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Antigen-antibody interaction on microcapsule surface
Antigen-Antibody Interaction on Microcapsule Surface Microcapsules are a useful tool in the basic research of colloid science. They have provided us with valuable information on such problems as polyelectrolyte-polyelectrolyte interactions (1, 2), polyelectrolyte-surfactant interactions (3), and permeability of capsular membranes toward low-molecular-weight solutes (4). This note adds another example to the application of microcapsules. That is, microcapsules carrying antigen or antibody on their surface were prepared and their aggregation by the corresponding antibody or antigen was investigated. Polyamide microcapsules containing water were prepared by an interracial polymerization technique (5, 6) making use of the interfacial polycondensation reaction between 1,11-(3,6,9-triazaundecane)diamine dissolved in water and sebacoyl chloride dissolved in an organic solvent. The polyamide microcapsules obtained were transferred to an aqueous phase and treated with gtutaraldehyde to crosslink terminal amino groups of the constituent polyamide molecules. Rabbit antihuman IgG was then covalently immobilized to the crosslinked polyamide microcapsules by the carbodiimide method using 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide hydrochloride and E-amino-n-caproic acid as spacer (7). The immunoglobulin-carrying polyamide microcapsules thus prepared are designated hereafter as immunomicrocapsules.
The immunomicrocapsules were aggregated by human IgG, the corresponding antigen of rabbit antihuman IgG on the microcapsule surface, while no aggregation was observed when rabbit IgG was added to a suspension of the immunomicrocapsules. In Fig. 1 fluorescence photomicrographs of immunomicrocapsules carrying FICT-labeled rabbit antihuman IgG in saline in the absence and presence of human IgG are shown. Aggregation of the immunomicrocapsules is clearly seen in Fig. lb. It was found that the rate of aggregation by human IgG of immunomicrocapsules with rabbit antihuman IgG is strongly dependent on temperature in a way which appears to be specific to the antigen-antibody interaction. As temperature rose the rate increased until a temperature of about 20°C was reached and slowed down thereafter. No appreciable pH effect was observed on the aggregation in the pH range of 6.68.3. Although poly(styrene sodium sulfonate) and poly(vinyl potassium sulfate) caused aggregation of the immunomicrocapsules the rate was almost independent of temperature in the aggregation by the former and it decreased with temperature when the latter was used as aggregant. These are shown in Fig. 2, where the logarithm of the rate of aggregation as evaluated from turbidity measurement is plotted against the reciprocal of absolute temperature. Poly(diallyldimethylammonium chloride), a cationic
FIG. 1. Fluorescence photomicrographs of immunomicrocapsules carrying FITC-labeled rabbit antihuman IgG in saline in the absence (a) and presence (b) of human IgG. 652 0021-9797/81/100652-03502.00/0 Copyright © 1981by AcademicPress, Inc. All rightsof reproductionin any form reserved.
Journal of Colloid and Interface Science,
Vol. 83, No. 2, October 1981
NOTES
_//3.3
1/ 103 3.4 TX
3.5
653 1.0
0.5
0.5 × 10 3
--1.0 FIG. 2. Aggregation rate as a function of temperature of immunomicrocapsules carrying rabbit antihuman IgG by human IgG (©), poly(styrene sodium sulfonate) (A), and poly(vinyl potassium sulfate) (O) at pH 6.6 in the presence of NaCI (0.1 M) and BSA (1 mg/ml). Mean capsule diameter: 3.5/~m. polyelectrolyte, failed to cause aggregation of the immunomicrocapsules, suggesting that the microcapsule surface has a net positive charge due presumably to the basic groups of the immobilized antibody. As human IgG bears a net negative charge in the pH region used in this work it is highly likely that the specific binding of the antigen occurs to the antibody on the microcapsule surface through specific 1.0
0.5
~i3
--0.
