhodamine B and lissamine RB 200 conjugates, when activated at 300 rnp or 350 rnp, gave rise to two emission peaks: one weaker (between 590 and 610 mp), and another stronger (between 650 and 700 mp). According to Emmart (lo), fluorescein conjugates when activated at 495 mp emit at 550 rn+ The intensity of fluorescence of the rosamine B-protein conjugates was found to be comparable to, and in some instances higher than, that of rhodamine B and tetramethylrhodamine B conjugates. The lissamine RB 200 conjugates were found to be more intensely fluorescent, but at the same time they also showed more tendency for nonspecific staining of tissue sections. Like all other available orange-fluorescent dyes, rosamine B shows fluorescence of a lower order of intensity than fluorescein. However, while fluorescein stains are known to fade completely after a few minutes of exposure to ultraviolet light, the diazorosamine B stains merely change their color from orange to yellow after approximately 5 min. of exposure, but retain most of their intensity even after being exposed for 30 min. to strong ultraviolet light. DISCUSSION
The data in Table II indicate that the optimal range of the initial dye-protein ratios for the diazorosamine B conjugate, when it is used to stain bacterial smea.rs, lies between 16: 1 and 32: 1. The use of diazotization for the coupling of the dye to protein has a considerable advantage.3 The procedure is simple and time tested, and does not involve the use of toxic reagents. Moreover, the addition of diazonium salts to proteins is known to be a mild reaction proceeding without appreciable protein denaturation, even upon introduction of 30 or more groups per molecule of protein (ll), and to a large extent without a significant change in the immunologic properties of the y-globulins (12). In contrast, the coupling of isocyanate derivatives has been found by Coons to result in a considerable denaturation of protein, and to be limited in the number of substituent groups that may be introduced into the protein molecule without causing its denaturation 3 As found by Coons, diazotization fluorescein and subsequent coupling results in a nonfluorescent conjugate
of aminoto protein (2).
FLUORESCENT
LABEL
FOR
(1). The use of a sulfonyl chloride derivative was found by Chadwick et al. to lead to a marked decrease in the antibody titer (5). The results of the experiments in bacterial staining indicate that although rosamine B is not suitable as a substitute for fluorescein, it can be used as a second label in conjunction with the latter. The fact that fluorescein is an anionic dye, while rosamine B is of the cationic type, may account for the observation that the fluorescent intensities of their respective conjugates change in opposite direction with varying pH. ACKNOWLEDGMENT We wish to thank Colonel W. C. Eveland, MSC, U.S.A., of the Bacteriology Branch of the Armed Forces Institute of Pathology for supplying us with antisera and bacterial smears.
A. H., AND KAPLAN, 91, 1 (1950).
297
PROTEINS
2. COONS, A. H., Intern. Rev. Cytol. 6, 1 (1956). 3. HIRAMOTO, R., ENGEL, K., AND PRESSMAN, D., Proe. Sot. Ezptl. Biot. Med. 97, 611 (1958). 4. SILVERSTEIN, A. M., J. Histochem. and Cytothem. 6, 94 (1957). 5. CHADWICK, C. S., MCENTEGART, M. G., NAIRN, R. C., Immunology 1,315 (1958). 6. CLAYTON,
R. M.,
Nature
7. RIGGS, J. L., SEIWALD, J. H., DOWNS, C. M., Am. J. Pathol. 34,108l 8. RIGGS, J. L., MASTER’S 1957.
M.
H.,
J. Exptl.
AND
174, 1059 (1954). R. J., BURCKHALTER, AND METCALF, T. G., (1958). THESIS,
Univ.
Kansas,
9. MILLER, F. A., in “Organic Chemistry” (Gilman, H., ed.), Vol. III, p. 145. John Wiley & Son, Inc., New York, 1953; cf. Ref. (7) or (8). 10. EMMART, E. W., 1 (1958).
Arch.
Biochem.
11. BOYD, W. C., AND HOOKER, Chem. 104, 329 (1934).
REFERENCES 1. COONS, Med.
ANTIBODY
Biophys. S. B.,
12. EAGLE, H., SMITH, D. E., AND J. Exptl. Med. 63, 617 (1936).
J.
VICKERS,
73, Biol. P.,