Proteins at Interfaces II - American Chemical Society

Chapter 38

Formation and Properties of Surface-Active Antibodies 1

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Shlomo Magdassi , Oren Sheinberg , and Zichria Zakay-Rones 1

Casali Institute of Applied Chemistry, School of Applied Science, and FacuIty of Medicine, Hebrew University of Jerusalem, 91904 Jerusalem, Israel

Downloaded by UNIV LAVAL on April 24, 2016 | http://pubs.acs.org Publication Date: May 5, 1995 | doi: 10.1021/bk-1995-0602.ch038

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Surface active antibodies were formed by covalent attachment of hydrophobic groups to the IgG molecule. The modified antibodies reduced surface tension and adsorbed onto emulsion droplets at surface concentrations higher than the native antibody. The chemical modification led to a decrease in the biological activity; however, at specific conditions, surface-active antibodies, which retained their recognition ability, could be formed. By using these antibodies, a new emulsion, which has a specific recognition ability for HSV-1 infected cells, was formed.

Chemical modifications of proteins may lead to significant changes in their surface activity and functional properties. For example, when hydrophobic chains are covalentiy attached to ovalbumin, the modified protein becomes more surface active than the native protein: it can adsorb at higher surface concentrations at hydrophobic surfaces(I), it can reduce surface and interfacial tensions (2) and may be used as a better emulsifier than native ovalbumin (3). The modified protein is, therefore, functioning as a polymeric surfactant, which has various properties, depending on the structure of the native protein, the number of chains and chain length of the attached hydrophobic groups. Since proteins may have specific biological activity, it could be possible to form surface active proteins, which also have a specific biological activity, while the protein is spontaneously adsorbed at various interfaces. We used such an approach for antibodies which after modification were able to reduce surface tension, adsorb onto oil droplets in oil-in-water emulsions and still retain their specific biological activity. This biological activity, which is the specific recognition, might be used for unique applications such as drug targeting and immunodiagnostics, both based on emulsions (4), clusters (5) and liposomes (6), containing a suitable probe molecule. The use of various colloidal systems, which contain the surface active antibodies, may lead to significant advantages, such as eliminating the need for covalent attachment of the probe molecule to the antibody as suggested for drug targeting (7), or achieving a very high load of probe molecules per antibody molecule, which then can be used for enhanced immunoassays.

0097-6156/95/0602-0533$12.00/0 © 1995 American Chemical Society Horbett and Brash; Proteins at Interfaces II ACS Symposium Series; American Chemical Society: Washington, DC, 1995.

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The present report will focus on the preparation of surface-active IgG molecules by attachment of various hydrophobic groups, and on the surface activity at oil-water interface, while retaining the recognition ability of the IgG molecules.


Experimental The active ester was prepared by reacting N-hydroxysuccinimide with fatty acids having chain length Cg-Cig, by a procedure described earlier(2). The octanoic acid was covalentiy attached to the IgG by adding its active ester, which is dissolved in dioxane, into IgG solution (6.5 mg/ml) in phosphate buffer (pH=10), in the presence of 2% WAV sodium deoxycholate. The amount of added ester could vary according to the expected degree of modification. The reaction mixtures were shaken in a water bath (25°C) for 3 hours, and then were filtered by 0.45pm and 0.2pm filters. The clear solutions were then dialyzed against phosphate buffer (pH=8) for 72 hours, while changing the buffer each 8 hours. The degree of modification was evaluated by determining the concentration of free lysine groups present in the IgG before and after modification, by using the TNBS method (8, 77). Adsorption studies were performed by mixing 2.5 ml IgG solution (native or modified) at various concentrations, with commercial oil-in-water emulsion (Intralipid, Kabi Pharmacia), which contained 10% soybean oil and was stabilized by egg phospholipids. The mixture was shaken in a water bath (25°C) for a period of time described in the text. Then, the mixture was filtered by 0.2pm filter and the concentration of protein in the clear solution was determined by the Lowry method (72). The adsorbed amount was calculated by the difference in protein concentration before and after adsorption. The surface tension measurements were performed by the Wilhelmy plate method, using a Lauda tensiometer, which allows a continuous measurement of surface tension until a constant value is obtained within a few hours, depending on IgG concentration. The biological activity was determined by a commercial ELISA diagnostic kit (Enzygnost anti HSV-l/IgG, Behring) suitable for detection of antibodies for Herpes Simplex 1 virus (HSV-1). The IgG prepared by suitable ammonium sulphate precipitation of human serum, had a high titer for HSV-1. The specific recognition by emulsion was performed by incubating the various antibody solutions for 45 minutes with HSV-1 infected BSC-1 cells on a microscope slide. BSC-1 cells (originating in African green-monkey's kidneys) were infected by HSV-1 with multiplicity of infections 0.1-0.01 TCID 50 per cell. After the incubation, the cells were rinsed by phosphate buffered saline to remove the emulsion droplets, which were not adhered. The cells and presence of attached oil droplets, were viewed by a Nikon Optiphot microscope. A l l modifying reagents were purchased from Sigma and were used without further purification. Results and Discussion Since the IgG molecule is water soluble, it was expected that the main parameter to influence its surface activity would be the number of hydrophobic groups covalentiy attached to the antibody molecule. The modification was performed on the lysine residues of the protein and, since each molecule contains 90 lysine groups, we could form surface-active antibodies with a very large number of hydrophobic groups. The modification was performed by various hydrophobic groups, but because of space