FIG. 4. Effect of urea on aggregation by human IgG of immunomicrocapsules carrying rabbit antihuman IgG at pH 6.6 in the presence of NaC1 (0.1 M ) and BSA (1 mg/ml). Urea concentration: 0 (0); I M (O). Mean capsule diameter: 3.5/zm. interactions including an electrostatic one between the proteins as the first stage of the aggregation. The second stage will be the formation of aggregates through nonspecific interactions among the immunomicrocapsules with the antigen bound on their surfaces. This argument leads to an expectation that there will be a remarkable valency effect of cation on the aggregation of the immunomicrocapsules. Figure 3 confirms the expectation; aluminum ion is the most effective, followed by magnesium and potassium ions. The rate of aggregation by human IgG of the immunomicrocapsules was also noticeably affected by the presence of urea. When compared with the control system in which no urea was added, the immuno-
IITXI0 3
--0.5
--1.0 FIG. 3. Valency effect of cation on aggregation by human IgG of immunomicrocapsules carrying rabbit antihuman IgG at pH 6.6 in the presence of BSA (1 mg/ml), Mean capsule diameter; 3.5 txm. Cation concentrations are adjusted to give an identical ionic strength of 0.1. Cation: aluminum ion (ZX);magnesium ion (O); potassium ion (0).
0.5 ~
0
~
3
0
0.5 FXG. 5. Effect of capsule size on aggregation by human IgG of immunomicrocapsules carrying rabbit antihuman IgG at pH 6.6 in the presence of NaC1 (0.1 M) and BSA (1 mg/ml). Mean capsule diameter: 3.5 /xm (©); 5.5/xm (O); 7.5 txm (A). Journal of Colloid and Interface Science,
Vol.83,No.2,October1981
654
NOTES
microcapsules were quickly aggregated by the antigen in the presence of urea at all temperatures examined, as shown in F!g. 4. This implies an important role of hydrophobic interactions between the antigen and the antibody in the first and/or second stages of the aggregation of the immunomicrocapsules since urea is well known as a potential structurebreaking agent of water and it may alter the tertiary structures of both antigen and antibody through the changes in water structure. Interestingly enough, an increase in the capsule size brought about a marked rise in the rate of aggregation by human IgG of the immunomicrocapsules. This is illustrated in.Fig. 5, where the temperature dependency characteristic of the aggregation of the immunomicrocapsules is retained, irrespective of capsule size. As the introduction of the antibody to the microcapsule surface was made under the condition of a constant ratio of the antibody concentration to the capsule volume concentration, the surface concentration of the antibody immobilized will be higher for the immunomicrocapsules of larger size. In view of this, it will be natural to think that aggregation is easier to take place among larger immunomicrocapsules owing to their stronger interactions. In conclusion, it is possible to accelerate the aggregation reaction between antigen and antibody by immobilizing either the former or the latter on the microcapsule surface. Thus, the antigen-antibody interaction makes its appearance very quickly and in a greatly amplified fashion taking the form of capsule aggregation because the concentration of antigen or antibody on the microcapsule surface is very high as compared with that in solution. It is our hope, therefore, that immunomicrocapsules will be used as a novel tool in the study of antigen-antibody inter-
Journal of CoUoidand Interface Science, Vol. 83, No. 2~October 1981
action from the viewpoint of colloid science. Immunomicrocapsules are also expected tofind many applications in clinical diagnosis as a detecting agent due to their advantages mentioned a b o v e . REFERENCES 1. Suzuki, S., and Kondo, T., J. Colloid Interface Sci. 67, 441~(1978). 2. Suzuki, S., and Kondo, T., J. Colloid Interface Sci. 77, 280 (1980). 3. Suzuki, S., Nakamura, T., Arakawa, M., and Kondo, T., J. Colloid Interface Sci. 71, 141 (1979). " 4. Ishizaka, T.,- Koishi, M., and Kondo, T., J. Membrane Sci. 5, 283 (1979). 5. Chang, T. M. S., Macintosh, F. C., and Mason, S. G., Canad. J. Physiol. Pharmacol. 44, 115 (1966). 6. Kondo, T., in "Surface and Colloid Science" (E. Matijevic, Ed.), Vol. 10, Chap. I. Plenum, New York, 1978. 7. Marumoto, K., Suzuta, T., Noguchi, H., and Uchida, Y., Polymer 19, 867 (1978). KIMIKO MAKINO MASAYUKI ARAKAWA TOMOTSU KONDO1
Faculty of Pharmaceutical Sciences Science University of Tokyo Shinjuku-ku, Tokyo 162, Japan Receive d March 25, 1981; accepted February 2 7, 1981 1 Author to whom all correspondence should be addressed.