Horbett and Brash; Proteins at Interfaces II ACS Symposium Series; American Chemical Society: Washington, DC, 1995.


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MAGDASSI ET AL.

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Surface-Active Antibodies

limitations we shall concentrate on the Cg modifications only. The modification degree, m, will define the number of hydrophobic groups which are linked to each IgG molecule. A series of IgGs at various degrees of modification was prepared by changing the molar ratio of active ester to native protein in the reaction mixture. As shown in Fig. 1, increasing this ratio led to an increase in the number of attached hydrophobic groups. However, we could not obtain a modification degree higher than about 30, and die number of attached groups was always lower than expected, according to the initial molar ratio. This is a result of the rapid hydrolysis of the active ester in the aqueous solution, and the precipitation of the highly modified proteins, which became too hydrophobic. One indication for the presence of surface-active antibody is its ability to reduce surface and interfacial tension, as was indeed observed for all the modified IgGs. As demonstrated in Fig. 2, both native and modified IgG can reduce surface tension; however, the modified IgG (modification degree, m=32) reduces more rapidly the surface tension to a lower equilibrium value than the native IgG. Since these measurements were performed at low protein concentrations (0.01 mg/ ml) the differences in the behaviour can be attributed to the presence of hydrophobic anchoring groups in the modified protein. TTie next step was to evaluate the possibility of adsorption of the modified antibodies onto O/W emulsion droplets. In this case, the adsorption does not take place at a simple oil-water interface, but at such interface which is already covered with a phospholipid monolayer (it should be noted that we found that the modified antibodies could be used as emulsifiers by themselves). As shown in Fig. 3, when a native IgG or modified IgG (m=9), is mixed with an O/W emulsion, both proteins are adsorbed onto the emulsion droplets within two hours, as reflected by the decrease in protein concentration in the aqueous solution with time. Since the initial concentration of the two proteins was the same, 0.75 mg/ml, it is obvious that the modified IgG is more rapidly adsorbed than the native IgG. It is important to note that the adsorption of native IgG on emulsion droplets has not been previously reported. In itself it is of great importance from the viewpoint of possible interactions of injectable emulsions with blood components, and possible applications, which could be based on these observations. Once the adsorption onto the emulsion droplets was proved, we performed experiments which could yield adsorption isotherms. Such a typical experiment was performed by mixing IgG at various concentrations, with a constant emulsion concentration, for 12 hours, which is above the time required to reach equilibrium. Typical adsorption isotherms are presented in Fig. 4. As can be seen, the native IgG is adsorbed until it reaches a plateau value, at about 0.7 mg/m (the available area for adsorption was calculated from the average droplet size in the emulsion). In comparison, the attachment of 30 hydrophobic groups leads to a significant increase of the adsorbed amount, up to 4 mg/m , without reaching a saturation. At lower degree of modification (m=5), a plateau value is observed, which is about twice the one obtained for the native IgG. Comparing these results to the surface concentrations of native IgG adsorbed onto various solid surfaces, shows that the adsorption onto emulsion droplets is much lower. For example, adsorption of IgG on anionic polyrinyltoluene surface reaches a monolayer value at about 7 m g / m (9). This significant lowering of adsorbed amount is probably due to the presence of the phospholipids, which are the emulsion stabilizers. In this case, the IgG molecules have to overcome the electrical repulsion caused by the negatively charged emulsifier (the IgG is also negatively charged), and penetrate through the emulsifier monolayer. This observation is in 2

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MAGDASSI ET AL.

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Figure 6. Photomicrograph of emulsion droplets adhered to HSV-1 infected cells. A Cg-modified IgG (m=9) was previously adsorbed on the emulsion droplets.