The immunomicrocapsules were aggregated by human IgG, the corresponding antigen of rabbit antihuman IgG on the microcapsule surface, while no aggregation was observed when rabbit IgG was added to a suspension of the immunomicrocapsules. In Fig. 1 fluorescence photomicrographs of immunomicrocapsules carrying FICT-labeled rabbit antihuman IgG in saline in the absence and presence of human IgG are shown. Aggregation of the immunomicrocapsules is clearly seen in Fig. lb. It was found that the rate of aggregation by human IgG of immunomicrocapsules with rabbit antihuman IgG is strongly dependent on temperature in a way which appears to be specific to the antigen-antibody interaction. As temperature rose the rate increased until a temperature of about 20°C was reached and slowed down thereafter. No appreciable pH effect was observed on the aggregation in the pH range of 6.68.3. Although poly(styrene sodium sulfonate) and poly(vinyl potassium sulfate) caused aggregation of the immunomicrocapsules the rate was almost independent of temperature in the aggregation by the former and it decreased with temperature when the latter was used as aggregant. These are shown in Fig. 2, where the logarithm of the rate of aggregation as evaluated from turbidity measurement is plotted against the reciprocal of absolute temperature. Poly(diallyldimethylammonium chloride), a cationic
FIG. 1. Fluorescence photomicrographs of immunomicrocapsules carrying FITC-labeled rabbit antihuman IgG in saline in the absence (a) and presence (b) of human IgG. 652 0021-9797/81/100652-03502.00/0 Copyright © 1981by AcademicPress, Inc. All rightsof reproductionin any form reserved.
Journal of Colloid and Interface Science,
Vol. 83, No. 2, October 1981
NOTES
_//3.3
1/ 103 3.4 TX
3.5
653 1.0
0.5
0.5 × 10 3
--1.0 FIG. 2. Aggregation rate as a function of temperature of immunomicrocapsules carrying rabbit antihuman IgG by human IgG (©), poly(styrene sodium sulfonate) (A), and poly(vinyl potassium sulfate) (O) at pH 6.6 in the presence of NaCI (0.1 M) and BSA (1 mg/ml). Mean capsule diameter: 3.5/~m. polyelectrolyte, failed to cause aggregation of the immunomicrocapsules, suggesting that the microcapsule surface has a net positive charge due presumably to the basic groups of the immobilized antibody. As human IgG bears a net negative charge in the pH region used in this work it is highly likely that the specific binding of the antigen occurs to the antibody on the microcapsule surface through specific 1.0
0.5
~i3
--0.
FIG. 4. Effect of urea on aggregation by human IgG of immunomicrocapsules carrying rabbit antihuman IgG at pH 6.6 in the presence of NaC1 (0.1 M ) and BSA (1 mg/ml). Urea concentration: 0 (0); I M (O). Mean capsule diameter: 3.5/zm. interactions including an electrostatic one between the proteins as the first stage of the aggregation. The second stage will be the formation of aggregates through nonspecific interactions among the immunomicrocapsules with the antigen bound on their surfaces. This argument leads to an expectation that there will be a remarkable valency effect of cation on the aggregation of the immunomicrocapsules. Figure 3 confirms the expectation; aluminum ion is the most effective, followed by magnesium and potassium ions. The rate of aggregation by human IgG of the immunomicrocapsules was also noticeably affected by the presence of urea. When compared with the control system in which no urea was added, the immuno-
IITXI0 3
--0.5
--1.0 FIG. 3. Valency effect of cation on aggregation by human IgG of immunomicrocapsules carrying rabbit antihuman IgG at pH 6.6 in the presence of BSA (1 mg/ml), Mean capsule diameter; 3.5 txm. Cation concentrations are adjusted to give an identical ionic strength of 0.1. Cation: aluminum ion (ZX);magnesium ion (O); potassium ion (0).
0.5 ~
0
~
3
0
0.5 FXG. 5. Effect of capsule size on aggregation by human IgG of immunomicrocapsules carrying rabbit antihuman IgG at pH 6.6 in the presence of NaC1 (0.1 M) and BSA (1 mg/ml). Mean capsule diameter: 3.5 /xm (©); 5.5/xm (O); 7.5 txm (A). Journal of Colloid and Interface Science,
Vol.83,No.2,October1981
654
NOTES
microcapsules were quickly aggregated by the antigen in the presence of urea at all temperatures examined, as shown in F!g. 4. This implies an important role of hydrophobic interactions between the antigen and the antibody in the first and/or second stages of the aggregation of the immunomicrocapsules since urea is well known as a potential structurebreaking agent of water and it may alter the tertiary structures of both antigen and antibody through the changes in water structure. Interestingly enough, an increase in the capsule size brought about a marked rise in the rate of aggregation by human IgG of the immunomicrocapsules. This is illustrated in.Fig. 5, where the temperature dependency characteristic of the aggregation of the immunomicrocapsules is retained, irrespective of capsule size. As the introduction of the antibody to the microcapsule surface was made under the condition of a constant ratio of the antibody concentration to the capsule volume concentration, the surface concentration of the antibody immobilized will be higher for the immunomicrocapsules of larger size. In view of this, it will be natural to think that aggregation is easier to take place among larger immunomicrocapsules owing to their stronger interactions. In conclusion, it is possible to accelerate the aggregation reaction between antigen and antibody by immobilizing either the former or the latter on the microcapsule surface. Thus, the antigen-antibody interaction makes its appearance very quickly and in a greatly amplified fashion taking the form of capsule aggregation because the concentration of antigen or antibody on the microcapsule surface is very high as compared with that in solution. It is our hope, therefore, that immunomicrocapsules will be used as a novel tool in the study of antigen-antibody inter-
Journal of CoUoidand Interface Science, Vol. 83, No. 2~October 1981
action from the viewpoint of colloid science. Immunomicrocapsules are also expected tofind many applications in clinical diagnosis as a detecting agent due to their advantages mentioned a b o v e . REFERENCES 1. Suzuki, S., and Kondo, T., J. Colloid Interface Sci. 67, 441~(1978). 2. Suzuki, S., and Kondo, T., J. Colloid Interface Sci. 77, 280 (1980). 3. Suzuki, S., Nakamura, T., Arakawa, M., and Kondo, T., J. Colloid Interface Sci. 71, 141 (1979). " 4. Ishizaka, T.,- Koishi, M., and Kondo, T., J. Membrane Sci. 5, 283 (1979). 5. Chang, T. M. S., Macintosh, F. C., and Mason, S. G., Canad. J. Physiol. Pharmacol. 44, 115 (1966). 6. Kondo, T., in "Surface and Colloid Science" (E. Matijevic, Ed.), Vol. 10, Chap. I. Plenum, New York, 1978. 7. Marumoto, K., Suzuta, T., Noguchi, H., and Uchida, Y., Polymer 19, 867 (1978). KIMIKO MAKINO MASAYUKI ARAKAWA TOMOTSU KONDO1
Faculty of Pharmaceutical Sciences Science University of Tokyo Shinjuku-ku, Tokyo 162, Japan Receive d March 25, 1981; accepted February 2 7, 1981 1 Author to whom all correspondence should be addressed.