38. MAGDASSI ET AL.

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agreement with the results obtained by Arai et al., who measured the adsorption onto phospholipid-coated latex particles (70). They have found that the amount of IgG adsorbed decreased with increase of lipid coverage of the latex surface. At about 90% lipid coverage, the surface concentration of IgG was about 0.5 mg/m^, which is similar to the present results obtained for native IgG on emulsion droplets (it is most likely that the lipid coverage in the emulsion is close to 100%). In view of these results, obtaining surface concentrations of several mg/m^ by the modified IgG, seems to be more impressive. Furthermore, the chemical modification leads to a shift of the iso electric point of the modified IgG, towards lower pH values, meaning that the tested antibodies are more negatively charged than the native ones, at pH 7.4. In general, it should be expected that the adsorption is decreased when the protein is below or above the iso electric point, about pH=7, as is also shown by Arai et al.(20). Therefore, it can be concluded, that the modification leads to the formation of antibodies which have enhanced tendency to adsorb at oil-water interface and are probably capable of replacing previously adsorbed surfactants. The main purpose of this research was to form surface-active proteins, combining surface activity and biological activity. Therefore, the biological activity was evaluated using two methods: (a) a conventional ELISA measurement of IgG, specific to Herpes Simplex virus (HSV-1), and (b) a method designed for the evaluation of specific recognition by emulsion droplets. The ELISA test was performed for solutions of native and modified IgG, which were tested specifically for HSV-1. As shown in Fig. 5, the activity of the modified proteins decreased with increase in the number of hydrophobic groups attached to the IgG molecule. This effect is not surprising since the chemical modification can cause denaturation of the IgG, or the presence of many hydrophobic groups can block the recognition site of the antibody (it should be emphasized that the chemical modification was not selective only toward the F fragment of the antibody). Another possibility for the decrease in biological activity is the formation of clusters of IgG molecules, due to hydrophobic modification (5). However, as expected, at low degree of modification, m=8, the biological activity was similar to that of the native IgG. Therefore, the specific recognition by emulsion droplets was evaluated for low degree of modification: a sample of O/W emulsion was mixed with the modified antibody for 10 hours. This emulsion was added dropwise to a microscope slide, which contained HSV-1 infected cells, and was incubated for 45 minutes to allow adhesion and interaction between the IgG and the HSV-1 antigens present on the cell walls. Then the slide was rinsed by phosphate buffer saline to remove all droplets which were not attached. As shown in Fig. 6, we could identify, by microscope, the adhesion of emulsion droplets to the infected cells, this observation means that the emulsion droplets have the ability to recognize specific antigens. Suitable control experiments, in which non-infected cells, emulsion without antibodies and emulsion with native antibody were used, revealed that the adhesion of emulsion droplets was indeed specific to HSV-1 infected cells. In conclusion, we have shown that hydrophobically modified antibodies become surface-active, and that under certain conditions they can retain biological activity, while being adsorbed at an oil-water emulsion interface. With this concept established, it should be possible now to continue with the research, aimed at both understanding the mechanism of adsorption-desorption, formation of micelle-like structures and applying the methods to development of new drug targeting and diagnostic systems. c

Literature Cited 1. 2. 3.

Maagdassi, S.; Leibler, D. and Braun, S. Langmuir, 1990, 6, 376. Magdassi, S.; Stawski, A. and Braun, S. Tenside, 1991, 28, 264. Magdassi, S. and Stawski, A . J. Disp. Sci. Technol. 1989, 10, 213.


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Magdassi, S.; Rones-Zakay, Z.; Lineritz, M. and Sheinberg, O. Israel Pat. Appl. No. 102718, 1992. 5. Magdassi, S.; Rones-Zakay, Z. and Toledano, O. Israel Pat. Appl. No. 106887, 1993. 6. Huang, L.; Huang, A. and Stephan, T. In Liposome Technology III; Gregoriadis, G., Ed.; 1985, pp. 52. 7. Trail, P.A.; Wilner, D.; Lasch, S.J.; Henderson, A.J.; Hofstead, S.; Casazza, A.M.; Firestone, R.A.; Hellstrom, I. and Hellstrom, K.E. Science, 1993, 261, 212. 8. Habeeb, S.A. Anal. Biochem. 1966, 14, 328. 9. Baghi, P. and Birnbaum, S.M. J. Colloid Interface Sci., 1981, 83, 460. 10. Arai, T.; Mishiro, R. and Kitamara, H. A paper presented at 201 ACS meeting, 1991. 11. Alder-Nissen, J. J. Agric. Food Chem., 1979, 27, 1256. 12. Peterson, G.L. Methods Enzymology, 1983, 91, 95. Downloaded by UNIV LAVAL on April 24, 2016 | http://pubs.acs.org Publication Date: May 5, 1995 | doi: 10.1021/bk-1995-0602.ch038

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Proteins at Interfaces II - American Chemical Society

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